Oil Adjuvant Vaccine Research Articles

Vaccination improves survival of Baltic salmon ( Salmo solar) smolts in delayed release sea ranching (net-pen period)

Baltic salmon ( Salmo salar) of the Finnish Iijoki stock were hatched and reared in freshwater in a salmon hatchery on the Danish island of Bornholm in the Baltic sea. Salmon parr were divided in three groups each comprising 22 000 fish. One group was vaccinated by intraperitoneal injection with a non-mineral oil-adjuvanted vaccine consisting of formalin killed Aeromonas salmonlcida, Yersinia ruckeri and Vibrio anguillarum (serotype 01 and 02). A second group was vaccinated by l h bathing in a corresponding vaccine without adjuvant. A third group was left as untreated control. Subsequently, presmolt groups were transferred to three identical net-pens located next to each other in the Baltic Sea (salinity 8 ppt), 500 m from the north-eastern coast of Bornholm, where they were studied for four months until tagging and release for restocking purposes. Mortality during this period in the ip vaccinated group was minimal (0.02%, RPS (relative per cent of survival) 99.80) and significantly lower compared to 10.13% mortality in the control group and 2.51% mortality (RPS 75.2) in the bath vaccinated group. Specific disease outbreaks were not observed during the four months. Growth was significantly enhanced in the injection vaccinated group compared to both the unvaccinated control and the bath vaccinated group. The humoral antibody response to the various bacteria was significantly elevated in the injection vaccinated group showing 4–5 fold titre increases three and four months after immunization. In contrast, no increase of titres was seen in the bath vaccinated and untreated groups. Marked cellular reactions in the abdominal cavity of injected fish were registered. A total of 3000 fish have been tagged and released to evaluate the effect of vaccination on the recapture rate. The implications of immunoprophylactic measures in the restocking programme with Baltic salmon are discussed.

Protection against haemorrhagic septicaemia induced by vaccination of buffalo calves with an improved oil adjuvant vaccine

Protection against haemorrhagic septicaemia induced by vaccination of buffalo calves with an improved oil adjuvant vaccine

Adjuvant activity of polar glycopeptidolipids fromMycobacterium chelonaein experimental vaccines againstAeromonas salmonicidain salmonid fish

In order to evaluate the adjuvant activity in fish of polar glycopeptidolipids fromMycobacterium chelonae(pGPL-Mc), salmonids were injected with pGPL-Mcalone or in combination withAeromonas salmonicidabacterin. Side-effects, antibody production againstA. salmonicidaand lymphocyte stimulation were studied. Twelve weeks after vaccination, the antibody responses of fish injected with 0·25mg kg−1pGPL-Mcin addition to bacterial antigen were significantly stronger than those in fish injected with a non-adjuvanted bacterin, and insignificantly lower than the antibody titres achieved with an oil-adjuvanted reference vaccine. Higher doses of pGPL-Mcreduced the levels of specific antibodies. The lymphocyte responses to lipopolysaccharide (LPS), a potent B-cell mitogen, were higher in rainbow trout injected with pGPL-Mcalone, compared to the fish injected with saline, suggesting B-cell activation as a mechanism. pGPL-Mcshowed a weak dose-dependent mitogen effect on fish leucocytes. No significant side-effects were observed after intraperitoneal injection of pGPL-Mc. The relatively long-term immunostimulation in fish with no side-effects makes pGPL-Mcan interesting candidate adjuvant for incorporation in future vaccine formulations.

Post-vaccination intra-abdominal adhesions as a marker to identify Atlantic salmon, Salmo salar L., escaped from commercial fish farms

