A historical review of the key bacterial and viral pathogens of Scottish wild fish

Thousands of Scottish wild fish were screened for pathogens by Marine Scotland Science. A systematic review of published and unpublished data on six key pathogens (Renibacterium salmoninarum, Aeromonas salmonicida, IPNV, ISAV, SAV and VHSV) found in Scottish wild and farmed fish was undertaken. Despite many reported cases in farmed fish, there was a limited number of positive samples from Scottish wild fish, however, there was evidence for interactions between wild and farmed fish. A slightly elevated IPNV prevalence was reported in wild marine fish caught close to Atlantic salmon, Salmo salar L., farms that had undergone clinical IPN. Salmonid alphavirus was isolated from wild marine fish caught near Atlantic salmon farms with a SAV infection history. Isolations of VHSV were made from cleaner wrasse (Labridae) used on Scottish Atlantic salmon farms and VHSV was detected in local wild marine fish. However, these pathogens have been detected in wild marine fish caught remotely from aquaculture sites. These data suggest that despite the large number of samples taken, there is limited evidence for clinical disease in wild fish due to these pathogens (although BKD and furunculosis historically occurred) and they are likely to have had a minimal impact on Scottish wild fish.     

Effects of temperature on Renibacterium salmoninarum infection and transmission potential in Chinook salmon,Oncorhynchus tshawytscha (Walbaum)

Renibacterium salmoninarum is a significant pathogen of salmonids and the causative agent of bacterial kidney disease (BKD). Water temperature affects the replication rate of pathogens and the function of the fish immune system to influence the progression of disease. In addition, rapid shifts in temperature may serve as stressors that reduce host resistance. This study evaluated the effect of shifts in water temperature on established R. salmoninarum infections. We challenged Chinook salmon with R. salmoninarum at 12 °C for 2 weeks and then divided the fish into three temperature groups (8, 12 and 15 °C). Fish in the 8 °C group had significantly higher R. salmoninarum‐specific mortality, kidney R. salmoninarum loads and bacterial shedding rates relative to the fish held at 12 or 15 °C. There was a trend towards suppressed bacterial load and shedding in the 15 °C group, but the results were not significant. Bacterial load was a significant predictor of shedding for the 8 and 12 °C groups but not for the 15 °C group. Overall, our results showed little effect of temperature stress on the progress of infection, but do support the conclusion that cooler water temperatures contribute to infection progression and increased transmission potential in Chinook salmon infected with R. salmoninarum.     

Bench‐top validation testing of selected immunological and molecular Renibacterium salmoninarum diagnostic assays by comparison with quantitative bacteriological culture

No gold standard assay exhibiting error‐free classification of results has been identified for detection of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non‐culture assays in three matrices (phosphate‐buffered saline, ovarian fluid and kidney tissue). Non‐culture assays included polyclonal enzyme‐linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane‐filtration FAT, nested polymerase chain reaction (nested PCR) and three real‐time quantitative PCR assays. Injection challenge of specific pathogen‐free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test.     

Monitoring of the in-river migration of smolts from two groups of spring chinook salmon, Oncorhynchus tshawytscha (Walbaum), with different profiles of Renibacterium salmoninarum infection

Abstract Broodstock segregation based on the measurement of maternal Renibacterium salmoninarum infection levels by the enzyme-linked immunosorbent assay (ELISA) and the membrane filtration-fluorescent antibody technique (MF-FAT) was previously shown to affect the prevalence and levels of bacterial kidney disease (BKD) in progeny of chinook salmon, Oncorhynchus tshawytscha (Walbaum), during hatchery rearing. Subgroups of fish from that study were marked with passive integrated transponder (PIT) tags, and monitored by PIT-tag detectors during the first 342km of their migration to the Pacific Ocean. Differences in the recovery of tagged fish were significant (P≤ 0·01) at each detection point and became more pronounced as the fish moved downstream. Cumulative recoveries of fish from the low-BKD group and the high-BKD group, respectively, were 31% and 28% after 116km, 44% and 37% after 176km, and 51% and 42% after 342km. There were no apparent differences in the migration timing of the two groups to the first detection point. The data suggested that in-river survival was higher in the progeny group from parents that had low R. salmoninarum infection levels or tested negative for R. salmoninarum (low-BKD group) than in the group female parents with high infection levels (high-BKD group).     

