Developments in Primatology: Progress and Prospects

Series Editor: Louise Barrett

Jessica F. Brinkworth
Kate Pechenkina Editors

Primates,
Pathogens, and
Evolution
Developments in Primatology:
Progress and Prospects

Series Editor: Louise Barrett

For further volumes:

http://www.springer.com/series/5852
Jessica F. Brinkworth • Kate Pechenkina
Editors

Primates, Pathogens,
and Evolution
Editors
Jessica F. Brinkworth Kate Pechenkina
Department of Pediatrics Department of Anthropology
CHU Sainte-Justine Research Center Queens College
University of Montreal City University of New York
Montreal, QC, Canada Flushing, NY, USA

ISBN 978-1-4614-7180-6 ISBN 978-1-4614-7181-3 (eBook)

DOI 10.1007/978-1-4614-7181-3
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For Jordan Aria and Jeremy
Acknowledgements

First, we must thank the 30+ authors who accepted our invitation to contribute to
this book. This volume unites researchers from a wide range of biological
fields including Anthropology, Biochemistry, Evolutionary Biology, Genetics,
Immunology, Medicine, Veterinary medicine, Virology, and Zoology. These authors
provided the collection of papers that examine the molecular interactions between
primates and pathogens within the context of evolution contained within, and did so
while juggling many other responsibilities. The publication of an edited book is a
long process. Many of the authors represented here agreed to work with us as
early as 2009, when we were first recruiting speakers for a symposium at the annual
meeting of the American Association of Physical Anthropologists. We thank these
authors for contributing their time and effort to write interesting works and for
allowing us to curate such works here. Above all, we thank them for their sup-
port and patience.
A very special thanks to our colleagues who offered anonymous review of the
chapters and who must go unnamed. Each chapter in this collection was critically
examined by 2–3 researchers. Many of our peers provided detailed reviews on
tight deadlines. Some even sent reviews from the field!
Thanks to our editor Janet Slobodien who approached us and encouraged us to
pursue this book. Melissa Higgs, editorial assistant, guided the assembly of this
book, and often helped us with the fine details of assembling figures and permis-
sions. Thanks to Lesley Poliner and Hedge Ritya, who oversaw the production and
copy editing of this volume and its supporting materials. Yurii Chinenov provided
critical comments on multiple chapters. Thank you Jeremy Sykes for editorial assis-
tance and for proofing drafts.
The initial idea to develop a collection of papers that would discuss molecular
host–pathogen interactions came to us while attending a number of talks and posters
across scattered presentation sections at the annual meeting of the American
Association of Physical Anthropologists in Columbus, Ohio, in 2008. We wanted
to provide a forum for researchers interested in the evolution of primate immunity
to meet, discuss findings and brainstorm. At the 2009 AAPA meetings, we began to

vii
viii Acknowledgements

approach potential contributors to a symposium and edited volume that would focus
on the functional outcomes of evolutionary primate–pathogen interactions. We were
very fortunate to be met with great enthusiasm by our future contributors, in particu-
lar George Armelagos who immediately suggested we include the research of
Graham Rook, Jenny Tung, and Kristin Harper. The original participants of the
“Pathogens and evolution of human and non-human primates” symposium, held in
Albuquerque, New Mexico, in April of 2010, presented some truly interesting inter-
disciplinary works that day. We thank those researchers for sharing their ideas and
enthusiasm - George Armelagos, Nels C. Elde, Harmit Malik, Cedric Feschotte,
Charlie Nunn, Kristin Harper, Jayne Raper, Jenny Tung, Susan C. Alberts, Gregory A.
Wray, Felicia Gomez, Wen-Ya Ko, Sarah Tishkoff, Ajit Varki, Melanie Martin,
Caleb Finch, Fabian Crespo, Rafael Fernandez-Botran, Manuael Casanova, and
Christopher Tilquist. Thank you to the American Association of Physical
Anthropology and the Human Biology Association who hosted this symposium at
their annual meetings in Albuquerque, New Mexico, in April 2010. A very special
thanks to the donors, particularly members of the law firm Labaton Sucharow, who
provided funds and consideration associated with the needs of this specific sympo-
sium. A special thanks to Kelly Zieman, Leslie and Sharon Brinkworth, Cheryl and
Charles Brinkworth, Deirdre O’Boy, Emerson McCallum and Michael Donnelly.
Thank you to Chris Brinkworth, Mary Wong, Eva and JoAnn Brinkworth and my
parents, Cheryl and Charlie, for help and support during key stages of this book.
Special mention to Jenny Tung, who was an early supporter of and contributor to
this project. Thanks Luis Barreiro who offered assistance at an important stage of
production and who, with Jenny, has provided me the opportunity to develop a
career working on these questions of primate evolution and immunity. My deepest
gratitude to my husband, Jeremy, for his patience, energy and unreserved enthusi-
asm for all things book, career, life. Thank you for finding symposium donors,
batting around ideas, reviewing many proposal drafts and working extra hard on all
other tasks so that I could complete this one. Very special thanks must be given to
my daughter, Jordan, who was a considerate traveling companion and made this
process rather easy. My dearest dear, thank you for all days past, present and future.
Over the course of production Brinkworth and Pechenkina’s studies were sup-
ported by the Wenner-Gren Foundation Grant (7845 and 8702, JFB), the National
Science Foundation (0752297, KP and JFB), the Réseau de Médicine Génétique
Appliquée (JFB) and the National Institutes of Health (1R01-GM102562 to Luis
Barreiro, which supports JFB). Thank you also to the City University of New York,
Queens College, City College of New York, Sophie Davis School of Biomedical
Education, the New York Consortium in Evolutionary Primatology, Centre
Hospitalier Universitaire Sainte-Justine Research Center, and the University of
Montreal for their support and the opportunity to pursue our interests in the evolu-
tion of disease and immune system function.

Montreal, QC, Canada Jessica F. Brinkworth

Contents

Primates, Pathogens and Evolution: An Introduction ................................. 1

Jessica F. Brinkworth and Kate Pechenkina

Part I Immunity and Primate Evolution

Vertebrate Immune System Evolution and Comparative

Primate Immunity........................................................................................... 17
Jessica F. Brinkworth and Mitchell Thorn
Genetic Variation in the Immune System of Old World Monkeys:
Functional and Selective Effects .................................................................... 65
Dagan A. Loisel and Jenny Tung
Toll-Like Receptor Function and Evolution in Primates ............................ 91
Jessica F. Brinkworth and Kirstin N. Sterner
Impact of Natural Selection Due to Malarial Disease
on Human Genetic Variation ......................................................................... 117
Felicia Gomez, Wen-Ya Ko, Avery Davis, and Sarah A. Tishkoff
Parasitic Lice Help to Fill in the Gaps
of Early Hominid History............................................................................... 161
Julie M. Allen, Cedric O. Worman, Jessica E. Light, and David L. Reed

Part II Emergence and Divergent Disease Manifestation

Treponema pallidum Infection in Primates: Clinical Manifestations,

Epidemiology, and Evolution of a Stealthy Pathogen .................................. 189
Kristin N. Harper and Sascha Knauf

ix
x Contents

Molecular Mimicry by γ-2 Herpesviruses to Modulate Host Cell

Signaling Pathways ......................................................................................... 221
Lai-Yee Wong, Zsolt Toth, Kevin F. Brulois, Kyung-Soo Inn,
Sun Hwa Lee, Hye-Ra Lee, and Jae U. Jung
Neotropical Primates and Their Susceptibility to Toxoplasma gondii:
New Insights for an Old Problem .................................................................. 253
José Luiz Catão-Dias, Sabrina Epiphanio,
and Maria Cecília Martins Kierulff
The Evolution of SIV in Primates and the Emergence
of the Pathogen of AIDS ................................................................................. 291
Edward J.D. Greenwood, Fabian Schmidt, and Jonathan L. Heeney

Part III Primates, Pathogens and Health

Microbial Exposures and Other Early Childhood Influences

on the Subsequent Function of the Immune System.................................... 331
Graham A.W. Rook
Make New Friends and Keep the Old? Parasite Coinfection
and Comorbidity in Homo sapiens ................................................................ 363
Melanie Martin, Aaron D. Blackwell, Michael Gurven, and Hillard Kaplan
Primates, Pathogens, and Evolution: A Context for Understanding
Emerging Disease ............................................................................................ 389
Kristin N. Harper, Molly K. Zuckerman, Bethany L. Turner,
and George J. Armelagos

Index ................................................................................................................. 411

Primates, Pathogens and Evolution:
An Introduction

Jessica F. Brinkworth and Kate Pechenkina

Introduction

Primate immune systems have evolved to interact with pathogens in different ways
(Mandl et al. 2008, 2011; Pandrea et al. 2007; Sawyer et al. 2004; Song et al. 2005;
Soto et al. 2010). Human and nonhuman primate immune systems have diverged
with some species exhibiting strong differences in immune response to certain
pathogens including immunodeficiency viruses [reviewed by Pandrea and Apetrei
(2010), Toxoplasma gondii (Epiphanio et al. 2003), herpesviruses (Estep et al. 2010;
Huang et al. 1978), and trypanosomes (Thomson et al. 2009; Welburn et al. 2001)].
Why closely related primates have evolved such divergent pathogen interaction
strategies is not well understood. Despite strong public interest in human/nonhuman
primate evolutionary history and the importance of various primate species as bio-
medical models, the current picture of interspecies differences in immunity remains
fairly incomplete. Our understanding of how primate immunity evolved is hindered
by disconnected research on primate–pathogen molecular interaction, an uneven
focus on primate coevolution with a limited number of pathogens, and the discon-
nect between research on primate molecular phylogeny and primate physiology. The
objective of the present collection of papers is to integrate research on the evolution
of primate genomes, primate immune function, primate–pathogen biochemical
interaction, and infectious disease emergence to provide a knowledge base for future
research on human and nonhuman primate speciation, immunity, and disease.

J.F. Brinkworth (*)

Department of Pediatrics, CHU Sainte-Justine Research Center, University of Montreal,
Montreal, QC, Canada
e-mail: [email protected]
K. Pechenkina
Department of Anthropology, Queens College, City University of New York,
Flushing, NY, USA
e-mail: [email protected]

J.F. Brinkworth and K. Pechenkina (eds.), Primates, Pathogens, and Evolution, 1

Developments in Primatology: Progress and Prospects,
DOI 10.1007/978-1-4614-7181-3_1, © Springer Science+Business Media New York 2013
2 J.F. Brinkworth and K. Pechenkina

The Role of Long-Term Evolutionary Processes in Shaping

the Primate Immune System

Evolution of the immune system is tied to the evolutionary history of a species and
intertwined with the evolution of physiological functions and developmental stages
of an organism. The majority of cold-blooded vertebrates appear to experience
functional shifts in their immune response depending on external temperatures,
resulting, in some cases, in impaired immune responses such as the inhibition of
immunoglobulin class switching (Jackson and Tinsley 2002). The importance of a
graft-rejection-like immune response during tadpole to adult morphogenesis in the
frog genus Xenopus and the failure of novel proteins associated with lactation to
stimulate “nonself” immune responses in mammals, for example, both suggest that
the immune system has likely evolved in parallel with the evolution of species
developmental stages (Izutsu 2009; Matzinger 1994, 2002).
To interpret variation in primate immune response within and between species,
the evolutionary forces that shaped the underlying molecular differences need to be
examined. Of these forces, pathogen-mediated natural selection has likely been the
leading factor in increasing the frequency of pathogen resistance in host popula-
tions. Factors such as an organism’s environment, diet, postural behavior, and soci-
ality led to interspecies differences in exposure to specific pathogens and the
frequency with which such pathogens were encountered. Complete or partial resis-
tance of specific human genotypes to certain pathogenic strains and the patterned
distribution of these genotypes among indigenous populations around the globe is
fairly well documented (Hamblin and Di Rienzo 2000; Hraber et al. 2007; Leffler
et al. 2013; Marmor et al. 2001; Tishkoff et al. 2001). While the genetic variability
of nonhuman primate immune factors is comparatively less well studied, specific
disease resistance genotypes in some of these species have been identified. Ecological
differences between savanna dwelling Guinea baboons (Papio papio) and chimpan-
zees (Pan troglodytes) are likely responsible for resistance on the part of the former
species to the savannah-based pathogen Trypanosoma brucei gambiensis, and high
susceptibility to fatal T. brucei-caused sleeping sickness in the latter. Baboons are
more involved in grassland ground foraging than chimpanzees, which also exploit
savannah resources but are tied to forested regions and tend to retreat to the forest
for sleep (Kageruka et al. 1991). In the course of their evolution in open habitats,
baboons have likely been continuously exposed to the low flying tsetse fly—the
vector for T. brucei (Lambrecht 1985; Welburn et al. 2001). The selective pressure
generated by T. brucei is thought to have favored fixation of two nonconserva-
tive mutations in the baboon trypanosomal lytic factor ApoLI that have been linked
to sleeping sickness resistance and are not shared with either humans or chimpan-
zees (Thomson et al. 2009). In humans, two unrelated mutations in ApoLI that
increase sleeping sickness resistance are geographically restricted to Africa and
absent in European populations. Interestingly, trypanolytic variants of ApoLI in
humans contribute to an increased risk of kidney disease, providing an example of
Primates, Pathogens and Evolution: An Introduction 3

heterozygous advantage (Genovese et al. 2010) parallel to the classic case of the
HbS variant of the HBB globin locus and other hemoglobinopathies that confer
heterozygous advantage by means of increasing resistance to malaria(Allison 1956;
Haldane 1949; Hedrick 2004; Kwiatkowski 2005; Lapoumeroulie et al. 1992; Oner
et al. 1992).
The pathogen-directed evolutionary mechanisms that contribute to the divergence
of immune systems remain hypothetical. In the simplest case, an epidemic of a highly
virulent infectious disease eliminates a large number of susceptible organisms very
quickly, thereby favoring the disproportional reproductive success of resistant indi-
viduals. In such a scenario pathogen-mediated selection is assumed to be strong.
However, such a model of host–pathogen coevolution is pertinent in extreme cases
only. Pathogens of moderate-to-low virulence can also affect host immune allele
frequencies by, for example, contributing to lowered fertility in the form of physical
inability to produce offspring after being infected or causing a lesser ability to acquire
mates as a result of decreased mobility (Cheney et al. 1988; Levin et al. 1988).
Pathogens that do not kill a host, therefore, can affect sexual selection and gene flow.
Of course, the consequences of a deadly epidemic may not be limited to removal of
the alleles that contribute to disease susceptibility, such as those of surface antigens
recruited by a pathogen to penetrate the host. Such strong selective pressure may also
affect alleles that contribute to increased susceptibility to coinfections or to overt
immune activity that leads to the development of secondary conditions (e.g., sepsis).
Pathogen pressure may also select for resistance traits and in doing so affect the
frequency of linked alleles in a population through selective sweeps.
The most popularized perception of host–pathogen evolutionary interaction is the
concept of a host–pathogen evolutionary arms race. This idea is derived from the
application of Leigh Van Valen’s 1973 Red Queen hypothesis to hosts and pathogens
(Van Valen 1973). The Red Queen hypothesis proposes that closely associated organ-
isms may coevolve so tightly that the likelihood of extinction for one or the other is
constant over geological time. Changes in one species affect the tightly coevolved
interface with another species, threatening either species with extinction. As stated by
the Red Queen in Lewis Carroll’s Through the Looking Glass: “Now, here, you see, it
takes all the running you can do, to keep in the same place.” Tightly coevolved hosts
and pathogens have to “run”—that is, evolve quickly—just to maintain a balance and
avoid extinction. Within this framework, it is tempting to characterize host–pathogen
relationships as beneficial for the host to recognize and either tolerate or eliminate a
pathogen, with a pathogen’s main recourse being to evade a host’s defensive mecha-
nisms. These interactions are thought to result in head-to-head collisions between a
host’s immune system and a pathogen, leading to selective pressure on the immune
system and the pathogen and culminating in coadaptation.
However, some pathogens actually co-opt normal mechanisms of primate
immune recognition and the subsequent responses, such as cytokine release, cell
trafficking, and tissue destruction, using them to their advantage. Mycobacterium
tuberculosis (tuberculosis) infection is enhanced by the release of host anti-
inflammatory cytokine IL-10 (Redford et al. 2011). Once consumed by a macro-
phage, Yersinia pestis (plague) migrates to its main point of dissemination, the
4 J.F. Brinkworth and K. Pechenkina

lymph nodes, while acquiring phagocytosis resistance (Oyston et al. 2000; Pujol
and Bliska 2003; Zhou et al. 2006). Similarly, HIV can disseminate to the lymph
nodes and other regions through antigen-presenting cells (Koppensteiner et al.
2012). Moreover, exaggerated and uncontrolled immune cell responses may result
in host death—such is the case of severe sepsis and septic shock triggered by strong
innate immune cell recognition of immune system insult (e.g., blood stream infec-
tions and injury) (Brown et al. 2006; Murdoch and Finn 2003; Zemans et al. 2009).
Rather than acquiring permanent and costly adaptations, some pathogens can escape
host immune defenses through transient amplification of a resistance gene that
creates tandem arrays aptly named “genomic accordions.” Poxviruses encode two
factors, E3L and K3L, which inhibit the host antiviral factor protein kinase R (PKR).
In viruses lacking E3L, K3L rapidly becomes amplified 10–15-fold via serial dupli-
cation, which increases viral fitness. However, the tradeoff for increased genome
size is less efficient replication. Remarkably, an expanded genomic array of identi-
cal resistance genes in a pathogen increases the probability of emergence, fixation,
and spreading of additional K3L mutants with improved host avoidance; these
viruses subsequently lose the K3L duplicated array but retain the novel resistance
mutation (Elde et al. 2012).
Two limiting factors for pathogen-driven evolution of the immune system are
disadvantages rendered to the host by hyper-responsiveness, leading to autoimmune
disorders, and inadvertent pressure on symbiotic and commensal organisms. An
overactive immune system is responsible for the development of chronic conditions
mediated by the immune system itself, such as systemic lupus erythematosus,
antiphospholipid syndrome, polycystic ovary syndrome, and diabetes, among oth-
ers, that negatively affect reproductive fitness and may therefore contribute to
immune system evolution (Carp et al. 2012).
Consequently, immune system divergence between species is not driven by host–
pathogen interaction alone but is profoundly affected by the living environment,
which includes a complex network of interspecies interactions between hosts, sym-
bionts, commensals, and pathogens (Klimovich 2002; Lee and Mazmanian 2010).
A species, with its associated microorganisms, can be considered a “holobiont,” an
evolutionary unit encompassing the totality of organisms involved in commensal-
istic, symbiotic, and parasitic relations (Zilber-Rosenberg and Rosenberg 2008).
Changes in the fitness of any holobiont-involved species affect the system as a
whole, rather than just the fitness of the host species. Indeed, changes in the compo-
sition of intestinal microbiota may promote outgrowth or invasion by pathogenic
microorganisms or induce an exaggerated host response that results in the onset of
various autoimmune disorders such as inflammatory bowel disease (Maynard et al.
2012). Alternatively, dietary and environmental changes may cause a shift in
species-associated microbiota that results in the appearance of new pathogens or
symbionts. Establishing whether new species-specific pathogens are effectors or
consequences of speciation is a daunting task.
Primates, Pathogens and Evolution: An Introduction 5

The Role of Pathogens in Primate Speciation

Interaction with pathogens likely played an important role in primate speciation in

several ways. Pathogens have contributed to primate genome divergence through
direct integration of microorganismal genomes into the genomes of primate germ-
line cells. Over millions of years, viral integration into host genomes has changed
genome sequences and affected multiple biological functions (Arnaud et al. 2007;
Hunter 2010). For instance, Dunlap et al. (2006) proposed that effective placenta
formation in mammals is impossible without a gene coding for an envelope protein
that was initially introduced by a retrovirus (HERV-W) (Dunlap et al. 2006). Once
incorporated, viral genomes were inherited by offspring. Past retroviral infections
of human ancestors now represent approximately 8 % of the human genome
(Bannert and Kurth 2006). Due to different histories of pathogen exposure, primate
genomes differ from one another in terms of the types and numbers of integrated
viral sequences (Horie et al. 2010; Kim et al. 2008). As such, viral pathogens have
contributed to the divergence of primate genomes and the divergent functions of
primate genes (Gogvadze et al. 2009; Wang et al. 2007; Yohn et al. 2005).
While portions of primate genomes have diverged because of species-specific
viral integration, some loci appear to have evolved under pathogen-driven selection.
Multiple pathogens have been identified as having exerted selective pressure on pri-
mate immune factors for millions of years [i.e., retroviruses and apolipoprotein
B-editing catalytic polypeptide 3G (APOBEC3G) (Sawyer et al. 2004), retroviruses
and Tripartite Motif 5 alpha (TRIM5α) (Sawyer et al. 2005; Song et al. 2005),
Plasmodium falciparum and glycophorin C (Maier et al. 2003), and Mycobacterium
tuberculosis an granulysin (Stenger et al. 1998)]. Primate immune genes, proteins,
and cells have structurally and functionally diverged. Primate immune factors show
evidence of selection [CC-motif receptor 5 (Wooding et al. 2005), Toll-like Receptors
1 and 4 (Nakajima et al. 2008; Wlasiuk and Nachman 2010), TRIM5α (Sawyer et al.
2005), Cluster of Differentiation-45 (Filip and Mundy 2004), and Protein kinase R
(Elde et al. 2009)] or interspecies divergence in function [Major-histocompatibility
Complexes, Killer cell Immunoglobulin-like Receptors (Abi-Rached et al. 2010;
Moesta et al. 2009), Toll-like Receptor 7 (Mandl et al. 2008, 2011), and ApoLI
(Thomson et al. 2009)]. Reconstructions of primate evolutionary relationships based
on the regulatory and coding sections of immune system genes deviate significantly
from generally accepted primate phylogenies [Toll-like receptor 2 (Yim et al. 2006),
CXC-motif receptor 4 (Puissant et al. 2003), and Major Histocompatability-DQA1
(Loisel et al. 2006)]. Although it appears that pathogens have directly contributed to
the evolution of individual loci, we primarily understand these changes in the context
of the primary structures of individual genes or proteins and not in the context of
immune function. Until the functional effects of these changes are considered, it is
impossible to appreciate how they contributed to speciation or disease susceptibility
and progression. A goal of this volume is to integrate available information on struc-
tural and functional differences in primate immunity with data on the evolutionary
analysis of gene sequences, pathogen life cycles, and evolutionary history.
6 J.F. Brinkworth and K. Pechenkina

Clinical Implications of Primate–Pathogen Coevolution

As a consequence of primate–pathogen evolutionary interactions, primates exhibit

strong interspecies and interpopulation differences in immune response to a broad
range of pathogens, some of which are major agents of human disease. Many lin-
eages of African nonhuman primates have hosted immunodeficiency viruses (IV)
over millions of years and their extant descendents do not develop the overt
immune activation and white blood cell loss that typifies late stage IV infection
(AIDS) in comparatively new hosts such as humans or Asian monkeys [reviewed in
Pandrea and Apetrei 2010; see also Greenwood et al. 2013]. Nonhuman primate
Herpes simian B virus infections in their natural hosts are fairly asymptomatic or
manifest mildly, in a manner similar to human herpes simplex mucosal blisters.
When transmitted to naïve primate hosts, including humans, these herpes infections
can progress to encephalopathy (Artenstein et al. 1991; Chellman et al. 1992; Estep
et al. 2010; Vizoso 1975). Unless severely immunocompromised, humans infected
with the brain and muscle parasite Toxoplasma gondii are asymptomatic or develop
a self-limited disease characterized by fever and enlarged lymph nodes (Jones et al.
2007). By contrast, T. gondii infections in New World monkeys are characterized
by loss of strength, respiratory difficulty, and high mortality (Epiphanio et al. 2003;
Catão-Dias et al. 2013). Research on the molecular mechanisms responsible for
such variation in disease manifestation among different primate species involves
multiple, often disparate areas of study, which contributes to gulfs between research
on immune function, research on primate–pathogen evolution, and the clinical
application of the resultant findings.
Arguably, the molecular mechanisms of immunodeficiency virus infection in
primates are the best understood primate–pathogen interactions. The severity of the
HIV pandemic and a strong interest in developing viable therapies have encouraged
the examination of disease susceptibility and progression in primates, but mainly in
a limited selection of catarrhine species. Primate-IV studies often use a comparative
evolutionary approach as a starting point for the analysis of primate immunity and
disease progression. An important advance in HIV therapy research has been the
finding that IVs have emerged multiple times in the course of primate evolution and
have closely coevolved with their hosts over millions of years (Pandrea and Apetrei
2010; Santiago et al. 2002; Switzer et al. 2005; Van Heuverswyn et al. 2006; Zhu
et al. 1998). Comparative studies of primate-IV interactions have led to the identifi-
cation of several immune factors thought to be under selective pressure from IVs
and might serve as therapeutic targets including TRIM5α (Ortiz et al. 2006),
APOBEC3G (Sawyer et al. 2004, 2005), Tetherin/BST2 (Jia et al. 2009), IL-4
(Koyanagi et al. 2010; Rockman et al. 2003), TLR7 (Mandl et al. 2008), TRAIL
(Kim et al. 2007), CCR5 (Wooding et al. 2005), and MHC I (de Groot et al. 2002).
A limited number of broader interspecies differences in the proportion of immune
cell types, activation of immune cells, expression of immune genes, and stimulation
of cell death that may affect disease progression have also been noted (Kim et al.
2007; Mandl et al. 2008; Soto et al. 2010). While primates serve as models for the
Primates, Pathogens and Evolution: An Introduction 7

study of other diseases and have been examined as xenotransplantation subjects,

what we currently know about interspecies differences in primate immune function
is largely derived from comparative IV-primate research.
Although the genetic and functional differences identified through IV research
may also contribute to a clearer picture of general immune responses to pathogens,
we do not know how many of these immune factors are activated across primate
species or whether this activation is stimulated in a similar way by other pathogens.
This gap points to fundamental problems in our understanding of primate–pathogen
interactions. First, current research is biased toward a limited number of species
such as rhesus macaques (Macaca mulatta), humans, and sooty mangabeys
(Cercocebus atys). Second, these studies typically use a challenge model. As such,
cross-species examinations of resting/baseline primate immunity are extremely
limited. How the activation and coordination of multiple immune factors
coevolved with pathogens has yet, therefore, to be thoroughly investigated. To
develop a better understanding of how pathogens affect primate speciation, conser-
vation, and health, considerable additional information is needed on the differences
in resting/baseline immune function and non-IV pathogen–host interactions across
a greater number of primate species. Given the expense and special care consider-
ations inherent in acquiring experimental data from primate species, it is particu-
larly important that these efforts are comprehensive and that the resulting reports are
made readily accessible.

The Effects of Increasing Human and Wild Nonhuman

Primate Contact on Primate–Pathogen Interaction

In areas where human settlements and nonhuman primate habitats overlap, the
potential for interspecies disease transmission increases dramatically (Chapman
et al. 2005; Daszak et al. 2000; Duval and Ariey 2012; Reynolds et al. 2012; Stothard
et al. 2012; Wheatley and Harya Putra 1994). Such transmission events can decimate
wild primate populations, as new infectious diseases may profoundly affect animal
survival, sociality, and reproduction (Berdoy et al. 2000; Nunn et al. 2008; Nunn
2012). Increased ecotourism and residential/agricultural contact has led to height-
ened transmission of anthroponotic pathogens to wild primates and subsequently to
increased mortality in primate populations [e.g., chimpanzees and Polio virus (Wallis
and Lee 1999), gorillas and respiratory disease (Palacios et al. 2011), baboons and
Schistosoma mansoni (Farah et al. 2003; Murray et al. 2000), chimpanzees and para-
myxoviruses (Kondgen et al. 2008), chimpanzees and Schistosoma mansoni (Stothard
et al. 2012), and baboons and Mycobacterium (Keet et al. 2000)]. Plasmodium falci-
parum may have been introduced anthropolonotically to the neotropical primates
during the colonial era, possibly through the forced migration of African slaves dur-
ing the slave trade. Neotropical primate P. simium has been proposed to have emerged
from Asian P. vivax during the nineteenth century, and perhaps introduced to South
America by laborers from East Asia (reviewed in Cormier 2010).
8 J.F. Brinkworth and K. Pechenkina

The transmission of pathogens from nonhuman primates to humans has also had
a profound effect on human health. The current HIV-1/AIDS pandemic likely origi-
nated with human consumption of SIV-contaminated nonhuman primate bushmeat
(Gao et al. 1999). The emergence of other diseases in humans has likewise been
attributed to human and nonhuman primate contact [i.e., Human T-Lymphotropic
virus 1 (Vandamme et al. 1998), Monkeypox (Mutombo et al. 1983; Reynolds et al.
2012), and Malaria (Liu et al. 2010)]. As humans encroach even farther onto nonhu-
man primate ranges, the need for veterinary and medical intervention will increase.
To be able to develop appropriate modes of intervention, it is very important to have
good information on the broad differences and similarities of primate immune systems
as well as the biochemical mechanics of specific primate–pathogen interactions.

Research on Primate–Pathogen Interaction Remains

Scattered and Incomplete

Despite the importance of pathogen-primate interactions over the course of primate

evolution, research on the functional outcomes of pathogen-mediated primate
evolution is incomplete and scattered across many disciplines. A unified approach
to the evolution of primate immunity will help better define the mechanisms of
disease emergence, immune function, resistance, and ecology.
To better understand the differences in human and nonhuman primate immunity
and help guide future research, it is important to integrate information from research-
ers who study the effects of pathogens on the evolution of primate genome diversity,
cell function, immune response, and gene expression. This volume is one attempt at
such a synthesis, incorporating contributions from a multidisciplinary group of
authors who:
1. Provide a compilation of current baseline information about primate–pathogen
interaction and comparative primate immunity.
2. Describe and analyze infectious disease emergence and pathogen escape of host
defense mechanisms in the context of primate–pathogen coevolution.
3. Explore divergent primate immune functions and the pathogen-mediated molec-
ular evolution of primates.
4. Discuss the human health implications of primate–pathogen evolutionary
interaction.

Overview

The first section, Immunity and Primate Evolution, includes chapters that discuss major
elements of the primate immune system (Brinkworth and Thorn; Brinkworth and
Sterner), the use of primates as models of immune system evolution (Loisel and Tung)
Primates, Pathogens and Evolution: An Introduction 9

and the pathogen-mediated evolution of primates (Gomez et al. and Allen et al.).
The second section, Emergence and Divergent Disease Manifestation, provides data
on the emergence, biochemical mechanics, and interspecies differences in immune
response to immunodeficiency viruses, as well as a range of clinically important, but
otherwise neglected pathogens. Attention is focused on how some primate pathogens
have emerged (Harper and Knauf; Greenwood et al.), coevolved with and escaped the
defenses of their hosts (Wong et al.), and triggered divergent responses in different
primate species (Catao-Dias et al. and Greenwood et al.). The third and final section,
Primates, Pathogens, and Health, focuses on how primate–pathogen coevolution
affects the health of modern primates. Three papers on the health and evolutionary
impact of disruptions to human and microorganism association (Rook, Martin and
Blackwell, and Harper et al.) review and test the hygiene hypothesis. The volume
closes with an analysis of major human and nonhuman primate cross-species pathogen
transmission events, the social and biological factors that contributed to those events,
and what the evolutionary, public health, and conservation consequences of these
events might be (Harper et al.). We thought it a wonderful chapter with which to close.

Conclusion

Primate immune systems have been formed through complex evolutionary pro-
cesses, within which pathogens have played an important role. The evolution of
primate immunity has likely been more nuanced than natural selection driven by
coevolutionary arms races with pathogens or large pathogen-mediated selective
sweeps of hosts. Rather, the evolution of the primate immune system has likely been
considerably shaped by, amongst other possibilities, moderately virulent pathogens,
pathogen strategies that co-opt normal immunity, viruses integrated into the primate
genome, commensal microorganism maintenance, autoreactivity, and overt immune
responses, as well as the evolution of primate developmental stages. As close rela-
tives, animals within the order Primates can be comparatively studied to not only
clarify how primate immune systems have functionally diverged and highlight
therapeutic targets for disease, but also to help define how such divergence contrib-
utes to disease emergence and interspecies disease transmission. As such, mapping
the evolution of the primate immune system can improve our understanding of
primate speciation, primate conservation, and human health. This volume is one
effort to unite information on the evolutionary interactions between primate immune
systems and pathogens. The chapters in this volume represent research from a broad
range of disciplines involved in the study of primate–pathogen molecular interac-
tion, primate immune function, and primate–pathogen coevolution. The work pre-
sented here discusses primate interactions with both major and neglected pathogens,
attempts to bridge research on molecular evolution and primate immune function,
and illustrates the impact of primate–pathogen evolutionary interactions on
human and nonhuman primate health. With this effort we aim to provide a sound
base of knowledge for future investigation of human and nonhuman primate evolu-
tion, immunity, and disease.
10 J.F. Brinkworth and K. Pechenkina

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 Part I
Immunity and Primate Evolution
Vertebrate Immune System Evolution
and Comparative Primate Immunity

Jessica F. Brinkworth and Mitchell Thorn

Introduction

Molecular and cellular responses as diverse as RNA interference against viral

infections in plants, antimicrobial peptides production in insects, and macrophage
phagocytosis of Listeria monocytogenes bacterium in mammals are all manifesta-
tions of the immune system—an array of defense mechanisms against pathogens,
cellular debris, and cancerous and dying cells that can secure the survival of host.
It is an exquisitely organized and regulated system of defenses that has diversified
over hundreds of millions years and, yet, is suitably conserved such that multiple
organisms (e.g., insects, lamprey, dogs, rodents, and primates) can serve as immu-
nological models of human health.
The practice of studying the immune system within the context of evolution, or
“comparative immunology”, emerged in the late nineteenth century. The most
famous early example of a comparative immunological approach is Elie
Metchnikoff’s 1882 discovery of leukocyte phagocytosis. When Metchnikoff
inserted rose thorns into starfish larvae to determine if the “wandering” cells (leuko-
cytes) of starfish responded to bodily invasion by foreign matter (Metchnikoff
1893), he found the cells aggregated around the thorns. He then reiterated his exper-
iment through the application of microorganisms to increasingly derived species
(e.g., flies, rabbits) known to maintain leukocytes to find that, universally, a subset
of these cells ingested microbes and offered host protection (Metchnikoff 1887).

J.F. Brinkworth (*)

Department of Pediatrics, Faculty of Medicine, CHU Sainte-Justine Research Center,
University of Montreal, Montreal, QC, Canada
e-mail: [email protected]
M. Thorn
Roger Williams Medical Center, Providence, RI, USA
e-mail: [email protected]

J.F. Brinkworth and K. Pechenkina (eds.), Primates, Pathogens, and Evolution, 17

Developments in Primatology: Progress and Prospects,
DOI 10.1007/978-1-4614-7181-3_2, © Springer Science+Business Media New York 2013
18 J.F. Brinkworth and M. Thorn

Thus, the innate immune strategy of phagocytosis was discovered. The comparative
immunological approach Metchnikoff employed goes a step beyond simply applying
a representative animal model to a question of immunity. Comparative immunology
involves comparing differences and similarities in organisms’ immune responses to
draw broader conclusions about immune system function and its evolution. This
approach can be useful for the identification of immune system components respon-
sible for particular disease phenotypes, as well as the discovery of novel immune
mechanisms. The use of other vertebrate animals such as jawless vertebrates,
amphibians, and rodents as biomedical models can shed light on overriding princi-
ples of immunity. Comparisons of primate immunity in the context of vertebrate
immune system evolution can be useful for the understanding of biomedical model
use, primate evolution, and human health.
Primates are a recent addition to the comparative exploration of animal immune
responses. Direct interspecies comparisons of primate immune system function
emerged in the 1920s but did not become common until decades later. The under-
standing of comparative primate immunity mainly developed through nonhuman
primate/human xenotransplantation studies in the 1960s, as well as immunodefi-
ciency virus research from the late 1980s onwards (Benveniste et al. 1986; Daniel
et al. 1984; Hardy et al. 1964; Hitchcock et al. 1964; Murphey-Corb et al. 1986;
Reemtsma et al. 1964a, b; Starzl et al. 1964). Until the adoption of catarrhine (Old
World monkey, apes, and humans) species for HIV research, few immunological
studies using different primate species compared interspecies differences. It is
now well established that primates exhibit within-order variation and, sometimes,
biologically unique manifestations of infectious diseases (Epiphanio et al. 2003;
Ngampasutadol et al. 2008; Pandrea and Apetrei 2010; Thomson et al. 2009;
Walker 1997). Still, monkey and ape species are commonly used in immunologi-
cal research as corollaries for human disease progression due to their biochemi-
cal, physiological, and genetic similarity to humans. Because there are so many
immune system similarities between primate species, there is a tendency in non-
HIV literature to make the assumption that what is represented in one primate
species is represented in other primate species and, possibly, other nonprimate
models. By making this assumption a researcher risks overlooking aspects of
primate immune systems that are unique and using primates unnecessarily to
explore immunological traits that they share with many other model organ-
isms. Comparative information on baseline primate immunity, particularly the
anatomy and function of major immune organs and cell types, is rare and scat-
tered across many fields. In certain cases it is entirely unexplored. The goal of
this chapter is to illustrate the place of primates in immune system evolution
by (1) putting the emergence of major primate immune system components in
the context of the evolution of vertebrate immunity as a whole and (2) illustrating
how baseline primate immunity has diversified by uniting and highlighting the
available information on interspecies functional differences in baseline primate
immune system structures and components.
Vertebrate Immune System Evolution and Comparative Primate Immunity 19

Overview of the Mammalian Immune System

As jawed vertebrates, mammals maintain an immune system that can be broken into
two major arms based on function. The innate immune response is the more ancient
of these two arms, having invertebrate origins (Leulier et al. 2003; Yoshida et al.
1986). Innate immune defenses are inherited, germline encoded, nonspecific, and
typified by barriers (e.g., mucosa, skin), antimicrobial peptides, phagocytosis (initi-
ated by cells such as macrophages and neutrophils), and inflammation (Janeway
and Medzhitov 2002; Kumar et al. 2009). This kind of immunity limits initial infec-
tions by recognizing “nonself”, and damage through a variety of sophisticated but
generalized mechanisms, including inherited pattern recognition receptors (e.g.,
Toll-like receptors, NOD-like receptors) that detect foreign material through molec-
ular patterns associated with pathogens or cellular damage. These patterns can be
shared broadly by microorganisms or may signal tissue damage. They are conven-
tionally and somewhat imprecisely referred to as pathogen- or danger- associated
molecular patterns (PAMPS or DAMPS) (Seong and Matzinger, 2004).
By contrast, adaptive immunity is highly specific, not immediate, key to immu-
nological memory, modulated by innate immunity, and acquired over a lifetime.
While phagocytosis is an important tool in innate immune defenses, the targeting
of matter bearing specific epitopes by lymphocytes (e.g., T and B cells) and the
retention of some of these target-specific lymphocytes is key to adaptive immunity.
Lymphocytes express membrane receptors (T-cell receptors for T cells and B-cell
receptors for B cells) that recognize antigens. Unlike innate immunity, which makes
use of germline encoded receptors, adaptive immunity has been traditionally viewed
as reliant on receptors and immunoglobulins that are made highly variable through
recombination activating gene (RAG)-mediated gene rearrangement/somatic
recombination that occurs during lymphocyte development. From a limited number
of receptor genes is borne a broad repertoire of specific receptors. As a result, rather
than recognizing pathogens through PAMPS, the lymphocytes and immunoglobu-
lins of the adaptive system recognize and “remember” distinct epitopes [reviewed in
(Hardy 2003)].
The simplified view of the vertebrate immune system function is one of immedi-
ate recognition of invading pathogens by the innate immune system and subsequent
initiation of a specific adaptive immune response. In mammals, for example, when
innate immune cells recognize foreign antigens, they initiate the release of reactive
signaling proteins known as cytokines. Cytokines degrade pathogens nonspecifi-
cally and initiate activation of an epitope/pathogen-specific T and B cell-mediated
adaptive immune response. T and B cells can become activated when receptors they
bear belonging to the immunoglobulin receptor superfamily, T- and B-cell receptors
(TCRs and BCRs), recognize specific epitopes that are presented to them via major
histocompatibility complexes (MHC) on phagocytic cells (e.g., macrophages and
dendritic cells). B cells can also become activated through direct encounters with
pathogens bearing these epitopes. Activated T and B cells then clonally replicate in
20 J.F. Brinkworth and M. Thorn

secondary lymphoid tissues, to be released as cytotoxic, phagocytic, or antibody

producing cells that recognize and attempt to eliminate a specific epitope target.
Engagement of the adaptive immune response typically occurs after the 4th hour of
infection. The first clonal adaptive immune cells are released ~96 hours from the
point of initial T- or B-cell activation. The first minutes and days of infection, there-
fore, are mainly mediated by innate immunity. As an infection is cleared over suc-
cessive hours, most clonal T and B cells die off. A small percentage, however,
remain in circulation as memory T and B cells. Memory cells speed the adaptive
response to reencountered foreign epitopes and are the basis for immunological
memory of past infections (Davis and Chien 2003; Jenkins 2003; Paul 2003).
Even this simplified description of mammalian immune system function only
partially represents the immune system of other vertebrate classes as certain key
components (e.g., lymph nodes, spleen components, and particular immunoglobu-
lins/BCRs) did not appear until the emergence of recent vertebrate classes such as
birds and mammals. The traditional paradigm of adaptive immunity is that it
emerged in the last common ancestor of jawed vertebrates (gnathostomata) approxi-
mately 625 million years ago (mya). All lower vertebrates were thought to survive
microbial assault only by initiating a generalized innate immune response. However,
in the last decade it has become apparent that some components we associate with
adaptive immunity emerged much earlier than previously assumed. The first evi-
dence of lymphocyte-derived cytokines and receptors with immunoglobulin (Ig)-
like domains, for example, can be found in sponges (Blumbach et al. 1999). In
2007, first evidence of somatic diversification of antigen receptors in lamprey came
to light, supporting the existence of an “adaptive” immune system in jawless verte-
brates (agnatha) (Guo et al. 2009; Rogozin et al. 2007). While these receptors,
known as variable lymphocyte receptors (VLRs, discussed further below), are not
related to the immunoglobulin receptor superfamily and are therefore not precursors
to TCRs and BCRS, they appear to serve a similar function as antigen receptors
(reviewed in Boehm et al. 2012b). Since the discovery of VLRs, it now seems pos-
sible that adaptive immune systems based on antigen receptor diversity mediated by
gene rearrangement/somatic recombination may have evolved more than once in
Metazoan history (Boehm et al. 2012b). The possibility that such sophisticated
immune strategies in diverse vertebrate clades may be the outcome of convergent
evolution attests to the immense evolutionary pressures that have shaped the verte-
brate immune system.

The Evolution of Vertebrate Immunity:

Major Lymphoid Tissues and Organs

Lymphoid tissues function as the sites of lymphoid cell development, selection of

antigen–receptor repertoires, and effector cell coordination. These structures can be
divided into primary or secondary lymphoid tissues based on their main function.
Primary lymphoid tissues are sites of lymphocyte effector cell poiesis and
Vertebrate Immune System Evolution and Comparative Primate Immunity 21

Fig. 1 Major lymphoid structures and their emergence in vertebrate clades over time. The sym-
bols “+” and “-” indicate the presence/occurrence of and the absence of lymphoid structure,
respectively. “*” while most birds do not appear to maintain lymph nodes, they are present ducks
(Anatidae). Molecular divergence dates from Blair and Hedges (2005)

development, as well as antigen–receptor selection (e.g., thymus, bone marrow).

The secondary lymphoid tissues are mainly coordination sites for mature immune
effector cell interactions between antigen-presenting cells and adaptive lympho-
cytes during the immune response (e.g., spleen, lymph nodes) (reviewed in Boehm
et al. 2012a; Karrer et al. 1997). The suite of major lymphoid tissues we see in
primates—thymus and bone marrow (primary), gut-associated lymphoid tissue,
spleen, and lymph nodes (secondary)—emerged in vertebrates in a complex pattern.
Some of these structures are certainly very ancient and were already established
before the emergence of jawed vertebrates (gnathostomata), while others appear
sporadically in later extant clades, only becoming well established in extant
mammals (Fig. 1) (e.g., lymph nodes). Here, the evolution of these tissues and
organs as they emerged in geological time is described.

GALT and Peyer’s Patches

Gut-associated lymphoid tissue (GALT) is a type of mucosa-associated lymphoid tis-

sue (MALT) and secondary lymphoid structure found in the gut tube of all vertebrates.
It is an ancient type of tissue dedicated to immune defense within an organ that is
subject to constant antigen exposure from both food and microbial organisms. The
primary role of mammalian MALT and GALT structures is the production of
22 J.F. Brinkworth and M. Thorn

immunoglobulin A (IgA) in support of T helper 2 cell-dependent responses, though

other T-cell activities occur in these regions (Kiyono and Fukuyama 2004). GALT can
also be a site of other important immune functions such as immunoglobulin class
switching and B-cell clonal expansion (Pospisil and Mage 1998a, b; Shikina et al.
2004). In primates, GALT serves as a site of Th2, Th1, and cytotoxic T-cell reactions
(Cesta 2006a; Kiyono and Fukuyama 2004; Kwa et al. 2006).
Prenatally, GALT is mainly organized by lymphoid cell activities (Pearson et al.
2012; Veiga-Fernandes et al. 2007). However, much of its development appears to
occur after birth, with exposure to microorganisms and development of gut flora
leading to the aggregation of T cells, the development and expansion of T-cell areas,
and the subsequent development of follicular germinal centers for B-cell develop-
ment (Cesta 2006a). Exposure to food-sourced antigens appears to affect GALT
structure as well, with intravenous feeding leading to a significant decrease in the
number of lymphocytes residing in rodent GALT (King et al. 1997a; Tanaka et al.
1991). Individual environment and ecology, therefore, likely contribute to intraspe-
cies variation in structure.
GALT organization and location is flexible and varies considerably between ani-
mal classes and species. The earliest example of GALT is found in cold-blooded
vertebrates and is not an organized tissue. It appears as aggregates of lymphoid cells
and tissues in the lamina propria, mesentery and, sometimes, in intraepithelial
regions (Bernard et al. 2006; Mussmann et al. 1996a; Zapata and Amemiya 2000).
The first organized GALT structure emerged in the submucosa of the ileum and the
lamina propria of the small intestine of birds and mammals (Hofmann et al. 2010).
The best characterized of these GALT structures are Peyer’s patches (PP), which
appear as ovular lymphoid follicles with germinal centers containing many B cells
that are surrounded by T cells (Haley 2003). PPs are found in their highest concen-
trations in the jejunum or ileum of most mammalian species (Cornes 1965; Haley
2003; HogenEsch and Hahn 2001; Owen et al. 1991). They are initially formed dur-
ing fetal development, but the maturation and development of important PP struc-
tures, such as germinal centers, is dependent on exposure to microbial organisms
(Adachi et al. 1997; Cornes 1965; Hooper 2004; Kyriazis and Esterly 1971; Spencer
et al. 1986). It should not be surprising, then, that the number, location, and size of
PPs differ considerably between mammalian species. Dogs, for example, maintain
26–29 patches that can be categorized as two types - small patches in the jejunum
and superior ileum and a large patch with dome-shaped regions in the inferior ileum
(HogenEsch and Felsburg 1992; HogenEsch and Hahn 2001). Rabbits, on the other
hand, have a cluster of lymphoid nodules in an ileal–caecal appendix and maintain
a single, large Peyer’s patch that covers the terminal end of the ileum (Haley 2003).
GALT represents the majority of the lymphoid tissue in the primate body, yet inter-
species differences in GALT structure are neither well characterized nor are the func-
tional ramifications of such differences well understood. In primates GALT appears to
be an important location for immunodeficiency virus (IV) replication (Mehandru et al.
2004; Veazey et al. 1998). In AIDS-susceptible/naïve IV host primates, such as humans
and macaques (Macaca sp.), immunodeficiency virus infection leads to the loss of
CD4+ T cells in this region as well as Th17 CD4+ T-cell dysfunction, threatening the
Vertebrate Immune System Evolution and Comparative Primate Immunity 23

integrity of the gut barrier (Brenchley and Douek 2008; Favre et al. 2009; Raffatellu
et al. 2008). This process does not appear to occur in AIDS-resistant/natural IV host
species such as sooty mangabeys (Cercocebus atys), suggesting that the regulation of
GALT components (including T cell subsets) has diverged since these species last
shared a common ancestor approximately 30 mya and that these differences can affect
infectious disease progression (Brenchley et al. 2008; Fabre et al. 2009; Steiper and
Young 2006).

The Caecal Appendix

The caecal appendix is a controversial manifestation of GALT in mammals. A nar-

row, terminal extension at the end of the caecum, this tissue occurs in a handful of
mammals, including primates, opossums, wombats, and rabbits, and is rich in lym-
phoid structures (Chivers and Hladik 1980; Dalquest et al. 1952; Fisher 2000;
Mitchell 1916). It has been proposed to have evolved at least twice within mam-
mals, appearing independently in diprotodont marsupials (e.g., the wallaby) and
again in Euarchontoglires (e.g., rabbits, primates) (Smith et al. 2009). Variations on
an appendix-like structure have been noted in animals lacking caecums (e.g., mono-
tremes and some birds), suggesting that organs of similar function may have
emerged multiple times over the evolution of animal life and possibly preceded the
canonical caecum (Laurin et al. 2011; Smith et al. 2009). An analysis of cellulose
content in lemur diet and caecal morphology suggests that, at least in the case of
primates, the presence of an appendix is likely under evolutionary pressures imposed
by diet as well as immune function (Campbell et al. 2000).
Amongst primates, a vermiform caecal appendix is found in all extant hominoids,
and variably described appendices are noted for Nycticebus, Loris, Perodicticus,
Euoticus, Eulemur, Varecia, Daubentonia, Saguinus, Callithrix, Callicebus, Aotus,
Cercopithecus, Chlorocebus, Macaca, Papio, Procolobus, and Piliocolobus (reviewed
in Fisher 2000). A relative reduction in caecum length and an increase in appendix
length have been noted to occur over the course of primate evolution, with strepsir-
rhines maintaining comparatively longer caecums relative to gut length than monkeys
or apes. The appendix, in turn, tends to be small and variably present in strepsirrhines,
while it is larger and more consistently present in catarrhines (Fisher 2000).
Long described as a digestive tract vestige, the mammalian appendix is now
thought to fulfill a subset of GALT-related immune functions including the mainte-
nance of normal gut flora (Gorgollon 1978; Randal Bollinger et al. 2007). Not sur-
prisingly, given that appendectomies in humans do not lead to immune dysfunction
or notable shifts in gut flora, the necessity of the appendix in primates remains ques-
tionable (Laurin et al. 2011; Smith et al. 2009). In animals that maintain a caecum but
lack a caecal appendix, the caecal apex tends to contain a high concentration of
GALT and microbial biofilms which may compensate for the absence of an appendix
(Smith et al. 2009). Frogs, which lack caecums entirely, share a very similar pattern
of microbial biofilm distribution in the rest of the gut with mammals that maintain an
appendix. This suggests that maintenance of flora by GALT in the primate large
bowel has very ancient origins (Marjanovic and Laurin 2007; Smith et al. 2009).
24 J.F. Brinkworth and M. Thorn

Thymus

The thymus is the oldest primary lymphoid organ, the earliest example of which can
be found in lampreys (Lampetra planeri and Petromyzon marinus) (Bajoghli et al.
2011). While in most jawed vertebrates both B- and T-cell poiesis occur in the bone
marrow, thymocytes (immature T cells) move to the thymus to mature (reviewed in
Boehm and Bleul 2007). There, they undergo T-cell receptor rearrangement, posi-
tive selection for MHC interaction, and negative selection for self-recognition.
These processes lead to the development of an enormous repertoire of T cells that
communicate with other immune cells and bear receptors that recognize individual
and specific epitopes.
Lampreys bear a thymus precursor at the tips of their gill filaments that maintains
T-like cells and expresses genes associated with the thymus in other animals (i.e.,
FOXN4L, a FOXN1 homologue; CDA1) (Bajoghli et al. 2011). All extant jawed
vertebrates with a canonical thymus also have T cells, TCRs, and MHCs, so it is
difficult to assess how these immune components evolved together. Considerable
vertebrate extinctions over the last 650 million years give the impression that this
entire system of receptor repertoire expansion and immune cell development simply
appeared, fully formed in cartilaginous fish (class Chrondrichthyes). This complex
system did not, of course, arise in this manner, but there is very little information
about the intermediate steps in its evolution. The appearance of the canonical thy-
mus and this system of T-cell development coincides with the acquisition of two
genes responsible for immunoglobulin domain receptor recombination (RAG1 and
RAG2) as well as with two rounds of whole genome duplication preceding the diver-
gence of jawed and jawless vertebrates (round 1: 652 mya) and the divergence of
bony and cartilaginous fish (round 2: 525 mya), respectively (Blair and Hedges
2005; Flajnik and Kasahara 2010; Ohno 1970; Rast et al. 1997). Boehm and Bleul
(2007) have proposed that the acquisition of the thymus is the outcome of natural
selection for a specialized site to eliminate autoreactive lymphocytes that might be
generated through RAG-mediated Ig domain receptor recombination. The discov-
ery of thymoid tissue in lampreys that predates the emergence of the TCR/MHC
system, however, conflicts with their hypothesis (Bajoghli et al. 2011; Boehm and
Bleul 2007).
The vertebrate thymus is derived from pharyngeal arches and pouches, the num-
ber of which differs across vertebrates. Unsurprisingly, the number, anatomical
position, and structure of thymi differ between species as well. Some bony fish
(superclass Osteichthyes), for example, have one thymus composed of two lobes,
whereas other cold-blooded vertebrates, birds, and placental mammals have multi-
ple thymi (reviewed in Rodewald 2008). By the time bony fish emerge, thymi
derived from the 3rd and 4th pharyngeal pouches with an adult location ranging
from the cervical to the thoracic region, like those in extant mammals, are estab-
lished (Grevellec and Tucker 2010; Rodewald 2008). While some mammals, such
as mice, have recently been described as having more than one thymus, primates
have a single thoracic thymus (Dooley et al. 2006; Terszowski et al. 2006; Wong
et al. 2011). Unlike fish and birds, whose thymi are attached to the pharynx for the
Vertebrate Immune System Evolution and Comparative Primate Immunity 25

whole of their lives, the mammalian thymus tends to migrate during development,
introducing the possibility of intra- and interspecies variation in its location (Dooley
et al. 2006; Grevellec and Tucker 2010; Lam et al. 2002). In humans, variation in
thymus position is rare and tends to be associated with disease (Shah et al. 2001;
Sturm-O’Brien et al. 2009; Tovi and Mares 1978).
Interspecies comparisons of thymus development and function in primates are
not common, however, some differences in development have been suggested by the
findings of Buse et al. (2006). Thymus development in cynomolgus/crab-eating
macaques (Macaca fascicularis) appears to be accelerated in comparison to humans,
with macaque lymphoid cells preceding the appearance and functional maturation of
human lymphoid cells by approximately four gestational weeks (Buse et al. 2006). By
comparison, the infiltration of other cells into the thymus occurs later in cynomolgus
macaques than in humans, with macrophages appearing at week 10 in the former and
week 8 in the latter. By parturition, however, the thymi of these species appears to be
similarly developed. Some of these differences in gestational development are likely
associated with a near 120-day discrepancy in gestation between these species.

Bone Marrow

The presence of bone marrow in some species of cartilaginous fish suggests that mar-
row emerged in some species in the absence of bone (Tavassoli 1986). In mammals,
bone marrow becomes a site of lymphopoiesis and takes on the role of a primary
lymphoid organ. In other animal classes, lymphocyte development can occur in other
locations. In adult amphibians, for example, bone marrow expresses genes important
for lymphocyte development (e.g., RAG genes), but B cells mainly undergo differen-
tiation in the spleen and liver (Du Pasquier et al. 2000; Greenhalgh et al. 1993).
Amongst mammals, strong interspecies differences in haematopoiesis have been
noted between rodents and hominoid primates. A simple indication of potential dif-
ferences in bone marrow function between mice and hominoids is the disparity in
the relative proportions of circulating neutrophils between species. Neutrophils
emerge from bone marrow as terminally differentiated granulocytes and only circu-
late for a few hours. They represent 50–70 % of the circulating blood leukocytes in
hominoids such as humans, and only 10–25 % in mice, suggesting an interspecies
difference in the rate of production (Doeing et al. 2003; Mestas and Hughes 2004).
Similarly, Old World monkeys have been noted to have lower percentages of circu-
lating neutrophils (10–42 %) than humans, though these numbers vary depending
on the living conditions of the animals (reviewed in Haley 2003). More complex
differences between haematopoietic stem cell receptors have been noted between
primates and other mammals, with humans exhibiting low CD117 and high CD135
expression on stem cell progenitors and mice exhibiting the inverse pattern (Sitnicka
et al. 2003). Both of these stem cell markers are tyrosine kinase receptors.
Differences in their expression suggest interspecies differences in tyrosine kinase
activity and possibly signal transduction in these cells in the marrow.
26 J.F. Brinkworth and M. Thorn

Spleen

The spleen is a highly vascularized organ that filters dead/damaged cells and foreign
material. Its primary immune function in mammals is to protect the host from
blood-borne pathogens (Diamond 1969; Evans 1985). In the healthy primate spleen,
this is achieved through either the filtering of antigens by macrophages and den-
dritic cells located in marginal zones surrounding arterioles, migration of activated
dendritic cells to splenic T cells regions and presentation of antigen to T cells, or
trapping of activated B cells in the splenic T-cell zone and their subsequent interac-
tion with antigen-specific T helper cells and proliferation (reviewed in Cesta 2006b).
Though lampreys maintain a primordial spleen, the organ is found in cartilagi-
nous fish and first emerged as an independent structure in the last common ancestor
to extant jawed vertebrates 652–525 million years ago (reviewed in Boehm et al.
2012a)(Blair and Hedges 2005). Splenic function and structure has changed consid-
erably over evolutionary time and varies strongly from animal class to animal class.
In mammals, for example, the spleen is also a very important site of B-cell develop-
ment. Mammalian B cells initially develop in the bone marrow, but typically mature
in the spleen (Brendolan et al. 2007; Drayton et al. 2006). This is not the case for
other animal classes. In cartilaginous fish B-cell development starts in the liver and
migrates to the kidneys before the final stages occur in the spleen (Du Pasquier
1973). In bony fish, B-cell development bypasses the spleen all together and occurs
mainly in the kidneys (or pronephros). In birds, B cells spend the earliest stages of
their development in the spleen before migrating to the bursa of Fabricius, located
near the cloaca (Boehm et al. 2012a; Pickel et al. 1993). While a perpetual blood
filter, the precise role a spleen plays in the immune system of an animal varies con-
siderably across the animal kingdom.
The complex structure of the primate spleen, with red pulp with a white pulp
compartmentalized into peri-arteriolar lymphoid sheath (PALS), follicular dendritic
cell clusters, and a marginal zone with germinal centers for B-cell proliferation,
only emerged with the last common ancestor of mammals (Fig. 2) (Cesta 2006b;
Hofmann et al. 2010). The spleen of cartilaginous fish bears a familiar structure of
blood filtering and component storing red pulp containing lymphocyte organizing
white pulp, with well-defined lymphocyte zones (Rumfelt et al. 2002). The white
pulp has become more complex over evolutionary time, with the spleens of more
recently emerged animal classes taking on progressively more complex white pulp
structure and function (Jeurissen 1991; Zapata and Amemiya 2000).
In the simplest of terms, mammalian white pulp is comprised of lymphoid tissue
that surrounds arterioles, with a centralized area containing resting or proliferating
B cells surrounded by a peripheral area where T cells congregate. This is, however,
a highly derived structure. The white pulp of cartilaginous fish is structured in the
opposite fashion of more recently emerged species, with centralized zones of T
cells, dendritic cells, and immunoglobulin-producing cells, encircled by B-cell clus-
ters (Rumfelt et al. 2002). Bony fish maintain spleens with these same basic com-
ponents, but have developed end capillary structures known as ellipsoids, covered
in macrophages and involved in antigen capture. Because of the position of
Vertebrate Immune System Evolution and Comparative Primate Immunity 27

Fig. 2 Evolution of the splenic white pulp. Positioning of Reptilia, Aves, and Mammalia based on
Blair and Hedges (2005) and Shedlock and Edwards (2009). GC germinal centers, FDCs follicular
dendritic cells, PELS peri-ellipsoid lymphocyte sheath, PALS peri-arteriolar lymphoid sheath

macrophages that capture antigens, ellipsoids bear a resemblance to the marginal

zone that serves this same function in mammals. They have, however, also been
posited to function as protogerminal centers where lymphocytes might proliferate
(Hofmann et al. 2010).
As the site where foreign antigens are captured and presented to lymphocytes,
the marginal zone is an extremely important acquisition for the mammalian spleen.
The marginal zone is found within the PALS, which encircles arterioles and func-
tions as a site where T cells interact with passing B cells and antigen-containing
dendritic cells (reviewed in Mebius and Kraal 2005). The white pulp of teleost fish
(Teleostei) and anuran amphibians (Anura), though less developed than that of rep-
tiles and birds, is known to antigen trap in ellipsoid structures physically associated
with capillaries and arterioles (Sailendri and Muthukkaruppan 1975; Secombes and
Manning 1980; Turner and Manning 1973). The current best first evidence of a
marginal zone-like structure is found in the increasingly compartmentalized white
pulp of reptiles (Murata 1959). Both reptiles and birds share a structure possibly
analogous to, or a precursor of the marginal zone, known as the peri-ellipsoid
lymphocyte sheath (PELS). The PELS covers ellipsoids, appears to antigen
trap, and contains cells similar to those found in the mammalian marginal
28 J.F. Brinkworth and M. Thorn

zone—lymphocytes and cells resembling follicular dendritic cells (reviewed in

Olah and Vervelde 2008). Unlike the mammalian marginal zone, however, reptile
PELS do not contain traditional germinal centers, where activated B cells undergo
proliferation and differentiation (Zapata and Amemiya 2000). Birds are the earliest
animals known to exhibit germinal centers in follicular sites (Jeurissen 1991).
With mammals, a macrophage-rich marginal zone specifically involved in anti-
gen capture emerged, along with germinal centers for B-cell proliferation (Zapata
and Amemiya 2000). How the development of these structures over time precisely
relates to differences in splenic function between animal classes is difficult to dis-
cern. Comparative information on amphibian and mammalian B-cell development
suggests that the absence of splenic structures such germinal centers can affect
immune function. In frogs, an absence of such centers has been associated with
lowered antibody affinity (Hsu 1998; Marr et al. 2007). Unfortunately, there is little
comparative information available on the structure and function of the primate
spleen, though interspecies differences in shape have been noted (von Krogh 1936).

Lymph Nodes

Lymph nodes are a relatively new immune innovation in the evolution of verte-
brates. They are found only in mammals and some birds [e.g., ducks (Anatidae)],
though reptiles maintain clusters of lymphoid nodules that bear some resemblance
to lymph nodes (Lawn and Rose 1981; Sugimura et al. 1977; Zapata and Amemiya
2000). The canonical lymph node consists of encapsulated lymphoid lobules, ves-
sels, and sinuses and acts as a local station of leukocyte responses to infection.
Lymph nodes mainly function to filter local lymph of antigens, act as sites for acti-
vated antigen-presenting cells to present antigens to naïve lymphocytes, and to host
and organize the clonal expansion of reactive lymphocytes (Kaldjian et al. 2001;
Karrer et al. 1997).
Lymph node number and location is highly variable between species. Mice, for
example, have 22 identified lymph nodes, while primates are prolific developers of
lymph nodes and maintain hundreds of these structures (e.g., humans have ~450
lymph nodes) (Van den Broeck et al. 2006; Willard-Mack 2006). Moreover, persis-
tent inflammation can lead to de novo synthesis of lymph nodes and contribute to
variation between individuals (Drayton et al. 2006).
Mammals also vary in their expression of specific types of lymph nodes. Rodents,
for example, do not exhibit tonsils (nasopharyngeal lymph nodes), unlike many
other mammals. It has been suggested that the extensive nasal-associated lymphoid
tissue (NALT) found in rodents is analogous to tonsils (Heritage et al. 1997). Many
nonhuman primates, however, maintain more extensive NALT than rodents and
maintain tonsils (Haley 2003). It is tempting to assume that many rodents, with their
heads a few millimeters from the ground, may have lost nasopharyngeal lymph
nodes due to the selection pressure of complications arising from increased lymph
node infections. However, the current information on the importance of tonsils in
Vertebrate Immune System Evolution and Comparative Primate Immunity 29

other species is sufficiently murky and incomplete that it does not support this
notion. The effect of pharyngeal tonsillectomy in both child and adult humans with
persistent pharyngeal infections has not been systematically studied and reviews of
case studies have argued that the procedure does not affect the occurrence of these
infections or lead to immune dysfunction (Blakley and Magit 2009; Burton and
Glasziou 2009; Burton et al. 2000).
The absence of tonsils in rodents does, however, suggest a significant alteration
to the embryonic structure from which they originate, the 2nd pharyngeal pouch
(Grevellec and Tucker 2010; Heritage et al. 1997). Similarly, second pouch devel-
opment has undergone modification in different primate species. Cesta (2006) has
noted that humans have four sets of tonsils (lingual, palatine, pharyngeal, and tubal),
while only three sets of tonsils have been described for other primates (lingual, pala-
tine, and pharyngeal). The acquisition of tubal tonsils in humans suggests a depar-
ture in second pouch development from that other primate species in the last 5–6
million years. The functional ramifications of the acquisition of these tonsils, how-
ever, are not known.

Major Cellular Components of Innate Immunity:

The Phagocytes

Monocytes/Macrophages

Phagocytic immune cells form a critical link between the mammalian innate and
adaptive immune systems by engulfing, processing, and presenting fragments of
pathogens to T cells and subsequently initiating adaptive responses. The phagocytes
include monocytes/macrophages, dendritic cells, and neutrophils (both a phagocyte
and granulocyte). Phagocytosis is likely the oldest technique of sequestering and
destroying foreign material in the interest of host defense. Indeed, phagocyte-like
cells have been identified in many lifeforms, suggesting that the phagocytes may
originate at the base of the Eukaryota domain and represent the earliest immune
cells in multicellular life (Waddell and Duffy 1986). Social acrasid amoebae, for
example, are often referenced as sharing a last common ancestor with all organisms
that either participate in or maintain cellular components that identify nonself and
phagocytose for nutrition or defense (Dzik 2010). Similarities in cellular organiza-
tion, phagocytosis, and the migration of unicellular amoebic organisms, such as
Acanthamoeba and human macrophages, have also been noted (Anderson et al.
2005). Several genera of amoebae and human macrophages share subsets of cell
receptors used for nonself detection and phagocytosis, experience manipulation of
similar cellular cascades by the same intracellular pathogens, and share a similar
mode of shifting ectoplasm and hyaloplasm to “crawl” (Al-Khodor et al. 2008;
Allen and Dawidowicz 1990; Yan et al. 2004). Particularly compelling evidence
that the origins of primate phagocytes might be rooted in single-celled eukaryotic
30 J.F. Brinkworth and M. Thorn

organisms is the tendency of Acanthamoeba to phagocytose a wide range of viruses,

bacteria, and fungi and maintain an oxidase respiratory burst system to degrade
phagocytosed material similar to that found in human macrophages (Allen and
Dawidowicz 1990; Davies et al. 1991). Of course, how these amoebic activities are
evolutionarily linked to primate phagocytes is not clear. As Siddiqui and Khan
(2012) point out, despite the similarities between some amoebae genera and human
macrophages, amoebae have evolved into organisms that live either as singletons or
as colonies and, in some cases, can become cysts when resources are in short supply
(Siddiqui and Khan 2012). These are important characteristics not shared with
human macrophages.
By the emergence of invertebrate life, phagocytosis became an essential compo-
nent of immune defense, and multiple phagocytic cell types had emerged (reviewed
in Wood and Jacinto 2007). Monocytes/macrophages are likely the oldest of these
cells and multiple authors have compared their morphology and function to that of
amoebae (Al-Khodor et al. 2008; Chen et al. 2007; Cooper 2010; Davies et al. 1991;
Siddiqui and Khan 2012). Typically derived from circulating monocytes that ven-
ture into tissues and differentiate there, vertebrate macrophages act as body “house-
keepers”, as well as immune system sentinels. Both cell types identify “nonself”
and phagocytose foreign material and cellular debris, eliminate tumour cells, secrete
a vast array of both proinflammatory and anti-inflammatory cytokines, modulate
other immune cells, and act as antigen-presenting cells (APCs) (Mosser and
Edwards 2008).
Macrophage-like cells have been noted in a wide variety of invertebrate species,
within which they identify nonself agents and initiate phagocytosis and cytotoxic
activities (Dzik 2010; Ehlers et al. 1992; Fautin and Mariscal 1991; Kurtz 2002;
Moita et al. 2005; Pech and Strand 1996; Rhodes et al. 1982). By the emergence of
cartilaginous fish macrophages became antigen-presenting cells expressing major
histocompatability complexes (MHC) (Flajnik and Kasahara 2001). MHC mole-
cules are polymorphic APC receptors, capable of binding a variety of both phago-
cytosed host and pathogen-derived antigenic peptides, presenting them to
lymphocytes, and subsequently initiating adaptive responses. From the appearance
of cartilaginous fish onwards, macrophages tend to distribute throughout bodily tis-
sues, accumulate at areas of pathogen entry, phagocytose invading pathogens, and
load MHC receptors bearing peptides from these pathogens onto the cell surface,
initiating T-cell responses through TCR interaction (reviewed in Flajnik and
Kasahara 2010).
How macrophage morphology and “behavior” has changed across vertebrate
classes over time is difficult to assess, primarily because phagocytic cell and mac-
rophage subsets have historically been difficult to accurately identify using
antibody-based technologies in major classes of animals such as fish and birds and
few markers have been identified (Kaspers et al. 2008). There is reason to believe
that while macrophage morphology and behaviour is fairly well conserved across
vertebrates, the cell type functions differently in certain classes. Macrophages
appear to reside in different body regions across animal classes. For example, they
are persistently present in the peritoneal cavity of mammals but appear to be
Vertebrate Immune System Evolution and Comparative Primate Immunity 31

completely absent from this region in healthy birds and must be recruited from
circulation or tissues (Conrad 1981; Rose and Hesketh 1974; Sabet et al. 1977).
Baseline comparative studies of primate monocytes/macrophages and examina-
tions of stimulated monocytes suggest the monocyte function of closely related pri-
mates differs considerably. An examination of baseline sialic acid-recognizing
Ig-superfamily lectins (siglec) – immune cell surface molecules that can downregu-
late immune cell responses – expression across human, rhesus macaque (Macaca
mulatta), and sooty mangabey (Cercocebus atys) monocytes found marked inter-
species differences in both type and level of siglec constitutively expressed
(Jaroenpool et al. 2007). The same study also found that monocytes from SIV+
rhesus macaques more strongly expressed two siglecs (1 and 7) compared to mono-
cytes from SIV+ sooty mangabeys. The authors suggest that these differences may
contribute to the strong immune activation and pathology typically seen in SIV+
macaques and typically absent in SIV+ mangabeys. They also suggest lineage-
specific adaptation to IVs in sooty mangabeys since they shared a last common
ancestor with rhesus macaques approximately 9 million years ago (Fabre et al.
2009). Barreiro et al (2010) found that human, chimpanzee (Pan troglodytes), and
rhesus macaque monocytes stimulated with lipopolysaccharide (LPS) initiated
species-specific transcription of genes associated with immune responses to viral
infection and cancer. Specifically, human and chimpanzee responses have diverged
from one another. Human responses were enriched for genes associated with
apoptosis and cancer pathology, while the responses of chimpanzees, a natural
immunodeficiency virus host thought to also have a low incidence of cancer, were
enriched for HIV-interacting genes (Barreiro et al. 2010). Such interspecies differ-
ences in monocyte/macrophage responses suggest lineage-specific adaptation has
occurred in catarrhines since cercopithecoids and hominoids last shared a common
ancestor ~30 million years ago (Steiper and Young 2006).

Dendritic Cells

Like all phagocytes, dendritic cells (DCs) are ancient in origin, though likely
younger than macrophages. DCs maintain a tree-like shape, with multiple dendrites
as “branches.” They tend to be found in tissues that have a direct interface with the
external environment (e.g., mucosa) and lymphoid organs, where they participate in
pathogen detection and antigen presentation. Canonical dendritic cells first emerged
in teleost fish, which suggests that DCs have existed since at least the Devonian
period ~420–360 million years ago (Setiamarga et al. 2009; Wolfle et al. 2009).
Mixed leukocyte reactions in jawless fish such as hagfish and lampreys, however,
suggest that these animals maintain similar antigen-presenting cells that recognize
nonself, introducing the possibility of a much earlier emergence date for a DC pre-
cursor (Raison et al. 1987). Once emerged DC morphology and function has
remained conserved in subsequent animal classes. Mammalian and teleost DCs, for
example, are derived from similar precursor cells, maintain dendrites, express
32 J.F. Brinkworth and M. Thorn

similar cell markers, are activated by TLR ligands, participate in phagocytosis, and
are found in similar lymphoid organs (e.g., the spleen) (Bassity and Clark 2012).
In primates, DCs are a heterogeneous group of sentinel cells that arise from a
haematopoietic or peripheral blood mononucleocyte progenitor, are present through-
out body tissues and organs (skin, intestines, liver, lungs, etc.), survey for patho-
gens, and engage bacteria and viruses through a variety of receptors (reviewed in
Wu and Liu 2007). Unlike other phagocytes, however, mammalian DCs are not
directly involved in early clearance of pathogens. While the primary function of
monocytes/macrophages is to destroy nonself, the primary function of DCs appears
to be antigen presentation via MHC I and MHC II molecules. Through antigen pre-
sentation DCs are exceptional stimulators of T-cell responses (reviewed in
Nussenzweig et al. 1980; Savina and Amigorena 2007). DCs mature through this
process of antigen presentation, switching from a phagocytosing cell to an antigen-
presenting cell that migrates to secondary lymphoid tissues, presents MHC bound
antigen to T cells, and initiates T-cell responses through a two-signal system
(reviewed in Gilliet et al. 2008; Kushwah and Hu 2011).
In primates DCs represent a small proportion of leukocytes (e.g., less than 1 % of
circulating leukocytes), so most comparisons of primate DC phenotype and function
are comparisons of DCs cultured from monocytes, bone marrow, or peripheral blood
stem cells (Ashton-Chess and Blancho 2005; Gabriela et al. 2005; Ohta et al. 2008;
Prasad et al. 2010; Soderlund et al. 2000). Such in vitro culture methods can differ
across studies and species, making interspecies comparisons of these DCs difficult.
Interspecies comparisons of DC phenotypes are also frustrated by inconsistent anti-
body affinity for DC markers across species. In vivo examinations of DCs have found
that nonhuman catarrhine species maintain the two major subsets of DCs found in
humans—the monocyte-like myeloid DCs (mDCs) and plasma cell-like plasmacytoid
(pDCs) (Coates et al. 2003; Diop et al. 2008; MacDonald et al. 2002; Malleret et al.
2008; Pichyangkul et al. 2001). However, markers for these cells differ between pri-
mate species. Rhesus macaque monocyte-derived DC (moDCs) markers CD11b,
CD16, and CD56 are expressed on human monocytes and natural killer cells, rather
than DCs (Brown and Barratt-Boyes 2009; Carter et al. 1999). Such differences in
protein expression can frustrate interspecies analyses by antibody-based flow cytom-
etry and may have consequences for immune function. Moreover, antibody affinity
for human DC markers can be depressed in other primates, making the identification
of pDCs particularly difficult for more distantly related primates such as platyrrhines
(specific antibodies discussed in Jesudason et al. 2012).
There are other notable interspecies differences in catarrhine DC phenotype that
suggest the function of these cells have diverged multiple times since cercopithecoids
and hominoids last shared a common ancestor ~30 million years ago. Human, African
green monkey (Chlorocebus sp.), and cynomolgus macaque moDCs strongly express
DC-SIGN/CD209, a receptor involved in pathogen recognition, cell migration, and
T-cell activation (Geijtenbeek et al. 2000a, b, Geijtenbeek et al. 2003). The weak
expression of this receptor on rhesus macaque moDCs suggests that these activities
are mediated differently between catarrhine species (Wu et al. 2002). Information on
primate DC baseline function is very limited, with most data on interspecies
Vertebrate Immune System Evolution and Comparative Primate Immunity 33

differences in function having been collected via a challenge model. An in vitro com-
parison of moDC responses to TLR3 ligand and common vaccine adjuvant Poly I:C,
for example, found that despite similar TLR expression levels, human moDCs initiate
stronger anti-inflammatory and antiviral responses (IL-12, IFNα) to the ligand than
rhesus macaque moDCs (Ketloy et al. 2008). As Poly I:C is a common adjuvant, these
differences may have implications for vaccine studies using rhesus macaques as a
model. During in vivo infection catarrhine DCs exhibit notable differences in func-
tion. Sooty mangabeys, in particular, exhibit signs of lineage-specific adaptation of
pDC function to viral infection. Unlike the pDCs of naïve hosts, such as macaques
and humans, pDCs from natural immunodeficiency virus (IV) host sooty mangabey
infected with IVs fail to express CCR7, an important receptor for cell homing to lym-
phoid tissue. Sooty mangabey pDCs stimulated with ligands that are viral mimetics do
not migrate to the lymph nodes (Mandl et al. 2008). Given that pDCs are implicated
in the dissemination of IVs in lymph nodes, it is possible that the absence of
IV-mediated homing of pDCs to the lymph nodes in sooty mangabeys partially
explains AIDS resistance in this species. Similarly, sooty mangabey pDCs have been
found to produce less IFNα in response to yellow fever virus 17D vaccine than pDCs
isolated from rhesus macaques and humans, suggesting that antiviral responses of
pDCs differ between catarrhine species (Mandl et al. 2011).

Neutrophils

Neutrophils are the most recently emerged phagocyte, first appearing in mammals.
Neutrophils are both phagocytes and granulocytes. Granulocytes are immune cells
that maintain intracellular/membrane-bound vesicles containing biologically active
compounds that can be quickly released in response to stimulus. While neutrophils
are unique to mammals, birds, and reptiles, amphibians and fish maintain a related
and considerably more granular phagocytic cell known as a heterophil. Both cell
types engulf and destroy microbes, maintain granules that contain antimicrobial
substances, circulate in the blood, and initiate tissue repair (Benoit et al. 2008;
Robert and Ohta 2009) (reviewed in Harmon 1998; Yoder 2004). Neutrophils/het-
erophils differ considerably from other phagocytic cells in a number of functionally
important ways. Unlike macrophages and dendritic cells, neutrophils/heterophils
emerge from bone marrow myeloblasts terminally differentiated and do not differ-
entiate further when they phagocytose material or migrate to and from tissues.
While macrophages and dendritic cells tend to traverse tissues in low numbers,
neutrophils/heterophils circulate in blood and cross into tissues in very large num-
bers. Neutrophils/heterophils are also some of the shortest lived immune cells,
maintaining lifespans of hours or a few days, rather than weeks (reviewed in Amulic
et al. 2012).
The primary function of neutrophils appears to be to limit bacterial infections
(Borregaard and Cowland 1997). They are often the first innate immune cells at the site
of an infection, migrating in large numbers to sites of inflammation in the tissues by
34 J.F. Brinkworth and M. Thorn

rolling along endothelium until they are arrested and trafficked to these sites by
expressed chemokines and selectins (Ley et al. 1998; Smith et al. 2004; Zhang et al.
2001). There they amplify inflammation through the release of cytokines and che-
moattractants and are implicated in overt immune responses that lead to tissue
injury or systemic inflammation (Ear and McDonald 2008; Kobayashi 2008;
Zemans et al. 2009). It is perhaps due to the potential of these cells to cause tissue
injury that neutrophils have evolved several mechanisms that limit their antimicro-
bial activities. They undergo apoptosis after phagocytosis and can initiate suicidal
pathogen trapping. They also participate in a unique feedback mechanism that con-
trols exuberant neutrophils. Mass migration of neutrophils to a site is followed by
the recruitment of monocytes (Scapini et al. 2001; Soehnlein et al. 2008). These
monocytes differentiate into macrophages, which then limit neutrophil activities
through lipoxin-triggered phagocytosis (Godson et al. 2000).
Neutrophils participate in several antimicrobial activities that the other phago-
cytes do not. While phagocytosis in macrophages occurs through an endocytic path-
way, neutrophil phagosomes become active upon granule–phagosome fusion
(reviewed in Amulic et al. 2012; Lee et al. 2003; Savina and Amigorena 2007).
Subsequent degranulation serves as an antimicrobial mechanism, nonspecifically
destroying both pathogens and host tissue as well as enhancing phagocytosis by
other phagocytes (Soehnlein et al. 2009). Neutrophils are known to swarm lymph
nodes during parasitic infection, an immune strategy that other phagocytes do not
appear to use (Chtanova et al. 2008). In an interesting potential adaptation to large
pathogens (e.g., helminths), neutrophils participate in a kind of antimicrobial “hara-
kiri” by releasing decondensed chromatin into the environment and creating extra-
cellular traps (NETS) (Brinkmann et al. 2004; Fuchs et al. 2007). This action
inevitably kills the neutrophil, but nets and kills the pathogens as well.
Neutrophils and heterophil granular contents differ, with neutrophils relying
more heavily on an oxidative burst response during phagocytosis (Penniall and
Spitznagel 1975; Stabler et al. 1994). Heterophils and neutrophils differ in how they
contribute to lesion pathology as well. In birds and reptiles, heterophils that invade
a lesion tend to form granulomas, while in mammals neutrophils may form granu-
lomas but can also liquefy and form abscesses (Harmon 1998; Montali 1988). The
number of circulating neutrophils/heterophils differ strongly between animal classes
and orders as well, with neutrophils representing between 50 and 70 % of circulat-
ing blood leukocytes in primates, while they represent only 15–20 % of the circulat-
ing population in rodents and zebrafish (Haley 2003; Hawkey 1985; Martin and
Renshaw 2009).
Interspecies comparisons of neutrophil phenotypes are difficult to complete for
many reasons. In vitro comparisons are hard to undertake as the cells are very dif-
ficult to immortalize and primary neutrophils have a very short life span (Amulic
et al. 2012). In vivo interspecies comparisons of neutrophil activities are made dif-
ficult by how widely the proportions of neutrophils to other circulating leukocytes
differ between mammalian species. It is apparent that neutrophils phenotypically
differ within the mammalian order. Mouse neutrophils, for example, lack defensins,
while human neutrophils maintain these antimicrobial proteins (Eisenhauer and
Vertebrate Immune System Evolution and Comparative Primate Immunity 35

Lehrer 1992). Human neutrophils tend to maintain large doses of histamine, a pro-
inflammatory amine that increases blood vessel permeability, triggers cytokine
release by neighboring leukocytes, triggers smooth muscle constriction, and is
active in allergic reactions. Rabbits and guinea pigs tend to maintain lower quanti-
ties of this molecule (Haley 2003).
Even within the context of specific infections, comparative functional studies
of neutrophils in primates are rare. Through an SIV challenge model Elbim et al
(2008) found some evidence that neutrophil function differs between primate
species. Rhesus macaques exhibit increased expression of an SIV co-receptor on
neutrophils, as compared to African green monkeys. This difference in expres-
sion is correlated with increased neutrophil death in SIV+ rhesus macaques dur-
ing the early stages of infection, suggesting that neutrophil function has diverged
since these catarrhine lineages last shared a common ancestor ~9 million years
ago (Elbim et al. 2008; Fabre et al. 2009; Steiper and Young 2006). More broadly,
primates exhibit interspecies differences in the proportions of circulating neutro-
phils. Neutrophils represent 50–70 % of circulating leukocytes in humans and
other apes and 10–42 % in cercopithecoid primates (Doeing et al. 2003; Haley
2003). An explanation for the intensely neutrophil-rich blood of hominoids is
difficult to determine. A number of primate traits appear to be correlated with
increased proportions of basal neutrophils, including traits that may increase
exposure to bacterial pathogens such as greater terrestriality/body mass and
female mating promiscuity (Nunn 2002). However, there are few comparative
interspecies studies of primate neutrophil function to supplement these findings.
As such, the functional ramifications of these differences in neutrophil propor-
tions are not known.

Major Cellular Components of Innate Immunity:

Other Granulocytes

Along with neutrophils (described above), vertebrate granulocytes include mast

cells, basophils, and eosinophils. Granulocytes are important combatants of invad-
ing bacteria and mediators of inflammation, roles they are able to complete through
degranulation and sometimes phagocytosis. While the evolutionary roots of phago-
cytosis have been postulated to predate multicellular eukaryotic life, the granulo-
cyte activity of “degranulation,” releasing compounds from internal vesicles known
as “granules,” has been postulated to have appeared with an expansion of the Meta-
zoa during the pre-Ediacaran periods of the Neoproterozoic era, between 1,000 and
600 mya (reviewed in Crivellato et al. 2010) (Peterson and Butterfield 2005). Key
functions of primate immune system granulocytes are rooted in the origins of piece-
meal degranulation of specific granule components, a function known to occur in
the immune cells of teleost fish that may have emerged in invertebrate life (reviewed
in Crivellato et al. 2010).
36 J.F. Brinkworth and M. Thorn

Mast Cells

A mast cell progenitor, sharing qualities with basophils, appears to have emerged in
tunicata during the Paleozoic (de Barros et al. 2007). Mast cells have subsequently
been found in all vertebrate chordates. Mast cells are innate immune effector cells
that also modulate the activities of innate and adaptive immune cells. They are a
heterogeneous population of bone marrow-derived granulocyte cells that store bio-
logically active compounds in membrane-bound granules. Mast cell precursors
emerge from bone marrow and differentiate into mast subsets when they migrate to
mucosal and connective tissues (Galli et al. 2008; Galli et al. 2005). At these sites,
mast cells often directly interface with the environment and can live for weeks or
months (Church and Levi-Schaffer 1997). Similar to other granulocytes, mast cells
release various granular proteins upon activation, as well as proinflammatory mol-
ecules such as prostaglandins and leukotrienes, cytokines, and chemokines. The
original function of mast cells was likely inflammation and host defense against
bacteria and parasites, though they now also play a very important role in immune
regulation, tissue repair, and blood vessel development (Malaviya et al. 1996)
(reviewed in Crivellato and Ribatti 2010). In mammals, mast cell cytokine produc-
tion can also induce a subset of dendritic cells called Langerhans cells to migrate to
the local lymph nodes, where antigen presentation takes place (Jawdat et al. 2004).
Intriguingly, mast cells are major mediators of detrimental hypersensitivity reac-
tions such as anaphylaxis and yet are maintained in all vertebrate chordates
(Krishnaswamy et al. 2001). Their persistence, despite their contribution to sudden
and fatal immune reactions suggests that mast cells must also play a very beneficial
role in immune protection.
Mast cell size, granule contents, and granule numbers vary considerably between
vertebrate classes and species (Dvorak 2005). A sharp divide in histamine content in
the mast cells of warm-blooded and some cold-blood vertebrates has been well noted
(Reite 1965; Takaya 1969; Takaya et al. 1967). All descendants of early reptiles store
large amounts of histamine in mast cells. Mammalian and avian mast cells are particu-
larly prolific producers of histamine, while histamine is either absent or present in
very low levels in amphibians and most fish. The stark contrast in histamine produc-
tion between animals that emerged before and after reptiles has led to the conclusion
that mast cell granule storage of histamine emerged in the last common ancestor of
extant reptilia, possibly as early as the Permian period (300–250 mya) (Chieffi Baccari
et al. 1998; Mulero et al. 2007; Shedlock and Edwards 2009). In fact, many fish seem
unable to respond to injections of histamine, suggesting that a histamine receptor sys-
tem is absent in much of this animal class (Mulero et al. 2007; Reite 1972). Mulero
et al. (2007) have concluded that the presence of histamine in the mast cells of perci-
form fish (e.g., trout), the largest order of teleost fish, suggests mast cell storage of
histamine evolved twice in vertebrate history (Mulero et al. 2007). This view has been
contested, as some amphibians and earlier emerged classes such as ascidians (e.g., sea
squirts) have been found to maintain low levels of histamine in mast/immune cell
granules (reviewed in Crivellato and Ribatti 2010).
Vertebrate Immune System Evolution and Comparative Primate Immunity 37

Mammalian mast cells have acquired at least one trait that sets them apart from
the mast cells of other vertebrate classes. They uniquely become activated in
response to immunoglobulin E (IgE) (reviewed in Crivellato and Ribatti 2010). This
reaction appears to be very important for defense against helminth parasites. Mice
bearing an IgE deletion, for example, experience increased Schistosoma sp. worm
loads and inflammation (King et al. 1997b). It was long thought that IgE and its
receptors on mast cells represented a system of mast cell activation that newly
emerged in mammals, but this view has recently been challenged by detection of an
IgE receptor-like protein on zebrafish mast cells (Da’as et al. 2011). While the roots
of an IgE receptor-based mast cell activation system may be considerably older than
previously thought, it appears mammals alone use IgE in antiparasitic strategies.
As with many leukocyte subtypes, primate mast cells have not been well investi-
gated for baseline functional differences. Some processes, such as the cleavage of
particular granule contents (e.g., chymases) appear to be conserved in cercopithe-
coids and hominoids (Thorpe et al. 2012). There are, however, indications that the
granular contents of primate mast cells differ between species. Tryptases, for exam-
ple, are serine peptidases stored in mast cell granules and implicated in pathological
immune conditions such as anaphylaxis and asthma (Clark et al. 1995; Schwartz
et al. 1987). δ-trypase (TPSD1) is functional and active in cercopithecoids but has
undergone several nonsense mutations and truncations in hominoids over the last 30
million years, and the human–chimpanzee lineage over the last 6 million years. As
a result δ-trypase is almost nonfunctional in humans and chimpanzees (Trivedi et al.
2008). Rather β-tryptases appear to be the more active tryptase in humans and
chimpanzees, suggesting that cercopithecoid models of mast cell disorders such as
allergy and anaphylaxis may not accurately reflect human tryptase activity (Trivedi
et al. 2008, 2007).

Basophils

Basophils are granulocytes and the rarest of circulating leukocytes in vertebrates.

Typically representing less than 0–0.3 % of circulating leukocytes, basophils share a
number of functions with mast cells, including contributing to antiparasitic activities
and allergic reactions (reviewed in Haley 2003; Karasuyama et al. 2011). Basophils
appear to share a common cellular ancestor with mast cells that first arose in the
hemolymph of tunicata (de Barros et al. 2007). Like mast cells, mammalian basophils
express IgE receptors and produce considerable levels of histamine (Ohnmacht and
Voehringer 2009; Stone et al. 2010). However, basophils are a distinct, short-lived cell
type that emerges from bone marrow terminally differentiated, circulates in blood
perennially, and fails to proliferate after maturity (reviewed in Hida et al. 2005;
Karasuyama et al. 2011). Recently, it has been found that mammalian basophils pro-
mote Th2-cell differentiation, specifically participating in antigen presentation and
stimulating T cells via IL-4 secretion (Sokol et al. 2009; Yoshimoto et al. 2009). This
represents a considerable departure from mast cell function.
38 J.F. Brinkworth and M. Thorn

Basophils are exceptionally rare in most vertebrate species, with only turtles and
rabbits reported to maintain comparatively high numbers in circulation (Canfield
1998; Haley 2003). The very low circulating numbers of basophils in vertebrates
has been a significant hindrance to the comparative functional study of this cell
type. Comparative studies of primate basophil function have not been completed.

Eosinophils

Eosinophils are terminally differentiated granulocytes with phagocytic capabilities

that circulate in the blood, traverse tissues easily to attack parasites and other patho-
gens, degranulate rapidly, and modulate Th2 responses (reviewed in Hamann et al.
1991; Shamri et al. 2011; Teixeira et al. 2001). Like basophils, they are implicated
in hypersensitivity reactions such as asthma and atopic dermatitis. Their granule
contents are distinct from those of other granulocytes and include cationic proteins
such as eosinophil-associated RNases and ribonuclease-3, which are antihelminthic
and are only expressed in very small quantities by other immune cells (Hogan et al.
2008; Olsson and Venge 1974). Eosinophil degranulation releases these cytotoxic
molecules in response to a variety of pathogenic challenges (viral, bacterial, and
fungal) (Butterworth 1977; Rosenberg and Domachowske 2001; Yoon et al. 2008;
Yousefi et al. 2008). Similar to neutrophils and basophils, eosinophils tend to be
short lived, spending 8–12 hours in circulation and possibly 8–12 days in tissues if
left unstimulated (reviewed in Uhm et al. 2012). In all vertebrate species they repre-
sent a very small proportion of leukocytes (1–4 %) (Uhm et al. 2012).
It is thought that eosinophils arose from a heterophil/eosinophil-like ancestor in
invertebrates that maintained both amoeboid-like locomotion and phagocytic capa-
bilities as well as granulocytic secretion and tissue remodeling capabilities (Lee and
Lee 2005). By the time jawless fish emerged, eosinophils and heterophils had
diverged into two different cell types (Kelenyi and Nemeth 1969; Rowley and Page
1985). Given the propensity of eosinophils to cluster at sites of tissue stress and that
the emergence of current eosinophil pathogen targets (e.g., helminths) postdate the
appearance of eosinophils themselves, Lee and Lee (2005) have proposed that het-
erophil/eosinophil divergence was driven by nonimmune system factors. Specifically,
they hypothesize that eosinophils arose to fulfill a need for a cell that mediated the
tissue repair and modeling needs created by an increase in body size and complexity
seen in emerging vertebrates. This divergence then led to a heterophil/neutrophil
cell lineage dedicated to host defense and an eosinophil lineage taking on tissue
remodeling, as well as defense responsibilities.
Though eosinophil phenotypes appear evolutionarily conserved across vertebrates,
there are a few differences worth noting. Avian eosinophils tend to be involved in
delayed hypersensitivity reactions, while mammalian eosinophils are typically not
involved in this sort of response (e.g., allograft rejection and contact dermatitis)
(reviewed in Campbell 2004; Lind et al. 1990). Eosinophil degranulation can also
have a species-specific phenotype. Human eosinophils, for example, are capable
Vertebrate Immune System Evolution and Comparative Primate Immunity 39

of very quickly and extensively degranulizing in response to both specific and nonspe-
cific stimulation, whereas direct evidence that the eosinophils of other mammalian
species, such as mice, can do the same is very limited (Lee et al. 2012). If tissue
remodeling is a primary function of eosinophils, interspecies differences in degranula-
tion would be expected, as tissue remodeling mechanisms would likely differ between
species (Lee and Lee 2005).
Eosinophil granule content also differs markedly between mammalian species,
which may reflect differences in cell function. While many primates appear to main-
tain two types of ribonucleases of varying functions in their eosinophil granules,
mice maintain six, all of which are paralogous to primate ribonucleases and exhibit
strong ribonuclease activity (Lee et al. 2012). Eosinophil granule content differs
strongly between major primate clades, suggesting lineage-specific evolution of
eosinophil function. Catarrhines, for example, store ribonucleases ribonuclease A
family 3 (RNASE3) and ribonuclease A family 2 (RNASE2) at high levels in their
granules, while Platyrrhines only store a ribonuclease homologous to RNASE3
(Olsson et al. 1977; Rosenberg and Dyer 1995; Rosenberg et al. 1995). RNASE3 is
particularly toxic to host tissue cells. These differences in ribonuclease production
and storage suggest that ribonuclease-mediated eosinophil apoptotic, cytotoxic, and
antimicrobial activities have changed in these clades since they last shared a com-
mon ancestor 40 million years ago (Fabre et al. 2009; Steiper and Young 2006).

Major Cellular Components of Innate Immunity:

Natural Killer Cells

Natural Killer (NK) cells are granular lymphocytes. NK cells recognize “altered”
self (e.g., tumours) and nonself and have acquired their name because of their abil-
ity to destroy cells spontaneously without the need to be primed as other lympho-
cytes require (Herberman et al. 1975). Through direct contact mammalian NK cells
can target and lyse tumour cells or virally infected cells that downregulate MHC I
expression as a result of oncogenesis or infection (reviewed in Purdy and Campbell
2009). MHC I class molecules found on other immune cells are NK ligands and
interactions between MHCs and NK killer cell immunoglobulin-like receptors
(KIR) control NK effector functions (reviewed in Lanier 2008). Target cell killing
is initiated when activated NK cells release perforin and granzyme that induce
apoptosis (Lieberman 2003). For their importance, NKs are a rare cell population.
In humans, NK cells represent 10–15 % of blood lymphocytes, or approximately
2–3 % of all circulating leukocytes (Purdy and Campbell 2009).
Cells exhibiting NK-like activities, such as direct killing of foreign materials via
perforin-like proteins, have been noted in invertebrate life (Kauschke et al. 2001).
NK-like cells are present in all tunicata, suggesting that this cell type was established by
the time this clade emerged between 520 and 794 mya (Blair and Hedges 2005; Chen
et al. 2000). As the earliest evidence of MHC molecules is found in cartilaginous fish, it
seems that NK adoption of MHC class I molecules as ligands occurred long after the
40 J.F. Brinkworth and M. Thorn

emergence of NK cells (Azumi et al. 2003; Khalturin et al. 2003). Thus, the origins of
NK lymphocytes appear to predate the adaptive immune system as a whole.
The role of NK-like cells in nonmammalian classes is not well investigated but
appears to be conserved. In tunicata NK-like cells appear to mainly participate in
allorecognition and killing nonself materials via granule release of perforin and
granzyme-like proteins (Bielek 1988; Shen et al. 2004). NK-like cells with cyto-
toxic, and allogeneic and tumor-killing capabilities have been found in amphibians
and avian species as well (Goyos and Robert 2009; Horton et al. 1996; Jansen et al.
2010; Wainberg et al. 1983). While interclass comparisons of NK-cell distribution
and characteristics have led to conclusions that mammalian but not avian NK cells
can circulate in blood, it is very likely that a dearth of cross-reactive NK marker
antibodies for nonmammals has frustrated these studies (Rogers et al. 2008b).
While mammalian NK cells are the focus of intense study, the comprehensive set of
markers that can reliably identify NK cells in all mammalian species has yet to be
determined (Walzer et al. 2007). Even very closely related species such as humans
and rhesus macaques exhibit sharp differences in NK marker expression. Human
NKs strongly express CD56, a marker mainly expressed by monocytes in rhesus
macaques (Webster and Johnson 2005). The primate species for which markers are
well defined tend to be those species important to HIV research (e.g., rhesus
macaques and sooty mangabeys). Interspecies comparisons of primate NK function
also tend to be limited to these two species. Rhesus macaque and sooty mangabey
NK-cell functional comparisons do indicate the evolution of baseline interspecies
differences since these species last shared a common ancestor ~9 million years ago
(Fabre et al. 2009). The NK cells of healthy sooty mangabeys appear to be twice as
cytotoxic as rhesus macaque NK cells when activated, suggesting that sooty mang-
abeys may be able to more quickly mount a strong NK-based response than rhesus
macaques when viral infection occurs (Pereira and Ansari 2009).
An interesting feature of primate NK cells is the acquisition and rapid evolution
of primate killer-cell immunoglobulin-like receptors (KIRs). Primate NK cells
alone express KIRs. Like KIRs, the NK-cell receptors of other mammals [e.g., killer
cell lectin-like receptor, subfamily A, member 2 (KLRA2/Ly49) lectin receptors of
rodents] bind with MHC class I molecules or their homologues and activate the NK
cell (Abi-Rached and Parham 2005; Barten et al. 2001; Colucci et al. 2002; Lanier
2008). The KIR family of receptors appears to have expanded from a single gene,
killer cell immunoglobulin-like receptor three domain long cytoplasmic tail
(KIR3DL), over the last ~50 million years. In that time, KIR genotypes have rapidly
diversified between and within haplorrhine species. Humans, for example, encode
over 130 genotypes for approximately 14 KIRs, while the grey mouse lemur encodes
only one functional KIR (Averdam et al. 2009; Bimber et al. 2008; Hollenbach et al.
2010). Human KIRs exhibit lineage-specific evolution of receptors that specifically
recognize epitopes of MHC-B and MHC-C receptors, as well as an increased num-
ber of KIR “activating receptors” as compared to chimpanzees (Abi-Rached et al.
2010). The divergence of haplorrhine and strepsirrhine NK receptors and subse-
quent lineage-specific diversification of these receptors have likely affected NK
interactions with similarly rapidly evolving MHC molecules. Strepsirrhines appear
Vertebrate Immune System Evolution and Comparative Primate Immunity 41

to have a different NK receptor/ligand system in place, with lectin receptors CD94

and KLRC1/NKG2 representing the primary NK receptors, and an absence of a
functional equivalent of the MHC-E molecules that serve as lectin receptor ligands
in haplorrhines (Averdam et al. 2009). Moreover, CD94 and KLRC1 receptors seem
to functionally diversify by recombination, as opposed to duplication as haplorrhine
NK receptors do. If true, the occurrence of combinatorial diversification in strepsir-
rhine NK cells introduces an interesting wrinkle in our understanding of the evolu-
tion of primate NK cells. It suggests that NK cells may share recent common
ancestry with cytotoxic T cells (Averdam et al. 2009).

