ISSN 1726-5274

35
Globally, the Vibrio parahaemolyticus and Vibrio vulnificus represent
important human pathogens associated with the consumption of seafood.
In response to the requests for scientific advice from Codex Committee on
Food Hygiene (CCFH), risk assessments for the pathogens V. vulnificus, V.
cholerae, V. parahaemolyticus and guidance on methods for the detection of
Vibrio spp. with seafood have been conducted and published previously by
JEMRA. In order to provide an update on the state-of-the-art advice regarding
risk assessment for V. parahaemolyticus and V. vulnificus in seafood, an
expert meeting was convened.
Advances in science

V. vulnificus associated with seafood

Advances in science and risk assessment tools for Vibrio parahaemolyticus and
Several critical developments in the last decade were subsequently noted
by the expert working group: 1) The emergence of highly pathogenic strains; and risk assessment tools
forAttributing illness
2) In response to climate change, there has been a significant geographical
spread regarding when and where these seafood-associated Vibrio Vibrio parahaemolyticus
infections; 3) Demographic considerations are very important; 4) A range
of new approaches for best practice; and 5) A range of new methods, such
as those utilising genomics and satellite imagery. This report describes the
andcaused by Shiga
V. vulnificus associated
output of that expert meeting.
with seafood
toxin-producing
Escherichia coli (STEC)
MEETING REPORT

to specific foods
REPORT

Food Systems and Food Safety - Economic and Social Development

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MICROBIOLOGICAL RISK

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 35
MICROBIOLOGICAL RISK
ASSESSMENT SERIES

Advances in science
and risk assessment tools
for Vibrio parahaemolyticus
and V. vulnificus associated
with seafood
MEETING REPORT

Food and Agriculture Organization of the United Nations

World Health Organization
Rome, 2021
Required citation:
FAO and WHO. 2021. Advances in science and risk assessment tools for Vibrio parahaemolyticus
and V. vulnificus associated with seafood. Meeting report. Microbiological Risk Assessment Series
No. 35. Rome. https://doi.org/10.4060/cb5834en

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Contents
Acknowledgements v
Contributors vi
Declarations of interest vii
Abbreviations vii
Executive summary ix

1 Introduction 1
1.1 Background – pathogenic vibrios and seafood safety 2
1.2 Vibrio parahaemolyticus 7
1.3 Vibrio vulnificus 9
1.4 Factors relevant to the fate of V. parahaemolyticus and V. vulnificus 12

2 Risk assessment for Vibrio spp. in seafood 12

2.1 Background to risk assessments in seafood 12
2.2 2011 Risk assessment of V. parahaemolyticus in seafood 13
2.3 2005 Risk assessment of V. vulnificus in raw oysters 14
2.4 Recent FAO/WHO activities and expert meetings on vibrios 15

3 New advances in vibrio science 16

3.1 2019 FAO/WHO meeting 16
3.2 Recently available data 21
3.3 Newly available risk assessment models 25
3.4 Recommendations for best practice 27
3.5 Microbiological testing methods 31
3.6 Climate change 36
3.7 Demographic drivers 38

4 Conclusions and recommendations 40

References 43

ANNEXES
Annex 1 Selected available data on the incidence of
V. parahaemolyticus infections in China 58
 TABLES
Table 1. Selected available data on the incidence of V. parahaemolyticus
infections. 6
Table 2. Selected available data on the incidence of V. vulnificus
infections in different countries. 9
Table 3. Currently available risk assessment models. 25
Table 4. Commonly used microbiological and molecular methods applied
in the isolation and characterisation of Vibrio parhaemolyticus
and Vibrio vulnificus. 32
Table 5. Climate suitability for vibrio outbreaks. Changes observed
in the percentage of suitable areas from the 1980s to the
present data. 38

FIGURES
Figure 1. Recent evolution of V. parahaemolyticus strain typing
approaches. 23
Figure 2. Major microbiological, environmental and human-related
factors responsible for driving vibriosis risks associated with
bivalve shellfish. 42

iv
Acknowledgements
The Food and Agriculture Organization of the United Nations (FAO) and the
World Health Organization (WHO) would like to express their appreciation to
all those who contributed to the preparation of this report through the provision
of their time and expertise, data and other relevant information before, during
and after the meeting. Special appreciation is extended to all the members of the
Expert Panel for their dedication to this project and to Dr Rachel Hartnell for her
expert chairing of the Panel, and Craig Baker-Austin for his excellent support in
preparing the final document. All contributors are listed in the following pages.

In addition, FAO and WHO would also like to appreciate those who contributed for
the data collection, which includes: Annick Robert-Pillot and Marie-Laure Quilici,
Institute Pasteur, France; Eckhard Strauch, Robert Koch Institute, Germany;
Saara Salmenlinna, National Institute for Health and Welfare, Finland; Rodríguez
Iglesias, Spain; Bo Pang, Chinese Center for Disease Control and Prevention,
China; and Ronnie Gavilan, National Institute of Health, Peru.

The preparatory work and expert meeting convened to prepare this report was
coordinated by the Secretariat of the Joint FAO/WHO Expert Meetings on
Microbiological Risk Assessment (JEMRA).

We would like to pay our special gratitude and respect to Dr Mitsuaki Nishibuchi.
After helping to discuss and initiate this meeting report, Dr Mitsuaki Nishibuchi
passed away in June 2019.

v
 Contributors
EXPERTS
Carmen Amaro González, Universidad de Valencia, Spain
Enrico Buenaventura, Health Canada, Canada
Viviana Cachicas, Instituto de Sáalud Pública de Chile. (ISP-CH), Chile
Rachel Hartnell, FAO Reference Centre for Bivalve Mollusc Sanitation, Centre
for Environment, Fisheries and Aquaculture Science (Cefas), the United Kingdom
Dominique Hervio-Heath, Institut Français de Recherche pour L’exploitation de
la Mer (Ifremer), France
Dorothy Jean McCoubrey, Dorothy-Jean & Associates Ltd, New Zealand
Iddya Karunasagar, Nitte University, India
Francesca Leoni, Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche
“Togo Rosati” (IZSUM), Italy
Mitsuaki Nishibuchi, Kyoto University, Japan
Erin Stokes, Centers for Disease Control and Prevention (US CDC), the United
States of America

RESOURCE PERSONS
Angelo DePaola, Angelo DePaola Consulting, LLC, the United States of America
Jaime Martinez-Urtaza, FAO Reference Centre for Bivalve Mollusc Sanitation,
Centre for Environment, Fisheries and Aquaculture Science (Cefas), the United
Kingdom
James D. Oliver, The University of North Carolina at Charlotte, the United States
of America

SECRETARIAT
Craig Baker-Austin, FAO Reference Centre for Bivalve Mollusc Sanitation in
Centre for Environment, Fisheries and Aquaculture Science (Cefas), the United
Kingdom
Haruka Igarashi, Department of Nutrition and Food Safety, World Health
Organization, Switzerland
Jeffrey LeJeune, Food Systems and Food Safety Division, Food and Agriculture
Organization of the United Nations, Italy
Satoko Murakami, Department of Nutrition and Food Safety, World Health
Organization, Switzerland
Kang Zhou, Food Systems and Food Safety Division, Food and Agriculture
Organization of the United Nations, Italy
vi
Declarations of interest
All participants completed a Declaration of interests form in advance of their
involvement in in this work.

Two of the Experts declared interest in the topic under consideration.

Angelo DePaola and James D. Oliver declared that they had consulting income.
Upon detailed review of the declaration, it was considered that the activities of
Angelo DePaola and James D. Oliver represent a potential conflict of interest.
Therefore, they were invited to the meeting, but did not participate in the final
adoption of the conclusions and recommendations of the meeting.

All of the declarations, together with any updates, were made known and available
to all the participants at the beginning of the meeting.

All the Experts participated in their individual capacities and not as representatives
of their countries, governments or organizations.

vii
 Abbreviations
°C Degree Celsius
APPCR Arbitrarily primed polymerase chain reaction
CDC Centers for Disease Control and Prevention
Cefas Centre for Environment, Fisheries and Aquaculture Science
CCFH Codex Committee on Food Hygiene
COVIS Cholera and Other Vibrio Illness Surveillance
FAO Food and Agriculture Organization of the United Nations
FDA Food and Drug Administration of the United States of America
HPP High pressure processing
IQF Individual quick freezing
ISO International Organization for Standardization
JEMRA Joint FAO/WHO expert meetings on microbiological risk assessment
kGy Kilogray
ILSI International Life Science Institute
L Litre
LAMP Loop-mediated isothermal amplification
LOD Limit of detection
MLST Multi-locus sequence typing
Mpa MegaPascal
MPN Most probable number
NSSP National shellfish sanitation programme
PBS Phosphate Buffered Saline
PCR Polymerase chain reaction
PFGE Pulsed field gel electrophoresis
PNW Pacific Northwest
Ppt Parts per thousand
PSU Practical salinity units
qPCR Quantitative polymerase chain reaction
RS Remote sensing
SNP Single nucleotide polymorphism
SST Sea surface temperature
ST3 Sequence type 3
ST36 Sequence type 36
TDH Thermostable direct hemolysin
TRH TDH-related hemolysin
T3SS Type III secretion system
USDA United States of America Department of Agriculture
USFDA United States of America Food and Drug Administration
VPRA V. parahaemolyticus risk assessment
VVRA V. vulnificus risk assessment
WGS Whole genome sequencing
WHO World Health Organization

viii
Executive summary
Globally, the bacterial species V. parahaemolyticus, V. cholerae and V. vulnificus
represent important human pathogens associated with the consumption of
seafood. In response to the requests for scientific advice from Codex Committee
on Food Hygiene (CCFH), risk assessments for the pathogens V. vulnificus, V.
cholerae, V. parahaemolyticus and guidance on methods for the detection of Vibrio
spp. in seafood have been conducted and published previously by FAO/WHO
Joint Expert Meeting on Microbiological Risk Assessment (JEMRA) (e.g. the
2005 Risk assessment of V. vulnificus in raw oysters (VVRA) and the 2011 Risk
assessment of V. parahaemolyticus in seafood (FAO/WHO, 2005a, 2011). In order
to provide an update on the state-of-the-art advice regarding risk assessment for
V. parahaemolyticus and V. vulnificus in seafood, an expert meeting was convened
at Centre for Environment Fisheries and Aquaculture Science (Cefas), Weymouth,
the United Kingdom, on 13-15 May 2019. This report is the output of that expert
meeting.

Raw shellfish products such as oysters and clams are the most common foodborne
source of vibriosis; thus this document outlines key areas where recent risk
assessment has been carried out with regards to assessing, understanding and
reducing potential human health risks in these food commodities. Experts reviewed
the draft outputs of the expert meeting in 2010 on the risk assessment tools for V.
parahaemolyticus and V. vulnificus associated with seafood. It was agreed that the
basic information of pathogenicity (including virulence markers), major factors
relevant to the fate of V. parahaemolyticus and V. vulnificus (water temperature
and salinity) and other main contents had not changed substantially; however,
several new models and methods have become available in the last decade which
were worthy of inclusion. In addition, several new developments were discussed,
including the emergence of highly pathogenic strains of V. parahaemolyticus,
as well as the pandemic spread of associated infections which represented key
challenges to the seafood industry, risk managers, clinicians and public health. The
expert group considered a number of topics where significant new information
had emerged in the last decade (and since the publication of the 2010 meeting
workshop draft report). These included (1) recent epidemiological data, (2)
approaches on remote sensing-based risk assessment models, (3) improvements to
detection and molecular methods, (4) aspects related to best practice for reducing
risk, (5) new information on climate change, and (6) demographics; all of which
represented key aspects in terms of modulating human health risks associated with
these pathogens.

Several critical developments in the last decade were subsequently noted by the
expert working group: 1) The emergence of highly pathogenic strains, in particular

ix
 the Pacific Northwest (PNW) V. parahaemolyticus strain (ST36), which have
spread to the East coast of the United States of America, Europe, South America
and New Zealand. The pandemic spread of these highly pathogenic strains is
of global concern for seafood safety. 2) In response to climate change, there has
been a significant geographical spread regarding where seafood-associated vibrio
infections have been reported, with a general trend in the poleward spread of V.
parahaemolyticus and V. vulnificus cases. Over the last decade in particular, there
has been an increase in reported illnesses as well as the geographical spread of
foodborne infections associated with these bacteria into regions where reported
infections were previously absent. 3) The expert group noted that demographic
considerations are also important. Globally, an increased at-risk population,
increased population densities in coastal regions and improvements in diagnosis of
infections may also have played a role in accentuating reported cases. 4) The expert
group identified that a range of new approaches for best practice, such as high-
pressure treatment, harvesting curfews, relaying and temperature controls appear
to offer effective and cost-effective approaches for reducing human health risks
postharvest associated with these pathogens. Finally, 5) the expert group identified
that a range of new methods, such as those utilising genomics and satellite
imagery, provide novel means of complementing approaches outlined in previous
risk assessment exercises for these globally important foodborne pathogens. The
expert group noted, however, that a range of critical data gaps exist. These include
approaches to infer further characterization of strains (for instance serotyping,
MLST, genotyping, APPCR, WGS); virulence testing; gene expression levels; strain
phylogeny and phylogeography. In particular, the paucity of high quality data from
geographically diverse regions (other than the United States of America) probably
represents the most pressing limitation for risk assessment efforts in this arena.

This work has been greatly facilitated by contributions from experts around the
world, with expertise in microbiology, risk assessment, molecular biology, remote
sensing, epidemiology and modelling among others.

x
 1
Introduction

1.1 BACKGROUND – PATHOGENIC VIBRIOS AND

SEAFOOD SAFETY
Vibrio spp. are responsible for the majority of human diseases attributed to the
natural flora of aquatic environments and seafood (Faruque and Nair, 2006).
Vibrio spp. are a group of common, Gram-negative rod-shaped bacteria that are
natural constituents of freshwater, estuarine and marine environments. Vibrio spp.
represent a diverse group of human pathogens, and the disease manifestation and
epidemiology associated with these bacteria is similarly complex (Baker-Austin et
al., 2018). Seafood is part of healthy and balanced diet, yet this food commodity
is responsible for a significant proportion of foodborne illnesses and outbreaks
globally. Consumption of raw or insufficiently cooked seafood can result in human
illness due to the presence of pathogenic microorganisms. Outbreaks of shellfish-
associated infection have been reported for more than a century (Potasman et al.,
2002). In particular, bivalve shellfish such as oysters, clams and mussels greatly
concentrate bacteria present in the surrounding water. Various historical studies
have shown that vibrio densities in oysters can exceed 100 times than that observed
in water (DePaola et al., 1990). The accumulation of vibrios in seafood, and in
particular bivalves, followed by consumption of that product either raw or not
fully cooked is an established route of human exposure to these pathogens. Other
contributing factors may include storage and transportation at inappropriate
temperatures (e.g. outside of cold-chain), contamination by an infected food
handler, or cross-contamination through contact with contaminated seafood or
seawater (Iwamoto et al., 2010). There is a wealth of historical data implicating
the pathogenic vibrios with the consumption of seafood, and in particular

1
 bivalve shellfish such as oysters, clams and mussels. Both V. vulnificus and V.
parahaemolyticus have a longstanding history with seafood risk. Clinical strains
associated with V. vulnificus were first isolated by the United States of America
Centers for Disease Control and Prevention (CDC) in the mid to the 1960s (Hollis
et al., 1976), whilst the first outbreak associated with V. parahaemolyticus was
identified in 1950 in Japan (Fujino et al., 1953).

