A dual-mode colorimetric and electrochemical strategy for sensitive detection of PRRSV using imidazole-copper nanozymes.

We characterized the effect of 1) temperature × time, 2) freeze-thaw cycles, and 3) high porcine reproductive and respiratory syndrome virus (PRRSV) RNA concentrations on the detection of PRRSV and a porcine-specific internal sample control (ISC) in serum, oral fluid, and fecal samples using a commercial PRRSV RT-rtPCR assay (Idexx). In study 1, the effect of temperature × time on PRRSV and ISC detection was shown to be specimen dependent. In serum stored at 4, 10, or 20°C, PRRSV detection was consistent for up to 168 h, but storage at 30°C reduced detectable PRRSV RNA. ISC RNA was stable in serum held at 4 and 10°C, but not at 20 and 30°C. In contrast, PRRSV and ISC RNAs in oral fluid and fecal samples continuously decreased at all temperature × time treatments. Based on these data, serum samples should be stored at ≤ 20°C to optimize PRRSV RNA detection. Oral fluid and fecal samples should be frozen in a non-self-defrosting freezer until tested. In study 2, freeze-thaw cycles had little impact on PRRSV and ISC detection, but more so in oral fluids than serum or fecal samples. Thus, freeze-thaw cycles in oral fluids should be minimized before RT-rtPCR testing. In study 3, the ISC was not affected by high concentrations of PRRSV RNA in serum, oral fluid, or fecal samples. It should not be assumed that data from our PRRSV study are applicable to other pathogens; additional pathogen-specific studies are required.

The objective of the present study was to determine the prevalence of porcine reproductive and respiratory syndrome virus (PRRSV) antigen-positive uterine tissue in gilts culled due to reproductive disturbance in relation to age at culling, reasons for culling, herds, and PRRSV vaccination. Uterine tissues of 100 gilts from six swine herds in Thailand were collected. The immunohistochemistry was performed to detect the PRRSV antigen using a polymer-based non-avidin-biotin technique. PRRSV was detected in the cytoplasm of the macrophages in the subepithelial connective tissue layers of the endometrium in 33.0% of the culled gilts. The detection of PRRSV antigen varied among the herds from 14.3% to 80.0% (P = 0.018). The detection of PRRSV in the uterine tissues at different ages was not statistically different (29.6%, 39.4%, and 40.9% in gilts culled at 6-8, 9-10, and 11-16 months of age, respectively, P = 0.698), similar to the reasons for culling (P = 0.929). PRRSV antigen was found in 24.5% of the gilts vaccinated against the EU-strain-modified-live PRRSV vaccine and in 23.1% of the gilts vaccinated against the US-strain-modified-live PRRSV (P = 0.941). The level of antibody titers against PRRSV had no impact on PRRSV antigen detection in the uterine tissues. Similarly, the detection of PRRSV antigen did not differ between the virgin gilts (35.4%) and the gilts mated before culling (30.8%) (P = 0.622). It can be concluded that PRRSV remains in the uterine tissue of the infected gilts for several months even though vaccinations and acclimatization have been carried out.

