Search Results (9)

This study aimed to evaluate the effectiveness of combining load–close–homogenize (LCH), test and removal (T&R), and rollover strategies for PRRSV elimination in breeding herds using PRRSV-positive weaned gilts. Here, a novel strategy was explored for PRRSV elimination from more than 1500 weaned gilts, and we documented the process from PRRSV natural infection to elimination at the herd level. With LCH implementation, the herd achieved PRRSV-positive stability within 8 months. Consequently, by rolling in self-breeding PRRSV-naive gilts to replace PRRSV-positive weaned sows batch by batch, the time from being positive stable to negative was 13 months. A PRRSV-positive farm intending to retain its genes in its repopulate farrow to become a finished breeding farm can initiate PRRSV elimination from its weaned gilts; this will result in the first farrowing batch of piglets aged 8–10 weeks becoming PRRSV-negative after 8 months of herd closure. This approach offers a viable pathway for genetic retention and PRRSV elimination in breeding farms. Full article

Foot and mouth disease virus (FMDV) is a highly pathogenic virus that mainly infects cloven hooved animals, such as pigs. The establishment of a rapid, sensitive and accurate point-of-care detection method is critical for the timely identification and elimination of infected pigs for controlling this disease. In this study, a RT-RAA-CRISPR/Cas13a method was developed for the detection of FMDV serotype O in pigs. Six pairs of RT-RAA primers were designed based on the conserved gene sequence of FMDV serotype O, and the optimal amplification primers and reaction temperatures were screened. The CRISPR-derived RNA (crRNA) was further designed based on the optimal target band sequence and the most efficient crRNA was screened. The results revealed that FMDV-O-F4/R4 was the optimal primer set, and the optimal temperature for the RT-RAA reaction was 37 °C. Moreover, crRNA4 exhibited the strongest detection signal among the six crRNAs. The established RT-RAA-CRISPR/Cas13a method demonstrated high specificity and no cross-reactivity with other common swine pathogens such as Senecavirus A (SVA), porcine reproductive and respiratory virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), and pseudorabies virus (PRV), additionally, it was observed to be highly sensitive, with a detection limit of 19.1 copies/µL. The repeatability of this method was also observed to be good. This method could produce stable fluorescence and exhibited good repeatability when three independent experiments yielded the same results. A validation test using three types of simulated clinical samples (including swab, tissue, and serum samples) revealed a 100% concordance rate. The detection results could be visualized via a fluorescence reader or lateral flow strips (LFSs). Thus, a highly specific and sensitive RT-RAA-CRISPR/Cas13a detection method was developed and is expected to be applied for the rapid detection of FMDV serotype O in situ. Full article

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a major threat to the global swine industry, causing significant economic losses. To address this, we developed a scalable recombinant adeno-associated virus (rAAV)-based strategy for the delivery of soluble viral receptors (SVRs) to treat and potentially eliminate PRRSV infections. This strategy involves fusing the virus-binding domains of two key cellular receptors, sialoadhesin (Sn4D) and CD163 (SRCR5-9), with an Fc fragment. We then used an insect cell–baculovirus expression vector system to produce the rAAV-SRCR59-Fc/Sn4D-Fc vector. Through a series of optimizations, we determined the best conditions for rAAV production, including a baculovirus co-infection ratio of 0.5:1.0, an initial insect cell density of 2.0 × 10⁶ cells/mL, a fetal bovine serum concentration of 2%, and a culture temperature of 30 °C. Under these optimized conditions, we achieved a high titer of rAAV-SRCR59-Fc/Sn4D-Fc in a 2 L bioreactor, reaching 5.4 ± 0.9 × 10⁹ infectious viral particles (IVPs)/mL. Notably, in vitro neutralization assays using a Transwell co-culture system demonstrated a 4.3 log reduction in viral titers across genetically diverse PRRSV-2 strains, including VR2332, JXA1, JS07, and SH1705. Collectively, this study provides a robust platform for large-scale rAAV production and highlights the potential of SVR-based gene therapy to address the antigenic diversity of PRRSV-2. Full article

