Phenotypic,molecular and pathological characterization of motile aeromonads isolated from diseased fishes cultured in Uruguay

Information about motile aeromonads from aquaculture systems of the Neotropical region is scarce. The aim of this study was to characterize motile Aeromonas isolated from ornamental and consumable fishes cultured in Uruguay. Biochemical and molecular methods were used for species identification. Antimicrobial susceptibility and the presence of virulence genes were evaluated. Genetic diversity was analysed by rep‐PCR, and virulence of the most representative isolates was determined by calculating the fifty lethal dose in experimentally challenged fish (Australoheros facetus). Aeromonas hydrophila and A. veronii were the most prevalent identified species (38.2% and 32.4%, respectively), whereas A. allosacharophila, A. bestiarium, A. caviae and A. punctata were less prevalent. This study constitutes the first report of these last four species in Uruguay. All isolates were resistant to at least three antimicrobials, and 82.3% of them showed multidrug resistance. Virulence genotypes were correlated with the Aeromonas species and haemolytic activity. The genotype act+/alt+/ast+/ela+/lip+ was the most prevalent (26.5%). A correlation between virulence genotypes and Aeromonas species was found. A. punctata showed a clonal structure according to rep‐PCR analysis, whereas other species showed high genetic diversity. The number of virulence genes of the isolates was related with virulence according to the experimental challenge assays.     

Diagnostic methods for identifying different Aeromonas species and examining their pathogenicity factors,their correlation to cytotoxicity and adherence to fish mucus

Aeromonas spp. are ubiquitous in the aquatic environment, acting as facultative or obligate pathogens for fish. Identifying Aeromonas spp. is important for pathogenesis and prognosis in diagnostic cases but can be difficult because of their close relationship. Forty‐four already characterized isolates of Aeromonas spp. were analysed by 16S rRNA gene sequencing, by gyrase B sequencing, by analysing their fatty acid profiles, by biochemical reactions and by MALDI‐TOF MS. To determine their pathogenicity, cytotoxicity, adhesion to mucus and the expression of 12 virulence factors were tested. The susceptibility of the isolates towards 13 different antibiotics was determined. MALDI‐TOF MS was found to be an acceptable identification method for Aeromonas spp. Although the method does not detect all species correctly, it is time‐effective and entails relatively low costs and no other methods achieved better results. A high prevalence of virulence‐related gene fragments was detected in almost all examined Aeromonas spp., especially in A. hydrophila and A. salmonicida, and most isolates exhibited a cytotoxic effect. Single isolates of A. hydrophila and A. salmonicida showed multiple resistance to antibiotics. These results might indicate the potentially pathogenic capacity of Aeromonas spp., suggesting a risk for aquatic animals and even humans, given their ubiquitous nature.     

Limited performance of MALDI‐TOF for identification of fish Aeromonas isolates at species level

The aim of this study was to evaluate the usefulness of the MALDI‐TOF MS to identify 151 isolates of Aeromonas obtained mostly from diseased fish. MALDI‐TOF MS correctly identified all isolates to the genus level but important differences in the percentage of isolates correctly identified depending on the species were observed. Considering exclusively the first identification option, Aeromonas bestiarum, Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas veronii and Aeromonas sobria were the best identified with results >95%. However, considering the first and second identification options, the only species that showed values >90% was A. hydrophila. Overall, when the database was supplemented with 14 new spectra, the number of accurate identifications increased (41% vs. 55%) and the number of inconclusive identifications decreased (45% vs. 29%), but great differences in the success of species‐level identifications were found. Species‐distinctive mass peaks were identified only for A. hydrophila and A. bestiarum (5003 and 7360 m/z in 95.5% and 94.1% of their isolates, respectively). This work demonstrates the utility of MALDI‐TOF MS for Aeromonas identification to the genus level, but there is no consistency for the accurate identification of some of the most prevalent species implicated in fish disease.     

