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.

Time Series Transcriptomic Analysis of Bronchoalveolar Lavage Cells from Piglets Infected with Virulent or Low-Virulent Porcine Reproductive and Respiratory Syndrome Virus 1.

Porcine reproductive and respiratory syndrome virus (PRRSV) has evolved to escape the immune surveillance for a survival advantage leading to a strong modulation of host's immune responses and favoring secondary bacterial infections. However, limited data are available on how the immunological and transcriptional responses elicited by virulent and low-virulent PRRSV-1 strains are comparable and how they are conserved during the infection. To explore the kinetic transcriptional signature associated with the modulation of host immune response at lung level, a time-series transcriptomic analysis was performed in bronchoalveolar lavage cells upon experimental in vivo infection with two PRRSV-1 strains of different virulence, virulent subtype 3 Lena strain or the low-virulent subtype 1 3249 strain. The time-series analysis revealed overlapping patterns of dysregulated genes enriched in T-cell signaling pathways among both virulent and low-virulent strains, highlighting an upregulation of co-stimulatory and co-inhibitory immune checkpoints that were disclosed as Hub genes. On the other hand, virulent Lena infection induced an early and more marked "negative regulation of immune system process" with an overexpression of co-inhibitory receptors genes related to T-cell and NK cell functions, in association with more severe lung lesion, lung viral load, and BAL cell kinetics. These results underline a complex network of molecular mechanisms governing PRRSV-1 immunopathogenesis at lung level, revealing a pivotal role of co-inhibitory and co-stimulatory immune checkpoints in the pulmonary disease, which may have an impact on T-cell activation and related pathways. These immune checkpoints, together with the regulation of cytokine-signaling pathways, modulated in a virulence-dependent fashion, orchestrate an interplay among pro- and anti-inflammatory responses. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the major threats to swine health and global production, causing substantial economic losses. We explore the mechanisms involved in the modulation of host immune response at lung level performing a time-series transcriptomic analysis upon experimental infection with two PRRSV-1 strains of different virulence. A complex network of molecular mechanisms was revealed to control the immunopathogenesis of PRRSV-1 infection, highlighting an interplay among pro- and anti-inflammatory responses as a potential mechanism to restrict inflammation-induced lung injury. Moreover, a pivotal role of co-inhibitory and co-stimulatory immune checkpoints was evidenced, which may lead to progressive dysfunction of T cells, impairing viral clearance and leading to persistent infection, favoring as well secondary bacterial infections or viral rebound. However, further studies should be conducted to evaluate the functional role of immune checkpoints in advanced stages of PRRSV infection and explore a possible T-cell exhaustion state.

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 visual detection of porcine reproductive and respiratory syndrome virus via recombinase polymerase amplification combined with a lateral flow dipstick.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating infectious diseases in the global swine industry. A rapid and sensitive on-site detection method for PRRS virus (PRRSV) is critically important for diagnosing PRRS. In this study, we established a method that combines reverse transcription recombinase polymerase amplification (RT-RPA) with a lateral flow dipstick (LFD) for detecting North American PRRSV (PRRSV-2). The primers and probe were designed based on the conserved region of all complete PRRSV-2 genomic sequences available in China (n = 512) from 1996 to 2020. The detection limit of the assay was 5.6 × 10-1 median tissue culture infection dose (TCID50) per reaction within 30 min at 42 °C, which was more sensitive than that of reverse transcription polymerase chain reaction (RT-PCR) (5.6 TCID50 per reaction). The assay was highly specific for the epidemic lineages of PRRSV-2 in China and did not cross-react with pseudorabies virus, porcine circovirus 2, classical swine fever virus, or porcine epidemic diarrhea virus. The assay performance was evaluated by testing 179 samples and comparing the results with those of quantitative RT-PCR (RT-qPCR). The results showed that the detection coincidence rate of RT-RPA and RT-qPCR was 100% when the cycle threshold values of RT-qPCR were < 32. The assay provides a new alternative for simple and reliable detection of PRRSV-2 and has great potential for application in the field.

