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Pseudoloma neurophilia ( Pn ), the causative agent of the most commonly reported disease of zebrafish, is a microsporidian parasite that confounds research by inducing behavioral and physiologic changes in zebrafish. Although a treatment for P. neurophilia has not been documented in zebrafish, albendazole (ALB) and fumagillin (FUM) have been used to treat microsporidian infections of other fish species. To investigate the efficacy of oral ALB and FUM in the treatment of Pn, we performed a pilot study that demonstrated the safety and palatability of novel gel-based diets containing FUM or ALB in adult AB zebrafish. In a subsequent study, approximately 250 adult AB zebrafish (previously infected with Pn ) were treated with these medicated diets for 4 wk. At 4 different time points (weeks 0, 5, 10, and 16 of the study), fish were euthanized and whole-body qPCR was performed to assess Pn prevalence across treatment and control groups. There was no statistically significant association between treatment group and Pn prevalence at any time point, although potential biologically relevant reductions in Pn prevalence occurred in the combination therapy group at weeks 5 and 16 and in the ALB group at week 5. Based on high-performance liquid chromatography analyses, the medicated diets contained less ALB and more FUM than expected, highlighting the importance of validating medicated feed concentrations to ensure safety, efficacy, and consistency. While Pn remains challenging to eradicate and control, results of this study warrant further investigation into the utility of ALB and FUM as potential treatments for this pathogen.
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Microsporidios , Pez Cebra , Animales , Albendazol/uso terapéutico , Proyectos Piloto , Microsporidios/fisiologíaRESUMEN
Alfaxalone, a synthetic neuroactive steroid, has been tested as an immersion anesthetic in ornamental fish, but its safety and efficacy in sport fish have not been investigated. In the current study, we compared the physiologic and behavioral effects of alfaxalone with those of tricaine methanesulfonate (MS222) for anesthesia of rainbow trout (Oncorhynchus mykiss) via water immersion. We also analyzed alfaxalone-exposed tissues to determine residue clearance times. Fish were anesthetized for 10 min by immersion in low-dose alfaxalone (Alow; 5 mg/L induction, 1 mg/L maintenance), high-dose alfaxalone (Ahigh; 5 mg/L induction, 2 mg/L maintenance), or MS222 (MS; 150 mg/L induction, 100 mg/L maintenance). Fish received all 3 treatments, separated by a washout period of at least 18 d in a blinded, complete crossover design. We hypothesized that immersion in Alow or Ahigh would provide a stable plane of anesthesia in rainbow trout, with dose-dependent time to recovery, and that opercular rates and depths of anesthesia would be equivalent to that of MS222. The time to anesthesia induction was longer for alfaxalone than MS222 but averaged less than 100 s. The time to recovery from anesthesia was also longer for alfaxalone than MS222, with significantly shorter recovery time for Alow than for Ahigh. All treatments decreased opercular rate and response to noxious stimuli. Alfaxalone residue clearance was greater than 80% from all tissues within 1 h, greater than 99% from muscle within 4 h, and 100% from all tissues within 36 h after exposure. We conclude that alfaxalone immersion at 5 mg/L for induction and 2 mg/L for maintenance provides a safe, viable alternative to MS222 for the anesthesia of rainbow trout.
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Anestésicos , Oncorhynchus mykiss , Aminobenzoatos , Anestesia General , Anestésicos/farmacología , Animales , Inmersión , Mesilatos , PregnanodionasRESUMEN
Introduction: Gastrointestinal illnesses associated with the consumption of shellfish contaminated with Vibrio parahaemolyticus have a negative impact on the shellfish industry due to recalls and loss of consumer confidence in products. This bacterial pathogen is very diverse and specific sequence types (STs), ST631 and ST36, have emerged as prevalent causes of Vibrio foodborne disease outbreaks in the US, though other STs have been implicated in sporadic cases. We investigated whether bacteriophages could be used as a proxy to monitor for the presence of distinct V. parahaemolyticus STs in coastal waters. Methods: For this purpose, bacteriophages infecting V. parahaemolyticus were isolated from water samples collected on the Northeast Atlantic coast. The isolated phages were tested against a collection of 29 V. parahaemolyticus isolates representing 18 STs, including six clonal complexes (CC). Four distinct phages were identified based on their ability to infect different sets of V. parahaemolyticus isolates. Results and Discussion: Overall, the 29 bacterial isolates segregated into one of eight patterns of susceptibility, ranging from resistance to all four phages to susceptibility to any number of phages. STs represented by more than one bacterial isolate segregated within the same pattern of susceptibility except for one V. parahaemolyticus ST. Other patterns of susceptibility included exclusively clinical isolates represented by distinct STs. Overall, this study suggests that phages populating coastal waters could be exploited to monitor for the presence of V. parahaemolyticus STs known to cause foodborne outbreaks.
