Development of an enzyme-linked immunosorbent assay using a monoclonal antibody to a dominant epitope in non-structural protein 4 of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) caused by the PRRS virus (PRRSV) is one of the most severe swine diseases causing great economic losses for the international swine industry. Non-structural protein 4 (NSP4) is critical to the life cycle of PRRSV and contains dominant B cell epitopes. This study prepared a monoclonal antibody against Nsp4, and 2D11, which contained the sequence 138KQGGGIVTRPSGQFCN153, was confirmed as the epitope. A 2D11-based double antibody sandwich enzyme-linked immunosorbent assay (dasELISA) was next developed with a cut value of 0.1987. A total of 1354 pig serum samples were detected by dasELISA and compared to a commercial ELISA kit (N-coated iELISA), resulting in a positive coincidence rate of 98.8% and negative coincidence rate of 96.9%. A total of 119 sera were positive by dasELISA while negative by iELISA. Higher positive rates by dasELISA were found in pig farms where PRRSV antibody levels varied widely. These results indicated that the dasELISA was a useful tool to detect PRRSV antibody in clinical samples.

Quantum dot fluorescent microsphere-based immunochromatographic strip for detecting PRRSV antibodies.

Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). Current vaccine prevention and treatment approaches for PRRS are not adequate, and commercial vaccines do not provide sufficient cross-immune protection. Therefore, establishing a precise, sensitive, simple, and rapid serological diagnostic approach for detecting PRRSV antibodies is crucial. The present study used quantum dot fluorescent microspheres (QDFM) as tracers, covalently linked to the PRRSV N protein, to develop an immunochromatography strip (ICS) for detecting PRRSV antibodies. Monoclonal antibodies against PRRSV nucleocapsid (N) and membrane (M) proteins were both coated on nitrocellulose membranes as control (C) and test (T) lines, respectively. QDFM ICS identified PRRSV antibodies under 10 min with high sensitivity and specificity. The specificity assay revealed no cross-reactivity with the other tested viruses. The sensitivity assay revealed that the minimum detection limit was 1.2 ng/mL when the maximum dilution was 1:2,048, comparable to the sensitivity of enzyme-linked immunosorbent assay (ELISA) kits. Moreover, compared to PRRSV ELISA antibody detection kits, the sensitivity, specificity, and accuracy of QDFM ICS after analyzing 189 clinical samples were 96.7%, 97.9%, and 97.4%, respectively. Notably, the test strips can be stored for up to 6 months at 4 °C and up to 4 months at room temperature (18-25 °C). In conclusion, QDFM ICS offers the advantages of rapid detection time, high specificity and sensitivity, and affordability, indicating its potential for on-site PRRS screening. KEY POINTS: • QDFM ICS is a novel method for on-site and in-lab detection of PRRSV antibodies • Its sensitivity, specificity, and accuracy are on par with commercial ELISA kits • QDFM ICS rapidly identifies PRRSV, aiding the swine industry address the evolving virus.

Development and application of a blocking ELISA based on a N protein monoclonal antibody for the antibody detection against porcine reproductive and respiratory syndrome virus 2.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most widespread illnesses in the world's swine business. To detect the antibodies against PRRSV-2, a blocking enzyme-linked immunosorbent assay (B-ELISA) was developed, utilizing a PRRSV-2 N protein monoclonal antibody as the detection antibody. A checkerboard titration test was used to determine the optimal detection antibody dilution, tested pig serum dilution and purified PRRSV coated antigen concentration. After analyzing 174 negative pig sera and 451 positive pig sera, a cutoff value of 40 % was selected to distinguish between positive and negative sera using receiver operating characteristic curve analysis. The specificity and sensitivity of the assay were evaluated to equal 99.8 % and 96 %, respectively. The method had no cross-reaction with PCV2, PRV, PPV, CSFV, PEDV, TGEV, and PRRSV-1 serum antibodies, and the coefficients of variation of intra-batch and inter-batch repeatability experiments were both <10 %. A total of 215 clinical serum samples were tested, and the relative coincidence rate with commercial ELISA kit was 99.06 %, and the kappa value was 0.989, indicating that these two detection results exhibited high consistency. Overall, the B-ELISA should serve as an ideal method for large-scale serological investigation of PRRSV-2 antibodies in domestic pigs.

