Establishment And Preliminary Application Of Real-time PCR And ELISA For Detection Of Pseudorabies From Oral Fluid

Pseudorabies(PR),which is caused by pseudorabies virus(PRV),is an economically important viral disease in pigs and other animals.PRV has a broad host range in domestic and wild animals,with the pig being its primary natural host and reservoir.PRV usually establishes a life-long latent infection in the host’s peripheral nervous system.Latently infected pigs can be a source of re-infections when the latent viral genome is reactivated spontaneously.PRV causes economically significant illness in domestic pigs.Oral fluid is a mixture of saliva and mucosal transudate which contains specific antibodies derived from both serum and salivary glands.Oral fluid samples for disease surveillance are gaining importance due to ease of collection and low-cost.In the present study,the real-time PCR and the indirect enzyme-linked immunosorbent assay(ELISA)method were first optimized and evaluated for PRV virus quantification and specific antibodies detection using pen-based pig oral fluid samples.The real-time PCR assays was developed and evaluated for their capability in the detection and differentiation of field and vaccine strains of PRV.The indirect ELISA method can be used to detect anti-PRV gB antibody and anti-PRV gE antibody induced by wild in oral fluid.To evaluate the diagnostic performance of PRV laboratory testing methods,both oral fluid and paired serum samples from different age groups of known vaccination histories were collected and tested,from a commercial pig farm,between 2015 and 2016.Our results demonstrated that the use of oral fluid for pathogen and antibody detection shows lower sensitivity than serum,which may be related to the inhibitory factor in oral fluid.However,PRV specific antibodies in oral fluid specimens are agreement with those in serum samples.In conclusion,we have standardized and validated the pig oral fluid based PRV pathogen and specific antibodies assay,the assay can be used by further research of herd oral fluid testing system,provides data and evidence for pre-clinical investigation.