Persistence of heterologous Flavobacterium psychrophilum genetic variants in microcosms simulating fish farm and hatchery environments.

Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease, causes substantial economic losses in salmonid farms and hatcheries. Some multilocus sequence types (ST) of F. psychrophilum are more likely to be associated with fish farms and hatcheries, but it is unclear if these patterns of association represent genetic lineages that are more adapted to aquaculture environments. Towards elucidating the disease ecology of F. psychrophilum, the culturability of 10 distinct F. psychrophilum STs was evaluated for 13 weeks in three microcosms including sterilized well water, sterilized well water with commercial trout feed, or sterilized well water with raceway detritus. All STs remained culturable in each of the microcosms for at least 8 weeks, with bacterial concentrations often highest in the presence of raceway detritus. In addition, most (e.g., 90%) STs remained culturable for at least 13-weeks. Significant differences in log10 cfus were observed among STs, both within and between microcosms, suggesting potential variability in environmental persistence capacity among specific variants. Collectively, results highlight the ability of F. psychrophilum to not only persist for weeks under nutrient-limited conditions but also thrive in the presence of organic substrates common in fish farms and hatchery-rearing units.

Immersion challenge of three salmonid species (family Salmonidae) with three multilocus sequence typing variants of Flavobacterium psychrophilum provides evidence of differential host specificity.

Bacterial coldwater disease (BCWD), caused by Flavobacterium psychrophilum, results in significant losses among multiple salmonid (family Salmonidae) species. Molecular epidemiology and serotyping studies have suggested that some variants are host specific; however, these associations have not been evaluated by cross-challenging fish species with putatively host-associated F. psychrophilum isolates via more natural (i.e. immersion) exposure routes. To this end, F. psychrophilum isolates US19-COS, US62-ATS and US87-RBT, each originally recovered from diseased coho salmon (Oncorhynchus kisutch), Atlantic salmon (Salmo salar) or rainbow trout (O. mykiss), and belonging to a host-associated multilocus sequence typing clonal complex (e.g. CC-ST9, CC-ST232 or CC-ST10), were PCR-serotyped, evaluated for proteolytic activity, and used to challenge adipose fin-clipped 4-month old Atlantic salmon, coho salmon and rainbow trout via immersion. Findings showed US87-RBT caused disease and mortality only in rainbow trout (e.g. 56.7% survival probability). US19-COS and US62-ATS caused more mortality in coho salmon and Atlantic salmon but also caused disease in both other host species, albeit to a lesser extent. Observed survival differences may be due to variant antigenic/virulence determinants as differences in serotype and proteolytic activity were discovered. Collectively, results highlight the intricacies of F. psychrophilum-host interactions and provide further in vivo evidence that some F. psychrophilum MLST variants are host specific, which may have implications for the development of BCWD prevention and control strategies.

First isolation of Carnobacterium maltaromaticum from farmed Rainbow Trout in Virginia.

OBJECTIVE: Carnobacterium maltaromaticum is considered an emerging pathogen of salmonids in the United States and around the world. METHODS: Bacterial cultures obtained from the posterior kidney and skin of moribund Rainbow Trout Oncorhynchus mykiss from a commercial aquaculture facility in Virginia, USA, grew C. maltaromaticum, which was confirmed by additional phenotypic and molecular characterization. RESULT: A presumptive diagnosis based on the clinical signs, necropsy observations, histopathology, and bacterial cultures was bacterial septicemia due to C. maltaromaticum. CONCLUSION: This represents the first documentation of C. maltaromaticum in Rainbow Trout from Virginia.

First isolation of Flavobacterium psychrophilum from wild adult Great Lakes lake whitefish (Coregonus clupeaformis).

