Background: The aquaculture in Nigeria
Nigeria is enormously blessed with land capacity averaging over 900,000 square kilometers with abundance of rain, rich groundwater resources and water surface system. Rainfall characteristically increases from the west to the east and from the north to the south with annual rainfall of 1778 mm, 4318 mm, 1270 mm and 508 mm in the Western, eastern, central and northern regions, respectively. These attributable rainfall and environmental characteristics are most suitable for a successful and flourishing aquaculture. Fish represents an important dietary element and one of the few animals’ protein source available for an average Nigerian (
FAO, 2017). Despite these innumerable land and water qualities, Nigeria spends an average of 97 billion Naira ($630 million) annually on the importation of about 700,000 tons of fish and fish products from USA, Europe (
Ozigbo et al., 2014). The increase in the importation is due to the failure of the production system to meet up the average 2.6 million tons required market demand of Nigeria as the total production in Nigeria currently approximates only about 680,000 tons per annum. These short falls in the production can be attributed to the insufficient large-scale fry production, lack of modern-day equipment supply, unreasonable farm design and settlement and most importantly, emerging and/or remerging infectious diseases. The aquaculture sector in Nigeria is presently driven by the private sector, owning to the current trend and growth in the Nigerian aquaculture sector. In spite the great potentials of the environment, lack of resources, knowledge and expertise of identification and control infectious agents and some other managemental defaults continues to flaw the aquaculture sector. Disease outbreaks are the primary limiting factor in catfish production. Cumulative losses in catfish production cycles are regularly between 15-20% of total fish stocked. Producers report infectious disease accounts for the largest percentage of losses, with approximately 65% of fry and fingerlings lost during production (
USDA, 2006). Approximately 45% of inventory losses on catfish farms are attributable to infectious diseases, of which 60% are associated with single or mixed bacterial infections (
Hawke and Khoo, 2004). The most important pathogens responsible for these loses are
Edwardsiella, Aeromonas, Vibrio, Flavobacterium and Streptococcus (
Xu and Zhang, 2014).
Edwardsiella tarda has recently been identified to cause a new upsurge in the incidence and prevalence of septicemia in different fish species in the US as well as causing human infections (
Park et al., 2012) making the organism an economic and zoonotic important.
Edwardsiella tarda- The organism, disease and pathology
The genus Edwardsiella includes two species of bacteria that cause major diseases in fish:
Edwardsiella tarda (
Ewing et al., 1965) infects fish and other animals and
Edwardsiella ictaluri (Hawke, 1979) infects fish only. A third species,
Edwardsiella hoshinae, infects birds and reptiles.
Edwardsiella tarda produces the disease commonly known as fish gangrene, emphysematous putrefactive disease of catfish or red disease of eels and hereafter known in this text as Edwardsiella septicaemia (ES) and
E. ictaluri causes ‘enteric septicaemia of catfish’ (ESC) (Fig 1). Because
E. tarda and
E. ictaluri produce distinctively different diseases, they are discussed separately.
Edwardsiella tarda is a Gram-negative bacterium responsible for enteric septicemia in both marine and freshwater fish species (
Buján et al., 2018) and culminates in severe economic losses in aquaculture worldwide (
Wang et al., 2005). A wide range of fish are affected by
Edwardsiella tarda, including, Japanese eel (Anguilla japonica)
(Egusa, 1979), channel catfish (
Ictalurus punctatus) (
Meyer and Bullock, 1973), Japanese flounder (
Paralichthys olivaceus) (
Nakatsugawa, 1983), tilapia (
Oreochromis spp.), tropical ornamentals (
Bullock and McCraran, 1989;
Dixon and Contreras, 1992), chinook salmon (
Oncorhynchus tshawytscha) (
Amandi et al., 1982), spawning Atlantic salmon (
Salmo salar) (
Martin, 1984), farmed rainbow trout (
Onchorhynchus gairdneri) in Australia (
Reddacliff et al., 1996), brook trout (
Salvelinus fontinalis) (
Uhland et al., 2002). Diseases due to
Edwardsiella tarda are not limited to fish species. Infections have also been reported in reptiles, birds, swine, ruminants, marine mammals, warm blooded animals and importantly, humans.
