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The channel catfish (Ictalurus punctatus, Rafinesque) ovary (CCO) cell line is the standard cell line used for channel catfish diagnostics. Next-gen sequencing studies of a virus cultured in the CCO cells revealed mitochondrial sequences matching those of brown bullhead (Ameiurus nebulosus, Lesueur). Therefore, we systematically performed partial cytochrome oxidase 1 gene sequencing of several sources of the CCO cell line and all matched the brown bullhead and not the channel catfish.

In 2008, viral hemorrhagic septicemia virus (VHSV) genotype IVb, the causative agent of viral hemorrhagic septicemia (VHS) in a wide range of fish, was isolated from sea lamprey (Petromyzon marinus) in the Great Lakes Basin. To better understand the implications of the reported isolation, we utilized four virus detection methods to follow the pathogenesis on an experimental infection of VHSV IVb in 60 wild, adult lamprey. Thirty lamprey were infected by intracoelomic cavity injections with 106 plaque forming units (pfu) of the VHSV IVb MI03 isolate, and 30 control lamprey were injected with sterile culture media. All media-injected controls tested negative by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and cell culture. Twenty-six of the 30 infected lamprey were positive for VHSV by qRT-PCR, and nine were positive by cell culture. In-vivo replication was demonstrated by a significant temporal increase in the mean concentration of VHS viral RNA copy number equivalents in pooled viscera measured by qRT-PCR from day 7 to day 28 and an increase in cell culture positive pools at day 28. Microscopically, infected lamprey did not develop lesions typical of virulent VHSV. Despite this, the virus was identified by immunohistochemistry in multiple organs, most readily in the kidney, where localization progressed from the interstitium to the tubular lumens. The combined results from qRT-PCR, cell culture, histopathology, and immunohistochemistry suggest that VHSV causes a sublethal infection in lamprey characterized by depletion of renal interstitial cells and in-vivo viral replication with maintenance in the brain at later stages of infection.

Additional file 1: Figure S1. ZO-1 immunofluorescence was performed on control (n = 4) and ALS CP (n = 5) and staining was imaged under 20x using confocal microscopy. DAPI denotes nuclei and DIC (differential interference contrast) images were taken to show tissue morphology. Scale bar: 50um. Red arrowheads point to areas of loss of ZO-1 staining in between CP epithelial cells. Figure S2. A) Immunohistochemistry for Claudin 5 was performed on control (n = 3) and ALS CP (n = 5). Pictures were taken at 40X magnification. Scale bar: 20 μm. Black Arrows point to staining in endothelial cells and red arrowheads point to staining within the lumen of blood vessels. B) Western blot analysis was performed on 9 controls and 18 SALS CP lysates and membranes were probed for Claudin 3 and 5, as well as Occludin. GAPDH was used as a loading control. Figure S3. A) Quantification of immunofluorescence signal intensity for CD13 and PDGFRbeta on n = 3 control and n = 5 ALS CP samples was performed using Imaris. Image stacks were taken at 63x magnification and ten different Z-stack fields per sample were captured and quantified. Tissues boundaries were delineated and signal intensity was normalized to section volume. The asterisk denotes significance with a p-value of 0.0095. Values for PDGFRbeta were not found to be significant. B) CD13/ANPEP immunohistochemistry in ALS (n = 5) and control (n = 4) CP. Black arrows point to CD13 stain around blood vessels on controls and red arrowheads denote vascular areas of CD13 loss in ALS. Figure S4 A) CD3 staining in ALS-CP tissues. Scale bar: 20 μm. B) MERTK immunohistochemistry in ALS and Control CP. Pictures were taken at low magnification to show extent of MERTK expression. Scale bar 100 μm C) p-TDP43 (S409/410) staining in CP from ALS and Control postmortem tissues. Only two ALS cases showed any inclusions, and these were located in the choroid stroma. Scale bar: 20 μm. Figure S5 Heatmap showing Z-scores across all samples for all gene targets that had reliable MSD signal. Samples are presented in their respective groups (control vs ALS), ranked by cumulative sum of all analytes measured. Figure S6 All cytokines and chemokines analyzed by MSD immunoassays in n = 22 controls and n = 44 ALS-CP lysates. None of the shown analytes showed significant differences between the two groups through a logistic regression analysis that accounts for patient age (under 65 vs above 65), as well as the plate on which the sample was run to avoid batch effect bias. Both IFN-γ and TNF-α had low signal to background ratio. Supplemental Table 1. ToppGene Analysis showing the top 30 enriched Biological Processes. Supplemental Table 2. MSD immunoassay analysis of CP lysates from ALS and non-neurological disease controls. Supplemental Table 3. Demographics for post-mortem tissue samples. Supplemental Table 4. CP samples used for RNA-seq. Supplemental Table 5. List of antibodies used in analysis of CP samples.

Emerging pathogens in wildlife are being described at an increasing rate, but the methods used to describe their dynamics in wildlife populations have been slow to develop. Understanding pathogen prevalence and risk factors for infection are critical first components of developing wildlife disease management. However, the estimation of these attributes can be biased by imperfect detection of the pathogen. In this study, we adopt a multi-scale site occupancy model to estimate the probability of pathogen detection when the diagnostic test is imperfect, host detection is imperfect, and detection can be measured at multiple scales. In addition, this model allowed the comparison of different diagnostic tests for pathogen presence. We then applied this model to the detection of viral hemorrhagic septicemia virus (VHSV) in wild fish populations in the Great Lakes. We show that VHSV is still widely distributed in the Great Lakes, and that a multi-scale model can identify additional risk factors to those identified by previous logistic regression approaches. We also estimate detection probabilities using molecular and traditional virological methods. Although our approach has several limitations, it has important implications in the management and modeling of VHSV and other emerging pathogens in aquatic wildlife.