Lake whitefish (Coregonus clupeaformis; LWF) is an economically and ecologically valuable native species to the Great Lakes, but recent declines in their recruitment have generated significant concern about their future viability. Although studies have sought to identify factors contributing to declining recruitment, the potential role(s) of infectious diseases has not been thoroughly investigated. In 2018 and 2019, adult LWF were collected from Lakes Superior, Michigan, and Huron for clinical examination and bacteriological analyses. Herein, we describe the first isolation of Flavobacterium psychrophilum, aetiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), from systemically infected adult LWF. Bacterial isolates were yellow-orange, Gram-negative, filamentous bacilli that were oxidase and catalase positive, and produced a flexirubin-type pigment in 3% potassium hydroxide. Isolate identity was confirmed via F. psychrophilum-specific PCR, and multilocus sequence typing revealed three new singleton sequence types (STs) that were distinct from all previously described F. psychrophilum STs. The prevalence of F. psychrophilum infections was 3.3, 1.7, and 0.0% in Lakes Superior, Michigan and Huron respectively. Findings illustrate the potential for F. psychrophilum to cause systemic infections in adult LWF and highlight the need for future studies to investigate the bacterium's potential role in declining LWF recruitment.

Genetic characterization of Flavobacterium columnare isolates from the Pacific Northwest, USA.

Flavobacterium columnare is the causative agent of columnaris disease. Previous work has demonstrated a high degree of genetic variability among F. columnare isolates, identifying 4 genetic groups (GGs) with some host associations. Herein, a total of 49 F. columnare isolates were characterized, the majority of which were collected from 15 different locations throughout the US Pacific Northwest. Most isolates were collected from 2015-2018 and originated from disease outbreaks in salmonid hatcheries and rearing ponds, sturgeon hatcheries and ornamental fish. Other isolates were part of collections recovered from 1980-2018. Initial identification was confirmed by F. columnare species-specific qPCR. Study isolates were further characterized using a multiplex PCR that differentiates between the 4 currently recognized F. columnare GGs. Multiplex PCR results were supported by repetitive sequence-mediated PCR fingerprinting and gyrB sequence analysis. F. columnare GG1 was the most prevalent (83.7%, n = 41/49), represented by isolates from salmonids (n = 32), white sturgeon (n = 2), channel catfish (n = 1), ornamental goldfish (n = 1), koi (n = 3), wild sunfish (n = 1) and 1 unknown host. Six isolates (12.2%, n = 6/49) were identified as GG3, which were cultured from rainbow trout (n = 3) and steelhead trout (n = 3). Two isolates were identified as GG2 (4.1%, n = 2/49) and were from ornamental fish. No GG4 isolates were cultured in this study. The biological significance of this genetic variability remains unclear, but this variation could have significant implications for fish health management. The results from this study provide baseline data for future work developing strategies to ameliorate columnaris-related losses in the US Pacific Northwest.

Host-specific preference of some Flavobacterium psychrophilum multilocus sequence typing genotypes determines their ability to cause bacterial coldwater disease in coho salmon (Oncorhynchus kisutch).

Flavobacterium psychrophilum causes bacterial coldwater disease (BCWD) in salmonids, resulting in significant losses worldwide. Several serotyping and genetic studies of F. psychrophilum have suggested some geno-/serotypes may be either host-specific or generalistic in nature; however, this association has not been adequately explored in vivo using more natural exposure routes. Herein, F. psychrophilum isolate US19-COS, originally recovered from coho salmon (Oncorhynchus kisutch) and belonging to multilocus sequence typing clonal complex (CC) CC-ST9, and isolate US53-RBT, recovered from rainbow trout (Oncorhynchus mykiss) and belonging to CC-ST10, were serotyped via PCR, evaluated for proteolytic activity and utilized to determine their median lethal dose in immersion-challenged coho salmon fingerlings. US19-COS belonged to serotype 0, hydrolysed casein and gelatin but not elastin, led to fulminant multiorgan infections and elicited severe gross and microscopic pathology. In contrast, US53-RBT, belonging to serotype 2, hydrolysed all three substrates, but did not lead to detectable infections, disease signs or mortality in any exposed coho salmon despite proving virulent to rainbow trout in previous experiments. This study provides in vivo evidence for potential host specificity of some F. psychrophilum genotypes that can also be serologically distinct, a matter of importance towards better understanding F. psychrophilum disease ecology and epidemiology.