The objectives of the present study were to determine the prevalence of porcine reproductive and respiratory syndrome virus (PRRSV) in Thailand between 2005 and 2010. The study was conducted by retrospectively investigating the detection of PRRSV from different pig types including boars, sows, piglets, nursery pigs, and fattening pigs from six regions of Thailand, i.e., the northern, eastern, northeastern, central, western, and southern parts. The data were obtained from cases submitted to the Chulalongkorn University Veterinary Diagnostic Laboratory for PRRSV detection between 2005 and 2010. Frequency analyses and generalized linear models were used to evaluate the prevalence of PRRSV in relation to various factors. In total, 2,273 tissues (n = 636), semen (n = 210) and serum (n = 1,427) samples were included. PRRSV was detected in 32.6% (740/2,273) of the pigs. The virus was found in 43.1%, 15.7%, and 30.3% in the tissues, semen, and serum samples, respectively (P < 0.001). The prevalence of PRRSV was highest in 2005 (43.6%) and lowest in 2009 (23.6%) (P < 0.001). The prevalence of PRRSV was highest in nursery pigs (43.7%) and lowest in boars (15.4%) (P < 0.001). The prevalence of PRRSV in the hot season (34.9%) was higher than that found in the cool season (28.1%, P = 0.018) but did not differ significantly compared to rainy season (34.0%, P = 0.486). The strain of PRRSV isolated in the present study was genotype 2 (54.5%), genotype 1 (31.0%), and mixed genotypes (14.5%). It can be concluded that PRRSV was detected in the tissue samples more frequently than the semen and serum samples. The prevalence of PRRSV was high in the nursery pigs. A high prevalence of PRRSV was found in the hot season, indicating that climatic factors may also contribute to the prevalence of PRRSV in Thailand. Of all the PRRSV detected, 31.0%, 54.5%, and 14.5% belonged to genotype 1, genotype 2, and mixed genotypes, respectively.

To document shedding of porcine reproductive and respiratory syndrome (PRRS) virus in mammary gland secretions of experimentally inoculated sows, to evaluate effects of vaccination during gestation on virus shedding during the subsequent lactation, and to evaluate shedding of PRRS virus in milk of sows in commercial herds. 6 sows seronegative for PRRS virus were used for experiment 1, and 2 sows were retained for experiment 2. For experiment 3, 202 sows in commercial herds were used. In experiment 1, 2 sows were inoculated with PRRS virus, 2 sows were vaccinated with modified-live PRRS virus vaccine, and 2 sows served as control pigs. Mammary gland secretions were assayed for PRRS virus. In experiment 2, pregnant vaccinated sows from experiment 1 were vaccinated with another modified-live PRRS virus vaccine. Mammary gland secretions were assayed in the same manner as for experiment 1. For experiment 3, milk collected from 202 sows in commercial herds was assayed for PRRS virus. In experiment 1, PRRS virus was detected in mammary gland secretions of both vaccinated and 1 of 2 virus-inoculated sows. In experiment 2, virus was not detected in samples from either vaccinated sow. In experiment 3, all samples yielded negative results. Naïve sows inoculated late in gestation shed PRRS virus in mammary secretions. Previous vaccination appeared to prevent shedding during the subsequent lactation. Results for samples obtained from sows in commercial herds suggested that virus shedding in mammary gland secretions of such sows is uncommon.

Virulence and genotype-associated infectivity of interferon-treated macrophages by porcine reproductive and respiratory syndrome viruses

Porcine reproductive and respiratory syndrome (PRRS) is the most economically significant disease affecting swine production in the United States (US). The etiologic agent of PRRS is an RNA virus named as PRRS virus (PRRSV). Substantial economic losses attributed to PRRS are due to reduced reproductive performance in sows and reduced growth rate and increased mortality in growing animals. In the US, PRRSV activity is routinely monitored by production systems and/or veterinary clinics. A group of swine production systems in the US voluntarily share their PRRSV breeding herd incidence to a program that gathers, aggregates, and reports the weekly incidence and prevalence among participants. As of February of 2020, most of the participants are from large production systems that represented 2.8 million sows. Currently, there is no information on PRRSV detection in the broader industry which totals of 6.46 million sows. Additionally, American Association of Swine Veterinarians (AASV) guidelines for PRRSV monitoring (from 2011) was based on the use of serum samples. However, there is a recent description of processing fluid as a population-based sample type to monitor PRRSV herd status more efficiently. The validation of this new sample type raises the need to better understand the practical applications of this sample type to monitor PRRSV. Considering (a) the high economic importance of PRRS to the US swine industry, (b) the current gap of information about PRRSV activity in most breeding herds, and in growing animals, and the (c) gap in knowledge about applications of processing fluids to monitor and surveil PRRSV, there is the critical need to develop additional tools for improvement in the monitoring and surveillance systems for PRRSV in the US swine industry. Therefore, the overall objectives of this dissertation were: a) to develop a capability to reliably and consistently track PRRSV detection over time, age group, geographical space, and specimen in the US swine industry; and b) to develop monitoring and surveillance systems to enable veterinarians to make science-driven decisions to support disease prevention, detection, and control strategies. To that end, a capability to monitor and report the detection of PRRSV RNA by RT-PCR at the four major US swine-centric veterinary diagnostic laboratories was built. A procedure to receive data, a process to compile to the same format, aggregate, report, and a capability for continuous automated updates was developed. This information was used to describe the macroepidemiological aspects of PRRSV RNA detection by RT-PCR in the US

