Date Full Report Received

06/24/2015

Date Abstract Report Received

06/24/2015

Investigation

Institution:
Primary Investigator:
Co-Investigators: Jane Christopher-Hennings, Travis Clement, S. Lawson, A.. Singrey

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.

Therefore, the overall objective of this study was to complete the development, optimization and validation of selected serological assays for improved monitoring and control of PEDV. These assays were then used to assess the best methods to measure exposure and the development and duration of “protective” immunity in pigs. Specific objectives addressed included:
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.
The PEDV FFN-based virus neutralization assay was optimized and validated for application with serum, milk and colostrum samples. Virus neutralization titers of serum samples from PEDV naïve pigs as determined by the FFN assay are generally <1:20, while most infected animals typically demonstrate titers of 1:40 to 1:1280 by 3 to 4 weeks post-exposure. Colostrum samples from recently infected sows tend to show substantially higher neutralizing antibody titers than serum samples from the same animals. Milk samples typically show neutralizing antibody levels similar to those observed in serum samples from the same sows. The PEDV FFN is now routinely offered by the South Dakota Animal Disease Research & Diagnostic Laboratory with over 10,000 samples tested to date.
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.
In summary, the FFN assay allows relative quantitation of functional neutralizing antibodies in serum, milk or colostrum samples. Serum antibody levels detected by the FFN show good correlation with neutralizing antibody levels present in milk and colostrum. Measurement of neutralizing antibody responses using the FFN assay should provide a valuable tool for assessment of vaccine candidates and assessment of herd immunity. Well-validated, high throughput indirect ELISA, blocking ELISA and FMIA assays for the detection of PEDV antibodies were also developed and validated, showing good correlation with IFA and each other. Each assay format has advantages that dictate how they will be used in the field.
Contact Information:
Eric A. Nelson
Veterinary & Biomedical Sciences Department
Animal Disease Research & Diagnostic Laboratory
Box 2175
South Dakota State University
Brookings, SD 57007-1396
eric.nelson@sdstate.edu