Piglet diarrhea poses significant economic losses to the pig industry, posing a worldwide challenge that urgently needs to be addressed in pig breeding practices. Porcine rotavirus (PoRV) is an important viral diarrhea pathogen in piglets, with a high incidence rate and a tendency to cause growth retardation. To enhance the sensitivity and specificity of PoRV detection, we sequenced the NSP3 gene of G5 and G9 genotypes of rotavirus A (RVA), enabling simultaneous detection of the two serotypes. Subsequently, we developed a rapid PoRV detection method using a combination of recombinase-aided amplification (RAA) and CRISPR/Cas12a. In this method, Cas12a binds to RAA amplification products, guided by CRISPR-derived RNA (crRNA), which activates its cleavage activity and releases fluorescence by cutting FAM-BHQ-labeled single-stranded DNA (ssDNA). In the optimized reaction system, the recombinant plasmid PoRV can achieve a highly sensitive reaction within 30 min at 37 °C, with a detection limit as low as 2.43 copies/μL, which is ten times higher in sensitivity compared to the qPCR method. Results from specificity testing indicate that no cross-reactivity was observed between the RAA-CRISPR/Cas12a analysis of PoRV and other viral pathogens, including PoRV G3, PoRV G4, porcine epidemic diarrhea virus (PEDV), porcine epidemic diarrhea (PDCoV), and porcine reproductive and respiratory syndrome virus (PRRSV). In the clinical sample detection using the RAA-CRISPR/Cas12a method and qPCR, Cohen’s Kappa value reached as high as 0.952. Furthermore, this approach eliminates the need for large-scale instrumentation, offering a visual result under an ultraviolet lamp through fluorescence signal output. Full article

Specimens collected from dead pigs are a welfare-friendly and cost-effective active surveillance. This study aimed to evaluate the accuracy of different postmortem specimens from dead piglets for disease detection, using PRRSV as an example. Three farrow-to-wean farms undergoing PRRSV elimination were conveniently selected. Samples were collected at approximately 8- and 20-weeks post-outbreak. Postmortem specimens included nasal (NS), oral (OS), and rectal (RS) swabs, tongue-tip fluids (TTF), superficial inguinal lymph nodes (SIL), and intracardiac blood. These were tested individually for PRRSV by RT-PCR. Sensitivity, specificity, negative and positive predictive values, and agreement of postmortem specimens were calculated using intracardiac sera as the gold standard. OS and SIL had the best overall performance, with sensitivities of 94.6–100%, specificities of 83.9–85.1%, and negative predictive values of 97.3–100%. TTF had high sensitivity (92.2%) but low specificity (53.9%) and positive predictive value (48.3%). While challenges in meeting sampling targets due to variable pre-weaning mortality were noted, PRRS was detected in all postmortem specimens. OS and NS showed promising results for disease monitoring, though TTF, despite their sensitivity, had lower specificity, making them less suitable for individual infection assessment but useful for assessing environmental contamination. Full article

This experiment aimed to evaluate commercially available disinfectants and their application methods against porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) on truck cab surfaces. Plastic, fabric, and rubber surfaces inoculated with PEDV or PRRSV were placed in a full-scale truck cab and then treated with one of eight randomly assigned disinfectant treatments. After application, surfaces were environmentally sampled with cotton gauze and tested for PEDV and PRRSV using qPCR duplex analysis. There was a disinfectant × surface interaction (p < 0.0001), indicating a detectable amount of PEDV or PRRSV RNA was impacted by disinfectant treatment and surface material. For rubber surfaces, 10% bleach application had lower detectable amounts of RNA compared to all other treatments (p < 0.05) except Intervention via misting fumigation, which was intermediate. In both fabric and plastic surfaces, there was no evidence (p > 0.05) of a difference in detectable RNA between disinfectant treatments. For disinfectant treatments, fabric surfaces with no chemical treatment had less detectable viral RNA compared to the corresponding plastic and rubber (p < 0.05). Intervention applied via pump sprayer to fabric surfaces had less detectable viral RNA than plastic (p < 0.05). Furthermore, 10% bleach applied via pump sprayer to fabric and rubber surfaces had less detectable viral RNA than plastic (p < 0.05). Also, a 10 h downtime, with no chemical application or gaseous fumigation for 10 h, applied to fabric surfaces had less detectable viral RNA than other surfaces (p < 0.05). Sixteen treatments were evaluated via swine bioassay, but all samples failed to produce infectivity. In summary, commercially available disinfectants successfully reduced detectable viral RNA on surfaces but did not eliminate viral genetic material, highlighting the importance of bioexclusion of pathogens of interest. Full article