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.

Development of a Nanobody-Based Competitive Enzyme-Linked Immunosorbent Assay for Efficiently and Specifically Detecting Antibodies against Genotype 2 Porcine Reproductive and Respiratory Syndrome Viruses.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes considerable economic loss to the global pig industry. Efficient detection assay is very important for the prevention of the virus infection. Nanobodies are the advantages of small molecular weight, simple genetic engineering, and low production cost for promising diagnostic application. In this study, to develop a nanobody-based competitive ELISA (cELISA) for specifically detecting antibodies against PRRSV, three nanobodies against PRRSV-N protein were screened by camel immunization, library construction, and phage display. Subsequently, a recombinant HEK293S cell line stably secreting nanobody-horseradish peroxidase (HRP) fusion protein against PRRSV-N protein was successfully constructed using the lentivirus transduction assay. Using the cell lines, the fusion protein was easily produced. Then, a novel cELISA was developed using the nanobody-HRP fusion protein for detecting antibodies against PRRSV in pig sera, exhibiting a cut-off value of 23.19% and good sensitivity, specificity, and reproducibility. Importantly, the cELISA specifically detect anti-genotype 2 PRRSV antibodies. The cELISA showed more sensitive than the commercial IDEXX ELISA kit by detecting the sequential sera from the challenged pigs. The compliance rate of cELISA with the commercial IDEXX ELISA kit was 96.4%. In addition, the commercial IDEXX ELISA kit can be combined with the developed cELISA for the differential detection of antibodies against genotype 1 and 2 PRRSV in pig sera. Collectively, the developed nanobody-based cELISA showed advantages of simple operation and low production cost and can be as an assay for epidemiological investigation of genotype 2 PRRSV infection in pigs and evaluation after vaccination.

Porcine reproductive and respiratory syndrome virus genetic variability a management and diagnostic dilemma.

As genetic analysis becomes less expensive, more comprehensive diagnostics such as whole genome sequencing (WGS) will become available to the veterinary practitioner. The WGS elucidates more about porcine reproductive and respiratory syndrome virus (PRRSV) beyond the traditional analysis of open reading frame (ORF) 5 Sanger sequencing. The veterinary practitioner will require a more complete understanding of the mechanics and consequences of PRRSV genetic variability to interpret the WGS results. More recently, PRRSV recombination events have been described in the literature. The objective of this review is to provide a comprehensive outlook for swine practitioners that PRRSV mutates and recombines naturally causing genetic variability, review the diagnostic cadence when suspecting recombination has occurred, and present theory on how, why, and where industry accepted management practices may influence recombination. As practitioners, it is imperative to remember that PRRS viral recombination is occurring continuously in swine populations. Finding a recombinant by diagnostic analysis does not ultimately declare its significance. The error prone replication, mutation, and recombination of PRRSV means exact clones may exist; but a quasispecies swarm of variable strains also exist adding to the genetic diversity. PRRSV nonstructural proteins (nsps) are translated from ORF1a and ORF1b. The arterivirus nsps modulate the hosts' immune response and are involved in viral pathogenesis. The strains that contribute the PRRSV replicase and transcription complex is driving replication and possibly recombination in the quasispecies swarm. Furthermore, mutations favoring the virus to evade the immune system may result in the emergence of a more fit virus. More fit viruses tend to become the dominant strains in the quasispecies swarm. In theory, the swine management practices that may exacerbate or mitigate recombination include immunization strategies, swine movements, regional swine density, and topography. Controlling PRRSV equates to managing the quasispecies swarm and its interaction with the host. Further research is warranted on the frequency of recombination and the genome characteristics impacting the recombination rate. With a well-defined understanding of these characteristics, the clinical implications from recombination can be detected and potentially reduced; thus, minimizing recombination and perhaps the emergence of epidemic strains.

