Protection of Chinook Salmon Smolts with Oral Doses of Erythromycin against Acute Challenges of Renibacterium salmoninarum

Abstract

We challenged duplicate groups of yearling smolts of chinook salmon Oncorhynchus tshawytscha held in seawater with an intraperitoneal inoculation of the kidney disease bacterium Renibacterium salmoninarum 1 d before and at intervals of 1, 11, and 29 d after a 21-d oral administration of erythromycin thiocyanate at 0.1 g/kg body weight per day. Most mortality attributable to bacterial kidney disease (BKD) in fish challenged with R. salmoninarum but not administered erythromycin occurred 2–3 weeks after challenge; the average survival 35 d after challenge was only 9%. Nearly 70% of the fish challenged 1 d before the erythromycin feeding were alive 35 d after the 21-d treatment, and more than 98% of the fish challenged the day after the 21-d erythromycin treatment survived a further 35 d. Fish challenged 29 d after the erythromycin treatment were not significantly protected against BKD. Chinook salmon that were not challenged with the bacterium but were injected with sterile phosphate-buffered saline and then fed a ration with erythromycin survived at a significantly higher rate than unchallenged fish injected with saline but not fed erythromycin. Fish that were not injected with saline or R. salmoninarum survived at a significantly higher rate than fish handled and injected with saline or pathogen. Because erythromycin protected fish challenged just before and immediately after treatment, the antibiotic should be useful early in an outbreak of BKD and as a prophylactic when stresses are expected.     

Stress Protein Expression in Coho Salmon with Bacterial Kidney Disease

Abstract

The expression of stress protein-70 (SP-70), also known as heat shock protein-70 (HSP-70), was measured by enzyme-linked immunosorbent assay in kidney and liver from coho salmon Oncorhynchus kisutch with bacterial kidney disease (BKD) experimentally induced by injection with Renibacterium salmoninarum. Fish with BKD had more SP-70 in both kidney and liver than did the control fish. The SP-70 measured was derived from the host tissue, not from the pathogen. In fish without clinical disease, SP-70 was not significantly elevated. Elevated stress protein (SP) levels in fish with BKD raise the possibility that BKD or other infectious diseases may interfere with the use of SP induction as a marker of environmental stress in fish and lead to statistical artifacts when the prevalence of disease is not considered.     

Comparison of Methods Used to Detect Renibacterium salmoninarum,the Causative Agent of Bacterial Kidney Disease

Abstract

Various diagnostic methods used to detect Renibacterum salmoninarum, the causative agent of bacterial kidney disease (BKD), were compared. The most sensitive method was the enzyme immunoassay (the indirect dot blot assay), which could detect 10² bacterial cells per gram of kidney tissue. The minimum bacteria concentrations showing positive reactions to the indirect fluorescent antibody test (IFAT) and the coagglutination test were 10³ and 10⁴ cells/g kidney, respectively. The sensitivities of the Gram stain, immunodiffusion procedure, and latex agglutination test were low, and these methods could only be applied to fish with overt BKD symptoms. Altogether, 656 coho salmon Oncorhynchus kisutch were examined for R. salmoninarum antigen with the direct and indirect dot blot assays (DDBA and IDBA) and the IFAT. Among the fish sampled, positive reactions were obtained in 11.8% by the DDBA, 28.2% by the IDBA, and 12.9% by the IFAT.     

Development and Evaluation of a Monoclonal-Antibody-Based Enzyme-Linked Immunosorbent Assay for the Diagnosis of Renibacterium salmoninarum Infection

