Disruption of the Francisella noatunensis subsp. orientalis pdpA Gene Results in Virulence Attenuation and Protection in Zebrafish.

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.

Differential susceptibility of Yukon River and Salish Sea stocks of Chinook salmon Oncorhynchus tshawytscha to ichthyophoniasis.

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.

Consequences of Piscine orthoreovirus genotype 1 (PRV-1) infections in Chinook salmon (Oncorhynchus tshawytscha), coho salmon (O. kisutch) and rainbow trout (O. mykiss).

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.

Testing of candidate non-lethal sampling methods for detection of Renibacterium salmoninarum in juvenile Chinook salmon Oncorhynchus tshawytscha.

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.

Genetic variation in bacterial kidney disease (BKD) susceptibility in Lake Michigan Chinook Salmon and its progenitor population from the Puget Sound.

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 (VP ), additive genetic variation (VA ) 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.

Comparative evaluation of molecular diagnostic tests for Nucleospora salmonis and prevalence in migrating juvenile salmonids from the Snake River, USA.

Nucleospora salmonis is an intranuclear microsporidian that primarily infects lymphoblast cells and contributes to chronic lymphoblastosis and a leukemia-like condition in a range of salmonid species. The primary goal of this study was to evaluate the prevalence of N. salmonis in out-migrating juvenile hatchery and wild Chinook salmon Oncorhynchus tshawytscha and steelhead O. mykiss from the Snake River in the U.S. Pacific Northwest. To achieve this goal, we first addressed the following concerns about current molecular diagnostic tests for N. salmonis: (1) nonspecific amplification patterns by the published nested polymerase chain reaction (nPCR) test, (2) incomplete validation of the published quantitative PCR (qPCR) test, and (3) whether N. salmonis can be detected reliably from nonlethal samples. Here, we present an optimized nPCR protocol that eliminates nonspecific amplification. During validation of the published qPCR test, our laboratory developed a second qPCR test that targeted a different gene sequence and used different probe chemistry for comparison purposes. We simultaneously evaluated the two different qPCR tests for N. salmonis and foundthat both assays were highly specific, sensitive, and repeatable. The nPCR and qPCR tests had good overall concordance when DNA samples derived from both apparently healthy and clinically diseased hatchery rainbow trout were tested. Finally, we demonstrated that gill snips were a suitable tissue for nonlethal detection of N. salmonis DNA in juvenile salmonids. Monitoring of juvenile salmonid fish in the Snake River over a 3-year period revealed low prevalence of N. salmonis in hatchery and wild Chinook salmon and wild steelhead but significantly higher prevalence in hatchery-derived steelhead. Routine monitoring of N. salmonis is not performed for all hatchery steelhead populations. At present, the possible contribution of this pathogen to delayed mortality of steelhead has not been determined.

Pathological and immunological responses associated with differential survival of Chinook salmon following Renibacterium salmoninarum challenge.

Chinook salmon Oncorhynchus tshawytscha are highly susceptible to Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD). Previously we demonstrated that introduced Chinook salmon from Lake Michigan, Wisconsin (WI), USA, have higher survival following R. salmoninarum challenge relative to the progenitor stock from Green River, Washington, USA. In the present study, we investigated the pathological and immunological responses that are associated with differential survival in the 2 Chinook salmon stocks following intra-peritoneal R. salmoninarum challenge of 2 different cohort years (2003 and 2005). Histological evaluation revealed delayed appearance of severe granulomatous lesions in the kidney and lower overall prevalence of membranous glomerulopathy in the higher surviving WI stock. The higher survival WI stock had a lower bacterial load at 28 d post-infection, as measured by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR). However, at all other time points, bacterial load levels were similar despite higher mortality in the more susceptible Green River stock, suggesting the possibility that the stocks may differ in their tolerance to infection by the bacterium. Interferon-gamma, inducible nitric oxide synthase (iNOS), Mx-1, and transferrin gene expression were up-regulated in both stocks following challenge. A trend of higher iNOS gene expression at later time points (> or = 28 d post-infection) was observed in the lower surviving Green River stock, suggesting the possibility that higher iNOS expression may contribute to greater pathology in that stock.