Assessment of Flavobacterium psychrophilum-associated mortality in Atlantic salmon (Salmo salar) and brook trout (Salvelinus fontinalis).

Salmonid diseases caused by infections of Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease, remain difficult to manage as novel, pathogenic strains continue to emerge in aquaculture settings globally. To date, much of the research regarding treatment options and vaccine development has focused on rainbow trout (Oncorhynchus mykiss), but other inland-reared salmonids are also impacted by this Gram-negative bacterium. As such, Atlantic salmon (Salmo salar) and brook trout (Salvelinus fontinalis) were injection-challenged with a variety of previously reported F. psychrophilum strains isolated from disease diagnostic cases in salmonids, as well as a standard and well-studied F. psychrophilum strain (CSF 259-93) known to be virulent in rainbow trout. In three separate virulence assessments (Trials A, B and C), strains US063 (isolated from lake trout; Salvelinus namaycush) and US149 (isolated from Atlantic salmon) caused a significantly higher cumulative per cent mortality (CPM) relative to other strains in Atlantic salmon (p <.001 for all trials), with US149 causing significantly greater mortality than US063 in Trials A (CPM 97% vs. 65%, p =.008) and B (CPM 96% ± 2.3% vs. 81.33% ± 4.8%, p =.014). Trial C used a lower dose (1.86 × 108  CFU/mL) for US149, resulting in a lower mortality (78.67% ± 9.33%) relative to Trials A and B. CSF259-93 did not cause significant mortality in any trials. In brook trout, the strain 03-179 (originally isolated from steelhead trout; Oncorhynchus mykiss) was significantly more virulent than any other (CPM 100% ± 0%, p <.001), followed by US063 (73% ± 3.8%) and US149 (40% ± 6.1%,) respectively. Again, CSF259-93 did not cause significant mortality relative to a mock challenge treatment. Results provide information about the applicability of strain selection in F. psychrophilum virulence testing in Atlantic salmon and brook trout, demonstrating the high virulence of US063 and US149 for these salmonid species. This information is applicable for the development of therapeutics and vaccines against F. psychrophilum infections and demonstrates the reproducibility of the experimental challenge model.

In Vivo Experiments Provide Evidence That Flavobacterium psychrophilum Strains Belonging to Multilocus Sequence Typing Clonal Complex ST191 Are Virulent to Rainbow Trout.

Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (BCWD), causes significant economic losses worldwide, particularly in farmed Rainbow Trout Oncorhynchus mykiss. Over the last decade, multilocus sequence typing has revealed >30 clonal complexes (CCs) globally, comprised of >320 F. psychrophilum sequence types (STs). Despite the large number of CCs worldwide, CC-ST10, which is currently the largest CC affecting Rainbow Trout, has been the primary focus of F. psychrophilum virulence studies, leaving the role of other CCs as primary causes of BCWD epizootics unclear. To this end, fingerling Rainbow Trout were experimentally challenged with F. psychrophilum strains belonging to the CC now recognized as the second largest in the world (CC-ST191) alongside CC-ST10 strains. Cumulative percent mortality was 100% in 7-month-old Rainbow Trout and between 27.8% and 61.1% in 8-month-old Rainbow Trout. All examined F. psychrophilum STs were virulent to Rainbow Trout, and no significant differences in virulence between CC-ST10 and CC-ST191 were detected. Due to their wide distribution and high pathogenic potential, both CC-ST191 and CC-ST10 F. psychrophilum strains are excellent candidates for further research aimed at preventing and controlling BCWD.

Henneguya michiganensis n. sp. (Cnidaria: Myxosporea) from the gills of muskellunge Esox masquinongy Mitchill(Esociformes: Esocidae).