Porcine respiratory coronavirus (PRCoV), porcine reproductive and respiratory syndrome virus (PRRSV), and swine influenza virus (SIV) are important pathogens of significant infectious diseases. They cause similar clinical respiratory symptoms, including fever, cough, runny nose, and respiratory distress, which makes these diseases difficult to distinguish from each other. In this study, three pairs of specific primers and TaqMan probes were designed for the conserved regions of the PRCoV S gene, PRRSV N gene, and SIV M gene, respectively. The annealing temperature, primer and probe concentrations, and reaction cycle were optimized, and a triplex crystal digital PCR (cdPCR) assay was established for the detection of PRCoV, PRRSV, and SIV. According to the test results, the assay was capable of specifically detecting PRCoV, PRRSV, and SIV, and there was no cross-reaction with other control swine viruses. Based on the Poisson distribution analysis, the limits of detection (LODs) for PRCoV, PRRSV, and SIV were 6.00, 5.75 and 6.00 copies/reaction, respectively, and the sensitivity was 26 times higher than those of the corresponding multiplex RT-qPCR. The coefficients of variation (CVs) of the intra-assay and inter-assay ranged from 0.19 to 1.84%. The assay was used to test 1,657 clinical samples, and the positivity rates of PRCoV, PRRSV, and SIV were 1.15, 12.79, and 2.05%, respectively. It showed diagnostic sensitivity and specificity of 100 and 99.82% for PRCoV, 100 and 99.24% for PRRSV, and 100 and 99.69% for SIV, respectively. These results indicated that the triplex cdPCR assay has strong specificity, high sensitivity, and excellent repeatability, which provides a valuable tool for the detection and differentiation of PRCoV, PRRSV, and SIV.

Comparison of virus detection, productivity, and economic performance between lots of growing pigs vaccinated with two doses or one dose of PRRS MLV vaccine, under field conditions

Many aspects of the porcine reproductive and respiratory syndrome virus (PRRSV) between-farm transmission dynamics have been investigated, but uncertainty remains about the significance of farm type and different transmission routes on PRRSV spread. We developed a stochastic epidemiological model calibrated on weekly PRRSV outbreaks accounting for the population dynamics in different pig production phases, breeding herds, gilt development units, nurseries and finisher farms, of three hog producer companies. Our model accounted for indirect contacts by the close distance between farms (local transmission), between-farm animal movements (pig flow) and reinfection of sow farms (re-break). The fitted model was used to examine the effectiveness of vaccination strategies and complementary interventions such as enhanced PRRSV detection and vaccination delays and forecast the spatial distribution of PRRSV outbreak. The results of our analysis indicated that for sow farms, 59% of the simulated infections were related to local transmission (e.g. airborne, feed deliveries, shared equipment) whereas 36% and 5% were related to animal movements and re-break, respectively. For nursery farms, 80% of infections were related to animal movements and 20% to local transmission; while at finisher farms, it was split between local transmission and animal movements. Assuming that the current vaccines are 1% effective in mitigating between-farm PRRSV transmission, weaned pigs vaccination would reduce the incidence of PRRSV outbreaks by 3%, indeed under any scenario vaccination alone was insufficient for completely controlling PRRSV spread. Our results also showed that intensifying PRRSV detection and/or vaccination pigs at placement increased the effectiveness of all simulated vaccination strategies. Our model reproduced the incidence and PRRSV spatial distribution; therefore, this model could also be used to map current and future farms at-risk. Finally, this model could be a useful tool for veterinarians, allowing them to identify the effect of transmission routes and different vaccination interventions to control PRRSV spread.

