PhD

This study was undertaken to develop biofilm vaccines against duck pasteurellosis using oil, saponin and aluminium hydroxide as adjuvants and to experimentally evaluate their immunogenicity in ducks.
Identity of P. multocida serotype A: 1 (DP1) used for study was confirmed by biochemical tests and by PM-PCR and pathogenicity was established in Swiss albino mice before vaccine production.
The LD50 (11 w) of the DP1 isolate determined was 10 CFU / bird and MDT was 23.75 h in 11 week old ducklings when a high dose of 3 X 10 6 CFU of P. multocida per bird was given. At 11 Weeks age MDT gradually increased as the dose of inoculum decreased. In 21 week old ducks, the LD50 (21w) of the isolate DP1 was estimated to be 3 ×108 CFU of P. multocida and it showed that the Kuttanad duck had decreased susceptibility to pasteurellosis with age.
On light microscopic studies planktonic cells appeared to be Gram negative coccobacillary, while biofilm cells were Gram negative and pleomorphic. Electron microscopic studies revealed that P. multocida could form well differentiated classic biofilm and 0.32 per cent TSB media supplemented 0.5 per cent chitin seemed to be excellent medium for biofilm formation by P. multocida.
Four different vaccines viz. OV, OBV, SV and SAV were prepared and all of them were found to be sterile and safe. The oil adjuvanted vaccines (OV and OBV) offered better protection compared to saponin and aluminium hydroxide adjuvanted vaccine groups (SV and SAV) following primary vaccination, up to seven weeks. The modified oil adjuvanted vaccine prepared was not only found to be homogenous, but also more efficient in stimulating a humoral immune response and hence may be recommended.

The oil adjuvanation gave better protection than saponin and aluminium hydroxide adjuvanation. The SAV gave better protection than SV which might be due to the presence of aluminium hydroxide which potentiated the immunostimulating ability of saponin. The combined vaccine (SAV) although was found to be better than single vaccine (SV) they cannot be used as a substitute to oil adjuvanted vaccines. The booster vaccination was found to have added advantageous effect on protection and is a must, to prevent losses. Pasteurella biofilms although found to be weak in inducing a primary immune response had the potency to evoke a more powerful secondary response compared to planktonic cells. Vaccination done at six weeks age followed by booster vaccination at 16 weeks age seemed to be a better modification of existing schedule and may be recommended.
In histopathological studies, lymphoid hyperplasia was observed in spleen in survived control birds and in SV and SAV vaccine groups that did not survive challenge test, which indicated the persistence of Pasteurella organisms through mild infection in them following experimental challenge. Lymphoid depletion was observed in caecal tonsil in experimental pasteurellosis as in spleen. As the survived vaccinated birds following challenge test showed normal intact caecal tonsil, the course of disease and lesions might be less prominent in vaccinated birds during infection process. The well developed bursa observed in OV and OBV birds that survived challenge test indicated that the humoral immune response was well induced in them compared to other groups.
The designed primers E1 and E2 amplified the gene E and hence, this pair of primers could be used for the production of amplified Gene E sequences for further studies on recombinant ghost system.

In conclusion, 0.32 per cent TSB media supplemented 0.5 per cent chitin seemed to be an excellent medium that support classical biofilm formation by P. multocida. Booster vaccination definitely had added advantageous effect on protection. Immunization at 6 weeks of age with OV followed by booster vaccination at 16 weeks age with OBV seemed to be a better modification of existing schedule and may be recommended. In histopathological studies, the lesions were less prominent in vaccinated birds than control birds which indicated that the vaccines were effective.