Henneguya Thélohan, 1892 is the second most species rich genus of myxozoans, with reports from freshwater and marine fish worldwide. In the Great Lakes region of North America, muskellunge Esox masquinongy is an important game fish species that serves as an apex predator in the ecosystems of many inland lakes. The myxozoan fauna of esocid fish, especially muskellunge, remains largely understudied. During fish health assessments, muskellunge were examined for parasitic infections and myxozoan pseudocysts were observed on gill clip wet mounts. When ruptured under pressure, the intralamellar pseudocysts released thousands of myxospores consistent with those of the genus Henneguya. The myxospores were 67.3-96.6 (79.1 ± 5.9) µm in total length. The spore body was 18.6-22.6 (20.9 ± 1.0) µm × 5.4-6.9 (6.3 ± 0.4) µm in valvular view and 3.5-4.0 (3.8 ± 0.3) µm wide in sutural view. The two pyriform polar capsules positioned at the anterior of the spore body were 6.4-7.7 (7.0 ± 0.4) µm × 1.8-2.1 (2.0 ± 0.1) µm and each contained a tightly coiled polar filament with 9-10 turns. Two tapering caudal processes extended from the posterior of the spore body and were 47.3-75.6 (58.3 ± 5.8) µm in length. Histologically, large intralamellar polysporic plasmodia were surrounded by plump pillar cells and a distinct layer of plasma. Mild inflammation was present peripherally, with small numbers of necrotic germinative cells and intraplasmodial phagocytes internally. Ribosomal 18S rRNA gene sequence data were obtained from three gill pseudocysts. The three ~2000-bp sequences were identical, but shared no significant similarity with any publicly available sequence data. Phylogenetic analyses demonstrated sequence data from this Henneguya fell within a well-supported clade of Henneguya spp. reported from northern pike Esox lucius in Europe. Based on the distinct morphological, histological and molecular data, this species is designated as Henneguya michiganensis n. sp. from muskellunge in Michigan, USA.

Disease Progression in Lake Trout (Salvelinus namaycush) Experimentally Infected With Epizootic Epitheliotropic Disease Virus (Salmonid Herpesvirus-3).

Epizootic epitheliotropic disease virus (salmonid herpesvirus-3; EEDV) is responsible for the death of millions of hatchery-raised lake trout (Salvelinus namaycush) in the Laurentian Great Lakes Basin. However, little is known about its biology, pathology, tropism, and host interactions. In this study, the presence and disease progression of EEDV were evaluated following exposure of naïve juvenile lake trout to EEDV via bath immersion under controlled laboratory conditions (n = 84 infected; n = 44 control). Individual tissues (n = 10 per fish), collected over 6 weeks, were analyzed for viral load by quantitative polymerase chain reaction, gross and histopathologic changes, and virus cellular targets using in situ hybridization. Skin, fin, and ocular tissues were the earliest viral targets and yielded the highest viral loads throughout the course of infection. Early gross lesions included exophthalmia, ocular hemorrhage, fin congestion, and hyperemia of visceral blood vessels. Advanced disease was characterized by multifocal to coalescing erosions and ulcerations of the skin, and congestion of visceral organs. Microscopically, there was cellular degeneration and necrosis in the epidermis and spleen, and lymphohistiocytic perivasculitis of the dermis, omentum, and the epicardium. EEDV DNA was first detected by in situ hybridization in epithelial cells of the epidermis, with subsequent labeling in the epithelial lining of primary and secondary gill lamellae. During advanced disease, EEDV was detected in endothelial and dendritic cells as well as blood monocytes. This study characterized EEDV tissue tropism and associated pathologic features, to guide research aimed at understanding EEDV disease ecology and improving strategies for disease control.

Cross-protection of a live-attenuated Flavobacterium psychrophilum immersion vaccine against novel Flavobacterium spp. and Chryseobacterium spp. strains.

