Unravelling the genomic secrets of bacterial fish pathogens: a roadmap to aquaculture sustainability.

In the field of aquaculture, bacterial pathogens pose significant challenges to fish health and production. Advancements in genomic technologies have revolutionized our understanding of bacterial fish pathogens and their interactions with their host species. This review explores the application of genomic approaches in the identification, classification, and characterization of bacterial fish pathogens. Through an extensive analysis of the literature, we have compiled valuable data on 79 bacterial fish pathogens spanning 13 different phyla, encompassing their whole genome sequences. By leveraging high-throughput sequencing techniques, researchers have gained valuable insights into the genomic makeup of these pathogens, enabling a deeper understanding of their virulence factors and mechanisms of host interaction. Furthermore, genomic approaches have facilitated the discovery of potential vaccine and drug targets, opening up new avenues for the development of effective interventions against fish pathogens. Additionally, the utilization of genomics in fish disease resistance and control in aquaculture has shown promising results, enabling the identification of genetic markers associated with disease resistance traits. This review highlights the significant contributions of genomics to the field of fish pathogen research and underscores its potential for improving disease management strategies and enhancing the sustainability of aquaculture practices.

The pathology of Lates calcarifer herpesviral disease-Disseminated intravascular coagulation explains mortality spikes.

Lates calcarifer herpesvirus (LCHV) causes an emerging serious disease in aquaculture. Sudden drops in feed rates and mortality spikes exceeding 40%-50% often accompany LCHV infections in juvenile L. calcarifer, soon after transfer into sea cages. Affected fish have patchy white skin and fins, corneal opacity and frequently hang in surface water column like 'ghost' or 'zombie' fish. Fish have pale gills, fluid-filled intestines with yellowish casts, lipid depleted liver, enlarged spleen and kidney and reddened brain. Epithelial hyperplasia, apoptosis, marginated nuclear chromatin, amphophilic intranuclear inclusion bodies and the occasional multinucleated cells are observed in gills, skin, intestines, liver and kidney. These are often accompanied by lymphocytic-monocytic infiltration and extensive necrosis in gills, skin, kidney and intestines. Martius scarlet blue stains indicate presence of fibrin in vasculature in brain, gills, intestines, kidney and liver, or disseminated intravascular coagulation (DIC). DIC has been reported in human herpesviral infections. Multifocal lifting of intestinal epithelium with proteinaceous exudate and necrosis of several adjacent villi often progress to involve entire gut sections. Atrophied livers with accentuated lobules may progress to marked loss of hepatic acini. Multifocal dilated attenuated renal tubules are often accompanied by casts and marked protein loosing renopathy. This study on LCHV demonstrates that it can cause significant pathology and mortality.

HRFSVM: identification of fish disease using hybrid Random Forest and Support Vector Machine.

Aquaculture fish diseases pose a serious threat to the security of food supplies. Fish species vary widely, and because they resemble one another so much, it is challenging to distinguish between them based solely on appearance. To stop the spread of disease, it is important to identify sick fish as soon as possible. Due to a lack of necessary infrastructure, it is still difficult to identify infected fish in aquaculture at an early stage. It is essential to promptly identify sick fish to stop the spread of disease. The purpose of this work is to suggest a machine learning technique based on the DCNN method for identifying and categorizing fish diseases. To solve problems involving global optimization, this paper suggests a brand-new hybrid algorithm called the Whale Optimization Algorithm with Genetic Algorithm (WOA-GA) and Ant Colony Optimization. In this work, for classification, the hybrid Random Forest algorithm is used. To facilitate the increased quality, distinctions between both the proposed WOA-GA-based DCNN architecture and the presently used methods for machine learning have been made. The effectiveness of the proposed detection technique is done with MATLAB. Performance metrics like sensitivity, specificity, accuracy, precision, recall, F-measure, NPV, FPR, FNR, and MCC are compared to the performance of the proposed technique.

Distribution and prevalence of the myxozoan parasite Tetracapsuloides bryosalmonae in northernmost Europe: analysis of three salmonid species.

