John K. Johnson

Detection of Porcine reproductive and respiratory syndrome virus (PRRSV) antibodies in oral fluid specimens using a commercial PRRSV serum antibody enzyme-linked immunosorbent assay

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.

Prolonged Detection of PCV2 and Anti-PCV2 Antibody in Oral Fluids Following Experimental Inoculation

The onset, level and duration of PCV2 and anti-PCV2 antibody in oral fluid were evaluated using samples collected from experimentally inoculated pigs for 98 days post-inoculation (DPI). Pigs (n = 24) were obtained at 3 weeks of age and randomly allocated to 4 treatment pens of 6 pigs each: (i) negative control group; (ii) inoculated with PCV2a (strain ISU 40895) on DPI 0; (iii) inoculated with PCV2a (strain ISU-40895) on DPI 0 and re-challenged at DPIs 35 and 70; (iv) inoculated with PCV2a (ISU-40895), PCV2b (PVG4072) and PCV2a (ISU-4838) on DPIs 0, 35 and 70, respectively. Serum was collected from each animal, and one oral fluid sample was collected from each pen (group) every other day from DPI 2 through DPI 14 and weekly through 98 DPI. Oral fluid samples were assayed for the presence of PCV2 by quantitative polymerase chain reaction (qPCR) and anti-PCV2 IgG, IgA and IgM antibody isotypes by enzyme-linked immunosorbant assay (ELISA). Serum was assayed for anti-PCV2 IgG by ELISA. Anti-PCV2 antibodies (IgG, IgM and IgA) were detected in oral fluid from experimentally inoculated pigs from DPI 14 with IgG and IgA clearly present at 98 DPI. PCV2 was detected in oral fluid samples from all pens of inoculated pigs at 2 DPI. Thereafter, PCV2 was detected in oral fluid throughout 98 DPI. Overall, the data indicated that PCV2 infection in swine is prolonged, persists in the face of an active antibody response and can be efficiently monitored using oral fluid specimens.

Comparison of three enzyme-linked immunosorbent assays to detect Porcine circovirus-2 (PCV-2)-specific antibodies after vaccination or inoculation of pigs with distinct PCV-1 or PCV-2 isolates

Porcine circovirus-2 (PCV-2) serology is frequently used to determine PCV-2 status and optimal timing of PCV-2 vaccination in the field. The objectives of the current study are to determine the diagnostic accuracy of 3 currently available commercial anti-immunoglobulin G (IgG) PCV-2 enzyme-linked immunosorbent assays (ELISAs) and to compare the ability of the 3 assays to detect and differentiate between anti—PCV-2a and anti—PCV-2b antibodies, as well as anti—PCV-2 and anti—PCV-1 antibodies. Fifty-five 3-week-old, conventional pigs were randomly allocated to 7 groups: 1) negative controls (n = 7), 2) PCV-2a (n = 8; inoculated with PCV-2 ISU-40895), 3) PCV-2b (n = 8; inoculated with PCV-2 NC-16845), 4) PCV-1 (n = 8), 5) vaccine A (n = 8; Ingelvac® CircoFLEX™), 6) vaccine B (n = 8; Circumvent® PCV2), and 7) vaccine C (n = 8; Suvaxyn® PCV2 One Dose). Blood samples were collected weekly, and all sera were tested by 3 different anti-PCV-2 IgG ELISAs. The results indicated that all ELISAs had area under the receiver operating curve values greater than 0.94, detected both anti-PCV-2a and −2b antibodies with no differentiation, and did not detect anti-PCV-1 antibodies in infected animals. One of the ELISAs was able to distinguish pigs vaccinated with vaccine B from pigs inoculated with either PCV-2a or PCV-2b.

Interlaboratory comparison of Porcine circovirus-2 indirect immunofluorescent antibody test and enzyme-linked immunosorbent assay results on experimentally infected pigs

A blinded interlaboratory assessment of the diagnostic agreement and accuracy of serologic tests for routine detection of antibodies against Porcine circovirus-2 (PCV-2), including indirect fluorescent antibody tests (IFATs) and enzyme-linked immunosorbent assays (ELISAs) was conducted in 7 North American laboratories. Serum samples were collected weekly, on trial days 0, 7, 14, 21, 28, 35, 42, and 49, from the following groups of animals: 1) negative controls (n = 7), 2) PCV-2a (n = 8), 3) PCV-2b (n = 8), 4) PCV-1 (n = 8), 5) PCV-2 vaccine A (n = 8; Ingelvac® CircoFLEX™), 6) PCV-2 vaccine B (n = 8; Circumvent® PCV2), and 7) PCV-2 vaccine C (n = 8; Suvaxyn® PCV2 One Dose). Results from each laboratory were analyzed by kappa and receiver operating characteristic (ROC) analysis. Kappa analysis indicated that, by trial day 49, IFATs had almost perfect agreement, in-house ELISAs had fair to almost perfect agreement, and commercially available anti–PCV-2 immunoglobulin G ELISAs (I or S) had moderate to substantial agreement. From trial days 14–49, the area under the ROC curve for the 2 laboratories that offered IFATs, the 4 laboratories that offered in-house ELISAs, and the 3 laboratories that used commercially available ELISAs ranged from 0.94 to 1.00, 0.72 to 1.00, and 0.95 to 1.00, respectively. However, test sensitivities varied based on laboratory-specific cutoffs that were used to dichotomize test results.