Abstract

A monoclonal-antibody-based enzyme-linked immunosorbent assay (ELISA) was developed for the diagnosis of bacterial kidney disease (BKD). This ELISA can detect Renibacterium salmoninarum antigen at concentrations as low as 0.05–0.1 μg/mL. During the 1988–1989 spawning season, 60 coho salmon Oncorhynchus kisutch, 60 chinook salmon O. tshawytscha, and 60 steelhead O. mykiss (Great Lakes rainbow trout) were caught and screened for BKD with the developed ELISA and a direct fluorescent antibody technique (FAT). Serum agglutination titers for R. salmoninarum were measured to determine any relationship between presence of antigen (R. salmoninarum) and humoral antibody to R. salmoninarum. Twelve of the coho salmon (20%), 48 of the chinook salmon (80%), and 7 of the steelhead (11.7%) were BKD-positive according to the ELISA. Only one steelhead (1.7%) was BKD-positive by FAT, whereas none of the coho salmon or chinook salmon were BKD-positive. It was concluded that the monoclonal-antibodybased ELISA was more sensitive than FAT. Antibody titers of these asymptomatic fish were variable. There was no correlation between antigen level and antibody titer.     

Demonstration of the Specificity of the Salmonid Hurnoral Response to Renibacterium salmoninarum with a Monoclonal Antibody against Salmonid Immunoglobulin

Abstract

The specificity of the antibody response of salmonids to Renibacterium salmoninarum antigens was demonstrated by western blotting techniques that utilized a monoclonal antibody against salmonid immunoglobulin. In this study, the specificity of the response in immunized chinook salmon Oncorhynchus tshawytscha was compared with the response in naturally infected chinook salmon and coho salmon O. kisutch, and immunized rabbits. The antibody response in immunized salmon and rabbits and the naturally infected fish was primarily against the 57–58kilodalton protein complex. In addition to recognizing these proteins in the extracellular fraction and whole-cell preparations, antibody from the immunized salmon and rabbits detected four proteins with lower molecular masses. Western blotting techniques allow identification of the specific antigens recognized and are a useful tool for comparing the immunogenicity of different R. salmoninarum preparations. Immunofluorescent techniques with whole bacteria were less sensitive than western blotting in detecting salmonid anti-R. salmoninarum antibody.     

Occurrence of Aeromonas salmonicida,Renibacterium salmoninarum,and Infectious Pancreatic Necrosis Virus in Ontario Salmonid Populations

Abstract

The results of samples collected from private and government fish farms and wild and feral fish populations in Ontario from 1981 to 1995 were synthesized to obtain prevalence estimates in salmonids at both the fish and site levels for three pathogens. Renibacterium salmoninarum and Aeromonas salmonicida were both detected on at least one site for every year investigated. Ontario Ministry of Natural Resources (OMNR) culture stations had the highest percentages of sites with infected fish for R. salmoninarum. Natural water bodies had the highest percentages of sites with infected fish for A. salmonicida. Infectious pancreatic necrosis virus (IPNV) was only detected sporadically on some commercial farms and never in OMNR hatcheries or in wild or feral fish. Although screening for any virus that would yield cytopathological effect was carried out during all the years surveyed, no virus other than IPNV was isolated. The low prevalence and “source-specific” presence of IPNV in Ontario demonstrates the necessity of representative sampling for the detection of rare pathogens. It was estimated that, overall, less than 1% of all fish in the sampled populations were infected with each of the three pathogens for almost every year studied. The importance of summarizing pathogen-testing data and the possible implications on disease control policy planning and assessment are discussed.     

Efficacy of Enrofloxacin for the Treatment of Salmonids with Bacterial Kidney Disease,Caused by Renibacterium salmoninarum

Abstract

Efficacy of enrofloxacin (Baytril®, Bayer) was evaluated for control of Renibacterium salmoninarum infection in salmonids. Minimum inhibitory concentration studies indicated efficacy at 0.25–0.5 μg/mL. In laboratory challenge studies with rainbow trout Oncorhynchus mykiss, mortality offish receiving enrofloxacin daily at a dosage of 1.25–2.5 mg/kg for 10 d was significantly lower than that of nonmedicated fish. A general trend of increased percent survival with increasing dose was also observed.     