1.2 VIBRIO PARAHAEMOLYTICUS

Vibrio parahaemolyticus is a ubiquitous Gram-negative marine bacterium, and
an inhabitant of temperate and tropical coastal areas around the world (Baker-
Austin et al., 2018). Globally, V. parahaemolyticus is the leading cause of bacterial
gastroenteritis associated with the consumption of seafood products. An
established route of transmission includes the consumption of raw bivalve shellfish
species, such as oysters and clams; however cross contamination of seafood is
another established route of human infections (Martinez-Urtaza et al., 2016).
While the majority of environmental strains are innocuous members of the marine
microbiota are opportunistic pathogens of humans (Johnson et al., 2008). Clinical
characteristics of V. parahaemolyticus infections include abdominal cramps,
diarrhoea, nausea, headaches, fever, and chills (Honda and Iida, 1993). Infections
tend to be self-limiting, with the vast majority of cases not requiring medical
interventions (Baker-Austin et al., 2018). The presence of V. parahaemolyticus in
the marine environment is closely related to water temperature, with strains readily
isolated when environmental temperatures exceed 15oC (Baker-Austin et al., 2010).

Clinical strains of V. parahaemolyticus isolated from ill patients tend to produce a

variety of recognized virulence factors. Of these, the thermostable direct haemolysin
(TDH) (Nishibuchi and Kaper, 1995), responsible for the Kanagawa haemolysis,
and the TDH-related haemolysin (TRH) (Honda et al., 1988) are currently the
most predictive overall indicators of potential virulence (Baker-Austin et al.,
2018; Jones et al., 2012; Pazhana et al., 2014). Most infections are associated with
strains that possess these genes, although there are notable published exceptions
(Ottaviani et al., 2012). Detection of tdh-trh- strains among clinical strains has
been the source of debate on the pathogenic roles of the tdh+ and/or the trh+
genes (FAO/WHO, 2020). Bhoopong et al. (2007) provided solid evidence for
the possibility that has long been suspected among clinical microbiologists: the
colonies on thiosulphate citrate bile salts sucrose agar (TCBS agar) that are derived
from clinical samples may consists of virulent (tdh+ and/or the trh+) and avirulent
(tdh-trh-) strains of V. parahaemolyticus and accidental isolation of an avirulent
(tdh-trh-) strain(s) is actually causing a misleading interpretation of the avirulent
(tdh-trh-) strain(s) (FAO/WHO, 2020). Recently, type III secretion systems (T3SS),
of which there are two types, have received attention. In particular, those located in
the pathogenicity islands associated with the tdh and trh genes are named T3SS2,

2 ADVANCES IN SCIENCE AND RISK ASSESSMENT TOOLS FOR VIBRIO PARAHAEMOLYTICUS AND V. VULNIFICUS
ASSOCIATED WITH SEAFOOD
and are considered to be possible virulence markers (Ceccarelli et al., 2019; Okada
et al., 2009). Whole genome sequencing efforts have confirmed that pathogenic
isolates of V. parahaemolyticus also encode two type III secretion systems (T3SS)
(Makino et al., 2003; Richie et al., 2012; Okada et al., 2009) which are multiprotein
structures that mediate the translocation of bacterial effector proteins directly into
eukaryotic cells (Baker-Austin et al., 2018).

Until the late 1960’s V. parahaemolyticus cases were geographically restricted to

Japan, but since 1969 infections have been reported from geographically diverse
locations, including the Atlantic, Pacific and Gulf States and Hawaii in the
United States of America. The geographical spread of these infections continued
throughout the 1970s with sporadic cases and outbreaks reported in Europe, Africa,
New Zealand and most of the Asian countries, thereby turning V. parahaemolyticus
into a major seafood-borne pathogen and a global public health concern (Joseph
et al., 1982; Baker-Austin et al., 2018). The epidemiology of V. parahaemolyticus is
frequently characterized by sporadic cases of infection as well as large outbreaks
in coastal areas, mostly associated with the consumption of raw or undercooked
seafood over the summer months (Faruque and Nair, 2006). In the United States of
America, Scallen et al. (2011) indicated that there are on average 34,664 infections
each year caused by this bacterium (range 18,260-58,027 cases/year). Data from
two independent epidemiological datasets in the United States of America (Cholera
and Other Vibrio illness surveillance-COVIS and FoodNet) also indicated that V.
parahaemolyticus infections were the most prevalent vibrio infections reported in
the United States of America in the period 1996-2010, and represented the main
pathogen associated with the consumption of seafood. (Newton et al., 2012; Anon
2020a, 2020b). Publications from the CDC using these datasets (Newton et al.,
2012) have shown that there have been over recent years, a clear increase in V.
parahaemolyticus infections in the United States of America. This study showed an
increase in the incidence of V. parahaemolyticus in the United States of America
between 1996 and 2010 with cases per 100,000 population raising from 0.01 to 0.13
(COVIS) and 0.06 to 0.23 (FoodNet). Of all bacterial foodborne diseases studied,
data from CDC (FoodNet) indicated that vibriosis is the only disease group that
has increased in incidence during the early 2000s (Anon, 2009). Although the
Gulf Coast has historically been the region most frequently associated with V.
parahaemolyticus infections, over recent years numerous reports have highlighted
outbreaks occurring outside this region. These include outbreaks associated with
oyster production in the Pacific Northwest in 1997 (Anon, 1998), Alaska in 2004,
(McLaughlin et al., 2005) and more recently, and since the last risk assessment
(FAO/WHO, 2011), a new clonal complex has emerged along the Eastern seaboard
of the United States of America (Martinez-Urtaza et al., 2013; Newton et al.,
2014). The strains that have emerged recently are termed “PNW” isolates (“Pacific
Northwest”), and demonstrate enhanced pathogenicity compared to other
pathogenic strains (Martinez-Urtaza et al., 2010). Recent studies have shown that
PNW strains have been successfully introduced in the Northeast United States of

CHAPTER 1 - INTRODUCTION 3
 America waters, and have become a prevalent source of infections in the Northeast
United States of America (Martinez-Urtaza et al., 2017; Xu et al., 2017). Critically,
shellfish-transmitted V. parahaemolyticus infections have recently increased in
locations with historically low disease incidence, such as the Northeast United
States of America. This change coincided with a bacterial population shift towards
a greater proportion of human pathogenic variants occurring in the environment,
in part because of the presence of several Pacific native lineages (ST36, ST43 and
ST636) occurring in nearshore areas off the Atlantic coast of the Northeast United
States of America (Xu et al., 2017). One of these instances was the result of the
introduction of an unusual ‘Pacific Northwest’ complex clonal strains (“PNW”
sequence type “ST36” strains) (Martinez-Urtaza et al., 2013). This same strain
resulted in the 2012 V. parahaemolyticus outbreak in Spain associated with use of ice
produced from local seawater contaminated with V. parahaemolyticus (Martinez-
Urtaza et al., 2016a). More recently, this ST36 strain was identified in Peru where
it was associated with locally-acquired infections as well as in the environment,
emphasizing the exceptional epidemic potential of the PNW complex (Abanto et
al., 2020). ST36 strains have also emerged in New Zealand, again highlighting the
rapid and pandemic expansion of these strains (New Zealand Ministry of Health,
2019). Elsewhere, V. parahaemolyticus infections have been reported across the
entire globe, including largescale outbreaks in China, Chile, India, Japan and the
Republic of Korea (Nair et al., 2007), mainly associated with the global spreading
of the pandemic clone O3:K6 (ST3).

In contrast to Asian countries and the United States of America, non-cholera vibrio
infections are less often reported in Europe (Baker-Austin et al., 2010). In Europe,
reflecting the infrequency of identified cases, V. parahaemolyticus is not a notifiable
illness and hence systematic epidemiological data are not available. However, a
rise in V. parahaemolyticus cases has been identified in some “hotspots” of disease
emergence. One of these regions is the northwest of Spain, where cases associated
with V. parahaemolyticus have been reported since 1998. Global genome-wide
phylogenetic analysis revealed that most of the pathogenic strains isolated from
infections in Galicia were associated with globally diverse isolates, indicating frequent
episodic emergence of these pathogens from disparate and remote sources. Examples
of these introductions includes several instances of ST3 and one outbreak associated
with the ST36, the two major epidemic clones of this pathogen (Martinez-Urtaza
et al., 2018). Cases abruptly emerged in 1998 and infections were associated with
large outbreaks caused by a single strain in 1999 (Lozano-León et al., 2003), 2004
(Martinez-Urtaza et al., 2005) and 2012 (Martinez-Urtaza et al., 2013). In 2015-2016,
a shift in the epidemiological pattern associated with this disease was documented in
this region, with the concurrent detection of cases scattered over the region linked
to different and unrelated strains. These major switches in the epidemic dynamics
of V. parahaemolyticus in the region have been associated with the increasing sea

4 ADVANCES IN SCIENCE AND RISK ASSESSMENT TOOLS FOR VIBRIO PARAHAEMOLYTICUS AND V. VULNIFICUS
ASSOCIATED WITH SEAFOOD
surface temperature trend experienced in coastal areas of northwest of Spain, which
has been suggested as a fundamental contributing factor in the emergence of illness
linked to these introduced pathogenic strains (Martinez-Urtaza et al., 2018). Similar
to the situation in Spain, several domestic and travel-associated V. parahaemolyticus
infections have been reported in the United Kingdom over the last 20 years (Baker-
Austin et al., 2019). A large genetic diversity of V. parahaemolyticus strains was
observed, with ST3 (pandemic group) strains the most common sequence type. ST3
strains were also identified in environmental sources in the United Kingdom (Powell
et al., 2013) which indicate the successful establishment in local environmental
reservoirs. Several sporadic but noteworthy outbreaks have also been reported over
the last 20 years in northern European countries. These include several cases involving
seafood in Norway in 2011 as well as more recent sporadic infections in France and
the United Kingdom (Baker-Austin et al., unpublished). Wound infections associated
with these bacteria have also been reported with at least one fatality in Europe (Baker-
Austin et al., 2012). Elsewhere V. parahaemolyticus strains isolated during an outbreak
of acute enteric disease in Vladivostok, the Russian Federation, in 1997 were identified
as belonging to serotype O3:K6 (Smolikova et al., 2001).

Globally, one of the most important areas in terms of disease emergence over recent
years is China, where V. parahaemolyticus has been the leading cause of foodborne
outbreaks and bacterial infectious diarrhoea since the 1990s, especially in coastal
regions (Lin et al., 2010; Liu et at., 2004). Between 2007–2012, V. parahaemolyticus
was the dominant bacterial cause of acute diarrhoea in the southern coastal region
of China, surpassing any other foodborne pathogen (Li et al., 2014). A total of
322 gastroenteritis outbreaks involving 9 041 illnesses and 3 948 hospitalizations
due to V. parahaemolyticus infection were reported in China from 2003 to 2008
(Wu et al., 2014). Analysis of all the V. parahaemolyticus cases captured through
surveillance established in Shenzhen City in the southern coastal region of China
during 2007–2012 identified 1 488 V. parahaemolyticus infections over this period
(Li et al., 2014). All these studies indicated the serotypes O3:K6 (ST3) and O4:K8
(ST88) as the most common among strains from clinical infections in China.

A reoccurring pattern of introduction of epidemic clones and disease emergence

was also identified recently in Latin America. Several instances of introductions of
epidemic clones have been documented in Peru over the last 30 years with infections
emerging over the course of consecutive events of El Niño, which typically implies
the incursions of warm oceanic waters along coastal areas of Peru coinciding with
heavy rainfall events (Martinez-Urtaza et al., 2016b). Interestingly, these strains
were identified previously in Asia, which supports the hypothesis a recurrent flow
of strains from Asia to South America and the subsequent emergence of infections
concurrently with the arrival of the warm conditions associated with El Niño.

TABLE 1. Selected available data on the incidence of V. parahaemolyticus infections.

CHAPTER 1 - INTRODUCTION 5
 tdh/trh
Country/ No. of cases Attributed presence
Period Symptoms Origin of data
Region (outbreaks) food (where
reported) (%)
Canada 2015 82 Raw oysters N/A N/A Public Health
Canada, 2020,
Taylor et al.
2018
Canada 2020 21 Raw oysters N/A N/A Public Health
Canada, 2020
Chile 2011-2019 431 (typically < Molluscs 9/35 cases Diarrheal cases Abanto et al.
100 year) ST 3 (tdh+) & 2020
8/35 ST 36
(tdh+ & trh+)
China 2010-2020 Average 313.5 N/A N/A Diarrheal cases Data courtesy
(South)a cases, (19) Bo Pang 2020.
China 2010-2020 Average 61.4 N/A N/A Diarrheal cases Data courtesy
(Central)a cases, (6) Bo Pang 2020.
China 2012-2020 Average 148.6 N/A N/A Diarrheal cases Data courtesy
(East)a cases, (13.6) Bo Pang 2020.
China 2010-2020 Average 5.6 N/A N/A Diarrheal cases Data courtesy
(South Bo Pang 2020.
West)a
England 2010-2020 ~22 cases/yrb N/A tdh+ & trh+, Diarrheal cases Baker-Austin
tdh+, trh+ et al. 2020.
Finland 2010-2020 11c N/A N/A N/A Data courtesy
Saara
Salmenlinna
2020.
France 2010-2019 91 Seafood - Gastroenteritis Data courtesy
(95%) Annick
Robert-Pillot,
Dominique
Hervio-Heath
& Marie-Laure
Quilici 2020
India 2008-2011 29 N/A 27 tdh+ (five Diarrheal cases Guin et al.
were also 2019.
trh+), 1 trh+
Peru 2015-2016 ~100 Seafood 100% tdh+ & Diarrheal cases Caro-Castro et
trh+ al. 2020.
Spain 2010-2020 7 cases N/A Diarrheal cases, Data courtesy
(Cadiz) external otitis Rodríguez
Iglesias 2020.
Spain 2010-2020 69 cases (51, N/A 100% tdh+ Diarrheal cases, Martinez-
(Galicia) 10, 2 & 6) & trh+ from external otitis Urtaza et al.
outbreak 2016.
with 6 cases
The United 2010-2018 4 116d N/A N/A N/A CDC, 2021.
States of
America
a Detail in annex 1.
b Vast majority of reported cases are derived from foreign-associated travel (Baker-Austin et al. 2020).
c Includes both domestic and foreign-associated infections.
d Includes only cases reported to the Cholera and Other Vibrio Illness Surveillance (COVIS) System and classified as confirmed or probable foodborne illness based
on the reported clinical specimen type and seafood consumption.
N/A, not available.

