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.

Pigs lacking the SRCR5 domain of CD163 protein demonstrate heritable resistance to the PRRS virus and no changes in animal performance from birth to maturity.

Porcine reproductive and respiratory syndrome (PRRS) is one of the world's most persistent viral pig diseases, with a significant economic impact on the pig industry. PRRS affects pigs of all ages, causing late-term abortions and stillbirths in sows, respiratory disease in piglets, and increased susceptibility to secondary bacterial infection with a high mortality rate. PRRS disease is caused by a positive single-stranded RNA PRRS virus (PRRSV), which has a narrow host-cell tropism limited to monocyte-macrophage lineage cells. Several studies demonstrated that the removal of CD163 protein or, as a minimum, its scavenger receptor cysteine-rich domain 5 (SRCR5) precludes the viral genome release, conferring resistance to PRRSV in live animals. Today, very limited information exists about the impact of such edits on animal performance from birth to maturity in pigs. Using CRISPR-Cas9 with dual-guide RNAs and non-homologous end joining (NHEJ), first-generation (E0) pigs were produced with a deletion of exon 7 in the CD163 gene. The selected pigs were bred to produce the next three generations of pigs to establish multiple lines of pigs homozygous for the edited allele, thereby confirming that the CD163 gene with removed exon 7 was stable during multiple breeding cycles. The pigs were evaluated relative to non-edited pigs from birth to maturity, including any potential changes in meat composition and resistance to PRRSV. This study demonstrates that removing the SRCR5 domain from the CD163 protein confers resistance to PRRSV and, relative to unedited pigs, resulted in no detected differences in meat composition and no changes in the growth rate, health, and ability to farrow. Together, these results support the targeted use of gene editing in livestock animals to address significant diseases without adversely impacting the health and well-being of the animals or the food products derived from them.

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.

Adapting a porcine reproductive and respiratory syndrome virus (PRRSV) oral fluid antibody ELISA to routine surveillance.

Distinct from tests used in diagnostics, tests used in surveillance must provide for detection while avoiding false alarms, i.e., acceptable diagnostic sensitivity but high diagnostic specificity. In the case of the reproductive and respiratory syndrome virus (PRRSV), RNA detection meets these requirements during the period of viremia, but antibody detection better meets these requirements in the post-viremic stage of the infection. Using the manufacturer's recommended cut-off (S/P ≥ 0.4), the diagnostic specificity of a PRRSV oral fluid antibody ELISA (IDEXX Laboratories, Inc., Westbrook, ME, USA) evaluated in this study was previously reported as ≥ 97 %. The aim of this study was to improve its use in surveillance by identifying a cut-off that would increase diagnostic specificity yet minimally impact its diagnostic sensitivity. Three sample sets were used to achieve this goal: oral fluids (n = 596) from pigs vaccinated with a modified live PRRSV vaccine under experimental conditions, field oral fluids (n = 1574) from 94 production sites of known negative status, and field oral fluids (n = 1380) from 211 sites of unknown PRRSV status. Based on the analysis of samples of known status (experimental samples and field samples from negative sites), a cut-off of S/P ≥ 1.0 resulted in a diagnostic specificity of 99.2 (95 % CI: 98.8, 99.7) and a diagnostic sensitivity of 96.5 (95 % CI: 85.2, 99.2). Among 211 sites of unknown status, 81 sites were classified as antibody positive using the manufacturer's cut-off; 20 of which were reclassified as negative using a cut-off of S/P ≥ 1.0. Further analysis showed that these 20 sites had a small proportion of samples (18.0 %) with S/P values just exceeding the manufacturer's cut-off (x̄ = 0.5). Whereas the remainder of positive sites (n = 61) had a high proportion of samples (76.3 %) with high S/P values (x̄ = 6.6). Thus, the manufacturer's cut-off (S/P ≥ 0.4) is appropriate for diagnostic applications, but a cut-off of S/P ≥ 1.0 provided the higher specificity required for surveillance. A previously unreported finding in this study was a statistically significant association between unexpected reactors and specific production sites and animal ages or stages. While beyond the scope of this study, these data suggested that certain animal husbandry or production practices may be associated with non-specific reactions.

Effects of sample handling on the detection of porcine reproductive and respiratory syndrome virus in oral fluids by reverse-transcription real-time PCR.

