Effect of pooled tracheal sample testing on the probability of Mycoplasma hyopneumoniae detection.

Tracheal pooling for Mycoplasma hyopneumoniae (M. hyopneumoniae) DNA detection allows for decreased diagnostic cost, one of the main constraints in surveillance programs. The objectives of this study were to estimate the sensitivity of pooled-sample testing for the detection of M. hyopneumoniae in tracheal samples and to develop probability of M. hyopneumoniae detection estimates for tracheal samples pooled by 3, 5, and 10. A total of 48 M. hyopneumoniae PCR-positive field samples were pooled 3-, 5-, and 10-times using field M. hyopneumoniae DNA-negative samples and tested in triplicate. The sensitivity was estimated at 0.96 (95% credible interval [Cred. Int.]: 0.93, 0.98) for pools of 3, 0.95 (95% Cred. Int: 0.92, 0.98) for pools of 5, and 0.93 (95% Cred. Int.: 0.89, 0.96) for pools of 10. All pool sizes resulted in PCR-positive if the individual tracheal sample Ct value was < 33. Additionally, there was no significant decrease in the probability of detecting at least one M. hyopneumoniae-infected pig given any pool size (3, 5, or 10) of tracheal swabs. Furthermore, this manuscript applies the probability of detection estimates to various real-life diagnostic testing scenarios. Combining increased total animals sampled with pooling can be a cost-effective tool to maximize the performance of M. hyopneumoniae surveillance programs.

Pen-Based Swine Oral Fluid Samples Contain Both Environmental and Pig-Derived Targets.

Laboratory methods for detecting specific pathogens in oral fluids are widely reported, but there is little research on the oral fluid sampling process itself. In this study, a fluorescent tracer (diluted red food coloring) was used to test the transfer of a target directly from pigs or indirectly from the environment to pen-based oral fluid samples. Pens of ~30, ~60, and ~125 14-week-old pigs (32 pens/size) on commercial swine farms received one of two treatments: (1) pig exposure, i.e., ~3.5 mL of tracer solution sprayed into the mouth of 10% of the pigs in the pen; (2) environmental exposure, i.e., 20 mL of tracer solution was poured on the floor in the center of the pen. Oral fluids collected one day prior to treatment (baseline fluorescence control) and immediately after treatment were tested for fluorescence. Data were evaluated by receiver operating characteristic (ROC) analysis, with Youden's J statistic used to set a threshold. Pretreatment oral fluid samples with fluorescence responses above the ROC threshold were removed from further analysis (7 of 96 samples). Based on the ROC analyses, oral fluid samples from 78 of 89 pens (87.6%), contained red food coloring, including 43 of 47 (91.5%) pens receiving pig exposure and 35 of 42 (83.3%) pens receiving environmental exposure. Thus, oral fluid samples contain both pig-derived and environmental targets. This methodology provides a safe and quantifiable method to evaluate oral fluid sampling vis-à-vis pen behavior, pen size, sampling protocol, and target distribution in the pen.

Generation of a Commercial-Scale Founder Population of Porcine Reproductive and Respiratory Syndrome Virus Resistant Pigs Using CRISPR-Cas.

Disease resistance genes in livestock provide health benefits to animals and opportunities for farmers to meet the growing demand for affordable, high-quality protein. Previously, researchers used gene editing to modify the porcine CD163 gene and demonstrated resistance to a harmful virus that causes porcine reproductive and respiratory syndrome (PRRS). To maximize potential benefits, this disease resistance trait needs to be present in commercially relevant breeding populations for multiplication and distribution of pigs. Toward this goal, a first-of-its-kind, scaled gene editing program was established to introduce a single modified CD163 allele into four genetically diverse, elite porcine lines. This effort produced healthy pigs that resisted PRRS virus infection as determined by macrophage and animal challenges. This founder population will be used for additional disease and trait testing, multiplication, and commercial distribution upon regulatory approval. Applying CRISPR-Cas to eliminate a viral disease represents a major step toward improving animal health.

