Bath Challenge Research Articles

Vibriosis and atypical furunculosis vaccines; efficacy, specificity and side effects in Atlantic cod, Gadus morhua L.

Atlantic cod, Gadus morhua L., vaccinated by injection with vaccines containing Vibrio anguillarum serotype O2 or atypical Aeromonas salmonicida bacterin, alone or with oil adjuvant, was highly protected against homologous challenge (relative percent survival [RPS]>94 and >77, respectively). Furthermore, the A. salmonicida bacterin also elicited significant protection against bath challenge with V. anguillarum (RPS>30). Oil adjuvant appeared to be unnecessary for obtaining short-term protection in cod against vibriosis and furunculosis. Neither the oil-adjuvanted vaccine nor the bacterin resulted in significantly reduced weight gain 12 weeks postvaccination, but intra-abdominal adhesions were observed in fish injected with oil-adjuvanted vaccines.

Vaccination of market-size eels against vibriosis due to Vibrio vulnificus serovar E

Vaccination with Vulnivaccine at eel farms has been previously shown to protect cultured eels against vibriosis caused by Vibrio vulnificus serovar E for more than 1 year. The reported protocol included an initial vaccination by triple prolonged immersion at the glass-eel stage together with one optional oral booster at the elver stage. However, eels at the market-size stage (around 150 g body weight) can suffer stress-related vibriosis after handling and transport to the selling facilities, which implies a serious risk for consumer health. The main objective of this work was therefore to develop an effective re-vaccination procedure, useful for preventing stress-related vibriosis and zoonosis in market-size eels. To this end, eels, primed 2 years before, were reimmunised by prolonged immersion with a very low dose of Vulnivaccine, a procedure commercially applicable to large fish. The protection, the immune response in plasma, skin mucus and bile as well as the bactericidal activity of the collected body fluids were evaluated in reimmunised and control animals (non-reimmunised) during a 30-day period. Reimmunisation significantly increased antibody titres in plasma, mucus and bile, as well as the bactericidal activity of these fluids, and reduced the mortality rate after bath challenge with the pathogen. In conclusion, the prolonged immersion in a very high dilution of Vulnivaccine can protect market-size eels against stress-related vibriosis and, consequently, significantly reduce additional risks of zoonosis.

Immunomodulation and Disease Resistance in Postyearling Rainbow Trout Infected with Myxobolus cerebralis, the Causative Agent of Whirling Disease

AbstractMyxobolus cerebralis, the myxosporean parasite that causes whirling disease, has a number of deleterious effects on its salmonid host. Although it is well established that juvenile salmonids in the active stages of whirling disease mount an immune response to the pathogen, the occurrence and longevity of any related immunomodulatory effects are unknown. In this study, postyearling rainbow trout Oncorhynchus mykiss infected with M. cerebralis were examined for leukocyte functions and for resistance to Yersinia ruckeri, a bacterial pathogen of salmonids. Compared with uninfected controls, M. cerebralis‐infected fish showed lower proliferative lymphocyte responses to four mitogens (concanavalin A, pokeweed mitogen, phytohemagglutinin, and lipopolysaccharide). Conversely, M. cerebralis‐infected fish displayed greater bactericidal activity of anterior kidney macrophages than did uninfected fish. After bath challenges with Y. ruckeri, M. cerebralis‐infected fish had slightly lower survival and a more rapid onset of mortality than did the control fish. Renal tissue and fecal samples from M. cerebralis‐infected and uninfected survivors were cultured for the presence of Y. ruckeri, and no difference in prevalence was noted between the two groups. Because immunomodulatory changes in the M. cerebralis‐infected fish involved functional enhancement and suppression of different leukocyte populations, disease resistance among M. cerebralis‐infected fish in the later stages of whirling disease will probably vary with the secondary pathogen and the nature of immune response the pathogen evokes.

Influences of temperature, salinity and starvation on the motility and chemotactic response of Vibrio anguillarum.

