Cytochrome oxidase gene sequencing reveals channel catfish ovary cell line is contaminated with brown bullhead cells.

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.

Complete sequences of 4 viral hemorrhagic septicemia virus IVb isolates and their virulence in northern pike fry.

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.

Safety of Strontium Chloride as a Skeletal Marking Agent for Pacific Salmon.

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.

Biochemical, Histopathologic, Physiologic, and Behavioral Effects of Nonsteroidal Antiinflammatory Drugs in Rainbow Trout (Oncorhynchus mykiss).

Because the number of fish being used in research is increasing rapidly, evaluating the analgesic and pathologic effects of NSAID in fish is essential. To determine the biochemical, histopathologic, physiologic and behavioral effects of 3 NSAID, 48 rainbow trout underwent anesthesia with tricaine methanesulfonate and exploratory celiotomy and were randomly assigned to receive flunixin (0.5 mg/kg IM), ketorolac (0.5 mg/kg IM), ketoprofen (2 mg/kg IM), or saline. Clinical pathologic variables were assessed 1 wk before surgery and 48 h after surgery. Histopathology was performed to evaluate the healing of the incision, tissue reaction at the injection site, and potential organ toxicity. Physiologic and behavioral parameters, including weight, feeding, opercular rate, and vertical position in the water, were measured to establish parameters for identifying pain in fish. The difference between the pre- and postoperative phosphorus concentrations was greater in the flunixin group than the saline group and was the only pathologic difference between treatment groups. Histopathology of incision site, injection site, and internal organs appeared normal, and healing did not appear to be inhibited by the drugs used. The physiologic parameters of opercular rate and weight were consistent and may be helpful in identifying pain in fish in future studies, whereas feeding and vertical position in the water were unhelpful as indicators of pain in this rainbow trout surgical model. Overall, according to clinical pathology and histopathology, the use of ketoprofen, ketorolac, and flunixin at the dosages used in this study lack negative effects in rainbow trout undergoing surgery.

Histologic and molecular characterization of Edwardsiella piscicida infection in largemouth bass (Micropterus salmoides).

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.

Goldfish Carassius auratus susceptibility to viral hemorrhagic septicemia virus genotype IVb depends on exposure route.

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.

Development and characterization of a largemouth bass cell line.

Abstract The development and characterization of a new cell line, derived from the ovary of Largemouth Bass Micropterus salmoides, is described. Gonad tissue was collected from Largemouth Bass that were electrofished from Oneida Lake, New York. The tissue was processed and grown in culture flasks at approximately 22°C for more than 118 passages during an 8-year period from 2004 to 2011. The identity of these cells as Largemouth Bass origin was confirmed by sequencing a portion of the cytochrome b gene. Growth rate at three different temperatures was documented. The cell line was susceptible to Largemouth Bass virus (LMBV) and its replication was compared with that of Bluegill Lepomis macrochirus fry (BF-2), one of the cell lines recommended for LMBV isolation by the American Fisheries Society Fish Health Section Blue Book. Quantitative PCR results from the replication trial showed the BF-2 cell line produced approximately 10-fold more LMBV copies per cell than the new Largemouth Bass cell line after 6 d, while the titration assay showed similar quantities in each cell line after 1 week. Received February 18, 2014; accepted April 16, 2014.

In vivo and in vitro phenotypic differences between Great Lakes VHSV genotype IVb isolates with sequence types vcG001 and vcG002.

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 an in vitro and in vivo approach. 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 102 plaque forming units ml-1 of vcG001. These experiments suggest that there are phenotypic differences between Great Lakes isolates of VHSV genotype IVb.

Experimental transmission of VHSV genotype IVb by predation.