Pluck-pools as diagnostic samples for detecting porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 in porcine abortion material and stillbirths.

Investigating infectious agents in porcine abortion material and stillborn piglets poses challenges for practitioners and diagnostic laboratories. In this study, pooled samples of individual reference organs (thymus and heart) from a total of 1000 aborted fetuses and stillborn piglets were investigated using quantitative PCR protocols for porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) and porcine circovirus type 2 (PCV2). Simultaneously, a pluck-pool containing equivalent portions of fetal thymus, heart, and lung tissue was collected, frozen at - 20 °C, and re-analyzed when a certain amount of either PRRSV-1 RNA or PCV2 DNA was detected in individual reference organs. Thirteen pluck-pools were assessed for PRRSV-1, all being PCR-positive. For PCV2, 11 of 15 pluck-pools investigated were PCR-positive. In all pluck-pools testing negative, viral loads in individual pools were low. This study indicates that pluck-pools can be valuable diagnostic material and the consolidation of multiple organs through a single RNA/DNA extraction optimizes the utilization of available laboratory resources. Additional research is required to assess the feasibility of follow-up investigations and to accurately define criteria for interpretation of viral loads in a clinical context.

Comparison of a novel rapid sampling method to serum and tonsil scraping to detect PRRSV in acutely infected sows.

Few practical methods are available to monitor the PRRSV status of the sows. Common sampling methods for sows like serum sampling, and tonsil scraping involve restraining individual sows and are labor-intensive, time-consuming, relatively invasive, and therefore, have limited use in large-scale production settings. Thus, a practical and rapid method of sampling large numbers of sows is needed. This study aimed to develop a new sampling method, named tonsil-oral scraping (TOSc) and compare TOSc to serum and tonsil scraping in terms of PRRSV qPCR detection rate and Ct values in thirty matched sows, thirty days after PRRSV outbreak. TOSc recovered a mixture of oral fluids and tonsil exudates from the sow oral cavity within seconds without restraining the animals. Results showed that, numerically, the TOSc samples had higher PRRSV qPCR detection rate (100 %) compared to serum (16.8 %) and tonsil scraping (73.1 %). Moreover, TOSc samples had lower average Ct values (29.7) than tonsil scraping (30.7) and serum (35.2). There was no significant difference in the detection rate between TOSc and tonsil scraping (Tukey test, p = 0.992), while there was a significant difference between serum and tonsil scraping (Tukey test, p < 0.001), as well as between serum and TOSc (Tukey test, p < 0.001). In terms of Ct values, there was no statistically significant difference between TOSc and tonsil scrapings (Dunn Test, p > 0.05), while there was a significant difference between tonsil scraping with serum (Dunn Test, p < 0.01), and TOSc with serum (Dunn Test, p < 0.01). Our results suggest great potential of the TOSc as a novel, practical, and rapid tool for PRRSV RNA detection in sows to assess sow herd status.

Normalizing real-time PCR results in routine testing.

Normalization, the process of controlling for normal variation in sampling and testing, can be achieved in real-time PCR assays by converting sample quantification cycles (Cqs) to "efficiency standardized Cqs" (ECqs). We calculated ECqs as E-ΔCq, where E is amplification efficiency and ΔCq is the difference between sample and reference standard Cqs. To apply this approach to a commercial porcine reproductive and respiratory syndrome virus (PRRSV) RT-qPCR assay, we created reference standards by rehydrating and then diluting (1 × 10-4) a PRRSV modified-live vaccine (PRRS MLV; Ingelvac) with serum or oral fluid (OF) to match the sample matrix to be tested. Sample ECqs were calculated using the mean E and reference standard Cq calculated from the 4 reference standards on each plate. Serum (n = 132) and OF (n = 130) samples were collected from each of 12 pigs vaccinated with a PRRSV MLV from -7 to 42 d post-vaccination, tested, and sample Cqs converted to ECqs. Mean plate Es were 1.75-2.6 for serum and 1.7-2.3 for OF. Mean plate reference standard Cqs were 29.1-31.3 for serum and 29.2-31.5 for OFs. Receiver operating characteristic analysis calculated the area under the curve for serum and OF sample ECqs as 0.999 (95% CI: 0.997, 1.000) and 0.947 (0.890, 1.000), respectively. For serum, diagnostic sensitivity and specificity of the commercial PRRSV RT-qPCR assay were estimated as 97.9% and 100% at an ECq cutoff ≥ 0.20, and for OF, 82.6% and 100%, respectively, at an ECq cutoff ≥ 0.45.

