Experimental birds
Day-old Vanaraja chicks were obtained from Hatchery, ICAR-Directorate of Poultry Research and were housed in battery brooders in the farm. Birds were fed with basal diet and water
ad libitum. All experiments were carried out at Directorate of Poultry Research (ICAR-DPR), Hyderabad during 2017-18 with the approval of Institute Animal Ethics Committee (IAEC/DPR/18/5), by following all the guidelines of CPCSEA.
Bacterial culture and propagation
Pasteurella multocida A:1 isolated from fowl cholera outbreak and maintained at avian health lab, ICAR-Directorate of Poultry Research was used in the study. Brain heart infusion (BHI) agar (Himedia, India) was used for isolating single colonies and counting colony forming units (CFU). Purity of the culture was checked by colony morphology, Gram’s stains and by PCR using specific primers. Live
Pasteurella isolate was stored in BHI broth with 50% glycerol at -20°C for a period of not more than 4 months. For bulk propagation of
P.
multocida, BHI broth was used for culture and incubated for 24 hrs at 37°C. For iron- supplementation of BHI, stock solution of 1 M FeCl
3 was added drop- by- drop to media up to a final concentration of 100 µM
(Herath et al., 2010). The virulence of the culture was confirmed by inoculating in 12-week-old Vanaraja chicks. An inoculum of 0.5 ml of 1.9 ×10
5 CFU/ml was given by intraperitoneal route and mean death time was observed
(Reed and Muench, 1938).
Formalin-inactivated vaccine
Scaled up culture from seed culture of
P.
multocida isolate was prepared in BHI broth. Formalin inactivated vaccine was prepared as per the method described by
Jones et al., (1988). Briefly, formaldehyde was added to bacterial culture harvested from bulk broth culture from 1 liter of 18h culture, washed and resuspended in 20 ml of normal saline up to the final concentration of 0.5%. After incubating for 24h at 37°C, the inactivated culture was washed and resuspended in normal saline. This was adjuvanted with 0.5% aluminum potassium sulfate (Merck) for the preparation of formalin inactivated with aluminum potassium sulfate vaccine (FIA). The formalin inactivated antigen was combined with Freund’s complete and incomplete adjuvant (Sigma-Aldrich) and designated as formalin inactivated with Freund’s adjuvant vaccine (FIF).
Iron-inactivated vaccine
BHI broth was enriched with 100 µM FeCl
3 was used to propagate the isolate. The bacterial culture harvested both from basal BHI and iron-enriched media were used for the preparation of iron-inactivated vaccines and designated as Iron inactivated (II) and Iron enriched iron inactivated (IEI) respectively. Iron inactivation of the culture was done as per the method described previously
(Kumar and Singh, 2005; Herath et al., 2010). Briefly, 1 M FeCl
3 was added drop by drop to the bacterial culture with intermittent shaking up to the final concentration of 100 mM. After incubating at 37°C for 4 hrs, the residual FeCl
3 Was removed by washing the bacterial pellet twice with normal saline.
Sterility and safety
The prepared vaccines were checked for sterility by inoculating on BHI plates and incubating for 24hrs at 37°C. The absence of bacterial growth on the plates indicated the sterility of prepared vaccines. Safety was assessed by inoculating the experimental vaccines at 0.5 ml dose through subcutaneous route and observed for survivability and adverse reactions
(Nangia et al., 1966).
Immunization trial
A total of 120 two-weeks old Vanaraja chicks were randomly divided into six groups (n=20 each). Antigenic mass in each dose was adjusted 2.5 mg of wet weight of
P.
Multocida A:1 harvested from culture of 5 × 10
8 CFU/ml. At two weeks of age, the birds were immunized with formalin inactivated with freund’s adjuvant vaccine (FIF), Formalin inactivated with aluminum potassium sulfate vaccine (FIA), Iron inactivated vaccine (II), Iron enriched iron inactivated vaccine (IEI) and Commercial oil emulsion vaccine (CV). One group was kept as unimmunized control. All vaccines were given by subcutaneous route with 0.5ml per dose. First and second booster were given at 3 and 6 weeks after first immunization with their respective vaccines by same route. After vaccination, birds were monitored for any post vaccine reactions either local or systemic
viz., reactions at the site of injection, increase in body temperature
etc. Sera from each group (n=6 at each interval) were collected at weekly interval from first immunization till 9 weeks. The sera were kept at -20°C until further use.
Humoral antibody response
Specific humoral antibody response to
P.
multocida was measured in serum samples collected at weekly interval from all groups by indirect ELISA. The antibody titers were quantified with IDEXX commercial kit (# 99-09251 FC antibody test kit, IDEXX, Westbrook, USA) by following the manufacturer’s instructions. Briefly, the test was performed on 96-well ELISA plate precoated with
P.
multocida antigen, provided in the kit by manufacturer. Diluted (1:500) test sera were dispended (100 µl/well) in duplicates. Undiluted positive and negative controls (each 100 µl/well) provided along with the kit were also dispended on the coated wells. After incubating for 30 min at 25°C, the plates were washed with distilled water to remove any unbound material and followed by the addition of 100 µl conjugate. After 30 min at 25°C, the unbound conjugate was washed away and TMB substrate (100 µl) was added. The subsequent color developed was measured by spectrophotometer at 650nm and corresponding OD values were recorded. The respective antibody titers were calculated as given below:
Wherein S/P = [Mean OD of test sample-Mean OD of negative control] / [Mean OD of positive control- Mean OD of negative control]
The OD values were converted into titers using their software (xChekPlus, IDEXX). The titer greater than 396 is considered positive.
Protective efficacy
Six chicken from each group were challenged with 0.2 ml of 5 × 104 CFU /ml culture of virulent P. multocida A:1 isolate through intraperitoneal route at 3 weeks after second booster vaccine. The birds were observed at 6 hrs interval for morbidity. mortality pattern and gross lesions for up to 7 days post-challenge. Liver and spleen tissue from necropsied birds were collected and fixed in 10% neutral buffered formalin for histopathology. Sections were made and stained with hematoxylin and eosin (H & E).
Statistical analysis
The ELISA antibody titers were expressed as mean ± SEM. The titers of different groups were analyzed by one-way analysis of variance (ANOVA) to test for significance at overall effect followed by Duncan’s post-hoc test to identify significant difference among groups. P values < 0.05 were considered significant.