Juvenile rainbow trout, Oncorhynchus mykiss, were fed diets containing 0, 0.5, 1.0, and 2.0% of a garlic extract, challenged with a modified 50% lethal dose of Aeromonas salmonicida and monitored for 28 d. There were significant increases in survival of trout fed 0.5 and 1.0% garlic extract as compared to the control and 2.0% garlic extract groups. A target animal safety study was performed at varying increments using the target dose of 0.5% garlic extract at 0× (0% garlic extract), 1× (0.5% garlic extract), 3× (1.5% garlic extract), and 5× (2.5% garlic extract) for 3× (6 wk) the duration of the original study. There was a significant increase in the level of circulating lymphocytes and a significant decrease in the level of circulating monocytes. The latter correlated to an increased level of pigment-containing macrophage centers within the renal tissue as garlic extract dosing increased, denoting a potential deleterious inflammatory effect as macrophage infiltration became severe at the highest dose. These studies suggest that feeding low-dose (0.5% or 1.0%) garlic extract improves survivability in rainbow trout when challenged with A. salmonicida and appears safe; however, higher levels do not appear to be effective and may cause deleterious effects on health.

A series of four isomeric 1,2,3-triazolium-based ionic liquids (ILs) with vary degree of branching were synthesized and characterized to investigate the effect of ion branching on thermal and physical properties of the resulting IL. It was found that increased branching led to a higher ionicity and higher viscosity. The thermal properties were also altered significantly and spectral changes in the near edge X-ray absorption fine structure (NEXAFS) spectra show that branching affects intermolecular interaction. While the ionicity and viscosity varying linearly with branching, the MDSC and NEXAFS measurements show that the cation shape has a stronger influence on the melting temperature and absorptive properties than the number of branched alkyl substituents.

Four viral hemorrhagic septicemia virus (VHSV) genotype IVb isolates were sequenced, their genetic variation explored, and comparative virulence assayed with experimental infections of northern pike Esox lucius fry. In addition to the type strain MI03, the complete 11183 bp genome of the first round goby Neogobius melanostomus isolate from the St. Lawrence River, and the 2013 and 2014 isolates from gizzard shad Dorosoma cepedianum die-offs in Irondequoit Bay, Lake Ontario and Dunkirk Harbor, Lake Erie were all deep sequenced on an Illumina platform. Mutations documented in the 11 yr since the MI03 index case from Lake St. Clair muskellunge Esox masquinongy showed 87 polymorphisms among the 4 isolates. Twenty-six mutations were non-synonymous and located at 18 different positions within the matrix protein, glycoprotein, non-virion protein, and RNA polymerase genes. The same 4 isolates were used to infect northern pike fry by a single 1 h bath exposure. Cumulative percent mortality varied from 42.5 to 62.5%. VHSV was detected in 57% (41/72) of the survivors at the end of the 21-d trial, suggesting that the virus was not rapidly cleared. Lesions were observed in many of the moribund and dead northern pike, such as hemorrhaging in the skin and fins, as well as hydrocephalus. Mean viral load measured from the trunk and visceral tissues of MI03-infected pike was significantly higher than the quantities detected in fish infected with the most recent isolates of genotype IVb, but there were no differences in cumulative mortality observed.

Walleyes Stizostedion vitreum were challenged with a topical application of a dilution series of cell-free dermal sarcoma tumor filtrates to determine the minimum dose of virus needed to induce these walleye tumors. A series of six 10-fold dilutions of the filtrate were applied to the side of the fish, which were allowed to develop grossly visible tumors at 15°C for 20 weeks. Quantification of the virus in the filtrates was accomplished by quantitative (real-time) reverse transcriptase-polymerase chain reaction. We determined that there are approximately 10(10) viral RNA copies in 100 μL of walleye dermal sarcoma inoculum. The minimum dose of walleye dermal sarcoma virus that could induce tumors by the topical challenge method was the 1,000-fold dilution of this 10(10) inoculum, or approximately 10(7) viral RNA copies.

The purpose of this study was to evaluate the biological effects associated with administering strontium chloride as a marking agent to age-0 Chinook Salmon Oncorhynchus tshawytscha fry. Fish were held in a 0× (0 mg/L), 1× (3,000 mg/L; current standard dosage), 3× (9,000 mg/L), or 5× (15,000 mg/L) solution of strontium chloride for 72 h (three times the standard duration of 24 h). The mortality among fish in the 5× strontium chloride exposure group was significantly higher than that observed in the other groups. A dose-related effect on general fish behavior and on feeding behavior was observed. Fish in all test tanks appeared to feed to satiation, except for fish in the 5× tanks during days 2 and 3. Fish in all other test tanks behaved normally. No dose-related effect on fish growth was detected. Histopathological evaluations showed that fish in the 5× exposure group had a significantly higher number of gill lesions than the 0× group. Our mortality, behavioral, and histological assessments suggested that juvenile Chinook Salmon could be safely immersed for three consecutive days in a 9,000-mg/L solution of strontium chloride. This finding potentially expands the present 1,000-3,000-mg/L dosage and 24-h holding period that can be used to mark juvenile fish with strontium chloride solutions. The research also provides necessary target animal safety data for U.S. Food and Drug Administration approval of strontium chloride as an alternative marking method that is suitable for fish with a short holding time. Received February 19, 2017; accepted July 16, 2017.

Viral hemorrhagic septicemia virus (VHSV) is an aquatic rhabdovirus first recognized in farmed rainbow trout in Denmark. In the past decade, a new genotype of this virus, IVb was discovered in the Laurentian Great Lakes basin and has caused several massive die-offs in some of the 28 species of susceptible North American freshwater fishes. Since its colonization of the Great Lakes, several closely related sequence types within genotype IVb have been reported, the two most common of which are vcG001 and vcG002. These sequence types have different spatial distributions in the Great Lakes. The aim of this study was to determine whether the genotypic differences between representative vcG001 (isolate MI03) and vcG002 (isolate 2010-030 #91) isolates correspond to phenotypic differences in terms of virulence using both in vitro and in vivo approaches. In vitro infection of epithelioma papulosum cyprini (EPC), bluegill fry (BF-2), and Chinook salmon embryo (CHSE) cells demonstrated some differences in onset and rate of growth in EPC and BF-2 cells, without any difference in the quantity of RNA produced. In vivo infection of round gobies ( Neogobius melanostomus ) via immersion exposure to different concentrations of vcG001 or vcG002 caused a significantly greater mortality in round gobies exposed to 10 2 plaque forming units ml − 1 of vcG001. These experiments suggest that there are phenotypic differences between Great Lakes isolates of VHSV genotype IVb.

