Sensitive glycoprotein gIII blocking ELISA to distinguish between pseudorabies (Aujeszky's disease) -infected and vaccinated pigs

Abstract

A blocking enzyme-linked immunosorbent assay (ELISA) test has been developed to distinguish pseudorabies virus (PRV) (Aujeszky's disease virus) -infected pigs from those immunized with a glycoprotein g92 (gIII) deletion mutant, PRV (dlg92dltk) [OMNIMARK-PRV]. This blocking ELISA test utilizes an anti-PRV gIII monoclonal (mAbgIII)-horseradish peroxidase (HRPO) conjugate, TMB for color development and a cloned PRVg92 (gIII) antigen to coat wells of microtiter test plates. Undiluted sera are used to block the binding of the mAbgIII-HRPO conjugate to the antigen. The gIII blocking ELISA is specific and has a sensitivity comparable to screening ELISA and latex agglutination tests. PRV-negative sera and sera from pigs vaccinated once, twice, or four times with the gIII-negative vaccine all showed negative S/N values of >0.70 (S/N defined as the optical density at 630 nm of test sera/optical density at 630 nm of negative control sera.) Sera from PRV-infected herds, sera from pigs experimentally infected with virulent PRV, and sera from pigs vaccinated with modified-live or inactivated gIII + vaccines were positive for gIII antibodies (S/N < 0.7). Sera from pigs experimentally infected with 200 PFU virulent PRV seroconverted to gIII + antibodies 7–10 days postinfection. Sera from pigs vaccinated with gpX − and gI − vaccines seroconverted to gIII + antibodies 7–8 days after vaccination. The gIII antibodies persisted after gIII + vaccination for at least 376 days postvaccination. Sera from pigs protected by vaccination with PRV (dlg92dltk) and then challenge exposed to virulent PRV at 21 days postvaccination showed gIII + antibodies by 14 days postchallenge. The specificity and sensitivity of the gIII blocking ELISA assay was further demonstrated on United States Department of Agriculture-National Veterinary Services Laboratory (USDA-NVSL) sera from the 1988 PRV check set and the 1989 gIII PRV check set by comparing the gIII blocking ELISA assay with virus neutralization, screening/verification ELISA and latex agglutination assays.