Peng Li

Porcine reproductive and respiratory syndrome virus (PRRSV) is a significant cause of economic loss in the swine industry, yet its control remains challenging in wean-to-harvest sites. This prospective observational study followed 95 wean-to-harvest sites across six U.S. states for one production cycle. Sites were required to be PRRSV-negative or vaccinated with a modified live virus (MLV) and free of major coronaviruses. Outbreaks were defined as RT-qPCR-positive in unvaccinated sites or detection of ORF5 sequences distinct from the MLV strain. Biosecurity data were collected through a survey, and oral fluids were tested every four weeks. PRRS outbreaks occurred in 14/42 nurseries (33.3%), 8/12 wean-to-finish (66.7%), and 35/41 finishers (82.4%), with lineage 1C.5 most frequently detected. In univariate models, higher odds of outbreaks were associated with transporting pigs of unknown status (OR 9.80, 1.73–55.37), rendering (OR 6.47, 1.62–25.84), and employee cohabitation (OR 6.15, 1.51–25.09). Protective factors included exclusive pumping equipment (OR 0.07, 0.01–0.43) and overnight downtime for multi-site workers (OR 0.15, 0.04–0.56). In multivariable models, finisher sites (OR 17.47, 2.44–125.19) and greater swine site density within one mile (OR 1.62, 1.09–2.41) significantly increased outbreak odds. These results support targeted biosecurity practices, helping farmers and the swine industry reduce PRRS outbreaks.

Tonsil oral scrubbing (TOSc) is a simple and practical method recently developed for detecting PRRSV RNA in sows. This study evaluated how pooling and freeze-thaw procedures affect PRRSV RT-rtPCR results in TOSc samples. To assess pooling effects, 22 PRRSV-positive samples were divided into three Ct categories and diluted with negative TOSc samples at various ratios before testing. To assess freeze-thaw effects, 90 TOSc samples were split into two equal aliquots: one tested immediately, and the other tested after one freeze-thaw cycle. Results showed that PRRSV RNA detection probability decreased and Ct values increased as dilution (pooling) ratios rose. Samples with Ct values > 35 had significant drops in probability of detection beginning at a 1:3 dilution (≤71%). In contrast, samples with Ct values 35. Similarly, the freeze-thaw process should be minimized during TOSc sample handling.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a major challenge for the swine industry, can be transmitted both vertically and horizontally. Common sample types, including processing fluid, serum, and family oral fluid, can neither determine whether PRRSV infection originates vertically or horizontally nor directly reflects the sow’s PRRSV status. At around 90 days post live-virus inoculation (LVI), 555 sows were sampled by Tonsil-oral-scrubbing (TOSc) 2 weeks pre-farrowing and tested for PRRSV RNA. From these, 59 PRRSV-positive sows, and 88 PRRSV-negative sows matched by parity were conveniently selected. TOSc from sows, blood swabs from live piglet, and tongue fluid (TF) plus serum from dead piglet were collected individually from all study litters within 12 h post-farrowing. The pre-farrowing TOSc samples had significantly higher PRRSV positivity than TF, serum and blood swab pools, while dead piglet serum had significantly lower mean Ct values than all other sample types. TOSc samples had 25% positive predictive value (PPV) for “live litter” PRRSV status while the pre-farrow and post-farrow TOSc had 87.2% and 89.0% negative predictive value (NPV), respectively. In conclusion, we characterized PRRSV RNA detection among all sub-populations within a litter with easy-to-use TOSc samples and neonatal litter samples, suggesting the occurrence of vertical transmission 90 days post-LVI in sows. TOSc samples from sows had low PPV and high NPV for their respective litter’s PRRSV status.

Porcine epidemic diarrhea virus (PEDV), Transmissible gastroenteritis virus (TGEV), Porcine deltacoronavirus (PDCoV) and Swine acute diarrhea syndrome coronavirus (SADS-CoV) rank among the most frequently encountered swine enteric coronaviruses (SECoVs), leading to substantial economic losses to the swine industry. The availability of a rapid and highly sensitive detection method proves beneficial for the monitoring and surveillance of SECoVs. Based on the N genes of four distinct SECoVs, a novel detection method was developed in this study by combining recombinant enzyme polymerase isothermal amplification (RPA) with clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) 12a. Results showed that the cut-off value of CRISPR-Cas12a assay for SADS-CoV, PEDV, PDCoV and TGEV was 2.19 × 10⁴ Relative Fluorescence Units (RFU), 1.57 × 10⁴ RFU, 3.07 × 10⁴ RFU and 1.64 × 10⁴ RFU, respectively. The coefficient of variation (CV) of within and between runs by CRISPR-Cas12a assay for 6 clinical diarrhea samples were both less than 10%. The CRISPR-Cas12a assay demonstrated high specificity for TGEV, PEDV, PDCoV, and SADS-CoV with no cross-reactivity to other common swine viruses. This method also exhibited a low limit of detection of 2 copies for each virus. Additionally, the results demonstrated a perfect agreement (100%) between the CRISPR-Cas12a assay and the RT-qPCR assay. Finally, a total of 494 pig samples from the field tested by CRISPR-Cas12a assay showed that positive rate for SADS-CoV, TGEV, PDCoV and PEDV was 0, 0, 1.2% and 48.6%, respectively. The results suggested the great potential of CRISPR-Cas12a assay to detect SECoVs in the field.