Although viral hemorrhagic septicemia virus (VHSV) is known to affect at least 28 species of Great Lakes fish, round gobies ( Neogobius melanostomus ) appear to be particularly affected. The first report of VHSV in New York State waters occurred in round gobies and in subsequent surveillance efforts a disproportionately high proportion of round gobies were infected with VHSV compared with other species tested. In this study, we tested the experimental susceptibility of round gobies to infection with VHSV in the laboratory, using naive and previously exposed fish. Naive fish were significantly more susceptible than previously exposed fish, however previously exposed fish experienced a mortality of 35% over 45 days suggesting that previous exposure did not result in complete protection. Field studies at two sites showed a significant change in prevalence over 10 weeks in the spring based on non-lethal fin and gill samples, suggesting that great care must be taken when interpreting prevalence from single sampling efforts during VHSV surveillance. There was no difference in the observed diversity of sequence types of virus from fish that tested positive during times of low or high prevalence, or during a confinement-induced laboratory epidemic. These results show that round gobies are experimentally susceptible to VHSV and that the field prevalence of VHSV in this species can vary greatly within a short period of time; these results also provide a preliminary exploration of the role round gobies may be playing in the dynamics of VHSV in the eastern Great Lakes and St. Lawrence River.

The Materials Genome Approach (MGA) aims to accelerate development of new materials by incorporating computational and data-driven approaches to reduce the cost of identification of optimal structures for a given application. Here, we use the MGA to guide the synthesis of triazolium-based ionic liquids (ILs). Our approach involves an IL property-mapping tool, which merges combinatorial structure enumeration, descriptor-based structure representation and sampling, and property prediction using molecular simulations. The simulated properties such as density, diffusivity, and gas solubility obtained for a selected set of representative ILs were used to build neural network models and map properties for all enumerated species. Herein, a family of ILs based on ca. 200,000 triazolium-based cations paired with the bis(trifluoromethanesulfonyl)amide anion was investigated using our MGA. Fourteen representative ILs spreading the entire range of predicted properties were subsequently synthesized and then characterized confirming the predicted density, diffusivity, and CO2 Henry's Law coefficient. Moreover, the property (CO2, CH4, and N2 solubility) trends associated with exchange of the bis(trifluoromethanesulfonyl)amide anion with one of 32 other anions were explored and quantified.

The emergence of a new sublineage of viral hemorrhagic septicemia virus (VHSV) within the Laurentian Great Lakes has caused concern for aquaculture in the United States. Because of the occurrence of VHSV in a new geographic location, new host species have been identified and the complete host range has not been determined. This study confirmed the high resistance of rainbow trout, Oncorhynchus mykiss, to VHSV type IVb infection. In addition, the experimental susceptibility of hybrid striped bass, Morone chrysops ♂ × Morone saxatilis ♀, to VHSV type IVb infection was examined but determined to be highly dependent on age of fish and exposure temperature. No mortality was observed in adult fish infected via intraperitoneal (IP) injection at 15 C, whereas yearling fish infected via IP injection under the same conditions experienced 20.8% mortality. Among yearling fish infected via IP injection, mortality increased to 100% when exposure to VHSV occurred at 10 C. An LD50 for yearling hybrid striped bass exposed to VHSV at 10 C by IP injection was determined to be 1.4 × 104 pfu (SE = 2.1). Thus, at 10 C, yearling hybrid striped bass experience a high mortality when exposed to VHSV IVb by IP injection.

This article documents an epizootic of inflammation and neoplasia selectively affecting the lateral line system of lake trout ( Salvelinus namaycush) in 4 Finger Lakes in New York from 1985 to 1994. We studied more than 100 cases of this disease. Tumors occurred in 8% (5/64) of mature and 21% (3/14) of immature lake trout in the most severely affected lake. Lesions consisted of 1 or more neoplasm(s) in association with lymphocytic inflammation, multifocal erosions, and ulcerations of the epidermis along the lateral line. Lesions progressed from inflammatory to neoplastic, with 2-year-old lake trout showing locally extensive, intense lymphocytic infiltrates; 2- to 3-year-old fish having multiple, variably sized white masses up to 3 mm in diameter; and fish over 5 years old exhibiting 1 or more white, cerebriform masses greater than 1 cm in diameter. Histologic diagnoses of the tumors were predominantly spindle cell sarcomas or benign or malignant peripheral nerve sheath neoplasms, with fewer epitheliomas and carcinomas. Prevalence estimates did not vary significantly between sexes or season. The cause of this epizootic remains unclear. Tumor transmission trials, virus isolation procedures, and ultrastructural study of lesions failed to reveal evidence of a viral etiology. The Finger Lakes in which the disease occurred did not receive substantially more chemical pollution than unaffected lakes in the same chain during the epizootic, making an environmental carcinogen an unlikely primary cause of the epizootic. A hereditary component, however, may have contributed to this syndrome since only fish of the Seneca Lake strain were affected.

Viral hemorrhagic septicemia virus (VHSV) type IVb has a wide host range that includes at least three cyprinid species: Fathead Minnow Pimephales promelas, Emerald Shiner Notropis atherinoides, and Bluntnose Minnow P. notatus. To date, VHSV IVb has only been found in wild fish. However, the possibility of infection in culture facilities remains. Koi Carp Cyprinus carpio are a major ornamental aquaculture species in the United States; however, their potential to become infected with VHSV IVb has not yet been examined. In this study, we exposed Koi to 3 × 10(6) PFU VHSV Great Lakes isolate MI03 by intraperitoneal injection. While we observed low mortality (0-5%), VHSV was isolated in cell culture from the majority of fish up to 28 d postexposure (DPE) and was detected by a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay up to 90 DPE, when the trial was terminated. The results of this study strongly suggest that Koi are at risk for VHSV infection, although their susceptibility by intraperitoneal injection appears to be low. This study also provides more evidence of the sensitivity of qRT-PCR for detection of VHSV IVb.