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Eleven viral haemorrhagic septicaemia virus (VHSV) genotype IVb isolates were sequenced, and their genetic variation explored to determine the source of a VHS outbreak on the eastern shore of Cayuga Lake. An active fish kill of round gobies (Neogobius melanostomus, Pallas) was intensively sampled at King Ferry, NY and nearby Long Point State Park in May 2017. Gross lesions observed on 67 moribund round gobies and two rock bass (Ambloplites rupestris, Rafinesque) included moderately haemorrhagic internal organs and erythematous areas on the head, flank, and fins. RT-qPCR tests for VHSV were positive for all 69 fish. Viral isolation on epithelioma papulosum cyprinid cells showed cytopathic effect characteristic of VHSV for six round goby samples from King Ferry. The complete nucleotide sequence of the VHSV IVb genomes of five Cayuga Lake round goby isolates were derived on an Illumina platform along with 2017 VHSV IVb isolates from round gobies collected from the following: Lake Erie near Dunkirk, NY; the St. Lawrence River near Clayton and Cape Vincent, NY; and Lake St. Lawrence near Massena, NY. The phylogenetic tree created from these aligned sequences and four other complete VHSV IVb genomes shows Cayuga Lake isolates are closely related to the Lake Erie isolates.
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Brotes de Enfermedades/veterinaria , Enfermedades de los Peces/virología , Peces/virología , Septicemia Hemorrágica Viral/epidemiología , Novirhabdovirus/genética , Animales , Encéfalo/virología , Femenino , Enfermedades de los Peces/epidemiología , Variación Genética , Genoma Viral , Genotipo , Lagos/virología , Masculino , New York/epidemiología , Novirhabdovirus/aislamiento & purificación , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
The aquaculture industry is growing rapidly to meet the needs for global protein consumption. Viral diseases in aquaculture are quite challenging due to lack of treatment options as well as limited injection-delivery vaccines, which are costly. Thus, water-immersion antiviral treatments are highly desirable. This study focused on broad-spectrum, light-activated antivirals that target the viral membrane (envelope) of viruses to prevent viral-cell membrane fusion, ultimately blocking viral entry into cells. Of the tested small-molecules, JL122, a new broad-spectrum antiviral previously unexplored against aquatic viruses, blocked infection of three aquatic rhabdoviruses (IHNV, VHSV and SVCV) in cell culture and in two live fish challenge models. Importantly, JL122 inhibited transmission of IHNV from infected to uninfected rainbow trout. Further, the effective antiviral concentrations were not toxic to cells or susceptible fish. These results show promise for JL122 to become an immersion treatment option for outbreaks of aquatic enveloped viral infections.
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Antivirales/uso terapéutico , Enfermedades de los Peces/virología , Virus de la Necrosis Hematopoyética Infecciosa , Novirhabdovirus , Oncorhynchus mykiss , Infecciones por Rhabdoviridae/veterinaria , Animales , Antivirales/clasificación , Línea Celular Tumoral , Enfermedades de los Peces/tratamiento farmacológico , Estructura Molecular , Infecciones por Rhabdoviridae/prevención & control , Infecciones por Rhabdoviridae/virología , Relación Estructura-ActividadRESUMEN
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.
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Enfermedades de los Peces/virología , Septicemia Hemorrágica Viral/virología , Novirhabdovirus/patogenicidad , ARN Viral/genética , Animales , Peces , Técnicas de Amplificación de Ácido Nucleico , Filogenia , VirulenciaRESUMEN
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.
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Salmón , Estroncio/farmacología , Animales , Enfermedades de los Peces , Branquias/efectos de los fármacos , Branquias/patología , Estroncio/efectos adversosRESUMEN
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.
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Lubina , Edwardsiella/aislamiento & purificación , Infecciones por Enterobacteriaceae/veterinaria , Enfermedades de los Peces/epidemiología , Animales , Edwardsiella/genética , Infecciones por Enterobacteriaceae/epidemiología , Enfermedades de los Peces/diagnóstico , Enfermedades de los Peces/microbiología , Florida/epidemiología , New York/epidemiología , Reacción en Cadena de la Polimerasa/veterinaria , ARN Ribosómico 16S/análisis , Especificidad de la EspecieRESUMEN
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.
