Epidemiological Investigation Of Piglet Diarrhea Virus Disease And Study Of Transmissible Gastroenteritis Virus Lactobacillus Edible Vaccine

Viral diarrhea piglets, mainly caused by porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV) and porcine rotavirus(PoRV) single or mixed infection, produce the greatest harm to piglets with the highest fatality. Since the winter of2010, some farms’(households’) piglets had appeared the main symptom of diarrhea in some districts of Shandong PoRVince, which caused enormouse economic losses to them. TGEV is the main cause of piglet diarrhea, which can cause the mortality up to 100 for piglet under the age of 10 days. The disease shows the incidence of intestinal partial that mainly produces mucosal immune, and edible vaccine can stimulate mucoantibody. TGEV recombination eukatyotic expression plasmid(pRc/CMV2-SAD-Rep.8014) had been constructed. The recombinant plasmid was transfected into L.acidophilus SW1 by electrical transformation to immune experimental animals. In order to investigate epidemiologie situation of PEDV,TGEV and PoRV as well as study the immune function of edible vaccine, the following researches were done in this experiment.1. Epidemiological investigation of piglet diarrhea virusIn this study,a total of 226 clinical samples were collected from Jinan, Weifang and Jining et al ten cities in Shandong PoRVince and subjected to detection ofPEDV, TGEVand PoRVby RT-PCR. The results showed that positive rates of PEDV, TGEV and PoRV were34.96%, 2.21% and 28.76%, respectively, and the infection rate of PoRV was significantly higher than that of average rate in other PoRVinces. The prevalence of PEDV and PoRV were different with seasons, which indicated that PEDV and PoRV were the major pathogens for the piglet diarrhea. Compared with free-range reared pigs, the large-scale pig farms had low positive rate of the three viruses. Meanwhile, after with the vaccination practices, the piglet infected rates of the three viruses were lower than that in non-immunized piglet, which indicated that reasonable feeding management and vaccination practices have added benefit of the prevention of the piglet diarrhea.2. The prokaryotic expression for the SA gene of TGEV and the establishment ofindirect ELISA methodThe laboratory saved plasmid pUC-S was used as a template to amplify the sequence of SAgene by PCR through designing specific primers. The recombinant plasmid was built by connecting the SA gene with pET-30 a, and imported into the E.coli BL21. The SA protein was expressed efficiently through optimization of various reaction conditions(the concentration of IPTG, temperature, speed and time). Then the high purity protein was obtained after passing the Ni-Agarose column. Tested by Western blot, it was showed that the recombinant protein had a good specific reaction with the positive serum of TGEV, while not had a reaction with positive serum of other diseases. Therefore, this protein had a good reactogenicity. An indirect ELISA was established using the protein above as coating antigen. By checkerboard method,the optimal solution of antibody was confirmed 1:100 and the optimal concentration of coating antigen was 0.75ug/ml.3. Study of transmissible gastroenteritis virus lactobacillus edible vaccineBALB/c mouses were vaccinated by L.acidophilusTGEV SAD with lactobacillus and commodity vaccine as controls. Blood samples were taken and collected, and sera were isolated to detect the specific IgG antibody, IL-4 and IFN-γ before the first immune, the second immune and after the second immune of 7th, 14 th, 21 thand 28th, respectively. At the time of pre-immunizationand post-immunizationexecuted two mice in each interval of two weeks to extract spleen to detect the number of CD4+and CD8+T-cells, meanwhile to prepare intestinal fluid to detect specific SIgA antibody. The test results showed that there were no significant differences between edible vaccine group and commodity vaccine group in the degrees of IgG antibody, IFN-γ and the number of CD4+and CD8+T-cells, and both of them were higher than lactobacillus control.There were no much difference of the cotent of IL-4except the time of the second post-immune of 14 th.The content of intestinal juice specific SIgA antibody in edible vaccine group was much higher than that in commodity vaccine group(P<0.05). Results showed that the edible vaccine had a certain immune effect, and can stimulate the body to produce higher mucosal immune antibody.