Mansoura University Faculty of veterinary medicine Department

of Fish diseases General program -Fish diseases -4th level student

Name: Anas Ashraf Mohamed

Section: 2

Title: Septicemic Bacterial Diseases of fish

Abstract
Egypt is the world’s ninth-largest fish producer with a total production of 1.5
billion tons per year, and farmed fishes comprise almost 79.6% of the total
production. Massive mortalities in market-sized farmed fish (freshwater and
marine species) were documented all over fish farms in Egypt leading to serious
economic losses. The poor biosecurity practices and awareness among
smallholder fish farmers accompanied with a long period of temperature
fluctuation may predispose farmed fish to be simultaneously coinfected with
multiple pathogens. Moribund fishes usually exhibited various septicemic
clinical signs and post-mortem lesions indicating that one or more septicemic
bacterial pathogens were involved in these outbreak reports. Therefore, rapid
and accurate identification of pathogens in the asymptomatic fish population is
important for preventing the occurrence of infectious diseases and protecting
consumers from seafood-borne illnesses. Molecular techniques provide
sensitive, quick, and accurate data for identifying specific pathogens without the
need for time-consuming traditional techniques. In addition, the development of
cost-effective molecular techniques facilitates their wide implementation in
routine clinical diagnostic approaches. Molecular diagnostic methods are useful
for simultaneously identifying multiple bacterial pathogens that were
challenging to recognize by commercial biochemical techniques. Genetic
testing procedures could determine the genetic diversity between closely related
strains at the subspecies level. Consequently, these techniques are required for
the development of detecting methods for specific strains and for
epidemiological investigations in bacterial diseases. This review documented a
variety of molecular techniques, including amplification of nucleic acids,
polymerase chain reaction (PCR), and nucleotide sequencing assays, that are
commonly used to identify fish pathogens.
Introduction

Water is an environment in which other organisms live besides fish, including

many species of saprophyte bacteria inhabiting sediments and plants, as well as
phyto- and zooplankton. Some of them colonise the skin, gills, and digestive
tract of fish, living there as commensals, supporting digestion and having a
beneficial effect on the immune system of these animals. These microorganisms
could also threaten fish health and therefore are referred to as conditionally
pathogenic. Interactions between fish, bacteria, and diseases are the subject of
many studies conducted around the world. The great interest aroused by these
topics indicates their important role in fish pathology. However, the
development of disease is a complicated process, dependent not only on bacteria
being capable of causing health disorders, but also on the immune status of fish,
environmental conditions, and virulence of the disease agent. Therefore,
changes occurring in freshwater ecosystems seem to be fundamental in the
development of any disease, including emerging ones (18).

The development of a particular fish disease depends largely on the climate

conditions, prevailing in a given zone, region, or country. This means that
dissimilar health problems occur in fish cultured in the Mediterranean Sea, in
continental Europe and in Northern European countries. For example, in
Scandinavian countries, one of the biggest health problems is posed by invasion
of copepods and amoebic gill disease (AGD). Meanwhile, in Central Europe,
infections caused by Aeromonas spp. are the most common among bacterial fish
diseases, causing motile aeromonas septicaemia (MAS), motile aeromonas
infection (MAI), and furunculosis. Infections caused by Flavobacterium spp.
are also very often observed (30). In Poland, the epizootic situation regarding
bacterial diseases in fish does not significantly differ from those presented by
other continental European countries. Nevertheless, during recent years
dynamic changes in the pathology of bacterial freshwater fish diseases have
been observed. Gram-negative bacteria established in fact as pathogenic to fish,
like Aeromonas spp., are displaced by infections caused by other species,
hitherto not known to be pathogenic or even conditionally pathogenic to
ichthyic species. Fish exhibiting clinical signs of the diseases often harbour the
following microorganisms: Acinetobacter spp., Kocuria spp., Plesiomonas
shigelloides, Sphingomonas paucimobilis, and Stenotrophomonas maltophilia.

Main body

Aeromonas infections. Almost every year health disorders in freshwater fish

are recorded on many farms in Poland. The causes of the diseases may be
various factors; however, the most important among bacterial infections are
those caused by motile Aeromonas: A. hydrophila, A. sobria, and A. caviae.
Clinical symptoms observed during health disorders vary depending on the type
of disease. In cases of MAI, skin ulceration and gill and fin lesions are most
frequently noticed. The disease can also become a systemic infection, and then
it is called MAS. The other psychrophilic Aeromonas, Aeromonas
salmonicida subsp. salmonicida, causes furunculosis in salmonids, which is
manifested in ulcers on the skin. During infection, high mortalities reaching
about 80% of stock are also observed (3).

