MVSc

A research work was undertaken to prepare effective vaccines against P. multocida grown under different conditions and assess their immunopotency in one month old ducklings.
The purity of the Pasteurella multocida A: 1 strain (DP1) and A: 4 strain (PA4) was confirmed as per standard procedures. Pathogenicity of DP1 and PA4 was assessed in six to eight week old mice. The isolates killed the intraperitoneally inoculated mice within eight hours and within 24 h when injected by subcutaneous route.
Pasteurella multocida A: 1 was used for the preparation of different vaccines. The organism was grown in BHIB for preparation of ordinary bacterin. The in vitro biofilm formation of the organism was assessed by growing it under nutrient restricted conditions. For this, the organism was grown in TSB (0.32 per cent) supplemented with 0.3 per cent bentonite clay. For preparation of OMP suspension, the organism was grown in iron restricted condition viz., BHIB supplemented with 100 µM 2, 2’ Dipyridyl and the OMPs were extracted using sodium lauryl sarcosinate. The protein concentration of OMP suspension was estimated to be 3 mg/ml.
Median lethal dose (LD50) of DP1 was 10-7.5, which contained 32 viable cells/ ml and that of PA4 was 10-7.38, which contained 24 viable cells/ ml when determined in one month old ducklings.
Oil adjuvant vaccines were prepared using ordinary bacterin, bacterin made from biofilm and OMP suspension and performed the sterility, safety and potency tests of the vaccine employing standard procedures.
A total of 260 four week old ducklings were divided into four groups with 65 birds in each group and the first three groups were vaccinated with ordinary bacterin, OMP vaccine and biofilm vaccine respectively. The fourth group served as control. The birds were vaccinated with 0.5 millilitre of vaccine intramuscularly in the thigh region. Blood was collected from all the ducks pre-vaccination, at weekly intervals upto 3 weeks post vaccination (PV) and then at 15 days interval upto 60 days, by cardiac puncture or by jugular venipuncture. Passive haemagglutination using GA-SRBC sensitized with sonicated antigen of DP1 was used to measure the humoral immune response. The IHA titres obtained for biofilm vaccine group on day 14 was very much higher than the other two groups. The antibody titre was observed from day seven onwards for all the groups. All the vaccine groups have shown significant difference from the control group at all the stages of the study.
On homologous challenging, biofilm vaccine gave higher protection rates of 80 per cent than the 70 and 50 per cent protection rates of ordinary bacterin and OMP vaccine respectively, when challenged with 100 LD50 dose on day 20 PV. On day 60 PV, biofilm vaccine gave higher protection rate of 70 per cent than the 60 and 50 per cent protection rates respectively of ordinary bacterin and OMP vaccine, when challenged with 100 LD50 dose.
On heterologous challenging, biofilm vaccine gave higher protection rates of 70 per cent, while only 50 per cent protection was afforded by both bacterin and OMP vaccine, when challenged with 100 LD50 dose on 20 day PV. On day 60 PV, biofilm vaccine gave higher protection rate of 60 per cent while both the other vaccines gave only 50 per cent protection, when challenged with 100 LD50 dose.
All the birds challenged on day 40 PV, either with homologous and heterologous organisms died. In most cases, the death occurred due to coliform infection along with stressful factor such as increased atmospheric humidity due to heavy rainfall at that time. In few cases, birds died due to pasteurellosis which might be due to lack of protective level of antibody titre.
Biofilm vaccine proved to be the best among the three vaccines tried. Further field trials are to be done before advocating this vaccine for commercial use.