Global climate change is altering the abundance and spread of many aquatic parasites and pathogens. Proliferative kidney disease (PKD) of salmonids caused by the myxozoan Tetracapsuloides bryosalmonae is one such emerging disorder, and its impact is expected to increase with rising water temperature. Yet, the distribution and prevalence of T. bryosalmonae in Northern Europe remain poorly characterized. Here, we studied 43 locations in 27 rivers in northernmost Norway and Finland to describe T. bryosalmonae infection frequency and patterns in 1389 juvenile salmonids. T. bryosalmonae was discovered in 12 out of 27 rivers (44%) and prevalence ranged from 4.2 to 55.5% in Atlantic salmon and from 5.8 to 75% in brown trout among infected rivers. In sympatric populations, brown trout was more frequently infected with T. bryosalmonae than was salmon. Age-specific parasite prevalence patterns revealed that in contrast to lower latitudes, the infection of juvenile fish predominantly occurs during the second summer or later. Temperature monitoring over 2 yr indicated that the mean water temperature in June was 2.1 to 3.2°C higher in rivers containing T. bryosalmonae compared to parasite-free rivers, confirming the important role of temperature in parasite occurrence. Temporal comparison in T. bryosalmonae prevalence over a 10 yr period in 11 rivers did not reveal any signs of contemporary parasite spread to previously uninfected rivers. However, the wide distribution of T. bryosalmonae in rivers flowing to the Barents Sea indicates that climate change and heat waves may cause new disease outbreaks in northern regions.

Molecular and morphological characterization of Dollfustrema bagarii (Digenea: Bucephalidae) metacercariae from aquaculture channel catfish (Ictalurus punctatus) in northern Vietnam.

White grub metacercariae were found in the livers and kidneys of diseased specimens of an introduced channel catfish, Ictalurus punctatus (Rafinesque), in Vietnam. Based on morphological features and 28S rDNA sequence analysis, the isolated metacercariae were identified as Dollfustrema bagarii (Digenea: Bucephalidae) Moravec & Sey. Histopathological examination shows that encysted metacercariae can change the tissue structure of the infected organs and is often accompanied by haemorrhaging and the presence of eosinophilic granular cell infiltration. Degenerative changes were also observed.

Kinetics of the invasion of a non-phagocytic fish cell line, RTG-2 by Yersinia ruckeri serotype O1 biotype 1.

Yersiniosis, caused by the fish pathogen Yersinia ruckeri, is a serious bacterial septicaemia affecting mainly salmonids worldwide. The acute infection may result in high mortality without apparent external disease signs, while the chronic one causes moderate to considerable mortality. Survivors of yersiniosis outbreaks become carriers. Y. ruckeri is able to adhere to, and to invade, phagocytic and non-phagocytic fish cells by using unknown molecular mechanisms. The aim of this study was to describe the kinetics of cell invasion by Y. ruckeri serotype O1 biotype 1 in a fish cell line (RTG-2) originating from rainbow trout gonads. The efficiency of invasion by Y. ruckeri was found to be temperature dependent, having a maximum at 20 °C. The bacterium was able to survive up to 96 h postinfection. The incubation of the cells at 4 °C and the pre-incubation of the bacteria with sugars or heat-inactivated antiserum significantly decreased the efficiency of invasion or even completely prevented the invasion of RTG-2 cells. These findings indicate that Y. ruckeri is capable of adhering to, entering and surviving within non-phagocytic cells, and that the intracellular environment may constitute a suitable niche for this pathogen that can favour the spread of infection and/or the maintenance of a carrier state of fish.

Effects of dietary marjoram, Origanum majorana extract on growth performance, hematological, antioxidant, humoral and mucosal immune responses, and resistance of common carp, Cyprinus carpio against Aeromonas hydrophila.

