Evaluation of cross protection by vaccines against atypical and typical furunculosis in Atlantic salmon, Salmo salar L.

In Iceland, farmed salmonids are vaccinated against A. salmonicida ssp. achromogenes (Asa), which causes atypical furunculosis and is endemic in local waters . Classical furunculosis, caused by A. salmonicida ssp. salmonicida (Ass), was not diagnosed in this country until June 1995. In the present study, protection in experimental challenges against atypical and classical furunculosis in Atlantic salmon vaccinated with an autogenous Asa bacterin (Iceland Biojec.OO, IBOO), a commercial furunculosis vaccine (Biojec.1500), or a mixture of both vaccines was compared. The results showed that both vaccines gave protection against an injection challenge with Asa. However, better protection was obtained with the IBOO (homologous) vaccine. Infection of Asa by cohabitation could not be established in fresh water. Fish vaccinated with Biojec.1500 or with both vaccines simultaneously were equally well protected against Ass in a cohabitation challenge. On the other hand, no protection against classical furunculosis was achieved in fish vaccinated by IBOO alone.     

Retention of furunculosis vaccine components in Atlantic salmon, Salmo salar L., following different routes of vaccine administration

The retention of vaccine components was studied in Atlantic salmon, Salmo salar L., following different routes of vaccination against Aeromonas salmonicida. Frozen tissue was collected from the spleen, head kidney, hind gut and liver of fish that had been vaccinated by intraperitoneal injection (monovalent and trivalent vaccines), immersion and oral administration 2,6,8 and 16 weeks previously. The trivalent injection group showed the highest levels of specific antibodies and was the only group to show protection following challenge with virulent A. salmonicida. Following intraperitoneal injection, there was an initial widespread distribution of Aeromonas lipo-polysaccharide, but by 16 weeks lipopolysaccharide was predominantly found in macrophage populations in the spleen, head kidney and abdominal granulomas. Only small amounts of lipopolysaccharide were retained in the head kidney of the immersion group and no lipopolysaccharide was retained in the oral group. Small and inconsistent amounts of A-layer protein were present in the meianomacrophages of the head kidney of all groups. The relative prominence of A-layer protein in the spleen of the trivalent injection group 8 weeks after vaccination was linked to the high levels of specific antibodies, and possible immune-complex trapping and the enhancement of immunological memory.     

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.     

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.     

Antibiotic protection against recrudescence of latent Aeromonas salmonicida during furunculosis vaccination

Problems of temporary immunosuppression following vaccination against Aeromonas salmonicida infection had to be overcome in the development of a furunculosis vaccine. Empirical observations have indicated that immunosuppression persists for some time after vaccination, rendering fish, especially subclinical carriers of A. salmonicida, highly vulnerable to bacterial invasion. The efficacy of simultaneous application of furunculosis vaccine and a long-lasting amoxycillin preparation to a population of Atlantic salmon smolts was evaluated. Control groups were treated with either vaccine alone, amoxycillin alone or were untreated. Moderate stress, simulating smolt transfer with a 5°C temperature rise, resulted in a rapid outbreak in mortalities reaching 100% in the vaccinates. Losses among the untreated controls were more gradual and rose to about 50%. Both amoxycillin-treated groups survived well. Further severe stress resulted in total mortalities among the untreated fish but no further losses in the amoxycillin groups. Four months after vaccination, evidence of a specific immune response was confirmed by ELISA, demonstrating circulating antibodies in the blood of vaccinates. In a severe and in a moderate challenge with A. salmonicida., the relative specific protection was 63 and 86%, respectively. Thus, effective protection against furunculosis was achieved without jeopardizing the stock during the vaccination process and with elimination of the carrier state.     

Comparison of different challenge methods to evaluate the efficacy of furunculosis vaccines in Atlantic salmon, Salmo salar L.

