Clinical Investigation Of Streptococcus Suis, Haemophilus Parasuis And Construction And Application Of Trivalent Genetic Engineering Vaccine

Streptococcus suis （SS）serotype2is an important zoonotic pathogen, bringing not only a huge loss to China’s pig industry, but also high risks to human public health. Haemophilus parasuis disease （Glasser’s disease） is also a common disease in pig farms which is characterized by high mortality, seriously restricting the development of the global swine industry.In this study, the clinical investigation results revealed that SS and Haemophilus parasuis （HPS） were the two major bacterial pathogens in the swine farms, which brought great losses and harm to the swine industry. So it is important to prevent these two pathogens. At present, the inactivated vaccine is more often used to prevent the bacterial pathogens in the clinical practice, but it has the disadvantage that a large dose and more times are needed, which may increase the stress reaction of pigs. What’s more, multiple pathogens mean that multiple immunizations are needed, which can increase the cost of manpower and resources. Hence, it is of great significance to develop a cheaper and more efficient new vaccine, which will prevent more pathogenic organisms at the same time.In this study, we used the pseudorabies gene deletion strain TK-/gE-/LacZ+as a vector and selected the novel antigen gene-Sao, HP0197, HPS₀6257and palA, which were screened for good protection for the SS and HPS by our laboratory earlier. Then we constructed recombinant pseudorabies viruses carrying the antigenic gene of SS and HPS with the method of homologous recombination. Next, we made an evaluation of immune effects on the mice, which could provide a new idea for a better prevention against the two pathogens. Finally, lyophilized vaccines were produced and the plan of clinical application was carried out on pig farms.1.Clinical investigation of Streptococcus suis and Haemophilus parasuis. In this study, isolation of Streptococcus suis and Haemophilus parasuis in the large-scale pig farms in South China was investigated. The result show that the separation rates of SS and HPS were25%and14.9%in2012, indicating that these two pathogens were the major pathogens in pig farms; Clinical cases also showed that both the pathogens were harmful for the different day-old pigs and brougt great losses to the pig farms. So the prevention of these two pathogens is quite important.2.Construction and identification of recombinant pseudorabies virus strains expressing the protective antigen of Streptococcus suis and Haemophilus parasuis.According to the reported Sao, HP0197gene sequences of streptococcus suis type2, HPS₀6257, pal A gene sequences of Haemophilus parasuis type5and the multi-clone sites of the Vector pIECMV’, primers were designed to amplify these genes by PCR. The genome of swine streptococcus suis type2SC19strain and Haemophilus parasuis SH0165strain were used as the template respectively. Next these genes were cloned into the vector and then the transfering plasmids pIE/06257/0197and pIE/palA/Sao were successfully constructed.Subsequently, the PRV TK-/gE-/LacZ+genome and transfering plasmids were co-transfected into PK-15cells with Lipofectamine Reagent2000. Then two recombinant viruses, PRV/06257/0197and PRV/palA/Sao were selected by several plaque purifications. Expression of these antigen genes were confirmed by Western blotting and indirect immunofluorescence assay （IFA）. Meanwhile, we studied the biological characteristics of the recombinant virus, such as the proliferation characteristics and the genetic stability of the exogenous genes. The results showed that there was no significant difference in the ability of proliferation in PK-15cells between the recombinant viruses and the parental strain. Additionally, the exogenous genes were able to be identified even after eighteen passages on the cells. In summary, all of these results indicated that the recombinant pseudorabies vaccine virus strains were successfully constructed which could express the protective antigens and had both favorable proliferation characteristics and genetic stability.3. Immunological evaluation of the recombinant vaccine strains on Balb/c mice.In this step, immune effects of the recombinant strains on Balb/c mice were explored. In the experiment, the mice were injected with each recombinant virus of106TCID50respectively and boosting immunization was performed with the same dose28days later. These mice were bled every two weeks after the initial immunization and the serum was collected for the determination of neutralizing antibody against PRV and ELISA antibodies against each antigen protein. Finally, the mice were challenged with the lethal dose of swine Streptococcus type2and Haemophilus parasuis type5virulent strains （SC19and SH0165） to test the protective capacity of the recombinant vaccine strains. The results showed that:1. Recombinant virus strains induced a high titer of neutralizing antibodies against PRV and ELISA antibodies against each protein;2. Recombinant virus strains enabled the mice to resist the Streptococcus suis and Haemophilus parasuis virulent strain.The conclusion could be made from the mice experiments that the recombinant vaccine strains had a considerable immunogenicity and provided the foundation for the swine experiments, as well as a theoretical basis for the further development of the new vaccine research on Streptococcus suis and Haemophilus parasuis.4.Clinical application of recombinant virus vaccineIn the present study, the effect of the vaccine was evaluated. The vaccine safety experiment was carried out on piglets. The results showed that the recombinant virus vaccine strain was quite safe even when pigs were excessively immunized. Additionally, the immunization of this recombinant vaccine had no side synergetic effect on the immunization of attenuated classical swine fever virus vaccine, and both the daily feed intake and the body temperature of the immunized piglets remained at the normal level.