Molecular Epidemiological Investigation,Isolation And Identification Of Two Viruses In Neonatal Piglets With Epidemic Diarrhea

Servere neonatal diarrhea has occurred in multiple provinces of China since the end of2011. The major clinic effects on diseased pigs were diarrhea, dehydration, anorexia and rapid emaciation. The causual pathogens included porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis of swine, rotavirus and boca virus. In order to study the characteristics of epidemic strains, this study collected clinical intestinal fecal samples from piglets in lactation during February2012to March2013, detecting of PEDV and two isolated strains of reovirus (REOV), clearly elucidated the molecular epidemiological features of PEDV, and laid a certain base for prevention and control of the disease. The main research contents were as following:1. Detection for porcine epidemic diarrhea virus and S gene molecular epidemiological investigationTo investigate pathogeny of diarrhea piglets, a total of102diarrheal samples were collected from31different pig farms during February2012to March2013, in Jiangsu, Shanghai, Zhejiang and other areas of China, Reverse transcription-PCR was carried out for detection of the PEDV antigen. Results showed that PEDV-positive rates were67.74%and46.08%among farms and samples, respectively. Eight pandemic strain S full genetic sequences were obtained. They were95.3%-99.5%homologous and with94.2%-95.7%homology to European representative strains CV777gene. Compared with attenuated DR13vaccine strain, the homology was94.0%-95.8%; compared with the2006domestic popular LZC strain, the homology was93.6%to95.1%. Phylogenetic analysis divided these PEDV strains into3groups. Seven epidemic strains obtained in this study belonged to group2, which were quite different from vaccine strains in China, while only one strain belonged to group1with South Korea:vaccine strains and the European strain CV777. Research results showed that PEDV is one of the pathogens responsible for the diarrhea of piglets in China and this Chinese strain phylogenetically distinct to strains previously described in other countries.2. Isolation and identification of porcine reovirus and piglets pathogenic study on reovirus102diarrheal disease samples from31pig farms were collected for RT-PCR detection of REOV, the results were as following:the REOV-positive rate of pig farms was45.16%, the REOV-positive rate of samples was26.47%. Two tissue samples were chosen inoculating Vero cells after aseptic processing,5μg/mL of trypsin was added to the maintaining fluid. After2-3passages, CPE were detected. They were proved as reovirus strains, detection by RT-PCR amplification, gene sequences analysis and electron microscopy of virus particles, and named ZJ120101and AH120411. Two-day health piglets were inoculated with these2REOV strains respectively by oral,5mL per head, control group were inoculated with DMEM. Piglets were separated in different cages and artificial feeded in experiment and control groups. By observation of clinical symptoms, pathological changes, and detection of pathogens5-7days after inoculation could get the results:ZJ120101and AH120411strains could cause a short-term appetite drops of piglet in a different period without diarrhea. No obvious clinical symptoms were seen in control. Pathologic observation showed that AH120411strain group of small intestine had mild pep, tissue lesions observation found that epithelial cells of cecum intestinal villus were damaged and fell off. No obvious pathological changes were seen in the rest of the intestinal segment, ZJ120101group had no obvious pathological changes as well as the control group. Intestinal tissue were collected for RT-PCR detection for REOV, the results were:positive for ZJ120101group and AH120411group, negative for control group. The results showed that two isolated strains had mild pathopoiesis, and the pathogenicity of different REOV strains was varied.