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.

Isolation and molecular characterization of a novel picornavirus from baitfish in the USA.

During both regulatory and routine surveillance sampling of baitfish from the states of Illinois, Minnesota, Montana, and Wisconsin, USA, isolates (n = 20) of a previously unknown picornavirus were obtained from kidney/spleen or entire viscera of fathead minnows (Pimephales promelas) and brassy minnows (Hybognathus hankinsoni). Following the appearance of a diffuse cytopathic effect, examination of cell culture supernatant by negative contrast electron microscopy revealed the presence of small, round virus particles (∼ 30-32 nm), with picornavirus-like morphology. Amplification and sequence analysis of viral RNA identified the agent as a novel member of the Picornaviridae family, tentatively named fathead minnow picornavirus (FHMPV). The full FHMPV genome consisted of 7834 nucleotides. Phylogenetic analysis based on 491 amino acid residues of the 3D gene showed 98.6% to 100% identity among the 20 isolates of FHMPV compared in this study while only 49.5% identity with its nearest neighbor, the bluegill picornavirus (BGPV) isolated from bluegill (Lepomis macrochirus). Based on complete polyprotein analysis, the FHMPV shared 58% (P1), 33% (P2) and 43% (P3) amino acid identities with BGPV and shared less than 40% amino acid identity with all other picornaviruses. Hence, we propose the creation of a new genus (Piscevirus) within the Picornaviridae family. The impact of FHMPV on the health of fish populations is unknown at present.

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.

Rapid quantitative detection of Aeromonas hydrophila strains associated with disease outbreaks in catfish aquaculture.

A new strain of Aeromonas hydrophila has been implicated in significant losses in farm-raised catfish. Outbreaks attributable to this new strain began in Alabama in the summer of 2009 and have spread to Arkansas and Mississippi in subsequent years. These outbreaks mostly afflicted market-sized fish and resulted in considerable losses in short periods of time. The present research was designed to develop an expeditious diagnostic procedure to detect the new strains of A. hydrophila due to the rapid onset and biosecurity concerns associated with this new disease. A discriminatory quantitative polymerase chain reaction assay was developed using gene sequences unique to the virulent strains identified in a related comparative genomic study. Using this assay, suspect colonies on a culture plate can be positively identified as the new strain within 2 hr. The assay is repeatable and reproducible with a linear dynamic range covering 8 orders of magnitude and a sensitivity of approximately 7 copies of target DNA in a 15-µl reaction. In addition, the assay is able to detect and quantify the virulent strain from catfish tissues (0.025 g), pond water (40 ml), and sediments (0.25 g) with a sensitivity limit of approximately 100 bacteria in a sample. This assay provides rapid discrimination between the new virulent strain and more common A. hydrophila and is useful for epidemiological studies involving the detection and quantification of the virulent strain in environmental samples and fish tissues.

Comparison of treatments to inactivate viral hemorrhagic septicemia virus (VHSV-IVb) in frozen baitfish.

Current US state and federal fish health regulations target the spread of viral hemorrhagic septicemia virus-IVb (VHSV-IVb) through movement restrictions of live fish; however, they largely ignore the potential for the virus to be spread through commercial distribution and use of frozen baitfish from VHSV-IVb-positive regions. Some state laws do require treatment of frozen baitfish to inactivate VHSV, and additional methods have been proposed, but few scientific studies have examined the efficacy of these treatments. In this study, bluegills Lepomis macrochirus were challenged with VHSV-IVb and frozen to represent standard industry methods, disinfected by various treatments, and tested for infectious VHSV-IVb using virus isolation. The virus was isolated from 70% of fish subjected to 3 freeze/thaw cycles. All other treatment methods were effective in inactivating the virus, including treatment with isopropyl alcohol, mineral oil, salt and borax, and dehydration. Dehydration followed by rehydration is rapid and effective, and therefore, seems to be the best option for inactivating VHSV-IVb present in frozen baitfish while maintaining their usefulness as bait.

Molecular and immunohistochemical diagnosis of Francisella noatunensis subsp. orientalis from formalin-fixed, paraffin-embedded tissues.

