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.

Molecular epidemiology of porcine reproductive and respiratory syndrome virus in Central China since 2014: The prevalence of NADC30-like PRRSVs.

Porcine reproductive and respiratory syndrome (PRRS), characterized by respiratory disorders in piglets and reproductive failure in sows, is still the great threat of swine industry. Recently, Emergence of the novel NADC30-like PRRS viruses (PRRSVs) has caused widespread outbreaks of PRRS. To investigate the epidemic characteristics of PRRSVs in Central China since 2014, 6372 clinical serum samples were tested by ELISA, 250 tissue samples were tested by RT-PCR, and among these, 30 ORF5 and 17 Nsp2 genes sequences were analyzed. Phylogenetic tree based on ORF5 revealed that, 17 isolates were clustered into subgroup 1, represented by the NADC30. And for the Nsp2, The strains which had a discontinuous 131-amino-acid deletion in Nsp2, called NADC30-like strains, were clustered into subgroup 2. Our data suggested that the NADC30-like PRRSV strains spread quickly and are now circulating and prevalent in Central China as well as the classical HP-PRRSV strains. In addition, amino acid variation analysis of GP5 revealed that the amino acid sequences of NADC30-like PRRSV strains underwent rapid evolution and contained extensive amino acid substitutions in important motifs, such as potential neutralization epitope and the N-glycosylation sites. In summary, our data would provide a large amount of detailed information on molecular variation and genetic diversity of PRRSV in central China.

Characterisation of the nucleic acid binding features of the PRRSV 7ap and its ability to induce antinuclear antibodies.

A short alternative open reading frame named ORF7a has recently been discovered within the nucleocapsid gene of the porcine reproductive and respiratory syndrome virus (PRRSV) genome. Proteins (7ap) translated from the ORF7a of two divergent strains - a type I and a type II - are able to completely reduce the motility of nucleic acids at relatively high molar charge ratios in gel retardation assays indicating strong dsDNA- and ssRNA-binding capability. Conserved RNA- and DNA-binding properties suggest that nucleic acid binding is a functional property of the divergent 7aps, and not an arbitrary consequence of their net positive charge. Sera from Hu7ap-immunised pigs and mice did not react with Hu7ap or Hu7ap-GFP; however, antinuclear antibodies were detected in the sera of the immunised animals, suggesting an ability of Hu7ap to interact with or mimic autoantigenic macromolecules.

Elevated plasma-soluble CD16 levels in porcine reproductive and respiratory syndrome virus-infected pigs: correlation with ADAM17-mediated shedding.

Soluble CD16 (sCD16) is closely correlated with chronic diseases in humans. Here, plasma sCD16 levels in pigs were increased by infection with porcine reproductive and respiratory syndrome virus (PRRSV) but not with porcine epidemic diarrhea virus, porcine circovirus type 2 and pseudorabies virus. Of interest, PRRSV attached to blood neutrophils and reduced surface CD16 expression on neutrophils. In vitro data confirmed that PRRSV caused CD16 shedding in neutrophils. Further analyses revealed that ADAM17 was involved in porcine CD16 shedding. Thus, our findings suggest that increase in sCD16 levels may be an indicator of PRRSV infection.

Pathogenicity comparison between highly pathogenic and NADC30-like porcine reproductive and respiratory syndrome virus.

The pathogenicity of HNjz15, an NADC30-like strain of porcine reproductive and respiratory syndrome virus (PRRSV), was investigated and compared to that of a highly pathogenic PRRSV JAX1 strain. Six-week-old pigs infected with each virus showed typical clinical symptoms, including high fever and respiratory disorders. Pigs infected with JXA1 had more-severe clinical manifestations than pigs infected with HNjz15. HNjz15 replicated in vivo with kinetics similar to those of JXA1 but induced a lower level of PRRSV-specific antibody at the beginning of virus infection. Histopathologically, JXA1 infection led to more-severe lung lesions and broader organ tropism than HNjz15 did. Different from what was observed with the previously reported NADC30-like PRRSV JL580 strain, all HNjz15-infected pigs survived until the end of the study. All of these results indicated that NADC30-like PRRSV HNjz15 is virulent to pigs but is less pathogenic than the JXA1 and JL580 PRRSV strains.

