A cohabitation challenge to compare the efficacies of vaccines for bacterial kidney disease (BKD) in chinook salmon Oncorhynchus tshawytscha

The relative efficacies of 1 commercial and 5 experimental vaccines for bacterial kidney disease (BKD) were compared through a cohabitation waterborne challenge. Groups of juvenile chinook salmon Oncorhynchus tshawytscha were vaccinated with one of the following: (1) killed Renibacterium salmoninarum ATCC 33209 (Rs 33209) cells; (2) killed Rs 33209 cells which had been heated to 37 degrees C for 48 h, a process that destroys the p57 protein; (3) killed R. salmoninarum MT239 (Rs MT239) cells; (4) heated Rs MT239 cells; (5) a recombinant version of the p57 protein (r-p57) emulsified in Freund's incomplete adjuvant (FIA); (6) the commercial BKD vaccine Renogen; (7) phosphate-buffered saline (PBS) emulsified with an equal volume of FIA; or (8) PBS alone. Following injection, each fish was marked with a subcutaneous fluorescent latex tag denoting its treatment group and the vaccinated fish were combined into sham and disease challenge tanks. Two weeks after these fish were vaccinated, separate groups of fish were injected with either PBS or live R. salmoninarum GL64 and were placed inside coated-wire mesh cylinders (liveboxes) in the sham and disease challenge tanks, respectively. Mortalities in both tanks were recorded for 285 d. Any mortalities among the livebox fish were replaced with an appropriate cohort (infected with R. salmoninarum or healthy) fish. None of the bacterins evaluated in this study induced protective immunity against the R. salmoninarum shed from the infected livebox fish. The percentage survival within the test groups in the R. salmoninarum challenge tank ranged from 59% (heated Rs MT239 bacterin) to 81% (PBS emulsified with FIA). There were no differences in the percentage survival among the PBS-, PBS/FIA-, r-p57- and Renogen-injected groups. There also were no differences in survival among the bacterin groups, regardless of whether the bacterial cells had been heated or left untreated prior to injection.     

Efficacy of cellular vaccines and genetic adjuvants against bacterial kidney disease in chinook salmon (Oncorhynchus tshawytscha)

DNA adjuvants and whole bacterial cell vaccines against bacterial kidney disease (BKD) were tested in juvenile chinook salmon. Whole cell vaccines of either a nonpathogenic Arthrobacter spp. or an attenuated Renibacterium salmoninarum strain provided limited prophylactic protection against acute intraperitoneal challenge with virulent R. salmoninarum, and the addition of either synthetic oligodeoxynucleotides or purified R. salmoninarum genomic DNA as adjuvants did not increase protection. However, a combination of both whole cell vaccines significantly increased survival among fish naturally infected with R. salmoninarum, and the surviving fish treated with the combination vaccine exhibited reduced levels of bacterial antigens in the kidney. This is the first demonstration of a potential therapeutic effect of a whole cell vaccine against BKD.     

Mortality and kidney histopathology of chinook salmon Oncorhynchus tshawytscha exposed to virulent and attenuated Renibacterium salmoninarum strains

An isolate of Renibacterium salmoninarum (strain MT 239) exhibiting reduced virulence in rainbow trout Oncorhynchus mykiss was tested for its ability to cause bacterial kidney disease (BKD) in chinook salmon Oncorhynchus tshawytscha, a salmonid species more susceptible to BKD. Juvenile chinook salmon were exposed to either 33209, the American Type Culture Collection type strain of R. salmoninarum, or to MT 239, by an intraperitoneal injection of 1 x 10(3) or 1 x 10(6) bacteria fish(-1), or by a 24 h immersion in 1 x 10(5) or 1 x 10(7) bacteria ml(-1). For 22 wk fish were held in 12 degrees C water and monitored for mortality. Fish were sampled periodically for histological examination of kidney tissues. In contrast to fish exposed to the high dose of strain 33209 by either injection or immersion, none of the fish exposed to strain MT 239 by either route exhibited gross clinical signs or histopathological changes indicative of BKD. However, the MT 239 strain was detected by the direct fluorescent antibody technique in 4 fish that died up to 11 wk after the injection challenge and in 5 fish that died up to 20 wk after the immersion challenge. Viable MT 239 was isolated in culture from 3 fish that died up to 13 wk after the immersion challenge. Total mortality in groups injected with the high dose of strain MT 239 (12%) was also significantly lower (p < 0.05) than mortality in groups injected with strain 33209 (73 %). These data indicate that the attenuated virulence observed with MT 239 in rainbow trout also occurs in a salmonid species highly susceptible to BKD. The reasons for the attenuated virulence of MT 239 were not determined but may be related to the reduced levels of the putative virulence protein p57 associated with this strain.     