The present study aimed to investigate the potential effects of dietary marjoram extract on growth performance, health, and disease resistance in common carp, Cyprinus carpio. To this purpose, the fish were assigned into four treatments and fed by diets supplemented with 0 (control), 100, 200, and 400 mg marjoram extract kg-1 over eight weeks and then challenged with Aeromonas hydrophila. According to the results, 200 mg kg-1 dietary marjoram extract inclusion showed the highest final weight, weight gain, and specific growth rate, and lowest feed conversion ratio (FCR). White blood cell number, Red blood cells, hematocrit, hemoglobin, mean corpuscular volume and mean corpuscular hemoglobin were markedly increased particularly at 200 mg kg-1 treatment. Marjoram extract significantly increased plasma superoxide dismutase activity and decreased malondialdehyde level compared to the control treatment. Plasma complement and lysozyme activities and total immunoglobulin levels, mucosal complement, lysozyme and alkaline phosphatase activities and immunoglobulin levels were significantly increased compared to the control group. The lowest post-challenge survival rate was observed in the control treatment, whereas the highest value was related to the 200 mg kg-1 marjoram treatment. In conclusion, the present study demonstrated that, marjoram extract is a suitable feed supplements for common carp, as it stimulates the fish growth, antioxidant, and immune systems, which suppress the fish mortality during Aeromonas septicemia. According to the results, 200 mg marjoram extract kg-1 is recommended for carp feed formulation.

Application of the FTA elute card coupled with visual colorimetric loop-mediated isothermal amplification for the rapid diagnosis of Streptococcus agalactiae in farmed tilapia (Oreochromis niloticus).

A method combining the FTA Elute card and visual colorimetric loop-mediated isothermal amplification (FTA-e/LAMP) was tested to diagnose Streptococcus agalactiae infections in vitro and in vivo. FTA-e/LAMP consists of two main steps: first, the FTA card is used to extract DNA and then a colorimetric loop-mediated isothermal amplification (LAMP) reaction is carried out on the extracted DNA. In vitro sensitivity was 1.9 x 102  CFU/mL, and regarding specificity, all nine S. agalactiae strains tested positive. All Streptococcus spp. tested negative, except for S. dysgalactiae, thereby indicating the need for another set of primers to distinguish this species from S. agalactiae. To diagnose S. agalactiae infections using FTA-e/LAMP in vivo, two experimental trials on juvenile Oreochromis niloticus infected with bovine or piscine strains were carried out. Sensitivity in symptomatic fish was 100%, and 50.7% of fish without signs were positive. All negative control fish tested negative (n = 28). No bacteria were detected after 16 days post-infection (dpi). Accuracy during the first week (1-7 dpi) was 89% and decreased to 44% thereafter (10-22 dpi). FTA-e/LAMP results suggest that this method is a promising tool for early and fast diagnosis of S. agalactiae on tilapia farms.

PCR, in-situ hybridization, and phylogenetic analysis suggest that 'big belly' disease in barramundi, Lates calcarifer (Bloch), is caused by a novel Vibrio species.

'Big belly' disease is a chronic, granulomatous bacterial enteritis and peritonitis, first reported in 3- to 4-week-old Asian seabass or barramundi, Lates calcarifer Bloch fry. Affected fry are emaciated and have a swollen abdomen, and the condition is referred to as 'skinny pot-belly' or 'big belly' disease. In this study, histopathological examinations of diseased fish from a batch of 2-month-old, 6- to 8-cm L. calcarifer fingerlings, kept in seawater recirculating aquaculture systems, showed pathology resembling 'big belly' disease. Ethanol-fixed tissues were tested positive using specific PCR primers based on 16S rRNA genes. In situ hybridization using dioxygenin-labelled positive PCR products on formalin-fixed paraffin-embedded tissues showed positive reactions with intralesional, clusters of the large, 'big belly' coccobacilli. A phylogenetic tree constructed based on analyses of these 16S rRNA gene PCR products from five positive fish suggests that the 'big belly' bacterium is most likely a novel Vibrio species.

Climate change-driven disease in sympatric hosts: Temporal dynamics of parasite burden and proliferative kidney disease in wild brown trout and Atlantic salmon.