Serological evidence of type 2 (North American genotype) porcine reproductive and respiratory syndrome virus in Nepal.

Porcine reproductive and respiratory syndrome virus (PRRSV) has spread throughout Asia, causing significant losses to commercial farmers and smallholders. However, little is known about PRRS in Nepal, a South Asian country with a gradually increasing pig industry. In 2011, a pilot project was initiated to identify the status of PRRSV in pigs of the Kathmandu Valley of Nepal. Out of 98 serum samples, 31 (32 %; 95 % CI 23-42 %) were found positive by ELISA. All positive samples belonged to the type 2 (North American) genotype. Molecular evaluation by real-time PCR however did not yield positive results. At the herd level, seropositivity was associated with a history of abortion and premature birth. Veterinarians, farmers and government should be aware of this threat to the Nepalese pig industry and initiate an appropriate response.

Identification of a putative quantitative trait nucleotide in guanylate binding protein 5 for host response to PRRS virus infection.

BACKGROUND: Previously, we identified a major quantitative trait locus (QTL) for host response to Porcine Respiratory and Reproductive Syndrome virus (PRRSV) infection in high linkage disequilibrium (LD) with SNP rs80800372 on Sus scrofa chromosome 4 (SSC4). RESULTS: Within this QTL, guanylate binding protein 5 (GBP5) was differentially expressed (DE) (p < 0.05) in blood from AA versus AB rs80800372 genotyped pigs at 7,11, and 14 days post PRRSV infection. All variants within the GBP5 transcript in LD with rs80800372 exhibited allele specific expression (ASE) in AB individuals (p < 0.0001). A transcript re-assembly revealed three alternatively spliced transcripts for GBP5. An intronic SNP in GBP5, rs340943904, introduces a splice acceptor site that inserts five nucleotides into the transcript. Individuals homozygous for the unfavorable AA genotype predominantly produced this transcript, with a shifted reading frame and early stop codon that truncates the 88 C-terminal amino acids of the protein. RNA-seq analysis confirmed this SNP was associated with differential splicing by QTL genotype (p < 0.0001) and this was validated by quantitative capillary electrophoresis (p < 0.0001). The wild-type transcript was expressed at a higher level in AB versus AA individuals, whereas the five-nucleotide insertion transcript was the dominant form in AA individuals. Splicing and ASE results are consistent with the observed dominant nature of the favorable QTL allele. The rs340943904 SNP was also 100 % concordant with rs80800372 in a validation population that possessed an alternate form of the favorable B QTL haplotype. CONCLUSIONS: GBP5 is known to play a role in inflammasome assembly during immune response. However, the role of GBP5 host genetic variation in viral immunity is novel. These findings demonstrate that rs340943904 is a strong candidate causal mutation for the SSC4 QTL that controls variation in host response to PRRSV.

Concentration and pattern changes of porcine serum apolipoprotein A-I in four different infectious diseases.

Apolipoprotein A-I (Apo A-I) is a major protein in lipid/lipoprotein metabolism and decreased serum levels have been observed in many species in response to inflammatory and infectious challenges. Little is known about the porcine homologue, therefore in this work we have characterized it through biochemical and proteomic techniques. In 2DE, porcine serum Apo A-I is found as three spots, the two more acidic ones corresponding to the mature protein, the more basic spot to the protein precursor. Despite high sequence coverage in LC-MS/MS, we did not find a sequence or PTM difference between the two mature protein species. Besides this biochemical characterization, we measured overall levels and relative species abundance of serum Apo A-I in four different viral and bacterial porcine infectious diseases. Lower overall amounts of Apo A-I were observed in Salmonella typhimurium and Escherichia coli infections. In the 2DE protein pattern, an increase of the protein precursor together with a lower level of mature protein species were detected in the porcine circovirus type 2-systemic disease and S. typhimurium infection. These results reveal that both the porcine serum Apo A-I concentration and the species pattern are influenced by the nature of the infectious disease.

Complete genomic characterization and genetic diversity of four European genotype porcine reproductive and respiratory syndrome virus isolates from China in 2011.