On-site differential diagnostic detection of HP-PRRSV and C-PRRSV using EuNPs-mAb fluorescent probe-based immunoassay.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused worldwide economic losses in the swine industry. Pigs infected with highly pathogenic (HP)-PRRSV display more severe symptoms than those infected with classical (C)-PRRSV. A rapid, sensitive, and reliable detection method to distinguish between HP-PRRSV and C-PRRSV is needed. In this study, we prepared a monoclonal antibody from a hybridoma that can distinguish HP-PRRSV(including TP, QJ, LQ, JN-HS, and TY strain) from C-PRRSV (CH-1A strain) using cell surface-fluorescence immunosorbent assays (CSFIA). Based on this monoclonal antibody (4D5), we developed a europium microsphere-based lateral flow immunochromatographic strip (EuNPs-LFICS) for the differential diagnostic detection of HP-PRRSV and C-PRRSV. Under optimized conditions, the method was rapid (15 min), sensitive (LOD: 2.57 ng mL-1, 606 TCID50/0.1 mL), selective for HP-PRRSV detection, and quantitative (DLR: 3.56-228 ng mL-1). In clinical samples, the EuNPs-LFICS assay was largely consistent with PCR results, indicating its practical clinical application.

Establishment of a peptide-based enzyme-linked immunosorbent assay for detecting antibodies against PRRSV M protein.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating diseases affecting the swine industry globally. Evaluation of antibody responses and neutralizing antibody titers is the most effective method for vaccine evaluation. In this study, the B cell line epitopes of PRRSV M protein were predicted, and two peptide ELISA assays were established (M-A110-129 ELISA, M-A148-174 ELISA) to detect antibodies against PRRSV M protein. Field serum samples collected from pig farms were used to validate the peptide ELISA and compare it with an indirect immunofluorescence assay. RESULTS: The sensitivity and specificity of M-A110-129 ELISA and M-A148-174 ELISA were (111/125) 88.80%, (69/70) 98.57% and (122/125) 97.60%, (70/70) 100%, relative to indirect immunofluorescence assay. This peptide ELISA could detect antibodies against different genotypes of PRRSV including type 1 PRRSV, classical PRRSV, HP-PRRSV, and NADC30 like PRRSV, but not antibodies against other common swine viruses. The results of ROC analysis showed that the area under the curve (AUC) of the M-A110-129 ELISA and M-A148-174 ELISA were 0.967 and 0.996, respectively. Compared the concordance of results using two peptide ELISA assays, the IDEXX PRRSV X3 Ab ELISA and a virus neutralization test, were assessed using a series of 147 sera from pigs vaccinated with the NADC30-like PRRSV inactivated vaccine. The M-A148-174 ELISA had the best consistency, with a Cohen's kappa coefficient of 0.8772. The concordance rates of the Hipra PRRSV ELISA kit, M-A110-129 ELISA and M-A148-174 ELISA in the field seropositive detection results were 91.08, 86.32 and 95.35%, relative to indirect immunofluorescence assay. CONCLUSIONS: In summary, compared with M-A110-129 ELISA, the PRRSV M-A148-174 ELISA is of value for detecting antibodies against PRRSV and the evaluation of the NADC30-like PRRSV inactivated vaccine, but the advantage is insufficient in serological early diagnosis.

Development of luciferase-linked antibody capture assay based on luciferase immunoprecipitation systems for antibody detection of porcine reproductive and respiratory syndrome virus.