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Enfermedades de los Peces/virología , Genotipo , Carpa Dorada , Novirhabdovirus/genética , Infecciones por Rhabdoviridae/veterinaria , Animales , Susceptibilidad a Enfermedades , Infecciones por Rhabdoviridae/virologíaRESUMEN
Streptococcus iniae, the etiological agent of streptococcosis in fish, is an important pathogen of cultured and wild fish worldwide. During the last decade outbreaks of streptococcosis have occurred in a wide range of cultured and wild fish in the Americas and Caribbean islands. To gain a better understanding of the epizootiology of S. iniae in the western hemisphere, over 30 S. iniae isolates recovered from different fish species and geographic locations were characterized phenotypically and genetically. Species identities were determined biochemically and confirmed by amplification and sequencing of the 16S rRNA gene. Repetitive-element palindromic PCR fingerprinting as well as biochemical and antimicrobial susceptibility profiles suggest that a single strain of S. iniae was responsible for two different disease outbreaks among reef fishes in the Caribbean, one in 1999 and another in 2008. Interestingly, a majority of the isolates recovered from cultured fish in the Americas were genetically distinct from the Caribbean isolates and exhibited a trend toward higher minimal inhibitory concentration with respect to several antibiotics as well as greater genetic variability. The biological significance of this genetic variability is unclear, but it could have implications for future vaccine development and treatment.
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Enfermedades de los Peces/microbiología , Infecciones Estreptocócicas/veterinaria , Streptococcus intermedius/clasificación , Streptococcus intermedius/genética , Américas/epidemiología , Animales , Enfermedades de los Peces/epidemiología , Peces , Filogenia , Infecciones Estreptocócicas/epidemiología , Infecciones Estreptocócicas/microbiología , Indias Occidentales/epidemiologíaRESUMEN
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.
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Lubina , Susceptibilidad a Enfermedades/veterinaria , Animales , Línea Celular , Infecciones por Virus ADN , Enfermedades de los Peces , PerciformesRESUMEN
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.
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Septicemia Hemorrágica Viral/virología , Novirhabdovirus/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Animales , Peces , Genotipo , Novirhabdovirus/genética , Vigilancia de la Población , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
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.
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Septicemia Hemorrágica Viral/diagnóstico , Novirhabdovirus/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Animales , Peces , Genotipo , Septicemia Hemorrágica Viral/virología , Novirhabdovirus/genética , Vigilancia de la Población , ARN Viral/genética , ARN Viral/aislamiento & purificación , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Especificidad de la EspecieRESUMEN
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.
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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 ( â 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.
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Cyprinidae/virología , Esocidae , Genotipo , Septicemia Hemorrágica Viral/virología , Novirhabdovirus/genética , Conducta Predatoria/fisiología , Animales , Septicemia Hemorrágica Viral/transmisiónRESUMEN
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.
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Aletas de Animales/patología , Cyprinidae , Branquias/patología , Novirhabdovirus/genética , Novirhabdovirus/aislamiento & purificación , Infecciones por Rhabdoviridae/veterinaria , Aletas de Animales/virología , Animales , Biopsia/métodos , Biopsia/veterinaria , Genotipo , Branquias/virología , Infecciones por Rhabdoviridae/patología , Sensibilidad y EspecificidadRESUMEN
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.
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Carpas , Septicemia Hemorrágica Viral/virología , Novirhabdovirus/clasificación , Animales , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
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.
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Carpas/virología , Enfermedades de los Peces/epidemiología , Infecciones por Herpesviridae/veterinaria , Animales , Enfermedades de los Peces/virología , Great Lakes Region/epidemiología , Herpesviridae/aislamiento & purificación , Infecciones por Herpesviridae/epidemiología , Infecciones por Herpesviridae/virología , Reacción en Cadena de la Polimerasa/veterinaria , Prevalencia , Vigilancia de Guardia/veterinaria , Microbiología del AguaRESUMEN
Real-time, or quantitative, polymerase chain reaction (qPCR) is quickly supplanting other molecular methods for detecting the nucleic acids of human and other animal pathogens owing to the speed and robustness of the technology. As the aquatic animal health community moves toward implementing national diagnostic testing schemes, it will need to evaluate how qPCR technology should be employed. This review outlines the basic principles of qPCR technology, considerations for assay development, standards and controls, assay performance, diagnostic validation, implementation in the diagnostic laboratory, and quality assurance and control measures. These factors are fundamental for ensuring the validity of qPCR assay results obtained in the diagnostic laboratory setting.