Preliminary surveillance of wild baitfish during the 2006 viral hemorrhagic septicemia virus genotype IVb (VHSV IVb) outbreaks indicated Emerald Shiners Notropis atherinoides and Bluntnose Minnow Pimephales notatus were infected with high levels of VHSV without showing clinical signs of disease. The movement and use of baitfish was recognized as the most probable vector for the introduction of VHSV to inland waters, such as Conesus Lake and Skaneateles Lake in New York, Budd Lake in Michigan, and Little Lake Butte des Morts and Lake Winnebago in Wisconsin. While numerous government agencies implemented restrictions to stop the movement of potentially infected baitfish into new waters and prevent the spread of VHSV IVb, until now, studies to investigate whether these initial introductions were by an oral route of infection have not occurred. Our studies identified infected Fathead Minnow Pimephales promelas as suitable vectors for transmitting VHSV IVb when fed to Tiger Muskellunge ( ♂ Northern Pike Esox lucius × â™€ Muskellunge Esox masquinongy) during laboratory trials. Six of 16 Tiger Muskellunge were infected with VHSV IVb after consumption of infected Fathead Minnows when assayed with quantitative reverse transcriptase polymerase chain reaction and viral isolation in cell culture. Weekly sampling of water and feces from these Tiger Muskellunge individually reared showed intermittent shedding of VHSV IVb. Those exposed to similarly VHSV IVb-inoculated fathead minnows by cohabitation only became infected in 1 case out of 16. A similar trial of 12 Tiger Muskellunge fed Round Goby Neogobius melanostomus that survived a VHSV IVb immersion challenge did not result in infection. Overall, our findings imply that consumption of infected wild baitfish may be a risk factor for introduction of VHSV.

Fin and gill biopsies are effective nonlethal samples for detection of viral hemorrhagic septicemia virus genotype IVb.

Nonlethal sampling is becoming a common method to diagnose fish diseases, especially with the availability of molecular testing. Viral hemorrhagic septicemia virus (VHSV) is a viral pathogen of finfish distributed worldwide. Although VHSV has been known to occur in some parts of the world for decades, a new genotype, IVb, recently emerged in the Laurentian Great Lakes of northeastern North America. Golden shiners (Notemigonus crysoleucas; Mitchill, 1814) and fathead minnows (Pimephales promelas; Rafinesque, 1820) were exposed to VHSV-IVb doses between 10(2) and 10(6) plaque forming units per fish by intraperitoneal injection at 10°C. Both species experienced significant mortality after exposure, ranging from 38% to 52% in golden shiners and from 35% to 95% in fathead minnows. In golden shiners, a fin or gill sample was somewhat less sensitive at detecting VHSV-IVb by quantitative reverse transcription polymerase chain reaction (qRT-PCR) than a pooled organ sample (consisting of liver, anterior and posterior kidney, spleen, and heart), however the relative sensitivity increased when a fin and gill sample were tested in parallel. In fathead minnows, a fin or gill sample tested alone or in parallel was relatively more sensitive than a pooled organ sample by qRT-PCR. Specificity was 100% for all sample types in both species. The results suggest that fin and gill biopsies are useful tools to test for VHSV in live fish.

Experimental Infection of Koi Carp with viral hemorrhagic septicemia virus type IVb.

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.

Low prevalence of Cyprinid Herpesvirus 3 Found in common carp (Cyprinus carpio carpio) collected from nine locations in the Great Lakes.

Cyprinid herpesvirus 3 (CyHV3) is a viral disease of fish first detected in the United States in 1998. Since that time, mortality events in common carp (Cyprinus carpio carpio) have occurred in several locations within the Great Lakes basin, but not within the Great Lakes themselves. We sampled 675 carp from 20 sites across the Great Lakes and Lake St. Clair, Michigan, USA, between 19 July and 26 September 2010. We tested the gill and a pooled internal organ sample from each fish for CyHV3 with the use of a quantitative polymerase chain reaction (qPCR) assay. Virus was detected in 18 fish from nine sites in four lakes (Lakes Michigan, Huron, St. Clair, and Ontario). Tissues from these 18 fish were also tested for CyHV3 with the use of the PCR assay recommended by the World Organization for Animal Health; amplification was achieved from two fish and confirmation by sequencing of CyHV3 from one fish collected in Lake St. Clair. The results of this study suggest that CyHV3 is present in the Great Lakes.