BACKGROUND: Early detection of porcine reproductive and respiratory syndrome virus (PRRSV) infection of swine is necessary to control this devastating disease. By monitoring host serum antibodies to viral antigens, early virus detection within herds is feasible. In this study, recombinant antigens were generated using recombinant DNA techniques to fuse PRRSV structural protein (N) or nonstructural protein 1α (nsp1α) with the Rellina luciferase gene. Next, fused genes were cloned into plasmids and transfected into HEK-293 T cells for transient expression. Upon co-incubation of lysates with pig sera, antigen-antibody complexes formed that bound to Protein-G coated onto microplates. By further measurement of luminance value, a modified form of Luciferase Immunoprecipitation Systems, namely luciferase-linked antibody capture assay (LACA) was developed for detection of PRRSV-specific antibodies. RESULTS: Known anti-PRRSV antibody-positive or -negative serum samples (125 and 122 samples, respectively) were used to validate the LACA and compared it with IDEXX PRRS ×3 ELISA. Based on the result, N-Rluc and nsp1α-Rluc LACA results were 95.3 and 94.4% in agreement with IDEXX ELISA, suggested a similar specificity of LACA to IDEXX ELISA. Moreover, when both LACA and IDEXX ELISA were used to evaluate sequential serum samples obtained from PRRSV experimentally infected pigs, the PRRSV-specific antibody response was detectable as early as 3 days post-inoculation (dpi) using N-Rluc LACA, but undetectable until 7 dpi using IDEXX ELISA, suggesting an improved sensitivity of LACA. Meanwhile, antibodies specific for nsp1α were detected at higher levels overall, but were undetectable until 10 dpi. Furthermore,. Notably, one IDEXX ELISA positive result was not confirmed by LACA or IFA and was thus considered a false-positive result. CONCLUSION: The LACA exhibited similar specificity but improved sensitivity to that of the commercial IDEXX PRRS ×3 ELISA kit for detection of PRRSV-specific antibodies in pig serum. Importantly, LACA could be adapted for detecting antibodies against other PRRSV targets, such as nsp1α, to achieve earlier detection of PRRSV infection.

A novel HRM assay for differentiating classical strains and highly pathogenic strains of type 2 porcine reproductive and respiratory syndrome virus.

Differentiation of classical strains and highly pathogenic strains of porcine reproductive and respiratory syndrome virus is crucial for effective vaccination programs and epidemiological studies. We used nested PCR and high resolution melting curve analysis with unlabeled probe to distinguish between the classical and the highly pathogenic strains of this virus. Two sets of primers and a 20 bp unlabeled probe were designed from the NSP3 gene. The unlabeled probe included two mutations specific for the classical and highly pathogenic strains of the virus. An additional primer set from the NSP2 gene of the highly pathogenic vaccine strain JXA1-R was used to detect its exclusive single nucleotide polymorphism. We tested 107 clinical samples, 21 clinical samples were positive for PRRSV (consistent with conventional PCR assay), among them four were positive for the classical strain with the remainder 17 for the highly pathogenic strain. Around 10 °C difference between probe melting temperatures showed the high discriminatory power of this method. Among highly pathogenic positive samples, three samples were determined as positive for JXA1-R vaccine-related strain with a 95% genotype confidence percentage. All these genotyping results using the high resolution melting curve assay were confirmed with DNA sequencing. This unlabeled probe method provides an alternative means to differentiate the classical strains from the highly pathogenic porcine reproductive and respiratory syndrome virus strains rapidly and accurately.

Serodiagnosis, targeting nonstructural protein 4, of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes great economic losses to the swine industry worldwide. Typically, an N protein-coated indirect enzyme-linked immunosorbent assay (N-coated iELISA) is used to detect PRRSV antibodies. Non-structural protein (NSP) 4 is essential to the PRRSV life cycle and contains B-cell epitopes. Yet, no specific antibody against NSP4 has been detected in clinical samples. In this study, we developed an NSP4-coated iELISA and compared its effectiveness with the N-coated iELISA. The NSP4-coated iELISA was developed with a cut-off value of 0.406 at an optical density of 450 nm by testing a panel of 70 PRRSV positive and 80 PRRSV negative pig serum samples, which generated a specificity and sensitivity of 100%. Agreement between the NSP4-coated and N-coated iELISAs was 92.2%. Interestingly, 50 serum samples, mostly from pigs vaccinated with the HP-PRRSV live strain, tested positive for PRRSV antibodies with the NSP4-coated iELISA, but were negative with the N-coated iELISA. These results were further confirmed by western blot analysis and another iELISA based on the N-terminus of NSP2 (NSP2-1-coated iELISA). The agreement between the results of western blot analysis with the NSP4-coated and NSP2-1-coated iELISA analyses were 92% and 96.1%, respectively, showing that the developed NSP4-coated iELISA is a useful tool to discriminate a false negative from a true negative response to the HP-PRRSV vaccine.

