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.     

Protection against carp erythrodermatitis following bath or subcutaneous exposure to sublethal numbers of virulent Aeromonas salmonicida subsp. nova

Abstract. A bath challenge system was used to infect carp. Cyprinus carpio L., with Aeromonas salmonicida subsp. nova , the causative agent of carp cruthrodermatitis. Bath-challenged fish became infected with the bacterium exihibitinng typical signs of the disease, Carp that were sublethally bath exposed became infected and exhibited some skin lesions, but after one week, these quickly healed and the animals fully recovered from the infection, Naive fish that had not been previously exposed to the bacterium had mortalities of 100% when infected by the subcutaneous route and 40–60% by the bath route of infection. Carp that received sublethal infections were able to withstand subsequent lethal infection and recover regardless of the route of infection. Sublethally bath-exposed carp were protected from subsequently lethal challenges of A. salmonicida subsp. nova for at least 5 months.     

Immunological responses in Atlantic salmon, Salmo salar L., against purified serine protease and haemolysins from Aeromonas salmonicida

Abstract. A method for purification of the 70-KDa extracellular serine protease of Aeromonas salmonicida by hydrophobic chromatography and ion exchange is described. The purified protease, adsorbed onto mineral particles, was used for immunization of salmon. Other groups of salmon were immunized with particles coated with purified glycerophospholipid:cholesterol acyltransferase (GCAT) or with the acyltransferase complexed with LPS (GCAT-LPS). Humoral immune responses assayed after 6 weeks by ELISA, showed relatively good responses against GCAT-LPS, while titres of antisera against the purified protease and GCAT were scarcely above those of control sera. However, the antibody responses to each toxin were shown by Western blotting to be specific and qualitatively similar to responses seen in rabbits. The toxin preparations were also used (in combination with whole bacteria) for vaccination of salmon. On challenge 3 months later, only GCAT-LPS elicited significant immunological protection. However, a more convincing protection was seen when total extracellular product was present in the vaccine.     

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.     

Experimental infection of turbot, Scophthalmus maximus (L.), by Aeromonas salmonicida subsp. achromogenes and evaluation of cross protection induced by a furunculosis vaccine

Turbot was shown to be sensitive to injection challenges by Aeromonas salmonicida subsp. achromogenes (Asa). A systemic disease was induced and the bacterium was isolated from various internal organs. Histopathological changes involved haemorrhages, necrosis and degeneration in skin and muscle, haemorrhages and necrosis in kidney, degeneration in the heart muscle, and fusion of the secondary gill lamellae. A polyvalent commercial salmon vaccine, containing A. salmonicida subsp. salmonicida as one of five antigens, did not confer protection in turbot against an experimental Asa infection 13 weeks post-vaccination. Vaccination induced a significant antibody response against Asa cells but not against extracellular products of the bacterium. The results of the study indicate that Asa may be a potential threat to turbot farming and that the development of new turbot vaccines is needed.     

Immune protection in carp, Cyprinus carpio L., after immunization with Aeromonas hydrophila crude lipopolysaccharide

Abstract. Vaccination with crude lipopolysaccharide (LPS) induced better protection against infection with Aeromonas hydrophila in carp than vaccination with formalin killed vaccine. Dipping fish in vaccine for 2 h at 25°C was more effective than intraperitoneal injection of the vaccine in procedural simplicity, lower stress loading and the degree of protection acquired. In carp immunized with crude LPS by the dip method, antibodies were not detected by bacteriai agglutination, passive haemagglutination and the agar diffusion tests. The results indicate that the protection against A. hydrophila infection in carp is not dependent on humoral immunity.     

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.     

Efficacy studies of passive immunization against Aeromonas salmonicida infection in brook trout, Salvelinus fontinalis (Mitchill)

Abstract. Aeromonas salmonicida , the aetiologic agent of furunculosis, causes high mortality in cultured salmonids. Experiments were conducted to determine the therapeutic and prophylactic efficacy of passive immunization against furunculosis in brook trout, Salvelinus fontinalis (Mitchill), infected by immersion. Rabbit hyperimmune serum was produced against a virulent strain of A. salmonicida and an aliquot of this serum was absorbed with cells of a non-virulent strain of A. salmonicida. Immunoglobulins from aliquots of the absorbed and non-absorbed serum were purified using affinity chromatography. Each serum or immunoglobulin preparation was tested in passive immunization experiments. Brook trout were infected by immersion in a suspension of virulent A. salmonicida , and passively immunized by intraperitoneal injection at the time of experimental infection, or at various periods after experimental infection. Passive immunization of brook trout against furunculosis was therapeutically efficacious when effected either at zero, 24 or 48h post-infection, but not at 72 or 96h. Purified rabbit immunoglobulins specific to virulent A. salmonicida were as protective as the initial rabbit hyperimmune serum in protecting brook trout against furunculosis. To determine the prophylactic efficacy of this treatment, the groups of fish passively immunized at the time of the experimental infection were challenged a second time at either 14, 35, 41 or 56 days after passive immunization. Brook trout were protected against a second experimental bath challenge with virulent A. salmonicida for a period of 35–41 days.     