Monoclonal Antibody-Based Analysis of the Renibacterium salmoninarum p57 Protein in Spawning Chinook and Coho Salmon

Abstract

Renibacterium salmoninarum, causative agent of bacterial kidney disease of salmonid fish, produces large amounts of soluble proteins during infection and broth culture. An enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies was developed for the precise quantification of p57, a major component of these proteins. Kidney, spleen, blood, and reproductive fluids of adult Pacific salmon Oncorhynchus spp. were examined by means of the assay. Kidney and spleen harbored the highest concentrations of this antigen. In the populations of returning salmon tested, 5–25% of the fish had p57 concentrations above a baseline level of 3 ng antigenig tissue, and antigen concentrations as high as 200 μg/g tissue were detected in kidneys of individual fish. The ELISA was compared to direct fluorescent antibody analysis, in which rabbit anti-R. salmoninarum antiserum was used to identify infected fish. There was 99% agreement (199 of 201 examined fish) between the two methods. Western blot analysis was used to demonstrate that antigenic proteins present in infected fish were similar to those seen from antigen prepared from broth culture of R. salmoninarum, although less degradation of p57 appears to occur in vivo.     

Effects of Practices Related to Catch-and-Release Angling on Mortality and Viral Transmission in Juvenile Largemouth Bass Infected with Largemouth Bass Virus

Abstract

Largemouth bass virus (LMBV; family Iridoviridae) has recently emerged as a causative agent in fish kills of largemouth bass Micropterus salmoides. Little is known about how the virus is transmitted or what factors predispose fish to mortality subsequent to infection. Concern has nevertheless arisen that activities related to recreational angling may affect transmission dynamics and may alter the susceptibility of infected fish to clinical disease. This study examined the separate effects of two angling-related factors on the susceptibility of juvenile largemouth bass to mortality from LMBV infection and on the transmission of LMBV from infected to uninfected fish. The first factor was hook-and-line angling. Infected fish that underwent a simulated angling treatment did not experience higher mortality or have higher viral loads in their tissues than those that were not angled. The second factor was direct contact between infected and uninfected fish, as would occur in live wells and holding tanks. The LMBV was transmitted from infected to uninfected fish through water, even when direct contact was prevented. Transmission of LMBV between infected and uninfected fish separated by a fenestrated barrier was nearly as efficient as LMBV transmission between infected and uninfected fish that were allowed direct contact. These results imply that angling itself may have only minimal effects on the survival of largemouth bass infected with LMBV but that angling-related practices that place infected and uninfected fish together in a limited water volume may facilitate viral transmission. Partitioning or cooling of live wells and holding tanks, as well as limiting their use, could reduce LMBV-associated mortality and viral transmission.     

The Gill Pathogen Dermocystidium salmonis in Oregon Salmonids

Abstract

Intense infections of the gill pathogen Dermocystidium salmonis were associated with mortality of prespawning chinook salmon Oncorhynchus tshawytscha in several Oregon rivers in 1988. The occurrence of the pathogen in returning adult chinook salmon was monitored in several coastal Oregon stocks from 1989 to 1993. Although the prevalence of the pathogen was high in these fish (up to 66.6%), infection intensities were generally low, and no mortality attributable to D. salmonis was observed. In 1988, the pathogen was associated with a lethal epizootic among juvenile chinook salmon smolts at the Trask State Fish Hatchery near Tillamook, Oregon. Histological examination of gills from heavily infected fish revealed hyperplasia of gill epithelium and fusion of gill lamellae. When naturally infected smolts were transferred from fresh to salt water, the most heavily infected fish died within 10 d, and the number of D. salmonis cysts declined and disappeared from previously infected salmon after 21–42 d.     

Influence of Infection with Renibacterium salmoninarum on Susceptibility of Juvenile Spring Chinook Salmon to Gas Bubble Trauma