The role of growth factors for the motility and chemotaxis of the fish pathogen Vibrio anguillarum was determined. Cells of V. anguillarum were chemotactic to serine in the temperature range 5-25 degrees C and in 0.8-2.7 % NaCl. The chemotactic response was significantly higher at 25 degrees C than at 5 or 15 degrees C. Growth in medium with 1.5 % NaCl gave a higher response than growth with 3 % NaCl; when the salinity of the chemotaxis buffer was raised, the chemotactic response was reduced. The role of starvation was also studied; V. anguillarum showed a high chemotactic response after starvation for 2 and 8 days. Motility and chemotaxis are important virulence factors for this bacterium. Not only was the ability to perform chemotactic motility maintained after starvation, but also it was shown that starvation does not interfere with the ability of the organism to cause infection in rainbow trout after a bath challenge. The swimming speed was reduced at lower temperatures. Within the range of salinity and starvation studied, the motile cells swam with the same velocity, indicating that V. anguillarum under all the examined conditions has a functional flagellum and rotates it with constant speed. Phenamil, a specific inhibitor of Na(+)-driven flagella, reduced the motility of both starved and non-starved cells of V. anguillarum indicating that, in both cases, a Na(+) motive force drives the flagellum.

Dietary oligonucleotides from yeast RNA influence immune responses and resistance of hybrid striped bass ( Morone chrysops × Morone saxatilis) to Streptococcus iniae infection

Three feeding trials were conducted to evaluate potential immunomodulatory effects of nucleotides in the diet of hybrid striped bass. A basal diet was formulated from menhaden fish meal to contain 40% crude protein and 10% lipid. An oligonucleotide product (Ascogen P ®) from brewer's yeast was added to the basal formulation at the manufacturer's recommended rate of 0.5% to produce the experimental diet. Each diet was fed to four replicate groups of juvenile hybrid striped bass for seven or eight weeks in two separate trials. After Trials 1 and 2, a Streptococcus iniae bath challenge was executed to test the effects of diet on disease resistance. No significant difference in growth performance was observed between fish fed the basal and experimental diets. Body composition of whole fish, hematocrit and serum lysozyme levels were observed to be within normal ranges and not influenced by dietary nucleotides. Neutrophil oxidative radical production of fish fed the nucleotide-supplemented diet was significantly ( P=0.011) higher than in fish fed the basal diet. Significantly ( P<0.05) enhanced survival after exposure to S. iniae also was generally observed in fish fed the nucleotide-supplemented diet. In addition, fish fed the nucleotide-supplemented diet tended to have a higher antibody response based on microtitration agglutination; however, the difference was not statistically significant because of high variation between individual fish. Long-term (16 weeks) administration of oligonucleotides in Trial 3 failed to show enhancement of immune responses between treatments. It is concluded that dietary oligonucleotides positively influenced immune responses and resistance of juvenile hybrid striped bass to S. iniae infection.

Atypical furunculosis in spotted wolffish ( Anarhichas minor O.) juveniles: bath vaccination and challenge

Farmed spotted wolffish ( Anarhichas minor O.) is susceptible to infections with atypical Aeromonas salmonicida, and experimental oil-adjuvanted vaccines are previously reported to induce protection against the disease. For prophylactic treatment of juveniles, an immersion vaccine is the preferred alternative, and to test the efficacy of different bath vaccination strategies, a waterborne challenge model has to be established. Exposing the wolffish juveniles to 2×10 7–10 8 live bacteria ml −1 for 60–90 min resulted in 70–85% mortality. Bath vaccinating wolffish juveniles (∼25 mm) for 1 h in a bacterin containing 10 8 inactivated cells ml −1 did not induce specific antibody responses although the bacterial antigens were localised in the muscle, kidney and liver tissues. No protection was established in the bath vaccinated fish after waterborne challenge, nor did a second immersion boost improve the efficacy. For comparison, fish were i.p. vaccinated with an oil-emulsified bacterin and significant protection against the pathogen was induced both after injection and bath challenge. The protection was significantly higher when both the vaccine and the pathogen were administered by injection as compared to i.p. vaccination and challenge by bath.

Protection against atypical furunculosis in Atlantic halibut, Hippoglossus hippoglossus (L.); comparison of a commercial furunculosis vaccine and an autogenous vaccine.