Global climate change is altering the abundance and spread of various parasites, which has important consequences not only for host-parasite interactions but also for the relationships between different host species. Here, we focus on the myxozoan endoparasite Tetracapsuloides bryosalmonae that causes temperature-dependent proliferative kidney disease (PKD) in salmonids. We characterized the temporal changes in the parasite load and the severity of PKD signs (renal hyperplasia, haematocrit) in two sympatric populations of wild brown trout (Salmo trutta) and Atlantic salmon (Salmo salar). We found that both the parasite load and disease signs vary considerably between individuals, species, rivers and sampling periods. We showed that Atlantic salmon was able to slow down the initial parasite proliferation rate and subsequently tolerate high parasite burden without obvious disease signs. In contrast, the initial parasite proliferation rate was much higher in brown trout, which was followed by the development of severe PKD signs. Thus, the speed of parasite proliferation, rather than the absolute number of the parasites in the host kidney, may play an important role in interspecific variation in PKD susceptibility. To conclude, this study illustrates the usefulness of temporal perspective for understanding host defence mechanisms and climate change-mediated impacts in the wild.

Molecular identification and phylogenetic analysis of fungal pathogens isolated from diseased fish in Xinjiang, China.

The outbreaks of fungal diseases in cultured fish have been severe in recent years, which is harmful to the healthy and sustainable development of fish farming. In this study, an investigation was conducted for significant fungal infections of 12 species of fish in four regions in Xinjiang, China, to understand the distribution of local fish fungal pathogens. Twenty-six fungal strains with pathogenicity were isolated, and the challenge experiment showed that eight strains from Changji area had high infection rate to fish eggs. Based on internal transcribed spacer sequence data and molecular analysis, the 26 strains were classified into nine different species of six fungal genera. Phylogenetic analysis showed that all strains were divided into two clades, namely Cluster 1 (contains only the genus Mucor) and Cluster 2 (consists of five small branches), and the distribution of strains from the same region was scattered in two clusters. There is no strict host selectivity for these fungi to infect fish. Mucor sp. are the main fungal pathogen of fish in these four regions, whereas Hypophthalmichthys molitrix and Carassius auratus are two types of fish that were susceptible to pathogen. In addition, the environmental adaptability experiments showed that eight highly pathogenic strains have different adaptability to the environment, and their optimum temperature and pH were 25°C and 7.0, respectively, whereas the concentration of NaCl was negatively correlated with the growth of strains. Therefore, these results indicated that the coinfection of multiple fungal pathogens in a culture region should be considered in the future study.

Epitheliocystis in wild marine fishes and its relation with anthropogenic pollution.

Epitheliocystis is a fish disease with recorded in increasing numbers globally. Here we show that (a) pollution-exposed wild marine fishes have higher infection prevalence than nonexposed individuals from reference sites, suggesting that anthropogenic pollution predisposes individuals to this disease in natural systems, (b) the effect of pollution on infection status did not vary significantly between two sympatric fish species, and (c) infected and noninfected individuals from both species did not differ significantly in selected immune and general-health parameters, a result that deserves further study with increased sample sizes.

Effects of thymol supplementation on performance, mortality and branchial energetic metabolism in grass carp experimentally infected by Aeromonas hydrophila.

We determined whether thymol supplementation of would minimize the negative effects of Aeromonas hydrophila infection on branchial energy metabolism, weight loss and mortality in grass carp (Ctenopharyngodon idella). We found that the infected fish all died, while 62.5% of those supplemented with 100 mg/kg thymol survived. Cytosolic and mitochondrial creatine kinase (CK) activities, as well as adenylate kinase (AK) and pyruvate kinase (PK) activities were significant lower in gills of A. hydrophila-infected fish than those of the control group, and adenosine triphosphate (ATP) levels were significant lower in the infected group. Finally, branchial reactive oxygen species (ROS) were significant higher in A. hydrophila-infected fish than in the control group. Supplementation with 100 and 300 mg thymol/kg diet prevented inhibition of branchial cytosolic and mitochondrial CK activities caused by infection, and also inhibited the reduction of branchial ATP levels. Supplementation with 100, 200 and 300 mg thymol/kg prevented the inhibition of branchial AK and PK activities induced by aeromonosis. Supplementation of 100 mg thymol/kg prevented weight loss after A. hydrophila infection. These data suggest that supplementation with 100 mg thymol/kg exerts potent bactericidal properties and augments longevity. Supplementation at all concentrations of thymol prevented A. hydrophila-induced branchial bioenergetics; nevertheless, higher concentrations were associated with side-effects.

