Detection of influenza A virus nucleoprotein antibodies in oral fluid specimens from pigs infected under experimental conditions using a blocking ELISA.

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.

Differences in capabilities of different enzyme immunoassays to detect anti-hepatitis E virus immunoglobulin G in pigs infected experimentally with hepatitis E virus genotype 3 or 4 and in pigs with unknown exposure.

Hepatitis E virus (HEV), a major cause of acute viral hepatitis in humans in many developing countries, is highly prevalent in the pig population worldwide. The objective of this study was to assess the capability of three porcine prototypes of a human enzyme-linked immunosorbent assay (ELISA), an in-house ELISA and a line-immunoassay (LIA) to detect anti-HEV antibodies in pigs infected experimentally with HEV (n = 57), known to be negative for HEV infection (n = 27), or with unknown exposure to HEV infection (field samples, n = 90). All 27 samples from non-infected pigs were negative with all five assays. The earliest detection of anti-HEV antibodies occurred at 14 days post-inoculation (dpi) with four of five assays. From 42 dpi, all samples from infected pigs were detected correctly as anti-HEV positive. Kappa analysis demonstrated substantial agreement among tests (0.62-1.00) at 14 dpi and complete agreement (1.00) at 56 dpi. The overall area under the curve for all quantitative tests as determined by receiver operator characteristic analysis ranged from 0.794 to 0.831 indicating moderate accuracy. The results showed that all five assays can detect anti-HEV IgG antibodies accurately in pigs infected experimentally with HEV. In field samples, a higher prevalence of anti-HEV IgG was found in breeding herds than in growing pigs (100% versus 66.7-93.9%). These serological assays should be very useful in veterinary diagnostic labs for HEV diagnosis in swine.

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.

Effect of porcine circovirus type 2 (PCV2) vaccination on porcine reproductive and respiratory syndrome virus (PRRSV) and PCV2 coinfection.

The objectives were to determine if PCV2 vaccination is effective in reducing disease and lesions associated with PRRSV and PCV2 coinfection and if there is a difference between intradermal (ID) and intramuscular (IM) route of PCV2 vaccination. Seventy-four, 21-day-old pigs were randomly allocated into one of six groups. On day 0, pigs were vaccinated with 2ml Suvaxyn PCV2 One Dose (Fort Dodge Animal Health, Inc.) by intramuscular (VAC-M-COINF) or intradermal (VAC-D-COINF) routes. On day 28, pigs were either singularly (PRRSV-only, PCV2-only) or coinfected (COINF) with PRRSV and PCV2. All pigs in all groups were necropsied on day 42. All vaccinated pigs seroconverted (IgM, IgG, and neutralizing antibodies) to PCV2 between 14 and 28 days post-vaccination. After challenge, all groups inoculated with PRRSV had reduced average daily gain compared to CONTROLS and PCV2-only (P<0.001). COINF pigs had significantly (P<0.05) reduced anti-PCV2-IgG antibody levels and neutralizing antibody levels compared to both vaccinated groups. COINF pigs had more severe lung lesions compared to VAC-M-COINF (P<0.05). COINF pigs had higher amounts of PCV2 DNA in serum samples and feces (P<0.05) and increased amounts of PCV2 in lymphoid tissues (P<0.05) compared to both vaccinated groups. In summary, PCV2 vaccination was effective at inducing a neutralizing antibody response and significantly reducing PCV2-associated lesions and PCV2 viremia in pigs coinfected with PCV2 and PRRSV. Differences between intradermal and intramuscular routes of vaccine administration were not observed.