Plasmid‐mediated antimicrobial resistance in motile aeromonads from diseased Nile tilapia (Oreochromis niloticus)

For the sustainable farming of tilapia, proper maintenance of their health and adequate treatment for infections at appropriate time are inevitable. The indiscriminate use of antibiotics in aquaculture, as a part of treatment and as growth promoters, accelerates antimicrobial resistance (AMR) among the fish pathogens. In the present study, we have isolated diverse aeromonads from Nile tilapia and studied their antibiogram and plasmid profiling. Aeromonas hydrophila, Aeromonas veronii, Aeromonas sobria, Aeromonas dhakensis, Aeromonas caviae, Aeromonas jandaei and Aeromonas aquatica were isolated from infected tilapia (n = 150), and their Shannon wiener diversity index was calculated as 1.926. A. veronii was found to be the most multiple antibiotic‐resistant pathogen with the MAR index of 0.46, and A. aquatica was noticed as the least resistant isolate. The minimum inhibitory concentration of resistant antibiotics was shown as >256 mcg/ml for most of the isolates. The virulent genes such as aerolysin and hemolysin were identified in all the isolates except A. aquatica. The detection of class 1 integrons, plasmid profiling and plasmid curing studies confirmed that AMR exhibited by most of the Aeromonas species is of plasmid mediated. This challenges the risk of wide spread of AMR among the pathogens and subsequent treatment of the infection.     

The effects of soybean, linseed and marine oils on aerobic gut microbiota of Arctic charr Salvelinus alpinus L. before and after challenge with Aeromonas salmonicida ssp. salmonicida

Populations of heterotrophic bacteria present in the hindgut region of Arctic charr Salvelinus alpinus L. fed dietary soybean, linseed and marine oils before challenge with Aeromonas salmonicida ssp. salmonicida and marine oil after challenge were estimated using the dilution plate technique. There were differences in bacterial composition between the rearing groups before and after challenge, as well as interindividual variations. For example, carnobacteria were only isolated from the hindgut region of fish fed soybean oil and linseed oil before challenge, whereas Carnobacterium spp. and Carnobacterium funditum‐like species were isolated from fish fed the same oils after challenge. Three non‐motile Aeromonas spp. were isolated from infected fish fed marine oil. One of these isolates was identified as identical to A. salmonicida ssp. salmonicida used in&the challenge test by microbial fingerprinting (amplified fragment length polymorphism). Electron microscopic examinations of hindgut regions demonstrated substantial numbers of bacterial cells associated with enterocytes, but bacterial colonization of the enterocyte surface varied between different rearing groups. The potential of bacteria found associated with the hindgut region to inhibit the fish pathogens A. salmonicida, Vibrio salmonicida and Vibrio anguillarum differed between rearing groups.     

Health status of two salmonid aquaculture facilities in North Portugal: characterization of the bacterial and viral pathogens causing notifiable diseases

A comparative bacteriological and virological survey was conducted in two fish farms in the North of Portugal. The fish species examined included cultured rainbow trout, Oncorhynchus mykiss (Walbaum), and brown trout, Salmo trutta L., as well as wild fish captured near both facilities. The microbial load in the internal organs of apparently healthy fish was nonitored over a year, an all the disease problems occurring during this period were investigated. Although both farms presented intermediate levels of infection(30–40% infected fish), farm B showed the poorest microbiological quality since constant but low mortalities were observed throughout the year. Flavobacterium and Psedomonas-Xanthomonas were the predominant bacterial groups, comprising around 40–50% of the isolates from each farm. In farm B, members of the Enterobacteriaceae and mortile Aeromonas also showed significant prevalence (about 20%). The only outbreak of a notifiable disease was an occurrence of furunculosis, caused by Aeromonas salmonicida subsp. salmoncida, in farm A. However, Yersinia ruckeri was isolated not only from diseased fish, but also from asymptomatic fish, usually in mixed infections with motile Aeromonas or infections with motile Aeromonas or infectious pancreatic necrosis virus (IPNV). While Y. ruckeri isolates associated with mortalities belonged to the serotype O1 (subgroup a), those isolated from asymtomatic fish corresponded to serotype O3. Two strains of IPNV (serotype Ab) were isolated in farm B, which represents the first viral agent detected in Portuguese aquaculture. Qualitative and quantitative differences in microbial load were observed between cultured and wild fish. No notifiable bacterial or viral pathogens were detected in any of the feral species studied.     