For salmonid producers, a common threat is Flavobacterium psychrophilum. Recent advancements in bacterial coldwater disease (BCWD) management include the development of a live-attenuated immersion vaccine that cross-protects against an array of F. psychrophilum strains. Emerging family Flavobacteriaceae cases associated with clinical disease have been increasing, including pathogenic isolates of Flavobacterium spp. and Chryseobacterium spp. The cross-protective ability of a live-attenuated F. psychrophilum vaccine was determined against three virulent Flavobacteriaceae isolates. Juvenile rainbow trout were vaccinated, developed high F. psychrophilum-specific antibody titres and were challenged with Chryseobacterium spp. isolates (S25 and T28), a Flavobacterium sp. (S21) isolate, a mixed combination of S21:S25:T28, and a standard virulent F. psychrophilum CSF259-93 strain. Results demonstrated strong protection in the CSF259-93 vaccinated group (relative per cent survival (RPS)=94.44%) when compared to the relevant CSF259-93 controls (p < .001). Protection was also observed for vaccinated fish challenged with the S21:S25:T28 mix (RPS = 85.18%; p < .001). However, protection was not observed with the S21, S25 or T28 isolates alone. Analysis of whole-cell lysates revealed differences in protein banding by SDS-PAGE, but conserved antigenic regions by Western blot in S25 and T28. Results demonstrate that this live-attenuated vaccine provided protection against mixed flavobacterial infection and suggest further benefits against flavobacteriosis.

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.

Identification of Chryseobacterium spp. isolated from clinically affected fish in California, USA.

Chryseobacterium spp. (Family Flavobacteriaceae) are emergent fish pathogens in Europe, Asia and North America. In 2016-2017, 7 bacterial isolates were recovered from posterior kidney or spleen of cultured diseased rainbow trout Oncorhynchus mykiss (n = 1), green sturgeon Acipenser medirostris (n = 1), white sturgeon A. transmontanus (n = 2), blue ram cichlid Mikrogeophagus ramirezi (n = 1), and returning fall Chinook salmon O. tshawytscha (n = 2) from different freshwater systems. Bacterial colonies were visible after 24-48 h incubation at 20°C on agar media. Isolates were Gram-negative, rod-shaped, catalase and oxidase positive. Amplification and partial sequence analysis of the 16S rRNA and gyrB genes allocated the microorganisms to the genus Chryseobacterium sharing 97.2-99.6% similarity to 6 described Chryseobacterium spp. at the 16S rRNA locus, and 87.8-99.1% similarity at gyrB. Phylogenetic analyses in conjunction with percent sequence identity suggest some of the recovered isolates may represent novel Chryseobacterium subspecies or species. The pathogenicity of 5 isolates was evaluated experimentally in rainbow trout (n = 60), brown trout Salmo trutta (n = 60) and white sturgeon (n = 36) in flow-through freshwater at 18°C. Approximately 107 CFU fish-1 was injected in the epaxial musculature of anesthetized animals. Limited mortality was observed and no bacteria were recovered from dead or moribund fish post-challenge. Thirty days post-challenge, survivors were euthanized and multiple tissues were collected and fixed for histological analysis. No consistent histopathological changes were observed in challenged or control fish. While results suggest the recovered Chryseobacterium spp. may be opportunistic pathogens, further research is warranted to better understand the role of these bacteria in fish disease.

Assessment of cross-protection to heterologous strains of Flavobacterium psychrophilum following vaccination with a live-attenuated coldwater disease immersion vaccine.

Bacterial coldwater disease, caused by Flavobacterium psychrophilum, remains one of the most significant bacterial diseases of salmonids worldwide. A previously developed and reported live-attenuated immersion vaccine (F. psychrophilum; B.17-ILM) has been shown to confer significant protection to salmonids. To further characterize this vaccine, a series of experiments were carried out to determine the cross-protective efficacy of this B.17-ILM vaccine against 9 F. psychrophilum isolates (representing seven sequence types/three clonal complexes as determined by multilocus sequence typing) in comparison with a wild-type virulent strain, CSF-259-93. To assess protection, 28-day experimental challenges of rainbow trout (Oncorhynchus mykiss) fry were conducted following immersion vaccinations with the B.17-ILM vaccine. F. psychrophilum strains used in challenge trials were isolated from several fish species across the globe; however, all were found to be virulent in rainbow trout. The B.17-ILM vaccine provided significant protection against all strains, with relative percent survival values ranging from 51% to 72%. All vaccinated fish developed an adaptive immune response (as measured by F. psychrophilum-specific antibodies) that increased out to the time of challenge (8 weeks postimmunization). Previous studies have confirmed that antibody plays an important role in protection against F. psychrophilum challenge; therefore, specific antibodies to the B.17-ILM vaccine strain appear to contribute to the cross-protection observed to heterologous strain. The ability of such antibodies to bind to similar antigenic regions for all strains was confirmed by western blot analyses. Results presented here support the practical application of this live-attenuated vaccine, and suggest that it will be efficacious even in aquaculture operations affected by diverse strains of F. psychrophilum.