6 ADVANCES IN SCIENCE AND RISK ASSESSMENT TOOLS FOR VIBRIO PARAHAEMOLYTICUS AND V. VULNIFICUS
ASSOCIATED WITH SEAFOOD
1.3 VIBRIO VULNIFICUS
The Gram-negative bacterium V. vulnificus is a naturally occurring and common
inhabitant of estuarine and coastal environments. This bacterium is a zoonotic
agent linked to fish farms whose life cycle inside and outside its main hosts as well
as its phylogeny have been recently reviewed by Hernández-Cabanyero and Amaro
(2020). As a fish pathogen, this species causes outbreaks of a septicemic disease
known as warm-water vibriosis (Amaro et al., 2015). As a human pathogen,
this bacterium can infect either by consumption of raw seafood, in particular
molluscan shellfish, or by contact of wounds with seawater or fish (mainly diseased
fish) (Amaro et al., 2015; Ceccarelli et al., 2019). In the first case, the pathogen
causes gastroenteritis or primary septicaemia and in the second case, severe wound
infections and secondary septicaemia (Baker-Austin et al., 2018; Ceccarelli et
al., 2019). Although some zoonotic cases after diseased fish handling have been
described, V. vulnificus is mainly recognized as a significant foodborne pathogen
since it is a leading cause of seafood-related mortality (Ceccarelli et al., 2019).
The pathogen is present in high numbers in filtering organisms, such as oysters,
especially in warmer months (Oliver et al., 2006). Vibrio vulnificus abundance and
risk differs from that of V. parahaemolyticus as it prefers warmer and less saline
conditions and is generally less widely distributed. Growth in oysters ceases below
13 oC and abundance plateaus above 25 oC; illnesses are rarely associated with
shellfish harvested from waters with temperatures below 20oC. The VVRA found
a broad salinity peak around 17 ppt (5-25ppt) with typically non-detectable levels
associated with negligible risk above 30ppt. Incidence of infection is related to
environmental distribution, and V. vulnificus exhibits quite distinct temperature
and salinity tolerances, being restricted to warm (13-30oC) and low salinity
(2-25ppt) waters (Hernández-Cabanyero and Amaro, 2020). Of some note, V.
vulnificus-associated primary septicaemia carries the highest fatality rate of any
studied foodborne pathogen (Rippey, 1994). The United States of America reported
the mortality rate in cases of V. vulnificus infection due to shellfish consumption
is approximately 53% (Anon, 1993). Most cases of infection (~95%) occur in
males. Vibrio vulnificus infections are characterised by a short incubation period
between the onset of symptoms and subsequent clinical outcome (Baker-Austin
et al., 2009; Baker-Austin and Oliver, 2018), typically within 24 hours of exposure
(Jones and Oliver 2009). Numerous studies on the virulence factors involved in
human vibriosis have been conducted. Of all the described virulence factors,
those involved in sepsis, the capsule and the MARTX toxin (Multifunctional
Autoprocessing Repeat in Toxin), also known as RtxA1, are the most relevant.
The capsule protects against the bactericidal action of serum complement and
phagocytosis (Carda-Dieguez et al., 2018) and the RtxA1 toxin, in conjunction
with hemolysin VvhA, promotes invasion from the intestine into the bloodstream

CHAPTER 1 - INTRODUCTION 7
 (Jeong and Satchell, 2012) as well as the activation of an early blood cytokine storm
(Murciano et al., 2016). Interestingly, the production of the capsule and the RtxA1
toxin are increased under iron excess, which could explain the rapid death from
sepsis of patients with high levels of iron in their blood (Hernández-Cabanyero et
al., 2019).

Several studies have utilised different molecular markers in V. vulnificus as a proxy

for potential human virulence, with varying degrees of success. Differences in the
sequence of the small subunit 16S rRNA gene, as correlating with either clinical
(pathogenic) and environmental (non-pathogenic) origin, have been utilised
previously (Aznar et al., 1994; Nilsson et al., 2003). More recently, an exploration
of genes associated with virulence in humans focused on the virulence correlated
gene (vcg) (Rosche et al., 2005) and pilus-type IV-related gene pilF (Roig et al.,
2010) which have both shown promise as molecular targets to identify virulent
strains.

Vibrio vulnificus is a rare cause of infection (~100 cases per year in the United
States of America), but published studies demonstrate an increase in disease in the
United States of America and also in Europe (Baker-Austin and Oliver, 2018). As
with V. parahaemolyticus, the availability of both FoodNet and COVIS datasets in
the United States of America helps to provide a highly useful national overview
of disease burden associated with V. vulnificus. Using V. vulnificus data obtained
from the CDC (COVIS) from recent collaborative work, the rate of infection has
increased from 0.029 cases per 100 000 of the population in 2007/2008 to 0.0502 in
2015/2016, an almost 2-fold increase in reported infections in less than a decade.
However, most of the rise in infections have been attributable to wound infections,
with shellfish-associated cases plateauing and, in some areas, reducing in incidence,
particularly in the last two decades in the United States of America (Baker-Austin
2020, unpublished). Since 1988 when the CDC started to systematically record
vibrio disease in the United States of America, there have been over 2 600 V.
vulnificus cases nationally, with over 700 associated deaths, averaging around 30
fatalities a year which are predominantly from seafood sources. Overall, numbers
of V. vulnificus cases have increased in the United States of America since 1988
(Baker-Austin et al., unpublished). Data published by the CDC (Newton et al.,
2012) indicates increases in V. vulnificus infections reported in the United States of
America between 1996 and 2010 in both the COVIS and FoodNet datasets, with
a marked increase in the FoodNet data (0.01 infections per 100 000 population
in 1996, increasing to 0.05 cases per population in 2010). Globally, surveillance
data regarding V. vulnificus infections are not gathered systematically, making
wider geographical and epidemiological comparisons problematical (Table 2). In
Europe, V. vulnificus infections are considered rare, and generally associated with

8 ADVANCES IN SCIENCE AND RISK ASSESSMENT TOOLS FOR VIBRIO PARAHAEMOLYTICUS AND V. VULNIFICUS
ASSOCIATED WITH SEAFOOD
bathing water exposure (Baker-Austin et al., 2012), with low salinity waters such
as the Baltic Sea a hotspot for reported infections during heatwave events. A small
number of seafood associated infections have occurred however, in Italy in the
early 2000s and more recently in France (Baker-Austin et al., unpublished data).
Where only fragmentary surveillance data exists (for example, published peer
reviewed reports of infections) studies and available grey literature have shown
infections reported in Europe, China, Uruguay, Japan and the Republic of Korea
(Baker-Austin et al., 2018).

TABLE 2. Selected available data on the incidence of V. vulnificus infections in different

countries.

Country Period No. of cases Symptoms Origin of data

Public Health England
England 2011-2018 8* N/A
(PHE), 2020
Data courtesy Saara
Finland 2010-2020 3** N/A
Salmenlinna 2020.
Data courtesy
Annick Robert-Pillot,
Dominique
France 2010-2019 10 Wound infections
Hervio-Heath &
Marie-Laure Quilici
2020
Robert Koch Institute,
Germany 2002-2019 <20*** N/A
2020
Data courtesy
Spain 2
2010-2016 External otitis Rodríguez Iglesias
(Cadiz) (2011,2016)
2020.
The United
States of 2010-2018 245**** N/A CDC, 2021.
America
*Vast majority of reported cases are derived from foreign-associated travel
**Includes both domestic and foreign-associated infections
***There was no mandatory reporting of vibrio infections before 2020. Increased numbers of infections apparent during heatwave
years, such as 2003, 2006, 2010, 2018. 8 V. vulnificus cases were reported in Germany in 2020.
****Includes only cases reported to the Cholera and Other Vibrio Illness Surveillance (COVIS) System and classified as confirmed or
probable foodborne illness based on the reported clinical specimen type and seafood consumption

1.4 FACTORS RELEVANT TO THE FATE OF

V. PARAHAEMOLYTICUS AND V. VULNIFICUS

To more fully understand risks associated with V. vulnificus and V. parahaemolyticus

it is necessary to understand factors that drive the abundance of these bacteria
in raw/undercooked seafood. The most recent MRA document focusing on V.
parahaemolyticus and V. vulnificus outlined a variety of factors that were deemed
important with regards to modulating these risks. Risk models (outlined in
section 2) draw heavily on factors that likely increase risk. Since the abundance
of V. vulnificus and V. parahaemolyticus is a critical parameter to estimate the

CHAPTER 1 - INTRODUCTION 9
 risk of infections, predictive models based on environmental variables were
developed by FAO/WHO VPRA and VVRA that were validated by market data
underpin V. parahaemolyticus and V. vulnificus Control Plans implemented by the
Interstate Shellfish Sanitation Conference (ISSC) in 2007 and 2010, respectively.
These tools estimate V. parahaemolyticus and V. vulnificus at harvest based on
environmental studies that derived relationships between abundance with water
temperature. Shellfish authorities employ these risk tools for scenario analysis of
proposed controls to achieve mandated acceptable levels of risk. Inputs include
water temperature to estimate concentrations of bacteria at the time of harvest, air
temperatures for post-harvest growth rates, times to first refrigeration and time to
reach no-growth temperatures. Outputs include abundance and risk per 100 000
meals. Several factors have previously been outlined as critical in this regard:

Temperature. Seawater temperature has been reported as one of the principal

environmental factors increasing the abundance of V. vulnificus and V.
parahaemolyticus in many areas of the world (FAO/WHO, 2020). Their abundance
in the natural environment tends to mirror ambient environmental temperatures
(Baker-Austin et al., 2016; Pfeffer and Oliver 2003). Recent outbreaks associated
with climatic anomalies, such as those observed in the NE United States of America
(Newton et al., 2014), Spain (Martinez-Urtaza et al., 2013), and Chile (Martinez-
Urtaza et al., 2010), and non-cholera vibrio infections reported in Northern
Europe during heatwave events (Baker-Austin et al., 2012; Baker-Austin et al.,
2016) outline the importance of temperature in modulating risk (Baker-Austin et
al., 2016). Recently, Hernandez-Cabanyero et al. (2020) demonstrated that warm
temperatures (over 22 oC) activate adaptive traits that would prepare the bacteria
for host colonization such as metabolism, motility, chemotaxis, protease activity,
iron-uptake and production of O-antigen of high molecular weight.

Salinity. Salinity has also been shown to play an important role in the survival
and subsequent ecology of V. vulnificus and V. parahaemolyticus, and subsequently
deemed critical to fully understand risk. Indeed, V. parahaemolyticus grows
preferentially in warm (>15oC), low-salinity marine waters (<25ppt NaCl) (Baker-
Austin et al., 2010). Vibrio vulnificus occupies an ecological niche similar to Vibrio
parahaemolyticus and its distribution is also governed by variations in salinity. V.
vulnificus does not tolerate high salinity, and its distribution is mostly restricted to
brackish water environments of temperate and tropical areas (Parvathi et al., 2004;
Rivera et al., 1989). In areas of moderate salinity (from 1 to 25 ppt) and temperate or
warm waters (e.g. Gulf of Mexico, Chesapeake Bay, the United States of America),
seawater temperature is the major factor influencing the abundance. In areas with
salinity close to oceanic waters (from 25 to 35 ppt) and temperate waters (e.g.
Atlantic coasts of Europe), V. parahaemolyticus is detected during periods of lowest

10 ADVANCES IN SCIENCE AND RISK ASSESSMENT TOOLS FOR VIBRIO PARAHAEMOLYTICUS AND V. VULNIFICUS
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salinity, whereas seawater temperature influences the concentration. In tropical
areas with minor changes in seawater temperature (e.g. India), no influence of
salinity and temperature has been reported.

Other factors. In addition to seawater temperature and salinity, some additional

abiotic and biotic factors have been identified modulating the presence and
abundance of V. vulnificus and V. parahaemolyticus in coastal water around the
world (FAO/WHO, 2020). Most studies have focussed on particular geographical
areas; however additional factors such as chlorophyll A (Martinez-Urtaza et al.,
2008; Urquhart et al., 2016; Oberbeckmann et al., 2012), nitrogen and carbon
(Froelich et al., 2019), oceanic microplastics (Bowley et al., 2020), and the
abundance of bacteriophages in the surrounding environment (Bastias et al., 2010)
have been shown to modulate concentrations of vibrios.

CHAPTER 1 - INTRODUCTION 11
 Risk assessment for Vibrio spp. in
2
seafood
2.1 BACKGROUND TO RISK ASSESSMENTS IN SEAFOOD
The food safety concerns associated with vibrio pathogens has led to the need for
microbiological risk assessments to support risk management practices. Previous
FAO/WHO risk assessments regarding V. parahaemolyticus and V. vulnificus have
been published (see below), and form the basis of international control measures
frequently adopted to reduce risks from seafood. Previous expert consultation
concluded that three species, V. parahaemolyticus, V. vulnificus, and choleragenic
V. cholerae were the species responsible for most cases of human illness caused by
vibrios, and several seafood vehicles associated with these illnesses were identified
(FAO/WHO 2005). In 2001 FAO/WHO initiated a more comprehensive series of
vibrio risk assessments and guidance, including:
• 2005: Risk assessment of V. vulnificus in raw oysters (FAO/WHO, 2005a)
• 2005: Risk assessment of choleragenic V. cholerae O1and O139 in warm water
shrimp in international trade (FAO/WHO, 2005b)
• 2011: Risk assessment of V. parahaemolyticus in seafood (FAO/WHO, 2011)
• 2016: Selection and application of methods for the detection and enumeration
of human pathogenic Vibrio spp. in seafood (FAO/WHO, 2016)
• 2020: Risk assessment tools for V. parahaemolyticus and V. vulnificus associated
with seafoods (FAO/WHO, 2020)
A short overview of these risk assessments and their associated outcomes is
outlined below.