Infections caused by this organism is generally presented as both gastrointestinal (gastroenteritis) and extra-intestinal infections (such as: myonecrosis, bacteremia and septic arthritis) (
Leung et al., 2012) in fishes while it is known to cause extra-intestinal manifestation such as biliary tract infection, bacteremia, skin and soft tissue infection, liver abscess, peritonitis, intra-abdominal abscess, tubo-ovarian abscess and mycotic aneurysm mostly in other immunocompromised animals (
Ebisawa et al., 2018). Infection is known to occur through the protein-protein interactions between the outer membrane proteins of
E. tarda and proteins in the gills of fishes, followed by intracellular replication in the intestinal phagocytic cells of the fish before progressing to systemic infection where they reach a critical mass and spread deeper inside the host’s body (
Leung et al., 2012). Signs associated with disease caused by this organism is usually in forms of petechial hemorrhage, poor pigmentation, protrusion and opacity of eyes, lesions on the skin, liquefaction and necrosis of tissues and organs such as kidney, spleen and liver
(Devi et al., 2016; Park et al., 2012) (Fig 1).
Epidemiology of Edwardsiella tarda
Edwardsiella tarda is a ubiquitous organism that has been isolated from both animals and environments of most continents. It is believed so far that the intestinal contents of infected or carrier animal is the most common source of the organism which often are aquatic animals.
E. tarda is more prevalent in environments with high temperature, poor water quality and high organic content which allow its adherence to and replication in cell lines (
Park et al., 2012). Occurrence of
E. tarda in both Nile tilapia and African catfish has been reported with prevalence ranging from low to high. In Uganda; 5% and 50% in tanks (
Walakira et al., 2014), Egypt; 10-70% (
Sedeek, 2017) in Nigeria, from
C. gariepinus; 3.5% (
Efuntoye et al., 2012). The bacterium is also known to occur in other continents as reported by
Kumar et al., (2016) in Korea and in the Mediterranean (
Katharios et al., 2015). In the United States of America, it has been isolated from water samples, pond-mud samples and from the animals that dwells in the environments (
Wyatt et al., 1979). Introduction of infection to a new environment may be associated with the introduction of asymptomatic infected live fish, or animal faeces with bacteria, contaminated feeds, or water (
Park et al., 2012). Transmission of Edwardsiella bacteria between fishes in the farm is from faecal shedding from infected fish or from the carcasses of a dead fish (
Park et al., 2012). Vertical transmission from infected brood stock to fry has not been demonstrated (
Park et al., 2012). Ciliated protozoans such as Trichodina and Tetrahymena pyriformis can act as a vector for
E. tarda transmission to farmed fish.
Edwardsiella tarda is transmitted to humans through contamination with infected water and the infections normally manifests as gastrointestinal (Fig 2).
The threat to Nigerian aquaculture industry
Aquaculture represents one of the fastest growing sources of animal protein production in Nigeria which principally have been geared towards extermination of hunger and poverty. Because of the health benefits attributed to fish products over other sources such as beef, as well as the affordability of fish, majority of citizens have found solace in fish consumption. In response to this, there is an increase demand and a consequential increase in the production and expansion of aquaculture industry in Nigeria (
Charles et al., 2020).
Edwardsiella infections cause mortalities in infected fish leading to a serious economic loss in aquaculture (
Park et al., 2012,
Charles et al., 2020). The economic impact of
E. tarda as mortality and morbidity in fish ranges from 5% to 70% globally. The disease course is usually characterized by mild to severe conditions, whilst clinical signs can vary between fish species. In channel catfish for example, it starts with small cutaneous lesions at the dorsolateral aspect of the fish that soon progresses to large necrotic abscesses within the flank muscle or caudal peduncle where the form convex swollen areas with loss of pigmentation. This can also be characterized with putrid smelling emphysematous diseases (
Park et al., 2012). In some other fish species, acute infections have been characterized with severe hyperemia, congested fins, ecchymosis or petechia haemorrhages on various body surfaces, gas-filled pockets in the skin and large necrotic lesions in the muscle and swollen and hyperemic anal regions. Internally, the infection can be characterized by extensive septicaemia and hyperaemia of the peritonium; oedematous, abscessed and mottled kidney and liver. A variety of clinical signs occur in other species of fish. For example,
E. tarda causes exophthalmia and cataracts in tilapia and striped bass, as well as abscesses in internal organs (
Baya et al., 1997). Japanese flounders, naturally infected with
E. tarda, develop ulcerative lesions and loss of skin, which expose underlying muscle, haemorrhage in fins, rectal protrusion and swelling of the spleen. Infected cage-cultured largemouth bass developed necrotic lesions on the caudal peduncle.