Major Cellular Components of Innate Immunity:

Small Lymphocytes

The adaptive immune system allows vertebrate organisms to recognize specific

antigens, develop a targeted clonal response, and retain an immunological memory,
so that a rapid and targeted immune response can be mounted when the antigen is
next detected. Key to this process in jawed vertebrates (gnathostomata) are T and B
lymphocytes, which bear highly diversified immunoglobulin domain-based recep-
tors (Ig) that recognize specific antigenic epitopes. It is through recognition of for-
eign molecules presented to lymphocytes by other cells via Ig receptors, such as
TCRs, BCRs, and MHCs, that lymphocytes initiate antigen-specific responses
(reviewed in Neefjes et al. 2011).
While the adaptive immune system we recognize in primates is found in cartilagi-
nous fish and emerged in the last common ancestor of extant jawed vertebrates ~
652–525 mya, some of its components appeared earlier. Lymphocyte-like cells are
found in jawless vertebrates (agnatha) and notochord-bearing animals, such as lance-
lets (cephalochordata), suggesting that the progenitor of this cell type could have
emerged as early as 715 mya, during the Cryogenian period (Blair and Hedges 2005;
Huang et al. 2007; Mayer et al. 2002; Nagata et al. 2002). All vertebrate life main-
tains cells that are functionally similar to T and B cells, suggesting that the roots of
adaptive immune system lymphocytes are actually invertebrate (Boehm 2011). It is,
however, in jawless vertebrates that the first lymphocyte-like cells with antigen
receptors diversified through somatic assembly are found (Alder et al. 2005; Pancer
et al. 2004).
For many years the conventional wisdom was that adaptive immunity was a
unique and complex arm of the immune system that emerged with jawed verte-
brates. However, in 2007, Rogozin et al. found receptors that undergo somatic rear-
rangement on lymphocyte-like cells in lampreys (Rogozin et al. 2007). This system
of adaptive immunity in jawless vertebrates has been extensively reviewed by other
authors and will only be touched upon briefly here (see reviews by Boehm et al.
2012b; Dzik 2010). The discovery of these leucine-rich repeat-containing variable
lymphocyte receptors (VLRs) profoundly changed our understanding of vertebrate
42 J.F. Brinkworth and M. Thorn

immune system evolution. VLRS are assembled and rearranged through the activi-
ties of APOBEC-like cytidine deaminases (AID) (Rogozin et al. 2007). This system
of expanding receptor repertoires through gene rearrangement/somatic recombina-
tion bears a strong resemblance to RAG-mediated TCR and immunoglobulin rear-
rangement. It is not, however, a homologous system and appears to be outcome of
convergent evolution. VLRs are not rearranged by RAG and they are not related to
the Ig receptor superfamily to which TCRs, BCRs, and MHC molecules belong
(Rogozin et al. 2007). Canonical T and B cells emerged with the last common
ancestor of jawed vertebrates (Flajnik and Kasahara 2001). The adaptive immune
systems of jawed and jawless vertebrates bear some remarkable similarities for
potentially being the products of convergent evolution. VLR expressing lympho-
cytes, for example, can be divided into three functional categories (VLRA+,
VLRB+, and VLRC+ protolymphocytes) that maintain similar gene expression
profiles and respond to antigens in a manner similar to that of vertebrate T and B
cells (Guo et al. 2009).

B cells

B cells represent the arm of the adaptive immune system responsible for the produc-
tion of soluble antibodies, or immunoglobulins (Ig), which recognize and bind epi-
topes expressed on a variety of microbial pathogens. In mammals, naïve B cells exit
the bone marrow and migrate to the secondary lymphoid organs where an encounter
with an antigen or antigen-specific T helper cells could lead to B-cell activation,
proliferation, Ig isotype class switching, and soluble antibody secretion. Canonical
B cells first emerged with the TCR/BCR/MHC adaptive immune components in
jawed vertebrates, 652–525 mya (Boehm and Bleul 2007). However, it seems likely
that B cells emerged earlier than T cells, as T cells require the additional step of
NOTCH signaling to differentiate from the lymphocyte progenitor cell and B cells
do not (Benne et al. 2009). Of the two cell types, B cells have not been as intensely
studied in primates as T cells. However, baseline interspecies differences in B-cell
populations have been noted between primates. For example, cynomolgus macaques
(M. fascicularis) maintain different proportions of B cell subsets than humans,
which may translate as interspecies differences in immune response. Specifically,
cynomolgus macaques retain higher percentage of B cells carrying the T cell
costimulatory molecule CD80 and a lower percentage of B cells carrying CD21, a
molecule that interacts with the complement system, than humans (Vugmeyster
et al. 2004).
Much of the comparative research on baseline B cell function has focused on
immunoglobulins (antibodies). Immunoglobulins may be secreted by B cells or
associate with accessory molecules to form the membrane-bound B cell receptor
complex (BCR). Secreted Igs can recognize and neutralize an antigen, while the
BCR complex can recognize an antigen and initiate cell signaling and/or internal-
ization and presentation of antigen to T cells. The genes that encode Igs in B cells
Vertebrate Immune System Evolution and Comparative Primate Immunity 43

and TCRs in T cells are arranged in a similar way: multiple variable and constant
gene segments that recombine and assemble in a largely random manner to generate
a vast and almost limitless repertoire of antigenic specificities. The Ig molecule is
made up of two identical Ig heavy and light chains, both of which contain variable
regions. Multiple variable (V), diversity (D), and junction (J) gene segments are
rearranged by V(D)J recombination to form an Ig heavy chain, while the light
chains are formed from V and D gene segments. The diversity of the various gene
segments is further amplified by the random nucleotide additions and subtraction at
the ends of Ig gene segments before they are joined (Thai et al. 2002). This system
of immunoglobulin diversification appeared by the emergence of cartilaginous fish,
652–525 million years ago (Blair and Hedges 2005; Rast et al. 1997). It is estimated
that the human B cell repertoire is capable of generating about 1011 unique immu-
noglobulins based on these rearrangements (Schatz et al. 1992).
The immunoglobulins produced by B cells are divided into several classes distin-
guished by constant regions, which impart specific effector properties to each class.
Mammals maintain immunoglobulins belonging to the IgM, IgG, IgA, IgE, and IgD
classes and have lost the IgY found in other animals (Kapetanovic and Cavaillon
2007; Mussmann et al. 1996b; Vollmers and Brandlein 2006; Zhao et al. 2006). Of
the immunoglobulins found in primates, IgM is oldest, having emerged with the last
common ancestor to all jawed vertebrates. It is highly conserved in function and
structure. All jawed vertebrates appear to produce the IgM class of secreted immu-
noglobulin ahead of all others in response to immune system stimulation (reviewed
in Flajnik 2002). Compared to other immunoglobulins, IgM has a lower affinity for
antigens, is often polyreactive, and has the ability to bind a variety of microbial and
viral antigens (Boes 2000).
Like IgM, an IgD homolog (e.g., IgW) appears to have been present in the last
common ancestor of jawed vertebrates (Ohta and Flajnik 2006). The evolution of
IgD is enigmatic, as many different isotypes are found in jawed fish, yet IgD is vari-
ably present in bird and mammalian species (Butler et al. 1996; Zhao et al. 2002).
In primates IgD is expressed on naïve and memory B cells; however, the function of
this immunoglobulin is not well understood (Messaoudi et al. 2011). It seems to be
involved in lymphocyte and granulocyte activities. In its secreted form in bony fish,
for example, it interacts primarily with granulocytes. In humans the membrane-
bound form of IgD may be important in the activation of B cells, while the secreted
form activates basophils (Flajnik and Kasahara 2010).
IgA is found in extant reptiles, which suggests it emerged at least ~320 million
years ago (Mussmann et al. 1996a). In mammals, IgA is commonly secreted in
mucosal sites by specialized B cells, where it binds and shields bacteria to maintain
gut homeostasis (Strugnell and Wijburg 2010). It is the most commonly secreted
immunoglobulin isotype in mammals, frequently found in GALT structures, where
its production is regulated by innate and T cell pathways in germinal centers (Suzuki
et al. 2010). IgA prototypes and analogs fulfill similar functions in nonmammalian
species. In teleost fish, for example, Ig-based mucosal immunity is accomplished
through IgT, an IgA analog (Zhang et al. 2010). Amphibian mucosal Ig immunity is
fulfilled by IgX, a possible prototype of IgA (Deza et al. 2007). IgA is also an
44 J.F. Brinkworth and M. Thorn

important neutralizer of bacteria, toxins, and viruses at the mucosal barrier and may
also facilitate antigen take up by the mucosal dendritic cells (Corthesy 2007; Stubbe
et al. 2000). When IgA does not regulate gut microflora appropriately, inflammatory
responses become dysregulated. Dysfunctional IgA is implicated in variety of
inflammatory gut disorders (reviewed in Sutherland and Fagarasan 2012).
In primates, IgA function appears to be very conserved. A comparison of IgA-
encoding genes (IGHA and IGCA) and IgA molecules of closely related nonhuman
primates [baboons (Papio sp.), sooty mangabeys, rhesus macaques, and pig-tailed
macaques (M. nemestrina)] found that despite high intraspecies variation in
sequence, the IgA receptor (CD89) of macaques could readily bind with the IgA of
other primates (i.e., humans). (Rogers et al. 2008a). The conservation of gut micro-
flora “enterotypes” across distant human populations has also been interpreted as
conservation of IgA activities (Arumugam et al. 2011).
IgE and IgG diverged from the ancestral IgY in the last common ancestors of
amphibians and mammals, respectively (Flajnik and Kasahara 2010; Warr et al. 1995;
Zhao et al. 2006). IgE is a proinflammatory immunoglobulin and a potent stimulator
of granulocytes. It is associated with Th2 responses to helminthic infections and
allergens, playing a key role in the atopic hypersensitivity response (i.e., allergic
asthma and food allergies) (Broide et al. 2010; Capron et al. 1987; Hagel et al. 2004;
Stone et al. 2010). As such, it has been the subject of intense investigation.
As IgE is only found in mammals, IgE-based mechanisms at play in allergies and
helminthic infections are unique to this class and comparatively recent in the con-
text of vertebrate evolution. The essential nature of IgE, however, has been ques-
tioned as it has been maintained by mammals for over 250 million years but is
frequently suppressed in humans by modern medications without ill effect (Cooper
et al. 2008; Cruz et al. 2007; Vernersson et al. 2004, 2002).
In humans, helminthic infections and IgE-mediated allergic responses have been
assumed to have had a complicated selective affect on the species, with high levels of
IgE considered beneficial against helminthes and yet key to atopic hypersensitivity
(Hagel et al. 2004). Moreover, under the hygiene hypothesis, human IgE responses have
coevolved with certain helminth species which are thought to dampen IgE-mediated
atopic hypersensitivity reactions (see Rook 2013; Martin et al. 2013 for reviews of this
hypothesis). Loss of these species in the modern human microbiota, due to increased
hygiene/use of antihelminthic medications, is thought to contribute to overt IgE
responses to allergens and subsequent hypersensitivity (Cardoso et al. 2012). However,
a recent finding of cross-reactivity between allergenic extracts from the helminth Ascaris
lumbricoides and mite allergens suggests the interplay between IgE responses to hel-
minthes and allergens may be more complicated and sometimes involve cases of “mis-
taken identity” (Acevedo and Caraballo 2011; Acevedo et al. 2009).
In primates, IgE appears to be functionally diverse. Analysis of membrane-
bound IgE gene-encoding regions suggests that tarsiers do not produce membrane-
bound IgE (Wu et al. 2012). Wu et al. (2012) note that mouse strains unable to
produce membrane-bound IgE exhibit very low IgE production during parasitic
infections, which suggests a functional difference between tarsier IgE and the IgE
of other primates (Achatz et al. 2008). While catarrhines produce both long and
Vertebrate Immune System Evolution and Comparative Primate Immunity 45

short isoforms of IgE, platyrrhines do not appear to produce a short isoform, and
lemurs, lorises and nonprimate mammals do not appear to produce a long. This
suggests that the ancestral condition for IgE is the short isoform and that the long
isoform was acquired sometime after the divergence of haplorrhines and strepsir-
rhines ~77 mya, but before the divergence of platyrrhines from catarrhines 43 mya
(Steiper and Young 2006 isoform; Wu et al. 2012).
IgE-mediated atopic hypersensitivity reactions appear to be increasing across the
globe (Rorke and Holgate 2004). Using data collected through the International
Study of Asthma and Allergies in Childhood (ISAAC), Asher et al (2010) have
noted a positive correlation between ecological factors associated with developed
nations, such as gross national product per capita, trans fatty acids and tylenol use,
and the incidence of asthma, eczema, and rhinoconjunctivitis (Asher et al. 2010).
Other studies have noted a positive association between lower socioeconomic status
and asthma/allergic reactions (Hagel et al. 2004; Litonjua et al. 1999; Von Behren
et al. 1999). It is worth noting, however, that an association between these factors
and conditions may be an outcome of increased “hygiene” in developed nations, but
may also be influenced by increased disease surveillance in wealthier countries.
While potential data bias should be considered, these data propose an interesting
connection between human cultural behaviors and human IgE expression.
IgG is the most abundant antibody type found in mammalian serum and has high
affinity and specificity for target antigens. The key function of IgG is to bind to
target antigens and activate effector cells, such as NK cells or monocytes, to destroy
immunoglobulin-coated targets (Schroeder and Cavacini 2010). Complement-
mediated cytotoxicity is another function modulated by IgG that depends on the
C1q complement component binding to the constant portion of IgG antibody bound
to a target (Schroeder and Cavacini 2010). IgG and IgG receptor interactions are
known to differ between primate species, with some cynomolgus macaque IgG sub-
classes exhibiting stronger effector function and greater binding affinity for IgG
receptors, known as Fc receptors, than human IgG (Warncke et al. 2012).
Additionally, the IgG receptor CD16 binds to different IgG subclasses in sooty
mangabeys than it binds in humans (Rogers et al. 2006). These results suggest that
IgG function has diverged in the 25–30 million years since these species shared a
last common ancestor (Fabre et al. 2009; Steiper and Young 2006).

T Cells

T cells, so named because they mature in the thymus, are lymphocytes that contrib-
ute to adaptive immune defense through cytotoxic activities and the regulation of
other immune cells. In jawed vertebrates, T cells are activated indirectly through
antigen presentation by MHC I and MHC II molecules of professional antigen-
presenting cells, such as DCs and macrophages, to the T-cell receptors (TCRs) of T
cells. This activity constitutes a bridge between innate immune responses and adap-
tive immunity, and initiates the adaptive immune response. Both TCRs and MHC
46 J.F. Brinkworth and M. Thorn

molecules are highly variable and capable of binding a wide variety of both host and
pathogen-derived antigenic peptides. In humans, for example, human lymphocyte
antigen (HLA) encodes about 2,000 MHC alleles according to the ImMunoGeneTics
HLA database (http://www.ebi.ac.uk/imgt/hal/atats.html) (MHC/HLA diversity in
Old World primates is discussed in Loisel and Tung 2013). It was previously thought
that TCRs, MHCs, and T and B lymphocytes emerged in the immunological equiva-
lent of a “big bang”, all at once, with the emergence of jawed vertebrates. With the
discovery of VLR+ lymphocytes in jawless fish, it appears that the origins of T-cell
lymphocytes are considerably more ancient than the Ig receptor superfamily
system.
The discovery of specialized lymphocytes in jawless vertebrates significantly
challenged a long-held wisdom that Ig receptors such as T-cell receptors (TCR)
drove the divergence of T and B cells. Specialized VLRs (VLRA, VLRB, and
VLRC) are not related to Ig receptors, but they are found in jawless fish on lympho-
cyte-like cells that are already proficient in different functions reminiscent of T and
B cells. VLRB molecules are mainly secreted by and found in the membrane of
cells whose functions and other receptors best resemble those of B cells (Alder et al.
2008; Herrin et al. 2008). Cells bearing VLRA and VLRC tend to respond to similar
stimuli and produce the same cytokines and receptors as T cells (Guo et al. 2009).
This suggests that lymphocyte subsets became specialized before the “big bang” of
Ig receptors. Moreover, lamprey have a TCR-like gene, along with VLRs, suggest-
ing that the foundations of both systems of somatic rearrangement/recombination
may have existed in some earlier animals (Pancer et al. 2004). As Hsu (2011) has
pointed out, these receptors suggest that rather than lymphocyte differentiation
being based on the inherent functions of the receptor, lymphocyte receptors have
been selected for based on the benefit they brought to lymphocyte function.
Within jawed vertebrates, both TCR gene organization and T-cell development are
highly conserved (Flajnik and Kasahara 2010; Rast et al. 1997). TCR gene segment
rearrangement takes place in the thymus of all jawed vertebrates and produces a
unique antigen-specific TCR for each T cell. Task-specific TCR subtypes known in
mammals (alpha/beta and gamma/delta) have been found in cartilaginous fish, which
suggests that TCRs had already differentiated into subtypes by the the time that jawed
vertebrates emerged (Hirano et al. 2011; Kreslavsky et al. 2010). Despite conserva-
tion, TCRs do differ across vertebrate classes and species. Two unique TCR genes
arose in marsupials and sharks (TCR mu and NAR-TCR) and are expressed as atypical
TCRs in these animals (Criscitiello et al. 2006; Parra et al. 2007).
In mammals, T cells can be divided into task-specific categories, the two largest
subsets being CD8+ cytotoxic T cells and CD4+ helper T cells (Th). Cytotoxic T
cells can directly kill infected cells, while helper T cells differentiate into Th subsets
(e.g., Th1, Th2, and Th17), after interacting with antigen-presenting cells, to regu-
late other T cells, B-cell antibody production, and Ig class switching. It is perhaps
out of a historical emphasis on adaptive immune responses that many subsets of T
cells have been defined and characterized [e.g., Th1, Th2, Th17, regulatory T cells
(Tregs), and additional within group subsets]. When adaptive immune features such
as Th cells or memory T-cell retention emerged in vertebrate immunity is very
Vertebrate Immune System Evolution and Comparative Primate Immunity 47

difficult to determine. In the most catholic interpretation, these traits appear with the
TCR/MHC/lymphocyte system in jawed vertebrates. Whether or not progenitors of
such T-cell subtypes exist in the repertoire of lymphocyte-like cells found in earlier
lifeforms is matter for further investigation.
Almost all comparative studies of primate T cells are based on a challenge model.
Under these experimental circumstances, primate T cells do exhibit unique charac-
teristics. Human T cells have been described as “overreactive” to stimulation in
comparison to chimpanzee T cells, a functional difference that Soto et al. (2010)
attribute to increased levels of inhibitory sialec acid-recognizing Ig-superfamily
lectin 5 (Siglec 5) on chimpanzee T and B cells. Siglec 5 can suppress immune cell
activation (Soto et al. 2010).
As nonhuman primates are important models of immunodeficiency virus infec-
tion and T-cell activities are key to HIV infection progression, most comparative
information on primate T cells has been captured in the context of SIV/HIV research.
As the primary targets of immunodeficiency virus infection, CD4+ T cells have
been particularly well examined for interspecies functional differences. Available
information suggests that lineage-specific adaptations have evolved in this subset of
T cells. The proportions of CD4+ T cells readily infected by HIV/SIV, that is those
CD4+ T cells that also express CCR5, differ between catarrhine species that are
natural and naïve hosts of IVs. Healthy sooty mangabeys, for example, maintain
fewer CD4+ CCR5+ T cells than humans and macaques. Moreover, these cells do
not upregulate CCR5 when stimulated and appear to be less susceptible to IV infec-
tion as a result (Paiardini et al. 2011). This trait may help explain sooty mangabey
resistance to IV pathogenesis. CD4+ T cells also proliferate more quickly in IV
infected humans and macaques than they do in IV natural hosts such as mandrills
(Mandrillus sphinx) and sooty mangabeys, suggesting that the haemopoietic mech-
anisms leading to the production T cells differ between species (Chan et al. 2010;
Engram et al. 2010). Healthy macaques have been found to maintain more CD4+ T
cells that produce a protease, granzyme B, that induces apoptosis in virus-infected
cells (granzyme B) in the lamina propria of the gut than African green monkeys, a
natural IV host (Hutchison et al. 2011). Granzyme B is a highly potent protease that
may contribute to the translocation of microbes from within the gut into the perito-
neal cavity during IV infection in naïve hosts. Taken together, these differences
suggest pathogen-mediated selection of CD4+ traits in natural IV hosts over the 30
million years since all catarrhines last shared a common ancestor.

Conclusions

The major components of the primate immune system represent hundreds of mil-
lions of years of evolution. Here, we have outlined how the vertebrate immune sys-
tem evolved and how primates have become specialized within that system, providing
the first summary of functional differences in primate baseline immunity within the
context of vertebrate immune system evolution. Where possible, we have offered a
48 J.F. Brinkworth and M. Thorn

comparative analysis of resting primate immune component function. Reflection on

vertebrate immune system evolution is important, not only for understanding how
primate immune systems have diverged from the immune systems of other animals,
but also for understanding aspects of primate immunity that are unique. An apprecia-
tion of the distinctive qualities of nonhuman primate immune systems will help
ensure the appropriate and efficient use of animal biomedical models, so that nonhu-
man primates are not applied to studies for which another animal could be success-
fully applied. In these efforts, however, comparative immunology of primates faces
challenges. Specifically, the data presented here is mainly limited to examinations of
baseline immunity in animals that are common biomedical models, which in the
case of primates disproportionately biases our information to several catarrhine spe-
cies (i.e., rhesus macaques, humans, and sooty mangabeys). Additionally, any
researcher seeking a better understanding of primate immunity faces the challenges
of working with a model that is very expensive, comes with special ethical consider-
ations and is subject to care and use guidelines not applied to other mammals used
in experimentation. Several nonhuman primate species housed in National Institutes
of Health facilities, for example, are now only available for access opportunistically
and for ex vivo experimentation, if available at all (i.e., chimpanzees and sooty
mangabeys). An understanding of how other immune systems differ and have
evolved can help bridge gaps in our knowledge about primate immune system func-
tion. As such, considering how specific immune components have evolved in other
vertebrates can be useful in forming testable hypotheses on immune component
function in primates when experimental data has been previously absent or difficult
to acquire. As a backdrop for the chapters that follow, this chapter serves as a refer-
ence for the evolution of major immune components and, we hope, as background
for discovering principles governing primate immune function and its evolution.

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Genetic Variation in the Immune System
of Old World Monkeys: Functional
and Selective Effects

Dagan A. Loisel and Jenny Tung

Introduction

The selective pressures exerted by pathogens and parasites have played a significant
role in human and nonhuman primate evolution, especially in shaping the form and
function of immune defenses (Barreiro and Quintana-Murci 2010; Nunn and Altizer
2006; Stearns and Koella 2008). While the evolutionary processes that historically
influenced the primate immune system cannot be directly observed today, the exten-
sive phenotypic diversity observed in contemporary primate populations reflects
these changes. Specifically, although many aspects of basic biology are conserved
among primates, both pathogen susceptibility and disease progression upon infec-
tion can greatly vary among species, subspecies, and populations. The adaptive and
mechanistic origins of this variation are therefore of great interest, including as a
source of insight into mechanisms of disease in humans.
Recent advances in genetic and genomic techniques, as well as steadily decreas-
ing costs, have enabled functional and evolutionary studies of intraspecies and inter-
species genetic variation in the primate immune system that would have been

D.A. Loisel
Department of Human Genetics, University of Chicago, 920 E 58th Street,
Chicago, IL 60637, USA
Department of Biology, Saint Michael’s College, Colchester, VT 05439
J. Tung (*)
Department of Evolutionary Anthropology, Duke University, Box 90383,
Durham, NC 27708, USA
Department of Human Genetics, University of Chicago, 920 E 58th Street,
Chicago, IL 60637, USA
Duke Institute for Population Research, Duke University, Box 90420,
Durham, NC 27708, USA
e-mail: [email protected]

J.F. Brinkworth and K. Pechenkina (eds.), Primates, Pathogens, and Evolution, 65

Developments in Primatology: Progress and Prospects
DOI 10.1007/978-1-4614-7181-3_3, © Springer Science+Business Media New York 2013
66 D.A. Loisel and J. Tung

prohibitive only a few decades ago. Thus, while interest in immune genetic varia-
tion is long-standing, research on this topic has become increasingly sophisticated.
It is now possible to not only identify which immune loci may have evolved under
selection (Bustamante et al. 2005; Haygood et al. 2007; Nielsen et al. 2005) but also
infer the type of selection that occurred, the time scale on which it occurred, and the
type of functional change (e.g., coding or regulatory) that was its target (Haygood
et al. 2010; Kosiol et al. 2008). At the same time, it is becoming increasingly routine
to use functional data to investigate the mechanistic and adaptive consequences of
sequence-level genetic variation.
The purpose of this chapter is to highlight recent development in these areas,
focusing specifically on research in Old World monkeys (OWMs). In what follows,
we briefly discuss the rationale for studying genetic variation in the OWM immune
system. We then review recent research on natural selection and functional genetic
variation in this clade pertaining to OWM immune function.

Why Is Immune System Variation in OWMs

an Interesting Topic to Study?

OWMs (family Cercopithecidae) represent a diverse and species-rich group of

primates that radiated approximately 27 million years ago (Purvis 1995) and that
currently occupy a large geographic range across much of Africa and Asia (Wilson
and Reeder 2005). OWMs retain close evolutionary ties to humans (93–94 % DNA
sequence identity: Gibbs et al. 2007; Silva and Kondrashov 2002) and, outside of
the apes, are our closest extant relatives. In some cases, they may also serve as
appropriate models for the ecological and environmental circumstances of ancestral
hominins (Behrensmeyer 2006; Jolly 2001; Potts 1998). OWMs are also important
in medical research. For instance, rhesus macaques are major medical models for
caloric restriction and aging, alcoholism, and infectious disease resistance, while
baboons have been utilized as models for steroid hormone signaling (in both captiv-
ity and in the wild) and cardiovascular disease (Comuzzie et al. 2003; Gardner and
Luciw 2008; Rogers and Hixson 1997; Roth et al. 2004). In addition to their utility
as models for humans, OWMs also exhibit several specific characteristics that moti-
vate studies of phenotypic and genetic variation.

Phenotypic Diversity in Disease Response

Many OWMs exhibit an unusually large geographic range relative to other primates
(macaques and baboons cover the largest geographic range of any primate genera,
other than humans) and also exhibit a high level of taxonomic diversity. As a result,
the OWM clade exhibits substantial ecological and phenotypic diversity. This diver-
sity extends to variation in susceptibility to infectious disease, including malaria
Genetic Variation in the Immune System of Old World Monkeys… 67

(Schmidt et al. 1977) and tuberculosis (Langermans et al. 2001), and is perhaps best
illustrated by the case of SIV/HIV. Surveys across OWM species indicate major
differences between the response to SIV in its natural African hosts and the response
to SIV in species that are not naturally exposed. Sooty mangabeys (Cercocebus
atys), a natural host for SIVsm (the strain from which HIV-2 in humans arose: Lemey
et al. 2003; Sharp et al. 1995), do not develop AIDS-like symptoms, despite robust
replication of the virus in the host upon infection. Comparisons between sooty
mangabeys and much more susceptible hosts, such as rhesus macaques, suggest that
the key to this difference may be reduced immune activation and/or reduced reli-
ance on T-cell-mediated immunity in the mangabeys (Silvestri et al. 2007). Infected
rhesus macaques also exhibit more dramatic T-cell depletion and SIV-specific anti-
body production in response to SIV infection than members of their sister species,
cynomolgus macaques (M. fascicularis) (Monceaux et al. 2007; Trichel et al. 2002).
Finally, variation in the response to SIV extends to the population level as well:
Chinese-origin rhesus macaques exhibit a slower rate of progression and a reduced
degree of immune activation compared to Indian-origin rhesus macaques (Ling
et al. 2002; Reimann et al. 2005).

Diversity in the Composition of Immune System Gene Families

Many of the components of primate immunity, such as Toll-like receptors,

immunoglobulin-like receptors, and major histocompatibility loci, can be grouped
into related classes of genes (Box 1). While the existence of these broad classes
tends to be conserved across primates, the number of genes per class and the levels
of polymorphism within these genes vary substantially between populations, spe-
cies, and species groups. This variation may contribute to observed species-specific
differences in immunity and disease. For example, the classical MHC class I genes,
HLA-A, HLA-B, and HLA-C, function in antigen presentation as part of the adap-
tive immune response to intracellular pathogens and are among the most polymor-
phic genes in the human genome (Shiina et al. 2009). Genetic variation at these loci
has been implicated in susceptibility to a broad suite of diseases, including HIV/
AIDS, hepatitis B, psoriasis, type 1 diabetes, and rheumatoid arthritis (Burton et al.
2007; Fellay et al. 2007; Fernando et al. 2008; Kamatani et al. 2009; Liu et al.
2008; Pelak et al. 2010). Compared to humans, the class I region of rhesus macaques
contains only orthologs for HLA-A and HLA-B and exhibits lower levels of poly-
morphism (Otting et al. 2005). In rhesus macaques, it appears that repeated seg-
mental duplications in this region (Daza-Vamenta et al. 2004; Kulski et al. 2004),
together with the presence of different combinations of HLA-A and HLA-B ortho-
log gene copies on a given haplotype, result in the expression of a diverse reper-
toire of class I antigens in the absence of very high levels of polymorphism
(Wiseman et al. 2009). The macaque example illustrates how structural polymor-
phism and sequence polymorphism can therefore serve as alternative solutions for
similar evolutionary challenges.
68 D.A. Loisel and J. Tung

Behavioral, Life History, and Ecological

Data from Field Studies of OWMs

OWMs are important behavioral and evolutionary models for humans and other spe-
cies that exhibit highly sophisticated social systems. Consequently, detailed longitudi-
nal and comparative data about the social structure, life history, and ecology of OWMs
in natural populations are available for many OWM species (for a partial summary of
these field studies, see Tung et al. 2010). These data permit the comparative study of
immune system evolution and its genetic effects in an ecological context, for example,
by relating socioecological characteristics that may influence exposure to, and spread
of, disease with molecular changes at immune loci. For instance, Nunn et al. (2000)
used such an approach in OWMs and other primates to show that species with more
promiscuous female mating patterns tended to have higher basal white blood cell
counts. They interpreted this result as a consequence of selection on the primate
immune system due to sexually transmitted disease. Indeed, in a follow-up study of 15
immune genes, Wlasiuk and Nachman (2010) identified stronger signatures of selec-
tion on branches leading to more promiscuous primate species, most notably for the
subset of genes whose function involves direct interactions with pathogens. Other
studies have focused on variation in the incidence of parasites and pathogens at differ-
ent geographic locations (Fooden 1994; Phillips-Conroy et al. 1988; Tung et al. 2009),
including how genetic effects contribute to these differences (Tung et al. 2009). In
humans, immune loci exhibit high Fst levels (a measure of the degree of overall genetic
variation explained by between-population differences) relative to the genome-wide
average, suggesting a history of local adaptation and selection (Akey et al. 2002;
Barreiro et al. 2008). Understanding when and whether this holds true in OWMs
could provide insight into how ecological differences between populations spur the
process of genetic differentiation between geographically separated groups.

Natural Selection in the Immune System of OWMs

Rapid and Repeated Evolution at Immune Loci

Parasites and pathogens have long been considered to be primary drivers of evolu-
tion. Indeed, parasite or pathogen infection can have severe consequences for the
health of wild primates and can exert strong selective effects (Keele et al. 2009;
Knauf et al. 2012; Sapolsky and Else 1987). Thus, genetic variation that increases
an individual’s ability to evade, overcome, or minimize the consequences of infec-
tion is also likely to be adaptive. Genes involved in the immune system, which often
evolve rapidly (including in, but not only in, primates: Hughes et al. 2005; Schlenke
and Begun 2003), are a natural place to look for such variants.
In primates, this idea has primarily been supported by studies of a small number
of candidate loci, such as those in the major histocompatibility complex (MHC)
Genetic Variation in the Immune System of Old World Monkeys… 69

(see Table 1 for examples of these genes). In the past decade, however, the avail-
ability of genome sequences for multiple primate species, including the rhesus
macaque, has facilitated larger-scale genome-wide studies that provide insight into
the general importance of adaptive change in the primate immune system. Typically,
these studies attempt to infer the presence of lineage-specific positive selection by
comparing rates of synonymous and nonsynonymous site evolution in a gene’s cod-
ing sequence. When more than two species are included in such a comparison,
mutations in genes that appear to have evolved under selection can be localized to
specific branches within the species tree (Yang 1997). Two recent studies—a three-
species analysis of selection in primates (human, chimpanzee, and macaque: Gibbs
et al. 2007) and a six-species analysis of selection in these three primates and three
additional mammals (mouse, rat, and dog: Kosiol et al. 2008)—demonstrated that
positive selection has indeed played an unusually important role, relative to other
functional groups of genes, in the evolution of immune response genes in OWMs,
as well as in other mammals. Genes involved in the “immune response” were sig-
nificantly enriched among positively selected genes in both studies relative to other
classes of genes, whether considering selection over the entire three-species primate
tree (Gibbs et al. 2007; Kosiol et al. 2008) or on the individual branches leading to
each species, which included rhesus macaque (Kosiol et al. 2008). Many of these
genes fell into subclasses that have been the target of previous candidate gene stud-
ies, including those whose protein products interact with MHC class I (e.g., LILRB1,
LAIR) and those involved in T-cell-mediated immunity (e.g., CD3E, TCRA) (Gibbs
et al. 2007; Kosiol et al. 2008). Interestingly, the overall rate of nonsynonymous to
synonymous changes on the macaque lineage appears to be somewhat slower than
the comparable rate in humans and chimpanzees (Gibbs et al. 2007), perhaps reflect-
ing more efficient negative selection for genes evolving under constraint. The abil-
ity of selection to influence phenotypic variation is correlated with effective
population size, which is larger in macaques than in humans or chimpanzees
(Hernandez et al. 2007). Potentially, therefore, positive selection may also be more
efficient in macaques and other OWMs with large effective population sizes. If true,
such a scenario would increase power to detect selective events in OWMs once a
larger number of genome sequences become available.
Interestingly, genes that have undergone selection somewhere in the primate tree
often have evolved under selection across more than one lineage. Sequencing of the
rhesus macaque genome, for example, identified 67 genes that carried signatures of
positive selection on all three branches (rhesus macaque, human, and chimpanzee:
Gibbs et al. 2007). Similarly, Kosiol et al. (2008) estimated that the majority of the
genes they identified as positively selected in any lineage (including rodents and
dogs) were identified as positively selected in more than one lineage. For instance,
glycophorin C (GYPC), an erythrocytic cell surface antigen, is believed to have
evolved under recent selection in humans (Wilder et al. 2009) in response to patho-
gen pressure from the malarial parasite Plasmodium falciparum. However, GYPC
also appears to have been positively selected on all examined branches of the pri-
mate tree, suggesting that this gene has been the target of pathogen-mediated adap-
tive evolution for a much longer period of time (Kosiol et al. 2008). Several
70 D.A. Loisel and J. Tung

Table 1 Summary of genes showing signatures of selection in Old World Monkeys

Immune Timescale of
Gene Function function Type of selection selection Reference
GYPA Erythrocyte Adaptive? Positive selection Macaque Baum et al.
surface lineage (2002)
receptor
MHC Class II Antigen Adaptive Balancing selection Intra-specific Alberts (1999);
gene DQA1 presentation (baboons) Loisel et al.
(2006)
MHC Class II Antigen Adaptive Balancing selection Inter-specific Loisel et al.
genes presentation (trans-species (baboons (2006)
(DQA1, polymorphism) and rhesus
DQB1, macaques)
DRB1, Inter-specific Doxiadis et al.
DPB1) (rhesus (2006)
macaques
&
cynomol-
gus)
PTPRC B- and T-cell Adaptive Positive selection Across Old Filip and Mundy
maturation World (2004)
and activation Monkeys
APOBEC3G Antiviral Innate Positive selection Across Old Sawyer et al.
DNA-editing World (2004);
enzyme Monkeys Zhang and
Webb (2004)
APOBEC3H Antiviral Innate Positive selection Across Old OhAinle et al.
DNA-editing World (2006)
enzyme Monkeys
APOL genes Parasite Innate Positive selection Across Old Smith and Malik
resistance World (2009)
Monkeys
β Defensin 2 Anti-microbial Innate Positive selection Across Old Sawyer et al.
defense World (2007);
Monkeys Boniotto
et al. (2003)
Lysozyme Anti-bacterial Innate Positive selection Across Old Messier and
activity World Stewart
Monkeys (1997)
MEFR Inflammatory Innate Positive selection Across Old Schaner et al.
response World (2001)
Monkeys
PKR Viral response Innate Positive selection Across Old Elde et al. (2009)
World
Monkeys
(continued)
Genetic Variation in the Immune System of Old World Monkeys… 71

Table 1 (continued)
Immune Timescale of
Gene Function function Type of selection selection Reference
Tetherin Antiviral Innate Positive selection Across Old Liu et al. (2010);
restriction World Lim et al.
factor Monkeys (2010c)
TLR4, TLR5, Pathogen Innate Positive selection Across Old Nakajima et al.
and other recognition World (2008);
TLR genes Monkeys Wlasiuk
et al. (2009);
Wlasiuk and
Nachman
(2010)
TRIM5 Antiviral Innate Balancing selection Rhesus Newman et al.
restriction macaques (2006)
factor and Sooty
mangbeys

nonhuman primate species are also susceptible to Plasmodium infection (Prugnolle

et al. 2010), raising the possibility that adaptive change in orthologous genes in
response to hematoprotozoan parasites may be common. As in the case of the gen-
eral pervasiveness of selection on the immune system, these findings reinforce the
suggestion from single-gene studies that selection in the primate immune system
may often be recurrent and episodic (Elde et al. 2009; Messier and Stewart 1997;
Schaner et al. 2001).

Biases in Current Studies of Selection: Where and How Does

Selection Operate?

Evidence from genome-wide analysis and studies of individual genes and gene
families suggests that positive selection has helped shape the evolution of gene
protein-coding sequences in OWMs. However, protein-coding sequence accounts
for only a small percentage of the genome. Both theoretical arguments and empiri-
cal evidence suggest that other components of the genome, especially those involved
in gene regulation, may also have been targets of selection (King and Wilson 1975;
Wray 2007; Wray et al. 2003). In particular, a recent meta-analysis of six scans for
selection (all of which included human, chimpanzee, and macaque data; some
included sequence from additional mammalian species as well) revealed a signifi-
cant enrichment for selection on genes involved in T-cell-mediated immunity in
both the coding and noncoding regions of these genes (Haygood et al. 2010; see also
Torgerson et al. 2009).
72 D.A. Loisel and J. Tung

Box 1 Major Targets of Natural Selection in Old World Monkeys

a Signatures of selection in patterns of genetic diversity b Evolution by duplication and divergence

Initial genetic diversity Genetic diversity after selection Ancestral gene
Positive
selection
Gene duplication

Balancing
selection

Diversification
via selection
No selection
(neutral)
No changes Changes in Changes in
in gene cis-regulatory coding
sequence sequence sequence

Episodes of natural selection leave characteristic signatures in the sequence

of gene targets (panel A). Many of the most compelling examples of natural
selection in OWMs involve genes that fall in one of a few well-studied fami-
lies (note that although genes involved in immunity commonly evolve under
selection across the tree of life, these families represent those that are both
present and well studied in primates). In addition to MHC class I and II genes
(discussed elsewhere in this chapter), these include:
APOBEC Proteins: APOBEC genes encode a family of proteins that inhibit
replication of HIV and other retroviruses (Neil and Bieniasz 2009). Their
antiretroviral function makes them prime candidates for coevolution with rap-
idly evolving viral pathogens. The evolutionary history of these genes is char-
acterized by gene duplication (e.g., the seven functional primate genes found
at the APOBEC3 locus resulted from a series of duplications from a single
ancestral APOBEC3 gene: Jarmuz et al. 2002) and by diversification via posi-
tive selection (panel B). Human–chimpanzee sequence comparisons revealed
that six APOBEC genes showed evidence for positive selection, while two
other evolved under purifying selection (Sawyer et al. 2004). In comparisons
involving OWMs, two of these genes (APOBEC3G and APOBEC3H) also
exhibited a significant excess of nonsynonymous changes, indicating a history
of positive selection (OhAinle et al. 2006; Sawyer et al. 2004; Zhang and
Webb 2004). The rapid expansion of this gene family in primates, combined
with evidence for recurrent selection, suggests that these genes may play a
particularly important role in primate antiviral immune defenses.
Defensins: Defensins are antimicrobial peptides that function in the innate
immune response against a broad spectrum of bacterial, fungal, and viral
invaders (Lehrer 2004). Like KIRs, the evolution of primate defensins is char-
acterized by repeated tandem gene duplication followed by gene sequence
diversification due to episodic positive selection (Das et al. 2010; Patil et al.

(continued)
Genetic Variation in the Immune System of Old World Monkeys… 73

Box 1 (continued)
2004; Semple et al. 2003), in contrast to many other innate immune system
genes (Mukherjee et al. 2009). In primates, evidence of positive selection has
been observed in genes from both the alpha and beta defensin families
(Boniotto et al. 2003; Das et al. 2010; Lynn et al. 2004; Semple et al. 2003),
and changes at positively selected sites have been shown to alter antimicrobial
activity in vitro (Antcheva et al. 2004).
Killer Cell Immunoglobulin-Like Receptors (KIRs): KIR genes encode
cell-surface receptors that interact with MHC class I genes to modulate natu-
ral killer cell and T-cell function, thus playing a central role in both innate and
adaptive immunity. Radiation of the KIR family, as well as genetic diversity
within specific KIR genes, may have evolved in response to either direct
interactions with pathogens or as a result of coevolution with their MHC class
I ligands (Hao and Nei 2005; Parham 1997). In OWMs, KIR family genes are
marked by extensive intraspecific allelic diversity and substantial interspecific
differences in KIR gene number and haplotype structure (Bimber et al. 2008;
Hershberger et al. 2005; Kruse et al. 2010; Palacios, et al. 2011).
Toll-Like Receptors (TLRs): TLRs are innate immune system genes that
are necessary for recognizing conserved patterns associated with pathogen
infection (e.g., lipopolysaccharide (LPS) in gram-negative bacteria). Although
TLRs share a general functional role, only some primate TLRs are associated
with strong signature of selection. In OWMs, for example, interspecific com-
parisons of TLR sequences in 10 genes indentified evidence for positive
selection at only three loci (TLR4, TLR1, and TLR8: Nakajima et al. 2008;
Wlasiuk and Nachman 2010), echoing variation in the selective histories of
this family in human populations (Barreiro et al. 2009).

The potential importance of immune-related selection on noncoding sequence is

reinforced by the case of the OWM MHC. Population genetic diversity at the MHC
DQA1 cis-regulatory region in wild baboons revealed a strong signature of balanc-
ing selection, with a deep split between two major and ancient allelic lineages
(Loisel 2007). Comparisons of sequence diversity in this region across multiple
OWM species indicated that the history of balancing selection identifiable in
baboons extends even deeper into primate evolution, likely predating the split of
baboons and macaques from their common ancestor (Loisel et al. 2006). This pat-
tern is unusual and strongly suggestive of a functional role for these differences.
Studies in other primates also indicate an important role for noncoding evolution in
the immune system. For instance, high levels of noncoding sequence diversity have
been characterized in a number of primate species at CCR5, the main entry point for
HIV-1 and poxviruses (Lalani et al. 1999; Mummidi et al. 2000), and changes sug-
gestive of clade-specific selection have been identified in the promoter sequence of
74 D.A. Loisel and J. Tung

TNF, a cytokine involved in acute inflammation (Baena et al. 2007). Recently, stud-
ies in cell lines derived from various primate species demonstrated widespread dif-
ferences in gene regulatory responses after LPS stimulation, some of which may be
a consequence of selection on gene regulation (Barreiro et al. 2010). Investigating
the role of regulatory sequence in the adaptive evolution of the immune system,
including how regulatory and coding changes may act in concert, remains an impor-
tant topic for future studies. Similarly, although maintenance of genetic diversity at
immune loci by means of balancing selection or frequency-dependent selection is
likely to be important (Charlesworth 2006; Dean et al. 2002; Garrigan and Hedrick
2003), we know relatively little about the extent to which this and other alternative
modes of selection play a role in OWM immune system evolution (genome-wide
studies have focused largely on positive selection; however, see Andres et al. 2009).
The generation of large-scale sequence data sets will be useful for addressing these
gaps in our current knowledge. For instance, relatively recent selective sweeps can be
localized using information about linkage disequilibrium (LD: the degree to which
genetic variants segregate non-independently due to physical linkage) across the
genome: recently selected loci exhibit more extensive LD with neighboring regions than
is typical genome-wide (Sabeti et al. 2006, 2007; Voight et al. 2006). Similarly, local
adaptation can be investigated by identifying genetic markers that show unusually high
levels of genetic differentiation between populations, relative to the genomic distribu-
tion of the same metric (Akey et al. 2004; Barreiro et al. 2008; Sabeti et al. 2006, 2007;
Voight et al. 2006). Both of these methods have identified a large number of candidates
for selection among human populations, including many immune-related loci (Barreiro
and Quintana-Murci 2010). Selective change on these time scales may also be common
in OWMs, especially among geographically widespread species. Indeed, functionally
important population-specific differences at MHC class I genes in rhesus macaques
(Otting et al. 2007) and at MHC class I (Kita et al. 2009) and class II genes in cynomol-
gus macaques (Ling et al. 2011; Sano et al. 2006) have already been described.
Additionally, in a broader comparison including a number of OWMs, Garamszegi and
Nunn (2011) found that both levels of MHC DRB allelic variation and rates of nonsyn-
onymous substitutions at this locus were correlated with parasite species richness
(defined as the total number of parasite species per host species) in 41 primates.

Functional Variation at Selectively Relevant Loci

A signature of selection implies the presence, either in the past or in contemporary popu-
lations, of functional genetic variation at the same locus. Thus, data that establish the
molecular- and organism-level effects of selected variants act as important corroborating
evidence for the adaptive history of a given region. Perhaps more importantly, they also
provide biological insight into the fitness-related effects of specific allelic variants.
Although establishing the functional relevance of specific genetic variants
remains challenging, a number of methods are now well established, particularly
those that focus on the molecular mechanisms that act as intermediate links between
genotype and organism-level phenotypes (Box 2). The combination of selection and
Genetic Variation in the Immune System of Old World Monkeys… 75

Box 2 Functional Characterization of Sequence Diversity

a Using reporter assays to test ability to drive transcription in vitro

Clone region of interest
into reporter construct
putative reporter
regulatory gene 1
region ele
All e2
el
reporter All ol
ntr
Transfect protein Co
into cells
plasmid Measure amount
vector of reporter protein

b Using allele-specific expression assays c Reconstructing the ancestral form of modern

to quanity mRNA expresion in vivo genes
Sequence gene from modern species
No functional
regulatory mRNA levels Modern
variant C equal for both species
1 HPILMFRNDAGMIL
upstream alleles (no 1 2 HPIRMFRSDAGMIL
C
of gene imbalance) 3 HPILMFRSDAGMIQ
mRNA 2 4 HPRLMFRSDAGWIL
Ancestral
species Computationally infer
3
Putative
regulatory
Increased
expression
A * ancestral sequence

variant
T
of one of the 4 A* HPILMFRSDAGMIL
A alleles (allelic
imbalance) Assess functional consequences
mRNA of ancestral sequence

Studies of natural selection or genotype-phenotype associations identify

regions of the genome harboring (or in strong linkage disequilibrium with)
putatively functional genetic variation. Genetic variants in coding regions
may affect protein structure, while those in regulatory regions can affect the
developmental timing, magnitude, and tissue distribution of expression. To
validate these effects, molecular experimental approaches are necessary.
Common approaches include:
Linking Cis-Regulatory Sequence Variation and Gene Expression In Vitro:
Reporter assays test the ability of regulatory sequences to drive transcription
in vitro (Chorley et al. 2008; Knight 2003). Allelic variants of a putative regu-
latory region are inserted into plasmid vectors containing a reporter gene that
lacks endogenous promoter activity (panel A). The resulting “constructs” are
introduced into an appropriate cell line and protein expression of the reporter
gene is measured, thus providing an indirect estimate of the ability of indi-
vidual sequences to drive gene transcription. See, for example, Loisel et al.
2006; Tung et al. 2009; Vallender et al. 2008a; Vallender et al. 2008b.
Strengths: Can be used to isolate the effects of specific alleles or mutations,
environmental and genetic backgrounds are controlled, and can be combined
with experimental stimuli to test for differences in basal expression among
alleles versus differences in expression after induction. Limitations: In vitro

(continued)
76 D.A. Loisel and J. Tung

Box 2 (continued)
behavior may not always reflect in vivo behavior (particularly when using
immortalized cells), reporter constructs are evaluated outside of the context of
normal chromatin architecture, cell lines that match the tissue and/or species
of interest may not be available, assays are low throughput.
Measuring Cis-Regulatory Effects via Allele-Specific Gene Expression
Assays In Vivo: Allele-specific expression assays quantify the relative abun-
dance of mRNA from the two alleles of a gene found within the same indi-
vidual (panel B) (Pastinen 2010; Yan et al. 2002). Differences in the expression
levels of these alleles imply the presence of variation in the cis-regulatory
region controlling that gene, which can be localized by testing for an associa-
tion between putative regulatory variants and the magnitude of allele-specific
gene expression across individuals (e.g., Tung et al. 2011).
Strengths: In vivo measurements reflect the natural behavior of cis-regula-
tory functional variants, amenable to studies of natural populations, environ-
mental and genetic background effects are controlled because comparisons
are conducted between alleles within individuals, potentially feasible on a
genome-wide scale (Fontanillas et al. 2010; Pastinen 2010). Limitations:
Putative regulatory variants are identified by association and are not experi-
mentally isolated, RNA samples may be difficult to obtain for the population
or tissue of interest, the magnitude of allele-specific effects may be modified
by epistatic or gene-environment interactions (de Meaux et al. 2005; Tung
et al. 2011; von Korff et al. 2009).
Reconstructing the Ancestral Forms and Functional Properties of
Modern Genes: Ancestral state reconstruction, which focuses on the infer-
ence and synthesis of sequences no longer found in contemporary popula-
tions (panel C), can be used to compare the functional consequences of
evolutionary changes on a longer time scale (Harms and Thornton 2010;
Thornton 2004). This approach permits the recreation of likely mutational
pathways leading to extant genes. See, for example, Goldschmidt et al. 2008;
Zhang and Rosenberg 2002.
Strengths: Permits experimental tests of the relationship between ancient
sequence changes and phenotypic change, series of reconstructed proteins can
indicate the probable sequence of mutations necessary to evolve novel func-
tions. Limitations: Requires gene-appropriate functional tests that do not nec-
essarily generalize well (i.e., assays for steroid hormone binding affinity work
only for steroid hormone receptors: Bridgham et al. 2009), dependent on the
accuracy of the inference method used to determine ancestral states, does not
take into account dependence of the focal gene’s function on other genes in the
ancestral genome or on the ancestral environment (Harms and Thornton 2010).
Genetic Variation in the Immune System of Old World Monkeys… 77

functional analysis provides a far more comprehensive perspective on the evolution

of immune genes than either approach alone and is therefore likely to become the
standard to meet. From an applied perspective, such analysis may identify immuno-
logically important genetic variation relevant to disease and pathogen resistance.
Here, we review examples that reveal the potential for such work in OWMs.

MHC Diversity and Molecular and Disease-Related Phenotypes:

Widespread and Repeated Evolution of an Immune Gene
Cluster Within and Between Species

Genes of the MHC function in the presentation of self and nonself peptides to T
cells and thus play a central role in the recognition of and response to pathogens and
parasites. Extensive study of MHC sequence diversity suggests that selection has
often contributed to shaping genetic variation at these loci (reviewed in Apanius
et al. 1997; Garrigan and Hedrick 2003; Hughes and Yeager 1998; Klein 1986;
Meyer and Thomson 2001). Evidence for adaptive evolution at OWM MHC loci
takes several different forms. At the population level, researchers have observed
allele frequency distributions that are inconsistent with those predicted by neutral
evolution (cynomolgus macaques: Bonhomme et al. 2007). On a deeper time scale,
comparisons of closely related species have revealed that some MHC polymor-
phisms seem to have been maintained since prior to divergence from the species’
common ancestor. These “trans-species” polymorphisms are unusual among dis-
tinct species but are relatively common among both class I (Otting et al. 2007) and
class II MHC genes (Doxiadis et al. 2006; Huchard et al. 2006; Loisel 2007), sug-
gesting that selection has acted to maintain segregating variation over long periods
of time (Klein et al. 2007). Finally, molecular evolution studies have shown that the
exons encoding the antigen-binding regions in MHC proteins consistently exhibit
elevated nonsynonymous to synonymous substitution ratios; this hallmark of strong
positive selection has been observed at several MHC class II genes in baboons
(Huchard et al. 2008; Loisel 2007) and in rhesus macaque class I genes (Urvater
et al. 2000). As MHC diversity on all three of these levels may contribute to
adaptively relevant phenotypic variation, these signatures of selection may harbor
important clues to the location of functionally important genetic variants.
For instance, Loisel et al. (2006) demonstrated that MHC population genetic
diversity is associated with functional consequences for gene regulation. The pro-
moter region of the classical MHC class II gene DQA1 is highly polymorphic in
many primate species, including yellow baboons, rhesus macaques, and pigtail
macaques, and shows evidence of trans-species polymorphism (Loisel et al. 2006).
As a result, the phylogenetic relationships among alleles for this region (the “gene
tree”) are not congruent with the phylogenetic relationships between OWM species
(the “species tree”), a pattern that is particularly unusual over such extended periods
of evolutionary time (~27 million years; Fig. 1). To assess the functional effects of
DQA1 promoter region variation, Loisel et al. (2006) cloned 12 of the alleles found
78 D.A. Loisel and J. Tung

Fig. 1 Evolutionary relationships of MHC-DQA1 exon 2 coding sequence in Old World monkeys.
(a) The allelic genealogy for MHC-DQA1 exon 2 sequences (containing the antigen-binding
region) in 12 species of Old World monkeys (OWMs) is characterized by the long-term mainte-
nance of ancestral allelic lineages across speciation events, a phenomenon known as trans-species
polymorphism. The incongruity between the DQA1 exon 2 gene tree and the OWM species tree
(shown in b.) reflects the presence of this trans-species polymorphism, in contrast to the typical
expected relationship between gene and species trees (shown in inset c.). The relationship between
DQA1 exon 2 sequences was inferred using neighbor-joining with evolutionary distances com-
puted using the Tamura-Nei method. The percentage of replicate trees in which the associated taxa
clustered together in the bootstrap test (1,000 replicates) is shown next to the branches. DQA1 exon
2 sequences were obtained from the IPD-MHC nonhuman primate database (Robinson et al. 2003)
Genetic Variation in the Immune System of Old World Monkeys… 79

in a contemporary yellow baboon population into a luciferase reporter construct and

introduced these constructs into a baboon lymphoblast cell line. The strongest pro-
moter allele, as reflected by increased production of the luciferase reporter gene,
drove gene expression twice as strongly as the weakest promoter allele—an effect
on DQA1 expression that is paralleled in humans (Fernandez et al. 2003; Haas et al.
2009; Morzycka-Wroblewska et al. 1997).
In addition to intracellular effects, OWM MHC variation may also influence
intercellular regulation of tissue composition. For example, the MHC class II DRB
region in cynomolgus macaques is characterized by gene duplication, high levels of
interspecies divergence, trans-species polymorphism, and extensive intraspecific
variation, which together strongly point to a history of natural selection (Leuchte
et al. 2004). Although the phenotypic outcomes of these effects are not well charac-
terized, at least one possible consequence includes an effect on the composition of
the peripheral blood, resulting from the role MHC molecules play in thymic lympho-
cyte (T cell) maturation. In support of this hypothesis, a recent study in captive cyno-
molgus macaques indicates that genetic variation at DRB is significantly associated
with interindividual variation in basal CD4+ T-cell counts (Aarnink et al. 2011).
By acting through regulatory, developmental, and structural intermediates such as
these, MHC genetic variation may contribute to variation in OWM organism-level
traits, particularly disease susceptibility (although MHC polymorphism has also been
suggested to influence behavior and fertility-related traits as well: Ober 1999; Ober
et al. 1998; Penn and Potts 1999; Ruff et al. 2011). Associations between MHC poly-
morphisms and diseases are common in humans, including a robust relationship
between MHC class I genetic variation and HIV infection and disease progression
(Fellay et al. 2007; Goulder and Watkins 2008; Pelak et al. 2010). Interestingly, work
on macaque models of HIV/SIV—a primate that, like humans, progresses to poten-
tially fatal AIDS-like symptoms—has revealed a parallel association between MHC
class I genes and SIV control and progression (Bontrop and Watkins 2005; Goulder
and Watkins 2008). In particular, specific alleles of the class I loci MHC-B and MHC-
A (orthologs of human HLA-B and HLA-A) were found to significantly influence the
viral set point and progression to disease in experimental SIV infections involving
rhesus macaques, cynomolgus macaques, and pig-tailed macaques. This parallelism
is remarkable considering the contrasting evolutionary histories of this region in
humans and macaques. Whereas macaque MHC class I diversity has largely been
driven by gene duplication events and relatively low allelic diversity within genes,
human MHC class I loci exhibit extraordinarily high levels of allelic variation. In both
cases, however, this genetic diversity appears to influence HIV/SIV control.

Functional Evolution at TRIM5: Interspecific and Intraspecific

Diversity in Response to Retroviral Threat

While the spread of HIV/SIV in humans is relatively recent, infection by retrovi-

ral pathogens in general has been ongoing for millions of years. Retroviral
sequences can be identified as “fossils” in the genomes of all fully sequenced
80 D.A. Loisel and J. Tung

primates (Gibbs et al. 2007; Han et al. 2007), suggesting that retroviruses have
exerted significant selective pressures on primate evolution for a very long time.
The evolution of the antiretroviral gene, tripartite motif protein 5 (TRIM5), illus-
trates the possible effects of these pressures. TRIM5 encodes a cytosolic protein
that restricts infection by HIV-1 and other retroviruses by interacting with retro-
virus capsid lattice (Stremlau et al. 2006). Through this interaction, TRIM5
restricts retroviral infection by acting as a pattern recognition receptor for the
capsid lattice and activating general innate immune signaling pathways (Pertel
et al. 2011). The TRIM5 gene is marked by numerous signatures of ancient and
recurrent natural selection within primates (Liu et al. 2005; Ortiz et al. 2006;
Sawyer et al. 2005, 2007), including both lineage-specific positive selection
(Johnson and Sawyer 2009) and long-term balancing selection, indicated by the
presence of trans-specific polymorphism between sooty mangabeys and rhesus
macaques (Newman et al. 2006) and between humans and chimpanzees (Cagliani
et al. 2010).
Research on natural selection at TRIM5 has been instrumental for understanding
the function of genetic diversity in the gene, which is high in both OWMs and
humans (Cagliani et al. 2010; Dietrich et al. 2010; Newman et al. 2006). For
instance, efforts to localize the specific nucleotide targets of positive selection in
TRIM5 indicated that two specific regions of protein, known as the B30.2 and
coiled-coil domains, were likely essential to its antiviral activity (Sawyer et al.
2005; Song et al. 2005b). Indeed, the ability of the human TRIM5α protein to
restrict the replication of retroviruses (including HIV-1, SIVagm, and murine leuke-
mia viruses) in vitro was significantly enhanced by mutating only a few amino
acids in these regions to the version found in rhesus macaques (Sawyer et al. 2005;
Yap et al. 2005). Thus, detailed analysis of selection at TRIM5 identified at least
some of the sites likely associated with species-specific differences in its ability to
restrict retroviral infection (Sawyer et al. 2005; Song et al. 2005a). Indeed, long-
term maintenance of TRIM5 alleles in rhesus macaques and sooty mangabeys may
have roots in retroviral resistance: allelic variation segregating within these species
also explains differences in the ability to restrict in vitro retroviral infection
(Newman et al. 2006; Wilson et al. 2008). This effect likely explains the finding
that specific TRIM5 alleles in rhesus macaques are associated with differences in
plasma viral load and disease progression following experimental SIV infection
(Lim et al. 2010a, b).

Genetic Variation at the Baboon FY Locus: Intraspecific

Functional Variation in Contemporary Populations

Finally, immune-related genetic variation may also have selective and functional
consequences observable within or between contemporary primate populations.
For instance, a recent study of a natural baboon population in Kenya identified a
significant association between genetic variation at the FY (DARC) locus and the
probability of infection by the hematoprotozoan parasite Hepatocystis (a close
Genetic Variation in the Immune System of Old World Monkeys… 81

relative of the malarial Plasmodium parasites) as well as unusually high levels

of population genetic differentiation at the locus among three East African
baboon populations (Tung et al. 2009). This association raised the possibility
that the evolution of FY in baboons might parallel that documented for humans,
in which a cis-regulatory genetic variant (i.e., a variant that influences the
expression levels of a linked gene: Wittkopp et al. 2004; Wittkopp et al. 2008)
upstream of the FY gene confers protection from Plasmodium vivax via eliminat-
ing expression of the gene on the erythrocyte surface. This variant has likely
been a target of selection in some human populations (Hamblin and Di Rienzo
2000; Hamblin et al. 2002). A comparable association in baboons also fell in the FY
cis-regulatory region, suggesting that a regulatory mechanism explained the relation-
ship between FY genetic variation and Hepatocystis incidence. Measurements of
allele-specific gene expression from blood-derived RNA samples from the same
baboon population indicated that functional cis-regulatory variation did indeed seg-
regate at this locus (see Box 2 for an explanation on inferring cis-regulatory effects
through allele-specific gene expression measurements). Specifically, in individuals
heterozygous for particular cis-regulatory variants, the two FY alleles for those indi-
viduals were expressed at different levels, suggesting that these variants (or nearby
linked variants) were responsible for driving FY expression at different rates. Using
promoter-based luciferase reporter constructs in cell culture, the authors were able to
confirm that a few base pair changes within this region were sufficient to alter gene
expression in the direction predicted by the allele-specific expression data. However,
unlike in humans, the variants identified through these screens did not completely
eliminate FY expression in erythrocytes nor did the association between FY variation
and Hepatocystis yield complete protection against the parasite (Tung et al. 2009).
Thus, the parallelism between FY and parasite infection in baboons and humans is
incomplete: instead of different mechanisms contributing to a shared association, as
in the case of MHC class I loci in human and rhesus macaque SIV/HIV, in this case
similar mechanisms yielded associations of different strengths.

Conclusions

Taken together, studies thus far on the genetics of OWM immunity reinforce a gen-
eral theme in evolutionary immunogenetics: adaptive change in the immune system
is pervasive, is recurrent, and takes a variety of forms. Specifically, studies of indi-
vidual immune-related genes reveal that selective regimes include both directional
selection and selection for maintenance of genetic variation (sometimes at the same
locus); that functional and/or selective divergence are apparent on multiple levels of
comparisons, including within populations, between populations, between species,
and between larger taxonomic groups; and that functional and selective change can
target both coding and noncoding regions.
We anticipate that newly developed high-throughput sequencing approaches will
make important contributions to extending this work, particularly functional anno-
tation of selectively important genetic changes. These methods allow data on
sequence variation and quantitative measurements of genome function, including
82 D.A. Loisel and J. Tung

gene expression levels, chromatin accessibility, and epigenetic patterns, to be col-

lected in tandem. For instance, comparative high-throughput sequencing efforts in
humans, chimpanzees, and rhesus macaques have already identified species-
specific, genome-wide changes in gene expression and isoform usage in the brain,
liver, heart, and kidney (Babbitt et al. 2010; Blekhman et al. 2010), as well as differ-
ences in histone methylation in immortalized cell lines (Cain et al. 2011). Combining
these assays with experimental exposure to immune-stimulating agents could also
yield insight into the basis for genetic differences in the response to particular
pathogens (e.g., Barreiro et al. 2010), a subject of great interest given the diversity
among OWMs in susceptibility to disease. The results of such work would allow
maps of functional changes to be overlaid on top of maps of targets of selection,
providing the first genome-wide maps of both functionally variable and selectively
relevant loci in the same species.
Ultimately, however, understanding genetic evolution and adaptation also
involves understanding the phenotypic variation that is the direct target of natural
selection. Thus, we wish to conclude by again highlighting the importance of the
extensive field data available for natural OWM populations. In particular, the natu-
ral ecological conditions that characterize particular OWM species and populations
have an important influence on the pathogens and parasites to which those individu-
als are exposed and thus on the evolution of the immune response. A second level
of integration—combining lab-based studies of functional variation and its conse-
quences with field-based studies of organism-level traits—therefore offers the
opportunity to understand not only the mechanistic consequences of selectively rel-
evant sequence variation but also its implications for ecologically relevant pheno-
typic variation. Research already underway illustrates the opportunities afforded by
combining field data with genetic approaches. These studies include research test-
ing for fine-grained correlations between pathogen and host genetic diversity (e.g.,
Schad et al. 2005; Schwensow et al. 2007 in lemurs); examining the relationship
between sociobehavioral characteristics, such as dominance rank, on the immune
response, including how these characteristics influence genotype-environment
interactions (Tung et al. 2011); and investigating the consequences of pathogen-
mediated selection on immune loci for other traits, such as mate choice (Ruff et al.
2011; Sommer 2005). Given the longitudinal nature of many primate field studies,
these systems also present important opportunities to test for trade-offs between
different arms of the immune system, as well as trade-offs between investment in
immune defense and growth, reproduction, and other forms of maintenance—two
major questions in ecological immunology (McDade 2003). Together, the integra-
tion of increasingly sophisticated genetic approaches into field-based ecological
studies promises to provide a compelling picture of OWM immune evolution in the
past and in the present day.

Acknowledgments J.T. was supported by a Chicago Fellows postdoctoral fellowship and

National Institutes of Health grants 1R01-GM102562 and P30-AG03442 (Center for Aging grant).
D.A.L. was supported by NIH grants F32HL095268 and T32HL007605. We thank J. Brinkworth
and two anonymous reviewers for helpful comments on an earlier version of this manuscript.
Genetic Variation in the Immune System of Old World Monkeys… 83

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Toll-Like Receptor Function and Evolution
in Primates

Jessica F. Brinkworth and Kirstin N. Sterner

Introduction

Despite close genetic relatedness, many primate species respond to infectious

pathogens (e.g., viruses, bacteria, protozoa, and fungi) differently (Table 1). Perhaps
most striking, many pathogen-induced diseases result in significant dysregulation
of immune responses in humans when compared to similar diseases in closely
related nonhuman primates (e.g., herpesviruses, Estep et al. 2010; Neisseria gonor-
rhoea, McGee et al. 1990; immunodeficiency viruses, Pandrea and Apetrei 2010;
Gram-negative bacterial sepsis, Redl et al. 1993; von Bulow et al. 1992; Fischer
et al. 1991; and hepatitis, Walker 1997). The most well-characterized example of
disparate primate disease progression is the discrepancy observed between the typi-
cal human and African Old World monkey responses to immunodeficiency viruses
(e.g., SIVs and HIVs). A long-term immunodeficiency virus infection in most
humans is characterized by massive loss of CD4+ T cells, immune cell irregulari-
ties, and overt immune activation associated with acquired immune deficiency syn-
drome (AIDS). Most African Old World monkeys, on the other hand, remain
comparatively symptomless despite high viral loads (Pandrea and Apetrei 2010;
Vodros and Fenyo 2004). Recent evidence suggests that simian immunodeficiency
virus may also be pathogenic in chimpanzees (Keele et al. 2009). The disparity in
immunodeficiency virus associated disease progression observed between African
cercopithecoids and hominoids is iterated in other pathogen-mediated diseases as
well, suggesting lineage-specific immune system evolution in primates. Humans

J.F. Brinkworth (*)

Department of Pediatrics, CHU Sainte-Justine Research Center, University of Montreal,
Montreal, QC, Canada
e-mail: [email protected]
K.N. Sterner (*)
Department of Anthropology, University of Oregon, Eugene, Oregon, USA
e-mail: [email protected]

J.F. Brinkworth and K. Pechenkina (eds.), Primates, Pathogens, and Evolution, 91

Developments in Primatology: Progress and Prospects,
DOI 10.1007/978-1-4614-7181-3_4, © Springer Science+Business Media New York 2013
 92

Table 1 Well-described disparities in primate responses to TLR-detected pathogens

Pathogen Human response and symptoms Nonhuman primate responses TLRa References
Neisseria gonorrhoeae Gonorrhea (urethral infection Chimpanzees: similar to human TLR2 Brown et al. (1972), Lucas et al.
with urinary pain, dis- response (1971), Massari et al.
charge, increased urination, Cercopithecoids (and other (2002), McGee et al. (1990)
fever, swollen testicles, and mammals): no symptoms
severe abdominal pain)
Schistosoma mansoni Schistosomiasis (diarrhea, Chimpanzees: similar to human TLR2 Farah et al. (2001), Farah et al.
abdominal pain, hematuria, response (2000), van der Kleij et al.
urinary pain, liver dysfunc- Baboons (Papio anubis): less (2002)
tion, and fibrosis) intense pathology, longer
term infections develop
periportal fibrosis
Toxoplasma gondii Toxoplasmosis (usually Capuchin monkey: mild, little TLR2 & TLR4 Bouer et al. (2010), Debierre-
subclinical, however mortality Grockiego et al. (2007),
immunosuppressed Callitrichids: 100 % mortality, Epiphanio et al. (2003),
individuals develop fever, anorexia, foaming nares, Poltorak et al. (1998),
anorexia, myalgia, lethargia, labored breathing Werner et al. (1969)
vomiting, and swollen Atelids: 20–80 % mortality, see Catão-Dias et al. (2013)
lymph nodes) chills, vomiting, lethargia,
and labored breathing
Gram-negative bacteria Severe sepsis/septic shock Chimpanzees: severe sepsis/ TLR4 Redl et al. (1993), Shi et al.
(e.g., Escherichia (high mortality, fever, septic shock (2010), von Bulow et al.
coli) neutropenia, organ Cercopithecoids: insensitive to (1992), Zurovsky et al.
dysfunction, hypotension, LPS, blunted febrile (1987)
and hypoperfusion) responses; requires biologi-
cally improbable levels of
LPS to initiate symptoms
Plasmodium falciparum Malaria (fever, chills, headache, Chimpanzees and gorilla: less TLR 2/1 Rayner et al. (2011), Zhu et al.
sweats, fatigue, nausea, susceptible; susceptible to (2011)
cough, enlarged spleen, and Plasmodium reichenowi but
J.F. Brinkworth and K.N. Sterner

pain) do not develop disease

Dengue virus Dengue (can be asymptomatic; Rhesus macaques: lower viral TLR7 Diebold et al. (2004),
or can cause dengue fever loads and no symptoms Onlamoon et al. (2010)
or the more life-threatening (although hemorrhage has
dengue hemorrhagic fever/ been induced with higher
dengue shock syndrome) doses)
Hepatitis C virus (HCV) Chronic liver disease (ranges Chimpanzees: often acute and TLR7 Reviewed in Bettauer (2010),
from mild to severe; can spontaneously cleared; De Vos et al. (2002),
lead to progressive liver self-limited; similar to mild Diebold et al. (2004), Major
fibrosis, cirrhosis, end-stage human cases (no chronic et al. (2004), Thomson et al.
liver disease, and cancer) active hepatitis, cirrhosis, or (2003)
cancer)
Macaques: not susceptible
Immunodeficiency AIDS (long-term infection Asian macaques: similar to TLR7 Benveniste et al. (1986), Daniel
viruses (SIV/HIV) associated with low viral humans et al. (1984), Diebold et al.
load, high HIV-specific Chimpanzees (Pan troglodytes (2004), Etienne et al.
cytotoxic T cells and schweinfurthii and P.t. (2011), Keele et al. (2009),
antibodies; followed by troglodytes subspecies): reviewed in Pandrea and
increasing viral load and increased likelihood of Apetrei (2010)
Toll-Like Receptor Function and Evolution in Primates

CD4+ T cell depletion, mortality; AIDS-like disease

immune cell irregularities, recently identified in wild
inflammation, and overt SIV+ animals; few captive
immune activation) P.t. verus cases.
Sooty mangabey and vervets:
long-term infection with high
viral load and without
AIDS-like symptoms
a
associated Toll-like receptor
93
94 J.F. Brinkworth and K.N. Sterner

and chimpanzees (Pan troglodytes) also share a higher susceptibility to Gram-

negative bacterial sepsis (Redl et al. 1993; van der Poll et al. 1994), schistosomiasis
(Abe et al. 1993; Sadun et al. 1970), and gonorrhea (Brown et al. 1972; Lucas et al.
1971; McGee et al. 1990), while Old World monkeys and other primates exhibit
reduced or null susceptibility. In addition, when infected with monkey B virus
(Cercopithecine herpesvirus 1/McHV1) macaques sometimes manifest mild muco-
sal blisters, whereas infected humans can quickly progress to encephalopathy
(Estep et al. 2010). Published observations of interspecies differences in the pro-
gression of these and other infections suggest lineage-specific immune responses
and potential divergence in immune function along major branches of Primates. As
immune system function plays a profound role in host survival and fitness, it is
important to not only characterize how these responses differ but also consider why
such variation exists.
Dysregulation of a host’s innate immune responses to infectious pathogens can
lead to increased pathogenicity. Such dysregulation can negatively influence host
function by causing either a reduction in innate immune activation or by allowing
for more prolonged/heightened immune responses (Bosinger et al. 2011; Hagberg
et al. 1984; Hoshino et al. 1999; Qureshi et al. 1999). Toll-like receptors (TLRs)
play an important role in the activation and regulation of innate immune responses
and profoundly affect host survival. As such, they make compelling targets when
studying the evolution of primate disease responses. Indeed, there is a growing body
of research on human and nonhuman primate immunity that suggests TLR-triggered
responses have not only diverged in these species but may also contribute to differ-
ing patterns of disease susceptibility and progression observed between primates
(Barreiro et al. 2010; Bochud et al. 2008; Feterowski et al. 2003; Hawn et al. 2005,
2007; Johnson et al. 2007; Mandl et al. 2008, 2011; Mir et al. 2012; Schroder et al.
2005; Vasl et al. 2008; Yim et al. 2004). In addition, intraspecific differences in
TLR-triggered responses have also been shown to influence disease progression and
severity in several human infections (e.g., Johnson et al. 2007; Oh et al. 2009). This
chapter reviews our current understanding of Toll-like receptor evolution in pri-
mates and discusses how an evolutionary perspective can help explain inter- and
intraspecies disparities in disease susceptibility.

TLRs and TLR-Triggered Innate Immune Responses

The immune system of vertebrate animals can be divided into the innate and the
adaptive immune responses. The innate immune system is an ancient, genetically
inherited and nonspecific response that forms the host’s first line of defense against
immune insult. The innate immune response is immediate, functions continuously,
requires no previous antigen exposure to be activated and is characterized by gener-
alized immune tactics such as inflammation, barriers (e.g., skin and mucosa), and
phagocytosis (reviewed in Janeway and Medzhitov 2002; Kumar et al. 2009). As the
immediate response to invading antigens and a modulator of adaptive immune
Toll-Like Receptor Function and Evolution in Primates 95

responses, innate immunity appears to exert great control over the course of infec-
tion. When excessive, the innate immune response can cause lethal pathologies in
the host (e.g., septic shock) (reviewed in Murdoch and Finn 2003). When weak or
disabled, initial infections grow unabated and adaptive responses exhibit dysfunc-
tion (Hagberg et al. 1984). As such, the innate immune system is an important
determinant of immune function and host survival.
Toll-like receptors represent one type of innate immune receptor and play a key
role in regulating innate immunity. The number of TLR types varies across animal
classes. Like some mammals, primates have ten functional TLRs. TLRs are horse-
shoe shaped, type 1 membrane proteins that contain three major functional domains:
an ecto- (extracellular) domain, a transmembrane domain, and a cytoplasmic
domain (Fig. 1). The ectodomain interacts directly with TLR ligands through a
series of leucine-rich repeats that facilitate protein–protein binding (Matsushima
et al. 2007). As type 1 membrane proteins, TLRs are bound to plasma membranes
and traverse either the cellular membrane (referred to as extracellular TLRs and
include TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10) or endosomal membranes
(referred to as intracellular TLRs and include TLR3, TLR7, TLR8, and TLR9). The
cytoplasmic region of all TLRs, however, is located in the cytoplasm and contains
an ultra-conserved region referred to as the Toll/IL-1 receptor (TIR) domain that is
important for intracellular signaling.
The recognition of self from nonself is a primary function of normal mammalian
immunity. TLRs are immune system sentinels that enable phagocytic cells (i.e.,
moncytes/macrophages, neutrophils, and dendritic cells) to detect nonself or
foreign antigens and respond accordingly. Specifically, TLRs recognize many
molecular motifs (referred to as pathogen-associated molecular patterns or PAMPS)
that are generally absent in vertebrate hosts but broadly shared across microbial
organisms (Jin and Lee 2008; Kawai and Akira 2007). Although TLRs have been
traditionally viewed as specialized sensors of broadly shared microbial molecules
(Table 2), some receptors appear to be more promiscuous and use co-receptors to
recognize a wider range of motifs. This is particularly true of extracellular TLRs.
For example, when TLR2 and TLR4 form heterodimers with either other TLRs or
other co-receptors (e.g., LY96) they can recognize motifs (e.g., lipomannan from
Mycobacterium or lipopolysaccharide from Escherichia coli) that they cannot rec-
ognize as homodimers or single receptors (Gioannini et al. 2004; Shimazu et al.
1999; Zahringer et al. 2008).
TLRs become activated upon TLR–ligand binding. Once activated, the TIR
domain of the cytoplasmic region recruits adaptor proteins and triggers signaling
cascades that initiate downstream transcription factor binding (e.g., NF-κB and
IFR7) (Kawai and Akira 2007, 2010). These transcription factors then induce the
production of cytokines and costimulatory molecules (reviewed in Kawai and Akira
2007; Palm and Medzhitov 2009). Cytokines nonspecifically degrade antigens as
well as initiate the activation of the epitope-specific T and B cell-mediated adaptive
immune response. The engagement of the adaptive immune response generally
occurs after the 4th hour of infection, with clonal expansion of T and B cells initi-
ated more than 96 hours post-infection. TLR signaling pathways appear to help
96 J.F. Brinkworth and K.N. Sterner

Fig. 1 Toll-like receptor and co-receptor cellular locations in primates. TLR2 is shown here het-
erodimerized with co-receptors TLR1 and TLR6, TLR4 with co-receptor LY96. Some (i.e., TLR2
and TLR4), possibly all TLRs, habitually exist as homodimers

regulate this arm of adaptive immunity (e.g., T-helper 1 and 2 type responses)
(Dabbagh et al. 2002; Krieg 2007). TLRs, therefore, are not only key components
in the early recognition of microbial invaders but also play a role in modulating the
adaptive immune response. As a result, they have the potential to strongly influence
and shape disease outcomes.
Table 2 Toll-like receptor location, ligands, and representative organisms
Cellular location TLR PAMP type PAMP Organisms
Cell membrane TLR1/2 heterodimer Lipoproteins/lipopeptides/GPI anchors/ Pam3Cys lipopeptides (Aliprantis et al. bacteria, mycobacteria,
lipoglycans 1999), Lipomannan (Quesniaux et al. Gram-positive bacteria
2004; Vignal et al. 2003),
TLR2 Peptidoglycan, Gram-positive bacteria,
glycosylphosphatidylinositol-anchored trypanosomes, yeast
proteins, lipoteichoic acid, porins,
zymosan (Campos et al. 2001; Massari
et al. 2002; Schwandner et al. 1999;
Takeuchi et al. 1999; Underhill et al.
1999)
TLR2/6 heterodimer Pam2CYs lipopeptides, MALP-2, PSM, Mycobacteria, Borrelia
(Bulut et al. 2001; Buwitt-Beckmann burgdorferi, Gram-
et al. 2005) positive bacteria
TLR4 Lipopolysaccharide (LPS), fungal Gram-negative bacteria
Toll-Like Receptor Function and Evolution in Primates

mannans, heat-shock proteins (Ohashi

et al. 2000; Poltorak et al. 1998; Tada
et al. 2002)
TLR5 Protein Flagellin (Hayashi et al. 2001) protists
TLR10 Unknown Profilin-like molecule (Yarovinsky et al. Toxoplasma gondii
2005)
Endosome TLR3 Nucleic acids Double-stranded RNA (Alexopoulou et al. dsRNA viruses
2001)
TLR7 Single-stranded RNA, Guanosine analogs ssRNA viruses
(Hemmi et al. 2002; Lund et al. 2004)
TLR8 Single-stranded RNA, imidazoquinolines ssRNA viruses
(Heil et al. 2004)
TLR9 CpG DNA motifs (Rutz et al. 2004) Bacterial DNA
97
98 J.F. Brinkworth and K.N. Sterner

Variation in TLR-Triggered Immune Responses

Between and Within Primate Species

Intra- and interspecies differences in TLR pathway function may explain within
and between species differences in susceptibility to TLR-detected pathogens.
Primates exhibit interspecies differences in immune response to a wide selection
of TLR-detected pathogens that are major agents of human diseases (Table 1).
Our current understanding of primate immune responses to such TLR-detected
pathogens is biased towards inferences drawn from DNA sequence data
(e.g., clinical association studies and phylogenetic analyses) and clinical/veteri-
narian observation, when available. Relatively few studies have directly exam-
ined primate TLR pathway function through controlled immunological challenge
experiments. Due to the clinical importance of such diseases as bacterial sepsis
and HIV/AIDS, most of these studies investigate primate TLR2, TLR4, and
TLR7. Moreover, controlled experiments almost entirely use catarrhine species
with studies biased towards the use of macaque, sooty mangabey, common chim-
panzee, baboon, and human models. Despite these limitations, however, current
data suggest both species- and family-level divergence in TLR pathway function.
Interestingly, a combination of controlled experimentation and veterinary/clinical
observations suggests a significant divergence between hominoid and cercopi-
thecoid species in response to TLR-detected pathogens in particular (Barreiro
et al. 2010; Brinkworth et al. 2012). Divergence in immune response has been
observed for a subset of TLR2-, TLR4-, and TLR7-detected pathogens dis-
cussed below.

Interspecies Divergence in TLR2 and TLR4 Function

A divergence between cercopithecoid and hominoid responses to TLR2- and TLR4-

detected pathogens is supported by a recent study (Brinkworth et al. 2012) that found
early chemokine and cytokine transcriptional responses of human and chimpanzee
total blood leukocytes to LPS (which is detected by TLR4, amongst other receptors),
Lipomannan from Mycobacterium smegmatis (TLR2/1 ligand), and Pam3CSK4
(TLR2/1 mimetic ligand) tend to be more similar to each other than to baboon
responses ex vivo. Perhaps the best-established evidence for divergence in hominoid
and cercopithecoid immune responses is to LPS and Gram-negative bacteria (Fischer
et al. 1991; Haudek et al. 2003; Shi et al. 2004, 2010; von Bulow et al. 1992; Zurovsky
et al. 1987). Humans and common chimpanzees are very sensitive to stimulation by
LPS/endotoxin and Gram-negative bacteria and are, therefore, highly susceptible to
Gram-negative bacterial sepsis, severe sepsis, and septic shock (reviewed in
Blackwell and Christman 1996; Redl et al. 1993). Severe sepsis is a systemic,
Toll-Like Receptor Function and Evolution in Primates 99

harmful, and unregulated proinflammatory innate immune response to immune

trauma (e.g., systemic bacterial infection, injury) that can involve the TLR4 pathway
(reviewed in Murdoch and Finn 2003). Severe sepsis in humans can be initiated by
4–10 ng/kg of LPS and is characterized by high fever, blood clots, organ dysfunction,
and, in severe cases, hypoperfusion and hypotension (septic shock). These symp-
toms are not observed in baboons or macaques under biologically probable condi-
tions. These Old World monkeys are comparatively less sensitive to LPS and
Gram-negative bacteria and fail to manifest fevers or broader symptoms of severe
sepsis/septic shock, even when very high doses are applied intravenously or to tis-
sues. For example, baboons require doses of LPS that are not likely to occur in nature
to establish minimal symptoms (100 ng/kg) and doses that are likely impossible to
establish via natural infection (6–25 mg/kg) to trigger deadly symptoms (Haudek
et al. 2003; Redl et al. 1993; Shi et al. 2004, 2010; von Bulow et al. 1992; Zurovsky
et al. 1987). Even when such high doses are artificially applied and baboons develop
severe sepsis or septic shock, they do not typically develop febrile responses.
Similarly, opportunistic observations of primate manifestations of TLR2-
detected pathogens suggest a divide between hominoid and cercopithecoid
responses, though these manifestations in nonhuman primates are comparatively less
well understood than sepsis. Neisseria gonorrhoeae, the causative agent of
gonorrhoeae, is a TLR2-detected pathogen that uniquely establishes itself in the
fallopian mucosa of humans and chimpanzees but not in cercopithecoids and other
mammals (Arko 1989; Massari et al. 2002; McGee et al. 1983, 1990). The species
specificity of N. gonorrhoeae-mucosa binding appears to be associated with
increased human and chimpanzee complement binding affinity to N. gonorrhoeae
porins (Ngampasutadol et al. 2008). Similarly, the TLR2-detected pathogen
Schistosoma mansoni causes diarrhea, liver dysfunction, and fibrosis in humans but
less intensively affects Papio anubis (Farah et al. 2000, 2001; van der Kleij et al. 2002).
TLR2 also detects Mycobacterium species. Some studies suggest that
Mycobacterium sp. infections progress more rapidly in cercopithecoids than in
hominoids, though these reports conflict and are affected by differences in colony
disease surveillance methods and long latency periods of such infections (Flynn
et al. 2003; Montali et al. 2001; Payne et al. 2011; Sapolsky and Else 1987; Tarara
et al. 1985; Walsh et al. 1996, 2007). For most case reports, the date of infection is
not known and is difficult to estimate within a margin of weeks. The few case stud-
ies available, in combination with a handful of deliberate Mycobacterium inocula-
tions suggest complex divergence in hominoid and cercopithecoid responses to this
genus of bacteria. Baboon and macaque species seem to exhibit rapid disease
progression, developing pulmonary inflammation and significant lung nodules
within 6 months of M. bovis or M. tuberculosis exposure (Garcia et al. 2004; Payne
et al. 2011; Sapolsky and Else 1987; Tarara et al. 1985). Rhesus macaques (Macaca
mulatta) have been described as being highly susceptible to very virulent manifesta-
tions of both M. bovis and M. tuberculosis compared to baboons and cynomolgus
macaques (Macaca fascicularis), respectively (Garcia et al. 2004; Langermans
et al. 2001; Walsh et al. 1996).
100 J.F. Brinkworth and K.N. Sterner

Interspecies Divergence in TLR7 Function

While some TLR2- and TLR4-detected pathogens appear to manifest very

differently in hominoids vs. cercopithecoids, there is also evidence suggesting
TLR7-viral interactions may vary considerably between African cercopithecoid
species. The sooty mangabey (Cercocebus atys), in particular, exhibits very diver-
gent TLR-initiated pathway function compared to other examined cercopithecoids.
Perhaps best known in biomedical research for being a natural host of the simian
precursor to HIV-2 (SIVsm), sooty mangabeys are well known to sustain lifelong
infections with high viral load without experiencing overt immune activation or
progressing to AIDS. Mandl et al. (2008) has provided ELISA data that suggests
that when the plasmacytoid dendritic cells (pDCs) of sooty mangabeys are stimu-
lated with SIV in vivo or TLR7 and TLR9 mimetic ligands ex vivo, they may
express dampened antiviral/anti-inflammatory IFNα responses. The pDCs from
SIV naïve hosts, such as rhesus macaque (Macaca mulatta), exhibit strong responses
that are more comparable to those seen in humans (Mandl et al. 2008). In the same
paper, these authors provide real-time PCR estimates of sooty mangabey IFNα
expression that are similar to rhesus macaque, suggesting that either the ELISA
capture antibody avidity differs between the IFNα of these species, or stark differ-
ences exist between sooty mangabey IFNα transcriptional activity and IFNα expres-
sion. Further examination of the reliability of the IFNα capture antibody used to
analyze sooty mangabey IFNα in this study and more tests for sooty mangabey-
specific mechanism of IFNα post-transcriptional regulation are needed.
While these results are conflicted, the observation that sooty mangabey and rhe-
sus macaque TLR7-triggered responses may differ is recapitulated in interspecies
differences in response to yellow fever virus (YFV). Yellow fever virus is a TLR7-
detected pathogen for which many African cercopithecoids serve as a natural reser-
voir including mangabey species (Cercocebus sp.) (World Health Organization
2004). YFV causes hemorrhagic fevers in humans and Asian monkeys (e.g.,
macaques) but does not pathogenically manifest in sooty mangabey hosts (Mandl
et al. 2011; Woodall 1968). When pDCs from sooty mangabeys, rhesus macaques,
and humans were stimulated ex vivo with live attenuated yellow fever virus 17 day
strain vaccine, the sooty mangabey pDCs expressed significantly less IFNα (Mandl
et al. 2011). When both monkey species were inoculated in vivo, sooty mangabeys
mounted a weaker innate immune response to YFV.
This pattern of weaker TLR-mediated overt innate immune activation in sooty
mangabeys appears to hold for viral/bacterial co-infection models and other cell
types as well. Monocytes from immunodeficiency virus infected mangabeys express
very low levels of pro-inflammatory cytokine TNFα in response to LPS ex vivo. By
contrast, monocytes from immunodeficiency virus infected humans and rhesus
macaques exhibited a strong TNFα response (Mir et al. 2012). Taken together, these
results suggest that TLR7-initiated signaling in the sooty mangabey can produce
dampened immune responses in comparison to humans and rhesus macaques.
Whether or not this trend is found in other African Old World monkeys remains to
be determined.
Toll-Like Receptor Function and Evolution in Primates 101

Intraspecific Variation

Taken together, these studies suggest that differences observed in primate immune
responses may be influenced by divergent TLR pathway signaling, but further con-
trolled studies of TLR-mediated immune function are needed in this area. It is also
important to consider that intraspecific variation in TLRs and TLR signaling plays an
important role in mediating immune responses between individuals (Table 3). Host
genetics have been shown to affect infection susceptibility, strength of inflammation
and infectious disease progression (Allikmets et al. 1996; Hawn et al. 2007; Jallow
et al. 2009; Kang et al. 2002; Ogus et al. 2004). Polymorphisms in both TLR genes
(Table 3) and TLR pathway genes (e.g., Orange and Geha 2003; Puel et al. 2004) have
been associated with alterations in primate infectious disease manifestation, although
most association studies connecting infectious disease progression and TLR signaling
focus on human TLR genes and their co-receptors specifically. While these data are
invaluable, there are less comparable data for nonhuman primates. One recent excep-
tion examined the impact of TLR7 polymorphisms on the degree of AIDS-like symp-
toms in immunized and unimmunized SIV-infected rhesus macaques and found that
unimmunized rhesus macaques that were carriers for two TLR7 polymorphisms (c.13G
and c.-17C alleles) survived SIV infection longer than unimmunized noncarriers
(Siddiqui et al. 2011). These findings suggest TLR7 polymorphisms may influence not
only disease progression but also vaccine efficacy in rhesus macaques and humans.

Understanding Why TLR-Triggered Responses

Differ Between and Within Primate Species

In order to explain why TLR-dependent responses vary in primates (see examples

above), it is necessary to examine species-specific patterns of pathogen exposure in
the context of those genetic and environmental factors that may drive or contribute
to divergent immune responses to shared pathogens. In addition, when building an
evolutionary framework for understanding disease susceptibility, it is important to
consider that these factors are likely interrelated and may be dynamic, varying in
both space and time. In the following section we review what is known about this
history of TLR-detected pathogen exposure in primates and how evolutionary pro-
cesses have shaped Toll-like receptor genes.

Evidence of Both Adaptation and Constraint

in Primate Toll-Like Receptors

Genes that encode proteins involved in the immune system are considered strong
targets of natural selection because of their relationship to an individual’s reproduc-
tive fitness and mortality (Barreiro and Quintana-Murci 2010; Nielsen et al. 2005).
102 J.F. Brinkworth and K.N. Sterner

Table 3 Polymorphisms in human Toll-like receptors with associated functional data

Receptor Polymorphismsa Corresponding pathogen-associated disease phenotypes
TLR1 N248S Associated with increased tuberculosis risk (Ma et al. 2007a)
and susceptibility to leprosy (Schuring et al. 2009)
P315L Impaired receptor function (Omueti et al. 2007)
Y554C Reduced NF-κB activation (Ben-Ali et al. 2011)
S602I Associated with increased tuberculosis risk (Ma et al.
2007a); 602S deficiencies in TLR1 trafficking to cell
surface and impaired cytokine and NF-κB responses
(Johnson et al. 2007); 602I increased lipopeptide-
triggered and basal NF-κB signaling (Hawn et al. 2007);
combination of 248S and 602S leads to impaired NF-κB
signaling, 602I increased signaling (Barreiro et al. 2009)
V651A Reduced NF-κB activation (Ben-Ali et al. 2011)
H720P Reduced NF-κB activation (Ben-Ali et al. 2011)
TLR2 T411I Reduced NF-κB activation (Ben-Ali et al. 2011)
P631H Reduced NF-κB activation (Ben-Ali et al. 2011)
R753Q Reduced NF-κB activation (Ben-Ali et al. 2011); abnormal
TLR2 signaling in Borrelia burgdorferi infection
(Schroder et al. 2005).
TLR3 P554S Causes TLR3 deficiency and predisposition to herpes
simplex encephalitis (Zhang et al. 2007)
TLR4 D299G Endotoxin hyporesponsiveness (Arbour et al. 2000);
T399I Endotoxin hyporesponsiveness (Arbour et al. 2000)
TLR5 Deletion 392–858 Increased susceptibility to Legionnaires’ disease and
inability to mediate flagellin signaling (Hawn et al. 2003)
TLR6 L128V Reduced NF-κB activation (Ben-Ali et al. 2011)
L194P Reduced NF-κB activation (Ben-Ali et al. 2011)
249S Associated with increased tuberculosis risk and reduced
NF-κB activation (Ma et al. 2007a)
A474V Reduced NF-κB activation (Ben-Ali et al. 2011)
N690T Reduced NF-κB activation (Ben-Ali et al. 2011)
Q708H Reduced NF-κB activation (Ben-Ali et al. 2011)
TLR7 Q11L Associated with increased HIV-1 susceptibility, higher viral
loads and hastened progression to late stage infection
(Oh et al. 2009)
SNP c.1-120T>G Associated with lower frequency of liver inflammation and
fibrosis in individuals chronically infected with hepatitis
C virus (Schott et al. 2007)
TLR8 SNP A1G (alters start; Protective effect on HIV disease progression and reduced
results in shorter NF-κB activation (Oh et al. 2008)
isoform)
TLR9 SNP G1174A (located Associated with rapid progression of HIV (Bochud et al.
in intron 1) 2007)
SNP T1237C (located Associated with increased susceptibility to malaria (Esposito
in 5′ region) et al. 2012)
SNP A1635G Associated with rapid progression of HIV (Bochud et al.
(synonymous 2007)
substitution)
a
amino acid changes unless noted as SNP
Toll-Like Receptor Function and Evolution in Primates 103

Because TLRs play an integral part in the innate immune response, amino acid
substitutions that are deleterious to receptor function and decrease a host’s fitness
should be selected against, resulting in strong signals of purifying selection at the
DNA level (Roach et al. 2005). Using population genetics and evolutionary genom-
ics, it is possible to identify patterns of sequence divergence within and between
species, respectively, and infer what role evolutionary processes (e.g., natural selec-
tion, genetic drift, and gene flow) have played in shaping these differences. A num-
ber of recent studies have examined the protein-coding sequences of primate
Toll-like receptors for evidence of adaptive evolution (Areal et al. 2011; Nakajima
et al. 2008; Ortiz et al. 2008; Wlasiuk and Nachman 2010a). Briefly, statistical tests
of neutrality are used to examine if patterns of genetic variation observed between
species deviate from those expected given more neutral models of sequence change
(Barreiro and Quintana-Murci 2010). In order to do this, a ratio of nonsynonymous
(dN) to synonymous (dS) nucleotide substitution rates (dN/dS) is used as an indica-
tor of selective pressure acting on a protein-coding gene. Using maximum
likelihood-based approaches, it is possible to test alternative models that allow
dN/dS to vary across sites and/or lineages (Delport et al. 2010; Kumar et al. 2009;
Pond and Frost 2005; Yang 2007). Once indicated, natural selection can be classi-
fied as either purifying selection (i.e., selection for the removal of deleterious
alleles), or positive selection (i.e., selection for the fixation of advantageous alleles).
When dN/dS is equivalent to 1, neutral evolution is assumed. Purifying or positive
selection is indicated by dN/dS < 1 and dN/dS > 1, respectively. These types of inter-
specific tests are useful for detecting older evolutionary events (such as those rele-
vant to differences observed between species), whereas intraspecific tests of
neutrality (reviewed in Barreiro and Quintana-Murci 2010) are better able to detect
more recent selective events. Both approaches have been used to study the molecu-
lar evolution of primate Toll-like receptors.
Although primate TLR genes are generally conserved and show functional
constraint, individual TLR domains show different degrees of underlying genetic
variation (Areal et al. 2011; Nakajima et al. 2008; Ortiz et al. 2008; Wlasiuk
et al. 2009; Wlasiuk and Nachman 2010a). It is important to note these studies
include different primate species, which may explain some inconsistencies in
their findings. That being said, patterns emerge from these and similar studies
that further our understanding of TLR molecular evolution in primates. For
example, the TIR domain of TLRs is highly conserved across primates and is
under strong purifying selection due to its role in intracellular signaling (Areal
et al. 2011; Nakajima et al. 2008; Ortiz et al. 2008; Wlasiuk et al. 2009; Wlasiuk
and Nachman 2010a). The extracellular or ectodomain domain of many TLRs,
on the other hand, is more divergent due to its role in ligand binding (Areal et al.
2011; Wlasiuk et al. 2009; Wlasiuk and Nachman 2010a). These findings are not
necessarily primate specific and may reflect more general trends of TLR evolu-
tion observed throughout mammalian (Areal et al. 2011) and avian (Alcaide and
Edwards 2011) evolution.
When more lineage-specific models of sequence evolution are applied, the distri-
bution of positively selected sites does not vary significantly across primates except
104 J.F. Brinkworth and K.N. Sterner

in the case of TLR4, where there is an increase in positively selected sites in

catarrhines (Nakajima et al. 2008; Wlasiuk and Nachman 2010a). Interestingly,
some of the substitutions considered targets of positive selection in TLR4 might
affect receptor function (i.e., an amino acid is replaced by another amino acid that
has a different charge, polarity, size, etc.). Moreover, several of the sites identified
by Wlasiuk and Nachman (2010a) as being under selection occur in the LPS and
LY96 co-receptor binding region identified by Park et al. (2009) and surround a
polymorphism (D299G) associated with resistance to Legionella pneumophila
infection and, less consistently, with an increased likelihood of developing Gram-
negative bacterial sepsis (Agnese et al. 2002; Barber et al. 2006; Feterowski et al.
2003; Hawn et al. 2005; Lorenz et al. 2002).
Population level genetic variation of Toll-like receptors has also been examined
in humans (including Barreiro et al. 2009; Ferrer-Admetlla et al. 2008; Mukherjee
et al. 2009; Wlasiuk et al. 2009; Wlasiuk and Nachman 2010a) and to a lesser
extent, chimpanzees (Wlasiuk et al. 2009; Wlasiuk and Nachman 2010a). Similar to
the interspecific tests described above, statistical tests of neutrality are used to
examine if allele frequencies observed within or between populations deviate from
those expected given more neutral models of sequence change (reviewed in Barreiro
and Quintana-Murci 2010). Such intraspecific data can be used to characterize the
level of genetic variation in human TLRs and reveal the type and degree of selective
pressures acting on these important immune receptors. These data in combination
with functional studies also help identify mechanisms of host defenses that may
vary between human populations or individuals. In general, patterns of within spe-
cies variation suggest purifying selection has had the greatest effect on the evolution
of human and chimpanzee TLRs (Wlasiuk and Nachman 2010a). Intracellular TLRs
(TLR3, TLR7, TLR8, and TLR9) have been subject to stronger purifying selection
than TLRs expressed on the cell surface, suggesting these receptors in particular
play essential and nonredundant roles in host survival (Barreiro et al. 2009; Wlasiuk
and Nachman 2010a). Alternatively, extracellular TLRs show more variation and
less constraint, which may reflect the ability of TLR2, TLR4, and their co-receptors
to identify multiple ligands (Kurt-Jones et al. 2000; Termeer et al. 2002; Zahringer
et al. 2008). When compared to chimpanzees, humans have a higher proportion of
deleterious mutations consistent with recent relaxation of selective constraint,
although greater sampling of chimpanzee population variation is needed (Wlasiuk
and Nachman 2010a).
Two haplotypes in the TLR10–TLR1–TLR6 gene cluster (i.e., these genes are
located on the same chromosome and found to be in strong linkage disequilibrium)
showed evidence suggesting positive selection (Barreiro et al. 2009). Interestingly,
one of the three nonsynonymous variants found in a common European haplotype
(H34; present in ~ half the population sampled) resulted in impaired NF-κB signal-
ing, which may limit damaging inflammatory responses (TLR1 602S; see Table 3).
Understanding patterns of genetic variation in TLRs within and between primate
species is of interest because variation observed today may reflect past relation-
ships between primates and their pathogens and explain why some individuals,
Toll-Like Receptor Function and Evolution in Primates 105

populations, or species are more/less vulnerable to certain pathogen-induced

diseases. While some have argued that variation in TLRs is evidence of long-term
coevolution between hosts and pathogens (Areal et al. 2011), Wlasiuk and Nachman
(2010a) suggest that such change is more episodic in nature and does not necessar-
ily reflect a continuous relationship between host and pathogen that persists over a
long time period. As sites within the extracellular domain (or ectodomain) are vari-
able in primates, it is tempting to speculate that selection may favor substitutions in
regions important for ligand binding that increase host fitness by improving the
recognition of host-specific pathogens. This variation may reflect adaptive responses
of hosts to species-specific pathogens or differences in species-specific pathogen
pressures. While sequence-based approaches are widely used to infer adaptive evo-
lution, whether or not these species-specific differences result in species-specific
TLR pathway function and actually increase fitness need to be tested experimen-
tally (as discussed in Barrett and Hoekstra 2011; and see Dean and Thornton 2007).
As a result, combining studies that address how TLR-dependent responses differ
between primates with those that seek to understand the context in which these
disparities exist will provide the most complete picture of the evolution of TLR-
depended responses in primates. In addition, while it has long been assumed that
variation in human TLRs is an adaptive response driven by pathogens (e.g.,
Fumagalli et al. 2011), genetic drift and geographic factors have also played an
important role in shaping genetic diversity in TLRs (TLR4, Ferwerda et al. 2008;
TLR4, Plantinga et al. 2012; and TLR5, Wlasiuk et al. 2009).

