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.

Detection and surveillance of viral hemorrhagic septicemia virus using real-time RT-PCR. I. Initial comparison of four protocols.

Eight laboratories worked collectively to evaluate 4 real-time RT-PCR (rRT-PCR) protocols targeting viral hemorrhagic septicemia virus (VHSV) being considered for deployment to a USA laboratory testing network. The protocols utilized previously published primers and probe sets developed for detection and surveillance of VHSV. All participating laboratories received and followed a standard operating protocol for extraction and for each of the rRT-PCR assays. Performance measures specifically evaluated included limit of detection (defined as the smallest amount of analyte in which 95% of the samples are classified as positive), analytical specificity, assay efficiency across genotype representatives, within- and between-plate variation within a laboratory, and variation between laboratories using the same platform, between platforms, and between software versions. This evaluation clearly demonstrated that the TaqMan®-based assay developed by Jonstrup et al. (2013; J Fish Dis 36:9-23) produced the most consistent analytical performance characteristics for detecting all genotypes of VHSV across the 8 participating laboratories.

Detection and surveillance of viral hemorrhagic septicemia virus using real-time RT-PCR. II. Diagnostic evaluation of two protocols.

Two real-time reverse transcription polymerase chain reaction (rRT-PCR) assays under consideration for deployment to multiple testing laboratories across the USA were evaluated for diagnostic sensitivity and specificity on tissue homogenates obtained from natural and experimental viral hemorrhagic septicemia (VHS)-infected fish. Estimates for diagnostic specificity using virus isolation as the reference method were similar between laboratories regardless of the assay. Diagnostic sensitivity estimates of 0.96 (95% CI: 0.95, 0.97) for Jonstrup et al. (2013)'s assay (J Fish Dis 36:9-23) exceeded the diagnostic sensitivity of 0.85 (95% CI: 0.83, 0.87) for Phelps et al. (2012)'s assay (J Aquat Anim Health 24:238-243). The Jonstrup rRT-PCR assay is robust as demonstrated by high sensitivity and specificity estimates across laboratories and can be used as a valuable tool for targeted surveillance and for testing of suspect VHSV samples.

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.

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.

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.