In May 2006 a large mortality of several thousand round gobies Neogobius melanostomus (Pallas, 1814) occurred in New York waters of the St. Lawrence River and Lake Ontario. Necropsies of sampled fish from these areas showed pallor of the liver and gills, and hemorrhagic areas in many organs. Histopathologic examination of affected tissues revealed areas of necrosis and hemorrhage. Inoculations of fathead minnow Pimephales promelas (Rafinesque, 1820) cell cultures with dilutions of tissue samples from the necropsied gobies produced a cytopathic effect within 5 d postinoculation. Samples of cell culture supernatant were tested using RT-PCR and confirmed the presence of viral hemorrhagic septicemia virus (VHSV). Sequence analysis of the VHSV isolate resulted in its assignment to the type-IVb subgroup. The detection of VHSV in a relatively recent invasive fish species in the Great Lakes and the potential impact of VHSV on the ecology and economy of the area will require further investigation and careful management considerations. KEY WORDS: Viral hemorrhagic septicemia · VHSV · Round goby · New York State Resale or republication not permitted without written consent of the publisherDis Aquat Org 76: [187][188][189][190][191][192] 2007 lege of Veterinary Medicine, Cornell University. The fish arrived at Cornell in moribund or freshly dead condition and were processed for diagnostic evaluation. The second sample consisted of 9 dead round gobies collected on 15 May 2006 from Sandy Creek, Lake Ontario, west of Rochester, New York. These fish were transported on ice to the AAHP at Cornell University.Cape Vincent, St. Lawrence River samples. All moribund fish were euthanized with an overdose of MS-222 (tricaine methanesulfonate, Western Chemical) in water. Eight fish were processed for diagnostic evaluation. The remaining 15 fish were frozen whole at -20°C for future evaluation if necessary. The procedure for diagnostic evaluation is described by Noga (1996) and included collecting skin scrapings and gill clip samples, sterile collection of posterior kidney samples for bacteriology, gross pathology and collection of tissues for histopathology and virology. Samples of liver, kidney, spleen and gonad were collected in 2 pooled samples for detection of viral agents. The first pooled sample (Sample A) included tissues from gobies that were dead at the time of presentation. The second pooled sample (Sample B) included fish that were moribund at the time of presentation. These samples collected for virus isolation were processed as described in the following sections. Attempts at bacterial isolation consisted of cultures taken from the posterior kidneys that were streaked onto blood agar (TSA II 5% SB, BBL™, Becton Dickinson). These cultures were incubated for 7 d at 21°C. Samples of intestines, gut contents and liver were collected to test additionally for type-E botulism by PCR (Getchell et al. 2006).Irondequoit Bay, Lake Ontario samples. Five fish were processed for diagnostic evaluation. Skin scrapings, gill ...

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.

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.

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