Publications by authors named "Thomas P Loch"

37 Publications

Phenotypic and Genetic Characterization of Flavobacterium psychrophilum Recovered from Diseased Salmonids in China.

Microbiol Spectr 2021 Sep 15:e0033021. Epub 2021 Sep 15.

Department of Fisheries and Wildlife, College of Agriculture and Natural Resources, Michigan State Universitygrid.17088.36, East Lansing, Michigan, USA.

Flavobacterium psychrophilum, the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome, causes great economic losses in salmonid aquaculture worldwide. Recent molecular studies have uncovered important epidemiological and ecological aspects of this pathogen; however, such data are lacking for F. psychrophilum populations affecting aquaculture in China. Herein, F. psychrophilum phenotype, genotype, and virulence were characterized for isolates recovered from epizootics in multiple salmonid aquaculture facilities across China. Thirty-one F. psychrophilum isolates, originating from four provinces and three host fish species, were predominantly homogeneous biochemically but represented 5 sequence types (STs) according to multilocus sequence typing (MLST) that belonged to clonal complex CC-ST10 or 3 newly recognized singleton STs. PCR-based serotyping classified 19 and 12 F. psychrophilum isolates into molecular serotypes 1 and 0, respectively, showing an obvious relationship with host species. Antimicrobial susceptibility analysis via broth microdilution revealed reduced susceptibility to enrofloxacin, flumequine, and oxolinic acid, moderate susceptibility to gentamicin, erythromycin, and florfenicol, and variable susceptibility to ampicillin and oxytetracycline. challenge experiments confirmed the ability of two representative Chinese F. psychrophilum isolates to induce typical signs of BCWD and mortality in 1-year-old rainbow trout (Oncorhynchus mykiss). Findings collectively demonstrate (i) that BCWD outbreaks in China studied thus far are caused by F. psychrophilum lineages that are common on other continents (e.g., CC-ST10) and others that have not been reported elsewhere (e.g., ST355, ST356, ST357), (ii) that F. psychrophilum molecular serotypes distinguish isolates from different host fish species, even within STs, and (iii) reduced F. psychrophilum antimicrobial susceptibility against compounds used for BCWD control in China. Flavobacterium psychrophilum causes substantial economic losses in salmonid aquaculture worldwide. Although this bacterium is also believed to be a disease source in China, published reports of its presence do not yet exist. Herein, F. psychrophilum was linked to multiple disease outbreaks in several salmonid aquaculture facilities within four Chinese provinces, and polyphasic characterization revealed that most isolates were genetically distinct from strains recovered on other continents. Analyses further revealed the predominating molecular serotypes, antimicrobial susceptibility profiles, and pathogenic potential of two representative recovered isolates. Collectively, the results presented here provide important data on the epidemiology and disease ecology of F. psychrophilum in China and pave the way for targeted prevention and control methods to be pursued in the future.
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http://dx.doi.org/10.1128/Spectrum.00330-21DOI Listing
September 2021

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

J Aquat Anim Health 2021 Sep 30;33(3):190-195. Epub 2021 Aug 30.

Department of Fisheries and Wildlife, College of Agriculture and Natural Resources and Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan, 48824, USA.

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.
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http://dx.doi.org/10.1002/aah.10140DOI Listing
September 2021

Experimental Evidence of Epizootic Epitheliotropic Disease Virus (Salmoid Herpesvirus-3, ) Transmission via Contaminated Fomites and Subsequent Prevention Using a Disinfectant.

Pathogens 2021 Jun 9;10(6). Epub 2021 Jun 9.

Comparative Medicine and Integrative Biology, College of Veterinary Medicine, Michigan State University, 1129 Farm Lane, Room 340G, East Lansing, MI 48824, USA.