Atlantic halibut, Hippoglossus hippoglossus (L.), was shown to be sensitive to infection by three different isolates of Aeromonas salmonicida ssp. achromogenes in pre-challenge tests using intraperitoneal (i.p.) and intramuscular (i.m.) injections as well as bath challenges. A commercial furunculosis vaccine, Alphaject 1200, and an autogenous vaccine, AAS, based on the challenge strain, induced immune protection as shown in challenge tests 8 weeks post-immunization. The survival rate of vaccinated fish after i.p. challenge was 100%, whereas mortality of control fish was 61%. Employing i.m. challenge, relative percentage survival induced by the furunculosis vaccine and the AAS vaccine was 47 and 44, respectively. Mortality of i.m. injected controls was 68%. Vaccinated fish behaved normally following vaccination but the weight gain was significantly reduced in vaccinated fish 8 weeks post-vaccination compared with control fish receiving phosphate-buffered saline. At the same time, intra-abdominal adhesions were observed in fish injected with either of the two vaccines or adjuvant alone. Antibody response against A. salmonicida ssp. achromogenes was detected in sera from fish receiving either vaccine.

Evaluation of brewers yeast ( Saccharomyces cerevisiae) as a feed supplement for hybrid striped bass ( Morone chrysops× M. saxatilis)

Two separate feeding trials were conducted to evaluate graded levels of dried brewers yeast in the diet of hybrid striped bass. A basal diet was formulated to contain 40% protein, 10% lipid and an estimated digestible energy level of 3.5 kcal/g. In Trial 1, three incremental levels of dried brewers yeast (1%, 2% and 4% of diet) were added to the basal diet in place of cellulose. In Trial 2, the same levels of brewers yeast were added to the basal diet, but menhaden fish meal and menhaden oil were adjusted to provide isonitrogenous and isolipidic diets. Each diet was fed to three (Trial 1) or four (Trial 2) replicate groups of juvenile hybrid striped bass twice daily at rates approximating apparent satiation for 6 or 8 weeks. After the second feeding trial, a Streptococcus iniae bath challenge was executed to test the effects of diet on disease resistance. Enhanced weight gain and feed efficiency were generally observed in fish fed the diets supplemented with yeast compared to the basal diet in both trials. In the second trial, body composition of whole fish, hemocrit and serum lysozyme levels were observed to be within normal ranges and not influenced by the various dietary treatments. After 9 weeks of feeding in the second trial, exposure to S. iniae resulted in no mortality and reduced signs of disease in fish fed diets supplemented with 2% and 4% brewers yeast, while 20% mortality was observed in fish fed the control diet ( P=0.1). In the second trial, blood neutrophil oxidative radical production, extracellular and intracellular superoxide anion production of head kidney macrophages and serum lysozyme were measured after 16 weeks of feeding each diet. Fish fed the diet with 2% brewers yeast were found to have significantly ( P<0.01) higher blood neutrophil oxidative radical and extracellular superoxide anion production of head kidney macrophages than control fish. However, no significant differences in intracellular superoxide anion and serum lysozyme were observed among the treatments. Based on the result of this study, it is concluded that brewers yeast positively influenced growth performance and feed efficiency of hybrid striped bass as well as resistance to S. iniae infection. In addition, results of immune response assays demonstrate that brewers yeast can be administered for relatively long periods without causing immunosuppression.

Experimental Streptococcus iniae Infection in Japanese Flounder Paralichthys olivaceus.

The capability of Streptococcus iniae to cause disease in Japanese flounder was investigated by oral and bath (30 min) challenges with low, medium and high inoculation doses. Only with the highest inoculation dose, 9.9t107 CFU/100 g body weight, the oral challenge induced deaths. In the bath challenge deaths were induced even at the lowest inoculation dose, 2.9t103 CFU/mL water. Hemorrhagic lesions on the fins were observed in dead fish challenged by both methods. It is suspected that S. iniae entered through the body surface such as abrasive sites of the fins to cause disease.