Which Pathogens Have Played the Largest Role

in Shaping TLR Gene Evolution?

While it is possible to target potentially adaptive genetic variation in primate TLRs,

it will be harder to link specific pathogens to specific genetic adaptations. Because
individual TLRs often bind different pathogens and there is some redundancy in
TLR signaling among cell surface receptors (Bafica et al. 2006; Kurt-Jones et al.
2000, 2004; Means et al. 2001; Ohashi et al. 2000; Ozinsky et al. 2000; Poltorak
et al. 1998), it is difficult to say which pathogen in particular has played the largest
role in shaping primate TLR genes and pathways. In addition, as we learn more
about TLR expression in the developing brain (Ma et al. 2007b; Sterner et al. 2012)
and gain a better appreciation of the interaction of TLRs and a host’s microbiome
(Cerf-Bensussan and Gaboriau-Routhiau 2010), it may be equally difficult to dif-
ferentiate what drives selection on these important, multi-purpose receptors.
Nevertheless, understanding the history of pathogen exposure in primates in the
context of the genetic variation observed today in TLR genes has the potential to
shed light on important aspects of human evolution (e.g., major shifts in diet, mating
strategies, social group systems, and habitat) and species-specific disparities in the
susceptibility to TLR-dependent diseases (e.g., HIV/AIDS).
106 J.F. Brinkworth and K.N. Sterner

Several TLR-detected pathogens appear to have been unevenly distributed

amongst primate species over the course of primate evolution due to geographic
barriers and primate behavioral ecology (e.g., diet, landscape use, and mating sys-
tems). Some authors have argued that these differences in pathogen exposure may
provide the basis for pathogen or PAMP-specific immune responses in different
primate species and populations (Galvani and Slatkin 2003; Martin 2003; Vitone
et al. 2004; Wlasiuk and Nachman 2010b). Increased exploitation of grasslands and
meat consumption, for example, have been proposed to have contributed to the
emergence of the progenitor of Mycobacterium tuberculosis in hominins 3 million
years ago (Gutierrez et al. 2005; Martin 2003). Habitual grassland use by species in
the Homo and Papio lineages may have routinely exposed these species to standing
watering pools contaminated with other ungulate pathogens such as the precursor
to the modern TLR2-detected Bacillus anthracis (anthrax) (Martin 2003;
Triantafilou et al. 2007).
The geographic distribution- of primate populations also influences primate–
pathogen interactions, as highlighted by Yersinia pestis. The causative agent of
bubonic plague, Y. pestis interacts with both TLR2 and TLR4 (Haensch et al. 2010;
Matsuura et al. 2010; Schuenemann et al. 2011; Sing et al. 2002). Now pandemic
and less virulent, historical Y. pestis potentially represents one of the strongest
agents of pathogen-mediated selection in human history. It is estimated to have
emerged in Asian human populations >2,600 years ago. Its spread to Europe via
trade routes killed nearly 30 % of the population by the year 1357, and approxi-
mately 15–20 % of the population again in 1665–1666 (Biraben 1975; Gottfried
1983; Morelli et al. 2010). A 3–5-day infection course, broad age range of hosts,
and high mortality suggests that historical plague epidemics have had a profound
effect on the genetic composition of modern Eurasian populations (reviewed in
Gage and Kosoy 2005). Since that time, it has re-emerged sporadically throughout
Eurasia, though with less virulence than the 1357 epidemic. Yersinia pestis does not
appear to have breeched Eurasian boundaries until very recently, which suggests
that the Y. pestis strain of the Black Death epidemic in the 1300s bypassed African
primate populations, African humans included (Morelli et al. 2010; Pollitzer 1951).
While not well investigated and inconsistently supported by functional studies
examining Y. pestis and host receptor interactions, selection by Y. pestis has been
pointed to as possible agent that “preselected” for immune receptor anomalies that
contribute to resistance to other infections in Eurasian populations (e.g., CCR5-
delta32, a deletion in C-C chemokine receptor type 5 that, when homozygous in
humans, confers resistance to HIV-1 infection; CCR5-delta32 allele is found in
~10 % of all Europeans) (Dean et al. 1996; Elvin et al. 2004; Galvani and Slatkin
2003; Lucotte 2001; Mecsas et al. 2004; Samson et al. 1996; Stephens et al. 1998).
The virulence of the pathogen, the significant biosafety requirements for its han-
dling, along with the an effective course of treatment for very early plague infec-
tions have meant that functional research into plague–receptor interactions has been
very limited. However, it seems an interesting candidate for examining the evolu-
tionary effects of uneven pathogen exposure across and within primate species to
virulent TLR-detected pathogens.
Toll-Like Receptor Function and Evolution in Primates 107

Conclusions and Future Directions

In this chapter we review our current understanding of TLR function and evolution
in primates and discuss how an evolutionary perspective can help explain why some
primate species differ in susceptibility to certain pathogen-driven diseases. As we
move forward, it is important to combine studies that address how TLR-dependent
responses differ between primates with those that seek to understand the genetic and
environmental context in which these disparities exist. Currently, there is little evi-
dence that directly links species-specific immune responses (e.g., those described
above) to fixed, species-specific TLR sequence differences identified as adaptive in
certain primate lineages. While this may suggest that sequence-based approaches
lack power to detect adaptive substitutions, it may also suggest that a more holistic,
pathway-based approach is needed. This is especially important when trying to
determine how and why the human innate immune response can become dysregu-
lated following TLR activation.

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Impact of Natural Selection Due to Malarial
Disease on Human Genetic Variation

Felicia Gomez, Wen-Ya Ko, Avery Davis, and Sarah A. Tishkoff

Introduction

An examination of malaria from an evolutionary perspective is important to under-

stand its etiology and effects on human genetic variation, including disease suscep-
tibility. In 1949, JBS Haldane proposed that individuals who are heterozygous for

F. Gomez
Department of Genetics and Biology, School of Medicine and School of Arts and Sciences,
University of Pennsylvania, Philadelphia, PA 19104, USA
Department of Anthropology, Hominid Paleobiology Doctoral Program, The George
Washington University, Washington, DC 20052, USA
Department of Anthropology, Center for the Advanced Study of Hominid Paleobiology,
The George Washington University, Washington, DC 20052, USA
Division of Biostatistics and Statistical Genomics, Washington University School
of Medicine in St Louis, 4444 Forest Park Blvd St. Louis, MO 63108, USA
W.-Y. Ko
Department of Genetics and Biology, School of Medicine and School of Arts and Sciences,
University of Pennsylvania, Philadelphia, PA 19104, USA
CIBIO, Research Center in Biodiversity and Genetic Resources, University of Porto,
4485–661 Vairão, Portugal
A. Davis
Department of Genetics and Biology, School of Medicine and School of Arts and Sciences,
University of Pennsylvania, Philadelphia, PA 19104, USA
Department of Biology, Swarthmore College, Swarthmore, PA 19081, USA
Graduate Program in Biological and Biomedical Sciences, Harvard Medical School,
Boston, MA 02115, USA
S.A. Tishkoff (*)
Department of Genetics and Biology, School of Medicine and School of Arts and Sciences,
University of Pennsylvania, Philadelphia, PA 19104, USA
e-mail: [email protected]

J.F. Brinkworth and K. Pechenkina (eds.), Primates, Pathogens, and Evolution, 117
Developments in Primatology: Progress and Prospects,
DOI 10.1007/978-1-4614-7181-3_5, © Springer Science+Business Media New York 2013
118 F. Gomez et al.

thalassemic traits may be more fit in malaria-endemic environments than wild-type

individuals (e.g., “The Malaria Hypothesis”; Haldane 1949). Since that time, many
scholars have shown that the distribution of thalassemias and other hemoglobin
variants is consistent with Haldane’s idea that resistance to malaria can maintain
otherwise deleterious mutations in malaria-endemic environments. Haldane’s foun-
dational hypothesis has led to the widely accepted notion that malaria is one of the
most important selective pressures in recent human evolution.
The purpose of this chapter is to provide an overview of genetic variants that may
confer resistance to malarial disease. These include variants that affect the red blood
cell (RBC), variants within endothelial receptors involved in malaria pathogenesis,
as well as genetic variants that affect immune functions involved in resistance to
malarial disease. For each of the genes discussed here, we review patterns of genetic
variation at these loci and signatures of natural selection. We also briefly review
some of the pathways and mechanisms that are thought to provide resistance or
protection from disease. Finally, we argue that studying our genome as a whole—
rather than looking at specific candidate loci—holds potential for the discovery of
new variants that confer resistance to malarial disease.

Malaria and Its Life Cycle

Malaria is a parasitic disease caused by species in the genus Plasmodium, which is

a member of a large phylum called Apicomplexa that includes parasites responsible
for many life-threatening human and animal diseases including toxoplasmosis,
cryptosporidiosis, coccidiosis, and babesiosis (Roberts and Janovy 2005). Several
Plasmodium species—including P. falciparum, P. ovale, P. malariae, and P. vivax—
infect humans. There have also been reports that P. knowlesi, a species that gener-
ally infects monkeys, can infect humans. However, infections from P. knowlesi are
rare (Van den Eede et al. 2009; van Hellemond et al. 2009).
Plasmodium parasites are transmitted by female Anopheles mosquitoes (Fig. 1).
When a female mosquito takes a blood meal from an infected human, the mosquito
ingests the malaria parasites along with its blood meal. The parasites subsequently
develop in the mosquito, and when the mosquito takes its next blood meal the devel-
oped parasites are injected with the mosquito’s saliva into a new individual, thus
transmitting the disease from one human to another. Once the parasites have entered
the bloodstream, they enter hepatic (liver) cells. In hepatic cells, the parasites
undergo a brief cycle of asexual reproduction and develop further into preerythro-
cytic parasites. The hepatic cells then rupture, and the parasites go on to infect the
host’s erythrocytes (red blood cells). At this point, the host has entered the erythro-
cytic stage of infection. This is the stage of infection when common symptoms
associated with malaria, including high fevers and chills, occur. Additionally, when
in the red blood cell, the parasite completes the asexual portion of its life cycle.
It produces gametes, which are ultimately taken up by a mosquito to begin the
infective process again (Fig. 1).
Impact of Natural Selection Due to Malarial Disease on Human Genetic Variation 119

Fig. 1 P. falciparum life cycle in a human and Anopheles mosquito. A Plasmodium-infected mos-
quito injects parasites into the human host during a bite that initiates a blood meal. Following the
initial bite, the parasites migrate to the liver where they invade hepatic cells. The parasites develop
within hepatic cells and are then released into the bloodstream, where they invade erythrocytes.
During the erythrocytic stage of infection, the parasites develop and replicate. Some parasites will
go on to infect other red blood cells (merozoite form). Other intraerythrocytic parasites will
develop into male and female gametes. Gametes are taken up by a mosquito during feeding and
will fuse in the mosquito gut to become a zygote. The zygote develops within the mosquito and
eventually migrates to the mosquito salivary gland, from which it will be injected into a human
host during the next blood meal (adapted from Cowman and Crabb 2006)

Phenotypically, malarial disease symptoms include fever, headaches, and vomit-

ing, which typically occur between a week and a half and 2 weeks after infection via
mosquito bite (WHO 2010). Malaria infection is heterogeneous, manifesting itself
differently among patients and at different points in the life cycle of the parasite.
“Severe malaria” is characterized by high fever and anemia due to an accumulation
of parasitized erythrocytes and subsequent oxygen deprivation of vital organs.
120 F. Gomez et al.

“Cerebral malaria” is a particular form of severe malaria characterized by reduced

oxygen supply to the brain resulting in coma (Chen et al. 2000).
Severe malaria is also characterized by parasite cytoadherence and sequestration.
Cytoadherence occurs when parasitized RBCs (PRBCs) adhere to specific host
proteins (e.g., ICAM-1, CD36, CD31, E-selectin, or/and VCAM) on microvascular
endothelium. Cytoadherence can also occur between PRBCs and noninfected red
blood cells—this process is called “rosetting.” Sequestration is the result of cytoad-
herence. It occurs when PRBCs adhere to the vascular endothelium and the cells are
sequestered from peripheral circulation. Sequestration and rosetting cause accumu-
lation of both infected and noninfected red blood cells, resulting in blocked blood
flow and limited oxygen supply.
Plasmodium falciparum is the most virulent of the Plasmodium species that
infect humans and it causes the largest number of deaths worldwide (Wahlgren
1999; WHO 2010). Although a cosmopolitan disease at one time, falciparum
malaria is now mainly concentrated in Sub-Saharan Africa, Central, South, and East
Asia, and parts of Central and South America (Hay et al. 2009). It has been esti-
mated that there are 2.37 billion people at risk for infection by P. falciparum world-
wide; ~47.48 % of those individuals are in Africa, ~49.58 % are in Central, South,
and East Asia, and the remaining ~2.94 % are in the Americas (Guerra et al. 2008).

Genes Involved in Malaria Resistance

In the sections to follow, we discuss specific genes that play potential roles in resis-
tance to malaria. We describe the genes, discuss the patterns of genetic variation at
these loci, and describe evidence for natural selection at each locus. We also review
some of the suggested mechanisms for protection or resistance that are associated
with genetic variants at these genes.

Erythrocyte Genetic Variation and Malaria

Hemoglobin Variants

Adult hemoglobin is comprised of four globin chains (two α and two β chains). These
chains are encoded by the α-globin genes, HBA1 and HBA2, and by the β-globin
gene, HBB. Hundreds of polymorphisms that cause hemoglobinopathies (disorders
of hemoglobin) have been identified worldwide. There are two broad categories of
hemoglobinopathies. These categories include pathologies that affect the structure of
hemoglobin, namely HbS, HbC, and HbE, and pathologies that are related to the
production of hemoglobin or the regulation of hemoglobin production (i.e., thalas-
semias, which will not be discussed here). Hemoglobinopathies were among the first
genetic disorders to be associated with malarial disease because of the strong correla-
tion between the global distribution of many of these diseases (and the genetic vari-
ants that cause them) and the current or historic distribution of malarial disease.
Impact of Natural Selection Due to Malarial Disease on Human Genetic Variation 121

HbS

HbS is the mutation that causes sickle-cell anemia, which is one of the most com-
mon hemoglobinopathies associated with protection from malaria. HbS is caused
by a point mutation in the HBB gene that results in a glutamic acid to valine substi-
tution at codon 6. Erythrocytes with this form of hemoglobin are prone to deform
into a characteristic sickle shape. When this mutation is in its homozygous form
(HbSS), the formation of sickled red blood cells is quite serious and can often be
fatal or extremely debilitating (Ashley-Koch et al. 2000). However, in the heterozy-
gous form, HbAS, there are very limited clinical symptoms. These individuals are
relatively healthy and benefit from protection from malaria. Indeed, studies have
confirmed that HbAS is protective against severe and lethal malaria (Aidoo et al.
2002; Williams et al. 2005; Williams 2006).
The HbS allele is widespread over areas of the world where malaria is, or was,
historically endemic. It is especially common in Sub-Saharan Africa, where it
reaches frequencies of 15–20 % (Weatherall 2001; Allison 2009; Verra et al. 2009).
The mechanisms by which HbS confers protection against P. falciparum malaria are
not well understood. Some common hypotheses postulate that HbS hemoglobin may
interfere with parasite growth in RBCs (Nagel and Roth 1989) or that HbS could
enhance splenic clearance of PRBCs (Shear et al. 1993; Roberts and Williams 2003;
Kwiatkowski 2005). It has also been suggested that HbS is protective against malaria
because the mutation causes an increase in the concentration of heme and conse-
quently heme oxygenase-1 (HO-1). HO-1 is an enzyme that catabolizes free heme
and has been shown to be protective against a number of diseases including cerebral
malaria (Pamplona et al. 2007; Ferreira et al. 2011). Recently, using a mouse model,
Ferreira et al. (2011) confirmed the protective effects of HO-1 against malaria and
showed that the protective effect of HbS occurs irrespective of parasite load. In
another recent study, Glushakova et al. (2010) showed that sickled RBCs may be
protective against malaria because they do not support parasite replication. In their
study, they showed that infected HbS RBCs have inefficient parasite egress or an
aborted parasite life cycle. It has also been hypothesized that the protective mecha-
nism of the HbS allele may involve the disruption of cytoadherence and sequestra-
tion. Compared to HbAA-infected erythrocytes, HbAS-infected erythrocytes show a
54 % reduction in adherence to vascular endothelium (Cholera et al. 2008).
The HbS mutation is associated with five “classical” haplotypes that are defined
by different RFLP (restriction fragment length polymorphism) patterns across a
70-kb region surrounding HBB. These haplotypes have different geographic distri-
butions and are named accordingly—Benin, Bantu (central Africa), Cameroon,
Senegal, and Arab (Hanchard et al. 2007). Because the HbS mutation is known to
occur on several haplotype backgrounds, some have suggested that this mutation
may have arisen multiple times (Pagnier et al. 1984; Nagel et al. 1985; Chebloune
et al. 1988). However, recombination and gene conversion have also been suggested
as alternative explanations for the occurrence of HbS on multiple haplotype back-
grounds (Orkin et al. 1982; Webster et al. 2003; Hanchard et al. 2007).
Aside from the well-documented correlation between the distribution of HbS
and P. falciparum malaria (Fig. 2), there is additional evidence of recent positive
122 F. Gomez et al.

Fig. 2 A global map of the spatial limits and endemic levels of P. falciparum malaria and the
geographic distribution of malaria susceptibility alleles. Hyper-holoendemic=areas where childhood
infection prevalence is 50 % or more of population; mesoendemic = areas where childhood infection
prevalence ranges between 11 and 50 %; hypoendemic = areas where childhood infection prevalence
is ≤10 %. Areas where only P. vivax is prevalent and the spatial limits of malaria transmission are
also shown. (Adapted from Snow et al. 2005.) Allelic distribution of HbS, HbC, HbE, Fy*O allele,
Southeast Asian Ovalocytosis (SAO), and Pyruvate Kinase (PK) deficiency overlay the map of
malaria endemicity

selection at this locus. Using 20 high-frequency SNP (single nucleotide polymor-

phism) markers across a 414-kb region of the HBB locus in Gambian, Jamaican, and
Yoruban individuals, Hanchard et al. (2007) demonstrated a high degree of haplo-
type similarity among HbS haplotypes. This extended haplotype homozygosity was
primarily observed for the Benin haplotype but was also observed for other HbS
haplotypes. The same pattern of extended haplotype homozygosity on HbS haplo-
types was observed by Liu et al. (2009). In addition to evidence of recent selection,
there is evidence for a recent origin of the HbS allele (Table 2), which suggests that
this allele could have arisen ~10,000 years ago when selection due to malaria is
expected to have increased (Livingstone 1958; Wiesenfeld 1967; Livingstone 1971).
Indeed, the selection coefficient is inferred to be ~0.15, which is amongst the high-
est in humans (Currat et al. 2002; Hedrick 2011).

HbC

HbC is another mutation that causes a structural change in hemoglobin. The HbC
allele causes a glutamic acid to lysine substitution in codon 6 of HBB, the same
codon at which the HbS substitution occurs (Table 1). This mutation is primarily
found in the malaria-endemic regions of West Africa and is much less common than
Table 1 Genetic polymorphisms, adaptive consequences, and associated pathologies
Sequence alteration/ Adaptive Associated pathology
Alleles haplotype consequences and severity References
Erythrocyte
Hemoglobin structural variants
HBB
HbS Glutamic acid/valine HbS heterozygotes HbS homozygotes Kwiatkowski (2005),
substitution at codon 6 have >90 % have sickle-cell Williams et al.
protection lethal disease, which can (2005), Williams
malaria (study be fatal and (2006)
conducted in debilitating; HbS
Kenya) heterozygotes have
mild to minimal
pathologies
HbC Glutamic acid/lysine HbC heterozygotes HbC homozygotes Modiano et al. (2001b),
substitution at codon 6 have 29 % have mild clinical Diallo et al. (2004)
reduction in risk symptoms; HbC
for clinical malaria; heterozygotes are
HbC homozygotes relatively healthy
have 93 %
reduction in risk
(study conducted in
Burkina Faso)
HbE Glutamic acid/lysine HbE heterozygotes HbE homozygotes Rees et al. (1998),
substitution at codon have less parasitic usually have Chotivanich et al.
26 invasion than HbE symptomless (2002)
homozygotes and anemia; HbE
Impact of Natural Selection Due to Malarial Disease on Human Genetic Variation

other red blood heterozygotes are

cells (study generally
conducted in asymptomatic
Thailand)
(continued)
123
 124

Table 1 (continued)
Sequence alteration/ Adaptive Associated pathology
Alleles haplotype consequences and severity References
Erythrocyte surface variants
Spectrin (SPTA1) Many alleles Missense mutations; In vitro studies show Elliptocytosis—elliptic Palek (1987), Gallagher
insertion/deletion mutation SPTA1 in RBCs that can and Forget (1996),
polymorphisms; that cause result in hemolysis Dhermy et al.
frameshift mutations elliptocytosis (2007)
resulted in 42 %
reduction in
parasite invasion
SLC4A1 SAO (Southeast 27-base pair deletion In vitro studies show Ovalocytosis—uncom- Cortes et al. (2004),
Asian (SLC4A1D27) causes reduced parasite mon form of Cortes et al. (2005)
ovalocytosis) a nine amino acid invasion; also elliptocytosis;
deletion protection from thought to be lethal
cerebral malaria
DARC (Duffy FY*A/FY*B; Glycine/aspartic acid Fy*O prevents P. vivax Occasional clinical Miller et al. (1976),
antigen Promoter SNP substitution at codon erythrocyte significance but no Mercereau-Puijalon
receptor for 42; T/C SNP at −33 invasion significant and Menard (2010)
chemokines) disrupts erythroid pathologies
expression (Fynul)
ABO O (O01/O02) 1-Base deletion in exon 6 Rosetting is signifi- No significant Carlson and Wahlgren
(D261) shared in O01 cantly reduced in pathologies (1992), Olsson and
a O02;O01 and O02 type O individuals Chester (1996),
differ by 10 mutations Rowe et al. (2007),
in exons 6 and 7 and Calafell et al.
F. Gomez et al.

intron 6 (2008)
 Sequence alteration/ Adaptive Associated pathology
Alleles haplotype consequences and severity References
Erythrocyte enzyme variants
G6PD A- Asparagine/aspartic acid In vitro studies show A− variant cause a Cappellini and Fiorelli
at codon 156; valine/ that parasite 10–15 % reduction (2008), Johnson
methionine at codon98 growth is slower in in G6PD enzyme et al. (2009)
G6PD deficient activity, which
RBCs reduces RBC
ability to counteract
oxidative stress
PKLR Many alleles SNPs, insertion/deletion In vitro studies show RBC PKLR deficiency Zanella et al. (2005),
polymorphisms; frame reduced invasion of can cause Ayi et al. (2008)
shift; splice site PKLR deficient hemolytic anemia;
mutations RBCs there is a wide
variety in clinical
severity of PKLR
deficiency
Endothelial receptors for cytoadhesion and sequestration
ICAM-1 ICAMKilifi/rs5491 Lysine/methionine at In vitro studies show No significant Fernandez-Reyes et al.
codon 29 that ICAM-1Kilifi can pathologies (1997), Adams
alter the binding et al. (2000), Tse
ability of some et al. (2004)
strains of P.
falciparum;
association studies
are inconsistent
(studies in Africa
and Asia)
(continued)
Impact of Natural Selection Due to Malarial Disease on Human Genetic Variation
125
 126

Table 1 (continued)
Sequence alteration/ Adaptive Associated pathology
Alleles haplotype consequences and severity References
rs5498 Lysine/glutamic acid at Association studies are No significant Amodu et al. (2005),
codon 469 inconsistent pathologies; Ma et al. (2006),
possible associa- Puthothu et al.
tions with Type 1 (2006)
diabetes and
asthma
CD36 T1264G/ Tyrosine/stop Association studies are CD36 deficiency Aitman et al. (2000),
rs3211938 inconsistent Fry et al. (2009)
Immune system variants
HLA
HLA-B (MHC- Bw53 haplotype Associated with No significant Hill et al. (1991), Hill
class I) reduced risk for pathologies et al. (1992)
severe malaria
(study conducted in
The Gambia)
HLA-DR (MHC DRB1*1302 haplotype Associated with No significant Hill et al. (1991)
class II) reduced risk for pathologies
severe malaria
(study conducted in
F. Gomez et al.

The Gambia)
 Sequence alteration/ Adaptive Associated pathology
Alleles haplotype consequences and severity References
TNF
−238 G/A SNP at −238 Multiple association Associations with Knight et al. (1999),
studies with many autoimmune Ubalee et al.
conflicting results; (e.g., rheumatoid (2001), Bayley
some suggest arthritis, asthma, et al. (2004), Flori
protection some and multiple et al. (2005)
−308 G/A SNP at −308 suggest increased sclerosis) and McGuire et al. (1994),
susceptibility infectious diseases Wattavidanage et al.
but no consistency (1999), Aidoo et al.
across studies (2001), Ubalee
et al. (2001), Meyer
et al. (2002),
Bayley et al.
(2004), Flori et al.
(2005)
−376 G/A SNP at −376 Knight et al. (1999),
Bayley et al. (2004)
Impact of Natural Selection Due to Malarial Disease on Human Genetic Variation
127
128 F. Gomez et al.

HbS. As described by Diallo et al. (2004), HbC is associated with phenotypes that
are much less severe than HbS. HbCC homozygotes are described as having mild
anemia and heterozygotes (HbAC) are relatively healthy. Like HbS, this structural
variant of hemoglobin is thought to provide protection from malaria. In a large
case–control study conducted in the Mossi of Burkina Faso, ~90 % of HbCC indi-
viduals and ~29 % of HbAC individuals showed a reduction in clinical malaria
(Modiano et al. 2001b; Min-Oo and Gros 2005). Other studies conducted in Ghana
(Mockenhaupt et al. 2004) and Mali (Agarwal et al. 2000) support the protective
effect of the HbC allele against malarial infection (Min-Oo and Gros 2005). It has
been suggested that HbC may inhibit the invasion or growth of the parasite in HbC
RBCs (Fairhurst et al. 2003). Fairhurst et al. (2005) also reported that HbC-positive
PRBCs showed 25 % reduced adhesion to vascular endothelium. They also showed
rosetting occurred 33 % less often in HbAC PRBCs and they showed rosetting
rarely occurred in HbCC PRBCs.
There have been a limited number of studies that focus on evidence for natural
selection on HbC haplotypes. Wood et al. (2005) estimated the age and strength of
selection associated with the HbC allele by sequencing a ~5.2 kb region of the HBB
gene and analyzing the extent of LD (linkage disequilibrium) (Table 2). Based on
allele frequencies, they estimated that the HbC allele is <5,000 years old and that
the selection coefficient at HbC is 0.04–0.09. However, despite this relatively strong
selection, they observed very little LD upstream from the HbC allele. They sug-
gested that either recombination or gene conversion may have weakened signatures
of recent selection. Similar results were also reported by Modiano et al. (2008).
They demonstrated that HbC haplotypes displayed more evidence of recombination
than did HbS Benin haplotypes and suggested that HbC is likely to be older than the
HbS Benin haplotype.

HbE

An additional variant that affects the structure of hemoglobin is HbE, which is prev-
alent in Southeast Asia. This variant is caused by a glutamic acid to lysine substitu-
tion in codon 26 of HBB (Table 1). Like HbC, HbE is relatively benign. HbEE
individuals usually have symptomless anemia, and HbAE individuals are generally
asymptomatic (Rees et al. 1998). A retrospective study of malaria patients con-
ducted in Thailand found that severe malaria complications were less acute and less
prevalent in individuals with the HbE allele (Hutagalung et al. 1999). In vitro exper-
iments suggest that HbAE RBCs showed reduced parasitic invasion compared to
HbEE RBCs, normal RBCs, and RBCs with other hemoglobinopathies (Chotivanich
et al. 2002).
To examine whether natural selection has affected the pattern of LD on haplo-
types that contain the HbE variant, Ohashi et al. (2004) sequenced a portion of the
HBB locus in Thai patients with mild malaria. They reported that there is extended
LD associated with the HbE variant extending >100 kb. Additionally, their haplo-
type analysis showed that the HbE variant was found on one haplotype background
in the Thai patients they surveyed. They suggested that the age of the HbE variant
Table 2 Age of selected polymorphisms described here, selection coefficients, and corresponding evidence of selection
Gene and Age
protective allele Generations Years Selection coefficient Evidence for natural selection References
HBB
S 45–70 1,125–1,750 0.152* Extended LD (>60 kB); long Currat et al. (2002),
range haplotype similarity Hanchard et al.
(>400 kb); evidence of (2007), Liu et al.
positive directional selection (2009), Hedrick
(2011)
10–28 250–700 – Modiano et al. (2008),
Hedrick (2011)
C 75–150 1,875–3,750 0.04–0.09 Evidence for selection is not Wood et al. (2005)
38–120 950–3,000 – strong; very little LD is Modiano et al. (2008),
associated with HbC perhaps Hedrick (2011)
due to age, recombination or
gene conversion
E 100.3 2,006(1,240–4,440) 0.079 (0.035–0.099) Extended LD (>100 kb); positive Ohashi et al. (2004),
(62–222) directional selection Hedrick (2011)
DARC
ES (null) 1323 33,075 (6,500–97,200) – No clear cut pattern; fixation in Hamblin and Di Rienzo
Africa implies a selective (2000)
ES (null) 490/310 12,250(4,250–26,500)/7,750 – advantage; reduced haplotype Seixas et al. (2002),
(3,625–13,125) variability in Africa also Hedrick (2011)
suggests positive selection in
Africa
ABO
Impact of Natural Selection Due to Malarial Disease on Human Genetic Variation

O01 1.15 million – Strong population differentiation; Calafell et al. (2008), Fry
O02 2.5 million – significantly positive Tajima's et al. (2008b)
D and Fu and Li's F suggests
balancing selection
(continued)
129
Table 2 (continued)
130

Gene and Age

protective allele Generations Years Selection coefficient Evidence for natural selection References
G6PD
A- 254.3 6,357 (3,840–11,760) 0.044 Constrained variability and Tishkoff et al. (2001)
extended LD; evidence of
positive directional selection
93 2,325 (1,200–3,862) Extended LD at least 413 kb Sabeti et al. (2002)
1800 4,500 (25,000–65,000) – Observed an excess of nonsyn- Verrelli et al. (2002)
onymous substitutions and
date of A- allele suggest
balancing selection
100 2,500–3,750 0.1 Extended LD >1.6 Mb; positive Saunders et al. (2002)
directional selection
HLA-B
B53 86 2,150 0.041* Extraordinary nucleotide Garrigan and Hedrick
polymorphism; evidence of (2003), Hedrick
balancing selection (2011)
The age of the allele generally corresponds to the time when strong selection for malaria resistance (due to high mortality) resulted in an increase of the fre-
quency of a given variant; “—” indicates that no selection (neutrality) is assumed in the estimation. Selection coefficients with an * are assumed, not estimated,
values (adapted from Hedrick 2011)
F. Gomez et al.
Impact of Natural Selection Due to Malarial Disease on Human Genetic Variation 131

is between 1,240 and 4,440 years old (Table 2). The results from Ohashi et al. (2004)
support the conclusion that the HbE mutation occurred recently and rose to high
frequency because of selection due to malarial disease.
As a whole, the population genetic data described here suggest that variants
involved in changing the structure of hemoglobin are relatively new mutations
(Table 2). All of the variants discussed here are all likely to have risen to high fre-
quency within the past 10,000 years due to strong selective pressures from falci-
parum malaria. Because these are new mutations and the selection pressures on
these mutations were strong, most loci exhibit long range LD on the haplotypes that
contain the functional alleles (i.e., a classic selective sweep). One exception is the
HBC haplotypes with shorter tracts of LD due to a recombination hotspot. These
haplotypes, however, show other signatures of recent strong selection.

Erythrocyte Membrane Proteins and Surface Antigens

Alleles that cause erythrocyte membrane abnormalities are another class of RBC
polymorphisms that confer resistance to P. falciparum malaria. These mutations
likely perturb the complex process of parasite invasion into erythrocytes, and there-
fore play an important role in providing protection from disease.

Variants Associated with Elliptocytosis and Ovalocytosis

One type of RBC membrane abnormality that can affect malaria pathogenesis is
elliptocytosis. Just as it sounds, elliptocytosis is a condition in which RBCs are
elliptical, rather than round, in shape. This condition is considered pathologic
because individuals affected by this condition suffer from anemia and hemolysis
(Palek 1987). The most common form of elliptocytosis is hereditary elliptocytosis.
Hereditary elliptocytosis (HE) includes a heterogeneous group of disorders and a
number of variants in different genes are associated with these disorders (Roux
et al. 1989).
Well-known mutations that cause HE are polymorphisms in the α and β chains of
spectrin, a structural protein that supports the cell membrane and regulates move-
ments of membrane proteins in erythrocytes. A number of these mutations have
been reviewed in Gallagher and Forget (1996). Within African populations there are
two common spectrin mutations, SpαI/65 and SpαI/46. These mutations result in
abnormal 46- or 65-kDa amino acid variants in Spectrin αI (SPTA1). Studies show
that in vitro invasion and growth of P. falciparum is reduced in both the SpαI/65 and
SpαI/46 RBCs (Dhermy et al. 2007).
In addition to spectrin mutations, deletions in the gene SLC4A1 have been stud-
ied extensively and may confer resistance to P. falciparum malaria. The primary
mutation that confers protection from malaria is a 27-bp deletion that causes what
is commonly known as Southeast Asian Ovalocytosis, or SAO (Table 1). As with
132 F. Gomez et al.

the spectrin mutations, this deletion causes the formation of oval-shaped RBCs. The
oval shape of the RBC is thought to be the result of modifications to band 3, a major
anion transporter of erythrocytes (Liu et al. 1990). This deletion is generally not
found in the homozygous state, and it is therefore thought to be lethal when an indi-
vidual inherits two copies (Genton et al. 1995; Allen et al. 1999; Patel et al. 2004;
Williams 2006). SAO is found at high frequencies (up to 30 %) in many parts of
Southeast Asia and in Papua New Guinea (Verra et al. 2009). Like the spectrin
mutations, it is thought that SAO confers protection from malaria because the struc-
tural change in the RBC membrane reduces parasite invasion. It has also been shown
that SAO is associated with reduced in vitro invasion by some strains of P. falci-
parum (Cortes et al. 2004). Upon further investigation, Cortes et al. (2005) showed
a marked increase in SAO RBCs infected with P. falciparum that adhere to CD36,
an adhesion receptor that is generally not associated with cerebral malaria. They
suggested that this increased adhesion to CD36 may prevent the parasite from
adhering to neurovascular adhesion molecules, such as ICAM-1, thereby protecting
the carriers of the SAO-causing deletion from cerebral malaria.
The population genetics of the SLC4A1 deletion in Japan, Taiwan, and Indonesia
were studied by Wilder et al. (2009), who addressed whether SAO chromosomes
differ from wild-type SLC4A1 chromosomes and whether or not these chromosome
show a pattern of variation that is consistent with natural selection. They rese-
quenced ~5 kb of the SLC4A1 gene and typed all individuals for the SAO deletion.
They found significantly negative values of Fay and Wu’s H in all populations,
which implies a larger than expected number of high-frequency derived alleles in
these populations. They also showed that the SAO deletion is on one haplotype
background and the non-SAO haplotype that is closest to the SAO haplotype is rare
and found only in Japan and Indonesia. The Indonesian sample showed the highest
level of diversity, and this was the only population in which the SAO deletion was
polymorphic. From these data, they concluded that the SAO deletion is relatively
recent and that it occurred on a rare haplotype that segregates in Asian
populations.

Glycophorin A and B Antigen Receptors

P. falciparum has multiple pathways to invade erythrocytes through interacting with

different erythrocyte surface receptors (Cowman and Crabb 2006). Among them are
glycophorin A and B, which are common glycoproteins on the erythrocyte surface.
The carbohydrate groups on the extracellular domains of these glycoproteins are
recognized by P. falciparum and are essential for parasite invasion of the erythro-
cyte (Pasvol et al. 1982; Sim et al. 1994; Mayer et al. 2009; Crosnier et al. 2011).
Although recent studies have revealed some evidence of adaptive evolution at the
glycophorin A and B genes (GYPA/GYPB), there are conflicting interpretations of
the patterns of genetic diversity at glycophorin A. Baum et al. (2002) observed an
excess of intermediate-frequency alleles (signatures of balancing selection) and
rapid rates of protein evolution at GYPA in humans. They suggested that the observed
Impact of Natural Selection Due to Malarial Disease on Human Genetic Variation 133

pattern of selection is consistent with the decoy hypothesis, which argues that
surface proteins like glycophorin A can act as decoy receptors to attract various
pathogens to nonnucleated cells (Gagneux and Varki 1999). Wang et al. (2003)
observed only accelerated rates of protein evolution at GYPA and suggested that
these patterns might have evolved adaptively to escape pathogen recognition during
erythrocyte invasion (evasion hypothesis).
Ko et al. (2011) studied sequence evolution of GYPA, GYPB, and another homo-
logue (GYPE) across diverse African ethnic groups residing within a broad geo-
graphic range. They observed signatures of balancing and positive selection on
different extracellular domains of GYPA. Ko et al. (2011) observed accelerated rates
of adaptive protein evolution at exons 3 and 4, which encode O-sialoglycan-poor
protein domains that are likely to be selected for maintaining the stability of the
protein structure. They also observed a skewed frequency spectrum toward an
excess of intermediate-frequency alleles (evidence of balancing selection) at exon
2, which encodes an O-sialoglycan-rich domain that can modify the binding affinity
of P. falciparum ligands. Ko et al. (2011) further showed that the magnitude of
skewness in the frequency spectrum at exon 2 is significantly correlated with malaria
exposure. Ko et al. (2011) suggested that the observed selection patterns at GYPA
can be better explained by the evasion hypothesis rather than the decoy hypothesis
because the decoy hypothesis usually predicts both fast evolutionary rates and high
genetic diversity at a genetic region, which Ko et al. (2011) did not observe.
Additionally, because the glycophorin genes are highly homologous to each
other at the nucleotide level (>95 %), high levels of gene conversion have been
observed among these homologues (Blumenfeld and Huang 1995; Wang et al. 2003;
Ko et al. 2011). In particular, Ko et al. (2011) showed that two GYPA nonsynony-
mous SNPs that code for the N allele of the MN blood group polymorphism were
introduced from GYPB through gene conversion. These two SNPs were also identi-
fied as candidate variants targeted by balancing selection. Thus, gene conversion
may be a mechanism for introducing high levels of variation upon which natural
selection may act (Wang et al. 2003; Ko et al. 2011).

Duffy Antigen Receptor for Chemokines

Another example of an erythrocyte surface antigen showing variability that may be

related to malaria resistance is the Duffy surface antigen (Duffy Antigen Receptor
for Chemokines, or DARC). The Duffy system consists of two principle antigens,
Fya and Fyb. These antigens are encoded by two co-dominant alleles called FY*A
and FY*B, which differ by a single nonsynonymous SNP that causes an amino acid
change from glycine to aspartic acid at codon 42. Another functionally important
polymorphic site at this locus, a T/C substitution at position −33 in the FY promoter
(the FY*O allele), disrupts a GATA box and silences expression of the FY antigen
in erythroid lineages without affecting transcription or expression in other cells
(Mercereau-Puijalon and Menard 2010). Most commonly, the FY*O alleles occur
on a FY*B haplotype background. These chromosomes are referred to as FY*BES
134 F. Gomez et al.

(erythroid silent) or FY*Bnull. An FY*Anull allele called FY*AES has been identified
in Papua New Guinea (Zimmerman et al. 1999); however, these haplotypes are gen-
erally rare (Mercereau-Puijalon and Menard 2010).
There are strong differences in the global geographic distribution patterns of the
Duffy alleles. For example, the FY*O allele is almost fixed in West and Central
Africa; thus, most Africans do not express the Duffy antigen (Hamblin and Di
Rienzo 2000; Fig. 2). The FY*BES allele is rare among Europeans, American
Indians, and Asian populations. Recently, Howes et al. (2011) used an extensive
collection of literature-based surveys of FY allele frequencies and a Bayesian geo-
statistical model to generate a continuous surface map of the FY alleles across the
globe. Their model showed that the FY*BES allele is fixed in West, Central, and East
Africa and at high frequencies in Madagascar and the Arabian Peninsula. They also
demonstrate that the most prevalent allele outside of Africa is FY*A.
The driving force behind fixation of the Duffy null allele in Africa is a long-
standing question within the fields of anthropology and human genetics. In a land-
mark study, Miller et al. (1976) reported that African Americans who do not express
the Duffy antigen on their RBCs are resistant to malarial infections caused by
Plasmodium vivax. Miller et al. (1976) suggested that because Duffy negativity
offers a selective advantage to people of African descent, the alleles that are respon-
sible for Duffy negativity have reached extremely high frequencies in African popu-
lations. It is now well understood that Duffy-negative individuals are resistant to
malaria caused by P. vivax because the Duffy antigen is the receptor that P. vivax
uses to enter the RBC and is necessary to begin the blood stage of a P. vivax
infection.
Studies of the FY (DARC) locus in a population genetics context loosely support
the idea that directional selection has influenced the patterns of genetic variation
and LD at this locus in African populations (Hamblin and Di Rienzo 2000; Hamblin
et al. 2002). In 2000, Hamblin and Di Rienzo showed that the DARC locus is two- to
threefold more diverse in Europeans than in Africans, which is expected if positive
directional selection has occurred in Africa. However, they also observed two major
FY*O haplotypes, which is not expected if Duffy negativity recently evolved and
quickly gained a selective advantage. Additionally, Hamblin et al. (2002) observed
patterns of genetic variation that departed from neutral evolution on chromosomes
with FY*A alleles in Italian and Chinese populations, suggesting that selection has
acted at this locus in regions other than Africa.
Using sequence data from the FY locus, Hamblin and DiRienzo proposed that
the time of fixation of the FY* O allele is 33,000 years ago, with a confidence inter-
val of 6,500–97,200 years (Table 2), which is inconsistent with recent selection
(Hamblin and Di Rienzo 2000; Hamblin et al. 2002). However, when Seixas et al.
(2002) used microsatellites to date the fixation of the FY*O allele, they suggested a
date of either 7,750 or 12,250 years ago, depending on the haplotype used for esti-
mation (see Hedrick 2011), which is more consistent with recent selection at DARC.
Because P. vivax infections are generally considered to be benign, or at a mini-
mum less serious than infection caused by P. falciparum, some have wondered
whether the strength of selection from P. vivax infection is sufficient to cause the
Impact of Natural Selection Due to Malarial Disease on Human Genetic Variation 135

FY*O allele to fix across the African continent (Livingstone 1984). When global
variation within the genome of P. vivax has been examined, an Asian origin for this
parasite has been suggested (Garnham 1966; Carter 2003; Escalante et al. 2005;
Cornejo and Escalante 2006). Additionally, using samples obtained from South and
Central America, Africa, Southeast Asia, and Melanesia, Mu et al. (2005) estimated
that the time to the most recent common ancestor (TMRCA) of P. vivax is 53,000–
265,000 years ago. Although these observations do not necessarily remove P. vivax
as a possible selective agent underlying the FY*O allele fixation in Africa, an old
TMRCA combined with an Asian origin of P. vivax does suggest that this parasite
was not present in Africa with enough time to cause a single SNP to sweep across
West and Central Africa. Thus, it has been suggested that some other selective pres-
sure besides P. vivax may have caused the FY*O allele to fix across most of Sub-
Saharan Africa (Mu et al. 2005).

ABO

Human blood group antigens have long been considered one of the many human
polymorphisms that are associated with resistance to malaria (Athreya and Coriell
1967; Martin et al. 1979; Kassim and Ejezie 1982; Cavalli-Sforza and Feldman
2003). The ABO blood antigens are coded for by one gene (ABO) on chromosome
9 (Table 1). There are four amino acid changes—Arg176Gly, Gly235Ser, Leu266-
Met, and Gly268Ala—that determine whether or not the A or B antigens are
expressed on the red blood cell surface (Type A—Arg176, Gly235, Leu266, and
Gly268; Type B—Gly176, Ser235, Met266, and Ala268; Seto et al. 1997). Type O
is the result of a single nucleotide deletion that causes a reading frame shift, which
creates a premature stop codon, resulting in the expression of a shortened protein
that lacks a necessary catalytic site for determining a blood cell surface antigen
(Yamamoto et al. 1990; Daniels 2005). There are two common O haplotypes, O01
and O02, which differ at exons 6 and 7 (Yamamoto et al. 1990; Olsson and Chester
1996; Roubinet et al. 2004; Calafell et al. 2008).
ABO blood types are differently distributed in global human populations—an
intriguing pattern that has made this a classic human genetic polymorphism of inter-
est to geneticists and anthropologists (reviewed in Uneke 2007). Over the years, a
number of studies have shown that there is a high prevalence of the O allele in tropi-
cal areas of the world and in places where malaria was historically prevalent—and
that blood group A is typically found in colder parts of the world, where malaria is
not historically prevalent (Cserti and Dzik 2007). In addition, numerous case–con-
trol studies have investigated the relationship between P. falciparum malaria and
ABO blood groups. These studies are comprehensively reviewed in Cserti and Dzik
(2007) and Cserti-Gazdewich et al. (2010). For example, Fry et al. (2008b) con-
ducted a case–control study with patients from The Gambia, Kenya, and Malawi
and showed that A, B, and AB individuals appear to be at significantly greater risk
for severe malaria than are individuals with blood group O. They also suggest that
individuals with type B have a slightly lower risk than blood group A, and blood
136 F. Gomez et al.

group AB has the greatest risk of the non-O blood types. Therefore, it has been
hypothesized that type O confers protection from P. falciparum malaria, the A group
confers a disadvantage for protection from malarial disease, and the B antigen has
an intermediate effect (Cserti and Dzik 2007).
The mechanism by which the O blood group confers protection from malaria is
not well understood. It has been shown that red blood cells with A, B, and O pheno-
types show differing amounts of cytoadherence to endothelial receptors and unin-
fected RBCs (Cserti and Dzik 2007; Cserti-Gazdewich et al. 2010). Chen et al.
(2000) reviewed the data that describe differences in rosette formation among the
blood groups and suggested that because A and B antigens are found in abundance
on the surface of non-O RBCs, they are important rosetting receptors. Carlson and
Wahlgren (1992) showed that rosettes can be formed with O RBCs, but they found
the greatest rosetting among A, B, and AB RBCs. Recently, Rowe et al. (2007)
conducted a matched case–control study of 567 Malian children and showed that
type O was least frequent among the patients with severe malaria. They were also
able to show that rosetting was significantly reduced in patients with type O RBCs,
suggesting that the protective effect of type O may be related to a reduced level of
rosetting.
The ABO locus has long been considered a target of natural selection (Akey et al.
2004; Sabeti et al. 2006), and polymorphisms at ABO are considered to be main-
tained due to balancing selection that preceded the divergence of modern humans
(Saitou and Yamamoto 1997; Calafell et al. 2008; Fry et al. 2008b). Thus, there may
be ancient selection maintaining genetic diversity at the ABO locus. In fact, in addi-
tion to malaria, ABO blood groups have also been implicated in resistance to other
infections, including Escherichia coli (Blackwell et al. 2002), Helicobacter pylori
(Boren et al. 1993), Campylobacter jejuni (Ruiz-Palacios et al. 2003), and Norwalk
virus (Lindesmith et al. 2003). Infections from these organisms could also have
acted as selective forces influencing variation at ABO (Calafell et al. 2008). Fry
et al. (2008b) analyzed SNP data from the International HapMap Project and
observed significantly low levels of population differentiation, measured by FST,
when the YRI (Yoruban population), ASN (combined Chinese and Japanese popu-
lation), and CEU (northwestern European population) were compared across the
ABO locus. Their observations are consistent with a model of long-standing balanc-
ing selection at this locus across all HapMap populations. In another study of pat-
terns of genetic diversity at ABO, Calafell et al. (2008) also found evidence for
balancing selection at this locus. They estimated the age of the two primary O lin-
eages (O01 and O02) to be >1 million years old, consistent with a signature of long-
standing balancing selection (Table 2). Thus, it is likely that genetic variation at
ABO has been maintained due to multiple selective forces acting over different time
periods.
Studies of genetic variation at loci that code for erythrocyte membrane proteins
and surface antigens show differing patterns of variation. The SLC4A1 locus shows
evidence of recent positive selection, while GYPA and DARC show mixed signa-
tures of balancing and positive selection depending on the gene region that is exam-
ined. By contrast, the ABO locus shows evidence of long-standing balancing
Impact of Natural Selection Due to Malarial Disease on Human Genetic Variation 137

selection. Although most of these loci are likely to participate in the process of
Plasmodium entry into the RBC, they play fundamentally different roles in normal
host biology, which affects the patterns of natural selection and evolution observed
at each locus.

Erythrocyte Enzymes and Enzyme Deficiency

G6PD

The gene that encodes the enzyme glucose-6-phosphate dehydrogenase (G6PD) is

an important human housekeeping gene. G6PD is the first important and rate-
limiting enzyme in the pentose phosphate metabolic pathway. It plays a critical
role in the metabolism of glucose and maintaining the balance of reduced, or oxi-
dized, states of glutathione, which is important for handling oxidative stress. The
G6PD locus, which is located on the X chromosome, is known to harbor many
mutations (>140) that are associated with enzyme deficiency (Cappellini and
Fiorelli 2008). Indeed, G6PD-enzyme deficiency is one of humanity’s most com-
mon enzymopathies, affecting >400 million people worldwide (Ruwende and Hill
1998; Nkhoma et al. 2009). Interestingly, some of the polymorphisms that cause
G6PD deficiency are highly correlated with the distribution of P. falciparum
malaria. This observation has led to the well-established hypothesis that G6PD
deficiency confers reduced risk of infection by Plasmodium falciparum (Beutler
1994; Ruwende and Hill 1998).
The A- variant, which is defined by two nonsynonymous point mutations, A376G
and G202A, is the most common deficiency mutation (10–50 % of normal enzyme
activity) in Sub-Saharan Africa and is believed to be associated with resistance to
malaria (Ruwende et al. 1995). A study conducted in Mali suggests that hemizy-
gous males and homozygous females with the A- variant have protection against
severe malaria. However, no protection was observed in heterozygous females
(Guindo et al. 2007).
Evidence for natural selection at G6PD-deficiency alleles has been addressed by
a number of studies. Tishkoff et al. (2001) examined RFLP and microsatellite varia-
tion in African and non-African individuals at G6PD. They identified low microsat-
ellite haplotype variation and high LD on haplotypes with the A- variant, which is
consistent with natural selection affecting variation at this locus. In addition, through
the use of coalescent analyses Tishkoff et al. (2001) inferred the age of the A- allele
to be approximately 6,357 years old (3,840–11,760 range), consistent with malaria
being the selective force that caused the A- variants to increase in frequency in
Africa. Sabeti et al. (2002) also found evidence of long-range haplotype homozy-
gosity at G6PD associated with the A- allele and estimated the age of the allele to
be 2,325 (1,200–3,862 range). These results were also confirmed by Saunders et al.
(2002), who used resequencing analyses to show that African chromosomes with
the A- allele have overall low level of variation and high levels of LD. They also
138 F. Gomez et al.

inferred the A- variant to be between 2,500 and 3,750 years old. To examine SNP
variation at G6PD in African populations, Verrelli et al. (2002) sequenced a total of
5.2 kb of the G6PD locus in eight African and five non-African populations. Similar
to what Tishkoff et al. (2001) reported, Verrelli et al.’s (2002) results show that there
is less variation associated with A- haplotypes than is expected by chance alone.
However, Verrelli et al. (2002) also suggested that there is an excess of amino acid
polymorphism and that the A- variant is much older than 2,000 years, which may be
consistent with a model of balancing selection maintaining diversity (Table 2).

Pyruvate Kinase

Pyruvate kinase (PK) is an enzyme that is important for the reactions involved in
glucose metabolism. It catalyzes the conversion of phosphoenolpyruvate to pyru-
vate, which results in the generation of one molecule of ATP. This reaction is con-
sidered to be the rate-limiting step of glycolysis, which is critically important for
energy production in red blood cells because they lack mitochondria and rely exclu-
sively on glycolysis for the production of ATP (Ayi et al. 2008; Berghout et al.
2012). First identified in 1960 (Valentine et al. 1961; Zanella et al. 2005), RBC PK
deficiency is known to be the most frequently inherited enzymatic disorder of the
glycolytic pathway and is one of the most common causes of nonspherocytic hemo-
lytic anemia (Ayi et al. 2008; Durand and Coetzer 2008; Tekeste and Petros 2010).
Similar to G6PD, many mutations in the human PKLR gene cause PK deficiency.
For example, Zanella et al. (2005) reported that there are at least 158 known muta-
tions in PKLR that cause enzyme deficiency. However, unlike G6PD, mutations that
cause PK deficiency are generally not in areas where malaria is prevalent (Fig. 1).
Although, PK-deficiency mutations do not show a general pattern of global dis-
tribution characteristic of genetic variation that is correlated with malarial disease
(Fig. 2), some data suggest that variation at this gene could confer protection against
malaria. The first line of evidence in this regard has come from mouse models.
Min-Oo et al. (2003) identified PKL (mouse PLKR gene) using QTL mapping in
mouse strains resistant to malaria. At PKL, Min-Oo et al. (2003) showed that a non-
synonymous SNP that is known to cause PK deficiency in humans, is significantly
associated with decreased parasitemia, and is associated with malarial infection sur-
vival in mice. (Min-Oo et al. 2003; Min-Oo et al. 2004; Min-Oo and Gros 2005).
Since this discovery in the mouse, further studies in humans have suggested that PK
deficiency is related to protection from malaria (Ayi et al. 2008; Durand and Coetzer
2008). Ayi et al. (2008) showed that invasion of RBCs by P. falciparum is signifi-
cantly lower in subjects with homozygous mutations for PK deficiency and phago-
cytosis of PRBCs in patients who are homozygous for PK-deficiency mutations is
markedly higher than phagocytosis in control PRBCs.
Patterns of genetic diversity at PKLR in humans are also suggestive of selection
acting at this locus. In a study of SNPs at the PKLR and neighboring GBA loci,
Mateu et al. (2002) showed that there is strong LD over about 90 kb in this region.
Additionally, Machado et al. (2010) examined SNP and STR variation in
Impact of Natural Selection Due to Malarial Disease on Human Genetic Variation 139

individuals from Angola, Mozambique, and Portugal. In this study they showed,
using FST, that there is strong population differentiation between Africans and
Portuguese for SNPs at PKLR. These data suggest selection could have resulted in
significantly different alleles frequencies among Africans and Europeans.
In a recent resequencing study of PKLR in 387 individuals from the Human
Genome Diversity Project (HGDP; Berghout et al. 2012), seven nonsynonymous
SNPs were described, three of which have been reported in PK-deficient individu-
als. Using the algorithms in the computer program SIFT (Henn et al. 2012), Berghout
et al. (2012) showed that these sites are likely to affect the function of PK. The
authors also identified potential signatures of recent positive selection at the PKLR
locus in a pooled population from Pakistan and Sub-Saharan Africa; they observed
significantly negative value of Tajima’s D in the Pakistani population and signifi-
cantly negative values of Fu and Li’s D* and F* in the combined Sub-Saharan popu-
lation and the population from Pakistan. It should be noted that there is likely to be
some substructure among the pooled populations examined by Berghout et al.
(2012), which can also result in negative values of Tajima’s D and Fu and Li’s D*
and F* (Simonsen et al. 1995; Ptak and Przeworski 2002).

Endothelial Receptors, Cytoadhesion, and Sequestration

As discussed above, one of the unique attributes of P. falciparum malaria pathogen-

esis is parasite sequestration caused by cytoadherence (Chen et al. 2000). There are
several human receptors known to mediate cytoadherence phenotypes (Rowe et al.
2009). ICAM-1 and CD36 are two important and well-studied receptors. ICAM-1 is
thought to be a key neurovascular receptor that mediates the pathogenesis of cere-
bral malaria, a syndrome that causes a number of serious complications that can be
fatal (Turner et al. 1994; Rowe et al. 2009). CD36 is a receptor known to adhere to
surface antigens from many different parasite lines but is generally not associated
with cerebral malaria (Newbold et al. 1997; Rowe et al. 2009). It is thought that
cytoadherence and sequestration are adaptive strategies that the parasite has evolved
to escape immune clearance and to maintain a chronic infection (Pasloske and
Howard 1994; Craig and Scherf 2001; Sherman et al. 2003).

ICAM-1

ICAM-1 (CD54) is a transmembrane glycoprotein that is expressed at low levels on

the surface of endothelial cells and leukocytes (Chakraborty and Craig 2004). Its
expression is markedly upregulated in response to many cytokines including TNF-α
and IL-1β (Chakraborty and Craig 2004; Amodu et al. 2005). ICAM-1 on the endo-
thelium of vascular tissue plays an important role in adhering to and facilitating the
migration of activated leukocytes to sites of infection. Because ICAM-1 is known to
140 F. Gomez et al.

be involved in severe malaria pathogenesis, a number of studies have tested whether

genetic variation at ICAM-1 provides protection from severe malaria (Fernandez-
Reyes et al. 1997; Bellamy et al. 1998; Kun et al. 1999; Tse et al. 2004; Fry et al.
2008a). In 1997, Fernandez-Reyes et al. described a nonsynonymous allele that
interacts with PfEMP-1 (Plasmodium falciparum Erythrocyte Membrane Protein-1),
which is the Plasmodium surface protein associated with cytoadherence and seques-
tration. In this study it was shown that this allele is associated with increased
susceptibility to malaria and occurs at a relatively high frequency (minor allele
frequency >30 %) within the study population. Fernandez-Reyes et al. (1997)
named this allele ICAM-1Kilifi, after the area in Kenya where they were working.
Since this initial paper, several other scholars (e.g., Bellamy et al. 1998; Kun
et al. 1999; Ndiaye et al. 2005) have studied association of ICAM-1Kilifi with malaria
susceptibility and obtained conflicting results. Bellamy et al. (1998) were unable to
show a significant association between ICAM-1Kilifi and severe malaria in The
Gambia, whereas in a study of children from Gabon, Kun et al. (1999) showed that
the ICAM-1Kilifi homozygous and heterozygous genotypes confer protection against
severe malaria. Fry et al. (2008a) examined the association of common SNPs in
ICAM-1 with severe malaria phenotypes in families and unrelated individuals from
Kenya, The Gambia, and Malawi. In their family-based analyses, Fry et al. (2008a)
were unable to show a significant association between ICAM-1 SNPs and severe
malaria phenotypes. In their population-based analysis, they showed a potential
association between cerebral malaria susceptibility and ICAM-1Kilifi homozygotes in
The Gambia, but the authors interpreted this result cautiously because this SNP was
at relatively low frequency in The Gambia and because previous association studies
conducted in The Gambia have failed to show a significant correlation between
ICAM-1 and severe malaria. These studies and others (Ohashi et al. 2001; Amodu
et al. 2005; Ndiaye et al. 2005) exemplify the uncertainty behind the role that nucle-
otide diversity at ICAM-1 plays in susceptibility to malaria.
The global pattern of allele frequency at ICAM-1Kilifi is consistent with the pos-
sibility that it plays a role in malaria resistance. The ICAM-1Kilifi allele is at moderate
frequency in African and Asian populations with high prevalence of malaria (Ohashi
et al. 2001; Fry et al. 2008a) and is absent or rare in European and European
American populations (Zimmerman et al. 1997; Vijgen et al. 2003; Register et al.
2004; Ma et al. 2006). Ryan et al. (2006) showed, using publicly available rese-
quencing data of African and European Americans, that the ICAM-1Kilifi allele has an
unusually high FST value when compared to SNPs in genes that encode cytokines,
adhesion molecules, cytokine receptors, and Toll-like receptors. These data suggest
that the allele frequency differences at ICAM-1Kilifi between African and European
Americans are quite large. In a recent study of genetic diversity at ICAM-1 in Africa,
Gomez et al. (2013) showed that the ICAM-1Kilifi allele is significantly correlated
with malaria endemicity in Africa. They also demonstrate through haplotype analy-
ses that the ICAM-1Kilifi allele potentially arose independently in African and Asian
populations. These data suggest that the ICAM-1Kilifi allele may confer a selective
advantage in malaria-endemic environments.
Although association studies have provided conflicting results, functional
studies suggest that the ICAM-1Kilifi allele could play a role in malaria resistance.
Impact of Natural Selection Due to Malarial Disease on Human Genetic Variation 141

The ICAM-1Kilifi allele is located in the exon that codes for the Ig-like domain that
interacts with PfEMP-1. Experiments have shown that proteins containing the ICAM-
1Kilifi variant amino acid (29M) are less adherent to certain laboratory strains of P. falci-
parum. Adams et al. (2000) showed, under static and flow conditions, that the P.
falciparum strain A4 is dramatically less adherent to proteins with the 29M mutation
than the reference protein. However, they also showed that adherence to the ItG P. falci-
parum strain was less affected by the ICAM-1Kilifi mutation. These data suggest that the
changes in adhesion efficacy due to the ICAM-1Kilifi variant may be strain specific.

CD36

CD36 is a transmembrane glycoprotein within the class B scavenger receptor family

(Armesilla and Vega 1994). Also known as platelet glycoprotein IV, or GP IIIb, CD36
is found on the surface of many different cell types including platelets, adipocytes,
endothelial cells, macrophages, dendritic cells, striated muscle cells, and hematopoi-
etic cells (Rac et al. 2007). CD36 has a variety of functions depending on the cell type
and tissue in which the protein is expressed. When expressed on macrophages, CD36
binds to oxidized low-density lipoproteins (ox-LDL) that can no longer interact with
their usual LDL receptors (Abbas and Lichtman 2005). In the literature, the removal
of ox-LDL is described as one of the primary functions of CD36.
As with ICAM-1, several groups (Aitman et al. 2000; Gelhaus et al. 2001; Pain
et al. 2001; Fry et al. 2009) have examined genetic variation at CD36 to test whether
alleles at this locus confer protection from malaria. Aitman et al. (2000) showed that
a T/G nonsense mutation in exon 10 (T1264G) and a compound mutation in exon
12 (G/C SNP with a frame-shift deletion) are associated with increased susceptibil-
ity to severe malaria. However, Pain et al. (2001) also examined the T1264G variant
and suggested that the G allele may confer protection against, rather than increased
susceptibility to, severe malaria.
The T1264G SNP is of particular interest because it is the most common SNP in
Africa responsible for CD36 deficiency. CD36 deficiency is a condition in which
CD36 is not expressed on platelets and monocytes/macrophages (type I), or when
CD36 is not expressed on platelets but shows a normal expression profile on mono-
cytes/macrophages (type II) (Imai et al. 2002). CD36 deficiency has been reported
in East Asians, Africans, and African Americans. The pathologies associated with
CD36 deficiency (types I and II) include hereditary hypertrophic cardiomyopathy,
increased serum LDL and triglycerides, high blood pressure, and insulin resistance
(Yamamoto et al. 1994; Kashiwagi et al. 2001; Tanaka et al. 2001; Ma et al. 2004;
Corpeleijn et al. 2006). Because CD36 deficiency can be deleterious and is also
common in some African and Asian populations, it has been suggested that this is
another selected trait because it confers protection from malaria (Sabeti et al. 2006;
Fry et al. 2009). Sabeti et al. (2006) used HapMap phase I data to show that the
1264G allele is associated with extended haplotype homozygosity in the Yoruba
HapMap population. Fry et al. (2009) genotyped the T1264G polymorphism in
3,420 individuals from 66 ethnic groups including several African populations.
Their results showed that the G allele is at relatively high frequency in the Yoruba
142 F. Gomez et al.

population and is at lower frequencies in other African groups. They also observed
unusually long extended haplotypes for the Yoruba but not for the populations in the
HGDP panel and the Gambia. Overall, Fry et al. (2009) did not find strong evidence
for an association between SNPs at CD36 and malarial phenotypes but suggested
that there was a nonsignificant trend toward heterozygous advantage for the 1264 G
allele. In addition, Bhatia et al. (2011) examined levels of population differentiation
across the genomes of African Americans, Nigerians, and Gambians and identified
strong signal of excessive population differentiation at CD36, which could be inter-
preted as a signal of natural selection at this locus.
These population genetics results are supported by functional studies that sug-
gest CD36 plays a role in malaria susceptibility. McGilvray et al. (2000) examined
whether CD36 may be involved in monocyte/macrophage-mediated malaria clear-
ance. Through several in vitro experiments, they showed that CD36 plays a signifi-
cant role in nonopsonic (nonmediated) phagocytosis of PRBCs and therefore is an
important component leading to infection clearance. Using a mouse model, Patel
et al. (2007) showed that CD36 contributes significantly to the success of an innate
inflammatory response to Plasmodium infections and that CD36 also influences the
duration and severity of malarial disease. These studies imply that CD36 has a com-
plex involvement in Plasmodium infections; it can facilitate infection and pathogen-
esis through adherence to PfEMP-1, but it may also play a critical role in mounting
an efficient immune response to disease.
The patterns of genetic variation at ICAM-1 and CD36 do not show a consistent
signature of strong recent natural selection. Although cytoadherence is an important
and fatal component of malarial disease, there are multiple host receptors that the
parasite exploits. This is an important advantage for the parasite, because it creates
flexibility in the ways in which cytoadherence can be achieved and limits the host’s
ability to protect itself. Thus, interpreting patterns of genetic variation and identify-
ing a classic “selective sweep” at these loci can be difficult. However, at ICAM-1
Gomez et al. (2013) showed that the frequency of the ICAM-1Kilifi allele is correlated
with malaria endemicity. These data suggest correlation analyses can be used to
detect potentially functional alleles in the absence of other signatures of natural
selection. With regard to CD36, it is difficult to explain the inconsistent signatures
of selection at this locus across Africa. Further investigation of sequence variation
at this locus (including regulatory regions) in diverse African populations may help
to reveal the evolutionary history of this locus and its potential role in malaria sus-
ceptibility (Gomez 2012).

Genetic Variation at Genes of the Immune System

The immune response to P. falciparum infection is an interesting and complex part

of the coevolution of the human and P. falciparum genomes (Langhorne et al. 2008).
The immune response to P. falciparum is complex because it is often considered to
be both beneficial (i.e., in the elimination and clearance of parasites) and
Impact of Natural Selection Due to Malarial Disease on Human Genetic Variation 143

detrimental (i.e., in the excessive release of proinflammatory cytokines) to the

human host. Doolan et al. (2009) comprehensively reviewed the literature that
discusses immunity to malaria and suggested that there are various types of acquired
and adaptive immunity to malaria. They described “antidisease immunity,” which is
immunity that affects the level of morbidity and mortality associated with an indi-
vidual’s infection; “antiparasite immunity,” which describes protection against
overall infection, parasitemia, or both; and “premunition,” which describes protec-
tion against new infections through persistent low levels of parasitemia.
Unfortunately, despite a sizable amount of literature describing the immune response
to P. falciparum and experimental model systems to explore the functional basis of
genetic variants that confer an immune response, we still have an inadequate under-
standing of mechanisms that provide immunity to malaria, especially the level of
immunity that Doolan et al. (2009) described as “premunition.”
However, it has been shown that there is an important heritable component to
malaria susceptibility (Mackinnon et al. 2005) and specifically to the immune
response that is mounted against this parasite (Sjoberg et al. 1992; Jepson et al.
1997; Stirnadel et al. 1999; Stirnadel et al. 2000; Phimpraphi et al. 2008; Duah et al.
2009). Therefore, it is informative to examine nucleotide diversity at genes impli-
cated in malarial immune response. These data will help to uncover the heritable
and potentially functional variants involved in disease protection.

Human Leukocyte Antigen

The major histocompatibility complex (MHC) system (HLA, or human leukocyte

antigen in humans) has been studied over a number of years in relation to malarial
disease susceptibility (reviewed in Kwiatkowski 2005; Verra et al. 2009; Hedrick
2011). In humans the HLA locus is located on chromosome six and encodes cell-
surface proteins that bind to and present intracellular and extracellular peptide frag-
ments, which are often pathogen derived, to immune effector cells. The presentation
of pathogen-derived proteins to the immune system is a fundamental component of
a successful immune response to most human pathogens. In humans, this process is
driven by HLA genes, which are divided into two classes, class I and class II. The
HLA region is very polymorphic (reviewed in Traherne 2008). It has been hypo-
thesized that the high degree of variability at the HLA locus has evolved through
natural selection in response to infectious disease (Potts and Wakeland 1993; Prugnolle
et al. 2005). A number of polymorphisms within the HLA region have been shown to
be associated with malarial disease susceptibility (reviewed in Ghosh 2008).
One of the first studies to show an association between genetic diversity at HLA
and malarial disease was conducted in Sardinia by Piazza et al. (1985). In this study,
they found large allele frequency differences at HLA-B (one of several HLA class I
genes) between highland regions of Sardinia, which is relatively malaria free, and
lowland areas where malaria was highly prevalent, analogous to patterns of varia-
tion observed at well-known malaria resistance loci (i.e., G6PD).
144 F. Gomez et al.

Hill et al. (1991) conducted a very influential study of HLA variation and malaria
in The Gambia. In this study of over 2,000 malaria cases and controls, they showed
that the HLA-Bw53 allele and the DRB1*1302-DQBB1*0501 haplotype (an HLA
class II gene) are associated with reduced susceptibility to severe malaria. Hill et al.
(1991) also highlight the fact that these alleles are common in West Africans and are
less common in other global populations, suggesting that natural selection may have
played a role in shaping the allele frequency distribution at these genes.
Although these studies and those reviewed by Ghosh et al. (2008) show that
variation at HLA is potentially important for protection against malaria, the extent
and relevance of this protection is unclear. Once the parasite has entered a human
host, it spends some time in liver cells, but it causes the most serious symptoms
and spends the remainder of its life cycle in the red blood cell. This is an important
point because erythrocytes do not express HLA molecules after enucleation,
which severely limits the ability of the erythrocyte to present antigens to the
immune system (de Villartay et al. 1985; Cserti-Gazdewich et al. 2010).
Furthermore, when Jepson et al. (1997) compared the humoral and cellular
immune response to malaria antigens in dizygotic and haploidentical dizygotic
twins, they found that genes in the HLA class II region did not significantly con-
tribute to the heritable component of an immune response to malaria. This result
suggests that variance in HLA genes does not greatly contribute to variance in
immune response to malarial antigens.

Inflammatory Cytokines and Antibody Response

Many other genes involved in immune function, especially those that encode cyto-
kines, have been suggested to harbor variants that influence susceptibility to malaria.
Unfortunately, many of the associations uncovered at cytokine genes have inconsis-
tent results depending on study location and the malarial phenotypes investigated.
One locus that is consistently shown to have polymorphisms that influence a num-
ber of different malarial phenotypes is the gene that encodes tumor necrosis factor
(TNFα), an important proinflammatory cytokine. TNFα is among the initial inflam-
matory cytokines that are released in response to infection. The gene, TNF, which
encodes this cytokine has several promoter polymorphisms (TNF-308, TNF-238,
and TNF-376) that have been shown in several ethnic groups to be related to suscep-
tibility to severe malaria, symptomatic reinfections with P. falciparum, and parasite
density (McGuire et al. 1994; Meyer et al. 2002; Flori et al. 2005). However, despite
some evidence that genetic variation at TNF may confer protection against some
malarial phenotypes, the exact function of these promoter SNPs and how those
functions relate to malaria susceptibility remains unclear (reviewed in Bayley et al.
2004; Kwiatkowski 2005; Smith and Humphries 2009).
Additionally, a number of other cytokine genes have been shown to contain
mutations that may affect malaria susceptibility. These include IL1A (Walley et al.
Impact of Natural Selection Due to Malarial Disease on Human Genetic Variation 145

2004), IL1B (Walley et al. 2004), IL4 (Gyan et al. 2004), IL12B (Morahan et al.
2002; Marquet et al. 2008), and IL10 (Wilson et al. 2005; Ouma et al. 2008).
However, as discussed above, many of these associations require replication and
further study to understand the functional mechanisms of these genes that are related
to malaria.

Fulani Immunity to Malaria

When compared to neighboring populations, the Fulani people, who inhabit North
Sudan, Central Africa, and much of West Africa, demonstrate markedly lower
susceptibility to malarial infection despite facing similar exposure levels com-
pared to neighboring populations (Modiano et al. 1995; Modiano et al. 1996;
Luoni et al. 2001; Dolo et al. 2005). Thus, the Fulani are speculated to have dis-
tinct genetic traits that confer increased protection against P. falciparum infection.
However, many of the known genetic traits that influence infection rate (e.g., HbS,
HbC, and G6PD deficiency) are not overly represented in the Fulani (Modiano
et al. 2001a). It has been proposed that the functional basis of the increased pro-
tection is the result of a more effective immune response (Luoni et al. 2001; Dolo
et al. 2005). When Torcia et al. (2008) studied the expression profiles of a panel
of genes involved in immune response in the Fulani and the Mossi (a neighboring
ethnic group), it was shown that peripheral blood mononuclear cells (PBMCs)
from the Fulani have higher expression of genes related to Th1 and Th2 immune
response compared to the Mossi. Additionally, they also showed a decrease in the
expression of genes related to T-cell regulatory activity in both PBMCs and T
regulatory cells. The authors suggest these data point to decreased activity of T
regulatory cells as the underlying factor mediating the Fulani’s immunity to
malaria.
More recent studies continue to demonstrate that, when compared to other neigh-
boring ethnic groups, the Fulani show differences in immune response to malaria
infection and different patterns of genetic variation at genes involved in the immune
response to malaria (Cserti and Dzik 2007; Senga et al. 2007; Carvalho et al. 2010;
Arama et al. 2011; Henn et al. 2011). For example, Israelsson et al. (2011) showed
that the Fulani have a higher frequency of SNPs associated with a proinflammatory
immune response to malaria compared to neighboring groups. Specifically, their
study showed Fulani have a higher frequency of SNPs that increase the expression
of IL1B (a proinflammatory cytokine) and SNPs that decrease the expression of
IL10 (an inhibitory cytokine). Additionally, Israelsson et al. (2011) showed that
when compared to other ethnic groups, the Fulani have a higher frequency of SNPs
in TNF that are associated with decreased severity of malarial infection (Israelsson
et al. 2011). It should be noted that cultural practices (i.e., pastoralism) and lifestyle
have also been suggested to contribute to the observed difference in malarial disease
in Fulani communities (Wallace and Wallace 2002).
146 F. Gomez et al.

Genome-Wide Association Studies

Several genome-wide linkage and association studies have been conducted to

identify the genes that contribute to protection against malaria. For example,
Timmann et al. (2007) used 10,000 SNPs from all the autosomes to conduct a link-
age study in families from Ghana. The results of the linkage analysis revealed three
regions associated with several malarial phenotypes (p < 10−4). These include loci
mapped to10p15.3–10p14, which was linked to malaria fever episode; 13q, which
was linked to parasite density; and 1p36, which was linked to both parasite preva-
lence and the level of anemia. Sakuntabhai et al. (2008) also conducted a genome-
wide linkage analysis in two Senegalese villages using 400 genome-wide
microsatellite markers and 66 SNPs in regions known to influence malaria suscep-
tibility (i.e., markers near TNF, ICAM-1, and CD36). This study showed notable
linkage results at markers associated with 5q31 (p < 10−4), a region that was previ-
ously shown to be associated with parasitemia and asymptomatic parasite infection
(Garcia et al. 1998; Rihet et al. 1998; Flori et al. 2003). One of the interesting
components of this study is that it was conducted in two Senegalese villages (Dielmo
and Ndiop) where exposure to malaria differs, and villagers’ ethnicities differ. This
study design allowed the authors to look for associations between genotype and
phenotype in different genetic backgrounds as well as in different environmental
contexts. The significant linkage that was identified at 5q31was found in Dielmo,
the village with intense and perennial (potentially lasting year-round) malaria trans-
mission, but they did not find the same association in Ndiop, the village with sea-
sonal malaria transmission. The most recent genome-wide linkage study was
conducted in Senegalese families (626 individuals—249 parents and 377 children)
using 250k SNPs (Milet et al. 2010). The results of their linkage analyses showed
an association between mild malaria attack and SNPs located at 6p25.1 and 12q22,
between parasite prevalence in asymptomatic infection and SNPs located at 20p11–
q11, and between the intensity of parasite infection and SNPs located at 9q34.
To date, the two largest genome-wide association studies involving malaria phe-
notypes were conducted by Jallow et al. (2009) and Timmann et al. (2012). Jallow
et al. examined >400k SNPs in 2,500 children from The Gambia. After correcting
for population structure, the strongest signal of association they achieved was close
to the HBB gene (p < 10−6). Additionally, after genotyping the HbS allele in a greater
number of samples they achieved a much stronger association signal (p = 1.3 × 10−28).
However, this study failed to identify any of the other commonly accepted genes
that affect malaria susceptibility and also did not find significant overlap with any of
the genome-wide studies mentioned above.
Timmann et al. (2012) examined 1,325 severe malaria cases and 828 unaffected
controls from Ghana using the Affymetrix Genome-Wide Human SNP Array 6.0
(>900k SNPs) and SNPs imputed from the 1,000 Genomes Project (http://
www.1000genomes.org) to create a panel of >5 million SNPs for analysis. After
analyses of the initial sample and replication experiments, four loci showed genome-
wide significance level of association (p < 5 × 10−8). These loci included HBB and
Impact of Natural Selection Due to Malarial Disease on Human Genetic Variation 147

ABO, as well as SNPS at two novel loci—the ATP2B4 (ATPase, Ca2+-transporting,

and plasma membrane 4) gene and an intergenic region of chromosome 16 between
the TAT (Tyrosine Aminotransferase) gene and MARVELD3 (MARVEL domain-
containing protein 3 gene). The authors suggested that both novel loci are interest-
ing genome-wide hits because ATP2B4 encodes the main calcium pump for RBCs
and MARVELD3 encodes part of the tight junction structures of epithelial and vas-
cular endothelial cells. Besides the large sample size and number of SNPs used by
Timmann et al. (2012), part of their success can also be attributed to their focus on
well-defined severe malaria phenotypes. The Timmann et al. (2012) results suggest
that with enough samples and enough power (i.e., genomic variation), new discov-
eries can be made about the underlying genetic architecture of susceptibility and
resistance to malaria.
Taken as a whole, these studies demonstrate that genome-wide investigations are
still in their infancy in terms of identifying causal variants that are associated with
malarial phenotypes. These studies generally do not overlap with each other, aside
from the consistent identification of HBB, and they do not replicate most associa-
tions found at previously identified candidate genes. There are a number of reasons
for the inconsistencies. These include the possibility that different genes or genetic
variants confer protection to malaria in different populations. Additionally, the cur-
rent commercially available chips that assay genome-wide variation do a very poor
job at assaying genetic variability in Africa (Albrechtsen et al. 2010). Recent whole
genome sequencing studies in Africa (Abecasis et al. 2012; Lachance et al. 2012)
indicate that common SNP arrays are missing much of the underlying genomic
variation in Africa, much of which may be population or region specific. Therefore,
it is quite likely that current genome-wide studies simply have not assayed enough
of the genetic variability in Africa to understand what portions of the genome are
involved in susceptibility to malaria. Additionally, studies have shown that there are
low levels of LD in African populations (Tishkoff and Williams 2002), which may
hamper conventional genotype-phenotype association tests. Lastly, even with large
sample sizes and large number of genetic markers, it is likely that there will still be
a large amount of “missing heritability” to be discovered, some of which may be the
result of complex gene–gene and gene–environment interactions as well as complex
regulatory networks.

Conclusions and Future Directions

Human genetic studies have identified a number of genetic variants that play a role
in malaria resistance and susceptibility. Because malaria is a strong selection pres-
sure, mutations that are potentially deleterious can be maintained in populations and
will evolve adaptively if they confer protection from malarial infection. Genes that
carry these adaptive mutations will exhibit signatures of natural selection that vary
depending on the age and functional consequences of a particular variant.
Several of the examples discussed above include malaria-protective variants that
have arisen relatively recently and have strong deleterious effects or result in obvious
148 F. Gomez et al.

clinical abnormalities (Table 2). The HbS mutation at HBB and the A- allele at G6PD
are examples of mutations that fall into this category. These genes are generally char-
acterized by distinct haplotypes on which the protective mutation occurred and the
adaptive haplotypes tend to have low haplotype variability and high LD. Other loci,
such as ABO, GYPA, ICAM-1, and CD36 have pleiotropic effects and have complex
signatures of selection resulting from different selective forces acting over different
time periods. These loci may not exhibit classic signatures of recent positive selec-
tion. However, studies that examine the correlation of patterns of variation at these
loci with malaria endemicity (e.g., GYPA and ICAM-1) may be informative for iden-
tifying signatures of selection that are associated with malaria susceptibility.
Looking forward, as the cost of genomic sequencing becomes more affordable
and population genomic studies become a reality, we will be able to examine many
diverse African populations with varying risk for malaria. We can use these data to
identify new candidate loci that influence susceptibility to malaria and test whether
genetic variation is correlated with malaria endemicity. These data may help to
explain the prevalence of deleterious genetic variants in specific ethnic groups and
populations of recent African descent. Low-cost whole-genome sequencing will
also create the opportunity to study genomic variation in Plasmodium genomes.
These data combined with human genomic sequences will provide the means to
explore host–pathogen coevolution and will help us better characterize malaria as a
foundational selective pressure in human evolution.
In summary, malaria has been and continues to be an important selective pres-
sure in modern human evolution. When we are able to combine our improved tech-
nology with access to all human populations at risk for malaria, we will better
understand how our genes play a role in susceptibility to malaria and the role malaria
has played in shaping the human genome.

Acknowledgments We would like to thank the two anonymous reviewers for their critiques and
helpful suggestions. We also thank Dr. Katrina Van Heest for her editorial assistance. S.A. Tishkoff
is supported by a National Institutes of Health grants R01GM076637 and DP1-OD-006445-01,
and NSF Hominid grant (BCS0827436). A Doctoral Dissertation Improvement Grant from the US
National Science Foundation (NSF) (BCS0925802) was given to F. Gomez An NSF IGERT grant
(9987590) to F. Gomez and S.A. Tishkoff supported this research. F. Gomez was also supported by
a Ford Foundation Pre-doctoral fellowship, a Cosmos Club research award, a Sigma Xi (GWU)
Grant-in-Aid of Research (GIAR), and an American Anthropological Association Minority
Dissertation Writing Fellowship.

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Parasitic Lice Help to Fill in the Gaps
of Early Hominid History

Julie M. Allen, Cedric O. Worman, Jessica E. Light, and David L. Reed

Introduction

Hearing the word “lice” will immediately terrify parents and cause school nurses to
spring into action. Pediculosis (a louse infestation) is not a new problem—lice have
coevolved with humans over millions of years, and at this moment in human history,
head lice are a worldwide epidemic. Although they can infect anyone, they are most
common among children aged 3–12 and are widely spread throughout our school
systems. According to the World Health Organization, it is thought that around
10–20 % of children are infested worldwide. In the USA alone, approximately 6–12
million infestations occur every year (Frankowski and Weiner 2002). Parents have
attacked this problem using every method from shaving their child’s head to cover-
ing the entire scalp with petroleum jelly, vinegar, and even toxic chemicals like
kerosene (Meinking 1999; Frankowski and Weiner 2002). Even though we have
been evolving with lice for millions of years, we are still struggling to understand
and eradicate these parasites.

J.M. Allen (*)

Florida Museum of Natural History, University of Florida, Museum Rd. and Newell Dr.,
Gainesville, FL 32611, USA
e-mail: [email protected]
C.O. Worman
Biology Department, University of Florida, 223 Bartram Hall, Gainesville, FL 32611, USA
J.E. Light
Department of Wildlife and Fisheries Sciences, Texas A&M University, 210 Nagle Hall,
College Station, TX 77843, USA
D.L. Reed
Florida Museum of Natural History, University of Florida, Museum Rd. and Newell Dr.,
Gainesville, FL 32611, USA

J.F. Brinkworth and K. Pechenkina (eds.), Primates, Pathogens, and Evolution, 161
Developments in Primatology: Progress and Prospects,
DOI 10.1007/978-1-4614-7181-3_6, © Springer Science+Business Media New York 2013
162 J.M. Allen et al.

Fig. 1 Male human head

louse (Pediculus humanus)
left and female chimpanzee
louse (Pediculus schaeffi)
right. The circle outlines the
modified tibia with extended
claw for hanging onto and
climbing up and down hair
shafts

Although it is the most common, the head louse (Pediculus humanus capitis) is
just one of three types of lice that infest humans. Most similar to head lice are body
lice (Pediculus humanus humanus), perhaps more aptly called “clothing lice”
because they live principally in the clothing. The third and more distantly related
louse is the pubic louse (Pthirus pubis), which lives primarily in the pubic region.
Taxonomically, pubic lice belong to a different louse family, Phthiridae, whereas
head and clothing lice belong to the family Pediculidae. These insects are host-
specific obligate parasites that do not spend any part of their life cycle off their host.
They live around 30 days and females attach eggs to the base of a hair shaft, laying
around 3–5 eggs per day. Eggs hatch in 5–9 days, and in about 10 days nymphs
become reproductively active. These insects feed on the blood of their hosts several
times a day (Buxton 1947). Because lice have secondarily lost their wings, they can-
not fly. Instead, lice move by climbing up and down hair shafts. Their tibiae are
modified with claws that are adapted specifically for holding onto hair (Fig. 1). If a
louse is removed from its host for a long period of time, it does not survive. In fact,
most lice become so dehydrated that they cannot move after 21 h off the host
(Burgess 2004).
Although we have been studying lice for hundreds of years (Darwin 1871; Hooke
1665), research slowed significantly in the 1940s when DDT was introduced and
seemed likely to eradicate lice. In the 1990s, however, prevalence of lice increased
worldwide due to pesticide resistance, and in 1997 there was a massive outbreak of
typhus, which is transmitted by clothing lice (Raoult and Roux 1999), and louse
research found a new beginning (Burgess 2004). This research reached a new peak
in 2010 when the first louse genome (Pediculus humanus humanus) was sequenced
(Kirkness et al. 2010). The genome work revealed a number of fascinating charac-
teristics about lice. For example, clothing lice have the smallest insect genome
sequenced to date (only 108 megabases), and they do not have many of the genes
related to environmental sensing, which may be a result of their highly specialized
lifestyle as an obligate parasite. Here we review the biology and latest research on
head, clothing, and pubic lice including what these parasites can teach us about our
own evolutionary history.
Parasitic Lice Help to Fill in the Gaps of Early Hominid History 163

Head Lice: Pediculus humanus capitis

Head lice have long been considered an economical and societal problem rather
than a dangerous infectious disease (Hansen and O’Havier 2004; Stafford 2008).
The estimated cost of head louse infestation ranges from $367 million to $1 billion
per year. Spending on over-the-counter chemical treatments, loss of school days,
and loss of workdays for parents contribute to this high cost (Hansen and O’Havier
2004; Frankowski and Bocchini 2010). There are a number of myths and stigmas
associated with head lice that cause undue stress to parents and family members of
children infested with lice (Frankowski and Weiner 2002; Gordon 2007; Frankowski
and Bocchini 2010). For example, head lice can be found in all social classes and
are not correlated with cleanliness (Frankowski and Bocchini 2010). Head lice do
not appear to spread disease in natural populations. Although, some studies have
detected infectious bacteria in head lice (Sasaki et al. 2006a,b; Bonilla et al. 2009;
Angelakis et al. 2011 but see Parola et al. 2006), and other research has suggested
that head lice even have the capacity to transmit the infectious bacteria (Goldberger
and Anderson 1912; Murray and Torrey 1975) to date, there has not been a known
outbreak of disease caused by head louse transmission. The most common symp-
tom from a head louse infestation is pruritis (itching) caused by a bite from the
louse. In extreme cases, scratching the bite can cause an infection from common
skin bacteria (Meinking 1999), but for the most part head lice are a mere annoyance
rather than a dangerous parasite.
Manual removal of lice is a common means of treating head louse infestations,
an activity which has shown up in artwork for centuries (the term “nit-picking”
actually refers to the physical removal of lice). Recently, the most popular treatment
of head lice has been chemical. Unfortunately, pesticide resistance has increased
tremendously over the last few decades, (Burgess 2004) and in some places lice are
even resistant to more than one chemical. Currently no pesticide is 100 % effective
(Frankowski and Weiner 2002; Tebruegge et al. 2011). Other nonchemical methods
(such as manual removal) have been suggested; however, these methods have been
met with mixed success likely due to the effort required to remove all the lice
(Frankowski and Bocchini 2010). One new method of louse eradication focuses on
the temperature sensitivity of the lice. This sensitivity to temperature has been
known for some time (Buxton 1947); in fact studies have shown that lice are likely
to leave a person with a fever for a healthy person (Lloyd 1919 cited in Buxton
1947), and it has been suggested for some time that hot temperatures may be a way
to kill lice. Following this literature, a modified prototype of the LouseBuster™ has
recently been released. This appliance uses hot air to kill the lice by dehydrating
them at high temperatures. In clinical trials, this method had a 94 % success rate in
killing lice and 100 % success killing eggs on infected individuals. Thus, the
LouseBuster™ may prove to be a faster, more effective, nonchemical method for
treating head lice (Bush et al. 2011).
Head lice are most commonly transmitted from human to human by direct con-
tact (Canyon and Speare 2010). It has been long suggested that lice can be
164 J.M. Allen et al.

transmitted via fomites, objects such as combs, and pillows (Burkhart and Burkhart
2007). However, this idea has been strongly challenged, and little to no evidence of
fomite transmission has been found (Canyon and Speare 2010). Furthermore, head
lice removed from the head die quickly due to dehydration (Burgess 2004), making
fomite transmission unlikely. Unfortunately, methods to eradicate lice from schools
have been difficult due to these types of misinformed ideas about how lice move
from child to child.
An extremely controversial societal issue with lice is the “no nit” policies adopted
by some schools. These policies require children to stay out of school until they are
free of detectable nits (louse eggs). One issue with this policy is that empty nit cas-
ings (those from which the louse has already hatched) can remain on the hair long
after a louse outbreak has ended. Furthermore, eggs are incubated by body heat so
nits more than 1 cm from the scalp are unlikely viable (Frankowski and Weiner
2002). This means that nits that are farther from the scalp are likely empty and left
over from a cured infestation and may remain in the hair until it grows out. Even
more problematic, there is evidence that cases of head lice are frequently misdiag-
nosed (Pollack et al. 2000). Because of this, many children, particularly those with
longer hair, miss school unnecessarily (Gordon 2007), which inflates the total cost
of pediculosis per year. Not only are these children missing valuable class time, they
are likely to face bullying by their classmates upon return due to the stigma associ-
ated with head lice.

Clothing Lice: Pediculus humanus humanus

Clothing lice (Pediculus humanus humanus), which are also called body lice, live in
clothing fibers where they attach eggs to cloth fibers rather than hair (Buxton 1947).
They look very similar to head lice, although there are some important differences
between them. Clothing lice are generally larger than head lice and can consume a
larger blood meal (Busvine 1978; Meinking 1999; Reed et al. 2004). Clothing lice
only go to the skin to feed a few times a day, much less than head lice and likely why
they consume a larger blood meal. Additionally, unlike head lice, clothing lice are
associated with conditions of poor hygiene and are commonly found on those forced
to live in crowded situations where they lack the ability to change or wash clothes
regularly such as refugees, the homeless, soldiers, and victims of war or natural
disasters (Meinking 1999).
It has been unclear for quite some time whether head lice and clothing lice are
one species or two, and many studies have found conflicting results (Busvine 1978;
Amevigbe et al. 2000; Burgess 2004; Leo and Barker 2005; Leo et al. 2005; Light
et al. 2008). However, recent studies with more comprehensive sampling are finding
that clothing lice and head lice are in fact the same species and that clothing lice
evolve from head lice in certain conditions (such as in situations where individuals
have a considerable head louse infestation and poor hygiene; Li et al. 2010). It is
now clear that throughout our history clothing lice have opportunistically evolved
Parasitic Lice Help to Fill in the Gaps of Early Hominid History 165

repeatedly from head lice to fill a different ecological niche from that of their head
louse counterparts (Reed et al. 2004; Light et al. 2008; Li et al. 2010).
Finally, and perhaps most importantly, clothing lice are the known vectors of three
human pathogens: Rickettsia prowazekii (agent of epidemic typhus; Andersson and
Andersson 2000), Borrelia recurrentis, and Bartonella quintana (the agents of relapsing
fever and trench fever, respectively; Buxton 1947). These transmissions occur when
louse feces are unintentionally rubbed into an open wound caused by the louse bite,
most generally occurring when scratching the area of the bite (Buxton 1947). These
diseases have had a devastating impact throughout human history. For example, epi-
demic typhus may have been largely responsible for the demise of Napoleon’s Grand
Army around 1812 (Raoult et al. 2006). These diseases have been a problem not only
historically but also recently. In 1999, a large outbreak of endemic typhus broke out in
Burundi, infecting more than 100,000 people. With the growing worldwide problem of
lice due to pesticide resistance, these diseases will need to be carefully monitored.

Pubic Lice: Pthirus pubis

The third type of louse that parasitizes humans is Pthirus pubis, commonly known
as the “crab” or pubic louse. Pubic lice are a sexually transmitted disease (STD),
and their presence has often been found in combination with other STD infections
(Anderson and Chaney 2009). Pubic lice are also a worldwide phenomenon.
Although it is much more difficult to calculate the level of prevalence because infec-
tions are not often reported, recent estimates suggest that 2 % of the world’s adult
population are infected with pubic lice (Anderson and Chaney 2009). Pubic lice are
found in all levels of society and among all ethnic groups (Meinking 1999).
Pubic lice live primarily in the androgenic hairs (hair that begins to grow at sex-
ual maturity) around the groin; however, these lice have been found on children in
the eyelashes, eyebrows, and edges of the hairline. In rare cases the presence of
Pthirus on children has alerted authorities to incidents of child abuse; however, this
is not common (Chosidow 2000). It is thought that Pthirus prefer hair that is more
widely spaced due to the wider spacing of their claws (Waldeyer 1900; Nuttall
1918; Fisher and Morton 1970). Interestingly, it is thought that fomite transmission
is more important in pubic lice, which may explain how children get an infestation
in their eyebrows and eyelashes without sexual contact (Meinking 1999). Similar to
human head lice, pesticides and manual removal are considered to be the primary
treatment for pubic lice (Orion et al. 2004).
The genus Pthirus has two species of lice: the human pubic louse and the gorilla
louse, Pthirus gorillae. The host associations of this genus of louse have puzzled
researchers for some time: why are humans and gorillas, but not chimpanzees, para-
sitized by Pthirus? Furthermore, although lice are very host specific, why do humans
have two genera of lice (Pediculus and Pthirus), whereas chimpanzees and gorillas
each have one (Pediculus and Pthirus, respectively; Fig. 2)? In 2007, Reed et al.
conducted molecular dating analyses on gorilla and human pubic lice and found that
166 J.M. Allen et al.

Fig. 2 The coevolutionary history of humans (Homo), chimpanzees (Pan), and gorillas (Gorilla)
and their lice. The primate lineages are indicated by thin black lines and black boxes that depict the
longevity (box height) and the species richness (box width) of the primate genera known from
physical evidence (either the fossil record or extant species). Parasite lineages are indicated with
thick gray bars with the lighter gray representing Pediculus and the darker gray Pthirus. Dotted
lines indicate possible coevolutionary scenarios that remain unclear due to lack of data. The most
recent estimated divergence between gorillas and the lineage leading to humans and chimpanzees
is shown at 9.2 mya; however, we also show the possible divergence (and consequently the cospe-
ciation event) between these lineages at 13 mya incorporating the gorilla-like fossil Chororapithecus
abyssinicus (dashed lines at 13 mya). We further show the host switch event 3–4 mya by lice in the
genus Pthirus from the gorilla lineage onto the hominin lineage. The extinct hominin genus
Paranthopus and its possible association with both Pediculus and Pthirus are shown at approxi-
mately 2 mya. Events on the right represent our current knowledge of hominin history including
time ranges for the origin of clothing use by Homo sapiens, the first putative shelters, the earliest
known butcher marks, and the development of bipedality (as indicated from the louse data).
Asterisks mark isolated fossil finds. Species abbreviations are as follows: Ar. ka = Ardipithecus
kaddaba, Ar. ra = Ardipithecus ramidus, Au. ana = Australopithecus anamensis, Au.
afa = Australopithecus afarensis, and Au. afr = Australopithecus africanus. O = Orrorin tugenensis,
S = Sahelanthropus tchadensis, K = Kenyanthropus platyops, and A = Australopithecus bahrel-
ghazali. (1) McBrearty and Jablonski (2005). (2) Foley (2002). (3) Pickford et al. (1988). (4) Suwa
et al. (2007). (5) Strait et al. (1997). (6) Leakey et al. (2001). (7) Brunet et al. (1995). (8) Kimbel
et al. (2006). (9) WoldeGabriel et al. (2009). (10) Haile-Selassie (2001). (11) Pickford et al. (2002).
(12) Brunet et al. (2002). (13) Toups et al. (2011). (14) Rantala (1999). (15) McPherron et al.
(2010). (16) Reed et al. (2007). (17) Pickford et al. (2002)
Parasitic Lice Help to Fill in the Gaps of Early Hominid History 167

these two species were sister taxa. Additionally, these two taxa diverged only 3–4
million years ago (mya), much more recently than the gorilla/human-chimp split,
which is estimated at around 9 mya (Wilkinson et al. 2011). This finding was
extremely interesting as it shed light onto two details of human evolutionary history
that were previously unknown. Here we go into more detail about the biology of
Pthirus to discuss what this host switch tells us about our ancestors 3–4 mya.

Lice Tell Us About Our Past

Sucking lice have been coevolving with their hosts for at least the last 65 million
years and likely much longer (Light et al. 2010). Due to their obligate nature and the
fact that they mostly move between hosts via direct contact, these blood-feeding lice
have been used to give us clues about their hosts’ evolutionary history not easily
gleaned from the fossil record (e.g., behavior). Interestingly, this idea dates back to
Darwin (1871) who in On the Decent of Man wrote:
“…and the fact of races of man being infested with parasites which appear to be specifically
distinct might fairly be urged as an argument that the races themselves ought to be classed
as distinct species.”