Members of the genus Francisella (viz., F. noatunensis subsp. orientalis [Fno] and F. noatunensis subsp. noatunensis) have been described as causative agents of chronic granulomatous and pyogranulomatous lesions in wild and cultured fish species. In the present study, 68 archived formalin-fixed, paraffin-embedded (FFPE) tissues from several fish species, collected at different geographical locations from 2000 to 2011, were analyzed using a real-time polymerase chain reaction assay for the detection of the Fno intracellular growth loci C (iglC) gene and by immunohistochemistry for the demonstration of Fno antigens. The results revealed a high correlation between these 2 diagnostic techniques validating their use for the diagnosis of Fno infection in archived FFPE tissues and confirming the presence of Fno in fish species from the Cari y years of the present century.

Genetic analysis of a novel nidovirus from fathead minnows.

A bacilliform virus was isolated from diseased fathead minnows (Pimephales promelas). Analysis of the complete genome coding for the polyprotein (pp1ab), spike (S), membrane (M) and nucleocapsid (N) proteins revealed that the virus was most like white bream virus (WBV), another bacilliform virus isolated from white bream (Blicca bjoerkna L.) and the type species of the genus Bafinivirus within the order Nidovirales. In addition to similar gene order and size, alignment of deduced amino acid sequences of the pp1ab, M, N and S proteins of the fathead minnow nidovirus (FHMNV) with those of WBV showed 46, 44, 39 and 15 % identities, respectively. Phylogenetic analysis using the conserved helicase domain of the replicase showed FHMNV was distinct from WBV, yet the closest relative identified to date. Thus, FHMNV appears to represent a second species in the genus Bafinivirus. A PCR assay was developed for the identification of future FHMNV-like isolates.

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.

Replication and persistence of VHSV IVb in freshwater turtles.

With the emergence of viral hemorrhagic septicemia virus (VHSV) strain IVb in the Great Lakes of North America, hatchery managers have become concerned that this important pathogen could be transmitted by animals other than fish. Turtles are likely candidates because they are poikilotherms that feed on dead fish, but there are very few reports of rhabdovirus infections in reptiles and no reports of the fish rhabdoviruses in animals other than teleosts. We injected common snapping turtles Chelydra serpentine and red-eared sliders Trachemys scripta elegans intraperitoneally with 10(4) median tissue culture infectious dose (TCID50) of VHSV-IVb and 21 d later were able to detect the virus by quantitative real-time reverse transcriptase PCR (qrt-RTPCR) in pools of kidney, liver, and spleen. In a second experiment, snapping turtles, red-eared sliders, yellow-bellied sliders T. scripta scripta, and northern map turtles Grapetemys geographica at 14 degrees C were allowed to feed on tissues from bluegill dying of VHSV IVb disease. Turtle kidney, spleen, and brain pools were not positive by qrt-RTPCR on Day 3 post feeding, but were positive on Days 10 and 20. Map turtles on Day 20 post-feeding were positive by both qrt-RTPCR and by cell culture. Our work shows that turtles that consume infected fish are a possible vector for VHSV IVb, and that the fish rhabdoviruses may have a broader host range than previously suspected.

Thelohanellus toyamai (syn. Myxobolus toyamai) infecting the gills of koi Cyprinus carpio in the eastern United States.

A myxozoan resembling species of Thelohanellus was isolated from the gills of koi (Cyprinus carpio) cultured in North Carolina. Plasmodia measuring ∼200 µm in diameter contained tear-shaped myxospores containing a single pyriform polar capsule. The spore body was concave on one side, measuring 16.2 (14.7-16.8) µm long and 5.6 (4.5-6) µm wide. The polar capsule was 6.4 (5.8-7.2) µm long and 4.2 (3.4-4.6) µm wide, containing a polar filament coiled perpendicular to the longitudinal axis of the spore body making 8 turns. Occasionally, an oblong, irregularly shaped mass of protoplasm was observed between the polar capsule and spore capsule. Analysis of 18S small-subunit ribosomal DNA sequence demonstrated this isolate as a 99.9% match with Myxobolus toyamai from gills of C. carpio. However, the case isolate lacked the characteristic second polar capsule of Myxobolus, morphologically placing it within the Thelohanellus. Here we supplement genetic sequence data with histopathology, an amended morphological description of the agent, and a review of the original classification. For future reference, we suggest this organism be referred to as Thelohanellus toyamai Kudo, 1933, in accordance with the original classification and the nomial M. toyamai be avoided because it is at best outdated and, at worst, incorrect.

The pathology associated with visceral toxicosis of catfish.

