| In recent years,the scale of pig production in Guangdong has been expanding and developing rapidly.However,the three prevalent pathogens of Haemophilus parasuis（HPS）,Streptococcus suis type 2（SS2）and Pasteurella multocida（Pm）still cause great damage to the pig industry and restrict the healthy development of the pig industry.Therefore,it is particularly important to study the epidemic situation of HPS,SS2 and Pm,establish a rapid and simple detection method,and develop a new inactivated vaccine of HPS,SS2 and Pm with high cross-immune protection.In view of the problems of long time consuming and complicated steps in traditional identification methods,by comparing the synthetic gene clusters of three bacterial capsular polysaccharide antigen regions,three pairs of primers were designed according to conserved interval of HPS’s tbp B,SS2’s CPS2J and Pm’s KMT1 to establish a PCR amplification.HPS,SS2 and Pm were able to detect the lowest DNA concentration of 2.0pg/μL,2.6 pg/μL and 4.2 pg/μL,respectively,with high sensitivity and specificity,and did not react with other bacteria.Based on the successful establishment of PCR,the triple PCR method of 25μL system was established by diluting three pairs of primers of HPS,SS2 and Pm with different concentration,screening different primers and annealing temperature.The triple PCR identification are not specific for Actinobacillus pleuropneumoniae,Escherichia coli,Mycoplasma hyopneumoniae,Staphylococcus aureus and Bordetella bronchiseptica.In the sensitivity test,the lowest content of DNA concentration detected by triple PCR was 20pg/μL,and the sensitivity was high.In the repetitive experiment,the target bands of three bacterium could be detected,the success rate was 100%,which proved that the repeatability was good.According to the epidemiological survey of three kinds of bacteria,serotypes with strong virulence and good immunogenicity were selected as candidate strains for vaccines in Guangdong Province.Among them,HPS selected serotypes 4;SS chose SS2;Pm chose A groups that cause Porcine pasteurellosis.The basic research on HPS4,SS2 and Pm A shows that HPS4 is cultured in a constant temperature oscillator at 37℃for 12 hours with200 r/min oscillation,and the number of viable bacteria reaches the maximum value.At this time,OD600 is about 0.46,and the number of viable bacteria is about 1.32×109 CFU/m L;while the culture time of SS2 is 10 hours,the corresponding OD600 is about 0.33,the number of viable bacteria is about 5.0×108 CFU/m L;the culture time of Pm A is 8 hours,and the corresponding OD600 is about 0.39,the number of viable bacteria is 2.8×109CFU/m L.The OD600 and the number of live bacteria at the corresponding time points of the three bacteria were used as the time basis for the cultivation of vaccine strains.Balb/c mice were selected as animal models to carry out toxicity and stability experiments.The virulent strains HPS4-HZ,SS2-SH and Pm A-YJ were screened out,and the LD50 of HPS4-HZ was2.03×109 CFU/m L;that of SS2-SH was 8.9×108 CFU/m L;and that of Pm A-YJ was 3.98CFU/m L.The cultured HPS4-HZ,SS2-SH and Pm-YJ bacterial solutions were centrifuged for10 minutes at 5000 r/min,precipitated with 0.85%normal saline,inactivated by 0.2%formaldehyde solution for 24 hours,and finally mixed with sterilized aluminium hydroxide gel adjuvant to prepare a inactivated vaccine.Balb/c mice were subcutaneously injected with the inactivated vaccine according to the immunization procedure.The mice did not die,they were in good mental state,and there was no inflammatory reaction in the immune site,which proved that the vaccine had good safety.The immune protective effect of inactivated vaccine was tested in mice.The data showed that the protective rate of HPS4 inactivated vaccine was 90%,that of SS2 inactivated vaccine was 100%,and that of Pm A inactivated vaccine was 90%.The protective rate of inactivated vaccine for HPS4 and SS2 and Pm A reached 90%and 80%,which proved that the inactivated vaccine had good immunity and could provide high immune protection for mice. |
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