PRRS elimination strategies often rely on depopulation-repopulation. However, this approach is accompanied by a long-term loss of production. With adequate control measures, such as well-designed immunization programs and technological changes along with prevalence-based laboratory testing, the virus-free status of the most vulnerable age groups in swine herds can be achieved. The most common reason for acquiring PRRSV at large farrow-to-finish swine farm units is that the previously settled fattening pigs serve as a source of infection for the newly reared PRRS-free animals. Following such unwanted events, PRRSV may persist in an affected establishment for several years. In this observational study, we selected four farrow-to-finish type swine herds. We implemented different laboratory testing protocols to find the most optimal solution for a successful PRRS elimination program. To aid our objectives, we used a DIVA PCR technique. The PRRS DIVA PCR assay is a fast, reliable method to identify sows shedding farm-specific PRRSV strain(s). As a result of elimination efforts at the sentinel pig herds, we found that reliable detection of wild-type PRRSV shedding among sows requires sampling at least three weaned piglets per litter. The strict adherence to this sampling protocol, the systematic use of laboratory methods that quickly detect the presence of wild virulent virus in the herd during the rearing period and the culling of DIVA PCR positive litters and their sows decreased the presence of the resident virus markedly. These procedures at Hungarian farrow-to-finish type farms successfully inhibited the wild-type PRRSV infection of different age groups. The results of this study demonstrate that applying this methodology together with strict biosecurity measures enabled us to reach PRRS-vaccinated-free status in large, farrow-to-finish herds within two years. Full article

It is well established that PRRSV elimination is an effective strategy for PRRS control, but published reports concerning successful PRRSV elimination cases in farrow-to-finishing herds are rare. Here, we have reported a successful PRRSV elimination case in a farrow-to-finish herd by employing a “herd closure and rollover” approach with some modifications. Briefly, the introduction of pigs to the herd was stopped and normal production processes were maintained until the herd reached a PRRSV provisional negative status. During the herd closure, strict biosecurity protocols were implemented to prevent transmission between nursery pigs and sows. In the current case, introducing gilts before herd closure and live PRRSV exposure were skipped. In the 23rd week post-outbreak, the pre-weaning piglets started to show 100% PRRSV negativity in qPCR tests. In the 27th week, nursery and fattening barns fully launched depopulation. In the 28th week, nursery and fattening houses reopened and sentinel gilts were introduced into gestation barns. Sixty days post-sentinel gilt introduction, the sentinel pigs maintained being PRRSV antibody negative, manifesting that the herd matched the standard of the provisional negative status. The production performance of the herd took 5 months to bounce back to normal. Overall, the current study provided additional information for PRRSV elimination in farrow-to-finish pig herds. Full article

Background: Hesitation on eliminating Porcine Reproductive and Respiratory Syndrome virus (PRRSV) from breeding herds exists since it is difficult to predict how long the herd will remain virus-free. We aimed to estimate the time that breeding herds remained virus-free (naïve) after PRRSV elimination was achieved. Methods: Production systems voluntarily shared their breeding herds’ health status weekly between July 2009 and October 2021. PRRSV incidence rate and the total number of days a breeding herd remained virus-free were estimated. Results: A total of 221 (17%) herds reached the naïve status 273 times. The median time sites remained in this status was approximately two years. The overall PRRS incidence rate after sites achieved a naïve status was 23.43 PRRS outbreaks per 100 farm years. Conclusion: Estimates obtained here provide insights on how frequently and for how long sites remain naïve, which contribute to informing management practices for PRRS control. Full article