Furunculosis Vaccines Research Articles

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.

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.

Vibrioantigens of polyvalent vaccines enhance the humoral immune response to Aeromonas salmonicidaantigens in Atlantic salmon ( Salmo salarL.)

Seven experimental oil-adjuvanted vaccines containing Aeromonas salmonicidaand one or more of the following components: Vibrio salmonicida, Vibrio anguillarumserovar O1 and V. anguillarumserovar O2, were compared to a monovalent A. salmonicidavaccine regarding their ability to induce antibody production against A. salmonicidain Atlantic salmon. Enzyme-linked immunosorbent assay (ELISA) tests were used to measure the humoral immune response against various antigen preparations from A. salmonicida; bacterial cell-sonicate, A-layer protein and lipopolysaccharide. The effects of the different Vibriospp. on the generation of specific antibodies were estimated through a factorial statistical design. The results showed that stronger humoral immune responses to A. salmonicidaantigens were induced in fish vaccinated with the various polyvalent vaccines, compared to fish vaccinated with the monovalent furunculosis vaccine. V. salmonicidatended to be the most important component for this enhancement, although V. anguillarumalso contributed. Immunological cross-reactions between A. salmonicidaand V. salmonicidais a possible mechanism.

A field study on intraperitoneal vaccination of Atlantic salmon ( Salmo salarL.) against furunculosis

Protection and side-effects after intraperitoneal (i.p.) vaccination of Atlantic salmon ( Salmo salarL.) against furunculosis were studied in a field trial. Pre-smolts held in fresh water and salmon growers held in sea water were immunised with different monovalent furunculosis vaccines. In both cohorts, disease attack rates and cumulative mortalities after field challenge were significantly lower in populations given a mineral oil adjuvanted vaccine compared to other vaccination groups included in the study, demonstrating that tentatively pathogen-free as well as furunculosis carrier populations can be protected by vaccination. Injection-site lesions of significance for the commercial quality of harvested salmon were observed, being more severe in fish given the mineral oil adjuvanted vaccine. In spite of the side-effects, i.p. vaccination with mineral oil adjuvanted vaccine is strongly recommended for effective furunculosis prophylaxis in commercial farming of Atlantic salmon.

Antibiotic protection against recrudescence of latent Aeromonas salmonicida during furunculosis vaccination

AbstractProblems 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.

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

AbstractThe 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.

Detection of Aeromonas salmonicidaby polymerase chain reaction in Atlantic salmon vaccinated against furunculosis

Furunculosis vaccine DNA was demonstrated by polymerase chain reaction (PCR) in head kidney and spleen cell preparations of Atlantic salmon. DNA primers transcribed from sequences of 16SrRNA were used, and the PCR products were verified by an internal, radioactively labelled 16SrDNA oligonucleotide probe. Intraperitoneally, single and double vaccinated pre-smolts were sampled 8 and 16 weeks after vaccination and found to contain vaccine DNA. Unvaccinated fish acted as negative controls. The spleen cell preparation of the double vaccinated fish were negative after 16 weeks.

Capsulated cells ofAeromonas salmonicidagrown in vitro have different functional properties than capsulated cells grown in vivo

When grown in vivo in the peritoneal cavity of rainbow trout, Aeromonas salmonicida produces a clearly defined capsule with virulence-related functions. Aeromonas salmonicida grown in vitro in a glucose-rich medium (GRM) has also been reported to reproduce capsular material. Because in vitro mimicry of in vivo induced traits is highly desirable in vaccine design, the extent to which growth in GRM mimicked in vivo growth was examined. Antibodies specific to in vivo grown cells partially labeled the surface of GRM-grown cells, as well as two distinct proteins (81,700 and 41,000 Mr) in immunoblots of mutants with S-layer or lipopolysaccharide defects. GRM-grown strains showed an increased sensitivity to trout serum in contradistinction to the complete serum resistance of in vivo grown cells; as well, GRM-grown cells were more adherent to trout macrophages. Thus in spite of possessing some surface antigens normally expressed in vivo, cells grown on solid GRM did not possess all functional properties of in vivo grown cells.

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.

Vaccination in European salmonid aquaculture: A review of practices and prospects

Disease control by vaccination is widely used in European salmonid aquaculture against vibriosis (Vibrio anguillarum), cold-water vibriosis (Vibrio salmonicida), yersiniosis or enteric redmouth disease (Yersinia ruckeri) and furunculosis (Aeromonas salmonicida subsp. salmonicida). The vaccines against the Vibrio spp. and Y. ruckeri have proven effective especially when administered by injection. Furunculosis vaccines have been less successful and have relied on combination with potent adjuvants to achieve acceptable protection. Application of modern molecular techniques to furunculosis research has delivered a crop of experimental vaccines that incorporate purified virulence factors and have shown increased protection during challenge. Gene technology has also been used to create a defined, nonreverting mutation in a strain of A. salmonicida, which has enhanced the feasibility of attenuated live vaccines. The development of experimental subunit vaccines against the viral infections and the continued advances in the field of immunostimulants, adjuvants and antigen carriers provide considerable promise for the future development of commercial vaccines for use in salmonid aquaculture.

