RESUMEN
Nuclear inclusion X (NIX), the etiological agent of bacterial gill disease in Pacific razor clams Siliqua patula, was associated with host mortality events in coastal Washington State, USA, during the mid-1980s. Ongoing observations of truncated razor clam size distributions in Kalaloch Beach, Washington, raised concerns that NIX continues to impact populations. We conducted a series of spatial and longitudinal NIX surveillances, examined archived razor clam gill tissue, and used population estimates from stock assessments to test whether (1) the prevalence and intensity of NIX infections is higher at Kalaloch Beach relative to nearby beaches, (2) infected gill tissue has features consistent with historical descriptions of NIX-associated histopathology, and (3) annual clam survival is inversely related to NIX infection prevalence and intensity. NIX prevalence exceeded 85% at all sampled locations, and infection intensity was the highest at Kalaloch Beach by 0.9-2.6 orders of magnitude. Kalaloch Beach clams revealed histopathology consistent with previous NIX epidemics, including enlarged and/or rupturing branchial epithelial cells, branchial necrosis, and high hemocyte densities. Estimated annual survival was 22% at Kalaloch Beach, and ranged between 57 and 99% at other study sites. NIX infection intensity (via quantitative PCR) was not significantly correlated with annual survival; however, annual survival was lowest at Kalaloch Beach, where infection intensities were highest, suggesting that clams can tolerate infections up to a lethal threshold. Collectively these data support the hypothesis that high NIX intensities are associated with host mortality. NIX-associated mortality appears to be more pronounced at Kalaloch Beach relative to other Washington beaches.
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Bivalvos , Cuerpos de Inclusión Intranucleares , Animales , Branquias , Washingtón/epidemiologíaRESUMEN
Several Francisella spp., including Francisella noatunensis, are regarded as important emerging pathogens of wild and farmed fish. However, very few studies have investigated the virulence factors that allow these bacterial species to be pathogenic in fish. The Francisella pathogenicity island (FPI) is a well-described, gene-dense region encoding major virulence factors for the genus Francisella. pdpA is a member of the pathogenicity-determining protein genes carried by the FPI that are implicated in the ability of the mammalian pathogen Francisella tularensis to escape and replicate in infected host cells. Using a sacB suicide approach, we generated pdpA knockouts to address the role of PdpA as a virulence factor for F. noatunensis. Because polarity can be an issue in gene-dense regions, we generated two different marker-based mutants in opposing polarity (the F. noatunensis subsp. orientalis ΔpdpA1 and ΔpdpA2 strains). Both mutants were attenuated (P < 0.0001) in zebrafish challenges and displayed impaired intracellular replication (P < 0.05) and cytotoxicity (P < 0.05), all of which could be restored to wild-type (WT) levels by complementation for the ΔpdpA1 mutant. Importantly, differences were found for bacterial burden and induction of acute-phase and proinflammatory genes for the F. noatunensis subsp. orientalis ΔpdpA1 and ΔpdpA2 mutants compared to the WT during acute infection. In addition, neither mutant resulted in significant histopathological changes. Finally, immunization with the F. noatunensis subsp. orientalis ΔpdpA1 mutant led to protection (P < 0.012) against an acute 40% lethal dose (LD40) challenge with WT F. noatunensis in the zebrafish model of infection. Taken together, the results from this study further demonstrate physiological similarities within the genus Francisella relative to their phylogenetic relationships and the utility of zebrafish for addressing virulence factors for the genus.