Oral fluid samples used for PRRSV acclimatization program and sow performance monitoring in endemic PRRS-positive farms.

An effective gilt acclimatization program is one of the most important management strategies for controlling porcine reproductive and respiratory syndrome virus (PRRSV) infection. Recently, oral fluid samples have been used as alternative diagnostic samples for various swine diseases. This study utilized oral fluids for PRRSV monitoring during the gilt acclimatization period in PRRSV endemic farms. The study was performed in two selected commercial breeding herds (farm A and farm B). PRRSV RNA and PRRSV-specific antibodies were monitored using oral fluid and serum samples. Sow performance parameters related to PRRSV infection were recorded and assessed. After PRRSV exposure during acclimatization, viral RNA was demonstrated in oral fluids from 1 to 10 weeks post-exposure (WPE). PRRSV RNA was detected in serum at 1 and 4 WPE in farm A and at 1, 4, 8, and 12 WPE in farm B. Prolonged viremia of gilts from farm B was possibly due to re-infection (within the herd) and later, reproductive problems were found in the breeding herd. The correlation of PRRSV RNA concentration in oral fluids and serum was evident. The S/P ratio values of PRRSV antibodies in oral fluid samples were higher and had similar patterns of antibody responses to the serum samples. The results suggest that the use of oral fluid samples for PRRSV monitoring during gilt acclimatization in endemic farms is effective, convenient, practical, and economical and would be most beneficial when used with other parameters.

Rapid detection of highly pathogenic porcine reproductive and respiratory syndrome virus by a fluorescent probe-based isothermal recombinase polymerase amplification assay.

A novel fluorescent probe-based real-time reverse transcription recombinase polymerase amplification (real-time RT-RPA) assay was developed for rapid detection of highly pathogenic type 2 porcine reproductive and respiratory syndrome virus (HP-PRRSV). The sensitivity analysis showed that the detection limit of RPA was 70 copies of HP-PRRSV RNA/reaction. The real-time RT-RPA highly specific amplified HP-PRRSV with no cross-reaction with classic PRRSV, classic swine fever virus, pseudorabies virus, and foot-and-mouth disease virus. Assessment with 125 clinical samples showed that the developed real-time RT-RPA assay was well correlated with real-time RT-qPCR assays for detection of HP-PRRSV. These results suggest that the developed real-time RT-RPA assay is suitable for rapid detection of HP-PRRSV.

Immunopotentiation of four natural adjuvants co-administered with a highly pathogenic porcine reproductive and respiratory syndrome virus glycoprotein 5 subunit.

To improve the efficacy of porcine reproductive and respiratory syndrome virus (PRRSV) subunit vaccine, the immunological enhancement effects of four natural adjuvants were compared, including Astragalus extract, Astragalus/Bacillus production, propolis, and Freund's adjuvant. The results showed that titers of IgG and neutralizing antibody against highly pathogenic PRRSV (HP-PRRSV) glycoprotein 5 (GP5) from the groups of Astragalus/Bacillus adjuvant and Freund's adjuvant co-administered with a recombinant HP-PRRSV GP5 subunit were the highest among all the experimental groups. In cellular immunity, as shown in T lymphocyte proliferation level, Astragalus/Bacillus adjuvant and Freund's adjuvant exhibited the most potent immunological enhancement effects. Exceptionally, pigs inoculated with Freund's adjuvant developed severely delayed humoral immune responses within 28 days post inoculation. The adjuvants of Astragalus extract and propolis also exhibited immunological enhancement effects on humoral immune and cell-mediated immune responses, but which were obviously lower than Astragalus/Bacillus adjuvant and Freund's adjuvant. Following challenge with HP-PRRSV, pigs inoculated with Astragalus/Bacillus adjuvant and Freund's adjuvant co-administered with GP5 subunit showed lower viremia, slighter clinical signs, and less pathological lung lesions compared with the groups inoculated using Astragalus extract and propolis. In conclusion, from immune responses and immunological protection against HP-PRRSV, the Astragalus/Bacillus adjuvant demonstrated the most potent immunological enhancement effect on HP-PRRSV GP5 subunit. Astragalus/Bacillus adjuvant showed promise as a candidate immune adjuvant, to more efficiently prevent and control PRRS.