Four challenge methods, intraperitoneal (i.p.) and intramuscular (i.m.) inoculation, bath and cohabitation exposure, were evaluated as methods for testing the efficacy of furunculosis vaccines in Atlantic salmon, Salmo salar L. Groups of fish vaccinated with one of two different vaccines containing aluminium phosphate or animal/vegetable oil as an adjuvant were challenged with Aeromonas salmonicida 6 and 12 weeks after vaccination. Relative per cent survival (RPS) was calculated daily during a 3-week observation period post-challenge. A large variation in protection measured by RPS, both between methods and between different time points for each method, was found. Towards the end of the observation period, RPS tended to be similar in the i.p. and cohabitation challenge groups. A high degree of protection was demonstrated for the oil adjuvanted vaccine. The i.m. challenge produced very low RPS numbers for both vaccines, but this was most marked for the aluminium-phosphate-adjuvanted vaccine. The bath exposure resulted in RPS values intermediate to the cohabitation and injection methods. The study also demonstrated that, after i.p. and i.m. challenges, the initial peak mortality caused by the inoculation was followed by a secondary increase in mortality, probably because of shedding of bacteria into the water during the first mortality phase and hence contributing to a superinfection state.     

Feeding Atlantic salmon Salmo salar L. soybean products: effects on disease resistance (furunculosis), and lysozyme and IgM levels in the intestinal mucosa

Two trials were initiated to investigate the consequences of including various soybean products in diets for Atlantic salmon Salmo salar L. on (1) mortality following infection by Aeromonas salmonicida ssp. salmonicida during a cohabitation challenge, and (2) the lysozyme and IgM content of the intestinal mucosa. Groups of salmon were fed control diets containing fishmeal as the sole protein source (Contr1 and Contr2, respectively), soy concentrate-containing diets (SoyConc1 and SoyConc2, respectively), or diets containing either solvent-extracted soybean meal (SoyMeal, trial 1) or soybean molasses (SoyMol, trial 2), an alcohol extract of soybean meal. Both SoyMeal and SoyMol caused enteritis-like changes in the distal intestine, which were not observed in fish fed the Contr1, Contr2, SoyConc1, or SoyConc2 diets. There were significant differences ( P  < 0.05) in mortality between feeding groups following the A.s. salmonicida challenge: these differences were greatest in fish fed SoyMeal (65.6%), least in fish fed SoyConc1 (60.5%), and intermediate in the fish fed the Contr1 diet (62.9%). The SoyMol diet caused significantly ( P  < 0.0001) increased levels of both lysozyme and IgM in the mid and distal intestinal mucosa. It is concluded that components of soybean meal and soybean molasses cause an inflammatory response in the distal intestine that may lead to increased susceptibility to furunculosis.     

Field testing of adjuvanted furunculosis vaccines in Atlantic salmon, Salmo salar L.

Abstract. Two different commercial vaccines against furunculosis, caused by Aeromonas salmonicida subsp. salmonicida , were tested in Atlantic salmon on seven fish farms. Both vaccines were based on formalin-inaclivated bacterins containing aluminium salts as adjuvants. The fish were vaccinated by intraperitoneal injection in the spring approximately one month prior to transfer to sea water, and they were challenged by natural outbreaks of furunculosis. During the first year, six of the farms experienced disease outbreaks. The overall mortality was 7·14% in vaccinated fish and 21·7% in unvaccinated controls, giving a relative percentage survival (RPS) of 67%. In the seventh farm, outbreaks of furunculosis more than one year after vaccination revealed that there was still a trend towards lower mortality in vaccinated fish, though the mean RPS fell to 22%. The use of adjuvants in the vaccines resulted in local lesions in the abdominal cavity of vaccinated fish. However, the severity of the lesions declined gradually, and they did not influence fish quality at the time of slaughtering. Vaccination also had a moderately adverse impact on fish weight gain in most cases.     

Florfenicol in Atlantic salmon, Salmo salar L., parr: tolerance and assessment of efficacy against furunculosis

Abstract. The efficacy of florfenicol against laboratory-induced infection with Aeromonas salmonicida was tested in Atlantic salmon, Salmo salar L., parr. Medication at three dose levels in the feed was started 24 h after bath challenge with A. salmonicida. The specific mortality rate in the unprotected infected control group was 75% compared with 5, 13 and 17% when florfenicol was given at dose levels of 20, 10 and 5 mg per kg body weight per day, respectively. Florfenicol was palatable to the fish at doses in excess of effective therapeutic levels and feeding for 10 days at 100 mg per kg, or for prolonged periods at 50 mg per kg and 10 mg per kg, resulted in no feeding problems or histopathological abnormalities. Florfenicol appears to have a good therapeutic index and considerable potential in the control of furunculosis in salmon.     