Evaluation of fast green FCF dye for non-lethal detection of integumental injuries in juvenile Chinook salmon Oncorhynchus tshawytscha.

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.

Decreased mortality of Lake Michigan Chinook salmon after bacterial kidney disease challenge: evidence for pathogen-driven selection?

In the late 1960s, Chinook salmon Oncorhynchus tshawytscha from the Green River, Washington, were successfully introduced into Lake Michigan. During spring from 1988 to 1992, large fish die-offs affecting Chinook salmon occurred in the lake. Multiple ecological factors probably contributed to the severity of the fish kills, but the only disease agent found regularly was Renibacterium salmoninarum, the causative agent of bacterial kidney disease. In this study, survival after challenge by R. salmoninarum was compared between two Chinook salmon stocks: a Lake Michigan stock from Wisconsin (WI) and the progenitor stock from the Green River. We found that the WI stock had significantly greater survival than the Green River stock. Next, the WI and Green River stocks were exposed to the marine pathogen Listonella anguillarum (formerly Vibrio anguillarum), one of the causative agents of vibriosis; survival after this challenge was significantly poorer for the WI stock than for the Green River stock. A close genetic relationship between the Green River and WI stocks was confirmed by analyzing 13 microsatellite loci. These results collectively suggest that disease susceptibility of Lake Michigan Chinook salmon has diverged from that of the source population, possibly in response to pathogen-driven selection.

Detection and quantification of Renibacterium salmoninarum DNA in salmonid tissues by real-time quantitative polymerase chain reaction analysis.

Renibacterium salmoninarum is an important salmonid pathogen that is difficult to culture. We developed and assessed a real-time, quantitative, polymerase chain reaction (qPCR) assay for the detection and enumeration of R. salmoninarum. The qPCR is based on TaqMan technology and amplifies a 69-base pair (bp) region of the gene encoding the major soluble antigen (MSA) of R. salmoninarum. The qPCR assay consistently detected as few as 5 R. salmoninarum cells per reaction in kidney tissue. The specificity of the qPCR was confirmed by testing the DNA extracts from a panel of microorganisms that were either common fish pathogens or reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA). Kidney samples from 38 juvenile Chinook salmon (Oncorhynchus tshawytscha) in a naturally infected population were examined by real-time qPCR, a nested PCR, and ELISA, and prevalences of R. salmoninarum detected were 71, 66, and 71%, respectively. The qPCR should be a valuable tool for evaluating the R. salmoninarum infection status of salmonids.

Both msa genes in Renibacterium salmoninarum are needed for full virulence in bacterial kidney disease.

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.

Protective immunity and lack of histopathological damage two years after DNA vaccination against infectious hematopoietic necrosis virus in trout.

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 microg 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.

Analysis of DNA-vaccinated fish reveals viral antigen in muscle, kidney and thymus, and transient histopathologic changes.

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.

Epizootiology and histopathology of Parvicapsula sp. in coho salmon Oncorhynchus kisutch.

The epizootiology and histopathology of the myxosporean Parvicapsula sp. was studied during monthly health surveys of 4 groups of coho salmon Oncorhynchus kisutch at a commercial farm in Puget Sound, Washington, USA, from 1984 to 1986. No Parvicapsula sp. was detected in histological samples taken from juvenile fish in fresh water, but the parasite was detected in fish from all groups 2 to 8 mo after transfer to seawater net pens. Groups placed in seawater net pens in November and December had a higher prevalence of infection, and a longer period of continuous detected infection, than those introduced into net pens in May. For the groups transferred to seawater in November and December, the highest infection prevalence (45 to 90%) was detected during the following March and April. Among 13 tissues examined histologically, only the pseudobranch and kidney were positive for Parvicapsula sp., with 26 (62%) of 42 positive fish showing infections only in the pseudobranch, 5 (12%) showing infections only in the kidney, and 11 (26%) showing infections in both organs. Both the pseudobranch and kidney were apparent primary infection sites, but pseudobranch infections appeared to persist longer in a population. Pseudobranch infections were frequently heavy and associated with extensive inflammation and necrosis of filament and lamellar tissues. The kidney had been the only infection site reported for Parvicapsula sp. in previous studies of coho salmon.