Development, Evaluation, and Clinical Application of PRRSV-2 Vaccine-like Real-Time RT-PCR Assays.

In this study, we developed and validated (1) singleplex real-time RT-PCR assays for specific detection of five PRRSV-2 MLV vaccine viruses (Ingelvac MLV, Ingelvac ATP, Fostera, Prime Pac, and Prevacent) and (2) a four-plex real-time RT-PCR assay (IngelvacMLV/Fostera/Prevacent/XIPC) including the internal positive control XIPC for detecting and distinguishing the three most commonly used vaccines in the USA (Prevacent, Ingelvac MLV, and Fostera). The singleplex and 4-plex vaccine-like PCRs and the reference PCR (VetMAXTM PRRSV NA&EU, Thermo Fisher Scientific, Waltham, MA, USA) did not cross-react with non-PRRSV swine viral and bacterial pathogens. The limits of detection of vaccine-like PCRs ranged from 25 to 50 genomic copies/reactions. The vaccine-like PCRs all had excellent intra-assay and inter-assay repeatability. Based on the testing of 531 clinical samples and in comparison to the reference PCR, the diagnostic sensitivity, specificity, and agreement were in the respective range of 94.67-100%, 100%, and 97.78-100% for singleplex PCRs and 94.94-100%, 100%, and 97.78-100% for the 4-plex PCR, with a CT cutoff of 37. In addition, 45 PRRSV-2 isolates representing different genetic lineages/sublineages were tested with the vaccine-like PCRs and the results were verified with sequencing. In summary, the vaccine-like PCRs specifically detect the respective vaccine-like viruses with comparable performances to the reference PCR, and the 4-plex PCR allows to simultaneously detect and differentiate the three most commonly used vaccine viruses in the same sample. PRRSV-2 vaccine-like PCRs provide an additional tool for detecting and characterizing PRRSV-2.

Development of a one-step reverse transcription-quantitative polymerase chain reaction assay for the detection of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is an important disease that severely affects the swine industry and, therefore, warrants rapid and accurate diagnosis for its control. Despite the progress in developing diagnostic tools, including polymerase chain reaction (PCR)-based methods such as reverse transcription quantitative PCR (RT-qPCR) to diagnose PRRSV infection, its diagnosis at the genetic level is challenging because of its high genetic variability. Nevertheless, RT-qPCR is the easiest and fastest method for diagnosing PRRSV. Therefore, this study aimed to develop an RT-qPCR assay for rapid and accurate diagnosis of PRRSV by encompassing all publicly available PRRSV sequences. The developed assay using highly specific primers and probes could detect up to 10 copies of PRRSV-1 and -2 subtypes. Furthermore, a comparison of the performance of the developed assay with those of two commercial kits widely used in South Korea demonstrated the higher efficiency of the developed assay in detecting PRRSV infections in field samples. For PRRSV-1 detection, the developed assay showed a diagnostic agreement of 97.7% with the results of ORF5 sequencing, while for commercial kits, it showed 95.3% and 72.1% agreement. For PRRSV-2, the developed assay showed a diagnostic agreement of 97.7%, whereas the commercial kits showed 93% and 90.7% agreement. In conclusion, we developed an assay with higher accuracy than those of the tested commercial kits, which will contribute markedly to global PRRSV control.