Two experiments were performed to determine the persistence of viral hemorrhagic septicemia virus (VHSV) genotype IVb on Walleye Sander vitreus eggs. Fertilized Walleye eggs were exposed for 30 min to 105 plaque-forming units/mL VHSV genotype IVb, and control eggs were exposed to phosphate-buffered saline. In the first experiment, the eggs were treated with 0 and 50 mg/L iodophor and incubated at 12±1°C until the first fry emerged. In the second experiment, a treatment of 100 mg/L iodophor was also tested. Periodic samples were taken during embryo development and tested for VHSV by viral isolation and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The effect of tannic acid, an inhibitor of qRT-PCR testing, was evaluated in the second experiment. In both experiments no virus was detected in any control eggs by either test. In the first experiment, virus was isolated in 0-mg/L iodophor-treated eggs up to 3 d postinfection (DPI). Virus was also isolated in the 50-mg/L iodoph...

Since the first reports of mortalities due to viral hemorrhagic septicemia virus (VHSV) type IVb in the Laurentian Great Lakes basin during 2005 (Lake St. Clair, USA and Bay of Quinte, Lake Ontario, Canada), many groups have conducted surveillance efforts for the virus, primarily in nearshore areas. The round goby ( Neogobius melanostomus ) has been identified as a key species to target for surveillance, because they have a very high probability of infection at a given site. Our objective in this study was to document and quantify VHSV in round gobies in offshore waters of Lake Ontario using molecular techniques. We collected 139 round gobies from depths ranging from 55 to 150 m using bottom trawls during the early spring of 2011 and detected VHSV in 4 individuals (1/26 fish at 95 m, 2/12 fish at 105 m, and 1/24 fish at 135 m). These results expand the known depth range of VHSV in the Great Lakes. They also have implications on the management of the spread of VHSV within infected bodies of water related to the mixing of populations of fish that would remain distinct in their breeding habitats, but then have the opportunity to mix in their overwintering habitats, as well as to increase overlap of predator and prey species in overwintering habitats.

Viral hemorrhagic septicemia virus (VHSV) is the etiologic agent of a disease of great concern to aquaculture. We performed experimental infections with intraperitoneal VHSV IVb injections at 106 pfu/fish in four species: tiger muskellunge, ♂Esox luciusA—♀Esox masquinongy; Atlantic salmon, Salmo salar; channel catfish, Ictalurus punctatus; and walleye, Sander vitreus. The fish were thermal shocked concurrent with infection to cause immunosuppression and increase the probability of eliciting disease. Each species was observed for morbidity and mortality and periodic samples taken. Virus was detected using virus isolation and real‐time quantitative reverse‐transcription polymerase chain reaction (qRT‐PCR). Histopathology confirmed lesions in tiger muskellunge and Atlantic salmon, and immunohistochemistry showed virus associated with some lesions. Tiger muskellunge were highly susceptible to VHSV IVb, and high viral levels were detected in exposed fish. A single mortality was observed in exposed Atlantic salmon with a short viral replication period. Channel catfish and walleye were resistant to developing clinical signs of VHS. The results show that these species vary in the development of VHS under these conditions, and qRT‐PCR testing enabled virus detection in subclinically affected fish. Basic concordance statistics between the qRT‐PCR test results and the virus isolation results were evaluated.

Viral hemorrhagic septicemia virus (VHSV) is the causative agent of a devastating disease in fish, viral hemorrhagic septicemia (VHS), which has recently spread to the Laurentian Great Lakes. In this paper, we report the results of infection surveillance conducted during 2009 for VHSV with a focus on yellow perch ( Perca flavescens ) and round goby ( Neogobius melanostomus ). We collected 1928 fish representing eight species at nine sites in Lake Ontario and the St. Lawrence River and found fish positive for VHSV by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) at every site. Prevalence of virus-positive fish at individual sites ranged from 0.7% to 27.5%. Yellow perch were significantly less likely to test positive than round goby in this study. In logistic regression models, maturity was a significant predictor of testing positive in yellow perch, but no evaluated factor was a significant predictor of testing positive in round goby. Virus isolation in cell culture was successful in seven out of 37 high titer fish samples, and partial glycoprotein gene sequences obtained from these virus isolates showed an increase in genetic diversity relative to the previously known diversity of Laurentian Great Lakes VHSV genotype IVb. These results highlight the importance of continued movement restrictions and surveillance for VHSV in wild fish in the Laurentian Great Lakes as well as suggest future work to further elucidate the infection dynamics of this virus in wild fish populations.

An adult walleye ( Sander vitreus) was submitted to Cornell University for evaluation of a hard pale-tan pharyngeal mass attached to the gill arches. Dozens of hard white conical structures radiated from the surface. Microscopically, conical structures were identified as denticles and rested on plates of dysplastic orthodentine, cementum, and acellular bone. A diagnosis of compound odontoma was made based upon the presence of proliferative epithelial and mesenchymal odontogenic tissues that recapitulated tooth structures normally present on gill rakers. Odontomas are classified as hamartomas and typically develop in immature diphyodont mammals. The pharyngeal location and lifelong regeneration of teeth in fish, however, both qualify the present diagnosis in the pharyngeal region of an adult teleost. Ontogenic and morphologic differences between mammalian and piscine dentition and differentials for tooth-bearing tumors in fish are presented within the context of a developmental anomaly.

Terramycin for Fish® (oxytetracycline, OTC) is one of three approved drugs for therapeutic treatment of fish in the United States. Nothing is known, however, of the effects of this therapeutic on drug metabolizing enzymes in fish post-treatment. The main purpose of the study was to examine whether the fish CYP1A and CYP3A enzymes would cross-react with antibodies to known mammalian cytochrome P-450 forms (CYP1A1 and CYP3A). Observational feeding studies of OTC effects were conducted in hybrid striped bass, channel catfish and Nile tilapia. Oxytetracycline was mixed into the feed to achieve a daily dose of 82.8 mg per kg body weight at a feeding rate of 1 % body weight per day. Hepatic microsomes of each fish were prepared and western blotting of CYP1A1 and CYP3A4 and enzyme assays of CYP1A2 and CYP3A4 were performed prior to OTC treatment and on post-treatment days 1, 6, 11 and 21. Both goat anti-rat CYP1A1 and rabbit anti-human CYP3A4 showed good cross-reactivity with all three species in this study. All three species exhibited distinct perturbations in one or more of the variables examined on day 1 post-treatment. Immediately following the 10-day medication period, relative liver weight (RLW) of hybrid striped bass was increased 44% and remained elevated through post-treatment day 21. Increased CYP3A4 enzyme activity and protein abundance were noted in channel catfish and Nile tilapia, respectively. This observational approach demonstrated species differences both in control activities and in the timing and extent of hepatic responses to OTC. The unique perturbations of hepatic CYP450 enzymes in different fish species to OTC treatment observed in this study may have relevance for the use of additional antibiotics or other therapeutics used in aquaculture.