Asunto(s)
Francisella/patogenicidad , Islas Genómicas , Pez Cebra/microbiología , Animales , Proteínas Bacterianas/genética , Enfermedades de los Peces/microbiología , VirulenciaRESUMEN
Preliminary evidence suggests that Chinook salmon Oncorhynchus tshawytscha from the Yukon River may be more susceptible to Ichthyophonus sp. infections than Chinook from stocks further south. To investigate this hypothesis in a controlled environment, we experimentally challenged juvenile Chinook from the Yukon River and from the Salish Sea with Ichthyophonus sp. and evaluated mortality, infection prevalence and infection load over time. We found that juvenile Chinook salmon from a Yukon River stock were more susceptible to ichthyophoniasis than were those from a Salish Sea stock. After feeding with tissues from infected Pacific herring Clupea pallasii, Chinook salmon from both stocks became infected. The infection was persistent and progressive in Yukon River stock fish, where infections sometimes progressed to mortality, and histological examinations revealed parasite dissemination and proliferation throughout the host tissues. In Salish Sea-origin fish, however, infections were largely transient; host mortalities were rare, and parasite stages were largely cleared from most tissues after 3-4 wk. Susceptibility differences were evidenced by greater cumulative mortality, infection prevalence, parasite density, proportion of fish demonstrating a cellular response, and intensity of the cellular response among fish from the Yukon River stock. These observed differences between Chinook salmon stocks were consistent when parasite exposures occurred in both freshwater and seawater. These results support the hypothesis that a longer-standing host-pathogen relationship, resulting in decreased disease susceptibility, exists among Salish Sea Chinook salmon than among Yukon River conspecifics.
Asunto(s)
Enfermedades de los Peces , Mesomycetozoea , Animales , Enfermedades de los Peces/epidemiología , Ríos , Salmón , El YukónRESUMEN
Nuclear inclusion X (NIX) is a gamma proteobacteria that infects the nuclei of gill epithelial cells in Pacific razor clams. NIX has been associated with clam die-offs in coastal Washington. A quantitative PCR (qPCR) assay was developed to detect NIX in Pacific razor clams, and assay specificity was confirmed by chromogenic in situ hybridization (CISH). Both tests were applied to evaluate NIX infections in wild Pacific razor clams collected during spring 2019. Consistent with results from earlier histopathological assessments, qPCR and CISH indicated 100% prevalence in razor clams from two Washington beaches and 0% prevalence from two Alaskan beaches.
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Bivalvos/microbiología , Pruebas Diagnósticas de Rutina/métodos , Proteobacteria/aislamiento & purificación , Animales , Infecciones Bacterianas/epidemiología , Branquias , Hibridación in Situ , Cuerpos de Inclusión Intranucleares/microbiología , Prevalencia , Reacción en Cadena en Tiempo Real de la Polimerasa , Washingtón/epidemiologíaRESUMEN
Piscine orthoreovirus genotype 1 (PRV-1) is the causative agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar L.). The virus has also been found in Pacific salmonids in western North America, raising concerns about the risk to native salmon and trout. Here, we report the results of laboratory challenges using juvenile Chinook salmon, coho salmon and rainbow trout injected with tissue homogenates from Atlantic salmon testing positive for PRV-1 or with control material. Fish were sampled at intervals to assess viral RNA transcript levels, haematocrit, erythrocytic inclusions and histopathology. While PRV-1 replicated in all species, there was negligible mortality in any group. We observed a few erythrocytic inclusion bodies in fish from the PRV-1-infected groups. At a few time points, haematocrits were significantly lower in the PRV-1-infected groups relative to controls, but in no case was anaemia noted. The most common histopathological finding was mild, focal myocarditis in both the non-infected controls and PRV-1-infected fish. All cardiac lesions were judged mild, and none were consistent with those of HSMI. Together, these results suggest all three species are susceptible to PRV-1 infection, but in no case did infection cause notable disease in these experiments.