Intraperitoneal vaccination of Atlantic salmon, Salmo salar L., against furunculosis using oil adjuvanted vaccines causes visible intra-abdominal markers of apparently permanent character. The feasibility of these side-effects for discrimination between fish from vaccinated and unvaccinated populations was evaluated in an open study using samples of vaccinated and unvaccinated farmed salmon, and in a blind study using individually tagged fish from a sea ranching vaccination trial. Vaccines were trivalent, oil adjuvanted bacterins extensively used in Norwegian aquaculture. Adhesions between internal organs and the gut wall were more sensitive and more specific for identifying fish from vaccinated populations than pigmentations on the peritoneum or on the viscera of the fish. Samples of farmed fish drawn from 3 to 35 months after vaccination yielded sensitivities of 93–100% and a specificity of 100% for this marker trait. In the blind study, intraperitoneal inoculation of saline as a placebo was found to induce minor adhesions, thus reducing the specificity of the adhesion marker to 75%. Using the observed sensitivities and specificities in a data simulation, the intra-abdominal adhesion marker proved feasible for the identification of fish from unvaccinated populations even under unfavourable sampling situations. Provided the current practices of vaccinating farmed salmon prevail, exclusion of individual fish carrying the intra-abdominal adhesion marker is recommended when estimating the number of wild salmon stocks, and when selecting broodfish for propagation of specific river populations of Atlantic salmon.

Evaluation of Immune Response and Protection in Animals Given Mink Parvovirus Vaccines

In orthogonal and pair design tests, mink, guinea-pigs and rabbits were inoculated with inactivated mink enteritis virus (MEV) vaccine prepared with mineral oil or Al(OH)3gel adjuvant. The animals were examined for serum haemagglutination-inhibition (HI) antibody during 6–55 days postvaccination (p.v.) and serum-neutralizing (SN) antibody for 11–31 days p.v. Differences between mineral oil and Al(OH)3gel adjuvant vaccines in the induction of HI and SN antibodies during 6–55 days p.v. were not significant. There was significant positive rank correlation between HI antibody level and SN antibody index from 11 to 31 days p.v. in serum of mink inoculated with the mineral oil or Al(OH)3gel adjuvant vaccine (P<0·05). The results of experimental challenge tests with vaccines in 29 mink showed a positive correlation between HI antibody titres, SNI and immune protective rates. Levels of HI antibodies in guinea-pigs, rabbits or mink during 11–40 days p.v. were reliable indices for monitoring the humoral immune response and immune protective rates. The criteria for virus vaccine potency and vaccine immune efficiency is an HA titre of 64 for the cell-cultured MEV and an HI antibody titre of 32 for guinea-pigs, rabbits or mink during 11–40 days p.v. The vaccine stimulated a protective response which lasts for a period of 12 months. The vaccine is stable for a period of 9 months at 4°C. Economic, simple and scientific means have been developed to monitor the quality of MEV vaccines, of which more than 70 000 doses of the mineral oil vaccine and more than 18 000 000 doses of the Al(OH)3gel vaccine have been used for the last 5 years.

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.

Pathology of Atlantic salmon Salmo salar intraperitoneally immunized with oil-adjuvanted vaccine. A case report

Gross and histopathological lesions in farmed Atlantic salmon Salmo salar L. immunized by intraperitoneal injections of oil-adjuvanted vaccines are reported. A few fish farms report serious side effects leading to poor feed uptake and conversion, retarded growth and downgrading at slaughtering due to severe adhesions between abdominal organs and body wall. The lesions consist of granulomatous tissue adhering to and embedding different visceral organs in such a way that normal functions may become severely affected. Histologically, the tissue is characterized by granulomas embedded in a fibrous granulation tissue with high numbers of eosinophilic granule cells. Possible explanations for the occurrence of severe lesions at some farms are discussed.

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.

Immunogenicity of outer membrane protein of Pasteurella multocida in buffalo calves

Outer membrane protein (OMP) from Pasteurella multocida serotype B:2 was extracted and characterized using SDS-PAGE. Ten major polypeptide bands of MW 88 to 25 kDa were observed. Immunoblotting suggested that the polypeptides of MW 44, 37 and 30 kDa were the major immunogens. Buffalo calves vaccinated with the OMP vaccine or a commercial haemorrhagic septicaemia oil adjuvant vaccine developed highest mean log 10 ELISA titres day 21 postvaccination (pv). Antibody titres detectable in these animals using an indirect haemagglutination assay were lower than the ELISA titres but the pattern of the antibody response was similar. A passive mouse protection assay revealed that the maximum protection against the challenge infection was conferred by the serum collected from both the groups on day 21 pv and 26 pv. Following challenge with virulent P. multocida cells, all the five OMP vaccinated animals survived whereas only 2 out of the 3 HS oil adjuvant vaccinated animals withstood the challenge. Results suggested that OMP was protective and could be used in vaccines against haemorrhagic septicaemia.