Viral hemorrhagic septicemia virus (VHSV) was first detected in the Laurentian Great Lakes in 2005 during a mortality event in the Bay of Quinte, Lake Ontario. Subsequent analysis of archived samples determined that the first known isolation of VHSV in the Laurentian Great Lakes was from a muskellunge Esox masquinongy collected in Lake St. Clair in 2003. By the end of 2008, mortality events and viral isolations had occurred in all of the Laurentian Great Lakes except Lake Superior. In 2009, a focused disease surveillance program was designed to determine whether VHSV was also present in Lake Superior. In this survey, 874 fish from 7 sites along the U.S. shoreline of Lake Superior were collected during June 2009. Collections were focused on nearshore species known to be susceptible to VHSV. All fish were dissected individually by using aseptic techniques and were tested for the presence of VHSV genetic material by use of a quantitative reverse transcription (qRT) polymerase chain reaction (PCR) targeting the viral nucleoprotein gene. Seventeen fish from two host species at two different sites tested positive at low levels for VHSV. All attempts to isolate virus in cell culture were unsuccessful. However, the presence of viral RNA was confirmed independently in five fish by using a nested PCR that targeted the glycoprotein (G) gene. Partial G gene sequences obtained from three fish were identical to the corresponding sequence from the original 2003 VHSV isolate (MI03) from muskellunge. These detections represent the earliest evidence for the presence of VHSV in Lake Superior and illustrate the utility of the highly sensitive qRT-PCR assay for disease surveillance in aquatic animals.

Outbreaks of type E botulism have affected fish and waterfowl in Lake Erie since 1998. We compared conditions in the eastern basin during a warm outbreak year (2002) and a cooler year with low mortality (2003) to determine the levels of Clostridium botulinum type E spore DNA present in sediments under both scenarios. Lake Erie warmed up earlier in 2002 with a mean summer temperature 2 °C higher than in 2003. A comparison between both years indicated that in 2002 there were anoxic episodes near the sediment and a decrease in redox potential and pH, conditions that favor C. botulinum growth. We found that in 2002, sediments were also warmer and contained more spores than in 2003. To determine the biotic sources of botulism for predators, we analyzed the benthic fauna for spore DNA. Benthic organisms (chironomids, oligochetes, nematodes, dreissenids and mayflies) carried higher levels of spores than sediments, and chironomids contained some of the highest levels reaching 820,000 copies DNA/mg. Dreissenid mussel tissue contained up to 2280 copies DNA/mg and their waste deposits had up to 275 copies DNA/mg. We conclude that there are numerous benthic pathways to transmit type E botulism to upper trophic levels given the right conditions. We present in this manuscript a hypothesis on the interplay of environmental conditions and the risk of outbreaks of type E botulism in the eastern basin of Lake Erie.

We investigated a newly introduced pathogen in a wild population of smallmouth bass Micropterus dolomieu in the St. Lawrence River to illuminate unknown temporal and demographic prevalence patterns. We used real-time quantitative reverse-transcription polymerase chain reaction to assess viral hemorrhagic septicemia virus (VHSV) genotype IVb (VHSV-IVb) viral N-gene levels. Our objectives were to characterize the distribution and abundance of this newly introduced pathogen within a naive population of economically valuable sport fish through an annual cycle. We also sought to describe VHSV-IVb levels across demographic groups (males and females; adults and subadults). Strong temporal variation in prevalence of VHSV-IVb was evident through the annual cycle; peaks in prevalence corresponded to the smallmouth bass spawning period and a temperature range of 10–14°C. No difference in VHSV-IVb prevalence between male and female smallmouth bass was detected. However, a markedly higher prevalence was obser...

From mid-2004 to mid-2005, several grass carp, Ctenophayngodon idella (Valenciennes), showing evidence of spinal deformity were presented to the Aquatic Animal Health Program, Cornell University. The carp were from three separate locations in New York State. The first case involved several fish from a natural body of water in the Catskill Mountain region of south-eastern New York State. The second was a single affected individual from a private pond in the Fingerlakes region of Central New York State. The third was a single individual from the Cold Springs Harbor Fish Hatchery, Cold Springs Harbor, Long Island. All fish were at least 7 years of age. Radiographs and computed tomography (CT) scans revealed the deformities to be of bony origin. The spinal deformities were characterized by variable amounts of kyphosis, scoliosis and rotation. While it is not possible to determine the specific cause(s) of the lesions, we consider a genetic component as a likely contributor to the observed pathology.

Surface de-adhesion and disinfection of eggs at hatcheries are critical steps in successful fry culture; however, little is known about the effects of the combination of these compounds on the efficacy of egg surface disinfection. We examined the effect of tannic acid on detection of viral hemorrhagic septicemia virus (VHSV) type IVb alone and in the presence of iodophor under in vitro conditions. We confirmed that tannic acid inhibits the quantitative reverse-transcriptase polymerase chain reaction assay developed in our laboratory and that it binds to VHSV, reversibly decreasing infectivity. Surprisingly, we also found that when combined with iodophor, tannic acid destroys the ability of either compound to inhibit infectivity. While we still strongly encourage the use of iodophor for its antiviral, bactericidal, and fungicidal properties, our results suggest that when tannic acid is used prior to disinfection with iodophor, it must be very thoroughly rinsed to ensure that it does not interfere ...

A 5-year-old male telescope goldfish (Carassius auratus) developed buphthalmia of the left eye. An enucleation was performed and a diagnosis of a neuroectodermal tumor was made on histological examination. Although the fish initially recovered, it was killed 49 days postsurgery due to a severe decline in its condition. On histological evaluation of postmortem tissue samples, it was determined that the fish also had a neuroectodermal tumor of the right eye with local invasion of the brain. On immunohistochemistry, the neoplastic cells were positive for S-100. To the authors' knowledge, this is the first published case of naturally occurring bilateral intraocular neuroectodermal tumors in a fish.