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.     

Antigen retention and enzyme reactivity in the spleen of Atlantic salmon, Salmo salar L., following administration of injectable furunculosis vaccines

Abstract. The retention of vaccine components and phenotypes of leucocyte populations were examined in the spleen of Atlantic salmon, Salmo salar L., 12 weeks after intraperitoneal administration of three different furunculosis vaccines. There were marked differences between the vaccine groups as judged by serum antibody response and survival following challenge with Aeromonas salmonicida. Abundant vaccine components were present in the spleen following administration of the two adjuvanted vaccines but not the non-adjuvanted vaccine. The non-adjuvanted group showed a disrupted pattern of silver staining in the splenic ellipsoids, suggesting possible toxic changes. Altered levels of enzyme reactivity in the spleens of vaccinated fish suggested activation of macrophages. Computer-assisted morphometric analysis was used to demonstrate that a significant ( P < 0.05) increase in acid phosphatase reactivity associated with the melanomacrophage accumulations only occurred in the group that had shown a good response to challenge (14% mortality when control group = 60%), and a high level of anti- A . salmonicida antibodies. The findings of the present study suggest that the retention of antigen and the activation of macrophages in melanomacrophage accumulations of Atlantic salmon are of significance in vaccination against furunculosis.     

鲤竖鳞病病原及其疫苗的研究

对发生在山东境内的鲤竖鳞病进行了病原学研究,经过病原分离,人工感染实验和细菌学鉴定,确定豚鼠气单胞菌（Ａｅｒｏｍｏｎａｓｃａｕｉａｅ）是鲤竖鳞病的病原菌,在此基础上,得到了免疫原性较高和生产工艺简单的鲤竖鳞病疫苗,同时,对该疫功的安全性,免疫保护力和效价等进行了系统研究,并进行了一定范围的口服免疫应用试验。     

Disease resistance of carp, Cyprinus carpio L.: identification of individual genetic differences by bath challenge with atypical Aeromonas salmonicida

Abstract. The present study was undertaken to identify female breeders, resistant or susceptible to disease, which might be used to obtain gynogenetically cloned carp lines differing in disease resistance. Experimentally induced erythrodermatitis was used as the disease model. Firstly, the effect of age on the resistance to bath challenge with atypical Aeromonas salmonicida was examined. These challenges indicated a shift from subacute to chronic infection with increasing age, as shown by a lower survival at 3 and 5 months (both 15%) compared to 10 months of age (60%). Then, to conserve and characterize breeder females, offspring of two females (nos 21 and 38), including F1, F2 and backcross (B1 and B2) progenies, were bath challenged at the age of 3 months. Comparison of the survival data showed a segregation into two groups of progenies: one resistant with nearly 100% survivors (W, F1 and B2), and one relatively susceptible group with 25% (R8 and F2) or 50% (B1) mortality. Analysis of inheritance indicated dominance of the resistant phenotype. Thus, the results identified two breeder females whose (gynogenetic) progeny might be expected to differ in resistance to bath challenge with atypical A. salmonicida.     

Immunological response of the Indian major carps to Aeromonas hydrophila vaccine

Abstract. Fingerlings of the three major types of Indian carp, i.e. Catla catla (Hamilton), Labeo rohita (Hamilton) and Cirrhunas mrigala (Hamilton), were immunized using a haemolysin-negative mutant of Aeromonas hydrophila . Very high titres of antibodies were induced in C. catla , followed by C. mrigala and L. rohita . Immunized fish showed good protection against homologous challenge. Moderate protection against heterologous challenge was observed in C. mrigala and L. rohita .     

Effect of three dietary oils on disease susceptibility in Arctic charr (Salvelinus alpinus L.) during cohabitant challenge with Aeromonas salmonicida ssp. salmonicida

Arctic charr ( Salvelinus alpinus L.) were fed diets based on a commercial recipe supplemented with either linseed, soybean or marine oil prior to cohabitant challenge with Aeromonas salmonicida ssp. s almonicida . Mortality varied significantly between the three dietary groups. Highest mortality (48%) was observed in fish fed the marine oil and the lowest mortality (20%) was in the group fed soybean oil. Transmission electron microscopy (TEM) examination of the digestive tract of uninfected fish demonstrated substantial numbers of bacterial cells between microvilli. However, only a few bacteria were recovered that were associated with the microvilli of infected fish. Immunocytochemical staining/labelling investigations using TEM and an immunogold method were performed on mid-gut segments of fish fed the marine oil diet and showed augmentation of goblet cells and the presence of A. salmonicida ssp. salmonicida in the gastrointestinal tract of diseased fish after challenge with the pathogen. It is suggested that the gastrointestinal tract could be an infection route of A. salmonicida ssp. salmonicida. The greater prevalence of goblet cells supports the suggestion that sloughing off mucus is a protective response against bacterial infections. These results make an important contribution to our understanding of how nutrition can affect the disease resistance of fish.     