Disease resistance and humoral immunomodulatory effects of vitamin C on grouper,Epinephelus awoara

The disease resistance and humoral immunomodulatory effects of vitamin C administered orally to grouper,Epinephelus awoara maintained on a frozen fish diet supplemented with vitamin C at 500, 1000, 1500 and 2000 mg/kg were investigated. After 20 weeks, the growth rates of the groups with high level of vitamin C apparently increased. The untreated fish had symptoms of vitamin C deficiency. The endogenous liver tissue vitamin C levels were found to reflect well the dietary treatments. After intraperitoneal injection or bath challenge with a virulent strain ofVibrio vulnificus, fish fed with high level vitamin C showed significantly higher survival rate compared with the normal control group. Vaccination with formalin inactivatedV. vulnificus significantly enhanced the specific antibody production in fish treated with vitamin C, and completely protected from strong bacterial challenge the groups fed on fish with vitamin C 1500 and 2000 mg/kg diet.

Susceptibility of juvenile and sub-adult Atlantic halibut (Hippoglossus hippoglossusL.) to infection byVibrio anguillarumand efficacy of protection induced by vaccination

Experimental bath challenge of juvenile and sub-adult Atlantic halibut with Vibrio anguillarum induced severe mortalities of 47 and 80%, respectively. However, animals vaccinated with a commercial V. anguillarum vaccine demonstrated excellent protection against the disease (100% RPS). This study also describes the gross pathology and histological changes associated with this infection. A loss of coordination, haemorrhage at the fin base and splenomegaly were frequent findings.Serum agglutinating activity demonstrated a rise following vaccination, the mean log2titre rising from 3·8 to 8·4. This was associated with a significant rise in antibody-mediated complement killing ability of immune serum when compared to non-immune serum. \\

Manganese requirement of Atlantic salmon (Salmo salar) fry

The present experiment was undertaken to establish the dietary manganese (Mn) requirement in Atlantic salmon fry and to examine the effect of Mn intake and status on disease resistance. Salmon fry were divided into six triplicate groups and fed a casein/gelatine diet (containing 1.1 mg Mn/kg) supplemented with 0, 3, 6, 9, 12 or 24 mg Mn/kg (as MnSO4) for 12 weeks. Growth, mortality and whole body Mn were recorded every 4 weeks. Concentrations of calcium, phosphorus, zinc and iron in whole fish and total hepatic superoxide dismutase activity were measured after 12 weeks of feeding. After the feeding experiment, the fish were challenged with Vibrio anguillarum. Dietary Mn levels did not affect growth and mortality. Whole body Mn concentration responded readily to dietary Mn supplementation and reached a plateau in fish receiving diets supplemented with more than 6 mg Mn/kg. Regression analysis showed that whole body Mn concentration was maintained at dietary Mn concentrations of 7.5-10.5 mg/kg dry feed. Supplementation of only 3 mg Mn/kg diet was enough to saturate the total hepatic superoxide dismutase activity. The dietary Mn levels did not affect the mortality after bacterial bath challenge with a virulent strain of V. anguillarum. In conclusion, a minimum Mn requirement of 7.5-10.5 mg/kg dry diet was needed to maintain Mn status in Atlantic salmon during start feeding.

Flavobacterium psychrophilum, invasion into and shedding by rainbow trout Oncorhynchus mykiss.

The infection route of Flavobacterium psychrophilum into rainbow trout Oncorhynchus mykiss was studied using bath and cohabitation challenges as well as oral challenge with live feed as a vector. Additionally, the number of bacterial cells shed by infected fish into the surrounding water was determined in the cohabitation experiment and in challenge experiments at 3 different water temperatures. The experiments showed that skin and skin mucus abrasion dramatically enhanced the invasion of F. psychrophilum into the affected fish in bath and cohabitation challenges. Disruption of the skin is discussed as an important invasion route for F. psychrophilum into the fish. The shedding rate of F. psychrophilum by infected fish was associated with water temperature and the mortality of the infected fish. High numbers of F. psychrophilum cells were released into the water by dead rainbow trout during a long time period compared to the numbers of cells shed by live fish. The results emphasise the importance of removing dead and moribund fish from rearing tanks in order to diminish the infection pressure against uninfected fish in commercial fish farms. In immunohistochemical examinations of organs and tissues of orally infected fish, F. psychrophilum cells were detected in only 1 fish out of 31 studied. Mortality of the orally challenged fish was not observed in the experiment.