Purinergic signaling and gene expression of purinoceptors in the head kidney of the silver catfish Rhamdia quelen experimentally infected by Flavobacterium columnare.

The head kidney is a lymphoid immune organ that plays a key role in the immune and inflammatory responses of teleost fish. It is associated with immunoglobulin G production and differentiation of B cells. The presence of a multi-enzymatic complex found anchored in the plasma membrane makes the head kidney an important purinergic-dependent tissue. Purinergic signaling has been associated with these responses under pathological conditions via regulation of extracellular adenosine triphosphate (ATP), the main damage molecular associated pattern agent released during bacterial infections. The aim of this study was to determine whether purinergic signaling is a pathway associated with impairment of immune responses in silver catfish (Rhamdia quelen) experimentally infected by Flavobacterium columnare, as well as to evaluate the role of P2 purine receptors in this response. Triphosphate diphosphohydrolase (NTPDase) activity in the head kidney was significantly lower in silver catfish experimentally-infected F. columnare 72 h post-infection (hpi) than in the control group, while no significant difference was observed with respect NTPDase activity on adenosine diphosphate, as well as on 5'-nucleotidase and adenosine deaminase activities. Extracellular ATP levels were significantly higher in the head kidney of experimentally-infected fish than in the control group at 72 hpi. Finally, p2ry11 and p2rx3 purine receptor levels were significantly higher in experimentally-infected fish than in the control group at 72 hpi. We conclude that purinergic signaling in the head kidney of silver catfish infected by F. columnare creates a pro-inflammatory profile that may contribute to impairment of immune and inflammatory responses via reduction of ATP hydrolysis and its accumulation in the extracellular milieu, accompanied by upregulation of p2ry11 and p2rx3 purine receptors, leading to pro-inflammatory status.

Current use and development of fish vaccines in China.

In the past decades, the aquaculture industry made great progress in China, which contributes more than 70% yield of the world's farmed fish. Along with the rapid growth of fish production, increased emergence and outbreak of numbers of diseases pose harm to the aquaculture industry and food safety. From the efficient, safe, environmental and ethical aspects, vaccines is definitely the most appropriate and focused method to control different kinds of fish diseases. In China, researchers have done huge works on the fish vaccines, and so far six domestic aquatic vaccine products along with one imported aquatic vaccine have obtained the national veterinary medicine certificate. More critically, some new vaccines have also entered the field experiment stage and showed broad market prospects. In the present review, authors summarize seven aquatic vaccines, including the live vaccine against grass carp hemorrhagic disease, the inactivated vaccine against Aeromonas hydrophila sepsis in fish, the live vaccine against Edwardsiella tarda in turbot, the anti-idiotypic antibody vaccine against Vibrio alginolyticus, V. parahaemolyticus, and E. tarda in Japanese flounder, the cell-cultured inactivated vaccine against grass carp hemorrhagic disease, the inactivated vaccine against fish infectious spleen and kidney necrosis virus (ISKNV), and the genetically engineered live vaccine against V. anguillarum in turbot. Moreover, different delivery routes of fish vaccines are also compared in this review, along with differential fish immune response after vaccination. All these efforts will ultimately benefit the healthy and sustainable development of aquaculture industry in China.