Resurgence of Salmonid Herpesvirus-3 Infection (Epizootic Epitheliotropic Disease) in Hatchery-Propagated Lake Trout in Michigan.

Over the past century, populations of Lake Trout Salvelinus namaycush have declined throughout the Great Lakes basin due to overfishing, habitat destruction, introduction of invasive species, and associated recruitment issues from high thiaminase, as well as emerging infectious diseases. To combat these declines, state and federal fishery management agencies undertook substantial stock enhancement efforts, including more stringent regulation of sport and commercial catch limits and increasing hatchery propagation of Lake Trout stocked into Great Lakes basin waterways. One state fish hatchery involved in these rehabilitation efforts experienced mass mortality events in 2012 and 2017. In 2012, following a period of abnormally heavy rain, hatchery staff observed abnormal behavior followed by increased mortalities in two strains of Lake Trout fingerlings, reaching upwards of 20% mortality and totaling a loss of approximately 100,000 fish. In 2017, following another heavy-rain season, 6-8% of 2-year-old Lake Trout experienced morbidity and mortality similar to that observed in 2012. During the 2012 event, Brook Trout Salvelinus fontinalis and splake (Lake Trout × Brook Trout hybrid) reared in flow-through systems receiving water from diseased Lake Trout remained clinically unaffected. Molecular analyses revealed all lots of affected Lake Trout were infected with the salmonid herpesvirus-3 (epizootic epitheliotropic disease virus [EEDV]), a disease that caused complete depopulation of this hatchery in the late 1980s and until 2012 was never again detected in this hatchery or in Michigan. Further sampling detected EEDV in apparently healthy 5-year-old Lake Trout and in wild Mottled Sculpin Cottus bairdii collected in the hatchery source water. The ability of the virus to replicate in tissues of infected fish was verified by exposing naïve Lake Trout to the filtered tissue homogenates of infected fish resulting in similar disease signs. Despite the virus going undetected for many years, these two EEDV episodes clearly demonstrate the continued presence of this deadly herpesvirus in the Great Lakes basin.

Laboratory investigation into the role of largemouth bass virus (Ranavirus, Iridoviridae) in smallmouth bass mortality events in Pennsylvania rivers.

BACKGROUND: Mortality episodes have affected young-of-year smallmouth bass (Micropterus dolomieu) in several river systems in Pennsylvania since 2005. A series of laboratory experiments were performed to determine the potential role of largemouth bass virus (Ranavirus, Iridoviridae) in causing these events. RESULTS: Juvenile smallmouth bass experimentally infected with the largemouth bass virus exhibited internal and external clinical signs and mortality consistent with those observed during die-offs. Microscopically, infected fish developed multifocal necrosis in the mesenteric fat, liver, spleen and kidneys. Fish challenged by immersion also developed severe ulcerative dermatitis and necrotizing myositis and rarely panuveitis and keratitis. Largemouth bass virus-challenged smallmouth bass experienced greater mortality at 28 °C than at 23 or 11 °C. Co-infection with Flavobacterium columnare at 28 °C resulted in significant increase in mortality of smallmouth bass previously infected with largemouth bass virus. Aeromonas salmonicida seems to be very pathogenic to fish at water temperatures < 23 °C. While co-infection of smallmouth bass by both A. salmonicida and largemouth bass virus can be devastating to juvenile smallmouth bass, the optimal temperatures of each pathogen are 7-10 °C apart, making their synergistic effects highly unlikely under field conditions. CONCLUSIONS: The sum of our data generated in this study suggests that largemouth bass virus can be the causative agent of young-of-year smallmouth bass mortality episodes observed at relatively high water temperature.

Retail Baitfish in Michigan Harbor Serious Fish Viral Pathogens.