Porcine reproductive and respiratory syndrome (PRRS) is an emerging disease that has caused serious economic losses to the swine industry worldwide. In 2011, a nation-wide surveillance program investigated the prevalence of PRRS viruses (PRRSV) in Chinese breeding swine farms, and four European genotype PRRSV (PRRSV-Type 1) were successfully isolated. To explore the genetic diversity of PRRSV-Type 1 in China, these 4 viral strains were subjected to genome sequencing and analysis. The four isolates shared 87.4-90.7 % nucleotide homology with the Lelystad strain (PRRSV-Type 1 stereotype strain). NSP2, ORF3, and ORF4 were the most variable regions and contained discontinuous deletions or insertions when compared to other PRRSV-Type 1 strains. All isolates fell into separate branches of the subtype 1 of PRRSV-Type 1 phylogenetic tree. This analysis of emerging PRRSV-Type 1 strains revealed previously unrecorded genetic diversity. Close attention should be paid to the prevention and control of this evolving virus.

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.

Cytokine profiles in pregnant gilts experimentally infected with porcine reproductive and respiratory syndrome virus and relationships with viral load and fetal outcome.

In spite of extensive research, immunologic control mechanisms against Porcine Reproductive and Respiratory Syndrome virus (PRRSv) remain poorly understood. Cytokine responses have been exhaustively studied in nursery pigs and show contradictory results. Since no detailed reports on cytokine responses to PRRSv in pregnant females exist, the objectives of this study were to compare host cytokine responses between PRRSv-infected and non-infected pregnant gilts, and to investigate relationships between cytokine levels in infected gilts and viral load or fetal mortality rate. Serum samples and supernatants of peripheral blood mononuclear cells (PBMC) either stimulated with PRRSv or phorbol myristate acetate/Ionomycin (PMA/Iono) were analyzed for cytokines/chemokines: interleukins (IL) 1-beta (IL1ß), IL4, IL8, IL10, IL12, chemokine ligand 2 (CCL2), interferon alpha (IFNα) and interferon gamma (IFNγ). Three cytokines (IFNα, CCL2, IFNγ) in gilt serum differed significantly in inoculated versus control gilts over time. In supernatants of PRRSv stimulated PBMC from PRRSv-infected gilts, levels of IFNα were significantly decreased, while IL8 secretion was significantly increased. PRRSv infection altered the secretion of all measured cytokines, with the exception of IFNα, from PBMC after mitogen stimulation, indicating a possible immunomodulatory effect of PRRSv. IFNα, CCL2, and IFNγ in serum, and IFNγ in supernatants of PMA/Iono stimulated PBMC were significantly associated with viral load in tissues, serum or both. However, only IFNα in supernatants of PRRSv stimulated PBMC was significantly associated with fetal mortality rate. We conclude that of the eight cytokines tested in this study IFNα was the best indicator of viral load and severity of reproductive PRRSv infection.

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.

Detection of total and PRRSV-specific antibodies in oral fluids collected with different rope types from PRRSV-vaccinated and experimentally infected pigs.

BACKGROUND: Oral fluid collected by means of ropes has the potential to replace serum for monitoring and surveillance of important swine pathogens. Until now, the most commonly used method to collect oral fluid is by hanging a cotton rope in a pen. However, concerns about the influence of rope material on subsequent immunological assays have been raised. In this study, we evaluated six different rope materials for the collection of oral fluid and the subsequent detection of total and PRRSV-specific antibodies of different isotypes in oral fluid collected from PRRSV-vaccinated and infected pigs. RESULTS: An initial experiment showed that IgA is the predominant antibody isotype in porcine saliva. Moreover, it was found that synthetic ropes may yield higher amounts of IgA, whereas all rope types seemed to be equally suitable for IgG collection. Although IgA is the predominant antibody isotype in porcine oral fluid, the PRRSV-specific IgA-based IPMA and ELISA tests were clearly not ideal for sensitive detection of PRRSV-specific IgA antibodies. In contrast, PRRSV-specific IgG in oral fluids was readily detected in PRRSV-specific IgG-based IPMA and ELISA tests, indicating that IgG is a more reliable isotype for monitoring PRRSV-specific antibody immunity in vaccinated/infected animals via oral fluids with the currently available tests. CONCLUSIONS: Since PRRSV-specific IgG detection seems more reliable than PRRSV-specific IgA detection for monitoring PRRSV-specific antibody immunity via oral fluids, and since all rope types yield equal amounts of IgG, it seems that the currently used cotton ropes are an appropriate choice for sample collection in PRRSV monitoring.