Epizootic epitheliotropic disease virus (EEDV) has caused considerable mortality in hatchery-reared lake trout in the Great Lakes Basin, and yet the routes of transmission and efficacious means of prevention remain poorly understood. To determine whether EEDV can be transmitted via contaminated fomites and clarify whether such transmission could be prevented via fomite disinfection, juvenile lake trout (n = 20 per treatment) were handled in nets previously soaked in an EEDV suspension (7.29 × 10-2.25 × 10 virus copies/mL of water) that were further immersed in either 1% Virkon Aquatic ("disinfected" treatment, in triplicate) or in sample diluent ("EEDV-contaminated" treatment). Negative control nets were soaked in sterile sample diluent only. Characteristic gross signs of EED developed in the "EEDV-contaminated" treatment group, which was followed by 80% mortality, whereas no gross signs of disease and 0-5% mortality occurred in the negative control and "disinfected" treatment groups, respectively. EEDV was detected via qPCR in 90% of the "EEDV-contaminated" treatment fish, however, it was not detected in any fish within the negative control or "disinfected" treatment groups. Study findings not only demonstrate that EEDV can be readily transmitted via contaminated fomites, but importantly suggest that Virkon Aquatic is an efficacious option for preventing EEDV contagion via the disinfection of hatchery tools, thereby highlighting a promising tool for improving lake trout hatchery biosecurity and minimizing EEDV-linked losses.
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http://dx.doi.org/10.3390/pathogens10060724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8227329PMC
June 2021

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

Dis Aquat Organ 2021 May 6;144:151-158. Epub 2021 May 6.

Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California Davis, California 95616, 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.
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http://dx.doi.org/10.3354/dao03588DOI Listing
May 2021

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

Syst Parasitol 2021 04 9;98(2):119-130. Epub 2021 Mar 9.

Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, Stoneville, MS, 38776, USA.

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.
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http://dx.doi.org/10.1007/s11230-021-09965-5DOI Listing
April 2021

HIGH PREVALENCE OF CIRCULATING ANTIBODIES TO RENIBACTERIUM SALMONINARUM IN SPAWNING ONCORHYNCHUS SPP. FROM LAKE MICHIGAN, USA.

J Wildl Dis 2021 01;57(1):19-26

Department of Fisheries and Wildlife, College of Agriculture and Natural Resources, Michigan State University, 1129 Farm Lane, Room 342, East Lansing, Michigan 48824, USA.

Bacterial kidney disease, caused by Renibacterium salmoninarum, threatens salmonids worldwide. Following devastating mortality episodes in Oncorhynchus spp. in Lake Michigan, US, in the 1980s and infection rates >90%, pathogen prevalence has steadily declined to <5% over three decades in the three state-managed stocks. In this study, we sought to determine if the declining infection rates were associated with heightened circulating antibodies in state-managed Oncorhynchus spp. residing in the Lake Michigan watershed. A single-dilution, indirect enzyme-linked immunosorbent assay (ELISA) was modified to detect circulating antibodies against R. salmoninarum. Baseline values were delineated from naive chinook salmon (Oncorhynchus tshawytscha) and rainbow trout (Oncorhynchus mykiss). The assay was first used to assess primary antibody production over a 4-wk period in chinook salmon experimentally infected with R. salmoninarum. Mean antibody response was detected as early as 2 wk postinfection and continued to increase to the end of the observation period. The modified ELISA was then used to detect antibodies in serum samples collected from feral adult chinook salmon, coho salmon (Oncorhynchus kisutch), and steelhead trout (O. mykiss) returning to spawn at Lake Michigan weirs in 2009 and 2013. Results demonstrated that about 80% of feral Oncorhynchus spp. had measurable titers of circulating antibodies to R. salmoninarum. The relative ease and reasonable costs of this modified ELISA makes it a valuable serosurveillance tool for assessing the humoral immune status of feral salmonid populations.
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http://dx.doi.org/10.7589/2019-04-098DOI Listing
January 2021

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

J Fish Dis 2021 May 9;44(5):645-653. Epub 2021 Feb 9.

Department of Fish and Wildlife Sciences, College of Natural Resources, University of Idaho, Moscow, ID, USA.

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 × 10  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.
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http://dx.doi.org/10.1111/jfd.13349DOI Listing
May 2021

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

J Fish Dis 2021 May 21;44(5):521-531. Epub 2021 Jan 21.

Department of Fisheries and Wildlife, College of Agriculture and Natural Resources, Michigan State University, East Lansing, MI, USA.

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.
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http://dx.doi.org/10.1111/jfd.13340DOI Listing
May 2021

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

Vet Pathol 2020 09 3;57(5):687-699. Epub 2020 Aug 3.

Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine,3078Michigan State University, East Lansing, MI, USA.

