Study On Differential Detection And Immune Prevention And Control Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome(PRRS),which caused by PRRS virus(PRRSV)and characterized by reproductive failure in sows and serious respiratory disease in pigs of all ages,can lead to serious economic losses to the pig industry.Clinically,it is very difficult to prevent and control PRRS because of the diversity and complexity of PRRSV.So,in order to effectively prevent and control PRRS,it is very important to establish differential detection method,to understand the characteristics of prevalent PRRSV strains,and to explore immune prevention and control measures.1.Establishment and application of a multiplex RT-PCR assay for differential detection of classical,highly pathogenic and vaccine strain of North American genotype PRRSVTo establish a rapid method for differential detection of classical,highly pathogenic and TJM-F92 vaccine strain of North American genotype PRRSV(NA-PRRSV),a multiple RT-PCR assay was established.In this assay,two pairs of primers were designed according to the genomic sequences of classical,highly pathogenic and TJM-F92 vaccine strain of NA-PRRSV.The assay could only detect NA-PRRSV,but not CSFV,FMDV,PRV and PCV2.The detection limit of the method was as little as 1.13×103 copies/pL of templates.The established assay were successfully used to detect 349 clinical samples and 119 samples were positive for PRRSV,of which 5 samples were positive for classical PRRSV(C-PRRSV),107 samples for highly pathogenic PRRSV(HP-PRRSV)and 7 samples for TJM-F92 vaccine strain(V-PRRSV),while 7 samples were positive for HP-PRRSV and V-PRRSV.The results showed that the established multiple RT-PCR assay could be used for differential detection and epidemiological investigation of NA-PRRSV.2.Genetic diversity of Nsp2 and ORP5 genes of PRRSV isolates in Guangxi provinceTo better understand the genetic diversity of PRRSV in Guangxi province,the Nsp2 and ORF5 genes were amplified from clinical samples in Guangxi from 2014 to 2016 and the gene sequences were analyzed.The results showed that all the acquired 34 Nsp2 and 45 ORF5 gene sequences belonged to North American genotype PRRSV(NA-PRRSV).Sequence analysis revealed that the homology identity of the nucleotide of Nsp2 genes was 91.8%-100%,and they shared 81.3%-84.3%,88.9%-92.1%,94.3%-99.3%,73.5%-75.1%identity with NA-PRRSV strain VR-2332,Ch-1a,JXA1 and NADC30,respectively;but shared only 51.5%-53.2%identity with European genotype PRRSV(EU-PRRSV)strain LV.The homology identity of the nucleotide of ORF5 genes was 91.8%-100%,and they shared 83.7%-99.5%,85%-95%,83.8%-99.7%,83.2%-86.4%identity with NA-PRRSV strain VR-2332,Ch-la,JXA1 and NADC30,respectively;but shared only 62.4%-64.5%identity with EU-PRRSV strain LV.The phylogenetic analysis showed that all PRRSV isolates were divided into four subgroups based on the deduced amino acid sequences of Nsp2 and ORF5 genes,and most isolates from Guangxi belonged to subgroup IV which was represented by JXA1 strain.The results indicated that the highly pathogenic PRRSV(HP-PRRSV)represented by JXA1 strain has become the dominant strain in Guangxi,and there exists genetic diversity among Nsp2 and ORF5 genes of isolated strains,while EU-PRRSV and NADC30-like strain of NA-PRRSV have not yet been found.3.Analysis of the immune efficacy of attenuated live vaccine in the pig herds previously infected with wild-type PRRSVTo evaluate the immune efficacy of PRRS attenuated live vaccine,this study selected 4 litters from two small pig farms(2 litters each farm)and vaccinated with PRRSV attenuated live vaccine.Sera were collected on different days post vaccination(dpv)to detect PRRSV nucleic acid by RT-PCR and PRRSV antibody by ELISA using two different ELISA kits,i.e.N-ELISA and G-ELISA.The results showed that wild-type strain PRRSV nucleic acid was positive on 0 dpv until 30 dpv in piglets and also positive on 0 dpv in the corresponding sows.All piglets were negative for PRRSV antibody on 0 dpv,but were positive on 30 dpv until 150 dpv.The positive rates detected by N-ELISA kit were higher than those of G-ELISA kit on 30 and 60 dpv,but lower than those of G-ELISA kit on 120 and 150 dpv.A total of 216 sera were detected respectively by two ELISA kits and the coincidence rate of the results was 95.83%.The P value of X2 test was more than 0.05,showing there was no significant difference between the results of two kits.The Kappa value was 0.87,showing there was strong consistence between them.The correlation coefficient was 0.605,showing there was significantly linear correlation between them.The results indicated that the wild-type PRRSV in the previously infected pig herds could be eliminated by vaccination with attenuated live vaccine and both N-ELISA and G-ELISA kits could be used to estimate the immune efficacy of the attenuated live vaccine effectively.4.Analysis of the efficacy of PRRSV attenuated live vaccine in the piglets with high level maternally-derived antibodySix litter piglets were selected from a large-scale pig farm(2 experiments,3 litters for each)and vaccinated with PRRSV attenuated live vaccine.Sera were collected on different days post vaccination to detect PRRSV nucleic acid by RT-PCR and detect PRRSV antibody by ELISA using two different ELISA kits,i.e.N-ELISA and G-ELISA.The results showed that vaccine strain nucleic acid were detected until 60 d after vaccination,and wild-type strain nucleic acid could not be detected during the experiment.All piglets had high level maternally-derived antibody and the positive rate was 100%on 0 d post vaccination,but the rate was gradually fall afterward and it fell down to the bottom on 28 d/60 d(2nd experiment/1st experiment),then it increased rapidly and became 100%from 60 d/90 d to the end of the experiment on 150 d post vaccination.A total of 329 serum samples were detected respectively by two ELISA kits and the coincidence rate of the results was 96.96%.The P value of X2 test was higher than 0.05,showing there was no significant difference between the results of two kits.The Kappa value was 0.81,showing there was strong consistence between them.The correlation coefficient was 0.794,showing there was significantly linear correlation between them.The results indicated that the piglets with high level of maternally-derived antibody could obtain good immune efficacy after vaccination with attenuated live vaccine,and N-ELISA and G-ELISA kits could be used to estimate the immune efficacy effectively.