We evaluated effects of handling procedures on detection of porcine reproductive and respiratory syndrome virus (PRRSV) in oral fluids (OFs) by reverse-transcription real-time PCR (RT-rtPCR). The experiments were conducted using a composite sample of PRRSV-positive OF collected from 5-wk-old pigs vaccinated 15 d earlier with a modified-live PRRSV vaccine. Five pre-extraction sample-handling steps and all combinations thereof were evaluated: 1) thaw temperature (4°C or 25°C); 2) sample diluent (1:1 dilution with nuclease-free water or guanidinium thiocyanate-phenol); 3a) sonication of the sample (yes or no); 3b) temperature (4°C or 25°C) at which step 3a was conducted; and 4) temperature at which the sample was maintained after step 3b and until RNA extraction was initiated (4°C or 25°C). All combinations of the 5 sample-handling steps (i.e., 32 unique treatments) were tested in a completely randomized factorial design with 4 replicates and 1 negative control for each treatment. The entire experiment was repeated on 5 separate days to produce a total of 800 PRRSV RT-rtPCR results. Binary (positive or negative) data were analyzed by logistic regression and results (Ct) were analyzed using a generalized linear model. Overall, 1 false-positive result was observed among 160 negative controls (99.4% specificity), and 85 false-negative results were observed among the 640 known-positive samples (86.7% sensitivity). The most significant factor affecting test outcome was thaw temperature (4°C or 25°C); samples thawed at 4°C had higher positivity rate (94% vs. 80%, p < 0.0001) and lower Ct (36.2 vs. 37.5, p < 0.0001).

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.

Porcine epidemic diarrhea virus (PEDV) detection and antibody response in commercial growing pigs.

BACKGROUND: Longitudinal samples from two production sites were used to (1) describe the pattern of PEDV shedding (rRT-PCR) in individual rectal swabs, pen fecal samples, and pen oral fluids (OF); (2) describe the kinetics of PEDV antibody by ELISA (IgA, IgG) testing of pig serum and pen oral fluid samples; and (3) establish cutoffs and performance estimates for PEDV WV ELISAs (IgA, IgG). Site One was PEDV positive; Site Two was PEDV negative. On Site One, pen samples (feces and oral fluids) and pig samples (rectal swabs and sera) were collected both before and after the population was exposed to PEDV. RESULTS: On Site Two, pen oral fluid samples and individual pig serum samples were negative for both PEDV antibody and nucleic acid. On Site One, PEDV was detected by rRT-PCR at 6 days post exposure (DPE) in all sample types. The last rRT-PCR positives were detected in rectal swabs and oral fluids on 69 DPE. IgG and IgA were detected in oral fluids and serum samples by 13 DPE. Analysis of the PEDV serum IgG WV ELISA data showed that a sample-to-positive (S/P) cutoff of ≥ 0.80 provided a diagnostic sensitivity of 0.87 (95% CI: 0.82, 0.91) and specificity of 0.99 (95% CI: 0.98, 1.00). Serum IgG results declined slowly over the monitoring period, with 60% of serum samples positive (S/P ≥ 0.80) at the final sampling on 111 DPE. Analysis of the PEDV oral fluid IgA WV ELISA found that a cutoff of S/P ≥ 0.80 provided a diagnostic sensitivity of 1.00 (95% CI: 0.92, 1.00) and a diagnostic specificity of 1.00 (95% CI: 0.99, 1.00). The oral fluid IgA response increased through 96 DPE and began to decline at the last sampling on 111 DPE. CONCLUSIONS: This study showed that oral fluid-based testing could provide an easy and "animal-friendly" approach to sample collection for nucleic acid and/or antibody-based surveillance of PEDV in swine populations.

Serological and Molecular Detection of Senecavirus A Associated with an Outbreak of Swine Idiopathic Vesicular Disease and Neonatal Mortality.

We performed a longitudinal field study in a swine breeding herd that presented with an outbreak of vesicular disease (VD) that was associated with an increase in neonatal mortality. Initially, a USDA Foreign Animal Disease (FAD) investigation confirmed the presence of Senecavirus A (SVA) and ruled out the presence of exotic agents that produce vesicular lesions, e.g., foot-and-mouth disease virus and others. Subsequently, serum samples, tonsil swabs, and feces were collected from sows (n = 22) and their piglets (n = 33) beginning 1 week after the onset of the clinical outbreak and weekly for 6 weeks. The presence of SVA RNA was evaluated in all specimens collected by reverse transcriptase quantitative PCR (RT-qPCR) targeting a conserved region of the 5' untranslated region (5'-UTR). The serological response (IgG) to SVA was evaluated by the weekly testing of sow and piglet serum samples on a SVA VP1 recombinant protein (rVP1) indirect enzyme-linked immunosorbent assay (ELISA). The rVP1 ELISA detected seroconversion against SVA in clinically affected and non-clinically affected sows at early stages of the outbreak as well as maternal SVA antibodies in offspring. Overall, the absence of vesicles (gross lesions) in SVA-infected animals and the variability of RT-qPCR results among specimen type demonstrated that a diagnostic algorithm based on the combination of clinical observations, RT-qPCR in multiple diagnostic specimens, and serology are essential to ensure an accurate diagnosis of SVA.