Influenza A Virus Surveillance Based on Pre-Weaning Piglet Oral Fluid Samples.

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.

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.

Comparison of specimens for detection of porcine reproductive and respiratory syndrome virus infection in boar studs.

Porcine reproductive and respiratory syndrome virus (PRRSV)-contaminated semen from boars is a route of transmission to females, and early detection of PRRSV infection in boars is a key component in sow farm biosecurity. The purpose of this study was to determine the optimum diagnostic specimen(s) for the detection of acute PRRSV infection in boars. Individually housed boars (n = 15) were trained for semen and oral fluid collection and then vaccinated with a commercial PRRSV modified live virus vaccine. Starting on the day of vaccination and for 14 days thereafter, oral fluid specimens were collected daily from all boars. The 15 boars were subdivided into three groups of 5, and serum, blood swabs and 'frothy saliva' were collected at the time of semen collection on a 3-day rotation. Frothy saliva, derived from the submandibular salivary gland, is produced by aroused boars. Semen was centrifuged, and semen supernatant and cell fractions were tested separately. All samples were randomly ordered and then tested by PRRSV real-time quantitative reverse-transcription polymerase chain reaction assay (rRT-PCR) and PRRSV antibody ELISA. In this study, a comparison of serum, blood swab, and oral fluid rRT-PCR results found no statistically significant differences in the onset of detection or proportion of positives, but serum was numerically superior to oral fluids for early detection. Serum and oral fluid provided identical rRT-PCR results at ≥ 5 day post-vaccination. Likewise, the onset of detection of PRRSV antibody in serum, oral fluid and frothy saliva was statistically equivalent, with serum results again showing a numerical advantage. These results showed that the highest assurance of providing PRRSV-negative semen to sow farms should be based on rRT-PCR testing of serum collected at the time of semen collection. This approach can be augmented with oral fluid sampling from a random selection of uncollected boars to provide for statistically valid surveillance of the boar stud.

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.

Kinetics of influenza A virus nucleoprotein antibody (IgM, IgA, and IgG) in serum and oral fluid specimens from pigs infected under experimental conditions.

Indirect influenza A virus (IAV) nucleoprotein (NP) antibody ELISAs were used to compare the kinetics of the NP IgM, IgA, and IgG responses in serum and pen-based oral fluid samples collected from 82 pigs followed for 42 days post inoculation (DPI). Treatment categories included vaccination (0, 1) and inoculation (0, 1) with contemporary H1N1 or H3N2 isolates. Antibody ontogeny was markedly affected by vaccination status, but no significant differences were detected between H1N1 and H3N2 inoculated groups of the same vaccination status (0, 1) in IgM, IgA, or IgG responses. Therefore, these data were combined in subsequent analyses. The correlation between serum and oral fluid responses was evaluated using the pen-based oral fluid sample-to-positive (S/P) ratios versus the mean serum S/P ratios of pigs within the pen. IgM responses in serum and oral fluid were highly correlated in unvaccinated groups (r=0.810), as were serum and oral fluid IgG responses in both unvaccinated (r=0.839) and vaccinated (r=0.856) groups. In contrast, IgM responses were not correlated in vaccinated groups and the correlation between serum and oral fluid IgA was weak (r∼0.3), regardless of vaccination status. In general, vaccinated animals exhibited a suppressed IgM response and accelerated IgG response. The results from this study demonstrated that NP-specific IgM, IgA, and IgG antibody were detectable in serum and oral fluid and their ontogeny was influenced by vaccination status, the time course of the infection, and specimen type.