Evaluation of the specificity of a commercial ELISA for detection of antibodies against porcine respiratory and reproductive syndrome virus in individual oral fluid of pigs collected in two different ways.

BACKGROUND: The monitoring of infectious diseases like the porcine reproductive and respiratory syndrome (PRRS) using pen-wise oral fluid samples becomes more and more established. The collection of individual oral fluid, which would be useful in the monitoring of PRRSV negative boar studs, is rather difficult. The aim of the study was to test two methods for individual oral fluid collection from pigs and to evaluate the specificity of a commercial ELISA for detection of PRRSV antibodies in these sample matrices. For this reason, 334 serum samples from PRRSV negative pigs (group 1) and 71 serum samples from PRRSV positive pigs (group 2) were tested for PRRSV antibodies with a commercial ELISA. Individual oral fluid was collected with a cotton gauze swab from 311 pigs from group 1 and 39 pigs from group 2. Furthermore, 312 oral fluid samples from group 1 and 67 oral fluid samples from group 2 were taken with a self-drying foam swab (GenoTube). The recollected oral fluid was then analysed twice with a commercial ELISA for detection of PRRSV antibodies in oral fluid. RESULTS: All serum samples from group 1 tested negative for PRRSV antibodies. The collection of oral fluid was sufficient in all samples. Sampling with GenoTubes was less time consuming than sampling with cotton gauze swabs. False positive results were obtained in 7 (measure 1) respectively 9 (measure 2) oral fluid samples recollected from cotton gauze swabs and in 9 and 8 samples from GenoTubes. The specificity of the oral fluid ELISA was 97.4% for cotton gauze swabs and 97.3% for GenoTubes. 70 out of 71 serum samples and all oral fluid samples from group 2 tested positive for PRRSV antibodies. The sensitivity of the oral fluid ELISA was 100%. According to the kappa coefficient, the results showed an almost perfect agreement between serum and oral fluid collected in both ways (kappa > 0.8). CONCLUSIONS: Both methods used for individual oral fluid collection proved to be practical and efficient and can be used for PRRSV antibody detection. It has to be considered, however, that false positive results may occur more often than in serum samples.

Comparison of serum and oral fluid antibody responses after vaccination with a modified live (MLV) porcine reproductive and respiratory syndrome virus (PPRSV) vaccine in PRRS endemic farms.

Porcine reproductive and respiratory syndrome (PRRS) is a disease that is both highly contagious and of great economic importance in Malaysia. Therefore, reliable and improved diagnostic methods are needed to facilitate disease surveillance. This study compared PRRSV antibody responses in oral fluid versus serum samples following PRRS modified live (MLV) vaccination using commercial antibody ELISA kits (IDEXX Laboratories, Inc.). The study involved two pig farms located in Perak and Selangor, Malaysia. Both farms were vaccinated with PRRS MLV 1 month prior to sample collection. Thirty-five animals were used as subjects in each farm. These 35 animals were divided into 7 different categories: gilts, young sows, old sows, and four weaner groups. Oral fluid and serum samples were collected from these animals individually. In addition, pen oral fluid samples were collected from weaner groups. The oral fluid and serum samples were tested with IDEXX PRRS Oral Fluid Antibody Test Kit and IDEXX PRRS X3 Antibody Test Kit, respectively. The results were based on sample to positive ratio (S/P ratio of the samples). Results revealed a significant and positive correlation between serum and oral fluid samples for both farm A (p = 0.0001, r = 0.681) and farm B (p = 0.0001, r = 0.601). In general, oral fluids provided higher S/P results than serum, but the patterns of response were highly similar, especially for the sow groups. Thus, the use of oral fluids in endemic farms is effective and economical, particularly for large herds. In conclusion, the authors strongly recommend the use of oral fluids for PRRS monitoring in endemic farms.

