Andrew E. Goodwin

First Report of Spring Viremia of Carp Virus (SVCV) in North America

Abstract Spring viremia of carp virus (SVCV) is an important viral pathogen of common carp Cyprinus carpio and one of only five fish pathogens listed as notifiable in the 2002 International Aquatic Animal Health Code of the Office International des Epizooties (OIE). Spring viremia is most problematic in Europe. The disease has been reported in many regions in the world but never in North America. In April 2002, an epizootic with clinical signs consistent with SVCV occurred on a fish farm on the East Coast of the United States that raises koi, a strain of common carp. A virus was isolated on epithelioma papillosum cyprini cells and subsequently demonstrated by immunocytochemistry to be SVCV. The OIE reference laboratory for SVCV (Centre for Environment, Fisheries, and Aquaculture Science, Weymouth, England) has confirmed the identity of the virus.

First Report of Spring Viremia of Carp Virus (SVCV) in Wild Common Carp in North America

Abstract In spring 2002, an estimated 1,500 common carp Cyprinus carpio in Cedar Lake, northwestern Wisconsin, died over a 6-week period from late April through the first week in June. Three moribund carp were necropsied and had signs consistent with spring viremia of carp (SVC) disease, including petechiae and ecchymotic hemorrhages on the skin, ascites, and edematous kidney and spleen. A virus was isolated on fathead minnow cells and shown to be a rhabdovirus by electron microscopy. Immunoassay results indicated a close serological relationship with spring viremia of carp virus (SVCV). This was confirmed by a reverse transcriptase–polymerase chain reaction assay and subsequent analysis of a subsection glycoprotein gene. Immunocytochemistry and serum neutralization tests indicated that the Cedar Lake isolate did not share complete antigenic identity with the European reference SVCV. Also, the isolate showed an inhibition of cytopathic effect after repeated subculture in epithelioma papulosum cyprini and ...

Goldfish Hematopoietic Necrosis Herpesvirus (Cyprinid Herpesvirus 2) in the USA: Molecular Confirmation of Isolates from Diseased Fish

Abstract Moribund goldfish Carassius auratus from private collections and breeding facilities were tested for hematopoietic necrosis herpesvirus 2 (also known as cyprinid herpesvirus 2 (CyHV-2)) by degenerate polymerase chain reaction (PCR) of the viral polymerase gene followed by sequencing. The degenerate PCR method produced a fragment with a DNA sequence that was more than 99% identical to the sequence of CyHV-2. Histology and electron microscopy showed that the moribund fish had severe necrosis in the hematopoietic tissues of the kidney and spleen and that herpesvirus particles were present. In the three cases studied, the presence of the virus was associated with high mortality (up to 80%) that could not be attributed to any other cause. The CyHV-2 is very difficult to isolate in cell culture, but our PCR and case studies demonstrate that this virus has a wide geographic distribution in the United States and that goldfish hematopoietic necrosis herpesvirus disease is likely to be an important, but ra...

Treatments for Ich Infestations in Channel Catfish Evaluated under Static and Flow-Through Water Conditions

Abstract In response to producer reports of poor efficacy using published treatments against Ichthyophthirius multifiliis (ich) infestations of channel catfish Ictalurus punctatus, we initiated a preliminary study to see which of the compounds that are permissible in food-fish aquaculture were efficacious under what we judged to be ideal laboratory conditions. We planned to use the results of this work as a basis for further study in the field. In our studies we used fingerling catfish in glass aquariums with stable water quality and daily treatments in both static and flow-through water systems. In some experiments, infested fish and healthy fish were stocked together so that the ability of the treatment to eliminate preexisting infestations could be examined separately from the treatments ability to block transmission. Malachite green and methylene blue were used as positive controls, and untreated fish were used as the negative control for efficacy. Treatments with sodium chloride, hydrogen peroxide, ...