Aeromonas spp. are commonly found in various environments including water,

and therefore fish are constantly exposed to bacteria. Interaction with bacteria is
especially dangerous under conditions of stress, which include unfavourable
environmental conditions as well as human intervention in catching, sorting,
and transporting of the fish. Aeromonas species are also part of the
physiological microflora of the fish intestine (3).

According to the data collected during the last five years in Poland, health
disorders caused by Aeromonas species were mostly observed in carp (Cyprinus
carpio L.) and were usually manifested by skin lesions (MAI) in the form of
ulceration (Fig. 1). Fish mortalities were also observed (26, data not published).
Co-infections with other microorganisms like Pseudomonas spp.

Fig. 1
Pseudomonas infections. Pseudomonas spp. are widespread in the
environment, forming a very large group of microorganisms. These
psychrophilic bacteria develop well at low temperatures and are the dominant
microflora. At higher temperatures (above 10ºC), they are quickly replaced by
competing, mesophilic microorganisms, including bacteria of the
genus Aeromonas.

P. fluorescens is the most important species in fish pathology and is very often
associated with skin (Fig. 2) and fin disease. In some trout farms, throughout the
year regardless of the water temperature, infections with P. fluorescens can
cause sudden mortality, reaching even 100% of the rainbow trout population
(data not published). Such a rapid disease course may be the effect of mutations,
resulting in the appearance of a new property allowing the bacteria to adapt to
new environmental conditions.

Fig. 2
Other Pseudomonas (P. putida or P. luteola) are often isolated from internal
organs of fish; however, these species are mostly an accompanying microflora
(23). Pseudomonas spp. can cause strawberry disease in rainbow trout
(Oncorhynchus mykiss, Walbaum, 1792) (Fig. 3) and tench (Tinca tinca).
Similar systemic infections with typical symptoms of septicaemia were also
observed in crucian carp (Carassius carassius) and silver carp (Carassius
gibelio) (1, 9, 23).

Fig. 3
Strawberry disease/infection with Pseudomonas spp. in rainbow trout (Oncorhynchus
mykiss, Walbaum, 1792) (photo: A. Kozińska, A. Pękala-Safińska)

Flavobacterium infections. Flavobacterium spp. naturally occur in the aquatic

environment. These bacteria are also a part of the physiological gill microflora
of healthy fish. However, the reservoirs of these bacteria as a source of potential
disorders for fish have not been yet established (3). Flavobacteriosis is caused
by three species of the genus Flavobacterium: F. columnare, which is the
aetiological agent of columnaris disease, F. branchiophilum causing bacterial
gill disease (BGD), and F. psychrophilum, which is associated with cold water
disease (CWD) or rainbow trout fry syndrome (RTFS). Health disorders in
cyprinids are caused by the first two species, while F. psychrophilum is mainly
isolated from salmonids, although this species may also cause dangerous
infections in cyprinids (28). Each of these diseases can be severe, and mortality
can be 50% or even occasionally 80% of the fish population.

Relatively often during the last ten years, Flavobacterium spp. have been
isolated from salmonids and cyprinids on Polish farms where clinical symptoms
of the disease were observed in the fish (25) (Figs 4 and and5).5). The first
documented outbreak of CWD in Poland was found in rainbow trout (4).

Fig. 4
Flavobacterium infection in rainbow trout (Oncorhynchus mykiss, Walbaum, 1792)
(photo: A. Kozińska, A. Pękala-Safińska)

Fig. 5
Rainbow trout fry infected with Flavobacterium psychrophilum (photo: A. Pękala-
Safińska)

Acinetobacter infections. Bacterial fish diseases are generally caused by

conditionally pathogenic microorganisms. One of them is Acinetobacter spp.,
which is widely dispersed in nature, including the aquatic environment. In
recent years in Poland, these microorganisms have been isolated from trout and
carp relatively often (Fig. 6). Detection was in fish in which disease symptoms
were observed in different seasons, most frequently in May and September (27).
Clinically, depigmentation of the skin, loss of scales, exophthalmia with
congestion of the eye, and gill petechiae were observed in infected trout.
Haemorrhages in the skin and gill congestion were noted in infected carp, and
post-mortem examination showed intestinal inflammation in both fish species.
Disease symptoms were accompanied by mortalities, ranging from 5% to 20%
(27).

