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.
Inclusion body disease (IBD) is a worldwide disease in captive boa constrictors (boa constrictor) and occasionally in other snakes of the families Boidae and Pythonidae. The exact causative agent(s) ...and pathogenesis are not yet fully understood. Currently, diagnosis of IBD is based on the light microscopic identification of eosinophilic intracytoplasmic inclusion bodies in hematoxylin and eosin stained tissues or blood smears. An antigenically unique 68 KDa protein was identified within the IBD inclusion bodies, called IBD protein. A validated immuno-based ante-mortem diagnostic test is needed for screening snakes that are at risk of having IBD. In this study, despite difficulties in solubilizing semi-purified inclusion bodies, utilizing hybridoma technology a mouse anti-IBD protein monoclonal antibody (MAB) was produced. The antigenic specificity of the antibody was confirmed and validated by western blots, enzyme-linked immunosorbent assay, immuno-transmission electron microscopy, and immunohistochemical staining. Paraffin embedded tissues of IBD positive and negative boa constrictors (n=94) collected from 1990 to 2011 were tested with immunohistochemical staining. In boa constrictors, the anti-IBDP MAB had a sensitivity of 83% and specificity of 100% in detecting IBD. The antibody also cross-reacted with IBD inclusion bodies in carpet pythons (Morelia spilota) and a ball python (python regius). This validated antibody can serve as a tool for the development of ante-mortem immunodiagnostic tests for IBD.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Several
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
pathogenicity island (FPI) is a well-described, gene-dense region encoding major virulence factors for the genus
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
suicide approach, we generated
knockouts to address the role of PdpA as a virulence factor for
. Because polarity can be an issue in gene-dense regions, we generated two different marker-based mutants in opposing polarity (the
subsp.
Δ
and Δ
strains). Both mutants were attenuated (
< 0.0001) in zebrafish challenges and displayed impaired intracellular replication (
< 0.05) and cytotoxicity (
< 0.05), all of which could be restored to wild-type (WT) levels by complementation for the Δ
mutant. Importantly, differences were found for bacterial burden and induction of acute-phase and proinflammatory genes for the
subsp.
Δ
and Δ
mutants compared to the WT during acute infection. In addition, neither mutant resulted in significant histopathological changes. Finally, immunization with the
subsp.
Δ
mutant led to protection (
< 0.012) against an acute 40% lethal dose (LD
) challenge with WT
in the zebrafish model of infection. Taken together, the results from this study further demonstrate physiological similarities within the genus
relative to their phylogenetic relationships and the utility of zebrafish for addressing virulence factors for the genus.
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.
The DNA vaccine pIHNw-G encodes the glycoprotein of the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV). Vaccine performance in rainbow trout was measured 3, 6, 13, 24, and 25 months ...after vaccination. At three months all fish vaccinated with 0.1
μg pIHNw-G had detectable neutralizing antibody (NAb) and they were completely protected from lethal IHNV challenge with a relative percent survival (RPS) of 100% compared to control fish. Viral challenges at 6, 13, 24, and 25 months post-vaccination showed protection with RPS values of 47–69%, while NAb seroprevalence declined to undetectable levels. Passive transfer experiments with sera from fish after two years post-vaccination were inconsistent but significant protection was observed in some cases. The long-term duration of protection observed here defined a third temporal phase in the immune response to IHNV DNA vaccination, characterized by reduced but significant levels of protection, and decline or absence of detectable NAb titers. Examination of multiple tissues showed an absence of detectable long-term histopathological damage due to DNA vaccination.
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.