Indigenous small cyprinid fish species play an important role in Great Lakes ecosystems and also comprise the backbone of a multimillion-dollar baitfish industry. Due to their widespread use in sport fisheries of the Laurentian Great Lakes, there are increasing concerns that baitfish may introduce or disseminate fish pathogens. In this study, we evaluated whether baitfish purchased from 78 randomly selected retail bait dealers in Michigan harbored fish viruses. Between September 2015 and June 2016, 5,400 baitfish divided into 90 lots of 60 fish were purchased. Fish were tested for the presence of viral hemorrhagic septicemia virus (VHSV), spring viremia of carp virus (SVCV), golden shiner reovirus (GSRV), fathead minnow nidovirus (FHMNV), fathead minnow picornavirus (FHMPV), and white sucker bunyavirus (WSBV). Using the epithelioma papulosum cyprini cell line and molecular confirmation, we demonstrated the presence of viruses in 18 of the 90 fish lots (20.0%) analyzed. The most prevalent virus was FHMNV, being detected in 6 of 30 lots of Fathead Minnow Pimephales promelas and 3 of 42 lots of Emerald Shiners Notropis atherinoides. We also confirmed GSRV in two fish species: the Golden Shiner Notemigonus crysoleucas (5 of 11 lots) and Fathead Minnow (3 of 30 lots). Two VHSV (genotype IVb) isolates were recovered from a single lot of Emerald Shiners. No SVCV, FHMPV, or WSBV was detected in any of the fish examined. Some of the infected fish exhibited clinical signs and histopathological alterations. This study demonstrates that live baitfish are a potential vector for the spread of viral pathogens and underscores the importance of fish health certifications for the Great Lakes baitfish industry.

Susceptibility of Representative Great Lakes Fish Species to the North Carolina Strain of Spring Viremia of Carp Virus (SVCV).

Spring viremia of carp virus (SVCV) is a notifiable pathogen of the World Organization of Animal Health. Since SVCV was isolated in Lake Ontario in 2007, concern has grown about its spread in the Great Lakes basin and its potential negative impacts on fish species of importance in stock enhancement programs basinwide. The susceptibility of representative fish species from the families Cyprinidae (Fathead Minnow Pimephales promelas, Golden Shiner Notemigonus crysoleucas, Spotfin Shiner Cyprinella spiloptera, and Creek Chub Semotilus atromaculatus), Centrarchidae (Largemouth Bass Micropterus salmoides), Percidae (Walleye Sander vitreus), Salmonidae (Rainbow Trout Oncorhynchus mykiss), and Esocidae (Muskellunge Esox masquinongy) to SVCV was evaluated by experimental infection under laboratory conditions. Morbidity and mortality were recorded, and virus re-isolation, seminested reverse transcription PCR, and histopathological assessments were performed. Using intraperitoneal (i.p.) injection, Fathead Minnows and Golden Shiners were highly susceptible to SVCV (40-70% mortality). All dead or moribund and apparently healthy surviving Fathead Minnows and Golden Shiners were SVCV positive. The SVCV was also detected in challenged but healthy Spotfin Shiners (30%) and Creek Chub (5%). However, noncyprinid species exhibited no morbidity or mortality and were free of SVCV following an observation period of 30 d. In a follow-up experimental challenge, Fathead Minnows and Golden Shiners were SVCV challenged at 103 and 105 PFU/mL by means of waterborne immersion. After immersion, Fathead Minnows and Golden Shiners exhibited characteristic SVCV disease signs, but mortality was less (30% and 10% mortality, respectively) than that in fish with i.p. injections. The SVCV was detected in all mortalities and a subset of healthy Fathead Minnows and Golden Shiners. Necrotic changes were observed in the kidneys, liver, spleen, ovaries, and heart, and other histopathological lesions also occurred. These findings suggest that two of the four cyprinids tested are susceptible to SVCV-induced disease and that all four can act as potential carriers of SVCV in the Laurentian Great Lakes. Received January 11, 2017; accepted July 17, 2017.

Genetic Diversity of Flavobacterium psychrophilum Isolates from Three Oncorhynchus spp. in the United States, as Revealed by Multilocus Sequence Typing.