Prevalence and Pathogenicity of a Heterophyid Trematode Infecting the Gills of an Endangered Fish, the Fountain Darter, in Two Central Texas Spring-Fed Rivers

Abstract Gills of 194 fountain darters Etheostoma fonticola collected from the Comal River in Texas from May 1997 through May 1998 were found to be parasitized with 8–1,524 metacercarial cysts of a heterophyid trematode tentatively identified as Centrocestus formosanus. The intensity of infection varied among three sites on the Comal River. In contrast, of 130 darters from the nearby San Marcos River that were examined, only 4 (3%) were infected, and these had 1–2 cysts per fish. Of 2,279 Melanoides tuberculata snails from the Comal River that were examined, 139 (6.1%) were infected with the trematode. Only 1 snail in 2,241 from the San Marcos River that were examined was infected. The presence of metacercariae in darters was associated with flared opercula, shortened or thickened gill filaments, epithelial hyperplasia, and engorged lamellae. The normal cartilage support of the filaments was distorted and displaced, leading to severe deformities of filament structure. Gill damage was severe and possibly l...

Comparison of multiple genes of spring viremia of carp viruses isolated in the United States

Five spring viremia of carp viruses (SVCV), Rhabdovirus carpio, were isolated in the United States (US) between 2002 and 2004. Single tube reverse transcription-polymerase chain reaction (RT-PCR) was used to generate overlapping cDNA fragments from the US isolates of SVCV. Multiple pairs of specific primers were designed to amplify a portion of the phosphoprotein gene, the matrix gene, and the glycoprotein gene of SVCV genogroup Id (corresponding to nucleotides 2174–4942 of GenBank accession NC_002803). Sequences were proofread and aligned to generate a consensus sequence for each isolate. Phylogenetic analysis of the 2705 nucleotide consensus sequence revealed that all five US isolates belong to SVCV genogroup Ia, Asian origin isolates, and a PCR primer binding site unique to SVCV genogroup Ia was identified.

Mortality and Carrier Status of Bluegills Exposed to Viral Hemorrhagic Septicemia Virus Genotype IVb at Different Temperatures

The emergence of the viral hemorrhagic septicemia virus (VHSV) genotype IVb (VHSV-IVb) in the Great Lakes of North America has led to concern that the virus might spread to natural fisheries and aquaculture in the southern USA. We exposed bluegills Lepomis macrochirus to VHSV-IVb by intraperitoneal injection at six temperatures from 10 degrees C to 30 degrees C and followed the disease course by quantitative real-time reverse transcriptase polymerase chain reaction (qrt-RT-PCR). Mortality of injected fish was 90% at 10 degrees C, 35% at 14 degrees C, and 10% at 18 degrees C; no mortality attributable to VHSV was observed at temperatures of 22-30 degrees C. In survivors tested at 21 d postchallenge, viral copies and prevalence determined by qrt-RT-PCR were inversely related to temperature, and VHSV-IVb could not be detected in fish held at temperatures above 22 degrees C. Similar results were obtained for bluegills that were exposed by cohabitation with the intraperitoneally injected fish. Acclimation of the fish to 12 degrees C after 21 d at higher temperatures did not appear to cause a re-emergence of the virus. Based on our findings, the temperature range of VHSV-IVb appears to be the same as published values for VHSV genotype I, which has an optimum of 9-12 degrees C and an upper limit of 18-20 degrees C.

Herpesviral haematopoietic necrosis virus (CyHV-2) infection: case studies from commercial goldfish farms

Herpesviral haematopoietic necrosis is a disease of goldfish, Carassius auratus, caused by Cyprinid herpesvirus-2 (CyHV-2) infection. Quantitative PCR was carried out on tissue homogenates from healthy goldfish fingerlings, broodfish, eggs and fry directly sampled from commercial farms, from moribund fish submitted to our laboratory for disease diagnosis, and on naturally-infected CyHV-2 carriers subjected to experimental stress treatments. Healthy fish from 14 of 18 farms were positive with copy numbers ranging from tens to 10(7) copies microg(-1) DNA extracted from infected fish. Of 118 pools of broodfish tested, 42 were positive. The CyHV-2 was detected in one lot of fry produced from disinfected eggs. Testing of moribund goldfish, in which we could not detect any other pathogens, produced 12 of 30 cases with 10(6)-10(8) copies of CyHV-2 microg(-1) DNA extracted. Subjecting healthy CyHV-2 carriers to cold shock (22-10 degrees C) but not heat, ammonia or high pH, increased viral copy numbers from mean copy number (+/-SE) of 7.3 +/- 11 to 394 +/- 55 microg(-1) DNA extracted after 24 h. CyHV-2 is widespread on commercial goldfish farms and outbreaks apparently occur when healthy carriers are subjected to a sharp temperature drop followed by holding at the permissive temperature for the disease.