Effects of dietary threonine and tryptophan supplementation on growing pigs induced by porcine respiratory and reproductive syndrome vaccination.

A total of 32 growing pigs were used in a 2 × 2 factorial arrangement of treatments with two different diets (conventional [CON] diet vs. threonine [Thr]- and tryptophan [Trp]-rich [TTR] diet) and two immunological challenge regimens (porcine respiratory and reproductive syndrome [PRRS] vaccine vs. phosphate buffer solution [PBS]) to study the hypothesis that dietary supplementation with Trp and Thr would benefit for growing pigs vaccinated with PRRS vaccine. After feeding the experimental diets for 21 d, the pigs were intramuscularly vaccinated with PRRS or PBS. Performance data were recorded over a period of 10 weeks and are presented for the pre-challenge period (3 weeks) and the challenge period (7 weeks, where on day 1, pigs were immunologically challenged). During the pre-challenge period, the growth performance was not different between dietary treatments. PRRS vaccination resulted in increased rectal temperature and decreased feed intake and growth rate (p < 0.05). In PRRS-vaccinated pigs, diet TTR enhanced the feed intake, especially during the first 2 weeks after the PRRS vaccination compared with diet CON (p < 0.05). PRRS vaccination also resulted in increased plasma concentration of urea nitrogen, essential and non-essential amino acids (p < 0.05) and porcine reproductive and respiratory syndrome virus specific antibodies (p < 0.05), but decreased concentration of immunoproteins including alpha-1-acylglycoprotein and immunoglobulin G (p < 0.05). The alleviation of the PRRS vaccination induced decrease in feed intake and growth rate by Thr and Trp supplementation, indicating that the PRRS-vaccinated pigs had a higher Thr and Trp requirement than non-vaccinated pigs.

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.

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.

Kinetics of the porcine reproductive and respiratory syndrome virus (PRRSV) humoral immune response in swine serum and oral fluids collected from individual boars.

BACKGROUND: The object of this study was to describe and contrast the kinetics of the humoral response in serum and oral fluid specimens during acute porcine reproductive and respiratory syndrome virus (PRRSV) infection. The study involved three trials of 24 boars each. Boars were intramuscularly inoculated with a commercial modified live virus (MLV) vaccine (Trial 1), a Type 1 PRRSV field isolated (Trial 2), or a Type 2 PRRSV field isolate (Trial 3). Oral fluid samples were collected from individual boars on day post inoculation (DPI) -7 and 0 to 21. Serum samples were collected from all boars on DPI -7, 0, 7, 14, 21 and from 4 randomly selected boars on DPI 3, 5, 10, and 17. Thereafter, serum and oral fluid were assayed for PRRSV antibody using antibody isotype-specific ELISAs (IgM, IgA, IgG) adapted to serum or oral fluid. RESULTS: Statistically significant differences in viral replication and antibody responses were observed among the three trials in both serum and oral fluid specimens. PRRSV serum IgM, IgA, and IgG were first detected in samples collected on DPI 7, 10, and 10, respectively. Oral fluid IgM, IgA, and IgG were detected in samples collected between DPI 3 to 10, 7 to 10, and 8 to 14, respectively. CONCLUSIONS: This study enhanced our knowledge of the PRRSV humoral immune response and provided a broader foundation for the development and application of oral fluid antibody-based diagnostics.