Genetic variation in susceptibility of Atlantic salmon, Salmo salar L., to furunculosis, BKD and cold water vibriosis

Genetic variation in susceptibility of Atlantic salmon, Salmo salar L., to furunculosis, bacterial kidney disease (BKD) and cold water vibriosis was studied by challenge testing one-year-old fingerlings. Fish from 81 full-sib families within 32 sire progeny groups were infected with Aeromonas salmonicida, Renibacterium salmoninarum and Vibrio salmonicida. Estimated heritabilities were relatively low, being highest for BKD (h²= 0.23) and lowest for cold water vibriosis (h²= 0.13). Genetic correlations between the ability to survive the diseases were all positive, but the magnitude of the genetic correlation between furunculosis and BKD may be biased upwards because some of the dead BKD fish were also infected with furunculosis. The application of selection to develop resistant populations of Atlantic salmon is advocated. Challenge testing seems to be a feasible method, with relatively low costs and easy management. The future response to selection will depend on the relationships between results from a challenge test and mortalities under farming conditions and between disease resistance and other traits in the breeding goal.     

Case definition for clinical and subclinical bacterial kidney disease (BKD) in Atlantic Salmon (Salmo salar L.) in New Brunswick,Canada

Bacterial kidney disease (BKD) is considered an important cause of loss in salmon aquaculture in Atlantic Canada. Causative agent of BKD is the Gram‐positive bacteria Renibacterium salmoninarum. Infected salmon are often asymptomatic (subclinical infection), and the disease is considered chronic. One of the challenges in quantifying information from farm production and health records is the application of a standardized case definition. Case definitions for farm‐level and cage‐level clinical and subclinical BKD were developed using retrospective longitudinal data from aquaculture practices in New Brunswick, Canada, combining (i) industry records of weekly production data including mortalities, (ii) field observations for BKD using reports of veterinarians and/or fish health technicians, (iii) diagnostic submissions and test results and (iv) treatments used to control BKD. Case definitions were evaluated using veterinarians’ expert judgements as reference standard. Eighty‐nine and 66% of sites and fish groups, respectively, were associated with BKD at least once. For BKD present (subclinical or clinical), sensitivity and specificity of the case definition were 75–100% varying between event, fish group, site cycle and level (site pen). For clinical BKD, sensitivities were 29–64% and specificities 91–100%. Industry data can be used to develop sensitive case definitions.     

Randomized clinical field trial of a bacterial kidney disease vaccine in Atlantic salmon,Salmo salar L.

A randomized, blinded clinical trial was performed to assess the relative effectiveness of five commercial and one experimental vaccine in a population of farmed fish experiencing a bacterial kidney disease (BKD) outbreak that occurred in one study cage that was part of a larger clinical field trial. A total of 6000 uniquely identified Atlantic salmon S1 presmolts were randomly assigned to vaccine groups in the hatchery and transferred to a commercial marine aquaculture site. Repeated sampling events to evaluate growth, inherent physical conditions and health status were carried out over the entire production cycle. During the second summer at sea, the study cage developed an outbreak of BKD that lasted approximately 240 days. The effectiveness of the selected vaccines was evaluated using survival analysis methods. The sole vaccine group offering protection for BKD was found to significantly decrease the hazard of dying (hazard ratio, HR = 0.68, P = 0.018) during the outbreak, compared to the industry standard, vaccine group. Additionally, during the outbreak, fish with a shortened operculum had a significantly decreased hazard (HR = 0.38, P = 0.033) compared to those fish with a normal operculum, while fish with jaw deformities had a significantly increased hazard (HR = 2.55, P = 0.001) compared to fish with normal jaw status.     