Identification of a third msa gene in Renibacterium salmoninarum and the associated virulence phenotype

Renibacterium salmoninarum, a gram-positive diplococcobacillus, causes bacterial kidney disease, a condition that can result in extensive morbidity and mortality among stocks of fish. An immunodominant extracellular protein, called major soluble antigen (MSA), is encoded by two identical genes, msa1 and msa2. We found evidence for a third msa gene, msa3, which appears to be a duplication of msa1. Unlike msa1 and msa2, msa3 is not present in all isolates of R. salmoninarum. The presence of the msa3 locus does not affect total MSA production in culture conditions. In a challenge study, isolates possessing the msa3 locus reduced median survival in juvenile chinook salmon (Oncorhynchus tshawytscha) by an average of 34% at doses of < or =10(5) cells per fish compared to isolates lacking the msa3 locus. In contrast, no difference in survival was observed at the highest dose, 10(6) cells per fish. The phenotype associated with the msa3 locus and its nonuniform distribution may contribute to observed differences in virulence among R. salmoninarum isolates.     

A deterministic model for the dynamics of furunculosis in chinook salmon Oncorhynchus tshawytscha

Studies were undertaken to determine the parameters of transmission of Aeromonas salmonicida in chinook salmon Oncorhynchus tshawytscha, and to develop a deterministic model of the dynamics of experimental furunculosis. For determination of disease transmission coefficient (beta), disease-related mortality rate (alpha) and natural mortality rate (gamma), fish in 70 tanks (approximately 42 fish tank(-1)) were each exposed to a single infectious donor fish, 7 tanks were randomly selected daily and all individuals were examined for the presence of A. salmonicida in the kidney. The proportion of susceptible (S), infected (I) and removed (R, dead) individuals were determined daily. The parameters beta, alpha, gamma, reproductive ratio (R0) and threshold density were estimated to be 0.0214 infected ind. d(-1), 0.29 infected ind. d(-1), 0.00015 ind. d(-1), 3.23 and 13.56 ind., respectively. Using these parameters, a deterministic disease model of A. salmonicida infection as a cause of furunculosis was constructed. The net rate at which new individuals became infected (the incidence rate) per unit time was proportional to S x I x beta. The model-produced data for S were significantly associated with experimental data (r2 = 0.92). In brief, a simple SIR (susceptible-infected-removed) model was successfully utilized to simulate observed data     

Isolation and characterization of Edwardsiella tarda from fall chinook salmon (Oncorhynchus tshawytscha).

A new bacterial pathogen of chinook salmon (oncorhynchus tshawytscha) was isolated from fish in Oregon's Rogue River. The bacteria are biochemically and serologically related to strains of Edwardsiella tarda. Initially isolated from chinook salmon, the bacteria were also pathogenic for steelhead and rainbow trout (Salmo gairdneri), and channel catfish (Ictalurus punctatus). The 50% lethal doses for chinook salmon, steelhead trout, and channel catfish injected intraperitoneally and maintained in 18 degrees C water were 4.1 x 10(6), 5.6 x 10(6), and 4.0 x 10(5) respectively. When chinook salmon and rainbow trout were injected intraperitoneally and held in 12 degrees C water, the mean lethal doses were 6.4 x 10(7) and 1.7 x 10(6), respectively. The invasiveness of the organism was low in steelhead trout exposed to the bacteria by the waterborne route. The optimum growth temperature of the bacteria in brain heart infusion broth was approximately 35 degrees C. The guanine plus cytosine content of DNA obtained from E. tarda isolated from salmon was 59 mol%.     

Different prevalences of Renibacterium salmoninarum detected by ELISA in Alaskan chinook salmon Oncorhynchus tshawytscha spawned from freshwater and seawater

Soluble antigen of Renibacterium salmoninarum (Rs) was detected by a polyclonal enzyme-linked immunosorbent assay (ELISA) at significantly higher prevalences in adult chinook salmon Oncorhynchus tshawytscha that matured in freshwater than in the same cohort of fish spawned after maturation in seawater. The cumulative results were consistent during 4 yr of comparison at the Little Port Walter Hatchery on Baranof Island, Alaska, USA. Possible causes for this difference are discussed. Maturation of chinook salmon broodstock in seawater has become a practical strategy at this hatchery to reduce the prevalence of Rs-positive parent fish and the numbers of culled eggs.     

