Serological Investigation Versus Isolation And Identification Of Wild-type Pseudorabies Virus In A Large-scale Pig Farm In Longyan

Pseudorabies（PR）is an acute infectious disease caused by Pseudorabies virus（PRV）in pigs,cattle and sheep.Pig is the reservoir of PRV,which can infect pigs of different ages.After the latent state of infection,pigs will carry the virus in their whole life.Since 2011,outbreaks of pseudorabies occurred in many provinces in China,and the wild mutant pseudorabies strains were isolated from many pig farms.In recent years,it was found that currently the wild pig pseudorabies virus infections are severe and which may tense the situation of prevention and control of the disease.This study explores the current situation and causes of wild virus infections in the large-scale breeding farm in Longyan city,to provides a basis for formulating some scientific and reasonable strategies about prevention and control.This study contained three aspects:1.Seroprevalence associated with wild type Pseudorabies virus infection in the pig farmTo master the immunization and wild virus infection status of swine pseudorabies in a large-scale breeding farm incapable of outputting10,000 pigs a year in Longyan,we conduct ELISA test on a total of 635serum samples from different pigs between 2015 and 2018.The results of the ELISA test showed that the over 97%pigs remained PRV-g B antibody titers at a high level for many years,and the positive rate of anti-PRV-g E antibody was higher than 40%from 2015 to 2017 but dropped to 17%in2018 when the vaccine and the immunization program was changed.This indicates that although the herds of the farm could have good immunity to the Batha-K61 vaccine used previously,it failed to provide complete protection among the pigs from the infections of the wild PRV.It suggested that the subsequently used vaccine（HB98 or HB2000）strain in2018 might shared higher homology with the prevalent wild virus.2.Development and application of a quantitative real-time PCR assay for detecting Pseudorabies virusTo determine porcine pseudorabies virus from suspicious samples,a real-time quantitative PCR method for detecting porcine pseudorabies virus was established.The primers were selected from the conserved regions of the g E gene of porcine Pseudorabies virus and the reaction system and amplification program were optimized for real-time PCR assay.The sensitivity,specificity,and reproducibility of this detection method were tested.After that,58 suspicious disease materials from a pig farm in Longyan were tested by this method and routine PCR.The results showed that the real-time PCR method had good sensitivity,specificity,and repeatability.A real-time fluorescent quantitative PCR method for detecting Pseudorabies virus was successfully established.Among the samples,82.75%and 77.58%of them were positive respectively by real-time PCR and routine PCR,and lower copy numbers could be detected by real-time PCR.That implied that the real-time PCR method for detecting Pseudorabies virus in this study showed better sensitivity than routine PCR.3.Isolation,identification and genetic evolution analysis of porcine Pseudorabies virusTo understand the characteristics of the epidemic strains in this farm,the positive samples were aseptically filtered and inoculated on Vero-E6cells for 3 generations.The g E and TK gene sequences of the isolated strain were then amplified by PCR and analyzed.The results showed that one sample could cause typical cytopathic effects like PRV infection in vero cells,and an isolated strain was successfully obtained and named PRV-FJ18 strain;the TCID₅₀ of this strain was 10^-4.29/100μL.The sequence of g E gene of the isolate PRV-FJ18 strain,shared 100%nucleotide homology with Guangdong GD0304 strain belonged to the same genetic evolutionary branch,and it fell into GⅠtype strain like other Asian strains;the nucleotide sequence of the TK gene is on the same genetic evolutionary branch as the Guangdong GD0304 strain,Japanese RC1 strain,Hubei Ea and China Fa strain,shared 100%homology with former three strains;although the gene homology between this isolated and Bartha vaccine is 99.7%,they are in two different branches of the TK gene’s phylogenetic tree.These findings indicated that this strain might share the same original source with GD0304..These findings indicated that this strain might share the same original source with GD0304.In summary,this scale-breeding pig farm was infected by wild porcine Pseudorabies virus.Among the positive samples detected by the real-time PCR method established in this paper,a pig pseudorabies virus was successfully isolated,which shared high similarity with the Guangdong GD0304 strain.It is recommended that the vaccine HB2000strain could be continually used in the farm vaccine for the immunization plan.