Although the idea that races of humans represent different species is no longer
entertained and even the concept of race has dramatically changed since Darwin’s
time, the idea that lice can tell us about our evolutionary history is now well accepted
and gaining momentum (Whiteman and Parker 2005; Hypsa 2006; Nieberding and
Olivieri 2007; Reed et al. 2009).
In 2003, it became apparent upon genetic examination of recently collected
human head and clothing lice that there were several distinct lineages of lice (Kittler
et al. 2003). Reed et al. (2004) used fossil calibrations for the split between humans
and chimpanzees (5.6 mya) and the split between great apes and Old World mon-
keys (22.5 mya) to estimate the divergence time between these human louse lin-
eages. They found that the youngest of these lineages splits around 1.18 mya, which
is similar in age to the ancestor of Homo sapiens and H. neanderthalensis. The other
louse lineage is even older, suggesting its origin may date back to a H. erectus-like
host. Because head lice are primarily transmitted through direct contact, this finding
suggests that modern humans came into contact with archaic hominin species and
picked up distinct lineages of head lice. Although we do not know which archaic
humans our ancestors came into contact with, the timing of the divergence of the
ancient head louse lineages is consistent with contact with H. neanderthalensis and
H. erectus. Furthermore, while the type of contact between different hominin spe-
cies is unknown, recent work sequencing the H. neanderthalensis genome found
evidence of interbreeding between H. neanderthalensis and H. sapiens (Green et al.
2010; Yotova et al. 2011). Similar types of contact between H. sapiens and H. erec-
tus would have been sufficient for the transfer of lice and may explain the existence
of ancient lineages of head lice on modern humans.
168 J.M. Allen et al.

Because clothing lice live exclusively in the clothing, they are thought to have
evolved only after humans began to wear clothes, and it has long been proposed that
dating the origin of clothing lice could give us a date by which H. sapiens must have
been wearing clothing (Kittler et al. 2003, 2004). Previous estimates of when
humans started wearing clothing were based on the emergence of eyed needles
(which suggests complex clothing had already been developed) around 40,000 mya
(Delson et al. 2000) and sometime after the loss of body hair as late as 1.2 mya
(Rogers et al. 2004; based on molecular evidence) and as early as 3 mya (Reed et al.
2007; detailed below). Recent molecular evidence from clothing lice suggests that
clothing use originated between 83,000 and 170,000 years ago, which is earlier than
previously proposed, and suggests that clothing use by H. sapiens likely originated
before they moved out of Africa (Toups et al. 2011). This clothing use may have
enabled modern humans to more readily move into colder climates as they migrated
out of Africa and eventually throughout the world.
The coevolutionary relationships between great apes and their lice have been
worked out morphologically and molecularly and are illustrated in Fig. 2. Humans
and chimpanzees share lice in the genus Pediculus as sister taxa, and humans and
gorillas share lice in the genus Pthirus (Fig. 2). The split between human and chim-
panzee lice was estimated to be 5–7 mya (Reed et al. 2004, using mitochondrial
genes; Light et al. 2008, using a multigenic approach with mitochondrial and
nuclear genes), strongly suggesting cospeciation between these two lice and their
primate hosts (humans and chimpanzees also are believed to have diverged at this
time; Wilkinson et al. 2011). On the other hand, Reed et al. (2007) found that the
Pthirus and Pediculus are sister taxa and they diverged ~13 mya, long before the
presumed 7 mya split between gorillas and the other African apes (Fig. 2). Reed
et al. (2007) hypothesized an evolutionary scenario in which there was a louse
duplication (or speciation) event on the African ape common ancestor to humans,
chimpanzees, and gorillas. In other words, one louse species would have diverged
into two on the ape common ancestor. More recent refinement of the somewhat
troublesome great ape molecular clock has pushed the gorilla divergence back to
9.2 mya (Wilkinson et al. 2011). Additionally, a recent fossil find of the gorilla-like
ape Chororapithecus abyssinicus dating to 10–11 mya (Suwa et al. 2007) suggests
that the great ape molecular clock may still be underestimating the divergence of the
gorillas by millions of years. If true, the presumed louse duplication event ~13 mya
suggested by Reed et al. (2007) could have actually been a cospeciation event, sug-
gesting that the divergence time between gorillas and the other African apes occurred
~13 mya, far earlier than currently thought (Fig. 2).
Reed et al. (2007) also examined the history of the two species of Pthirus, one on
gorillas and the other on humans, which diverged 3–4 mya. Reed et al. (2007)
hypothesized that after Pthirus and Pediculus diverged ~13 mya, the Pthirus lineage
remained on ancestral gorillas but went extinct on the common ancestor of humans
and chimpanzees, and the Pediculus lineage remained on the common ancestor of
humans and chimpanzees but went extinct on ancestral gorillas. Then, approxi-
mately 3–4 mya, the Pthirus lineage from ancestral gorillas switched to a human
ancestor (Fig. 2). This type of host switch is not uncommon; there have been a
Parasitic Lice Help to Fill in the Gaps of Early Hominid History 169

number of zoonotic transmissions of diseases from primates, as well as domesti-

cated animals, to humans (Wolfe et al. 2007). For example, HIV-1 is now known to
have come from a chimpanzee (Gao et al. 1999) possibly as a result of humans hunt-
ing chimpanzees for food. This particular host-switching event of Pthirus on ances-
tral gorillas to our human ancestors gives us clues about human evolutionary history
that we outline and detail below.

Human Hair Loss

Reed et al. (2007) postulated that a Pthirus-type louse switched hosts from archaic
gorillas to hominins approximately 3–4 mya. It is interesting to consider what was
necessary for this host switch to have been successful: there had to have been a
niche for Pthirus to occupy. Studies of chewing lice have found that their mouth-
parts (which grip the hair) are highly adapted and specialized to the hair of their
hosts (Reed et al. 2000), so it is likely that hair type is similarly important to suck-
ing lice. Rather than using their mouthparts to grip hair, sucking lice use their
claws, which are also highly specialized. Pthirus, in particular, is highly adapted
to hairs in the pubic regions as these hairs are more widely spaced, which match
the wider spacing of their claws (see below). As stated previously, pubic hair is a
type of androgenic hair—hair that grows in response to increased levels of andro-
gens circulating in the human body at sexual maturity (Randall 2008). Proposed
functions of pubic hair (pheromonal and visual signaling; Randall 2008) could
have only come into play after the loss of typical ape body hair. Additionally, the
invasion of a new host would have been far more likely if Pediculus had already
been confined to the head by the loss of functional body hair, leaving competitor-
free regions available to Pthirus. We hypothesize that the loss or reduction of
body hair as well as the development of androgenic hair would have facilitated the
success of this host switch and therefore suggest that human hair loss and the gain
of androgenic hair had occurred by 3–4 mya, a date that is much older than other
predictions.
Among primates, humans are unique in their apparent nakedness. Humans,
however, are not actually hairless. They have a similar number and density of
hair follicles as other great apes, but the hairs are much finer (i.e., smaller in
diameter) and shorter and offer little protection or insulation (Kushlan 1985;
Amaral 1996; Rantala 1999). There is a great variety of hypotheses ranging from
the bizarre to the pedestrian as to why humans had such a drastic reduction in
body hair (reviewed in Rantala 2007). Many of these hypotheses are directly
related to the Pthirus host switch because they either incorporate habitat (as dis-
cussed below) and thermodynamics (which is closely tied to habitat) or attempt
to establish the timing of hair reduction. Habitat and thermodynamics are impor-
tant to the cooling device, bipedality, hunting, vestiary, allometry, and other
hypotheses of why humans lost their body hair. The timing of hair loss is incor-
porated to some extent in any hair loss hypothesis, but it is particularly important
in the clothing, vestiary, and ectoparasite hypotheses.
170 J.M. Allen et al.

The reduction in body hair has obvious thermodynamic consequences. This

loss of insulation increases heat exchange with the environment. Several body
hair loss hypotheses (see Rantala 2007) are based on the need to shed increased
heat loads resulting from either a move from forest into hotter savanna habitats
(cooling device, bipedality, hunting, vestiary hypotheses), an increased activity
(hunting and vestiary hypotheses), or a large body size (allometry hypothesis).
The cooling device hypothesis states that the increased heat load was simply
caused by the move into open savannas from the forest (Rantala 2007). The biped-
ality hypothesis adds to this by examining how an upright stance decreases the
solar heat load experienced by an individual in a bipedal stance compared to a
quadrupedal stance (Wheeler 1992). Active hunting in the savanna and the excess
heat that must be shed from high levels of activity are incorporated into the hunt-
ing hypothesis (Brace and Montagu 1977). The additional need to retain heat
during the cool savanna nights (fulfilled by the use of clothing) while being able
to shed heat during the day, presumably through hairlessness and sweating, is the
basis of the vestiary hypothesis (Kushlan 1985). The allometry hypothesis is
based on the observation that larger primates have increasingly more widely
spaced hair (Schwartz and Rosenblum 1981).
As intuitive as it may seem to people from cooler climes that shedding insulation
increases heat loss, hot open savanna environments make body hair extremely valu-
able for decreasing heat gain from both solar radiation and the air (Newman 1970).
The upright stance central to the bipedality hypothesis reduces solar heat gain com-
pared to a quadrupedal stance, making it less detrimental to be hairless, but it does
not make hair loss beneficial in savanna environments (Amaral 1996). In addition,
it is now clear that bipedalism evolved in basal hominins by the time of Orrorin
tugenensis (Pickford et al. 2002; Galik et al. 2004) around 5.7–6 mya (Richmond
and Jungers 2008), long before the shift to dry open habitats by Homo (Elton 2008),
and that evaporative cooling is not prevented by body hair; the patas monkey
(Erythrocebus patas), a cursorial savanna monkey, has both thick fur and effective
sweating (Mahoney 1980). Another characteristic of savannas compared to forests
(addressed by the vestiary hypothesis) is colder nights unmitigated by heat-retaining
forest tree cover and humidity (Amaral 1996). This makes the insulation provided
by body hair even more valuable and makes loss of body hair in the savanna envi-
ronment doubly detrimental.
The allometry hypothesis is not a complete explanation of human hairlessness by
itself. Schwartz and Rosenblum (1981) reanalyzed Schultz’s (1931, 1969) measure-
ments of primate hair density and found that there are fewer hairs per unit of body
surface in larger primates compared to smaller primates. They reasoned that this
reduction in body hair was likely due to thermoregulatory constraints associated
with decreasing ratios of surface area to volume, which make shedding metabolic
heat difficult for larger animals. Because fossil data indicated that early australo-
pithecine hominins weighed between 45 and 70 kg (Pilbeam and Gould 1974),
Schwartz and Rosenblum (1981) hypothesized that substantial decreases in hom-
inin hair density likely occurred prior to human shifts from forest to grassland habi-
tats at the end of the Pliocene.
Parasitic Lice Help to Fill in the Gaps of Early Hominid History 171

However, the effective hairlessness of humans is not just a result of hair density
but also hair size. In contrast to humans, other similar-sized and larger apes (orang-
utans and gorillas) have substantial body hair. Thus, hominins appear to have exag-
gerated the typical primate strategy of shedding metabolic heat via reduced
insulative effectiveness of body hair (the heavy sweating of humans and patas mon-
keys does not appear to be typical of primates; Amaral 1996) by reducing hair size.
It seems likely that this was in answer to an additional metabolic heat load beyond
that experienced by typical apes. Although it is impossible to say with any degree of
certainty what this additional heat load was, the development of bipedalism, a more
energetically efficient mode of locomotion than knuckle walking (Sockol et al.
2007), hints that increased daily travel may have been important to the basal hom-
inin niche and that hair loss may have occurred very early on. The extra metabolic
heat produced by travel through forests could have been shed by decreasing body
hair insulation without the costs of nakedness associated with savanna environ-
ments. Based on the timing of the Pthirus host switch, the habitat in which this
switch likely occurred (see below), and the problems of hairlessness in open habi-
tats, hair loss in the hominin line almost certainly occurred in a forested habitat and
was complete and effectively irreversible by the time savanna habitats were fully
utilized. The human dependence on sweating as a cooling mechanism likely
occurred long after hair loss to deal with the additional heat loads in open habitats
as sweating is less effective in the humid still air of forests than in drier more open
habitats (Newman 1970; Montagna 1972).
Other than lice, the only line of evidence that helps establish the timing of hair
loss in the hominin line is genetic. The human melanocortin 1 receptor (MC1R)
gene is involved in human skin coloration. By looking at the neutral variation in this
gene, Rogers et al. (2004) estimated that human skin has been exposed to strong
sunlight for at least 1.2 my. Therefore, based on the MC1R data, human ancestors
became both hairless and began living in the savanna between 1.2 mya and 6–7 mya
(the chimpanzee/hominin split). The lice data are consistent with the MC1R data
but give a narrower range of 3–4 mya from the Pthirus switch to the 6–7 million
year split between the human and chimpanzee lineages.
The timing of hair loss is particularly central to the clothing, vestiary, and ecto-
parasite hypotheses. The clothing hypothesis (Glass 1966) is similar to the vestiary
hypothesis (Kushlan 1985) in that they both posit that clothing superseded the insu-
lative value of body hair and hair loss occurred with or after the invention of cloth-
ing. However, while the vestiary hypothesis holds (erroneously, as discussed above)
that the loss of body hair was advantageous during the hot days and that clothing
replaced the need for body hair during the cool nights, the clothing hypothesis
argues that after clothing was invented, body hair disappeared as it was no longer
needed (Glass 1966; however, Glass does not propose a reason for the invention and
use of clothing by hominins with functional coats of body hair). The louse and
MC1R data estimates for both hairlessness (Pthirus, >3–4 mya in Reed et al. 2007;
MC1R, >1.2 mya in Rogers et al. 2004) and the invention of clothing (Pediculus,
0.08–0.17 mya in Toups et al. 2011) indicate that clothing had nothing to do with
the evolution of hairlessness in hominins.
172 J.M. Allen et al.

The ectoparasite hypothesis states that when hominins first established long-term
habitations, they were beset with new types of ectoparasites, such as fleas, that com-
pleted their life cycles in the living space but off the body of the host (Rantala 1999).
Thus, the loss of body hair was a defense against increased parasite loads encouraged
by the establishment of a home base. In apparent conflict with the adaptation-
against-ectoparasites hypothesis is the presence of pubic hair (Pagel and Bodmer
2003). Pubic hair, however, may play an important role in sexual selection and thus
may have been selected for in spite of its ability to shelter ectoparasites. The moist
and humid environment of the pubic region (due to an increased density of sweat
glands; Stoddart 1990) is favorable to pheromonal signaling (Guthrie 1976), and
pubic hair could have initially functioned in pheromonal signaling (Randall 1994),
visual signaling (Randall 2008), or both. Rantala (1999) associates the beginnings
of long-term settlements with an increase in cooperative hunting and places both
developments at ~1.8 mya based on excavations of Homo habilis artifacts at Olduvai
Gorge. While this estimate is consistent with the hairlessness range provided by the
MC1R gene (>1.2 mya), it is far later than the hairlessness estimate provided by the
Pthirus host switch.

Pediculus and Pthirus

The two genera of human lice (Pediculus and Pthirus) occupy distinct niches on the
body (head/clothing and pubic region, respectively). Many researchers have won-
dered why Pediculus and Pthirus do not co-occur and are apparently isolated to
these different regions especially given their similar biology (Howlett 1917).
Hypotheses have included Pthirus having a preference for darkness and moist areas
(Nuttall 1918; however, this idea is not accepted as Pthirus survives on eyelashes
and eyebrows) and that differences in hair spacing have geographically restricted
these lice because Pediculus cannot adequately grasp the hairs of the pubic region,
and Pthirus cannot grasp the hairs on the head. Both genera of lice have been found
occasionally occupying and surviving in other regions of the body (see below), but
they do not seem to be successful in these areas. Of these hypotheses, hair spacing
seems to be the most likely explanation for restricting these two types of lice to their
respective habitats.
Schwartz and Rosenblum (1981) found that there are fewer hairs per unit of body
surface in larger primates compared to smaller primates. If the Reed et al. (2007)
hypothesis that the human pubic louse (Pthirus pubis) is a descendent of gorilla lice
is true, then based on Schwartz and Rosenblum’s (1981) findings, we can postulate
that Pthirus was adapted to living among widely spaced hairs because gorillas are
the largest extant primate. Early studies of Pthirus support this idea. Pthirus uses its
second and third pair of legs to cling to host hair, and these legs, when stretched
apart, span a distance of 2 mm (Waldeyer 1900; Nuttall 1918; Fisher and Morton
1970). It just so happens that hairs in the pubic region are also distributed 2 mm
apart (Waldeyer 1900; Nuttall 1918). Furthermore, the number of hairs present in
Parasitic Lice Help to Fill in the Gaps of Early Hominid History 173

the pubic region (34 hairs/cm2) is significantly less than the head (220 hairs/cm2;
Waldeyer 1900; Payot 1920). All in all, Pthirus appears to prefer body regions with
widely spaced hairs for better grasping as well as for ease of flattening itself against
the skin (Burgess et al. 1983; Burgess 1995; Nuttall 1918; Buxton 1947; Fisher and
Morton 1970). According to measurements made by Schultz (1931), chimpanzee
hair density is more similar to humans than to gorillas. That, in addition to the lack
of pubic-type hair on chimpanzees, may help explain why there are no Pthirus spe-
cies currently parasitizing chimpanzees.
The Pthirus preference for widely spaced hairs is likely why this genus can
be occasionally found in other sparsely haired areas on the human body, such as
the margins of the scalp, eyebrows, eyelashes, and areas of the trunk such as the
chest, stomach, and thighs (if sufficient body hair is present; Burgess 1995, and
references therein; Buxton 1941, 1947; Elgart and Higdon 1973). Pediculus, in
comparison, is rarely found in the pubic region of humans (Busvine 1944).
Pediculus schaeffi, the louse on chimpanzees louse, is more catholic in habitat
choice than Pediculus humanus and can be found almost anywhere on a chim-
panzee host but favors the groin, underarms, and head (D. Cox, pers. comm.). It
is likely that the Pediculus found on hominins before the loss of body hair was
similarly widely spread but was restricted to the head region during the hominin
denudation and subsequently prevented from spreading to androgenic hair
because the larger spacing of pubic hair made movement from one hair to
another difficult.
Differences in mobility also may prevent Pediculus from traveling to the pubic
region as often as Pthirus appears to move to other parts of the body. Although
several studies have found that Pediculus moves faster than Pthirus when displaced
from the body (Nuttall 1918; Busvine 1944), Pthirus does tend to wander more
(Burgess et al. 1983). Furthermore, head lice are recognized as being rather picky in
how they move from hair to hair, suggesting that they are unlikely to move readily
to foreign objects or fomites (Canyon et al. 2002). Closer examination of the first
tarsal claws (which may facilitate movement when lice are not in contact with hair)
of both Pediculus and Pthirus reveals why there may be differences in mobility
between these two genera (Ubelaker et al. 1973). The inner surface of the first tarsal
claw in Pthirus is serrated, allowing for traction even on smooth surfaces, whereas
in Pediculus the inner surface of the claw is smooth and the lice are unable to move
without hair follicles or roughened surfaces (Nuttall 1918; Ubelaker et al. 1973;
Burkhart and Burkhart 2000). This simple difference, along with preferential move-
ment patterns, may restrict Pediculus from moving easily on smooth, non-haired
substrates. Pthirus, on the other hand, with their serrated first tarsal claws, may be
able to move much more easily on non-haired substrates, thus allowing them to
reach other parts of the body such as the perimeter of the scalp. The biology of these
two parasites supports the idea that human ancestors had not only lost their body
hair by the host switch 3–4 mya (isolating Pediculus in the head region) but that
early hominins had also developed androgenic hair, providing a suitable environ-
ment for Pthirus.
174 J.M. Allen et al.

Habitats of Early Hominins

Given the fossil species currently known, the most parsimonious scenario explain-
ing the appearance of Pthirus in the human lineage is a host switch directly from
gorilla ancestors to human ancestors. This scenario strongly implies that the two
ancestral host species came into repeated and close contact, which further implies
significant overlap in habitat. By combining data from the fossil record, paleocli-
mate, extant species, and the Pthirus host switch, we can augment the current think-
ing of the habitat and habits of human ancestors.
The fossil record of nonhuman African apes is abysmal to say the least. There
is currently only one known chimpanzee fossil, which lived 0.5 mya (McBrearty
and Jablonski 2005); one gorilla fossil from 5 to 6 mya (Pickford et al. 1988);
and the very gorilla-like Chororapithecus abyssinicus from 10 to 11 mya (Suwa
et al. 2007; Fig. 2). The reasons for the dearth of these fossils are likely due to
several factors (Cote 2004). For one, apes were an uncommon component of the
fauna in any region, and African fossil sites commonly produce fewer speci-
mens than Eurasian fossil sites. Therefore, a site must produce a large number
of fossils if any apes are expected to be represented in the first place. Second,
with the exceptions of Samburupithecus kiptalami and Nakalipithecus nakay-
ami, which might have been adapted to drier forests (Kunimatsu et al. 2007),
nonhuman African apes are, and appear to always have been, tightly associated
with moist tropical forests. The wet acidic soil in these types of habitats is much
more conducive to quick bone decomposition than fossilization, so it is likely
that very few specimens were fossilized to begin with (Kingston 2007). Added
to those problems is the fact that sites currently under moist tropical forests are
seldom found or excavated, partly due to a lack of exposed strata and partly due
to the political insecurity that often inflames those regions, reducing safety for
international teams and handicapping intranational capacity for, and interest in,
research.
It is generally thought that the nonhuman African apes are conservative in body
form and habits contrasting with the hominins that stumbled upon a new behavior/
body form (bipedalism) that led to their subsequent radiation into a speciose and
relatively diverse group. That assumption is supported by the dearth of non-hominin
ape fossils in Africa, which indicates they were restricted to the wet forests that are
particularly hostile to fossil formation (Kingston 2007), and by the nature of the few
fossils that have been found. Additionally, the morphology and wear of the
Chororapithecus abyssinicus fossil from 11 to 10 mya (Suwa et al. 2007) and the
gorilla tooth from 5 to 6 mya (Pickford et al. 1988), as well as their respective faunal
assemblage contexts, suggest that gorillas have been conservative in diet and habitat
over the period of time during which the hominin group was rapidly developing
novel traits and habits.
Molecular work has also indicated the conservatism of the gorilla lineage.
Thalmann et al. (2007) have estimated that eastern and western gorillas (Gorilla
beringei and G. gorilla, respectively) diverged 0.9–1.6 mya with very little subse-
quent gene flow between those two species. Thalmann et al. (2007) also suggest that
Parasitic Lice Help to Fill in the Gaps of Early Hominid History 175

the genetic divergence between the eastern and western forms is small enough to
unite the two into a single species. The gene flow between the two groups is low
enough that it is likely not the cause of their genetic similarity but a result of it. With
the apparent conservatism of the gorilla lineage in mind, we can cautiously use the
natural history of extant gorillas to inform us of the probable habits of their ances-
tors and examine how that information fits into and expands the understanding of
our ancestors.
Gorillas today range across forested tropical Africa (with a large interruption
between the eastern and western species) from lowland rainforest to high-altitude
montane forest. In spite of these wide longitudinal and altitudinal ranges, both spe-
cies share similar diets based on succulent herbaceous vegetation (Kingdon 1974).
The diet often incorporates more fruit in areas where fruit is available, but herba-
ceous vegetation still forms a large portion of the diet, retaining its primary impor-
tance especially as a fallback food in times of fruit scarcity (Yamagiwa and Basabose
2006).
The importance of fibrous herbaceous foods makes gorillas more independent of
often unreliably fruiting trees than the two chimpanzee species (Pan troglodytes and
P. paniscus); however, it also limits their available habitat to moist forests with
enough sunlight penetrating the canopy to support a rank herbaceous understory
(Schaller 1963). While swidden agriculture produces ample areas of lush secondary
growth, prior to agriculture, suitable gorilla foraging areas would have been limited
to montane forests, river edges, treefall gaps, elephant tramples, and the like
(Schaller 1965a), with feeding and use by gorillas likely slowing succession and
extending the usable life and possibly the size of temporary clearings (Plumptre
1994). Even if the gorilla lineage had significantly different dietary preferences than
extant gorillas during the host switch of Pthirus 3–4 mya, it is unlikely that the dif-
ferences would have a meaningful impact on our analysis as the conservatism of
body and tooth form and lack of fossils indicate a folivorous/frugivorous diet in a
moist forest.
During the 3–4 mya range given for the Pthirus host switch, there were 1–4
hominin species present (Fig. 2) depending on the validities of species identifica-
tions: Australopithecus anamensis, A. afarensis, A. bahrelghazali, and Kenyanthropus
platyops (Fig. 2). K. platyops (3.5 mya) is known from only one locality and the
skull upon which the identification is based is severely fragmented and distorted
(Leakey et al. 2001). Therefore, the identity of the specimen as a new genus
(Kenyanthropus), a new species within Australopithecus, or another A. afarensis
specimen is controversial (White 2003; Spoor et al. 2010). However, if K. platyops
is a valid species, it is potentially ancestral to both Homo and Paranthropus (robust
australopithecines) and lived in a well-watered forest or woodland (Leakey et al.
2001; Strait and Grine 2004).
Australopithecus bahrelghazali is another species known only from a single fos-
sil from 3.5 mya (Brunet et al. 1995; Brunet 2010) and, like K. platyops, is contro-
versial as to whether it is a separate species or an unusual A. afarensis (Kimbel et al.
2006; Guy et al. 2008). This is a unique find because it is the only australopithecine
found in Chad rather than East or Southern Africa. Unfortunately, the state of the
176 J.M. Allen et al.

fossils makes establishing phylogenetic relationships difficult (Strait and Grine

2004), but it is likely that A. bahrelghazali lived in a gallery forest/wooded savanna/
grassland mosaic context (Brunet et al. 1995).
Australopithecus anamensis (3.9–4.2 mya) was probably the anagenetic ancestor
of A. afarensis (Kimbel et al. 2006) and therefore also an ancestor of Homo (Strait
et al. 1997). Because A. anamensis and A. afarensis are chronospecies, they appear
to be similar in habitat and diet. The habitat of A. anamensis was likely mosaic for-
est, woodland, grassland, bush, and riverine forest (Bonnefille 2010). In spite of the
variety of habitats postulated, A. anamensis seems to have been tied to the presence
of at least some trees and lived at a time of increasing tree cover in Africa (Bonnefille
2010). The diet has been postulated with many methods. Tooth morphology sug-
gests hard brittle items (Grine et al. 2006) but with the ability to exploit fleshy fruits
(Teaford and Ungar 2000). Microwear patterns suggest tough and fibrous foods
(Ungar et al. 2010). Finally, enamel microstructure suggests tough, hard, and abra-
sive foods with limited brittle and acidic foods (Macho and Shimizu 2010). It seems
likely that the majority of the A. anamensis diet was fibrous vegetation that required
grinding with brittle foods forming an important fallback food (Ungar et al. 2010).
The fourth species, Australopithecus (Praeanthropus) afarensis, is by far the
best understood of the four candidates and a presumed ancestor of Homo (Strait
et al. 1997). Sites containing A. afarensis fossils have been found all over East
Africa, which dated from 3.0 to 3.6 mya (Kimbel and Delezene 2009). The paleoen-
vironments of these sites, temporally and spatially, are extremely variable, ranging
from steppe to woodland to forest (Bonnefille et al. 2004), but not wet dense ever-
green forest (Bonnefille 2010). Because A. afarensis showed no apparent associa-
tion with any particular habitat in a single site that fluctuated between being
dominated by steppe and being dominated by forest, it has been described as a
generalist (Bonnefille et al. 2004).
The teeth of A. afarensis are thought to be adapted for crushing hard, brittle
foods such as seeds, hard fruits, and/or tubers (Luca et al. 2010); however, microwear
analysis of the teeth paints a different picture entirely. Compared to a variety of
other primates, including those that specialize in eating hard seeds and those that
consume substantial numbers of tubers, the microwear patterns on A. afarensis teeth
from a diversity of habitats actually most closely resemble those of the mountain
gorilla (Grine et al. 2006), the least frugivorous gorilla subspecies (Yamagiwa and
Basabose 2006). As Grine et al. (2006) make clear, this resemblance does not mean
that A. afarensis had the same diet as a gorilla, and the gross tooth morphology
makes it unlikely that they could eat the same foods in the same way. Rather, the
similarity means that they both ate fibrous foods with fine abrasiveness but none of
the hard brittle foods for which the A. afarensis teeth appear to be adapted, at least
not in the period before each of the individuals died. However, in areas of range
overlap, it seems likely that they may have often been attracted to some of the same
types of food, increasing the chances of interaction.
The tooth morphology seemingly at odds with wear patterns may indicate a dif-
ference between commonly eaten preferred foods and the ability to efficiently pro-
cess seasonally important fallback foods (Ungar 2004). Other authors suggest that
Parasitic Lice Help to Fill in the Gaps of Early Hominid History 177

wetland vegetation was an important A. afarensis food (also exploited by gorillas in

certain areas) that could explain the tooth wear patterns and the insensitivity of A.
afarensis to changes in upland habitat (Verhaegen et al. 2002). Significant for this
discussion is an additional A. afarensis food: meat scavenged from large animals
with the help of stone butchering tools (McPherron et al. 2010).
It is difficult to choose the hominin most likely to have first acquired Pthirus
given the similarities between the hominins alive 3–4 mya and the limited, vague,
and contradictory information available. However, the species with the most infor-
mation available, A. afarensis, appears to be a good candidate because it was a habi-
tat generalist that foraged on fibrous foods and scavenged large mammals (and
therefore could have come into repeated contact with gorilla ancestors, as discussed
below) and is a likely ancestor to Homo. However, nothing rules out other species
in hominin lineage, especially as use of wet forests would not be recorded in the
fossil record.
Whichever hominin species was actually first infested with Pthirus, the success-
ful transfer of a disease or parasite to another species is most likely when there is
relatively frequent contact between the two host species, which indicates that moist
forests were a far more important hominin habitat than previously realized. The
savanna/grassland model of the origin of bipedalism and hominins has been largely
rejected by careful examination of the context of hominin fossils (WoldeGabriel
et al. 1994; Reed 1997; WoldeGabriel et al. 2009; Luca et al. 2010; Brunet 2010).
These fossils tend to indicate the importance of wooded habitats including wood-
lands and dry forests or at the very least forests lining bodies of water or forest/
woodland/savanna/grassland mosaics (although mosaics can be an illusion created
by the coarse resolution of the paleontological record and time averaging of more
homogenous habitats changing over a period of time; Elton 2008). Though the
emphasis has shifted from the savanna to the woodland and dry forest, moist forests
have largely been ignored as potentially important habitats for hominins; however,
there is no reason to think hominins would be less flexible in habitat use than those
extant primates that use both wet forests and drier open habitats (Elton 2008). This
oversight has certainly been reasonable based on the context of fossil finds; in fact,
there has never been a hominin fossil found associated with rain forest habitat, more
likely due to the difficulty of fossilization rain forests (Kingston 2007 and see
above) than habitat specificity. However, the evidence of habitual contact between
hominins and gorilla ancestors given by the Pthirus host switch provides strong
support for wet forests playing a more important role in hominin evolution than
normally thought.
Additionally, the loss of body hair that is likely to have occurred before the
Pthirus switch from gorilla ancestors to human ancestors is much more likely to
have happened under a closed canopy forest than in a less wooded ecosystem
because the forest reduces the usefulness of body hair by reducing the solar heat
load and mitigating diurnal temperature changes as discussed above (Newman
1970). Thus, the loss of body hair likely occurred when hominins were restricted
exclusively to dense forests long before the evolution of the australopithecines as
habitat generalists that incorporated more open areas into their ranges. While
178 J.M. Allen et al.

australopithecines could presumably still utilize warm moist forests, use of montane
forests of australopithecines is not likely given the loss of body hair by this point
and the cold temperatures experienced in these forests. Unfortunately, fossil evi-
dence of wet forest use by hominins will likely be as difficult to come by as fossil
evidence of the other great apes that are restricted to wet forests. As mentioned
before, the fossil record is almost silent even on the subject of common chimpan-
zees, which venture into drier habitats (such as woodlands and scrublands) than
gorillas but remain largely tied to closed canopy forests. Thus, if restricted to fossil
evidence, our picture of hominin evolution and habits is severely limited by the
taphonomic processes in the moist forests that form the origin of African ape
diversity.
Our suggestion that australopithecines expanded their habitat from drier wooded
areas into wet tropical forests introduces several more possibilities. Habitat use
could have been seasonal with australopithecines predictably moving from drier
habitats to wet forest areas and back. Alternately, a widely spread generalist species,
as A. afarensis has been proposed to be, might have populations permanently inhab-
iting entirely different habitats. In this case, the question becomes which habitat was
preferred, i.e., which, if any, was able to support higher densities of hominins. A
third possibility is that one of the habitats was primary with the other being utilized
only in times of drought, etc. The importance of moist forests with poor fossiliza-
tion conditions to hominins raises the possibility that it was not the dry woodlands
that were the center of hominin radiation; rather the radiation occurred in the rain
forests with a minority of the species expanding out into more xeric areas to be fos-
silized and finally found. If this is true, we may still be missing a large portion of
hominin diversity.
Of course, for Pthirus to have switched hosts successfully, sharing habitats
would have been insufficient for transfer—far more intimate contact would have
been required. However, the contact would not have to be as intimate as most people
seem to gleefully assume. Although hybrids between different guenon monkey spe-
cies are known (Struhsaker et al. 1988; de Jong and Butynski 2010), there are far
more likely scenarios than two species as divergent as archaic gorillas and australo-
pithecines having sexual contact.
Pthirus are known to be transmitted via fomites (Meinking 1999) and therefore
could have potentially switched hosts if a hominin used an abandoned louse-infested
archaic gorilla nest (Reed et al. 2007). All extant great apes including orangutans
fashion nests (Schaller 1965b), so it is reasonable to assume that all apes 3–4 mya
also made nests. While this scenario is possible, it seems unlikely. Great ape nests
(aside from those of humans) are constructed swiftly for a single use and are simple
rudimentary structures. This is especially true for gorilla nests made on the ground,
which have better rims than bottoms, provide little if any padding, and are typically
constructed in 1 min (though the minority that are made in the trees are more sub-
stantial; Schaller 1965a). Unfortunately (but not unexpectedly), there is no surviv-
ing evidence of australopithecine nests or nest use. However, if they practiced the
single-use pattern typical of great apes, there would have been little reason for
individuals who did not reuse their own nests to reuse the nests of another species.
Parasitic Lice Help to Fill in the Gaps of Early Hominid History 179

On the other hand, if australopithecines reused nests like at least some of the later
Homo spp., the reuse would probably have been motivated by increased effort
required to build more complex and functional structures. Again, the reuse of old
slipshod gorilla-type nests would have been unlikely.
A more probable scenario for contact between human and gorilla ancestors is the
existence of mixed foraging groups. Though rare compared to multiple species
associations in monkeys (Yamagiwa and Basabose 2006), mixed foraging groups
have been observed containing gorillas and chimpanzees. Interactions from avoid-
ance (Yamagiwa et al. 1996) to obliviousness (Kuroda et al. 1996) have been seen
taking place in mixed groups of apes (even from a distance of 3 m; Stanford 2006).
The nature of the interaction likely depends on food availability and level of com-
petition as well as the individual personalities of those involved. Habituated gorilla
and chimpanzee troops tend to ignore human observers, but some physical interac-
tions (e.g., playing, bluffing, and testing) have occurred, normally with young ani-
mals. In primate mixed foraging groups, interactions are most frequent between
young animals and can involve play.
Mixed foraging groups of gorilla and human ancestors would have given oppor-
tunities for the Pthirus switch through play or grooming; however, primate interspe-
cific interactions are rare even where multispecies associations are common.
Aggressive interactions are the most numerous interactions, with play being rela-
tively unusual and grooming being extremely rare and of short duration (Ihobe
1990; Heymann and Buchanan-Smith 2000). Because juveniles are typically the age
group involved in interspecific interactions and young hominins would have not yet
developed the pubic hair that forms the current habitat of Pthirus on humans, the
ancestral lice would have to be passed to an adult host before they could become
established on the new host species. The possibility of a host switch through mixed
foraging groups does exist, but because aggression and play rarely involve physical
contact (Rose 1977), which occurs quickly and generally between juveniles who
would then have to pass Pthirus to an adult host, social interaction is a less likely
route of host switching than the last possibility: the consumption of archaic gorilla
meat by human ancestors.
Other than the gorillas, all the African great apes hunt and consume meat,
although only humans have managed to prey on animals of similar and larger
body sizes through the use of relatively sophisticated tools. Given the body size of
A. afarensis (♀, ~29 kg; ♂, ~45 kg; McHenry 1994) compared to that of gorillas
(♀, ~80 kg; ♂, ~169 kg; Smith and Jungers 1997) as well as the dangerous nature
of enraged gorillas and relatively simple tools used by the australopithecines, it
seems unlikely that hunting archaic gorillas by early hominins would be a particu-
larly effective or common food acquisition strategy. It is far more likely that scav-
enging on gorilla carcasses led to the kind of contact most conducive to a louse
host switch: repeated close contact over a substantial period of time. Additionally,
lice are extremely sensitive to environmental conditions and readily abandon dead
hosts. The desperate situation of lice on a dead or dying host makes the switch to
any available host, even one of the incorrect species, much more likely than casual
contact between a living native host and a potential novel host.
180 J.M. Allen et al.

The presence of likely butcher marks on the bones of large mammals contem-
porary with A. afarensis indicates that scavenging was likely an important and
effective component in their feeding repertoire (McPherron et al. 2010, 2011; but
see Domínguez-Rodrigo et al. 2010, 2011). Although the validity of these butcher
marks does not determine the level of carnivory by A. afarensis (Domínguez-
Rodrigo et al. 2010), simple tools would have enabled both more efficient pro-
cessing of meat than allowed by primate dentition alone and the transportation of
meat away from the main carcass to a location with less predation danger. Thus,
the most likely scenario is that a hominin habitually used moist tropical forests far
more than previously realized or shown by the fossil record and opportunistically
scavenged meat from gorilla carcasses. This feeding resulting in contact with
Pthirus that was frequent enough to establish a population of Pthirus on hominins
3–4 mya.

Conclusion

There has been much research into the biology, epidemiology, and the evolution-
ary history of lice. These parasites have bedeviled human and nonhuman primates
alike for millions of years, and the increase in louse prevalence over the last 20
years suggests they will continue to parasitize humans for some time yet. Due to
their obligate host-specific nature, we can use these parasites to inform us about
human evolutionary history and gather information that is not available in the host
fossil record, providing an unexpected benefit to an otherwise bothersome
parasite.
Although most great ape lice have strictly cospeciated with their great ape hosts,
Pthirus pubis (the human pubic louse) has a different evolutionary history. Pthirus
switched to the human lineage 3–4 mya from an archaic gorilla. The biology of
Pthirus suggests that for this host switch to have occurred, suitable habitat had to be
available, which indicates that hominins had not only lost their body hair but also
developed androgenic hair by 3–4 mya.
Because gorillas are conservative in their habitats and diet, we postulate that
this likely means that these archaic hominins were using similar habitat as goril-
las (moist forest habitat). The best candidate for this host switch was
Australopithecus afarensis (based on our current knowledge). A. afarensis pos-
sibly butchered large mammal carcasses during this time, presenting a scenario
of A. afarensis scavenging archaic gorilla meat and Pthirus likely switching to
A. afarensis from a dead gorilla host. Pthirus then continued to evolve with the
hominin lineage as a sexually transmitted disease due to their placement on the
body, explaining the presence of two genera of lice (Pthirus and Pediculus) on
extant humans today.
Parasitic Lice Help to Fill in the Gaps of Early Hominid History 181

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 Part II
Emergence and Divergent Disease
Manifestation
Treponema pallidum Infection in Primates:
Clinical Manifestations, Epidemiology,
and Evolution of a Stealthy Pathogen

Kristin N. Harper and Sascha Knauf

Introduction

Treponema pallidum is a pathogenic bacterium that causes a variety of debilitating

diseases in humans and nonhuman primates (NHPs). In humans, T. pallidum causes
the sexually transmitted disease syphilis and the nonsexually transmitted diseases
yaws and bejel. Its history in humans is mysterious and has been a source of great
controversy over the years. Did yaws affect our earliest ancestors, perhaps present
even in Homo erectus (Rothschild et al. 1995)? Where and when did syphilis
arise—did Columbus bring it from the New World to the Old (Crosby 1969; Diaz
de Isla 1539; Harper et al. 2008, 2011)? Its history and distribution in NHPs has
been much less studied, but novel data are accumulating. Although Treponema
infection has been reported in a Pleistocene bear (Rothschild and Turnbull 1987),
reports of naturally occurring T. pallidum infection in species outside of primates
have never been replicated. Here, we summarize what is known about T. pallidum
infection in both human and NHPs, considering diverse sources of evidence,
including serology of wild animals, clinical manifestations, and genetic character-
ization of strains. We focus on how our understanding of the history of the disease
in NHPs can inform our understanding of its transmission and evolutionary history
in humans and vice versa.

K.N. Harper (*)

Environmental Health Sciences, Columbia University, New York, NY 10032, USA
e-mail: [email protected]
S. Knauf
Pathology Unit, German Primate Center, Leibniz-Institute for Primate Research,
Goettingen, Germany

J.F. Brinkworth and K. Pechenkina (eds.), Primates, Pathogens, and Evolution, 189
Developments in Primatology: Progress and Prospects,
DOI 10.1007/978-1-4614-7181-3_7, © Springer Science+Business Media New York 2013
190 K.N. Harper and S. Knauf

Treponema pallidum in Humans

T. pallidum subsp. pallidum, the causative agent of syphilis, is the only treponemal
subspecies that is primarily transmitted via sex. An estimated 12 million new cases
of syphilis occur every year and can be found throughout the world (WHO 2001).
Certain socio-ecological risk factors (e.g., multiple sex partners and immunosup-
pression) are associated with a greater risk of contracting and transmitting the dis-
ease (Karp et al. 2009; Rolfs et al. 1990; Yahya-Malima et al. 2008). Yaws, caused
by subsp. pertenue, is a nonvenereal disease that is usually acquired during early
childhood. Transmission appears to occur primarily via skin-to-skin contact (Perine
et al. 1984), although flies may also serve as vectors (Cousins 1972; Kumm and
Turner 1936; Satchell and Harrison 1953). Yaws was once common in tropical
regions throughout the world. Although it has never been eradicated, it grew
increasingly rare after a WHO-sponsored eradication campaign mid-century (Arya
and Bennett 1976; Guthe et al. 1953, 1972). Surveillance is typically poor in the
areas most likely to be affected; accurate figures on the infection’s current preva-
lence are not available. However, recent foci of infection in the Republic of Congo
and the Central African Republic (Salomone 1999), the Democratic Republic of
Congo (Gersti et al. 2009), Papua New Guinea (Mitjà et al. 2011), East Timor
(Satter and Tokarz 2010), and Vanuatu (Fegan et al. 2010) have been documented.
The disease appears to have been eradicated in India (Lahariya and Pradhan 2007)
and has not been reported recently in South America either. Finally, bejel, caused
by subsp. endemicum, was once common in arid regions such as the Middle East
and the Balkans, but it has not been reported in the literature since the 1990s
(Yakinci et al. 1995). Like yaws, it is typically acquired during childhood and is
thought to be transmitted primarily via fomites such as utensils and drinking vessels
(Perine et al. 1984).
All three diseases are chronic, multistage infections in humans that are easily
treated by antibiotics in most cases, but they can be deadly if neglected (Table 1). It
should be noted that increasing macrolide resistance has begun to complicate treat-
ment worldwide, and antibiotic-resistant T. pallidum could one day represent a sub-
stantial health challenge (Stamm 2010). Syphilis typically begins with a hard,
painless chancre at the site where the bacterium entered the body. Untreated infec-
tion leads to a secondary stage, often characterized by a rash that appears weeks
later. The rash associated with secondary syphilis may take several forms, but it
generally affects the palms and soles and does not itch (Richens and Mabey 2009).
Mild fever, patchy hair loss, and weight loss are also common clinical signs in
humans. If left untreated, tertiary-stage disease may occur, though sometimes not
until decades after the infection was initially contracted. Destructive lesions called
gummata may appear in virtually any organ of the body; neurosyphilis may result in
psychiatric disorders, while cardiovascular syphilis killed many people in the pre-
antibiotic era (Holmes et al. 2007).
The disease progression is similar for infection by all three of the T. pallidum
subspecies, although a number of important differences have been reported (Table 1).
Table 1 Characteristics of natural infection with T. pallidum in different primate species
Humans Nonhuman primatesa
Baboons-West
Syphilis Yaws Bejel Africa Baboons-Tanzania Gorillas
Genetic characteris- T. pallidum subsp. pallidum T. pallidum subsp. pallidum T. pallidum subsp. Genetic Genetic sequences Unknown
tics of strain pallidum sequences from strains at
responsible from one Lake Manyara
strain National Park
collected in show most
Guinea similarity to
show most subsp.
similarity pertenue
to subsp.
pertenue
Primary-stage Hard, painless chancre Papillomatous “mother yaw” Lesions at site where When present Moderate to Scabby, dry raised
lesions develops at site where develops at site where bacterium enters the at all: mild, severe genital lesions
bacterium entered the bacterium entered the body are rare. When small, ulceration; primarily
body (usually anogeni- body (most often legs) present, they resemble keratotic enlargement of affecting lips,
tal region) chancres lesions and inguinal lymph nose, eyes, and
Secondary-stage Non-itchy rash affecting Crustopapillomatous skin Angular stomatitis, mucous ulcers nodes cheeks. Large
lesions palms and soles of feet; lesions, polydactylitis, patches in mouth, rashes affecting destructive
alopecia, mild fever, osteoperiostitis, nodules, with hypertrophic muzzle, lesions
weight loss, enlarge- plaques, and papules on lesions, pigmentary eyelids, primarily
ment of lymph nodes the skin changes, osteoperiostitis armpits affecting nose
Tertiary-stage Gummata can develop in Hyperkeratosis, ulceration Severe osteoperiostitis, None reported and mouth
lesions virtually any organ of around the nasal and sometimes resulting in areas; lesions
the body, neurological maxillary areas (gan- saber tibia, gummata of also found on
and cardiovascular gosa), saber tibia, gondou the skin, palate, and wrists and
involvement (hypertrophic osteitis of nasal septum, gangosa ankles
nasal process of maxilla)
(continued)
Table 1 (continued)
Humans Nonhuman primatesa
Baboons-West
Syphilis Yaws Bejel Africa Baboons-Tanzania Gorillas
Primary transmis- Sexual Skin to skin Thought to be mouth to Unknown Lesions targeting Unknown
sion mode mouth. Fomites such the genitals
as utensils and and involve-
drinking vessels may ment of only
be important sexually active
animals
suggests
sexual
transmission
Typical age of Sexually mature Childhood Childhood; 66 % acquired Unknown Sexually mature Lesions have been
infection individuals before the age of 16 in animals reported in
one study of 3,507 cases almost 5 % of
in Iraq infants in the
Parc National
d’Odzala-
Kokoua
(Republic of
Congo); peak
incidence
between infant
and juvenile
stages
Congenital Yes Rarely, if ever Rarely, if ever Unknown Unknown Unknown
transmission
Geographic range Global Once very common in hot, Once very common in hot, West Africa: Tanzania West Africa:
humid regions of the arid regions such as the Guinea, Republic of
world. Still reported in Middle East and the Senegal, Congo,
some countries in Eastern Mediterranean. Cameroon Cameroon,
Western Africa and the Last reported case in Democratic
South Pacific Turkey, during the 1990s Republic of
Congo
Sources Perine et al. (1984), Salazar Noordhoek et al. (1991), Yakinci et al. (1995), Pace Baylet et al. Wallis and Lee Cousins (1984),
et al. (2002a, b) Perine et al. (1984) and Csonka (1984), (1971), (1999), Karesh (2000),
Csonka (1953), Perine Fribourg- Mlengeya Levréro et al.
et al. (1984) Blanc and (2004), Knauf (2007)
Mollaret et al. (2012)
(1969),
Harper
et al.
(2008),
Smajs et al.
(2011)
a
Infection in NHPs is not well characterized enough to determine whether there are primary, secondary, and tertiary-stage lesions. Therefore, all lesions are described in one
category
194 K.N. Harper and S. Knauf

First, only syphilis is regularly transmitted congenitally. While there are several
reports of possible yaws infection via in utero transmission in the literature
(Engelhardt 1959; Wilson and Mathis 1930), it appears likely that congenital infec-
tion does not occur in the nonvenereal treponematoses (Antal et al. 2002). It has
been hypothesized that this may be because yaws and bejel are typically acquired
early in life, so active infection at sexual maturity is virtually nonexistent (Willcox
1955). It is also commonly believed that only subsp. pallidum, the agent of syphilis,
affects the central nervous system (CNS). However, in-depth study of the progres-
sion of subsp. pertenue and endemicum infections has not been carried out, and one
of the rare studies focusing on CNS involvement in yaws infection reported the pres-
ence of spirochetes in ocular fluid (Smith et al. 1971). In addition, Román and
Román (1986) draw attention to a possible association between CNS complications
and yaws infection. Finally, although for the most part syphilis transmission occurs
via a venereal route and the other treponemal diseases are nonsexually transmitted,
the potential for transmission via alternate routes is known for all T. pallidum infec-
tions. Cases of extragenital syphilis resulting from nonsexual contact have been
reported several times (Luger 1972; Taylor 1954), for example, by human bites (Oh
et al. 2008) or by mouth-to-mouth feeding in infants (Zhou et al. 2009). Similarly,
infectious yaws and bejel lesions have been reported on the genitalia (Turner and
Hollander 1957b; Wilson and Mathis 1930), suggesting that the opportunity for
sexual transmission might be present to some extent in all of the
T. pallidum subspecies. In summary, because so few in-depth studies have been
performed on the nonsexually transmitted T. pallidum subspecies, the exact nature
of the similarities and differences between the three diseases remains ambiguous.
Only recently have we gained the tools to differentiate between the three subspe-
cies. For decades, a diagnosis of syphilis, yaws, or bejel was given based on the clini-
cal presentation of the disease, the age of the patient, and the area of the world in
which they lived. In areas where multiple treponemal diseases were present, the dif-
ferential diagnosis could be quite puzzling (Lagarde et al. 2003). In the 1990s, single-
nucleotide polymorphisms that reliably differentiated subsp. pallidum strains from
the two nonsexually transmitted subspecies were identified for the first time
(Centurion-Lara et al. 1998; Noordhoek et al. 1990). Subsequently, patterns of genetic
variation that could be used to separate all three subspecies were identified (Centurion-
Lara et al. 2006; Harper et al. 2008). Thus, while we still have no serological test that
can differentiate between the three diseases, we can now use genetics to do so.
The multiple subspecies of T. pallidum present in humans raise the question:
which selective pressures led to the emergence of multiple transmission modes? In
the past, researchers have speculated that subsp. pertenue may represent the ances-
tral, nonsexually transmitted pathogen, perhaps infecting the earliest hominids
(Cockburn 1967; Harper et al. 2008). In this view, subsp. pallidum is a relatively
recent pathogen. It could have emerged in the Old World, as large cities began to
appear and novel sexual behavior fostered sexual transmission (Hudson 1963). Or,
according to a more conventionally held theory supported by recent genetic evi-
dence (Harper et al. 2008), it may have arisen in the New World during Pre-
Columbian times, introduced into the Old World by Columbus and his men during
Treponema pallidum Infection in Primates: Clinical Manifestations, Epidemiology… 195

the voyages of discovery (Crosby 1969). Some researchers believe that the skeletal
evidence indicates that subsp. pallidum may not have emerged until the voyages of
discovery themselves, when Columbus and his crew took a nonvenereal form of
T. pallidum back to the Old World, where it rapidly gained the ability to be sexually
transmitted in its new environment (Baker and Armelagos 1988). Alternatively, one
team of researchers has posited that all three T. pallidum subspecies are relatively
recent additions to the family of human pathogens and arose at similar times in
human history (Gray et al. 2006). Luckily, novel data have recently allowed us to
reject yet another theory. The “Unitarian hypothesis” states that all human trepone-
mal infections are caused by a single, protean pathogen with an opportunistic trans-
mission mode, resulting in dramatically different clinical manifestations depending
upon factors such as climate (Hudson 1963). Given the morphological and overall
genetic similarity between the T. pallidum subspecies, this view did not seem unrea-
sonable in the past. Recent genetic evidence stemming from intensive sequencing
efforts by multiple groups has convincingly shown, however, that the three trepone-
mal diseases, syphilis, yaws, and bejel, are caused by genetically distinct pathogens
(Centurion-Lara et al. 2006; Harper et al. 2008; Smajs et al. 2011). Since an essen-
tial tenet of the Unitarian hypothesis is that the strains that cause the three diseases
are interchangeable and thus cannot be distinguished, we can now discard this evo-
lutionary explanation for T. pallidum’s multiple transmission modes. As described
above, however, many competing hypotheses remain.
In the remainder of this chapter, we will investigate what is known about T. pal-
lidum in NHPs and will conclude by discussing what insights NHP infections may
provide in understanding the history of human infection.

Treponemal Infection in Nonhuman Primates

Thanks to observational and serological surveys of wild NHPs, we now know that
Treponema infection occurs naturally in many species. Based on the available
sequences from the Fribourg-Blanc strain, collected from a wild baboon captured in
Guinea in the 1960s (Fribourg-Blanc et al. 1966), we also know that the NHP strains
responsible are very closely related to human T. pallidum strains. This baboon strain
harbors 16S ribosomal subunit DNA that is 100 % identical to that found in T. pal-
lidum human strains,1 a level of identity which exceeds the threshold of 97–99 %
often used to distinguish between bacterial species (Gevers et al. 2005). However,
we do not yet know whether NHP strains represent a sister subspecies to pertenue
or whether human subsp. pertenue and NHP strains represent one large clade.

1
Sequences from the 16S ribosomal subunit (2nd operon) available from GenBank were obtained
and aligned in ClustalX. They included the subsp. pallidum strains Dallas-1 (NC016844.1),
Chicago (CP001752.1), Nichols (NC000919.1), Street Strain 14 (NC010741.1), and Mexico A
(HM585252.1), the subsp. pertenue strains CDC-2 (NC016848.1), Gauthier (NC016843.1),
Samoa D (NC016842.1), and the Fribourg-Blanc strain (HM165231.1).
196 K.N. Harper and S. Knauf

Thus, although treponemal infection in NHPs is often referred to as yaws, we would

like to raise the possibility that our understanding of the systematics involved may
change when we know more about the relationship between human and NHP strains.
Whole genome sequencing of different T. pallidum strains, especially the simian
ones, is expected to shed further light on the small but potentially significant genetic
differences between them.
Some background information on serological tests for T. pallidum is necessary
before we review what is known about treponemal diseases in wild NHPs. Most sur-
veys conducted in NHPs have relied heavily on serological screening methods, so the
interpretation of their results requires some knowledge about the sensitivity and speci-
ficity of different tests. Serological tests for T. pallidum are basically divided into two
types: non-treponemal and treponemal. Non-treponemal tests, often used for screen-
ing in humans, detect antibodies to phospholipid antigens, such as cardiolipid, that are
not specific to T. pallidum. Because these antigens occur in a number of diseases, such
as malaria, tuberculosis, viral fevers, leprosy, and trypanosomiasis, among others
(Herring et al. 2006), they have a lower specificity than the other available tests and
may not be particularly reliable when used on wild NHPs, especially those living in
regions where multiple infections are common. In contrast, treponemal tests, which
react to anti-T. pallidum antibodies, are quite specific (Herring et al. 2006). These tests
include the T. pallidum immobilization (TPI) test, the fluorescent treponemal antibody
absorption (FTA-ABS) test, and the microhemagglutination assay (MHA-TP).
Despite their greater specificity, treponemal tests also have their limitations (Binnicker
et al. 2011). Non-treponemal tests and the FTA-ABS test, which was used often in the
surveys of NHPs discussed below, have been reported to cross-react with sera from
patients with Lyme disease (Hunter et al. 1986; Magnarelli et al. 1990; Russell et al.
1984), which is caused by the Borrelia spec., spirochetes closely related to T. palli-
dum. Luckily, Lyme disease appears to be extremely rare in the Southern Hemisphere,
where most NHPs live (Jowi and Gathua 2005; Yoshinari et al. 1993), so false posi-
tives from this disease are unlikely to be a significant problem when interpreting the
results described here. In addition, autoimmune disorders are known to cause false-
positive results when using non-treponemal serological tests, as well as the FTA-ABS
assay when the latter is performed under certain conditions (Mackey et al. 1969; Seña
et al. 2010). Luckily, this limitation does not appear to apply to the MHA-TP and TPI
tests, which were also used in the surveys presented in this chapter, frequently in tan-
dem with the FTA-ABS test (Mackey et al. 1969; Russell et al. 1984).

African Monkeys

NHP treponemal infection has probably been studied best in wild baboons (Papio
spec.). Beginning in the 1960s, serological surveys demonstrated that treponemal dis-
ease was present at high prevalence in the baboons of Equatorial Guinea, Senegal, and
Cameroon (Table 2) (Baylet et al. 1971; Fribourg-Blanc and Mollaret 1969). As
described previously, one T. pallidum strain, the Fribourg-Blanc strain, was isolated
from an infected baboon during the course of these surveys (Fribourg-Blanc et al. 1966).
Table 2 T. pallidum infection in African monkeys
Seroprevalence in
Species Origin sample Test Source
Mangabey (Cercocebus, Laboratory animal caught in West Africa 0/1 (0.0 %) FTA-ABS Felsenfeld and Wolf (1971)
species not determined) Bangui region, Central African Republic 0/2 (0.0 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
Green monkey Laboratory animals caught in West Africa 0/3 (0.0 %) FTA-ABS Felsenfeld and Wolf (1971)
(Chlorocebus sabaeus)
Vervet monkey Laboratory animals caught in Kenya 1/2 (50.0 %), + result FTA-ABS Felsenfeld and Wolf (1971)
(Chlorocebus equivocal
pygerythrus)
Chlorocebus, species not Casamance region of Senegal 3/8 (37.5 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
determined Fouta Toro Fleuve region of Senegal 28/45 (73.0 %) FTA-ABS Baylet et al. (1971)
Niamey, Niger 0/1 (0.0 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
Bangui, Central African Republic 0/3 (0.0 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
Brazzaville region, Democratic Republic 0/3 (0.0 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
of Congo
Laboratory animals caught in East Africa 2/7 (28.6 %), + FTA-ABS Felsenfeld and Wolf (1971)
results equivocal
Colobus monkey (Colobus, Casamance region of Senegal 1/1 (100.0 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
species not determined)
Patas monkey (Erythrocebus Laboratory animals caught in West Africa 1/13 (7.7 %) FTA-ABS Felsenfeld and Wolf (1971)
patas) Casamance region of Senegal 6/26 (23.1 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
Mali 0/18 (0.0 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
Bobo-Dioulasso region of Burkina Faso 0/17 (0.0 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
Niamey, Niger 0/1 (0.0 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
Fort-Lamy, Chad 1/14 (7.1 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
Laboratory animals caught in East Africa 1/10 (10.0 %), + FTA-ABS Felsenfeld and Wolf (1971)
Treponema pallidum Infection in Primates: Clinical Manifestations, Epidemiology…

result equivocal
Fouta Toro Fleuve region, Senegal 1/4 (25.0 %) FTA-ABS Baylet et al. (1971)
Koussanar region of Senegal 1/38 (2.6 %) FTA-ABS Baylet et al. (1971)
197

(continued)
Table 2 (continued)
198

Seroprevalence in
Species Origin sample Test Source
Yellow baboon (Papio Kindia region of Guinea 164/216 (75.9 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
cynocephalus) Casamance region of Senegal 6/10 (60.0 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
Kaolack region of Senegal 80/171 (46.8 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
Bobo-Dioulasso region of Burkina Faso 0/159 (0.0 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
Yaounde, Cameroon 3/3 (100.0 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
Kenya 0/276 (0.0 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
Olive baboon (Papio Casamance region of Senegal 49/82 (59.8 %) FTA-ABS Baylet et al. (1971)
anubis) Koussanar region of Senegal 15/55 (27.3 %) FTA-ABS Baylet et al. (1971)
Falémé region of Senegal 0/111 (0.0 %) FTA-ABS Baylet et al. (1971)
Lake Manyara National Park, Tanzania 43/57 (75.4 %) Serodia TP*PA Knauf et al. (2012)
Baboon (Papio, species not Brazzaville region, Democratic Republic 0/2 (0.0 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
determined) of Congo
K.N. Harper and S. Knauf
Treponema pallidum Infection in Primates: Clinical Manifestations, Epidemiology… 199

Experiments have shown that this strain is capable of causing infection in humans
(Smith et al. 1971), and genetically it is closely related to, but possibly distinct from,
human subsp. pertenue strains (Centurion-Lara et al. 2006; Harper et al. 2008; Smajs
et al. 2011). Clinical signs in the affected populations were described as mild and
included small keratotic lesions and ulcers on the muzzle, eyelids, and armpits (Baylet
et al. 1971), though most infected animals did not appear to display any lesions at all
(Table 1). It is interesting that in these same surveys, not a single animal from countries
farther east, such as Burkina Faso (n = 159) or Kenya (n = 276), was found to be positive
(Fribourg-Blanc and Mollaret 1969).
In the late 1980s, however, a form of treponemal disease with strikingly different
clinical signs was described among the olive baboons (P. anubis) at Gombe Stream
National Park (Table 1). “Penelope,” an adult female baboon at Gombe, was the first
recorded case displaying what would come to be recognized as the genital-associated
lesions typical of the infection (Collins et al. 2011). Subsequently, researchers
noticed the skin disease in several baboon troops in the national park (Wallis 2000b;
Wallis and Lee 1999). At that time, laboratory analysis (dark field illumination) of
skin lesions confirmed the presence of T. pallidum. However, DNA-based verifica-
tion of the subspecies of T. pallidum involved was not performed, nor had other
pathogens that cause genital ulceration in humans and NHPs been ruled out defini-
tively. Because of the infection’s association with genital lesions and the observa-
tion that it appeared in sexually mature animals, it was hypothesized that the disease
might be sexually transmitted (Wallis and Lee 1999). Moreover, unlike the mild
lesions described in West Africa, it was reported that lesions in a small proportion
of the individuals affected at Gombe became so severe that urinary flow was
obstructed and death resulted, with autopsy of one young male revealing wide-
spread sepsis within the urogenital tract (Wallis and Lee 1999).
In the 1990s, similar lesions (Fig. 1) were reported for the first time in olive
baboons at Lake Manyara National Park, also in Tanzania but 700 km away from
Gombe (Mlengeya 2004). In contrast to other studies examining treponemal infec-
tion in NHPs, Knauf et al. (2012) were able to describe in detail the macroscopic
clinical manifestations and histological findings that characterize T. pallidum infec-
tion in baboons. In addition, for the first time, they were able to demonstrate simian
T. pallidum strains in situ, using immunohistochemistry (Fig. 2). Molecular biologi-
cal tests such as qualitative and quantitative PCR were performed using skin tissue
samples, and they demonstrated the presence of T. pallidum in infected animals
while ruling out other pathogens. Qualitative and quantitative serological tests,
including the Serodia TP*PA test, offered still more proof that T. pallidum infection
was responsible for the lesions observed. Four informative genetic polymorphisms
were used to demonstrate that, despite their predilection for causing anogenital
lesions similar to those found in human syphilis, the simian strains collected from
baboons at Lake Manyara National Park were most closely related to nonvenereal
human T. pallidum subsp. pertenue strains. Finally, the study showed that many
animals that looked healthy in the field had positive PCR, serological, or histologi-
cal results for T. pallidum. For example, of 20 baboons with no clinical signs of
infection, 15 showed histological abnormalities, of which six tested PCR positive
200 K.N. Harper and S. Knauf

Fig. 1 Photos depicting clinical signs associated with genitotropic T. pallidum infection in
baboons. The genitals of a severely affected female (left) and male (right) are shown

Fig. 2 Photos depicting T. pallidum in a skin tissue sample from the genitals of an Olive baboon
at Lake Manyara National Park, Tanzania. Immunohistochemistry utilizing rabbit polyclonal
antibodies against T. pallidum, was performed with epithelial cells counterstained using Mayer’s
hematoxylin. Bar 10 μm

for T. pallidum. Seven of the 20 clinically unaffected animals had anti-T. pallidum
antibody titers ≥1:80 (Knauf et al. 2012). This indicates that the prevalence of the
disease is much higher than originally suspected from field observations (Mlengeya
2004).
That this infection is prevalent in the baboons of Tanzania is also reinforced by a
small survey we performed of baboons imported into the United States by labora-
tory suppliers (Harper, data not published). In 2006, we assayed 17 serum samples
from olive baboons (Papio anubis) imported from Tanzania to the United States
using the Sero-DIA TPPA test (Fujirebio Diagnostics, Malvern, PA), a highly
Treponema pallidum Infection in Primates: Clinical Manifestations, Epidemiology… 201

sensitive and specific serological treponemal test. Sixteen samples came from adult
males imported in 2003 (n = 6) and 2005 (n = 10). Pooled serum from six adult
females imported in 2001 was also tested. Four of the 16 samples from adult males
tested positive for T. pallidum antibodies, as did the pooled serum from the adult
females. It is not clear where in Tanzania these animals were captured, but the
relatively high prevalence of infection in these samples suggests that the infection
has become established outside of Gombe and Lake Manyara National Parks.
Serological evidence of T. pallidum infection has also been found in other African
monkey species (Table 2), though no gross-pathological lesions that appear to result
from infection have yet been described. Antibodies have been detected in the
Chlorocebus species [vervet (C. pygerythrus) and green monkeys (C. sabaeus)],
patas monkeys (Erythrocebus patas), and in one Colobus monkey (Colobus spec.).

Gorillas

Reports that yaws infection is frequent in wild gorillas (Gorilla gorilla) in West
Africa have appeared for some time. For example, stories from indigenous people
as well as reports from the Service de Chasse in the former French Equatorial Africa
region told of gorillas in the Ewo, Kelle, and Mekambo regions that suffered from a
leprosy-like disease (Cousins 1984). In the 1950s, two researchers got the opportu-
nity to examine four young gorillas captured in this area that displayed the “lep-
rous” lesions described in earlier reports. The raised lesions, scabby and dry,
primarily affected the lips, nose, eyes, and cheeks, with one animal also exhibiting
lesions on the shoulder and forearm (Table 1). It should be noted that leprosy has
been confirmed in an Asian wild-born macaque (Valverde et al. 1998), two African
wild-born sooty mangabeys (Gormus et al. 1991), and wild-born chimpanzees from
West Africa (Hubbard et al. 1991; Leininger et al. 1978; Suzuki et al. 2010). It is not
yet clear whether these laboratory animals were infected while living in the wild or
via contact with infected handlers while being captured and cared for prior to export
in leprosy-endemic areas. However, in the case of the wild gorillas with “leprous”
lesions, the researchers reported that the infection was successfully cleared up in 8
days with penicillin injections, which was consistent with T. pallidum infection
rather than leprosy (Cousins 1984).
Mid-century, the scientist Armand Denis shared his observations on yaws in the
wild gorillas of the Republic of Congo (ROC). He described the first case he saw,
affecting an adult male, thus:
It was like a mask eaten into by some flesh-consuming disease. The lips were gone. The
nostrils were eaten almost away and the fangs of teeth were blackened and askew in what
remained of the creature’s lower jaw. Only the eyes were untouched… Whatever the disease
was the wretched animal had caught I had no idea… (Denis 1963, p 181).

Although the clinical signs described again appear to overlap with those caused
by leprosy, Denis learned that this disease was thought by the natives to be yaws, the
same disease that was common in human populations that lived along the coast.
202 K.N. Harper and S. Knauf

Table 3 T. pallidum infection in great apes

Seroprevalence
Origin in sample Tests Source
Chimpanzees (Pan troglodytes)
Laboratory animals caught in western 1/15 (6.7 %), + FTA-ABS Felsenfeld and
region of Central Africa result Wolf (1971)
equivocal
Bukavu region, Republic of Congo 3/9 (33.3 %) TPI and Fribourg-Blanc and
FTA-ABS Mollaret (1969)
Bangui region, Central African Republic 0/1 (0.0 %) TPI and Fribourg-Blanc and
FTA-ABS Mollaret (1969)
Laboratory animals, origin not specified 48/250 (19.2 %) FTA-ABS Kuhn (1970)
Gorillas (Gorilla gorilla)
Bukavu region, Republic of Congo 0/1 (0.0 %) TPI and Fribourg-Blanc and
FTA-ABS Mollaret (1969)
Laboratory animals, origin not specified 0/14 (0.0 %) FTA-ABS Kuhn (1970)
Orangutans (Pongo spec.)
Laboratory animals, origin not specified 0/39 (0.0 %) FTA-ABS Kuhn (1970)

Reports of yaws were not limited to gorillas living in the ROC. Lesions consistent
with yaws were also described in the young gorillas of Rio Muni, in Equatorial Guinea
(Cousins 1984). Similarly, yaws was diagnosed in two out of five young gorillas
imported from the French Cameroons (modern day Cameroon and Nigeria) to the
Lincoln Park Zoo, mid-century (Cousins 1972), though the method of T. pallidum
confirmation is not given in the report. Sensitive and specific serological tests have
confirmed that gorillas do indeed come into contact with T. pallidum (Table 3). Four
blood samples from solitary gorillas in the Parc National d’Odzala-Kokoua in the
Republic of Congo, two of which had skin lesions consistent with yaws, tested posi-
tive for treponemal antibodies (Karesh 2000).
Skeletal evidence consistent with (but not specific to) treponemal infection has
also been found in the remains of wild gorillas (Lovell et al. 2000). Lovell and col-
leagues examined 126 gorilla skeletons collected from the Democratic Republic of
Congo (DRC), the ROC, and Cameroon in the early twentieth century. Eighteen
percent of gorillas were found to exhibit possible signs of treponemal disease, with
active lesions found almost entirely among subadults. In addition, cranial deformi-
ties involving massive osseous tumors have been documented in gorillas, and some
researchers have speculated that these may correspond to goundou, a rare manifes-
tation of tertiary-stage yaws in humans (Cousins 2008). However, whereas in
humans the lesions grow out of the nose and upper jaw, in gorillas, growth of
goundou-like lesions seems to occur primarily over the cheekbones (Cousins 2008).
Recently, an evaluation of skin lesions in 377 gorillas living in the Parc National
d’Odzala-Kokoua in the ROC gave us a much better understanding of treponemal
disease in this population (Levréro et al. 2007). The macroscopic lesions identified
were found to be similar to yaws in humans (Fig. 3) and affected 17 % of animals
examined. However, since some animals that are infected do not display lesions
(Karesh 2000), the actual prevalence of the infection is likely to be higher. Infection
Treponema pallidum Infection in Primates: Clinical Manifestations, Epidemiology… 203

Fig. 3 Photos depicting skin lesions suspected to result from T. pallidum infection in gorillas.
Skin lesions are shown on (a) an adult male, (b) a juvenile female, and (c) an adult female gorilla
(Source: Levréro et al. 2007)

in the gorillas studied began early, with almost 5 % of infants displaying skin lesions
compatible with yaws and peak incidence occurring between the infant and juvenile
stages. Some animals were found to exhibit destruction of the nose and/or lips or to
display deep lesions on their wrists or ankles. Possible social consequences of
severe infection were hinted at in the observations of Levréro et al. One adult female
with a nose that was completely destroyed was forced to leave her group; during the
animal’s seven subsequent visits to the clearing before disappearing, the researchers
observed the males from groups she approached behaving antagonistically towards
her. As the researchers note, the consequences of such rejection must certainly have
implications for survival. Although lesions were also reported in gorillas at nearby
sites, including Maya, Moba, and Lossi, infection may not be universal, since no
such lesions were observed at the Mbeli clearing, also nearby.
Treponemal infection in gorillas has been identified with yaws, due to its gross-
pathological manifestations and also the fact that the areas in which the gorillas
reside tend to be places in which the human prevalence of this disease was quite high
historically, affecting the vast majority of some human groups inhabiting the rain
forest (Hackett 1953; Pampiglione and Wilkinson 1975) and still infecting more
than 10 % of residents in some areas (Gersti et al. 2009). Unfortunately, not a single
report of treponemal infection has been confirmed via molecular biological tests,
such as T. pallidum-specific PCR or immunohistochemistry, capable of demonstrating
the spirochetes in situ. Obtaining the samples needed to perform such tests in endan-
gered great apes is extremely challenging, both politically and technically, and this
explains why they have not yet been performed. However, without the information
these tests can provide, it is impossible to determine with certainty that T. pallidum
is responsible for the lesions and, if so, to which human subspecies the responsible
strains are most closely related.
Although most reports of treponemal infection in wild gorillas have described a
yaws-like disease, descriptions of animals with diseased sexual organs that may be
linked to treponemal infection have also surfaced. For example, in Cameroon, one
adult female exhibited genitalia covered with running sores, as well as deforming
sores on the hands and wrists (Cousins 1984). This animal was said by the nearby
residents to be suffering from “marjal” or “mebata,” a local word for yaws. As with
204 K.N. Harper and S. Knauf

the yaws-like cases, providing an explanation for the etiology of such lesions is
challenging, especially in the context of other sexually transmitted infections that
can cause genital ulceration in NHPs. Definitively assigning an etiological agent in
such cases will only be possible with advanced molecular biological techniques.

Chimpanzees

Descriptions of the manifestations of treponemal infection in chimpanzees (Pan

troglodytes) are less common. That contact with T. pallidum occurs is clear from
serological surveys of wild animals (Table 3). For example, one survey of more
than 250 wild-born chimps, drawn from unknown locations, found that 48 (19 %)
were positive for T. pallidum antibodies (Kuhn 1970). Serological evidence of
infection has also been found in wild chimps from the DRC and Sierra Leone
(Felsenfeld and Wolf 1971; Fribourg-Blanc and Mollaret 1969). In addition, the
physical anthropologists who found evidence consistent with treponemal disease in
gorillas also found that almost 20 % of 102 sets of wild chimp remains from Western
Africa exhibited osseous lesions that could be due to treponemal disease (Lovell
et al. 2000). However, only a single case of treponemal infection causing observ-
able clinical manifestations has been reported in the literature (Edroma et al. 1997).
This case occurred in a chimpanzee at Gombe, in Tanzania, who may have been
infected as a result of the ongoing epidemic in neighboring baboons. The means via
which T. pallidum was identified as the source of the disease was not described in
the report.

Asian Monkeys

Some of the first yaws experiments were performed in macaques (Macaca spec.).
However, it appears that natural infection of wild macaques is very rare, if it occurs
at all; over 1,000 animals have been tested without one robust seropositive reaction
(Table 4). Similarly, though Thivolet et al. found treponemal antibodies in 46 of 415
African monkeys, not a single one of the 152 Asian monkeys this research group
tested were positive (Kuhn 1970).
Only one serologically reactive animal captured in Asia has been reported. Kuhn
(1970) describes finding a seropositive reaction in a Celebes crested macaque
(Macaca nigra), from Sulawesi, Indonesia. Given the paucity of positive infections
among Asian monkeys, however, one must wonder whether this result represents
one of the rare false positives that can stem from the FTA-ABS test or an infection
acquired during the animal’s time in captivity vs. during its life in the wild.
Additional sampling of Asian NHPs will help answer this question.
Table 4 T. pallidum infection in Asian and South American monkeys
Species Origin Seroprevalence in sample Tests Source
Asia
Macaques (Macaca, species Laboratory animals caught in 1/6 (16.7 %), + results equivocal FTA-ABS Felsenfeld and Wolf (1971)
not determined) Southeast Asia
Crab-eating Macaque Phnom-Penh region of Cambodia 0/1236 (0.0 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
(Macaca fascicularis)
Crab-eating Macaque Asia 0/5 (0.0 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
(Macaca fascicularis)
Rhesus Macaque (Macaca Laboratory animals caught in 0/22 (0.0 %) FTA-ABS Felsenfeld and Wolf (1971)
mulatta) Southeast Asia
Rhesus Macaque (Macaca India 0/5 (0.0 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
mulatta)
Rhesus Macaque (Macaca Asia 0/30 (0.0 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
mulatta)
Pig-tailed Macaque (Macaca, Asia 0/5 (0.0 %) TPI and FTA-ABS Fribourg-Blanc and Mollaret (1969)
species not determined)

South America
Owl monkey (Aotus, species Laboratory animals, origin not 2/84 (2.4 %), + results equivocal FTA-ABS Levine et al. (1970)
not determined) specified 0/9 TPI
Owl monkey (Aotus Laboratory animals caught in South 0/3 (0.0 %) FTA-ABS Felsenfeld and Wolf (1971)
trivirgatus) America
Red-bellied titi (Callicebus Laboratory animals caught in South 5/25 (20.0 %), + results equivocal FTA-ABS Felsenfeld and Wolf (1971)
moloch) America
Squirrel Monkey (Saimiri Laboratory animals caught in South 0/18 (0.0 %) FTA-ABS Felsenfeld and Wolf (1971)
sciureus) America
Squirrel monkey (Saimiri Laboratory animals, origin not 4/63 (6.3 %), + results equivocal FTA-ABS Levine et al. (1970)
sciureus) specified 0/10 TPI
Marmoset (Callithrix spec.) Laboratory animals, origin not 0/24 (0.0 %) FTA-ABS Levine et al. (1970)
specified 0/23 TPI
206 K.N. Harper and S. Knauf

South American Monkeys

To date, not a single wild South American NHP has been found with a definite sero-
positive reaction to T. pallidum, although artificial infection of owl (Aotus) and
squirrel (Saimiri) monkeys is possible (Elsas et al. 1968; Smith 1969). However, as
is clear from Tables 2 and 4, sampling in African monkeys has been much more
thorough. In order to conclude definitively that natural infection of South American
monkeys does not occur, more extensive sampling would have to be performed.
Sampling areas of Central and South America the prevalence of treponemal disease
in human indigenous groups was once very high, such as French Guiana, Brazil,
Venezuela, and Colombia (Black 1975; Hopkins and Flórez 1977; St John 1985),
would be especially important.

Experimental Insights into T. pallidum Infection

and Host Factors in Primates

NHPs played a pivotal role in the discovery of T. pallidum. In the early years of the
twentieth century, Metchnikoff and Roux (1903, 1904, 1905) demonstrated that
syphilis could be transferred from humans to chimpanzees and from one chimpan-
zee to another. Thus, apes became the first reliable animal model for the study of T.
pallidum infection. Around the same time, Castellani (1907) demonstrated that
monkeys could be infected with T. pallidum subsp. pertenue. Even so, our under-
standing of the differences in how infection progresses in humans versus NHPs is
rudimentary. Here, we review what is known about NHP responses to experimental
infection with T. pallidum, focusing on between-species similarities and differences
(Table 5).
Turner and Hollander (1957b) did a series of experiments on rhesus macaques
and African green monkeys. After inoculating animals in the thighs, eyebrows, and
genital regions, the animals were followed for 14–17 months before postmortem
examination; no gross changes suggesting syphilitic infection were identified, and a
number of the animals remained seronegative throughout, though their organs con-
tained infective spirochetes. On this basis, Turner and Hollander suggested that
NHPs did not provide a suitable animal model for understanding T. pallidum patho-
genesis. Similarly, although humans typically develop a primary chancre or “mother
yaw” at the site where the bacterium enters the body, in a study utilizing ten
macaques, Sepetjian et al. (1972) reported that only two animals developed signifi-
cant lesions at the site of inoculation—the others exhibited no lesions at all or mild
macular discoloration—and these lesions disappeared quickly. Like Turner and
Hollander, Sepetjian et al. (1969) also observed no visceral lesions upon necroscopy
after 4 months, although in half the animals treponemes were present in various
organs.
Table 5 Comparison of NHP host response to experimental T. pallidum subsp. pallidum infection and human response to natural infection with syphilis
Humans Macaques African Green Monkeys Owl Monkeys (Aötus trivirgatus)
Geographic distribution Global Asia Africa South America
of host
Lesion at site of Yes Small, mild, and quick to Only in half the animals studied; No
inoculation disappear when present at small and disappeared within
all after subsp. pallidum several weeks
infectiona
Treponemes disseminate Yes Yes Yes Yes
through body
Lesions remote from Yes None observed None observed Yes
inoculation site
Serologic response to Yes Sometimes but not always; Appears rare (1 out of 4 animals Sometimes but not always
infection response is often slow to seroconverted during study
develop period)
Robust CD4+ T-cell Yes Yes Unknown Unknown
response
Sources Salazar et al. (2002a, Sepetjian et al. (1969, 1972), Turner and Hollander (1957b) Elsas et al. (1968), Smith et al. (1965)
b), Perine et al. Turner and Hollander
(1984) (1957b), Marra et al.
(1998)
a
Significant lesions at the site of inoculation were observed when the animals were injected with subsp. pertenue and Fribourg-Blanc strain (Sepetjian et al.
1969)
Treponema pallidum Infection in Primates: Clinical Manifestations, Epidemiology…
207
208 K.N. Harper and S. Knauf

Other studies suggest that the NHP response to infection may offer parallels to
that observed in humans, however. For example, experiments have demonstrated
that owl monkeys and macaques develop a chronic, systemic infection in response
to inoculation with strains of human-derived T. pallidum (Elsas et al. 1968; Marra
et al. 1998). In some of these animals, a long-lasting serological response develops
(Sepetjian et al. 1972), though, as Turner and Hollander and others have described,
others remain seronegative for months on end, despite harboring infective trepo-
nemes (Smith et al. 1965; Turner and Hollander 1957b; Wells and Smith 1967).
Similar to humans, NHPs can develop T. pallidum-related lesions weeks or months
after infection, distant from the site of inoculation (Elsas et al. 1968; Sepetjian et al.
1969; Smith et al. 1965). Clark and Yobs (1968) have opined that the variation in
terms of lesion development and serological response in NHP hosts such as the owl
monkey might be viewed as typical of the varied response found in humans; in their
view, the lesions observed are even consistent with primary and secondary stages of
disease, as in humans.
Immunological studies suggest further similarities between the human and NHP
response to T. pallidum infection. In humans, humoral, antibody, and CD8+ cyto-
toxic T-cell responses have been shown to be relatively ineffective at clearing syphi-
litic infection or curbing the progression of lesions (Carlson et al. 2011). Instead,
delayed-type hypersensitivity, which is mediated by CD4+ T cells, appears to play a
role of particular importance (Carlson et al. 2011). Similar findings have been
reported in macaques infected with a subsp. pallidum strain. As in humans, CD4+
T cells appear to be responsible for clearing T. pallidum from the central nervous
system during early infection (Marra et al. 1998). In terms of the components of the
bacterium that stimulate an immune response, T. pallidum lacks lipopolysaccha-
rides (LPS) in its cell wall and therefore does not cause an inflammation cascade by
activating the toll-like receptor (TLR) 4 (Schroder et al. 2008). TLR4 activation is a
common pathway induced by the LPS of Gram-negative bacteria and to a certain
extent by the lipoteichoic acids of Gram-positive bacteria. This mechanism repre-
sents a major immunological host defense against bacterial infection, which is virtu-
ally absent in T. pallidum infection. Instead, the T. pallidum infected host responds
to infection via the activation of TLR 2/1 by immunostimulatory lipoproteins in the
outer membrane of the spirochete (Lien et al. 1999; Schroder et al. 2008). There is
evidence that natural simian and human infections are similar in this respect. For
example, in our laboratory, we performed immunoblots upon sera from wild baboons
in Lake Manyara National Park infected with T. pallidum. The sera were tested for anti-
T. pallidum IgM and IgG antibodies to recombinant T. pallidum proteins Tp15, Tp17,
and Tp47, and Treponema membrane protein A (TmpA) obtained from human-infect-
ing T. pallidum strains. The results consistently showed that the animals were producing
antibodies against these proteins (Knauf unpublished data). On this basis, we predict
that simian T. pallidum strains express immunostimulatory lipoproteins similar to the
Tp15, Tp17, Tp47, and TmpA proteins of known human strains and that NHPs are
responding to these antigens in a similar way to humans.
In humans, the relative distribution of the four immunoglobulin G subclasses
changes over the course of infection (Baughn et al. 1988; Moskophidis 1989;
Treponema pallidum Infection in Primates: Clinical Manifestations, Epidemiology… 209

Salazar et al. 2002a, b). Whether similar changes occur in NHPs is not known, but
field research on animals displaying signs of early- and late-stage disease will help
answer this question.
Most of the results described above feature inoculation of NHPs with strains of T.
pallidum obtained from human infections. In one study, however, five macaques were
infected with the baboon-derived Fribourg-Blanc strain (Sepetjian et al. 1969). The
lesions developed by the macaques were characteristic of yaws rather than syphilis.
Rather than an indurated chancre, they displayed vegetative, hyperkeratotic papular
lesions. Interestingly, the TPI serology was weak in one animal and nonreactive in the
other three tested, one of which died 5 weeks into the study; the single weak reaction
did not even begin to develop until the eleventh week of infection. FTA-ABS serology
was similar, weak and slow to develop in two animals (one of which had the weak TPI
test) and nonreactive in the other two. It remains unclear whether the fact that the
antibody response of these macaques to an NHP-derived strain was so weak is signifi-
cant. Is the NHP response to T. pallidum strains native to their own species similarly
slow to their own species similarly slow to develop? At least for baboons naturally
infected with T. pallidum, Knauf et al. (unpublished data) were able to demonstrate
that anti-T. pallidum antibody titers in infected animals were generally high, with
mean anti-T. pallidum IgM+IgG titers of 1:2.94E+04 ± SE9.87E+03 in the initial clin-
ical stage of disease, 1:2.17E+05 ± SE1.83E+05 in the moderate stage, and
1.78E+06 ± SE1.38E+06 in the severe stage of infection. Moreover, comparison to a
PCR-based test demonstrated that sensitivity (Sen) and positive predictive values (PP) for
the antibody-based gelatin particle agglutination assay (Sen 100 %, PP 95 %), the FTA-
ABS IgG (Sen 100 %, PP 98 %), and the immunoblot IgG (Sen 100 %, PP 93 %) serologi-
cal tests in the baboons were reliable, although the specificity and negative prediction
values need more testing. Of course in studies of wildlife, the major disadvantage is the
paucity of information regarding the timing of infection. Thus, results regarding the
serological response of wild NHPs to infection need to be considered carefully.
There is one report of inoculating human “volunteers” with a strain of T. palli-
dum obtained from an NHP infection. In The Caracas Project, a study of late-stage
yaws and pinta carried out in 1969, five people were inoculated with the Fribourg-
Blanc strain (Smith et al. 1971). Two of the five patients developed reactive FTA-
ABS/TPI serology, while three had nonreactive tests. Furthermore, two of the three
with nonreactive serology had elevated IgG levels in their cerebrospinal fluid, indi-
cating that four of the five patients mounted some type of immunological response
to infection. Finally, abnormal ophthalmological results consistent with late-stage
yaws were found in three of the five patients, although it is not entirely clear that
these abnormalities were due to infection with the Fribourg-Blanc strain. Thus, the
Caracas Project showed that an NHP-derived T. pallidum strain could establish an
infection in humans and possibly cause ocular abnormalities. Unfortunately,
whether or not these infections yielded additional clinical signs in humans is
unknown, as the project focused only on neuro-ophthalmologic manifestations. Nor
was the length of experimental infection or a discussion of the ethics of infecting
healthy adults with T. pallidum and allowing them to develop late-stage infection
provided.
210 K.N. Harper and S. Knauf

In sum, experimental studies of response to inoculation suggest that primates

may mount an immune response against T. pallidum that shares some basic charac-
teristics with that of humans while differing in other aspects. In all species exam-
ined, infection appears to be chronic, and the host uses a predominately CD4+
response to control pathogen growth. NHPs on the whole, though, appear to present
much milder clinical signs than humans, often exhibiting a slow rise in antibody
titers. Visceral signs of experimental infection have not been reported, in NHPs yet.
Because most NHP studies have continued for only a few months or a year, it is not
clear whether lesions akin to tertiary-stage symptoms in humans would emerge,
given enough time. In addition, most experiments have been performed in species,
such as Asiatic macaques or New World monkeys, which, for whatever reason, do
not appear to be susceptible to infection in the wild. Therefore, it is not clear whether
the same results would be found in species that have presumably evolved in tandem
with their own T. pallidum strains. Finally, it should be noted that the results of the
experiments described here are probably contingent to some degree upon the strain
of T. pallidum used. For example, in one study, macaques inoculated with a subsp.
pallidum strain did not develop significant lesions at the site of the injection; how-
ever, all macaques infected with a subsp. pertenue strain did develop considerable
lesions (Sepetjian et al. 1969). While animal experiments studying T. pallidum
pathogenesis are on the wane, especially those utilizing NHP hosts, field studies of
treponemal infection in wild NHPs may help clarify the similarities and differences
in response to infection that have evolved in various host species over the years.

Some Open Questions on Host Specificity

and Strain Pathogenicity in Primates

As described above, even though T. pallidum infections have not been documented
in species such as macaques, owl, and squirrel monkeys in the wild (Table 4), labo-
ratory experiments show that these animals can be infected with T. pallidum. If
these species are susceptible to infection, what prevents them from serving as hosts
in the wild? Which is more important in explaining why NHP species in Africa but
not Asia and South America are infected with T. pallidum: host geography (Fig. 4)
or phylogeny (Fig. 5)? Is the reason for this inconsistency across primate species
rooted in biological differences, the varied environments they inhabit, or chance
playing out over evolutionary time? The genital ulcerative disease caused by T. pal-
lidum in olive baboons, which has been reported exclusively in Papio anubis (Knauf
et al. 2012; Wallis and Lee 1999), poses a similar puzzle. Olive and yellow baboons
are known to hybridize (Alberts and Altmann 2001), which means that a significant
level of sexual interaction occurs between the two species. However, even at
Treponema hot spots in Tanzania where olive and yellow baboon subspecies overlap
and transmission opportunities should arise frequently, yellow baboons have never
been observed with genital lesions consistent with T. pallidum infection. Could
Treponema pallidum Infection in Primates: Clinical Manifestations, Epidemiology… 211

Fig. 4 Geographic distribution of T. pallidum infection in nonhuman primates. Drawn from

sources cited in Tables 2–4; only areas in which more than ten animals had been tested were
included

some constellation of host genetics in yellow baboons provide them with physiolog-
ical protection against the disease? Or are differences in behavior between olive and
yellow baboons responsible for the presence of the infection in one subspecies but
not the other? The factors that determine whether or not an NHP clade serves as a
natural host remain mysterious at this point.
Parallel questions exist about the importance of strain vs. host characteristics in
determining clinical manifestations. For example, is the recently described infection
in Tanzanian baboons, characterized by genital ulceration, caused by a strain or
strains which are genetically distinct from the strains that cause milder, non-genitally
ulcerating infections in the baboons of West Africa? Or is some characteristic of the
Tanzanian baboons, or their environment, responsible for these novel manifesta-
tions? Previously, it has been demonstrated that climatic factors such as temperature
and humidity play an important role in modulating clinical lesions in T. pallidum
infection (Turner and Hollander 1957a). Could this important finding help explain
the disease dynamics observed in some of the naturally occurring NHP epidemics
in East Africa (Collins et al. 2011)? Detailed surveys of T. pallidum manifestations
in different NHP groups may help begin to answer some of these questions, allow-
ing us to begin to disentangle the effects of the evolution of host and pathogen from
environment.
212 K.N. Harper and S. Knauf

Fig. 5 Species distribution of T. pallidum infection in nonhuman primates. Drawn from sources
cited in Tables 2–4; only species in which more than ten animals had been tested were included

Conclusion

Surveys of T. pallidum infection in NHPs performed thus far have provided fasci-
nating insights into the ecology of treponemal disease in the wild. It appears that T.
pallidum infection in wild NHPs is common in many parts of Africa, but very rare,
if it occurs at all, in Asia and South America. Robust evidence of seropositive ani-
mals has only been found in Old World species of NHPs (Figs. 4 and 5). However,
more comprehensive testing of Asian and South American NHPs must be performed
before solid conclusions regarding the geographical and phylogenetic distribution
of infection may be drawn.
Because the history of T. pallidum infection in humans remains controversial,
studies of NHP strains may help elucidate the origin of our own. Thus far, only two
NHP strains have been characterized genetically. These two strains are both from
baboons, one from Guinea and the other from Tanzania, and both are closely related
Treponema pallidum Infection in Primates: Clinical Manifestations, Epidemiology… 213

to human T. pallidum subsp. pertenue strains (Harper et al. 2008; Knauf et al. 2012),
which are responsible for the disease yaws. However, it is not clear whether the
NHP strains are merely closely related to subsp. pertenue or whether they are actu-
ally members of the subspecies. Sampling strains from other NHP species and geo-
graphic regions and sequencing their entire genomes using next-generation methods
should help us better understand how NHP strains are related to human strains and
to one another. If T. pallidum strains tend to diverge in the same order as their host
species, this would indicate that the infection is ancient in primates, with each spe-
cies possessing its own genetically distinct type of pathogen. It could also indicate
that T. pallidum infected our earliest hominid ancestors. In contrast, if T. pallidum
strains diverge in patterns that do not reflect their hosts’ phylogenies, host switching
may be implicated—and it is possible that T. pallidum infection may be a more
recent addition to the human disease-scape.
The clinical signs that have been described in association with T. pallidum infec-
tion in NHPs are primarily consistent with mild, nonvenereal infection. However,
the T. pallidum infection described in baboons in Tanzania is interesting in that it
appears to be associated with the genitals and possibly spread via sexual transmis-
sion. Our field observations of hundreds of baboons at Lake Manyara (Knauf et al.
2012; Mlengeya 2004) echo the findings reported from Gombe (Wallis 2000a;
Wallis and Lee 1999), in that the clinical lesions we observed are almost exclusively
found in the anogenital region and appear to be limited to sexually mature individu-
als. Further epidemiological characterization of this infection should clarify the
means of spread.
Given the fact that the human T. pallidum subspecies, transmitted via different
modes, are genetically distinct (Centurion-Lara et al. 2006; Harper et al. 2008;
Smajs et al. 2011), the same may be true of NHP strains transmitted via alternate
routes. If future studies reveal that some NHP strains are consistently sexually trans-
mitted while others are not, then comparison of closely related strains transmitted
via different routes may help generate hypotheses about the genetic polymorphisms
underlying sexual transmission. It is possible that comparison of genetic polymor-
phisms in T. pallidum associated with sexual transmission in both humans and
NHPs could identify genetic regions that are especially important in adaptation to
this mode of transmission. In addition, the selective pressures that favor sexual
transmission of the pathogen in some NHPs but not others could be studied;
environmental, social, immunological, and other factors might all be investi-
gated as possible driving forces. In short, the spectrum of different transmission
routes and types of clinical manifestations in NHPs may provide a unique
opportunity to better understand the pathogenicity and evolution of T. pallidum
in our own species.
Current efforts to study T. pallidum infection in wild NHPs are hampered by the dif-
ficulty of gathering adequate biological samples, especially in endangered species such
as the great apes. A solid diagnosis of treponemal disease can only be obtained by dem-
onstrating that the spirochete is present, and carefully documenting microscopic and
macroscopic signs of infection, including molecular biological proof, is essential.
Serology can be used to screen large numbers of wild NHPs for T. pallidum infection,
214 K.N. Harper and S. Knauf

but it requires that blood samples be taken. Moreover, because serology cannot aid in
differentiating between the T. pallidum subspecies, nor can it provide information about
clinical manifestations, disease progression, or even whether an infection is active,
serum samples are not sufficient for a truly comprehensive examination. Though obtain-
ing specimens from NHPs for histological and molecular biological tests is often very
difficult, in order to reach a definitive diagnosis using the methods currently available,
access to these types of samples is a necessity.
In the future, it is hoped that noninvasive means of studying treponemal infection
in wild NHPs may be developed, which could greatly facilitate diagnosis and sur-
veillance. Antibody-based tests have been successfully used on urine and fecal
samples to study NHP pathogens such as simian immunodeficiency virus (SIV)
(Santiago et al. 2003), simian foamy virus (SFV) (Liu et al. 2008), hepatitis B
(Makuwa et al. 2005), and Cryptosporidium and Giardia (Salzer et al. 2007). In
addition, urine and fecal samples have been used to obtain DNA from NHP patho-
gens such as SIV (Santiago et al. 2003), SFV (Liu et al. 2008), respiratory viruses
(Köndgen et al. 2010), and Plasmodium spp. (Liu et al. 2010), paving the way for a
flurry of recent phylogeographic studies of unprecedented size. Because it is not
clear whether urine or feces are reasonable samples for T. pallidum antibody-based
tests, or whether pathogen DNA is shed in urine or feces at detectable levels during
the various stages of infection, the potential of noninvasive samples to revolutionize
studies of T. pallidum in NHPs is unclear and currently under investigation (Knauf
et al., unpublished data).
Understanding treponemal infection in NHPs is important for several reasons.
First, it is possible that morbidity and mortality associated with T. pallidum infec-
tion has important conservation implications, especially for species such as the
great apes (Levréro et al. 2007). Second, a dialogue about reinitiating the yaws
eradication campaign that began mid-century is currently active (Asiedu et al. 2008;
Rinaldi 2008). Before the feasibility of such a campaign may be assessed, we must
understand the relevant disease ecology. Frequent transmission from an animal res-
ervoir would have serious ramifications for an attempt to eradicate infection in
humans, and phylogenetic studies can help determine whether or not this occurs.
Third, understanding the evolution and transmission of T. pallidum among diverse
primate species may help us better understand the forces that mold its evolution and
transmission dynamics within our own species. Further study may shed light on the
geographic distribution, the mode of transmission, and the NHP species affected by
T. pallidum infection, as well as the relationship between human and NHP strains.

Acknowledgments We thank the Tanzania Wildlife Research Institute (TAWIRI), Tanzania

National Parks (TANAPA), the Lake Manyara National Park headquarter staff, the NCA authority,
and the Tanzania Commission for Science and Technology for making our study of T. pallidum
infection in baboons possible. We also thank Jim Thomas for the use of his laboratory, Worldwide
Primates and the Buckshire Corporation for providing samples, and Columbia University’s
Training Program in Cancer-Related Population Sciences (5-R25-CA 094061), the Robert Wood
Johnson Foundation Health & Society Scholars program, NSF (Grant 0622399), the Wenner-Gren
Foundation, and the Howard Hughes Medical Institute Predoctoral Fellowship program for their
financial support.
Treponema pallidum Infection in Primates: Clinical Manifestations, Epidemiology… 215

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Molecular Mimicry by γ-2 Herpesviruses
to Modulate Host Cell Signaling Pathways

Lai-Yee Wong, Zsolt Toth, Kevin F. Brulois, Kyung-Soo Inn,

Sun Hwa Lee, Hye-Ra Lee, and Jae U. Jung

Introduction

Herpesviruses are large double-stranded DNA viruses that can establish life-long
infection in their respective hosts and can undergo two different phases in their life
cycle: lytic or latent (Fig. 1a) (Pellett and Roizman 2007). Lytic replication is char-
acterized by the expression of most viral genes in an ordered cascade (immediate
early, early, and late), leading to the production of infectious virions. Latency is
marked by minimal viral gene expression and the maintenance of the viral genome
in the nucleus. Reactivation to lytic replication from latency can be triggered by
multiple factors, such as stress or chemical reagents. The ability of herpesviruses to
utilize these two very distinct modes of replication is an excellent survival strategy
as establishment of latency with periodic reactivation may facilitate persistent infec-
tion in the host while allowing evasion from the immune system.
Herpesviruses are prevalent in nature, with most animal species being infected
by at least one herpesvirus (Knipe and Howley 2007). Out of more than 200 herpes-
viruses identified to date, only eight are endemic to humans and are grouped into
three subfamilies (α, β, γ) based on their structural and biological properties
(Table 1). γ-Herpesviruses are lymphotropic and are further divided into two sub-
groups: γ-1 (Lymphocryptovirus) and γ-2 (Rhadinovirus) (Fig. 2). Epstein-Barr
virus (EBV) was the first γ-herpesvirus to be discovered and is the prototype mem-
ber of the γ-1 group (Diehl et al. 1968). Kaposi’s sarcoma-associated herpesvirus
(KSHV) is so far the only human virus assigned to the Rhadinovirus family, which
also includes herpesvirus saimiri (HVS), rhesus rhadinovirus (RRV), and mouse
herpesvirus 68 (mHV68) (Table 2) (Blaskovic et al. 1980; Chang et al. 1994;
Desrosiers et al. 1997; Melendez et al. 1968; Moore et al. 1996).

L.-Y. Wong (*) • Z. Toth • K.F. Brulois • K.-S. Inn • S.H. Lee • H.-R. Lee • J.U. Jung
Department of Molecular Microbiology and Immunology, University of Southern California,
Keck School of Medicine, Los Angeles, CA 90033, USA
e-mail: [email protected]

J.F. Brinkworth and K. Pechenkina (eds.), Primates, Pathogens, and Evolution, 221
Developments in Primatology: Progress and Prospects,
DOI 10.1007/978-1-4614-7181-3_8, © Springer Science+Business Media New York 2013
222 L.-Y. Wong et al.