Adult height is a risk factor in numerous human cancers that involve aberrant receptor tyrosine kinase (RTK) signalling. However, its importance is debated due to conflicting epidemiological studies and the lack of useful in vivo models. In Xiphophorus fishes (Platyfishes/Swordtails), a functional RTK, Xiphophorus melanoma receptor kinase (Xmrk), serves as the dominant oncogene and has been maintained for several million years despite being deleterious and in an extremely unstable genomic region. Here we show that the Xmrk genotype is positively correlated with standard length in male and female wild caught Xiphophorus cortezi sampled throughout their phylogeographic distribution. Histopathology confirms the occurrence of malignant melanomas in both sexes; however, melanoma incidence was extremely male biased. Furthermore, males collected with malignant melanomas in the field were significantly larger than both Xmrk males collected without melanomas and wildtype (Xmrk deficient) males. These results not only provide a novel selective mechanism for the persistence of the germline Xmrk oncogene but also create an innovative avenue of melanoma research within the Xiphophorus fishes. Wildlife cancer in natural systems is a growing concern, therefore, future research investigating life history characteristics associated with certain phenotypes and genotypes that predispose an individual to cancer will be fundamental to increasing our understanding of the evolutionary biology of cancer in nature as well as in humans.

Walleye dermal sarcoma (WDS) is a benign tumor of walleye fish that develops and completely regresses seasonally. The retrovirus associated with this disease, walleye dermal sarcoma virus, encodes three accessory genes, two of which, rv-cyclin (orfA) and orfb, are thought to play a role in tumor development. In this study, we attempted to recapitulate WDS development by expressing rv-cyclin in chimeric and stable transgenic zebrafish. Six stable transgenic lines expressing rv-cyclin from the constitutive CMVtk promoter were generated. Immunohistochemistry and quantitative reverse transcriptase polymerase chain reaction demonstrate that rv-cyclin is widely expressed in different tissues in these fish. These lines were viable and histologically normal for up to 2 years. No increase in tumors or tissue proliferation was observed following N-ethyl N-nitrosourea exposure or following tail wounding and subsequent tissue regeneration compared to controls. These data indicate that rv-cyclin is not independently sufficient for tumor induction in zebrafish.

Viral hemorrhagic septicemia virus (VHSV) is an important pathogen of cultured and wild fish in marine and freshwater environments. A new genotype, VHSV IVb, was isolated from a fish collected from the Great Lakes in 2003. Since the first isolation, VHSV IVb has been confirmed in 28 species, signaling the early invasion and continued spread of this Office International des Epizooties-reportable agent. For surveillance of this virus in both wild and experimental settings, we have developed a rapid and sensitive one-step quantitative real-time polymerase chain reaction (qRT-PCR) assay that amplifies a 100-base-pair conserved segment from both the genomic negative strand and the mRNA positive strand of the nucleoprotein (N) gene of VHSV IVb. This assay is linear over seven orders of magnitude, with an analytical capability of detecting a single copy of viral RNA and reproducibility at 100 copies. The assay is approximately linear with RNA input from 50 to 1000 ng per assay and works equally well with RNA prepared from a column-based or phenol-chloroform-based method. In wild-caught fish, 97% of the cases were found to be more than three orders of magnitude more sensitive using qRT-PCR than using cell culture. Of the 1,428 fish from the Great Lakes region tested in 2006 and 2007, 24% were positive by qRT-PCR whereas only 5% were positive by cell culture. All of the fish that were positive by cell culture were also positive by qRT-PCR. Importantly, qRT-PCR sensitivity is comparable to that of cell culture detection when comparing VHSV viral RNA levels with viral titer stocks, confirming that the high qRT-PCR signals obtained with diagnostic samples are due to the accumulation of N gene mRNA by transcriptional attenuation. The qRT-PCR assay is particularly valuable for rapid and high-throughput prescreening of fish before confirmatory testing by cell culture or sequencing tissue-derived amplicons and especially in detecting infection in fish that do not show clinical signs of VHS.

Fast, M. D., Sokolowski, M. S., Dunton, K. J., and Bowser, P. R. 2009. Dichelesthium oblongum (Copepoda: Dichelesthiidae) infestation in wild-caught Atlantic sturgeon, Acipenser oxyrinchus oxyrinchus. – ICES Journal of Marine Science, 66: 2141–2147. Atlantic sturgeon were sampled in autumn 2007 and 2008 along the New York Bight. The fish were examined for the presence of external parasites, and blood, fin, and opercula biopsies were collected for subsequent serum analysis and histopathological evaluation. Dichelesthium oblongum, a parasitic copepod, was observed on 93% of the sturgeon sampled (77 out of 83) between Rockaway and Jones Beaches, NY, and Sandy Hook, NJ. During the course of the examinations, grossly visible lesions associated with the attachment and feeding of juvenile stages of D. oblongum were noted on the operculum, pectoral, dorsal, and anal fins of infested fish. Significant differences were observed between the level of infestation and serum chemistry across sampling sites, such that the most heavily infested fish were caught off Jones Beach (15.8 ± 2.79 lice fish−1), higher infestations than at Rockaway Beach (8.86 ± 0.89 lice fish−1) and Sandy Hook (5.31 ± 1.40 lice fish−1). Animals from Jones Beach also indicated ion loading (i.e. sodium, calcium, and magnesium), possibly as a result of stress or water loss through D. oblongum infestation compromising the epithelial barrier. The interaction of the environment and parasite with host life-history characteristics are discussed in terms of their ecological significance to this threatened fish species.

Nile tilapia Oreochromis niloticus were medicated with florfenicol (AQUAFLOR type A medicated article; Schering-Plough Animal Health, Summit, New Jersey) via a medicated ration at 15 mg florfenicol x kg fish body weight(-1) d(-1) for 10 d to compare the elimination kinetics of the test article in different size fish held at 25 degrees C. The groups of fish used in the study had mean weights of approximately 100, 250, and 500 g. In each trial, the fish were provided the medicated ration and 15 fish were processed at each of seven time points postfeeding for determination of the florfenicol concentration in serum and the florfenicol residue in the edible portion (composite muscle and skin). There was a trend toward shorter half-lives of elimination in the smaller fish. The elimination times in muscle-skin (times to reach the established tolerance concentration for channel catfish Ictalurus punctatus and salmonids of 1.0 microg florfenicol residue/g) and half-lives were 9.2 and 1.2 d (100 g), 8.6 and 1.7 d (250 g), and 12.7 and 2.2 d (500 g), respectively.