12
2.2 2011 RISK ASSESSMENT OF V. PARAHAEMOLYTICUS
IN SEAFOOD
Quantitative risk assessments have previously been developed for V.
parahaemolyticus in oysters and followed the basic risk assessment structure
outlined by Codex Alimentarius in their guidance for microbiological risk
assessment: (1) hazard identification, (2) hazard characterization, (3) exposure
assessment, and (4) risk characterisation (FAO/WHO, 2020). The 2011 V.
parahaemolyticus risk assessment used an oyster harvest public health model
developed in one country (the United States of America) to assess risk in
oysters from harvesting areas in other regions. The 2011 risk assessment work
on V. parahaemolyticus in raw oysters used models developed during the United
States of America Quantitative Risk Assessment on the Public Health Impact
of Pathogenic V. parahaemolyticus in Raw Oysters (FDA VPRA) (FDA, 2005)
to estimate risk of illness from this pathogen due to consumption of oysters
in Australia, Canada, Japan and New Zealand. In short, that quantitative risk
assessment used factors influencing the presence of V. parahaemolyticus in oysters
and the flow of events that lead to human illnesses. The approach taken was
applied to other products, such as the blood clam and finfish to determine to what
extent such risk assessments could be adapted to other food commodities. When
the FAO and WHO initiated the above risk assessments, most of the available
data were generated in the United States of America. It was recognized early on
that there were key regional differences even within the United States of America
and the need to collect local data in order to calibrate predictions for accurate risk
assessment purposes. Applying risk models based on data of the United States of
America to other countries with different shellfish species and practices would
increase uncertainty. However, a range of positive outcomes were established
from the 2011 risk assessment work. The report represented an important body
of knowledge regarding risk assessments for these globally important foodborne
pathogens. Firstly, the model that was developed was successfully used to estimate
illness from different oyster species grown under various regimes and regulatory
management systems. Secondly, the framework of the model made available in
the report could be modified and subsequently used by risk assessors in other
countries. Finally, the model that was developed could be utilised as a useful tool
for assessing and determining the efficacy of mitigation strategies, both at harvest
(such as reduced cooling times) and postharvest by heating, freezing or high-
pressure treatment (FAO/WHO, 2011). A range of key deficiencies in the data
available to conduct the risk assessment were subsequently outlined in the report.
These included a lack of data on the abundance of pathogenic V. parahaemolyticus
in water and shellfish and the factors that drive the incidence of these bacteria in
the environment; the role of oysters in concentrating and retaining these bacteria;
the potential role of enterotoxin trh in driving infections; growth of pathogenic
and non-pathogenic strains across a wider temperature range as well as the

CHAPTER 2 - RISK ASSESSMENT FOR VIBRIO SPP. IN SEAFOOD 13

 need for an improved global public health surveillance of V. parahaemolyticus
to identify new epidemic strains as they emerge, and a greater clarity regarding
the reporting systems used in each country of study – particularly with regards
to underreporting of infections. The VPRA was useful in predicting exposure of
total V. parahaemolyticus in the United States of America’s oysters. However, flaws
in two critical assumptions have emerged. The proportion of pathogenic strains
(tdh+) was estimated at 0.2% in Atlantic and Gulf regions and 3% in Pacific areas;
these have been found to vary up to near 100% temporally and geographically.
More consequentially, all tdh+ strains were assumed to be equally virulent with
an infectious dose (LD50) between one and ten million tdh+ cells. An infection
rate >1 000-fold greater was observed in 2004 Alaska outbreak. Evidence of high
infections rates have been reported for certain outbreak strains such as ST3 and
ST36 (see section 1.2).

2.3 2005 RISK ASSESSMENT OF V. VULNIFICUS IN RAW

OYSTERS
Prior to the 2011 V. parahaemolyticus risk assessment, the pathogen V. vulnificus
was chosen as a target to establish the first quantitative risk assessment for a vibrio
pathogen in seafood. A major objective of the V. vulnificus risk assessment was to
determine the usefulness of adapting the FDA VPRA and FAO/WHO VPRA models
to assess the risk from V. vulnificus septicaemia associated with the consumption
of raw oysters (determined to be the major route of foodborne human infections)
(FAO/WHO, 2005a). A secondary objective of the V. vulnificus risk assessment
aimed to identify the most appropriate data, as well as gaps in the available dataset,
for modelling purposes (FAO/WHO, 2005a). An additional objective of this risk
assessment was to determine potential mitigation strategies in reducing human
infections (foodborne septicaemia). One key limitation to this risk assessment
endeavour was noted from the outset: for reasons of data availability, the risk
assessment was geographically limited to using data regarding primary septicaemia
cases associated with consumption of raw oysters from the Gulf Coast of the United
States of America. There is a paucity of data regarding primary septicaemia cases
elsewhere (e.g. outside of the Gulf coast states of the United States of America).
The risk assessment contained a number of key modules, including data derived
from environmental sources (for instance water temperature at harvest, number
of bacteria in oysters), key data on bacteria coupled to temperature postharvest,
predicted numbers of bacteria in oysters at consumption, with dose response analysis
coupled to the number of oyster servings and lastly an analysis of the risk of illness
coupled to observed clinical infections. Several key conclusions were noted from this
assessment. Firstly, a quantitative risk assessment could be successfully achieved for
V. vulnificus by utilizing the framework and parameters for the V. parahaemolyticus

14 ADVANCES IN SCIENCE AND RISK ASSESSMENT TOOLS FOR VIBRIO PARAHAEMOLYTICUS AND V. VULNIFICUS
ASSOCIATED WITH SEAFOOD
risk assessment model. It was noted that where additional data were available it
was possible to validate certain aspects of the risk assessment model (FAO/WHO,
2005a). Good agreement was noted between the exposure assessment predictions
compared to retail study data used in the risk assessment. Because human volunteer
data is not available for V. vulnificus (for obvious ethical reasons) even in the absence
of such dose-response data it was possible to develop a correlative relationship from
seasonal exposure predictions and the reported frequency of illness which was
effective for risk characterization and the evaluation of interventions (FAO/WHO,
2005a). It was also noted that the model used for the V. vulnificus risk assessment
provided a useful framework for other countries that required risk assessments on
this pathogen, particularly in oysters. However, to be able to use the model outlined
it was necessary to refine the approach using country-specific data, and to date this
remains elusive, in particular on the abundance of V. vulnificus in seafood associated
with primary septicaemia, at harvest and the point of consumption, as well as data
on the susceptibility of the population in that country. A key point made in the risk
assessment was that this model was developed for oysters, and to use this model
for other shellfish species such as clams (which have been recently implicated in
septicaemia in the United States of America, Baker-Austin et al. 2020, unpublished
data) would need to be adapted accordingly. A key data-gap is where outside of the
United States of America these shellfish-associated infections occur regularly.

2.4 RECENT FAO/WHO ACTIVITIES AND EXPERT

MEETINGS ON VIBRIOS
The FAO/WHO VPRA was released in 2011 and included sections on raw oysters
worldwide, blood clams in Thailand and raw fish in Japan. In addition, there
were a number of activities based on early drafts and expert consultations. While
finalising the Codex Guidelines in the Application of General Principles of Food
Hygiene for the Control of pathogenic Vibrio spp. in seafoods, the 41st session of
the CCFH recognized the need to provide countries with tools to assist them in
the implementation of the guidelines under the various conditions that exist in
different regions and countries. Such tools were envisioned to support countries
in their efforts to use risk-based approaches in the selection of control measures
appropriate for their seafood species, primary production and postharvest practices.
Such risk management tools (V. parahaemolyticus calculator and V. vulnificus
calculator tools) had already been developed for application in the United States of
America. However, as it was based on the conditions and data of the United States
of America, its broader application could not be recommended without a review of
its validity when applied to the non-United States of America scenarios. In light of
this, the CCFH requested FAO/WHO convene an Expert Meeting tasked with the
following terms of reference:

CHAPTER 2 - RISK ASSESSMENT FOR VIBRIO SPP. IN SEAFOOD 15

 • Conduct validation of the predictive risk models developed by the United
States of America based on FAO/WHO risk assessments, with a view toward
constructing more applicable models for wide use among member countries,
including adjustments for strain virulence variations and ecological factors.
• Review the available information on testing methodology and recommend
microbiological methods for Vibrio spp. in order to monitor the levels of
pathogenic Vibrio spp. in seafood or seawater, or both.
• Conduct validation of growth rates and doubling times for V. parahaemolyticus
and V. vulnificus in Crassostrea virginica (eastern or American oyster)
using strains isolated from different parts of the world and different bivalve
molluscan species.

The FAO/WHO convened this Expert Meeting on 13-17 September 2010, and a
meeting report based on this (MRA20) has been recently published (FAO/WHO,
2020). The outputs from this meeting included the following:

Risk calculators and models: Risk assessment comprises estimation of exposure

to the hazard, called “exposure assessment” and characterization of the relationship
between the amount or dose of the hazard that is ingested and the probability
of illness, or some other measure of harm (FAO/WHO, 2003). Rather than
exhaustively validating individual risk models, the working group decided that it
would be more appropriate to assess and evaluate existing models to be amended
and extended for other uses. In this regard, simplified calculator tools could
then be developed to answer separate risk questions routinely, and potentially
in different regions and utilising different shellfish species and pathogens. The
workshop noted that the V. parahaemolyticus calculator tool may be used to
estimate the relative risk reductions, primarily because of the linear dose-response,
associated with temperature controls (postharvest refrigeration) in areas in which
the strain virulence, initial concentration and growth rates of V. parahaemolyticus
in the bivalve spp. of concern are similar to the United States of America. The
expert group concluded that the V. vulnificus calculator tool is less likely to be
applicable to a broader region outside the United States of America due to potential
differences in environmental, harvesting and post harvesting parameters, but more
significantly due to the nature of the dose-response relationship that is derived
completely from the United States of America epidemiological data coupled with
estimated exposure levels and is nonlinear. Specific shellfish species might also
influence the risk estimate. In addition, the group noted the need to develop a
tool that is applicable to particular regions/or and other products, or to answer
risk management questions other than postharvest refrigeration, it was preferable
to first modify the Joint FAO/WHO Expert Meetings on Microbiological Risk
Assessment (JEMRA) risk assessment model used for the 2010 meeting, or develop
a new model, that considered and evaluated the influence of other factors including

16 ADVANCES IN SCIENCE AND RISK ASSESSMENT TOOLS FOR VIBRIO PARAHAEMOLYTICUS AND V. VULNIFICUS
ASSOCIATED WITH SEAFOOD
salinity, strain differences, temperatures, etc. A simplified calculator tool was then
be developed to answer that specific question routinely. Some key data limitations
were also noted by the group, notably evidence that the V. parahaemolyticus model
currently used overestimated its growth at higher temperatures (for instance >
25 °C) in live oysters. This phenomenon requires further investigation. Finally,
there was no data to evaluate the performance of the growth models in any other
oyster species or other filter feeding shellfish or other seafood and as such its
use in these products could not be supported but if used, should be done with
clear understanding of the associated uncertainty. Several key recommendations
emerged from this meeting:

Suggestions for improvement for the V. parahaemolyticus model:

• The available V. parahaemolyticus prediction model was is a linear model and
therefore may be useful to estimate relative change in risk (percent reduction
in risk) for different countries with more virulent strains, only providing that
the ranges of doses in that country are much less than the Infectious Dose 50
(ID50) for the more virulent strain (i.e. in the linear range of the dose response
relationship).
• There is a need for country-specific assessments (e.g. in Chile) which should be
undertaken and which should use an ecological dose-response approach like
V. vulnificus DR-assessment (but prospectively); studies should be undertaken
in other regions to obtain estimates of dose by sampling at retail.
• There is a need to develop dose-response models that incorporate multiple
strains and variations in virulence based on data of relative expression of
hemolysins plus differences in prevalence of pathogenic strains in clinical
versus environmental isolates.

Suggestions for improvement for the V. vulnificus model:

• Conversely, the use of the V. vulnificus prediction tool outside of the United
States of America was not advisable at that time (dose-response nonlinear so
not useful as relative risk reduction, different countries might have different
prevalence of C-type vs E-type strains in the environment, there may be
differences in the proportion of population susceptible, etc.).
• The United States of America V. vulnificus dose-response assessment should
be revisited using an approach that incorporates the effect of (a) seasonality
of the C vs E type & (b) relative virulence of the C vs E type e.g. for (a) try
logistic regression of type C vs type E versus water temperature or season;
for (b) 60-fold difference by comparison of prevalence in clinical versus
environmental strains.
• The concept of incorporating distributions of susceptibility to infection
among the at-risk population should be investigated (e.g. assess the reliability
of extrapolation to a country with different prevalence of predisposing
conditions).
CHAPTER 2 - RISK ASSESSMENT FOR VIBRIO SPP. IN SEAFOOD 17
 Microbiological approaches and techniques: Microbiological monitoring
methods, particularly molecular-based approaches, have rapidly evolved recently,
particularly since the risk assessment efforts for these pathogens began in the
early 2000s. The group noted that because of these rapid changes, it would not be
appropriate to make single recommendations regarding choice of molecular targets,
detection methodologies, etc. It was recommended, however, that a few options
could be considered, which would also address issues such as the type, extent and
sampling intensity of a vibrio monitoring programme alongside constraints such
as the cost, the speed with which results are required and the technical capacity of
testing laboratories. To facilitate data collection, the meeting recommended the
establishment of an internationally recognized system for developing criteria and
protocols for evaluation of methods and to elaborate performance parameters of
methods for detection and enumeration of pathogenic Vibrio spp. in seafood.

FAO/WHO convened an Expert Meeting in 2011 that produced a Guidance

document on methods for detection and enumeration of V. parahaemolyticus,
V. vulnificus, including performance characteristics of the methods and the
application of these methods for different end uses, ranging from harvest area
monitoring, postharvest process verification, end product testing, outbreak
investigation and growth studies (FAO/WHO, 2016). Following this, FAO, in
collaboration with International Life Science Institute (ILSI) organized two regional
training workshops, one in Asia and another in Latin America to disseminate the
information on methodology for vibrio studies.

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 3
New advances in vibrio science
3.1 2019 FAO/WHO MEETING
The FAO/WHO solicited Cefas to provide an update on key developments that had
taken place since the 2010 meeting report. The Joint FAO/WHO Expert Meeting
on Microbiological Risk Assessment on V. parahaemolyticus and V. vulnificus
took place at Cefas, Weymouth, the United Kingdom, on 13-15 May 2019. The
meeting reviewed and updated the existing risk assessment models/tools of V.
parahaemolyticus and V. vulnificus that could be used to address a range of risk
management questions in a number of different regions.

During the workshop, several key areas were identified by the expert working group
that had greatly advanced since the 2010 meeting. These are can be summarized
into several main areas:
1) Available epidemiology. There are now a variety of epidemiological
datasets available (from 2010 onwards) that greatly expand our knowledge
regarding the global distribution and clinical impact of V. parahaemolyticus
and V. vulnificus from seafood. The most recent epidemiological data
is included in Tables 1 and 2. The emergence of unusual species (e.g.
nontoxigenic V. cholerae) in seafood-related outbreaks was also noted.
2) Recently available scientific data, in particular information on new
pathogenic strains and their geographical spread and clinical incidence,
was outlined and discussed.
3) Methods for the detection and characterisation of vibrios of human
health relevance. These include new culturing methods as well as novel

19
 characterisation approaches such as those facilitated by next generation
sequencing-based techniques, LAMP, immunocapture approaches, etc.
4) Risk modelling advances. These include approaches for apportioning
risk using remote sensing based techniques to measure variables such as
temperature and salinity.
5) Advances in best practice. Since the 2010 meeting report, and previous
FAO/WHO risk assessment reports published on V. vulnificus (2004)
and V. parahaemolyticus (2011), a variety of studies have improved our
understanding of practical interventions that can be used to reduce
vibriosis risks associated with the consumption of seafood. These
include relaying, cooling, post-harvest treatments, etc. These have been
summarised here.
6) Climate, demographics and behaviour. There is a much greater
understanding of aspects related to human behaviour and also the
impact of climate change on risks associated with V. vulnificus and V.
parahaemolyticus. There are also notable changes in demography at a
national, regional and global level that may impact human health risk.
These have been summarised and discussed further below.

Some vibrio studies have identified that climate, handling practices, resident
and emergent vibrio strains and shellfish species may affect the growth of V.
parahaemolyticus and V. vulnificus - hence more representative data from other
regions are required, to determine whether it is appropriate to update these models
and tools or if indeed new risk assessments are needed. Since then, there have
been many developments in this area over the last decade, and understanding of
these organisms and their management continues to evolve. Taking into account
those continuous discussions, the meeting reviewed and updated the existing risk
assessment models/tools of V. parahaemolyticus and V. vulnificus that could be
used to address a range of risk management questions in a number of different
regions.