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Enfermedades de los Peces/virología , Genotipo , Hematócrito/veterinaria , Cuerpos de Inclusión Viral/fisiología , Oncorhynchus , Orthoreovirus/fisiología , Infecciones por Reoviridae/veterinaria , Animales , Oncorhynchus kisutch , Oncorhynchus mykiss , Orthoreovirus/genética , ARN Viral/análisis , Infecciones por Reoviridae/virologíaRESUMEN
A novel virus, rainbow trout orthomyxovirus (RbtOV), was isolated in 1997 and again in 2000 from commercially-reared rainbow trout (Oncorhynchus mykiss) in Idaho, USA. The virus grew optimally in the CHSE-214 cell line at 15°C producing a diffuse cytopathic effect; however, juvenile rainbow trout exposed to cell culture-grown virus showed no mortality or gross pathology. Electron microscopy of preparations from infected cell cultures revealed the presence of typical orthomyxovirus particles. The complete genome of RbtOV is comprised of eight linear segments of single-stranded, negative-sense RNA having highly conserved 5' and 3'-terminal nucleotide sequences. Another virus isolated in 2014 from steelhead trout (also O. mykiss) in Wisconsin, USA, and designated SttOV was found to have eight genome segments with high amino acid sequence identities (89-99%) to the corresponding genes of RbtOV, suggesting these new viruses are isolates of the same virus species and may be more widespread than currently realized. The new isolates had the same genome segment order and the closest pairwise amino acid sequence identities of 16-42% with Infectious salmon anemia virus (ISAV), the type species and currently only member of the genus Isavirus in the family Orthomyxoviridae. However, pairwise comparisons of the predicted amino acid sequences of the 10 RbtOV and SttOV proteins with orthologs from representatives of the established orthomyxoviral genera and a phylogenetic analysis using the PB1 protein showed that while RbtOV and SttOV clustered most closely with ISAV, they diverged sufficiently to merit consideration as representatives of a novel genus. A set of PCR primers was designed using conserved regions of the PB1 gene to produce amplicons that may be sequenced for identification of similar fish orthomyxoviruses in the future.
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Enfermedades de los Peces/virología , Genoma Viral , Oncorhynchus mykiss/virología , Infecciones por Orthomyxoviridae/veterinaria , Orthomyxoviridae/genética , ARN Viral/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Especiación Genética , Isavirus/clasificación , Isavirus/genética , Orthomyxoviridae/clasificación , Infecciones por Orthomyxoviridae/virología , Filogenia , Alineación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
UNLABELLED: The white sucker Catostomus commersonii is a freshwater teleost often utilized as a resident sentinel. Here, we sequenced the full genome of a hepatitis B-like virus that infects white suckers from the Great Lakes Region of the United States. Dideoxy sequencing confirmed that the white sucker hepatitis B virus (WSHBV) has a circular genome (3,542 bp) with the prototypical codon organization of hepadnaviruses. Electron microscopy demonstrated that complete virions of approximately 40 nm were present in the plasma of infected fish. Compared to avi- and orthohepadnaviruses, sequence conservation of the core, polymerase, and surface proteins was low and ranged from 16 to 27% at the amino acid level. An X protein homologue common to the orthohepadnaviruses was not present. The WSHBV genome included an atypical, presumptively noncoding region absent in previously described hepadnaviruses. Phylogenetic analyses confirmed WSHBV as distinct from previously documented hepadnaviruses. The level of divergence in protein sequences between WSHBV and other hepadnaviruses and the identification of an HBV-like sequence in an African cichlid provide evidence that a novel genus of the family Hepadnaviridae may need to be established that includes these hepatitis B-like viruses in fishes. Viral transcription was observed in 9.5% (16 of 169) of white suckers evaluated. The prevalence of hepatic tumors in these fish was 4.9%, and only 2.4% of fish were positive for both virus and hepatic tumors. These results are not sufficient to draw inferences regarding the association of WSHBV and carcinogenesis in white sucker. IMPORTANCE: We report the first full-length genome of a hepadnavirus from fishes. Phylogenetic analysis of this genome indicates divergence from genomes of previously described hepadnaviruses from mammalian and avian hosts and supports the creation of a novel genus. The discovery of this novel virus may better our understanding of the evolutionary history of hepatitis B-like viruses of other hosts. In fishes, knowledge of this virus may provide insight regarding possible risk factors associated with hepatic neoplasia in the white sucker. This may also offer another model system for mechanistic research.