Abstract

During experiments in our laboratory to assess the progression and severity of gas bubble trauma (GBT) in juvenile spring chinook salmon Oncorhynchus tshawytscha, we had the opportunity to assess the influence of Renibacterium salmoninarum (Rs), the causative agent of bacterial kidney disease, on the susceptibility of salmon to GBT. We exposed fish with an established infection of Rs to 120% total dissolved gas (TDG) for 96 h and monitored severity of GBT signs in the fins and gills, Rs infection level in kidneys by using an enzyme-linked immunosorbent assay (ELISA), and mortality. Mortality occurred rapidly after exposure to 120% TDG, with a LT20 (time necessary to kill 20% of the population) of about 37 h, which is at a minimum about 16% earlier than other bioassays we have conducted using fish that had no apparent signs of disease. Fish that died early (from 31 to 36 h and from 49 to 52 h) had significantly higher infection levels (mean ± SE ELISA absorbance = 1.532 ± 0.108) than fish that survived for 96 h (mean ± SE ELISA absorbance = 0.828 ± 0.137). Fish that died early also had a significantly greater number of gill filaments occluded with bubbles than those that survived 96 h. Conversely, fish that survived for 96 h had a significantly higher median fin severity ranking than those that died early. Our results indicate that fish with moderate to high levels of Rs infection are more vulnerable to the effects of dissolved gas supersaturation (DGS) and die sooner than fish with lower levels of Rs infection. However, there is a substantial amount of individual variation in susceptibility to the apparent cumulative effects of DGS and Rs infection. Collectively, our findings have important implications to programs designed to monitor the prevalence and severity of GBT in juvenile salmonids in areas like the Columbia River basin, and perhaps elsewhere.     

Survey of Pathogens in Juvenile Salmon Oncorhynchus Spp. Migrating through Pacific Northwest Estuaries

Abstract

Although the adverse impact of pathogens on salmon populations in the Pacific Northwest is often discussed and recognized, little is currently known regarding the incidence and corresponding significance of delayed disease-induced mortalities. In the study reported herein, we surveyed the presence and prevalence of selected micro- and macroparasites in out-migrant juvenile coho salmon Oncorhynchus kisutch and Chinook salmon O. tshawytscha from 12 coastal estuaries in the Pacific Northwest over a 6-year period (1996–2001). The major finding of this study was the widespread occurrence of pathogens in wild salmon from Pacific Northwest estuaries. The six most prevalent pathogens infecting both juvenile Chinook and coho salmon were Renibacterium salmoninarum, Nanophyetus salmincola, an erythrocytic cytoplasmic virus (erythrocytic inclusion body syndrome or erythrocytic necrosis virus), and three gram-negative bacteria (Listonella anguillarum, Yersinia ruckeri, and Aeromonas salmonicida). The most prevalent pathogen in both Chinook and coho salmon was N. salmincola, followed by the pathogens R. salmoninarum and the erythrocytic cytoplasmic virus. Statistically significant differences in the prevalence of R. salmoninarum and N. salmincola were observed between Chinook and coho salmon. Based on the prevalence of pathogens observed in this study, disease appears to be a potentially significant factor governing the population numbers of salmon in the Pacific Northwest. Development of a detailed understanding of the principal components influencing the ecology of infectious disease will aid in the development of management and control strategies to mitigate disease in and hence further the recovery of salmon stocks listed under the Endangered Species Act.     

Isolation of <Emphasis Type="Italic">Vagococcus salmoninarum</Emphasis> in Rainbow Trout, <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> (Walbaum), Broodstocks: Characterization of the Pathogen

Coldwater ‘streptococcosis’, caused by Vagococcus salmoninarum, is an emerging disease of rainbow trout in the European Union, causing mortality rates up to 50% in broodstock during the spawning period, with water temperature of 10–12°C. A study to determine the presence and role of this bacterium was undertaken using classical bacteriological techniques confirmed with polymerase chain reaction. This is the first report of isolation of V salmoninarum in relation to outbreaks of mortality in a rainbow trout farm devoted exclusively to broodstock rearing in Spain. A total of 10 isolates of V salmoninarum were characterized by their morphological, cultural, physiological, biochemical and enzymatic traits. Some differences were observed in parameters such as growth on MacConkey agar, H2S production, acid production from starch, and some other minor variations. Isolates were sensitive to erythromycin and oxytetracycline tested in vitro, but treatments conducted in the field were ineffective. An attempt at vaccination did not provide encouraging results.     

