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We evaluated the detection of pseudorabies virus (PRV) antibodies in swine oral fluid. Oral fluid and serum samples were obtained from 40 pigs allocated to 4 treatment groups (10 pigs/group): negative control (NC); wild-type PRV inoculation (PRV 3CR Ossabaw; hereafter PRV); PRV vaccination (Ingelvac Aujeszky MLV; Boehringer Ingelheim; hereafter MLV); and PRV vaccination followed by PRV inoculation at 21 d post-vaccination (MLV-PRV). Using a serum PRV whole-virus indirect IgG ELISA (Idexx Laboratories) adapted to the oral fluid matrix, PRV antibody was detected in oral fluid samples from treatment groups PRV, MLV, and MLV-PRV in a pattern similar to serum. Vaccination alone produced a low oral fluid antibody response (groups MLV and MLV-PRV), but a strong anamnestic response was observed following challenge with wild-type virus (group PRV). Analyses of the oral fluid PRV indirect IgG ELISA results showed good binary diagnostic performance (area under ROC curve = 93%) and excellent assay repeatability (intra-class correlation coefficient = 99.3%). The demonstrable presence of PRV antibodies in swine oral fluids suggests the possible use of oral fluids in pseudorabies surveillance.

Oral fluid (n = 564) samples collected longitudinally from twelve 14-week-old pigs vaccinated with a porcine reproductive and respiratory syndrome virus (PRRSV) modified live vaccine were used to evaluate and compare the diagnostic performance of three commercial PRRSV oral fluid (OF) ELISAs (ELISAs 1, 2, 3). Serum samples (n = 132) tested by a PRRSV serum ELISA (ELISA 'S') provided an antibody response baseline for comparison. The initial analysis comparing the rate of positivity between each OF ELISA versus ELISA 'S' and then pairwise among the three OF ELISAs determined that ELISA 2 (143 false negative results) was significantly different from ELISAs 1 and 3, and from ELISA 'S' (Cochran's Q test, p < 0.05). Receiver operating characteristic (ROC) analyses based on the manufacturers' recommended cutoff were used to estimate the diagnostic sensitivities and specificities of ELISA 1 (100%, 100%), ELISA 2 (62%, 97%), and ELISA 3 (94%, 100%). As an additional aid for interpreting results, the diagnostic sensitivities and specificities of each OF ELISA were also estimated over a range of cutoffs. Area under the curve comparisons found no significant difference between ELISAs 1 and 3, but ELISA 2 differed from both ELISA 1 and 3 (ROC Chi-square, p < 0.05). Based on these analyses, the overall diagnostic performance of the three OF ELISAs ranked ELISA 1 ≥ ELISA 3 > ELISA 2., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Routine collection and testing of oral fluid (OF) samples facilitates porcine reproductive and respiratory syndrome virus (PRRSV) surveillance in commercial swine herds in a cost-effective, welfare-friendly fashion. However, OFs often contain environmental contaminants that may affect liquid handling and test performance. Traditional processing methods (e.g., filtration or centrifugation) are not compatible with high-throughput testing because of the burden of additional processing costs and time. OF "clarification" using chemical flocculants is an alternative approach not widely explored. Therefore, we evaluated the effect of chitosan-based clarification treatment on a commercial PRRSV OF ELISA. Serum and individual OFs were collected from vaccinated pigs ( n = 17) at -7 to 42 d post-vaccination and subdivided into 4 aliquots. Each aliquot was clarified (treatment A, B, C), with the 4th aliquot serving as untreated control. All samples were tested by PRRSV OF ELISA immediately after treatment and then were held at 4°C to be re-tested at 2, 4, 6, and 14 d post-treatment. Quantitative and qualitative treatment effects were evaluated. A Kruskal-Wallis test found no significant difference in ELISA S/P responses among treatments by days post-treatment. No difference was detected in the proportion of positive PRRSV antibody samples among treatments (Cochran Q, p > 0.05). Treatment of swine OFs using chitosan-based formulations did not affect the performance of a commercial PRRSV OF ELISA. Chitosan (or other flocculants) could improve OF characteristics and could be adapted for use in the field or in high-throughput laboratories.

The ontogeny of PRRSV antibody in oral fluids has been described using isotype-specific ELISAs. Mirroring the serum response, IgM appears in oral fluid by 7days post inoculation (DPI), IgA after 7 DPI, and IgG by 9 to 10 DPI. Commercial PRRSV ELISAs target the detection of IgG because the higher concentration of IgG relative to other isotypes provides the best diagnostic discrimination. Oral fluids are increasingly used for PRRSV surveillance in commercial herds, but in younger pigs, a positive ELISA result may be due either to maternal antibody or to antibody produced by the pigs in response to infection. To address this issue, a combined IgM-IgA PRRSV oral fluid ELISA was developed and evaluated for its capacity to detect pig-derived PRRSV antibody in the presence of maternal antibody. Two longitudinal studies were conducted. In Study 1 (modified-live PRRS vaccinated pigs), testing of individual pig oral fluid samples by isotype-specific ELISAs demonstrated that the combined IgM-IgA PRRSV ELISA provided better discrimination than individual IgM or IgA ELISAs. In Study 2 (field data), testing of pen-based oral fluid samples confirmed the findings in Study 1 and established that the IgM-IgA ELISA was able to detect antibody produced by pigs in response to wild-type PRRSV infection, despite the presence of maternal IgG. Overall, the combined PRRSV IgM-IgA oral fluid ELISA described in this study is a potential tool for PRRSV surveillance, particularly in populations of growing pigs originating from PRRSV-positive or vaccinated breeding herds., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Formulas and software for calculating sample size for surveys based on individual animal samples are readily available. However, sample size formulas are not available for oral fluids and other aggregate samples that are increasingly used in production settings. Therefore, the objective of this study was to develop sampling guidelines for oral fluid-based porcine reproductive and respiratory syndrome virus (PRRSV) surveys in commercial swine farms. Oral fluid samples were collected in 9 weekly samplings from all pens in 3 barns on one production site beginning shortly after placement of weaned pigs. Samples (n=972) were tested by real-time reverse-transcription PCR (RT-rtPCR) and the binary results analyzed using a piecewise exponential survival model for interval-censored, time-to-event data with misclassification. Thereafter, simulation studies were used to study the barn-level probability of PRRSV detection as a function of sample size, sample allocation (simple random sampling vs fixed spatial sampling), assay diagnostic sensitivity and specificity, and pen-level prevalence. These studies provided estimates of the probability of detection by sample size and within-barn prevalence. Detection using fixed spatial sampling was as good as, or better than, simple random sampling. Sampling multiple barns on a site increased the probability of detection with the number of barns sampled. These results are relevant to PRRSV control or elimination projects at the herd, regional, or national levels, but the results are also broadly applicable to contagious pathogens of swine for which oral fluid tests of equivalent performance are available., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)