The purpose of the present study was to evaluate the diagnostic performance of a commercial serum antibody enzyme-linked immunosorbent assay (ELISA) modified to detect anti– Porcine reproductive and respiratory syndrome virus (PRRSV) antibodies in pen-based oral fluid specimens. Experimental and field oral fluid samples of defined status in reference to exposure of swine with PRRSV were used to derive the kinetics of detectable concentrations of antibody against PRRSV. Immunoglobulin (Ig)M and IgA were readily detected in oral fluid specimens from populations in which PRRSV infection was synchronized among all individuals but not in samples collected in commecial herds. In contrast, IgG was readily detected at diagnostically useful levels in both experimental and field samples for up to 126 days. Estimates of the IgG oral fluid ELISA performance were based on results from testing positive oral fluid samples ( n = 492) from experimentally inoculated pigs ( n = 251) and field samples ( n = 241) and negative oral fluid samples ( n = 367) from experimentally inoculated pigs ( n = 84) and field samples ( n = 283). Receiver operating characteristic analysis estimated the diagnostic sensitivity and specificity of the assay as 94.7% (95% confidence interval [CI]: 92.4, 96.5) and 100% (95% CI: 99.0, 100.0), respectively, at a sample-to-positive ratio cutoff of ≥0.40. The results of the study suggest that the IgG oral fluid ELISA can provide efficient, cost-effective PRRSV monitoring in commercial herds and PRRSV surveillance in elimination programs.
Pen-based oral fluid sampling has proven to be an efficient method for surveillance of infectious diseases in swine populations. To better interpret diagnostic results, the performance of oral fluid assays (antibody- and nucleic acid-based) must be established for pen-based oral fluid samples. Therefore, the objective of the current study was to determine the probability of detecting Porcine reproductive and respiratory syndrome virus (PRRSV) infection in pen-based oral fluid samples from pens of known PRRSV prevalence. In 1 commercial swine barn, 25 pens were assigned to 1 of 5 levels of PRRSV prevalence (0%, 4%, 12%, 20%, or 36%) by placing a fixed number (0, 1, 3, 5, or 9) of PRRSV-positive pigs (14 days post PRRSV modified live virus vaccination) in each pen. Prior to placement of the vaccinated pigs, 1 oral fluid sample was collected from each pen. Thereafter, 5 oral fluid samples were collected from each pen, for a total of 150 samples. To confirm individual pig PRRSV status, serum samples from the PRRSV-negative pigs ( n = 535) and the PRRSV vaccinated pigs ( n = 90) were tested for PRRSV antibodies and PRRSV RNA. The 150 pen-based oral fluid samples were assayed for PRRSV antibody and PRRSV RNA at 6 laboratories. Among the 100 samples from pens containing ≥1 positive pig (≥4% prevalence) and tested at the 6 laboratories, the mean positivity was 62% for PRRSV RNA and 61% for PRRSV antibody. These results support the use of pen-based oral fluid sampling for PRRSV surveillance in commercial pig populations.
