2.1. Experimental layout The study included vaccination of Salmonella bivalent vaccine followed by measurement of antibody titre by the passive haemagglutination (PHA) test, determination of lethal dose fifty (LD50) of the bacteria and performance of challenge test in the vaccinated and control birds.
2.2. Passive haemagglutination (PHA) test The test was used to determine the antibody titres in birds against Salmonella spp. following of vaccination as described by Carter (1979), Tripathy et al., (1970), Chowdhury et al., (1985), Sarker et al. (1976) and Siddque (1997) with slight modification.
2.3. Principle of the test The sensitivity and specificity of PHA test depends on the use of soluble antigens. In this case, somatic antigens of Salmonella spp. were coupled with chemically modified horse erythrocytes so that antigen-coated erythrocytes readily react with specific antibodies and results in haemagglutination.
2.4. Collection and preparation of 2.5% horse red blood cells (HRBC) Blood was collected from the right jugular vein of a normal adult horse with sterile syringe and needle containing 5 ml of Alsever's solution per 10 ml of blood. The blood was centrifuged in graduated centrifuge tube at 1500 rpm for 10 minutes. The supernatant was then pipetted off and the blood cells were resuspended with PBS and then centrifuged. The process was repeated for at least three times for washing the blood cells. During last washing the cells were maintained in PBS for 15 minutes and then centrifuged at 2000 rpm for 10 minutes to obtain the packed cells. The sediment blood cells were diluted with PBS to make 2.5% suspension of the blood cells and preserved at 4 to 80C.
2.5. Tannic acid solution (1:200) Tannic acid solution (1:200) was prepared by dissolving 1 gm of tannic acid powder in 200 ml of distilled water in a conical flask and the solution was sterilized by autoclaving at 121oC maintaining a pressure of 15 lb pressure per sq. inch for 15 minutes (1 kg/Cm2 ) and kept at 4°C to 8°C until used. Finally, tannic solution was prepared by mixing one ml (1:20,000) of the stock solution (1:200) with 99 ml of PBS taken in a conical flask and mixed thoroughly (Siddique, 1997)
2.6. Inactivation and preparation of 1% normal rabbit serum diluents (NRSD) Collected rabbit serum was inactivated at 56°C in hot water bath for 30 minutes and then one ml of the inactivated serum was added with 99 ml of PBS (pH 7.2) in a conical flask to obtain 1% solution. The serum solution then kept at 4 to 8oC.
2.7. Tannic acid treatment of horse red blood cells Five milliliters of 2.5% HRBC and 5 ml of 1: 20,000 dilution of tannic acid was taken in a test tube and mixed thoroughly. The mixture was then incubated at 37°C for 10 to 15 minutes in water bath according to the methods of Tripathy et al., (1970). The cells were centrifuged at 2000 rpm for 10 minutes; the sediment was then washed with PBS. Washed tanned HRBC was again diluted to make 2.5% suspension with PBS and used for the test.
2.8. Preparation of somatic antigen The isolates of S. gallinarum and S. pullorum organism were cultured on SS agar media. Incubated overnight at 37°C and selected a smooth colony and carried out slide agglutination test to ensure that the required somatic antigen is present. A pure culture on nutrient agar slope after incubation for 8-12 hours at 37oC was washed off the plate by using Pasteur pipette with 2 ml of absolute alcohol. It was then transferred into a sterile container. The antigen was left for 4-6 hours at room temperature to enable the alcohol to kill the bacteria. The container was spined for 5 minutes at 1000 rpm. The liquid was poured off and added enough phenol saline to make the antigen up to opacity. Standard titration was carried out with known serum to ensure that the antigen is positive for the required factor and stored at 4°C.
2.9. Sensitization of somatic antigen with tannic acid treated horse red blood cell Three ml of sensitized HRBC (2.5%), 1 ml of somatic antigen (1:10 dilution) and 8 m1 PBS were mixed together. This mixture was incubated at 37°C for 20 to 30 minutes. After sensitization, the cells were centrifuged at 1500 rpm for 10 minutes, then the supernatant fluid was discarded and the sedimented HRBC was collected and diluted with 1% normal rabbit serum diluents (NRSD) at the ratio of 1: 4. This was then mixed thoroughly and kept at room temperature for an hour and centrifuged for 10 minutes. The cells were resuspended in 1% NRSD to make 0.5% sensitized cells for use in microtitre plate method and stored at 4°C until used (Tripathy et al., 1970).