A study was designed to evaluate the hepatopancreas of the American lobster (Homarus americanus) as a potential indicator organ for early dietary stress. Seventy-five 7th stage lobsters were divided into 3 groups. Group 1 was fed live brine shrimp (Artemia salina), group 2 was fed a sub-optimal artificial diet (M4), and group 3 was starved. Three animals from each group were sacrificed at days 2, 4, 8, 12, 16, 22 and 29. The hepatopancreas from each animal was examined grossly and then prepared for histological evaluation. A general atrophy of especially the absorptive and secretory cells in the hepatopancreas of the starved animals was noticed as early as 4 days. In the hepatopancreas of the M4 fed juveniles, a progressive increase in the number of pycnotic nuclei and necrosis was evident by 8 days. Evidence of amyloid and ceroid were also observed. This ability to detect early cellular differences due to dietary deficiencies can be an important procedure for analyzing nutritional data.

During 2004 and 2005 a survey was conducted to investigate the presence and geographic distribution of largemouth bass virus (LMBV) in New York State. This iridovirus is widely distributed across the eastern United States; however, it had not previously been reported in New York State. Two hundred and eighty-three wild largemouth bass Micropterus salmoides and 8 smallmouth bass M. dolomieu were collected from 37 locations across the state. No clinical signs of LMBV or mortalities attributable to the virus were observed in the fish collected. Using a quantitative polymerase chain reaction (QPCR) method, we detected LMBV in 28 fish from 13 locations. Viral cytopathic effect in cell culture was observed in 5 fish from 3 locations. The virus isolated from cell culture was confirmed to be LMBV by an independent PCR method. Statistical analysis of the largemouth bass samples collected during 2005 revealed a wide difference in prevalence between the QPCR results and the cell culture results. Analysis of possible predictors, including age, sex, and month collected, showed no significant associations with the QPCR results. This survey confirms the presence and wide distribution of a potentially pathogenic form of LMBV in multiple water systems across New York State.

We report on the emergence of Cochlodinium polykrikoides blooms in the Peconic Estuary and Shinnecock Bay, NY, USA, during 2002–2006. Blooms occurred during late summer when temperatures and salinities ranged from 20 to 25 °C and 22 to 30 ppt, respectively. Bloom patches achieved cell densities exceeding 105 ml−1 and chlorophyll a levels exceeding 100 μg l−1, while background bloom densities were typically 103–104 cells ml−1. Light, scanning electron and ultrathin-section transmission electron microscopy suggested that cells isolated from blooms displayed characteristics of C. polykrikoides and provide the first clear documentation of the fine structure for this species. Sequencing of a hypervariable region of the large subunit rDNA confirmed this finding, displaying 100% similarity to other North American C. polykrikoides strains, but a lower similarity to strains from Southeast Asia (88–90%). Bioassay experiments demonstrated that 24 h exposure to bloom waters (>5 × 104 cells ml−1) killed 100% of multiple fish species (1-week-old Cyprinodon variegates, adult Fundulus majalis, adult Menidia menidia) and 80% of adult Fundulus heteroclitus. Microscopic evaluation of the gills of moribund fish revealed epithelial proliferation with focal areas of fusion of gill lamellae, suggesting impairment of gill function (e.g. respiration, nitrogen excretion, ion balance). Lower fish mortality was observed at intermediate C. polykrikoides densities (103–104 cells ml−1), while fish survived for 48 h at cell densities below 1 × 103 cells ml−1. The inability of frozen and thawed-, or filtered (0.2 μm)-bloom water to cause fish mortality suggested that the thick polysaccharide layer associated with cell membranes and/or a toxin principle within this layer may be responsible for fish mortality. Juvenile bay scallops (Argopecten irradians) and American oysters (Crassostrea virginica) experienced elevated mortality compared to control treatments during a 9-day exposure to bloom water (∼5 × 104 cells ml−1). Surviving scallops exposed to bloom water also experienced significantly reduced growth rates. Moribund shellfish displayed hyperplasia, hemorrhaging, squamation, and apoptosis in gill and digestive tissues with gill inflammation specifically associated with areas containing C. polykrikoides cells. In summary, our results indicate C. polykrikoides blooms have become annual events on eastern Long Island and that bloom waters are capable of causing rapid mortality in multiple species of finfish and shellfish.

Gross and histological evidence is presented to document two novel and distinct cases of non-hybrid melanomas in wild caught northern mountain swordtails Xiphophorus nezahualcoyotl. During initiation one showed increases in melanin expression from a micromelanophore pattern associated with the sword (sword stripes), and the other showed melanin enhancement associated with the spotted-side macromelanophore pattern on the flanks of the fish. Both cases have spindle cell type melanomas located in the epidermis, dermis, adipose tissue and underlying muscle in several locations on each fish.

The genus Edwardsiella is composed of a diverse group of facultative anaerobic, gram-negative bacteria that can produce disease in a wide variety of hosts, including birds, reptiles, mammals, and fish. Our report describes the isolation and identification of Edwardsiella piscicida associated with chronic mortality events in 2 separate captive largemouth bass ( Micropterus salmoides) populations in New York and Florida. Wet-mount biopsies of skin mucus, gill, kidney, and spleen from several affected largemouth bass contained significant numbers of motile bacteria. Histologic examination revealed multifocal areas of necrosis scattered throughout the heart, liver, anterior kidney, posterior kidney, and spleen. Many of the necrotic foci were encapsulated or replaced by discrete granulomas and associated with colonies of gram-negative bacteria. Initial phenotypic and matrix-assisted laser desorption ionization–time of flight mass spectrometric analysis against existing spectral databases of recovered isolates identified these bacteria as Edwardsiella tarda. Subsequent molecular analysis using repetitive sequence mediated and species-specific PCR, as well as 16S rRNA, rpoB, and gyrB sequences, classified these isolates as E. piscicida. As a newly designated taxon, E. piscicida should be considered as a differential for multiorgan necrosis and granulomas in largemouth bass.