The performance of southern African territories serotypes of foot and mouth disease antigen in oil-adjuvanted vaccines.

The performance of selected oil adjuvants containing Southern African Territories (SAT) serotypes of foot and mouth disease virus was assayed by testing antibody levels elicited in cattle, sheep and goats, and by testing protection of cattle on challenge. Various oil adjuvant formulations were tested initially in cattle and guinea pigs, and compared with a standard alhydrogel and saponin-based (AS) vaccine. A commercial double oil emulsion vaccine elicited higher antibody titres and a more prolonged antibody response than the conventional AS vaccine, than a commercial single emulsion oil formulation or an incomplete Freund's adjuvant formulation. Incomplete Freund's adjuvant was selected for the formulation of a trivalent vaccine which was assayed in cattle, sheep and goats for antibody response and local reactions. The double oil emulsion elicited a high level of antibodies, which was maintained for at least six months after a single inoculation. The product was easily injectable and did not cause local or systemic reactions.

International bank for foot-and-mouth disease vaccine: assessment of Montanide ISA 25 and ISA 206, two commercially available oil adjuvants

The International Vaccine Bank at Pirbright has recently installed large-scale vaccine formulation equipment for the preparation of oil-adjuvanted vaccines. Such vaccines are claimed to offer a number of advantages over Al(OH) 3, particularly their ability to raise better immunity in pigs. This paper reports on the potency in pigs, cattle and guinea-pigs of foot-and-mouth disease vaccines prepared with two novel oil adjuvants, Montanide ISA 25 and 206 (Seppic, Paris). The results indicate that vaccines adjuvanted with these oils retain potency for a longer period than our conventional aqueous formulation, following storage at +4°C, and elicit good antibody responses in both pigs and cattle regardless of injection route. In addition they gave no evidence of toxicity or prolonged pyrexia following their administration. Local reactions at the site of inoculation were not observed in cattle vaccinated intramuscularly, even following a booster dose. Pigs vaccinated intramuscularly only showed local reactions if the volume administered exceeded the 2.0 ml dose or the animals received a second vaccination. These observations on the efficacy of such oil formulated vaccines suggest that they have potential as an alternative to the current aqueous formulation.

Experimental studies on the efficacy and side-effects of intraperitoneal vaccination of Atlantic salmon ( Salmo salarL.) against furunculosis

Efficacy and side-effects of anti-furunculosis vaccination were studied in an experimental trial. One unadjuvanted and eight adjuvanted bacterins were administered by intraperitoneal injection to Atlantic salmon ( Salmo salarL.) presmolts held in fresh water. Adjuvant systems represented were mineral oil, aluminium salts, glucan and levamisole. Protection, plasma antibodies, and side-effects were determined six weeks, three months, and six months after vaccination. After six weeks, four vaccination groups were significantly protected compared to unvaccinated fish. Only two groups, being vaccinated with a trivalent and a monovalent mineral oil adjuvanted vaccine, respectively, were protected after three and six months. Fish from these two groups were increasingly protected with time. These two groups also displayed significant intra-abdominal lesions. Plasma antibody levels measured against A-layer protein and sonicated cells of Aeromonas salmonicidadiffered greatly between groups, though all vaccinated groups mounted a significant response in at least one of the samplings. High antibody levels increasing with time were found in the two long-term protected groups. At all sampling times, a positive association between antibody levels and survival was found. In conclusion, only use of mineral oil adjuvanted vaccines induced durable protective immunity against virulent waterborne furunculosis challenge.

Effect of storage on the prevalent alum-precipitated hemorrhagic septicaemia vaccine in Pakistan and preparation of a more efficient oil adjuvant vaccine using dense culture of Pasteurella multocida Roberts type 1 on an improved culture medium.