Epizootic epitheliotropic disease virus (salmonid herpesvirus-3; EEDV) is responsible for the death of millions of hatchery-raised lake trout () 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 ( = 84 infected; = 44 control). Individual tissues ( = 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.
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http://dx.doi.org/10.1177/0300985820941268DOI Listing
September 2020

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

J Fish Dis 2020 Aug 18;43(8):915-928. Epub 2020 Jun 18.

Department of Fish and Wildlife Sciences, College of Natural Resources, University of Idaho, Moscow, ID, USA.

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.
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http://dx.doi.org/10.1111/jfd.13201DOI Listing
August 2020

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

Dis Aquat Organ 2019 Nov;136(3):227-234

Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616, 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.
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http://dx.doi.org/10.3354/dao03409DOI Listing
November 2019

Shedding of the Salmonid Herpesvirus-3 by Infected Lake Trout ().

Viruses 2019 06 26;11(7). Epub 2019 Jun 26.

Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA.

Salmonid Herpesvirus-3, commonly known as the Epizootic Epitheliotropic Disease virus (EEDV), causes a disease of lake trout () that has killed millions of fish over the past several decades. Currently, most aspects of EEDV disease ecology remain unknown. In this study, we investigated EEDV shedding in experimentally challenged (intracoelomic injection) lake trout that were individually microchipped. In order to assess viral shedding, each infected fish was placed in individual static, aerated aquaria for a period of 8 h, after which the water was assessed for the presence of EEDV DNA using quantitative PCR. Water sampling was conducted every seven days for 93 days post-infection (pi), followed by additional sampling after one year. Results demonstrated that lake trout began shedding EEDV into the water as early as 9 days pi. Shedding peaked approximately three weeks pi and ceased after nine weeks pi. In contrast, mortalities did not occur until 40 days pi. Although mortality reached 73.9%, surviving fish ceased shedding and continued to grow. However, additional shedding was detected 58 weeks after infection in 66% of surviving fish. Findings of this study demonstrate that EEDV is shed into the water by infected lake trout hosts for extended periods of time, a mechanism that favors virus dissemination.
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http://dx.doi.org/10.3390/v11070580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6669692PMC
June 2019

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

J Aquat Anim Health 2019 03 25;31(1):31-45. Epub 2019 Jan 25.

Michigan Department of Natural Resources, Fisheries Division, Post Office Box 30446, Lansing, Michigan, 48909, USA.

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.
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http://dx.doi.org/10.1002/aah.10051DOI Listing
March 2019

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.

Appl Environ Microbiol 2019 03 6;85(6). Epub 2019 Mar 6.

Department of Fisheries and Wildlife, College of Agriculture and Natural Resources, Michigan State University, East Lansing, Michigan, USA

, 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 (). Prior studies have used multilocus sequence typing (MLST) to examine genetic heterogeneity within 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 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 CC identified was CC-ST10, within which 10 novel genotypes were discovered, most of which came from 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 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. 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 isolates that were recovered from diseased fish across North America. Results highlighted multiple 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.
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http://dx.doi.org/10.1128/AEM.02305-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6414368PMC
March 2019

Development of a loop-mediated isothermal amplification assay for the detection and quantification of epizootic epitheliotropic disease virus (salmonid herpesvirus-3).

J Virol Methods 2019 02 13;264:44-50. Epub 2018 Nov 13.

Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI, 48824, USA; Comparative Medicine and Integrative Biology, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA; Department of Fisheries and Wildlife, College of Agriculture and Natural Resources,Michigan State University, East Lansing, MI 48824, USA. Electronic address:

Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus-3) causes a serious disease hatchery-reared lake trout (Salvelinus namaycush), threatening restoration efforts of this species in North America. The current inability to replicate EEDV in vitro necessitates the search for a reproducible, sensitive, and specific assay that allows for its detection and quantitation in a time- and cost-effective manner. Herein, we describe a loop-mediated isothermal amplification (LAMP) assay that was developed for the quantitative detection of EEDV in infected fish tissues. The newly developed LAMP reaction was optimized in the presence of calcein, and the best results were produced using 2 mM MgCl, 1.8 mM dNTPs and at an incubation temperature of 67.1 °C. This method was highly specific to EEDV, as it showed no cross-reactivity with several fish viruses, including Salmonid Herpesvirus, -2, -4, and -5, Infectious Pancreatic Necrosis Virus, Spring Viremia of Carp Virus, Infectious Hematopoietic Necrosis Virus, Golden Shiner Reovirus, Fathead Minnow Nidovirus, and Viral Hemorrhagic Septicemia Virus. The analytical sensitivity of the EEDV-LAMP method was estimated to be as low as 16 copies of plasmid per reaction. When infected fish tissue was used, a positive reaction could be obtained when an infected gill tissue sample that contained 430 viral copies/μg was diluted up to five orders of magnitude. The sensitivity and specificity of the newly developed LAMP assay compared to the SYBR Green qPCR assay were 84.3% and 93.3%, respectively. The quantitative LAMP for EEDV had a correlation coefficient (R = 0.980), and did not differ significantly from the SYBR Green quantitative PCR assay (p > 0.05). Given its cost- and time-effectiveness, this quantitative LAMP assay is suitable for screening lake trout populations and for the initial diagnosis of clinical cases.
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http://dx.doi.org/10.1016/j.jviromet.2018.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7119762PMC
February 2019

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

J Fish Dis 2019 Jan 28;42(1):75-84. Epub 2018 Oct 28.

Department of Fisheries and Wildlife Sciences and the Aquaculture Research Institute, College of Natural Resources, University of Idaho, Moscow, Idaho.

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.
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http://dx.doi.org/10.1111/jfd.12902DOI Listing
January 2019

Retail Baitfish in Michigan Harbor Serious Fish Viral Pathogens.

J Aquat Anim Health 2018 12 12;30(4):253-263. Epub 2018 Oct 12.

Michigan Department of Natural Resources, Fisheries Division, Post Office Box 30466, Lansing, Michigan, 48909, USA.

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.
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http://dx.doi.org/10.1002/aah.10034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7159410PMC
December 2018

Identification of Four Distinct Phylogenetic Groups in With Fish Host Associations.

Front Microbiol 2018 13;9:452. Epub 2018 Mar 13.

MSD Animal Health Innovation Pte. Ltd., Singapore, Singapore.

Columnaris disease, caused by the Gram-negative bacterium , is one of the most prevalent fish diseases worldwide. An exceptionally high level of genetic diversity among isolates of has long been recognized, whereby six established genomovars have been described to date. However, little has been done to quantify or characterize this diversity further in a systematic fashion. The objective of this research was to perform phylogenetic analyses of 16S rRNA and housekeeping gene sequences to decipher the genetic diversity of . Fifty isolates and/or genomes of , originating from diverse years, geographic locations, fish hosts, and representative of the six genomovars were analyzed in this study. A multilocus phylogenetic analysis (MLPA) of the 16S rRNA and six housekeeping genes supported four distinct genetic groups. There were associations between genomovar and genetic group, but these relationships were imperfect indicating that genomovar assignment does not accurately reflect genetic diversity. To expand the dataset, an additional 90 16S rRNA gene sequences were retrieved from GenBank and a phylogenetic analysis of this larger dataset also supported the establishment of four genetic groups. Examination of isolate historical data indicated biological relevance to the identified genetic diversity, with some genetic groups isolated preferentially from specific fish species or families. It is proposed that isolates be assigned to the four genetic groups defined in this study rather than genomovar in order to facilitate a standard nomenclature across the scientific community. An increased understanding of which genetic groups are most prevalent in different regions and/or aquaculture industries may allow for the development of improved targeted control and treatment measures for columnaris disease.
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http://dx.doi.org/10.3389/fmicb.2018.00452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5859164PMC
March 2018

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

BMC Vet Res 2018 Mar 2;14(1):62. Epub 2018 Mar 2.

Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, 1129 Farm Lane, Room 174, East Lansing, MI, 48824, USA.

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.
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http://dx.doi.org/10.1186/s12917-018-1371-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834888PMC
March 2018

The Asian fish tapeworm Schyzocotyle acheilognathi is widespread in baitfish retail stores in Michigan, USA.

Parasit Vectors 2017 12 22;10(1):618. Epub 2017 Dec 22.

Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI, 48824, USA.

Background: The Asian fish tapeworm Schyzocotyle acheilognathi (Yamaguti, 1934) is an important fish pathogen because of its wide range of intermediate and definitive hosts and its pathological consequences. This study was designed to determine if baitfish are a likely vector contributing to the expansion of the invasive Asian fish tapeworm.