UNLABELLED: The use of a multilocus sequence typing (MLST) technique has identified the intraspecific genetic diversity of U.S. Flavobacterium psychrophilum, an important pathogen of salmonids worldwide. Prior to this analysis, little U.S. F. psychrophilum genetic information was known; this is of importance when considering targeted control strategies, including vaccine development. Herein, MLST was used to investigate the genetic diversity of 96 F. psychrophilum isolates recovered from rainbow trout (Oncorhynchus mykiss), coho salmon (Oncorhynchus kisutch), and Chinook salmon (Oncorhynchus tshawytscha) that originated from nine U.S. states. The isolates fell into 34 distinct sequence types (STs) that clustered in 5 clonal complexes (CCs) (n = 63) or were singletons (n = 33). The distribution of STs varied spatially, by host species, and in association with mortality events. Several STs (i.e., ST9, ST10, ST30, and ST78) were found in multiple states, whereas the remaining STs were localized to single states. With the exception of ST256, which was recovered from rainbow trout and Chinook salmon, all STs were found to infect a single host species. Isolates that were collected during bacterial cold water disease outbreaks most frequently belonged to CC-ST10 (e.g., ST10 and ST78). Collectively, the results of this study clearly demonstrate the genetic diversity of F. psychrophilum within the United States and identify STs of clinical significance. Although the majority of STs described herein were novel, some (e.g., ST9, ST10, ST13, ST30, and ST31) were previously recovered on other continents, which demonstrates the transcontinental distribution of F. psychrophilum genotypes. IMPORTANCE: Flavobacterium psychrophilum is the causative agent of bacterial cold water disease (BCWD) and rainbow trout fry syndrome (RTFS) and is an important bacterial pathogen of wild and farmed salmonids worldwide. These infections are responsible for large economic losses globally, yet the genetic diversity of this pathogen remains to be fully investigated. Previous studies have identified the genetic diversity of this pathogen in other main aquaculture regions; however, little effort has been focused on the United States. In this context, this study aims to examine the genetic diversity of F. psychrophilum from the United States, as this region remains important in salmonid aquaculture.

Isolation of the Fathead Minnow Nidovirus from Muskellunge Experiencing Lingering Mortality.

In 2011, the Fathead Minnow nidovirus (FHMNV; Genus Bafinivirus, Family Coronaviridae, Order Nidovirales) was isolated from pond-raised juvenile Muskellunge Esox masquinongy suffering from lingering mortality at the Wild Rose Hatchery in Wild Rose, Wisconsin. Moribund Muskellunge exhibited tubular necrosis in the kidneys as well as multifocal coalescing necrotizing hepatitis. The FHMNV was also isolated from apparently healthy juvenile Muskellunge at the Wolf Lake State Fish Hatchery in Mattawan, Michigan. The identity of the two syncytia-forming viruses (designated MUS-WR and MUS-WL from Wild Rose Hatchery and Wolf Lake State Fish Hatchery, respectively) as strains of FHMNV was determined based on multiple-gene sequencing and phylogenetic analyses. The pathogenicity of the MUS-WL FHMNV strain was determined by experimentally infecting naive juvenile Muskellunge through intraperitoneal injection with two viral concentrations (63 and 6.3 × 10(3) TCID50/fish). Both doses resulted in 100% mortality in experimentally infected fish, which exhibited severely pale gills and petechial hemorrhaging in eyes, fins, and skin. Histopathological alterations in experimentally infected fish were observed mainly in the hematopoietic tissues in the form of focal areas of necrosis. Phylogenetic analysis of concatenated partial spike glycoprotein and helicase gene sequences revealed differences between the MUS-WL FHMNV, MUS-WR FHMNV, and two other FHMNV originally isolated from moribund Fathead Minnows Pimephales promelas including the index FHMNV strain (GU002364). Based on a partial helicase gene sequence, a reverse transcriptase PCR assay was developed that is specific to FHMNV. These results give evidence that the risks posed to Muskellunge by FHMNV should be taken seriously. Received May 1, 2015; accepted February 8, 2016.