Fig. 1 (a) Diagram showing the two phases of a herpesvirus life cycle. Upon infection, the virus
can enter latency where the viral genome is typically kept in an episomal form in the cell nucleus
and only a few viral genes are expressed. Alternatively, the virus may undergo lytic replication to
produce infectious virus that go on to infect new cells. Viral genes are expressed in an ordered
cascade during lytic replication, starting with immediate-early genes, early genes, and late genes.
Common functions of the proteins encoded by these lytic genes are listed underneath each group.
Late gene expression only occurs after viral DNA replication. (b) A burst of lytic replication
occurs after infection by a herpesvirus, after which the virus will establish latency in the now
infected host. Periodically the virus will reactivate to produce new virus, followed by another
round of latency where no virus is produced

γ-Herpesviruses are oncogenic viruses capable of causing neoplasia in the

infected host (Table 2) and contain multiple open reading frames (ORFs) that con-
tribute to virus-induced tumorigenesis. In addition to being the only oncoviruses in
the herpesvirus family, the pathogenesis of γ-herpesviruses is largely associated
with their latency program (Fig. 1b). In contrast, the disease symptoms of α- and
β-herpesviruses are due to the lytic replication and the resulting host immune
responses to contain the virus.
A striking feature of γ-herpesviruses is the high degree of molecular piracy
among their viral genes that are postulated to have been “appropriated” from the
host cell during virus-host evolution. This chapter focuses on several intriguing
ORFs of KSHV, RRV, and HVS that are similar in structure (membrane localization
Molecular Mimicry by γ-2 Herpesviruses to Modulate Host Cell Signaling Pathways 223

Table 1 Human herpesviruses

Name Subfamily Synonym
HHV-1 α Herpes simplex virus 1 HSV-1
HHV-2 α Herpes simplex virus 2 HSV-2
HHV-3 α Varicella-zoster virus VZV
HHV-4 γ Epstein-Barr virus EBV
HHV-5 β Cytomegalovirus CMV
HHV-6 β Human herpesvirus 6
HHV-7 β Human herpesvirus 7
HHV-8 γ Kaposi’s sarcoma-associated herpesvirus KSHV

Fig. 2 Phylogenetic tree showing the three subfamilies of herpesviruses (α, β, γ). The tree was
constructed using sequences of the viral DNA polymerase catalytic subunit. The program
MUSCLE was used for alignment and PhyML for phylogeny analysis (Dereeper et al. 2008)

and signaling motifs), function (modulating lymphocyte activation events), and

genomic position despite not having any sequence homology. Furthermore, we will
also touch on the burgeoning field of virus-encoded microRNAs (miRNAs), which
is another mechanism used by herpesviruses to manipulate host cell signaling
through the repression of gene expression.

Kaposi Sarcoma-Associated Herpesvirus

KSHV was first discovered in 1994 as the etiological agent in Kaposi’s sarcoma
(KS) lesions and has since been further implicated in primary effusion lymphoma
(PEL) and multicentric Castleman’s disease (MCD) (Cesarman et al. 1995; Chang
et al. 1994; Soulier et al. 1995). KSHV DNA has been detected in all cases of KS
using multiple assays such as polymerase chain reaction and immunohistochemis-
try (Boshoff et al. 1995; Dupin et al. 1999). The first case of KS was described in
1872 by Dr. Moritz Kaposi as an indolent tumor affecting elderly men of
Mediterranean and Jewish origins (Iscovich et al. 2000; Kaposi 1872). This later
became known as classical KS after three other epidemiological forms were
224 L.-Y. Wong et al.

Table 2 The respective host and associated malignancies of γ-herpesviruses

Group Virus NameHost species Associated malignancies
γ-1 Lymphocryptovirus Epstein-Barr EBV Homo Burkitt’s lymphoma,
virus sapiens infectious mononucleo-
(human) sis, nasopharyngeal
carcinoma (NPC)
Kaposi’s KSHV Homo Kaposi’s sarcoma (KS),
sarcoma- sapiens primary effusion
associated (human) lymphoma (PEL),
herpesvirus multicentric Castleman’s
disease (MCD)
γ-2 Rhadinovirus Herpesvirus HVS Saimiri Lymphoma (nonnatural
saimiri sciureus hosta)
(squirrel
monkey)
Rhesus RRV Macaca Lymphoproliferative
rhadinovirus mulatta disorderb
(rhesus
monkey)
Murine herpesvi- mHV68 Mus
rus 68 musculus
(mouse)
a
HVS-C in common marmosets and rabbits
b
Naïve rhesus macaques

reported, which includes iatrogenic KS in immunosuppressed patients after organ

transplants, endemic KS in sub-Saharan Africa, and acquired immunodeficiency
syndrome (AIDS)-related epidemic KS (Ambroziak et al. 1995; Foreman et al.
1997; Gao et al. 1996; Kedes et al. 1996; Regamey et al. 1998; Schalling et al. 1995;
Simpson et al. 1996). Unlike the classical form, endemic KS in Africa is extremely
aggressive and rapidly fatal and occurs in both sexes with equal frequency
(Dourmishev et al. 2003; Hengge et al. 2002). It also strikes children and in fact
accounted for 4 % of all childhood cancer in Cameroon from 1986 to 1993 (Kasolo
et al. 1997; Wabinga et al. 1993). Likewise, AIDS-related epidemic KS is a very
aggressive, fulminant, and disseminated form of the disease (Schwartz 1996). It
occurs predominantly in young homosexual and bisexual men with AIDS and was
recognized as one of the first signs of the AIDS epidemic (Friedman-Kien 1981;
Gottlieb et al. 1981).
Despite the various epidemiological forms of KS with different clinical courses, all
KS lesions are characterized by spindle-shaped cells of endothelial origin with infil-
tration of inflammatory cells (Niemi and Mustakallio 1965). Analysis of KS lesions
showed that most of these spindle cells are latently infected, with a very low number
of cells undergoing lytic replication at any time (Staskus et al. 1997; Zhong et al.
1996). KS spindle cells are unique as these cells are not fully transformed as with
conventional tumor cells (Aluigi et al. 1996; Benelli et al. 1996). Explanted spindle
cells gradually lose viral episomes in vitro and cannot form tumors in nude mice
Molecular Mimicry by γ-2 Herpesviruses to Modulate Host Cell Signaling Pathways 225

(Aluigi et al. 1996; Ganem 2006; Salahuddin et al. 1988). These spindle cells secrete
many proinflammatory and angiogenic factors, which contribute to the inflammatory
and neovascular characteristics of KS (Salahuddin et al. 1988). The driving force
behind spindle cells has largely been attributed to the expression of a viral latent pro-
tein; FLICE-inhibitory protein (vFLIP); an antiapoptotic, anti-autophagy factor; and a
strong inducer of the nuclear factor kappa-light-chain-enhancer of activated B-cell
(NF-κB) pathway (Grossmann et al. 2006; Lee et al. 2009).
KSHV is also associated with PEL and MCD, rare lymphoproliferative disorders
of B-cell origin usually diagnosed in AIDS patients. Coinfection with EBV is com-
monly found in cases of PEL, an aggressive subtype of non-Hodgkin B-cell lym-
phoma. PEL is characterized by proliferation of cells that lack most B-cell markers
but have features reminiscent of plasmablasts (Jenner et al. 2003). MCD is a rare,
polyclonal lymphocyte hyperplasia in which KSHV-infected cells are found to be
IgM-λ positive B cells located in the mantle zone in the lymph node (Du et al.
2001). In vitro infection of human tonsillar B cells with KSHV revealed that the
virus has a propensity to infect IgM-λ positive B cells and subsequently acquire
characteristics similar to infected cells in MCD (Hassman et al. 2011). It is unclear
why KSHV shows favoritism towards a specific subtype of B cells or how this
specificity is linked to the pathogenesis seen in MCD.
In nature, KSHV infection is restricted to humans. In an experimental setting,
KSHV could infect a variety of primary cells and cell lines from different species
such as human, mouse, and rat (Lagunoff et al. 2002; McAllister and Moses 2007;
Renne et al. 1998). Despite the promiscuous ability of KSHV to infect multiple cell
types, these infected cells are not immortalized or transformed and, without any
selective pressure such as antibiotic selection, tend to lose the viral genome after
several passages (Lagunoff et al. 2002). Recently (2012), Jones et al. successfully
developed a KSHV-induced tumorigenesis in vitro model using primary rat mesen-
chymal cell (Jones et al. 2012).
The in vivo study of KSHV and its malignancies uses mostly nude, transgenic,
or humanized mouse model systems (Dittmer et al. 1999; Wu et al. 2006). KSHV-
positive PEL cells are transplantable into nude mice lacking functional B and T
cells, but the virus does not spread to murine cells (Picchio et al. 1997). The study
of vFLIP using transgenic mice showed increased incidence of lymphomas, and
when this viral protein expression is restricted to B cells, the mice acquired pheno-
types similar to MCD abnormalities (Ballon et al. 2011; Chugh et al. 2005). The
first nonhuman primate model of KSHV infection was established using common
marmosets (Callithrix jacchus) (Chang et al. 2009). Infection with recombinant
KSHV led to persistent infection in these animals, and one marmoset showed devel-
opment of KS-like lesion, which shared similar histopathological characteristics as
human AIDS-associated KS lesions (such as presence of spindle cells with detect-
able expression of KSHV proteins). It would be interesting to determine if the
KS-like lesion progresses similarly in this common marmoset animal model
compared to human KS.
226 L.-Y. Wong et al.

Table 3 KSHV genes and cellular homologues

KSHV ORF Name Function Cellular homologue
ORF4 KCP Complement CR2
K2 vIL-6 Cytokine IL-6
K4 vMIP-II Chemokine CCL3
K4.1 vMIP-III Chemokine CCL3
K6 vMIP-I Chemokine CCL3
K9 vIRF-1 Interferon IRF4 and 8
K10 vIRF-4 Interferon IRF4 and 8
K10.5 vIRF-3 Interferon IRF4 and 8
K11 vIRF-2 Interferon IRF4 and 8
ORF16 vBcl-2 Antiapoptotic and anti-autophagy Bcl2
ORF71 vFLIP Antiapoptotic and anti-autophagy cFLIP
ORF72 v-cyclin Cell cycle Cyclin D2
ORF74 vGPCR Signaling Interleukin-8 receptor
K14 v-OX2 Cytokine OX2

Molecular Piracy in KSHV

Sequencing of the complete KSHV genome revealed that KSHV encodes multiple
ORFs with noticeable homology to cellular genes (Neipel et al. 1997). These pirated
genes are involved in multiple pathways such as antiapoptosis, autocrine/paracrine
signaling, immune responses, and cell cycle regulation. Most of these “pilfered”
genes are also found in other Rhadinoviruses at the equivalent genomic position,
suggesting that the piracy event may have happened early during the virus evolu-
tion. KSHV v-cyclin, a latent gene, is a type D cyclin with 53 % similarity to both
HVS and cellular cyclin (Li et al. 1997). In addition, some of the genes first identi-
fied as unique to KSHV turned out to have cellular equivalents that were discovered
after the characterization of the viral counterparts. Two notable examples are the
KSHV K3 and K5 genes, also known as the modulator of immune recognition
MIR-1 and MIR-2, respectively. K3 and K5 are ubiquitin E3 ligases and potent
immune dysregulators that downregulate a variety of surface immune receptors,
such as MHC class I (Coscoy and Ganem 2000; Ishido et al. 2000). Studies on K3
and K5 led to the identification of a family of cellular ubiquitin E3 ligases called
membrane-associated RING CH domain (MARCH) proteins which share a com-
mon enzymatic motif and domain organization (Goto et al. 2003). A list of pirated
viral genes and their cellular homologues is provided in Table 3.
While the few examples of viral molecular mimicry discussed so far contain
cellular homologues that are conserved not only in function but also in sequence,
the γ-2-herpesviruses also contain unique genes that have no cellular counterparts
but functionally mimic a cellular protein. The first and last ORFs of these viruses
(but the first and second ORFs for HVS) encode transmembrane proteins involved
in modulating lymphocyte activation events and are unique to the respective viruses
(K1 and K15 for KSHV, R1 and R15 for RRV, Tip and STP for HVS) (Fig. 3).
Molecular Mimicry by γ-2 Herpesviruses to Modulate Host Cell Signaling Pathways 227

Fig. 3 Genomic location of the viral transmembrane proteins and viral pre-miRNAs of KSHV,
RRV, and HVS. In HVS strains A and B, STP-A and STP-B are the first ORFs of the genome, but
in HVS subgroup C, Tip is the first ORF followed by STP-C as the second ORF. The pre-miRNAs
are located in the latency-associated region. The U-rich RNAs of HVS are located at the left end
region of the viral genome. Dihydrofolate reductase (DHFR)

The ability of γ-herpesviruses to subordinate the lymphocyte activation pathways

is particularly important to ensure its survival and concealment from the host
immune surveillance since they establish a latent reservoir within circulating lym-
phocytes. These viral proteins control multiple key cellular pathways involved in
cellular transformation and thus are likely to be important contributors to viral-
dependent lymphoproliferation phenotypes seen in infected organisms.
Sequence comparison between these distinct ORFs show low sequence similar-
ity, but they share structural and functional conservation. Some of the proteins even
show a high level of sequence divergence between different strains of the same
virus. It is possible that the proximity of these genes to the terminal repeats, an area
of high homologous recombination activity, contributed to this sequence variation.
Despite not having cellular homologues, these viral proteins possess domains com-
monly found in cellular proteins. It is unclear whether these genes evolved from a
common ancestor that pirated a cellular gene and later acquired unique functions
due to their different tropisms and high mutation frequency, or whether the different
viruses acquired these genes independently during the course of their evolution.

KSHV K1

K1, the first ORF of KSHV, is located at the 5′ end of the KSHV genome after the
terminal repeats (Fig. 2) (Lagunoff and Ganem 1997; Lee et al. 1998b; Russo et al.
1996). K1 is expressed at low levels during latency, and its presence has been
228 L.-Y. Wong et al.

Fig. 4 Structures of the transmembrane signaling proteins of KSHV (K1 and K15), RRV (R1 and
RK15), and HVS (Tip, STP-C and STP-A). The pathways depicted by squares indicate they are
activated by the respective viral proteins, while those in octagons indicate inhibition of signaling.
P-Y denotes phosphorylated tyrosine residues

detected in PEL, MCD, and KS tumors (Lagunoff and Ganem 1997; Lee et al. 2003;
Samaniego et al. 2001). K1 encodes a type-I transmembrane glycoprotein that mim-
ics the signaling activity of the cellular B-cell receptor (BCR) (Fig. 4) (Lee et al.
1998a). Its extracellular domain is highly divergent between different KSHV strains,
yet they all maintain the ability to oligomerize via this region (Lagunoff et al. 1999;
Lee et al. 1998b). On the other hand, the cytoplasmic tails of K1 variants are highly
conserved and contain an immunoreceptor tyrosine-based activation (ITAM) motif,
which is involved in cell activation signals (Cambier 1995; Lee et al. 1998a). The
structure of K1 is similar to the BCR, and indeed K1 expression has been shown to
generate a signaling profile reminiscent of BCR activation. At the same time, K1
deregulates BCR by retaining the newly synthesized μ chain of the BCR complex in
the endoplasmic reticulum (ER) as well as promoting the internalization of surface
BCRs via clathrin-dependent endocytosis (Lee et al. 2000; Tomlinson and Damania
2008). Thus, K1 can be localized to the cell surface, ER, and endosomes (early and
recycling) (Tomlinson and Damania 2008). The presence of K1 in recycling endo-
somes strongly suggests that K1 is recycled after endocytosis; however, direct evi-
dence for this scenario has yet to be presented (Table 4).
Through its ITAM motif, K1 induces calcium mobilization and increases tyro-
sine phosphorylation of cellular proteins leading to the activation of nuclear factor
for activated T cells (NFAT) and NF-κB, events reminiscent of BCR activation
(Lagunoff et al. 1999; Lee et al. 1998a). A prerequisite step for initiation of BCR
signaling is the clustering of multiple BCRs, which only occurs after ligand bind-
ing. In contrast, K1 is constitutively active and does not require extracellular sig-
nals. It has been postulated that this intrinsic activation of K1 is due to its extracellular
domain, which mediates multimerization even in the absence of ligand binding
(Lagunoff et al. 1999). K1 interacts with a variety of cellular signal transduction
Table 4 Summary of the ORFs discussed
KSHV K1 KSHV K15 RRV R1 RRV RK15 HVS STP HVS Tip
Interaction partners Syk, Lyn, Vav, Src, Hck, Lyn, Fyn, Syk Src, TRAFs, Ras Lck, p80, STAT-1 and
PLCγ2, Grab, Yes (STP-C), STAT3 -3
PTK-1 and −2
Signaling motifs ITAM—phosphory- SH2—phosphory- ITAM—phosphory- SH3 only TRAF-binding motif SH3 and CSKH
lated by Src lated by Src lated by Syk (STP-A and (phosphorylated by
kinases, SH3, STP-C), Lck)
TRAF-binding collagen-like
motif repeats (STP-A
and STP-C), SH2
(STP-A and
STP-B)
Ligand dependent No. Multimerization No. Oligomerization
of extracellular of collagen-like
domain repeats
Ca2+ mobilization Increased upon αK1 Inhibited upon BCR Increased upon Decreased
stimulation stimulation stimulation
Cellular pTyr level Increased Decreased Increased Decreased
Activated pathways NFAT, NF-κB, AP-1, NF-κB (SH2 NFAT NF-κB (SH2 NF-κB, NFAT, NF-κB, NFAT
VEGF, PI3K/Akt dependent), dependent), MAPK, AP-1
AP-1 (K15P JNK
only), MAPK
(TRAF and SH2
dependent), JNK
(K15M only),
OncomiRs (only
Molecular Mimicry by γ-2 Herpesviruses to Modulate Host Cell Signaling Pathways

K15M tested)
Downregulate BCR surface levels BCR signaling and Lck activity, TCR and
surface levels CD4 surface levels
(continued)
229
Table 4 (continued)
230

KSHV K1 KSHV K15 RRV R1 RRV RK15 HVS STP HVS Tip
Transformation Yes. Rat-1 fibroblasts Yes. Rat-1 fibroblast STP-C—T cells from
and common and common rabbit, primates,
marmoset T cells marmoset T cells and human—
induces lym- induces lym-
phoma in phoma in rhesus
transgenic mice monkeys. STP-A
–lymphocytes
from common
marmosets
Consequences Cell survival, Angiogenesis, Antiapoptosis, Blocks TCR signaling
inflammatory metastasis IL-2-independent
cytokines, growth
angiogenesis,
blocks BCR
signaling, and
suppressing lytic
replication
L.-Y. Wong et al.
Molecular Mimicry by γ-2 Herpesviruses to Modulate Host Cell Signaling Pathways 231

molecules termed the K1 “signalosome” and characterized in a study by Lee et al.

(Lee et al. 2005). These cellular factors include SH2-binding factors such as spleen
tyrosine kinase (Syk), Lyn, phospholipase C-γ2 (PLCγ2), Vav, Grab2, and protein
tyrosine phosphatases 1 and 2 (Lee et al. 2005). These SH2-binding proteins partici-
pate in a variety of signaling pathways such as tyrosine phosphorylation, intracel-
lular calcium fluxes, and transcription factor activation, ultimately leading to the
upregulation of inflammatory cytokine and angiogenic factor genes (Lee et al.
2005). KSHV-infected cells are highly dependent on autocrine and paracrine signal-
ing, so a cytokine-rich environment is important for KSHV-induced oncogenesis
(Ensoli et al. 1989, 2000). K1 expression results in the production of inflammatory
cytokines and angiogenic factors such as vascular endothelial growth factor (VEGF),
highlighting the central role of K1 in promoting the paracrine model of KSHV
tumorigenicity (Lee et al. 2005; Prakash et al. 2002, 2005; Wang et al. 2004). In
addition, K1 also protects infected cells from apoptosis via multiple mechanisms
such as blocking the binding of Fas ligand to the Fas receptor and activation of the
phosphoinositide 3-kinase (PI3K) and AKT pathways (Berkova et al. 2009;
Tomlinson and Damania 2004). The ability of K1 to modulate multiple cellular
signal transduction pathways underlies its oncogenic potential, as shown by the
ability of rodent fibroblast cells expressing K1 to undergo transformation and foci
formation (Lee et al. 1998b).
Furthermore, when the saimiri transforming protein (STP) oncogene of HVS is
replaced by K1, this recombinant virus retains the ability to immortalize common
marmoset (Callithrix jacchus) T cells in vitro independent of exogenous cytokines
and induced lymphoma in infected common marmosets, albeit at a delayed time
point compared to HVS wild-type infected animals (Lee et al. 1998b). Results from
K1 transgenic mice highlight the transforming potential of K1 in vivo as these mice
develop spindle cell sarcoma and plasmablastic lymphoma after a year (Prakash
et al. 2002). The authors showed that K1 constitutively activated the NF-κB and
VEGF pathways, and, indeed, serums levels of VEGF were three times higher in K1
transgenic mice compared to control mice (Prakash et al. 2002). Thus, it is likely
that K1 is an important contributor to the angiogenic characteristic seen in KS
tumors, where most of the cells are latently infected.
Given the ability of K1 to activate so many different pathways, it is interesting to
consider its affect on viral lytic replication. Lagunoff et al. showed that inhibition of
K1 by expressing a dominant-negative mutant (with tyrosine to phenylalanine sub-
stitutions in the ITAM) actually reduces the efficiency of viral lytic replication
(Lagunoff et al. 2001).
Their data suggests that K1 contributes to efficient lytic replication (Lagunoff
et al. 2001). Since the ITAM is essential for the signaling role of K1, it is hard to tell
if the signaling activity of K1 is intimately linked to its role in enhancing viral lytic
replication.
232 L.-Y. Wong et al.

KSHV K15

K15 is another gene unique to KSHV and is found at the 3′-end of the KSHV
genome, adjacent to the terminal repeats (Fig. 3). This gene contains eight exons
and multiple splicing variants with the dominant spliced form encompassing all the
exons to express a protein with 12 transmembrane domains followed by a short
cytoplasmic tail (Fig. 4) (Brinkmann et al. 2003; Choi et al. 2000b; Glenn et al.
1999). While K15 is widely considered to be a lytic gene, low levels of expression
can be detected in KSHV-positive PEL cell lines explanted from patients (Sharp
et al. 2002; Wong and Damania 2006). It is uncertain if this low level of K15 expres-
sion is due to lytic reactivation in a minority of infected cells (1–5 %) or if the
latently infected cells periodically express a low amount of K15. Two variants of
K15 exist: the predominant (P) form and the minor (M) form, termed K15P and
K15M, respectively (Glenn et al. 1999; Poole et al. 1999). These two variants show
low sequence identity at the protein level, but are almost structurally (splicing pat-
tern and protein domains) and functionally identical (Glenn et al. 1999; Poole et al.
1999). The cytoplasmic tails of the K15 variants are highly conserved, suggesting
that loss of function in this domain is deleterious to the virus. So far, most studies
have been done with the K15P form.

K15P

Expression of K15P interferes with BCR signaling but induces the activation of
NF-κB, activator protein-1 (AP-1), and mitogen-activated protein kinase (MAPK)
pathways, which ultimately leads to expression of genes involved in cell survival,
proliferation, and inflammation. Specifically, K15P activates the extracellular
signal-regulated kinases (ERK), c-Jun amino-terminal kinases (JNK), but not the
p38 cascade of the MAPK superfamily (Brinkmann et al. 2003, 2007; Cho et al.
2008; Pietrek et al. 2010, Tsai et al. 2009; Wang et al. 2009). The cytoplasmic
domain of K15 contains multiple motifs important in cellular signaling cascades,
including a tumor necrosis factor (TNF), receptor-associated factors (TRAF)-
interacting site, a Src homology 2 (SH2) domain, and a proline-rich SH3 motif
(Choi et al. 2000b). These domains mediate interactions with a variety of cellular
signaling molecules to modulate the pathways mentioned above. The SH2 and SH3
motifs of K15 interact with multiple Src kinases such as Src, Hck, Lck, Fyn, and
Yes, with the Y481 in the SH2 motif being constitutively phosphorylated by tyro-
sine kinases (Brinkmann et al. 2003). This phosphorylation is required for activa-
tion of NF-κB and MAPK (Brinkmann et al. 2003).
The K15 cytoplasmic tail is sufficient to downregulate BCR signaling, as shown
in a study using a chimeric protein composed of the K15 cytoplasmic portion fused
to the CD8α transmembrane domain and expressed in a B-cell line (Choi et al.
2000b). The inhibition of the calcium cascade upon BCR stimulation requires intact
SH2 and SH3 motifs of K15 (Choi et al. 2000b). It is hypothesized that the
Molecular Mimicry by γ-2 Herpesviruses to Modulate Host Cell Signaling Pathways 233

recruitment of Lyn by K15 sequesters this major B-cell kinase from BCR signaling
events (Cho et al. 2008). Indeed, mutating the SH3 motif of K15 interferes with its
ability to deregulate BCR signaling (Cho et al. 2008). Furthermore, K15 increases
the rate of BCR internalization from the cell surface; thus, it is likely that K15 uses
multiple mechanisms to inhibit BCR activation (Lim et al. 2007).

K15P Versus K15M

Despite the conservation of signaling motifs in the cytoplasmic tails of the K15P
and K15M variants, there are phenotypical differences between these two variants.
For one, even though both are predominantly cytoplasmic, the P variant contains a
mitochondria localization sequence and can thus localize to the host mitochondria
and other organelles such as ER and Golgi, while K15M is found on lysosomal
membranes (Choi et al. 2000b; Sharp et al. 2002; Tsai et al. 2009; Wang et al. 2007).
Although both K15 variants activate NF-κB to the same degree, only the P form can
positively regulate AP-1 activity (Tsai et al. 2009). Whether this distinct effect is
due to the difference in localization remains to be tested.
The importance of the SH2 motif in K15 to initiate the expression of cellular
cytokines involved in angiogenesis and inflammation (e.g., IL-8, IL-6, CXCL2) also
varies slightly between the two K15 variants. While mutation of the amino acids
tyrosine-glutamic acid-glutamic acid-valine at position 481–484 (Y481EEV) of the
K15P SH2 domain abolishes this phenotype, the K15M Y490EEV mutant retained
residual activity in cytokine upregulation, which may be due to the ability of K15M
to activate the JNK pathway independently of its SH2 motif (Pietrek et al. 2010;
Wang et al. 2007). Furthermore, K15M can upregulate miR-21 and miR-23, cellular
cancer-related miRNAs (oncomiRs) that are involved in tumor cell proliferation,
metastasis, and angiogenesis (Tsai et al. 2009). This ability is dependent on the
tyrosine residue in the SH2 domain. It is unclear whether K15P, which also has an
SH2 motif, can upregulate the same oncomiRs.
Despite the higher prevalence of the K15P isoform in KSHV isolates, the two
variants seem to function equivalently in most of the in vitro assays used to study
them (Pietrek et al. 2010; Wang et al. 2007). Whether the P variant has an in vivo
survival advantage over the M isolates (due to activation of AP-1 pathway) remains
to be tested.

Rhesus Rhadinovirus

RRV was first discovered in rhesus monkeys (Macaca mulatta) housed at the New
England Primate Research Center (NEPRC) (Desrosiers et al. 1997). The animals
reacted to HVS antigens in serology tests, hinting that they were infected by an
agent related to γ-herpesviruses. A further survey after the characterization of RRV
revealed that at least 90 % of rhesus monkeys housed at both the NEPRC and
234 L.-Y. Wong et al.

Oregon Regional Primate Research Center (ORPRC) were seropositive for RRV
(Mansfield et al. 1999; Wong et al. 1999). This uniformly high prevalence of infec-
tion at both centers suggested that RRV is endemic in rhesus monkeys. Sequencing
of its viral genome revealed that RRV is a close cousin of KSHV with high sequence
conservation. Indeed, both these Old World Rhadinoviruses share homologous
genes that are not found in New World Rhadinoviruses like HVS (e.g., viral inter-
leukin 6 (vIL-6)) (Alexander et al. 2000; Searles et al. 1999). RRV has been detected
in B cells of rhesus macaques, suggesting that, similar to KSHV, lymphocytes are
the major reservoir of viral persistence (Bergquam et al. 1999; Desrosiers et al.
1997; Wong et al. 1999). Unlike KSHV, which primarily enters latency upon de
novo infection, RRV can undergo lytic replication and produce high viral titer in
primary rhesus fibroblasts (Desrosiers et al. 1997).
To recapitulate the pathogenesis of KSHV often seen in HIV-coinfected patients,
two groups from NEPRC and ORPRC have infected rhesus monkeys with both
RRV and simian immunodeficiency viruses (SIVmac239, a virus used to model
HIV-like infection in rhesus monkeys) (Mansfield et al. 1999; Wong et al. 1999).
One group reported that transient lymphadenopathy followed by persistent infection
has been detected in the rhesus macaques infected with RRV alone (Mansfield et al.
1999). At 3 months postinfection, these pathologies disappeared as the immune
systems of the rhesus macaques were able to dampen the pathogenesis of the virus
(Mansfield et al. 1999). However, when the animals were coinfected with both RRV
and SIVmac239, they showed weaker antibody response to both viruses and had
shorter survival times compared to monkeys infected with only one of the agents.
The other group found that coinfected animals displayed symptoms of lymphopro-
liferative disorder such as splenomegaly and hypergammaglobulinemia in coin-
fected animals, symptoms reminiscent of MCD induced by KSHV in humans
(Wong et al. 1999). These results demonstrate the utility of RRV/SIVmac239 coin-
fection in monkeys as a model to recapitulate the KSHV-related lymphoprolifera-
tive disorders in HIV-positive patients.

RRV R1

Like K1, the R1 gene encodes a transmembrane protein with an extracellular immu-
noglobulin domain. However, the cytoplasmic tail of R1 contains multiple potential
ITAM motifs and is considerably longer than K1 (171 and 38 amino acids for R1
and K1, respectively) (Fig. 4) (Damania et al. 1999, 2000). Nonetheless, R1 is able
to transduce a signaling profile similar to that of K1-activated B cells (Damania
et al. 2000). R1 specifically interacts with the tyrosine kinase Syk and is itself a
substrate of Syk (Damania et al. 2000). Fusion of the cytoplasmic tail of R1 to the
extracellular and transmembrane domains of CD8 results in increased cellular tyro-
sine phosphorylation, intracellular calcium influx, and NFAT activation upon stimu-
lation with an αCD8 antibody, events that are reminiscent of B-cell activation
(Damania et al. 2000). In fact, expression of R1 is sufficient to constitutively
Molecular Mimicry by γ-2 Herpesviruses to Modulate Host Cell Signaling Pathways 235

activate NFAT, a key family of transcription factors important for lymphocyte acti-
vation, without any stimulation (Damania et al. 2000). Thus R1 is a close cousin of
K1 with respect to its ability to control B-cell signaling independently of BCR
(Fig. 4). Interestingly, the duration of calcium signaling was longer and stronger
with R1 than K1 upon stimulation with αCD8 antibody (Damania et al. 2000).
Whether this difference in activity strength is due to the longer cytoplasmic tail with
more potential ITAM motifs is unknown.
Likewise, R1 displays oncogenic traits similar to K1 (Damania et al. 1999).
When expressed in Rat-1 cells, R1 can induce foci formation in vitro and tumors
in vivo when inoculated into nude mice (Damania et al. 1999). Furthermore, recom-
binant HVS with R1, expressed in place of STP, could still immortalize T cells from
common marmosets, highlighting the ability of R1 to activate cellular pathways
leading to cell growth and survival, even in the absence of exogenous signals
(Damania et al. 1999). The authors did not mention if there were any differences in
the transforming abilities of HVS/K1 or HVS/R1 versus wild-type HVS (Damania
et al. 1999).

RRV RK15

The RK15 gene is situated at the same genomic location as KSHV K15 and is the
homologue of K15 in RRV (Fig. 3) (Alexander et al. 2000). It is termed RK15 as not
to cause confusion with an RRV gene already named R15 of strain 17577 that is the
RRV homologue of cellular OX2 and KSHV ORF74 (Pratt et al. 2005; Wang et al.
2009). Similar to K15, RK15 displays a complex splicing pattern with the most
common transcript encoding all the 12 transmembranes (Wang et al. 2009). While
RK15 does not have the SH2 YEEV motif that is important for the signaling activi-
ties of K15, it retains the SH3 motif, albeit at a different location (K15P–P387PLP,
K15M–P396PLP versus RK15–P492PLP) (Brinkmann et al. 2003; Wang et al. 2007,
2009). Nonetheless, RK15 can still activate the NF-κB and JNK pathways to a simi-
lar degree as K15, but only weakly activates the ERK cascade compared to K15.
This suggests that RK15 utilizes an SH2-independent mechanism to target NF-κB
and JNK activity. Furthermore this also implies that RK15 require an SH2 domain
to robustly activate the ERK pathway. A study by Wang et al. using microarray
analysis to compare the spectrum of cellular genes regulated by K15 and RK15
found that there was little overlap between the cellular genes regulated by these two
viral proteins (Wang et al. 2009). While both induced the expression of proinflam-
matory cytokines and chemokines such as IL-8 and IL-6, the extent of induction
was more robust in the presence of K15 compared to RK15. On the other hand,
RK15 preferentially upregulates cellular factors such as fibroblast growth factor 21
(FGF21), Kruppel-like factor 15 (KLF15), and complement component 5a receptor
1 (C5aR1), genes whose expression is unchanged in the presence of K15 (Wang
et al. 2009). This differential display of gene induction activities between K15 and
RK15 could be due to the inability of RK15 to activate the ERK pathway, leading to
236 L.-Y. Wong et al.

weaker induction of proinflammatory signaling pathways by RK15. One possible

reason why K15 preferentially activates a wider range of cytokines and chemokines
genes compared to RK15 could be due to the proinflammatory signature of KS, and
so far, no KS-like symptoms have been seen in RRV and SIV infected macaques
(Mansfield et al. 1999; Wong et al. 1999).

Herpesvirus Saimiri

HVS is the prototype of γ-2-herpesviruses and belongs to the New World

Rhadinovirus group (Roizmann et al. 1992). This virus is nonpathogenic in its natu-
ral host, the squirrel monkey (Saimiri sciureus), but infection of other New World
primates such as common marmosets (Callithrix jacchus) results in fatal T-cell lym-
phoma and leukemia (Fleckenstein and Ensser 2007; Melendez et al. 1968, 1969).
In addition, certain strains of HVS are capable of immortalizing T cells from
humans, rhesus monkeys, common marmosets, and rabbits to interleukin-2 (IL-2)-
independent growth (Ablashi et al. 1985; Biesinger et al. 1992; Daniel et al. 1975a;
Duboise et al. 1998a). IL-2 is required for T-cell growth and function; thus, the abil-
ity of certain strains of HVS to induce IL-2-independent transformation demon-
strates its potent oncogenicity and gives these infected cells a growth advantage
when the cytokine supply is limited.
The genome organization of HVS is similar to other Rhadinoviruses, but HVS
lacks certain genes that are shared between the Old World Rhadinoviruses (such as
vIRFS encoded by KSHV and RRV). Three strains of HVS named A, B, and C have
been discovered so far, each of which display different oncogenic potentials
(Biesinger et al. 1990; Daniel et al. 1975b; Desrosiers and Falk 1982; Falk et al.
1972; Medveczky et al. 1984). Subgroup C is the most potent; it is able to immortal-
ize T cells from humans, rabbits, and primates in vitro and causes lymphoma in
rhesus monkeys upon inoculation (Duboise et al. 1998a). Strain A is able to induce
cytokine-independent transformation of lymphocytes from common marmosets.
Finally, subgroup B is the least oncogenic. The oncogenic dissimilarities between
these HVS strains are due to the 5′ ORFs at the location corresponding to K1 and
R1 of KSHV and RRV, respectively (Fig. 3) (Desrosiers et al. 1985; Koomey et al.
1984; Murthy et al. 1989). Similar to low sequence conservation of K1 and R1 in
different strains, these HVS ORFs also have low homology between the different
HVS strains (Fig. 4).

Saimiri Transforming Protein

In HVS subgroups A and B, the first ORF encodes a protein named STP A and B
(STP-A and STP-B), respectively (Fig. 3) (Murthy et al. 1989). In HVS subgroup C
(strain C488), however, STP-C is the second ORF from the left end of the genome,
Molecular Mimicry by γ-2 Herpesviruses to Modulate Host Cell Signaling Pathways 237

as the first ORF encodes a protein called tyrosine-interacting protein (Tip) (Fig. 3)
(Duboise et al. 1998a). STP-C and Tip are translated from a bicistronic mRNA;
thus, both genes are very likely to be expressed at the same time (Biesinger et al.
1995). The STPs and Tip are not required for viral replication but are essential for
the transformation of lymphocytes in vitro and the induction of lymphoma in vivo.
Expression of STP-A or STP-C alone in rat fibroblasts results in transformation of
the cells and tumor formation following inoculation in nude mice (Jung et al. 1991).
Even though the STPs are structurally similar, they do not share high sequence
homology, with STP-B being 28 % and 22 % identical to STP-A and STP-C, respec-
tively. STP-A and STP-C both have collagen-like repeats (glycine-X-Y with X or Y
being proline or glutamine) after a highly acidic amino terminus, while STP-B lacks
these repeats. STP-C has 18 of these repeats arranged in tandem, while STP-A only
has 9 repeats that are scattered throughout the protein. The importance of these
collagen-like repeats for STP oligomerization and ultimately the oncogenicity of
HVS has been shown in several studies. Indeed, a chimeric version of STP-B con-
taining the 18 copies of repeats from STP-C is now able to transform cells (Choi
et al. 2000a). Oligomerization of STP-C through its collagen repeats may mimic a
ligand-independent, constitutively active receptor, which then activates the NF-κB
pathway to induce expression of genes important for survival and transformation.
Not surprisingly, mutating the repeats in STP-C abolishes its oncogenic activity
(Choi et al. 2000a).
STP-C was the first reported virus-encoded protein to interact with cellular rat
sarcoma (Ras), a molecular switch for MAPK cascade important for oncogenesis
and cell growth (Jung and Desrosiers 1995). Expression of STP-C activates the Ras
signaling cascade, and disrupting the STP-C-Ras interaction interferes with the
transformation of cells by this viral oncogene (Fig. 4). In fact, a recombinant virus
that encodes Ras in lieu of STP-C can still transform lymphocytes, albeit with lower
efficiency compared to the wild-type virus (Jung and Desrosiers 1995). Thus, Ras is
an important player in the transformation of lymphocytes by STP-C.
STPs also contain other motifs involved in cellular signaling cascades, such as a
TRAF-interacting domain and an SH2 motif (in STP-A and STP-B only). TRAFs
are a family of cellular proteins involved in relaying signaling from initiation by the
external stimuli to downstream molecules and thus regulate diverse functions. The
TRAF-binding site of STP-C is required for its interaction with TRAF-1, TRAF-2,
and TRAF-3 and subsequent activation of NF-κB (Lee et al. 1999). Mutation of the
TRAF-binding motif abolishes the ability of STP-C to immortalize primary human
T cells in vitro. However, this STP-C TRAF-binding mutant protein can still trans-
form lymphocytes of common marmoset in vivo and in vitro, suggesting that while
host TRAFs are important for the oncogenicity of HVS STP-C, this multifunctional
protein can utilize other mechanisms to achieve the same result (Lee et al. 1999).
Residue Y115 in the SH2 motif of STP-A is responsible for binding to Src and is
itself a substrate for this tyrosine kinase (Chung et al. 2004; Garcia et al. 2007). This
interaction leads to strong activation of the transcription factors, AP-1 and NFAT,
while the binding of STP-A to TRAF-6 elicits a vigorous NF-kB response. The acti-
vation of all three pathways contributes to IL-2 promoter activity (Garcia et al. 2007).
238 L.-Y. Wong et al.

Another binding partner of STP-A is signal transducer and activator of transcription

3 (STAT3), and a triple complex is formed with STAT3, STP-A, and Src. Even though
the interaction between STP-A and STAT3 is independent of its interaction with Src,
the close proximity of these molecules results in phosphorylation and activation of
STAT3 by Src (Chung et al. 2004). STP-A-induced STAT3-mediated signal transduc-
tion leads to upregulation of proteins involved in antiapoptosis and cell cycle such as
Bcl-XL and cyclin D1, ultimately allowing STP-A-expressing cells to survive and
proliferate in the absence of serum (Chung et al. 2004). Together, these studies
suggest that STP activates these key cellular pathways to prevent apoptosis and
contribute to IL-2 -independent growth.

Tyrosine-Interacting Protein

Tip is encoded by the first ORF of HVS subgroup C (Fig. 3) (Biesinger et al. 1995).
Like all the viral proteins discussed so far, Tip also contains multiple motifs for
interacting with cellular proteins. First and foremost is the interaction of Tip with
Lck, the major Src tyrosine kinase in T cells (Biesinger et al. 1995). This binding is
mediated by the SH3 motif and the C-terminal Src-related kinase homology (CSKH)
domain of Tip (Jung et al. 1995a). The interaction between Tip and Lck results in
the phosphorylation of Tip by the kinase as well as deregulation of the downstream
T-cell receptor (TCR) signaling cascade, likely by sequestering Lck away from the
TCR (Cho et al. 2004).
In addition, Tip expression also decreases the cell surface level of TCR and CD4
molecules, which is dependent on the localization of Tip to the membrane rafts via
its transmembrane and an amphipathic helix motif just preceding the transmem-
brane domain (Cho et al. 2006; Min et al. 2008). Tip also interacts with a cellular
endosomal protein, p80, through its serine-rich (SR) domain (Park et al. 2002). This
interaction in turn mediates formation of large vesicles that help traffic the internal-
ized TCR and CD4 molecules to the lysosome for degradation (Park et al. 2003).
The functional consequences of Tip binding to Lck are controversial. In
certain contexts, expression of Tip suppresses the activation of Lck. For exam-
ple, expression of Tip in the human Jurkat T-cell line results in lowered levels of
tyrosine phosphorylation, while in fibroblasts Tip is able to inhibit the activity of
an oncogenic mutant of Lck (F505) (Jung et al. 1995b). On the other hand, infec-
tion of human peripheral blood T lymphocytes (PBL) with a Tip-deleted mutant
virus results in a significant decrease in Lck activation compared to wild-type
virus, strongly suggesting that the presence of Tip increases the activity of Lck
(Lund et al. 1997). The enzymatic activity of Lck is also enhanced by Tip in an
in vitro assay (Kjellen et al. 2002; Lund et al. 1997; Wiese et al. 1996). Duboise
et al. mutated the Lck-binding SH3 motif (proline to alanine mutations) of Tip
in the context of the virus and surprisingly found that this mutant virus can still
immortalize common marmoset lymphocytes in vivo and in vitro (Duboise et al.
1998b). This data suggests that deregulation of Lck by Tip is not required for its
Molecular Mimicry by γ-2 Herpesviruses to Modulate Host Cell Signaling Pathways 239

ability to immortalize. Interestingly, this mutant virus was even more pathogenic
compared to the wild-type virus, with increased lymphoid infiltration upon nec-
ropsy of the infected animals (Duboise et al. 1998b). However, it was reported
that mutating just one of the dual Lck-interacting sites in Tip does not com-
pletely abrogate the interaction between Tip and Lck (Heck et al. 2006). In sum-
mary, the oncogenic ability of HVS and the modulation of Tip on Lck are
complex and multifaceted.
Another function of Tip is its activation of several transcription factors including
NF-κB, NFAT, STAT1, and STAT3 (Merlo and Tsygankov 2001). The modulation
of STATs by Tip is Lck dependent, but STAT activation is dispensable for the trans-
formative function of Tip (Heck et al. 2005).

Summary of Viral Signaling Molecules

The studies on these six ORFs from KSHV, RRV, and HVS suggest conservation of
functions despite the lack of sequence homology and divergence of cell tropism. K1
and R1 both activate pathways that are also induced by BCR activation and both
have transforming potentials. In addition, the abilities of K1 and R1 to replace STP
in recombinant HVS and still immortalize T cells from common marmosets suggest
that these three viral proteins have a conserved capability to activate the same path-
ways leading to cell survival and transformation (Damania et al. 1999; Lee et al.
1998b). Comparison of K15 and RK15 revealed a weaker propensity for RK15 to
activate inflammatory pathways, perhaps reflecting the more cytokine-dependent
nature of KSHV pathogenesis as compared with RRV. Nonetheless, all of these viral
proteins activate cellular pathways important for cell survival. The in vivo contribu-
tion of K1, K15, R1, and RK15 to virus-induced tumorigenesis is still unknown and
hopefully will be elucidated in the near future.

γ-2-Herpesvirus miRNAs

Another mechanism used by herpesviruses to manipulate host cell signaling path-

ways is through the regulation of gene expression by virus-encoded miRNAs. miR-
NAs are noncoding RNAs 19–24 nucleotides in length that regulate gene expression
by binding to the 3′ untranslated region (UTR) of the target mRNAs to either repress
protein translation or induce mRNA degradation. The sequence of the 5′ nucleo-
tides from positions 2–7 of the miRNA determines the target and is termed the seed
sequence. Perfect complementation between the miRNA and the target mRNA
leads to proteolytic cleavage of the target, while imperfect complementation results
in repression of protein translation. The biogenesis pathway of miRNAs will not be
covered here, but more in depth reading on these regulatory RNAs can be found in
these reviews (Garzon et al. 2009; Krol et al. 2010; Winter et al. 2009).
240 L.-Y. Wong et al.

Herpesviruses, in general, prove to be abundantly rich in miRNAs (reviewed in

(Boss et al. 2009)). miRNAs are ideal tools for viruses to modulate gene expression
as they are non-immunogenic, economical (miRNAs require less coding capacity
compared to ORFs), capable of acting on multiple targets and can undergo rapid
evolution to target new transcripts (Skalsky and Cullen 2010). Furthermore, viruses
can utilize the highly conserved cellular pathway for miRNA biogenesis without
having to encode any specific viral factors dedicated for miRNA processing.

Herpesvirus miRNAs are Expressed During Latency

KSHV encodes 12 pre-miRNAs clustered between ORF K12 and ORF71, an area
termed the latency-associated region (Fig. 3) (Cai et al. 2005; Grundhoff et al. 2006;
Pfeffer et al. 2005; Samols et al. 2005). From these 12 pre-miRNAs, 17–25 mature
miRNAs can be predicted to form following maturation (Abend et al. 2010; Lin
et al. 2010, 2011). As these miRNAs are located within the major latency-associated
region of the viral genome, it is not surprising that these miRNAs are highly enriched
in latent PEL cells (Cai et al. 2005; Samols et al. 2005). With the exception of miR-
K10 and miR-K12, all other miRNAs levels remain constant during lytic replica-
tion, indicating that KSHV miRNAs primarily function during viral latency (Cai
et al. 2005; Grundhoff et al. 2006).
KSHV miRNAs have been shown to target both viral and cellular genes, exerting
control over both the viral life cycle and host immune responses. miR-K12-11 is an
ortholog of cellular miR-155, an important regulator of lymphocyte differentiation
and innate immunity (Faraoni et al. 2009; Gottwein et al. 2007; Skalsky et al. 2007).
miR-155 is a potent antiapoptotic miRNA when expressed in breast cancer cells and
has been implicated in tumorigenesis of lymphoid and myeloid cancers (Ovcharenko
et al. 2007). PEL cells lack the expression of miR-155, but express high levels of
miR-K12-11 (Skalsky et al. 2007). Since it has an identical seed sequence and regu-
lates the same set of genes as miR-155, miR-K12-11 may contribute to the KSHV-
induced oncogenesis (Gottwein et al. 2007). A recent study (2011) showed that
miR-K12-11 is indeed the viral mimic of the cellular miR-155 (Boss et al. 2011).
Ectopic expression of either miRNAs in humanized NSG (NOD-scid interleukin-2
receptor γ-chain null (IL2Rγnull)) mouse model led to hyperproliferation of human
B cells in the spleen. This phenotype could be traced to the dysregulation of CCAAT
enhancer-binding protein β (C/EBPβ), a regulator of IL-6 transcription, by these
miRNAs (Boss et al. 2011). Since IL-6 is a key driver of inflammation and prolifera-
tion of lymphocytes, activation of IL-6 by miR-K12-11 is likely to be an important
aspect of KSHV pathogenesis.
The example of miR-K1, which targets multiple signaling pathways, highlights
the versatility of miRNA-mediated regulation of gene expression. First, miR-K1 has
been shown to be an important regulator of latency by positively regulating the
NF-κB pathway through reducing the level of the inhibitor IκBα to maintain the
viral latency state (Lei et al. 2010). Furthermore, by manipulating this key pathway,
Molecular Mimicry by γ-2 Herpesviruses to Modulate Host Cell Signaling Pathways 241

KSHV can suppress host immunity and enhance cell survival. Second, miR-K1
specifically prevents the expression of p21, an important regulator of cell cycle
(Gottwein and Cullen 2010).
Lytic replication is critical for gammaherpesviruses to maintain a disseminated
latent infection in vivo but has unwanted consequences such as immune activation
and cell death to release infectious virion. Thus, tight control of lytic reactivation
may allow gammaherpesviruses to maintain persistent infection in the presence of
host immune surveillance. Rta, encoded by ORF50, is the master transcriptional
regulator of lytic replication in KSHV and is directly targeted by at least two viral
miRNAs (miR-K9, miR-K12-7) (Bellare and Ganem 2009; Lin et al. 2011; Lu et al.
2010b). As a consequence of miR-K3 targeting the nuclear factor I/B (NFIB), a
transcriptional activator for the Rta promoter, miR-K3 also has a role in repressing
expression of Rta and thus contributes to the maintenance of latency (Lu et al.
2010a). The number of viral miRNAs devoted to targeting Rta, either directly or
indirectly through inhibition of other mRNAs, suggests that KSHV tightly main-
tains latency to prevent inappropriate induction of lytic replication.
Sequencing of KSHV miRNAs from PEL cell lines and tissue samples from
patients with different KSHV-induced diseases revealed high conservation among
most of the viral miRNAs, suggesting the importance of miRNAs in KSHV biology
(Marshall et al. 2007). Interestingly, a few miRNAs (e.g., miR-K12) showed poly-
morphisms between the different isolates (Marshall et al. 2007). Since it has been
shown that a single nucleotide polymorphism (SNP) in a viral miRNA could affect
its maturation and function, it is tempting to speculate that these “minor” changes in
viral miRNA sequences may impact the virus biology in a significant manner
(Gottwein et al. 2006). These viral miRNA SNPs could reflect a relatively rapid
adaptation strategy by the virus to outwit the host response.
RRV also encodes 15 miRNAs processed from 7 pre-miRNAs located in the
same genomic region as the KSHV miRNAs (Fig. 3) (Schafer et al. 2007; Umbach
et al. 2010). Umbach et al. (2010) used a deep sequencing assay to analyze the
expression of viral miRNAs in RRV-induced tumors. They showed that all but one
RRV miRNA lack sequence homology with KSHV miRNAs. The targets and func-
tions of RRV miRNAs have yet to be characterized. The observation that the miR-
NAs of KSHV and RRV are located in the same viral genomic region but do not
share sequence homology suggests that these viruses may have adapted the use of
miRNA before evolutionary diverging, but have since altered the seed sequence to
target their respective host.
Analysis of the HVS genome failed to predict with sufficient certainty the pres-
ence of miRNAs (Walz et al. 2010). Several potential miRNA candidates are pre-
dicted to be encoded at the location where miRNAs are found in the KSHV and
RRV genomes, but have yet to be verified (Walz et al. 2010). HVS, however, does
encode seven viral U-rich RNAs called HSURs in its 3′ terminal L-DNA region
(Fig. 3) (Biesinger et al. 1990; Lee et al. 1988). These noncoding RNAs are not
required for viral replication in cell culture (Ensser et al. 1999). Steiz and colleagues
found that HSUR-1 and HSUR-2 bind to cellular miR-27, miR-142-3p, and miR-
16, leading to degradation of miR-27 while not affecting the other two miRNAs
242 L.-Y. Wong et al.

(Cazalla et al. 2010). The functional consequences of miR-27 downregulation and

miR-142-3p and miR-16 binding by HVS is an exciting area that warrants further
investigation.
As the examples described above illustrate, there is little conservation in the
sequence and function among the known miRNAs expressed by these γ-herpesviruses.
However, as this area is still under intense investigation, there may be more targets
that are yet to be discovered. Due to the flexibility of the miRNA seed sequence,
each miRNA may target many mRNAs, enabling these viruses to target the same
pathways either by using different seed sequences to bind to a different target site of
the same mRNA or by targeting different mRNAs that encode components of the
same pathway. That miR-K11 and miR-155 have the same seed sequence suggests
that KSHV may have pirated cellular miRNA for its own advantage.

Conclusion

In summary, herpesviruses are remarkably well adapted to their respective hosts as

even the oncogenic γ-2-herpesviruses rarely cause fatalities unless the host is immu-
nocompromised or contribution from other factors (e.g., genetic or environment).
As life-long “passengers” in their infected hosts, herpesviruses must strike a deli-
cate balance with the host immune response. To achieve this, γ-2-herpesviruses
devote multiple resources to manipulate the key signaling pathways described here,
ultimately promoting the survival of virus-infected cell, immune evasion, and
tumorigenesis. The molecular piracy strategy used by these viruses has been remark-
ably successful for their adaptation to specific niches. The abilities of the viral ter-
minal transmembrane proteins (K1 and K15 of KSHV, R1 and RK 15 of RRV, STP
and Tip of HVS) and miRNAs to manipulate host cell signaling pathways and drive
viral replication at appropriate times very likely contribute to the pathogenesis of
these viruses. Moreover, the high frequency of molecular piracy by γ-herpesviruses
allows researchers to study these viral mimics and gain insights into the mecha-
nisms viruses use to divert cellular pathways, even leading to the discovery of new
classes of cellular proteins.

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Neotropical Primates and Their Susceptibility
to Toxoplasma gondii: New Insights for an Old
Problem

José Luiz Catão-Dias, Sabrina Epiphanio,

and Maria Cecília Martins Kierulff

Introduction

Toxoplasmosis is a zoonosis caused by Toxoplasma gondii, an obligate intracellular

coccidium belonging to the Phylum Apicomplexa. This coccidium is considered one
of the most competent known parasites, and it is believed that it can virtually parasit-
ize all species of birds and mammals. In most immunocompetent hosts, infection with
T. gondii is subclinical or causes discrete clinical and pathological alterations.
However, in different groups of mammals, including strepsirrhines from Madagascar,
marsupials from Oceania, hares from Europe, and, in particular, neotropical nonhu-
man primates, toxoplasmosis can cause disease processes associated with high mor-
tality rates. In our experience, it is possibly the most important cause of acute infectious
disease death affecting Platyrrhini in captivity in Brazil. The reasons for the high
susceptibility of New World Primates (NWPs) to toxoplasmosis are not fully under-
stood, but it is assumed that platyrrhines have evolved for over 20 million years with-
out the presence of felines and, thus, these did not develop an efficient immune
response to the parasite. In addition, the arboreal habits of platyrrhines would have
contributed to minimize the contact of NWPs with the protozoan after its arrival to the
American continent during the Pleistocene, about one to three million years ago.

J.L. Catão-Dias (*)

Laboratório de Patologia Comparada de Animais Selvagens,
Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia,
Universidade de São Paulo, São Paulo, Brazil
e-mail: [email protected]
S. Epiphanio
Departamento de Ciências Biológicas, Universidade Federal de São Paulo,
Diadema, São Paulo, Brazil
M.C.M. Kierulff
Departamento de Ciências Biológicas e Agrárias, Centro Universitário Norte do Espírito
Santo, Universidade Federal do Espírito Santo, São Mateus, São Paulo, Brazil

J.F. Brinkworth and K. Pechenkina (eds.), Primates, Pathogens, and Evolution, 253
Developments in Primatology: Progress and Prospects,
DOI 10.1007/978-1-4614-7181-3_9, © Springer Science+Business Media New York 2013
254 J.L. Catão-Dias et al.

On the other hand, available clinical, serological, pathological, and experimental

data suggest that the response of NWPs to toxoplasmosis is not homogeneous.
There is a clear difference between the genus Cebus, which apparently reacts
mildly to the disease, and most other groups, for which toxoplasmosis can be
devastating. The understanding of the reason for this variability of responses of
NWPs to T. gondii is not clear. The aim of this chapter is to present a brief
summary of existing knowledge about toxoplasmosis in NWP and review some
possibilities that justify them.

New World Primate Ecology

New World primates (NWPs, Platyrrhini) are distributed from South and Central
America to North America, in Mexico. Table 1 presents a distribution of NWPs
according to their diet and forest strata preferences. They are all small- to medium-
sized primates, weighing from 120 g to approximately 10 kg. The different species
live in sympatry throughout most of their ranges with up to 13 species at some
Amazonian sites. One of the characteristics of Platyrrhini is the absence of ter-
restrial species, and few species occasionally forage on the ground or travel short
distances between trees, but none spend most of the day feeding on the ground
(Feagle 1999). New World primates include species that feed on gums, fruits,
leaves, seeds, a variety of invertebrates, and small vertebrates. Some of the smaller
species rely heavily on nectar during the dry periods of the year. Some NWPs
show morphological adaptations for more specialized diets, while others are
omnivorous, and most species show preference for one of the arboreal strata,
where they stay most part of the time.
Perelman et al. (2011) divided Platyrrhini into three families and listed 17 gen-
era, but did not include the two monotypic genera: Callibella (Aguiar and Lacher
2003; Van Roosmalen and Van Roosmalen 2003) and Oreonax (Groves 2001),
although some authors do not separate Oreonax from Lagothrix (Rosenberger and
Matthews 2008). According to Rylands and Mittermeier (2009), there are 19 genera
and 199 species and subspecies of primates in the Neotropics.
Pitheciidae (Cacajao, uakaris; Calicebus, titis; Chiropotes, bearded sakis;
Pithecia, sakis) have an unusual dental specialization for processing fruits and seeds
encased in a hard outer covering that are generally too hard for other monkeys to
bite through (Kinzey 1992). Compared to Cacajao and Chiropotes, Pithecia seems
to eat fruits with relatively softer outer covering, has a more diverse diet, and con-
centrates its activity in the middle and upper canopy but can use the lower canopy
for foraging (Feagle 1999).
Chiropotes prefers high rain forests, being usually in the middle and upper levels
of the main canopy (Feagle 1999). They feed on hard, often unripe fruits and on
seeds with very hard shells, which they open with their large canines. They occa-
sionally feed on insects (Norconk et al. 2009). Cacajao are also specialized on fruits
with hard outer shells and immature seeds and include in their diet small amounts
Table 1 Diet and forest strata preferences by Neotropical primates
Species and
Family Genusa subspeciesb Common names Diet Forest strata preference References
Cebidae Mico 14 Amazonian Fruits, arthropods, Middle of the canopy to the Bicca-Marques et al. (2011),
marmosets flowers, exudates ground, lower levels of the Feagle (1999)
forest
Cebuella 2 Pygmy marmoset Exudates, arthro- Middle of the canopy to the Norconk et al. (2009), Feagle
pods, fruits, and ground, lower levels of the (1999), Soini (1988),
small vertebrates forest Bicca-Marques et al. (2011).
Callithrix 6 Marmosets Exudates, arthro- Middle of the canopy to the Norconk et al. (2009), Feagle
pods, fruits, ground, lower levels of the (1999), Bicca-Marques et al.
small verte- forest (2011)
brates, eggs,
seeds, molluscs
Callimico 1 Goeldi’s monkey Arthropods, fruits, Middle of the canopy to the Norconk et al. (2009), Feagle
fungi, exudates ground, lower levels of the (1999)
forest
Leontopithecus 4 Lion tamarins Fruits, arthropods, Middle of the canopy to the Norconk et al. (2009), Feagle
exudates, ground, lower levels of the (1999), Kierulff et al. (2002)
flowers, small forest
vertebrates
Saguinus 33 Tamarins Arthropods, fruits, Middle of the canopy to the Norconk et al. (2009), Feagle
exudates, young ground, lower levels of the (1999), Snowdon and Soini
leaves, small forest (1988), Bicca-Marques et al.
Neotropical Primates and Their Susceptibility to Toxoplasma gondii…

vertebrates (2011)
Aotus 12 Owl monkeys Fruits, young leaves, No preference for a particular Norconk et al. (2009), Feagle
flowers, canopy level (1999), Cunha (2008),
arthropods Bicca-Marques et al. (2011)
Saimiri 10 Squirrel monkeys Arthropods, fruits, Middle and lower levels of the Norconk et al. (2009), Feagle
young leaves, forest, eventually come down (1999), Defler (2005), Baldwin
flowers, seeds, to the ground. and Baldwin (1981), Boinski
small verte- (1987), Ingberman et al. (2008),
255

brates, and eggs Bicca-Marques et al. (2011)

(continued)
 256

Table 1 (continued)
Species and
Family Genusa subspeciesb Common names Diet Forest strata preference References
Cebus 26 Capuchin monkeys Fruits, arthropods, Main canopy levels but Norconk et al. (2009), Izawa
young leaves, frequently come down to the (1979), Freese and
seeds, flowers, understory or to the ground Oppehheimer (1981), Fragaszy
small verte- during both travel and et al. (2004)
brates, eggs. In feeding
the wild, use
large stones as
tools to open
hard palm fruits
Atelidae Lagothrix 5 Woolly monkeys Fruits, insects, Upper levels of the main canopy, Norconk et al. (2009), Feagle
young leaves, rarely coming down to the (1999), Bicca-Marques et al.
flowers, seeds ground (2011)
Brachyteles 2 Muriquis Young leaves, fruits Upper levels of the main canopy, Norconk et al. (2009), Feagle
(pulp), flowers, rarely coming down to the (1999), Mendes et al. (2010)
mature leaves, ground
seeds
Ateles 15 Spider monkeys Fruits (pulp), young Highest levels of the forest but Norconk et al. (2009), Feagle
leaves, flowers eventually come down to the (1999), Zanon et al. (2008),
ground Bicca-Marques et al. (2011).
Alouatta 19 Howler monkeys Young leaves, fruits Most species prefer the main Norconk et al. (2009), Feagle
(pulp), mature canopy and emergent levels; (1999), Bicca-Marques et al.
leaves, flowers species from dry areas (2011)
regularly come down to the
ground
Pitheciidae Cacajao 6 Uakaris Seeds, fruits (pulp), Upper parts of the forest but Norconk et al. (2009), Rickli and
flowers eventually come down to the Reis (2008)
ground to forage
J.L. Catão-Dias et al.
 Species and
Family Genusa subspeciesb Common names Diet Forest strata preference References
Chiropotes 5 Bearded saki Seeds, fruit (pulp), Middle and upper levels of the Norconk et al. (2009), Feagle
monkeys arthropods, main canopy, rarely coming (1999), Bicca-Marques et al.
flowers down to the ground (2011).
Pithecia 9 Saki monkeys Seeds, fruits (pulp, Middle and upper canopy, Norconk et al. (2009), Feagle
whole, arils), eventually come down to the (1999)
young leaves, ground to forage
arthropods, and
flowers
Callicebus 29 Titi monkeys Fruits (pulp), seeds, Main canopy to the understory Norconk et al. (2009), Bordignon
young leaves, and rarely coming down to et al. (2008), Feagle (1999),
flowers, the ground Bicca-Marques et al. (2011)
arthropods
a
Taxonomy of Perelman et al. (2011)
b
Number of species and subspecies according to Rylands and Mittermeier (2009)
Neotropical Primates and Their Susceptibility to Toxoplasma gondii…
257
258 J.L. Catão-Dias et al.

of leaves, flowers, nectar, and insects (Rickli and Reis 2008). They use more
frequently the upper parts of the forest but can eventually come down to the ground
to forage (Rickli and Reis 2008).
Calicebus have very short canine teeth in comparison with other species of the
family Pitheciidae. They are mainly frugivorous but can supplement their diet with
leaves, seeds, flowers, and insects (Bordignon et al. 2008; Norconk et al. 2009). The
different species are distributed in different habitats from mature forest to the dry
scrub forest (caatinga) or bamboo thickets, where they use the main canopy or the
understory or low levels in the forest (Feagle 1999; Bordignon et al. 2008).
The family Atelidae includes two predominantly folivorous genera (Alouatta,
howler monkey, and Brachyteles, muriqui) that supplement their diet with fruits, flow-
ers, and seeds and two frugivorous genera, the spider (Ateles) and woolly monkeys
(Lagothrix and Oreonax), that feed mainly on fruits but also leaves, buds, flowers, and
insects (Groves 2001; Zanon et al. 2008; Norconk et al. 2009; Bicca-Marques et al.
2011). All atelines have a long, prehensile tail, and spider monkeys, woolly monkeys,
and muriquis are largely restricted to high primary rain forests where they prefer the
upper levels of the main canopy, rarely coming down to the ground (Mendes et al.
2010; Feagle 1999). Alouatta are found in a variety of habitats, including primary and
secondary forest, dry deciduous forest, and habitats containing patches of relatively
low trees in open savannah. Most species seem to prefer the main canopy and emer-
gent levels, but some species that live in drier areas (A. caraya) regularly come down
to the ground and cross-open areas between patches of forest (Feagle 1999).
The family Cebidae is composed of four subfamilies: Cebinae that includes
capuchin monkeys (Cebus); Saimirinae, represented by squirrel monkeys (Saimiri);
Callitrichinae, the smallest and most distinctive New World primates, separated in
Goeldi’s monkey (Callimico), tamarins (Saguinus and Leontopithecus), and mar-
mosets (Mico, Callithrix, Cebuella, Callibella); and the subfamily Aotinae (owl
monkeys), the only nocturnal Platyrrhini (Fernandez-Duque 2006; Norconk et al.
2009; Perelman et al. 2011).
Aotus are found in a variety of forest habitats, and there are no indications that
they prefer any particular canopy level. They are primarily frugivorous with a diet
that is supplemented by flowers, leaves, insects, and occasionally small vertebrates
and eggs (Wright 1981; Feagle 1999; Cunha 2008). Callitrichines use different
types of forest but seem to be characterized by the ability to exploit marginal and
disturbed habitat, and their diet is composed of fruits, flower, arthropods (mainly
insects), exudates, fungus, and small vertebrates (lizards, birds, frogs, and small
rodents). All species spend most of the time in the middle levels of the forest and
forage for fruits and insects in the middle of the canopy to the ground in the lower
levels of the forest (Snowdon and Soini 1988; Soini 1988; Feagle 1999; Kierulff
et al. 2002; Bicca-Marques et al. 2011).
Capuchin monkeys (Cebus) and squirrel monkeys (Saimiri) are the two most
omnivorous Platyrrhini (Feagle 1999). Saimiri occupy a variety of rain forest habi-
tats but seem to prefer riverine and secondary forests, where they are commonly
found in the middle and lower levels (Feagle 1999; Defler 2005). They are frugivo-
res and insectivores and supplement their diet with leaves, seeds, small vertebrates,
nectar, and eggs (Baldwin and Baldwin 1981; Boinski 1987; Feagle 1999; Defler
Neotropical Primates and Their Susceptibility to Toxoplasma gondii… 259

2005; Ingberman et al. 2008). The insect component of their diets is the highest of
any non- Callitrichinae, but the differences between these genera lie in mandibular
and dental robusticity, which is much stronger in Cebus and could determine differ-
ences in their diets (Norconk et al. 2009).
Capuchin monkeys are divided into tufted (Cebus apella, C. macrocephalus, C.
libidinosus, C. nigritus, C. robustus, C. cay, C. flavius, C. xanthosternos) and
untufted (C. albifrons, C. olivaceus, C. kaapori) (Bicca-Marques et al. 2011)1.
Cebus are found in virtually all types of neotropical forest including humid and dry
forest, swamp forests, seasonally flooded forest, as well as more open vegetation
types in savannahs and caatingas, where rainfall is absent for 5–6 months each year
(Fragaszy et al. 2004; Bicca-Marques et al. 2011). They seem to prefer the main
canopy levels but frequently come down to the understory or to the ground during
both travel and feeding (Feagle 1999).
Their diet includes many types of fruits and other vegetal parts and animal matter
(invertebrates and small vertebrates), and they are considered omnivores and also
classified as frugivore–insectivore (Izawa 1979; Freese and Oppehheimer 1981;
Feagle 1999). Fragaszy et al. (2004) characterized them as innovative and extreme
foragers due to their ability to acquire sustenance from a variety of potentially dan-
gerous sources that require special foraging skills and also for trying to eat almost
anything remotely edible. It gives them three types of adaptive advantages: first, the
flexibility to switch from more accessible foods such as fruits to more inaccessible
ones at time of food scarcity; second, the capacity to exploit habitats with different
structure and phenological characteristics (such as secondary or disturbed forests);
and third, it reduces the degree of dietary overlap between capuchins and other
arboreal vertebrates, mainly other primate species, more specialized in fruit or
insects (Brown and Zunino 1990; Fragaszy et al. 1990, 2004).

Toxoplasma gondii

Taxonomy and Trophism

Toxoplasmosis is a zoonosis of global occurrence caused by the protozoan Toxoplasma

gondii, an obligate intracellular coccidium belonging to Phylum Apicomplexa, Class
Sporozoasida. The first descriptions of T. gondii occurred almost simultaneously in
1908, and the history of the agent’s discovery reveals the independent work of research-
ers in two different continents. In Tunisia, Nicolle and Manceaux, investigating the

1
Recent phylogeographic analysis has shown that capuchins contain two well-supported mono-
phyletic clades, the morphologically distinct “gracile” (or untufted) and “robust” (or tufted)
groups, and placed the age of the split at 6.7 Ma (95 % highest posterior density 4.1–9.4 Ma)
(Alfaro et al. 2012a). Morphological and behavioral–ecological data also support a division of
capuchins into the same two distinct groups. As a consequence Alfaro et al. (2012b) have argued
for a division of capuchin monkeys into two genera: Sapajus Kerr, 1792, for the robust capuchins
and Cebus Erxleben, 1777, for the gracile capuchins.
260 J.L. Catão-Dias et al.

participation of rodent of Ctenodactylus gundi species in the epidemiological cycle of

leishmaniasis, described arc-shaped protozoa, which were given the name from the
Greek “toxo” (arc) “plasma” (life) (Nicolle and Manceaux 1909). One week later, in
Brazil, Alfonso Splendore, investigating histological lesions in rabbits resembling
human leishmaniasis, described a new protozoan, morphologically similar to that
reported by Nicolle and Manceaux (Splendore 1909). In subsequent years, several
reports describing similar parasites in other hosts were observed; however, immunobio-
logical evidence showed that the different strains isolated from humans and animals
represented the same agent (Innes 2010).
As the only species of the genus, until recently, it was believed that T. gondii was
a clonal organism with minimal genetic variation, a condition that allowed classify-
ing it into three lineages, namely, I, II, and III. However, newly published work has
shown that strains isolated from the free-living chicken (Gallus domesticus) from
different regions of Brazil have phenotypic and genetic characteristics markedly
different from strains from other countries, indicating that the genetic variability of
the parasite is higher than originally thought (Dubey 2009; Dubey and Su 2009).
In the 100 years since its discovery, T. gondii has proven to be a remarkably
competent parasite, since it appears to be able to parasitize virtually all species of
mammals and birds (Innes 2010). Recent work suggests that any cell of these verte-
brates can be infected by the protozoan (Elmore et al. 2010). Moreover, T. gondii
likely emerged in the manner that all other Coccidia have, using only one host and
being transmitted via fecal–oral route (Dubey 2009). However, the acquired skill of
being transmitted through other routes, such as ingestion of meat or through the
placenta, gave the parasite the opportunity to occupy new geographical areas and
move into new biomes and corresponding hosts (Dubey and Su 2009). These fea-
tures make T. gondii the archetype of the ideal parasite and be considered the most
successful existing parasite by some authors (Innes 2010).
On the other hand, the genetic basis of the virulence mechanisms involved in
Toxoplasma infection is only beginning to be unraveled. Recent data obtained from
the proteomic analysis of rhoptries show that several proteins in this organelle such
as ROP18 and ROP16 are released within parasitophorous vacuoles in the exact
moment of the host cell invasion by the protozoan and may represent, therefore, the
communication interface in the host–parasite relationship (Bradley et al. 2005;
Boothroyd 2009; Elmore et al. 2010).

Toxoplasma gondii Life Cycle

The life cycle of T. gondii was elucidated throughout the 1960s and 1970s of the
twentieth century with the identification of the parasite’s sexual cycle (Frenkel et al.
1969, 1970; Hutchison et al. 1970; Ferguson et al. 1974). Figure 1 summarizes the
T. gondii life cycle (Gardiner et al. 1998). Three infective forms are recognized:
sporozoites in sporulated oocysts and asexual fast- and slow-replicating forms
tachyzoites and bradyzoites. Felines are the only known natural hosts and become
infected through meat consumption or intake of water and/or other food contami-
nated with infective forms. However, there is a marked difference in competence
Neotropical Primates and Their Susceptibility to Toxoplasma gondii… 261

Fig. 1 Toxoplasma gondii life cycle (Adapted from Gardiner et al. 1998)

between the various types of zoites in terms of how effectively they infect suscep-
tible felines. In this sense, bradyzoites are exceptionally efficient when compared to
tachyzoites and sporozoites. The ingestion of only one bradyzoite is sufficient
to cause infection in felines, while the intake of at least 1,000 oocysts is required to
produce the same effect (Dubey 2009). Besides the domestic cat (Felis catus), 17
species of wild felines, including five New World species (Puma concolor, cougar;
Puma yagouaroundi, jaguarundi; Leopardus geoffroyi, Geoffroy’s cat; L. pardalis,
ocelot; and L. colocolo, pampas cat) have been identified as natural hosts (Silva
2007; Elmore et al. 2010).
Once ingested by cats, the infective form invades the enterocyte of the small
intestine and undergoes asexual reproduction cycles, followed by sexual reproduc-
tion with the formation of non-sporulated oocyst with eight sporozoites. These
oocysts are excreted in feces and, depending on environmental conditions, undergo
sporulation and become infective. Fecal excretion of non-sporulated oocysts by
immunocompetent felines occurs 1–2 weeks after infection and, once established,
generates large amounts of oocysts. However, recently published data showed that
the incidence of immunosuppressive events can promote new episodes of oocyst
excretion in affected animals (Malmasi et al. 2009). Sporulated oocysts are very
resistant to environmental conditions such as desiccation and freezing, and to the
action of disinfectants, and can survive in the environment for many months. It is
262 J.L. Catão-Dias et al.

believed that the favorable conditions of humidity and temperature, as those found
in several tropical biomes, such as Amazon and Atlantic forest, greatly improve the
viability of sporulated oocysts (Silva 2007).
The infection of intermediate hosts may occur in many ways, either through meat
consumption/ingestion of prey infected by asexual zoites, the consumption of other
food and water contaminated with sporulated oocysts, or congenitally (Dubey et al.
1998). However, there are reports of infection caused by blood transfusions, trans-
plantation, and laboratory accidents in humans (Dubey and Jones 2008). The pos-
sibility of transmission occurring through inhalation of aerosols or ingestion of
secretions containing T. gondii in Neotropical primates has also been suggested
(Furuta et al. 2001; Carme et al. 2009). In most vertebrate homeotherm hosts, zoites
multiply asexually within the infected cells through endodyogeny and tend to form
perennial cysts viable for many years in multiple tissues and organs, particularly in
skeletal and cardiac muscle, and in the central nervous system (Dubey 2009).
In summary, T. gondii life cycle is characterized by sexually reproducing and
developing oocysts in the small intestine of a natural feline host, before transmitting
to an intermediate homeotherm host. Within the intermediate host, the parasite
develops, forming cysts in various tissues.

Toxoplasma gondii and Host Immune Response

Although host immune responses to T. gondii have been studied since the pathogen’s
discovery more than a century ago, their interactions are not fully understood
(Boothroyd 2009; Tait and Hunter 2009). Experimental studies have shown that more
than 1,000 host genes are modulated in Toxoplasma-infected cells, among them genes
encoding proteins implicated in several processes including inflammation, apoptosis,
metabolism, and cell growth and differentiation (Blader and Saeij 2009).
The development of toxoplasmosis in mice and humans is determined by mecha-
nisms involving the pathogenicity of the parasite strain, in addition to the host
immune status (Hill et al. 2011; Pifer and Yarovinsky 2011). Surface antigen 1
(SAG1), the major surface protein of T. gondii, has been recognized as an essential
target of adaptive immune response. However, the parasite has developed strategies
to avoid the powerful immune response (Buzoni-Gatel and Werts 2006). In addition,
it is believed that three main T. gondii genotypes responsible for infection in humans
(types I, II, and III) may induce different patterns of the disease (Saeij et al. 2005).
Clonal lineages differ in growth, migration, and transmigration. In laboratory mice,
it is known that type I strains are very virulent (lethal dose – LD100 of one parasite),
while type II and III strains are much less virulent (lethal dose – LD50 ~103 and
~105). In humans, type I strains are frequently associated with postnatally acquired
ocular infections, whereas type II strains are more related with congenital infections
and toxoplasmic encephalitis (Blader and Saeij 2009; Sibley et al. 2009).
Typically, the natural and intermediate host response to T. gondii infection is
capable of holding back parasite dissemination, reducing mortality rates. The early
Neotropical Primates and Their Susceptibility to Toxoplasma gondii… 263

stimulation of the immune system following infection is an essential step in establishing

a balanced host–parasite relationship, once both host and parasite survive the initial
phases of infection and, in this way, the process progresses towards chronic disease
(Aliberti 2005).

Immune System: How Cells React

It is known that both humoral and cellular immune responses are involved in the reso-
lution of acute toxoplasmosis, but the cellular response is considered the most impor-
tant mechanism responsible for the host defense (Däubener and Hadding 1997; Innes
1997). When the parasites invade the intestinal mucosa of intermediate hosts (the
main infection route), the infected enterocytes suffer morphological and physiological
changes and secret chemokines and cytokines that attract polymorphonuclear leuko-
cytes, macrophages, and dendritic cells (DCs) (Buzoni-Gatel and Werts 2006).
Proinflammatory cytokines produced by lymphocytes, macrophages, DCs, and
neutrophils are crucial for controlling T. gondii. In the first stages of infection,
T. gondii activates cells such as macrophages, DCs, and neutrophils to produce high
levels of IL-12 (Gazzinelli et al. 1993b; Johnson and Sayles 1997). Mitogen-
activated protein kinase p38 is required for IL-12 production by macrophages in
response to soluble tachyzoite antigen (STAg) (Mason et al. 2004).
Many studies have demonstrated that IFN-γ, the hallmark of the inflammatory
response, is the major defense component against T. gondii infection that inhibits
parasite replication in various human and mouse cells (Suzuki et al. 1988; Sharma
1990; Däubener and Hadding 1997; Buzoni-Gatel and Werts 2006). Additionally,
IFN-γ, which is produced by natural killer cells (NK cells) in response to IL-12
secretion, contributes to the differentiation of lymphocytes into the Th1 phenotype.
IFN-γ, produced by activated T lymphocytes, natural killer cells, and natural killer
T cells, stimulates macrophages to produce reactive oxygen intermediates (ROI),
leading to the death of T. gondii (Nathan et al. 1983). Hence, IFN-γ is an essential
cytokine for resistance to acute and chronic Toxoplasma infections (Suzuki et al.
1988; Gazzinelli et al. 1993a). Similar to IFN-γ, TNF-α, IL-6, and IL-1 have syner-
gistic effects on the induction of an adequate immune response against T. gondii
(Lang et al. 2007).
In addition to IFN-γ, other cytokines, such as TNF-α, IL-2, and lymphotoxin-α, are
cofactors important to host responses to T. gondii infection. Natural killer cells and
CD4+ and CD8+ T cells are three lymphocyte subsets that produce these cytokines and
have been suggested to influence T. gondii immunity in mice and humans (reviewed in
Blanchard et al. 2008). IL-2, produced by CD4+ T cells, is an important T cell mitogen
(reviewed in Tait and Hunter 2009). CD4+ and CD8+ T cell activation prevent reactiva-
tion of infection, probably by IFN-γ production (Gazzinelli et al. 1992). CD8+ T cells
are known to be crucial for protection against the intracellular parasite T. gondii, because
they are involved in the capacity to induce apoptosis in infected cells (Däubener and
Hadding 1997). CD8+ T cells have also been reported to directly kill the extracellular
264 J.L. Catão-Dias et al.

and intracellular T. gondii, independent or dependent of major histocompatibility

complexes (MHC), respectively. In addition, perforin present in the granules of CD8+ T
cells and NK cells has been implicated in the parasite death, and it has been found to
play a critical role in chronic toxoplasmosis (Denkers et al. 1997).
The production of nitric oxide (NO) by macrophages limits the growth and replica-
tion of T. gondii, and this secretion depends on the expression of the inducible NO
synthase (iNOS). IFN-γ through signal transducer and activator of transcription 1
(STAT1) regulates effector mechanisms, including the iNOS overexpression. NO and
reactive nitrogen intermediates have been recognized as the main effector molecules
of microbicidal and microbiostatic activities in activated macrophages. Experimentally,
it has been shown that in acute infection, the lethality of the pathogen is associated
with high NO levels in serum (reviewed by Denkers and Gazzinelli 1998; Tait and
Hunter 2009). In addition, IFN-γ induces indoleamine 2,3-dioxygenase, resulting in
L-tryptophan depletion and inhibition of parasite growth (Fujigaki et al. 2002).
CD40 (in T cells) and CD154 (in macrophages) pathways promote killing of
T. gondii through induction of vacuole–lysosomal fusion. In addition, these path-
ways promote proinflammatory cytokines and active autophagic mechanisms
(Subauste 2009). The upregulation of p47 GTPases, in response to IFN-γ produc-
tion, is also involved in the autophagy process (reviewed by Tait and Hunter 2009).
In vivo studies indicate that the activation of the host innate immunity plays a
crucial role in the early resistance against infection and pathogenesis of toxoplas-
mosis (Gazzinelli et al. 1994). Toll-like receptors (TLR) are a family of innate
immune receptors focused on recognition of “pathogen-associated molecular pat-
terns” (PAMPs), which are molecules that are critical for microorganisms and natu-
rally not expressed by host cells (Pifer and Yarovinsky 2011). However, ligands
from the parasite that stimulate these receptors are not completely known. Toll-like
receptors (TLR) such as TLR2, TLR4, TLR9, and TLR11 bind to Toxoplasma-
derived factors, and TLR11 is considered to be a major innate immune receptor that
regulates IL-12 response to T. gondii infection (Yarovinsky et al. 2005). It has
already been reported that TLR9 is required for Th1 immune response after oral
infections with T. gondii (reviewed in Oykhman and Mody 2010).
Glycosylphosphatidylinositols (GPIs) of Toxoplasma, as well as other apicom-
plexan protozoa, stimulate the production of TNF-α in macrophages through NF-κB
activation, via both TLR4 and TLR2 (reviewed in Debierre-Grockiego and Schwarz
2010). However, only the loss of MyD88, an adaptor protein that mediates TLR
signaling, is essential for the survival in parasite-infected animals (Scanga et al.
2002; Yarovinsky et al. 2005; Debierre-Grockiego et al. 2007).

Humoral Immunity

Humoral immunity has been considered of minor importance in toxoplasmosis

protection and resistance (Sharma 1990). However, antigens from the parasite
induce the production of antibodies such as IgM, IgG, and IgA (Correa et al. 2007).
Neotropical Primates and Their Susceptibility to Toxoplasma gondii… 265

These immunoglobulins may lead to neutralization, inhibition of parasite cell

invasion, activation of the classical pathway of complement, and inflammation
(Däubener and Hadding 1997; Correa et al. 2007). Anti-T. gondii antibodies are
mainly specific to tachyzoites (reviewed in Hegab and Al-Mutawa 2003). However,
some studies have shown that IgA is important against cyst formation in mucosa
during oral infection (reviewed in Denkers and Gazzinelli 1998).
IgG is the most important immunoglobulin involved in the humoral immune
response against T. gondii infection. Specific IgG isotypes, IgG1 in humans and
IgG2a in mice, can play an important role in resistance to different pathogens
through mechanisms such as complement fixation, opsonization, or antibody-
dependent cell cytotoxicity. In humans, these antibodies reach a peak around 6–14
months postinfection, decreasing afterwards, with the host remaining reactive for-
ever. On the other hand, very high IgG titers are indicative of acute infection (Sharma
1990; Denkers and Gazzinelli 1998).
The detection of specific IgM in host serum indicates a recently acquired infec-
tion and does not indicate reinfection, because a minimum titer of IgG suppresses
IgM production (reviewed in Hegab and Al-Mutawa 2003). In the newborn, IgM is
diagnostic of congenital infection, since maternal IgM cannot cross the placental
barrier (reviewed in Hegab and Al-Mutawa 2003). Intriguingly, T. gondii IgM in
adults commonly persists well over 6 months. A microreactivation of cysts and the
generation of cross-reactive hetero- or autoantibodies can be involved in long-
lasting IgM (Correa et al. 2007).
In T. gondii infection, IgA is produced during the digestive stage. However,
IgA can be produced in the eye during intraocular disease in acute or recurrent
infection, but not in chronic toxoplasmosis (reviewed in Hegab and Al-Mutawa
2003). IgA response appears prior to any IgG production and is upregulated by
IL-10 and TGF-β (Correa et al. 2007). Moreover, IgA can be present in the colos-
trum, but does not cross the placenta (reviewed in Hegab and Al-Mutawa 2003).
In addition, a recent study has shown that IgE induces elimination of intracellular
parasites by human macrophages through its ability to trigger CD23 signaling,
and this capability is dependent on nitric oxide (NO) and controlled by IL-10
(Vouldoukis et al. 2011).

The Balance Between Proinflammatory and Anti-Inflammatory

Response

The balance between the production of proinflammatory (IFN-γ, TNF-α, IL-6, IL-1)
and anti-inflammatory (TGF- β and IL-10) cytokines appears to be decisive for the out-
come of T. gondii infection (reviewed by Lang et al. 2007). IL-10, an anti-inflammatory
cytokine, traditionally inhibits proinflammatory responses, controlling cytokine and
chemokine production. Neutralization of IL-10 in murine models increases central ner-
vous system inflammation. IL-10-deficient mice lose control of their immune response
and die in the acute phases of toxoplasmosis, after uncontrolled IFN-gamma and TNF-α
266 J.L. Catão-Dias et al.

production (reviewed by Aliberti 2005). In addition, IL-10 contributes to the suppression

of the T cell function, and it is considered to play a vital role in the control of
T. gondii immunopathology (reviewed by Tait and Hunter 2009).
Lipoxin, an anti-inflammatory eicosanoid, plays an important role in regulating the
immune response to T. gondii (reviewed by Machado and Aliberti 2009). Soluble
tachyzoites antigen (STAg) initiates lipoxin A4 production, inhibits dendritic cell
migration, and hinders in vivo and in vitro IL-12 production (Aliberti et al. 2002a, b).
Recently, it has been determined that IL-27 is a new anti-inflammatory cytokine
involved in the regulatory mechanism modulating infection-induced pathology
(reviewed by Tait and Hunter 2009). An in vivo study shows that IL-27R-deficient
mice generate aberrant IL-2 responses that are associated with fatal toxoplasmosis.
Depletion of IL-2 was found to prolong the survival of infected IL-27R−/− mice,
strongly suggesting that IL-27 limits IL-2 production during Th1 differentiation
(Villarino et al. 2006).
The P2X(7) receptor is a transmembrane receptor that is expressed on the surface
of a broad range of immune cells, but also in parenchymal cells. The activation of this
receptor by extracellular ATP in infected cells can kill intracellular pathogens or may
stimulate the production of proinflammatory cytokines in immune cells. The P2X(7)
receptor can mediate T. gondii death by human and murine macrophages (Jamieson
et al. 2010; Lees et al. 2010). However, T. gondii infection in the murine model showed
that the absence of the P2X(7) receptor did not affect IFN-γ, IL-12, IL-1β, monocyte
chemoattractant protein-1 (MCP-1), or TNF production. However, significant and
prolonged production of nitric oxide and delayed production of IL-10 in P2X(7)
R-deficient mice lead to more susceptibility and weight loss (Miller et al. 2011).

Immune Evasion Strategies

Protozoans have developed mechanisms to escape the immune system of immuno-

competent hosts. Some of these mechanisms include antigenic masking and variation,
serum factor blocking, intracellular location, and immunosuppression (Seed 1996).
Figure 2 summarizes the immune evasion mechanisms involved in T. gondii infection
in an immunocompetent host.
It has been demonstrated that T. gondii interferes with macrophages and the signal-
ing pathways of dendritic cells, where it blocks the nuclear import of transcription
factors such as Stat1 and NF-κB. This leads to, among other consequences, inhibition
of TNF-α and production of IL-12 by dendritic cells and macrophages, blockade of
MHC class II upregulation, and defects in the production of reactive oxygen interme-
diates (ROI), reactive nitrogen intermediates (RNI), and costimulatory molecules
such as CD80 and CD86 (Denkers and Butcher 2005; Luder et al. 2009).
T. gondii infection induces IL-10, lipoxin A4, TGF-β, and IFN-α and IFN-β
upregulation and inhibits NO production and p47 GTPases (Lang et al. 2007; Luder
et al. 2009). In addition, T. gondii could interfere with NO production at
Neotropical Primates and Their Susceptibility to Toxoplasma gondii… 267

NFKB translocation Upregulation of Prevention of fusion of lysossomes Formation of cysts

Stat1 translocation Lipoxin with phagocytic vacuole
MAPK re-phosphrylation

Anti-apoptotic effect
Supress NO production

IL-12, TNF-α, RNI, ROI

MHC class II • Interfere with mitocondrial cytochrome c
blocking • ↓ Caspase proteolitc activation
• ↓ Poly (ADP-ribose) polymerase
• Phosphatidylserine exposure
• Inhibition of granzyme B

Fig. 2 Different escape mechanisms of Toxoplasma-infected cells. Nuclear factor-κB (NF-κB),

signal transducers and activator of transcription 1 (Stat1), mitogen-activated protein kinase
(MAPK), interleukin 12 (IL-12), tumor necrosis factor-α (TNF-α), reactive nitrogen intermediate
(RNI), reactive oxygen intermediate (ROI), major histocompatibility complex class II (MHC class
II), nitric oxide (NO), and adenosine diphosphate (ADP) (Based on Seed 1996; Denkers and
Butcher 2005; Lang et al. 2007; Luder et al. 2009; Santos et al. 2011)

transcriptional level by reduction of mRNA and protein levels of iNOS. The reduc-
tion of NO production in serum has been implicated in triggering a conversion stage
in the parasite and protecting the host from immunopathological effects of infection
(reviewed in Lang et al. 2007).
In addition, the parasite can suppress the host immune response through induction
of apoptosis in CD4+ T cells while inhibiting the apoptosis of infected cells by inter-
fering with the mitochondrial cytochrome c protein, decreasing caspase proteolytic
activation, and exposing of phosphatidylserine, among others (reviewed by Denkers
and Butcher 2005; Lang et al. 2007; Luder et al. 2009; Santos et al. 2011). Finally,
T. gondii can avoid the fusion of lysosomes with the phagocytic vacuole and form
cysts, thus escaping the host immune system (Seed 1996; Denkers and Butcher 2005).

Toxoplasmosis and the Usual Response in Mammals

The tachyzoite, due to its rapid replication, is the protozoan form involved in
triggering Toxoplasma-induced tissue necrosis. Thus, the host’s ability to prevent,
inhibit, or minimize the spread of tachyzoites will determine, in large part, the clinical
changes observed in toxoplasmosis. In most hosts, particularly in immunocompe-
tent adult mammalians, T. gondii infection has no clinically relevant implications,
268 J.L. Catão-Dias et al.

since the immune response is usually effective in limiting the multiplication of

tachyzoites. In the natural feline host, infection is also predominantly subclinical,
and clinically important changes are mainly observed in congenitally infected off-
spring or in adults debilitated by immunosuppressive processes (i.e., animals posi-
tive for feline immunodeficiency virus). In these cases, the main clinical signs are
fever, anorexia, lethargy, abdominal pain, and neurological and ocular dysfunctions
(Elmore et al. 2010). In farm animals, toxoplasmosis is an important cause of abor-
tion in sheep and to a lesser extent in pigs and goats worldwide, particularly in
Europe, the UK, and Oceania (Brown and Barker 2007; Klevar 2007; Innes 2010).
Infected offspring from several domestic species, including cats, dogs, and pigs,
may present widespread interstitial pneumonia, myocarditis, necrotizing hepatitis,
meningoencephalomyelitis, lymphadenitis, and myositis (Brown and Barker 2007;
Klevar 2007).
Most immunocompetent humans that are infected after birth do not develop rel-
evant clinical symptoms, while few have fever and generalized lymphadenopathy.
In immunocompromised people, toxoplasmosis is an important cause of retinocho-
roiditis, encephalitis, and pneumonia (Elmore et al. 2010).

Toxoplasma gondii Infection, Toxoplasmosis,

and New World Primates

Toxoplasma Epidemiology in NWP

The first report of toxoplasmosis in New World primates was made in 1916, affecting
a specimen of Stentor seniculus (Alouatta seniculus) (reviewed by Nery-Guimaraes
et al. 1971). Since that time, many other reports of this disease in NWP have been
made, predominantly affecting captive animals (Hessler et al. 1971; Anderson and
McClure 1982; Borst and Vanknapen 1984; Cunningham et al. 1992; Dietz et al.
1997; Pertz et al. 1997; Juan-Salles et al. 1998; Bouer et al. 1999; Epiphanio et al.
2000, 2001, 2003; Andrade et al. 2007; Carme et al. 2009; Cedillo-Pelaez et al. 2011).
Although it has been known that NWPs are susceptible to toxoplasmosis for almost
100 years, very little is known about the immune response induced by T. gondii in
these animals, and only serological data are available in the literature.
Table 2 presents a comparison between major clinical and pathological manifesta-
tions of toxoplasmosis in NWP, felids, and humans. In general, toxoplasmosis in
NWPs is a disease with a hyperacute clinical course. In most cases, the animals are
found dead without prior clinical history. When present, the main clinical findings
reported are prostration, dyspnea, hypothermia, nasal foamy serum–bloody exuda-
tion, anorexia, and vomiting. At necropsy, in most animals, the macroscopic changes
are diverse and occur in multiple organs and tissues and are characterized by severe
pulmonary edema and congestion, hepatic congestion with hepatomegaly, spleno-
megaly, spleen lymphoid hyperplasia, mesenteric and mediastinal fibrin-hemorrhagic
Table 2 Major clinical and pathological manifestations of toxoplasmosis in New Word Primates, felids, and humans
New Word Primates Felids Humans
Pattern II (most
Pattern I Cebidae and Pattern III Immune
(Callitrichinae) Atelidae) (Cebus) competent Immune suppressed Immune competent Immune suppressed Congenitally infected
Clinical Hyperacute, Acute, severe, Subacute, Subclinical Congenitally or Most subclinical; Fever, headache, Asymptomatic to
manifes- markedly and mild, or mild lactationally few develop myalgia, anorexia, fatal. Prematurity,
tations severe; mortality with self- infected offspring; fever and fatigue, abdominal intrauterine
mortality from 20 % very limiting stillborn or lethargy lymphadenopa- pain, vomiting, growth retarda-
close to to 80 %. low diarrhea depression, thy. nausea, dyspnea, tion, debilitating
100 %. Prostration, mor- hypothermia, ascites, Debilitating arthralgia. ocular disease,
Prostration, anorexia, tality hepatomegaly, ocular disease Lymphadenopathy strabismus,
dyspnea, hypother- rate chorioretinitis, and has been psychomotor
nasal foamy mia, sudden death reported in few impairment,
exudation, dyspnea, Older cats with cases prostration,
anorexia vomiting immunosuppressive microcephalus,
processes: anorexia, hydrocephalus,
lethargy, dyspnea, convulsion,
persistent/intermit- icterus, hypotonia,
tent fever and hepatomegaly
Pathological Severe Severe Mild and Nonspecific Moderate to severe Retinochoroiditis Encephalitis Retinochoroiditis,
findings fibrin- fibrin- non- necrotizing hepatitis, anterior uveitis,
hemorrhagic hemorrhagic spe- lymphadenitis, and and encephalitis
necrotizing necrotizing cific pneumonia.
pneumonia, pneumonia, Neuronal necrosis
hepatitis, hepatitis, and meningitis
splenitis, splenitis,
enteritis, and enteritis, and
lymphadeni- lymphadeni-
tis tis
Based on Anderson and McClure (1982), Elmore et al. (2010) Epiphanio et al. (2003), Silva (2007), Epiphanio and Catão-Dias (unpublished data)
270 J.L. Catão-Dias et al.

lymphadenitis, gastric ulceration, hemorrhagic enteritis, brain edema, and congestion.

Microscopically, the main reported changes are fibrin-hemorrhagic interstitial pneu-
monia, fibrin-necrotic splenitis, multifocal necrotizing hepatitis, fibrin-hemorrhagic
lymphadenitis, and multifocal necrotic-hemorrhagic enteritis (Anderson and McClure
1982; Cunningham et al. 1992; Dietz et al. 1997; Juan-Salles et al. 1998; Epiphanio
et al. 2000, 2001, 2003; Andrade et al. 2007; Carme et al. 2009; Cedillo-Pelaez et al.
2011). However, detailed studies of the macro- and microscopic pathological findings
resulting from toxoplasmosis in NWPs showed that there is significant variation in the
types and intensity of lesions, depending on the Platyrrhini species affected.
Necroscopy, histopathological, histochemical, immunohistochemical, morphometry,
and ultrastructural analyses of 33 cases of toxoplasmosis in NWPs in captivity (11
Atelidae, i.e., 6 A. fusca and 5 L. lagotricha; 22 Cebidae, i.e., 18 Callitrichinae, 3
Saimirinae, and 1 Aotinae) showed that Atelidae presented pathological changes more
variable and pleomorphic than Cebidae, making the preliminary necroscopic diagno-
sis more difficult. In particular, jaundice was observed only in A. fusca, affecting 50 %
(3/6) of animals evaluated (Epiphanio et al. 2003).
A relevant aspect that stands out in the analysis of several reports of toxoplasmo-
sis in Platyrrhini is the scarcity of information on the occurrence of fatal clinical
outcomes involving the genus Cebus. The cases described include the death of one
specimen of C. apella2 and one of C. capucinus due to natural infection (De
Rodaniche 1954; Nery-Guimaraes and Franken 1971).
Serological investigation shows that, similar to other intermediate hosts,
Platyrrhini species apparently exhibit distinct humoral responses against infection
by T. gondii. Table 3 presents a summary of epidemiological surveys of NWPs for
Toxoplasma. When surveys conducted with apparently healthy animals are consid-
ered, and excluding data for a very small number of individuals from genera Aotus
and Ateles (Bouer et al. 2010), the frequency of animals with positive titer of anti-
Toxoplasma gondii antibodies is generally higher for genus Cebus. In Cebus kept in
captivity, the frequencies ranged from 28.7 % to 79 %, while a frequency of 30.2 %
in free-living Cebus has been reported (Garcia et al. 2005; Leite et al. 2008; Bouer
et al. 2010).

2
The capuchin monkeys are particularly complex in their taxonomy. For many years, taxonomic
arrangements reduced all tufted capuchin monkeys to just one species, Cebus apella, with 11 and
16 (Hill 1960) subspecies. The most recent revisions, by Groves (2001) and Silva (2001), both
based on morphology, differently recognized, as species, the following: apella and macrocephalus
in the Amazon and libidinosus, nigritus, robustus, cay, and xanthosternos to the south. Groves
(2001) presented an alternative as follows: Amazon forms C. apella apella, C. a. fatuellus, C. a.
macrocephalus, C. a. peruanus, and C. a. tocantinus and southern forms C. libidinosus libidinosus,
C. l. pallidus, C. l. paraguayanus, C. l. juruanus (Amazonian), C. nigritus nigritus, C. n. robustus,
C. n. cucullatus, and C. xanthosternos (see Fragaszy et al. (2004) and Rylands et al. (2005)). In
Brazil, captive capuchins (independently of origin) are generally named as Cebus apella, and in
many occasions and in different institutions, individuals of different subspecies or species are kept
together generating hybrid groups (M.C.M. Kierulff, personal observation). Because of all these
problems, we decided to maintain the original names used for Cebus species cited in the refer-
ences, mostly named just as Cebus apella with no distinction to subspecies, even known that it
refers to species other than the Amazonians.
Table 3 Seroprevalence of Toxoplasma gondii in New World Primates
Genus/species Local Animal tested (n) Test Captive/free ranging Positive test (%) Reference
S. oedipus oedipus South 100 SFR FR 0 Werner et al. (1969), Nery-Guimaraes and Franken
America (1971)
S. sciureus Brazil 17 SFR Captive/FR 17,6 Nery-Guimaraes and Franken (1971)
C. apella 26 15,3
A. belzebuth 1 0
Aotus sp. 1 0
A. geoffroyi 1 0
C. jacchus 2 0
C. penicillata 5 0
L. lagotricha 1 0
C. apella Brazil 5 SFR FR 60 Sogorb et al. (1972)
A. fusca 12 42,1
Saimiri spp. Brazil 49 IHT FR 63,3 Ferraroni and Marzochi (1980)
C. apella Colombia 10 NA Captive 0/0 Cadavid et al. (1991)
C. capucinus 15 13,3
C. albifrons 22 40,9
Saimiri sciureus England 4a IFA Captive 100/75 Cunningham et al. (1992)
11# 100/54,5
A. seniculus French 50 DA FR 4 De Thoisy et al. (2003)
Neotropical Primates and Their Susceptibility to Toxoplasma gondii…

S. midas Guiana 50 0
C. apella Brazil 43 MAT FR 30,2 Garcia et al. (2005)
A. caraya 17 17,6
L. lagotricha USA 2a LA Captive 100 Gyimesi et al. (2006)
13 IHT 0/0/0
MAT
C. apella Brazil 14 IFAT Captive 28.7 Leite et al. (2008)
13 MAT 30.8
(continued)
271
 272

Table 3 (continued)
Genus/species Local Animal tested (n) Test Captive/free ranging Positive test (%) Reference
Alouatta caraya USA 1 MAT Captive 0 de Camps et al. (2008)
Ateles geoffroyi 4 0
Callicebus moloch 5 0
donacophilus 3 0
Callimico goeldii 1 0
Callithrix kuhlii 4 0
L. lagotricha 1 0
L. chrysomelas 3 0
L. rosalia 8 0
Pithecia pithecia 6 0
Saguinus
geoffroyi
S. sciureus Israel 24a MAT Captive 83,3 Salant et al. (2009)
C. apella Brazil 105 IFAT Captive 79 Bouer et al. (2010)
Callithrix sp. 42 26,2
Alouatta sp. 20 50
Leontopithecus 15 20
sp. Ateles sp. 7 57,14
Saimiri sp. 6 33,33
Saguinus sp. 5 0
Aotus sp. 3 66,66
J.L. Catão-Dias et al.