Results: We collected live baitfish for examination from 78 retail stores in Michigan between September 2015 and June 2016. A total of 5400 baitfish (90 lots, 60 fish/lot) were examined, including 42 emerald shiners [Notropis atherinoides (Rafinesque, 1818)] lots, 30 fathead minnow [Pimephales promelas (Rafinesque, 1820)] lots, 11 golden shiners [Notemigonus crysoleucas (Mitchill, 1814)] lots, 3 sand shiners [Notropis stramineus (Cope, 1865)] lots, 1 lot each of spottail shiners [Notropis hudsonius (Clinton, 1824)], Northern redbelly dace [Phoxinus eos (Cope, 1861)], and blacknose dace [Rhinichthys atratulus (Hermann, 1804)] and 1 lot of mixed two species: weed shiners [Notropis texanus (Girard, 1856)] and sand shiners.

Conclusions: Based on its scolex and strobilar morphology combined with gene sequence analysis, S. acheilognathi was only found in emerald shiners, golden shiners and sand shiners. The mean within lot prevalence and abundance of infection was highest in emerald shiners (20.3 ± 14.0 and 1.15 ± 1.34), followed by golden shiners (8.3 ± 10.7 and 0.89 ± 1.27) and sand shiners (1.3 ± 2.6 and 0.02 ± 0.05). However, the mean intensity of S. acheilognathi in emerald shiners was lower (4.3 ± 2.6) than that of golden shiners (6.6 ± 6.7). S. acheilognathi-infected fish exhibited enlargement of the abdomen, distension of the intestinal wall, and intestinal occlusion and hemorrhage. This finding suggests that live baitfish are a likely vector by which the invasive Asian tapeworm's range is expanding.
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http://dx.doi.org/10.1186/s13071-017-2541-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5741914PMC
December 2017

The Emerging Fish Pathogen Isolated from Chinook Salmon: Comparative Genome Analysis and Molecular Manipulation.

Front Microbiol 2017 30;8:2339. Epub 2017 Nov 30.

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, United States.

strain T16 was isolated from a disease outbreak in hatchery-reared Chinook salmon () fingerlings. To gain insight into its genomic content, structure and virulence pathogenesis factors, comparative genome analyses were performed using genomes from environmental and virulent strains. shared low average nucleotide identity (ANI) to well-known fish-pathogenic flavobacteria (e.g., , and ), indicating that it is a new and emerging fish pathogen. The genome in T16 had a length of 5,359,952 bp, a GC-content 35.7%, and 4,422 predicted protein-coding sequences. core genome analysis showed that the number of shared genes decreased with the addition of input genomes and converged at 1182 genes. At least 8 genomic islands and 5 prophages were predicted in T16. At least 133 virulence factors associated with virulence in pathogenic bacteria were highly conserved in T16. Furthermore, genes linked to virulence in other bacterial species (e.g., those encoding for a type IX secretion system, collagenase and hemolysin) were found in the genome of T16 and were conserved in most of the analyzed pathogenic . was resistant to ampicillin and penicillin, consistent with the presence of multiple genes encoding diverse lactamases and the penicillin-binding protein in the genome. To allow for future investigations into virulence , a transposon-based random mutagenesis strategy was attempted in T16 using pHimarEm1. Four putative gliding motility deficient mutants were obtained and the insertion sites of pHimarEm1 in the genome of these mutants were characterized. In total, study results clarify some of the mechanisms by which emerging flavobacterial fish pathogens may cause disease and also provide direly needed tools to investigate their pathogenesis.
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http://dx.doi.org/10.3389/fmicb.2017.02339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5714932PMC
November 2017

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

J Aquat Anim Health 2017 Dec;29(4):214-224

a Department of Pathobiology and Diagnostic Investigation , College of Veterinary Medicine, Michigan State University , 1129 Farm Lane, Room 177K, East Lansing , Michigan 48824 , USA.

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 10 and 10 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.
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http://dx.doi.org/10.1080/08997659.2017.1360410DOI Listing
December 2017

Detection accuracy of Renibacterium salmoninarum in Chinook salmon, Oncorhynchus tshawytscha (Walbaum) from non-lethally collected samples: Effects of exposure route and disease severity.