Evaluation of a whole cell, p57- vaccine against Renibacterium salmoninarum.

A whole cell Renibacterium salmoninarum vaccine was developed using 37 degrees C heat treated cells that were subsequently formalin fixed; this treatment reduced bacterial hydrophobicity and cell associated p57. Coho salmon Oncorhynchus kisutch were immunized with the p57- vaccine by either a combination of intraperitoneal (i.p.) and intramuscular (i.m.) injections or per os. In the first experiment, i.p./i.m. vaccination of coho salmon with p57- cells in Freund's Incomplete Adjuvant (FIA) conferred a statistically significant increase in mean time to death after the salmon were i.p. challenged with 4.1 x 10(6) colony forming units (cfu) of R. salmoninarum. There was no significant difference in response between fish immunized with R. salmoninarum cell surface extract in FIA and those immunized with extracellular protein (ECP) concentrated from culture supernatant in FIA. The i.p. challenge dose resulted in complete mortality of all fish by Day 43. In a second experiment, fish were orally vaccinated with p57- R. salmoninarum cells encased in a pH protected, enteric-coated antigen microsphere (ECAM). Fish were bath challenged with 4.2 x 10(6) cfu ml-1 on Day 0 and sampled at time points of 0 (pre-challenge), 50, 90, or 150 d immersion challenge. Vaccine efficacy was determined by monitoring the elaboration of p57 in the kidneys of vaccinated and control fish. Fish vaccinated orally demonstrated a significantly lower concentration of p57 (p < 0.01) at Day 150 post challenge compared to fish receiving ECAMs alone. Fish receiving p57 cells without ECAM coating also showed a significantly lower p57 level (p < 0.03) versus control. In contrast, fish injected intraperitoneally with the p57- cells or fish fed p57+ R. salmoninarum cells in ECAMs demonstrated no significant difference (p > 0.05) versus controls. In summary, these studies suggest the preliminary efficacy of 37 degrees C treatment of R. salmoninarum cells as an oral bacterial kidney disease vaccine.     

Method for flow cytometric monitoring of Renibacterium salmoninarum inactivation

The slow growth of Renibacterium salmoninarum limits the usefulness of culture as a research tool. Development of a 2-color flow cytometric assay to quantify the proportions of live and dead R. salmoninarum in a test population is described. Bacteria were simultaneously stained with fluorescein isothiocyanate-conjugated immunoglobulin and exposed to the exclusion dye propidium iodide. Propidium iodide red fluorescence profiles of control groups of untreated and killed R. salmoninarum were compared with those for bacteria exposed to chlorine. Bacterial inactivation was based on mean red fluorescence intensity, and analyzed by high-red fluorescence intensity (HRFI) and curve subtraction (CS) analyses. When the concentration of R. salmoninarum was 8.65 x 10(6) bacteria ml(-1) and the bacteria exposed to chlorine at 1 mg l(-1) for periods from 1 to 20 min (high-Rs assessment), the mean red fluorescence intensity of the profile for each chlorine-exposure group was higher than that for the untreated control (p < 0.0001). When the concentration of R. salmoninarum was reduced to 1.76 x 10(6) bacteria ml(-1) and exposed to 0.8 mg l(-1) free chlorine level for periods from 20 s to 5 min (reduced-Rs assessment), the mean red fluorescence intensities of the exposure groups were higher than that for the untreated control only when the R. salmoninarum was exposed to chlorine for at least 1 min (p < or = 0.01). On the basis of red fluorescence intensity, the proportion of dead cells generally increased with the duration of chlorine exposure. Whereas the rates of inactivation derived from the HRFI and CS analyses did not correlate with the duration of exposure in the high-Rs assessment (r2 < or = 0.27), there was a correlation between these estimates and the duration of exposure in the reduced-Rs assessment (r2 > or = 0.92). Because of the rapid loss of culturable R. salmoninarum in both assessments following chlorine exposure, neither the duration of exposure nor the inactivation estimates correlated with bacteriological culture (r2 < or = 0.22). In both assessments, there was a correlation between the estimates of inactivation based upon HRFI and CS analyses (r2 > 0.99). These results suggest that flow cytometry can be used as a supplementary or alternative method to bacteriological culture for monitoring the inactivation of R. salmoninarum.     

