Effects of Intranasal Immunization and Mucosal Adjuvants on the Immunogenicity of Pasteurella multocida in Rats.

Abstract

This study was carried out to study the effects of intranasal immunization, mucosal adjuvants and vaccine-adjuvant ratio on the immunogenicity of Pasteurell multocida in rats. Forty-eight rats were kept in a conventional animal house and allowed to adapt to the environment, food and water for one week. They were assigned to one of five groups (G1 to G5). G1, G2 and G3 were further divided into subgroups (a) and (b) each composing 6 rats. Groups 4 and 5 were control groups of 6 rats each. The rats were immunized intranasally with P. multocida broth bactrin mixed with mucosal adjuvants at day one and day 15. Mucosal adjuvants used were olive oil for group 1, sesame oil for group 2 and vitamin D3 for group 3. The vaccine adjuvant ratio for all a subgroups was 1:1 and 2:1 for all subgroups b. Rats in group 4 received plain P. multocida broth bacterin and rats in group 5 were sham-vaccined with phosphate-buffered saline (PBS). All rats were sacrificed on day 21 post initial immunization. Lung and trachea from each rat were carefully removed and the trachea and bronchial tree were washed thoroughly with 5 ml PBS. The tracheobronchial (TB) lavage was collected, centrifuged and the supernatant was kept at -20 ºC for the detection of P. multocida specific mucosal Ab using gel diffusion precipitation test (GDPT) and indirect haemagglutination (IHA) assay. P. multocida whole cell lysate (WCL) was used as antigen for GDPT and for coating formalized tanned RBCs. In GDPT, precipitation lines were obtained using TB lavage from all Pasteurella multocida intranasally immunized rats, and relatively sharp precipitation lines were obtained in TB lavage of rats of G1b, G2a and G3a, indicating the presence of P. multocida specific Ab in their TB lavage. The IHA was performed using two-fold dilution of TB lavage in v-shaped microtitre plates, starting with 1/200 dilution. Significant iv (P<0.05) titre were obtained in TB lavage of G1b, G2a and G3a. The IHA titre was expressed as the reciprocal of the highest dilution of TB lavage showing a diffuse matt of erythrocytes as compared to negative control showing a clearly delineated spot of erythrocyte within the vertex of the well. Rats of G1b that received P. multocida broth bactrin in olive oil in a ratio of 2:1 gave high Ab activity in TB lavage as was determined by IHA assay compared to ratio of group 4 ( Adjuvant control) that received plain broth bactrin vaccine. This difference is highly significant (P=0.0082). Rats in this group G1b showed also higher Ab activity in their TB lavage than rats in group 2b that received P. multocida broth bactrin in sesame oil in a ratio of 2:1 (P=0.9991) as well as to rats in G3b (P=0.9661). On the other hand, rats in group 2a that received P. multocida broth bactrin in sesame oil in a 1:1 ratio showed high Ab activity in their TB lavage as determined by IHA assay. The Ab titre of this group 2a was significantly higher (P=0.0227) than in rats of G2b, G1a and G3b. Similarly, rats in G3a that received P. multocida broth bactrin in vitamin D3 in the ratio of 1:1 had significant Ab titres in their TB lavage, in comparison to G4 with P values equal to 0.0272. In addition, sesame oil and vitamin D3 in 1:1 ratio with P. multocida broth bactrin had induced high Ab activity at the respiratory tract, whereas olive oil vaccine-adjuvant ratio of 2:1 induced Ab activity of the same magnitude. It seems that the booster dose was necessary to ensure a prolonged and strong local immune response for P. multocida broth bactrin. Since the effectiveness of this broth bactrin vaccine was expected to be much lower than live vaccine, mucosal adjuvants as potent delivery systems for antigens were incorporated to enhance the uptake of the vaccine-antigen and ameliorate the induction of local Ab response at the respiratory tract as measured by IHA and GDPT assay.