Lagothrix sp. 3 0
Genus/species Local Animal tested (n) Test Captive/free ranging Positive test (%) Reference
C. jacchus Brazil 25 LA Captive 0 Epiphanio & Catão-Dias, unpublised data
C. penicillata 18 0
C. geoffroyi 8 0
C. aurita 2 0
C. kuhlii 1 0
Callithrix sp. 1 0
S. bicolor 2 0
S. midas 3 0
S. niger 2 0
L. chrysomelas 46 2,2
L. rosalia 1 0
L. chrysopygus 1 0
C. apella 100 73
A. marginatus 2 100
A. belzebuth 1 0
A. paniscus 5 20
Ateles sp. 3 100
Saimiri sciureus 6 0
L. lagotricha 2 0
A. caraya 7 42,9
A. fusca 5 0
Alouatta sp. 5 0
Aotus sp. 2 50
IFAT Indirect immunofluorescence, MAT Modified Agglutination Test, IHT indirect heamagglution test, LA latex agglutination, PCR polymerase chain reac-
Neotropical Primates and Their Susceptibility to Toxoplasma gondii…

tion, SFR Sabin-Feldman reaction, DA direct agglutination method

a
died; # survivors
273
274 J.L. Catão-Dias et al.

Moreover, the data linking the evolution of the serological profile with the occur-
rence of toxoplasmosis outbreaks in NWPs can provide interesting information for
understanding this process. At the London Zoo, one-third of the S. sciureus colony
died of toxoplasmosis, and serological surveys showed that most animals had titers
indicative of recent infection. IgG was detected in 11 surviving animals and IgM in
six individuals from this group (Cunningham et al. 1992). In another outbreak that
caused the death of 24 S. sciureus in Israel, 83.3 % of the animals had positive serol-
ogy, suggesting that humoral immunity is not an effective defense mechanism for
sudden toxoplasmosis or reinfection in this NWP species (Salant et al. 2009).
Similarly, Lagothrix lagotricha individuals who died of sudden toxoplasmosis
showed positive serology for anti-Toxoplasma gondii antibodies in three distinct
types of tests (latex agglutination, indirect hemagglutination, modified agglutina-
tion), besides identification of T. gondii by PCR (Gyimesi et al. 2006).
Experimental infections have been performed in an attempt to better understand
the role of the humoral response in the development of toxoplasmosis in NWPs. In
one experiment, 28 Saguinus sp. were infected and died within few days, without
the detection of specific antibodies anti-Toxoplasma gondii (Werner et al. 1969). In
another study, five C. apella individuals were infected with T. gondii (N strain, type
II tachyzoite suspension—1 × 105 mL—by intraperitoneal route) and exhibited non-
specific and mild clinical signs for only 3 days postinfection and were euthanized
102 days postinfection. Macroscopic and microscopic lesions observed were mild
and were not correlated with toxoplasmosis. However, anti-Toxoplasma gondii IgG
titers were detected by IFA and ELISA 9 days postinfection and lasted until the end
of the investigation (Bouer et al. 2010). In another experiment, S. sciureus orally
infected died approximately 1 week postinfection; however, anti-Toxoplasma
immunoglobulin titers were not detected by immunoblot (Furuta et al. 2001).

Response Patterns to T. gondii Infections in NWP

Serological, clinical, pathological, and experimental data available on toxoplasmo-

sis in NWPs suggest three distinct response patterns to T. gondii infection: patterns
I, II, and III. Pattern I is that observed mainly in Callitrichinae. In these animals
(Saguinus, Leontopithecus, Callithrix), the disease is markedly severe, with mortal-
ity close to 100 %, which makes the occurrence of animals with positive serology to
be very low or zero. Pattern II involves a diverse group of NWPs from families
Cebidae (Saimiri, Aotus) and Atelidae (Alouatta, Ateles, Lagothrix), being charac-
terized by the occurrence of severe outbreaks, with variable mortality, but with the
survival of a reasonable number of individuals with positive serology in the popula-
tion (from 15 % to 66 %), particularly Saimiri and Alouatta. In both patterns, despite
the pleomorphism observed in some Atelidae (Epiphanio et al. 2003), the lesions
are predominantly consistent and characterized by multifocal, coalescing, severe,
and multisystemic necrosis, associated with the presence of large amounts of
tachyzoites. In turn, pattern III is observed in the genus Cebus, which differs greatly
Neotropical Primates and Their Susceptibility to Toxoplasma gondii… 275

from those seen in most other platyrrhines. In Cebus, the infection tends to induce
high and persistent IgG titers, the animals rarely die, and the morphological changes
seen in experimental cases are mild and nonspecific (Bouer et al. 2010).
The variable prevalence of anti-Toxoplasma gondii antibodies in NWPs, and
especially the very low frequency of antibodies in Callitrichinae, may explain the
very high susceptibility of these animals to infection. Callitrichinae, specifically, die
rapidly during the acute phase of infection, before IgG production is initiated. On
the other hand, the high IgG titers observed in Cebus, associated with rare reports of
death in this genus, suggests that these animals are able to establish an efficient
antibody immune response against the infection.
Over the course of 20 years at the Laboratory of Comparative Pathology of Wildlife
(LAPCOM—Laboratório de Patologia Comparada de Animais Selvagens) of the
FMVZ–USP, the authors have witnessed at least six major toxoplasmosis outbreaks in
NWP. These outbreaks mainly occurred among the genera Callithrix, Leontopithecus,
Lagothrix, Alouatta, Saimiri, and Saguinus. In these outbreaks, all individuals who
exhibited clinical signs subsequently died. In at least two of the outbreaks, diseased
animals (Leontopithecus chrysopygus, L. rosalia, L. chrysomelas) were submitted to
recommended therapeutic procedures (Osborn and Lowenstine 1998), but did not sur-
vive. Significantly, Cebus specimens were not affected in any of the outbreaks fol-
lowed by LAPCOM, although these animals constitute the large majority of the
NWPs populations kept in captivity in the zoos involved.
The clinical and serological aspects of toxoplasmosis (described above) for most
NWPs, especially Callitrichinae, resemble those reported for the mountain hare
(Lepus timidus). These Eurasian animals are especially susceptible to the disease
and develop severe acute symptoms that are often fatal. Experimental studies have
shown that mountain hares have very low antibody titers and inefficient prolifera-
tion of T lymphocytes when exposed to T. gondii, compared with those observed in
domestic rabbits (Oryctolagus cuniculus) (Gustafsson et al. 1997), suggesting that
the hares are incompetent to establish an efficient adaptive immune response against
the parasite.
Other aspects that deserve to be addressed in understanding differing susceptibil-
ity of NWP species to T. gondii include the variability of strains, infective doses,
and the possibility of recrudescence of cysts. The recent characterization of differ-
ent T. gondii strains shows that there is significant genetic diversity that was previ-
ously unknown (Dubey 2009; Dubey and Su 2009). In addition, it is known that the
main three known strains can induce different patterns of the disease in humans
(Saeij et al. 2005).
Furthermore, studies investigating the types of strains involved in cases of toxo-
plasmosis in NWPs are rare and are restricted to four outbreaks in Saimiri: two in
French Guiana, with the identification of strains II and III and atypical alleles
(Carme et al. 2009); one in Israel involving strain III (Salant et al. 2009); and one in
Mexico involving strain I (Cedillo-Pelaez et al. 2011). The high susceptibility of
Saimiri to the three main T. gondii strains suggests that, at least for this NWP spe-
cies, the genetic variability of the parasite may not be a determining factor for dis-
ease manifestation.
276 J.L. Catão-Dias et al.

Fig. 3 Peripheral blood

mononuclear cells
proliferation in response to
soluble Toxoplasma gondii
tachyzoite antigen (STAg). C.
apella n = 2 and L.
chrysomelas n = 2; *p ≤ 0.05,
two-way ANOVA test

In humans, the immunocompromised host (patients with AIDS, organ transplan-

tation, cancer, or taking immunosuppressants) may experience cyst reactivation,
with bradyzoites transforming back into tachyzoites, leading to infection recrudes-
cence and life-threatening encephalitis (Montoya and Liesenfeld 2004).
Toxoplasmosis recrudescence has been demonstrated in other hosts, such as in
immunosuppressed Rattus norvegicus after experimental treatment with dexameth-
asone (Silva 2007) and in dogs and cats undergoing renal transplantation (Bernsteen
et al. 1999). It has already been suggested that captivity stress, as well as immuno-
suppression, may lead to reactivation of Toxoplasma cysts in NWPs (Gyimesi et al.
2006). Thus, one possibility to explain, at least partially, the existing data is that part
of Atelidae and Cebidae (except Cebus) with positive serology for T. gondii could
be chronically infected and, in response to diverse immunosuppressant stimuli,
could undergo infection reactivation with the development of clinical disease and
death. Another possibility is that animals were reinfected with a strain more virulent
than the previous one, or with higher infective doses, resulting in acute toxoplasmo-
sis. In these cases, it is possible to suggest that these animals would respond rapidly
with the production of antibodies, progressing to resistance or death.
To investigate differences in cellular immune response against T. gondii, prolif-
eration assays were performed with peripheral blood mononuclear cells (PBMC)
from two Cebus apella and two L. chrysomelas. These species were selected because
they represent the extremes of the NWP susceptibility to T. gondii, taking into
account the epidemiological, serological, clinical, and pathological records of
LAPCOM. PBMCs were stimulated with varying concentrations (3, 30, and 300 μg/
ml) of soluble antigens of T. gondii tachyzoites (STAg) and analyzed by liquid scin-
tigraphy. Positive controls were stimulated with phytohemagglutinin (PHA). Our
preliminary stimulation indices (SI) show that stimulation with STAg is dose depen-
dent in C. apella, but not in L. chrysomelas (Fig. 3). Moreover, the SI were 1.8-,
5.28-, and 6.64-fold higher in C. apella when compared with L. chrysomelas
(Epiphanio and Catão-Dias, unpublished data).
These results, although preliminary and involving a small number of samples, sug-
gest that the immune system of Cebus is more able to respond to infection by
Neotropical Primates and Their Susceptibility to Toxoplasma gondii… 277

T. gondii, by more efficiently controlling the spread of pathogens and allowing the
establishment of a more balanced host–parasite relationship similar to the vast major-
ity of warm-blooded hosts infected with T. gondii. Moreover, the inadequate prolifera-
tive response observed in L. chrysomelas would prevent the emergence of an efficient
cellular response against the infection, favoring the development of fatal infections.
Our results, associated with those published by other researchers, raise several
questions about the mechanisms involved in the unsatisfactory cellular immune
response shown by the vast majority of NWPs against T. gondii, such as:
1. Would the inflammatory response induced by T. gondii in NWPs be intensified to
the point of leading to the overproduction of IFN-γ, TNF-α, IL-6, IL-12, or IL-1β?
2. Could there be overstimulation of TLR, with consequent amplification of the
expression of the transcription factor NF-kB?
3. Could regulatory mechanisms, such as anti-inflammatory cytokines TGF-β,
IL-10, and lipoxins, be downregulated?
4. Could there be disturbances in the fusion of the parasitophorous vacuole with
lysosomes, favoring the survival of T. gondii in cells infected by T. gondii in
NWPs?
5. Could the apoptosis mechanisms of Toxoplasma-infected cells be deficient?
6. Could the mechanisms of NO production be lacking?
Currently, many sequences of genes encoding proteins related to the immune
system of platyrrhines are known (Table 4). Hopefully in the near future, these tools
can be used to clarify some of the many unanswered questions regarding the toxo-
plasmosis immunology in NWPs.

Toxoplasma gondii Infection and NWPs’ Response:

Approaching the Susceptibility Differences from Ecological
and Behavioral Perspectives

Approximately three million years ago, Panamanian land bridge formed and allowed
the immigration of many intermittent “invaders” from the North America into the
South America, including carnivores (e.g., felids, canids, and mustelids) over the
course of the Pleistocene (Webb 1976; Simpson 1980; Marshall 1988). At that time,
T. gondii may also have migrated into the continent with carnivore species (reviewed
in Sibley et al. 2009). In fact, T. gondii strains from North and South America share
a common ancestry, and it was estimated that they last shared a common ancestor
one million years ago (Sibley et al. 2009).
Platyrrhines, however, have a long evolutionary history preceding the migration
of carnivore species into South America. The early platyrrhine fossils come from
the Late Oligocene in Bolivia (24–28 million years ago). Indeed, species related to
the living Neotropical primates were present in Colombia, during the Middle to
Late Miocene, suggesting a common platyrrhine ancestor in the Late Oligocene or
Early Miocene (14–24 million years ago) (Fleagle and Tejedor 2002). Recent
Table 4 List of selected genes related to immune response in New World Primates
Species Gene GenBank accession
Aotus infulatus IL-12B DQ989359.1
Aotus lemurinus TNF-a AF097329
Aotus lemurinus IFN-γ AF097327.1
Aotus nancymaae CD4 FJ623078.1
Aotus nancymaae TLR9 AY788894.1
Aotus nancymaae IFN-γ AF014512.1
Aotus nigriceps TNF-a AF097328
Aotus trivirgatus CD40 ligand AF344860.1
Aotus trivirgatus MHCI AB113205.1
Aotus vociferans TNF-a AF014508
Aotus vociferans IL-10 AAD01532.1
Aotus vociferans IFN-γ AF014507.1
Ateles belzebuth TLR4 AB446521
Ateles belzebuth MHCI AB113112
Ateles geoffroyi IL-10 ABM65916.1
Ateles geoffroyi TLR4 AB446522
Callicebus moloch MHCII AF197231.1
Callithrix jacchus IL-12B AB539805.1
Callithrix jacchus iNOS AM712438
Callithrix jacchus CD4 AF452616.1
Callithrix jacchus CD8 DQ189217
Callithrix jacchus IL-1a AB539804
Callithrix jacchus TNF-a DQ520835
Callithrix jacchus TLR4 AB446516
Callithrix jacchus TLR9 XM_002758237
Callithrix jacchus IL-27 XM_002756059.1
Callithrix jacchus CD40 DQ189221.1
Callithrix jacchus p47 GTPase XM_002762275
Callithrix jacchus MyD88 XM_002759734
Callithrix jacchus P2X(7) XM_002753098
Callithrix jacchus MHCII AF197230.1
Cebus apella TLR4 AB446520.1
Leontopithecus rosalia TLR4 AB446518
Saguinus imperator TLR5 FJ542217
Saguinus labiatus MHCII JF414576.1
Saguinus mystax IFN-γ FJ598592.1
Saguinus oedipus TNF AY091968
Saguinus oedipus TLR4 AB446517.1
Saguinus oedipus TLR2 EU488857.1
Saguinus oedipus MHCII AF197226.1
Saimiri sciureus IL-12B DQ989358.1
Saimiri sciureus CD4 AF452617
Saimiri sciureus CD8 AJ130819
Saimiri sciureus IL-1b AF294754
Saimiri sciureus TNF-a AJ437697
Saimiri sciureus TNF DQ989365
Saimiri sciureus TLR4 AB446519
Saimiri sciureus IL-10 Q8MKG9.1
Saimiri sciureus IFN-γ AF414102.1
Neotropical Primates and Their Susceptibility to Toxoplasma gondii… 279

phylogenetic analysis using DNA samples of primate species showed that Platyrrhini
diverged from a last common ancestor with Catarrhini 43.5 million years ago during
the Eocene. The common ancestor to Pitheciidae originated 20.2 million years ago,
and the Cebidae radiation initiated with the emergence of Cebinae and Saimirinae
approximately 20 million years ago (Perelman et al. 2011). These data indicate that
nonhuman primates were already in South America at the time of the felid (possibly
infected with T. gondii) invasion, from the north hemisphere.
The high susceptibility of NWPs to toxoplasmosis has been known for almost
100 years. The main hypotheses proposed to explain this condition can be summa-
rized as follows: (a) NWPs have evolved for over 20 million years without the pres-
ence of felids and therefore would not have acquired adaptations to the pathogen
over this time, especially cellular responses to T. gondii (Cunningham et al. 1992),
and (b) even after the arrival of felids (possibly infected with T. gondii) to the
Neotropics, the arboreal habits of NWPs would have restricted their contact with the
feces of felids infected by the protozoan oocysts, limiting the development of effi-
cient immune response (Innes 1997). The available data compiled in this review
corroborates many of the assumptions covered above to justify the high susceptibil-
ity of some NWPs to toxoplasmosis. On the other hand, they do not explain the
significant differences observed between groups of NWPs, in particular the high
resistance reported for Cebus.
To some extent, it is possible to assess how a particular animal explores and
occupies an environment in relation to the intensity and diversity of the parasite load
it carries. A study conducted in Costa Rica showed C. capucinus with higher para-
site infestation in fecal samples than Alouatta and Ateles (Stuart et al. 1998).
Fragaszy et al. (2004) suggested that at least three characteristics of Cebus behavior
may lead to higher parasite infestation in these monkeys than in sympatric Alouatta
and Ateles: Cebus drinks from water holes, frequently forages on the ground, and
eats a wider variety of foods. These behaviors may bring Cebus into contact with
greater variety of parasites. Considering the epidemiological characteristics of
T. gondii, it is possible to speculate that the aspects described above could justify a
diverse exposure of NWPs to the protozoan.
Contaminated water can be a source of toxoplasmosis, and this behavior has
already been recorded for other Alouatta species that drink water in holes in
branches or trunks or in bromeliads (Glander 1978; Gilbert and Stouffer 1989;
Bicca-Marques 1992; Giudice and Mudry 2000) or go to the ground to drink water
(Almeida-Silva et al. 2005). Other Platyrrhini such as Aotus (Wright 1981), Saimiri
(Baldwin and Baldwin 1981), Cebuella (Soini 1988), Saguinus (Snowdon and Soini
1988), and Brachyteles (Mendes et al. 2010) have also been observed drinking on
the banks of streams and/or rivers during the dry season.
Cebus sp. comes down to the ground more frequently in comparison to other
NWPs where they may be more frequently exposed to excreted T. gondii. Recent
studies have reported the use of tools (stones used as hammers and anvils) to open
hard nuts by wild Cebus in places where the groups (from different species) use the
ground more frequently (Fragaszy et al. 2004; Canale et al. 2009). Robinson (1984)
found that many of the invertebrates consumed by C. olivaceus were found on the
280 J.L. Catão-Dias et al.

Fig. 4 Group of yellow-breasted capuchin monkeys (Cebus xanthosternos) eating a bristle-spined

rat pup (Chaetomys subspinosus) they had just caught, Una Biological Reserve, Bahia, Brazil
(Photo by Jean Marc Lernould)

ground. They visually search the leaf litter and sweep the leaves to reveal the insects
hidden underneath (Fragaszy et al. 2004).
Additionally, in our opinion, an important behavioral characteristic of Cebus is
that it is the most carnivorous of the platyrrhines. It has been often noted that the
consumption of invertebrates and small warm-blooded vertebrates plays a role in
the transmission of T. gondii to NWPs (Epiphanio et al. 2003; Carme et al. 2009;
Salant et al. 2009). Most of the protein in Cebus diets comes from invertebrates
(insects and other arthropods), but while hunting for invertebrates, they sometimes
find vertebrates that they capture and consume. It has been widely reported that they
capture and consume a variety of relatively large vertebrates that may weigh up to
one third the Cebus body weight and may constitute up to 3 % of their feeding time
(Fragaszy et al. 2004). Along with chimpanzees (Pan troglodytes), Cebus are one of
the few nonhuman primate species that have been reported to hunt vertebrate prey
in more than an occasional, incidental manner (Fragaszy et al. 2004).
The types of vertebrate prey that Cebus has been reported to consume include
birds and their nestlings and eggs, lizards, frogs, rodents, bats, squirrels (Sciurus
variegatoides), coati pups (Nasua narica), and infant titi monkeys (Callicebus
moloch) (Izawa 1978; Terborgh 1983; Fedigan 1990; Galetti 1990; Rose 1997;
Sampaio and Ferrari 2005). Cebus xanthosternos from the Una Reserve, Bahia,
Brazil, have been seen preying upon bristle-spined rat pups (Chaetomys subspi-
nosus) on four occasions (Priscilla G. Suscke, personal communication) (Fig. 4).
In a forest in Rio de Janeiro, a bamboo rat (Kannabateomys amblyonyx) was
found preyed upon immediately following a passage of a Cebus nigritus group
Neotropical Primates and Their Susceptibility to Toxoplasma gondii… 281

(M.C.M. Kierulff, personal observation). Ferreira et al. (2002) described preda-

tion on birds by a group of Cebus at the Tietê Ecological Park, São Paulo. Resende
et al. (2003) reported the same semi-free-ranging group eating an adult male rat
(Rattus rattus) and an infant opossum (Didelphis sp.). These and all other warm-
blooded vertebrates that may be consumed by Cebus are potential source of
T. gondii infection.
As previously described, other NWP species show a variety of items in their diet,
and sometimes they may even prey on small vertebrates such as birds and rodents.
Neotropical primates, despite their preference for forest strata, do forage or move
around for short distances on the ground. However, none have the sophisticated
hunting techniques, so carnivorous diet, and spend as much time on the ground as
the genus Cebus. Due to their behavior and ecology, Cebus seems to be the platyr-
rhine with the greatest access to T. gondii in nature. We believe that the hunting and
exploratory habits of Cebus promotes frequent interactions with the protozoa and
may have led this monkey genus to select and develop a more effective immune
response and consequent resistance to T. gondii.

Final Comments and Research Perspectives

Most NWPs are very susceptible to toxoplasmosis, and according to the authors’
experience, this is possibly the most important cause of acute death of infectious
origin affecting Platyrrhini in captivity in Brazil. However, the susceptibility of
NWPs to T. gondii is variable, with Callitrichinae (Callithrix, Saguinus, and
Leontopithecus) showing mortality rate close to 100 %, Atelidae and some Cebidae
(Saimiri, Aotus) showing variable mortality patterns, and genus Cebus showing
high resistance, with rare deaths reported due to toxoplasmosis.
The reasons for the high susceptibility of most NWPs to T. gondii are not clear.
We believe that it may be due, at least in part, to ecological and behavioral charac-
teristics of different NWPs that led to different degrees of exposure to T. gondii over
evolutionary time. It is possible to speculate that such variable exposure to the pro-
tozoa may have led, along the evolutionary process of NWPs, to differentiated
immunological features culminating in the relative ability to resist the infection.
Naturally, there are many unanswered questions to investigate and novel areas to
research regarding Toxoplasma–NWP interactions. To better understand
Toxoplasmosis manifestation in NWPs, we believe certain studies are very impor-
tant including further research on NWP cellular (proliferative assays, role and mea-
surement of cytokines) and humoral immune responses to the pathogen (more
comprehensive serological surveys, both in captivity and in the wild; use and valida-
tion of different techniques) as well as molecular epidemiology of T. gondii (char-
acterization of strains and their environmental distribution). Obtaining new
information in these areas will certainly help clarify questions about NWP–T. gon-
dii interactions.
282 J.L. Catão-Dias et al.

Finally, considering our laboratory has witnessed on several occasions the devas-
tating effect that toxoplasmosis can have on ex situ conservation programs for
NWPs, we would like to emphasize the importance of curatorial/zoological institu-
tions adopting the best management practices. We see such policies as the only
effective option, currently, for the prevention of new outbreaks that can, otherwise,
decimate genetically invaluable populations of NWPs.

Acknowledgments We are grateful to the staff at LAPCOM–FMVZ/USP, UNIFESP, and UFES;

without their efforts, this chapter would not have been possible. In particular, we would like to
acknowledge the continuous financial support from Fundação de Amparo à Pesquisa do Estado de
São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq). José Luiz Catão-Dias is a recipient of a scholarship by the CNPq (301517/2006-1).

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The Evolution of SIV in Primates and
the Emergence of the Pathogen of AIDS

Edward J.D. Greenwood, Fabian Schmidt, and Jonathan L. Heeney

Introduction

Three decades have passed since the first reports of opportunistic diseases in
previously healthy individuals and the description of acquired immunodeficiency
syndrome (AIDS) as a new human disease (CDC 1981a, b, c). Shortly after, the
causative agent was identified as a T-cell tropic retrovirus (Barre-Sinoussi et al.
1983) that eventually came to be termed human immunodeficiency virus 1 (HIV-1).
HIV-1 is a retrovirus of the Lentivirus genus and the first primate lentivirus to be
discovered. A second human lentivirus, HIV-2, was later discovered in patients
suffering from AIDS in West Africa (Clavel et al. 1986).
Lentiviruses have since been discovered to naturally infect over 40 different
African primate species, termed simian immunodeficiency viruses (SIVs). Study of
SIV infection of the majority of these species is difficult, as many are endangered
and (rightly) protected in the wild, with limited or no captive populations available
for study. However, some important facts have been established. Firstly, it is now
clear that HIV-1 has originated from SIVcpz of the common chimpanzee (Pan trog-
lodytes) and HIV-2 from SIVsmm of the sooty mangabey (Cercocebus atys).
Secondly, some species of SIV-infected African primates are present in European
and US research centers, and thus, the natural history of their infection has been
studied in detail. In particular, the SIV infection of two African green monkey spe-
cies (Chlorocebus sabaeus and C. pygerythrus) and sooty mangabeys has been stud-
ied intensively. In these species, it is clear that the vast majority of individuals do
not progress to AIDS.
In contrast, Asian macaques are not infected with SIV in the wild, but can
develop AIDS when experimentally infected with SIV from other species,

Edward JD Greenwood and Fabian Schmidt contributed equally to this work

E.J.D. Greenwood • F. Schmidt (*) • J.L. Heeney
Department of Veterinary Medicine, University of Cambridge, Cambridge, UK

J.F. Brinkworth and K. Pechenkina (eds.), Primates, Pathogens, and Evolution, 291
Developments in Primatology: Progress and Prospects,
DOI 10.1007/978-1-4614-7181-3_10, © Springer Science+Business Media New York 2013
292 E.J.D. Greenwood et al.

providing a model for disease in humans caused by HIV. Rhesus macaques (Macaca
mulatta) and pig-tailed macaques (Macaca nemestrina) are the two species princi-
pally used in these studies. These models were first established after macaques in
numerous American primate centers developed AIDS-like clinical signs and were
also found to be infected with T-cell tropic retroviruses, which collectively came to
be termed SIVmac (Benveniste et al. 1986; Daniel et al. 1984). Due to the similarity
between SIVmac and SIVsmm, it was hypothesized early on that SIVmac could
have its origins in SIVsmm infection of sooty mangabeys (Murphey-Corb et al.
1986), which has since been confirmed. It is likely that SIVsmm was unknowingly
transmitted from sooty mangabeys into macaques during invasive experiments for
the study of prion diseases in American primate centers and subsequently spread
within captive macaque populations (reviewed by Apetrei et al. 2006).
Comparison of the pathogenic infection of humans and macaques with HIV/SIV
with the nonpathogenic infection of sooty mangabeys and African green monkeys
has provided key insight into the pathways most important in the development of
AIDS in susceptible species and the host mechanisms that have evolved in African
primate species to avoid disease as a result of lentivirus infection.
In this chapter, we will first discuss the age and diversity of primate/human len-
tiviruses, the outcome of SIV transmission into humans, and the mechanisms pro-
posed to have promoted the pandemic spread of HIV-1. Next, we will compare the
natural history of pathogenic HIV infection of humans and SIV infection of Asian
macaques (the best available animal model of human HIV/AIDS) with the non-
pathogenic SIV infection of sooty mangabeys and African green monkeys. We will
discuss in depth the mechanisms that have been proposed to explain the dichoto-
mous outcome of lentivirus infection between these groups. We will then examine
the specific differences between HIV-1 and other primate lentiviruses, including
potential mechanisms for the high pathogenicity of HIV-1. Finally, we will review
what is known of the host–virus relationship in the species in which the HIV-1 lin-
eage evolved and suggest that examination of this relationship is of special impor-
tance to HIV-1 and SIV research.

Age and Diversity of the SIV Lineage

SIVs observed in wild African primates are generally species-specific: multiple iso-
lates of virus from one primate species generally form monophyletic lineages in
phylogenetic trees. The degree to which SIVs differ within one species varies, but is
largely biased by the number of isolates sequenced and their geographical distribu-
tion (Bibollet-Ruche et al. 2004; Liegeois et al. 2012). Species with highly diver-
gent SIVs have also been observed, which are usually the result of cross-species
transmissions, sometimes followed by recombination between distant SIVs
(Aghokeng et al. 2007; Liegeois et al. 2012; Souquiere et al. 2001). High frequen-
cies of recombination are a characteristic of primate lentiviruses (Chen et al. 2006),
but such recombination events are most apparent when heterologous viruses
The Evolution of SIV in Primates and the Emergence of the Pathogen of AIDS 293

recombine. These so-called mosaic or chimeric viruses are described for African
green monkeys, mandrills (Mandrillus sphinx), and chimpanzees (Bailes et al.
2003; Jin et al. 1994; Takemura and Hayami 2004) and can be identified when com-
paring phylogenetic trees created using alignments from different parts of the
genome, as in Fig. 1.
A precise age of primate lentiviruses has been difficult to ascertain, but has been
estimated in several studies. Such dating methods normally require the use of a
“molecular clock,” in which sequences are compared, and a known or estimated rate
of genetic change is applied to estimate the time to the most recent common ances-
tor. Estimates resulting from molecular clock methods are dependant on how this
rate of change is estimated. While this is relatively simple for eukaryotic species, as
the rate of genetic change is both slow and well established for different species,
calibrating a molecular clock for retroviruses, which change extremely rapidly and
often recombine, is much more challenging.
Using only relatively modern SIV and HIV sequences of known dates (from
1975 to 2005) to calibrate a molecular clock resulted in a estimate that the primate
lentivirus lineage is only centuries old (Wertheim and Worobey 2009). However,
this dating was controversial as it was already suspected that using only modern
sequences to extrapolate the history of a possibly ancient lineage would lead to
erroneous estimates (Sharp et al. 2000).
However, a recent study of SIV infection of primates on the African island of
Bioko has allowed for a new calibration of the molecular clock estimate of the age
of this lineage (Worobey et al. 2010). The island has been separated from the
African mainland for a period of 10,000–12,000 years and accommodates a num-
ber of primate species. Individuals from four of these species were found to be
infected with SIV. Most importantly, the Bioko drill (Mandrillus leucophaeus
poensis) is infected with an SIV similar to that isolated from the mainland drill
(Mandrillus leucophaeus leucophaeus). The time to the most recent common
ancestor of SIVdrl from Bioko and SIVdrl from mainland Africa is therefore
known to be at least 10,000 years old, providing a new method for calibrating the
molecular clock. The resulting estimate is that SIVs have been present in African
primates for 76,000 years.
Finally, evidence exists that the primate lentivirus lineage is ancient. The
genomes of a number of lemur species of genera Cheirogaleus and Microcebus
contain sequences of a lentivirus that has at some point infected germ line cells and
become integrated into the genome—an endogenous lentivirus. After integration
into the genome, the sequences of endogenous retroviruses are expected to be sub-
ject to the same rate of mutation as other host genomic sequences. This rate is well
established for eukaryotic species and is much slower than the rate of mutation of
exogenous retroviruses. Endogenous retroviruses are therefore ideal for estimating
dates on ancient timescales. It seems that there were two independent integration
events, both estimated to have occurred around 4 million years ago (Gifford et al.
2008; Gilbert et al. 2009). Unless SIV was introduced to Madagascar independently
and prior to the introduction of SIV to the African mainland, this would indicate that
the African primate lentivirus lineage is at least equally as ancient.
294 E.J.D. Greenwood et al.

Fig. 1 Phylogenetic trees demonstrating the relationships between different SIVs and HIV-1 and
HIV-2. Trees were created from alignments using nucleic acid sequences from the (a) gp41 and
(b) protease genes. Scale bar indicates 0.9 substitutions per site. SIVagmSab is excluded from the
protease tree as a recombination event has occurred in the region used for this alignment
The Evolution of SIV in Primates and the Emergence of the Pathogen of AIDS 295

In addition, in general, the relationship between SIVs of different species mirrors

the relationships between their primate hosts, with SIVs from closely related pri-
mates being the most similar to one another. It has therefore been suggested that
species-specific SIVs could be the result of concurrent host diversification, with
splits in the SIV lineage occurring at the same time as splits in the primate lineage
(host-dependant evolution) (Sharp et al. 2000). The SIVs of African green monkeys
show particularly strong evidence of this. Each of the four species of African green
monkey (Chlorocebus aethiops, C. tantalus, C. pygerythrus, C. sabaeus) is infected
with a species-specific SIV, and the genetic divergence between viruses mirrors the
divergence of the host species (Jin et al. 1994; Muller et al. 1993; Sharp et al. 2000).
For host-dependant evolution to occur, the most recent common ancestor of the four
African green monkey species must have been infected with SIV. The time to the
most recent common ancestor of these species is approximately 3 million years
(Fabre et al. 2009).

Distribution of Accessory Genes in the Primate

Lentivirus Lineage

HIV/SIV are retroviruses of the Lentivirus genus. The genomes of all retroviruses
include the genes gag, pol, and env, the major structural and enzymatic proteins of
the virus. All known members of the Lentivirus genus also possess the regulatory
proteins Rev and Tat, including the oldest recognized lentivirus, “RELIK,” an
endogenous retrovirus estimated to have integrated into the genome of the European
rabbit (Oryctolagus cuniculus) over 12 million years ago (Katzourakis et al. 2007;
Keckesova et al. 2009). All extant lentiviruses found in primates, cats, sheep, and
cattle possess an additional gene, vif.
In contrast, four genes are unique to extant primate lentiviruses; nef and vpr are
found in all primate lentiviruses, while vpx and vpu are found in nonoverlapping
subsets of these viruses. Our understanding of the functions of vif, nef, vpr, vpx, and
vpu has increased dramatically during recent years, and it can be concluded that all
factors have evolved to play a role in counteracting host defense mechanisms called
restriction factors (Ayinde et al. 2010; Kirchhoff 2010).
Vpx, one of the two genes unique to primate lentiviruses, evolved in the Papionini
tribe of primates, and the distribution of this gene within primate SIVs is likely to
have been increased as a result of several recombination events between SIVs of
different primate species (Takemura and Hayami 2004). It is found in SIVsmm (of
sooty mangabeys), SIVrcm (red-capped mangabey, Cercocebus torquatus),
SIVmnd-2 (mandrill), and SIVdrl (drill). The other gene unique to primate lentivi-
ruses, vpu, was acquired by an SIV within a subset of guenon species (Bailes et al.
2003). Guenons (tribe Cercopithecini) are a species-rich group of primates; how-
ever, only four guenon species, which associate in the wild, carry SIVs with the vpu
gene. These are the mona monkey (Cercopithecus mona), greater spot-nosed mon-
key (C. nictitans), and mustached monkey (C. cephus), infected with SIVmon,
296 E.J.D. Greenwood et al.

SIVgsn, and SIVmus, respectively. A fourth Cercopithecus species, Dent’s mona

monkey (C. mona denti), was found to be infected with an SIV harboring the vpu
gene (SIVden), but with a shorter coding region (Dazza et al. 2005; Schmokel et al.
2010). Two other nonhuman primate species are infected with a vpu-carrying
virus—the chimpanzee, Pan troglodytes, infected with SIVcpz, and the western
gorilla (Gorilla gorilla) infected with SIVgor. SIVcpz has been identified in two
subspecies of chimpanzees, Pan troglodytes troglodytes and P. t. schweinfurthii,
while surveys of wild P. t. verus and P. t. ellioti (previously known as P. t. vellero-
sus) have demonstrated with some confidence that they are not infected (Sharp and
Hahn 2011). SIVgor has been identified in the western lowland gorilla subspecies
(Gorilla gorilla gorilla) and has thus far been found exclusively in Cameroon (Neel
et al. 2010). SIVgor is highly related to SIVcpz and gorillas have most likely become
infected more recently through cross-species transmission of SIVcpz from Pan
troglodytes troglodytes.
The recombination events involved in the genesis of SIVcpz/SIVgor are of
particular interest. Phylogenetic analyses suggest that a vpx-expressing virus
found in red-capped mangabeys was transmitted into chimpanzees where it
recombined with an SIV expressing the accessory gene vpu. The chimpanzee
mosaic virus shows homology with SIVs found in a subset of guenons within
the 3′ half of its genome (Courgnaud et al. 2002). The 5′ region of the genome
and possibly the nef gene at the 3′ end are closely related to SIVrcm (Beer et al.
2001; Kirchhoff 2009). One recombination crossover is therefore likely to have
occurred in the short region between vpr and vpu and another between env and
nef (Kirchhoff 2009; Sharp et al. 2005). It is believed that the vpx gene was
transferred within the initial recombination, but must have subsequently been
lost as no remnant of this gene is apparent in contemporary SIVcpz. The vpu
gene however remained conserved.

Transmission of SIV into Humans and the Spread of HIV

There are four recognized HIV-1 groups: M, N, O, and P. As each of these HIV-1
groups is closest in sequence homology to different isolates of SIV in chimpanzees
or gorillas, it is clear that there have been at least four cross-species transmission
events into humans, with M and N group viruses having their source in chimpanzees
of the Pan troglodytes troglodytes subspecies and O and P group most closely
related to SIV of gorillas (Keele et al. 2006; Plantier et al. 2009; Van Heuverswyn
et al. 2006). To prevent confusion, it should also be noted that there is a separate
alphabetical nomenclature for describing different subtypes (also referred to as
clades) of HIV-1 group M viruses. Based on sequence homology, HIV-1 group M
viruses are assigned to subtypes from A to K or identified to have been generated
through recombination between viruses of previously identified subtypes.
Non-M HIV-1 viruses are mostly restricted to Cameroon. Group O infection
accounts for around 1 % of all HIV-1 infection in Cameroon (Vergne et al. 2003),
The Evolution of SIV in Primates and the Emergence of the Pathogen of AIDS 297

while N has been identified in less than 20 individuals (Vallari et al. 2010a) and
group P in only two cases so far (Plantier et al. 2009; Vallari et al. 2010b). Clinical
data regarding non-M infections is limited, but it is important to note that group O
and group N infections have been identified in patients with AIDS (Ayouba et al.
2000; Gurtler et al. 1994), while the first group P infection to be identified was asso-
ciated with depleted CD4+ T cells and a high viral load (Plantier et al. 2009). There
is therefore no evidence that HIV-1 non-M infections are less pathogenic than HIV-1
M infections, despite their more limited spread.
In addition to HIV-1, there is a second human immunodeficiency virus, HIV-2.
In contrast to HIV-1, HIV-2 infects globally only between 1 and 2 million individu-
als and is predominantly confined to West Africa (Campbell-Yesufu and Gandhi
2011). In common with HIV-1, HIV-2 consists of several groups, each the result of
a different cross-species transmission event. Eight groups, A–H, have been recog-
nized, with only groups A and B having spread more extensively within the human
population. The remaining groups are limited to infections of single individuals.
HIV-2 is the result of transmission of SIV from the sooty mangabey, and because of
this different source, HIV-2 has a different genetic structure to HIV-1. Viral genomes
of several primate lentiviruses, along with HIV-1 and HIV-2, are compared in Fig. 2.
There are critical differences between HIV-1 and HIV-2 infection of humans that are
discussed at the end of this chapter.
As with dating the age of the primate lentivirus lineage, dating the cross-species
transmission events leading to HIV has been difficult. However, the availability of
two HIV-1 group M sequences attained from archived samples from 1959 and 1960,
from Kinshasa (formally Leopoldville), Democratic Republic of Congo (DRC, for-
mally Belgian Congo and, later, Zaire), allows for greater confidence in dating the
origin of the HIV-1 group M pandemic. While the chimpanzees infected with
SIVcpz isolates most closely related to HIV-1 group M are found in Cameroon, it
seems that Kinshasa is a candidate for the epicenter of the HIV-1 pandemic. A study
conducted in 1997 indicated that there is unparalleled genetic diversity of HIV-1 M
in the DRC, with all subtypes represented, along with recombinant forms not repre-
sented outside of the DRC (Vidal et al. 2000). Notably, HIV-1 extracted from the
two samples from 1959 and 1960 show a high degree of genetic divergence—com-
parable to the genetic difference between two contemporary isolates of different
subtypes. This suggests that by 1960, HIV-1 had already circulated extensively in
humans in this region (Worobey et al. 2008).
Using sequences from these two early samples, along with later samples to cali-
brate a molecular clock, leads to an estimate that the cross-species transmission
event leading to HIV-1 group M most likely occurred near the start of the twentieth
century, between 1873 and 1924 (Worobey et al. 2008). Dating the cross-species
transmission events that resulted in the other HIV-1 and HIV-2 groups is more prob-
lematic due to the markedly fewer available sequences and the lack of sequences
from older achieved material. Estimates for the date of the four cross-species trans-
mission events leading to HIV-1 groups O and N and HIV-2 groups A and B are all
also within or near the beginning of the twentieth century (de Sousa et al. 2010;
Wertheim and Worobey 2009).
298 E.J.D. Greenwood et al.

Fig. 2 Annotated depiction of the genomes of HIV-1, HIV-2, and relevant SIVs. Diagrams are
based on annotated sequences, with the sequence used named in brackets. Numbers 1–3 at left
indicate reading frames

Pandemic Spread of HIV

Human exposure to SIV-infected primates is unlikely to be novel to the late nineteenth

and early twentieth century. Notably, some strains of another human retrovirus, human
T-cell lymphotropic virus (HTLV), present in Africa seem to be the result of cross-
species transmission events from primates occurring thousands of years ago (Switzer
et al. 2006; Van Dooren et al. 2001), also most likely through bushmeat. Several
authors have therefore attempted to identify mechanisms to explain why only the
cross-species transmission events in this recent time frame have resulted in the several
HIV-1 and HIV-2 epidemics and the HIV-1 group M pandemic—i.e., mechanisms
that promoted the spread of HIV that are unique to the twentieth century.
The Evolution of SIV in Primates and the Emergence of the Pathogen of AIDS 299

Firstly, Worobey and colleagues have suggested that the development of large
urban centers, not present in western Central Africa prior to 1900, facilitated the
spread of HIV-1 M. They assert that prior to 1910, there was not a single site with a
population greater than 10,000 people in western Central Africa (Worobey et al.
2008). Kinshasa underwent particularly rapid growth, from only a few thousand
people in 1905, to around 40,000 in 1940, and over 400,000 in 1961 (Chitnis et al.
2000).
However, this rapid urbanization, combined with the (often-forced) movement of
workers, led to disruption of social norms that may have been a greater contributing
factor than the size of the urban centers alone. De Sousa and colleagues carried out
extensive analysis of colonial medical articles and reports regarding Kinshasa from
the start of the twentieth century to the country’s independence (and general expul-
sion of Belgian authorities) in 1960 (Chin 2007; de Sousa et al. 2010). They note
that in 1929 there were a large number of commercial sex workers, possibly due to
the heavily male biased population, at 4:1 males to females. They also find that
sexually transmitted diseases, including syphilis and other genital ulcerative dis-
eases, were highly prevalent, with one survey in 1930–1932 finding that 5 % of the
female population had active genital ulcers (de Sousa et al. 2010). While the low
risk of HIV-1 transmission from heterosexual sex, estimated to be between 0.01 and
1.1 % per act (Boily et al. 2009), could be viewed as a barrier to early spread of
HIV-1 through purely sexual contact, the risk of transmission is considerably
increased when genital ulcers (and other non-ulcerative sexually transmitted dis-
eases) are present in either the HIV-positive or HIV-negative sexual partner, with
most studies finding a 2–5-fold increase in risk of infection, but some finding the
risk of transmission to be almost 20-fold higher (Boily et al. 2009; Fleming and
Wasserheit 1999).
The role of non-sterile injections throughout sub-Saharan Africa in the twentieth
century has also been proposed as a major factor allowing the early spread of HIV-1.
Marx and colleagues propose that initial “serial human passage” of the virus through
non-sterile injections allowed the adaptation to humans which they suggest would
be required for subsequent spread by sexual transmission (Marx et al. 2001). They
point to numerous mass injection campaigns in sub-Saharan Africa and evidence
for large numbers of injections involving the reuse of non-sterilized injection mate-
rials in the twentieth century, especially since the discovery of penicillin and its use
in Africa from the 1950s onwards.
The mechanisms reviewed above that are proposed to have facilitated the spread
of HIV-1 span the entirety of the twentieth century. Interestingly, in their 2008 anal-
ysis, Worobey et al. propose a model of the growth of HIV-1 that suggests relatively
slow growth prior to 1960, followed by much more rapid expansion after this date.
Although their analysis is likely to be subject to bias due to the limited availability
of samples available, this computational analysis has some support from the early
reports of AIDS in Africa, as it correlates with marked increases in the opportunistic
diseases that now define AIDS, such as Kaposi’s sarcoma and esophageal candidia-
sis, occurring in Kinshasa, Uganda, Zambia, and Rwanda from the late 1970s to the
early 1980s (Quinn et al. 1986). This would be consistent with aggressive spread of
300 E.J.D. Greenwood et al.

HIV-1 in the 1960s and early 1970s, due to the approximately decade-long incuba-
tion period between HIV-1 infection and the development of AIDS (see below).
Together, these lines of evidence tend not to favor extensive spread in urban cen-
ters in the early part of the twentieth century. May and colleagues have proposed an
elegant model of rural spread among “loosely interlinked villages” (May et al.
2001). One significant attraction of this model is that it predicts that HIV-1 could
persist at a very low level for decades within rural villages, with only limited spread
within each village compensated for by introduction to new villages, preventing the
virus from becoming extinct. In this model, the period of extremely slow spread is
then followed by much more rapid expansion of HIV, without requiring mecha-
nisms postulated above. Of course, this does not exclude a role for the other factors
reviewed, especially as urbanization, high levels of other sexually transmitted dis-
eases, and the reuse of non-sterilized injection materials could all have contributed
to the introduction of HIV-1 to urban centers and extensive spread within these
centers—which was almost certainly a requirement for HIV dissemination through-
out and beyond Africa.
While HIV-1 group M subtype B causes only a minority of HIV-1 infections
in Africa, it is the most prevalent subtype in a large number of countries outside
of Africa, including the USA and Europe. The disparity between the prevalence
of subtype B outside and within Africa could indicate that the pandemic beyond
Africa was the result of a single transmission from Africa. Gilbert and col-
leagues examined sequences from archived samples collected in 1982 and 1983
of Haitian nationals that had immigrated to the USA after 1975 and hospitalized
with AIDS prior to 1981 (Gilbert et al. 2007). They focused on these patients as
shortly after the recognition of AIDS as a novel syndrome firstly primarily in
homosexual men in the USA in 1981 (Gottlieb et al. 1981), and intravenous
drug users in 1982 (CDC 1982b), there were reports of Haitian nationals in the
USA suffering from AIDS (CDC 1982c). The majority of these Haitian patients
indicated they had practiced neither homosexual sex nor intravenous drug use,
suggesting that being a recent Haitian immigrant was an independent risk factor
for the development of AIDS.
In their analysis of sequences from these patients, in addition to other sequences
of HIV-1 isolates from Haiti, and sequences representing HIV-1 subtype B isolates
from multiple other countries, Gilbert and colleagues find that subtype B is the most
genetically diverse within Haiti, indicating an earlier introduction of HIV-1 subtype
B into this nation than any other, and that the HIV-1 epidemic in Haiti was the result
of a single introduction of the virus from Africa. They also show that with few
exceptions, the pandemic spread of HIV-1 subtype B beyond Haiti (including North
American, South American, European, and Asian countries) is the result of another
single founder event linked to Haiti, which they postulate to be most likely the intro-
duction of HIV-1 to the USA through immigration.
The authors estimate that the introduction of HIV-1 from Africa into Haiti
occurred between 1962 and 1970. Interestingly, it seems that there was a large
movement of Haitians to the DRC (then Zaire) in the early 1960s following two
events: firstly, political problems appearing in Haiti after Francois Duvalier
The Evolution of SIV in Primates and the Emergence of the Pathogen of AIDS 301

(‘Papa Doc’) took power in 1957 and, secondly, the vacancies for “executive”
positions (administrators, healthcare workers, and teachers) in Zaire following
the expulsion of the Belgian authorities in 1960. Many Haitians returned to Haiti
or to other nations in the late 1960s and early 1970s (Chin 2007; Molez 1998; Piot
et al. 1984), possibly indicating a potential mechanism by which HIV-1 was
introduced into Haiti. Gilbert et al. also estimate that the single founder event
leading to the spread of HIV-1 subtype B from Haiti occurred between 1966 and
1972 (Gilbert et al. 2007), which is constant with the earliest cases of AIDS in the
USA, retrospectively diagnosed as occurring in 1978 (CDC 1982a).

Natural History of HIV/SIV Infection of AIDS-Resistant

and AIDS-Susceptible Species

The clinical progression of HIV-1 infection of humans is normally monitored by

assessing the number of peripheral blood CD4+ T cells and the peripheral blood viral
load. In the acute stage of infection, the highest levels of virus replication are reached,
with viral loads frequently in the region of 106–107 copies of the RNA genome per ml
of blood (Kaufmann et al. 1998; Rieder et al. 2010). This occurs concurrently with a
sudden drop in the CD4+ T-cell count that is partly recovered after the first few months
of infection. In the chronic phase of infection, a lower, relatively stable “set point”
viral load is established, generally in the region of 104–106 copies/ml, with the chronic
viral load correlating with the rate of disease progression (Mellors et al. 1996;
Rodriguez et al. 2006). Levels of peripheral blood CD4+ T cells slowly decline, until
they are depleted to the point that the immune system can no longer function. This
depletion of CD4+ T cells is thought to be due to a number of factors, including, but
certainly not limited to, the direct infection of CD4+ T cells by HIV-1. The mean
CD4+ T-cell count in healthy HIV-negative adult humans has been found to be
between 700 cells/μl and 1,200 cells/μl in studies of numerous different populations
(Aina et al. 2005; Hazenberg et al. 2000; Kibaya et al. 2008; Tugume et al. 1995). The
risk of developing opportunistic infections—the clinical manifestations of AIDS—is
greatly increased when the CD4+ count drops below 200 cells/μl (Begtrup et al. 1997;
Phair et al. 1990; Phillips et al. 1989). The median survival time following untreated
HIV-1 infection is approximately 8–12 years (Collaborative Group on AIDS
Incubation and HIV Survival 2000; Morgan et al. 2002).
Two primate species are commonly used in modeling the pathogenesis of HIV:
the rhesus macaque and the pig-tailed macaque. SIVmac/SIVsmm infection of
rhesus macaques follows a similar, albeit accelerated, course to HIV-1 infection
of humans, with progression to AIDS normally occurring within 6 months to 3
years of infection (Brown et al. 2007). Pig-tailed macaques seem to have an even
higher susceptibility to developing AIDS. In addition to developing AIDS as a
result of SIVsmm or SIVmac infection, they also can develop AIDS following
infection with SIVagm, which does not cause disease in rhesus macaques (Favre
et al. 2009; Hirsch et al. 1995).
302 E.J.D. Greenwood et al.

The natural history of the SIV infection of African green monkeys or sooty
mangabeys with SIVagm and SIVsmm, respectively, is very different. These ani-
mals generally do not develop disease despite peripheral blood viral loads similar to
those of humans and rhesus macaques. A recent study of sooty mangabeys has
demonstrated that SIV infection does result in an extremely slow depletion of CD4+
T cells (Taaffe et al. 2010), but unlike in humans and rhesus macaques, the magni-
tude of CD4+ T-cell loss does not correlate with viral load. CD4+ T-cell loss has not
been reported in African green monkeys. However, in both African green monkeys
and sooty mangabeys, measurements of CD4+ T cells are complicated by factors
relating to expression of CD4 (see below). For both of these species, only a single
case of an AIDS-like condition in an SIV-infected animal has been described (Ling
et al. 2004; Traina-Dorge et al. 1992). The mechanisms by which these species cir-
cumvent disease development are of great interest and are discussed below.

Limiting Target Cell Availability

Entry of HIV-1 into the cell is mediated by the use of CD4 as a receptor, along with
a coreceptor. CD4 is expressed principally on CD4+ T cells, and it binds to MHC-II
on antigen-presenting cells. These T cells, upon activation, become “T helper cells,”
which direct the adaptive immune response primarily through cytokine production.
The coreceptor used by HIV-1 is principally the chemokine receptor C-C chemo-
kine receptor type 5 (CCR5), but in some cases a different chemokine receptor,
C-X-C chemokine receptor type 4 (CXCR4). Interestingly, humans lacking CCR5
expression through an inherited mutation are almost completely resistant to infec-
tion by both sexual and parenteral routes (Dean et al. 1996; Wilkinson et al. 1998).
Partly because of the restriction of CCR5 expression to memory T cells, memory
CD4+ T cells are preferentially infected compared to naïve CD4+ T cells. In humans
and rhesus macaques, quantitative PCR detection of the HIV-1 genome in different
CD4 T-cell subsets consistently finds that the majority of infected cells have a mem-
ory CD4+ cell phenotype, though infected naïve T cells are also found (Brenchley
et al. 2004a; Mattapallil et al. 2005; Ostrowski et al. 1999). Macrophages and den-
dritic cells, the primary antigen-presenting cells of the immune system, also express
CD4 and CCR5 and can be productively infected by HIV-1. An obvious potential
mechanism by which species may adapt to SIV infection would therefore be to
restrict the expression of the viral receptor and coreceptor.
Interesting observations have been made regarding CD4 expression in sooty
mangabeys and African green monkeys. Adult uninfected African green monkeys
have a much lower frequency of peripheral blood CD4+ T cells than other primates,
including humans, sooty mangabeys, and rhesus macaques (Beaumier et al. 2009).
They have a large peripheral blood T-cell population with a unique phenotype of
CD4-CD8αdim. Interestingly, it seems that while naïve T cells express normal levels
of CD4, activation and development of a subset of these into a memory phenotype
is associated with down-modulation of CD4 and upregulation of CD8α. Curiously,
The Evolution of SIV in Primates and the Emergence of the Pathogen of AIDS 303

these cells retain the functionality of T helper cells and are restricted to antigen
presented on MHC-II, despite the lack of CD4 expression (Beaumier et al. 2009).
As in humans and rhesus macaques, CD4+ T cells with a memory phenotype make
up the majority of SIVagm-infected CD4+ T cells. However, while the limited num-
ber of memory T cells that maintain CD4 expression has the highest level of infec-
tion, the CD4-CD8αdim population contains relatively low numbers of infected cells.
Thus, the loss of CD4 expression in differentiation from naïve to memory appar-
ently protects a subset of CD4+ memory T cells from infection.
Similarly, a large population of CD4-CD8- (“double-negative”) peripheral blood
T cells has been identified in both infected and uninfected sooty mangabeys (Milush
et al. 2011). These cells were first identified when it was discovered that some
strains of SIVsmm are in fact capable of causing very rapid and severe loss of CD4+
T cells in sooty mangabeys, to levels that would be associated with AIDS in humans
and macaques. Despite this, these animals maintain a competent immune system,
and this led to the discovery that the CD4–CD8- T cells seem to be capable of per-
forming functions normally associated with CD4+ T cells. In both species, the seg-
regation of functions normally carried out by CD4+ T cells to cells resistant to
infection means that regardless of the extent of CD4+ T-cell depletion, a minimum
level of immune function can be maintained.
Expression of the coreceptor CCR5 has also been examined in primate species,
in the context of SIV infection. One report has compared the frequency of CCR5+
CD4+ T cells in the blood and lymph nodes of uninfected individuals of multiple
primate species—comparing those species that are known to be SIV infected with
the wild (including African green monkeys and sooty macaques) with humans and
rhesus macaques. This report demonstrates that sooty mangabeys, African green
monkeys, and numerous other African species that are host to SIV have a much
lower frequency of CD4+ T cells that express CCR5 (Pandrea et al. 2007a). The low
frequency of CCR5 expression has therefore been assumed to be convergent evolu-
tion by SIV-exposed species to restrict target cell availability.
A recent study has further demonstrated that upon in vitro stimulation, naïve T
cells from sooty mangabeys show more restricted CCR5 upregulation compared to
naïve T cells from rhesus macaques. This failure to upregulate CCR5 is especially
pronounced in cells that take on a central memory phenotype after activation. This
may protect central memory T cells from infection; as in sooty mangabeys, this
population shows a much lower frequency of infection than the Tcm population of
rhesus macaques (Paiardini et al. 2011). However, it should be stressed that in this
comparison, different viral isolates were used to infect the two different species, and
the possibility of minor differences in viral tropism cannot be excluded.
Further examination of sooty mangabeys has demonstrated a relatively high fre-
quency of CCR5 deletion mutant alleles, with 8 % homozygous for CCR5 deletions
in a large captive population (Riddick et al. 2010). Interestingly, in contrast to HIV-
1-infected humans, these animals are not resistant to SIV infection. In the captive
population studied, prevalence of naturally acquired SIV infection was similar in
the CCR5 deletion homozygous animals as in heterozygous animals or those with
two functional CCR5 alleles (Riddick et al. 2010). Viruses isolated from both
304 E.J.D. Greenwood et al.

homozygous CCR5 deletion animals and those expressing CCR5 were able to use a
variety of additional coreceptors, including CXCR6, GPR15, and GPR1, in addition
to maintaining CCR5 utilization. Similarly, in red-capped mangabeys, two studies
have shown a high frequency of one CCR5 deletion (CCR5 Δ24), with approxi-
mately 60–70 % of animals being homozygous for this deletion (Beer et al. 2001;
Chen et al. 1998). Cells from red-capped mangabeys with the homozygous CCR5
deletion were completely resistant to infection by CCR5-using viruses from other
species. Interestingly, SIV of red-capped mangabeys has apparently adapted to this
selection pressure, as this virus uses principally CCR2b as a coreceptor and cannot
use CCR5 (Beer et al. 2001; Chen et al. 1998).

Damage to the Mucosal Immune System and Bacterial

Translocation

The clinical picture of HIV-1 infection of humans suggests that damage to the
immune system is a chronic, gradual process, resulting in the cumulative immuno-
logical collapse that allows the onset of opportunistic disease. In contrast, a number
of studies suggest that a great deal of irreversible damage occurs early in infection
in the secondary lymphoid organs and gut-associated immune system.
The gut-associated lymphoid system (GALT) can be divided into inductive sites
(gut-draining mesenteric lymph nodes and Peyer’s patches), at which T-cell
responses are generated through interaction with antigen-presenting cells and effec-
tor sites, primarily lymphocytes residing in the lamina propria (lamina propria lym-
phocytes, LPLs) and between the epithelial cells of the mucosal surface
(intraepithelial lymphocytes, IELs). There are a large number of CD4+ T cells in the
Peyer’s patches and the lamina propria, with expression of CCR5 at a much higher
frequency in these compartments than CD4+ T cells in the peripheral blood or other
secondary lymphoid tissues.
Several studies have demonstrated a dramatic depletion of CD4+ T cells in one
or more of these compartments from the very earliest stage of HIV-1 infection, with
little or no recovery of these cells in the chronic phase of infection (Brenchley et al.
2004b; Estes et al. 2008; Guadalupe et al. 2003; Mehandru et al. 2004). The result
of this T-cell loss has been the topic of much discussion in the field. Some authors
have stated that the gut mucosal immune system contains the majority of lympho-
cytes in the body (Guadalupe et al. 2003; Mehandru et al. 2004; Veazey et al. 1998),
with some further stating that the acute loss of CD4+ T cells in the gut therefore
“reflects the loss of most CD4+ T-cells in the body” (Brenchley and Paiardini 2011).
However, available literature on this subject (Ganusov and De Boer 2007) finds that
it is more likely that at most around 20 % of all lymphocytes are resident in the
human gut, with a similar percentage of the total CD4+ T cells found there.
Despite this, there does seem to be at least one important consequence of damage
to the mucosal immune system by HIV-1 infection, as it appears to result in the loss
of integrity of the mucosal barrier. This allows translocation of bacteria and
The Evolution of SIV in Primates and the Emergence of the Pathogen of AIDS 305

bacterial products and their systemic dissemination, which in turn causes the pro-
duction of pro-inflammatory cytokines by cells of the innate immune system. The
level of bacterial translocation, as measured by concentration of lipopolysaccharide
(LPS) in the peripheral blood, is significantly upregulated in chronically infected
HIV-1 patients when compared to uninfected controls in European and US cohorts
(Brenchley et al. 2006). LPS translocation also shows a significant correlation with
the level of peripheral blood activated CD8+ T cells, as measured by expression of
the markers CD38 and HLA-DR (Brenchley et al. 2006). This acute damage to the
gut mucosa therefore seems to be a major contributing factor to the chronic immune
activation that is thought to drive the progression from chronic HIV-1 infection to
AIDS (see below). The damage to the gut mucosal immune system in SIVmac-
infected rhesus macaques is similar to humans, with profound depletion of CD4+ T
cells occurring within weeks of infection at multiple sites of the GI tract and
increased levels of plasma LPS (Brenchley et al. 2006; Ling et al. 2007; Mattapallil
et al. 2005; Veazey et al. 1998).
The importance of LPS translocation in HIV-1 pathogenesis, supported by data
from HIV-1+ patients in the USA and Europe, is not entirely supported by two
studies carried out in sub-Saharan Africa. One study finds no difference in LPS
levels between patient samples taken from pre-infection to AIDS (Redd et al.
2009), while another finds a significant difference only between uninfected
patients and patients that have already progressed to AIDS—not earlier in the
disease process (Nowroozalizadeh et al. 2010). In this second study, there is how-
ever a nonsignificant trend for increased plasma LPS in HIV-1-positive patients
compared to negative control individuals and a significant inverse correlation
between peripheral blood CD4+ T-cell counts and plasma LPS. However, these
findings would be compatible with a theory that LPS translocation is allowed to
occur due to loss of CD4+ T cells by other mechanisms and becomes progres-
sively worse throughout the course of disease—rather than necessarily indicating
that LPS translocation is a driving factor in immune activation and loss of CD4+
T cells from the outset of infection.
However, the importance of loss of the integrity of the gut mucosal barrier in
pathogenic lentivirus infections is emphasized by the different outcome in African
nonhuman primates. Surprisingly, two studies have demonstrated that in nonpatho-
genic infection of African green monkeys and sooty mangabeys, there is also mas-
sive depletion of gut mucosal T cells, comparable with depletion seen in
SIVmac-infected rhesus macaques (Gordon et al. 2007; Pandrea et al. 2007b).
Recovery of CD4+ T cells in this compartment after acute infection is variable, with
some animals restoring numbers of cells to near pre-infection levels and some main-
taining very low levels throughout the chronic phase of infection. Despite this, the
mucosal barrier appears to remain intact, as SIV-infected African green monkeys
and sooty mangabeys do not show increased levels of plasma LPS (Brenchley et al.
2006; Gordon et al. 2007; Pandrea et al. 2007b).
The critical difference between the different outcomes may involve a specific
subset of CD4+ T cells, Th17 cells. These cells are postulated to be involved in
defense against bacterial pathogens through the production of the cytokines IL-17
306 E.J.D. Greenwood et al.

and IL-22 (Brenchley et al. 2008; Liang et al. 2006) and are found at higher fre-
quency in the gut mucosa compared with peripheral blood or lungs (Brenchley et al.
2008; Cecchinato et al. 2008). In HIV-1-infected humans and SIVmac-infected rhe-
sus macaques, in addition to the general destruction of gut CD4+ T cells, there is a
specific greater loss of Th17 cells—they are underrepresented in the residual CD4+
T cells (Brenchley et al. 2008; Cecchinato et al. 2008). In contrast, in the SIV infec-
tion of sooty mangabeys, total gut CD4+ T cells are depleted in SIV infection but
the percentage of Th17 cells within the remaining CD4+ cells is not altered
(Brenchley et al. 2008). Furthermore, in a study directly comparing SIVagm infec-
tion of pig-tailed macaques and African green monkeys, significant specific deple-
tion of Th17 only occurs in pig-tailed macaques, which progress to AIDS after
SIVagm infection (Favre et al. 2009). Thus, maintenance of the Th17 cell popula-
tion seems to play an important role in sustaining a competent mucosal barrier,
which in turn prevents systemic spread of bacteria from the gut and the subsequent
immune activation which follows in AIDS-susceptible species. How natural host
species have evolved to avoid this specific depletion of Th17 cells in the face of
massive general loss of CD4+ T cells in the gut has yet to be elucidated.

Persistent or Resolving Innate and Adaptive

Immune Activation

As mentioned above, elevated levels of immune activation in the chronic phase of

infection is seen as an integral part of HIV-1 pathogenesis. Numerous markers of
activation of the innate and adaptive immune system are elevated in HIV-1-infected
humans, with many showing correlation with the rate of disease progression, espe-
cially markers of activation of CD4+ and CD8+ T cells. High levels of immune
activation are thought to drive CD4+ T-cell depletion by inducing multiple rounds
of expansion, followed by activation-induced cell death (AICD), or infection of
activated cells by HIV-1 (as they are more susceptible than naive/resting cells). The
level of increased expression of markers of immune activation on CD8+ T cells such
as CD38 can be used to predict disease progression with power similar to that of set
point viral load measurement (Deeks et al. 2004; Hazenberg et al. 2003). These
activated cells are unlikely to represent exclusively CD8+ T cells that are respond-
ing to HIV-1 antigens, as the proportion of HIV-1-specific CD8+ T cells in the
peripheral blood is generally of a lower percentage than the proportion of CD8+ T
cells expressing immune activation markers (depending on the markers measured)
(Doisne et al. 2004; Gea-Banacloche et al. 2000; Saez-Cirion et al. 2007; Sieg et al.
2005). In addition, while increased frequency and level of CD38 expression on
CD8+ T cells correlates with increased viral load (Deeks et al. 2004) and predicts
faster disease progression in HIV-1-infected humans, the frequency of HIV-1-
specific CD8+ cells has been shown to either correlate inversely with viral load
(Edwards et al. 2002; Ogg et al. 1998) or show no correlation with viral load and
survival (Addo et al. 2003; Schellens et al. 2008).
The Evolution of SIV in Primates and the Emergence of the Pathogen of AIDS 307

Studies using both flow cytometry to measure levels of activated T cells and
microarray analysis to measure gene expression in the peripheral blood and lym-
phoid tissues, throughout the course of infection, have demonstrated striking differ-
ences in immune activation in SIV-infected African green monkeys and sooty
mangabeys compared to disease susceptible HIV-1-infected humans and SIV-
infected rhesus and pig-tailed macaques. In the acute stage of infection, in all spe-
cies, there is a dramatic increase in the number of circulating activated T cells,
expression of genes that are induced by type I interferon, and soluble markers of
immune activation in the plasma (Harris et al. 2010; Kornfeld et al. 2005; Li et al.
2009; Meythaler et al. 2009; Stacey et al. 2009). In all species, the level of immune
activation is reduced in the transition from acute to chronic phase of infection.
However, the key difference between pathogenic and nonpathogenic infections is
the magnitude of this reduction in immune activation after the acute phase. In
humans and macaques, the reduction is only partial, and multiple markers of
immune activation remain highly elevated compared to pre-infection levels or unin-
fected controls.
In contrast, in sooty mangabeys and African green monkeys, the reduction in
immune activation after acute infection is more complete. In sooty mangabeys, a
small number of markers remain slightly (but significantly) upregulated when
chronically SIV-infected animals are compared to uninfected controls (Meythaler
et al. 2009; Silvestri et al. 2003). Interestingly, the extent of low-level chronic
immune activation does correlate inversely with CD4+ T-cell counts (Silvestri et al.
2003). Chronically infected African green monkeys are indistinguishable from
uninfected animals in the majority of markers, as immune activation is completely
suppressed (Kornfeld et al. 2005; Lederer et al. 2009; Lozano Reina et al. 2009).
Of particular interest is the strong induction of interferon-sensitive genes in the
lymph nodes of all species, which are then reduced to near-normal levels in sooty
mangabeys and African green monkeys, while remaining high in humans and rhe-
sus and pig-tailed macaques. Continuous immune activation has severe conse-
quences for the secondary lymphoid environment in pathogenic infections (see
below). In addition, the type I interferon response is likely connected to the other
elements of immune activation. Purified activated CD4+ T cells from HIV-1-
infected humans show much higher expression of interferon-sensitive genes than
activated cells from uninfected humans (Sedaghat et al. 2008), suggesting that type
I interferon plays a role in the excessive T-cell activation seen in HIV-1 infection. In
addition, the down-modulation of a type I interferon receptor on monocytes (which
is likely to be a measure of previous type I interferon exposure) correlates with
lower CD4+ T-cell counts, higher viral loads, and higher expression of CD38 on
CD8+ T cells (Hardy et al. 2009).
The mechanism by which sooty mangabeys and African green monkeys restrict
the interferon response and other elements of immune activation after the acute
stage of infection has been difficult to elucidate and may well be different for each
species. Down-modulation of the interferon response in the lymphoid tissue
occurs in natural host species despite similar levels of viral replication in second-
ary lymphoid tissues (Gueye et al. 2004). One study has shown that in vitro,
308 E.J.D. Greenwood et al.

plasmacytoid dendritic cells (pDCs), a major interferon-producing cell population,

from sooty mangabeys produce much less interferon in response to a range of
stimuli, compared to humans or rhesus macaques, and suggested this results in
reduced immune activation in SIV infection (Mandl et al. 2008). However, this is
somewhat at odds with the high levels of expression of interferon-sensitive genes
in the acute stage of infection in this species, which does not suggest a reduced
capacity for interferon production (Bosinger et al. 2009). Furthermore, immuno-
histochemical stainings of lymph node sections from rhesus macaques, African
green monkeys, and sooty mangabeys during acute infection found pDCs to be
strongly producing type I IFNs in all three species (Harris et al. 2010). Crucially,
in sooty mangabeys and African green monkeys, the interferon response is down-
regulated after the acute phase of infection, while it remains elevated throughout
the infection of rhesus macaques.
Several studies have analyzed gene expression throughout infection, comparing
species with different outcomes. In African green monkeys, there is an early induc-
tion of the immunosuppressive cytokine IL-10 (Jacquelin et al. 2009; Kornfeld et al.
2005; Lederer et al. 2009) when compared to rhesus macaques and pig-tailed
macaques, but this is not found in sooty mangabeys. Several other immunosuppres-
sive genes are upregulated in sooty mangabeys, such as the enzyme indoleamine-
pyrrole 2,3-dioxygenase (IDO/INDO) (Bosinger et al. 2009). However, other studies
using similar methods of analysis at the gene expression level, in addition to immu-
nohistochemistry to analyze expression at the protein level, have found increased
expression of IL-10 and IDO, along with other immunosuppressive molecules pos-
tulated to restrict immune activation in natural host species, in pathogenic infections
of humans (Li et al. 2009) and rhesus macaques (Estes et al. 2006; Jacquelin et al.
2009). Thus, no convincing mechanism has been postulated by which the immune
system is consistently able to bring itself under control in nonpathogenic infections
of these African primates.

Destruction of the Lymph Node Environment

One consequence of high and persistent levels of immune activation appears to be

severe damage to the secondary lymphoid environment. The structure of secondary
lymphatic tissue is of vital importance in mediating interactions between antigen-
presenting cells, T cells, and B cells, which are required to induce an immune
response. The T-cell zones of secondary lymphoid tissues are also the major ana-
tomical site in which naïve T cells may reside. Pathological changes have been
recognized in the lymph nodes of HIV-1 patients since the first description of the
disease, with severe lymphadenopathy being a symptom of HIV-1 infection. Lymph
nodes from HIV-1-infected patients usually show hyperplasia and, in the later stages
of disease, follicular lysis and involution. Damage to the secondary lymph node
environment has long been postulated to be an important underlying factor in AIDS
pathogenesis (Heeney 1995).
The Evolution of SIV in Primates and the Emergence of the Pathogen of AIDS 309

Important changes occur in the T-cell zone, causing dramatic effects on the
whole body T-cell population. Firstly, infected humans and rhesus macaques show
significantly higher levels of collagen deposition in this area compared to unin-
fected controls (Diaz et al. 2010; Estes et al. 2007). As might be expected, the level
of collagen deposition correlates inversely with the number of naïve (and total)
CD4+ T cells resident in the lymph node, suggesting that collagen deposition
discourages habitation of this niche. In addition, the degree of collagen deposition
before the start of antiretroviral treatment inversely correlates with the increase in
peripheral blood T cells after 6 months of treatment (Schacker et al. 2002) or after
an even longer duration of treatment (Kumarasamy et al. 2009). Deposition of col-
lagen may therefore physically restrict the size of the niche available for CD4+ T
cells to occupy. In addition, a study in rhesus macaques has demonstrated that col-
lagen deposition restricts access of naïve T cells to the fibroblastic reticular cell
(FRC) network. This network provides the framework for migration of T cells
through the T-cell zone, and cells of this network also produce IL-7, a key stimulus
for naïve T-cell survival. Loss of access to this network in these animals leads to
apoptosis of naïve T cells in the node (Zeng et al. 2011).
The cause of this damage to the secondary lymphoid tissues has been
connected to two factors. First, in HIV-1-infected humans, there is abnormal
accumulation of CD4+ and CD8+ effector memory T cells in lymphoid tissues.
Effector memory cells are rare in the lymphoid tissue of uninfected humans, as
their normal role is to respond to antigen in non-lymphoid tissue. The extent of the
infiltration correlates with the area of collagen deposition in the tissue (Brenchley
et al. 2004b). These cells are presumably recruited to the lymph nodes as a result
of the pro-inflammatory environment induced by HIV-1, and so the presence of
such cells could either be a cause of damage or only a consequence of the inflam-
mation ongoing in the lymph node.
The second factor is the increased prevalence of TGFβ1-producing regulatory T
cells (Tregs) in the lymphatic tissue of infected humans and rhesus macaques. Tregs
are CD4+ T cells with an immunosuppressive function, including the production of
the cytokine TGFβ1. This cytokine has multiple functions, most prominently as an
immunosuppressant and in mediating wound repair. However, inappropriate expres-
sion is known to have a role in tissue fibrosis in other disease pathways (Branton and
Kopp 1999). In both humans and rhesus macaques, increased numbers of TGFβ1-
expressing cells and increased collagen deposition occur in the acute stage of infec-
tion and become exacerbated through the chronic stage of infection. Interestingly,
increased numbers of T cells producing the immunosuppressive factors IDO and
IL-10 are also found in infected rhesus macaques (Estes et al. 2006). It seems likely
that TGFβ1 is produced as part of a negative feedback response to the high levels of
immune activation, with the side effect of promoting damaging fibrosis in the
immune microenvironment.
The damaging effect of this immunosuppressive response is somewhat paradoxi-
cal, especially given that greater or similar levels of IL-10, IDO, and even TGFβ1
are found in gene expression analyses of SIV-infected African green monkeys and
sooty mangabeys when compared to rhesus and pig-tailed macaques (Bosinger
310 E.J.D. Greenwood et al.

et al. 2009; Jacquelin et al. 2009; Lederer et al. 2009). However, when using immu-
nohistology to analyze expression at the protein level, SIV-infected sooty mang-
abeys do not show increased numbers of TGFβ1-producing T cells in the lymph
node, and increased collagen deposition is not found in the T-cell zone. It seems
likely that the pathological aspect of TGFβ1 production in the lymph nodes of
humans and macaques is due to the failure, despite an immunosuppressive response,
to bring levels of immune activation down after acute infection. The ability men-
tioned above of sooty mangabeys and African green monkeys to resolve innate and
adaptive immune activation after chronic infection therefore likely saves the lymph
node environment from destruction.
The destruction of the lymph node environment in pathogenic infection leads to
loss of naïve T cells and the inability to generate de novo CD4+ and CD8+ T-cell
responses by preventing normal interactions between T cells and antigen-presenting
cells. Both naïve and memory T cells may become inappropriately activated due to the
high levels of interferon and are lost through subsequent apoptosis or direct HIV
infection. The combination of loss of memory T cells and the environment required to
generate new responses presumably drives the immune system to the critical point at
which AIDS occur (Heeney 1995). Existing memory cells to a specific antigen/patho-
gen are sufficiently depleted that there is no longer a meaningful memory response to
opportunistic infections, and the generation of new responses is sufficiently delayed
by the loss of proper lymph node environment and depletion of naïve T cells that an
immune response cannot be mounted in time to prevent illness and death. The key
pathways leading to disease in humans and macaques and how they are prevented in
African green monkeys and sooty mangabeys are outlined in Fig. 3.

The HIV-1 Viral Lineage Has Increased Pathogenic Potential:

Limitations of Available Animal Models

As discussed, SIVsmm generally does not cause disease in sooty mangabey but has
the potential to cause an AIDS-like disease upon direct inoculation of Asian
macaques (McClure et al. 1989; Silvestri et al. 2005). The disease caused in rhesus
macaques is a commonly used animal model for AIDS in humans caused by HIV-1.
SIVsmm has also been transmitted into humans, causing the HIV-2 epidemic.
Numerous lines of evidence demonstrate that in humans, HIV-2 is not as pathogenic
as HIV-1. In surveys taking place in Guinea-Bissau, Holmgren and colleagues
found only a twofold increase in mortality in individuals infected with HIV-2, com-
parable with previous findings within the same geographical region (Holmgren
et al. 2007; Poulsen et al. 1997; Ricard et al. 1994). In contrast, HIV-1 is associated
with a 10–15-fold increase in mortality rate (reviewed in (Jaffar et al. 2004).
Longitudinal investigations with statistically meaningful cohorts indicate that about
80 % of HIV-2-infected individuals do not develop disease and maintain a normal
life expectancy (van der Loeff et al. 2010). One-third of all HIV-2-infected
The Evolution of SIV in Primates and the Emergence of the Pathogen of AIDS 311

Fig. 3 Pathways leading to the development of AIDS in progressive SIV/HIV infection compared
with the nonprogressive SIV infection of African primates

individuals have an undetectable plasma viral load (van der Loeff et al. 2010).
Nevertheless, progressive HIV-2 infections show clinical features that are indistin-
guishable from AIDS caused by HIV-1 (Martinez-Steele et al. 2007).
312 E.J.D. Greenwood et al.

Given the considerable difference in the outcome of HIV-1 and HIV-2 infection
of humans, is it appropriate to use the disease of rhesus macaques infected with
viruses of the SIVsm/SIVmac/HIV-2 lineage as a model for disease caused by
HIV-1 in humans? If it is the case that there are specific aspects of the HIV-1 lineage
that make this virus more pathogenic than HIV-2 in humans, then this model
becomes less attractive in understanding HIV-1 pathogenesis. We will now examine
the differences between these viruses to establish if this is the case.
Despite their different origins, HIV-1 and HIV-2 are relatively closely related
retroviruses with 60 % similarity at the amino acid level in the capsid and poly-
merase proteins and 30 % similarity in the envelope protein (Guyader et al. 1987).
However, the genomes of both viral lineages differ by two genes. While vpx is pres-
ent in the HIV-2/SIVsmm lineage (in addition to SIVrcm, SIVdrl, and SIVmnd2),
the accessory gene vpu, not vpx, is found in HIV-1, in addition to SIVcpz and the
SIVs of four species of the Cercopithecus genus (Barlow et al. 2003; Courgnaud
et al. 2002, 2003; Huet et al. 1990; Strebel et al. 1988).
Both vpu and vpx seem to have evolved to allow lentiviruses to escape host
restriction mechanisms. In macaques, the accessory viral gene vpx has been directly
shown to contribute to virulence. Animals infected with an SIVmac mutant lacking
the vpx gene show lower virus burdens, delayed declines in CD4 lymphocytes, and
either a lack of disease or delayed progression to disease (Gibbs et al. 1995; Hirsch
et al. 1998). The Vpx of the SIVsmm/SIVmac/HIV-2 viral lineage enables the virus
to efficiently replicate in primate macrophages by antagonizing a cellular restriction
factor termed SAM domain and HD domain 1 (SAMHD1) that is expressed in this
cell type (Laguette et al. 2011; Sharova et al. 2008). That vpx can provide a fitness
advantage to the virus was further confirmed in vivo when a wild-type and a vpx-
deleted SIVsmm isolate were inoculated into pig-tailed macaques, resulting in the
vpx mutant showing a strong competitive disadvantage in the early virus dissemina-
tion (Hirsch et al. 1998).
vpu, the accessory gene uniquely expressed in the SIVcpz/HIV-1 lineage, includ-
ing the small subset of guenon SIVs, also seems to posess various functions that
may impact pathogenicity. Vpu helps HIV-1 to escape host restriction and enhances
release of virus particles (Klimkait et al. 1990). Vpu contributes to HIV-1-induced
CD4 receptor downregulation, which enhances virus replication (Willey et al.
1992). In addition, it increases the release of progeny virions from infected cells and
cell-to-cell spread of viral particles by antagonizing tetherin, an interferon-induced
host restriction factor that directly cross-links virions on the host cell surface (Neil
et al. 2008). However, both CD4 and tetherin downregulation are not exclusive to
the lineage of HIV-1 and are facilitated by the nef or env gene in other lentiviruses
(Gupta et al. 2009; Le Tortorec and Neil 2009; Zhang et al. 2009). However, two
functions of vpu have recently been discovered that are thus far unique to HIV-1.
Firstly, Vpu inhibits the recycling of CD1d receptor from endosomal compartments,
strongly inhibiting the ability of infected DC to activate CD1d-restricted natural
killer T cells (NKT cells) (Moll et al. 2010). Secondly, Vpu downregulates the NKT
and B cell coactivator (NTB-A) at the surface of infected cells and as a result inter-
feres with the degranulation of NK cells that recognize the infected cells (Shah et al.
The Evolution of SIV in Primates and the Emergence of the Pathogen of AIDS 313

2010). Importantly, HIV-2-infected cells do not downregulate NTB-A and are killed
more efficiently by NK cells than HIV-1-infected cells (Shah et al. 2010).
Furthermore, vpu seems to have influenced the evolution of the other viral genes.
A mechanism encoded in the viral gene nef is believed to have evolved to restrict
T-cell activation, through down modulation of the T-cell receptor (also referred to as
CD3) in infected cells. However, in all viruses that carry vpu this function of nef is
reported to be lost (Schindler et al. 2006). Immunodeficiency viruses depend on
activated T cells for replication, but accelerated immune activation induces a range
of host defense mechanisms. It has been suggested that acquisition of the vpu gene
by this viral lineage has allowed the virus to trigger immune activation, increasing
viral replication, without the consequence of host-mediated restriction (Kirchhoff
2009). There are therefore reasons to believe that vpu plays an important role in
HIV-1-induced disease, and models of human AIDS should include this factor.
While pig-tailed macaques can become transiently infected with HIV-1 (Agy
et al. 1992), the infection does not persist. Other macaque species are resistant to
infection, as macaque-encoded restriction factors provide dominant-acting blocks
the establishment of infection (Stremlau et al. 2004). Using the macaque model to
investigate influence of the vpu gene in vivo therefore requires more complex
approaches. Our increased understanding of host restriction factors has led to novel
approaches in which HIV-1 isolates have been artificially equipped with viral gene
variants capable of antagonizing the macaque’s host restriction factors (Hatziioannou
et al. 2009). However, to date, these artificial HIV variants are not pathogenic. An
alternative approach has been to infect macaques with partial recombinants between
SIVs and HIV-1 isolates, called SHIVs. The first SHIVs were created by exchang-
ing the SIV envelope for an HIV-1 envelope sequence in order to test HIV-1 vaccine
candidates (Stremlau et al. 2004). Some SHIVs are equipped with the HIV-1 vpu
gene in addition to the HIV envelope, and a few studies indicate that the presence of
vpu can influence the pathogenic outcome in pig-tailed macaques (Hout et al. 2005;
Singh et al. 2001, 2003; Stephens et al. 2002). However, the mechanistic back-
ground remains unknown, especially as the various described functions of vpu are
yet not characterized for these Asian primate hosts. The lack of a suitable model for
HIV-1 has driven the search for alternative animal models such as the rodent model
in which mice were “humanized” with bone marrow/liver/thymus grafts from
human donors (Melkus et al. 2006). To date, these models are still in a premature
state and only further research will establish whether insights into HIV-1 virulence
can be gained with such models.

Examining the Virus–Host Relationships

Responsible for Generating HIV-1

As stated previously, other than HIV-1, only SIVcpz of chimpanzees and SIVmon/
mus/gsn/den of four primate species of the guenon genus carry the vpu gene. Given
the difficulties in assessing the role of vpu in existing in vivo models described
314 E.J.D. Greenwood et al.

above, it would be extremely informative to understand the outcome of SIV infec-

tion in these species. Here, we will review what is known of SIV infection of chim-
panzees and in these guenon species.
Before the origin of HIV-1 was traced to chimpanzees, it was found that chim-
panzees can be persistently infected with HIV-1 (Alter et al. 1984). As no other
species can be persistently infected with HIV-1, the lack of alternatives made this
animal model initially attractive to study HIV-1. However, as the vast majority of
animals infected with HIV-1 failed to develop clinical signs, and due to cost and
ethical reasons, the use of the chimpanzee model for HIV-1 study has been gener-
ally abandoned. Nevertheless, by 1996, over 200 chimpanzees had been infected
with HIV-1 (Committee on Long-Term Care of Chimpanzees 1997). Many of these
animals have now been infected for over 20 years. Of these, only 5 animals, all at
the Yerkes Primate Centre, have been described as developing AIDS and subse-
quently euthanized (Juompan et al. 2008). The majority of these 200 HIV-1-infected
chimpanzees were infected with the CXCR4 coreceptor using HIV-1 virus isolate,
IIIb. It would be tempting to postulate that due to the expansion of this virus in vitro
and the artificial nature of infection with a CXCR4 using virus, this isolate is inher-
ently less able to induce disease. Unfortunately, data exists that this strain remains
able to induce disease in humans, as a laboratory worker was accidentally infected
with HIV-1IIIb and progressed to AIDS after 8 years of infection (Beaumont et al.
2001). In general, captive chimpanzees infected with HIV-1 rapidly control viral
load to undetectable levels. There is therefore direct evidence that HIV-1 is gener-
ally not pathogenic in chimpanzees.
The vast majority of chimpanzees held in European and US primate centers and
therefore the majority of HIV-1-infected chimpanzees were of the Pan troglodytes
verus subspecies. This subspecies is not naturally infected with SIVcpz—SIV infec-
tion has only been found in the troglodytes and schweinfurthii subspecies despite
extensive surveys of captive and wild populations of the verus subspecies. It is
worth noting, however, that while SIV infection has not been found in the verus
subspecies, these animals must have a significant history of exposure to the SIV of
the western red colobus monkey. The red colobus monkey is frequently hunted by
chimpanzees (Boesch and Boesch-Achermann 2000) and has a high prevalence of
SIV infection (Locatelli et al. 2008), though this virus has not been found in verus
chimpanzees (Leendertz et al. 2011). This could suggest that some resistance mech-
anisms shared with naturally SIV-infected primate species, such as low frequency of
CCR5 expression on CD4+ T cells (Pandrea et al. 2007a), have evolved in the verus
subspecies due to historic exposure to SIVwrc.
Given the close relationship between HIV-1 and SIVcpz, it would seem reason-
able to assume that SIVcpz would also not be pathogenic in the chimpanzee.
However, a recent report studying wild chimpanzees has found the opposite. A
study of wild, habituated animals (wild animals accustomed to being observed very
closely) of the P. t. schweinfurthii subspecies has found that SIVcpz-infected ani-
mals within the group showed greater risk of mortality of the study period (Keele
et al. 2009). In addition, the bodies of three infected animals were recovered for
necropsy, along with the bodies of two uninfected animals. Significant depletion of
The Evolution of SIV in Primates and the Emergence of the Pathogen of AIDS 315

CD4+ cells in the periarteriolar lymphoid sheaths (PALS) of the spleens of these
animals was shown in the SIVcpz-infected animals. Particularly severe depletion
was shown in an animal that had become infected during the study, three years prior
to death, whose death was attributed to an “AIDS-like” disease by the group report-
ing this finding. A more recent article reports also identified a Pan troglodytes trog-
lodytes chimpanzee with AIDS-like clinical signs in a Cameroonian sanctuary
(Etienne et al. 2011). A pathogenic outcome of SIVcpz infection would lend further
support to the importance of vpu in HIV-1 infection.
From 1989 to 2005, seven SIVcpz-infected chimpanzees were housed in primate
centers in Europe and the USA (Heeney et al. 2006): one naturally infected sch-
weinfurthii animal, two experimentally infected schweinfurthii animals, and four
experimentally infected verus animals. Of these seven animals, four are still alive,
including Noah, a naturally infected animal that first tested positive in 1989, aged
around 2 years old, and remains asymptomatic after over 20 years of infection
(Greenwood et al., unpublished data). Three animals have died of cardiac condi-
tions, which are common in captive chimpanzees (Seiler et al. 2009).
Two of these chimpanzees have already been studied in some detail as part of a
cohort of HIV-1-infected chimpanzees. As with the HIV-1-infected chimpanzees,
these two SIVcpz-infected chimpanzees seem to lack the profound changes to the
immune system that are found in HIV-1-infected humans (Gougeon et al. 1997;
Rutjens et al. 2008, 2010). Given the apparent pathogenic outcome of SIVcpz infec-
tion of wild chimpanzees, it should be possible to identify, in these captive animals,
disease mechanisms that are shared with HIV-1-infected humans. Further study of
samples from these and other captive SIVcpz-infected animals is therefore war-
ranted, especially if this is possible using previously archived samples and/or non-
invasive methods.
As previously mentioned, SIVcpz is the result of recombination between SIVrcm
and SIVgsn/mon/mus. The importance of the vpu gene seems to be emphasized by
the fact that it is retained in the chimeric virus in favor of vpx. The small subset of
Cercopithecus species infected with these vpu-carrying viruses have not been
investigated for their outcome of infection. The only knowledge we have of these
viruses is their genomic sequences and their prevalence in the wild. Interestingly,
the few studies that investigated significant numbers of these Cercopithecus ani-
mals found an exceptionally low prevalence of the vpu-harboring SIVs in mus-
tached and greater spot-nosed guenon populations in Cameroon (2–4 %). This is in
stark contrast to the seroprevalence seen in primate populations that carry an SIV
lacking the vpu gene, which reported seroprevalence of 50–90 % (Aghokeng et al.
2006, 2009; Ellis et al. 2004). It therefore seems that the outcome of SIVgsn/
SIVmon infection is likely to differ from other SIV infection of natural hosts. It is
possible to postulate two possible mechanisms for this low prevalence. SIV infec-
tion in these species could be much more pathogenic than in other species, perhaps
due to the presence of vpu—which leads to infected animals being rapidly removed
from the population. Alternatively, these species could have evolved novel mecha-
nisms to prevent the spread of the virus within the population, though presumably
the evolutionary pressure for this to occur would again have to be the result of
316 E.J.D. Greenwood et al.

increased pathogenicity of SIV infection of these specific species. The opportunity

of further study for research on these monkeys is restricted, as they are not found in
primate research centers and are (rightly) protected in the wild. While surveys of
bushmeat samples from African countries have allowed some limited analysis, the
low prevalence of SIV carrying will make further studies a major challenge.
Nevertheless, due to their possible key role in our understanding of HIV-1 pathoge-
nicity, such efforts may be justified.

Conclusion

The African primate origins of HIV-1 and HIV-2 are now well established, and a
relatively thorough history of how HIV-1 has spread from western Central Africa
throughout the world has been proposed. However, the mechanisms by which HIV
causes AIDS in humans remain heavily debated. It should be noted that it is not
possible to review here the entirety of the vast and conflicting literature describing
the observed alterations to the immune system in HIV infection and the various
mechanisms proposed to cause CD4+ T-cell loss and AIDS in HIV in infected
humans.
Instead, we have highlighted the key differences found throughout the course of
infection between the pathogenic HIV/SIV infection of humans and macaques and
the nonpathogenic SIV infection of two well-studied African primate species and
used these differences to propose a relatively straightforward model by which lenti-
virus infection induces AIDS in humans and macaques but not in sooty mangabeys
and African green monkeys.
Finally, we have reviewed the possible limitation of current animal models that
specifically the HIV-1/SIVcpz viral lineage may have greater potential to cause
disease than other primate lentiviruses, which is not well addressed by current pri-
mate models. Specifically, we have noted the functions of the vpu gene. While some
of the functions of vpu are carried out by other genes in primate lentiviruses (such
as down-modulation of CD4 and tetherin), other functions are so far described
uniquely for this gene. It is also noted that the virus–host relationship in primate
species infected with a vpu-harboring virus seems to differ from that of other African
primates. While further examination of primate species infected with vpu-carrying
viruses—chimpanzees, gorillas, and members of the Cercopithecus genus—is
clearly complicated by ethical and practical considerations, noninvasive studies of
these species have already proven to be fruitful. Expansion of such noninvasive
studies may lead to new insights in the subject of lentivirus pathogenesis and host
adaptation not available in other animal models.

Acknowledgments We would like to thank Joel Wertheim for creating the HIV/SIV alignments
used to create Fig. 1.
The Evolution of SIV in Primates and the Emergence of the Pathogen of AIDS 317

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 Part III
Primates, Pathogens and Health
Microbial Exposures and Other Early
Childhood Influences on the Subsequent
Function of the Immune System

Graham A.W. Rook

Introduction

The developed countries have undergone massive increases in the prevalence of a

wide range of chronic inflammatory disorders including allergies, autoimmune dis-
eases and inflammatory bowel disease. Rigorous meta-analyses that check the diag-
nostic criteria used have confirmed that these massive increases are real (discussed
detail in a later section) (Eder et al. 2006; Elliott et al. 2005). The increases were
seen first in Northern countries and so correlated with economic development and
standards of hygiene. In Europe this phenomenon can be traced back to the nine-
teenth century. In 1873 Charles Harrison Blackley, working in England, noted that
hay fever was associated with exposure to pollen, but he also remarked “farmers
rarely experience the condition” (Blackley 1873). Indeed hay fever began to be
regarded as a mark of wealth, education and sophistication. In the 1880 s Morell
Mackenzie, a British physician, went so far as to state “As, therefore, summer sneez-
ing goes hand-in-hand with culture, we may, perhaps infer that the higher we rise in
the intellectual scale, the more is the tendency developed” (Mackenzie 1887).
Recent studies have rediscovered and definitively confirmed the protective effect of
the farming environment (Riedler et al. 2001; von Ehrenstein et al. 2000; von Mutius
and Vercelli 2010) and shown that contact with animals such as dogs is also protec-
tive (Ownby et al. 2002). In addition to these observations on allergic disorders, a
link between hygiene and an autoimmune disease was explicitly suggested in 1966,
when it was reported that the prevalence of multiple sclerosis (MS) showed a posi-
tive correlation with sanitation in Israel (Leibowitz et al. 1966). However, it was not
until 1989 that the term “hygiene hypothesis” was coined following the observation
that in young adults, a history of hay fever was inversely related to the number of

G.A.W. Rook (*)

Department of Infection, University College London, London, UK
e-mail: [email protected]

J.F. Brinkworth and K. Pechenkina (eds.), Primates, Pathogens, and Evolution, 331
Developments in Primatology: Progress and Prospects,
DOI 10.1007/978-1-4614-7181-3_11, © Springer Science+Business Media New York 2013
332 G.A.W. Rook

siblings (especially older male siblings) in their family when they were 11 years old
(Strachan 1989). Then Matricardi and colleagues found that army recruits with evi-
dence of infections attributable to faecal–oral transmission were less likely to have
allergic manifestations (Matricardi et al. 1998). Such data were considered consis-
tent with a protective influence of postnatal infection that might be lost in the pres-
ence of modern hygiene (Matricardi et al. 1998; Strachan 1989; Strachan et al.
1996). A few years later it was pointed out that type 1 diabetes (T1D; caused by
autoimmune destruction of the insulin-secreting β-cells in the pancreas) is increas-
ing at the same rate and in the same countries (mostly rich and developed) as the
allergic disorders (Stene and Nafstad 2001). Similarly, a parallel rise in inflamma-
tory bowel diseases (IBD; Crohn’s disease and ulcerative colitis) had clearly started
at the beginning of the twentieth century, rising from rare and sporadic in 1900 to
400–500/100,000 by the 1990s in rich northern developed countries (the epidemio-
logical basis for these assertions is expanded below) (Elliott et al. 2005).
How can we make sense of all this? There are two obvious problems. First, aller-
gic disorders, autoimmune diseases and inflammatory bowel diseases involve dif-
ferent immune mechanisms. Allergic disorders are mediated largely by T helper 2
cells (Th2) secreting IL-4, IL-5 and IL-13, while the autoimmune disorders that are
increasing (multiple sclerosis (MS) and T1D) involve T helper 1 (Th1) and T helper
17 (Th17 cells) secreting IFN-γ, IL-17 and other mediators. The various forms of
IBD (Crohn’s disease, ulcerative colitis and celiac disease) involve a variety of
effector cell types. Is there an “umbrella” concept associated with hygiene or eco-
nomic development that can explain increases in pathologies that involve such
diverse effector cell types?
The second problem is the diversity of the epidemiology. What is the connection
between economic development, latitude, the farming environment, pet ownership
and dirty older siblings? Indeed, is there any possibility of finding a mechanism
with such broad explanatory power?
Since about 1998 it has been apparent that the first problem can be solved by the
view that the increase in the prevalence of these chronic inflammatory diseases is
due at least in part to defects in maturation of the immune system, leading notably
to defective immunoregulation (Rook and Stanford 1998). Thus, one cause of these
increases lies in a broad imbalance between immunoregulatory mechanisms and
effector mechanisms (whether Th1, Th17 or Th2). A failure of immunoregulatory
mechanisms can indeed lead to simultaneous increases in diverse types of pathol-
ogy. We know this because genetic defects of Foxp3, a transcription factor that
plays a crucial role in the development and function of regulatory lymphocytes,
leads to the X-linked autoimmunity–allergic dysregulation syndrome (XLAAD)
that includes aspects of allergy, autoimmunity and enteropathy (Wildin et al. 2002).
So although the recent increases in chronic inflammatory disorders are too rapid to
be due to genetic changes, there could be a lifestyle-associated factor that has a
broad detrimental effect on regulation of the immune system.
More recently the second problem (i.e. the factor that is common to economic
deprivation, farms, siblings, dogs and poor hygiene) has also been resolved. As will
be explained in detail below, we now know that the correct functioning of the
Microbial Exposures and Other Early Childhood Influences… 333

immune system is heavily dependent on the microbial input that it receives in utero,
in the neonatal period and also throughout life (Ege et al. 2008; Rook 2010).
Epidemiological and experimental studies have progressively identified organisms
that have appropriate immunoregulatory properties, are depleted from modern envi-
ronments and can downregulate chronic inflammatory disorders in experimental
models. Some of these are now entering clinical trials in humans. In the context of
this book, the interesting thing about the identified organisms is that not only do
they resolve the farm/dog/sibling dilemma, but they also take us back at least as far
as early hominins. The organisms identified are viruses, bacteria and helminths,
often harmless, that coevolved with humans but are depleted from the modern urban
environment. These can be divided into three overlapping groups: (1) organisms
that form part of the coevolved human microbiota that are altered by modern diets,
living conditions and antibiotics; (2) infections that will have been present in early
humans, usually harmless, transmitted by the faecal–oral route very early in life,
that have been depleted since the second epidemiological transition; and (3) harm-
less environmental organisms in mud, untreated water and fermenting vegetable
material that are eliminated by the modern city lifestyle. I refer to these organisms
collectively as “Old Friends”, to emphasise our long association with them and our
dependency on their presence (Rook 2010). Below, I discuss the evolutionary sig-
nificance and mechanisms of the immunoregulatory effects of these organisms.
As so often in medicine, a Darwinian approach has huge explanatory power. But
it is emphasised that this chapter is drawn from the medical literature and discusses
the effects on the immune system of recent human cultural and technological devel-
opments. I leave to others the interesting task of asking whether there were other
similar immunological turning points at earlier stages in hominoid evolution.
This chapter starts with a general account of the role of the immunoregulatory
“Old Friends”, in which immunology has been kept to a minimum. Those who want
more detail of the immunology and molecular mechanisms will find that in the later
section entitled “Mechanisms”.