Prev Vet Med 2017 Sep 6;145:110-120. Epub 2017 Jun 6.

Department of Fisheries and Wildlife, College of Agriculture and Natural Resources, Michigan State University, East Lansing, MI, 48824, USA; Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI, 48824, USA.

Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum, threatens salmonid populations throughout the Northern hemisphere. Many fishery regulatory authorities require ongoing disease monitoring in hatcheries and spawning runs prior to gamete collection to prevent BKD outbreaks and spread. According to diagnostic protocols of the American Fisheries Society-Fish Health Section, monitoring for R. salmoninarum generally consists of lethal sampling of visceral organs from fish. However, non-lethal sampling would be preferable, especially for valuable broodstock or endangered species. In this study, non-lethal sampling methods were evaluated for their ability to detect R. salmoninarum in Chinook salmon (Oncorhynchus tshawytscha) that were experimentally infected via two different routes (e.g., intraperitoneal injection and waterborne immersion) to mimic acute and chronic disease courses. Non-lethal (e.g., blood, mucus, and a urine/feces mixture) and lethal (e.g., kidney and spleen homogenate) samples were collected from challenged and mock-challenged Chinook salmon and the presence of R. salmoninarum was assessed by culture on modified kidney disease medium, nested polymerase chain reaction (nPCR), and semi-quantitative enzyme-linked immunosorbent assay (ELISA). Sensitivity, specificity, and accuracy of lethal and non-lethal samples in detecting R. salmoninarum were calculated using receiver operating characteristic (ROC) analyses. For ROC analyses, true disease status was evaluated under two different assumptions: 1) that lethal samples represented the true disease status and 2) that all experimentally challenged fish were truly infected. We found that sensitivity and specificity of non-lethal samples depended upon time of sampling after experimental infection, sample type, and R. salmoninarum exposure route. Uro-fecal samples had the greatest potential as non-lethal samples compared to mucus and blood. In terms of future monitoring, combining lethal samples tested by ELISA assay with uro-fecal samples tested by nPCR could be the best strategy for detecting R. salmoninarum prevalence in a population as it reduces the overall number of fish required for sampling.
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http://dx.doi.org/10.1016/j.prevetmed.2017.06.001DOI Listing
September 2017

Antimicrobial Susceptibilities of Flavobacterium psychrophilum Isolates from the Great Lakes Basin, Michigan.

Microb Drug Resist 2017 Sep 9;23(6):791-798. Epub 2017 Jan 9.

1 Department of Fisheries and Wildlife, College of Agriculture and Natural Resources, Michigan State University , East Lansing, Michigan.

Flavobacterium psychrophilum is a serious pathogen of salmonids worldwide, a matter that is compounded by the lack of effective vaccine preparations. As a result, biosecurity measures and antimicrobial agents remain the only available methods to control diseases caused by F. psychrophilum. It is feared that antimicrobial use may have led to the development of F. psychrophilum strains with reduced susceptibility. Therefore, the primary objective of this study was to determine the antimicrobial susceptibility profiles of 50 F. psychrophilum isolates from Michigan in response to 10 antimicrobial compounds. As recommended by the Clinical and Laboratory Standard Institute and the World Organization of Animal Health, a standardized microdilution broth assay was employed to determine the minimum inhibitory concentrations (MICs) for ampicillin (AMP), gentamicin (GEN), enrofloxacin (ENRO), oxolinic acid (OXO), flumequine (FLUQ), trimethoprim-sulphamethoxazole (SXT), ormetoprim-sulphadimethoxine (PRI), erythromycin (ERY), florfenicol (FFN), and oxytetracycline (OXY). Epidemiological cutoff values were calculated using the normalized resistance interpretation (CO) and the ECOFFinder analysis methods (CO). The MIC distributions in response to OXY exhibited bimodality, indicating the presence of isolates with reduced susceptibility in addition to the wild-type isolates. The OXY epidemiological cutoff values (CO <0.06 μg/ml; CO <0.12 μg/ml) demonstrated that 24% of Michigan isolates exhibited reduced susceptibility to this commonly used drug. No other antimicrobial exhibited a bimodal distribution of MICs. This study represents the first antimicrobial susceptibility assessment of F. psychrophilum strains recovered from Michigan and contributes valuable data to the worldwide validation efforts to determine universal epidemiological cutoff values of this deadly fish pathogen.
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http://dx.doi.org/10.1089/mdr.2016.0103DOI Listing
September 2017

Gamete-associated flavobacteria of the oviparous Chinook salmon (Oncorhynchus tshawytscha) in lakes Michigan and Huron, North America.

