| Since 2010, there appeared different degrees of diarrhea in pig farms in some areas of Guizhou Province.Some farms used porcine epidemic diarrhea and porcine transmissible gastroenteritis bivalent inactivated vaccine for emergency vaccination, but the effects were unreliable. In order to acknowlege swine virus diarrhea in the prevalence of Guizhou Province, the author carried out studies on establishing rapid detection methods for pathogenic porcine virus diarrhea, piglet viral diarrhea epidemiology investigation in Guizhou Province and analysis of gene ralativity, with the aim of providing the basis for its diagnosis and treatment and vaccine research.1. PEDV、TGEV and RV by RT-PCR and its clinical application:According to the PEDV, TGEV and RV gene sequence in Genebank, three pairs of primers were designed. PEDV-TGEV-RV DPT RNA reverse transcription c DNA was used as the template and amplified by PCR and optimized under reaction conditions. The PT-PCR detect method of PEDV, TGEV and RV was estblished. The results showed that: the amplification lengths of PEDV, TGEV and RV were 651bp(PEDV), 859bp(TGEV) and 309bp(RV) respectively and were same as expection. The optimum temperature of three primers were 53 ℃.Primer concentration was 10 nmol/μL. The lowest diluted concentration of PEDV, TGEV and RV in vaccine strains were 1.28×10-8g/L,0.36×10-7g/L and2.6×10-9g/L, and the results are good in specificity and sensibility,and can be applied into clinic detection.2. Epidemiological survey on viral diarrhea of piglets in Guizhou Province:(1)With the epidemiological methods, surveys on swine virus diarrhea incidence, immune status, clinical symptoms and pathological changes in some large pig farms in Guizhou Province were conducted. The results showed that:among 13 large pig farms in five cities and areas, there appeared a high morbidity from December to next May.The number of the cases was 3020, with 70.35%(3020/4293) incidence; 2300 pig were dead, with the mortality rate of 76.16%(2300/3020);(2) By ELISA method, 184 scaled pig serum samples were collected from 20 pig farms in seven cities and areas from 2013 to 2014 are tested with PEDV, TGEV and RV. The results showed: the total positive rates of serum in PEDV, TGEV and RV were 39.13%, 33.70% and 42.39% respectively. The positive antibody rates were 44.05%, 32.14%and 46.43% respectively. The positive rates of unvaccinated pigs were44.05%(37/84)、32.14%(27/84)、46.43%(39/84) respectively.(3) With RT-PCR method, 111 clinically suspecteddiarrhea samples were sent for PEDV, TGEV and RV pathogenic nucleic acid detection. The results showed the positive rates of PEDV, TGEV and RV were 82.88%(92/111), 0.02%(2/111) and 10.81%(12/111), with 0.02%(2/111) for PEDV / TGEV, 10.81%(12/111) for PEDV / RV. Those results sugested that there are inapparent PEDV, TGEV and RV infection in piggeries in Guizhou province.3. Porcine epidemic diarrhea virus(PEDV) molecular epidemiology: Based on the gene sequences of Belgium PEDV strains of CV777 strain(NO.AF353511) registered in Genbank, the design of specific priimers to S,M and ORF3 gene,the author adopted RT-PCR method to increase the target genes,and did the combination,transformation,clone and sequenced. Six strain of S, M and ORF3 genes had been successfully cloned.The S, M and ORF3 gene were analysised by DNAStar and MEGA5.0. The results showed that:(1) the total length of S genes of the 6 pandmic strain is 4161 bp,which coded 1387 amino acids. The homologies of nucleotides and amino acids were 98.0%~99.1% and 97.3%~100% respectively. Compared to referance PEDV strains, the homologies of nucleotides and amino acids were 91.8%~93.9% and 91.3%~99.3%respectively.Compared to CV777 strains, there were 420 nucleotide mutation.Pandemic strain nucleotide sequence inserts 15 bases in 175~186,417~419 bit and 5 nucletide deletion in 480~485 bit.(2) The total length of M genes of the 6 pandemic strain is 681 bp, which coded 226 amino acids. Homology of nucleotides and amino acids was99.9% to 100% and 99.6% respectively. Compared to PEDV strains, the homologies of nucleotides and amino acids were 97.8%~98.2% and 97.8%~98.2% respectively. The M sequence contained a few of point mutation,without inserion,deletion or change,which indicate that conservation of M gene is high.(3) the total length ORF3 gene of 6 pandmic strain is 675 bp, which coded 225 amino acids. Homology of nucleotides and amino acids was 99.0% to 100% and 98.2% to 99.6% respectively. Compared to referance PEDV strains, the homologies of nucleotides and amino acids were 96.1%~98.5% and 95.6%~99.1% respectively. Compared to CV777, there are 29 site mutations without insertion. According to the analysis of phylogenitic tree, there is a close relationship between 6 pandmic strain and CV777 strains, as well sa the American strains and Korean strains,but less relationship with LZC strains and Brl/87 strains.Conclusion1. The experiment successfully established the detection methods of PEDV, TGEV and RV RT-PCR,the results are good in specificity and sensibility,and can be applied into clinic detection.2. According to the survey,diarrhea appeared in some large-scale pig farms in Guizhou province, and the main pathogen is PEDV. The serological investigation showed that the PEDV vaccin was not effective, and therewere silent infections.3.The inheritance and development analyses on S, M and ORF3 of PEDV showed that pandmic strain of PEDV is divide into 2 groups,and is closely to Korean strains,Vietnam strains and American strains, but far from LZC strains and Brl/87 strains.According to the analyses of S and ORF3 genes, it can be predicted that the Guizhou epidemic strains are highly virulent strains. |
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