The impact of
E. tarda in wild fish populations is unknown due to the absence of routine spatial and temporal
E. tarda environmental disease monitoring and effects of
E. tarda on fish populations. While extensive infections and economic losses are common to Edwardsiella infections, little or no information is available regarding the rate of incidence/prevalence, the attributable morbidity and mortality, chemotherapeutics and its associated cost implications and the diagnostic materials and techniques in Nigeria. Furthermore, the extent of economic loss due to this infection is also unknown. The threat posed by this organism is further exacerbated by the progression of the diseases caused (
Charles et al., 2020). Usually, the disease caused by
Edwardsiella tarda is a chronic problem that that would not only increase fish morbidity and mortality, but also increase production cost, reduced feed conversion rates and delayed harvesting time. Infection is usually not confined to any age or size group, thereby, losses associated to
Edwardsiella tarda range from moderate to high resulting in reduced availability and productivity of fish to the market and public, as well as loss of revenue to the producer whose daily income depends on the fish markets.
Due to the unaesthetic appearance of fish infected with
E. tarda, a poor perception of fishery products from areas where infection has been reported may deter their purchase and consumption by humans for fear of sickness. Tourism may also be impacted because of human perception of an unclean environment (
Charles et al., 2020).
The public health and food safety threats
As
Edwardsiella tarda is a threat to fish, so is it a threat to humans. Transmission to human usually occur through the handling/or consumption of undercooked sub-clinically ill fish intended for human consumption. Contamination of equipment can occur when sub-clinically ill fish is processed and the equipment are not properly cleaned and disinfected. Human infections principally occur through digestive tract or muscle infection through puncture wounds by spines or bones of infected fish. This can be attributed we occupational and recreational exposure of human to
E. tarda infections (
Ogunleye et al., 2021).
E. tarda isolates from fish are like those of human origin, therefore, isolates from fish are potential human pathogens. Because
E. tarda has such a wide host susceptibility and environmental adaptation it may enter the human food chain through fish processing plants and/or poor sanitation
(Nucci et al., 2002). As far as human infection is concerned, some of the earliest isolation of
E. tarda were from human faeces (
Ewing et al., 1965). Human infections are often characterized as intestinal and extraintestinal infections. Intestinal infections are manifested as diarrhoea and gastroenteritis while extraintestinal infections includes may produce a typhoid-like illness, peritonitis with sepsis, cellulitis and meningitis. Occasionally,
E. tarda-induced abscesses have been seen in liver. The disease is more prevalent among the vulnerable groups of the human population (children and adult over 50 and those with pre-existing liver disease and underlying iron overload states (cirrhosis, red cell sickling and leukemia). Because
E. tarda is an emergent bacterium for food borne disease of humans, its potential impact on food production and safety must be significant. Based on the findings so far, there is sufficient evidence to indicate that
E. tarda can be a public-health problem.
The ability of
E. tarda to infect human has been demonstrated. Its ability relies on the invasion of the Hep-2 cell monolayers, production of cell- associated haemolysin and siderophores and express mannose-resistant hemagglutination against guinea-pig erythrocyte (
Janda et al., 1991). Principally, haemolytic activity was found as a major virulent factor in the pathogenesis of
E. tarda in humans.
Responding to various degrees of economic losses due to infectious diseases as well as preventing zoonoses, antimicrobial agents have been devised to combat the challenges emanating from microorganisms in aquaculture practices. The inappropriate usage of these agents by farmers without adequate prescription has contributed to the current global public health concerns associated with the antimicrobial resistance (AMR).
Edwardsiella tarda, like many other bacteria have been reported to have overtime developed resistance to several commonly used antibiotics (
Efuntoye et al., 2012,
Nagy et al., 2018, Ogunleye et al., 2021, Charles
et al., 2020) and drug resistant infections have been predicted to likely be associated with the mortality of up to 10 million people annually if the current trends of antimicrobial resistance persist (
Dadgostar, 2019). Overuse of antibiotics has been implicated as the major cause of resistance (
Johnson et al., 2006). Antimicrobial resistance strains have evolved to become a serious health problem worldwide (
Chiu et al., 2002).
E. tarda have been found show resistance to antibiotics such as Ceftazidime, Meropenem, Cefuroxime, Tetracycline and Cotrimoxazole (
Nagy et al., 2018;
Ishola et al., 2021).
Diagnosis
Clinical signs of
E. tarda infections vary between species of fish, therefore, they are generally of little use, except to indicate the likely presence of a bacterial infection. Edwardsiella infection in fish, like any other bacterial infection can be diagnosed by several methods including clinical signs, histopathology, haematology, bacterial isolation and biochemical characteristics (
Park et al., 2012;
Hemraj et al., 2013).