Visceral toxicosis of catfish (VTC) syndrome was recognized in the late 1990 s and recently has been associated with exposure to Clostridium botulinum type E neurotoxin. Tentative diagnosis is based on clinical presentation and gross findings, and is confirmed by bioassay. In April 2009, channel catfish (Ictalurus punctatus) from 2 different farms presented with abnormal swimming behavior and mortalities. Nine fish were submitted to the Aquatic Research and Diagnostic Laboratory (Stoneville, Mississippi) for evaluation. Bacterial cultures from these fish were negative. Necropsy findings included intestinal intussusceptions, ascites, pale proximal intestines with engorged serosal blood vessels, splenic congestion, and a reticular pattern to the liver. Significant histopathologic findings were limited to cerebral, splenic, and hepatic congestion, splenic lymphoid depletion and perivascular edema, vascular dilation and edema of the gastrointestinal tract, and perivascular edema in the anterior and posterior kidneys. Intoxication from C. botulinum type E neurotoxin was suspected based on the clinical signs and lack of gross and microbiological evidence of an infectious disease process. The toxicosis was confirmed with a positive bioassay using serum collected from the submitted fish.

Detection and prevalence of the nonsyncytial American grass carp reovirus Aquareovirus G by quantitative reverse transcriptase polymerase chain reaction.

The American grass carp reovirus (AGCRV) Aquareovirus G is not strongly associated with disease in fish, but it is often detected by cell culture during routine inspections of healthy fish. The cytopathic effect of AGCRV does not involve the typical syncytia associated with most aquareoviruses. Instead, the AGCRV produces a pattern of cell rounding that is very similar to that produced by rhabdoviruses, including those that are highly regulated. We have developed a quantitative polymerase chain reaction assay that can be used to identify AGCRV in cell cultures or directly on fish tissues. The assay detects as few as two copies of the plasmid template, has a coefficient of variation of 15% among assays performed on different days, and does not cross-react with any other aquareoviruses tested. Assays performed on tissues of cultured golden shiners Notemigonus crysoleucas and fathead minnow Pimephales promelas revealed a high prevalence of infection among healthy fish but no association with disease.

Are all koi ulcer cases associated with infection by atypical Aeromonas salmonicida? Polymerase chain reaction assays of koi carp skin swabs submitted by hobbyists.

Infection by atypical Aeromonas salmonicida is regarded as the cause of ulcer disease (KUD) in koi carp Cyprinus carpio and goldfish Carassius auratus. However, other causes--including parasites, viral infection, and fungi--have been proposed. In our diagnostic work, we often fail to isolate A. salmonicida even when clear clinical signs of KUD are present. This failure may be because these fastidious and slow-growing bacteria are difficult to isolate in culture or because the bacteria are not actually present in the lesions. In this study, we used polymerase chain reaction (PCR) to detect A. salmonicida in DNA samples swabbed from koi carp ulcers. These alcohol-preserved samples were collected and submitted by hobbyists and included 40 separate cases from 12 different states. We identified atypical A. salmonicida by PCR in 52 of 62 samples submitted and in 33 of 40 unique cases. The negative findings for A. salmonicida by PCR could all be attributed to high water temperatures, prior antibiotic use, poor sample quality, or misdiagnosis of columnaris disease as KUD. Tests for Aphanomyces invadans by PCR were negative in every case. This work confirms that A. salmonicda is still the predominant cause of KUD and that our negative culture results were most likely due to technical failures rather than an absence of A. salmonicda in the ulcer lesions.

The effects of largemouth bass virus on a quality largemouth bass population in Arkansas.

A 22.4-ha impoundment experienced an outbreak of Largemouth bass (Micropterus salmoides) virus (LMBV) disease in the summer of 2006. All dead or dying largemouth bass observed throughout the entire event were recorded and removed. In this study, we estimated mortality and examined size distribution, condition, and biomass following the outbreak. Boat-mounted electrofishing was used to collect largemouth bass for a mark-recapture population estimate and other population metrics. Fish samples were examined for evidence of LMBV, other infectious diseases, and physical abnormalities. Cell cultures inoculated with samples from moribund fish developed cytopathic effects typical of LMBV, and polymerase chain reaction (PCR) confirmed the presence of LMBV. The total number (N+/-95% confidence interval) of stock-size largemouth bass remaining was estimated to be 2,301+/-528 fish (103 bass/ha). The total observed mortality, including dead and dying individuals, during the LMBV outbreak was 176 largemouth bass (7% of the initial population). The total biomass remaining was estimated at 1,592 kg of stock-size bass and a relative biomass of 71.5 kg of stock-size largemouth bass per hectare. Largemouth bass size structure was dominated by quality and preferred (300-510 mm) size classes, with very few memorable-size or larger (>510 mm) fish, and the relative weight of largemouth bass was unusually variable. These results demonstrate that largemouth bass abundance and biomass in the reservoir remained very high despite mortalities attributed to a LMBV outbreak.

