Date Full Report Received06/24/2015
Date Abstract Report Received06/24/2015
InvestigationInstitution: South Dakota State University
Primary Investigator: Eric A. Nelson
Co-Investigators: Jane Christopher-Hennings, Travis Clement, S. Lawson, A.. Singrey
Funded ByNational Pork Board
Porcine epidemic diarrhea virus (PEDV) was first detected in the U.S. in May 2013 and spread rapidly across the country. PCR assays were quickly developed to detect the presence of PEDV RNA in intestinal contents or fecal material and these assays provide an important tool in control of the virus. The continuation of severe outbreaks of PEDV in North America highlights the need for additional well-validated diagnostic tests for the detection of recently infected animals and evaluation of their immune status to this virus. Specific serological tests can be used to detect the antibody response in animals following infection or vaccination. They can provide an important tool in the screening of replacement animals and certain tests, such as virus neutralization assays, may provide insight into the level of protective immunity in a given group of animals.
Various serological assays for PEDV have been previously described, but few were readily available in the U.S. Several U.S. laboratories quickly developed indirect fluorescent antibody (IFA) assays for the detection of antibodies to PEDV in swine serum, indicating prior exposure. However, the IFA has several disadvantages, including low throughput, moderate sensitivity and relatively subjective interpretation. Different serologic test formats have advantages and disadvantages, depending on the questions being asked, so a full repertoire of tests is useful.
1. Finalize validation of our new PEDV fluorescent focus neutralization (FFN) assay and assessment of its ability to detect and quantify neutralizing or “potentially protective” antibodies in milk/colostrum as well as in serum samples.
2. Complete development of next generation assays such as specific ELISAs and fluorescent multiplex immunoassays (FMIA) to detect “potentially protective” antibodies in milk/colostrum, oral fluids and serum and determine correlation with functional virus neutralization, measured by the PEDV FFN.
3. Assess different tests and sample types (serum, milk and oral fluids) to determine the most accurate and cost effective strategies to predict duration of immunity and protection of piglets.
Recombinant proteins including the PEDV nucleoprotein (NP) and the S1 region of the spike protein were expressed and purified for use in ELISA and FMIA tests. A variety of different assays were developed and validated by approved standards. Assessment involving over 1400 samples of known status demonstrated that the NP-based FMIA had the highest sensitivity and specificity at 98.2% and 99.2%, respectively, with a significant level of testing agreement among all assays. No cross-reactivity with the closely related coronaviruses, transmissible gastroenteritis virus (TGEV) or porcine respiratory coronavirus (PRCV) was noted. All assay formats detected seroconversion of naïve animals within 6-9 days post exposure.
Eric A. Nelson
Veterinary & Biomedical Sciences Department
Animal Disease Research & Diagnostic Laboratory
South Dakota State University
Brookings, SD 57007-1396