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.

Herd-level infectious disease surveillance of livestock populations using aggregate samples.

All sectors of livestock production are in the process of shifting from small populations on many farms to large populations on fewer farms. A concurrent shift has occurred in the number of livestock moved across political boundaries. The unintended consequence of these changes has been the appearance of multifactorial diseases that are resistant to traditional methods of prevention and control. The need to understand complex animal health conditions mandates a shift toward the collection of longitudinal animal health data. Historically, collection of such data has frustrated and challenged animal health specialists. A promising trend in the evolution toward more efficient and effective livestock disease surveillance is the increased use of aggregate samples, e.g. bulk tank milk and oral fluid specimens. These sample types provide the means to monitor disease, estimate herd prevalence, and evaluate spatiotemporal trends in disease distribution. Thus, this article provides an overview of the use of bulk tank milk and pen-based oral fluids in the surveillance of livestock populations for infectious diseases.

Detection of porcine reproductive and respiratory syndrome virus (PRRSV)-specific IgM-IgA in oral fluid samples reveals PRRSV infection in the presence of maternal antibody.

The ontogeny of PRRSV antibody in oral fluids has been described using isotype-specific ELISAs. Mirroring the serum response, IgM appears in oral fluid by 7days post inoculation (DPI), IgA after 7 DPI, and IgG by 9 to 10 DPI. Commercial PRRSV ELISAs target the detection of IgG because the higher concentration of IgG relative to other isotypes provides the best diagnostic discrimination. Oral fluids are increasingly used for PRRSV surveillance in commercial herds, but in younger pigs, a positive ELISA result may be due either to maternal antibody or to antibody produced by the pigs in response to infection. To address this issue, a combined IgM-IgA PRRSV oral fluid ELISA was developed and evaluated for its capacity to detect pig-derived PRRSV antibody in the presence of maternal antibody. Two longitudinal studies were conducted. In Study 1 (modified-live PRRS vaccinated pigs), testing of individual pig oral fluid samples by isotype-specific ELISAs demonstrated that the combined IgM-IgA PRRSV ELISA provided better discrimination than individual IgM or IgA ELISAs. In Study 2 (field data), testing of pen-based oral fluid samples confirmed the findings in Study 1 and established that the IgM-IgA ELISA was able to detect antibody produced by pigs in response to wild-type PRRSV infection, despite the presence of maternal IgG. Overall, the combined PRRSV IgM-IgA oral fluid ELISA described in this study is a potential tool for PRRSV surveillance, particularly in populations of growing pigs originating from PRRSV-positive or vaccinated breeding herds.

Sampling guidelines for oral fluid-based surveys of group-housed animals.

Formulas and software for calculating sample size for surveys based on individual animal samples are readily available. However, sample size formulas are not available for oral fluids and other aggregate samples that are increasingly used in production settings. Therefore, the objective of this study was to develop sampling guidelines for oral fluid-based porcine reproductive and respiratory syndrome virus (PRRSV) surveys in commercial swine farms. Oral fluid samples were collected in 9 weekly samplings from all pens in 3 barns on one production site beginning shortly after placement of weaned pigs. Samples (n=972) were tested by real-time reverse-transcription PCR (RT-rtPCR) and the binary results analyzed using a piecewise exponential survival model for interval-censored, time-to-event data with misclassification. Thereafter, simulation studies were used to study the barn-level probability of PRRSV detection as a function of sample size, sample allocation (simple random sampling vs fixed spatial sampling), assay diagnostic sensitivity and specificity, and pen-level prevalence. These studies provided estimates of the probability of detection by sample size and within-barn prevalence. Detection using fixed spatial sampling was as good as, or better than, simple random sampling. Sampling multiple barns on a site increased the probability of detection with the number of barns sampled. These results are relevant to PRRSV control or elimination projects at the herd, regional, or national levels, but the results are also broadly applicable to contagious pathogens of swine for which oral fluid tests of equivalent performance are available.