Development and Implementation of a Quadruple RT-qPCR Method for the Identification of Porcine Reproductive and Respiratory Syndrome Virus Strains.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS), leading to abortion in sows and respiratory distress in breeding pigs. In China, PRRSV1 and PRRSV2 are the two circulating genotypes in swine herds, with distinct virulence. PRRSV2 further consists of classical (C-PRRSV2), highly pathogenic (HP-PRRSV2), and NADC30-Like (N-PRRSV2) subtypes. The diversity of PRRSV poses challenges for control and eradication, necessitating reliable detection assays for differentiating PRRSV genotypes. METHODS: A new TaqMan-based RT-qPCR assay with four sets of primers and probes targeting conserved regions of the ORF7 and NSP2 genes of PRRSV was developed, optimized, and evaluated by us. Reaction conditions such as annealing temperature, primer concentration, and probe concentration were optimized for the assay. Specificity, sensitivity, repeatability, stability, limit of detection (LOD), concordance with the reference method were evaluated for the assay. RESULTS: The assay could detect and type PRRSV1, C-PRRSV2, HP-PRRSV2, and N-PRRSV2 simultaneously with 97.33% specificity, 96.00% sensitivity, 12 copies/µL LOD, 97.00% concordance with reference assays. We applied the assay to 321 clinical samples from swine farms in China. The assay successfully detected and typed 230 PRRSV-positive samples, with 24.78% (57/230) of them further confirmed by ORF5 gene sequencing. The prevalence of PRRSV subtypes among the positive samples was as follows: C-PRRSV2 (15.22%), HP-PRRSV2 (23.48%), and N-PRRSV2 (61.30%). Two samples showed coinfection with different PRRSV subtypes. CONCLUSION: The quadruple RT-qPCR assay is a powerful tool for detecting and typing the currently circulating PRRSV strains in Chinese swine populations. It can assist in the surveillance of PRRSV prevalence and the implementation of prevention and control strategies.

Critical evaluation of strategies to achieve direct real-time PCR detection of swine pathogens in oral fluids.

Based on publications reporting improvements in real-time PCR (rtPCR) performance, we compared protocols based on heat treatment or dilution followed by direct rtPCR to standard extraction and amplification methods for the detection of porcine reproductive and respiratory syndrome virus (PRRSV), influenza A virus (IAV), porcine epidemic diarrhea virus (PEDV), or Mycoplasma hyopneumoniae (MHP) in swine oral fluids (OFs). In part A, we subjected aliquots of positive OF samples to 1 of 4 protocols: protocol 1: heat (95°C × 30 min) followed by direct rtPCR; protocol 2: heat and cool (25°C × 20 min) followed by direct rtPCR; protocol 3: heat, cool, extraction, and rtPCR; protocol 4 (control): extraction and then rtPCR. In part B, positive OF samples were split into 3, diluted (D1 = 1:2 with Tris-borate-EDTA (TBE); D2 = 1:2 with negative OF; D3 = not diluted), and then tested by rtPCR using the best-performing protocol from part A (protocol 4). In part A, with occasional exceptions, heat treatment resulted in marked reduction in the detection of target and internal sample control (ISC) nucleic acids. In part B, sample dilution with TBE or OF produced no improvement in the detection of targets and ISCs. Thus, standard extraction and amplification methods provided superior detection of PRRSV, IAV, PEDV, and MHP nucleic acids in OFs.

Effect of extrinsic factors on the detection of PRRSV and a porcine-specific internal sample control in serum, oral fluid, and fecal specimens tested by RT-rtPCR.

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.

Identificationof a novel linear epitope on the porcine reproductive and respiratory syndrome virus nucleocapsid protein, as recognized by a specific monoclonal antibody.

Introduction: Porcine reproductive and respiratory syndrome virus (PRRSV) remains one of the most threatening pathogens of swine. The nucleocapsid (N) protein is the major structural protein of the virus and has been used as a PRRSV diagnostic antigen due to its high level of inherent immunogenicity. Methods: The recombinant PRRSV N protein was generated by the prokaryotic expressing system and used to immunized mice. Monoclonal antibodies against PRRSV were produced and validated by western blot analysis and indirect immunofluorescence analysis. In this study, the linear epitope of a specific monoclonal antibody mAb (N06) was subsequently identified by enzyme-linked immunosorbent assays (ELISA) using the synthesized overlapping peptides as antigens. Results: According to the results of western blot analysis and indirect immunofluorescence analysis, mAb (N06) was capable of recognizing the native form as well as the denatured form of PRRSV N protein. The results of ELISA showed that mAb N06 recognized the epitope NRKKNPEKPHFPLATE, which was consistent with BCPREDS predictions of antigenicity. Conclusion: All the data suggested that the mAb (N06) can be used as diagnostic reagents for PRRSV detection, while the recognized linear epitope can be useful in epitope-based vaccines development, which is helpful for the control of local PRRSV infections in swine.