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.

Vertical transmission of Ovipleistophora ovariae (microspora) within the eggs of the golden shiner.

Fertilized eggs collected from broodfish infected by Ovipleistophora ovariae were tested by quantitative polymerase chain reaction (PCR) and found to be positive for the O. ovariae genome at 7.77 X 10(2) to 3.26 x 10(7) copies per microgram of host DNA. Fry hatched from these eggs contained from 1.37 X 10(2) to 9.89 X 10(6) copies of the O. ovariae genome per microgram of host DNA. Surface treatments of fertilized eggs with 150 mg formalin/L (used by farms as a fungicide) or a 1.5% solution of sodium sulfite (which removes the adhesive egg matrix) did not reduce vertical transmission to fry. Treatment of eggs with a 10% solution of bleach or a proprietary commercial DNA denaturant did not reduce the number of egg-associated copies of the O. ovariae genome. Histology of ovaries of infected fish demonstrated spores within the oocytes. However, no spores were observed by histology in positive fry hatched from infected eggs. The PCR and histological demonstration of the presence of O. ovariae spores in oocytes and fry, and the failure of strong DNA denaturants to reduce egg-associated copies, give evidence that O. ovariae is vertically transmitted within eggs.

Complete characterisation of the American grass carp reovirus genome (genus Aquareovirus: family Reoviridae) reveals an evolutionary link between aquareoviruses and coltiviruses.

An aquareovirus was isolated from several fish species in the USA (including healthy golden shiners) that is not closely related to members of species Aquareovirus A, B and C. The virus, which is atypical (does not cause syncytia in cell cultures at neutral pH), was implicated in a winter die-off of grass carp fingerlings and has therefore been called 'American grass carp reovirus' (AGCRV). Complete nucleotide sequence analysis of the AGCRV genome and comparisons to the other aquareoviruses showed that it is closely related to golden ide reovirus (GIRV) (>92% amino acid [aa] identity in VP5(NTPase) and VP2(Pol)). However, comparisons with grass carp reovirus (Aquareovirus C) and chum salmon reovirus (Aquareovirus A) showed only 22% to 76% aa identity in different viral proteins. These findings have formed the basis for the recognition of AGCRV and GIRV as members of a new Aquareovirus species 'Aquareovirus G' by ICTV. Further sequence comparisons to other members of the family Reoviridae suggest that there has been an 'evolutionary jump,' involving a change in the number of genome segments, between the aquareoviruses (11 segments) and coltiviruses (12 segments). Segment 7 of AGRCV encodes two proteins, from two distinct ORFs, which are homologues of two Coltivirus proteins encoded by genome segments 9 and 12. A similar model has previously been reported for the rotaviruses and seadornaviruses.

Validation of a quantitative PCR diagnostic method for detection of the microsporidian Ovipleistophora ovariae in the cyprinid fish Notemigonus crysoleucas.

Microsporidian parasites are easily detected by light microscopy when infections are heavy and spores are present. However, early infections without spores, or light infections with low numbers of spores, are easily missed. This limitation has made it difficult to conduct investigations into microsporidian prevalence and transmission. In this study, we developed a quantitative TaqMan polymerase chain reaction assay to assess the presence of Ovipleistophora ovariae in the tissues of the cyprinid fish Notemigonus crysoleucas (golden shiner). The efficiency of the primer set was 100.8%, with a correlation coefficient of threshold position to copy number of 0.997 over 9 logs using a plasmid containing the cloned reaction product. No product was produced from other closely related microsporidian species (Nucleospora salmonis, Pseudoloma neurophila, Glugea stephani, Heterosporis sp., and O. mirandella). The coefficient of variation for replicate assays done on different days was 12.4%. The assay detects O. ovariae reliably at less than 10 genomic copies and 0.14 spores per reaction, but maximum sensitivity is only achieved when sonication is included as part of the DNA purification step. Using the assay, we found 4.44 x 10(1) to 7.91 x 10(6) copies microg(-1) host DNA in female golden shiners, with the spore density increasing during the spawning season. The parasite was also detected for the first time in the testes of male golden shiners at 2.60 x 10(1) to 8.62 x 102 copies microg(-1) host DNA.

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.