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.

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.

Immune response and protective immunity after vaccination of Atlantic salmon ( Salmo salar L.) against furunculosis

The development of protective immunity in Atlantic salmon ( Salmo salar L.) following intraperitoneal immunisation with an adjuvanted furunculosis vaccine was studied. The level of protection 6 weeks after vaccination was evaluated using cohabitation challenge. Serum antibody levels were measured weekly using an enzyme-linked immunosorbent assay (ELISA) against whole cells of Aeromonas salmonicida subspecies salmonicida , and against lipopoly-saccharide (LPS) and A-layer isolated from A. salmonicida . Six weeks after vaccination head kidney leucocytes were isolated and stimulated with the two mitogens, LPS from Echerichia coli and phytohaemagglutinin (PHA), and also with whole cells of A. salmonicida . Vaccinated fish were found to produce antibodies against all antigens tested, though some individual variation was seen. A significant increase in antibody level to whole cells of A. salmonicida was observed 3–8 weeks after vaccination. The antibody response to A-layer was not significant until 7 weeks after vaccination. Leucocytes from vaccinated fish were found to respond significantly stronger to whole A. salmonicida than did unvaccinated controls, while the responses to mitogens did not differ significantly. Vaccinated fish also showed a higher survival than unvaccinated, the relative percent survival (RPS) 21 days after challenge being 44%.

Bacterial inhibitors affecting proteases produced byAeromonas salmonicida, and the occurrence of such inhibitors in furunculosis vaccines

Abstract.Cultures ofAeromonas salmonicida, subspeciessalmonicidaandachromogenes, produced considerable quantities of inhibitors that affected extracellular bacterial proteases (endopeplidases), as well as some animal trypsins (from pig, salmon and trout) included in this study. Electrophoretic separation of these inhibitors revealed a complex of three to four single factors which were similar for the two subspecies. One of the factors only inhibited protease from the homologus subspecies, while another only affected enzyme from the other subspecies (cross‐wise inhibition). Both subspecies produced a factor which inhibited animal trypsins only. Subspeciessalmonicidaalso possessed a factor which inhibited most of the examined proteases unspecifically. In young cultures (2–3 days at 15°C), the inhibitors were demonstrable both in the disintegrated cell material and in cell‐free culture filtrate. The activity of the factor which only inhibited the protease from subspeciessalmonicidacould be increased considerably by moderate heating of the material. The effect of inhibitors produced by other relevant Gram‐negative bacteria on the proteases of theA. salmonicidasubspecies included was more limited. Considerable quantities of inhibitors against proteases of the subspeciessalmonicidaandachromogeneswere demonstrated in cell‐free filtrates of two commercially available vaccines against furunculosis in fish.

Surface-disorganized, attenuated mutants of Aeromonas salmonicida as furunculosis live vaccines

A slow-growing, aminoglycoside-resistant mutant and a rapidly-growing psuedorevertant were isolated from Aeromonas salmonicida, the causative agent of salmonid furunculosis. These mutants continued to elicit a variety of classical virulence factors associated with A. salmonicida pathogenesis. They differed morphologically from the wild-type and from one another with respect to A-layer organization, membrane antagonist sensitivity and particularly to aerobic metabolism. Both mutants were drastically altered in the architecture of the 2D crystalline surface array (A-layer), although both were similar to wild-type with respect to cell surface composition. The slow-growing, antibiotic-resistant mutant differed significantly from the wildtype by the apparent loss of virtually all aerobic metabolism; the pseudorevertant had partially recovered the ability to aerobically metabolize certain carbon sources. Both mutants were avirulent and incapable of tissue persistence. The rapidly-growing, antibiotic-sensitive pseudorevertant, when administered either intraperitoneally or by immersion, effectively protected salmonid fish from challenge by a heterologous virulent strain suggesting its candidature as a live, attenuated furunculosis vaccine.

Failure of anti-furunculosis vaccination of rainbow trout (Salmo gairdneri Richardson). Using extra-cellular products of Aeromonas salmonicida as an immunogen.

Furunculosis vaccines were prepared from virulent strains of Aeromonas salmonicida: 2 were formalin killed bacterins, and 3 were “cell surface extracts” prepared by stirring the bacteria in lithium acetate-EDTA buffer at different pH in presence of glass beads. Rainbow trout were vaccinated by intra-peritoneal injection and challenged with virulent strains injected intra-muscularly or added to water. Although the challenges were conducted at the wanted levels and provided good reliability, no protection could be demonstrated in any of the tested groups. Techniques and methods of work cannot explain these failures and it is concluded that rainbow trout were not protected. Discrepances of these results with other recent reports are outlined, and some comments are done about the meaning and bearing of furunculosis vaccination studies.