Identification of bacterial pathogens in cultured fish with a custom peptide database constructed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

BACKGROUND: The majority of infectious diseases of cultured fish is caused by bacteria. Rapid identification of bacterial pathogens is necessary for immediate management. The present study developed a custom Main Spectra Profile (MSP) database and validate the method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid identification of fish bacterial pathogens. Streptococcus agalactiae, Streptococcus iniae, Aeromonas hydrophila, Aeromonas veronii, and Edwardsiella tarda obtained from diseased fish were used as representative bacterial pathogens in this study. Bacterial peptides were extracted to create a Main Spectra Profile (MSP), and the MSPs of each bacterial species was added into the MALDI Biotyper database. Fifteen additional isolates of each bacterial species were tested to validate the utilized technique. RESULTS: The MSPs of all field isolates were clearly distinguishable, and the MSPs of the same species were clustered together. The identification methodology was validated with 75 bacterial isolates. The reliability and specificity of the method were determined with MALDI Biotyper log score values and matching results with 16 s rDNA sequencing. The species identification using the public MALDI Biotyper library (Bruker MALDI Biotyper) showed unreliable results (log score < 2.000) with 42.67% matching result with the reference method. In contrast, accurate identification was obtained when using the custom-made database, giving log score > 2.115, and a 100% matching result. CONCLUSION: This study demonstrates an effective identification of fish bacterial pathogens when a complete custom-made MSP database is applied. Further applications require a broad, well-established database to accommodate prudent identification of many fish bacterial pathogens by MALDI-TOF MS.

Prevalence, intensity and histopathology of Zeylanicobdella arugamensis infestation on groupers reared on different aquaculture systems.

The marine leech, Zeylanicobdella arugamensis, is a major threat to aquaculture in grouper-producing countries including Indonesia. This study aimed at investigating prevalence, intensity and histopathology of the ectoparasite in humpback and hybrid groupers cultured in different rearing systems. A total of 260 groupers (60 humpback groupers and 200 hybrid groupers) were used for samples. The marine leech was observed on skin, fins, gills and mouth, followed by histopathological assay on the skin tissue. The results showed that prevalence of the leech in both groupers was higher when they were cultured in the floating net cages compared with the hatchery, p < .05. Furthermore, humpback grouper had a higher prevalence than hybrid grouper when they were cultured in a similar system, p < .05. Meanwhile, there was no significant difference in intensity between the two groupers, p > .05. Within the hybrid groupers, the highest prevalence was obtained from hybrid groupers reared in the earthen ponds. Histopathological studies showed that the infected groupers exhibited inflammation, congestion and erosion of the epidermis layer. Hybrid grouper had more severe histopathological lesions in the skin tissues. These results suggested that species and type of aquaculture system had significantly determined the prevalence, intensity and severity of lesion in Z. arugamensis infestation.

Characterization of a novel Yersinia ruckeri serotype O1-specific bacteriophage with virulence-neutralizing activity.

A lytic bacteriophage (φNC10) specific to serotype O1 Yersinia ruckeri has been identified and evaluated as a model to assess the potential use of bacteriophages and their products for disease control in aquaculture. Electron microscopy of purified φNC10 revealed a virion particle with a small (70 nm) polyhedral head and short tail. φNC10 infected only serotype O1 strains of Y. ruckeri and failed to bind a defined Y. ruckeri mutant strain lacking O1 lipopolysaccharides (O1-LPS), suggesting that φNC10 uses O1-LPS as its receptor. In addition, spontaneous φNC10-resistant mutants of Y. ruckeri exhibited defects in O1-LPS production and were sensitive to rainbow trout serum. Purified φNC10 displayed a polysaccharide depolymerase activity capable of degrading Y. ruckeri O1-LPS and thereby sensitizing Y. ruckeri to the bactericidal effects of rainbow trout serum. The φNC10-associated polysaccharide depolymerase activity also reduced the ability of Y. ruckeri cells to cause mortality following intraperitoneal injection into rainbow trout. These data demonstrate a potential utility of φNC10 and its associated polysaccharide depolymerase activity for Y. ruckeri disease prevention.