Association of concurrent porcine circovirus (PCV) 2a and 2b infection with PCV associated disease in vaccinated pigs

Investigations were performed to characterize porcine circovirus (PCV) 2 infection in 10 week old pigs from a case of apparent vaccine failure. Thirty serum samples were collected from affected or non-affected pigs and tested for anti-PCV2 antibodies and PCV2 DNA. To address potential PCV2 vaccine compliance issues, samples were tested for antibodies against baculovirus and Mycoplasma hyopneumoniae antigens present in the PCV2 vaccine utilized in this herd. Both PCV2a and PCV2b DNA were detected in 76.6% (90% positive for PCV2a, 86.6% positive for PCV2b), anti-PCV2 IgG in 90%, anti-baculovirus IgG in 50%, and anti-M. hyopneumoniae IgG in 43.3% of the samples. Frequency of baculovirus and M. hyopneumoniae seropositive pigs was significantly lower in affected pigs. The finding that only 50% of the pigs developed a detectable immune response to vaccination suggests poor vaccine compliance or efficacy. Concurrent PCV2a and PCV2b infection was common and may have resulted in enhanced PCV2 replication.

Evaluation of Screening Assays for the Detection of Influenza A Virus Serum Antibodies in Swine

Increased surveillance of influenza A virus (IAV) infections in human and swine populations is mandated by public health and animal health concerns. Antibody assays have proven useful in previous surveillance programmes because antibodies provide a record of prior exposure and the technology is inexpensive. The objective of this research was to compare the performance of influenza serum antibody assays using samples collected from pigs (vaccinated or unvaccinated) inoculated with either A/Swine/OH/511445/2007 γ H1N1 virus or A/Swine/Illinois/02907/2009 Cluster IV H3N2 virus and followed for 42 days. Weekly serum samples were tested for anti-IAV antibodies using homologous and heterologous haemagglutination-inhibition (HI) assays, commercial swine influenza H1N1 and H3N2 indirect ELISAs, and a commercial influenza nucleoprotein (NP)-blocking ELISA. The homologous HIs showed 100% diagnostic sensitivity, but largely failed to detect infection with the heterologous virus. With diagnostic sensitivities of 1.4% and 4.9%, respectively, the H1N1 and H3N2 indirect ELISAs were ineffective at detecting IAV antibodies in swine infected with the contemporary influenza viruses used in the study. At a cut-off of S/N ≤ 0.60, the sensitivity and specificity of the NP-blocking ELISA were estimated at 95.5% and 99.6%, respectively. Statistically significant factors which affected S/N results include vaccination status, inoculum (virus subtype), day post-inoculation and the interactions between those factors (P < 0.0001). Serum antibodies against NP provide an ideal universal diagnostic screening target and could provide a cost-effective approach for the detection and surveillance of IAV infections in swine populations.

Ring test evaluation of the repeatability and reproducibility of a Porcine reproductive and respiratory syndrome virus oral fluid antibody enzyme-linked immunosorbent assay

The precision of a Porcine reproductive and respiratory syndrome virus (PRRSV) oral fluid antibody enzyme-linked immunosorbent assay (ELISA) was evaluated by calculating reliability coefficients for assay repeatability (within laboratory) and assay reproducibility (between laboratories). Randomly ordered oral fluid samples of known (n = 39) and unknown (n = 224) PRRSV antibody status were tested in 12 diagnostic laboratories. Each laboratory tested the samples twice, first using an antibody ELISA kit and reagents provided to them (phase 1) and then using an ELISA kit and reagents configured in their respective laboratory (phase 2). Repeatability (within laboratory) reliability coefficients calculated using results from samples of known PRRSV antibody status ranged from 0.724 to 0.997 in phase 1 and from 0.953 to 0.998 in phase 2. Reproducibility (between laboratories) reliability coefficients were calculated for 3 conditions: case 1—samples of unknown status (n = 224); case 2—samples of known status (n = 39), and case 3—all samples (n = 263). Among the 3 cases, reliability coefficients ranged from 0.937 to 0.964 in phase 1 and from 0.922 to 0.935 in phase 2. For case 3, it was estimated that 96.67% of the total variation in phase 1 and 93.21% in phase 2 could be attributed to the oral fluid samples themselves. Overall, the PRRSV oral fluid antibody ELISA was highly repeatable and reproducible. The current study supports the routine use of this test in laboratories providing diagnostic service to pig producers.