Experts reviewed the outcomes of the expert meeting in 2010 on the risk
assessment tools for V. parahaemolyticus and V. vulnificus associated with seafood.
Experts agreed that the basic information of pathogenicity (including virulence
markers), major factors relevant to the fate of V. parahaemolyticus and V. vulnificus
(water temperature and salinity) and other main contents have not been changed;
however, there are several models and methods that have become available in the
last decade.

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3.2 RECENTLY AVAILABLE DATA
Microbiology and microbiological risk assessment are constantly evolving fields,
and new developments such as changes in microbiological detection methods,
the emergence of new pathogens and pathogenic strains, coupled to a changing
environment, among others, provide constant challenges for risk assessors. A
variety of scientific developments have taken place in the last decade that have
significant ramifications for risk assessment purposes. Some of these highlight
scientific facts relevant for consideration within the existing risk assessment
model that were not available during earlier meetings or during the previous risk
assessment reports in 2005 and 2011. These findings include the following:

Emergence of highly pathogenic strains: New and highly pathogenic strains of

V. parahaemolyticus have emerged along the eastern seaboard of the United States
of America and Europe recently, and since the previous RA work was considered
(Martinez-Urtaza et al., 2013). These strains represent an additional concern
for risk assessment purposes as they have a significantly lower infectious dose
50 and different growth characteristics compared to “pathogenic” (e.g. tdh+) V.
parahaemolyticus strains used in the 2011 VPRA. Infections associated with these
clones have now been reported in Europe and the NE United States of America
(Martinez-Urtaza et al., 2013) but also now in Latin America, with cases confirmed
in Peru (Abanto et al., 2020). These findings are especially noteworthy, indicating
the potential pandemic spread similar to ST3 strains in the 1990’s (Nair et al., 2007).

The United States of America risk management tools outlined previously (e.g. in
previous MRA documents, MRA 20) did not consider the arrival of new pathogenic
strains in a geographical location mediated by human activity or natural events,
and how climatic or oceanographic factors, like El Niño and climate change, can
influence the dispersal or successful introduction and establishment in natural
reservoirs. Although the sources and routes of introduction of these foreign clones
remain yet undetermined, a growing body of evidence has linked the epidemic
dynamics and spreading of disease in particular regions such as the NE United States
of America and Spain to the movement of shellfish species, and in South America to
El Niño events. Strikingly, the long-term persistence and presence of environmental
isolates indicate the successful establishment of ST36 in environmental reservoirs
(Abanto et al., 2020), as evidenced by the overwintering of ST36 in the NE United
States of America from 2012-2013; this is especially relevant from a risk assessment
perspective. The presence of these strains now in NE United States of America, Latin
America, New Zealand and Europe, as well as its traditional host range in the Pacific
Northwest, is evidence of successful pandemic spread, and the potential for ongoing
risks associated with this particular clonal group. These findings require in-depth

CHAPTER 3 - NEW DEVELOPMENTS IN VIBRIOS 21

 and judicious consideration from a risk assessment perspective because of the
clinical capabilities of these strains as well as the precedent of rapid clonal expansion
of these pathogens, such as that seen by the O3:K6 group in the 1990s which caused
significant outbreaks globally (Nair et al., 2007).

Ecological variables: The United States of America risk management tool for
V. parahaemolyticus was based on the presumption that in a geographical area,
a certain proportion of V. parahaemolyticus strains are pathogenic and that
environmental factors like temperature and salinity affect all V. parahaemolyticus
strains equally. Some recent evidence indicates that certain ecological parameters
may favour the proliferation of pathogenic strains over non-pathogenic strains
(Baker-Austin et al., 2016). Alongside this, there is emerging data to show that a
larger proportion of strains in other regions may be toxigenic (e.g. in the United
Kingdom, trh+ are typically up to 50% of all tested V. parahaemolyticus during
summer months, Baker-Austin et al. published, FAO/WHO, 2020). Currently, the
risk potential of these strains is unknown.

Development of molecular tools: Most of the methods for detection of V.

parahaemolyticus and V. vulnificus were outlined and described in MRA Series
22 (FAO/WHO, 2016). A multitude of methods for characterization of strains
such as improvements in the isolation of these bacteria, serotyping, multi-locus
sequence typing, genotyping, and more recently whole genome sequencing have
been more widely introduced recently which has improved our understanding
and characterization of the risks associated with these bacteria. More recently,
the application of molecular approaches, and in particular the use of whole
genome analysis, has revolutionized our understanding of these pathogens, but
has provided key challenges regarding the importance of strain phylogeny and
phylogeography and thus requires more information. Genetic analysis of outbreak
strains in the United States of America indicate involvement of both endogenous
and nonendogenous emerging strains (Baker-Austin et al., 2018). There is some
evidence for global spread of pathogenic strains through oceanic currents, ballast
water and movement of shellfish commodities (Martinez-Urtaza et al., 2016;
Baker-Austin et al., 2016).

The recent emergence of genomic epidemiology approaches applying next-

generation sequencing, coupled to open access bioinformatic tools, can now be
used to reconstruct the emergence, dispersal, and evolution of these and other
important foodborne pathogens. These approaches are typically faster, less
expensive and provide greater resolution than traditional subtyping methods such
as PCR, multi-locus sequence typing, pulsed field gel electrophoresis and serotyping,
which were the approaches of choice available during the most recent (2011) V.
parahaemolyticus and V. vulnificus risk assessments. The use of sequencing in the

22 ADVANCES IN SCIENCE AND RISK ASSESSMENT TOOLS FOR VIBRIO PARAHAEMOLYTICUS AND V. VULNIFICUS
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 tnaA

Reference
sequence
gyrB
recA dnaE

pntA

Strain 1
dtdS
pyrC
1. Isolation of strain 2. PCR genes in MLST scheme 3. Comparative sequence analysis – 4. Phylogenetic analysis
SNP identfication

Identification
of SNPs in core
genome

1. Isolation of strain 2. NGS of strain – comparative analysis 3. Phylogenetic analysis

against ref. genome

FIGURE 1. Recent evolution of V. parahaemolyticus strain typing approaches.

Traditional methods, such as serotyping, pulsed field gel electrophoresis (PFGE), and
multi-locus sequence typing (MLST, top) are typically time consuming, expensive
and specialist methodologies. MLST using PCR analysis of a small number of core
housekeeping genes, coupled to sequencing and identification of mutations (single
nucleotide polymorphism, SNPs) were generally capable of distinguishing most strains
of clinical interest, such as those implicated in foodborne outbreaks. The advent of
whole genome sequence (WGS) based methods has facilitated the identification of
several hundred mutations – using the same basic approach at a fraction of the cost
and time of traditional methods. These WGS methods have dramatically increased the
granularity of analysis, revolutionising the ability to conduct rapid and precise outbreak
investigations.

routine analysis of vibrio strains along with the availability of genomic data from
vibrio strains collected all around the world have introduced a fundamental change
in the way that strains and associated epidemiological metadata are analysed.
The possibility of building large datasets of genomes with global coverage has
become a routine procedure for the analysis of strains within an epidemiological
context, detecting similar strains isolated anywhere in the world and using these
strains to infer the colonization history and finally draw the most probable routes

CHAPTER 3 - NEW DEVELOPMENTS IN VIBRIOS 23

 of dissemination from the original sites of emergence. This new framework for
the study of pathogenic populations allows for a precise reconstruction of the
migratory routes for the major epidemic clones and this information can be applied
to identify potential drivers of dispersal and introduction. The application of new
tools has successfully contributed to help reconstruct the colonization history
for the major epidemic clones of V. parahaemolyticus (Baker-Austin et al., 2018).
There are notable limitations to these methods, which can be expensive, technically
complex and require carefully-trained staff; however, there are now avenues for
support in their use in low and middle-income countries.

Development of satellite risk assessment approaches: In the last decade, a range

of risk assessment approaches have been developed utilising satellite measurement.
Satellite-based remote sensing for studying marine systems has become a useful
tool in predicting human health risks associated with marine bacterial pathogens
such as vibrios (Grimes et al., 2014). These globally applicable methods have been
used largely to analyse non-cholera Vibrio spp., particularly for bathing water
infections and outbreaks associated with seafood consumption (Semenza et al.,
2017). This data could be useful to assess (retrospectively) outbreaks and to provide
temperature and key environmental data into risk assessment approaches. Some
of the risk models developed also have forecasting capabilities, which could be
used for risk management purposes. These approaches have been used successfully
to analyse environmental conditions such as temperature and salinity, which are
well established variables that can modulate vibriosis risk. Numerous studies,
such as those focussing on bathing water (Baker-Austin et al., 2016) and shellfish-
associated infections (Martinez-Urtaza et al., 2010) have demonstrated the
usefulness of these methods to ascribe increased risk prior to and during outbreak
episodes. An overview of currently available risk models using these approaches is
outlined in Table 3.

Climate change: Because the growth of pathogenic vibrios in the natural

environment is largely dictated by temperature, this group of pathogens represent
an important and tangible barometer of climate change in marine systems
(Baker-Austin et al., 2017). The growth of V. parahaemolyticus and V. vulnificus is
proportional to environmental temperatures, and these pathogens grow extremely
well above 15 ⁰C. As such, and in a rapidly warming marine environment, there are
likely to be a greater number of vibrio-associated human infections. Since the 2004
and 2011 risk assessments a wide variety of studies and a general improvement
in analysing the link between vibrios and climate warming have been published
that provide a more coherent understanding regarding the role of climate change
and risk. A key recent finding is that there has been a significant expansion in
coastal regions where these bacteria can proliferate. An overview of how climate
change will likely modulate risk was considered in detail during the workshop and
is outlined in Section 3.6.

24 ADVANCES IN SCIENCE AND RISK ASSESSMENT TOOLS FOR VIBRIO PARAHAEMOLYTICUS AND V. VULNIFICUS
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 3.3 NEWLY AVAILABLE RISK ASSESSMENT MODELS
Table 3 summarizes the information on all available models. The V. parahaemolyticus
risk calculator and V. vulnificus risk calculator has been discussed in previous sections.
Information about other available models is provided in this section. The models provide
outputs ranging from:
1. Abundance of V. parahaemolyticus at harvest and doubling time of V. parahaemolyticus
in oysters at certain specified sites in the United States of America (e.g. NOAA model).
2. Occurrence of V. vulnificus in water in the Chesapeake Bay (e.g. NOAA model).
3. Environmental suitability for non-cholera Vibrio spp. (e.g. Vibrio Suitability Tool).

TABLE 3. Currently available risk assessment models

Model Parameters used in Output Availability Applicability and

the model limitations
V. parahaemolyticus Temperature Cases per Excel file Regional in the United
(Vp) risk and Vp levels at 100 000 serving, publicly States. Assumptions
harvest, growth risk reductions available (FAO tested in Connecticut.
rate prediction that can be WHO FS tools) Relative risk in regions
based on time and achieved by may vary. Different tdh
temperature, FAO/ implementing positive strains may have
WHO RA dose temperature different attack rates.
response model control The model assumes that
they are all same.
Emergence of new
strains with different
attack rates in a location
is not factored in.
Growth rates may vary in
different bivalve species
and this has not been
factored in this model.
V. vulnificus (Vv) Temperature Cases per Excel file Dose response based
risk calculator and Vv levels at 100 000 serving, publicly only on the United States
harvest, growth risk reductions available FAO epidemiological data,
rate prediction that can be WHO FS tools hence only applicable in
based on time and achieved by the United States
temperature, FAO/ implementing
WHO RA dose temperature
response model control
Vibrio Suitability Salinity, sea surface Predicts risk of ECDC E3 Management tool to
Tool temperature non-cholera vibrio Geoportal1 and facilitate harvest and use
infections based NOAA Atlantic of recreational water low
on exposure using OceanWatch risk period. Validated for
model described ERDDAP Baltic Sea, possibly, has
by Baker Austin et Server2 global application. The
al., 2013. output does not provide
information on the type
of Vibrio spp. that may
be involved. Considers
only water exposure,
though it is assumed
that wound exposure and
food exposure may result
in similar infection rates.
(cont.)

CHAPTER 3 - NEW DEVELOPMENTS IN VIBRIOS 25

 Model Parameters used in Output Availability Applicability and
the model limitations
NOAA National Temperature, Expected V. Web based3 The model predicts
Center for Coastal salinity and parahaemolyticus V. parahaemolyticus
Ocean Science growth rate of V. levels at harvest levels in certain specific
(NCCOS) vibrio parahaemolyticus and after geographical regions
predictive model and occurrence postharvest in the United States
of V. vulnificus in handling in (e.g. Chesapeake
Chesapeake Bay. Chesapeake Bay, Delaware, Pacific
Bay, doubling Northwest etc) and
time for V. occurrence of V.
parahaemolyticis vulnificus in Chesapeake
in specified Bay. Provides shellfish
regions, V. related guidance in
vulnificus specified regions.
occurrence in Needs to be adapted to
Chesapeake Bay. other regions. Can do
region specific scenario
analysis. The limitation
is that relation between
risk and abundance may
vary in different regions
based on strains, attack
rates etc.
V. vulnificus forecast Salinity and Presence and Published Relation between risk
model temperature density of V. (Oliver and and abundance may
vulnificus Kaper, 2001, vary in different regions.
2007) Does not factor in
abundance of clinical and
environmental strains.
1
https://e3geoportal.ecdc.europa.eu/SitePages/Vibrio%20Map%20Viewer.aspx
2
https://cwcgom.aoml.noaa.gov/erddap/search/index.html?searchFor=vibrio
3
https://coastalscience.noaa.gov/products/vibriopredictive-models/

NOAA model: The model available on the website of National Center for Coastal
Ocean Science (NCCOS) predicts abundance of V. parahaemolyticus in certain
specified locations in the United States of America based on temperature, salinity
and USFDA model for growth of this organism. The output is presented differently
for different locations. For example, V. parahaemolyticus levels in oysters at harvest
from Gulf of Mexico and Tampa Bay. For Delaware Bay, Pacific Northwest, and
Northeast V. parahaemolyticus doubling time is predicted hourly for first 36
hours, every 3 hours till 72 hrs and every 6 hrs for 7 days. This could be used by
shellfish farmers to plan harvesting and cooling strategies. For Chesapeake Bay, V.
parahaemolyticus levels at harvest and postharvest (for 10 hrs based on regionally
adjusted 1 KM air temperature) is predicted.

For V. vulnificus, only occurrence in water in Chesapeake Bay has been presented
and no shellfish guidance is indicated. This information may be used by those
likely to be exposed to water in Chesapeake Bay. Vibrio vulnificus levels in water
may also be used by shellfish harvesters since levels in water and oysters would be

26 ADVANCES IN SCIENCE AND RISK ASSESSMENT TOOLS FOR VIBRIO PARAHAEMOLYTICUS AND V. VULNIFICUS
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related. One of the limitations of the model is that risk of infection is not predicted.
These models cannot be directly used by other countries, but they may develop
their own model based on local data on temperature, salinity and growth of V.
parahaemolyticus in their own shellfish species.