An intranuclear microsporidium associated with acute anemia in the chinook salmon, Oncorhynchus tshawytscha

An intranuclear microsporidium is described from hemoblastic cells of the chinook salmon, Oncorhynchus tshawytscha. The infection is associated with an acute anemia in the fish. Up to 47% of the hemoblast nuclei were infected in anemic fish. The organisms, found only in spleen and kidney tissues, were 1-2 microns in diameter and consisted of vegetative and early sporulation forms. This microsporidium differs from known species which parasitize fish in its tissue location; however, the absence of mature spores and other life cycle stages precludes determination of its precise taxonomic identity.     

Vector potential of the salmon louse Lepeophtheirus salmonis in the transmission of infectious haematopoietic necrosis virus (IHNV)

To better understand the role of vector transmission of aquatic viruses, we established an in vivo virus-parasite challenge specifically to address (1) whether Lepeophtheirus salmonis can acquire infectious haematopoietic necrosis virus (IHNV) after water bath exposure or via parasitizing infected Atlantic salmon Salmo salar and if so, define the duration of this association and (2) whether L. salmonis can transmit IHNV to naive Atlantic salmon and whether this transmission requires attachment to the host. Salmon lice which were water bath-exposed to 1 x 10(5) plaque-forming units (pfu) ml(-1) of IHNV for 1 h acquired the virus (2.1 x 10(4) pfu g(-1)) and remained IHNV-positive for 24 h post exposure. After parasitizing IHNV-infected hosts (viral titer in fish mucus 3.3 x 10(4) pfu ml(-1)) salmon lice acquired IHNV (3.4 x 10(3) pfu g(-1)) and remained virus-positive for 12 h. IHNV-positive salmon lice generated through water bath exposure or after parasitizing infected Atlantic salmon successfully transmitted IHNV, resulting in 76.5 and 86.6% of the exposed Atlantic salmon testing positive for IHNV, respectively. In a second experiment, only salmon lice that became IHNV-positive through water bath exposure transmitted IHNV to 20% of the naive fish, and no virus was transmitted when IHNV-infected salmon lice were cohabitated but restrained from attaching to naive fish. Under laboratory conditions, adult L. salmonis can acquire IHNV and transmit it to naive Atlantic salmon through parasitism. However, the ephemeral association of IHNV with L. salmonis indicates that the salmon louse act as a mechanical rather than a biological vector or reservoir.     

Comparative sensitivities of diagnostic procedures used to detect bacterial kidney disease in salmonid fishes

Kidney and spleen homogenates from each of 60 coho salmon (Oncorhynchus kisutch) and steelhead trout (Salmo gairdneri) were examined for detection of Renibacterium salmoninarum. The proportions of positives differed widely with the detection procedures used: in coho salmon, 5% were positive by the Gram-stain procedure, 10% by the direct fluorescent antibody test, 48% by bacteriological isolation, 65% by staphylococcal coagglutination, and 73% by counterimmunoelectrophoresis; in steelhead trout, 3% were positive by Gram-stain, 8.3% by fluorescent antibody, 17% by bacteriological isolation, and 67% by counterimmunoelectrophoresis. Renibacterium salmoninarum was not detected in either coho salmon or steelhead trout by immunodiffusion analysis.     

Experimental Horizontal Transmission of Enterocytozoon salmonis to Chinook Salmon, Oncorhynchus tshawytscha

Enterocytozoon salmonis was transmitted to chinook salmon Oncorhynchus tshawytscha by feeding tissues infected with the parasite and by cohabitation of noninfected fish with experimentally infected fish. Affected fish (dead and survivors) in both transmission trials had gross and microscopic signs of the disease and merogonic and sporogonic stages of the parasite. There were no morbidities or mortalities, or evidence of the parasite among control fish in either study. Results suggest that the parasite may be contracted by indirect contact among healthy and infected fish held in crowded ponds or net pens or by direct ingestion of spores found in the water.     

Evidence for a carrier state of infectious hematopoietic necrosis virus in chinook salmon Oncorhynchus tshawytscha.