Comparison of viremia of type II porcine reproductive and respiratory syndrome virus in naturally infected pigs by zip nucleic acid probe-based real-time PCR.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a RNA virus with high genetic variation. This virus causes significant economic losses in most pig-producing countries. The clinical presentation of PRRSV ranges from asymptomatic to devastating. In this study, we developed a sensitive and specific zip nucleic acid probe-based real-time PCR assay to evaluate the viremia of natural PRRSV-infected pigs in Taiwan. Serum samples were collected from 577 pigs aged 5-12 weeks. These include 444 clinically healthy pigs and 133 symptomatic pigs were confirmed to have porcine respiratory disease complex (PRDC). RESULTS: Viremia was quantified in 79 of the 444 (17.8%) clinically healthy pigs and in 112 of the 133 (84.2%) PRDC cases. Viremias were significantly more common in pigs with PRDC compared with the clinically healthy pigs (P <0.0001). These results suggest that a high viral load is a major feature of PRRSV-affected pigs. CONCLUSIONS: ZNA probe-based real-time PCR can be a useful tool to diagnose symptomatic and asymptomatic PRRSV-infected pigs. The presence of this marker in a sample of animals with high PRRSV loads (>10(4.2) PRRSV genomes/µl of serum) seems to indicate that it correlates with the presence of PRDC in pigs.

Blood antioxidant enzymes (SOD, GPX), biochemical and haematological parameters in pigs naturally infected with porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) has become one of the most economically important diseases for the swine industry worldwide. The objective of the study was to determine selected blood antioxidant enzymes (glutathione peroxidase (GPX), superoxide dismutase (SOD)), biochemical and haematological parameters in PRRS positive and negative pigs of three different categories, mainly to test oxidative stress hypothesis in pigs naturally infected with PRRS virus. Ninety PRRS positive and 90 PRRS negative pigs were included in the study. The presence of PRRS was confirmed by serological detection of antibodies against PRRS virus (PRRSV) and detection of PRRS viral RNA by RT-PCR. Pigs were further divided into three groups of 30: piglets just before weaning (weaners), fatteners and finishers. Blood samples for determining selected blood parameters were collected from the vena cava cranialis. Significantly (P < 0.05) higher activities of SOD in weaners and fatteners and of GPX in weaners were determined in PRRS positive pigs than in corresponding groups of PRRS negative pigs. In contrast, significantly (P < 0.05) lower GPX activity was observed in finishers of PRRS positive pigs than in the corresponding group of PRRS negative pigs. Concentrations of serum total protein in PRRS positive weaners and fatteners were significantly (P < 0.05) higher than those found in PRRS negative pigs. Leukopenia was observed in all three groups of PRRS positive pigs. It has been demonstrated, for the first time, that oxidative stress might be increased in PRRSV naturally infected pigs, especially in weaners.

Serum protein profiles as potential biomarkers for infectious disease status in pigs.

BACKGROUND: In veterinary medicine and animal husbandry, there is a need for tools allowing the early warning of diseases. Preferably, tests should be available that warn farmers and veterinarians during the incubation periods of disease and before the onset of clinical signs. The objective of this study was to explore the potential of serum protein profiles as an early biomarker for infectious disease status. Serum samples were obtained from an experimental pig model for porcine circovirus-associated disease (PCVAD), consisting of Porcine Circovirus type 2 (PCV2) infection in combination with either Porcine Parvovirus (PPV) or Porcine Reproductive and Respiratory Syndrome virus (PRRSV). Sera were collected before and after onset of clinical signs at day 0, 5 and 19 post infection. Serum protein profiles were evaluated against sera from non-infected control animals. RESULTS: Protein profiles were generated by SELDI-TOF mass spectrometry in combination with the Proteominer™ technology to enrich for low-abundance proteins. Based on these protein profiles, the experimentally infected pigs could be classified according to their infectious disease status. Before the onset of clinical signs 88% of the infected animals could be classified correctly, after the onset of clinical sigs 93%. The sensitivity of the classification appeared to be high. The protein profiles could distinguish between separate infection models, although specificity was moderate to low. Classification of PCV2/PRRSV infected animals was superior compared to PCV2/PPV infected animals. Limiting the number of proteins in the profiles (ranging from 568 to 10) had only minor effects on the classification performance. CONCLUSIONS: This study shows that serum protein profiles have potential for detection and identification of viral infections in pigs before clinical signs of the disease become visible.