Peritoneal and head kidney cell response to intraperitoneally injected yeast glucan in Atlantic salmon, Salmo salar L.

Abstract. Previous studies have shown that intraperitoneal (i.p.) injection of a yeast cell wall glucan into Atlantic slamon, Salmo salar L., resulted in increased resistance to bacterial pathogens and that the glucan enhanced the protective effect of an i.p.-administered furunculosis vaccine in salmon. In the present work, the peritoneal cell responses to yeast glucan, glycogen, Freund's incomplete adjuvant (FIA) and saline were compared. Whereas saline gave no detectable peritoneal cell response, injection of the three other agents resulted in accumulation of macrophages, neutrophils and thrombocytes in the peritoneal cavity 2–22 days later. Glucan and glycogen gave maximum leucocyte responses 2 days after injection, whereas the number of leucocytes after FIA injection showed a continuous increase during the 22-day experimental period. Two days after injection, macrophages dominated in the leucocyte response to yeast glucan, whereas neutrophils dominated in response to glycogen and FIA. At 8 and 22 days post-injection of yeast glucan and FIA, thrombocytes represented the largest fraction of peritoneal cells. Except for an elevated number of neutrophils 3 weeks after injection, i.p. administration of yeast glucan gave no significant change in leucocyte numbers from the head kidney. On the other hand, head kidney macrophages from glucan-treated fish showed an increased ability to kill a virulent strain of Aeromonas salmonicida.     

Efficacy of furunculosis vaccines in turbot, Scophthalmus maximus (L.): evaluation of immersion, oral and injection delivery

The commercial furunculosis vaccine Aquavac Furovac 5 and an autogenous vaccine, based on the challenge strain, induced immune protection in turbot, Scophthalmus maximus (L.), as shown in challenge tests 120 days post-immunization by injection (relative percentage of survival, RPS = 72-99%). This protective effect lasted for at least 6 months post-immunization at appreciable levels (RPS = 50-52%). Neither the autogenous vaccine nor the commercial vaccine was able to induce significant levels of protection against Aeromonas salmonicida in turbot when administered by immersion. Antibody levels were high or moderate in fish vaccinated by injection with the different vaccines and very low in fish vaccinated by immersion. The field results show that delivering an oral boost after the primary vaccination by injection did not enhance protection of turbot against furunculosis and that water-based (autogenous vaccine) and oil adjuvanted (Alpha Ject 1200) vaccines administered by injection conferred similar levels of protection (RPS > 80%) in turbot.     

Survival and humoral antibody response of Atlantic salmon, Salmo salar L., vaccinated against Aeromonas salmonicida ssp. achromogenes

Atlantic salmon were vaccinated against Aeromonas salmonicida ssp. achromogenes (Asa) by injection with three vaccines developed in our laboratory and an autogenous bacterin (IcelandBiojec.OO, IBOO) produced by a commercial vaccine producer. The humoral antibody responses to bacterial antigens were monitored by ELISA and Western blotting. The fish were challenged by infection with Asa 6 and 12 weeks post-vaccination. Protection was induced in all groups of vaccinated fish. The protection achieved was time-dependent. The autogenous bacterin, IBOO, induced a protective immune response later than our experimental vaccines. All the vaccines tested induced specific antibody response that increased between 6 and 12 weeks after vaccination. The antibody response was mainly directed against the A-layer protein, but antibodies to other bacterial components were also detected. Significant correlation was obtained between the antibody titre to extracellular Asa antigens, induced by the different vaccine preparations, and survival of vaccinated fish challenged by a virulent Asa strain. Furthermore, the detection of antibodies directed against an extracellular toxic metallo-caseinase, AsaP1, in fish sera correlated with protection.     