The health status of the American lobster, Homarus americanus Milne-Edwards, in Long Island Sound (LIS) has been in decline, with seasonal mortality events occurring since 1998. In order to assess the potential effects of environmental conditions on lobster health via haemolymph analysis, lobsters collected from various sites in LIS were examined and sampled while concurrent environmental data (water temperature, salinity and dissolved oxygen) were recorded. The pH of the haemolymph of each lobster was tested, followed by a collection of haemolymph for serum biochemistry analysis and bacterial culture. This report focuses on the results of the bacterial sampling. The majority of bacteria cultured were opportunistic pathogens commonly found in the environment, including some that are associated with sewage and pollution. The prevalence of bacteraemia was correlated with the site of collection, the month in which the lobsters were sampled, and water temperature.

The use of quantitative polymerase chain reaction (QPCR) to test for largemouth bass virus (LMBV) was evaluated during a challenge experiment in which largemouth bass Micropterus salmoides were immersed in the type strain of LMBV. The real-time PCR and cell culture methods were both used to measure LMBV present in the inoculum. Additional samples tested by QPCR included gill, gonad, kidney, liver, mucus, spleen, and swim bladder. A plasmid clone containing a 248-base pair (bp) fragment of the major capsid protein gene (MCP*) was serially diluted and used as a standard to quantify the number of LMBV DNA copies present in the samples tested. A 62-bp fragment of DNA located in MCP* was amplified in the real-time PCR assay. This work has demonstrated the value of the QPCR assay in LMBV surveys.

Tissue depletion studies of antibacterials are an important part of data packages required to obtain a label from the U.S. Food and Drug Administration (FDA) in the United States for use of therapeutants in food fishes. Currently, withdrawal tima are set based on results of such studies obtained from healthy animals. Bacterial infection can lead to dramatic physiological changes in affected fish. In this investigation, the impact of bacterial infection on depletion kinetics of oxytetracycline (OTC) was examined in tilapia Oreochromis niloticus challenged with Streptococcus iniae (a model Gram positive bacterium) or Vibrio vulnificus (a model Gram negative bacterium). An additional group of fish injected with Brain Heart Infusion (BHI) broth was included as a non-infectious stimulus. Although some differences in elimination kinetics of OTC were observed between treated fish and non-treated controls, OTC was rapidly eliminated from tilapia in all groups. In all cases, mean concentration of OTC was below the current 2.0 ppm (μg/g) FDA tolerance for OTC in the edible portion (muscle plus skin) after day 3 postdosing.

Since 1999, eight adult Chinook salmon (Onchorhynchus tshawytscha) from Lake Ontario with large, focal, cavernous, fluid-filled muscle lesions have been examined in our respective laboratories. Gross and microscopic examination, cytology, and bacteriology were performed. Microscopically the lesions were consistent with chronic abscesses. Cytologic evaluation revealed diplomonad flagellate Spironucleus within these lesions. We provide a description of the gross and microscopic pathology associated with the cavernous lesions.

We assessed the susceptibility of goldfish Carassius auratus to infection by genotype IVb of the viral hemorrhagic septicemia virus. Goldfish were infected by intraperitoneal injections of 106 plaque-forming units (pfu) fish-1, single bath exposure of 105 pfu ml-1 for 24 h, or consumption of 0.4 g of commercial fish feed soaked in 107 pfu per 8 fish. The mortality rate of intraperitoneal-infected goldfish was 10 to 32%, although the virus was detected by quantitative RT-PCR in 77% (65/84) of the survivors at the end of the 42 d trial, suggesting a carrier state. Severe gross lesions were observed in many of the moribund and dead goldfish such as hemorrhaging in the skin, fin, liver, kidney, brain, intestine, and eye as well as abdominal distension, bilateral exophthalmia, and splenomegaly. There was minimal morbidity or mortality in the immersion, feeding, or control groups.

Avian and fish botulism outbreaks have been recorded since 1999 in eastern Lake Erie. These outbreaks are caused by Clostridium botulinum type E, a toxin-producing bacteria that is found in anoxic substrates rich in organic material. We studied the environmental conditions present in eastern Lake Erie during 2002, a year when several botulism outbreaks were observed. We also tested for the presence of C. botulinum type E in lake sediments. Samples were taken at six stations from two sites of different depths in the Dunkirk (New York, USA) area. The depth of the sampling sites influenced physico-chemical and biological processes in the sediments. We used the quantitative polymerase chain reaction (Q-PCR) to quantify the levels of C. botulinum type E in the samples. Sediment samples contained a patchy distribution of type E spore concentrations (from not detectable to 5520 DNA copies/mg). Samples of benthic invertebrates tested positive for C. botulinum type E spores in tissues (Gammarus 2028 DNA copies/mg, oligochaetes 428 DNA copies/mg, chironomids 148 DNA copies/mg and dreissenid mussels 715 DNA copies/mg). Principal components analysis (PCA) from inshore stations indicated that a decrease in dissolved oxygen, pH and redox potential near the sediment was associated to an increase in specific conductance and the type E toxin gene in sediments. We also found that C. botulinum type E spores are present in sediments at different depths and at different times through the ice-free season.

The first isolation of Vibrio vulnificus in southern Taiwan from hybrid tilapia Oreochromis sp. raised in freshwater and brackish water environments is documented in this report. The infection was only found in fish in ponds where the salinity was less than 10 ppt. Tilapia raised in water of higher salinities in the same region were not affected. Grossly visible signs of infection included dark coloration, lethargy, and external hemorrhage and ulceration of the skin. The most prominent internal signs of infection included splenomegaly and severe hemorrhagic lesions in the liver. Septicemia was documented in moribund fish. All bacterial isolates from moribund fish were tested by polymerase chain reaction, using V. vulnificus hemolysin/cytolysin gene-specific primers. Sequence data from the 16S ribosomal RNA gene suggest that these isolates were V. vulnificus. The isolates were indole and mannitol positive, characteristics shared by human clinical isolates and isolates from freshwater European eel, Anguilla anguilla. The isolates from tilapia were unique in that they were negative for ornithine decarboxylase and citrate.