Evolved Dependence and the Environment of Evolutionary

Adaptedness

The concept of “evolved dependence” provides essential background to this discus-

sion. This concept refers to situations where an organism has become adapted to the
presence of a partner and can no longer perform well without that partner (de
Mazancourt et al. 2005). It was originally used to describe endosymbiosis. A clas-
sical example was seen in the laboratory environment when an amoeba (Amoeba
discoides) became infected with a bacterium (Jeon 1972). Initially this infection
severely compromised the growth of the amoebae. However, after 5 years the
relationship between the two species had changed, and neither organism could sur-
vive without the other. This indicates genetic changes leading to dependence.
For instance, an enzyme that is encoded in the genome of both species might be
334 G.A.W. Rook

dropped from the genome of one of them. Access to that gene is now “entrusted” to
the other species. This idea is at first surprising to most readers, but it is in fact rather
commonplace. For instance, most mammals can synthesise vitamin C, but large
primates and guinea pigs have lost the relevant pathways. In effect, man and guinea
pig are now in a state of evolved dependence on fruit and vegetables. Of course the
same is true for many other genes involved in the synthesis of vitamins and other
essential nutrients that we have to consume after other organisms have created them
for us (Resta 2009).
The role of this evolved dependence is most clearly seen in germ-free animals,
propagated by caesarean section under sterile conditions and maintained in a sterile
environment. The immune systems of germ-free animals fail to develop correctly
and are functionally distorted. There is lack of cellularity and, above all, lack of
effective immunoregulation (discussed in detail later). In 2005 Mazmanian and col-
leagues showed that a single polysaccharide from an intestinal commensal,
Bacteroides fragilis, could partly correct these developmental abnormalities
(Mazmanian et al. 2005). More recently they have shown, using three different
models of intestinal inflammation, that the same polysaccharide, given by mouth,
can turn on crucial anti-inflammatory, immunoregulatory pathways (Mazmanian
et al. 2008). In the discussion of the latter paper they state:
We propose that the mammalian genome does not encode for all functions required for
immunological development but rather that mammals depend on critical interactions with
their microbiome (the collective genomes of the microbiota) for health.

To put it even more simply, some genes needed for the development and regula-
tion of the mammalian immune system might have been “entrusted” to microorgan-
isms: a clear example of “evolved dependence”. It is obvious that these organisms
have to be those with which mammals have coevolved for a very long time and that
were always present. They cannot be organisms that merely cause sporadic infec-
tions or high death rates. The latter can modify the human genome by elimination
of susceptible genotypes, but they cannot be entrusted with the role of supplying
genes and functions that we need.

Environment of Evolutionary Adaptedness

It may be useful to think about the state of evolved dependence seen in the mam-
malian immune system in the context of the Environment of Evolutionary
Adaptedness (EEA). The term EEA was first used in 1969 by John Bowlby, who
was concerned that those aspects of human behaviour that are genetically deter-
mined might be adapted to the hunter-gatherer existence rather than to modern city
life (Bowlby 1971 first published by Hogarth press in 1969). The basis for this was
the view that since the start of agriculture and pastoralism about 10,000 years ago,
much human adaptation to new environments has been cultural and technological
rather than genetic. For example, we have not adapted genetically to living in cold
Microbial Exposures and Other Early Childhood Influences… 335

places: we have learnt to make fur coats and electric heaters. Humans easily detect
problems within the physical environment and invent appropriate technological
adaptations. But humans are not equipped with a sense that tells us when the envi-
ronment is inappropriate for our immune systems. Only since the development of
modern biology have we begun to understand that we have an immune system and
that we need to think in a Darwinian and anthropological way about the nature and
timing of the inputs that it receives.

Epidemiological Transitions

It is possible to predict the identity of the organisms on which our immune systems
have evolved dependence (i.e. that were part of the immune system’s EEA) by con-
sidering the history of man’s changing microbial exposures. In 1971 Omran coined
the term “epidemiological transition” to describe the major watersheds in human
development that led to massive changes in mortality (discussed in Armelagos et al.
2005). Palaeolithic populations carried organisms inherited from primate ancestors
(“heirloom” species), including many viruses, as shown in Fig. 1 (Armelagos et al.
2005; Van Blerkom 2003). In addition they would have been exposed to zoonoses
that they picked up as they scavenged carrion (Armelagos et al. 2005; Despres et al.
1992; Hoberg 2006). Phylogenetic trees for these organisms confirm the extremely
ancient association with humans, probably for a million years or more (Despres
et al. 1992; Hoberg 2006). Finally, Palaeolithic populations will have consumed
several milligrams of harmless environmental saprophytes every day, since these are
ubiquitous in soil and untreated water (Delmont et al. 2011). We have called these
“pseudocommensals” because of their inevitable continuous presence until chlori-
nation and purification of water in the modern era. The organisms that have been
found to be important for the hygiene hypothesis belong within these three
categories.
About 10,000 years ago, the shift to agriculture and husbandry created the first
(Neolithic) epidemiological transition (Fig. 1) (Armelagos et al. 2005). This will
have had little effect on exposure to the “pseudocommensals” or to the heirloom
species. However, the more sedentary lifestyle increased faecal–oral transmission
and combined with husbandry caused prolonged contact with animals. The latter
led to adaptation to humans of a number of animal viruses shown in Fig. 1 (Van
Blerkom 2003). However, the viruses acquired during the Neolithic such as influ-
enza (B and C), smallpox, mumps and measles cannot have become endemic until
populations were large enough. This required communities of several hundreds of
thousands, which did not occur until the appearance of cities 2000–3000 years ago
(Armelagos et al. 2005). Since this represents only 100–150 generations from the
present, extremely strong selection pressure would have been required for evolved
dependence to appear, and this seems unlikely. Moreover, most humans did not live
in such large groups, and these viruses were, for example, absent from pre-Colum-
bian American populations. In any case, one would not expect “evolved
336 G.A.W. Rook

Fig. 1 Aspects of man’s microbiological history that are most relevant to the hygiene hypothesis.
Epidemiological data, laboratory and animal models and preliminary clinical trials investigating
the hygiene hypothesis implicate organisms that are thought to have accompanied mammalian and
human evolution. This relationship was long enough for the establishment of evolved dependence
on these organisms, that must be tolerated, and so have developed roles in the initiation of regula-
tory pathways. Organisms that evolved during the Neolithic are less likely to be relevant in this
context, and the first epidemiological transition did not reduce human contact with organisms
associated with animals, faeces and mud. On the other hand, the second epidemiological transition
has led to gene–environment misfit, as the “Old Friends” from the Palaeolithic were progressively
removed from the modern environment

dependence” on contact with organisms that were both dangerous and sporadic, and
as explained later, these “recently” acquired viruses tend to cause or exacerbate
chronic inflammatory disorders, rather than prevent them.
In short, there were dramatic changes to the human microbial environment after
the first epidemiological transition, but this did not result in loss of exposure to the
organisms that had coevolved with humans and are now implicated by epidemiology,
experimental models and clinical trials in the hygiene hypothesis, as detailed below.
Until the modern era more than 97 % of the population still lived in rural environ-
ments, close to mud, animals and faeces, which were the sources of these organisms.
The situation did not change until the mid-nineteenth century. Since then some
populations have undergone a second epidemiological transition in which concrete,
tarmac, diminished contact with animals, public health measures and, more recently,
antibiotics have resulted in diminished (or delayed) exposure to the “Old Friends”
that were present in earlier eras. The dramatic effects of the second epidemiological
Microbial Exposures and Other Early Childhood Influences… 337

transition are revealed by epidemiological studies of the allergic disorders. The

prevalence of symptoms of asthma, allergic rhinoconjunctivitis and atopic eczema
varied between 20-fold and 60-fold in different regions. For asthma symptoms, the
highest 12-month prevalences were in the UK, Australia, New Zealand and the
Republic of Ireland. The lowest prevalences were found in Eastern European coun-
tries, Indonesia, Greece, China, Taiwan, Uzbekistan, India and Ethiopia (ISAAC
Steering Committee 1998).

How Real Are the Modern Increases in Chronic

Inflammatory Disorders?

It must be emphasised that these progressive increases in the incidences of chronic

inflammatory diseases that start at the second epidemiological transition are real
and not merely due to changing awareness and diagnostic methods in different
countries.
The allergies are mainly due to unregulated and inappropriate immune responses
to trivial allergens in the air or food: a failure of immunoregulatory mechanisms
(Larche 2007; Strickland and Holt 2011). There are several types of study that con-
firm the recent increases in allergic disorders. First, we can perform studies in the
same developed countries at intervals of a decade or more, using standardised diag-
nostic methods. The very large increases in allergic asthma during the twentieth
century were confirmed in a massive meta-analysis that included more than 40 stud-
ies, most of them performed during the last 2 decades (Eder et al. 2006). Second, we
can look at urban–rural differences in developing countries. Allergies increase first
in hygienic urban settings (Robinson et al. 2011). A third approach is to look at
societies that are undergoing the second epidemiological transition and document
changing disease patterns. A striking example is Karelia, an area populated by a
genetically homogeneous group, but partitioned between ultra-modern Finland and
developmentally retarded Russia (Seiskari et al. 2007b). Allergic sensitisation was
lower in the Russian Karelian children, while their antibodies to microorganisms
and the abundance and diversity of microorganisms in their homes were much
higher (Pakarinen et al. 2008; Seiskari et al. 2007b). A fourth approach applicable
to the allergies is to document the effect of prolonged treatment of helminths so that,
at least as far as these organisms are concerned, the population begins to resemble
modern urban humans. Deworming Vietnamese schoolchildren for 12 months
(Flohr et al. 2010) and still more prolonged treatment of children in Venezuela or
Gabon all led to increased allergen sensitisation and skin prick test responses (Lynch
et al. 1993; van den Biggelaar et al. 2004).
In autoimmune diseases the immune system’s regulatory police force (such as
regulatory T lymphocytes (Treg)) is failing to stop the immune system from attacking
the host’s own tissues (Long and Buckner 2011). Here the Karelians are again inter-
esting. The autoimmune inflammatory disorder, type 1 diabetes (where the immune
system destroys the insulin-secreting β-cells in the pancreas), is six times more
338 G.A.W. Rook

prevalent in Finnish Karelians than in Russian Karelians despite the fact that the geno-
types associated with susceptibility are at the same frequency in the two populations
(Kondrashova et al. 2005). Meanwhile striking and repeatedly confirmed large global
variations in the incidence of multiple sclerosis (Rosati 2001) (an autoimmune inflam-
matory disorder where the immune system attacks the brain) are found to correlate
inversely with the prevalence of the helminth Trichuris trichiura, which is an excellent
surrogate marker of economic underdevelopment and of the likelihood of exposure to
other developing country infections (Fleming and Cook 2006).
Inflammatory bowel diseases (IBD) are at least partly due to inappropriate, unregu-
lated immune responses to bowel contents (Boden and Snapper 2008). The early
medical literature suggests that IBD had been extremely rare before the 1900s (Kirsner
1995). Then during the early twentieth century IBD was uncommon, sporadic and
usually seen in individuals from the upper classes, urban areas and northern latitudes
(Kirsner 1995). Later in the twentieth century, the incidence increased steadily, as
proven in repeat studies in individual countries (England, USA, Scotland, Wales,
Denmark, Sweden, Israel), using reproducible methods (reviewed and referenced in
Elliott et al. 2005). Meanwhile the greater incidence of IBD in hygienic urban settings
continues to be obvious, and the incidence increases (using standardised diagnostic
criteria) as hygiene and modernisation progress (Klement et al. 2008).

The Critical Organisms and Their Immunological Role

These considerations allow prediction of the organisms involved in the “Old

Friends” hypothesis—that is to say, organisms that triggered immunoregulatory
mechanisms in the past but are now lacking from the modern environment. From a
Darwinian perspective we would expect the relevant organisms to have been pres-
ent, inevitably and continuously, from relatively early in the evolution of the immune
system (“Old Friends”). One would also anticipate a reliable mode of transmission
such as the faecal–oral route, often accompanied by the ability to establish carrier
states that facilitate such transmission. The following section identifies the organ-
isms implicated as “Old Friends” that are necessary for the maturation of the regula-
tory pathways of the immune system (Figs. 1 and 2). Note that the mechanisms, and
the evidence that the immunoregulation-enhancing properties of these organisms
can be demonstrated in experimental models, are discussed later in a separate sec-
tion entitled “Mechanisms”.

Gut Microbiota

Proof, using modern methods, that the microbiota of city-dwelling European (EU)
children differs dramatically from that of rural Africans was obtained recently (De
Filippo et al. 2010). The faecal microbiota of children from Burkina Faso (BF) had
Microbial Exposures and Other Early Childhood Influences… 339

“Old Friends” that must be tolerated;

Evolved dependence on them to drive immunoregulation
Micro- and macro-organisms “Pseudocommensals”

Organisms to Waldeyer’s ring

Entero-mammary circulation

Fecal-oral transmission Organisms in mud and

carrier states water;
~ 109 mycobacteria/litre
Lung microbiota

Helminths (~100%) Fermented foods and

drinks; lactobacilli
Gut microbiota

Ectoparasites

Skin microbiota ?? “AOB”

Fig. 2 Locations of some of the organisms that can be regarded as “Old Friends” and that play a
role in setting up immunoregulatory circuits. AOB ammonia-oxidising bacteria

more Bacteroidetes and less Firmicutes and Enterobacteriaceae. Only the BF

children had organisms (Prevotella and Xylanibacter) known to express enzymes
for hydrolysis of cellulose and xylan which may have contributed to the fact that BF
children had more short-chain fatty acids (SCFA) than did EU children (De Filippo
et al. 2010). SCFA have a protective anti-inflammatory role in the gut (dos Santos
et al. 2010; Maslowski et al. 2009). There was also significantly greater microbial
richness and biodiversity in BF samples than in EU samples (De Filippo et al. 2010).
This is important because a decrease in the abundance and biodiversity of Firmicutes
has been observed repeatedly in Crohn’s disease patients (Sokol et al. 2008).
Interestingly, Faecalibacterium prausnitzii, an anti-inflammatory commensal bacte-
rium that also contributes to SCFA generation, was present in the microbiota of the
BF children (De Filippo et al. 2010) but is often lacking from European CD patients
(Sokol et al. 2008).
The virome of the microbiota is now also being studied. Most of the viruses are
bacteriophages, but how they vary or affect the immunoregulatory effects of the
bacterial microbiota is not yet known (Reyes et al. 2010).

Organisms Introduced by Pets

Prenatal exposure to household pets influences fetal immunoglobulin E production

(Aichbhaumik et al. 2008), and neonatal exposure to dogs reduces subsequent risk
of allergic sensitisation (Ownby et al. 2002). It is now known that owning a cat or
340 G.A.W. Rook

dog strikingly increases the abundance and diversity of bacterial taxa in house dust
(Fujimura et al. 2010). The role of prenatal exposure is supported by the observation
that pregnant women (not sensitised to dog) who had a dog or cat in the home had
higher circulating regulatory T cell (Treg) levels compared with women who did not
have pets (Wegienka et al. 2009). Treg readily suppress allergic responses (dis-
cussed in the mechanisms section below).

Chronic Infections Transmitted by the Faecal–Oral Route

Organisms highlighted in recent studies of the hygiene hypothesis that are transmit-
ted by the faecal–oral route include H. pylori, Salmonella, hepatitis A virus (HAV),
enteroviruses and Toxoplasma gondii (Fig. 1 and Table 1) (Matricardi et al. 2000;
Pelosi et al. 2005; Seiskari et al. 2007a; Umetsu et al. 2005).
A number of studies have demonstrated an association between infection with the
hepatitis A virus (HAV) and protection against the development of asthma (Matricardi
et al. 2000; Umetsu et al. 2005). This infection was probably universal and coevolved
with humans (Van Blerkom 2003). It was transmitted to the neonate by faecal–oral
contamination and harmless if transmitted in this way (it is of course dangerous in
the modern world when infection occurs in an adult). Its incidence rapidly declined
during the twentieth century. HAV directly infects lymphocytes via the receptor
TIM-1 (T-cell immunoglobulin and mucin domain containing 1). This is thought to
alter differential survival of T cell subsets and so bias the immune response away
from allergy-mediating Th2 and towards immunoregulation (Umetsu et al. 2005).

Viruses and Asthma: Induction Rather than Protection

Apart from HAV, mentioned in the previous paragraph, most studies implicate
childhood viruses as triggers of asthma rather than as protective organisms.
The viruses most frequently implicated as inducers of allergic disorders are human
rhinovirus (HRV) (Jackson 2010) and respiratory syncytial virus (RSV) (reviewed
inYoo et al. 2007). A prospective study of 95,310 children from birth through early
childhood showed that the risk of developing asthma was greatest for children born
approximately 4 months before the winter virus peak (Wu et al. 2008). This finding
implied a role for early exposure to winter viruses in the triggering of later asthma
(Wu et al. 2008). Animal models provide further support. It is possible to provoke a
chronic inflammatory Th2-biased airway disease in mice using Sendai virus. This is
a mouse parainfluenza-type I virus that is similar to RSV. In this mouse model acute
lung disease appears 3 weeks after infection when IL-13-producing CD4+ Th2 cells
are recruited to the airways. Seven weeks after infection there is a chronic phase
associated with continuing production of IL-13 and Th2-mediated inflammation
(Holtzman et al. 2009).
Microbial Exposures and Other Early Childhood Influences… 341

Table 1 Organisms inducing immunoregulation, identified by epidemiology and/or testing in

experimental models of chronic inflammatory disease
Organism or location Disease or model or effect References
Faecal–oral transmission
Helicobacter pylori Allergies (epidemiology) Matricardi et al. (2000)
Salmonella Allergies (epidemiology) Pelosi et al. (2005)
Toxoplasma Allergies (epidemiology) Matricardi et al. (2002), Matricardi
et al. (2000)
Viruses
Enteroviruses Allergies (epidemiology) Seiskari et al. (2007b)
Hepatitis A virus Asthma (epidemiology) Matricardi et al. (2002), Umetsu and
DeKruyff (2010)
Viruses protective if infected early but trigger disease if late (Filippi and von Herrath 2008)
Coxsackievirus B Type 1 diabetes (T1D) Serreze et al. (2000)
(mouse model)
Rotavirus T1D (mouse model) Harrison et al. (2008)
Helminths
Many species Allergies (human studies) Flohr et al. (2010), Huang et al.
(2002), Scrivener et al. (2001),
van den Biggelaar et al. (2004),
Yazdanbakhsh and Wahyuni
(2005)
Assorted natural infection Multiple sclerosis (MS) Correale et al. (2008), Correale and
(human studies) Farez (2011)
Trichuris trichiura MS (correlation) Fleming and Cook (2006)
Enterobius vermicularis T1D (correlation) Gale (2002)
Various species Inflammatory bowel Koloski et al. (2008), Weinstock and
disease (IBD) Elliott (2009)
(epidemiology)
Animal models treated with helminths
Heligmosomoides Allergy, T1D, colitis Reviewed in Osada and Kanazawa
polygyrus (2010)
Schistosoma mansoni Allergy, T1D, EAE, colitis, Reviewed in Osada and Kanazawa
arthritis (2010)
Strongyloides stercoralis Allergy Reviewed in Osada and Kanazawa
(2010)
Fasciola hepatica EAE Reviewed in Osada and Kanazawa
(2010)
Trichinella spiralis T1D, EAE Reviewed in Osada and Kanazawa
(2010)
Hymenolepis diminuta Colitis, arthritis Reviewed in Osada and Kanazawa
(2010)
Human clinical trials with helminths
Trichuris suis MS Fleming et al. (2011)
Trichuris suis IBD (Crohn’s disease, Summers et al. (2005a, b)
ulcerative colitis)
Necator americanus Asthma Feary et al. (2010)
(continued)
342 G.A.W. Rook

Table 1 (continued)
Organism or location Disease or model or effect References
Gut microbiota
Segmented filamentous Th17 cells (mice) Gaboriau-Routhiau et al. (2009),
bacteria Ivanov et al. (2009), Wu et al.
(2010)
Clostridia species Treg in lamina propria Geuking et al. (2011)
(mice)
Bacillus fragilis IL-10 and Treg (mice) Round et al. (2011)
Faecalibacterium Crohn’s disease (human Sokol et al. (2008)
prausnitzii and animal studies)
Other microbiota
Skin microbiota; ammonia- Nitrite, nitric oxide Whitlock and Feelisch (2009)
oxidising bacteria
Lung microbiota Asthma Huang et al. (2011)
Oral and periodontal IBD Singhal et al. (2011)
microbiota
Gut organisms transported ? immunoregulation Donnet-Hughes et al. (2010)
to breast milk
Ectoparasites
Various Response to TLR agonists Friberg et al. (2010)
in vitro
Environmental saprophyte
Mycobacterium vaccae Allergy (mouse, dog) Ricklin-Gutzwiller et al. (2007),
Zuany-Amorim et al. (2002)
KEY: T1D type 1 diabetes, IBD inflammatory bowel disease, EAE experimental autoimmune
encephalomyelitis, TLR toll-like receptor

Viruses and Autoimmunity: A Question of Timing?

Enteroviruses such as Coxsackievirus B (CVB) and other faecal–oral viruses such

as rotavirus have been implicated as triggers of type 1 diabetes (Filippi and von
Herrath 2008). Weaker evidence implicates mumps virus, cytomegalovirus and
rubella virus (Filippi and von Herrath 2008). However, timing is crucial, and
viruses such as Coxsackieviruses (Serreze et al. 2000) or rotaviruses (Harrison
et al. 2008) or lymphocytic choriomeningitis virus (LCM) that provoke autoim-
munity when mice are infected at weaning or later can be protective when given
very soon after birth (Filippi and von Herrath 2008; Harrison et al. 2008; Serreze
et al. 2000). These findings suggest another twist to the hygiene hypothesis.
Modern hygiene may cause delayed faecal–oral transmission, so that the virus
infection occurs later than was normal during early human evolution. Consequently
the immune system is at an inappropriate stage of maturation, and levels of anti-
body obtained transplacentally are lower.
Microbial Exposures and Other Early Childhood Influences… 343

Helminths

It is estimated that in 1947, about 36 % of the population of Europe carried hel-

minths such as Enterobius vermicularis, Trichuris trichiura and Ascaris lumbricoi-
des (Stoll 1947). Now even pinworm (E. vermicularis) has become a rarity in
Europe (Gale 2002). A number of studies have reported inverse correlations between
indicators of helminth burden and allergic sensitisation to environmental allergens
(Araujo et al. 2000; Cooper et al. 2003; Hagel et al. 1993; Lynch et al. 1983; Nyan
et al. 2001). More importantly, the risk of wheeze was reduced in individuals with
hookworm (Necator americanus) infection in Ethiopia (Scrivener et al. 2001), and
Enterobius infestation was negatively correlated with asthma and rhinitis in primary
school children in Taiwan (Huang et al. 2002). Similarly it was suggested that infec-
tion with Schistosoma mansoni was associated with milder forms of asthma
(Medeiros et al. 2003).
If helminths protect from chronic inflammatory disorders, the protection should
be lost when the helminths are eliminated by anti-helminthic treatments. In fact this
effect constitutes strong evidence for the protective role. Short periods of treatment
(<12 months) did not change the prevalence of atopy or clinical signs of allergy
(Cooper et al. 2006). By contrast, deworming Vietnamese schoolchildren for 12
months (Flohr et al. 2010) and still more prolonged treatment of children in
Venezuela or Gabon all led to increased allergen sensitisation and skin prick test
responses (Lynch et al. 1993; van den Biggelaar et al. 2004). These studies did not
reveal simultaneous increases in clinical allergies such as eczema, wheeze or rhini-
tis, but this would probably require still longer periods of treatment and follow-up
(Flohr et al. 2010).
The inverse relationship between the multiple sclerosis (MS) and the prevalence
of helminth infections was mentioned earlier (Fleming and Cook 2006; Rosati
2001). Correale and colleagues have shown that patients with MS who become
infected with helminths have a strikingly diminished rate of disease progression
(Correale and Farez 2007). Similarly, a recent study has also shown exacerbation of
MS after treatment of helminth infestation in patients suffering from this disorder
(Correale and Farez 2011). A Phase 1 clinical trial using ingestion of living eggs of
Trichuris suis (the pig whipworm) to treat MS has shown that this parasite is well
tolerated, and favourable trends were observed in exploratory magnetic resonance
imaging (MRI) and immunological assessments (Fleming et al. 2011).
As far as inflammatory bowel disease (IBD; Crohn’s disease and ulcerative coli-
tis) is concerned, the epidemiological data are less strong than for allergic disorders,
because IBD is less common. Nevertheless, analyses of the available data conclude
that exposure to helminths is one of the environmental factors most convincingly
associated with a low risk of IBD (Koloski et al. 2008; Weinstock and Elliott 2009).
Two well-documented anecdotes are also informative. A 12-year-old girl with
344 G.A.W. Rook

occasional gastrointestinal symptoms was found to be heavily infected with

Enterobius vermicularis. Curing the Enterobius infection led to severe active ulcer-
ative colitis (Buning et al. 2008). Interestingly, a patient suffering from severe ulcer-
ative colitis treated himself with the human whipworm (Trichuris trichiura) and
underwent a clear remission (Broadhurst et al. 2010). Similarly, clinical trials with
Trichuris suis have given significant results (Summers et al. 2005a, b).

Microbiota of the Skin

The role of recent changes to the microbial microbiota of the skin, lung and breast
has received almost no attention. Before the invention of modern soaps and deter-
gents, the skin was probably colonised by ammonia-oxidising bacteria (AOB)
(Fig. 2). These are ubiquitous in soil, but they are exquisitely sensitive to alkyl-
benzene sulfonate detergents (Whitlock and Feelisch 2009). AOB can convert the
high concentrations of urea and ammonia found in human sweat into nitrite and
NO which are efficiently absorbed via the skin, so this source of nitrite might have
been biologically significant for immunoregulation in which NO is known to play
a major role (Whitlock and Feelisch 2009).

Microbiota of the Lung

The lung is not sterile. It is estimated that there are about 2,000 bacterial genomes per
square centimetre of the surface of the bronchial tree, though there is a risk of con-
tamination from the bronchoscope used to gather the samples. Pathogenic
Proteobacteria, particularly Haemophilus spp., were more abundant in asthmatic air-
ways (Hilty et al. 2010). Interestingly bacterial concentrations and diversity were sig-
nificantly higher among asthmatic patients, as determined by assaying 16S ribosomal
RNA amplicons and the relative abundance of members of several bacterial families
correlated with bronchial hyperresponsiveness (Huang et al. 2011). Nothing is yet
known of differences between lung microbiota of hunter-gatherers and modern urban
man, but they must be striking (Fig. 2). This will be a difficult area to study because
truly sterile samples of material from healthy individuals probably cannot be obtained.

The Role of Milk

Breast milk is not sterile (Donnet-Hughes et al. 2010), and there appears to be an
“entero-mammary” circulation. Human peripheral blood mononuclear cells and
breast milk cells contain bacteria during lactation, due to increased translocation
from the gut to Peyer’s patches and thence into circulating dendritic cells. Bacteria
can also be seen in the glandular tissue of healthy breast and in mononuclear cells
in breast milk, which when “sterile” contains small numbers of cultivable organisms
Microbial Exposures and Other Early Childhood Influences… 345

(<103). The mononuclear cells seem to be partially matured dendritic cells that
might promote tolerogenic responses in the neonate (Donnet-Hughes et al. 2010). It
remains to be seen whether this is an important part of the colonisation and educa-
tion of the neonatal gut and immune system, but if it is, it must be severely disrupted
in many modern humans (Fig. 2).
Not only does human milk contain bacteria as described above, but it also con-
tains complex oligosaccharides that favour growth and establishment of selected
strains such as Bifidobacterium longum subsp. infantis in the infant gut (Zivkovic
et al. 2010). Thus, the decline in breastfeeding due to pressure to use formula milk
substitutes must be altering infant microbiota.

Environmental “Pseudocommensals”

Mud and untreated water contain milligrams of various saprophytic bacterial spe-
cies per litre. Therefore, milligram quantities were inevitably consumed by every-
one until modern chlorinated water supplies were developed. We have designated
these “pseudocommensals” because they were consumed regularly and inevitably
throughout mammalian evolution (Fig. 2). These too turn out to have immunoregu-
latory roles and are currently entering further clinical trials (Ricklin-Gutzwiller
et al. 2007; Zuany-Amorim et al. 2002). Some non-colonising lactobacillus strains
present in fermenting vegetable matter must have been encountered daily, and many
Lactobacillus strains are immunoregulatory (Smits et al. 2005) and are entering
clinical trials as probiotics (Sheil et al. 2007).

Ectoparasites

In a study of wild rodents, it emerged that various aspects of the innate immune
response were profoundly affected by the load of ectoparasites (i.e. fleas, lice, etc.)
(Friberg et al. 2010). It is perhaps not surprising that repeated “injections” of phar-
macologically active materials, many of them designed to modulate the host immune
response, can provoke lasting systemic effects. Such exposures must be greatly
diminished in modern society, but further work is needed to determine the impor-
tance of this factor (Friberg et al. 2010).

Malaria and Other Organisms Not Yet Implicated in the Old

Friends Hypothesis

Malaria clearly needs to be studied in this context, especially in view of its impor-
tance in human evolution. Plasmodium vivax induces Treg population expansion in
humans (Bueno et al. 2010). Similarly, placental malaria, whether active at delivery
346 G.A.W. Rook

or resolved, leads to an expanded population in cord blood of malaria-specific

FOXP3(+) Treg and a larger expansion of non-malaria-reactive Treg revealed by
stimulation in vitro (Flanagan et al. 2010). In a mouse model Plasmodium chabaudi
infection will attenuate the course of experimental autoimmune encephalomyelitis
(EAE), an experimentally induced autoimmune disease which is widely regarded as
a model of human MS (Farias et al. 2011).
Endemic malaria also causes selection of mutations within the immune system
that facilitate control of the parasite (Sotgiu et al. 2007). In the Sardinian population
this has included selection of proinflammatory variants of genes encoding tumour
necrosis factor (TNF) and the human lymphocyte antigens (HLA) that help to pro-
tect against malaria. Unfortunately, in the absence of malaria, these variants are risk
factors for MS. It has recently been suggested that the disappearance of malaria
might be relevant to increasing levels of MS noted in Sardinia immediately after
elimination of the parasite (Sotgiu et al. 2007).
Other protozoa have also received little or no attention in this context. For exam-
ple, what about the very ancient Entamoeba, Giardia and Trichomonas all of which
have lost their mitochondria and so become obligate parasites with a close
association with humans? This area needs to be explored.

Evidence from Animal Models and Clinical Trials

If the depletion of the organisms listed in the previous sections is contributing to the
increases in disorders of immunoregulation that have occurred since the second
epidemiological transition, it should be possible to demonstrate that administration
of these organisms can prevent and/or treat the same conditions in animal models
and in human clinical trials. A few examples have been cited in context above, and
a detailed analysis would be beyond the scope of this review, but this issue is
expanded briefly below.

Animal Models

There are numerous experimental models in which exposure to microorganisms that

were ubiquitous during mammalian evolutionary history but are currently “miss-
ing” from the environment in rich countries (or from animal units with Specific
Pathogen-Free facilities) will treat allergy (Ricklin-Gutzwiller et al. 2007; Zuany-
Amorim et al. 2002), autoimmunity (Farias et al. 2011; Osada and Kanazawa 2010;
Zaccone et al. 2003) or intestinal inflammation (Elliott et al. 1999, 2003). More
examples are listed in Table 1. The dominant mechanism revealed in these models
is the induction of immunoregulatory, anti-inflammatory cells, particularly Treg.
Microbial Exposures and Other Early Childhood Influences… 347

Clinical Trials

Clinical trials using hookworm (Necator americanus) (Blount et al. 2009) or

Trichuris suis have been completed (Summers et al. 2005a, b). The focus has been
on allergies and inflammatory bowel disease (Blount et al. 2009; Summers et al.
2005a, b) with significant benefit in IBD. In Brisbane a trial of hookworm has been
completed in celiac disease (http://clinicaltrials.gov/show/nct00671138).
Hookworm did not have much impact, if any, on clinicopathological measures of
celiac disease in the context of a robust gluten challenge. However, it does seem that
hookworm dampens the gluten-specific Th1 and Th17 responses (John Croese, per-
sonal communication). Trials in multiple sclerosis are also in progress. Trichuris
suis eggs by mouth were well tolerated, and favourable trends were observed in
exploratory MRI and immunological assessments during a Phase 1 study (Fleming
et al. 2011). Further efficacy studies are, therefore, in progress.
There is clearly also enormous scope for the modulation of gut microbiota with pro-
biotics or prebiotics, and this area is likely to develop rapidly. So far the most convincing
results have come from certain gastroenterological conditions, while in other clinical
areas results have been variable (Round and Mazmanian 2009; Yan and Polk 2010).

Mechanisms

How and why do the Old Friends modulate our immune systems? Numerous mech-
anisms exist, but there are two related underlying evolutionary principles that prob-
ably apply to all the “Old Friends”. First, the encounters with a broad range of
microbial molecules that trigger the innate immune system via pattern recognition
receptors (PRR) such as Toll-like receptors (TLR), and also experience of diverse
microbial antigens, may be a necessary maturation stimulus for the immune system.
Second, the “Old Friends” persist as commensals (or “pseudocommensals”), carrier
states or chronic subclinical infections. Therefore, the host–“Old Friend” relation-
ship evolved so that rather than provoking needless damaging aggressive immune
responses, an anti-inflammatory equilibrium is established. This translates into the
priming of immunoregulation. Maturation and immunoregulation are considered
separately below, though they are clearly overlapping concepts.

Maturation of the Immune System In Utero and in the Neonate

It is suggested that the neonatal immune system defaults to a Th2 bias, in the absence
of stimuli that drive maturation (Pfefferle et al. 2010). Since this is the arm of the
348 G.A.W. Rook

Anti-inflammatory
mechanisms
Helminths
IL-10+ regulatory B cells

TGF-β, RA IL-10+ regulatory

Multiple factors, macrophages
most not yet
DC
identified,
Modulation interact with Toll-
like receptors, (IDO
of the
microbiota epithelial cells aldh1a2) anti-inflammatory DC
and dendritic CD103
cells to regulate
Treg & effector T T cells
cell types Treg Foxp3+ & also
other TGF-β & IL-10+
Treg

Microbiota Short chain fatty acids

GPR43 anti-inflammatory
receptor

Fig. 3 Some mechanisms involved in the immunoregulatory properties of the “Old Friends” and
microbiota. A key pathway is the modification of DC so that they tend to drive Treg. Such DC also
process self-antigens, allergens, etc. and so drive crucial specific regulatory cell populations. The
intestinal helminths also exert indirect effects by modulating the microbiota. The individual path-
ways (some taken from experimental systems in the mouse, as illustrations) are referenced in the
main text: RA retinoic acid, DC dendritic cell, Aldh1a2 retinaldehyde dehydrogenase 2, GPR43
G-protein-coupled receptor 43, RegIIIγ regenerating islet-derived 3γ, CD103 an integrin and
marker of intestinal regulatory DC, IDO indoleamine 2,3, dioxygenase

immune response that drives allergic disorders, this point is of some interest. It now
seems that children exposed to high quantities and high diversity of microbial materi-
als are protected from subsequent allergic disorders (Ege et al. 2011). Interestingly
this exposure can take place before birth. Exposing pregnant women to barns and
farm animals during gestation reduced the risk of allergic disease in their offspring
(Ege et al. 2008; von Mutius and Radon 2008). Similarly, prenatal exposure to house-
hold pets reduces fetal immunoglobulin E production (Aichbhaumik et al. 2008).
Maturation following microbial exposures seems to push the system towards Th1,
with increased production of the Th1 signature cytokine, IFN-γ (Pfefferle et al. 2010).
Interestingly, this protective effect of prenatal exposure to microbial components
has been reproduced in a mouse allergy model and strongly suggests that epigenetic
factors are involved (Conrad et al. 2009). It is also possible that accelerated matura-
tion and Th1 bias induced by microbial exposure indirectly protects from allergy by
facilitating rapid removal of the viruses that are implicated in allergic disorders
(RSV and HRV discussed earlier) (Jackson 2010; Yoo et al. 2007).
These points all raise the interesting issue that it might take several generations
for the full consequences of loss of exposure to “Old Friends” to be manifested.
This greatly complicates the epidemiology.
However, maturation and Th1 bias is not the whole story and might in fact be less
important than priming of immunoregulation. For example, farm exposures during
pregnancy increased the number and function of cord blood Treg cells, in addition
to reducing the extent of Th2 cytokine secretion after in vitro stimulation (Schaub
et al. 2009). Immunoregulation is discussed in the next sections.
Microbial Exposures and Other Early Childhood Influences… 349

Initiation of Immunoregulation

The Old Friends persist as commensals, carrier states or chronic subclinical infec-
tions and so coevolved a role as inducers of immunoregulation. This avoids point-
less and damaging inflammatory attacks on organisms that are essential to our
health (such as gut microbiota) or impossible to remove (such as established hel-
minth infections). For instance, a futile effort to destroy Brugia malayi microfilariae
results in lymphatic blockage and elephantiasis (Babu et al. 2006). Thus, many
helminths induce expansion of Treg populations. For instance, the percentage of
circulating Treg was positively related to the level of infection with Schistosoma
haematobium in Zimbabwean children aged 8–13 years (Nausch et al. 2011). Some
mechanisms are summarised in Fig. 3.
A frequent mechanism is modulation of dendritic cells (DC) such that these drive
Treg rather than Th1, Th17 or Th2 effector cells (referenced in Rook 2009). These
modified DC can be regarded as DCreg. Then the constitutive presence of the “Old
Friends” causes continuous background activation of the DCreg and of Treg specific
for the Old Friends themselves, resulting in background bystander suppression of
inflammation. Meanwhile these DCreg inevitably sample self, gut contents and
allergens and so induce Treg specific for the illicit target antigens of the three groups
of chronic inflammatory disorder. Release of the anti-inflammatory cytokines,
IL-10 and TGF-β is often involved in the anti-inflammatory effects of these cells
(Zuany-Amorim et al. 2002).

Helminths and Immunoregulation

A striking example of this in human autoimmunity is a recent experiment of nature.

Patients in Argentina suffering from multiple sclerosis were followed up for 4.6
years. It was found that those who developed parasite infections (which were not
treated) had significantly fewer exacerbations than those who did not (Correale and
Farez 2007). Moreover, they also developed regulatory lymphocytes that specifi-
cally responded to myelin basic protein by releasing IL-10 and TGF-β. In other
words, the presence of the parasite appeared to drive the development of regulatory
cells that recognised the auto-antigen and inhibited the autoimmune disease pro-
cess. The parasites acted as “Treg adjuvants”.
Little is yet known about the precise molecular signals involved in immunoregu-
lation by helminths. For some helminths it seems that complex oligosaccharides are
important, especially those that mimic human oligosaccharides. Dendritic cells (DC)
express many different C-type lectin receptors on their membranes, which vary
within distinct DC subsets. For example, DC-SIGN (full name Dendritic Cell-
Specific Intercellular adhesion molecule-3-Grabbing Non-integrin), macrophage
galactose-type C-type lectin (MGL, CD301) and the mannose receptor (MR) are all
expressed by DCs and shown to interact with host-like glycans of helminths in ways
that are thought to provoke immunoregulatory changes that assist parasite persis-
tence (van Die and Cummings 2010). Heligmosomoides polygyrus (a helminth that
has shown immunoregulatory effects in several mouse models) secretes a molecule
350 G.A.W. Rook

that binds the TGF-βRΙΙ and causes FoxP3-negative T cells to become functional
Foxp3+ Treg (Grainger et al. 2010). But this helminth also modulates DC function in
an anti-inflammatory way (Hang et al. 2010) and may exert indirect immunomodula-
tory effects via induced changes in the bacterial microbiota (Walk et al. 2010).

Gut Microbiota and Immunoregulation

This topic was extensively reviewed recently, and Fig. 3 lists some of the known
mechanisms (Ehlers and Rook 2011; Round and Mazmanian 2009). In the 1980s it
was revealed that defined alterations to the microbiota could reproducibly either
increase or decrease susceptibility to autoimmune arthritis (Kohashi et al. 1985).
Similar findings have been published recently using experimental autoimmune
encephalomyelitis (EAE), a mouse model of multiple sclerosis (Lee et al. 2010).
Modulation of the bowel microbiota could alter susceptibility to EAE by mecha-
nisms that involved the ability of intestinal DC to prime Th1, Th17 or Treg responses
(Lee et al. 2010). Some bacteria have also been found to enhance numbers and
activity of Treg (Atarashi et al. 2011) or even to secrete single molecules that lead
directly to expansion of Treg populations (Round et al. 2011). Short-chain fatty
acids (SCFA) produced by many gut bacteria also have an anti-inflammatory role in
the gut. SCFAs bind the G-protein-coupled receptor 43 (GPR43, also known as
FFAR2) and exert an anti-inflammatory effect that proved relevant in models of
colitis, arthritis and asthma (Maslowski et al. 2009). Other mechanisms include
induction of regulatory B cells (Correale et al. 2008) and regulatory macrophages
(Schnoeller et al. 2008), modulation of Treg/Th17 balance (Ivanov et al. 2008) and
indirect effects via epithelial cell products that cause DC to drive Foxp3+ cells with
gut-homing properties (Iliev et al. 2009), and induced secretion of REGIIIγ, a
C-type lectin with bactericidal effects on Gram-positive bacteria (Cash et al. 2006).
Interestingly there is evidence that very clean Specific Pathogen-Free (SPF)
mice (i.e. animals with a “normal” microbiota, in theory lacking pathogens) have
abnormally functioning Treg that can fail to secrete IL-10 and can switch function
to an aggressive cell type (Erdman et al. 2010). Humans in rich Western cities are
not SPF, but some modern babies must be getting close to the SPF state, with less
diverse commensals and microbiota and little exposure to pathogens.

Viruses and Immunoregulation

As outlined earlier, there is strong epidemiological evidence that neonatal infection

with HAV can protect against allergic disorders. The cellular receptor for HAV is
TIM-1 (T-cell immunoglobulin domain and mucin domain). TIM-1 is an important
atopy susceptibility gene. Furthermore, recent studies indicate that TIM-1 is a
receptor for phosphatidylserine, a marker of apoptotic cells (Umetsu and DeKruyff
2010). It is not yet clear how this translates into effects on immunoregulatory path-
ways, but there seems to be an effect on the Th2/Treg balance.
It was stated above that viruses such as Coxsackieviruses (Serreze et al. 2000) or
rotaviruses (Harrison et al. 2008) or LCM that provoke autoimmunity when given
Microbial Exposures and Other Early Childhood Influences… 351

Fig. 4 Interaction of genetics and loss of the “Old Friends”. The Old Friends had to be tolerated
and so coevolved roles as triggers of immunoregulatory pathways. In areas with very high loads of
these and other organisms, particularly helminths, compensatory genetic variants accumulated, to
partially restore inflammatory responses. In the absence of the Old Friends, not only is immuno-
regulation inadequately primed, but also these genetic variants cause excessive inflammation and
become risk factors for chronic inflammatory disorders. Thus, genetic variants that were advanta-
geous and did not cause disease in the past start to do so in the absence of the Old Friends (refer-
enced in main text). Several aspects of modern life are potentially interacting with the lack of “Old
Friends” at the level of immunoregulation. Obesity is associated with altered gut microbiota and
excessive release of proinflammatory cytokines. Lack of vitamin D exacerbates immunodysregula-
tion, as does the triggering of Th17 cells by dioxins (changes in the microbiota also impact on
Th17 development). Diesel particulates drive Th2 cells. Social stressors also drive inflammation,
and delayed exposure to childhood viruses may cause them trigger allergy and autoimmunity

late (for instance, at weaning) can be protective when given very early (Filippi and
von Herrath 2008; Harrison et al. 2008; Serreze et al. 2000). The mechanism again
involves immunoregulation. Such viruses, at least in a mouse model, can activate
invariant natural killer T cells that induce TGF-β-producing plasmacytoid DCs
(pDC) in the pancreatic lymph nodes (Diana et al. 2011). These regulatory pDC
then drive development of CD4 + CD25+ Tregs that synergise with upregulated pro-
grammed cell death-1 ligand 1 (PD-L1) to shut off the autoimmune response (Filippi
et al. 2009).

Compensatory Genetic Variants

In parts of the world where there was a heavy load of organisms causing immuno-
regulation, there has been selection for single nucleotide polymorphisms (SNP) or
other variants that partially compensate for the immunoregulation. This is seen for
several proinflammatory cytokines (Fumagalli et al. 2009) and IgE (Barnes et al.
352 G.A.W. Rook

2005). There is also an increased frequency of a truncated form of the serotonin

transporter that also has a marked proinflammatory effect (Fredericks et al. 2010).
The problem here is clear (Fig. 4). As soon as the immunoregulation-inducing
organisms are withdrawn by the modern lifestyle, these genetic variants lead to
excessive inflammation and become risk factors for chronic inflammatory disorders
(Barnes et al. 2005; Fredericks et al. 2010; Fumagalli et al. 2009). The selection in
Sardinia of TNF and HLA variants that protect from malaria, but predispose to MS
in the absence of malaria, is another example of this that was quoted earlier (Sotgiu
et al. 2007). These effects constitute a second layer of evolved dependence on the
continuing presence of the “Old Friends” (Fig. 4).
This is important because work that identifies proximate “causes” for diseases
that were rare or nonexistent before the second epidemiological transition may
merely be unravelling a problem that would be irrelevant if the microbial status
could be returned to that seen in the Palaeolithic. For instance, gluten-associated
enteropathies might be an “artefact” of poorly immunoregulated guts. Similarly the
recent claim to have discovered that the “cause” of Crohn’s disease is a genetically
determined defect in the homing of neutrophils is difficult to reconcile with the fact
that 100 years ago the disease barely existed. It is the recent environmental changes
that have caused this phenotype to become a risk factor (Smith et al. 2009) (Fig. 4).

Interactions with Other Changes in Modern Lifestyles

It would be foolish to assume that decreased exposure to microbial “Old Friends” is

the only reason for the increasing frequency of chronic inflammatory disorders in
developed countries. Other aspects of modern life that deviate from the EEA of our
primate ancestors must contribute and are likely to interact with and amplify the
immunoregulatory deficit resulting from the altered microbial environment (Fig. 4).

Diminished Nonmicrobial Immunoregulatory Exposures

Modern children may also be deprived of relevant nonmicrobial immunoregulatory

exposures. For instance, the powerful protection from allergic disorders provided by
early exposure to cowsheds (Riedler et al. 2001) might be in part due to exposure to
immunomodulatory arabinogalactans derived from grass (Peters et al. 2010), rather
than to microorganisms. Exposure to such grass dust oligosaccharides will have
been inevitable during those phases of human evolution that took place on grass-
lands. Similarly the protective effect of unpasteurised farm milk direct from the cow
is not understood (von Mutius 2010), but is probably not entirely microbial.
Microbial Exposures and Other Early Childhood Influences… 353

Obesity and Diet

The modern human diet encourages obesity, and adipose tissue releases proinflam-
matory mediators that will exacerbate the effects of any immunoregulatory defects
(Collins and Bercik 2009). Moreover, obesity is associated with phylum-level
changes in the microbiota and reduced bacterial diversity (Turnbaugh et al. 2009),
which will have immunoregulatory consequences (Round and Mazmanian 2009).
Psychological stress also modulates gut microbiota and gut permeability, while both
obesity and stress result in greater release of proinflammatory cytokines (Collins
and Bercik 2009). This all leads to a vicious circle, because the tendency to develop
obesity is modulated by the nature of the microbiota. For example, low levels of
Bifidobacteria and high levels of Staphylococcus aureus in infant microbiota may
predict the development of obesity later in life (Kalliomaki et al. 2008).

Vitamin D

Humans need sunlight to drive formation of vitamin D3, which is rarely present at
adequate levels in the diet. Vitamin D is involved in driving regulatory cells such as
Treg (Xystrakis et al. 2006). In rich developed countries deficiency of vitamin D is
increasingly common, partly because of fears of melanoma. This deficiency is
implicated in the increases in chronic inflammatory disorders, some cancers and
allergic disorders (Brehm et al. 2010; Herr et al. 2011; Honeyman and Harrison
2009). Moreover, vitamin D may also protect from allergic disorders indirectly by
enhancing immunity to respiratory viruses (Sabetta et al. 2010), some of which are
implicated in the causation of allergic disorders (Jackson 2010; Yoo et al. 2007).
This area has become deeply controversial following a recent report from the
Institute of Medicine in the USA (Ross et al. 2011) that recommended much lower
levels of vitamin D than most workers in the field would want to see (Heaney and
Holick 2011). The recommended levels are certainly likely to be lower than those
that would have been found in scantily clad hunter-gatherer humans.

Pollution

Several modern environmental pollutants might increase the incidence of chronic

inflammatory disorders. Dioxins, which drive Th17 cells via the aryl hydrocarbon
receptor (Veldhoen et al. 2008), will also encourage inflammatory responses of the
type seen in autoimmune disease. Diesel particulates contribute to Th2 responses
354 G.A.W. Rook

and allergic sensitisation (Riedl and Diaz-Sanchez 2005). There must be many other
novel molecules in our environments that have significant though poorly docu-
mented proinflammatory effects.

Broader Clinical Implications

The realisation that the hygiene or “Old Friends” hypothesis is largely a matter of
immunoregulation leads to the possibility that several other groups of disease might
also be increasing as a consequence of diminished exposure to organisms with
which we coevolved (Rook 2010). Two examples are particularly worthy of men-
tion. First, chronically raised levels of proinflammatory cytokines cause symptoms
of depression and are often found in depressed patients (Raison et al. 2010). Second,
chronic inflammation is oncogenic, and once tumours have developed, ongoing
inflammation releases growth factors and angiogenic factors that encourage tumour
growth. The epidemiology of some human cancers that are increasing in frequency
is similar to that of the allergic disorders and of T1D, and it is likely that failed
immunoregulation is playing a role (Rook and Dalgleish 2011). Thus, the conse-
quences of a lifestyle that no longer resembles the EEA of the primate or even that
of early hominins may be very broad and still not fully revealed.

Conclusions

This chapter has attempted to describe the microbiological history of humankind

from the hunter-gatherer past to the modern city lifestyle. Clearly this is impossible,
and the result is a superficial overview. Nevertheless, some simple and important
principles emerge. The immune system evolved three overlapping types of interac-
tion with the microbial world. First it had to combat infection with pathogens.
Second it had to “manage” and stabilise complex interactions with organisms that
are part of our physiology and so essential for health (microbiota and pseudocom-
mensals). Third it had to evolve ways to coexist with chronic infections that could
not be eliminated (many helminths). The last two types of interaction involve immu-
noregulation and tolerance, and the immune system is not surprisingly in a state of
evolved dependence on these organisms to drive maturation and set up the correct
regulatory pathways. It is interesting that these microbial roles were not upset until
the second epidemiological transition, when organisms that had coexisted with
mammals from the very distant past—largely from faeces, mud and other animals—
became depleted. Thus, we now see massive increases in chronic inflammatory dis-
orders in rich countries, and the same increases are beginning to appear in developing
countries. There is of course a limitation to this approach. We can measure rather
accurately the changing patterns of disease and of microbial exposures during the
twentieth and early twenty-first centuries, and we can also observe what happens in
Microbial Exposures and Other Early Childhood Influences… 355

contemporary hunter-gatherer and subsistence farmer populations as they undergo

the second epidemiological transition, lose their “Old Friends” and start to develop
chronic inflammatory disorders. But extrapolation from these contemporary obser-
vations to our distant past is more uncertain. Nevertheless, phylogenetic trees are
increasingly confirming the antiquity of our association with the “Old Friends”, and
archaeology is strengthening the view that contemporary hunter-gatherers and sub-
sistence farmers do indeed reflect life as it was in Palaeolithic and Neolithic times.
The exciting conclusion is that it should be possible to reverse the increases in
chronic inflammatory disorders that plague developing countries by manipulating
the microbial environments and by devising drugs based on their immunoregulatory
components.

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Make New Friends and Keep the Old?
Parasite Coinfection and Comorbidity
in Homo sapiens

Melanie Martin, Aaron D. Blackwell, Michael Gurven,

and Hillard Kaplan

Introduction

Across species, the fitness costs of parasitic infection have been a major force
shaping host adaptations to avoid infection (Hart 2009; Schmid-Hempel 2003;
Sheldon and Verhulst 1996), diminish the cost of infection (Minchella 1985; Råberg
et al. 2009), and even advertise resistance to infection to possible mates (Hamilton
and Zuk 1982; Moller 1990). These adaptations, in turn, have shaped selection on
parasite transmission and virulence, leading to coevolved host–parasite systems.
Host–parasite interactions are further shaped by local environments and proximate
host factors that influence transmission risk and infectious outcomes, including age,
sex, and nutritional and immune status (Anderson and May 1981; Anderson 1991;
Quinnell et al. 1995; Schad and Anderson 1985; Woolhouse 1992).
Host–parasite interactions are often observed and modeled as hosts interacting
with a single parasite species. Yet as is increasingly observed in animal populations,
including humans, coinfection with two or more species (alternately termed
“multiple-species” or “polyparasitic” infection) may be the rule in nature (Howard
et al. 2001; Booth et al. 1998; Bordes and Morand 2009; Pullan and Brooker 2008).
Coinfecting species may include any number of “typical parasites” (e.g., helminths,
flukes, tapeworms) and/or pathogens (e.g., bacteria, viruses, protozoa), each with
different associated exposure risks, infectious sites, reproductive strategies, virulence,

M. Martin (*) • A.D. Blackwell • M. Gurven

Department of Anthropology, University of California Santa Barbara,
Santa Barbara, CA 93106-3210, USA
e-mail: [email protected]
H. Kaplan
University of New Mexico, Department of Anthropology, MSC01-1040,
Anthropology 1, 87131, Albuquerque, NM, USA
Tsimane Health and Life History Project

J.F. Brinkworth and K. Pechenkina (eds.), Primates, Pathogens, and Evolution, 363
Developments in Primatology: Progress and Prospects,
DOI 10.1007/978-1-4614-7181-3_12, © Springer Science+Business Media New York 2013
364 M. Martin et al.

and associated immune responses (Alizon 2008; May et al. 2009; Rigaud et al. 2010;
de Roode et al. 2005; Van Baalen and Sabelis 1995). Of particular interest is the role
of immune responses in mediating coinfection risk, as an immune response gener-
ated by one species may either increase or decrease a host’s susceptibility to infec-
tion with another (Christensen et. al. 1987; Cox 2001; Supali et al. 2010). At the
same time, infecting species may competitively inhibit establishment or replication
by other species (Lim and Heyneman 1972; Fredensborg and Poulin 2005). As such,
coinfection may increase, decrease, or have no effect on host fitness, depending on
the individual species involved (Fellous and Koella 2009).
For humans—whose habitats span from hot, humid jungles to dry deserts, frozen
tundras, and sterile office buildings—infection risk may be especially varied.
Differences in parasitic and pathogenic exposure appear to have been a major force
shaping genetic variation across human populations (Fumagalli et al. 2011). Given
its ubiquity in nature and among nonindustrialized populations (Howard et al.
2001), multiple-species infection was likely equally common and varied among
human and hominin ancestral populations. Along with more transient infections,
hominin ancestors would have harbored multiple symbiotic organisms common to
other mammals: commensal bacteria, pseudo-commensals, ectoparasites, and hel-
minths (Armelagos and Harper 2005; Rook 2008). As proposed by the “hygiene
hypothesis,” continuous exposure to these organisms during mammalian evolution
may have favored the evolution of immunoregulatory systems that required their
antigenic input to develop appropriately (Jackson et al. 2008). In modern industrial-
ized, hygienic environments, infection with many of these “old friends” (particu-
larly helminths) is exceedingly rare, and consequently, disorders of immunoregulation
(i.e., allergy, asthma, chronic inflammatory conditions) have become increasingly
common (Rook 2008).
However, these old friends are also not without costs. First, helminth-induced
immunoregulation, which downregulates proinflammatory responses, may decrease
resistance to other parasites and more virulent pathogens, resulting in increased
infection intensity or exacerbated immunopathology (Graham et al. 2005; Pullan
and Brooker 2008). Second, exposure to helminth coinfection may increase invest-
ment in immune function (Bordes and Morand 2009), which may divert energy
away from other fitness-enhancing allocations, such as growth and reproduction
(Sheldon and Verhulst 1996; Adamo 2001; Uller et al. 2006; Blackwell et al. 2010;
Muehlenbein et al. 2010). Given the risk of helminth coinfection in ancestral envi-
ronments, potentially divergent immune responses, and the costs of increased
investment in immune function, several questions arise. How does helminth coin-
fection risk and associated morbidity vary across environments and with different
interacting species? How costly are multiple-species infections involving helminths
in humans? What multiple-species infections would have been typical for ancestral
populations? Finally, how have recent environmental changes altered helminth
coinfection risk in modern populations, and what are the consequences for human
health?
In this chapter, we review known aspects of immune responses to helminths and
other parasitic and pathogenic threats and consider how coinfections involving
Make New Friends and Keep the Old? Parasite Coinfection and Comorbidity… 365

helminths may affect human immune function and health, both past and present.
We first review host, environmental, and parasitic characteristics that influence the
likelihood of helminth coinfection. We then examine helminth-protozoa coinfection
and helminth-associated morbidity among the Tsimane of lowland Bolivia. The
Tsimane are a subsistence-scale, forager-horticulturalist population afflicted with a
high burden of both parasites and pathogens. We examine coinfection involving
helminths and protozoa and interactions between helminths and the risk of other
infections and inflammatory conditions. Although many aspects of the Tsimane
environment are unlikely to match those of ancestral hunter-gatherer populations,
the Tsimane disease ecology is likely more representative of ancestral conditions
than that of a contemporary industrialized or transitioning population. Our intent is
to provide an example of the complex interactions between infecting species that
would likely have been present through much of human history.

Multiple-Species Infections in Humans

Across human populations, associations between coinfecting species and infec-

tious outcomes vary widely (Hagel et al. 2011; Howard et al. 2001; Walson and
John-Stewart 2007). This variation may result from (1) individual host and para-
site factors influencing transmission and infection risk and (2) direct and indirect
interactions between coinfecting species (Cox 2001; Karvonen et al. 2009; Lello
and Hussell 2008). First, factors that mediate the risk of single-species transmis-
sion influence the likelihood of multiple-species infection. Host factors influenc-
ing susceptibility to infection include age, sex, socioeconomic status, physical
condition, nutritional status, sanitation and hygiene, work and labor demands,
access to medical care, prior exposure or immunization to infecting species, water
supply, and interactions with infected or reservoir hosts (Esrey et al. 1991;
Haswell-Elkins et al. 1987; Sayasone et al. 2011). Local ecological features (e.g.,
seasonality, soil, streams, ponds, etc.) and the life cycles, growth requirements,
and density and distribution of parasites in a given environment further influence
transmission risk (Anderson 1991; Hall and Holland 2000; Holland 2009).
Parasites with similar transmission routes (e.g., soil transmitted or waterborne)
and microclimatic requirements for growth and replication are more likely to
coinfect hosts (Ellis et al. 2007; Fleming et al. 2006; Haswell-Elkins et al. 1987;
Supali et al. 2010).
Once transmitted, infecting species must also establish and replicate. Typical
parasites such as helminths do not replicate inside hosts; eggs are excreted and lar-
val life stages occur in soil or animal vectors. Consequently, infection intensity and
pathogenicity depend on the infectious dose of initial and secondary infections
(Anderson and May 1979; May and Anderson 1979; Lafferty and Kuris 2002). In
contrast, pathogens replicate asexually in hosts and effects on hosts are independent
of the initial infectious dose (Lafferty and Kuris 2002).
366 M. Martin et al.

For coinfecting species, the sequential order of establishment, infectious dose,

and density of established parasites influence infectious outcomes of secondarily
invading species (Fellous and Koella 2009). Coinfecting species may inhibit the
establishment of new parasites through direct competition or competitive inhibition
(e.g., monopolizing host resources), ultimately reducing infection intensity or
pathogenesis of one or more species (Lafferty et al. 1994; Fredensborg and Poulin
2005). Direct competition is more likely when species inhabit similar locales in the
host (e.g., skin, lung, gut, blood, or lymphatic system) (Karvonen et al. 2009).
Finally, individual species can indirectly influence establishment and clearance of
coinfecting species across locales through antagonistic or synergistic immune
responses.

Immune Responses to Parasites and Pathogens

Mammalian, and indeed all vertebrate, immune systems have evolved to counter
invasions from diverse parasites and pathogens. Responses may be optimized to
clear infection and/or minimize damage from infection, depending on the infec-
tious agent’s own evolved strategy to evade or exploit host immune pathways
(Allen and Maizels 2011). The immune system makes strong phenotypic com-
mitments in response to infection, which may lead to biased immune responses
that in turn influence coinfection outcomes (Bradley and Jackson 2008). Each
of the several different types of immune defense, therefore, has its own costs
and benefits, and organisms must allocate resources appropriately to invest in
defenses that are useful for local pathogens (Long and Nanthakumar 2004;
McDade 2005).
The vertebrate immune system is generally divided into two levels of response:
innate and adaptive immunity. Innate immunity is the first line of defense, found in
all plants and animals; it recognizes and responds to generic signals of invasion
(e.g., unchanging structures on bacteria cell walls) with nonspecific responses
including inflammation, induction of acute-phase proteins (e.g., C-reactive protein),
activation of the complement system (a cascade of proteins that assist antibodies
and phagocytic cells in pathogen clearance), and activation and recruitment of white
blood cells, or leukocytes, to target and clear infected host cells and extracellular
viruses, bacteria, and protozoa.
Adaptive immunity is found only in vertebrates and, compared to innate immu-
nity, is highly specific, highly flexible in its recognition capabilities, and capable of
antigen-specific memory. Importantly, helminth diversity—a proxy for coinfection
risk—may have selected for increased investment in adaptive immunity during
mammalian evolution (Bordes and Morand 2009). Adaptive immunity is activated
when particles from invading organisms (antigens) are engulfed and processed by
phagocytic cells of the innate immune system. These cells present the antigens to
effector cells of the adaptive system (T and B cells), which are then activated and
clonally expanded. Activated B cells release antibodies, which bind to antigen and
Make New Friends and Keep the Old? Parasite Coinfection and Comorbidity… 367

facilitate pathogen clearance. Activated B cells may also develop into memory B
cells, which are the basis of acquired immunity.
Activated T cells undergo further differentiation into various subgroups of T
cells that direct different immune responses. These subgroups include cytotoxic T
cells, helper T cells (TH), and regulatory T cells (Treg cells). Helper T cells are further
differentiated into TH1, TH2, and TH17 cells based on the cytokines they are associ-
ated with. In brief, TH1 cells and associated cytokines such as IFN-γ stimulate
inflammatory and cell-killing activity important in clearance of pathogens (e.g.,
protozoa, trypanosomes, bacteria, viruses). TH2-associated cytokines (primarily
IL-4, IL-5, IL-13) stimulate antibodies including immunoglobulin E (IgE), IgG1,
and (in humans) IgG4 production, as well as basophils, eosinophils, and mast cells,
which are important in mediating clearance and tissue repair associated with typical
parasites (e.g., helminths, flatworms) (Allen and Maizels 2011). TH1 and TH2
responses are directly antagonistic, with IL-4 inhibiting IFN-γ production and vice
versa (Maizels and Yazdanbakhsh 2003).
Clearance of coinfecting species that provoke similar immune responses may be
enhanced through cross-immunity (Lello and Hussell 2008; Supali et al. 2010),
which can also diminish the likelihood of future coinfection with other commonly
associated species (Karvonen et al. 2009). Conversely, immune responses directed
against one species may suppress responses against other species if the coinfecting
species invoke antagonistic immune responses. As is discussed below, increasing
evidence suggests that helminths may bias immune function in a manner that
increases susceptibility to viral and bacterial infections.

Helminth-Induced Immune Responses and Coinfection

in Humans

Helminths are a large category of parasite known to have significant effects on host
fitness. The term “helminth” refers collectively to wormlike parasites and encom-
passes two phyla of major human parasites: Platyhelminthes (flatworms), which
include tapeworms (e.g., Taenia spp. and Hymenolepis spp.) and flukes (e.g.,
Schistosoma mansoni and Schistosoma japonicum), and Nematoda (roundworms),
which include ascarids (e.g., Ascaris lumbricoides), filarial worms (e.g., Wuchereria
bancrofti), pinworm (e.g., Enterobius vermicularis), whipworm (Trichuris trichi-
ura), threadworm (Strongyloides stercoralis), and hookworm (referring to
Ancylostoma duodenale and Necator americanus, which are often undifferentiated
in microscopic identification). Helminths—which are long lived, grow to sexual
maturity in hosts, but do not replicate in hosts—evoke relatively gentle immune
responses in mammals, quite distinct from the strong inflammatory responses
evoked by transient microbial pathogens that present imminent threats to host fit-
ness (Jackson et al. 2008; Allen and Maizels 2011).
368 M. Martin et al.

Helminths shift T cell populations towards a TH2 immune response, with corre-
sponding decreases in TH1 and proinflammatory responses (Cooper et al. 2000;
Fallon and Mangan 2007; Fox et al. 2000; Hewitson et al. 2009; Maizels and
Yazdanbakhsh 2003; Yazdanbakhsh et al. 2002). TH2 cells activated by helminths in
mucosal tissues induce production of IL-13 and IL-4 cytokines that drive mucosal
and muscular responses to dislodge the parasites. In non-mucosal tissues, TH2-
induced pathways and innate immune cells such as eosinophils, basophils, and mast
cells help drive parasite killing. IgE secreted from B cells is important in protecting
hosts from extraintestinal and encysted stages of helminths and may facilitate
antibody-induced larval killing following concomitant or secondary infections
(Allen and Maizels 2011).
Many helminths (as well as commensal bacteria) also induce Treg activity in order
to enhance their own survival in the host (Maizels et al. 2009). Treg cells release
cytokines that suppress TH1 and TH2 responses in order to minimize immunopathol-
ogy and epithelial damage caused by immune activation (Rook 2008; Round and
Mazmanian 2009). Treg activity may also promote production of IgG4 over IgE and
reduce expulsion of worms from the host (Mingomataj et al. 2006). Enhanced and
spontaneous production of Treg cytokines has also been observed in children with
chronic A. lumbricoides or T. trichiura infection and A. lumbricoides/T. trichiura
coinfection, suggesting endemic exposure to multiple helminths promotes stronger
immunoregulation (Turner et al. 2008; Figueiredo et al. 2010).
As such, the prototypical TH2/Treg response induced by helminths may be better
characterized as a “tolerance” response that contains the extent of helminth infec-
tion while limiting damage to the host (Jackson et al. 2008). In this case, the inter-
ests of host and parasite may align. Chronic exposure to helminths, which present
low or intermediate threats to host fitness compared to pathogens, would favor a
continual tolerance response over successive, highly inflammatory responses that
would be energetically costly and highly immunopathogenic to hosts. A tolerance
response would also be favored by helminths, as the Treg response enhances their
own long-term survival in the host, while TH2 responses may limit establishment
and competition by secondary invaders, allowing established parasites to monopo-
lize host resources (Jackson et al. 2008).
The tolerance response suggests that mammals share a deep coevolutionary leg-
acy with helminths. The IgE antibody, which is integral to antihelminth responses,
is a derived innovation in the mammalian lineage (Jackson et al. 2008). However,
more recent coevolution is also apparent: in humans, helminths appear to have
played a major role in genetic population divergence since the appearance of ana-
tomically modern humans within the last 200,000 years (Fumagalli et al. 2010).
More recent evidence of helminth infection in human history comes from mummi-
fied remains dating to approximately 30,000 BP in the Old World (A. lumbricoides)
and 7,837 BP in the New World (E. vermicularis) (Gonçalves et al. 2003), as well
as historical writings from classical Egyptian and Greek physicians (Cox 2002).
Today, however, helminth exposure and associated immune phenotypes are var-
ied across human populations. Hygiene, medicine, and socioeconomic development
Make New Friends and Keep the Old? Parasite Coinfection and Comorbidity… 369

Fig. 1 Variation in human IgE by population. Values are geometric means for each published
population (Adapted from Blackwell et al. (2010), which contains the complete references for each
population)

have drastically reduced early helminth exposure in many industrialized popula-

tions. Average IgE levels in North America and Europe are as much as 200 times
lower than those observed in subsistence-scale populations (Fig. 1). The highest IgE
levels are found among lowland indigenous groups in Ecuador (Buckley et al. 1985;
Kaplan et al. 1980; Kron et al. 2000) and Venezuela (Hagel et al. 2006; Lynch et al.
1983), which have reported geometric mean IgE in excess of 10,000 IU/ml. In con-
trast, geometric mean IgE in the USA is 52 IU/ml (Blackwell et al. 2011). Although
genetic factors have been shown to influence IgE levels (Weidinger et al. 2008)
and IgE levels show relatively high heritability when parents and offspring experi-
ence similar environments (Grant et al. 2008), differences between populations
appear to be influenced largely by exposure to helminths (Cooper et al. 2008) and
other parasites and pathogens, including malaria (Perlmann et al. 1994, 1999).
Immigrants who move from areas with endemic helminth infections to those with
low endemicity show an eventual drop in IgE levels, although it may take a decade
or more for levels to fall significantly (Iancovici Kidon et al. 2005; Kalyoncu and
Stålenheim 1992).
While the low IgE levels observed in North America and Europe are consistent
with low levels of helminth exposure, individuals in these populations with allergic
370 M. Martin et al.

diseases such as asthma do show elevated IgE levels (e.g., Bergmann et al. 1995;
Holford-Strevens et al. 1984; Lindberg and Arroyave 1986). Variations on the
“hygiene” and “old friends” hypotheses posit that a major factor in the rise of aller-
gic, autoimmune, and inflammatory disorders in industrialized populations today is
a mismatch between a human immune system that coevolved with “old friends”
(e.g., helminths and commensals) and a modern hygienic environment in which
these “old friends” are largely absent (Rook 2008). Without early and regular anti-
genic input from helminths, immune system development is altered, resulting in
“inappropriate” TH2 immune responses to harmless environmental antigens. When
induced by helminths, those same immune responses may depress allergic reactions
(Maizels 2005; Wilson and Maizels 2004), to the extent that prescribed low-dose
infections may be effective clinical treatments (Blount et al. 2009; Feary et al.
2010). The downregulation of inflammatory pathways induced by low-grade hel-
minth infections may also protect against diabetes (Maizels et al. 2009), obesity
(Wu et al. 2011), and immunopathology associated with opportunistic bacterial
infections (Anthony et al. 2008).
At the same time, chronic helminth infection can be costly to hosts. Presently, the
most common human helminth infections involve intestinal nematodes, especially
the soil-transmitted helminths (STH) A. lumbricoides, T. trichiura, and hookworm.
Adult STH reside and replicate in the host’s gut and pass eggs through host feces.
A. lumbricoides and T. trichiura eggs are ingested by hosts, whereas hookworm
eggs penetrate the skin, generally the bottom of the feet (Hotez et al. 2008; Jackson
et al. 2008). STH are widespread but are most prevalent in tropic and subtropic
regions (Chan et al. 1994; Silva et al. 2003). In many nonindustrialized nations,
STH infections are endemic and—despite increased efforts to improve sanitary con-
ditions, access to health care, and implement large-scale control programs—remain
a significant cause of morbidity, particularly among children (Bethony et al. 2006).
Complications ensuing from STH infections in children and adults (e.g., anemia,
growth faltering, reduced work output, and impaired cognitive ability), compounded
with poor nutrition and poverty, likely contribute to poor economic growth in these
areas (Guyatt 2000).
Coinfections involving multiple STH, or at least one STH and another parasite or
pathogen, are exceedingly common in nonindustrialized populations (Hotez et al.
2008). Multiple STH infections are often associated with increased infection inten-
sity, egg output, and morbidity (Booth et al. 1998; Brooker et al. 2000; Ellis et al.
2007; Pullan and Brooker 2008). It is also increasingly documented that helminth
infection and helminth-typical immune biasing may diminish immune responses to
vaccines, viruses, and bacteria, resulting in increased susceptibility to other infec-
tious diseases (e.g., HIV/AIDS (Bentwich et al. 1995); BCG, typhoid, measles, and
polio vaccines (Labeaud et al. 2009); tuberculosis (Lienhardt et al. 2002)). In sum,
helminths may interact with human immune function and other host factors in a
myriad of complex ways that have varying implications for human health. The risks
and effects of helminth coinfection therefore, while of clear clinical and epidemio-
logical significance, are also of relevance to researchers working across evolution-
ary and ecological fields.
Make New Friends and Keep the Old? Parasite Coinfection and Comorbidity… 371

Helminth Coinfection and Morbidity in the Tsimane of Bolivia

Human immune pathways may have been selected to counter the disease ecologies
of our predecessors, which included constant exposure to multiple parasites and
pathogens. Unfortunately, much of our understanding of human immune function
and health has derived from populations living under evolutionarily novel condi-
tions in which common parasites and pathogens are largely absent. Wider surveying
of the patterns of helminth coinfection and comorbidity across a range of environ-
ments are needed to better understand the varying consequences of endemic hel-
minth exposure—and lack thereof—in both nonindustrialized and industrialized
populations.
In this section we present and review data on helminth coinfection and comor-
bidity patterns in an Amazonian, small-scale subsistence population, the Tsimane of
lowland Bolivia. We focus specifically on the risk of coinfection involving hel-
minths and Giardia lamblia (aka Giardia intestinalis, Giardia duodenalis), a com-
mon Tsimane intestinal protozoan. We then examine the links between helminth
infection and other medical diagnoses. This research provides an example of the
interactions that may occur when multiple species and conditions afflict a single
population (Table 1).

Overview of the Tsimane

The Tsimane are a subsistence-level Amerindian population (pop. ~10,000) scat-

tered across approximately 120 villages along the Maniquí River and surrounding
forest areas. Most Tsimane have minimal access to medical care, market foods, or
wage labor opportunities and subsist primarily on locally cultivated plantains,
rice, manioc, and corn, hunted game, and wild fish (Gurven et al. 2007). Tsimane
live in large family clusters in open-air huts with thatched-palm roofs. Few vil-
lages have wells or other clean water sources; water is generally obtained from
nearby rivers and streams and rarely boiled. As of yet, no village has electricity or
sewage. The Tsimane do not maintain outhouses but urinate and defecate privately
in surrounding foliage. Domestic animals (dogs, cats, pigs, and chickens) are
owned by individual families but are rarely penned and roam freely around vil-
lages and familial spaces. Despite economic impoverishment, the Tsimane are
food secure. Nearly 70 % of the average adult diet is comprised of locally culti-
vated rice, plantain, manioc, and maize, with the remaining 30 % of the diet com-
prised of hunted game, river fish, and cultivated or foraged fruits and nuts. There
is little wasting indicative of protein malnutrition in children (Foster et al. 2005),
and the prevalence of underweight (body mass index < 18.5) among reproductive
aged females is <2 %.
 372

Table 1 Characteristics of common Tsimane intestinal parasites

% infected Latin Immune
Parasite/type Am/Caribbean Transmission route Infection site response Age peak Associated morbidity
Hookworm 9% Skin penetration Oral Small intestine TH2/Treg Adulthood Intestinal blood loss;
(helminth)a iron-deficiency anemia;
protein malnutrition
Ascaris lumbricoides 15 % Fecal–oral Small intestine TH2/Treg 5–10 year Lactose intolerance; vitamin
(helminth) A deficiency; intestinal
obstruction; hepatopan-
creatic ascariasis
Trichuris trichiura 18 % Fecal–oral Cecum Colon TH2/Treg 5–10 year Colitis; Trichuris dysentery
(helminth) syndrome; rectal
prolapse; impaired
nutrition
Giardia lamblia Unknown Contaminated water Small intestine TH1/TH2 Weaning infants, Diarrhea, flatulence,
(protozoan) Fecal–oral children vomiting, intestinal
mucosal damage; fat,
sugar, and vitamin
malabsorption; lactose,
vitamin A deficiency
References: Bethony et al. (2006), Brooker et al. (2004), Hotez et al. (2008), Ortega and Adam (1997), Wolfe (1992)
a
Hookworm = Ancylostoma duodenale/Necator americanus
M. Martin et al.
Make New Friends and Keep the Old? Parasite Coinfection and Comorbidity… 373

Methods of Tsimane Data Collection

Since 2002, the Tsimane have been participants in the ongoing Tsimane Health and
Life History Project (THLHP). THLHP researchers have worked extensively in
Tsimane villages, collecting demographic, anthropological, and biomedical data
while also providing primary medical care. The data presented in this chapter was
collected from participants seen by a mobile team of THLHP physicians, who trav-
eled annually through Tsimane villages from 2007 to 2010.
Patients seen by THLHP physicians were given routine physical exams (patient
history, symptom investigation, blood pressure and temperature, height and weight).
Physicians administered vitamins, antibiotics, and antihelmintics as warranted, fol-
lowing on-site analysis of participant blood and fecal samples. Ethnographic and
epidemiological information on the Tsimane, methods for age estimation, subject
sampling, biomarker collection, and physician diagnostics have been described
elsewhere (Gurven et al. 2007, 2008, 2009).
Results of parasitic infection presented and reviewed in this chapter were
obtained through community sampling and patient diagnostics conducted from
2004 to 2010. Fecal samples collected by THLHP researchers were analyzed using
two methods. From 2004 to 2008, fecal samples were analyzed for the presence of
helminth eggs, larvae, and protozoa by direct identification on wet mounts.
Beginning in 2007, fecal samples were also preserved in 10 % formalin solution
following direct identification and later quantitatively analyzed using a modified
Percoll (Amersham Pharmacia) technique (Eberl et al. 2002). Methods of fecal
sample collection and parasite identification using both methods have been described
in greater detail elsewhere (Vasunilashorn et al. 2010; Blackwell et al. 2011). Data
presented here were aggregated from the two methods, with individuals coded as
either infected or not infected if helminths were detected by either method
(n = 3,628).

Helminth and Protozoan Infections Among the Tsimane

Tsimane exposure to multiple gastrointestinal parasites and pathogens is endemic

and lifelong. From fecal samples collected from 2004 to 2010, we estimate that
77 % of Tsimane are infected with at least one intestinal parasite (Table 2). The
most common infections are hookworm, G. lamblia, and A. lumbricoides, infecting
51 %, 37 %, and 15 % of Tsimane, respectively. Females are more likely to be
infected with A. lumbricoides than males (OR = 1.33, χ2 = 9.30, p = 0.002) and less
likely to be infected with S. stercoralis (OR = 0.70, χ2 = 4.57, p = 0.03), while other
infections do not vary significantly by sex. T. trichiura and S. stercoralis are rela-
tively uncommon, infecting only 3.6 % and 3.7 % of subjects. Hookworm infections
are less common in children 10 and younger than in Tsimane over age 10 (37 % vs.
55 %) and show a steady increase with age (Fig. 2). Nearly 1/3 of the Tsimane
374 M. Martin et al.

Table 2 Prevalence of Tsimane single- and multiple-species infections

All ages
(n = 3,628) ≤10 (n = 893) ≥10 (n = 2,735)
N % N % N %
Infection
Hookworm 1,842 50.8 328 36.7 1,514 55.4
G. lamblia 1,336 36.8 308 34.5 1,028 37.6
A. lumbricoides 544 15.0 132 14.8 412 15.1
T. trichiura 129 3.6 16 1.8 113 4.1
S. stercoralis 135 3.7 18 2.0 117 4.3
Any infection 2,801 77.2 581 65.1 2,220 81.2
Hookworm only 956 26.4 176 19.7 780 28.5
G. lamblia only 680 18.7 186 20.8 494 18.1
A. lumbricoides only 133 3.7 32 3.6 101 3.7
T. trichiura only 14 0.4 2 0.2 12 0.4
S. stercoralis only 12 0.3 2 0.2 10 0.4
Total single-species infections 1,795 49.5 398 44.5 1,397 51.1
Hookworm and G. lamblia 422 11.6 61 6.8 361 13.2
Hookworm and A. lumbricoides 194 5.3 47 5.3 147 5.4
Hookworm and S. stercoralis 67 1.8 5 0.6 62 2.3
A. lumbricoides and G. lamblia 65 1.8 17 1.9 48 1.8
Hookworm and T. trichiura 29 0.8 3 0.3 26 1.0
T. trichiura and G. lamblia 14 0.4 2 0.2 12 0.4
S. stercoralis and G. lamblia 12 0.3 7 0.8 5 0.2
A. lumbricoides and T. trichiura 12 0.3 1 0.1 11 0.4
A. lumbricoides and S. stercoralis 6 0.2 1 0.1 5 0.2
Total 2-species infections 821 22.5 144 16.1 677 24.8
Hookworm, A. lumbricoides, G. lamblia 79 2.2 26 2.9 53 1.9
Hookworm, S. stercoralis, G. lamblia 25 0.7 2 0.2 23 0.8
Hookworm, A. lumbricoides, T. trichiura 21 0.6 2 0.2 19 0.7
Hookworm, T. trichiura, G. lamblia, 20 0.6 3 0.3 17 0.6
Hookworm, A. lumbricoides, S. stercoralis 15 0.4 2 0.2 13 0.5
Hookworm, S. stercoralis, T. trichiura 5 0.1 0 0.0 5 0.2
A. lumbricoides, T. trichiura, G. lamblia 8 0.2 2 0.2 6 0.2
A. lumbricoides, S. stercoralis, G. lamblia 3 0.1 1 0.1 2 0.1
Total 3-species infections 176 4.9 38 4.1 138 5.0
Hookworm, A lumbricoides, T. trichiura, 4 0.1 1 0.1 3 0.1
G. lamblia
Hookworm, A. lumbricoides, S. stercoralis, 3 0.1 0 0.0 3 0.1
G. lamblia
Hookworm, S. stercoralis, T. trichiura, 1 0.0 0 0.0 1 0.0
G. lamblia
Hookworm, A. lumbricoides, S. stercoralis, 1 0.0 0 0.0 1 0.0
T. trichiura
Total 4-species infections 9 0.2 1 0.1 8 0.3
Total 2+ species infections 1,006 27.6 183 20.2 823 30.1
Make New Friends and Keep the Old? Parasite Coinfection and Comorbidity… 375

Fig. 2 Total prevalence of Tsimane intestinal parasites by age

population harbors a multiple-species infection, though prevalence rates of different

coinfectious combinations are varied (Table 2). Hookworm is present in 85 % and
G. lamblia present in 65 % of all coinfections, while 55 % of coinfections involve
both hookworm and G. lamblia.

Helminth Coinfection and Infection Intensity Risk Among

the Tsimane

Half of all multiple-species infections observed involved infection with at least two
helminth species. As has been shown elsewhere (e.g., Howard et al. 2001), we found
that individual helminth infection increases the risk of coinfection with other hel-
minths. The strongest positive association we observed was between A. lumbricoi-
des and T. trichiura, with T. trichiura-infected subjects nearly six times as likely to
be coinfected with A. lumbricoides (Table 3). Each helminth infection was associ-
ated with higher odds of infection with another helminth. A. lumbricoides (OR = 1.40,
p < 0.001), T. trichiura (OR = 1.47, p = 0.05), and S. stercoralis (OR = 3.28, p < 0.001)
were all predictive of hookworm infection, while hookworm was associated with
A. lumbricoides (OR = 2.32, p < 0.001), and A. lumbricoides was associated with
T. trichiura (OR = 2.54, p < 0.001). Howard et al. (2001), in a survey of 60 interna-
tional studies of helminth coinfection, found that in ~70 % of cases, the risk of
coinfection with A. lumbricoides and T. trichiura was significantly higher than
would be expected by independent transmission. In the same study, increased risks
376 M. Martin et al.

Table 3 Odds ratios for infection with one parasite given infection with another
Dependent
Independent G. lamblia Hookworm A. lumbricoides T. trichiura S. stercoralis
G. lamblia 0.54*** 0.64* 0.89ns 0.51t
Hookworm 0.54*** 2.32*** 1.14ns 1.77ns
A. lumbricoides 0.72** 1.40*** 2.54** 0.97ns
T. trichiura 1.13ns 1.47* 2.03t 1.11ns
S. stercoralis 0.83ns 3.28*** 1.08ns 1.10ns
Age (decades) 1.04* 2.22*** 0.87* 1.05ns 1.06ns
Sex (male) 1.03ns 1.10ns 0.44** 0.77ns 0.91ns
Odds ratios were calculated in binomial logistic mixed models with all independent variables in
one model, controlling for age, sex, and repeat observations
Parameter significance: tp ≤ 0.10; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; nsnonsignificant

for hookworm-A. lumbricoides and hookworm-T. trichiura coinfections were also

widely documented (Howard et al. 2001).
Significantly higher infection intensity associated with helminth coinfection has
been widely reported (Brooker et al. 2000; Howard et al. 2002); we observed a simi-
lar relationship in the Tsimane. Among infected Tsimane patients, hookworm egg
count was significantly higher in the presence of S. stercoralis (β = 170.7 eggs/g,
p <0.001). A. lumbricoides intensity was significantly increased by hookworm coin-
fection (β = 416.2 eggs/g, p = 0.004). S. stercoralis intensity was higher with A. lum-
bricoides (β = 397.4 eggs/g, p <0.001), but not if an individual was infected by both
A. lumbricoides and hookworm (β = −417.4, p <0.001). There were no significant
effects of coinfection on T. trichiura intensity. As a caution, the relationship between
coinfection and infection intensity is somewhat difficult to parse. In many studies,
high-intensity infection with one species is associated with increased coinfection
risk, but it is unclear if high-intensity infections predispose hosts to coinfection or
vice versa (Raso et al. 2004; Fleming et al. 2006). Host factors such as age or nutri-
tional status may also predispose hosts to coinfection and higher infection intensity
(Pullan and Brooker 2008).
Given the high prevalence of helminth infection among the Tsimane (77 %), the
prevalence of helminth coinfection (14 %) in the Tsimane is much lower than rates
reported for many other tropical, underdeveloped populations (e.g., 58 % Tanzania,
Booth et al. 1998; 49 % Kenya, Brooker et al. 2000; 68 % Rwanda, Mupfasoni et al.
2009). The lower helminth coinfection rates in the Tsimane may be due to the rela-
tively lower prevalence of helminths other than hookworm. As compared to the
referred African populations, the Tsimane may also be less negatively impacted by
recent environmental and socioeconomic changes (e.g., food insecurity, malnutri-
tion, increased population density, environmental degradation) that may increase
host susceptibility to coinfection and/or exposure to multiple parasites—including
more virulent parasites and pathogens such as Schistosoma and Plasmodium spp.
It is also worth nothing that even in the absence of malnutrition and more virulent
coinfecting species, coinfection in the Tsimane was associated with increased infec-
tion intensity.
Make New Friends and Keep the Old? Parasite Coinfection and Comorbidity… 377

Table 4 Odds of infection based on receipt of antihelmintic or antiprotozoal drugs at the previous
medical visit
Any helminth Hookworm A. lumbricoides G. lamblia
(Intercept) 0.41*** 0.33*** 0.05*** 0.77**
Received antihelmintic 1.15 1.12 0.41*** 1.29*
Received antiprotozoal 1.46** 1.61*** 0.83 0.93
Age (years) 1.02*** 1.02*** 1.00 1.00
Sex (male) 1.06 1.13t 0.72* 1.05
Dist. to San Borja (10 km) 1.14*** 1.09*** 1.19*** 0.92***
Parameter values are odds ratios estimated in separate generalized logistic mixed model for each
parasite or pathogen.
Parameter significance: tp ≤ 0.10; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001

Previously, we have also shown that early and chronic elevated IgE—characteristic
of endemic helminth exposure—is also associated with growth deficits (Blackwell
et al. 2010). Thus, endemic exposure to multiple helminths may be a significant
cause of morbidity in the Tsimane. Future research with the Tsimane will investi-
gate host factors that increase susceptibility to helminth coinfection and infection
intensity and will evaluate if certain helminth coinfections and higher infection
intensity are associated with increased morbidity.

Evidence of Hookworm and Giardia lamblia Antagonism

Among the Tsimane

In contrast to the higher odds of helminth coinfection, we have found that the risk
of helminth-giardia coinfection among the Tsimane is significantly less common
than would be predicted by independent transmission. G. lamblia infection was
associated with significantly lower odds of infection with both hookworm
(OR = 0.54, p < 0.001) and A. lumbricoides (OR = 0.64, p < 0.001), while hookworm
and A. lumbricoides were conversely associated with lower odds of G. lamblia
infection (OR = 0.54, p < 0.001; OR = 0.72, p < 0.001). To put the size of this effect
into perspective, 31 % of those infected with any helminth were also infected with
G. lamblia, compared to 45 % of those without helminth infection. Of those with G.
lamblia infection, 49 % were infected with at least one helminth, compared to 64 %
of those without G. lamblia.
Given the apparent antagonism between helminth and G. lamblia infection, we
examined how treatment for helminths affected later risk of infection with G. lam-
blia, and vice versa (Table 4). Receiving an antihelmintic had no effect on the odds
of being infected with any helminth (OR = 1.15, p = 0.28) or with hookworm
(OR = 1.12, p = 0.34) but did reduce the odds of A. lumbricoides infection (OR = 0.41,
p < 0.001). However, receipt of antihelmintics was also associated with increased
odds for G. lamblia infection (OR = 1.29, p = 0.03). Antiprotozoal agents had no
effect on the odds of G. lamblia infection 1 year later (OR = 0.93, p = 0.55) but were
378 M. Martin et al.

associated with increased odds of hookworm infection at the subsequent visit

(OR = 1.60, p < 0.001).
Our results are consistent with those reported by Rousham (1994), who observed
an increase in G. lamblia prevalence following mebendazole treatment for A. lum-
bricoides and T. trichiura infection. The apparent antagonism may reflect competi-
tive inhibition or cross-immunity. In a murine model, G. lamblia, which reside on
microvilli in the small intestine, were inhibited by Trichinella spiralis when these
helminths inhabited the small intestine but not at later stages when they moved to
muscular tissue, suggesting a physical rather than immune interaction between the
two species (Chunge et al. 1992). In our study, only A. lumbricoides and hookworm,
both of which inhabit the small intestine, were negatively associated with G. lam-
blia, whereas T. trichiura (located further down in the large intestine) was not, sup-
porting these earlier observations.
Studies have also shown that G. lamblia clearance and protective immunity are
mediated by mixed TH1 and TH2 cytokine production (characterized by both INF-γ
and IL-4), as well as TH2 antibody responses (IgA, IgG, IgE) (Abdul-Wahid and
Faubert 2008; Jiménez et al. 2009; Matowicka-Karna et al. 2009). Therefore, it is
possible that helminth-induced TH2 activity may provide some cross-immunity
against G. lamblia. However, Hagel et al. (2011) found a higher prevalence of
G. lamblia in association with A. lumbricoides infection—but only at moderate
intensity and in conjunction with increased Treg cytokine activity—suggesting
helminth-giardia coinfection risk may only be increased with helminth-induced Treg
activity. Future studies with the Tsimane will examine the range of immune param-
eters alternately associated with helminth and protozoan infection and may shed
further light on the role of immune responses in helminth-giardia antagonism.

Helminth Infection Is Associated with Altered Odds

for Respiratory and Inflammatory Diagnoses

It has been suggested that the immunomodulatory properties of helminths may pro-
tect against allergies, autoimmune, and inflammatory disorders. However, helminth-
induced immune biasing may increase susceptibility to other infectious diseases. To
examine potential interactions between helminths, G. lamblia, and other medical
conditions, we grouped THLHP patient disease diagnoses into broad categories rep-
resenting the most common types of complaint. Excluding diagnoses of helminthia-
sis and giardiasis, these included gastrointestinal problems (43 % of 3,391 patient
examinations), muscle or back pain (34 %), upper respiratory illnesses (28 %), uri-
nary tract infections (13 % cases), fungal infections (8 %), arthritis (6 %), skin
infections (3 % cases), and traumatic burns or injuries (2 %). For analysis, we
divided the sample by age into children ≤ 16 years of age and adults over age 16
since many diagnoses were not equally prevalent in children and adults (Table 5).
Controlling for age, sex, and village location, helminth infections were associated
with greater odds of upper respiratory infection in children (OR = 1.33, p = 0.04).
Table 5 Association between current helminth and giardia infection and likelihood of medical diagnosis during medical visit
Odds ratios
Giardia Dist. to town
Sample Medical diagnosis (cases) Cases (%) Helminth infected infected Age (years) Sex (male) (per 10 km)
Children ≤ 16 years Gastrointestinal problems 454 (42 %) 1.09 0.88 0.97t 1.01 0.91**
n = 894 Fungal infections 70 (6 %) 1.17 0.88 1.00 1.23 1.06
obs = 1,086 Upper respiratory infections 474 (44 %) 1.33* 0.99 0.93*** 0.73* 1.11***
Urinary tract infections 19 (2 %) 0.58 0.75 1.24 0.46 0.62
Skin infections 57 (5 %) 0.84 0.37 0.79t 0.46 1.13
Trauma 17 (2 %) 0.54 0.68 1.13 0.51 0.65
Muscle or back pain 10 (1 %) 0.50 0.28 1.54 1.45 1.33
Adults > 16 Gastrointestinal problems 989 (43 %) 1.31** 1.01 1.00 1.02 0.97
n = 1,439 Fungal infections 186 (8 %) 1.00 0.49* 0.99 1.09 1.02
obs = 2,305 Upper respiratory infections 474 (21 %) 1.09 0.84 0.99*** 0.97 0.99
Urinary tract infections 423 (18 %) 1.03 0.96 1.00 0.35*** 0.88***
Skin infections 35 (2 %) 0.20 0.53 0.99 1.01 1.07
Trauma 55 (2 %) 1.75 1.07 0.95 1.35 0.81
Arthritisa 198 (9 %) 0.68* 1.13 1.06*** 0.48*** 1.13**
Muscle or back pain 1,143 (50 %) 0.72*** 1.32** 1.00 2.01*** 1.07***
Parameter values are odds ratios estimated in separate generalized logistic mixed model for each medical diagnosis
Make New Friends and Keep the Old? Parasite Coinfection and Comorbidity…

Parameter significance: tp ≤ 0.10; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001

a
There were no children with arthritis
379
380 M. Martin et al.

In adults, helminths were associated with greater odds of non-giardia gastroin-

testinal problems (OR = 1.31, p < 0.01) and with reduced odds of both arthritis
(OR = 0.68, p = 0.04) and muscle or back pain (OR = 0.72, p < 0.001). G. lamblia was
associated with reduced odds of adult fungal infection (OR = 0.49, p = 0.02) and
greater odds of adult muscle or back pain (OR = 1.32, p < 0.01). Neither was signifi-
cantly associated with trauma, skin infection, or urinary tract infection.
The observation of increased odds of respiratory infection in children but reduced
odds of arthritis and muscle or back pain in older adults is consistent with predic-
tions based on the immunomodulatory effects of helminths and elaborated on in the
hygiene hypothesis. In the absence of helminth and commensal-induced immuno-
regulation, inflammation may be excessive, leading to autoimmune disorders such
as arthritis. Previously we have shown that IgE, a marker of past and current hel-
minth infection, is associated with lower inflammatory markers, such as CRP
(Blackwell et al. 2010), and lower total cholesterol (Vasunilashorn et al. 2010).
However, as observed in the Tsimane, helminth infection may have varying effects
on comorbidity during different life stages, with different clinical implications.
Helminths may be protective in preventing inflammatory disorders during adult-
hood, but their immunoregulatory effects may increase susceptibility to infectious
disease at younger ages.

Limitations and Future Directions

There are several limitations of our analysis that limit wider extrapolation of our
results and interpretations. First, infectious status among the Tsimane is diagnosed
on the basis of a single fecal sample only, which may underestimate the true preva-
lence of both single- and multiple-species infections in this population. Second,
THLHP community sampling does not permit us to conclusively discriminate
between acute, chronic, or resolving infections, which may influence our interpreta-
tions of parasite associations in hosts. We also as yet do not have data on the pos-
sible risk of coinfection involving helminths and more virulent diseases known to
afflict the Tsimane, including leishmaniasis, dengue fever, tuberculosis, leptospiro-
sis, and other common viral and bacterial infections. While we have shown that
endemic helminth infection in the Tsimane may be protective against a common
intestinal protozoan, helminth-induced TH2/Treg biasing may increase susceptibility
to coinfection with these more virulent pathogens.
Finally, although we have shown that the prevalence of helminth coinfection
among the Tsimane is lower than that of several African populations, Tsimane coin-
fection rates may have increased in recent decades and may continue to increase in
coming years. The Tsimane have only been permanently settled in their current
territory since the mid-twentieth century (Gurven et al. 2007; Huanca 2006).
Increased sedentism, population growth and density, interactions with domesticates,
fecal contamination of local water sources, and emerging social threats (e.g., pros-
titution and alcohol abuse) have likely increased the rate of exposure to existing and
Make New Friends and Keep the Old? Parasite Coinfection and Comorbidity… 381

novel parasites and other infectious diseases. Meanwhile, hygienic conditions,

access to medical care, vaccine coverage, and antibiotic usage—though improv-
ing—remain poor. Novel patterns of coinfection, and their effects on Tsimane
health and immune function, may yet be emerging. Moreover, while the Tsimane
environment may be more similar to a recent ancestral environment than that of a
contemporary industrialized population, it is by no means identical to the disease
ecology of our hominin ancestors.
Future research with the Tsimane will examine the role of additional host and
environmental factors on coinfection susceptibility, such as regional and seasonal
risks, and additional variation in immune and morbidity markers associated with
different coinfectious combinations and infection intensities. In particular, more
sensitive surveys of pathogen prevalence and helminth-pathogen risk are needed in
this population. Such research may help to identify current risk factors associated
with a wider range of multiple-species infections and may help predict how emerg-
ing infectious threats are likely to interact with current endemic intestinal parasites
and associated immune responses.

Conclusions

In humans, the prevalence and distribution of a range of multiple-species infections

has been increasingly well documented in epidemiological research but remains
understudied from an evolutionary or ecological perspective. Researchers interested
in human-pathogen coevolution should consider the additional challenges posed by
parasitic coinfection. There are many factors that influence parasite coinfection risk
and coinfectious outcomes in hosts. In this chapter, we focused on coinfections
involving STH, which are the most widespread human intestinal parasite and induce
characteristic immunoregulatory responses suspected to influence a range of coin-
fectious outcomes.
Chronic helminth infection, which may have been the norm during human ances-
try, is associated with varying risks and benefits in modern human populations.
While the prevalence of multiple helminth infection among the Tsimane is lower
than that reported for several African populations, infection with a single helminth
species in the Tsimane was associated with helminth coinfection risk, and multiple
helminth infections were associated with increased infection intensity.
Our results suggest that helminth infection during childhood increases risk of
respiratory illness, which may indicate increased susceptibility to bacterial and
viral infection due to helminth-induced immune biasing. These results have impli-
cations for vaccination programs and the spread of epidemic diseases among the
Tsimane and require further investigation. Other work has suggested that early and
chronic elevated IgE, characteristic of endemic helminth exposure, is also associ-
ated with growth deficits (Blackwell et al. 2010). Therefore, helminth infection
may still pose a substantial threat to health and well-being in the Tsimane and other
nonindustrialized populations.
382 M. Martin et al.

At the same time, helminth-giardia coinfection in the Tsimane occurs less fre-
quently than would be predicted by independent transmission. The lower risk of
helminth-giardia coinfection may be mediated by direct competition or controlled
by strong TH2 mechanisms invoked by chronic helminth challenge. Future research
is needed to elucidate the mechanisms of helminth-giardia antagonism, particularly
given the clinical implications of increased infection risk following antihelmintic or
antiprotozoal administration. The results reviewed here also suggest that helminth
infection and elevated IgE in adulthood is associated with lower incidence of
inflammatory-associated morbidity (Blackwell et al. 2010; Vasunilashorn et al.
2010). These findings are all consistent with the proposal that some inflammation-
linked “diseases of modernity,” such as obesity and heart disease, may be due to the
absence of “old friends” with which our immune systems coevolved.
We intend this discussion of the costs, benefits, and altered risks of coinfection
associated with helminths to illustrate the importance of considering multiple-
species infections and the role of infectious communities in affecting the evolution
of immune responses in hosts, the virulence of pathogens, and implications for
health and treatment. In sum, we have presented evidence of a complex trade-off in
the risks and benefits of helminth infection in humans. These trade-offs are likely to
vary by life stage, environment, and host factors influencing coinfection and
morbidity risk, which are constantly changing across the human landscape. These
factors may influence differences in patterns of coinfection prevalence and associ-
ated morbidity observed today and must be considered in models of ancestral
parasite and pathogen coinfection risk. Identifying and understanding both the
ancestral and emerging risks of coinfection pose an important challenge for research-
ers working across varied human populations, which will be best met by an inte-
grated cultural, epidemiological, ecological, and evolutionary approach.