Reverse transcription recombinase polymerase amplification assay for the rapid detection of type 2 porcine reproductive and respiratory syndrome virus

Simultaneous detection of classical PRRSV, highly pathogenic PRRSV and NADC30-like PRRSV by TaqMan probe real-time PCR

BackgroundAfrican swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV) are still prevalent in many regions of China. Co-infections make it difficult to distinguish their clinical symptoms and pathological changes. Therefore, a rapid and specific method is needed for the differential detection of these pathogens.ObjectivesThe aim of this study was to develop a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) for the simultaneous differential detection of ASFV, CSFV, and PRRSV.MethodsThree pairs of primers and TaqMan probes targeting the ASFV p72 gene, CSFV 5′ untranslated region, and PRRSV ORF7 gene were designed. After optimizing the reaction conditions, including the annealing temperature, primer concentration, and probe concentration, multiplex qRT-PCR for simultaneous and differential detection of ASFV, CSFV, and PRRSV was developed. Subsequently, 1,143 clinical samples were detected to verify the practicality of the assay.ResultsThe multiplex qRT-PCR assay could specifically and simultaneously detect the ASFV, CSFV, and PRRSV with a detection limit of 1.78 × 100 copies for the ASFV, CSFV, and PRRSV, but could not amplify the other major porcine viruses, such as pseudorabies virus, porcine circovirus type 1 (PCV1), PCV2, PCV3, foot-and-mouth disease virus, porcine parvovirus, atypical porcine pestivirus, and Senecavirus A. The assay had good repeatability with coefficients of variation of intra- and inter-assay of less than 1.2%. Finally, the assay was used to detect 1,143 clinical samples to evaluate its practicality in the field. The positive rates of ASFV, CSFV, and PRRSV were 25.63%, 9.36%, and 17.50%, respectively. The co-infection rates of ASFV+CSFV, ASFV+PRRSV, CSFV+PRRSV, and ASFV+CSFV+PRRSV were 2.45%, 2.36%, 1.57%, and 0.17%, respectively.ConclusionsThe multiplex qRT-PCR developed in this study could provide a rapid, sensitive, specific diagnostic tool for the simultaneous and differential detection of ASFV, CSFV, and PRRSV.

Porcine reproductive and respiratory syndrome (PRRS) virus infection often lead to infertility in gilts and sows. Nevertheless, the impact of PRRS virus on the endometrial function has not been fully elucidated. The present study aimed to determine the effect of PRRS virus infection on the expression of oestrogen receptor (ER) α in the endometrium of gilts. Uterine tissues from 54 gilts were classified into two groups according to PRRS virus detection using immunohistochemistry (26 positive and 28 negative samples). The reproductive status was classified as prepubertal, luteal and follicular phases. The expression of ERα in the epithelium, subepithelium and glandular tissue layers of the endometrium was determined by immunohistochemistry. ERα immunostaining was detected in 22.2, 13.5 and 33.6 % of the cells in the epithelial, subepithelial and glandular tissue layers, respectively. The ERα immunostaining in the glandular layer of the endometrium in follicular-phase gilts was higher than that in prepubertal gilts (46.1 and 15.2 %, respectively, P = 0.011). The ERα immunostaining in the glandular layer of the endometrium was positively correlated with body weight (r = 0.138, P = 0.020) and average daily gain (r = 0.169, P = 0.005) of the gilts. The ERα immunostaining in all tissue layers of the endometrium with PRRS virus detection did not differ significantly compared to that without PRRS virus (P > 0.05). However, in prepubertal gilts, the ERα immunostaining cells in the epithelial layer of PRRS virus negative (9.5 %) tended to be lower than PRRS virus positive (26.3 %, P = 0.196) uterine tissues. This tendency indicates some impact of PRRS virus infection on the ERα expression in prepubertal gilts.