The gills are an important site of iNOS expression in rainbow trout Oncorhynchus mykiss after challenge with the gram-positive pathogen Renibacterium salmoninarum.

Following injection challenge of rainbow trout with the Gram-positive pathogen Renibacterium salmoninarum, serum nitrate levels increased indicative of NO production. The timing and amount of nitrate produced varied with the virulence of the bacterial strain used, with the highest levels seen in fish challenged with the most virulent (autoaggregating) strain. Immunization with a killed R. salmoninarum preparation in Freund's incomplete adjuvant significantly increased nitrate levels after challenge. Inducible nitric oxide synthase (iNOS) transcript expression was detectable in rainbow trout tissues after injection challenge with R. salmoninarum, and its induction in the gills was both quick (between 3 and 6 hr) and relatively prolonged (lasting several days). iNOS expression in the kidney was also seen at a later stage (24 hr) but appeared to switch off relatively rapidly. Bath challenge with R. salmoninarum also induced iNOS expression in gill, and a variable expression in the gut and kidney also occurred. These results highlight the importance of the gills, not only as a point of entry of pathogens but also as a tissue capable of mounting an immune response.

Susceptibility of Atlantic halibut, Hippoglossus hippoglossus (L.) to infection with typical and atypical Aeromonas salmonicida

Halibut, Hippoglossus hippoglossus (L.) and Atlantic salmon, Salmo salar (L.) were challenged with virulent typical or atypical strains of Aeromonas salmonicida by both injection and bath models. The groups were injected intraperitoneally with 100 μl of logarithmically decreasing dilutions of A. salmonicida (range 10 8–10 3 cells/fish) or bathed in 10 5 CFUs/ml of A. salmonicida for 24 h. Halibut were significantly more resistant to infection, compared to Atlantic salmon, with 10 6 and 10 7 typical and atypical A. salmonicida cells/halibut being the minimum lethal dose. No halibut died in the bath challenge although approximately 80% of salmon died by this challenge method. All animals that died during the challenge were positive for A. salmonicida on culture A. salmonicida was cultured from internal organs of approximately 60% of surviving Atlantic salmon while only 2.3% of the surviving halibut were culture positive, but only from the intestinal lumen. All surviving salmon but none of the surviving halibut, showed histological evidence of infection with A. salmonicida. Stress tests of both halibut and Atlantic salmon showed that recovered salmon still had a high carrier rate of A. salmonicida while none of the surviving halibut were carrier test positive. A significant rise in agglutinating antibody titre was observed in surviving Atlantic salmon. However, no rise in antibody titre was observed in the surviving halibut, suggesting that the processing of the bacteria during an acute infection may be fundamentally different between these two species.

Tissue localization of Aeromonas salmonicida in Atlantic salmon, Salmo salar L., following experimental challenge

Three groups of Atlantic salmon, Salmo salar L., were exposed to live, colony‐forming, radiolabelled Aeromonas salmonicida bacteria in a bath challenge: (1) fish with artificial wounds; (2) fish with a reduced epidermal mucus layer caused by removal of the mucus layer on two occasions by a swabbing procedure; and (3) a control group of untreated fish. Fish were killed 2, 6 and 24 h after challenge, and radioactivity (cpm g–1) was measured in the blood, mucus, skin, wound area, gills, anterior kidney, posterior kidney, spleen, midgut and hindgut. The highest levels of radioactivity were measured in the wound areas and in the gills. There was a significant positive correlation between the levels of radioactivity in the gills and blood, and between the mucus and skin at 2 h post‐challenge. Two hours after the bath challenge, live A. salmonicida bacteria were found in the blood of fish in the ‘swabbed’ and ‘artificial wound’ groups, and not in the control group. Twenty‐four hours after the bath challenge, the kidney of fish from all groups contained viable bacteria, whereas the blood was negative.

Variables affecting the challenge pressure of Aeromonas salmonicida and Vibrio salmonicida in Atlantic salmon ( Salmo salar L.)