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.     

Plasma ladderlectin concentration following sterile inflammation and Aeromonas salmonicida subsp. salmonicida infection

Plasma samples obtained from rainbow trout either experimentally infected with Aeromonas salmonicida or injected with either A. salmonicida lipopolysaccharide (LPS) or a commercial A. salmonicida vaccine (Lipogen) were analysed by enzyme immunoassay to evaluate changes in rainbow trout ladderlectin (RTLL) concentrations during the acute phase response (APR). Plasma RTLL concentrations in fish injected with A. salmonicida LPS, vaccine or live A. salmonicida varied over a 10 day period, but did not significantly increase. In contrast, fish experimentally infected with A. salmonicida exhibited a modest, but statistically significant ( P  <   0.05), decrease in RTLL concentration. These studies demonstrate that RTLL is not detectably induced during the trout APR to sterile inflammation or A. salmonicida infection, but plasma concentration of this protein may be reduced during bacterial infection.     

Optimization by orthogonal array design and humoral immunity of the bivalent vaccine against Aeromonas hydrophila and Vibrio fluvialis infection in crucian carp (Carassius auratus L.)

Aeromonas hydrophila and Vibrio fluvialis are the causative agents of a serious haemorrhagic septicaemia that affects a wide range of freshwater fish in China. In order to develop a bivalent anti‐A. hydrophila and anti‐V. fluvialis formalin‐killed vaccine to prevent this disease, an orthogonal array design (OAD) method was used to optimize the production conditions, using three factors, each having three levels. The effects of these factors and levels on the relative per cent survival for crucian carp were quantitatively evaluated by analysis of variance. The final optimized formulation was established. The data showed that inactivation temperature had a significant effect on the potency of vaccine, but formalin concentration did not. The bivalent vaccine could elicit a strong humoral response in crucian carp (Carassius auratus L.) against both A. hydrophila and V. fluvialis simultaneously, which peaked at 3 or 5 weeks respectively. Antibody titres remained high until week 12, the end of the experiment, after a single intraperitoneal injection. The verification experiment confirmed that an optimized preparation could provide protection for fish at least against A. hydrophila infection, and did perform better than the non‐optimized vaccine judged by the antibody levels and protection rate, suggesting that OAD is of value in the development of improved vaccine formulations.     

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.     

Experimental infection of turbot, Scophthalmus maximus (L.), by Moritella viscosa, vaccination effort and vaccine-induced side-effects

Moritella viscosa is the causative agent of winter ulcers in farmed salmonids and Atlantic cod in countries around the North Atlantic. The bacterium has also been isolated from various marine fish species. Bacterial diseases have been a limiting factor in farming of turbot, but M. viscosa has not so far been isolated. In this study, turbot was shown to be sensitive to M. viscosa infection in experimental challenges. Pathological changes in infected turbot were comparable with those previously described for winter ulcers in salmon. A multivalent commercial salmon vaccine, containing M. viscosa as one of five antigens and a mineral oil adjuvant, did not protect turbot against challenge 13 weeks post-vaccination. Weight gain of vaccinated turbot compared with controls was not reduced 7 weeks post-vaccination. Vaccination did not induce a specific anti-M. viscosa response, while elevated anti-M. viscosa antibody levels were detected both in vaccinated and unvaccinated fish 5 weeks post-challenge. The vaccine did, however, induce an antibody response against Aeromonas salmonicida, another vaccine component. Minor intra-abdominal adhesions were detected in vaccinated fish and fish injected with a mineral oil adjuvant. The measurement of various innate humoral immune parameters did not reveal significant differences between vaccinated and control groups.     

Modified extracellular product antigens of Aeromonas salmonicida as potential vaccines for the control of furunculosis in Atlantic salmon, Salmo salar L.

Abstract. Extracellular product (ECP) antigens of Aeromonas salmonicida were modified in an attempt were tested on an antigen-induced proliferation assay, for the ability to induce antibodies as measured by dot blot dot assay and as vaccines in vaccination/challenge trials. Modifications tested included particularization on to polystyrene beads, coating on to sheep red blood cells, mixing with BCG vaccine as adjuvant, and attachment to the T-independent carrier Fieoll. The only modification that resulted in increased protection levels was the particularization on to polystyrene beads.