Instrument-free, visual and direct detection of porcine reproductive and respiratory syndrome viruses in resource-limited settings.

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is one of the most complicated and dangerous diseases in pigs with high mortality since it modulates the immune system of the lungs and has been closely associated with secondary infection of other lethal bacteria and viruses. The gold standard of molecular diagnosis for PRRSV, reverse transcription (RT)-PCR, is time-consuming, expensive and requires transportation of samples to a specialized laboratory. In this study, a direct colorimetric RT-loop-mediated isothermal amplification (RT-LAMP) method was developed to specifically and rapidly detect PRRSV. The RT-LAMP outcomes can be visualized by the naked eye after 45 min of incubation at 65˚C without any cross-reactivity recorded with the bacteria and other viruses tested. In particular, the mobile, non-instrumented, commercial pocket hand warmers were demonstrated to su-ccessfully provide constant temperature for consistent nucleic acid amplification throughout the RT-LAMP reactions. The limit of detection of the assay was defined as the genomic RNA concentration extracted from a known viral titer of 10-2.5 TCID50/ml. The direct use of clinical serum samples required a simple dilution to maintain the performance of the colorimetric RT-LAMP assay. Therefore, the direct colorimetric RT-LAMP assay developed is well-qualified for producing a ready-to-use kit for PRRSV diagnosis in the field. Keywords: porcine reproductive and respiratory syndrome; rapid testing; RT-LAMP; colorimetric; direct detection; instrument-free.

Rapid and Visual Detection of Type 2 Porcine Reproductive and Respiratory Syndrome Virus by Real-Time Fluorescence-Based Reverse Transcription Recombinase-Aided Amplification.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important diseases that has brought significant economic losses to the swine industry worldwide. Rapid and accurate PRRS virus (PRRSV) detection is one of the key factors for PRRS prevention and control. This study developed a real-time fluorescence-based reverse transcription recombinase-aided amplification (RF-RT-RAA) method for type 2 PRRSV (PRRSV-2) detection. The RF-RT-RAA assay could be performed at 42 °C for 20 min with the optimal primers and a probe. RF-RT-RAA results could be monitored using real-time fluorescence read-out or visually observed with the naked eye using a portable blue light transilluminator. The method had a strong specificity; no cross-reaction was identified with the detected common swine viruses. Moreover, the technique yielded high sensitivity with the lowest detection limit of 101 copies/µL and exhibited good repeatability and reproductively with the coefficients of variation (CV) less than 10%. Eighty-seven clinical samples were tested using RF-RT-RAA and a commercial PRRSV-2 RT-qPCR detection kit. The coincidence rate was 100% between RF-RT-RAA (real-time fluorescence read-out) and RT-qPCR, and 97.7% between RF-RT-RAA (visually observed) and RT-qPCR. The RF-RT-RAA assay provides a new method for rapid and visual detection of PRRSV-2.

Porcine Reproductive and Respiratory Syndrome virus (PRRSv): A Cross-Sectional Study on ELISA Seronegative, Multivaccinated Sows.