Implication of Lateral Genetic Transfer in the Emergence of Aeromonas hydrophila Isolates of Epidemic Outbreaks in Channel Catfish

To investigate the molecular basis of the emergence of Aeromonas hydrophila responsible for an epidemic outbreak of motile aeromonad septicemia of catfish in the Southeastern United States, we sequenced 11 A. hydrophila isolates that includes five reference and six recent epidemic isolates. Comparative genomics revealed that recent epidemic A. hydrophila isolates are highly clonal, whereas reference isolates are greatly diverse. We identified 55 epidemic-associated genetic regions with 313 predicted genes that are present in epidemic isolates but absent from reference isolates and 35% of these regions are located within genomic islands, suggesting their acquisition through lateral gene transfer. The epidemic-associated regions encode predicted prophage elements, pathogenicity islands, metabolic islands, fitness islands and genes of unknown functions, and 34 of the genes encoded in these regions were predicted as virulence factors. We found two pilus biogenesis gene clusters encoded within predicted pathogenicity islands. A functional metabolic island that encodes a complete pathway for myo-inositol catabolism was evident by the ability of epidemic A. hydrophila isolates to use myo-inositol as a sole carbon source. Testing of A. hydrophila field isolates found a consistent correlation between myo-inositol utilization as a sole carbon source and the presence of an epidemic-specific genetic marker. All epidemic isolates and one reference isolate shared a novel O-antigen cluster. Altogether we identified four different O-antigen biosynthesis gene clusters within the 11 sequenced A. hydrophila genomes. Our study reveals new insights into the evolutionary changes that have resulted in the emergence of recent epidemic A. hydrophila strains.

Development of a Polymerase Chain Reaction Assay to Detect Cyprinid Herpesvirus 2 in Goldfish

Cyprinid herpesvirus 2 (CyHV2) has been associated with epidemic mortality among cultured populations of goldfish Carassius auratus. As the principal target tissues are hematopoietic cells in the kidney and spleen, the disease is designated herpesviral hematopoietic necrosis (HVHN). Originally described from Japan, the virus is present in at least five other countries and probably has a global distribution in goldfish. Preventing the further spread of the virus via control programs that exploit sensitive viral detection methods is critical. We developed a conventional polymerase chain reaction (PCR) test based on unique sequences found in the putative helicase gene of CyHV2 and completed initial steps toward the validation of this test. The helicase CyHV2 PCR has an analytic sensitivity of at least 78 copies of the target sequence per reaction in serially diluted plasmid and 84 copies/microg of DNA from the kidney and spleen of goldfish experimentally infected with CyHV2. The analytic specificity of the helicase CyHV2 PCR was demonstrated by the lack of amplification of genomic DNA from cyprinid herpesvirus 1, cyprinid herpesvirus 3, and ictalurid herpesvirus 1 (IcHV1). The helicase CyHV2 PCR effectively detected DNA from CyHV2 from goldfish over a broad geographic range, including Japan, California, Ohio, and Pennsylvania. The performance of the helicase CyHV2 PCR was compared with that of the previously described real-time TaqMan PCR for CyHV2 on a set of 37 samples of DNA from goldfish after experimental or natural exposure to CyHV2. The two tests had very strong agreement (kappa coefficient = 0.907) in classifying fish as positive or negative for CyHV2. The helicase CyHV2 PCR therefore complements the real-time PCR test as a conventional diagnostic method for preventing the further spread of CyHV2.