Renibacterium salmoninarum iron‐acquisition mechanisms and ASK cell line infection: Virulence and immune response

Renibacterium salmoninarum is the aetiological agent of bacterial kidney disease (BKD) in salmonid farms. This pathogen possesses at least three iron‐acquisition mechanisms, but the link between these mechanisms and virulence is unclear. Therefore, this study used RT‐qPCR to assess the effects of normal and iron‐limited conditions on iron‐uptake genes controlled by IdeR and related to iron acquisition in Chilean R. salmoninarum strain H‐2 and the type strain DSM20767^T. Further evaluated was the in vitro immune‐related response of the Atlantic Salmon Kidney (ASK) cell line, derived from the primary organ affected by BKD. R. salmoninarum grown under iron‐limited conditions overexpressed genes involved in haemin uptake and siderophore transport, with overexpression significantly higher in H‐2 than DSM20767^T. These overexpressed genes resulted in higher cytotoxicity and an increased immune response (i.e., TNF‐α, IL‐1β, TLR1 and INF‐γ) in the ASK cell line. This response was significantly higher against bacteria grown under iron‐limited conditions, especially H‐2. These observations indicate that iron‐acquisition mechanisms are possibly highly related to the virulence and pathogenic capacity of R. salmoninarum. In conclusion, treatments that block iron‐uptake mechanisms or siderophore synthesis are attractive therapeutic approaches for treating R. salmoninarum, which causes significant aquaculture losses.     

Vagococcus salmoninarum II—qPCR,tropism and egg-associated transmission

Vagococcus salmoninarum was identified as the causative agent of a chronic epizootic in broodstock “coaster” brook trout (Salvelinus fontinalis) at the Iron River National Fish Hatchery. The epizootic spanned more than a year, was unresponsive to multiple florfenicol treatments, and resulted in >50% mortality of the affected fish. The decision was made to cull the remaining fish during spawning, which presented an opportunity to more thoroughly examine V. salmoninarum sampling methods, organ tropism and vertical transmission. A newly developed qPCR targeting the pheS gene was used in concert with bacterial culture to show that V. salmoninarum indeed disproportionately affects females and has a tropism for female reproductive tissues. The study demonstrates that some female reproductive tissues (e.g. ovarian fluid, unfertilized eggs) are also an effective option for non-lethal detection. Despite the widespread presence of V. salmoninarum in ovarian fluid and on egg surfaces, we found no evidence of intra-ova transmission.     

Content of synthetic astaxanthin in escaped farmed Atlantic salmon, Salmo salar L., ascending Norwegian rivers

Abstract  Isomeric ratios of astaxanthin in eggs and alevins of Atlantic salmon, Salmo salar L., have proven useful in identifying female spawners of farmed origin, but the method underestimates the proportion of fish of farmed origin. The rate of underestimation was studied by analysing astaxanthin content in tissue of 55 farmed Atlantic salmon ascending two Norwegian rivers in the autumn of 1991. The astaxanthin content fell into two distinct classes. Fifty-one per cent of the adult escaped salmon had isomeric ratios similar to salmon fed synthetic astaxanthin, whereas all the remaining fish had ratios similar to wild fish. Discriminant analysis classified 96.4% of the fish with known astaxanthin content into the correct astaxanthin class on basis of tail-fin erosion, length, weight and gill-cover damage. This discriminant function was used to estimate the astaxanthin classification of 1017 farmed salmon caught in nine rivers during 1989–1991. The classification success varied among years from 52 to 64%. Corresponding numbers for females and males were 45–48% and 54–70%, respectively. Thus, estimates of spawning rates of farmed female salmon via astaxanthin content in eggs or alevins from redds should be adjusted accordingly. The observed isomeric ratios of astaxanthin in the escaped farmed salmon and the relationship with morphology indicates that a significant proportion of the escapees ascending rivers have spent more than 1 year in the wild after escape.     