In British Columbia, Canada, infectious hematopoietic necrosis virus (IHNV) is prevalent in wild sockeye salmon Oncorhynchus nerka and has caused disease in seawater net-pen reared Atlantic salmon Salmo salar. In this study, chinook salmon Oncorhynchus tshawytscha experimentally exposed to an isolate of IHNV found in British Columbia became carriers of the virus. When Atlantic salmon were cohabited with these virus-exposed chinook salmon, IHNV was isolated from the Atlantic salmon. Identification of chinook salmon populations that have been exposed to IHNV may be difficult, as virus isolation was successful only in fish that were concurrently infected with either Renibacterium salmoninarum or Piscirickettisia salmonis. Also, IHNV-specific antibodies were detected in only 2 of the 70 fish experimentally exposed to the virus. Two samples collected from chinook salmon exposed to IHNV while at a salt water net-pen site had a seroprevalence of 19 and 22%; however, the inconsistencies between our laboratory and field data suggest that further research is required before we can rely on serological analysis for identifying potential carrier populations. Because of the difficulty in determining the exposure status of populations of chinook salmon, especially if there is no concurrent disease, it may be prudent not to cohabit Atlantic salmon with chinook salmon on a farm if there is any possibility that the latter have been exposed to the virus.     

A PCR-based assay for the identification of the fish pathogen Renibacterium salmoninarum

Abstract By means of a one-step one-tube extraction from less than 1 mg of tissue it is possible to identify, via the polymerase chain reaction, Renibacterium salmoninarum in salmon with bacterial kidney disease. A 149-bp DNA sequence unique to R salmoninarum was specifically amplified and its nature confirmed by Southern hybridization using a non-isotopically labelled probe. The sensitivity of the approach allowed the detection of 22 R. salmoninarum cells. The procedure was successfully applied in the identification of the causative agent of bacterial kidney disease in kidney tissue from infected fishes.     

Characterization of attenuated Renibacterium salmoninarum strains and their use as live vaccines

Two nutritionally mutant strains of Renibacterium salmoninarum (Rs) were isolated that grew on tryticase soy agar (Rs TSA1) or brain heart infusion agar (Rs BHI1). These 2 strains could be continuously cultured on these media, whereas typical R. salmoninarum would only grow on KDM-2 agar. We determined no other phenotypic difference that could be used to distinguish them from wild-type R. salmoninarum. Both strains were found to be avirulent when 5 x 10(6) bacteria were intraperitoneally (i.p.) injected into Atlantic salmon. Rs TSA1, Rs BHI1, and Rs MT-239 (a R. salmoninarum strain previously shown to be attenuated) were tested as live vaccines in 2 separate trials. The best protection was seen with Rs TSA1. Vaccinated Atlantic salmon had relative percent survival (RPS) of 50 at 74 d post-challenge in Trial 1 and 76 at 60 d post-challenge in Trial 2. In both trials, 100% of the control salmon died from bacterial kidney disease (BKD) (within 40 d for Trial 1 and 50 d for Trial 2) after i.p. challenge with 5 x 10(6) live cells of the virulent isolate Rs Margaree.     

Metabolic effects of amoebic gill disease (AGD) and chloramine-T exposure in seawater-acclimated Atlantic salmon Salmo salar

Our aim was to determine possible metabolic effects amoebic gill disease (AGD) on Atlantic salmon Salmo salar. Standard (R(S)) and routine (R(ROU)) metabolic rates were evaluated by continually measuring oxygen consumption in 2 independent tanks of fish (18.69 +/- 1.01 kg m(-3), mean +/- SE). Active metabolic rate (R(ACT)) and metabolic scope (R(ACT) - R(S)) were assessed using a chasing protocol and determined at 3 time periods: (1) pre-infection, (2) 3 d post-infection, and (3) 2 d post-treatment. On Day 3 of the study, the fish were infected with amoebae isolated from the gills of AGD-affected salmon (2300 cells l(-1)). No significant elevations in R(ACT) or metabolic scope were detected 3 d post-infection and 2 d post-treatment; however, significant elevations in R(S) and R(ROU) were detected 3 d post-infection and 2 d post-treatment. Assessment of R(ROU) data, especially for the light period, also indicated a rise in oxygen consumption rate over the course of the experiment. Treatment of AGD-affected Atlantic salmon with chloramine-T (CL-T) appeared to briefly mitigate the rise in R(S), as there was a 30% drop (though non-significant) in R(S) following treatment. Despite this, R(S) continued the upward trend 1 d following treatment. These results suggest that over the course of AGD development, R(S) in Atlantic salmon increases. Therefore, considering the physical conditions which constrain R(ACT), we expect that metabolic scope would become compromised in fish more heavily affected with AGD. Treatment with CL-T shows promise for mitigating the respiratory effects of AGD and potentially minimising the loss of metabolic scope.