Evaluation of diagnostic assays for the serological detection of Actinobacillus pleuropneumoniae on samples of known or unknown exposure

Accurate diagnosis of exposure to Actinobacillus pleuropneumoniae is important for maintaining negative farms. In the present study, the ability of a dual-plate complement fixation (CF) assay and 3 commercially available enzyme-linked immunosorbent assays (ELISAs; quad-plate ELISA-1, single-plate ELISA-2, and single-plate ELISA-3) in detecting serological evidence of A. pleuropneumoniae exposure was compared using serum samples of experimentally infected or vaccinated pigs, or field samples from the United States. Forty-two pigs were divided into groups of 2 pigs and were inoculated with 1 of 15 A. pleuropneumoniae strains representing all known serovars of A. pleuropneumoniae, or with Actinobacillus suis, or were vaccinated with a bacterin containing A. pleuropneumoniae serovar 1, 3, 5, or 7. Serum samples collected at the day of inoculation or vaccination and 7, 14, 21, and 28 days later were used to compare the assays. On samples from experimentally infected pigs, the dual-plate CF assay, quad-plate ELISA-1, single-plate ELISA-2, and single-plate ELISA-3 had sensitivities of 0.46, 0.74, 0.13, and 0.13 and specificities of 0.90, 1.0, 1.0, and 1.0, respectively. Vaccinated pigs were identified only by the dual-plate CF assay and the quad-plate ELISA-1. In addition, 90 serum samples with unknown A. pleuropneumoniae exposure collected under field conditions were tested with all assays. The agreement of the 4 assays on field samples was slight to fair. While several assays are available for demonstration of A. pleuropneumoniae exposure, differences in assay targets complicate test choices. Decisions on which assay or combination of assays to use depend on the specific reasons for running the assays.

Evaluation and use of a serological assay for the detection of antibodies to Lawsonia intracellularis in swine

Abstract Porcine proliferative enteropathy (PPE) is a common and economically important gastro-intestinal disease of swine caused by the intracellular bacterium, Lawsonia intracellularis. Conventional tests to detect antibody responses to L. intracellularis include the immuno-peroxidase monolayer assay (IPMA), immuno-fluorescent antibody test (IFAT) and a lipopolysaccharide ELISA (LPS-ELISA). These tests are not commercially available. Therefore, objective of this study is to evaluate the performance of a commercial L. intracellularis blocking ELISA. Performance of the commercial ELISA was compared to the IPMA and LPS-ELISA using serum from experimentally infected animals (N = 40). The prevalence of L. intracellularis sero-positive animals was assessed by comparing suspect and randomly selected sera (N = 394). The commercial ELISA, IPMA and a non-commercial lipopolysaccharide (LPS) LPS-ELISA showed a 95% correlation when tested using experimentally derived known status samples. When compared to the IPMA the sensitivity of the commercial ELISA was 91% while the specificity was 100%. Therefore, the diagnostic sensitivity and specificity of the commercial L. intracellularis ELISA was comparable to the LPS-ELISA and IPMA. A comparison of suspect and randomly selected field samples with the commercial ELISA indicated that L. intracellularis sero-positivity is widespread and does not correlate with possible disease status.

Serological Cross-Reactivity of the Novel H1N1 and Implications for Protection with a Commerical Vaccine

and Implications The recent emergence of the pandemic H1N1 viruses and their being labeled as ‘swine flu’ has had several detrimental effects on the pork industry. Novel H1N1 strains have been recently detected in swine populations in the United States and in other parts of the world. While the public health significance of these findings is unknown, it is important to determine whether existing vaccines or exposure to previously circulating strains of swine influenza will protect pigs against the novel H1N1. Using a partial two-way cross-hemagglutination inhibition (HI) assay, we have determined a swine H1N1 strain present in a commercial vaccine cross-reacted with antiserum specific to the novel H1N1. We have also shown that a field serum sample with a HI high titer to a ’99 H1N1 strain crossreacted strongly with the novel H1N1. Anti-sera which were specific to the non-pandemic H1N1 strains also crossreacted with the novel H1N1. Therefore, we conclude that current vaccines and circulating non-pandemic H1N1 field strains will provide at least partial protection against the novel H1N1 virus in pigs.