Influenza A virus (IAV) surveillance using pre-weaning oral fluid samples from litters of piglets was evaluated in four ˜12 500 sow and IAV-vaccinated, breeding herds. Oral fluid samples were collected from 600 litters and serum samples from their dams at weaning. Litter oral fluid samples were tested for IAV by virus isolation, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), RT-PCR subtyping and sequencing. Commercial nucleoprotein (NP) enzyme-linked immunosorbent assay (ELISA) kits and NP isotype-specific assays (IgM, IgA and IgG) were used to characterize NP antibody in litter oral fluid and sow serum. All litter oral fluid specimens (n = 600) were negative by virus isolation. Twenty-five oral fluid samples (25/600 = 4.2%) were qRT-PCR positive based on screening (Laboratory 1) and confirmatory testing (Laboratory 2). No hemagglutinin (HA) and neuraminidase (NA) gene sequences were obtained, but matrix (M) gene sequences were obtained for all qRT-PCR-positive samples submitted for sequencing (n = 18). Genetic analysis revealed that all M genes sequences were identical (GenBank accession no. KF487544) and belonged to the triple reassortant influenza A virus M gene (TRIG M) previously identified in swine. The proportion of IgM- and IgA-positive samples was significantly higher in sow serum and litter oral fluid samples, respectively (P < 0.01). Consistent with the extensive use of IAV vaccine, no difference was detected in the proportion of IgG- and blocking ELISA-positive sow serum and litter oral fluids. This study supported the use of oral fluid sampling as a means of conducting IAV surveillance in pig populations and demonstrated the inapparent circulation of IAV in piglets. Future work on IAV oral fluid diagnostics should focus on improved procedures for virus isolation, subtyping and sequencing of HA and NA genes. The role of antibody in IAV surveillance remains to be elucidated, but longitudinal assessment of specific antibody has the potential to provide information regarding patterns of infection, vaccination status and herd immunity.
BackgroundThe object of this study was to describe and contrast the kinetics of the humoral response in serum and oral fluid specimens during acute porcine reproductive and respiratory syndrome virus (PRRSV) infection. The study involved three trials of 24 boars each. Boars were intramuscularly inoculated with a commercial modified live virus (MLV) vaccine (Trial 1), a Type 1 PRRSV field isolated (Trial 2), or a Type 2 PRRSV field isolate (Trial 3). Oral fluid samples were collected from individual boars on day post inoculation (DPI) -7 and 0 to 21. Serum samples were collected from all boars on DPI −7, 0, 7, 14, 21 and from 4 randomly selected boars on DPI 3, 5, 10, and 17. Thereafter, serum and oral fluid were assayed for PRRSV antibody using antibody isotype-specific ELISAs (IgM, IgA, IgG) adapted to serum or oral fluid.ResultsStatistically significant differences in viral replication and antibody responses were observed among the three trials in both serum and oral fluid specimens. PRRSV serum IgM, IgA, and IgG were first detected in samples collected on DPI 7, 10, and 10, respectively. Oral fluid IgM, IgA, and IgG were detected in samples collected between DPI 3 to 10, 7 to 10, and 8 to 14, respectively.ConclusionsThis study enhanced our knowledge of the PRRSV humoral immune response and provided a broader foundation for the development and application of oral fluid antibody-based diagnostics.
In commercial swine populations, influenza is an important component of the porcine respiratory disease complex (PRDC) and a pathogen with major economic impact. Previously, a commercial blocking ELISA (FlockChek(™) Avian Influenza Virus MultiS-Screen(®) Antibody Test Kit, IDEXX Laboratories, Inc., Westbrook, ME, USA) designed to detect influenza A nucleoprotein (NP) antibodies in avian serum was shown to accurately detect NP antibodies in swine serum. The purpose of this study was to determine whether this assay could detect NP antibodies in swine oral fluid samples. Initially, the procedure for performing the NP-blocking ELISA on oral fluid was modified from the serum testing protocol by changing sample dilution, sample volume, incubation time and incubation temperature. The detection of NP antibody was then evaluated using pen-based oral fluid samples (n = 182) from pigs inoculated with either influenza A virus subtype H1N1 or H3N2 under experimental conditions and followed for 42 days post inoculation (DPI). NP antibodies in oral fluid were detected from DPI 7 to 42 in all inoculated groups, that is, the mean sample-to-negative (S/N) ratio of influenza-inoculated pigs was significantly different (P < 0.0001) from uninoculated controls (unvaccinated or vaccinated-uninoculated groups) through this period. Oral fluid versus serum S/N ratios from the same pen showed a correlation of 0.796 (Pearson's correlation coefficient, P < 0.0001). The results showed that oral fluid samples from influenza virus-infected pigs contained detectable levels of NP antibodies for ≥42 DPI. Future research will be required to determine whether this approach could be used to monitor the circulation of influenza virus in commercial pig populations.
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