Vibrio Suitability Tool

Link: https://e3geoportal.ecdc.europa.eu/SitePages/Vibrio%20Map%20Viewer.
aspx (Baltic Sea) and https://cwcgom.aoml.noaa.gov/erddap/search/index.
html?searchFor=vibrio (Global Fields)

The Vibrio Suitability Tool is intended to inform public health professionals about
environmental suitability for non-cholera vibrio to be used as a tool for adoption of
prevention measures and public awareness. The model is based on the methodology
described in Baker-Austin et al. (Baker-Austin et al., 2013) and uses salinity and sea
surface temperature (SST) to estimate the environmental suitability for vibrios in
coastal waters in relation to disease risk. The model is based on data of non-cholera
vibrio infections (vibriosis) from previous studies relating number of infections and
environmental variables. The algorithm is a general model with thresholds for salinity
and SST, 28 Practical Salinity Units (PSU) and 18oC respectively, that allows the
inclusion of areas suitable for the occurrence of all human pathogenic Vibrio spp. The
output of the model delineates coastal areas with environmental conditions suitable
for the occurrence of human pathogenic Vibrio spp. that can drive the emergence
of infections. The areas of environmental suitability are colour coded, ranging from
zero to a maximum which is determined by the highest sea surface temperature
value. The model has been developed for the Baltic Sea and is under evaluation
with epidemiological data from other parts of the world. However, preliminary
analyses of the outputs based on a review of Vibrio spp. and epidemiological data
and literature review have shown similar results for other regions (Semenza et al.,
2017). Environmental parameters used in the model are exclusively salinity and sea
surface temperature and no other critical abiotic and biotic factors governing the
occurrence of Vibrio spp. in the marine environment is considered. As the last layer of
complexity, there is a large amount of demographic, behavioural and epidemiological
information shaping the risk of exposure. These factors can of course leverage the
overall exposure of the population.

3.4 RECOMMENDATIONS FOR BEST PRACTICE

The efficacy of various postharvest treatment technologies, alone or in combination,
have been previously described (see MRA20). Below is a summary of some new
approaches and data regarding postharvest treatment technologies that have emerged
in the last decade and that were discussed at the most recent expert workshop.

CHAPTER 3 - NEW DEVELOPMENTS IN VIBRIOS 27

 Harvesting curfews: Harvesting curfews aim to ensure oysters are harvested under
conditions which coincides with the periods of lowest contamination and growth
of V. parahaemolyticus in oysters. Examples include early morning harvests (before
the maximum heat of the day) and within specific tidal periods. The latter example
is based on the scientific observation that V. parahaemolyticus levels increase when
oysters are exposed on the sunny mudflats by a receding tide, then decrease when
the tidal waters submerge the shellfish and filter-feeding recommences (Jones et
al., 2016). However, it must be recognized that the climatic and tidal conditions
in other countries are different around the world. Much of the United States of
America tidal V. parahaemolyticus research relates to situations where oysters are
grown directly on the substrate; the substrates materials vary; diurnal temperatures
are often higher than other countries daily maximum; and there are spatial and
temporal differences in the tidal cycles. Local research should be undertaken to
ascertain the harvesting and environmental conditions conducive to mitigating the
vibriosis food safety risk.

Harvesting cessation: Harvesting cessation can be used both as a reactive response

to reported illnesses and a proactive measure to prevent illness. Proactive measures
can be based on temperature or salinity levels in the environment, measured or
based on remote sensing models (reference to NOAA and EU models), or can be
based on restrictions for harvesting during months traditionally associated with
peak illness. The NSSP has also developed reactive controls including time to
refrigeration and closures based on reported cases over selected periods.

Resubmersion: Re-submerging is used in Canada and the United States of

America as a V. parahaemolyticus mitigation process and the USFDA has validated
this process. The resubmerging practice is usually defined as harvesting, culling
and placing oysters in larger cages for re-submerging in a deeper water body or for
re-submersion by the tide. Consideration of these practices alongside other control
measures (such as for biotoxin and classification status) should be also taken into
account. USFDA studies found that oysters grown on both the east and west coast,
containing elevated V. parahaemolyticus caused by pre-submerging conditions,
returned to background levels after one tidal cycle following re-immersion
(ISSC, 2017). This practice requires availability of water space deep enough for
re-submerging the harvested oysters. Industry would need to design systems
whereby the intertidal product is harvested, shifted and anchored in deeper water
or submerged in the incoming tidal waters on the lease, in a manner from which
they can be directly harvested. For example, a single commercial shellfish operator
uses a barge fitted with a crane could harvest the cages of re-submerged oysters.

Deep water suspension: Deep water suspension, or the movement of oyster nets
into cooler waters when water temperatures exceed 15°C should be considered as

28 ADVANCES IN SCIENCE AND RISK ASSESSMENT TOOLS FOR VIBRIO PARAHAEMOLYTICUS AND V. VULNIFICUS
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method of vibriosis risk reduction (McLaughlin et al., 2005). To date, little data
on this intervention exists, however it may represent a cost-effective management
option. As with other control measures, consideration of how these practices may
impact other factors (e.g. for biotoxin and classification status) should also be
taken into account.

Relaying: There is limited information on the success of relaying as a treatment

step to remove V. parahaemolyticus. However, recent USFDA studies confirm
that relaying to higher salinity and/or cooler waters shows promise for reducing
V. parahaemolyticus levels, with around seven days sufficient time to reduce V.
parahaemolyticus levels in oysters (ISSC, 2017). It should be recognized that there
is the potential for V. parahaemolyticus in the relayed lots to contaminate other
shellstock growing in the new water space.

Depuration: Depuration or purification is one of the major treatment processes

in controlling the public health risks associated with microbially-contaminated
shellfish. Depuration is the process by which shellfish naturally relinquish any
microbiological component bioaccumulated in the environment. According to the
Codex Code of Recommended Practice for Fish and Fishery Products, depuration
means the reduction of microorganisms to a level acceptable for direct consumption
by the process of holding live bivalve molluscs for a period of time under approved,
controlled conditions in natural or artificial sea water suitable for the process,
which may be treated or untreated. Historically shellfish depuration has not been
considered an appropriate process for mitigating or eliminating the vibriosis food
safety risk. Unfortunately, depuration has been shown to be ineffective in reducing
a range of pathogenic Vibrio spp., such as V. cholerae and V. parahaemolyticus
from bivalve matrices (Croci et al., 2002). However, a recent study (Shen et al.,
2019) demonstrated that optimal V. parahaemolyticus depuration occurred at a
temperature of 12.5°C and stocking density of two oysters/L of artificial seawater.
The mean depuration time to achieve a target reduction of 3.52 log10 (NSSP
reduction target) was 3.17 days, and five days to achieve a level of <30MPN/g.

Temperature Controls: Experiments with oysters artificially contaminated with

V. parahaemolyticus found that treatment of 50°C for 10 minutes was needed to
reduce the concentration by >5 log10 MPN/g (Ye et al., 2012). Treatment at 50°C
for only 5 minutes or treatment at 45°C for 20 minutes only achieved reductions
of 3.9 and 2.6 log10 MPN/g, respectively. An earlier study (Andrews et al., 2000)
had measured a 5 log reduction of V. parahaemolyticus in oysters after 5 minutes at
50°C. The difference may be due to different strains or methods (e.g. Andrews et al.
used a kettle at 55ºC to initially heat the oysters to 50°C, while Ye et al. (2012) used
a water bath at 50°C (Ye et al., 2012). Ice slurries were effective for rapidly cooling
oysters (24°C to 10°C within 12 minutes), but repeated dipping of oysters caused

CHAPTER 3 - NEW DEVELOPMENTS IN VIBRIOS 29

 the ice to become contaminated with faecal coliforms, Clostridium perfringens,
V. vulnificus and total V. parahaemolyticus (Lydon et al., 2015). However, the
concentrations of Vibrio spp. were unchanged in the flesh of the oysters after 15
minutes submersion in the contaminated ice slurry. Another study found that
onboard and dockside icing did not predictably reduce the concentration of V.
parahaemolyticus in oysters, and icing significantly and negatively affected oyster
survival (Melody et al., 2008). The impact of climate change on shellfish safety
may mean it is necessary for countries and regions undergoing increased risk to
assess and potentially implement enhanced control measures, such as cold-chain
interventions recently adopted in the United States of America to reduce illnesses
associated with V. parahaemolyticus.

Cryogenic individual quick freezing (IQF) with extended storage: IQF is an

USFDA-approved control for Vibrio spp. IQF involves the use of cryogenic or blast
freezing technology to rapidly lower the product temperature below freezing. This
process results in a reduction in the number of temperature sensitive pathogens.
Some pathogens are more sensitive to freezing than are others. For example, V.
parahaemolyticus and V. vulnificus are especially sensitive to colder temperatures.
To reduce V. parahaemolyticus and/ or V. vulnificus to nondetectable levels, the IQF
process is followed by a period of frozen storage, which may vary depending on
organism (USFDA, 2011).

High hydrostatic pressure: High pressure processing (HPP) is the application

of hydrostatic compression in the range of 14 500 to 145 000 pound per square
inch (100 to 1 000 megapascal (MPa)) which inactivates V. parahaemolyticus and
V. vulnificus by damaging the cell membrane, cell wall and degrading cellular
proteins (Berlin et al., 1999; Wang et al., 2013). These pressures are capable
of inactivating pressure-sensitive pathogens, especially vegetative forms. The
effectiveness of the process is dependent upon the amount of pressure applied, the
process temperature, and the duration of the process. However other organoleptic
changes, such as texture, viscous liquor and a “plumper” appearance have been
reported in shellfish. Additionally, the pressure facilitates oyster adductor muscle
changes; hence, HPP may result in a shucked oyster (USFDA, 2011). HPP is being
increasingly being used for minimising the risk of pathogens in seafood. Vibrio
spp. have been reported to be sensitive to high hydrostatic pressure. This approach
was previously outlined in MRA20 (FAO/WHO, 2020). In a recent meta-review of
post-harvest practices and processes (PHP) approaches to reduce risks associated
with Vibrio parahaemolyticus, high hydrostatic pressure consistently emerged as
the most effective PHP approach in reducing abundance of V. parahaemolyticus
(Spaur et al., 2020).

30 ADVANCES IN SCIENCE AND RISK ASSESSMENT TOOLS FOR VIBRIO PARAHAEMOLYTICUS AND V. VULNIFICUS
ASSOCIATED WITH SEAFOOD
Low dose gamma radiation: Vibrio spp. are among the most radiation-sensitive
bacteria. Irradiation involves exposing oysters to ionising energy, either gamma
rays, machine-generated electrons or X-rays. An ionising irradiation dose of 1.0
kGy reduced V. vulnificus artificially bioaccumulated in whole shell oysters from
107 MPN/g to nondetectable levels and had the same effect on naturally present
V. vulnificus (103 MPN/g) (Andrews et al., 2003; Ama, Hamdy and Toledo, 1994).

Mild heat treatment: New data on the effect of mild heat treatment on V.
parahaemolyticus and V. vulnificus concentration in shellfish have been published
since 2003 FAO/WHO risk assessment and some results are promising, but still it
would be preferable to perform validation at specific conditions/practices before
implementation of the PHP and its inclusion in the model.

Freezing: V. parahaemolyticus present in shucked and shell stock oysters does not
tolerate freezing well. Detrimental effects of freezing were greater at -18°C than at
-30°C, consistent with greater bacterial damage at the higher temperatures due to
the formation of larger intracellular ice crystal formation (Shen et al. 2009).

3.5 MICROBIOLOGICAL TESTING METHODS

A variety of approaches have been developed for the isolation, detection,
enumeration and characterisation of vibrios from environmental matrices such as
water (American association for wastewater 1997) and shellfish (Hartnell et al.,
2018). Since the 2005 and 2011 risk assessment exercises were completed a range
of advances in testing methods have been published. In particular, an international
standard method for the detection of V. parahaemolyticus and V. vulnificus in
seafood products has been established and published (ISO 21872-1), which
includes options for conventional and real-time PCR detection of these pathogens
(Hartnell et al., 2018; ISO, 2017). There has also been a significant amount of
work carried out in the arena of method standardization in the last decade, for
instance with underlying performance data to underpin choice of molecular
targets, such as for PCR and qPCR for pathogenic vibrios. Previous guidance was
developed in response to a request to FAO/WHO from the 42nd Session of the
Codex Committee on Food Hygiene to provide recommendations on a range
of test methods for quantifying V. parahaemolyticus (total and pathogenic) and
V. vulnificus in seawater and bivalves, and to facilitate performance evaluation
of the methods (FAO/WHO, 2016). A number of key recommendations were
subsequently outlined through this work and during the recent expert meeting:

CHAPTER 3 - NEW DEVELOPMENTS IN VIBRIOS 31

 • Establish an internationally recognized system for developing criteria and
protocols for evaluation of method performance parameters of molecular
detection methods for pathogenic Vibrio spp. in seafood (explore similar
approach for other pathogen/commodity pairs).
• Examine appropriateness of coupling methods performance and analyst
proficiency evaluation.
• Develop a collection of appropriate reference material including bacterial
strains and sample matrix.
• Explore the development of a PCR network patterned after Global FoodNet.
• Refer to MRA22 (2016): Selection and application of methods for the detection
and enumeration of human-pathogenic halophilic Vibrio spp. in seafood.
• Determine the importance and applicability of traditional virulence factors
(e.g. tdh/trh in V. parahaemolyticus) as targets for both the detection and
enumeration across clinical, environmental and food matrices.

Several key datagaps were also identified. These include approaches to infer further
characterization of strains (e.g. serotyping, MLST, genotyping, ATBR, WGS);
virulence testing; gene expression levels; strain phylogeny and phylogeography. A
selection of the most important methods pertaining to vibrios – in particular for
the isolations, enumeration and characterisation of these bacteria from a variety of
clinical, environmental and foodbased matrices was compiled by the expert group
(Table 4):

TABLE 4. Commonly used microbiological and molecular methods applied in the isolation
and characterisation of Vibrio parhaemolyticus and Vibrio vulnificus.

Diagnostic Relevant applications Limitations Relevant

Method reference

Vibrio enrichment Thiosulfatecitratebile salts (TCBS) Certain Vibrio spp. such as V. Hartnell et al.
on solid media agar is a standard medium used vulnificus can struggle to grow (2018).
commonly for the selective on TCBS media.
isolation and further subculturing
and purification of vibrios. Strains Food, especially seafood,
which are able to use sucrose will may contain large numbers
form yellow colonies on TCBS such of bacteria, including
as V. cholerae and V. alginolyticus, nonpathogenic Vibrio spp.
while the other pathogenic species which may grow through the
such as V. parahaemolyticus, selective culture process.
and V. vulnificus produce green Subculture of small numbers
colonies. Other media types such as of colonies may result in
blood agar and chromagar are used potentially pathogenic species
to isolate V. parahaemolyticus being missed.
and CPC (cellobiose-polymyxin
B-colistin agar) is frequently used
for isolation of V. vulnificus.

(cont.)