Vaccination against Porcine Reproductive and Respiratory Syndrome virus (PRRSv) is widely used to control clinical disease, but the effectiveness appears in some cases to be suboptimal. Field reports have stated the presence of routinely PRRSv-vaccinated but ELISA seronegative sows: the ELISA non-responders. The real extent of this phenomenon (prevalence-origin-consequences) was not yet investigated. In this study, the prevalence of ELISA non-responders was assessed by measuring PRRSv-specific antibodies in 1400 sows, originating from 70 PRRSv-vaccinating sow herds, using IDEXX ELISA (ELISA 1) and CIVTEST E/S ELISA (ELISA 2). Neutralizing antibodies (NAbs) were quantified in a virus neutralization assay. Univariable logistic regression was used to identify herd risk factors for the presence of ELISA non-responders. The global prevalence of non-responders varied from 3.5% (ELISA 1) to 4.1% (ELISA 2), the herd-level prevalence was 40% and the within-herd prevalence ranged from 5% to 20% (ELISA 1) and from 5% to 30% (ELISA 2). The ELISA non-responders had significantly lower NAbs than the ELISA responders. Herds using the combination of one modified live vaccine and one killed vaccine had a significantly reduced risk of having ELISA non-responders. A first assessment of the prevalence and possible consequences of ELISA non-responders has been provided by this study. The clinical importance, origin and underlying immunological mechanisms warrant further research.

Genetic diversity of porcine reproductive and respiratory syndrome virus and evaluation of three one-step real-time RT-PCR assays in Korea.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses in the global swine industry. Frequent genetic variations in this virus cause difficulties in controlling and accurately diagnosing PRRSV. METHODS: In this study, we investigated the genetic characteristics of PRRSV-1 and PRRSV-2 circulating in Korea from January 2018 to September 2021 and evaluated three one-step real-time reverse transcription polymerase chain reaction (RT-PCR) assays. RESULTS: A total of 129 lung samples were collected, consisting of 47 samples for PRRSV-1, 62 samples for PRRSV-2, and 20 PRRSV-negative samples. Nucleotide sequence analysis of open reading frames (ORFs) 5, ORF6, and ORF7 genes from PRRSV samples showed that PRRSV-1 belonged to subgroup A (43/47, 91.49%) and subgroup C (4/47, 8.51%), whereas PRRSV-2 was classified as lineage 1 (25/62, 40.32%), Korean lineage (Kor) C (13/62, 20.97%), Kor B (10/62, 16.13%), lineage 5 (9/62, 14.52%), and Kor A (5/62, 8.06%). Amino acid sequence analysis showed that the neutralizing epitope and T cell epitope of PRRSV-1, and the decoy epitope region and hypervariable regions of PRRSV-2 had evolved under positive selection pressure. In particular, the key amino acid substitutions were found at positions 102 and 104 of glycoprotein 5 (GP5) in some PRRSV-2, and at positions 10 and 70 of membrane protein (M) in most PRRSV-2. In addition, one-step real-time RT-PCR assays, comprising two commercial tests and one test recommended by the World Organization for Animal Health (OIE), were evaluated. CONCLUSION: The results revealed that two of the real-time RT-PCR assays had high sensitivities and specificities, whereas the real-time RT-PCR assay of the OIE had low sensitivity due to mismatches between nucleotides of Korean PRRSVs and forward primers. In this study, we genetically characterized recent PRRSV occurrences and evaluated three one-step real-time RT-PCR assays used in Korea.

Considerations in the use of processing fluids for the detection of PRRSV RNA and antibody.

Surveillance is mandatory for tracking the progress of porcine reproductive and respiratory syndrome virus (PRRSV) control and elimination efforts in breeding herds. Processing fluids, the fluid recovered from tissues collected at castration and/or tail docking, are used for breeding herd surveillance by large segments of the industry, but the basic diagnostic characteristics of processing fluids are largely undescribed. We undertook 3 studies to address this information gap. In study 1, we found no differences among the PRRSV RT-rtPCR results obtained with 4 commercial RNA extraction kits. In study 2, we found that PRRSV RNA was highly stable in processing fluid samples at -20°C or 4°C, but detrimental effects were observed at ≥22°C within 24 h. In study 3, using a modified PRRSV ELISA at a sample:positive cutoff of ≥0.5, we found excellent discrimination in the detection of PRRSV antibody (IgM, IgA, IgG) in processing fluids from herds of known PRRSV status. Judicious handling of processing fluid samples from sow herds, and the use of methods available in veterinary diagnostic laboratories, can provide a foundation for reliable PRRSV surveillance.

Effect of pooling family oral fluids on the probability of PRRSV RNA detection by RT-rtPCR.