Serine protease and glycerophospholipid:cholesterol acyltransferase of Aeromonas salmonicida work in concert in thrombus formation; in vitro the process is counteracted by plasma antithrombin and α2-macroglobulin

Abstract. Intravascular injection of purified 70-kDa protease or GCAT-LPS from Aeromonas salmonicida , or both, invariably led to consumptive coagulopathy within 2h in Atlantic salmon, Salmo salar L. By entering the coagulation cascade at two different levels, the two enzymes work in concert in thrombus formation, the significance being that circulatory failure is probably the major cause of death in acute furunculosis. Only intravascular injection of GCAT-LPS led to consumptive coagulopathy in rainbow trout, Oncorhynchus mykiss (Walbaum), despite the fact that trout received only half the GCAT dose, and/or four times the protease activity administered to salmon. These results indicate that salmon is, by far, the most susceptible to protease and suggest that rainbow trout is the most susceptible to GCAT-LPS. The substrate profile of the purified protease gave supporting evidence that it works as activated coagulation factor X. The protease is inhibited in vitro by antithrombin and by α₂-macroglobulin and both inhibitors are consumed in vivo in response to intravascular administration of the enzymes, thus showing a potential for AT and α₂M to inhibit the protease also in vivo. Provided such plasma antiprotease activities are correlated with resistance to the disease, and the inhibitors show genetic variation they would be promising candidates for indirect selection and hence a means to prevent furunculosis independently of vaccines. Circulating inhibitors of GCAT-LPS remain to be identified.     

Humoral immune response in Atlantic salmon, Salmo salar L., to cellular and extracellular antigens of Aeromonas salmonicida

Abstract. The putative virulence factors of Aeromonas salmonicida , the aetiological agent of furunculosis in salmonids, are candidates for protective antigens in effective vaeeines against furunculosis. In this report, the authors have compared the immunogenieily of eell-associated and extracellular antigens of A. salmonicida in Atlantic salmon, Salmo salar L., to that in rabbit. The animals were immunized with formalin-killed whole cells and formalin-inactivated extracellular products (ECP), either separately or in combination. The ability of the antigens to induce antibody production was studied by elisa and Western blotting techniques. These results confirm previous reports that far more structures are immunogenie in rabbit compared to the antibody responses elicited in salmon. However, in both species, some antigens were dominant, including a caseinolytic protease in addition to the A-protein and high and low MW LPS.     

Do extracellular products of Aeromonas salmonicida induce thrombosis by entering the fish coagulation system at factor X?

Abstract. Thrombosis of minute vessels is a common feature of acute furunculosis in Atlantic salmon. Crude extracellular products (F.CP) of Aeromans, salmonicida injected into the dorsal aorta of cannulated fish elicited an activation of the coagulations systems of the fish When screened with a number of chromogenic substrates, the ECP protease revealed a factor-X like activity. Thus, it may he hypothesized that the ECP induces thrombosis by entering the fish coagulation systems at factor X. and that thrombus formation is a way of creating a site of infection.     

Aeromonas salmonicida infectivity studies in goldsinny wrasse, Ctenolabrus rupestris (L.)

Goldsinny wrasse, Ctenolabrus rupestris (L.). were experimentally and naturally infected with Aeromonas salmonicida. Goldsinny wrasse were found to be susceptible to infection with A. salmonicida but significantly more resistant to infection than Atlantic salmon, Salmo salar L. The pathology of the acute infection is described. Following infection significantly higher levels of agglutinating antibody were detected in the sera of recovered wrasse than those observed in the Atlantic salmon controls. However, a large proportion of the recovered wrasse were culture positive for A. salmonicida and appeared to have entered a chronic stage of infection quite distinct from the carrier status that can develop in Atlantic salmon. This study indicates that, although goldsinny wrasse are susceptible to A. salmonicida, these fish are unlikely to become primarily infected, but contract furunculosis from infected Atlantic salmon. However, goldsinny wrasse may act as a reservoir for subsequent infections of cultivated Atlantic salmon because of the development of a chronic form of the disease. The potential for the vaccination of goldsinny wrasse against furunculosis is discussed.     