J Microbiol 2016 Jul 28;54(7):477-86. Epub 2016 Jun 28.

Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI, 48824, USA.

Flavobacterial diseases, caused by multiple members of the Family Flavobacteriaceae, elicit serious losses in wild and farmed fish around the world. Flavobacteria are known to be transmitted horizontally; however, vertical transmission has been suspected but proven only for one fish-pathogenic flavobacterial species (e.g., Flavobacterium psychrophilum). Herein, we report on the isolation and molecular identification of multiple Flavobacterium and Chryseobacterium taxa from the ovarian fluid and eggs of feral Great Lakes Chinook salmon (Oncorhynchus tshawytscha). Identified egg- and ovarian fluid-associated flavobacteria were either well-known flavobacterial fish pathogens (e.g., F. psychrophilum and F. columnare), most similar to emerging fish-associated flavobacteria (e.g., F. spartansii, F. tructae, F. piscis, C. piscium, C. scophthalmum), or were distinct from all other described Chryseobacterium and Flavobacterium spp., as determined by phylogenetic analyses using neighbor-joining, Bayesian, and Maximum Likelihood methodologies. The gamete-associated flavobacteria fell into three groups (e.g., those that were recovered from the ovarian fluid but not eggs; those that were recovered from the ovarian fluid and eggs; and those that were recovered from eggs but not ovarian fluid), a portion of which were recovered from eggs that were surface disinfected with iodophor at the commonly used dose and duration for egg disinfection. Some gamete-associated flavobacteria were also found in renal, splenic, and neurological tissues. Systemic polymicrobial infections comprised of F. psychrophilum and F. columnare were also detected at nearly an 11% prevalence. This study highlights the potential role that sexual products of female Great Lakes Chinook salmon may play in the transmission of fish-associated flavobacteria.
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http://dx.doi.org/10.1007/s12275-016-5629-3DOI Listing
July 2016

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

J Aquat Anim Health 2016 06;28(2):131-41

e U.S. Department of Agriculture, Animal and Plant Health Investigation Services , National Veterinary Services Laboratories, Diagnostic Virology Laboratory , 1920 Dayton Avenue, Ames , Iowa 50010 , USA.

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.
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http://dx.doi.org/10.1080/08997659.2016.1159620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7159395PMC
June 2016

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

Appl Environ Microbiol 2016 06 16;82(11):3246-3255. Epub 2016 May 16.

Department of Fisheries and Wildlife, College of Agriculture and Natural Resources, Michigan State University, East Lansing, Michigan, USA

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.
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http://dx.doi.org/10.1128/AEM.00411-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4959235PMC
June 2016

Flavobacterium psychrophilum Infections in Salmonid Broodstock and Hatchery-Propagated Stocks of the Great Lakes Basin.

J Aquat Anim Health 2015 Dec;27(4):192-202

a Department of Fisheries and Wildlife , College of Agriculture and Natural Resources, Michigan State University , 480 Wilson Road, Room 4, East Lansing , 48824 , Michigan , USA.