The role and mechanism of icmF in Aeromonas hydrophila survival in fish macrophages

Survival in host macrophages is an effective strategy for pathogenic bacteria to spread. Aeromonas hydrophila has been found to survive in fish macrophages, but the mechanisms remain unknown. In this paper, the roles and possible mechanisms of IcmF in bacterial survival in fish macrophages were investigated. First, a stable silencing strain icmF‐RNAi was constructed by shRNA and RT‐qPCR confirmed the expression of icmF was down‐regulated by 94.42%. The expression of Hcp, DotU and VgrG was also decreased in icmF‐RNAi. The intracellular survival rate of the wild‐type strain was 92.3%, while the survival rate of icmF‐RNAi was only 20.58%. The escape rate of the wild‐type strain was 20%, while that of the icmF‐RNAi was only 7.5%. Further studies indicated that the expression of icmF can significantly affect the adhesion, biofilm formation, motility and acid resistance of A. hydrophila, but has no significant effect on the growth of A. hydrophila even under the stress of H₂O₂. The results indicated that IcmF of A. hydrophila not only acts as a structural protein which participates in virulence‐related characteristics such as bacterial motility, adhesion and biofilm formation, but also acts as a key functional protein which participates in the interaction between bacteria and host macrophages.     

The effect of piscidin antimicrobial peptides on the formation of Gram-negative bacterial biofilms

Fish-derived antimicrobial peptides are an important part of the innate immune system due to their potent antimicrobial properties. Piscidins are a class of antimicrobial peptides first described in hybrid striped bass (Morone chrysops x Morone saxatilis) but have also been identified in many other fish species. Previous work demonstrated the broad antimicrobial activity of piscidins against Gram-negative and Gram-positive bacterial species. This study sought to determine the extent to which class I (striped bass piscidin 1, white bass piscidin 1 and striped bass/white bass piscidin 3) and class II (striped bass piscidin 4 and white bass piscidin 5) piscidins inhibit biofilm formation of different Gram-negative bacteria. In general, the class I and II piscidins demonstrate potent activity against Escherichia coli and Flavobacterium columnare biofilms. The class II piscidins showed more activity against E. coli and F. columnare isolates than did the class I piscidins. The piscidins in general were much less effective against inhibiting Aeromonas hydrophila and A. veronii biofilm growth. Only the class I piscidins showed significant growth inhibition among the Aeromonas spp. examined.     

Indigenous AHL‐degrading bacterium Bacillus firmus sw40 affects virulence of pathogenic Aeromonas hydrophila and disease resistance of gibel carp

Interrupting quorum sensing represents a novel anti‐infective strategy to combat bacterial pathogen, and biodegradation of quorum sensing signal AHLs has been proved to be an efficient way to control pathogenic Gram‐negative bacteria in aquaculture. In this study, the effect of Bacillus firmus sw40 as efficient AHL‐degrading strain on virulence of fish pathogen Aeromonas hydrophila and disease resistance of gibel carp Carassius auratus gibelio was investigated. The results demonstrated that in vitro the B. firmus sw40 extracellular production (ECP) was able to significantly decrease protease production, haemolytic activity and biofilm formation in A. hydrophila. Dietary administration of B. firmus sw40 (10⁹ CFU/g) for 4 weeks significantly reduced the inflammatory cytokines TNF‐1a, TNF‐2a and IFN‐γ genes expression, antioxidant parameter MDA and GSH levels in serum and increased antioxidant enzyme SOD activity. Besides, B. firmus sw40 could significantly increase the survival of gibel carp with pathogenic A. hydrophila infection.     

Intestinal bacterial flora of Mediterranean gilthead sea bream (Sparus aurata Linnaeus) as a novel source of natural surface active compounds

Microbial communities play different functions in the digestive tract of fish and represent a source of bioactive compounds. This study was aimed to detect the presence of biosurfactant (BS)‐producing bacteria in the intestine of gilthead sea bream (Sparus aurata L.) to select strains candidate for biotechnological applications in aquaculture. A total of 100 bacterial strains were isolated from the guts of three groups of fish and analysed by different qualitative screening tests and the tensiometric analysis. BSs were extracted, characterized by thin‐layer chromatography (TLC) and their antibacterial activity against bacterial pathogens of interest in aquaculture was determined. A total of 17 out of 100 strains, affiliated to the genera Pseudomonas (11 isolates), Acinetobacter (1 isolate), Sphingomonas (4 isolates) and Aeromonas (1 isolate), displayed a stable emulsion production with the E₂₄ index from 0% to 44%, a surface tension reduction from 8.3 to 30 mN/m and 11 of them exhibited antibacterial activity against fish pathogens. The TLC analysis indicated that the intestinal BSs consisted of compounds ascribed to the class of glycolipids–rhamnolipids. This is the first study reporting on the isolation and selection of BS‐producing bacteria from fish gut, a natural novel source of surface active compounds with potential biotechnological applications in aquaculture.     