Asunto(s)
Cipriniformes/virología , Enfermedades de los Peces/epidemiología , Enfermedades de los Peces/virología , Genoma Viral/genética , Virus de la Hepatitis B/genética , Neoplasias Hepáticas/veterinaria , Animales , Secuencia de Bases , Secuencia Conservada/genética , Evolución Molecular , Componentes Genómicos , Great Lakes Region , Virus de la Hepatitis B/clasificación , Neoplasias Hepáticas/epidemiología , Neoplasias Hepáticas/virología , Microscopía Electrónica/veterinaria , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN/veterinaria , Especificidad de la Especie , Virión/ultraestructuraRESUMEN
Non-lethal pathogen testing can be a useful tool for fish disease research and management. Our research objectives were to determine if (1) fin clips, gill snips, surface mucus scrapings, blood draws, or kidney biopsies could be obtained non-lethally from 3 to 15 g Chinook salmon Oncorhynchus tshawytscha, (2) non-lethal samples could accurately discriminate between fish exposed to the bacterial kidney disease agent Renibacterium salmoninarum and non-exposed fish, and (3) non-lethal samples could serve as proxies for lethal kidney samples to assess infection intensity. Blood draws and kidney biopsies caused ≥5% post-sampling mortality (Objective 1) and may be appropriate only for larger fish, but the other sample types were non-lethal. Sampling was performed over 21 wk following R. salmoninarum immersion challenge of fish from 2 stocks (Objectives 2 and 3), and nested PCR (nPCR) and real-time quantitative PCR (qPCR) results from candidate non-lethal samples were compared with kidney tissue analysis by nPCR, qPCR, bacteriological culture, enzyme-linked immunosorbent assay (ELISA), fluorescent antibody test (FAT) and histopathology/immunohistochemistry. R. salmoninarum was detected by PCR in >50% of fin, gill, and mucus samples from challenged fish. Mucus qPCR was the only non-lethal assay exhibiting both diagnostic sensitivity and specificity estimates>90% for distinguishing between R. salmoninarum-exposed and non-exposed fish and was the best candidate for use as an alternative to lethal kidney sample testing. Mucus qPCR R. salmoninarum quantity estimates reflected changes in kidney bacterial load estimates, as evidenced by significant positive correlations with kidney R. salmoninarum infection intensity scores at all sample times and in both fish stocks, and were not significantly impacted by environmental R. salmoninarum concentrations.
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Enfermedades de los Peces/microbiología , Infecciones por Bacterias Grampositivas/veterinaria , Micrococcaceae/aislamiento & purificación , Salmón/microbiología , Animales , Enfermedades de los Peces/diagnóstico , Infecciones por Bacterias Grampositivas/diagnóstico , Infecciones por Bacterias Grampositivas/microbiología , Riñón/microbiología , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Viral erythrocytic necrosis (VEN) is a condition that affects marine and anadromous fish species, including herrings and salmonids, in the Atlantic and Pacific oceans. Infection is frequently associated with severe anemia and causes episodic mortality among wild and hatchery fish when accompanied by additional stressors; VEN can be presumptively diagnosed by (1) light microscopic identification of a single characteristic-a round, magenta-colored, 0.8-µm-diameter inclusion body (IB) within the cytoplasm of erythrocytes and their precursors on Giemsa-stained blood films; or (2) observation (via transmission electron microscopy [TEM]) of the causative iridovirus, erythrocytic necrosis virus (ENV), within erythrocytes or their precursors. To better understand the kinetics of VEN, specific-pathogen-free Pacific herring Clupea pallasii were infected with ENV by intraperitoneal injection. At 1, 4, 7, 10, 14, 21, and 28 d postexposure, samples of blood, spleen, and kidney were collected and assessed (1) via light microscopy for the number of intracytoplasmic IBs in blood smears and (2) via TEM for the number of virions within erythrocytes. The mean prevalence of intracytoplasmic IBs in the blood cells increased from 0% at 0-4 d postexposure to 94% at 28 d postexposure. Viral load within circulating red blood cells peaked at 7 d postexposure, fell slightly, and then reached a plateau. However, blood cells observed within the kidney and spleen tissues demonstrated high levels of ENV between 14 and 28 d postexposure. The results indicate that the viral load within erythrocytes does not correlate well with IB prevalence and that the virus can persist in infected fish for more than 28 d.