Virulence of the aetiological agent of goldfish ulcer disease in Atlantic salmon, Salmo salar L.

Abstract. An atypical strain of Aeromonas salmonicida was isolated from goldfish. Carassitis auraius L., with cutaneous ulcerativc lesions. Atlantic salmon, Salmo salar L., weighing 30–40 g were challenged parentcrally and by hath immersion with this strain. It was found to be highly virulent in both forms of challenge and was capable of achieving an LD₅₀ of three colony-forming units in parenterally challenged fish. The gross lesions and histopathology of infected fish bore a close resemblance to classical furunculosis in salmonids. Goldfish ulcer disease is endemic to some regions of Australia and represents a potential threat to the emerging salmonid farming industry.     

A bath challenge model for furunculosis in rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, Salmo salar L.

Abstract. A natural bath challenge method has been developed for furunculosis using Atlantic salmon, Salmo salar L., and rainbow trout, Salmo gairdneri Richardson. Fish were placed in an enclosed, continuously circulating tank system, supplemented with additional oxygen, the temperature raised gradually (overnight) to 15–16°C, a low dose of Aeromonas salmonicida (strain 184/186) introduced into the tank and the challenge maintained for 14 days. The challenge strain was characterized with respect to possible virulence factors and possessed an A-layer, ability to auto-agglutinate, haemagglutinate, adhere to Atlantic salmon cells and resist destruction by serum. No caseinase activity was detected. LD₅₀ for salmon using this method was 1.8×10³ cells per millilitre while trout had an LD₅₀ of 9.5×10⁴ cells per millilitre. Onset of the disease appeared to depend on fish size with larger trout (50 g) taking up to 10 days to show signs of the disease while mortalities in smaller trout (8.5g) commenced on day 1 post-challenge.     

Immunogenicity of vaccines from a virulent and an a virulent strain of Aeromonas salmonicida

Abstract. Injected vaccines consisting of formalin-killed cells and extracellular antigens prepared from a virulent and an avirulent strain of Aeromonas salmonicida were tested for their efficacy in protecting juvenile coho salmon. Oncorhychus kisutch (Walbaum). Against experimental furunculosis following active and passive immunization. Sera used in the passive immunization experiments were raised in subadult coho salmon and in rabbits. Results indicated that the avirulent strain was inferior to the virulent strain in its immunogenicity for coho salmon. Thus, even though avirulent cells possessed at least one immunogen–an immunogen that was show in passive immunization experiments to be well recognized by rabbits–the immunogen was only inefficiently protection when vaccinated with avirulent cells. Furher, extracellular antigens of the avirulent strain were not protective for coho salmon even though they elicited the production of anti- A. salmonicida agglutinins. In contrast, the killed cells and extracellular antigens of the virulent strain were both immunogenic in coho salmon. By passively immunizing coho salmon with rabbit sera raised against the virulent and the virulent strain produced at least two immunogens, only one of which was shared in common with the avirulent strain. The extra immunogen possessed by the virulent strain is presumably responsible for its superior immunogenicity in coho salmon; the immunogen had the properties of a protein and is believed to be the A-protein.     

Influence of dietary vitamin B6 on tissue vitamin B6 contents and immunity in Atlantic salmon, Salmo salar L.

Atlantic salmon, Salmo salar L., (14 g) were fed a practical fish-meal-based diet supplemented with 0. 10, 20. 40, 80 and 160 mg pyridoxine (PN) per kg feed for 20 weeks. Tissue vitamin B₆ contents were significantly reduced in fish fed the non-supplemented diet compared with fish fed PN-supplemented diets. Serum haemolytic complement activity and head kidney lysozyme activity, and the specific antibody response following immunization with Vibrio salmonicida. were not influenced by the dietary regimes. Challenge with Aeromonas salmonicida showed that increasing the dietary levels of vitamin B₆ did not improve the resistance to furunculosis. Growth, mortality and haematology were not affected by supplementing a practical diet with vitamin B₆ In conclusion, feed levels of vitamin B₆ higher than the minimum dietary requirement did not enhance immune functions and disease resistance in Atlantic salmon.