Fig. 6
Infection with Acinetobacter spp. in carp (Cyprinus carpio L.) (photo: A. Pękala-Safińska)

Infection with Acinetobacter spp. in most cases is mixed with other bacterial
infection, mainly from the genus Aeromonas or Chryseobacterium spp.;
however, the dominant flora in bacteriological examinations belonged to the
genus Acinetobacter. It is worth noting that Acinetobacter spp. are generally
regarded as carriers of antimicrobial resistance genes. Therefore, they can be
very important in the spread of drug resistance in the environment (29).
Shewanella putrefaciens infections. For over 10 years, shewanelloses have
been serious diseases of freshwater cultured fish. The aetiological
agent, Shewanella putrefaciens, is a halophilic bacterium and very well known
as an important microorganism of the food spoilage process. It mainly putrefies
fish stored at low temperatures, but also poultry meat and beef products (6, 15).
This bacterium has also been isolated from marine and brackish waters, as well
as from marine fish (2). The first bacterium isolations from diseased freshwater
fish were described by Kozińska and Pękala (24). Infection rapidly spread and
health disorders were noticed among different fish species, both cultured and
ornamental: common carp (Cyprinus carpio L.), rainbow trout (Oncorhynchus
mykiss, Walbaum, 1792), European eel (Anguilla anguilla), brown trout (Salmo
trutta m. trutta), silver carp (Hypophthalmichthys molitrix), European whitefish
(Coregonus lavaretus), sander (Sander lucioperca), ide (Leuciscus idus),
common roach (Rutilus rutilus), zebrafish (Brachydanio rerio), slender krib
(Pelvicachromis taeniatus), least killifish (Heterandria formosa), and koi carp
(Cyprinus carpio L.) (36, 37).

Health disorders in cultured fish were noted mainly in spring, when water
temperature rose to 7ºC–10ºC (34). Clinical signs, prevalently lethargy,
darkening of the skin, skin lesions, and ulceration, have been observed in
infected fish (Fig. 7). In post-mortem examination haemorrhage in the kidneys
and spleen was noticed. The range of mortality varied from 40% to 85% in
different fish species (34, 36).

Fig. 7
Shewanella putrefaciens infection in carp (Cyprinus carpio L.) (photo: A. Kozińska, A.
Pękala-Safińska)

Fish infection with Gram-positive bacteria. In recent years, the number of

infections caused by Gram-positive bacteria, especially in rainbow trout, has
increased significantly in Poland (26). Among many species of Gram-positive
microorganisms, two of them are of particular importance in the pathology of
bacterial fish diseases: Lactococcus garviae and Streptococcus iniae. Both
bacterial species cause serious health disorders in different species of freshwater
and marine fish: rainbow trout and other salmonids from
the Oncorhynchus family, eels, and fish belonging to the Ictaluridae (catfish)
and Cichlidae (tilapia) families (3).
The source of bacterial infection for fish can be both water and sediments (32).
Although bacteria might be present in the environment and also in fish
throughout the year, their related disorders occur during summer when the water
temperature increases and reaches optimal values in the range of 18ºC–25ºC.
Therefore, it is assumed that the temperature as well as the sanitary state of the
aquatic environment are the most important factors conducive to the occurrence
of disease symptoms (3).

The clinical symptoms of L. garviae and Str. iniae infections are usually similar
and vary only slightly, depending on the fish species. In both infections, a
typical clinical sign is exophthalmia. Furthermore, darkening of the skin, anal
oedema, petechiae in the eyes, on the gill covers, and at the base of the fins are
observed. Anatomopathological examination showed haemorrhages in the swim
bladder, liver, spleen, and kidney, as well as stomach inflammation. During the
process of disease development, the initial phase of infection is very
characteristic. Then, fish exhibit nervous whirling rapid movements from the
bottom to the surface or in the opposite direction, resulting from meningitis or
encephalitis (12, 13).
Conclusion
Bacterial coinfections are often recorded in wild and farmed fish when more
than one bacterial pathogens coinfect fish. In Egypt, mixed infections may lay
behind the different clinical signs and are responsible for difficulties in
diagnosis and control régimes in closed aquatic environments. Therefore,
dependence on traditional methods only for diagnosis may mask the identity of
real causes.
On the other hand, molecular methods have improved the ability to identify
bacterial pathogens. The whole-genome sequencing of microorganisms has
made significant progress in understanding their biology and makes it possible
to increase the specificity, sensitivity, and speed of diagnosis by using these
nucleic acid techniques. These techniques also provide a way to examine the
connections between the genotype and phenotype of different micro-agents.
These advanced techniques may support epidemiological investigations and
help to pinpoint the origins of illness outbreaks or the existence of infections.
Therefore, these genetic technologies can be used frequently in the quest for
better ways to identify and manage fish infections and understand the spread of
infectious diseases in fish. Unfortunately, there are few diagnostic wet
laboratories in Egypt that use these techniques regularly. In addition, there are
several problems to be fixed before using these tests in aquatic laboratories,
such as the availability of specific equipment, cost-effectiveness for regular
operation, personnel training, accuracy, and reproducibility. Currently, PCR-
based techniques are widely used; however, alternative technologies needed to
be implanted in laboratories in the coming years as sequencing devices.
Therefore, this review shed the light on the importance of popular molecular
tools in the thrust of identification of bacterial pathogens in farmed fish.
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