Pseudorabies (Aujeszky's disease) virus DNA detection in swine nasal swab and oral fluid specimens using a gB-based real-time quantitative PCR

Authors: CHENG, TING-YU - Iowa State University; HENAO-DIAZ, ALEXANDRA - Iowa State University; POONSUK, KORAKRIT - Iowa State University; Buckley, Alexandra; VAN GEELEN, ALBERT - Animal And Plant Health Inspection Service (APHIS); Lager, Kelly; HARMON, KAREN - Iowa State University; GAUGER, PHILLIP - Iowa State University; WANG, CHONG - Iowa State University; AMBAGALA, ARUNA - Iowa State University; ZIMMERMAN, JEFFREY - Iowa State University; GIMENEZ-LIROLA, LUIS - Iowa State University

Submitted to: Preventive Veterinary Medicine
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/20/2021
Publication Date: 2/24/2021
Citation: Cheng, T., Henao-Diaz, A., Poonsuk, K., Buckley, A.C., Van Geelen, A., Lager, K.M., Harmon, K., Gauger, P., Wang, C., Ambagala, A., Zimmerman, J., Gimenez-Lirola, L. 2021. Pseudorabies (Aujeszky's disease) virus DNA detection in swine nasal swab and oral fluid specimens using a gB-based real-time quantitative PCR. Preventive Veterinary Medicine. 189. Article 105308. https://doi.org/10.1016/j.prevetmed.2021.105308.
DOI: https://doi.org/10.1016/j.prevetmed.2021.105308

Interpretive Summary: Pseudorabies virus (PRV) is a herpesvirus whose natural host is swine. Infection can result in central nervous system, reproductive, and respiratory clinical signs, which vary in severity with age of infection. PRV has been eradicated from the United States (US) domestic swine herd, but the virus can be found in some feral swine populations. Many other countries have also been successful in eradicating PRV from their domestic swine herds. However, in China PRV is endemic and there have been reports of highly pathogenic PRV isolates in swine. Thus, there has been renewed interest in ensuring there are validated diagnostic assays in preparation for control and elimination of PRV if it were to infect the US domestic swine herd. In order to sample large groups of swine quickly in an outbreak, oral fluids have emerged as an easy to collect aggregate sample. But, studies need to be performed to ensure that the current diagnostic platforms are validated for swine oral fluids. In this study, pigs were challenged with wild-type PRV, vaccinated with a commercial PRV vaccine or vaccinated and then challenged with wild-type PRV. Oral fluids and nasal swabs were collected from these animals of known PRV status along with negative control animals. Both sample types were tested for PRV DNA by a real-time PCR assay and had similar patterns of detection. Performance of the assay was also similar with a diagnostic specificity of 100% and diagnostic sensitivity of 53% and 70% for oral fluids and nasal swab samples respectively. This study demonstrated detection of PRV by real-time PCR in oral fluids could be a useful tool for PRV diagnosis and surveillance.

Technical Abstract: In this study, the detection of PRV DNA in nasal swab ( n = 440) and oral fluid ( n =1,545) samples collected over time from experimentally PRV vaccinated and/or PRV inoculated pigs ( n = 40) was comparatively evaluated by real-time PCR. Serum samples ( n = 440) were tested by PRV gB/gE blocking ELISAs (Pseudorabies Virus gB Antibody Test Kit and Pseudorabies Virus gpI Antibody Test Kit, IDEXX Laboratories, Inc., Westbrook, ME) to monitor PRV status over time. Following exposure to a gE-deleted modified live vaccine (Ingelvac ® Aujeszky MLV, Boehringer Ingelheim, Ridgefield, CT) and/or a wild-type virus (3 CR Ossabaw), PRV gB DNA was detected in oral fluid specimens in a pattern similar to that of nasal swabs. For quantitative analyses, PRV PCR quantification cycle (Cq) results were reexpressed as “efficiency standardized Cqs (ECqs)” as a function of PCR efficiency using plate-specific positive amplification controls. ROC analyses of the PRV gB PCR ECqs results showed a similar performance of the PRV gB PCR for nasal swab and oral fluid specimens (area under the ROC curve = 85% vs 83%) and, based on an ECq cutoff of 0.01 a diagnostic specificity of 100% and diagnostic sensitivities for oral fluid and nasal swab specimens of 53% and 70%, respectively. Thus, the results described herein demonstrated the detection of PRV gB DNA in swine oral fluid and supported the use of this specimen in PRV diagnosis and surveillance.