Edwardsiella septicemia in fish is characterized by the attributable lesions which include necrotic abscesses in the muscle that emit a putrid odor when incised. Skin lesions includes muscle abscess with or without petechia hemorrhages. Morbidity and mortality can be acute or chronic causing various degrees of fish losses. Commonly, the organism resides in the intestinal tract of the infected fish, thereby, adequate isolation is based on their recovery from the intestinal tract of the infected fish. Furthermore, they can also be recovered from the environmental samples such as water bodies and pond muds.
Edwardsiella tarda is identified in the laboratory as a motile, facultatively anaerobic, Gram-negative rod; categorized as a member of the family Enterobacteriaceae, first recognized by
Trabulsi et al., (1962), followed by a description of
E. tarda in the mid-1960s. Diagnosis involves identification of the bacterium using microbial culture techniques followed by biochemical characterization of the isolates. The bacteria can be isolated from infected tissues using general culture media like Brain Heart Infusion agar (BHI), Trypticase Soy Agar (TSA), MacConkey agar, Blood Agar, Xylose Lysine Deoxycholate (XLD) agar or a selective medium
(E. ictaluri medium or EIM) for the recovery of
E. ictaluri (
Charles et al., 2020).
E tarda is normally seen as small, circular, raised, clear colonies with black centers on XLD and pale on MacConkey agar. Alternative techniques to direct detection of the bacteria can also be used like serological methods which include using Enzyme immunosorbent assay (ELISA), loop-mediated isothermal amplification (LAMP), indirect fluorescent antibody (IFA) test, fluorescent
in situ hybridization (FISH) and use of molecular techniques like polymerase chain reaction (PCR).
Treatment strategies
The use of antimicrobials has become the most important step to controlling infections caused by
E. tarda especially, because vaccines against the organisms are not available. The use of antimicrobial medicated-feeds alongside feed restriction which causes selective pressure on the microbial flora in aquatic environments and often promotes antibiotic resistance and multidrug-resistant (MDR) species have become the proven strategy for controlling
E. tarda (
Buján et al., 2018). As antibiotic medicated feeds promote AMR and MDR, feed restriction also results in reduced growth rates and subsequently, delay in harvest. Sulfadimethoxine/ormetoprim (Romet-30™), florfenicol (Aquaflor®) and oxytetracycline (Terramycin®) have been approved by the FDA for treatment of bacterial infections in aquaculture. Resistance of
E. tarda to these approved antimicrobials have been confirmed by the presence of plasmid-mediated AMR genes in
E. tarda isolate obtained from infected and/or mortalities of fish (
Hirai et al., 2015). These therefore limits the control options available for
E. tarda. A prompt action against increase in the frequency rate of MDR-
E. tarda should be instituted to prevent ascent from the current 6.4% to an alarming rate.
Prevention strategies-
Virulence, vaccination and vaccine development
The
E. tarda is known to exhibit a two-component system (TCS) with which they survive stress impacted by the intracellular environment of the invaded hosts during infections. Several of these systems (TCS) have been found to play vital roles in the pathogenesis of the bacterium. The TCS is known to be comprised of a transmembrane histidine kinase sensor that interacts with the outward signals, internal signals and a cytoplasmic response regulator which facilitate proper change in the bacterial cell physiology (
Derzelle et al., 2004). In response to external stimuli, the histidine kinase sensor autophosphorylates at a conserved histidine residue and then transfers the phosphoryl group to the regulator component, promoting its binding to DNA to regulate transcription of virulence genes
(Capra and Laub, 2012). Importantly, these TCS are suitable targets for the development of vaccine to control the bacterial infections.
Efforts have been put in place severally to develop alternative methods to the control of infections caused by
E. pscicida. This is more imminent due to the few available and/or approved AMs used in aquaculture as well as the recent spate of AMR/MDR globally. Several vaccines ranging from mutant attenuated live vaccines as demonstrated by several researchers such as asdA (
Banikalyan and Roy, 2018), EsrA-EsrB (
Yin et al., 2019),
luxS /AI-2 (
Sun et al., 2020), uhpA (
Liu et al., 2020),
etc., as well as
E. ictaluri vaccines have been proven effective against the bacterium by several researchers through the administration at fingerling stage (
Griffin et al., 2020).