In the present study a cohabitation challenge with Aeromonas salmonicida in Atlantic salmon was investigated, with respect to the influence of water temperature and number of added cohabitants on mortality. Further, intra-peritoneal injection (i.p.) and bath challenges with Vibrio salmonicida were investigated with respect to different doses of the challenge organism. The results clearly demonstrated that specific cumulative mortality in the cohabitation challenge with A. salmonicida was highly dependent on both water temperature and number of cohabitants added. I.p. challenge with V. salmonicida demonstrated a clear dose response with respect to time from inoculation to onset of disease. All doses caused 100% mortality amongst test fish 21 days post challenge. Bath challenge revealed a dose response relationship to onset of disease, but failed to cause specific cumulative mortality above a certain minimum level within a reasonable time post challenge.

Development of a bath challenge for the marine shrimp Penaeus vannamei Boone, 1931

Despite the major financial losses caused by vibriosis in shrimp culture, no reliable laboratory model has yet been developed for this disease. A reproducible experimental model is therefore urgently required for further study. In this study, a bath challenge technique was developed for juveniles of the marine shrimp Penaeus vannamei. Four different treatments were applied to the shrimp: (1) shrimps were wounded on the third abdominal segment and were immediately exposed to a bath of Vibrio parahaemolyticus afterwards; (2) shrimps were only wounded; (3) shrimps were only exposed to V. parahaemolyticus; and (4) control, shrimps were neither wounded nor challenged. The experiment was repeated four times to verify the reproducibility of the technique. All the mortalities occurred within four days and the cumulative mortalities were significantly different between treatments: varying from 37 to 52% for the first treatment, 12 to 22% for the second, 0 to 13% for the third, and 0 to 3% for the control.

Infectious pancreatic necrosis virus: experimental infection of goldsinny wrasse, Ctenolabrus rupestris L. (Labridae)

The use of wrasse as cleaner fish in farming of Atlantic salmon, Salmo salar L., is now widespread in Scotland, Ireland and Norway, but little is known about the susceptibility of these fish to the common pathogens of cage-cultured salmon. The susceptibility of goldsinny wrasse, Ctenolabrus rupestris L., to infectious pancreatic necrosis virus (IPNV) was investigated. Captive-bred C. rupestris were infected with IPNV-Sp (Shetland strain) using a bath challenge method. Two infection doses were used, low (4.1 × 105 pfu mL−−1) and high (2.4 × 106 pfu mL−−1), with two replicates for each experiment. Bathing times were 1 h for the low dose and 5 h for the high dose. A maximum prevalence of infection (30%) was seen 2 weeks post-infection in replicate 1 of the low dose experiment and was accompanied by low tissue titres. The tissue titres dropped to an undetectable level by 4 weeks post-infection. In the high dose experiment, a high prevalence of infection was seen along with moderate titres in tissues as well as a marked pathological response. Both prevalence and intensity declined rapidly and the fish recovered. In the high dose experiment, faecal titres followed the same pattern as those in the tissues. The implications for the use of wrasse in the farming of Atlantic salmon are discussed.

Experimental infection with Vibrio salmonicida in Atlantic salmon (Salmo salar L.): an evaluation of three different challenge methods

In testing the efficacy of vaccines for cold water vibriosis in Atlantic salmon a reliable challenge method is essential. Three different challenge methods with Vibrio salmonicida in Atlantic salmon (Salmo salar L.) were evaluated. The results from 20 batch release tests for vaccines against cold water vibriosis in a test system using intraperitoneal (i.p.) injection were evaluated with respect to the EU guidelines for vaccines intended for fish. Nine bath challenges employed in the development of new vaccine candidates, and one pilot study using a cohabitation challenge, were also included. Review of the test results showed that the i.p. injection was the most reliable method of inducing cold water vibriosis with cumulative specific mortalities in controls above 70% during a three-week observation period post challenge. Nineteen out of 20 challenges fulfilled this criterium. For the bath challenges, only six out of nine challenges succeeded in reaching the same level. In the single pilot study using cohabitation challenge, the disease was transferred from cohabitants to the test fish, but cumulative specific mortalities did not reach the minimum levels stated in the EU guidelines.