Infectious porcine reproductive and respiratory syndrome virus (PRRSV) causes PRRS, but noninfectious PRRSV cannot. PCR and ELISA are commonly used for PRRSV detection but they cannot discriminate PRRSV infectivity. Virus isolation is a gold standard to determine virus infectivity. However, it is time-consuming. Therefore, we developed a propidium monoazide (PMA) qPCR assay for rapid and universal detection of infectious PRRSV in this study. After comparing the inactivation efficacies of distinct disinfectants, ultraviolet (UV) light, and heat, heat at 72°C for 15 min was determined as an effective strategy for PRRSV inactivation, which was confirmed by virus isolation and immunofluorescence assay (IFA) detection. In addition, PMA pretreatment parameters were optimized, including PMA concentration (5 μM), PMA binding time (25 min), PMA binding temperature (37°C), and photolysis time (25 min). The optimal concentration of primers and probes adapted from our previous study was redetermined. The optimized PMA-qPCR assay exhibited satisfied specificity, sensitivity, and reproducibility. Furthermore, the new PMA-qPCR was applied on the detection of 270 clinical samples (including 57 environmental feces, 177 lungs, 33 lymph nodes [LN], and 3 sera) and compared with previously developed qPCR. Eighty samples were qPCR positive, while only 63 samples were PMA-qPCR positive. No virus could be isolated in the 17 qPCR-positive but PMA-qPCR-negative clinical samples; meanwhile, PRRSV could be isolated in representative PMA-qPCR-positive samples, supporting that only live PRRSV isolates in distinct samples could be detected by this PMA-qPCR assay. In conclusion, this study provides the first PMA-qPCR assay for rapid and universal detection of infectious PRRSV, offering an alternative and effective method for PRRSV diagnosis, prevention, and control.

IntroductionThe porcine reproductive and respiratory syndrome virus (PRRSV) continues to challenge swine production in the US and most parts of the world. Effective PRRSV surveillance in swine herds can be challenging, especially because the virus can persist and sustain a very low prevalence. Although weaning-age pigs are a strategic subpopulation in the surveillance of PRRSV in breeding herds, very few sample types have been validated and characterized for surveillance of this subpopulation. The objectives of this study, therefore, were to compare PRRSV RNA detection rates in serum, oral swabs (OS), nasal swabs (NS), ear-vein blood swabs (ES), and family oral fluids (FOF) obtained from weaning-age pigs and to assess the effect of litter-level pooling on the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) detection of PRRSV RNA.MethodsThree eligible PRRSV-positive herds in the Midwestern USA were selected for this study. 666 pigs across 55 litters were sampled for serum, NS, ES, OS, and FOF. RT-qPCR tests were done on these samples individually and on the litter-level pools of the swabs. Litter-level pools of each swab sample type were made by combining equal volumes of each swab taken from the pigs within a litter.ResultsNinety-six piglets distributed across 22 litters were positive by PRRSV RT-qPCR on serum, 80 piglets distributed across 15 litters were positive on ES, 80 piglets distributed across 17 litters were positive on OS, and 72 piglets distributed across 14 litters were positive on NS. Cohen's kappa analyses showed near-perfect agreement between all paired ES, OS, NS, and serum comparisons (). The serum RT-qPCR cycle threshold values (Ct) strongly predicted PRRSV detection in swab samples. There was a ≥ 95% probability of PRRSV detection in ES-, OS-, and NS pools when the proportion of positive swab samples was ≥ 23%, ≥ 27%, and ≥ 26%, respectively.DiscussionES, NS, and OS can be used as surveillance samples for detecting PRRSV RNA by RT-qPCR in weaning-age pigs. The minimum number of piglets to be sampled by serum, ES, OS, and NS to be 95% confident of detecting ≥ 1 infected piglet when PRRSV prevalence is ≥ 10% is 30, 36, 36, and 40, respectively.