rstA mutant gene
rstA mutant gene has been found to be involved in the downregulation of bacteria adherence to HeLa cells thereby downregulating the pathogenicity of
E. coli. RstA mutant genes have been recorded to affect sporulation and toxin gene expression, as demonstrated by
Liu et al., (2019). Till date, little or nothing have been done to establish facts about the identification and the virulence properties of the rstA genes and its mutant in
E. tarda. This therefore serves as an integral justification for this study to ascertain the effects of this gene in the pathogenicity of
E. tarda.
Quorum sensing regulation (QSR) system
Quorum Sensing Regulation (QSR) is known to cause autoinduction in bacteria cells through two conserved regulatory gene products: LuxItype acyl-HSL synthase and LuxR-type transcriptional activator whose activity requires a particular acyl-HSL made by the cognate LuxI enzyme (
James, 2002). These genes are known to facilitate virulence, biofilm development and many other processes in
P. aeruginosa (
James, 2002).
Two-component crp/Dcrp system
Peng et al., 2019 found
crp gene to be associated with the capacity of the bacterium to utilize maltose and impaired motility due to the lack of flagella synthesis. This evidencing that the deletion of this gene impairs the ability of the bacterium to evade host immune clearance, thereby, conferring immunity on the fish and the infections caused by
E. pscicida.
Two-component asdA system
Banikalyan and Roy, 2018 demonstrated the recombinant attenuated
Edwardsiella vaccine (RAEV) useful for the providing immunity against the infections caused by the bacterium. This group found asd genes to be associated with obligate requirement for diaminopimelic acid (DAP) or a plasmid vector with the wild-type asdA gene to grow, thereby enabling the synthesis of recombinant antigens that induces protective immunity against the bacterium.
Two-component EsrA/EsrB system
EsrA-EsrB have been found to play vital roles in the pathogenicity of the bacterium by controlling the expression of the T3/T6SS machineries of the bacterium as well as some effector cells (
Liu et al., 2017,
Yin et al., 2019). The systematic review and studies of the operations and activities of the regulatory networks of the EsrA-EsrB operations will therefore be important for the understanding of
E.
piscicida pathogenicity (
Yin et al., 2019). Based on this, The Mutation of
esrA-esrB has therefore been found to be a fruitful strategy for development of live attenuated vaccines against edwardsiellosis in fish (
Yin et al., 2019).
Two-component LuxS/AI-2 system
LuxS/AI-2 is known to be an important quorum sensing system which affects the growth, biofilm formation, virulence and metabolism of the bacterium (
Sun et al., 2020). The
luxS /AI-2 gene mutant strains were found to decrease
E. piscicida growth (
Sun et al., 2020).
UhpA/UhpB-UhpC mutant
UhpA/UhpB-UhpC is known to enable cell to acquire phosphorylated sugars from its environment that can be used as carbon or energy sources (
Liu et al., 2020). The deletion of the uhpA gene deletion decreased the metabolic level
E. piscicida, increases flagellar-related gene expression in
E. pscicida and increased the T3SS- and T6SS-related gene expression in
E. piscicida (
Liu et al., 2020).
Use of E. ictaluri vaccine
It has also been reported that the use of
E. ictaluri vaccine provides a cross protection against
E. pscicida. This was demonstrated by
Griffin et al., (2020), who administered the
E. ictaluri live attenuated vaccine orally through feed mix and then by immersion bath. It was found to provide suitable protection immunity against infections caused by the bacterium. One very important achievement of this vaccination is the use of both the oral and immersion bath methods ensuring that all the fishes are vaccinated, as fishes that could not be vaccinated orally will most certainly be vaccinated through the bath immersion. Presently, these vaccines have not been commercialized on large scale and made available for use by the farmers.
Edwardsiella tarda-
what we know so far in Nigeria
So far, little or nothing is known about the incidence/ prevalence of
E. tarda both in fish and in humans, the spatio-temporal distribution, the economic impacts and losses. The earliest report of
E. tarda was by
Gugnani et al. (1986) who isolated the organism from wall gecko. In the recent time,
Ogunleye et al., 2021 reported a 62.5% prevalence of the bacterial from a study in Ibadan, Oyo State Nigeria and Ogbonne
et al., 2018 described the antibiotic resistance pattern and plasmid profiling of the organism. These three reports being the only available reports indicates a dearth of knowledge and information on
E. tarda and an indication of fish infections due to
E. tarda as well as human exposure to the infections. According to our previous report on the detection and antibiogram of
Edwardsiella tarda from
O. niloticus sold in Ibadan, Oyo State, we have been able to establish based on bacteriological and morphological evidence that
E. tarda is present and culturable from tilapia and could serve as a source of infection for fish consumers and handlers suggesting human and public health risks associated with the bacterium.
