All colostrum samples were tested with the S1 ELISA for PEDV IgG and IgA antibodies. for the animal study was approved by the Iowa State University Institutional Animal Care and Use Committee (approval number 1-11-7071S, date of approval November 2013). Serum samples from 239 pigs from 19 commercial USA herds were selected from routine diagnostic cases submitted to the VDL-ISU for PEDV investigation due to the occurrence of diarrhea or to monitor PEDV contamination status from September to December 2013. In addition, 133 E 2012 colostrum samples from sows were selected, including 102 PEDV unfavorable colostrum samples with unknown TGEV and PRCV status collected in 2010 2010 (O’Neill et?al., 2012). The remaining 31 samples were collected in November 2013 from a PEDV positive farm known to E 2012 be PRCV positive and TGEV unfavorable. Testing strategy and farm status All serum samples were tested for anti-PEDV IgG antibodies by IFA and S1 ELISA, and for anti-TGEV and anti-PRCV IgG antibodies by a commercial ELISA (Swinecheck TGEV/PRCV ELISA; Biovet). All colostrum samples were tested with the S1 ELISA for PEDV IgG and IgA antibodies. The PEDV, PRCV and TGEV contamination status was decided based on a duplex real-time reverse-transcriptase (RT)-PCR for PEDV and TGEV in fecal samples using a commercially available assay test (Tetracore) and PRCV serology. A farm was considered to be positive when antibodies and/or viral RNA were detected in 50% of the submitted samples in at least two consecutive submissions. A farm was considered to be unfavorable when all tested samples of at least two consecutive submissions were antibody and/or viral RNA unfavorable. Farms that could not be classified as unfavorable and had <50% of positive samples for a given pathogen were excluded. This 50% cut-off was adopted arbitrarily to ensure the reliability of the classification considering that some farms had a submission size as low as three samples. Instead of establishing different parameters depending on the number of samples submitted, a unique conservative value of 50% was used for all cases. ELISA cut-off values and assay performance The cut-off value for the serum-based S1 ELISA was calculated by using the mean OD TSPAN4 value of 40 PEDV unfavorable serum samples plus three standard deviations (SDs). This value was further evaluated by receiver operator characteristic (ROC) analysis using 60 PEDV positive serum samples in addition to the E 2012 cumulative data from all samples using the IFA as the reference method for sample classification. The cut-off value for the colostrum-based ELISA was calculated using ROC analysis on 102 PEDV unfavorable samples and 31 samples from a positive PEDV farm. Diagnostic sensitivity and specificity were estimated for different S1 ELISA cut-offs using a maximum likelihood method considering a sensitivity of 95.6% and a specificity of 98.1% for the IFA. McNemar’s test for pairwise comparisons was used to determine whether the proportions of positive samples were significantly different by assay. Differences between groups were considered to be significant at P?<?0.05. The Kappa () index was calculated to determine the agreement between assays. Statistical analyses were performed using SAS version 9.2. Results ELISA cut-off values and assay performance The mean OD value of the 40 unfavorable serum samples was 0.08 (x), with a SD of 0.04. Therefore, samples with an OD value 0.20 (x?+?3 SD) were considered to be positive. Based on the maximum likelihood method, the sensitivity was 100% and the specificity was 88.9% for a cut-off of 0.2, and the sensitivity was 100% and the specificity was 94.0% for cut-off of 0.3. Therefore, a final cut-off 0.3 was selected and samples with OD values between 0.2 and 0.3 were considered to be indeterminate (Fig.?2 ). Open in a separate windows Fig.?2 Distribution of anti-IgG porcine.
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