Antibiotics modulate biofilm formation in fish pathogenic isolates of atypical Aeromonas salmonicida

Atypical Aeromonas salmonicida causes furunculosis infections of non-salmonid fish, which requires antibiotic therapy. However, antibiotics may induce biofilm in some bacteria, which protects them against hostile conditions while allowing them to persist on surfaces, thus forming a reservoir for infection. The aim of this study was to determine whether atypical isolates of A. salmonicida increased biofilm in the presence of two antibiotics, florfenicol and oxytetracycline. A microtitre plate assay was used to quantify biofilm in the presence and absence of each antibiotic. Fifteen of 28 isolates formed biofilms under control conditions, while 23 of 28 isolates increased biofilm formation in the presence of at least one concentration of at least one antibiotic. For oxytetracycline, the most effective concentration causing biofilm to increase was one-quarter of that preventing visible bacterial growth, whereas for florfenicol it was one-half of this value. This is the first study to demonstrate that a bacterial pathogen of fish increases biofilm in response to antibiotics. Biofilm formation may increase the risk of re-infection in culture systems and this lifestyle favours the transmission of genetic material, which has implications for the dissemination of antibiotic-resistance genes and demonstrates the need for enhanced disease prevention measures against atypical A. salmonicida.     

Maltoporin (LamB protein) contributes to the virulence and adhesion of Aeromonas veronii TH0426

Aeromonas veronii is one of the main pathogens causing freshwater fish sepsis and ulcer syndrome. More and more cases have shown that it has become an important zoonotic and aquatic agent. In this study, a A. veronii TH0426 mutant strain (ΔlamB) with an in‐frame deletion removed nucleotides 10–1,296 of the lamB gene was firstly constructed to investigate its functions. The results showed that the LD₅₀ value of the mutant ΔlamB to zebrafish and mice was 13.7‐fold and 5.6‐fold higher than those of the wild‐type strain, respectively. The toxicity of wild‐type strain to EPC cells was 2.1‐fold and threefold higher than those of ?lamB when infected for 1 and 2 hr. Furthermore, the ability of biofilm formation and the adhesion and invasion to EPC cells of ?lamB significantly decreased for 5.6‐fold and 1.8‐fold separately. In addition, motility detection result indicated that ?lamB lost the swimming ability. The results of flagellar staining and TEM demonstrated that the flagella of ?lamB were shed. In general, the deletion of lamB gene caused a significant decrease in the virulence and adhesion of A. veronii TH0426, and it can be known that the lamB gene of A. veronii plays a crucial role in the pathogenesis.     

Polyphasic characterization of Aeromonas salmonicida isolates recovered from salmonid and non‐salmonid fish

Michigan's fisheries rely primarily upon the hatchery propagation of salmonid fish for release in public waters. One limitation on the success of these efforts is the presence of bacterial pathogens, including Aeromonas salmonicida, the causative agent of furunculosis. This study was undertaken to determine the prevalence of A. salmonicida in Michigan fish, as well as to determine whether biochemical or gene sequence variability exists among Michigan isolates. A total of 2202 wild, feral and hatchery‐propagated fish from Michigan were examined for the presence of A. salmonicida. The examined fish included Chinook salmon, Oncorhynchus tshawytscha (Walbaum), coho salmon, O. kisutcha (Walbaum), steelhead trout, O. mykiss (Walbaum), Atlantic salmon, Salmo salar L., brook trout, Salvelinus fontinalis (Mitchill), and yellow perch, Perca flavescens (Mitchill). Among these, 234 fish yielded a brown pigment‐producing bacterium that was presumptively identified as A. salmonicida. Further phenotypic and phylogenetic analyses identified representative isolates as Aeromonas salmonicida subsp. salmonicida and revealed some genetic and biochemical variability. Logistic regression analyses showed that infection prevalence varied according to fish species/strain, year and gender, whereby Chinook salmon and females had the highest infection prevalence. Moreover, this pathogen was found in six fish species from eight sites, demonstrating its widespread nature within Michigan.     