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Eritrocitos , Enfermedades de los Peces/virología , Cuerpos de Inclusión , Necrosis/veterinaria , Carga Viral , Virosis/veterinaria , Animales , Enfermedades de los Peces/patología , Peces , Necrosis/virología , Virosis/virologíaRESUMEN
A rapid staining procedure for detection of recent skin and fin injuries was tested in juvenile Chinook salmon Oncorhynchus tshawytscha. Immersion of anesthetized fish for 1 min in aerated aqueous solutions of the synthetic food dye fast green FCF (Food Green 3) at concentrations of 0.1 to 0.5% produced consistent and visible staining of integumental injuries. A 0.1% fast green concentration was satisfactory for visual evaluation of injuries, whereas a 0.5% concentration was preferable for digital photography. A rinsing procedure comprised of two 30 s rinses in fresh water was most effective for removal of excess stain after exposure of fish. Survival studies in fresh water and seawater and histopathological analyses indicated that short exposures to aqueous solutions of fast green were non-toxic to juvenile Chinook salmon. In comparisons of the gross and microscopic appearance of fish exposed to fast green at various times after injury, the dye was observed only in areas of the body where epidermal disruption was present as determined by scanning electron microscopy. No dye was observed in areas where epidermal integrity had been restored. Further comparisons showed that fast green exposure produced more consistent and intense staining of skin injury sites than a previously published procedure using trypan blue. Because of its relatively low cost, ease of use and the rapid and specific staining of integumental injuries, fast green may find widespread application in fish health and surface injury evaluations.
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Enfermedades de los Peces/diagnóstico , Colorantes Verde de Lisamina , Salmón , Piel/lesiones , Animales , Relación Dosis-Respuesta a Droga , Extremidades/lesiones , Piel/ultraestructura , Coloración y Etiquetado , Azul de TripanoRESUMEN
A highly efficacious DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was mutated to introduce two stop codons to prevent glycoprotein translation while maintaining the plasmid DNA integrity and RNA transcription ability. The mutated plasmid vaccine, denoted pIHNw-G2stop, when injected intramuscularly into fish at high doses, lacked detectable glycoprotein expression in the injection site muscle, and did not provide protection against lethal virus challenge 7 days post-vaccination. These results suggest that the G-protein itself is required to stimulate the early protective antiviral response observed after vaccination with the nonmutated parental DNA vaccine.
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Codón de Terminación/genética , Glicoproteínas/inmunología , Virus de la Necrosis Hematopoyética Infecciosa/inmunología , Vacunas de ADN/inmunología , Vacunas Virales/inmunología , Animales , Enfermedades de los Peces/mortalidad , Enfermedades de los Peces/prevención & control , Genes Virales/genética , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Virus de la Necrosis Hematopoyética Infecciosa/genética , Mutación/genética , Mutación/inmunología , Oncorhynchus mykiss , Infecciones por Rhabdoviridae/mortalidad , Infecciones por Rhabdoviridae/prevención & control , Infecciones por Rhabdoviridae/veterinaria , Organismos Libres de Patógenos Específicos , Análisis de Supervivencia , Vacunas de ADN/genética , Vacunas Virales/genéticaRESUMEN
A highly efficacious DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was used in a systematic study to analyze vaccine tissue distribution, persistence, expression patterns, and histopathologic effects. Vaccine plasmid pIHNw-G, containing the gene for the viral glycoprotein, was detected immediately after intramuscular injection in all tissues analyzed, including blood, but at later time points was found primarily in muscle tissue, where it persisted to 90 days. Glycoprotein expression was detected in muscle, kidney, and thymus tissues, with levels peaking at 14 days and becoming undetectable by 28 days. Histologic examination revealed no vaccine-specific pathologic changes at the standard effective dose of 0.1 mug DNA per fish, but at a high dose of 50 mug an increased inflammatory response was evident. Transient damage associated with needle injection was localized in muscle tissue, but by 90 days after vaccination no damage was detected in any tissue, indicating the vaccine to be safe and well tolerated.