Immunohistochemical evaluation of experimental Vagococcus salmoninarum infection in rainbow trout (Oncorhynchus mykiss,Walbaum 1792)

The aim of this study was to investigate the pathogenesis and histopathological and immunohistochemical findings in rainbow trout (Oncorhynchus mykiss) following experimental vagococcosis. For this purpose, 60 rainbow trout were used. The experimental study used the pathogen Vagococcus salmoninarum. The fish were intraperitoneally (IP) administered with an inoculate containing 0.1 mL of the bacteria, resulting in a dose of 1.2 × 10⁹ cfu mL^?1 per fish. For histopathological observations, tissue samples were taken from fish that died during the experiment and fish that survived until the end of the trial (60th day). All the tissue samples were immunohistochemically stained by the avidin–biotin–peroxidase complex and immunofluorescence methods using polyclonal antibody to detect V. salmoninarum antigens. In immunoperoxidase staining, positive reactions to bacterial antigens were most commonly seen in the kidney, heart and liver. In the immunofluorescence analysis, the distribution of antigens in the tissue and organs was similar to that observed with the immunoperoxidase staining. The results reveal an important correlation between histochemical and immunohistochemical staining in demonstrating the distribution of V. salmoninarum antigens in the affected tissues.     

Utility of genetically based health indicators for selection purposes in captive-reared chinook salmon, Oncorhynchus tshawytscha, Walbaum

Three health indicators, plasma lysozyme activity, PCR‐based detection of Renibacterium salmoninarum (a causative agent of bacterial kidney disease), and a necropsy‐based Health Assessment Index (HAI), were used to examined genetically based variation in a captive population of chinook salmon (Oncorhynchus tshawytscha W.). The study group consisted of four distinct genetic cross‐types: two purebred cross‐types originating from mating wild parents (Big Qualicum River, BC, Canada) and domestic parents (Yellow Island Aquaculture, Ltd, Quadra Island, BC, Canada) and two reciprocal hybrid cross‐types from the mating of wild and domestic parents. Narrow‐sense heritability estimates for plasma lysozyme activity and the incidence of R. salmoninarum were calculated, and the genetic correlation of health indicator response with survival and growth was estimated. Significant differences among cross‐types were found for plasma lysozyme activity, HAI, survival after a natural outbreak of vibriosis (but not after a vibriosis disease challenge), relative growth rate, size‐at‐age (420 and 615 days post fertilization), and R. salmoninarum presence. Despite a significant sire component of heritability for plasma lysozyme activity, the lack of significant heritability estimates for R. salmoninarum presence, and non‐significant genetic correlations with performance variables indicates that selection to improve the health status of fish stock using the three health indicators examined here would likely not result in a measurable correlated response in survival or growth.     

The immune response of Atlantic salmon, Salmo salar L., to the causative agent of bacterial kidney disease, Renibacterium salmoninarum

Abstract. Both under-yearling and post-yearling Atlantic salmon parr produced high agglutinating antibody titres in response to a single intraperitoneal injection of killed bacterial kidney disease (BKD) cells emulsified in. Freund's complete adjuvant (FCA), Low or no response was observed in animals injected with BKD cells in saline or in animals vaccinated by hyperosmotic immersion. Immunological duration was insufficient in fish vaccinated as under-yearling parr to provide protective immunity 2 years later when the fish had become smolts. Atlantic salmon post-yearling parr injected with BKD cells in FCA demonstrated a reduced prevalence of BKD lesions compared to control animals when both were observed as smolts 1 year after vaccination.     

Mercury comparisons between farmed and wild Atlantic salmon (Salmo salar L.) and Atlantic cod (Gadus morhua L.)

Wild and farmed Atlantic salmon ( Salmo salar L.) and Atlantic cod ( Gadus morhua L.) were collected to assess changes in mercury with size in wild vs. farmed fish. Mercury concentrations were compared with Health Canada and United States Environmental Protection Agency consumption guidelines. Lipid dilution of mercury was examined by comparing lipid-extracted (LE) and non-lipid-extracted (NLE) flesh samples in both farmed and wild fish. Mercury concentrations in the flesh and liver of farmed salmon were significantly lower than concentrations in wild salmon of similar fork length ( P <0.001), possibly due to growth dilution in rapidly growing farmed fish. Mercury concentrations were higher in LE tissue compared with NLE ( P <0.05), suggesting lipid dilution of mercury in farmed fish with a high lipid content. Farmed cod, which do not grow more rapidly than wild cod, did not have significantly different flesh and liver concentrations compared with wild cod of similar fork length ( P >0.05). Between species of farmed fish, cod had significantly higher mercury concentrations than salmon ( P <0.05), but neither farmed nor wild salmon mercury concentrations exceeded federal consumption guidelines. These results suggest that rapid growth rates and a high lipid content may play important roles in regulating concentrations of contaminants such as mercury.     