Characterization of phenotype variations of luminescent and non‐luminescent variants of Vibrio harveyi wild type and quorum sensing mutants

Vibrio harveyi, a luminescent Gram‐negative motile marine bacterium, is an important pathogen responsible for causing severe diseases in shrimp, finfish and molluscs leading to severe economic losses. Non‐luminescent V. harveyi obtained by culturing luminescent strains under static and dark condition were reported to alter the levels of virulence factors and metalloprotease gene and luxR expression when compared to their luminescent variants. Presently, we conducted an in vitro study aiming at the characterization of virulence‐related phenotypic traits of the wild‐type V. harveyi BB120 strain and its isogenic quorum sensing mutants before and after switching to the non‐luminescent status. We measured the production of caseinase, haemolysin and elastase and examined swimming motility and biofilm formation. Our results showed that switching from the bioluminescent to the non‐luminescent state changed the phenotypic physiology or behaviour of V. harveyi resulting in alterations in caseinase and haemolytic activities, swimming motility and biofilm formation. The switching capacity was to a large extent independent from the quorum sensing status, in that quorum sensing mutants were equally capable of making the phenotypic switch.     

Sickeningly Sweet: L‐rhamnose stimulates Flavobacterium columnare biofilm formation and virulence

Flavobacterium columnare, the causative agent of columnaris disease, causes substantial mortality worldwide in numerous freshwater finfish species. Due to its global significance and impact on the aquaculture industry continual efforts to better understand basic mechanisms that contribute to disease are urgently needed. The current work sought to evaluate the effect of L‐rhamnose on the growth characteristics of F. columnare. While we initially did not observe any key changes during the total growth of F. columnare isolates tested when treated with L‐rhamnose, it soon became apparent that the difference lies in the ability of this carbohydrate to facilitate the formation of biofilms. The addition of different concentrations of L‐rhamnose consistently promoted the development of biofilms among different F. columnare isolates; however, it does not appear to be sufficient as a sole carbon source for biofilm growth. Our data also suggest that iron acquisition machinery is required for biofilm development. Finally, the addition of different concentrations of L‐rhamnose to F. columnare prior to a laboratory challenge increased mortality rates in channel catfish (Ictalurus punctatus) as compared to controls. These results provide further evidence that biofilm formation is an integral virulence factor in the initiation of disease in fish.     

Simple and direct detection of Aeromonas hydrophila infection in the goldfish, Carassius auratus (L.), by dot blotting using specific monoclonal antibodies

A combination of eight isolates of Aeromonas hydrophila was used to produce monoclonal antibodies (MAbs). Ten different groups of MAbs specific to Aeromonas were selected. The first five groups of MAbs demonstrated high specificity and bound to only one or two isolates of A. hydrophila. The sixth and the seventh groups of MAbs were A. hydrophila specific. They recognized seven of eight A. hydrophila isolates (AH1, 2, 3, 4, 5, 6, 8); however, the MAb in the seventh group also showed cross‐reactivity to one isolate of Aeromonas caviae (AC3). The eighth MAb group recognized two isolates of A. hydrophila (AH2 and AH5) and demonstrated cross‐reactivity to one isolate of Aeromonas sobria (AS1) and one isolate of A. caviae (AC3). The tenth group of MAbs bound to all isolates of Aeromonas spp. tested (AH1‐8, AS1‐6, AC1‐5, Aeromonas veronii and Aeromonas jandaei) without cross‐reactivity to any of the other bacteria tested. MAbs in the ninth group showed similar specificity to those in the tenth group but did not recognize two isolates of A. sobria (AS4 and AS6) or A. jandaei. All the MAbs could be used to identify Aeromonas by dot blotting with a sensitivity ranging from 10⁵ to 10⁷ CFU mL^?1. However, the sensitivity of detection was increased to 10²–10³ CFU mL^?1 after inoculation of the sample in tryptic soy broth for 3–6 h before performing the dot blotting. The dot blot method can be used for the direct detection of A. hydrophila infection in symptomatic and asymptomatic goldfish. This study demonstrated a convenient immunological tool that can be used for the direct detection of A. hydrophila and Aeromonas infections in a complex sample without the requirement for separation of the bacteria or isolation and biochemical tests.     