Bacterial coldwater disease (BCWD), caused by Flavobacterium psychrophilum, threatens wild and propagated salmonids worldwide and leads to substantial economic losses. In addition to being horizontally transmitted, F. psychrophilum can be passed from infected parents to their progeny, furthering the negative impacts of this pathogen. In Michigan, both feral and captive salmonid broodstocks are the gamete sources used in fishery propagation efforts. A 5-year study was initiated to follow the prevalence of systemic F. psychrophilum infections in feral broodstocks of four species (steelhead Oncorhynchus mykiss [potadromous Rainbow Trout]; Coho Salmon O. kisutch; Chinook Salmon O. tshawytscha; and Atlantic Salmon Salmo salar) residing in three Great Lakes watersheds. Additionally, captive broodstocks of four species (Rainbow Trout, Brown Trout Salmo trutta, Lake Trout Salvelinus namaycush, and Brook Trout Salvelinus fontinalis) maintained at two facilities were assessed for the presence of F. psychrophilum. The resultant offspring from each broodstock population were sampled for F. psychrophilum infections multiple times throughout hatchery residency. Using selective flavobacterial culture and PCR confirmation, F. psychrophilum was detected in all broodstocks except the captive Lake Trout and Brook Trout. Logistic regression analysis demonstrated that among the infected feral broodstocks, Chinook Salmon from the Lake Michigan watershed had the highest prevalence of systemic F. psychrophilum infection (mean = 63.2%). Among the captive broodstocks, the Gilchrist Creek strain of Brown Trout had the highest infection prevalence (mean = 5%). Collectively, the captive broodstocks were found to have significantly lower infection prevalence than the feral broodstocks. Despite the high prevalence of systemic F. psychrophilum infections in many broodstock populations, the bacterium was rarely detected in their progeny during hatchery rearing. However, heavy losses associated with clinical BCWD outbreaks did occur. Collectively, our results reinforce that BCWD continues to threaten Great Lakes basin salmonids.
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http://dx.doi.org/10.1080/08997659.2015.1088488DOI Listing
December 2015

Emerging flavobacterial infections in fish: A review.

J Adv Res 2015 May 7;6(3):283-300. Epub 2014 Nov 7.

Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, 174 Food Safety and Toxicology Building, Michigan State University, East Lansing, MI 48824, USA ; Department of Fisheries and Wildlife, College of Agriculture and Natural Resources, Natural Resources Building, Room 4, Michigan State University, East Lansing, MI 48824, USA.

Flavobacterial diseases in fish are caused by multiple bacterial species within the family Flavobacteriaceae and are responsible for devastating losses in wild and farmed fish stocks around the world. In addition to directly imposing negative economic and ecological effects, flavobacterial disease outbreaks are also notoriously difficult to prevent and control despite nearly 100 years of scientific research. The emergence of recent reports linking previously uncharacterized flavobacteria to systemic infections and mortality events in fish stocks of Europe, South America, Asia, Africa, and North America is also of major concern and has highlighted some of the difficulties surrounding the diagnosis and chemotherapeutic treatment of flavobacterial fish diseases. Herein, we provide a review of the literature that focuses on Flavobacterium and Chryseobacterium spp. and emphasizes those associated with fish.
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http://dx.doi.org/10.1016/j.jare.2014.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4522593PMC
May 2015

Polyphasic characterization reveals the presence of novel fish-associated Chryseobacterium spp. in the Great Lakes of North America.

Dis Aquat Organ 2015 Mar;113(2):113-25

Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, 174 Food Safety and Toxicology Building, Michigan State University, East Lansing, MI 48824, USA.

Recent reports suggest an emergence of novel Chryseobacterium spp. associated with aquaculture-reared fish worldwide. Herein, we report on multiple Chryseobacterium spp. infecting Great Lakes fishes that are highly similar to previously detected isolates from Europe, Africa, and Asia but have never before been reported in North America. Polyphasic characterization, which included extensive physiological, morphological, and biochemical analyses, fatty acid profiling, and phylogenetic analyses based upon partial 16S rRNA gene sequences, highlighted the diversity of Great Lakes' fish-associated chryseobacteria and also suggested that at least 2 taxa represent potentially novel Chryseobacterium spp. Screening for the ability of representative chryseobacteria to elicit lesions in experimentally challenged fish showed that they induced varying degrees of pathology, some of which were severe and resulted in host death. Median lethal dose (LD50) experiments for the isolate that elicited the most extensive pathology (Chryseobacterium sp. T28) demonstrated that the LD50 exceeded 4.5 × 108 cfu, thereby suggesting its role as a facultative fish-pathogenic bacterium. Histopathological changes in T28-infected fish included epithelial hyperplasia of the secondary lamellae and interlamellar space that resulted in secondary lamellar fusion, monocytic infiltrate, and mucus cell hyperplasia, all of which are consistent with branchitis, along with monocytic myositis, hemorrhage within the muscle, liver, adipose tissue, and ovaries, spongiosis of white matter of the brain, multifocal edema within the granular cell layer of the cerebellar cortex, and renal tubular degeneration and necrosis. The findings of this study underscore the widespread presence of chryseobacteria infecting Great Lakes fish.
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http://dx.doi.org/10.3354/dao02819DOI Listing
March 2015
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