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Primates, Pathogens, and Evolution:
A Context for Understanding Emerging
Disease

Kristin N. Harper, Molly K. Zuckerman, Bethany L. Turner,

and George J. Armelagos

Introduction

Nonhuman primates (NHPs) are an important source of human infectious disease,

likely due to our close phylogenetic relationship (Wolfe et al. 2007). Major human
diseases that appear to have originated in NHPs include malaria (Liu et al. 2010),
AIDS (Chen et al. 1997; Gao et al. 1999), and perhaps even hepatitis B infection
(Chen et al. 1997). In addition, NHPs serve as a reservoir for infections such as yel-
low fever, monkeypox, and Ebola; indeed, it has been estimated that while primates
constitute only 0.5 % of all vertebrate species, they have contributed approximately
20 % of major infectious diseases among humans (Wolfe et al. 2007). Conversely,
human pathogens1 can have devastating effects on NHPs, especially the great apes,
all of which are listed as endangered species and some of which, like gorillas, are
critically endangered. Infectious diseases have had substantial negative impacts on

1
Here we use the term “pathogen” broadly, to include both microparasites (viruses, bacteria, and
fungi) and macroparasites (such as worms).
K.N. Harper (*)
Department of Environmental Health Sciences, Columbia University, Black Building, Rm
1618, 650 W 168th Street, New York, NY 10032, USA
e-mail: [email protected]
M.K. Zuckerman
Department of Anthropology and Middle Eastern Cultures, Mississippi State University,
Mississippi State, MS 39762, USA
e-mail: [email protected]
B.L. Turner
Department of Anthropology, Georgia State University, Atlanta, GA 30303, USA
e-mail: [email protected]
G.J. Armelagos
Department of Anthropology, Emory University, Atlanta, GA 30322, USA
e-mail: [email protected]

J.F. Brinkworth and K. Pechenkina (eds.), Primates, Pathogens, and Evolution, 389
Developments in Primatology: Progress and Prospects,
DOI 10.1007/978-1-4614-7181-3_13, © Springer Science+Business Media New York 2013
390 K.N. Harper et al.

wild great ape populations, exacerbating existing threats posed by habitat loss,
human encroachment, and hunting (Boesch and Boesch-Achermann 2000; Leendertz
et al. 2004; Wolfe et al. 1998). Whether occurring as epidemics, small outbreaks, or
single deaths, infectious diseases can negatively affect the viability of small or iso-
lated NHP populations, due to the characteristically low reproductive rate of many
NHP species, especially great apes (Boesch and Boesch-Achermann 2000; Ferber
2000; Goodall 1970, 1983; Nishida 1990; Wallis 2000; Wolfe et al. 1998).
Understanding how pathogens move between human and NHP populations and
sometimes become established in a novel host requires knowledge of a pathogen’s
evolutionary history in its natural host, as well as the potential for transmission in
both its natural and novel hosts. However, establishing these evolutionary and epi-
demiological relationships has been complicated by methodological missteps and
conceptual pitfalls. Molecular methods allow us to construct a better picture of the
past disease-scape of primates, and the past few years have witnessed a veritable
explosion of information regarding major pathogen transmission events between
humans and NHPs. These data have helped to elucidate pathogens’ histories, though
much remains to be done. In this chapter, we discuss some of the theoretical issues
and methodological advances involved in reconstructing the evolutionary relation-
ships between pathogenic organisms, humans, and NHPs. Using examples of major
human diseases and their causative agents, specifically malaria (Plasmodium spp.)
and HIV (human immunodeficiency virus), we discuss the implications for under-
standing emerging infectious diseases in both humans and our closest relatives.
First, we explore the differentiation of the human disease-scape, attempting to
reconstruct which pathogens diverged with their human hosts over evolutionary
time. Next, we outline which human pathogens are believed to have resulted from
cross-species transmission from NHPs. Finally, we discuss examples of cross-
species parasite transmission from humans to NHPs and consider their ramifications
upon conservation biology.

Differentiation of the Human Disease-Scape

We begin this chapter by exploring the differentiation of the human disease-scape

from the pathogen profiles of our closest NHP relatives. We frame this discussion in
terms of “heirloom” pathogens and “souvenir” species—the former representing
those that we inherited from our most recent common ancestor with chimpanzees and
the latter those that entered human populations via a host species switching event.

Theoretical Considerations

Studies of human disease ecology suggest that infectious diseases affecting human
populations can generally be grouped into two broad categories: those with a long-
standing, millennial relationship with humans and the latter those that have been
Primates, Pathogens, and Evolution: A Context for Understanding Emerging Disease 391

more recently acquired. Sprent (1969a, b) was the first to make this distinction,
identifying two distinct classes of microbes that afflicted hunter-gatherers in the
Paleolithic: “heirloom species” and “souvenir species.” Heirloom species are patho-
gens that originated in our anthropoid ancestors and continued to infect hominins
and eventually modern humans. In contrast to heirloom species, souvenir species
are newer evolutionary acquisitions that are typically “picked up” via exposure to
zoonotic reservoirs or vectors (Kilks 1990). These diseases are generally zoonoses,
i.e., infectious diseases that can be transmitted from animals to humans. Zoonoses
can be contracted through a number of routes: from sympatric reservoir host species
through cross transfer, through insect or animal bites, or through the preparation and
consumption of contaminated animal flesh. It appears that many of these souvenir
species are capable of temporary host switches without significant levels of adapta-
tion, as long as the novel host is sufficiently similar to the natural host in terms of
resources available to the pathogen (Kellog 1896, in Brooks and Ferrao 2005, p.
1292). Thus, these microbes can remain specialized in their natural hosts while
infecting multiple hosts, including humans, across a wider ecological niche. In
some cases, however, a souvenir species enters the human population and stays
there permanently, adapting to its novel host and becoming established; indeed, as
will be discussed, this category of souvenir species includes some of the major
pathogens that affect human societies.
Given the complexity of pathogen host specificity and adaptation, it should
come as no surprise that reconstructing the history of human pathogens is a diffi-
cult endeavor. Often, in studying the origins of human pathogens, it is necessary to
gather samples from our close NHP relatives to assess the presence or absence of
a pathogen in a given species as well as to determine its genetic relationship to
human variants. As the examples ahead will show, this process frequently repre-
sents a limiting factor in our search for disease origins due to the difficulty inher-
ent in collecting biological samples from wild NHPs, many of which live in remote
areas of the world as well as being endangered and enjoying special protections. In
order to illustrate the effect that host switching and limited sampling of NHPs can
have on our understanding of a pathogen’s history in humans, we present the cau-
tionary tale of malignant malaria. By beginning our discussion of human pathogen
origins with this example, we hope to demonstrate the careful attention to sam-
pling necessary when considering the evolutionary history of even intensively
studied pathogens such as Plasmodium falciparum, never mind the myriad less-
studied ones discussed in this volume (e.g., Treponema, Toxoplasma, Pediculus,
and Pthirus lice).

Malaria: From Heirloom to Established Souvenir Species

Malignant malaria, which is caused by P. falciparum, is the most dangerous form

of the disease, with the highest rates of complications and mortality. This species
of Plasmodium alone is responsible for an estimated 515 million episodes of
392 K.N. Harper et al.

illness (Snow et al. 2005) and nearly one million deaths annually (WHO 2010).
The pathogen has exerted tremendous selective pressure upon the human genome in
recent history (Hamblin et al. 2002; Sabeti et al. 2002; Tishkoff et al. 2001), but
fundamental questions persist about the Plasmodium’s history in humans.
Early molecular studies indicated that the protozoan’s closest relative was
P. reichenowi (Escalante and Ayala 1994), a parasite species found in chimpanzees.
This species was, for many years, represented by only a single strain isolated from
a chimp captured in the Democratic Republic of Congo several decades ago (Collins
et al. 1986). For this reason, many researchers believed that humans and chimpan-
zees both harbored their own distinct malaria strains; humans were infected by
P. falciparum, while chimps carried P. reichenowi. This was consistent with a sce-
nario in which each host species possessed its own heirloom Plasmodium species.
More intensive sampling, enabled by advances in noninvasive techniques, has
recently generated a novel twist on the origins of this pathogen by suggesting that
P. falciparum should instead be considered a souvenir species in humans. By exam-
ining blood as well as noninvasive samples collected from many wild chimpanzees,
researchers identified many new Plasmodium isolates among chimpanzee popula-
tions (Prugnolle et al. 2010; Rich et al. 2009). Sequence analysis showed that the
global genetic diversity of human P. falciparum was very low relative to the diver-
sity of chimpanzee P. reichenowi strains, indicating that the human species had
diverged more recently than the chimpanzee species. This finding was inconsistent
with P. falciparum being an heirloom pathogen in humans. Moreover, a more com-
prehensive study of fecal samples from nearly 3,000 wild great apes, including
chimpanzees, gorillas, and bonobos from throughout Central Africa, prompted
consideration of an alternative scenario regarding P. falciparum’s original host
(Liu et al. 2010). This study demonstrated that the gorilla branch of the Plasmodium
phylogeny included the strains most closely related genetically to human falci-
parum strains. Thus, it would appear that all circulating P. falciparum strains might
be the result of a single, very successful cross-species transmission event from
gorillas to humans.
Given the propensity of Plasmodium to switch primate hosts (Garamszegi 2009)
and the numerous twists and turns in the history of malaria thus far, it has been
noted that only further in-depth sampling of wild animals will confirm that no still-
closer relative is going undetected and that the current story is the correct one
(Prugnolle et al. 2011). Even with regard to a well-studied disease such as malaria,
our understanding may well change with future developments, which hinge on sam-
ple availability. As Wolfe et al. (2007) note, while resolving the debate surrounding
the origin of malaria will not necessarily assist with global eradication of the dis-
ease, it may contribute to our broader understanding of the dynamics of disease
emergence. With the potential importance of such knowledge in mind, as well as the
complexity involved in determining a pathogen’s history, we now turn to a discus-
sion of which human infections appear to be due to heirloom pathogens and which
appear to be caused by souvenir species instead.
Primates, Pathogens, and Evolution: A Context for Understanding Emerging Disease 393

Heirloom Pathogens and Human Paleolithic Ecology

During the Paleolithic, small human population sizes and, to a lesser extent, low
population density would have limited the diversity of possible heirloom species
affecting human and hominin populations by preventing sustained transmission of
many viruses and bacteria (Dunn et al. 2010, p. 2590). However, some species of
parasites and bacteria appear to have thrived in this setting. Characteristically, heir-
loom pathogens are organisms that are able to persist in small, dispersed popula-
tions; generate incomplete or short-lived immunity; or have a chronic course,
enabling prolonged transmission to new hosts. Several potential heirloom species
have been identified, including macroparasites such as head and body lice (Pediculus
humanus) (Reed et al. 2004) and pinworms (Enterobius vermicularis) (Hugot et al.
1999) as well as Staphylococci (Cockburn 1967, 1971; Sprent 1962, 1969a). Other
possible heirloom pathogens include the causative agents of yaws (Treponema pal-
lidum subsp. pertenue) (Harper et al. 2008) and typhoid (Salmonella typhi)
(Roumagnac et al. 2006).
The identification of extant pathogen species that belong to heirloom lineages
has been controversial, however, as the example of P. falciparum, above, illustrates.
Novel genetic data have assisted in this process, helping to clarify which species
most likely belong in the heirloom category by providing information on pathogen
divergence times and host histories. In addition, studies of host specificity in patho-
gens that infect wild NHP populations suggest that some major classes of parasites,
such as helminths, are more likely to be heirlooms due to their species specificity
than are protozoa and viruses, which are typically able to infect a wider swathe of
hosts (Pedersen et al. 2005).
Unique patterns of human behavior have no doubt guided the adaptation of heir-
loom pathogens. Weiss and Wrangham (1999), for example, note that while chim-
panzees and humans share 95–99 % of their DNA (Olson and Varki 2003), we only
share approximately 50 % of our pathogens. More specifically, pathogens and para-
sites affecting wild NHP species primarily include helminths, viruses, and protozoa,
while pathogens affecting humans are dominated by fungi and bacteria (Pedersen
et al. 2005) (Fig. 1). The reasons for these differences are poorly understood but
likely reflect divergent characteristics of host ecology and behavior.
The evolution of human herpesviruses may offer one example of an heirloom
pathogen molded over time by uniquely human behaviors. Phylogenetic studies
indicate that all eight members of the human herpesvirus family (Herpesviridae)
likely derive from an ancestral viral genome which infected the last common
ancestor of hominins and great apes (Gentry et al. 1988). Herpes simplex virus
(HSV) spreads from sites of initial infection in skin or mucosal surfaces to neuronal
cell bodies in order to establish latent infection, forming a long-term relationship
with its host. This long latency would have allowed HSV to persist in small, low-
density Paleolithic populations. There are two types of herpes simplex: HSV-1 pri-
marily produces oral herpes infection, while HSV-2 primarily produces genital
infection. HSV-2 appears to be the only type of herpesvirus among primates that is
394 K.N. Harper et al.

Fig. 1 Comparison of the taxonomic distribution of parasites in (a) free-living nonhuman pri-
mates and (b) humans. This data was based on a survey of 369 nonhuman primates and 1,415
humans (Figure reproduced from Pedersen et al. (2005))

transmitted primarily via sexual contact, and some have suggested that it may owe
its existence to uniquely human sexual practices. The divergence of HSV-1 and
HSV-2 dates back approximately 8–10 million years (McGeouch et al. 1995) and
presumably reflects the development of species-specific tropisms for the epithelium
of the oropharynx and the urogenital tract, respectively. For HSV-1 and HSV-2 to
take on these distinct tropisms, oral and genital sites had to become microbiologi-
cally isolated from each other, while oral–oral and genital–genital contact between
the hosts had to be maintained. McGeouch et al. (1995) have suggested that the
evolution of continual sexual attractiveness of hominin females throughout the
entire menstrual cycle, with an expected attendant increase in the frequency of sex-
ual intercourse, and the adoption of close face-to-face mating among hominins,
which may have facilitated the practice of kissing, provided the necessary condi-
tions for the evolutionary divergence of HSV-1 and HSV-2. This hypothesis, of
course, awaits rigorous testing, and more generally, the issue of how the behaviors
of particular primate species provide niches conducive to sexual transmission
remains a subject for further study.
Even absent the unique behavioral practices that characterize humans, however,
millions of years of evolution in different hosts ensures divergence among the
microbes that inhabit the bodies of humans and NHPs. For instance, the gut micro-
bial communities of the great apes, including humans, have evolved independently
with their hosts, diverging over the years in a manner consistent with host specia-
tion patterns (Ochman et al. 2010). Common shared ancestors are hypothesized to
exist between gut microflora species of humans and chimpanzees (Ushida et al.
2010) and also strepsirrhines (Bo et al. 2010). This suggests that some of the sym-
biotic microbes in human and NHP guts could be heirlooms. Understanding the
intersecting roles of physiological similarity, behavioral divergence, environmen-
tal context, and microbial colonization is therefore crucial when reconstructing
the evolutionary histories of heirloom microbes, both beneficial and pathogenic, in
humans and NHPs.
Primates, Pathogens, and Evolution: A Context for Understanding Emerging Disease 395

Souvenir Species and Changing Human Ecology

Hominins, particularly the genus Homo, became increasingly generalized as they

evolved new strategies for inhabiting and manipulating new environments. Modern
humans, in particular, have increased the scope of their exposure to pathogens via
the environmental modification and plant and animal domestication associated with
the adoption and intensification of agropastoralism. Numerous souvenir pathogens
have come to infect human populations, some of which have their origins among
NHPs. Many of these primarily remain residents of their reservoir animal hosts. For
example, though limited human-to-human transfer may occur in pathogens such as
SARS or Ebola, they must be considered what Weiss (2009) designates “temporary
exhibits” in humans. There are cases, though, in which a souvenir species adapts to
its novel host so well that it becomes established and flourishes within human popu-
lations. One example, already discussed, is P. falciparum. Another major example
comes in the form of HIV/AIDS, which we discuss here.

HIV: A Souvenir Species with a Complicated Past

Similar to the example of malaria discussed above, investigation into the NHP
origins of HIV/SIV has demonstrated that continued research on even the most
well-studied disease agents can yield important and surprising insights into their
origins and evolution. HIV represents the best-known example of an NHP pathogen
transmitted to and then sustained within humans, and it remains one of the most
serious pandemics in history. The UNAIDS Report on the Global AIDS Epidemic
2010 estimates that 33.3 million people are infected with HIV worldwide, among
them 2.6 million children. It is estimated that some 25 million people worldwide
have died from HIV-/AIDS-related diseases (UNAIDS 2010). The majority of indi-
viduals with HIV live in sub-Saharan Africa (UNAIDS 2010). Adding to the dis-
ease burden in sub-Saharan Africa and other regions with high rates of HIV is the
fact that this infection disproportionately affects young adults (Patton et al. 2009),
leading to high morbidity and mortality among those individuals who would other-
wise be among the most economically active in their societies. A result of this pattern
of infection is a demographic crisis in which young children and older adults carry
the burden of looking after themselves, each other, and those who are infected. The
fact that a disproportionate burden of infection is found in countries with high rates
of political and economic inequality compounds this crisis even further (Fox 2012).
There are two types of HIV: HIV-1 and HIV-2. Scholars have understood for
some time that both types evolved from the simian immunodeficiency viruses (SIVs),
with HIV-1 deriving from the SIV variant of chimps (SIVcpz) and HIV-2 from the
SIV of sooty mangabeys (SIVsmm) (Gao et al. 1999; Hahn et al. 2000; Hirsch et al.
1995; Peeters et al. 2002; Weiss and Wrangham 1999). SIV infection is quite com-
mon in NHPs over 40 species-specific SIV variants have been documented in
396 K.N. Harper et al.

African monkeys (Sharp and Hahn 2010). Moreover, cross-species transmission of

SIV has been postulated from African green monkeys to both patas monkeys
(Bibollet-Ruche et al. 1996) and yellow baboons (Jin et al. 1994). In a survey of 788
wild-caught NHPs from Cameroon, serological evidence of SIV infection was pres-
ent in 13 of the 16 primate species tested, with about 20 % of total samples testing
seropositive (Peeters et al. 2002). SIV has also been demonstrated in sooty mang-
abey bushmeat samples from rural Sierra Leone, underscoring how the initial trans-
fer of HIV-2 may have occurred (Apetrei et al. 2005). Therefore, NHPs represent a
substantial potential reservoir of continued SIV transmission to humans.
Our understanding of where human HIV strains originated has become more
nuanced with time. For example, it has long been understood that the HIV-1 lineage,
as a whole, originated from chimpanzees; however, one group within the lineage
may have a slightly different history than the others. HIV-1 group O (named for its
“outlier” status) accounts for a relatively small proportion of HIV cases and is rarely
found outside of Cameroon. Intensive sampling of wild chimpanzees and gorillas
has demonstrated that HIV-1 group O appears to be most closely related to an SIV
strain circulating in gorillas (SIVgor) (Van Heuverswyn et al. 2006). Thus, it is pos-
sible either that humans initially contracted the group O virus from gorillas or that
chimpanzees independently transmitted the virus to both gorillas and humans.
How often does SIV take hold in humans, establishing sustained infection in its
new host and spreading to other people? Determining the answer to this question
requires extensive and sensitive surveillance of a sort that has yet to be conducted,
but it appears that SIV infection in humans in close contact with NHPs is relatively
common. In Cameroon, one man was found to be infected with a virus serologically
related to SIVmnd, a version of the virus found in mandrills (Souquière et al. 2001),
demonstrating that NHPs other than the great apes and sooty mangabeys are capable
of transmitting SIV to humans. In another instance, a Cameroonian woman who
reported no contact with great apes or bushmeat was found to harbor a novel HIV
virus closely related to SIVgor (Plantier et al. 2009). The virus’s high replication
rate in this patient and the ease of its isolation in culture suggest that this novel variant
had adapted to human cells and may have spread from human to human at some low
level. Finally, 23 individuals out of a sample of 2,436 people at high risk for expo-
sure through poaching and bushmeat consumption tested seropositive for SIV,
though no active infections were demonstrated in this group (Djoko et al. 2012).
Thus, it appears that SIV transmission to humans may not be a rare event, and a low
level of human-to-human transmission may even occur at times.
What is the fate of individuals infected with SIV? Strains of SIV that infect
mangabeys and macaques appear to be very closely related to HIV-2, which is less
virulent than HIV-1. Accidental infection of two different laboratory workers with
these SIV strains did not result in AIDS-like symptoms, despite the presence of SIV
and HIV-2 antibodies (Khabbaz et al. 1994). This suggests that SIV infections in
humans may not necessarily be harmful, which could prove lifesaving for individu-
als in Central Africa who have tested seropositive. However, Peeters et al. (2002)
Primates, Pathogens, and Evolution: A Context for Understanding Emerging Disease 397

have noted that recombination between SIVs and circulating HIVs may pose a threat
to human health if it results in novel strains with an increased ability to exploit
our species.
In conclusion, though the sequence of events that allowed for permanent
establishment and spread of HIV-1 and HIV-2 in humans remains uncertain
(Pepin 2011), molecular and epidemiological studies are providing a richer context
for understanding these souvenir pathogens. As discussed, it has been known for
some time that at least two host switches, one from chimpanzees and one from sooty
mangabeys, resulted in HIV-1 and HIV-2, respectively. Recent research, though, has
demonstrated that a third host switch from gorillas may have resulted in one rare
HIV subtype (HIV-1 group O) (Van Heuverswyn et al. 2006). Additionally, molecular
epidemiological studies have demonstrated that cross-species exposure to SIV is
ongoing (Djoko et al. 2012) and may at times even result in low-level transmission
among humans (Plantier et al. 2009). Thus, the potential for novel forms of HIV to
take root in humans appears to be present, though most SIV transmission events
seem to fizzle out quickly.

Major Factors Facilitating Adaptation of NHP Pathogens

to Humans

Malaria and HIV, both discussed extensively in this chapter, are examples of patho-
gens that originally came from NHPs but found a permanent home in humans.
There are multiple reasons why a given NHP host species may or may not become
an established source of infection for humans. Phylogenetic relatedness and physi-
cal and environmental proximity are two primary—and tightly interwoven—factors
which are likely to affect this dynamic. In an assessment of the animal origins of 25
major human infectious human diseases, Wolfe et al. (2007) partially attributed the
finding that the majority of tropical infectious diseases arose in the Old World rather
than the New World to the greater genetic distance separating New World monkeys
and humans. It represents roughly twice the genetic distance between Old World
monkeys and humans and many times that between humans and Old World apes. At
the same time, physical proximity and shared habitats are likely to play a substantial
role. For example, Wolfe et al. (2007) partially attributed the high number of souve-
nir infections arising in the Old World to the greater evolutionary time available for
transfers between primates and humans there (c. 7–8 million years) as compared to
the New World (c. < 14,000 years).
On a regional level, some populations have maintained fairly frequent exposure
to NHPs. In Africa, South America, and Asia, in particular, communities living in
close proximity to NHPs often become involved in activities associated with a high
risk of exposure to NHP pathogens. Bushmeat handling and consumption provides
one of the most effective means for the spread of pathogens from NHPs to humans
398 K.N. Harper et al.

Fig. 2 Nonhuman primate

bushmeat confiscated in the
Lomako Forest of the
Equateur province in the
Democratic Republic of
Congo. (a) Fresh remains of
the lower half of a black
mangabey (Lophocebus
aterrimus) found at the Iyema
study site in 2010. It lies atop
a pile of leaves that were used
to wrap it for easier carrying.
(b) A pile of smoked
bushmeat confiscated in 2007
by the members of the Institut
Congolais pour la
Conservation de la Nature,
who work as park guards.
The pile includes bonobos,
monkeys, antelope,
crocodiles, and red river
hogs. (c) Close-up of a
skewered and smoked
monkey from the pile, most
likely a black mangabey
(Photo Credits: Amy Cobden)

(Fig. 2). Almost 100 % of villagers in rural forested areas of Cameroon have
reported eating NHPs, with over 70 % involved in hunting and 30 % active in butch-
ering (Wolfe et al. 2004a). Both activities involve repeated contact with potentially
infective body fluids and tissues. They also generate opportunities for pathogen
transmission to other individuals and communities linked by the bushmeat trade. A
market survey of two cities in Equatorial Guinea recorded 4,222 primate carcasses
on sale over 424 days (Fa et al. 1995), illustrating the great importance of NHP
hunting to local economies.
Primates, Pathogens, and Evolution: A Context for Understanding Emerging Disease 399

The prevalence of human infections derived from NHPs suggests that these
pathogens are able to exploit between-species interactions effectively. For example,
Wolfe et al. (2005) found that bushmeat hunters in rural Cameroon are infected with
a wide variety of human T-lymphotropic viruses (HTLVs), which are linked to leu-
kemia, lymphoma, and HTLV-associated myelopathy, as well as multiple simian
T-lymphotropic virus (STLV)-1-like viruses. The high diversity of these viruses in
Cameroon indicates ongoing cross-species transmission from NHPs to humans.
Similarly, at least three independent examples of NHP-to-human transmission of
simian foamy virus (SFV), which to date has not been linked to any signs of disease
in humans, have been confirmed in hunters (Wolfe et al. 2004b). Each event derived
from distinct NHP lineages—De Brazza’s guenons, mandrills, and gorillas—indi-
cating that many species can potentially infect humans. Bites and scratches by
NHPs, in particular, appear to be a very efficient means of transmitting viruses.
In one study, over 35 % of hunters reporting such wounds were seropositive for SFV
infection, and almost 2 % of serum samples belonging to adults from Cameroon
were found to be seropositive as well, underscoring the high prevalence of cross-
species transmission opportunities (Calattini et al. 2007).
While the factors leading to introduction of NHP pathogens into human popula-
tions are increasingly well characterized, the conditions that facilitate their estab-
lishment in our species are poorly understood. For example, though studies of
Cameroonian hunters suggest that exposure to STLVs via NHP blood contributes to
a greater diversity of HTLVs than is found in other populations (Wolfe et al. 2005),
only HTLV-1 and HTLV-2 appear to have established themselves worldwide.
Similarly, reports of the presence of dual HIV-1 and SFV infections in a commercial
sex worker from the Democratic Republic of Congo and in a blood donor from
Cameroon suggest the potential for SFV to be transmitted from human to human via
sex or blood, as is HIV (Switzer et al. 2008). Such human-to-human transmission
events appear to occur relatively rarely in the case of SFV, however. At present, our
ability to predict which souvenir pathogens will “take off,” flourish, and become
established within human populations is poor. Nonetheless, continued attention to
the movement of pathogens across the NHP-human interface may contribute to our
knowledge of this process.
It is worrisome that the trend of increased contact between NHPs and humans
seems to be intensifying. Population pressure, expanded ecotourism and conserva-
tion programs, and ever more powerful technological advances are enabling humans
to encroach further and further into NHP habitats (Auzel and Hardin 2001). For
instance, the villages surrounding logging concessions in Equatorial Africa have
grown rapidly, often having increased from a few hundred individuals to several
thousand. Political instability and forced migration, from Liberia into Sierra Leone,
for example, have also played a role in the dramatic redistribution of human popula-
tions into areas of increased contact with NHPs (Hodges and Heistermann 2003).
This new proximity, paired with the desirability of NHPs as prey, has increased
opportunities for cross-species transmission events. In addition, people in these
areas can utilize new and improved roads to transport bushmeat from remote vil-
lages to major cities, greatly increasing the number of humans who come into
400 K.N. Harper et al.

contact with NHP carcasses. In truth, contact with wild NHPs now spans continents,
reaching consumers who have never set foot in wild NHP habitat (Ellicott 2011);
examination of bushmeat samples confiscated at US airports has revealed NHP tis-
sue infected with SFV and herpesviruses (Smith et al. 2012). It is expected that
these trends will intensify in the future, in the absence of decisive community-level
and governmental actions to regulate ecotourism and constrain environmental
destruction and the bushmeat trade.

Examples of Major Diseases Transmitted from Humans

to Nonhuman Primates

Naturally, the increasing proximity between humans and NHPs also leads to greater
opportunity for human pathogens to infect both captive and wild NHPs. In general,
as the level of interaction between humans and NHPs increases, so does the risk of
transmission of diseases such as measles and tuberculosis (Wolfe et al. 1998). Not
surprisingly, there are many well-documented examples of captive NHPs becoming
infected with human pathogens, with “immunologically naïve” great apes proving
especially susceptible. For instance, there are frequent reports of tuberculosis infec-
tions of human origin among captive NHPs (Montali et al. 2001). Poliovirus can
also infect chimpanzees and gorillas, as well as more distantly related anthropoids,
like Colobus monkeys (Brack 1987; Suleman et al. 1984). As such, accidental expo-
sure to infected laboratory workers has led to poliovirus infections of chimpanzees
and gorillas since the 1940s (Ruch 1959). In another example, Arcobacter butzleri,
which is a member of the same bacterial family as Campylobacter
(Campylobacteraceae) and is associated with chronic diarrhea in humans, was
implicated in a spate of cases of chronic diarrhea among captive primate popula-
tions at a research center (Andersen et al. 1993).
There is accumulating evidence for similar episodes of disease transmission in
the wild (Adams et al. 1999; Homsy 1999; Wallis 2000; Wolfe et al. 1998; Woodford
et al. 2002). There are a number of “likely” instances of human-to-NHP transmis-
sion. In perhaps the most infamous instance, in 1966, six chimpanzees at Gombe
Stream National Park in Tanzania died from a polio-like virus, and six others were
paralyzed for life, shortly after a polio epidemic swept through neighboring human
settlements (Wallis 2000). Unfortunately, as no biological samples were collected
from the animals, it was impossible to verify if the epidemic was due to a poliovirus
introduced by local human populations or researchers (Wolfe et al. 1998). Confirmed
examples of human to wild NHP transmission are relatively rare, due to the diffi-
culty inherent in collecting samples but include Cryptosporidium infections in
mountain gorillas (Nizeyi et al. 2002) as well as the cases discussed below.
Respiratory diseases, in particular, have been recognized as a major source of
morbidity and mortality among free-living NHPs. This class of diseases is widely
regarded as the most important cause of morbidity and mortality among wild great
Primates, Pathogens, and Evolution: A Context for Understanding Emerging Disease 401

apes habituated to the presence of humans, whether due to research, tourism, or

human communities living in close proximity (Goodall 1986; Hanamura et al.
2007; Homsy 1999; Nishida 1990; Woodford et al. 2002). For example, a serologi-
cal survey demonstrated that 100 % of macaques at a temple in Katmandu were
seropositive for antibodies to the measles virus (Jones-Engel et al. 2006). Of more
potential significance for NHP conservation efforts, about half of long-term chim-
panzee research populations have shown major population declines that are likely
a consequence of respiratory disease (Hill et al. 2001). For instance, Köndgen
et al. (2008) have documented transmission of two common strains of human para-
myxoviruses—human respiratory syncytial virus (hRSV) and human metapneu-
movirus (hMPV)—from humans to chimpanzees at a research station in the Taï
Forest in Côte d’Ivoire. These two viruses are common causes of respiratory dis-
ease in humans. They are the leading causes of lower respiratory disease in chil-
dren and, in developing countries, are a major source of infant mortality (Boivin
et al. 2003; Weber et al. 1998). Transmission of the viruses from humans to the
chimps of the Taï Forest resulted in five discrete epidemics during the study period,
each accompanied by high morbidity, with an average of 92.2 % of individuals
showing clinical symptoms. In three of the epidemics, 18–34 % of chimpanzees in
the study population succumbed to the infection. Viral strains sampled from the
deceased chimpanzees were found to be closely related to strains circulating in
contemporaneous, worldwide human epidemics, indicating a link between the epi-
demic and the continuous flow of outside ecotourists and researchers at the station
(Köndgen et al. 2008).
Unfortunately, such epidemics are not unique to the Taï Forest. hMPV was also
identified in association with acute and fatal respiratory illness outbreaks in the
chimpanzees of Mahale Mountains National Park, in Tanzania (Kaur et al. 2008).
Additionally, hMPV was documented in association with two mountain gorilla
deaths in Rwanda (Palacios et al. 2010). Thus, molecular epidemiology has con-
firmed that human pathogens are responsible for many of the “mysterious” ail-
ments currently driving population declines in NHPs. Not surprisingly, fatal
outbreaks of respiratory disease at Mahale Mountains National Park have coin-
cided with peak tourist season (Kaur et al. 2008). Similarly, wild chimpanzees in
Kibale National Park, Uganda, have been found to harbor E. coli strains genetically
similar to those carried by the humans they come into proximity with via research
or tourism. This underscores the fluid transmission of microbes from humans to
NHPs made possible by high rates of ecotourism and conservation-oriented
research (Goldberg et al. 2007).
As noted, while it is clear that human-derived pathogens have been responsible
for swift epidemics and dramatic declines in NHP populations, often the identity of
the agent responsible for a given epidemic remains merely suspected or wholly
unknown. For example, outbreaks of gastrointestinal illness (Goodall 1983) and
respiratory disease (Ferber 2000) suspected to originate in human populations have
been recorded among chimpanzees in Tanzania from the 1960s onwards. Similarly,
suspected cases of measles among gorillas were documented in Rwanda in 1988.
Finally, suspected but unconfirmed cases of scabies among gorillas have been
402 K.N. Harper et al.

reported in multiple regions (Kalema-Zikusoka et al. 2002). Our inability to deter-

mine the etiological agent responsible for most NHP diseases is due in large part to
the difficulty of acquiring samples for diagnostic testing from wild NHPs. Systematic
screening for the pathogens involved in NHP fatalities is performed infrequently,
even though such investigations can reveal both the causal agents and, given the
right molecular data, their transmission dynamics (Leendertz et al. 2006).
There is no evidence—yet—of sustained transmission of human pathogens
among NHPs. It is probable that many human pathogens that have adapted to large
populations with constantly replenished pools of susceptible hosts (i.e., crowd dis-
eases) “burn out” after rapidly infecting small groups of NHPs. The possibility of
sustained transmission of human pathogens in NHPs is certainly possible, however,
especially for infections with long latent periods. Kaur et al. (2008) note that persis-
tent infection with hMPV in the absence of respiratory symptoms has been demon-
strated in humans; if the same is true in NHPs, then the potential of individual
animals to carry the disease from one group to another via emigration could have
devastating consequences. Implementing rigorous, systematic monitoring of infec-
tious disease outbreaks as part of modern conservation practice is being strongly
encouraged (e.g., Leendertz et al. 2006) and gradually implemented (e.g., the Great
Ape Health Monitoring Unit (http://www.eva.mpg.de/primat/GAHMU/ index.htm)
and the Mountain Gorilla Veterinary Project (http://www.gorilla doctors.org/)).
It seems reasonable that given increased surveillance of NHP infections, examples
of human microbes capable of sustained transmission among NHPs will be
identified.

Conclusion

The surveillance of humans living in close proximity to NHPs has revealed tantaliz-
ing clues about the process of disease transmission from NHPs to humans. Perhaps
intensive study of circulating strains of HIV/SIV, HTLV/STLV, and SFV will
increase our knowledge of the features which characterize cross-species transmis-
sion events that result in subsequent sustained transmission between humans. In
addition, the explosion of knowledge surrounding pathogens of NHP origin, such as
P. falciparum and HIV, in the last few years underscores the need to delve into the
disease-scape of closely related NHPs to better understand our own infections. For
instance, it was not until 2010 that researchers demonstrated that wild chimps in the
Taï Forest appeared to be naturally infected with five different Plasmodium species
(Kaiser et al. 2010). Casting a wider net for pathogens may lead to similar advances
in our understanding of the other pathogens that make up the NHP disease-scape.
It is likely that the rapid development of sophisticated, noninvasive means of
NHP sampling will provide insight into the existence and/or prevalence of various
pathogens. These noninvasive methods include approaches similar to those devel-
oped in primatology to study endocrinology (e.g., Deschner et al. 2003; Hodges and
Heistermann 2003), characterize NHP genetics (e.g., Boesch et al. 2006; Bradley
Primates, Pathogens, and Evolution: A Context for Understanding Emerging Disease 403

et al. 2004; Vigilant et al. 2001), and perform urine assessment (e.g., Knott 1996;
Krief et al. 2005). For instance, fecal samples have already been used to assay SIV
and malaria in NHPs. In addition, a recent retrospective study of the epidemics in
the Taï Forest suggests that it is possible to monitor infections caused by paramyxo-
viruses, such as hMPV and respiratory syncytial virus, using fecal samples (Köndgen
et al. 2010). A pioneering study using aDNA techniques to study curated early-
twentieth-century NHP skeletal material even suggests that we can explore the his-
tory of viruses such as STLV using museum specimens (Calvignac et al. 2008).
Such noninvasive approaches may also help address the role of human pathogens
in NHP demographic declines. There are many unresolved questions. Do human
pathogens associated with epidemics in NHPs tend to rapidly infect small groups
before burning out? Or are some human-derived pathogens circulating continuously
and even adapting to their new hosts? In some cases, whether or not the pathogens
sweeping through NHP populations derive from humans is not clear. For example,
S. pneumoniae was recently found to be responsible for clusters of sudden death in
the chimpanzees of the Taï Forest (Chi et al. 2007). However, comparison to
sequences obtained from people living nearby suggested that the pathogen might
not be of human origin. Chimpanzees in different areas, but in frequent contact with
one another, were also found to harbor distinct S. pneumoniae clones. If not from
humans, from what reservoir did this pathogen arise? Similarly, adenoviruses were
isolated from two chimpanzees with signs of acute respiratory disease in Mahale
Mountains National Park (Tong et al. 2010). Sequence analysis identified two dis-
tinct viruses, but their origin was unclear; they could have been acquired from
humans, acquired from another species, or circulating among chimpanzees for some
time. Further investigation may shed light on the processes underlying such
outbreaks.
Continued study of the relationship between humans, pathogens, and NHPs con-
fers several substantial benefits. First, knowledge in this area is fundamental when
assessing the impact of pathogen exchange between species (Wolfe et al. 2007),
including research on the footprint of natural selection imposed by various infec-
tious diseases upon the human genome (e.g., Hamblin et al. 2002; Sabeti et al. 2002;
Tishkoff et al. 2001). Second, understanding more about how NHP pathogens are
introduced into human populations and then spread has practical implications.
According to Wolfe et al. (2007), benefits include a better understanding of disease
emergence and the potential for novel laboratory models helpful in studying public
health threats. Applications might include the development of indicators useful in
monitoring pathogen transmission between NHPs and high-risk individuals, such as
hunters and wildlife veterinarians; predicting which NHP pathogens might repre-
sent a future threat; and detecting and even controlling local human outbreaks
before they become epidemics (Wolfe et al. 2007). Third, some scholars have argued
that wild NHPs can serve as “sentinel species” for predicting disease outbreaks
among humans (Leendertz et al. 2006; Rouquet et al. 2005).
Finally, studying the relationship between pathogens, NHPs, and humans has
important conservation implications. Köndgen et al. (2008) and others have argued
that the close proximity between NHPs and humans, which is critical to both
404 K.N. Harper et al.

research and ecotourism programs, represents a serious threat to the existence of

wild primate populations. Obviously, this represents a dilemma, as both of these
activities have clear benefits for conservation efforts, whether via suppressing
poaching, generating income for local communities, or creating additional knowl-
edge about primate biology and behavior. Do the benefits associated with conserva-
tion efforts outweigh the health costs for apes and other NHPs wrought by increased
contact between NHPs and humans? Research efforts should be directed towards
reducing deleterious health outcomes, and an improved understanding of the evolu-
tionary trajectory and dynamics of pathogen exchange between humans and NHPs
stands to make a substantial contribution to this effort. For instance, research on
disease transmission from humans to NHPs can be used to perfect targeted strate-
gies for preventing infection, including close monitoring of the health and behaviors
of human observers and workers in conservation, scientific, and veterinary contexts
(Homsy 1999; Nizeyi et al. 2002; Woodford et al. 2002).
Findings from the studies discussed above have already been used to generate
specific recommendations for reducing the negative effects of tourists, local com-
munities, and researchers upon NHPs, especially endangered great ape communi-
ties (see Ryan and Walsh 2011). These guidelines include a variety of strategies for
limiting disease spillover into NHPs via the use of facemasks, minimum approach
distances, limited-duration visits, and strict hygiene protocols. They also encom-
pass the education of and collaboration with local stakeholders to determine optimal
rates of tourism for preventing disease transmission while maximizing tourism rev-
enues (maximum sustainable yield concept); vaccinating NHPs and treating infec-
tions when they arise; prohibiting human access to restricted areas in order to
minimize both direct and indirect contact, such as through human feces, between
local humans and NHPs; and establishing health programs for local communities
and staff involved in habituating NHPs for tourism and research (Ryan and Walsh
2011). An active area of research focuses on how these disease-mitigating measures
can be carried out with the full participation of local communities throughout Africa
and other regions home to endangered NHP populations, in order to make these
endeavors sustainable, practical, and desirable for the people involved (Ryan and
Walsh 2011).
In terms of more research-intensive interventions, observations stemming from
invasive and noninvasive tests on chimpanzees involved in the Taï Forest epidemics
have been used to generate demographic, clinical, and diagnostic monitoring sys-
tems which could potentially enable humans to intervene quickly in future NHP
epidemics there. Researchers involved in this ongoing project have strongly encour-
aged other investigators to implement similar systems at NHP research centers and
parks (see Ryan and Walsh 2011). Such efforts would not only protect NHP popula-
tions but also objectively document the negative effects of research or ecotourism
on NHPs (Köndgen et al. 2008).
In summary, the rapid and extensive destruction of forest ecosystems and chang-
ing patterns of contact between humans and NHPs, both stemming from the
increase in size and changing distribution of human populations, have changed the
disease-scape of all species involved. Someday, as detection and surveillance
Primates, Pathogens, and Evolution: A Context for Understanding Emerging Disease 405

improves, we may be able to perform comprehensive analyses of the different

disease-scapes of humans and NHP species. When this happens, we will be able to
explicitly test hypotheses such as whether host genetic similarity correlates neatly
with the proportion of pathogens shared between two given species. Moreover, in
the future we may learn more about how different primate species react to identical
pathogens, which will yield important information on how immune responses and
transmission dynamics differ within and between populations. The relationships
between NHPs, humans, and pathogens are fluid. Targeted research may help us
prevent the worst possible consequences of these constantly shifting associations
by allowing us to learn some general lessons about the processes underlying patho-
gen host switches.

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Index

A AIDS. See Acquired immunodeficiency

ABO syndrome (AIDS)
and Campylobacter jejeuni, 136 Allergies/allergy/allergic disorders, 37, 44, 45,
and Escherichia coli, 136 331, 332, 336–337, 340–343,
geographic distribution of groups in 346–348, 350–354, 364, 378
humans, 135 basophils, 37
and malaria, 135–136, 147, 148 helminthes, 44, 337, 343, 349, 351, 364,
natural selection, 135, 136, 146–147 369–370
Acanthamoeba, similarity to macrophages, IgE, 37, 44–45, 369–370
29, 30 increase in prevalence, 44–45, 331, 332,
Acquired immunodeficiency syndrome 337–338
(AIDS) mast cells, 37, 38
in Africa, 292, 299 response, 44, 337, 340, 343, 347–348,
AIDS-resistant and AIDS-susceptible 351, 364
species, 301–302 Alouatta
AIDS-resistant/natural IV host species, 23 A. caraya
AIDS-susceptible/naïve IV host Toxoplasma gondii, 268, 271–273
primates, 22 A. fusca
Asian macaques, 291, 292, 310 Toxoplasma gondii, 258, 268,
clinical importance, 98 270–275, 279
HIV-1 (see Human immunodeficiency A. seniculus
virus-1 (HIV-1)) Toxoplasma gondii, 268, 271
HIV-2, 291, 311 Amphibian/amphibia, immunity, 18, 25, 27,
and Kaposi’s sarcoma, 224, 225, 299 28, 33, 36, 40, 43, 44
lentivirus infection, 316 Ancylostoma duodenale, 367, 372
overt immune activation, 91, 100 Anopheles (mosquito), 118, 119
secondary lymph node environment, 308 Aotus (owl monkey)
SIVs (see Simian immunodeficiency Aotus trivigatus
viruses (SIVs)) Treponema pallidum, 205, 206
TLR7 polymorphisms, 101, 102 appendix, 23
in USA, 300, 301 Toxoplasma gondii, 255, 258, 270–274,
Acute infectious disease, 253 279, 281
Adaptation Treponema pallidum, 205–208
and genetic evolution, 82 ApoLI, 2, 5
genetic markers, 74 Apolipoprotein B-editing catalytic polypeptide
Agriculture and health, 334 3G (APOBEC3G), 5, 6, 70, 72

J.F. Brinkworth and K. Pechenkina (eds.), Primates, Pathogens, and Evolution, 411
Developments in Primatology: Progress and Prospects,
DOI 10.1007/978-1-4614-7181-3, © Springer Science+Business Media New York 2013
412 Index

Appendix, caecal IgE and IgG, 43–45

mammalian, 23 immunoglobulins, 19, 22, 42, 43
in primates, 23 inter-primate differences in proportion, 42
vermiform, 23 BCR. See B-cell receptor (BCR)
Arcobacter butzleri, 400 Behavior
Ascaris lumbricoides (roundworms), 44, 343, characteristics, 280
367, 372 and ecological data, 259
Asthma New World monkeys and Toxoplasma
Eosinophils, 38 gondii, 6
helminths, 343 T. gondii (see Toxoplasma gondii)
Hepatitis A virus (HAV), 343 toxoplasmosis, 277–281
ICAM, 126 Bifidobacterium longum subsp. Infantis, 345
Immunoglobulin E (IgE), 44–45, 370 Bird/Aves, immunity, 20–28, 30–31, 33, 34, 43
Increased prevalence, 337 Body lice, 162, 164, 393
microbiota, 344, 350 Bone marrow
Second epidemiological transition, 337 cartilaginous fish, 25
TNF, 127 emergence, 25
Tryptases, 37 tyrosine kinase activity, 25
viruses, 340, 342 Borrelia burgdorferi, TLR2, 102
Haemophilus spp., 344 Brachyteles (muriqui), 256, 258, 279
and human rhinovirus, 340 Breast milk, 342, 344–345
and respiratory syncytial virus, 340 Bubonic plague. See Yersinia pestis
Ateles (spider monkey)
A. belzebuth
immune genes, 278 C
Toxoplasma gondii, 258, 271, 273, 278 Cacajao, (uakaris), 254, 256
A. geoffroyi Callibella, 254, 258
immune genes, 278 Callicebus (titis), 23, 205, 257, 272,
Toxoplasma gondii, 258, 272, 278 278, 280
A. marginatus C. donacophilus
Toxoplasma gondii, 273 Toxoplasma gondii, 272
A. paniscus C. moloch
Toxoplasma gondii, 273 Toxoplasma gondii, 272
ecology, 277 Treponema pallidum, 205
Toxoplasma gondii, 256, 258, 270, Callimico goeldii
272–274, 278, 279 Toxoplasma gondii, 272
Atelidae Callithrix
Toxoplasmosis, 258, 270, 274 Callithrix sp.
ATP2B4 and malaria, 147 Toxoplasma gondii, 255, 258,
Autoimmunity, 4, 127, 196, 331, 332, 337, 338, 272,–275, 281
342, 346, 349–351, 353, 370, 378, 380 Treponema pallidum, 205
C. aurita
Toxoplasma gondii, 273
B C. jacchus (common marmoset)
Bacillus anthracis, 106 Fatal infection of herpesvirus saimiri
Bacteroides fragilis, 334 (HVS), 236
Bacteroidetes, 338–339 Kaposi’s sarcoma-associated
Basophils, 35–38, 43, 367, 368 herpesvirus, 225
B-cell receptor (BCR), 19, 20, 41, 42, 228, Toxoplasma gondii, 271, 273, 278
232–233, 235, 239 C. kuhlii
B cells, 19, 20, 22, 25–28, 46, 47, 95, 225, Toxoplasma gondii, 272, 273
232–235, 312, 350, 366–368 C. penicillata
function, 42, 43, 46 Toxoplasma gondii, 271, 273
gene segments, 42–43 Callitrichinae
IgA, 43–44 description, 258
Index 413

Toxoplasma gondii, 258, 259, 268–277, 281 as HIV model, 310

Campylobacter, 136, 400 SIV/SIVsm, 67, 80, 100, 291, 292,
Cartilaginous fish 295, 297, 302, 303, 305–310,
emergence of MHC and TCRs, 24, 30, 39, 46 316, 395–397
RAG, 24, 25 TLR function, 93, 100
TCRs, 24, 30, 46 yellow fever virus, 100
C-C chemokine receptor type 5 (CCR5), 6, 47, Yellow fever virus, 100
73, 106, 302–304, 314 Cercocebus sp.
CCR5delta24, 304 Treponema, 197
CCR5delta32, 106 Cercopithecus
HIV coreceptor, 47, 73, 302–304, 314 C. cephus, SIV, 295–296
CCR5. See C-C chemokine receptor type 5 C. mona denti SIV, 296
(CCR5) C. mona, SIV, 295, 296
CD4+, 22, 46, 47, 79, 91, 93, 207, 208, 210, C. nictitans, SIV, 295–296
229, 238, 263, 264, 267, 278, 297, Cheirogaleus, 293
301–307, 309, 310, 312, 314–316, Chiropotes (bearded sakis), 254, 257
340, 351 Chlorocebus sp. (African green monkey)
CD8+, 46, 208, 232, 234, 263, 264, 278, 303, C. pygerythrus
305–307, 309, 310 SIV, 291, 295
CD16, 32, 45 C. sabaeus
CD36 SIV, 291, 295
function, 141, 142 SIV, 35, 291, 292, 295, 302, 303, 305–309,
polymorphisms and malaria, 120, 132, 316, 396
139, 141, 142, 146, 148 Treponema, 201, 206, 207
Cebidae, 270, 279 Chororapithecus abyssinicus, 166, 168, 174
and Atelidae, 270, 274, 276, 281 Chronic inflammatory disorders, 336, 355
families, 258, 274 allergies, 331, 337
Toxoplasma gondii, 255, 258, 268–277, autoimmune diseases, 331, 337–338
279, 281 colitis, 341
Cebus, 254, 281 Crohn’s disease, 341, 342, 352
C. albifrons genetic variants, 352
Toxoplasma gondii, 271 helminths, 343
C. apella IBD, 338
Toxoplasma gondii, 259, 270–274, immunoregulation, 340–342
276, 278 infections, faecal-oral route, 333, 340
C. capucinus modern environments, 333, 353
Toxoplasma gondii, 270, 271, 279 pollutants, 353–354
Cebus sp. (capuchin monkeys) prevalence, 331, 332
Toxoplasma gondii, 256, 258, 259, 270, vitamin D, 353
279, 280 Clothing use
Cebus sp. (capuchin monkeys), 258–259 Homo sapiens (human), 166–168
body weight, 280 origin, 168
C. apella, 259, 276 Coinfection. See Polyparasitic infections
Toxoplasma gondii, 270–274 Colobus
captivity, 270 Treponema, 197, 201
C. capucinus Conservation, primate, 398, 401–404
Toxoplasma gondii, 270, 279 Coxsackievirus B (CVB), 341, 342
C. nigritus, 259, 280–281 Cross-species transmission, 292, 296–298,
C. olivaceus, 259, 279–280 390, 392, 396, 399
C. xanthosternos, 259, 280 CVB. See Coxsackievirus B (CVB)
description, 258 C-X-C chemokine receptor type 4 (CXCR4),
Toxoplasma gondii, 268–277, 279–281 302, 314
tufted, 259 Cytokines, 3, 19, 20, 30, 34, 36, 46, 74, 95, 98,
Cephalochordata, 41 144, 145, 231, 233, 263, 265, 281,
Cercocebus atys (sooty mangabey), 7, 31, 67, 291 302, 367
414 Index

D Enteroviruses, 340–342
DAMPS. See Danger-associated molecular Environment of evolutionary adaptedness
patterns (DAMPS) (EEA), 333–335, 352, 354
Danger-associated molecular patterns Eosinophils, 35, 367, 368
(DAMPS), 19 granule content, 38, 39
DARC. See Duffy antigen receptor for and heterophils, 38
chemokines (DARC) origins, 35
Daubentonia (aye-aye), 23 phenotypes, 38
Dendritic cells (DCs), 26–29, 36, 44, 141, 263, role in immunity, 35
266, 344, 345, 349, 350 tissue remodeling, 38, 39
description, 31 Epidemiological transitions, 333, 335–337,
immunodeficiency viruses, 33 346, 352, 354–355
inter-primate differences, 26, 32 Epidemiology, 180, 268–274, 336, 341–342,
markers for primates, 32 354, 401
morphology and function, 31–32 leishmaniasis, 260
myeloid, 32 malaria, 118, 120
plasmacytoid, 32, 100, 307–308, 351 toxoplasma, NWP (see New World
TLR expression, 33 primates (NWPs))
Dengue Treponema, 189–214
inter-primate differences, 93 Episodic selection, 71, 73
Diabetes, type 1 (T1D), 126, 332, 337, 341, Erythrocebus patas (Patas monkey), 170, 171,
342, 354 197, 201, 396
Disease susceptibility, 3–6, 79, 94, 101, and Treponema sp., 197, 201
143, 307 Erythrocyte, 81, 118–130, 144
Duffy antigen receptor for chemokines enzymes and deficiency
(DARC), 80, 136 G6PD, 137–138
alleles, 134 PK, 138–139
antigens, 133–134 genetic variation and malaria
diversity in humans, 133–135 enzymes and deficiency, 137–139
haplotypes, 134 hemoglobin variants (see Hemoglobin)
Hepatocystis, 81 membrane proteins and surface
Plasmodium, 81, 133–134, 137 antigens, 131–137
Plasmodium vivax, 134–135 membrane proteins and surface antigens
ABO, 135–137
DARC, 133–135
E elliptocytosis and ovalocytosis,
Early humans, 333, 342 131–132
and chimpanzees, 168 glycophorin A and B receptors,
gorillas, 168 132–133
and lice, 168 Escherichia coli, 92, 95, 136, 401
and pathogens, 165 Eulemur (Brown lemur), 23
Ebola, 389, 395 Euoticus (needled-clawed bushbaby), 23
Ecology, NWPs. See New World primates
(NWPs)
Ecotourism, 7, 399–401, 403–404 F
Ectoparasite hypothesis, 169, 172. See also Faecalibacterium prausnitzi, 339, 342
Lice Fasciola hepatica, 341
Eczema, 45, 337, 343 Firmicutes
EEA. See Environment of evolutionary and Crohn’s disease, 339
adaptedness (EEA) Flukes, 363, 367
E3L, 4 Fluorescent treponemal antibody absorption
Enterobacteriaceae, 338–339 (FTA-ABS), 196, 204, 209
Enterobius vermicularis (pinworm), 341, 343, Fossil apes
344, 367, 393 Nakalipithecus nakayami, 174
Index 415

Samburupithecus kiptalami, 174 appendix, function, 23

FTA-ABS. See Fluorescent treponemal caecal appendix, 23
antibody absorption (FTA-ABS) and immunodeficiency virus (IV)
Fulani damage, 22
and malaria, 145 location and structure, 22
and LPS translocation, 304–305
and MALT, 21–22
G organization and location, 22
GALT. See Gut-lymphoid tissue (GALT) origins, 23
Genome scans and peyer’s patches (PP), 21–23, 304
function, 82 Th17 cells, 306
Giardia lamblia, 371–378, 380
antihelmintics, 377
Ascaris lumbricoides, 373, 377 H
clearance and, 378 Habitat, 2, 7, 106, 170–180, 258, 259, 309,
helminth coinfection, 371, 376, 377 364, 390, 397, 399, 400
Treg cytokine activity, 378 Alouatta, 258
Trichinella spiralis, 378 Aotus, 258
Glucose-6-phosphate dehydrogenase (G6PD), baboons, 2, 7
145, 148 C. albifrons, 259
African and non-African populations, 138 Calicebus, 258
A376G and G202A, 137 C. cay, 259
enzyme deficiency, 137 Cebus, 258
and malaria, 137, 143, 145 Cebus apella, 259
natural selection, 137 C. flavius, 259
Glycophorin A and B Antigen Receptors C. kaapori, 259
GYPA/GYPB, 132–133, 136, 148 C. libidinosus, 259
Glycophorin C (GYPC), 5, 69 C. macrocephalus, 259
Gorilla gorilla (gorilla) C. nigritus, 259
Cryptosporidium, 400 C. olivaceus, 259
HTLV/STLV, 399, 402 C. robustus, 259
Plasmodium, 392 C. xanthosternos, 259
poliovirus, 400 early hominins, 170–180
SIV, 296, 316, 396, 397 Saimiri, 258
yaws, 201–204 savanna, 2, 170, 171, 177, 258
Gorillas, 7, 167, 169, 173–180, 392, 399–402 and thermodynamics, 169, 170
lice, 165–166, 168, 172 Haemophilus spp., 344
ROC, 201, 202 Hagfish, 31
SIV, 296, 316, 396, 397 Hair loss, human
skin lesions, 202, 203 allometry hypothesis, 169, 170
T. pallidum/treponemal infection, 201–204 body hair, 169–172, 190
yaws, 201–204 ectoparasite hypothesis, 169, 172
G6PD. See Glucose-6-phosphate MC1R, 171, 172
dehydrogenase (G6PD) Pediculus, 169, 171
Gram-negative bacteria, 73, 91, 92, 94, 98, 99, Pthirus, 169, 171, 172
104, 208 timing, 169–171
Granulocytes, 25, 33, 43, 44 HBB. See Hemoglobin
basophils, 37–38 HCV. See Hepatitis C virus (HCV)
description, 35 Head lice, 161–165, 167, 173
eosinophils, 38–39 description, 163
mast cells, 36–37 manual removal, 163, 165
neutrophils, 35, 38 “no nit” policies, 164
Gut-lymphoid tissue (GALT), 43, 304 transmission, 163–164, 167
AIDS, 22, 23, 304–306 Heirloom species, 335, 390–394
416 Index

Helicobacter pylori, 136, 340, 341 Hepatitis C virus (HCV), 93, 102
Heligmosomoides polygyrus, 341, 349 interprimate differences, 93
Helminths, 34, 37, 38, 44, 333, 337, 338, TLR7 polymorphism, 102
341, 343–344, 348–351, 354, Herpes simplex virus (HSV), 102, 223,
363–382, 393 393, 394
adults, 370, 378–380, 382 HSV emergence, 393–394
affect, 44, 365, 377, 382, 393 HSV emergence, 393–394
chronic infection, 343, 349, 354, 364, 368, γ-Herpesviruses, 91, 221–242
370, 377, 380–382 cancer, 222–224
cost of infection to host, 370 Epstein-Barr virus (EBV), 221, 223–225
definition, 367 herpesvirus saimiri (HVS), 221, 222, 224
diminish immune responses, 370 and IL-2, 236
and G. lamblia infection, 371, interprimate differences in
373–378, 380 manifestation, 236
human IgE, 44, 367–370 human, 221, 223–225, 230, 234,
and IgE, 37, 44, 368–369, 377, 380 236–238, 240
immune phenotypes, 368–369 Herpesvirus saimiri (HVS), 221, 222, 224,
immunological role, 343 226–228, 231, 233–239, 241, 242
immunomodulatory effects, 350, 380 Kaposi sarcoma (see Kaposi’s sarcoma-
and immunoregulation, 333, 348–351, 364, associated herpesvirus (KSHV))
368, 380 Kaposi’s sarcoma-associated herpesvirus
induced immune responses in humans, 367 (KSAV), 221, 223–233
induce Treg activity, 368, 378 Callitrix jacchus (marmoset) model,
and inflammatory disease, 44, 343–344, 225
378, 380 control of lymphocyte activation,
morbidity, 364, 365, 370, 371 226–228
multispecies infections in humans, and HIV, 224
364–366, 375 interference with BCR signaling, 232
and neutrophils, 34 and multicentric Castleman’s disease
prototypical TH2/Treg response, 368 (MCD), 223–225, 228
shift T cell populations, 368 and primary effusion lymphoma (PEL),
soil transmitted helminth (STH) 223–225, 232
infections, 370 subordination of apoptosis, 231
T cell responses, 368 life cycle, 221–222, 240
TH2 immune response, 367, 368, 370 Lymphocryptovirus, 221, 224
tolerance response, 368 lytic replication, 221, 222, 224, 230, 231,
treatment, 163, 165, 337, 341, 343, 349, 234, 240, 241
370, 377, 378, 382 miRNAs (see microRNAs (miRNAs))
Tsimane of Bolivia, 371–381 molecular piracy, 222–223, 226–233, 242
Hemoglobin, 118, 120–131 mouse herpesvirus 68 (mHV68), 221, 224
geographic distribution of variants, phylogenetic tree, 221, 223
121–122 phylogeny, 223
geographic distribution of variants, respective host and malignancies, 221,
121–122 222, 224
HBB alleles and malaria, 120–131, 146 Rhadinovirus, 221, 224, 226, 236
HBC, malaria resistance, 120, 122, 123, rhesus rhadinovirus (RRV), 221, 222, 224,
128, 129, 131 233–236
HbC, malaria resistance, 122, 123, 128, 129 prevalence, 234
HBE, malaria resistance, 120, 122, 123, similarity to KSHV, 234, 235
128–131 STP, 226–231, 236–239, 342
HbE, malaria resistance, 122, 123, Tip, 226–230, 237–239, 242
128–129, 131 viral signaling molecules, 239
HBS, malaria resistance, 120–123, 128 virus-induced tumorigenesis, 222, 239
HbS, malaria resistance, 121–123, 129 Herpesvirus saimiri (HVS), 221, 222, 224,
Hepatitis A virus (HAV), 340, 341, 350 226–231, 233–239, 241, 242
Index 417

Herpesvirus saimiri U RNAs (HSUR), 241 HSUR. See Herpesvirus saimiri U RNAs
HERV-W, 5 (HSUR)
HIV. See Human immunodeficiency virus HSV. See Herpes simplex virus (HSV)
(HIV) HTLVs. See Human T-lymphotropic viruses
HIV-1. See Human immunodeficiency virus-1 (HTLVs)
(HIV-1) Human disease-scape, 213, 390–400
HIV-2, 67, 100, 291, 294, 297, 298, 310–313, heirloom pathogens and human Paleolithic
316, 395–397 ecology, 390, 393–394
HLA. See Human leukocyte antigen (HLA) HIV, 395–397
hMPV. See Human metapneumovirus (hMPV) malaria, 390–392
Holobiont, 4 pathogens to humans
Hominins bushmeat handling and consumption,
Ardipithecus kaddaba, 166 397–398
Ardipithecus ramidus, 166 ecotourism and constrain
Australopithecus afarensis, 166, 175, environmental destruction, 400
177–180 genetic distance, 397
Australopithecus africanus, 166 HTLVs, 399
Australopithecus anamensis, 166, 175 SFV, 399–400
Australopithecus bahrelghazali, 166, 175 STLV, 399
Homo erectus, 167 transmission, individuals and
Homo habilis, 172 communities, 398
Homo neanderthalensis, 167 souvenir species and changing human
Kenyanthropus platyops, 166, 175 ecology, 395
Orrorin tugenensis, 166 Human immunodeficiency virus-1 (HIV-1),
Paranthropus, 175 8, 73, 80, 102, 106, 169, 291,
Sahelanthropus tchadensis, 166 292, 294, 296–302, 304–316,
Homo sapiens 395–397, 399
Dengue virus, 93 in Africa, 291, 297–300, 305, 316, 396
gram-negative bacterial sepsis, 92, 94, 98 animal models
hepatitis C virus (HCV), 93 HIV-2, 310–313
historical exposure to pathogens, 106 rhesus macaques, 310, 312
HIV, 92, 291–292, 296–315, 395–397 SHIVs, 313
Kaposi’s sarcoma-associated herpesvirus T cells, 312–314
(KSHV), 224, 225 vpu and vpx, 312
lice and loss of hair, 169, 171 annotated depiction, genomes, 298
monkey B virus, 94 CCR5, 75, 106, 302, 314
Neisseria gonorrhoeae, 92, 99 CD8+ T cells, 305–307
Plasmodium falciparum, 92 CD4+ T cells and disease progression,
Schistosoma mansoni, 92, 99 301, 306
TLR polymorphisms, 102 chimpanzees, 80, 169, 291, 296, 297,
Toxoplasma gondii, 92, 262, 267–268 313–316, 396, 397
Hookworm, 343, 347, 367, 370, 372–378 cross-species transmission, 296–298, 397
and antagonism, 377–378 discovery of AIDS and HIVs, 291
and Ascaris lumbricoides, 367, 370, emergence, 8, 291, 292, 294, 296–302,
373–378 304–316
infections, 343, 370, 373–378 groups, 292, 296–298, 300, 301, 314, 315,
odds ratios, infection, 376 396, 397
Tsimane intestinal parasites, 371–373 humanized mice, 313
Tsimane single-and multiple-species immune activation, 306–308
infections, 373, 374 infection, 80, 106, 291, 292, 296, 297,
Host–parasite interactions, 260, 363 299–302, 304–308, 311–316, 395,
Host-pathogen coevolution, 2–3, 105, 148 396, 399
hRSV. See Human respiratory syncytial virus LPS, 305
(hRSV) lymph node environment, 308–310
418 Index

Human immunodeficiency virus-1 (HIV-1) HVS. See Herpesvirus saimiri (HVS)

(cont.) Hygiene hypothesis, 9, 44, 331, 335, 336, 340,
M,N,O groups, 296 342, 364, 380
mucosal immune system, 304, 305 definition, 331–332
origins and distribution of strains, 297–298 germ-free animals, 334
origins/SIVcpz, 291, 296–267, 312, “Old Friends”, 333
314–316, 395 Hymenolepis spp. (tapeworm)
phylogenetic trees, 294 Hymenolepis diminuta, 341
SIVs (see Simian immunodeficiency
viruses (SIVs))
spread from Haiti, 300–301 I
TLR polymorphisms, 102 IBD. See Inflammatory bowel diseases (IBD)
virus–host relationships ICAM-1. See Intercellular adhesion molecule
CD4+ cells, 314–315 1 (ICAM-1)
chimpanzees, 314, 315 Immune dysregulation, 91, 94, 107, 226
SIV infection, 314–316 Immune system
surveys, bushmeat samples, 316 allergic disorders, 331, 332, 337, 340, 343,
vpu gene, 313, 315 348, 350, 352, 353
Human immunodeficiency virus (HIV), 4, 6, animal models, 18, 346
18, 31, 40, 47, 67, 72, 79–81, 98, asthma (see Asthma)
102, 106, 214, 292, 293, 295–303, chronic infections transmitted, faecal-oral
310, 311, 313, 316, 390, 395–397, route, 340–342
399, 402 chronic inflammatory disorders, 331–333,
and AIDS (see Acquired immunodeficiency 337–338, 341, 343, 349, 351–355
syndrome (AIDS)) clinical implications, 354
HIV-1 (see Human immunodeficiency clinical trials, 333, 336, 343–347
virus-1 (HIV-1)) ectoparasites, 345, 364
HIV-2, 67, 298, 316, 395, 397 epidemiological and experimental
infection, 6, 31, 47, 67, 79, 81, 93, 234, studies, 333
292, 301, 303, 310, 311, 316, epidemiological transitions, 333, 335–337,
395–397 346, 354, 355
pandemic spread, 292, 298–301 evolved dependence and EEA, 333–335
and SIV transmission, 292, 296, 297 evolution of mammalian immune system,
souvenir species 17–48
molecular and epidemiological farming environment, 331, 332
studies, 397 genetic variants, 65–82, 142–143, 351–352
NHP pathogen, 395, 399 helminths, 333, 337, 338, 341, 343–344,
SIV transmission, 396, 397 348–351, 354, 366, 370, 382
types, 395 “hygiene hypothesis”, 331–332, 335, 336,
TLR, 98, 102, 106 340, 342
Human leukocyte antigen (HLA), 46, 67, 79, immunoregulation (see
126, 130, 143–144, 305, 346, 352 Immunoregulation)
allele frequency differences, 143 malaria, 8, 143, 144, 345–346, 352
and malarial disease, 126, 143–144 maturation, 332, 338, 342, 347–348, 354
Human lymphocyte antigen (HLA), 46, 126, microbiota (see Microbiota)
143–144, 346, 352 milk, 344–345, 352
diversity, 46, 143 modern lifestyles
and malaria, 126, 143, 144, 346 diminished nonmicrobial
Human metapneumovirus (hMPV), 401–403 immunoregulatory exposures, 352
Human respiratory syncytial virus obesity and diet, 353
(hRSV), 401 pollution, 353–354
Human T-lymphotropic viruses (HTLVs), 8, vitamin D, 353
298, 399, 402 “Old Friends”, 333, 338, 347, 355,
Hunting, 169, 170, 172, 179–180, 280, 370, 382
281, 390, 398 organisms identification, 333
Index 419

pets, 332, 339–340 mechanisms, 332, 333, 337, 338, 346–350

phylogenetic trees, 335, 355 obesity, influence of, 353
“Pseudocommensals”, 335, 345, 347 pollution, influence of, 353–354
regulatory pathways, 338, 339, 354 and viruses, 333, 339, 350–351
tolerance and immunoregulation, 354 viruses, by, 350–351
type 1 diabetes, 67, 332, 337, 342 vitamin D, influence of, 353
viruses and autoimmunity, 342 Infectious disease emergence, 1, 8, 392, 403
Immune system dysregulation, 91, 94. Inflammatory bowel diseases (IBD), 4, 331,
See also Human immunodeficiency 332, 338, 341–343, 347
virus (HIV); Simian Innate and adaptive immune activation,
immunodeficiency viruses (SIVs) 306–308
Immune system variation, OWMs Innate immune system
behavioral, life history, and ecological cellular location, primates, 95–96
data, 68 cytokines, 95, 305
description, 66 granulocytes (see Granulocytes)
gene families, 67 location, ligands and representative
natural selection targets, 72–73 organisms, 95, 97
parasites and pathogens, 68 lymphocytes, 41–47
phenotypic diversity, disease response, NK cells (see Natural Killer (NK) cells)
66–67 origins, 19
population genetic diversity, MHC, phagocytes (see Phagocytes)
73–74, 77 vertebrate animals, 94
rhesus macaque, 66, 69 Intercellular adhesion molecule 1 (ICAM-1)
selection signatures, 69–71 endothelium, vascular tissue, 139–140
sequence diversity, 73–75 function, 139–140
T-cell, 67, 71 malaria resistance, 140–141
Immunity and development polymorphisms and malaria, 140, 146
disease progression, 6 SNPs, 140, 141, 146
evolution, 8 Interleukins
immune system maturation, 347–348 and malaria, 144–145
primate, 1 Interspecies divergence
Immunodeficiency viruses (IV), 1, 6, 9, 22, 33, on disease manifestation, 101
47, 91, 93, 291, 313 polymorphisms, 79, 100, 102
Immunoglobulins (Ig) TLR2 and TLR4 function, 98–99
emergence and differences TLR7 function, 100
IgA, 21–22, 43–44, 264, 265, 378 ITAM motif. See Immunoreceptor tyrosine-
IgE, 37, 43–45, 265, 351, 367–370, based activation (ITAM) motif
378, 380
IgG, 43–45, 208, 209, 264, 265, 274,
275 J
IgM, 43, 208, 264, 265, 274 Jawed vertebrates (gnathostomata), 19–21, 24,
Toxoplasma gondii infection, 265 26, 41–43, 45–47
Treponema pallidum infection, 209 Jawless vertebrates (agnatha), 18, 24, 41,
Immunoreceptor tyrosine-based activation 42, 46
(ITAM) motif, 228, 229, 231,
234, 235
Immunoregulation K
Brugia malayi microfilariae, 349 Kaposi’s sarcoma-associated herpesvirus
dendritic cells (DC), 348–351 (KSHV)
and gut microbiota, 349–351, 353 AIDS, 224, 225
gut microbiota, by, 350 description, 223
and helminths, 333, 341, 348–351, genes and cellular homologues, 226
364, 368 genomic location, 226, 227, 235
helminths, by, 349–350 in vivo study, 225
420 Index

Kaposi’s sarcoma-associated herpesvirus divergence of pubic and clothing/head lice,

(KSHV) (cont.) 172–173
infection, 225 emergence of human clothing,
K15P, 232–233 168, 170
K15P vs. K15M, 233 evolution of human hair loss, 169–172, 177
KSHV K1 Hominoid-specific lineages, 167
BCR, 228 host switch and hominin meat
cellular signal transduction, 228, 231 consumption, 179–180
description, 227–228 Lineage-specific selection, 69
ITAM motif, 228 Lipopolysaccharide (LPS), 31, 73, 74, 95, 100,
lytic replication, 231 104, 305
recycling endosomes, 228 and Gram-negative bacteria, 73, 98,
STP, 231 99, 208
transmembrane signaling proteins, 228 LY96 co-receptor, 104
VEGF, 231 TNF α, 100
viral lytic replication, 231 Loris (loris), 23
KSHV K15, 232 lorises, 45
ORFs, 222, 226, 227, 229–230, 239 LPS. See Lipopolysaccharide (LPS)
PEL and MCD, 223, 225, 228, 241 Lymph nodes
spindle cells, 224, 225, 231 emergence, 20
viral molecular mimicry, 226 environment
Killer-cell immunoglobulin-like receptors HIV-1 infection, 308
(KIRs), 5, 39, 40, 72, 73 immune activation, 308–310
K3L, 4 immunosuppressive, 309, 310
KSHV. See Kaposi’s sarcoma-associated pathways, disease in humans and
herpesvirus (KSHV) macaques, 310, 311
T-cells, 308–310
mammals, 21, 28
L NALT, 28–29
Lagothrix (wooly monkey) number and location, 28
L. lagotricha tonsils, 28, 29
Toxoplasma gondii, 271–273 Lymphocytes
Lamprey (Lampetra planeri and Petromyzon adaptive immune system, 41
marinus), 24, 26, 31, 41, 46 B cells (see B cells)
Legionella pneumophila (Legionnaire’s emergence, 24, 41–42
disease) immune response, 21, 41
TLR5 polymorphisms, 102 and immunoglobulins, 19
TLR4 polymorpisms, 104 mammalian marginal zone, 28
Leontopithecus (tamarin) sp. NK cells, 39, 263
L. chrysomelas signaling pathways, 227
Toxoplasma gondii, 273 T-cells (see T-cells)
L. rosalia VLRs, 20, 41, 42, 46
Toxoplasma gondii, 272
Toxoplasma gondii, 272
Leprosy (Mycobacterium leprae), TLR1, 102, M
196, 201 Macaca fascicularis (crab-eating macaques,
Leukocytes (white blood cells) cynomolgus macaques)
evolution, 6, 25, 366 Mycobacterium, 99
major types, 37, 98 SIV/HIV progression, 67, 291
proportions, interspecies differences in, 25, Treponema pallidum, 205
32, 38 Macaca mulatta (rhesus macaques)
Lice. See also Parasitic lice; Pediculus; as an HIV model, 310–313
Pthirus (pubic lice) Dengue virus, 93
ape and early hominin habitats, 174–177 HLA/MHC diversity, 67
Index 421

rhesus rhadinovirus (RRV), 221, 224, infective process, 118, 119

233–236 inflammatory cytokines and antibody
SIV/HIV progression, 67, 291–292, 302, response, 144–145
309, 310 PKLR (Pyruvate kinase), 138–139
TLR function, 100–101 Plasmodium
TLR7 polymorphisms, 101, 102 P. chabaudi, 346
Treponema pallidum, 205, 206 P. falciparum, 5, 69, 92, 118–122,
yellow fever virus, 33, 100 131–145, 391–393, 395, 402
Macaca nemestrina (pig-tailed macaque) P. knowlesi, 118
HIV/SIV, 292, 301, 306, 313 P. reichenowi, 92, 392
IgA, 44 P. vivax, 81, 118, 122, 124,
Treponema pallidum, 205 134–135, 345
Macaca nigra (Celebes crested macaque) RBC cytoadherence, 120, 136
Treponema, 204 RBC elliptocytosis, 131
Macaca sp. (macaque), 22 resistance, 69, 80–81, 120, 130, 133, 140,
Chinese macaques and SIV/HIV, 67 143, 147
Monkey B virus, 94 selection pressure on host RBCs, 136–138
Mycobacterium, 99 symptoms, 118, 119
Treponema pallidum, 205, 207 TLR polymorphisms, 102
Macrophages, 25–27, 32–34, 45, 95, 141, 263, Malaria hypothesis, 118
264, 302 MALT. See Mucosa-associated lymphoid
baseline comparative studies, 31 tissue (MALT)
comparison to amoebae, 29, 30 Mammalian immune system
invertebrate species, 30 adaptive immunity, 19, 20
MHC, 19, 30, 266 appendix, 23
morphology and behavior, 30–31 GALT, 21–22
origins, 29 IgE, 37, 43–45
phagocytic immune cells, 29 innate immunity, 19, 20
Major histocompatibility complexes (MHC), lymph nodes, 20
5, 19, 143, 264 phagocytosis, 19
cartilaginous fish, 24, 30, 39 T and B cells, 19–20
diversity in old world monkeys, 73, 77–79 Mandrillus sphinx (mandrill)
emergence, 24, 30, 32, 39–41, 46 HTLV/STLV, 399
evolutionary relationships, 77–78 SIV, 47, 293
genes, 67, 69, 77, 79 MARVELD3 and malaria, 147
and HIV/SIV, 79 Mast cells
as ligand for NK cells, 39, 40, 264 cytokine production, 36
and OWMs, 69, 73, 74, 77–79 histamine production, 36, 37
Malaria mammalian, 37
Anopheles mosquitoes, 118, 119 origins, 36
CD36, 141–142, 146 primate, 37
DARC, 80, 133–135 MCD. See Multicentric Castleman’s disease
description, 117–118 (MCD)
erythrocyte genetic variation MC1R. See Melanocortin 1 receptor (MC1R)
(see Erythrocyte) Melanocortin 1 receptor (MC1R), 171, 172
Fulani immunity, 145 Metchnikoff, Elie, 17, 206
genetic variation, immune system, MHC. See Major histocompatibility
142–143 complexes (MHC)
genome-wide association studies, 146–147 Microbiome, 5, 105–106, 334
genome-wide linkage, 146–147 Microbiota, 4, 334, 353, 354
G6PD, 137–138, 143, 145 gut, 338–339, 347, 349, 350
hemoglobin alleles, 120–131 lung, 339, 342, 344
HLA, 126, 143–144, 346 skin, 339, 342, 344
ICAM-1, 139–142, 146, 148 Microcebus (mouse lemur), 40, 293
infection, 119–120, 145 MicroRNAs (miRNAs), 223, 233
422 Index

flexibility, 242 Natural selection and immunity

γ-2-herpesvirus, 239–240 inter-primate differences in rate, 69
and HSUR, 241 lineage-specific adaptation, 47
and KSHV, 240 promiscuous species, 68
latency-associated region, 227, 240 Necator americanus (hookworm), 341, 343,
lytic replication, 241 347, 367, 370, 372–378
and RRV, 241 Neisseria gonorrhoea, 91, 92, 99
and SNP, 241 hominoid response, 91, 99
Mimicry, 221–242 interprimate differences, 91, 92, 99
miRNAs. See microRNAs (miRNAs) Neisseria gonorrhoeae, 92, 99
Molecular clock methods, 293 Neonatal immune system, 347–348
Monkey B virus, 94 Neotropical primate
Monocyte/macrophages curatorial/zoological institutions, 282
inter-primate differences, 29–32 diet and forest strata preferences, 254–257
role in Toxoplasma gondii infection, immune evasion strategies, 266–267
262–264 natural killer (NK) cells, 263, 264
Monocytes. See Macrophages NWPs (see New World primates (NWPs))
Mosaic/chimeric viruses, 292–293 susceptibility, 253, 281
Mucosa-associated lymphoid tissue Toxoplasma-NWP interactions, 281
(MALT), 21 toxoplasmosis, mammals, 267–268
Mucosal immune system and bacterial Neutrophils, 19, 25, 29, 34, 95, 263, 352
translocation, 304–306 antimicrobial activities, 34
Multicentric Castleman’s disease (MCD), 223, emergence, 38
225, 228, 234 function, 33–35
Mycobacterium, 99 granulocytes, 33, 35
in cercopithecoids, 99 and heterophil granular, 34
leprosy, 102 interspecies comparisons, 34–35
Mycobacterium bovis, 99 NETS, 34
Mycobacterium smegmatis, 98 rhesus macaques, 35
Mycobacterium sp., 99 New World primates (NWPs)
Mycobacterium tuberculosis, 3, 5, adaptive advantages, 259
99, 106 Aotus, 258, 270, 274, 278, 279, 281
TLRs, 95, 99, 102 Atelidae family, 258, 270, 274, 276, 281
Calicebus, 254, 258
capuchin monkeys, 258–259
N Cebidae family, 258, 274
NALT. See Nasal-associated lymphoid tissue Cebus, 279, 281
(NALT) Chiropotes, 254, 257
Nasal-associated lymphoid tissue (NALT), diet and forest strata preferences, 254–257
28–29 Pitheciidae, 254
Natural hosts, 6, 47, 67, 100, 211, 260, 261, Platyrrhini, 253, 254, 270, 281
306–308, 315, 390, 391 T. gondii infections (see Toxoplasma gondii)
Natural killer (NK) cells, 32, 45, 263, 264, toxoplasma epidemiology (see Toxoplasma
312, 313 epidemiology, NWP)
emergence, 40 NF-kB
HIV, 40, 312–313 TLR polymorphisms affecting function, 102
inter-primate differences, 40, 41 NHPs. See Nonhuman primates (NHPs)
KIRs, 40–41 Nonhuman primates (NHPs)
markers, 40 African monkeys, 196–201
MHC I, 39–40 Asian monkeys, 204–205
Natural selection. See Immune system chimpanzees, 204, 394, 403
variation, OWMs; Malaria diseases transmission
Arcobacter butzleri, 400
Index 423

hRSV and hMPV, 401 Tip, 238

human-derived pathogens, 401 virus-induced tumorigenesis, 222
interaction, humans and NHPs, 400 Oreonax, 254, 258
morbidity and mortality, 400–401 ORFs. See Open reading frames (ORFs)
poliovirus, 400
systematic screening, 402
FTA-ABS, 196, 204 P
gorillas, 201–204, 389, 400 Paleolithic and disease, 391, 393–394
human disease-scape (see Human PAMPs. See Pathogen-associated molecular
disease-scape) patterns (PAMPs)
infectious diseases, 389–390 Pan troglodytes (chimpanzee), 2, 7, 31, 37, 40,
invasive and noninvasive tests, 47, 48, 69, 71, 72, 80, 82, 91–94,
chimpanzees, 404 98, 99, 104, 162, 165–169, 171,
molecular methods, 390 173–175, 179, 201, 202, 204, 206,
noninvasive methods, 402, 403 280, 291, 293, 296, 297, 313–316,
pathogen exchange, species, 403 390, 392, 394, 396, 397, 400, 401,
preventing infection, 404 403, 404
serological surveys, 195 AIDS-like disease, 93, 315
South American monkeys, 206 gram-negative bacterial sepsis, 91, 94, 98
Nonself (recognition), 2–4, 19, 20, 24, 29–32, Hepatitis C virus (HCV), 93
39–42, 47, 71, 73, 77, 95, 96, 105, HIV/SIV, 31, 72, 80, 91, 93, 98, 169, 291,
132, 133, 173, 199, 224, 226, 260, 293, 296, 297, 313–316, 396, 397
262, 264, 270, 295, 296, 300, 308, Neisseria gonorrhoeae, 91, 92, 99
312, 347, 349, 366, 400 poliovirus, 7, 400
NWPs. See New World primates (NWPs) Schistosoma mansoni, 7, 92, 99
Nycticebus, 23 Pan troglodytes (common chimpanzee), 296,
314, 315
Papio anubis (olive baboon), 92, 99, 198–200,
O 210
“Old Friends”, 333, 336, 338, 339, 345–349, Treponema, 198–200, 210
351, 352, 354, 355, 364, 370, 382 Papio cynocephalus (yellow baboon)
autoimmunity and viruses, 342, 351 Treponema, 198, 210–211
chronic infections, 340–342 Papio Papio (Guinea baboon), 2
ectoparasites, 345 Papio sp. (baboon)
environmental “pseudocommensals”, 345 gram-negative bacterial sepsis, 98
epidemiological transition, 333, historical exposure to pathogens, 2, 196
336, 355 Mycobacterium, 7, 98, 99
gut microbiota, 338–339, 351 Treponema, 196, 198
helminths, 343–344, 364, 370 Parasitic lice
immunoregulation (see Immunoregulation) clothing lice, 162, 164–165, 167, 168
loss of, 336, 348, 351 description, 161
malaria, 345–346 early hominins, 173–180
microbiota, 344, 348 hair loss, 169–173
milk, 344–345 head lice (see Head lice)
pets, 339–340 male human head louse, 162
viruses and asthma, 340 prevalence, 162, 180
Open reading frames (ORFs) pubic lice, 162, 165–169
HVS, 236 Pathogen-associated molecular patterns
KSHV (see Kaposi’s sarcoma-associated (PAMPs), 19, 95, 264
herpesvirus (KSHV)) Pathogens
pre-miRNAs, 240 evolution, 5–6
RRV RK15 gene, 235 genomes, 5
STP, 236–237 primate speciation, 5, 7
424 Index

Pattern recognition receptors (PRR), 19, coinfecting species, 363–367, 376

80, 347 disease ecology, Tsimane, 365
PBMCs. See Peripheral blood mononuclear helminth-giardia coinfection, 377, 378, 382
cells (PBMCs) helminth-induced immune responses,
Pediculosis, 161, 164 367–370
Pediculus (head lice) host–parasite interactions, 363
life cycle, 162 humans, 363, 364, 367, 368, 381
P. humanus capitis, 162–164 humans-whose habitats span, 364
P. humanus humanus, 162, 164–165 “hygiene hypothesis”, 364, 380
and bacterial disease, 162, 165 immune function, 364
and hygiene, 164 immune responses, parasites and
transmission, 164–165 pathogens, 366–367
PEL. See Primary effusion lymphoma (PEL) immunoregulation, 364, 368
Peripheral blood mononuclear cells (PBMCs), morbidity risk, 382
145, 276, 344 multiple-species infections, 364–366, 374,
Perodicticus, 23 375, 380, 381
Peyer’s patches (PP), 21–29, 304, 344 Tsimane (see Tsimane)
Phagocytes Prevotella, 339
DCs, 31–33 Primary effusion lymphoma (PEL), 223, 225,
monocytes/macrophages, 29–31 228, 232, 240, 241
neutrophils, 33–35 Primary lymphoid organs/tissues
Piliocolobus (red colobus), 23, 314 lymphocyte effector cell poiesis, 20
Pithecia (sakis) lymphopoiesis, 25
P. pithecia thymus, 24
Toxoplasma gondii, 272 Primate conservation, 9
PK. See Pyruvate kinase (PK) Primate lentivirus lineage, 293, 295–297
PKLR (Pyruvate kinase) Procolobus (olive colobus), 23
diversity in humans, 139 PRR. See Pattern recognition receptors (PRR)
Plague (Yersinia pestis), 3, 106, 107, 355 Pseudocommensals, 335, 345, 347
Plasmodium, 391–392 Pthirus (pubic lice), 165–175, 177–180
life cycle, 118 and ape and early hominin habitats,
mosquito vector (Anopheles), 118, 119 174–180
P. chabaudi, 346 ape nesting, 178–179
P. falciparum, 5, 69, 92, 118–122, host switch, 168–169, 171–175, 177,
131–145, 391–393, 395, 402 179, 180
P. knowlesi, 118 life cycle, 162
P. malariae, 118 P. gorillae, 165
P. ovale, 118 P. pubis, 162, 165, 172, 180
P. reichenowi, 92, 392 primate species divergence, 167
P. simium, 7 P. schaeffi, 162, 173
P. vivax, 81, 118, 122, 124, 134–135, 345 transmission, 165
Duffy antigens, 133, 134 Pyruvate kinase (PK)
hygiene hypothesis, 345 deficiency mutations, 122, 138
Treg cells, 345–346 genetic diversity, PKLR, 138, 139
as souvenir organism, 391–392 SNP and STR variation, 138–139
Platyrrhine, 32, 39, 45, 253, 275, 277, 280
Poliovirus, 400
Polymorphism, 67, 77–80, 101, 102, 104, 120, R
121, 123–127, 131, 133, 135–138, RAG. See Recombination activating gene
141, 143, 144, 194, 199 (RAG)
Polyparasitic infections Recombination activating gene (RAG), 19, 24,
adulthood, 382 25, 42
childhood, 381 RAG 1 and 2 emergence in cartilaginous
chronic helminth infection, 370, 381 fish, 24
Index 425

RAG mediated gene rearrangement, 19, interprimate differences, 92

24, 42 Old World monkeys (cercopithecoids), 99
Red Queen hypothesis, 3 TLR4 and endotoxin responsiveness, 102
Reptile/reptilia, immunity, 27, 28, 33, 34, Septic shock, 4, 92, 95, 98, 99
36, 43 Serology
Rhesus rhadinovirus (RRV), 221, 222, NHPs, 213–214
226–228, 239, 241, 242 Treponema pallidum subspecies, 214
B cells, 234 Sexually transmitted disease (STD), 68, 165,
coinfected animals, 234 180, 189, 299
Macaca mulatta, 233 Sexually transmitted infection, 199, 204
R1 gene, 234–235 SFV. See Simian foamy virus (SFV)
RK15 gene, 235–236 SHIV. See Simian-human immunodeficiency
transient lymphadenopathy, 234 virus (SHIV)
Rotavirus, 341, 342, 350, 351 Siglec 1, 31
RRV. See Rhesus rhadinovirus (RRV) Siglec 5, 47
Siglec 7, 31
Simian foamy virus (SFV), 214, 399, 400, 402
S De Brazza’s guenons, 399
Saguinus (tamarin), 23, 255, 258, 272, 274, gorillas, 399
275, 278, 279, 281 mandrills, 399
S. bicolor Simian-human immunodeficiency virus
Toxoplasma gondii, 273 (SHIV), 313
S. geoffroyi Simian immunodeficiency viruses (SIVs), 35,
Toxoplasma gondii, 272 214, 236
S. midas African primate, 291, 316
Toxoplasma gondii, 271, 273 age and diversity of strains, 292–295
S. niger AIDS-resistant and-susceptible species,
Toxoplasma gondii, 273 301–302
Saimiri (squirrel monkey) Cheirogaleus, 293
Saimiri sp. description, 291–292
Toxoplasma gondii, 271, 272, 274, 275, destruction, lymph node environment,
281 308–310
S. oedipus oedipus and HIV (see Human immunodeficiency
Toxoplasma gondii, 271 virus (HIV))
S. sciureus and HIV-1 viral lineage (see Human
herpesvirus saimiri (HVS), 236 immunodeficiency virus-1 (HIV-1))
Toxoplasma gondii, 271–274 human consumption, 8
Treponema pallidum, 205, 206 immune activation, 306–308
Saimiri transforming protein (STP), 226, 231, infection, 67, 79, 80, 101, 291–293, 302,
235–239, 242 303, 306, 308, 311, 314–316, 395,
Salmonella, 340, 341 396
Schistosoma japonicum, 367 innate and adaptive immune activation,
Schistosoma mansoni (fluke), 7, 92, 99, 341, 306–308
343, 367 interprimate differences, 301–310
and allergies, 341 limiting target cell availability, 302–304
interprimate differences, 99 Microcebus, 293
Secondary lymphoid organs/tissues, 20, 21, mucosal immune system and bacterial
32, 304, 308, 309 translocation, 304–306
bone marrow, 42 primate lentivirus lineage, 293, 295–297
T and B cells, 19, 42 relationship of strains, 294, 295
Sepsis, severe sepsis SIVcpz, 291, 296, 297, 312–315, 395
Gram-negative bacterial sepsis, 91, 92, 94, origins, 297–298
98–99, 104 SIVgor
hominoid susceptibility, 98–99 origins, 297–298
426 Index

Simian immunodeficiency viruses (SIVs) (cont.) T-cells

SIVmac origins, 292 CD4+ CCR5+ T cells
SIVsmm, 292, 302, 303, 310, 312, 395 interprimate differences, 47,
T-cell depletion, 67 302–304, 314
therapies, 6 role in Toxoplasma gondii infection,
TLR7 and TLR9 mimetic ligands, 100 262–266
transmission, humans, 296–298 differentiation, 42, 46, 303, 340, 367
vpu gene, 295–296, 312, 316 evolution, 24, 69, 71, 313
Simian T-lymphotropic virus (STLV), granzyme B production, differences, 47
399, 403 and human Treponema infection, 208
Single nucleotide polymorphisms (SNP), inter-primate differences proportion, 42, 47
122, 133, 135, 136, 140, 141, 146, mammals, 19, 21, 22, 26, 27, 29, 32, 37,
147, 241, 351 42, 43, 46, 47, 69, 71, 366
PK deficiency, humans, 138 NOTCH signaling, 42
and STR variation, 138–139 primate, 22, 26, 29–30, 32, 42, 46, 47, 69,
SIVs. See Simian immunodeficiency viruses 91, 225, 230, 236, 291, 298, 302,
(SIVs) 303, 305, 314, 316
SLC4A1, 124, 131, 132, 136 TCRs and MHC molecules, 24, 42, 45–46
SNP. See Single nucleotide polymorphisms TCRs. See T-cell receptors (TCRs)
(SNP) T1D. See Diabetes, type 1 (T1D)
Soil-transmitted helminths (STH), 370, 381 Tetherin/BST2, 6, 71, 312, 316
Souvenir species, 390 Thymoids, 24
HIV, 395–397 Thymus
human ecology, 395 interspecies comparisons, 25
malaria, 391–392 lampreys, 24
Spectrin (SPTA1), 124, 131–132 vertebrate, 21, 24, 45, 46
Spleen Tip. See Tyrosine-interacting protein (Tip)
secondary lymphoid tissue, 21 TLRs. See Toll-like receptors (TLRs)
description, 26 Toll-like receptors (TLRs)
evolution, 26, 27 cellular location, 95–97
function and structure, 26 detected-pathogens, 92, 98–101, 106, 107
function in birds, 20, 26, 27 dysregulation, 91, 94
function in fish, 26 function, 32–33, 73, 91–107
function in mammals, 20, 21, 26–28 genetic variation
marginal zone, mammalian, 27–28 population level, 104
Starfish, 17 primate species, 104–105
STD. See Sexually transmitted disease (STD) innate immune system (see Innate immune
STH. See Soil-transmitted helminths (STH) system)
STLV. See Simian T-lymphotropic virus interprimate differences in function, 98–101
(STLV) interspecies divergence (see Interspecies
STP. See Saimiri transforming protein (STP) divergence)
Strongyloides stercoralis (roundworm), 341, ligands, 31–33, 95, 97, 98, 100, 103–105,
367, 373, 375, 376 264
Susceptibility, T. gondii. See Toxoplasma molecular evolution, 103
gondii natural selection, 71, 73, 103–105
pathogen-mediated evolution of, 91,
105–106
T pathogens, 71, 73, 91–95, 98–102,
Taenia spp. (tapeworm), 367 105–107, 264
Tapeworms, 363–364, 367 pathway function, 98, 100, 105
Tarsius (tarsier), 44 polymorphisms affecting function,
T-cell receptors (TCRs), 19, 20, 24, 30, 101–102
41–43, 45–47, 229, 230, structure, 95
238, 313 Toxoplasma gondii infection, 264
Index 427

Treponema pallidum, 208 fatal, 266, 269, 270, 275

Toxoplasma epidemiology, NWP in mammals, 253, 267–268
captivity, 270 mice and humans, 262, 263
clinical and pathological manifestations, NWP (see New World primates (NWPs))
268, 269 protection and resistance, 264–265
experimental infections, 274 zoonosis cause, 253, 259
Platyrrhini, 270 TPSD1, 37
seroprevalence, 270–273 TRAIL, 6
toxoplasmosis, 268 Treg cells
Toxoplasma gondii, 1, 6, 92, 97, 253–282, 340 allergies, 337, 340, 350, 353
and allergies, 340, 341 autoimmune disorders, 337, 346, 349
Aloutta seniculus, 268, 271 gut microbiota, 349, 350
Callitrichid responses, 92 helminthic infection, 349–350, 368
Capuchin (Cebus) monkey responses, 92 Plasmodium, 345–346
clinical responses in New World Primates, SIV, 309
268–277 viruses, 350–351, 353
in felids, 269, 277, 279 vitamin D, 353
host immune response, 262–263, 267 Treponema, 89, 195, 208, 210, 391, 393
humoral immunity to, 264–265, 274, 281 in African green monkeys (Chlorocebus
immune evasion, 266–267 sp.), 197, 201, 206, 207
immune system, 1, 262–264, 266, 267, distribution in nonhuman primates,
276–277 189–194, 211, 212
infections, 1, 6, 253, 262–281 in patas monkeys (Erythrocebus
anti-Toxoplasma gondii antibodies, patas), 201
270, 274, 275 serological test problems, 195
Cebus sp., 274, 276–277, 279–281 in wild baboons, 195–200, 208
cellular immune response, 263, 276 Treponema pallidum
clinical and serological aspects, 275 antibody, 196, 200, 208–210
description, 281 description, 189
humans, 1, 6, 262–266, 276 geographic distribution, 207, 210–212, 214
hypotheses, 279 humans
immune response, 1, 253, 262–268, CNS, 194
277, 278 disease progression, 190–193
intensity and diversity, parasite, 279 natural infection, 190–193, 206, 207
patterns I, II and III, 274–275 subspecies, 190, 194, 195, 199,
PBMCs, 276 203, 213
platyrrhines, 253, 275, 277 immunological studies, 208
strains, types, 275 lesion development, 207–209
susceptibility, 253, 266, 275, 277–281 NHPs (see Nonhuman primates (NHPs))
inflammation response to, 266 species distribution, 212
interprimate differences, 92, 268–277 species similarities and differences,
life cycle, 260–262 206–207
NWP (see New World primates (NWPs)) subsp. pertenue strain, 199, 210, 212–213
NWP immune response and ecology, Treponema pallidum (Bejel, Yaws, Syphillus),
277–281 189–214
proinflammatory and anti-inflammatory apes as model, 202, 203, 206, 213, 214
response, 265–266 in Asian monkeys, 204–205
taxonomy and trophism, 259–260 CD4+ T cells, 208, 210
TLR detection of, 92, 264 in chimpanzees, 201, 202, 204, 206
typical mammalian host response to, in gorillas (yaws), 191–193, 201–204
262–268 immunoglobulin response, 209
Toxoplasmosis interprimate differences in disease,
acute, 253, 263, 265, 269, 275, 276, 281 206–208
chronic, 264, 265, 276 origins of T. pallidum in humans, 194–195
428 Index

Treponema pallidum (Bejel, Yaws, Syphillus) Variable lymphocyte receptors (VLRs),

(cont.) 20, 41–42, 46
in South American monkeys, 204–206 lymphocytes, 20, 41, 42, 46
symptoms, 190–194, 210 molecules, 42, 46
TLRs, 208 Vascular endothelial growth factor (VEGF),
transmission, 189, 190, 194, 195, 210, 229, 231
213, 214 VEGF. See Vascular endothelial growth factor
Trichinella spiralis, 341, 378 (VEGF)
Trichuris suis (whipworm), 341, 343, 344, 347 Vertebrate immunity
Trichuris trichiura (whipworm), 338, comparative immunology, 17, 18, 48
341, 343, 344, 367, 368, 370, description, 17
373–376, 378 GALT and PP, 21–23
TRIM5 (TRIM5a) lymphoid tissues function, 19–22,
antiviral activity, 80 26, 32
natural selection on, 80 mammalian immune system, 19–20
Trypanosoma brucei gambiensis, 2 primates, 17–48
Tsimane, 365, 371–382 Viral signaling molecules, 239
adult diet, 371 Virulence, effect on host evolution,
Ascaris lumbricoides and Trichuris 2–3, 382
trichiura, 375–376 VLRs. See Variable lymphocyte receptors
characteristics, intestinal parasites, (VLRs)
371, 372
data collection, 373
domestic animals, 371 W
endemic exposure, 377 Wuchereria bancrofti (filarial worm), 367
environmental and socioeconomic
changes, 376
Giardia lamblia, 371 X
helminth and protozoan infections, Xenopus (frog), 2
373–375 Xenotranplantation, 6–7, 18
hookworm and antagonism, 377–378 XLAAD. See X-linked autoimmunity-
human immune pathways, 371 allergic dysregulation syndrome
intensity risk, 375–376 (XLAAD)
limitations and future directions, 380–381 X-linked autoimmunity-allergic
odds ratios, infection, 375, 376 dysregulation syndrome
respiratory and inflammatory diagnoses, (XLAAD), 332
378–380 Xylanibacter, 339
Tumour necrosis factor (TNF)
and malaria, 139–140, 144, 146, 346, 352
polymorphisms, 127, 144 Y
Tunicata, 36, 37, 39, 40 Yaws infection, 194, 201
Tyrosine-interacting protein (Tip), 226–230, Yellow fever, 33, 100
236–239, 242 Yersinia pestis, 3–4, 106

V Z
Varecia, (ruffed lemur), 23 Zoonotic transmissions, 168–169, 391