Family oral fluids (FOFs) are an aggregate sample type shown to be a cost-efficient and convenient option for determining the porcine reproductive and respiratory syndrome virus (PRRSV) status of weaning age pigs. This study investigates the effect of pooling PRRSV-positive FOF samples with PRRSV-negative FOF samples at different levels (1/3, 1/5, 1/10, 1/20) on the probability of PRRSV RNA detection by reverse-transcription real-time polymerase chain reaction (RT-rtPCR). Mathematical models were built to assess how much the probability of RT-rtPCR PRRSV detection changed with increasing proportion of PRRSV-positive samples present within pools and how partially sampling a farrowing room influenced the probability of RT-rtPCR detection of PRRSV RNA in pooled samples at different prevalence scenarios. A general example of a guideline for FOF-based sampling under different prevalence scenarios to detect PRRSV RNA by RT-rtPCR with at least 95 % certainty is presented. At the sample level, the probability of detecting PRRSV RNA by RT-rtPCR decreased from 100 % to 87 %, 68 %, and 26 % when diluting up to 1/20 for PRRSV positive FOF having an initial Cycle threshold (Ct) below 34, between 34 and 36, or above 36, respectively. When PRRSV prevalence is near-zero (1 or 2 litters positive out of 56), the most cost-efficient farrowing room sampling strategy to detect PRRSV RNA with at least 95 % certainty was pooling FOF samples up to 1/10; at higher prevalence (≥ 3 of 56 litters positive), the most cost-efficient strategy was submitting samples in pools of 20. Subsampling a farrowing room for FOF pools was also demonstrated to be a valuable cost-saving strategy. Overall, based on the conditions of this study, pooling FOFs up to 1/20 is a valid option in situations of cost constraint and regardless of pooling level chosen, capturing as many litters as possible improves the probability of PRRSV detection.

Veterinary syndromic surveillance using swine production data for farm health management and early disease detection.

The use of syndromic surveillance (SyS) has grown in animal health since the 2010s, but the use of production data has been underexplored due to methodological and practical challenges. This paper aimed to tackle some of those challenges by developing a SyS system using production data routinely collected in pig breeding farms. Health-related indicators were created from the recorded data, and two different time-series types emerged: the weekly counts of events traditionally used in SyS; and continuous time-series, where every new event is a new observation, and grouping by time-unit is not applied. Exponentially Weighted Moving Average (EWMA) and Shewhart control charts were used for temporal aberration detection, using three detection limits to create a "severity" score. The system performance was evaluated using simulated outbreaks of porcine respiratory and reproduction syndrome (PRRS) as a disease introduction scenario. The system proved capable of providing early detection of unexpected trends, serving as a useful health and management decision support tool for farmers. Further research is needed to combine results of monitoring multiple parallel time-series into an overall assessment of the risk of reproduction failure.

Detection of Porcine Respirovirus 1 (PRV1) in Poland: Incidence of Co-Infections with Influenza A Virus (IAV) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in Herds with a Respiratory Disease.

Porcine respirovirus 1 (PRV1) is also known as porcine parainfluenza virus 1 (PPIV1). The prevalence and the role of PRV1 infections for pig health is largely unknown. In order to assess the PRV1 prevalence in Poland, nasal swabs and oral fluids collected from pigs from 30 farms were examined with RT real-time PCR. Additionally, IAV and PRRSV infection statuses of PRV1-positive samples were examined. The results showed that the virus is highly prevalent (76.7% farms positive) and different patterns of PRV1 circulation in herds with mild-moderate respiratory disease were observed. Co-infections with IAV and PRRSV were infrequent and detected in 8 (23.5%) and 4 (11.8%) out of 34 PRV1-positive nasal swab pools from diseased pens, respectively. In one pen PRV1, IAV, and PRRSV were detected at the same time. Interestingly, PRV1 mean Ct value in samples with co-infections was significantly lower (29.8 ± 3.1) than in samples with a single PRV1 infection (32.5 ± 3.6) (p < 0.05), which suggested higher virus replication in these populations. On the other hand, the virus detection in pig populations exhibiting respiratory clinical signs, negative for PRRSV and IAV, suggests that PRV1 should be involved in differential diagnosis of respiratory problems.