Different transcriptional response between susceptible and resistant common carp (Cyprinus carpio) fish hints on the mechanism of CyHV-3 disease resistance.

BACKGROUND: Infectious disease outbreaks form major setbacks to aquaculture production and to further development of this important sector. Cyprinid herpes virus-3 (CyHV-3) is a dsDNA virus widely hampering production of common carp (Cyprinus carpio), one of the most farmed fish species worldwide. Genetically disease resistant strains are highly sought after as a sustainable solution to this problem. To study the genetic basis and cellular pathways underlying disease resistance, RNA-Seq was used to characterize transcriptional responses of susceptible and resistant fish at day 4 after CyHV-3 infection. RESULTS: In susceptible fish, over four times more differentially expressed genes were up-regulated between day 0 and 4 compared to resistant fish. Susceptible and resistant fish responded distinctively to infection as only 55 (9%) of the up-regulated genes were shared by these two fish types. Susceptible fish elicited a typical anti-viral response, involving interferon and interferon responsive genes, earlier than resistant fish did. Furthermore, chemokine profiles indicated that the two fish types elicited different cellular immunity responses. A comparative phylogenetic approach assisted in chemokine copies annotation pointing to different orthologous copies common to bony-fishes and even carp-specific paralogs that were differentially regulated and contributed to the different response of these two fish types. Susceptible fish up-regulated more ccl19 chemokines, which attract T-cells and macrophages, the anti-viral role of which is established, whereas resistant fish up-regulated more cxcl8/il8 chemokines, which attract neutrophils, the antiviral role of which is unfamiliar. CONCLUSIONS: Taken together, by pointing out transcriptional differences between susceptible and resistant fish in response to CyHV-3 infection, this study unraveled possible genes and pathways that take part in disease resistance mechanisms in fish and thus, enhances our understanding of fish immunogenetics and supports the development of sustainable and safe aquaculture.

Large-Scale Analysis of Flavobacterium psychrophilum Multilocus Sequence Typing Genotypes Recovered from North American Salmonids Indicates that both Newly Identified and Recurrent Clonal Complexes Are Associated with Disease.

Flavobacterium psychrophilum, the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), causes significant economic losses in salmonid aquaculture, particularly in rainbow trout (Oncorhynchus mykiss). Prior studies have used multilocus sequence typing (MLST) to examine genetic heterogeneity within F. psychrophilum At present, however, its population structure in North America is incompletely understood, as only 107 isolates have been genotyped. Herein, MLST was used to investigate the genetic diversity of an additional 314 North American F. psychrophilum isolates that were recovered from ten fish host species from 20 U.S. states and 1 Canadian province over nearly four decades. These isolates were placed into 66 sequence types (STs), 47 of which were novel, increasing the number of clonal complexes (CCs) in North America from 7 to 12. Newly identified CCs were diverse in terms of host association, distribution, and association with disease. The largest F. psychrophilum CC identified was CC-ST10, within which 10 novel genotypes were discovered, most of which came from O. mykiss experiencing BCWD. This discovery, among others, provides evidence for the hypothesis that ST10 (i.e., the founding ST of CC-ST10) originated in North America. Furthermore, ST275 (in CC-ST10) was recovered from wild/feral adult steelhead and marks the first recovery of CC-ST10 from wild/feral fish in North America. Analyses also revealed that at the allele level, the diversification of F. psychrophilum in North America is driven three times more frequently by recombination than random nucleic acid mutation, possibly indicating how new phenotypes emerge within this species.IMPORTANCEFlavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), both of which cause substantial losses in farmed fish populations worldwide. To better prevent and control BCWD and RTFS outbreaks, we sought to characterize the genetic diversity of several hundred F. psychrophilum isolates that were recovered from diseased fish across North America. Results highlighted multiple F. psychrophilum genetic strains that appear to play an important role in disease events in North American aquaculture facilities and suggest that the practice of trading fish eggs has led to the continental and transcontinental spread of this bacterium. The knowledge generated herein will be invaluable toward guiding the development of future disease prevention techniques.