Investigation On Epidemic Status Of Five Porcine Reproductive Disorder Related Disease And Genetic Variation Of Porcine Circovirus Type 2 In Pig Farms In East China

Classical swine fever virus(CSFV),Porcine reproductive and respiratory syndrome virus(PRRSV),Pseudorabies virus(PRV),Porcine circovirus type 2(PCV2)and Porcine parvovirus(PPV)are the main pathogens causing the disease of porcine reproductive disorder related disease and have caused serious economic losses to the pig industry.These five viruses often show mixed infection and the symptoms are similar,which cause great difficulties to the clinical testing.In order to achieve rapid and accurate detection of mixed infection of the five pathogens,multiplex PCR was established in this study.The method was applied to detect the clinical samples,and the genetic variation of PCV2 was analyzed.The main research contents are as follows:1.Establishment of the multiple PCR method of CSFV,PRRSV,PRV,PCV2 and PPVAccording to the CSFV,PRRSV,PRV,PCV2 and PPV genesequences published in Genbank,designed 5 pairs of primers to amplify CSFV E2,PRRSV Nsp2,PRV gB,PCV2 ORF2 and PPV VP2.Reaction conditions were optimized to establish the multiple PCR detecting CSFV,PRRSV,PRV,PCV2 and PPV on the basis of the single PCR.The results showed great sensitivity and specificity.Clinical results showed that multiple PCR and single PCR positive coincidence rate and was 90%and above.2.Application of multiplex PCR method in clinical samplesA total of 335 samples from different pig farms in eastern China from 2014 to 2015 were detected by multiplex PCR,the results showed that the positive rate of the CSFV,PRRSV,PRV,PCV2 and PPV was3.58%、40.90%、5.67%、43.58%、13.73%,while the mixed infection rate of PCV2 and PRRSV was 23.58%,and the mixed infection rate of PCV2 and PPV was 6.87%.It should be noted that 79 PCV2 can be detected in 137 PRRSV positive samples,the mixed infection rate of them was pretty high.3.The genotyping of PCV2 and genetic variation analysis of ORF2The positive samples of PCV2 were identified by genotyping while the complete ORF2 sequences were amplified from 15 positive samples.The genotyping results showed that among 146 positive samples 11 samples were PCV2a(7.53%),127 were PCV2b(86.99%),and the co-infection rate was 5.48%,meaning that genotype 2b was the majorgenotype in East China.For ORF2 sequences,89.9%~99.9% and86.2%~99.7% similarities were found between the examined isolates and references respectively,indicating there was a high homology between them.The Cap protein encoded by ORF2 had different degree of amino acid variation,and there were sequence recombination or drift between different genotypes.Among 15 ORF2 sequences,there were 9PCV2 b,4 PCV2 d and 2 PCV2 a,no PCV2 c and PCV2 d was found.This study established a rapid and accurate method for the detection of porcine reproductive disorder related diseases and provided useful information for epidemiological studies.The genetic variation analysis of PCV2 could provide a reference for vaccine development in the future.