Effect of testing protocol and within-pen prevalence on the detection of Mycoplasma hyopneumoniae DNA in oral fluid samples.

Combinations of 2 nucleic acid extractions and 3 Mycoplasma hyopneumoniae (MHP) PCRs (namely Protocol 1, 2, 3, and 4) were compared in terms of the probability of detecting DNA in pen-based oral fluid samples as a function of within-pen MHP prevalence. Oral fluid samples were created by randomly assigning 39 7-week old pigs to one of 5 pens, i.e., negative control pen (3 pigs) and 4 pens of 9 pigs each that differed in the proportion of MHP-inoculated pigs (1, 3, 6, or 9). Deep tracheal swabs were collected twice weekly to establish individual pig MHP infection status and derive within-pen prevalence estimation. On DPI 3, tracheal swabs from 15 of 19 inoculated pigs were MHP DNA positive. Oral fluids (n = 320) were collected daily from - 4 to 59 days post inoculation (DPI). Using a piecewise exponential model to account for within-pen transmission dynamics followed by a mixed-effect logistic regression, the probability of detecting MHP DNA in oral fluids was positively associated with within-pen prevalence (P < 0.0001) and differed among test protocols. MHP DNA was detected in 173 oral fluid samples with Protocol 3 versus 148, 134, and 101 with Protocols 4, 2, and 1, respectively. At 100% within-pen prevalence, the probability of detecting MHP DNA in oral fluids was highest using Protocol 3 (95.7%), followed by Protocols 4 (70.1%), 2 (60.1%), and 1 (34.0%). The fact that PCR protocols performed differently suggests that further improvements in extraction methods and MHP PCRs are possible. In the field, the dynamics of MHP infections should be taken into account if using oral fluid samples in surveillance.

An Assessment of Diagnostic Assays and Sample Types in the Detection of an Attenuated Genotype 5 African Swine Fever Virus in European Pigs over a 3-Month Period.

African swine fever virus causes hemorrhagic disease in swine. Attenuated strains are reported in Africa, Europe, and Asia. Few studies on the diagnostic detection of attenuated ASF viruses are available. Two groups of pigs were inoculated with an attenuated ASFV. Group 2 was also vaccinated with an attenuated porcine reproductive and respiratory syndrome virus vaccine. Commercially available ELISA, as well as extraction and qPCR assays, were used to detect antibodies in serum and oral fluids (OF) and nucleic acid in buccal swabs, tonsillar scrapings, OF, and blood samples collected over 93 days, respectively. After 12 dpi, serum (88.9% to 90.9%) in Group 1 was significantly better for antibody detection than OF (0.7% to 68.4%). Group 1's overall qPCR detection was highest in blood (48.7%) and OF (44.2%), with the highest detection in blood (85.2%) from 8 to 21 days post inoculation (dpi) and in OF (83.3%) from 1 to 7 dpi. Group 2's results were not significantly different from Group 1, but detection rates were lower overall. Early detection of attenuated ASFV variants requires active surveillance in apparently healthy animals and is only reliable at the herd level. Likewise, antibody testing will be needed to prove freedom from disease.

Mycoplasma hyopneumoniae Surveillance in Pig Populations: Establishing Sampling Guidelines for Detection in Growing Pigs.

Antemortem detection of Mycoplasma hyopneumoniae infection in swine production systems has relied on antibody testing, but the availability of tests based on DNA detection and novel diagnostic specimens, e.g., tracheal swabs and oral fluids, has the potential to improve M. hyopneumoniae surveillance. A field study was performed over a 14-week period during which 10 pigs in one pen at the center of a room with 1,250 6-week-old pigs housed in 46 pens were intratracheally inoculated with M. hyopneumoniae Thereafter, one tracheal sample, four serum samples, and one oral fluid sample were collected from every pen at 2-week intervals. Tracheal and oral fluid samples were tested for M. hyopneumoniae DNA and serum samples for M. hyopneumoniae antibody. Test results were modeled using a hierarchical Bayesian model, based on a latent spatial piecewise exponential survival model, to estimate the probability of detection by within-pen prevalence, number of positive pens in the barn, sample allocation, sample size, and sample type over time. Analysis showed that tracheal samples provided the earliest detection, especially at large sample sizes. While serum samples are more commonly collected and are less expensive to test, high probability of detection estimates were only obtained 30 days postexposure at large sample sizes. In all scenarios, probability of detection estimates for oral fluids within 30 days were significantly lower than those for tracheal and serum samples. Ultimately, the choice of specimen type, sample number, and assay will depend on testing objectives and economics, but the estimates provided here will assist in the design of M. hyopneumoniae surveillance and monitoring programs for different situations.

Detection of anti-influenza A nucleoprotein antibodies in pigs using a commercial influenza epitope-blocking enzyme-linked immunosorbent assay developed for avian species.

Influenza virus causes acute respiratory disease in pigs and is of concern for its potential public health significance. Many subtypes of influenza virus have been isolated from pigs, and the virus continues to evolve in swine populations. Current antibody assays have limited antigenic recognition, and accurate, broad-spectrum, high through-put screening tests are needed to detect infections in swine herds and to aid in the implementation of control measures. In the current study, a commercial blocking enzyme-linked immunosorbent assay (ELISA) developed for the detection of Influenza A virus nucleoprotein antibodies in avian species was evaluated for the detection of anti-influenza serum antibodies in swine. Serum samples used to evaluate the test were archived samples from influenza research conducted at the U.S. Department of Agriculture-Agricultural Research Service-National Animal Disease Center and included samples from influenza-inoculated pigs (H1N1, H1N2, H2N3, and H3N2), contact-infected pigs, vaccinated pigs, and negative controls. Based on samples of known status (n = 453), a receiver operating characteristic (ROC) curve analysis of the ELISA results estimated the optimized diagnostic sensitivity and specificity at 96.6% (95% confidence interval [CI]: 92.3, 98.9) and 99.3% (95% CI: 97.6, 99.9), respectively. By using the cutoff established in the ROC analysis, the assay was evaluated in pigs infected with 2 isolates of the 2009 pandemic H1N1 virus. Overall, the assay showed excellent diagnostic performance against the range of influenza subtypes investigated and could serve as a useful screening assay for swine.