An effective vaccine needs not only good antigens but also preferable adjuvant to enhance the immunogenicity of antigen. The adjuvant was used to enhance humoral and cellular immune responses, but adjuvant would also lead to side-effect to bodies, such as inflammation, tissue damage and pain [10
]. Oil emulsion vaccine could promote antibody titre and extend immunity period. However, mineral oil long term stand at the injection site, and caused inflammation and local tissue necrosis, lead commercial value of the birds lower.
In present experiment, mineral oil, 70M, and 206 adjuvant were used to evaluate adjuvant effects. 70M adjuvant and 206 adjuvant were oil in water (W/O) and water in oil (W/O/W) adjuvant based on purified mineral, respectively. The previous studies showed that the vaccine combined 70M adjuvant, Freund's complete adjuvant, or incomplete Freund's adjuvant were able to induce similar antibody responses, but 70M adjuvant vaccine caused obviously much less inflammatory response after inoculation [11
]. The avian influenza vaccine emulsified with 70M adjuvant prepared provided a good protection to immunized chicken [15
]. Vaccine conjugated 70M adjuvant induced not only humoral immune responses, but also strong cellular immune responses after immunizing mice [18
]. 70M and 206 adjuvant could provide the immune enhancing effects on Eimeria acervulina vaccine [19
In this experiment, the antibodies of SPF chicken induced by 70M adjuvant vaccine or mineral oil adjuvant vaccine were higher than that of protective threshold two weeks post-inoculation, but 206 adjuvant vaccine and antigen without adjuvant did not. The antibody levels elicited by 206 adjuvant group achieved protective threshold three weeks later and reached the highest level after four weeks then began to decline, but it was no longer able to provide theoretical protection 13 weeks after immunization, which is consist with porcine circle virus disease inactive vaccine [20
]. In part I and II experiments, the antibody titres of 70M adjuvant group were higher than that of the mineral oil adjuvant group, but the differences between two groups were not significant (P > 0.05) statistically, while the two groups showed significant differences (P < 0.01) compared to 206 adjuvant group in part I but not in part II experiments. In part II, 206 adjuvant group induced the similar immune responses to 70M adjuvant group and mineral oil adjuvant group of ducks.
We speculated that 206 adjuvant was a kind O/W type adjuvant, it released into the lymphoid tissue rapidly after immunization and leaded to inefficiency to chickens, while 70M adjuvant and mineral oil adjuvant were a type of W/O adjuvant and could be stored at the injection site and release slowly. Another reason maybe due to the chickens, ducks and pigs are different species, their immune systems possessed differences leading to the distinguishing immune responses elicited by 206 adjuvant vaccines. However, the real reason was unknown and to be further studied.
Since 70 M adjuvant induced better immune effects to the chickens and ducks, the French Seppic company optimized 70M adjuvant and prepared eight 70M Series of adjuvant, Montanide™ ISA 70 essai Mi (i = 1-8) adjuvants. Immunity and challenge experiments to SPF chickens showed that the antibody induced by 70M adjuvant group surpassed protective threshold two weeks after immunization, while the other groups were lower than that. Three weeks later, only 70M6 adjuvant group achieved that level. All the other vaccine groups (70 M1, 70M3-5, 70M7, and 70M8), the titres of antibody stay low from two weeks to thirteen weeks post-vaccination, and to 13 weeks each groups antibody titre was lower than the threshold while 70M2 and 70M6 adjuvant groups antibody levels remained at 7log2 above, 70M adjuvant group reached to 9log2 above. 70M2 and 70M6 adjuvant groups achieved to the highest levels of antibody at six weeks post-immunization, and the other groups were at eight weeks post-immunization. Challenge experiment results showed that 70M, 70M2, and 70M6 adjuvant groups could provide 100% protection. The results suggested that 70M adjuvant and 70M6 adjuvant groups had good effects in all groups, and 70M adjuvant group was the best one with statistically significant differences (P < 0.01) to all other groups.
There were no visible clinical and pathological signs at birds inoculation sites two weeks after immunization of the 70M adjuvant vaccine, 70Mi Series adjuvant vaccines, and 206 adjuvant vaccines, suggesting that all these Seppic adjuvant had smaller side reactions. 70M adjuvant could be best as a new type of adjuvant applied in the production of avian influenza vaccines.