32 ADVANCES IN SCIENCE AND RISK ASSESSMENT TOOLS FOR VIBRIO PARAHAEMOLYTICUS AND V. VULNIFICUS
ASSOCIATED WITH SEAFOOD
Diagnostic Relevant applications Limitations Relevant
Method reference

Toxin testing Commercial kits are available Will not identify non-tdh Vibrio Wagatsuma S.
to test for the thermostable parahaemolyticus strains, (1968).
direct hemolysin (TDH) of V. so limited applicability for
parahaemolyticus. Screening routine testing of clinical and
colonies of V. parahaemolyticus on environmental strains.
Wagatsuma agar to detect strains
that are TDH-positive are termed
“Kanagawa positive” via the
identification of hemolysis around
tdh+ colonies.

Serotyping Usually, the in-depth Agglutination using Sakazaki et al.

identification and subtyping commercially available testing (1968).
of V. parahaemolyticus is approaches can be subjective,
performed by serological tests. and require experience and
V. parahaemolyticus is classified training in interpretation. Kits
by serotyping and the serotypes are often expensive, with a
of V. parahaemolyticus are limited shelf life. No scheme
determined by the combination of for V. vulnificus.
somatic (O) antigens and capsular
(K) antigens. Testing typically
involves serological analysis
of all strains determined by
agglutination using commercially
available V. parahaemolyticus
antisera. Clinically relevant V.
parahaemolyticus infections
are usually associated with
pathogenic strains of several
important serotypes (clinical
e.g. O3:K6, O4:K12 etc); whereas
nonpathogenic strains comprise a
broader array of serotypes.

Immunomagnetic Immunomagnetic separation is Some issues with nonspecific Tomoyasu et al.

Separation after generally used to improve the reactivity of antigens. For (1992).
Enrichment yield of a particular strain after broad testing applications
enrichment. The process is based requires the generation of
on the use of a specific antiserum many antigens which can be
for the strain being sought (for expensive to produce.
example V. parahaemolyticus
O3:K6) which is added to uncoated
immunomagnetic beads.
Immunomagnetic separation (IMS)
generally consists of two steps: the
incubation of immunomagnetic
beads (IMBs) with an enriched
bacterial culture followed by a
washing step to remove nontarget
bacteria in a given sample.

(cont.)

CHAPTER 3 - NEW DEVELOPMENTS IN VIBRIOS 33

 Diagnostic Relevant applications Limitations Relevant
Method reference

Biochemical Most biochemical tests use a Clinical isolates of Martinez-Urtaza

identification variety of specific miniaturized certain species such as V. et al. (2006).
growth substrates to identify parahaemolyticus can show
bacterial strains. Typically, significant differences in
dehydrated media with specific up to five biochemical tests
chemically-defined compositions used. Certain standardized
are used for each test. Growth biochemical tests can struggle
of bacteria is used to detect to differentiate vibrio species.
enzymatic activity, mostly related Some testing approaches may
to fermentation of carbohydrate be subjective and open to
or catabolism of proteins or amino operator bias.
acids. Generally quick and simple
to use, and inexpensive.

PCR testing Widely used method for the PCR requires that sequence Bej et al. (1999).
detection of isolated bacteria information is available for andHill et al.
utilizing specific primer sets that at least a part of the DNA (1991).
are amplified using repeated PCR that is to be amplified, and
cycling. Primer sets for all major as such requires sequencing
Vibrio species and pathotypes now data which may limit uses
well established. More in-depth for new or emerging strains.
molecular characterization, such Because of the sensitivity
as PCR of the virulence genes for of PCR, contamination and
vibrio pathogens, e.g. tdh/trh false-positives can be an issue.
analysis of V. parahaemolyticus, Does not provide quantitative
as well as a variety of virulence- information.
associated PCR tests for V.
vulnificus can be carried out
for further analyses. Extremely
sensitive, rapid, easy to use and
inexpensive. Well standardized and
protocols freely available.

Realtime PCR Real-time PCR utilizes probes that Although real-time PCR is Nordstrom.
are labeled with two fluorescent quantitative, multiplexing (2007). and
dyes that emit at different reactions can be difficult Campbell &
wavelengths during hydrolysis to optimize. As with Wright. (2003).
catalyzed during the PCR cycle. The conventional PCR, because
emission is subsequently detected real-time PCR is extremely
during PCR cycling using optical sensitive, contamination and
sensors. The probe sequence is falsepositives can be an issue.
intended to hybridize specifically Requires the use of a real-time
to the DNA target region of PCR machine which can be
interest, which is located between costly. Probes for certain
the two PCR primers. A rapid, multiplex reactions can also be
specific and quantitative method, expensive.
real-time PCR is also inexpensive
and allows highthroughput testing
of strains derived from clinical,
environmental and food sources.
Primer and probe sets for all major
Vibrio species and pathotypes now
well established.

(cont.)

34 ADVANCES IN SCIENCE AND RISK ASSESSMENT TOOLS FOR VIBRIO PARAHAEMOLYTICUS AND V. VULNIFICUS
ASSOCIATED WITH SEAFOOD
Diagnostic Relevant applications Limitations Relevant
Method reference

Multi-locus MLST is a technique used for Limited scheme for V. González-

sequence typing the subtyping of multiple loci, vulnificus. Approach being Escalona et al.
(MLST) typically derived via PCR or from refined and superseded by (2008). and
whole genome sequencing-based WGS-enabled approaches.
analyses. Typically, the procedure Databases required regular Bisharat et al.
subtypes isolates using the DNA updating and maintenance. Hybrid Vibrio
sequences of a small number vulnificus.
(5-8) of highly conserved genes, (2005). 040440
such as housekeeping genes.
Schemes have been developed
for V. parahaemolyticus. Methods
are rapid and robust and are
amendable for larger and more
comprehensive analyses using
WGS-derived datasets.

Whole genome Whole genome sequencing Start up costs of purchasing Chen et al.
sequencing-based is the process of determining a next generation sequencer (2003).
methods the complete DNA sequence can be expensive. Requires Makino et al.
of a specific genome. Typically, dedicated staff trained in the (2003).
bacterial isolates are DNA use of methods. Required Gonzalez-
extracted, and the DNA is sheared assistance with interpretation Escalona et al.
into smaller fragments where for in-depth bioinformatic (2017).
it can be sequenced using a analysis, as well as computing
variety of chemical methods. support for analyses.
Following sequencing, the DNA is
assembled, and strains of interest
can be analyzed using a variety
of bioinformatic approaches to
infer phylogenetic relationships,
evolutionary history as well as
the presence of virulence and
antibiotic resistance genes. Whole
genome sequencing methods
offer unparalleled resolution and
information regarding the genome
of analyzed isolates. The process
is rapid and now extremely cost
effective.

Colony This method is based upon direct Methods require significant Suffredini et al.
hybridization plating of sample material on a training. Colony hybridization (2014).
nutrient medium. The resulting is a slow and technically
colonies are transferred onto demanding method.
nylon membranes and hybridized
with DNA digoxigenin-labeled
oligoprobes to detect genes
associated to pathogenicity and/or
species-specific. It allows bacterial
species enumeration.
(cont.)

CHAPTER 3 - NEW DEVELOPMENTS IN VIBRIOS 35

 Diagnostic Relevant applications Limitations Relevant
Method reference

LAMP LAMP assays (loop-mediated Requires specialist training Yamazaki et al.

isothermal amplification) are for use. Primer design can (2008).
a singlestep procedure that be technically challenging.
requires four to six primers that Because LAMP is a highly Han et al. (2011).
bind laterally to distinct sites sensitivity method and
using stranddisplacement DNA produces very large amounts
polymerases. Typically, LAMP takes of amplified DNA it is
place under isothermal conditions. susceptible to false positive
Because of the numbers of primers reactions because of cross-
used, they permit extremely contamination events in the
specific amplification. Amplified laboratory. LAMP can be less
DNA products can be detected by sensitive than PCR to inhibitors
turbidimeter, gel electrophoresis, in case of complex samples.
lateral flow dipstick, as well as
the naked eye. Assay for LAMP
have been developed for both V.
vulnificus and V. parahaemolyticus.
LAMP is a sensitive, rapid, and
cost-effective method that can be
deployed in the field using portable
equipment.

MALDI-TOF MALDI-TOF (matrix-assisted laser Start-up costs of purchasing a Dieckmann,

desorption ⁄ ionization time MALDI-Tof can be expensive. Strauch, & Alter.
of-flight) mass spectrometry Requires specialist training for (2010).
for the rapid identification of Vibrio use. Requires databases for
spp. Sample preparation is simple species identification.
and bacterial species identification
is rapid and automated.

3.6 CLIMATE CHANGE

There is increasing concern regarding the role of climate change in mediating the
spread of waterborne and foodborne infectious diseases, such as those caused by
vibrios. A number of physical manifestations of climate change are likely to play
a significant role in increasing risks associated with pathogenic vibrios (Baker-
Austin et al., 2012), and in particular non-cholera vibrios such as V. vulnificus, V.
parahaemolyticus, and non-O1 V. cholerae (Baker-Austin et al., 2017). The recent
change in sea temperature is considered as the most pervasive and severe cause
of impact in coastal ecosystems worldwide (Halpern et al., 2008), particularly in
light of recent observations demonstrating significant warming in over 70% of the
world’s coastlines (Lima and Wethey, 2012). Climate change plays a significant
role in determining the prevalence as well as growth dynamics of many bacterial
pathogens, and many diseases are expected to increase in range and severity with
projected climate change (Koelle et al., 2009). Non-cholera vibrios such as V.

36 ADVANCES IN SCIENCE AND RISK ASSESSMENT TOOLS FOR VIBRIO PARAHAEMOLYTICUS AND V. VULNIFICUS
ASSOCIATED WITH SEAFOOD
vulnificus and V. parahaemolyticus grow in warm, low salinity waters, and their
growth is proportional to ambient environmental temperatures. Vibrios have some
of the fastest replication times of all known and studied bacteria, making them
highly responsive to favourable environmental stimuli (Baker-Austin et al., 2016).
There is now much greater understanding regarding the role of climate change,
and in particular anomalous warming events in driving vibriosis outbreaks and
associated human health risks. Worldwide, oceanic warming has significantly
increased the areas suitable for pathogenic vibrios to proliferate and cause human
health illness (Watts et al., 2019). Infections from these pathogens are now being
reported in areas with little or no previous incidence, with clear implications for
future risk. These include shellfish-associated outbreaks of V. parahaemolyticus in
Alaska (McLaughlin et al., 2005), and Chile (Martinez-Urtaza et al., 2010), and
non-cholera vibrio infections reported in Northern Europe during heatwave events
(Baker-Austin et al., 2012; Baker-Austin et al., 2016). In 2018, the first outbreak
of non-O1/nonO139 Vibrio cholerae infections linked to consumption of herring
eggs were reported in British Columbia, Canada. Other extreme climatic events
have also been linked to an increase in reported vibrio infections. Data from the
US CDC revealed a sharp increase in vibrio wound infections following Hurricane
Katrina making landfall on the Gulf coast of the United States of America in August
2005 (Anon, 2005).

There is a growing body of evidence to indicate that climatic warming may allow
certain vibrio strains to emerge in new areas. For instance, a highly pathogenic
variant of V. parahaemolyticus belonging to the clonal complex ST36 and termed
the Pacific Northwest strain emerged on the Northeast coast of the United States of
America during the unusually warm spring of 2012 (Newton et al., 2014). As such,
these rapid and localised warming events may represent an important epidemic
ignition that allows particular strains to emerge from environmental sources,
leading to outbreaks. Oceanic warming has also been linked to the successful
dissemination of outbreak strains of V. parahaemolyticus in Galicia, NW Spain
over the last 2 decades (Martinez-Urtaza et al., 2018). Few long-term studies in this
area exist, somewhat limiting our understanding, thus the emergence of clinical
cases is often interpreted as a sporadic event due to exceptional conditions, rather
than a response to long-term environmental change (Baker-Austin et al., 2012).
However, recent analysis of the long-term plankton datasets in the North Sea have
demonstrated a clear transition during the 1980’s, with the bacterial community
becoming dominated by vibrios (Vezzulli et al., 2012). These data are striking as
the transition appears to correspond closely with the temporal warming trends
in the area, and further corroborates the emergence of marine-borne vibriosis,
in particular at high latitudes. Future ocean acidification and warming linked to
climate change is another physiochemical stressor to oceanic organisms. Ocean
acidification has been implicated in increased V. parahaemolyticus growth in

CHAPTER 3 - NEW DEVELOPMENTS IN VIBRIOS 37

 certain shellfish species (Hernroth et al., 2015), potentially increasing public health
risks.

TABLE 5. Climate suitability for vibrio outbreaks. Changes observed in the percentage
of suitable areas from the 1980s to the present data.

Country Warming trend Ranking Region

Lebanon 1.50351 1 Mediterranean

Israel 1.4186 2 Mediterranean

Syrian Arab 1.38938 3 Mediterranean

Republic

Netherlands 1.03714 7 North Sea

Belgium 0.988717 9 North Sea

Portugal 0.971618 10 Atlantic

Lithuania 0.907568 11 Baltic Sea

Germany 0.852456 13 Baltic Sea

Poland 0.832356 15 Baltic Sea

France 0.693102 23 Atlantic

The emergence of non-cholera vibrio diseases, particularly in geographical regions

with a lack of longterm epidemiological datasets provides startling practical
challenges to the vibrio research community. Improvements in the epidemiology, and
in particular the surveillance and reporting of these pathogens is critical, principally
in geographical regions that lack a centralised and coordinated monitoring and
surveillance system for vibrios. Countries with enhanced and well embedded
surveillance systems, such as the COVIS system in the United States of America
(Newton et al., 2012), may represent a useful blueprint for other countries to use.

3.7 DEMOGRAPHIC DRIVERS

Population changes in industrialized countries may modulate certain risks related
to the likelihood of vibrio infections from seafood. According to the United
Nations, a significant ageing of the world population is expected in the next several
decades and is projected for most regions of globe. An aging demographic may
increase the likelihood and severity of infections for a larger percentage of the

38 ADVANCES IN SCIENCE AND RISK ASSESSMENT TOOLS FOR VIBRIO PARAHAEMOLYTICUS AND V. VULNIFICUS
ASSOCIATED WITH SEAFOOD
population globally. Globally, the fraction of the population aged 65 years or over
increased from 6 per cent in 1990 to 9 per cent in 2019. That proportion is projected
to rise further to 16 per cent by 2050, so that one in six people in the world will
be aged 65 years or over (United Nations, 2019). All continental regions will see
an increase in the size of their older population between 2019 and 2050 (United
Nations, 2019). Importantly, infections with V. vulnificus septicaemia show greatest
risk for individuals with pre-existing conditions, often linked to age (Jones and
Oliver 2009). Previous studies have indicated that individuals with compromised
immune systems or chronic liver disease such as cirrhosis are up to 80 times more
likely than healthy individuals to develop V. vulnificus primary septicaemia (Anon,
1993; Baker-Austin and Oliver 2018). Recent epidemiological data, such as liver
cirrhosis trends in the United States of America (Scaglione et al., 2015), show a
similar correlation between gender and age to that of observed V. vulnificus cases,
with a disproportionate rate of cirrhosis in males and in older age groups (e.g.
> 40 years old). Projections to 2025, the alcohol per capita consumption (15+
years) is expected to continue to increase in half of the WHO regions, potentially
greatly increasing risk. Likewise increasing liver cirrhosis in women (Scmucke et
al., 2005), increasing liver disease in the elderly and hepatitis infections (Scaglione
et al., 2015) should also be considered. Other demographic drivers that can be
considered include the changing middle class (WHO, 2014) arising from rapidly
rising economies in China and India where food preference may be changing and
thus influencing the demand of aquaculture production, both domestically and
globally. These increasing exposures are likely drivers of risk for vibriosis in the
future. Considerations may also include age-dependent behaviours influencing
risk of infections.