Significantly drastic effects of storage on the potency of the alum-precipitated haemorrhagic septicaemia (APHS) vaccine are reported. The APHS vaccine, studied through challenge infection of vaccinated rabbits (post-60 days of vaccination), showed 100% potency when stored at 4 degrees C for 30 days. The potency dropped to 20% when storage period was extended to 60 or more days. At 30 degrees C, the potency reduced by 40, 40 and 60%, respectively, after 30, 60 and 90 days of storage, while, at 37 degrees C, the decrease was 60, 60 and 100% after 30, 60 and 90 days of storage, respectively. In view of this, the oil-adjuvant (OA) HS vaccine was developed by culturing Pasteurella multocida on a medium comprising yeast extract, sucrose, trypticase and sodium bicarbonate, under continuous aeration at 37 degrees C. This gave a far better bacterial count (maximum count 15 x 10(8)/ml) than the conventional APHS vaccine (maximum count 6 x 10(8)/ml). The OAHS vaccine-carrying water-in-oil emulsion remained stable at room temperature for 1 year. The log protection values of the two batches of the OAHS vaccine, studied in mice, were 5.2 and 5.3, as against 1.9 of the APHS vaccine.

Detection of Newcastle disease virus in poultry vaccines using the polymerase chain reaction and direct sequencing of amplified cDNA

In order to develop an in vitro method for the control of poultry vaccines for identity and the absence of extraneous agents, reverse transcription-polymerase chain reaction (RT-PCR) was applied for the detection of Newcastle disease virus (NDV). NDV vaccines were employed for the establishment of the method, using two primer pairs spanning the cleavage site of the F0 fusion protein coding sequence. Amplification of a specific cDNA segment was possible from live and inactivated, oil-adjuvanted NDV vaccines without prior treatment. The cDNA was characterized by restriction endonuclease digestion as well as by direct nucleotide sequencing. The RT-PCR was able to detect between 5 × 10 2 EID 50 (in live vaccine preparations) and 10 5 EID 50 or 0.056 haemagglutinating units of NDV (in inactivated vaccine preparations). In addition, live vaccine preparations were inactivated with β-propiolactone (β-PL). Amplified cDNA was obtained after treatment with 0.1% β-PL, whereas at a concentration of 1% or 10% no specific bands were visible in the agarose gel. These results demonstrate the applicability of the method for the control of poultry vaccines for identity and for the absence of extraneous agents, and additionally allow a rapid characterization of the respective NDV strain.

Relationship between active protection in vaccinated buffaloes against haemorrhagic septicaemia and passive mouse protection test or serum antibody titres

The relationship between the standard passive mouse protection test or serum antibody titres measured by indirect haemagglutination or enzyme-linked immunosorbent assays and active protection in buffaloes immunized with different types of haemorrhagic septicaemia bacterins was investigated. Groups of 2–3 buffaloes were immunized with the bacterius currently in use in Asia, viz., broth bacterin (BB), alum precipitated vaccine (APV) and oil adjuvant vaccine (OAV) either subcutaneously (BB, APV) or intramuscularly (OAV) and challenged subcutaneously with virulent organisms at different periods post-immunization. Although the passive mouse protection and indirect haemagglutination tests carried out with the pre-challenge sera from vaccinated buffaloes revealed no relationship with active protection in buffaloes, a relationship was observed between the ELISA antibody titres and protection. In contrast, a dose-response relationship was observed between the homologous active and passive mouse protection test.

Characterization of immune response and duration of protection in buffaloes immunized with haemorrhagic septicaemia vaccines

Two of the three buffaloes immunized with a non-adjuvanted broth bacterin were found to be protected against experimental challenge at 6 weeks but not at 3 months post-challenge. Similarly all buffaloes (4/4) immunized with alum-precipitated vaccine were protected at 6 months but only 1 of the 2 vaccinated animals were protected at 12 months post-immunization. On the other hand, buffaloes immunized with an oil adjuvant and a double emulsion vaccine were completely protected at 12 months post-immunization. Statistically significant differences between immunized versus non-immune animals became evident at 3 months post-immunization, although analysis of cumulative antibody titres of pre-challenge sera of vaccinated buffaloes surviving versus those succumbing to experimental challenge revealed significant by higher antibody titres in the former as compared to the latter group. These results suggested that there was a relationship between ELISA antibody titres and active protection in buffaloes. There also appeared to be a relationship between cutaneous delayed-type hypersensitivity and active protection in buffaloes. Preliminary analysis of the antibody isotype distribution in the pre-challenge sera of 2 buffaloes vaccinated with the oil adjuvant vaccine revealed predominance of IgG1 and IgG2 subclasses whose role in protection against haemorrhagic septicaemia was not eludicated.

