Investigation Of Infection And Immune Status And Its Media Of Prrs In Guizhou Prevince

Porcine reproductive and respiratory syndrome(PRRS),is an acute and highly contagious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV),which is characterized by sow reproductive disorders and respiratory tract symptoms of piglets,and have high morbidity and mortality.In recent years,there has been a suspected PRRS epidemic in pig farms in some areas of Guizhou province.In order to prevent and control of PRRS,the following research works have been carried out.1.Investigation of the immune status of PRRS in Guizhou provinceTo understand the immune status of PRRS in pig farms,88146 of pig serum samples were collected in pig farms from 2015 to 2017,and detected by the ELISA method.The results showed that the immune coverage rate of PRRS was 68.9% in the 9 regions,in which were respectively 100%,82.8% and 47.1% in pig breeding farms,commodity farms and scattered farms.And the average rate of antibody of PRRS was 82.2%,in which were respectively 98.9%,81.9% and 59.2% in pig breeding farms,commodity farms and scattered farms,and respectively 81.6%,83.0%,81.9% and 82.2% in spring,summer,autumn and winter,and 81.1%,82.8% and 81.1% respectively in altitudes under 800 m,800m~1600m and above 1600 m.These results showed that the immunization of PRRS was more important and the immune effect was better,but different in the different pig farms,and there were no obvious difference in the different seasons and altitude.2.Investigation of PRRS infection situation in Guizhou provinceTo understant the infection situation of PRRS in Guizhou province,the suspected cases were observed by the clinical symptoms and pathological dissection,and 9751 of the tissue samples were collected from 2015 to 2017 and detected by RT-PCR method.The results showed that the infected pigs had obvious PRRS symptoms,which is 41.5 degrees temperature,respiratory symptoms and reproductive disorders.The pathological changes showed the freckle kidney,the haemorrhage of the lymph nodes and so on.The average detection rate of PRRSV was 26.5% in pig farms from 2015 to 2017,in which 13.0%,26.5% and 31.0% respectively in pig breeding farms,commodity farms and scattered farms.And the detection rate were respectivel11.3% and 15.2% with the classic PRRSV stains and the variant PRRSV strains.And The detection rates were respectively 17.9%,29.3%,28.2% and 26.6% in spring,summer,autumn and winter.In the PRRSV infection cases,there was a double or multiple infection of pathogens,of which the main pathogens were mainly porcine circovirus 2 and CSFV and swine pseudorabies virus,which the infection rates were respectively 28.4%,25.5% and 13.9%.These results indicate that PRRSV infection is also prevalent in pig farms in Guizhou province.3.Isolation,identification and gene mutation analysis of PRRSV Guizhou strainTo solve the variation status of PRRSV Guizhou strain,the tissue material were collected from the suspected PRRS cases,and inoculated into Marc-145 cells for virus isolation,and identified by fluorescent antibody staining and RT-PCR technology,then the GP5 and NSP2 gene were respectively cloned and analyzed.The results showed that the obvious CPE were appeared in Marc-145 cells after sample inoculation.The obvious yellowish green fluorescence were observed in the infected cells,and the expected amplification fragment(254bp)were found by RT-PCR amplification.The GP5 gene fragment of 603 bp was obtained,which the similarity were 97.8% to 99.9% with the newly prevalent PRRSV strains,such as JXA1 strain,HUN4 strain,CG strain and LC0810 strain,while only 88.6%.94.5% with the classical PRRSV strains,such as HB-2 strain,CH-1a stain and VR-2332 strain.There is some point mutation in the amino acid sequence of GP5.The NSP2 gene fragment of 1064 bp were obtained,which the similarity were 90.1% to 99.5% with the newly prevalent PRRSV strains,such as JXA1 strain,HUN4 strain,CG strain and LC0810 strain,while only 78.1% to 88.7% with the classical PRRSV strain,such as HB-2 strain,Ch-1a strain and VR-2332 strain.There are 31 aa and 59 aa deletion on the amino acid sequence of NSP2.These results indicated that the PRRSV Guizhou strain were successfully isolated and identified,with some point mutation in GP5 and the deletion of amino acid in NSP2,which is a highly pathogenic PRRSV.4.Investigation on biological media of PRRSV in Guizhou provinceTo find out the biological vector of PRRSV in the pig farms,the mosquitoes,flies and rats were collected from the clinical infected pig farms and the clinical health pig farms,and used for identification of species,then extracted for virus RNA and amplificed by RT-PCR,which the positive samples were took for sequencing of the Nsp2 gene.These results were showed as followed:(1)The mosquito species were mainly Culex tritaeniorhynchus(50.1%),Aedes albopictus(25.9%)and Culex quinquefasciatus(14.1%),and the fly species was Musca domestica(77.2%)and the rats species were Rattus norvegicus(100%);(2)The positive rates of detection for PRRSV N gene were respectively 62.5%,62.5%,50%,25% and 12.5% of Culex tritaeniorhynchus,Aedes albopictus,Culex quinquefasciatus,Musca domestica and Rattus norvegicus,which in 5,5,4,2 and 1 of pig farms infected PRRSV,but not detected in clinical health farms.(3)The PRRSV Nsp2 gene fragment were detected from these samples of Culex tritaeniorhynchus,Aedes albopictus,Culex quinquefasciatus,Musca domestica and Rattus norvegicus,which were consistent from the infected pig.These results shows that the mosquito,flies and rats can carry PRRSV,which may play an important role on prevalence of PRRSV.5.Studies on proliferation possibility of PRRSV in Aedes albopictus in vivo To verify whether Aedes albopictus is the biological media of PRRSV,the cell cultures of PRRSV GY strain were selected to mix with an equivalent amount of fresh pig blood,then used for infecting Aedes albopictus,and these mosquitoes were collected in the different time and different generations and used for detection for PRRSV N gene.At the same time,these mosquitoes were fixed,dehydration and ultrathin sectioning,and observation of virus morphology by electron microscopy.The results were showed as following:(1)The PRRSV N gene were detected from the mosquito samples of 0h,6h,12 h,18h and 24 h infected by PRRSV,but from the mosquito samples of 30 h,36h,42 h,48h,5d and 7d.(2)The PRRSV N gene were not detected from larva of incubation of 1d,2d,4d and 7d,and pupa worm,and their adult mosquito of 1d and 3d,which incubated from eggs of the adult mosquito infected by PRRSV.(3)The PRRSV particles were not observed in these adult mosquito of 12 h,24h,48 h and 5d infected by PRRSV.These results showed that PRRSV is not proliferated and passed in Aedes albopictus in vivo,and Aedes albopictus is only a mechanical media of carrying PRRSV.