Bacterial infections of Chinook salmon, Oncorhynchus tshawytscha (Walbaum), returning to gamete collecting weirs in Michigan

Herein, we describe the prevalence of bacterial infections in Chinook salmon, Oncorhynchus tshawytscha (Walbaum), returning to spawn in two tributaries within the Lake Michigan watershed. Ten bacterial genera, including Renibacterium, Aeromonas, Carnobacterium, Serratia, Proteus, Pseudomonas, Hafnia, Salmonella, Shewanella and Morganella, were detected in the kidneys of Chinook salmon (n = 480) using culture, serological and molecular analyses. Among these, Aeromonas salmonicida was detected at a prevalence of ～15%. Analyses revealed significant interactions between location/time of collection and gender for these infections, whereby overall infection prevalence increased greatly later in the spawning run and was significantly higher in females. Renibacterium salmoninarum was detected in fish kidneys at an overall prevalence of >25%. Logistic regression analyses revealed that R. salmoninarum prevalence differed significantly by location/time of collection and gender, with a higher likelihood of infection later in the spawning season and in females vs. males. Chi‐square analyses quantifying non‐independence of infection by multiple pathogens revealed a significant association between R. salmoninarum and motile aeromonad infections. Additionally, greater numbers of fish were found to be co‐infected by multiple bacterial species than would be expected by chance alone. The findings of this study suggest a potential synergism between bacteria infecting spawning Chinook salmon.     

Diagnosis of bacterial kidney disease by detection of Renibacterium salmoninarum by real-time PCR

Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum (Rs), is a serious threat to salmon in aquaculture as well as to wild populations. We have developed a real-time polymerase chain reaction (PCR) for detection of Rs in kidney samples. The PCR is based on detection of unique parts of the 16S rRNA gene of Rs and DNA equivalent to 1-10 Rs genomes was detected per reaction. No cross-reactivity with other fish pathogenic or related bacteria could be demonstrated. Analysis of individual kidney samples collected from BKD classified populations identified 39.9% of the fish as positive by real-time PCR compared with 28.0% by polyclonal enzyme-linked immunosorbent assay (ELISA). The real-time PCR assay was found to be well suited for complementary use with ELISA for diagnosis of BKD, with the ability to detect clinical as well as covert Rs infections. The infection level determined by the polyclonal ELISA and by real-time PCR was significantly correlated.     

Fecundity of the sea louse Caligus rogercresseyi on its native host Eleginops maclovinus captured near salmon farms in southern Chile

The sea louse (Caligus rogercresseyi) is the most significant parasitic pathogen in Chilean salmon farms, and it infects farmed salmon and native host fish. Fecundity is one of the most important parameters for understanding the population dynamics of a species; however, this information is scarce for this parasite. The fecundity of C. rogercresseyi females collected from native hosts (Eleginops maclovinus) captured near salmon farms in southern Chile was measured to evaluate the reproductive output of this parasite on this host fish. From June 2008 to May 2009, 212 specimens of E. maclovinus were examined. Each fish was measured, and all its parasites were collected, sorted and counted. Seventy‐nine ovigerous C. rogercresseyi females (OFs) were measured. Total body length, egg string length and total number of eggs per string were recorded for each parasite. Ovigerous females body length varied between 3.9 and 5.0 mm. Fecundity varied between 12 and 56 eggs string^?1, and it was correlated with OF body length. Temporal variations in OF fecundity were explained by co‐variation in OF body length, but not by month. Ovigerous females on E. maclovinus were smaller and showed lower fecundity than OFs on farmed salmon. Our results suggest that native hosts play a secondary role in C. rogercresseyi egg production in Codigue bay.