CHAPTER 3 - NEW DEVELOPMENTS IN VIBRIOS 39

 4
Conclusions and
recommendations
Globally, raw shellfish products, especially oysters, represent the most common
foodborne source of vibriosis (Newton et al., 2012; Baker-Austin et al., 2018). It
is clear that in the last decade our understanding of these organisms, the risks
that they pose, as well as their management continues to evolve. Considering these
continuous discussions, a JEMRA expert meeting on V. parahaemolyticus and V.
vulnificus was held at Cefas, Weymouth, the United Kingdom, 13-15 May 2019.
The meeting reviewed and updated the existing risk assessment models/tools of
V. parahaemolyticus and V. vulnificus that could be used to address a range of
risk management questions in a number of different regions. Several important
areas where addressed and discussed during this expert workshop (summarised
in Figure 2). The experts concurred that the basic scientific outputs underpinning
previous risk assessment endeavours were still valid; however, a range of important
development have occurred in the last decade that are noteworthy and have direct
impacts on the risk assessment of these important human pathogens.

These developments are complex and often multifaceted, and draw on aspects
related to microbiology, genomics, risk assessment, epidemiology, climate
sciences and oceanography. Firstly, the emergence of highly pathogenic strains, in
particular the PNW (ST36) V. parahaemolyticus complex have caused infections in
new areas and in regions where these diseases have not been observed before. The
pandemic spread of these bacteria provides new challenges to the risk assessment
community. Secondly, in response to climate change, there has been a significant

40
geographical spread regarding when and where these seafood-associated vibrio
infections have been reported, with a general trend in the poleward spread of V.
parahaemolyticus and V. vulnificus cases. We are now observing seafood-related
infections in traditionally non-endemic areas such as the NE United States of
America, Spain, and South America, among others. Demographic considerations
are also important. Globally, an increased at-risk population, increased population
densities in coastal regions and improvements in diagnosis of infections may
also have played a role in accentuating reported cases. A number of important
datagaps still exist, notably the availability of high quality epidemiology data from
geographically diverse regions (other than the United States of America), which
probably represents the most pressing current limitation. New approaches for
best practice, such as high-pressure treatment, harvesting curfews, relaying and
temperature controls appear to offer cost effective approaches for reducing human
health risks postharvest. Finally, a range of new methods, such as those utilising
genomics and satellite imagery provide novel means of building on previous risk
assessment exercises for these important foodborne pathogens. The remote-
sensing based approaches have proved invaluable in understanding the conditions
that can drive outbreaks of foodborne disease, and potentially offer the capability
to predict future outbreak conditions in near real-time. The development of WGS
allows us to understand the genomic and biological architecture of pathogens that
can cause disease, reconstruct the emergence, dispersal, and even the evolution of
these bacteria. The unparalleled resolution of sequencing methods has enormous
practical applications whilst inferring mechanisms of transmission, unravelling
the evolution of strains, as well as pinpointing outbreaks for risk management
purposes.

Several recommendations were noted during this meeting. These include the
following: 1) The establishment of systems for epidemiological data collection at
a regional, national and international level; 2) A systematic review of the efficacy
of post-harvest processing treatments and pre and post-harvest interventions in
risk mitigation – including appropriate cost/benefit analysis; 3) verification of
the efficacy of remote sensing, satellite approaches and WGS to predict periods
of elevated risks and to better control those risks. The translation and integration
of these novel methods into practical and tractable risk control measures is also
required; 4) a thorough assessment of laboratory methods utilised to study these
bacteria is urgently required. This could take the form of guidance documents or
good practice documents that outline and describe in detail the most appropriate
methods applicable for the isolation, growth, detection and enumeration of these
bacterial pathogens across environmental, clinical and food samples. Consensus
on appropriate molecular targets and testing approaches such as those applied to
shellfish (for example tdh/trh in V. parahaemolyticus) potentially represents the
most important technical issue that should be considered at his point.

CHAPTER 4 - CONCLUSIONS AND RECOMMENDATIONS 41

 FIGURE 2. Major microbiological, environmental and human-related factors responsible
for driving vibriosis risks associated with bivalve shellfish.

Relevant subjects in Major factors in the human and natural environment possibly affecting emergence
MRA 20 and spread of Infections by Vibrio parahaemolyticus and Vibrio vulnificus
2. Pathogenicity
3. Factors relevant to the fate
of V. parahaemolyticus HUMAN ENVIRONMENT NATURAL ENVIRONMENT
Microbes
and V. vulnificus Infection
3.1. Water temperature Humans Pathogenic Pathogens
3.2. Salinity mechanism 2
Immunity Vibrio parahaemolyticus
3.3. Other environmental Vibrio vulnificus
factors Resistance Changed by
3.4 Seafood of concern mutation
3.5 Preharvest and post
Acquired by
harvest practices Infection 4.4 Other
gene transfer
Eating/
4. Evaluation and application cooking microbes
of existing risk assessment habit
tools
Infectious disease

3.2
4.2 Evaluation of the Contatmination Spread, persistence and propagation Salinity
Hygenic 3.1
models for vibrio practice 3.5, 4.2, 4.3 Water temperature
concentration at harvest 3.3
Climate change
4.3 Evaluation of the vibrio
3.4, 5
growth rate models Seafood
4.4. Evaluation of dose Seafood trade
response models
International
5. Method for isolation, travelers
identification, and Tidal currents
determination of
Migratory birds
pathogenic potentials of
Spread
shellfish-associated Vibrio
parahaemolyticus, Vibrio
Note: Sections of MRA20 are referenced (left) to indicate the corresponding position on the figure
vulnificus

42 ADVANCES IN SCIENCE AND RISK ASSESSMENT TOOLS FOR VIBRIO PARAHAEMOLYTICUS AND V. VULNIFICUS
ASSOCIATED WITH SEAFOOD
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Annexes

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 Annex 1

Selected available data on the incidence

of V. parahaemolyticus infections in
China
tdh / trh presence
(where reported)
No. of cases Attributed
Regions Period (%) (Note: confirm Symptoms Origin of data
(outbreaks) food
all figures are given
in %)
2011 367(17) N/A N/A diarrhea Data not published
2012 193(17) N/A N/A diarrhea Data not published
2013 145(7) N/A N/A diarrhea Data not published
2014 353(27) N/A N/A diarrhea Data not published

South 2015 143(10) N/A N/A diarrhea Data not published

China
2016 246(12) N/A N/A diarrhea Data not published
2017 508(26) N/A N/A diarrhea Data not published
2018 371(23) N/A N/A diarrhea Data not published
2019 512(30) N/A N/A diarrhea Data not published
2020 297(21) N/A N/A diarrhea Data not published
2010 62(5) N/A N/A diarrhea Data not published
2011 46(3) N/A N/A diarrhea Data not published
2012 34(3) N/A N/A diarrhea Data not published
2013 8(2) N/A N/A diarrhea Data not published
2014 42(4) N/A N/A diarrhea Data not published
Central
2015 62(4) N/A N/A diarrhea Data not published
China
2016 41(5) N/A N/A diarrhea Data not published
2017 70(7) N/A N/A diarrhea Data not published
2018 76(6) N/A N/A diarrhea Data not published
2019 147(10) N/A N/A diarrhea Data not published
2020 26(1) N/A N/A diarrhea Data not published

58
 tdh / trh presence
(where reported)
No. of cases Attributed
Regions Period (%) (Note: confirm Symptoms Origin of data
(outbreaks) food
all figures are given
in %)
2010 NA N/A N/A diarrhea Data not published
2011 NA N/A N/A diarrhea Data not published
2012 32(2) N/A N/A diarrhea Data not published
2013 16(1) N/A N/A diarrhea Data not published
2014 77/(1) N/A N/A diarrhea Data not published
East China 2015 26(4) N/A N/A diarrhea Data not published
2016 194(23) N/A N/A diarrhea Data not published
2017 250(24) N/A N/A diarrhea Data not published
2018 422(31) N/A N/A diarrhea Data not published
2019 294(34) N/A N/A diarrhea Data not published
2020 26(2) N/A N/A diarrhea Data not published
2010 0 N/A N/A diarrhea Data not published
2011 1 N/A N/A diarrhea Data not published
2012 4 N/A N/A diarrhea Data not published
2013 3 N/A N/A diarrhea Data not published
2014 2 N/A N/A diarrhea Data not published
Southwest
2015 6 N/A N/A diarrhea Data not published
China
2016 13 N/A N/A diarrhea Data not published
2017 17 N/A N/A diarrhea Data not published
2018 5 N/A N/A diarrhea Data not published
2019 8 N/A N/A diarrhea Data not published
2020 3 N/A N/A diarrhea Data not published

ANNEX 1 - SELECTED AVAILABLE DATA ON THE INCIDENCE OF V. PARAHAEMOLYTICUS INFECTIONS IN CHINA. 59

 FAO/WHO Microbiological Risk Assessment Series
1 Risk assessments of Salmonella in eggs and broiler chickens: Interpretative
Summary, 2002

2 Risk assessments of Salmonella in eggs and broiler chickens, 2002

3 Hazard characterization for pathogens in food and water: Guidelines, 2003

4 Risk assessment of Listeria monocytogenes in ready-to-eat foods: Interpretative

Summary, 2004

5 Risk assessment of Listeria monocytogenes in ready-to-eat foods: Technical

Report, 2004

6 Enterobacter sakazakii and microorganisms in powdered infant formula:

Meeting Report, 2004

7 Exposure assessment of microbiological hazards in food: Guidelines, 2008

8 Risk assessment of Vibrio vulnificus in raw oysters: Interpretative Summary and

Technical Report, 2005

9 Risk assessment of choleragenic Vibrio cholerae 01 and 0139 in warm-water

shrimp in international trade: Interpretative Summary and Technical Report,
2005

10 Enterobacter sakazakii and Salmonella in powdered infant formula: Meeting

Report, 2006

11 Risk assessment of Campylobacter spp. in broiler chickens: Interpretative

Summary, 2008

12 Risk assessment of Campylobacter spp. in broiler chickens: Technical Report,

2008

13 Viruses in food: Scientific Advice to Support Risk Management Activities:

Meeting Report, 2008

14 Microbiological hazards in fresh leafy vegetables and herbs: Meeting Report,

2008

15 Enterobacter sakazakii (Cronobacter spp.) in powdered follow-up formula:

Meeting Report, 2008

16 Risk assessment of Vibrio parahaemolyticus in seafood: Interpretative Summary

and Technical Report, 2011

17 Risk characterization of microbiological hazards in food: Guidelines, 2009.

18 Enterohaemorragic Escherichia coli in beef and beef products: Approaches for

the provision of scientific advice, Meeting Report, 2010

19 Salmonella and Campylobacter in chicken meat: Meeting Report, 2009

60 DEVELOPMENTS OF SCIENCE AND RISK ASSESSMENT TOOLS FOR VIBRIO PARAHAEMOLYTICUS AND V. VULNIFICUS
ASSOCIATED WITH SEAFOOD
20 Risk assessment tools for Vibrio parahaemolyticus and Vibrio vulnificus
associated with seafood: Meeting Report, 2020

21 Salmonella spp. In bivalve molluscs: Risk Assessment and Meeting Report, In

press

22 Selection and application of methods for the detection and enumeration of

human pathogenic halophilic Vibrio spp. in seafood: Guidance, 2016

23 Multicriteria-based ranking for risk management of food-borne parasites, 2014

24 Statistical aspects of microbiological criteria related to foods: A risk managers

guide, 2016

25 Risk-based examples and approach for control of Trichinella spp. and Taenia
saginata in meat: Meeting Report, 2020

26 Ranking of low moisture foods in support of microbiological risk management:

Meeting Report and Systematic Review, In press

27 Microbiological hazards associated with spices and dried aromatic herbs:

Meeting Report, In press

28 Microbial safety of lipid based ready-to-use foods for management of moderate

acute malnutrition and severe acute malnutrition: First meeting report, 2016

29 Microbial safety of lipid based ready-to-use foods for management of moderate

acute malnutrition and severe acute malnutrition: Second meeting report, 2021

30 Interventions for the control of non-typhoidal Salmonella spp. in beef and pork:
Meeting Report and Systematic Review, 2016

31 Shiga toxin-producing Escherichia coli (STEC) and food: attribution,

characterization, and monitoring, 2018

32 Attributing illness caused by Shiga toxin-producing Escherichia coli (STEC) to

specific foods, 2019

33 Safety and quality o

 f water used in food production a
 nd processing, 2019

34 Foodborne antimicrobial resistance: role of the environment, crops and biocides,

2019.

35 Advances in science and risk assessment tools for Vibrio parahaemolyticus and
V. vulnificus associated with seafood, 2021.

FAO/WHO MICROBIOLOGICAL RISK ASSESSMENT SERIES 61

 ISSN 1726-5274

35
Globally, the Vibrio parahaemolyticus and Vibrio vulnificus represent
important human pathogens associated with the consumption of seafood.
In response to the requests for scientific advice from Codex Committee on
Food Hygiene (CCFH), risk assessments for the pathogens V. vulnificus, V.
cholerae, V. parahaemolyticus and guidance on methods for the detection of
Vibrio spp. with seafood have been conducted and published previously by
JEMRA. In order to provide an update on the state-of-the-art advice regarding
risk assessment for V. parahaemolyticus and V. vulnificus in seafood, an
expert meeting was convened.
Advances in science

V. vulnificus associated with seafood

Advances in science and risk assessment tools for Vibrio parahaemolyticus and
Several critical developments in the last decade were subsequently noted
by the expert working group: 1) The emergence of highly pathogenic strains; and risk assessment tools
forAttributing illness
2) In response to climate change, there has been a significant geographical
spread regarding when and where these seafood-associated Vibrio Vibrio parahaemolyticus
infections; 3) Demographic considerations are very important; 4) A range
of new approaches for best practice; and 5) A range of new methods, such
as those utilising genomics and satellite imagery. This report describes the
andcaused by Shiga
V. vulnificus associated
output of that expert meeting.
with seafood
toxin-producing
Escherichia coli (STEC)
MEETING REPORT

to specific foods
REPORT

Food Systems and Food Safety - Economic and Social Development

35
[email protected]
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Food and Agriculture Organization of the United Nations
Viale delle Terme di Caracalla
00153 Rome, Italy
MICROBIOLOGICAL RISK

32
ASSESSMENT SERIES
Department of Nutrition and Food Safety ISBN 978-92-5-134739-3 ISSN 1726-5274
[email protected]
FAO/WHO

https://www.who.int/health-topics/food-safety/
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