Swimbladder Leiomyosarcoma in Atlantic salmon (Salmo salar) in North America.

Leiomyosarcoma with associated retrovirus were found in North America for the first time in adult Atlantic salmon (Salmo salar) held in a quarantine facility at the North Attleboro National Fish Hatchery (NANFH), Massachusetts, USA. The fish had been collected as age 1-2 yr animals from the Pleasant River, Maine, and were to be used as brood stock in a population augmentation program for that river. Neoplastic disease was observed at NANFH initially in older (age 4 yr) fish, followed by age 3 yr fish. Disease was not observed in age 2 yr fish. The mortality pattern was chronic.

Transgenic expression of walleye dermal sarcoma virus rv-cyclin (orfA) in zebrafish does not result in tissue proliferation.

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.

Detection of viral hemorrhagic septicemia virus by quantitative reverse transcription polymerase chain reaction from two fish species at two sites in Lake Superior.

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.

Bilateral intraocular malignant neuroectodermal tumors in a telescope goldfish (Carassius auratus).

Abstract 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.

Selection for a dominant oncogene and large male size as a risk factor for melanoma in the Xiphophorus animal model.

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.

Comparison of quantitative RT-PCR with cell culture to detect viral hemorrhagic septicemia virus (VHSV) IVb infections in the Great Lakes.

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.

Distribution of an invasive aquatic pathogen (viral hemorrhagic septicemia virus) in the Great Lakes and its relationship to shipping.

Viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus found in fish from oceans of the northern hemisphere and freshwaters of Europe. It has caused extensive losses of cultured and wild fish and has become established in the North American Great Lakes. Large die-offs of wild fish in the Great Lakes due to VHSV have alarmed the public and provoked government attention on the introduction and spread of aquatic animal pathogens in freshwaters. We investigated the relations between VHSV dispersion and shipping and boating activity in the Great Lakes by sampling fish and water at sites that were commercial shipping harbors, recreational boating centers, and open shorelines. Fish and water samples were individually analyzed for VHSV using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and cell culture assays. Of 1,221 fish of 17 species, 55 were VHSV positive with highly varied qRT-PCR titers (1 to 5,950,000 N gene copies). The detections of VHSV in fish and water samples were closely associated and the virus was detected in 21 of 30 sites sampled. The occurrence of VHSV was not related to type of site or shipping related invasion hotspots. Our results indicate that VHSV is widely dispersed in the Great Lakes and is both an enzootic and epizootic pathogen. We demonstrate that pathogen distribution information could be developed quickly and is clearly needed for aquatic ecosystem conservation, management of affected populations, and informed regulation of the worldwide trade of aquatic organisms.

Finfish and aquatic invertebrate pathology resources for now and the future.

Utilization of finfish and aquatic invertebrates in biomedical research and as environmental sentinels has grown dramatically in recent decades. Likewise the aquaculture of finfish and invertebrates has expanded rapidly worldwide as populations of some aquatic food species and threatened or endangered aquatic species have plummeted due to overharvesting or habitat degradation. This increasing intensive culture and use of aquatic species has heightened the importance of maintaining a sophisticated understanding of pathology of various organ systems of these diverse species. Yet, except for selected species long cultivated in aquaculture, pathology databases and the workforce of highly trained pathologists lag behind those available for most laboratory animals and domestic mammalian and avian species. Several factors must change to maximize the use, understanding, and protection of important aquatic species: 1) improvements in databases of abnormalities across species; 2) standardization of diagnostic criteria for proliferative and nonproliferative lesions; and 3) more uniform and rigorous training in aquatic morphologic pathology.