From the mid‐1980s through the early 1990s, outbreaks of bacterial kidney disease (BKD) caused by Renibacterium salmoninarum continued in Chinook salmon Oncorhynchus tshawytscha in Idaho Department ...of Fish and Game (IDFG) hatcheries despite the use of three control methods: (1) injection of returning adult fish with erythromycin to reduce prespawning BKD mortality and limit vertical transmission of R. salmoninarum, (2) topical disinfection of green eggs with iodophor, and (3) prophylactic treatments of juvenile fish with erythromycin‐medicated feed. In addition, programs to manage BKD through measurement of R. salmoninarum antigen levels in kidney tissues from spawning female Chinook salmon by an enzyme‐linked immunosorbent assay (ELISA) were tested over 13–15 brood years at three IDFG hatcheries. The ELISA results were used for either (1) segregated rearing of progeny from females with high ELISA optical density (OD) values (usually ≥0.25), which are indicative of high R. salmoninarum antigen levels, or (2) culling of eggs from females with high ELISA OD values. The ELISA‐based culling program had the most profound positive effects on the study populations. Mortality of juvenile fish during rearing was significantly lower at each hatchery for brood years derived from culling compared with brood years for which culling was not practiced. The prevalence of R. salmoninarum in juvenile fish, as evidenced by detection of the bacterium in kidney smears by the direct fluorescent antibody test, also decreased significantly at each hatchery. In addition, the proportions of returning adult females with kidney ELISA OD values of 0.25 or more decreased 56–85% for fish reared in brood years during which culling was practiced, whereas the proportions of ELISA‐negative adults increased 55–58%. This management strategy may allow IDFG Chinook salmon hatcheries to reduce or eliminate prophylactic erythromycin‐medicated feed treatments. We recommend using ELISA‐based management of BKD in Chinook salmon hatcheries where it is a concern.
Mass mortality events in wild fish due to infectious diseases are troubling, especially given the potential for long-term, population-level consequences. Evolutionary theory predicts that populations ...with sufficient genetic variation will adapt in response to pathogen pressure. Chinook Salmon Oncorhynchus tshawytscha were introduced into Lake Michigan in the late 1960s from a Washington State hatchery population. In the late 1980s, collapse of the forage base and nutritional stress in Lake Michigan were thought to contribute to die-offs of Chinook Salmon due to bacterial kidney disease (BKD). Previously, we demonstrated that Lake Michigan Chinook Salmon from a Wisconsin hatchery have greater survival following BKD challenge relative to their progenitor population. Here, we evaluated whether the phenotypic divergence of these populations in BKD susceptibility was due to selection rather than genetic drift. Comparison of the overall magnitude of quantitative trait to neutral marker divergence between the populations suggested selection had occurred but a direct test of quantitative trait divergence was not significant, preventing the rejection of the null hypothesis of differentiation through genetic drift. Estimates of phenotypic variation (V P), additive genetic variation (V A) and narrow-sense heritability (h ²) were consistently higher in the Wisconsin relative to the Washington population. If selection had acted on the Wisconsin population there was no evidence of a concomitant loss of genetic variation in BKD susceptibility. The Renibacterium salmoninarum exposures were conducted at both 14°C and 9°C; the warmer temperature accelerated time to death in both populations and there was no evidence of phenotypic plasticity or a genotype-by-environment (G × E) interaction. High h ² estimates for BKD susceptibility in the Wisconsin population, combined with a lack of phenotypic plasticity, predicts that future adaptive gains in BKD resistance are still possible and that these adaptive gains would be stable under the temperature range evaluated here.
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.
Renibacterium salmoninarum, a gram-positive diplococcobacillus that causes bacterial kidney disease among salmon and trout, has two chromosomal loci encoding the major soluble antigen (msa) gene. ...Because the MSA protein is widely suspected to be an important virulence factor, we used insertion-duplication mutagenesis to generate disruptions of either the msa1 or msa2 gene. Surprisingly, expression of MSA protein in broth cultures appeared unaffected. However, the virulence of either mutant in juvenile chinook salmon (Oncorhynchus tshawytscha) by intraperitoneal challenge was severely attenuated, suggesting that disruption of the msa1 or msa2 gene affected in vivo expression.
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