Experimentally Induced Whirling Disease I. Dose Response of Fry and Adults of Rainbow Trout Exposed to the Triactinomyxon Stage of Myxobolus cerebralis

Abstract

The intensity and prevalence of whirling disease was tested by exposure of 2-monthold fry and 1-, 2-, and 3.5-year-old adults of rainbow trout Oncorhynchus mykiss to a known number of laboratory-produced Myxobolus cerebralis at the actinosporean triactinomyxon stage. Fry exposed to graded concentrations of infectivity (triactinomyxons) for 3 h were individually examined for spores of Myxobolus cerebralis 5 and 6 months later. Exposure of fish to the lowest doses, 1 and 10 triactinomyxons per fish, did not result in detectable myxosporean spores. Fish that became lightly infected by a dose of 100 triactinomyxons per fish experienced a decrease in the incidence of infection between 5 and 6 months after exposure. A linear relationship was found between the numbers of recovered myxosporean spores and doses of 100–10,000 triactinomyxons per fish, and the spore burden appeared to plateau at doses of 10,000–100,000 triactinomyxons per fish. Adult fish continuously exposed to the highest dose of triactinomyxons for 3.5 months were infected and asymptomatic, however, the severity of myxosporean infection decreased with increased age of fish. This information may help in controlling whirling disease in salmonids.     

Experimental Infection of Rainbow Trout with Saprolegnia parasitica

Abstract

A method was developed to experimentally induce saprolegniasis in rainbow trout Oncorhynchus mykiss. The development of a reliable method to produce infected fish is essential to efforts to determine the efficacy of various antifungal treatments. Three methods for inducing saprolegniasis were evaluated in waters containing known concentrations of Saprolegnia parasitica zoospores. These methods included application of the following stressors to fish: (1) abrasion and dewatering, (2) water temperature increase, and (3) a combination of abrasion, dewatering, and temperature increase. Neither physical abrasion nor temperature increase stress alone was effective for inducing saprolegniasis. Only 25.9% of fish stressed by abrasion and dewatering alone became infected. Application of both abrasion and temperature stress, however, induced saprolegniasis in 77.8% of fish tested. Most of these fish became infected after 5 d of stress treatments. No fish became infected or died in the positive control group (not stressed but exposed to S. parasitica zoospores) or the negative control group (not stressed or challenged). This method should enable researchers to induce saprolegniasis in rainbow trout to study its pathogenesis or to test the efficacy of antifungal treatments. In conducting efficacy studies, it is important that therapeutic treatments begin promptly after the first signs of saprolegniasis are observed because the disease can progress very quickly and often results in mortality.     

Rate of Disappearance of Gas Bubble Trauma Signs in Juvenile Salmonids

Abstract

To assess the rate of disappearance of gas bubble trauma (GBT) signs in juvenile salmonids, we exposed spring chinook salmon Oncorhynchus tshawytscha and steelhead O. mykiss to water containing high levels of dissolved gas supersaturation (DGS) for a time period sufficient to induce signs of GBT, reduced the DGS to minimal levels, and then sampled fish through time to document changes in severity of GBT. Because of the tendency of GBT signs to dissipate at different rates, we conducted trials focusing on emboli (bubbles) in the gill filaments and lateral line and separate trials that focused on bubbles in the external surfaces (fins, eyes, and opercula). Bubbles in gill filaments dissipated almost completely within 2 h after transfer of fish to water of nearly normal DGS (104%), whereas bubbles in the lateral line dissipated to negligible levels within 5 h. Bubbles on external surfaces were more persistent through time than they were in gill filaments and the lateral line. Although typically dissipating to low levels within 48 h, external bubbles sometimes remained for 4 d. Assuming a direct relation exists between easily observable signs and direct mortality, our results suggest that fish can recover quickly from the potentially lethal effects of DGS once they move from water with high DGS to water of almost normal gas saturation. These results should be of fundamental importance to fishery managers interpreting the results of monitoring for the severity and prevalence of GBT in juvenile salmonids in the Columbia River system and perhaps elsewhere.     