A sensitive multiplex real-time PCR panel for rapid diagnosis of viruses associated with porcine respiratory and reproductive disorders.

The objective of this study was to develop a multiplex real-time PCR panel using TaqMan probes for the detection and differentiation of porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus North American type (PRRSV-NA), pseudorabies virus (PRV), classical swine fever virus (CSFV), porcine parvovirus type 1 (PPV1) and Japanese encephalitis virus (JEV). Specific primer and probe combinations for PCV2, PRRSV, PRV, CSFV, PPV1 and JEV were selected within the conserved region of each viral genome. The multiplex real-time PCR panel which was run in two separate tubes was capable of specific detection of the six selected pig viruses, without cross-reactions with other non-targeted pig viruses. The detection limit of the assays was 10 copies/µL for PCV2, PRV, CSFV and PRRSV and 100 copies/µL for PPV and JEV. The two-tube multiplex real-time PCR panel showed 99.2% concordance with conventional PCR assays on 118 field samples. Overall, the multiplex real-time PCR panel provides a fast, specific, and sensitive diagnostic tool for detection of multiple viral pathogens in pigs and will be useful not only for diagnostics, or ecological, epidemiological and pathogenesis studies, but also for investigating host/virus or virus/virus interactions, particularly during coinfections.

Comparison of different commercial ELISAs for detection of antibodies against porcine respiratory and reproductive syndrome virus in serum.

BACKGROUND: In recent years, several new ELISAs for the detection of antibodies against the porcine reproductive and respiratory disease virus (PRRSV) in pig serum have been developed. To interpret the results, specificity and sensitivity data as well as agreement to a reference ELISA must be available. In this study, three commercial ELISAs (INgezim PRRS 2.0 - ELISA II, Priocheck® PRRSV Ab porcine - ELISA III and CIVTEST suis PRRS E/S PLUS - ELISA IV, detecting PRRSV type 1 antibodies) were compared to a standard ELISA (IDEXX PRRS X3 Ab Test - ELISA I). The serum of three pigs vaccinated with an attenuated PRRSV live vaccine (genotype 2) was tested prior to and several times after the vaccination. Furthermore, serum samples of 245 pigs of PRRSV positive herds, 309 pigs of monitored PRRSV negative herds, 256 fatteners of assumed PRRSV negative herds with unknown herd history and 92 wild boars were tested with all four ELISAs. RESULTS: ELISAs II and III were able to detect seroconversion of vaccinated pigs with a similar reliability. According to kappa coefficient, the results showed an almost perfect agreement between ELISA I as reference and ELISA II and III (kappa > 0.8), and substantial agreement between ELISA I and ELISA IV (kappa = 0.71). Sensitivity of ELISA II, III and IV was 96.0%, 100% and 91.5%, respectively. The specificity of the ELISAs determined in samples of monitored PRRSV negative herds was 99.0%, 95.1% and 96.4%, respectively. In assumed negative farms that were not continually monitored, more positive samples were found with ELISA II to IV. The reference ELISA I had a specificity of 100% in this study. CONCLUSIONS: All tested ELISAs were able to detect a PRRSV positive herd. The specificity and sensitivity of the tested commercial ELISAs, however, differed. ELISA II had the highest specificity and ELISA III had the highest sensitivity in comparison to the reference ELISA. ELISA IV had a lower sensitivity and specificity than the other ELISAs.

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.