Tilapia recirculating aquaculture systems as a source of plant growth promoting bacteria

A recirculating aquaculture system with farmed tilapia is the most popular combination in aquaponics, an integration of aquaculture and hydroponics. Despite nutrient‐rich fish‐rearing water being regarded as a valuable resource for aquaponics, the quality and value of inhabitant microorganisms are certainly understudied. Our present research illustrates the feasibility of the tilapia‐rearing water as a valuable source of beneficial microorganisms called plant growth promoting bacteria (PGPB). Microbial communities were examined with a combination of culture‐independent high‐throughput 16S rRNA gene sequencing and cultivation methods. Microbial communities determined using high‐throughput sequencing indicated the usefulness of Bacteroidetes and Alphaproteobacteria as beneficial microbial indicators to assess the health condition of recirculating aquaculture systems. Siderophore production, ammonia production and phosphate solubilization assays were used for screening and 41% of isolates were identified as plant growth promoting bacteria. These bacteria were classified as Actinobacteria (eight strains [32% in total], Dietzia, Gordonia, Microbacterium, Mycobacterium and Rhodococcus), Bacilli (six strains [24%], Bacillus and Paenibacillus), Flavobacteriia (one strain [4%], Myroides), Betaproteobacteria (two strains [8%], Acidovorax and Chromobacterium) and Gammaproteobacteria (eight strains [32%], Aeromonas, Plesiomonas and Pseudomonas). We found that the tilapia‐rearing water naturally contained various lineages of PGPB and could be esteemed as a worthy seed bank of PGPB. Because aquaponics is a difficult system to use pesticides and herbicides, the role of PGPB to prevent plant pathogens and maintain healthy root system may be more important than traditional agricultural settings.     

Effect of quorum quenching bacteria on growth,virulence factors and biofilm formation of Yersinia ruckeri in vitro and an in vivo evaluation of their probiotic effect in rainbow trout

Five N‐acyl homoserine lactone‐degrading bacteria (quorum quenching (QQ) strains) were selected to evaluate their impacts on growth, virulence factors and biofilm formation in Yersinia ruckeri in vitro. No difference was observed among the growth pattern of Y. ruckeri in monoculture and coculture with the QQ strains. To investigate the regulation of virulence factors by quorum sensing in Y. ruckeri, cultures were supplemented with 3oxo‐C8‐HSL. The results indicated that swimming motility and biofilm formation are positively regulated by QS (p < 0.05), whereas caseinase, phospholipase and haemolysin productions are not influenced by 3oxo‐C8‐HSL (p > 0.05). The QQs were able to decrease swimming motility and biofilm formation in Y. ruckeri. QQ bacteria were supplemented to trout feed at 10⁸ CFU/g (for 40 days). Their probiotic effect was verified by Y. ruckeri challenge either by immersion or injection in trout. All strains could significantly increase fish survival with Bacillus thuringiensis and Citrobacter gillenii showing the highest and lowest relative percentage survival (RPS) values (respectively, 85% and 38%). Besides, there was no difference between the RPS values by either immersion or injection challenge expect for B. thuringiensis. The putative involvement of the QQ capacity in the protection against Yersinia is discussed.     

The influence of salmon surface mucus on the growth of Flavobacterium columnare

Flavobacterium columnare is the causative agent of columnaris disease. The presence of lesions on the gills, skin and fins of diseased fish suggests that F. columnare is able to utilize fish skin mucus as a substrate for growth and that exposure to this material would alter the expression of genes involved in the colonization of the outer surfaces of the fish. Growth, biofilm formation, extracellular protease production and changes in protein expression of F. columnare strain C#2 cultured in media supplemented with juvenile Atlantic salmon skin mucus were compared with the same media without mucus. C#2 was able to grow by using mucus as the sole nutrient source. Growth in mucus-containing media induced cells to grow as a biofilm and extracellular protease activity increased in mucus-containing cultures. SDS-PAGE protein profiles showed that expression of six extracellular proteins increased in mucus-containing media. These results demonstrate that salmon surface mucus promotes the growth of F. columnare and that exposure to mucus alters the growth characteristics of this bacterium with regard to protease production and biofilm formation. Further characterization of mucus-induced physiological changes will increase our understanding of the basis of virulence of this economically important fish pathogen.