Technique for Enumeration of Renibacterium salmoninarum in Fish Kidney Tissues

Abstract

Effects of prefiltration and enzyme treatment on the filtrability of kidney tissue of chinook salmon Oncorhynchus tshawytscha were examined. The clarification of kidney suspensions with a 5-μm prefilter did not affect microbial recovery from the samples examined. Samples of kidney suspensions inoculated with Renibacterium salmoninarum (10²-10⁵ cells/g) were prefiltered and then tested for their filtrability through a 0.2-μm membrane filter. Less than 50% of the samples could be filtered without enzyme treatment, but all samples were rendered filtrable by enzyme treatment. Inoculum levels of R. salmoninarum as low as 100 cells/g could be detected and quantified. The application of these procedures greatly enhances membrane filtration for the certification of R. salmoninarum-free fish.     

Présence de la maladie bactérienne du rein chez les salmonidés au Québec

Presence of the bacterial kidney disease in salmonid fish in Quebec During the summers of 1979 and 1980, wild and hatchery fish were analysed for the presence of the bacterial kidney disease (BKD) agent in salmonid fish in Quebec by the indirect immunofluorescence technique. The causative agent of BKD was detected in all hatcheries tested. Ten to 25% of the fish were positive. The presence of this agent was independent of age and species. We were unable to detect the BKD in fish from the rivers in the northern part of Quebec (over the 50th parallel).     

Chinook Salmon Epizootics in Lake Michigan: Possible Contributing Factors and Management Implications

Abstract

Stability of the Lake Michigan fishery for chinook salmon Oncorhynchus tshawytscha at high levels became questionable after stocks declined dramatically following spring epizootics in which bacterial kidney disease (BKD) was a major factor. Initially stocked in 1967, favorable survival and growth of chinook salmon through the 1970s led to increases in abundance and in popularity with anglers. Returns of chinook salmon improved annually until the late–1980s, when, with little warning, spring epizootics reduced the abundance of adult salmon by 50% or more. Reduced abundance of alewives (Alosa pseudoharengus), coupled with an increase in chinook salmon density and heavy parasite infection rates were hypothesized to have reduced chinook salmon growth and fitness and to have increased their susceptibility to BKD. Evidence of slower growth exists and low food availability may be the stressor that triggered the epizootics. Chinook salmon were a major component of the economic development and subsequent hardship of the sportfishing industry on Lake Michigan. Sustaining the chinook salmon fishery at previous levels may require managing for high abundance of alewives, which may be inconsistent with overall fish community management goals. The future sustainability and role of chinook salmon needs to be reevaluated in the context of the entire Lake Michigan fish community.     

Susceptibility of Fish and Turtles to Three Ranaviruses Isolated from Different Ectothermic Vertebrate Classes

Abstract

Ranaviruses have been associated with mortality of lower vertebrates around the world. Frog virus 3 (FV3)-like ranaviruses have been isolated from different ectothermic vertebrate classes; however, few studies have demonstrated whether this pathogen can be transmitted among classes. Using FV3-like ranaviruses isolated from the American bullfrog Lithobates catesbeianus, eastern box turtle Terrapene carolina carolina, and Pallid Sturgeon Scaphirhynchus albus, we tested for the occurrence of interclass transmission (i.e., infection) and host susceptibility (i.e., percent mortality) for five juvenile fish and three juvenile turtle species exposed to each of these isolates. Exposure was administered via water bath (10³ PFU/mL) for 3 d and survival was monitored for 28 d. Florida softshell turtles Apalone ferox experienced no mortality, but 10% and 20% of individuals became infected by the turtle and fish isolate, respectively. Similarly, 5% of Mississippi map turtles Graptemys pseudogeographica kohni were subclinically infected with the turtle isolate at the end of the experiment. Channel Catfish Ictalurus punctatus experienced 5% mortality when exposed to the turtle isolate, while Western Mosquitofish Gambusia affinis experienced 10% mortality when exposed to the turtle and amphibian isolates and 5% mortality when exposed to the fish isolate. Our results demonstrated that interclass transmission of FV3-like ranaviruses is possible. Although substantial mortality did not occur in our experiments, the occurrence of low mortality and subclinical infections suggest that fish and aquatic turtles may function as reservoirs for FV3-like ranaviruses. Additionally, our study is the first to report transmission of FV3-like ranaviruses between fish and chelonians.

Received October 22, 2013; accepted January 8, 2014.