A comparative study of the immune responses of sheep against foot-and-mouth disease virus types Asia-1 and O PEG-concentrated aluminium hydroxide gel and oil-adjuvanted vaccines

Foot-and-mouth disease (FMD) vaccines prepared against types Asia-1 and O PEG-concentrated and adjuvanted with aluminium hydroxide gel were compared with oil-adjuvanted types Asia-1 and O vaccines in sheep. The study was conducted by inoculating 0.5 ml of monovalent vaccine under laboratory conditions and 1 ml dose of bivalent vaccine in field conditions. The antibody responses were monitored by serum neutralization and ELISA tests. The results indicate that PEG-concentrated gel vaccine was of comparable immunogenicity to oil-adjuvanted vaccine over a study period of 8 weeks. No significant difference was observed in the antibody response with monovalent or bivalent vaccine. However, to substantiate the present findings and reach a final conclusion, further studies with both vaccines may have to be carried out over a longer period of time.

Large-scale use of liquid-phase blocking sandwich ELISA for the evaluation of protective immunity against aphthovirus in cattle vaccinated with oil-adjuvanted vaccines in Argentina

Specific serum activity levels against four reference strains of foot-and-mouth disease virus (FMDV) were evaluated from 1634 animals vaccinated with commercial quadrivalent oil vaccines and from 746 unvaccinated, naive animals, using the liquid-phase blocking sandwich ELISA (lpELISA) test. Cows from the FMDV-free area of Argentina were tested for the absence of specific FMDV antibodies (sp FMDV Abs) and those showing lpELISA titres <1.0 were grouped in lots of 16 animals. They were vaccinated and challenged at 90 days postvaccination (DPV) with one of four virus strains used for vaccine production and control (prototype strains). Serum samples from vaccinated and control cattle were collected 60 and 90 DPV and the level of sp FMDV Abs was determined by lpELISA. Animals were examined for clinical signs of disease. Results show that serum lpELISA titre levels directly correlate with the percentage of protected animals. It was seen that 100, 98, 93 and 87% of the vaccinated cattle with antibody titre levels ⩾2.1 were protected against challenge with serotypes C85, A87,01 Cas and A79, respectively. Evidence is also presented of seroconversion in a sample of 3–5-month-old calves vaccinated in the field, showing lpELISA titres compatible with protection against the four vaccine viruses as long as 150 DPV. Results reported in this paper strongly support the use of the lpELISA test for a rapid and reliable evaluation of the efficacy of FMDV commercial vaccines as well as for the assessment of the immunological status of cattle in FMDV-free and enzootic regions of South America.

Development and testing of a bovine trichomoniasis vaccine

Eighteen culled dairy cows were randomly allocated into 1 of 5 treatment groups. Six cows were vaccinated twice (2V), 21 days apart, 3 with whole cell (2WC) and 3 with fragmented cell membrane (2FC) containing 1 × 10 9 Trichomonas fetus organisms or protein equivalent in a commercial mineral oil adjuvant vaccine. Six more cows were vaccinated once (1V), 3 with whole cell (1WC) and 3 with fragmented cell vaccine (1FC), using the same vaccine, while 6 cows were used as the unvaccinated controls. All cows were challenged with 1 × 10 5 organisms 4 weeks after the second or the only vaccination. After challenge, cervico-vaginal mucus (CVM) samples were cultured for T . fetus weekly for 9 weeks. Whole cell vaccines were superior to fragmented cell vaccines, and both performed better than no vaccination for apparent elimination of trichomonad infections in dairy cows. In addition, 2V was superior to 1V, which, in turn, was superior to no vaccination. Furthermore, clearance time was reduced most by 2V and whole cell vaccination compared with 1V and fragmented cell vaccination. Clearance time was decreased significantly in all vaccinated cows compared with that in unvaccinated cows.