Increased prevalence of torque teno viruses in porcine respiratory disease complex affected pigs.

The role of swine torque teno sus viruses (TTSuVs) as co-factors in disease syndromes involving porcine circovirus strain 2 (PCV2) and porcine reproductive and respiratory disease syndrome virus (PRRSV) has been a debatable subject. In this study, the prevalence of TTSuVs in Iowa, the leading pork producing state in the U.S., was estimated by a duplex PCR. The PCR is capable of simultaneously detecting both teno sus viruses 1 and 2 (TTSuV1 and 2). Based on an analysis of 300 random samples representing six major geographical regions of the state, the overall prevalence rates for TTSuV1 and 2 were 47.34% and 24.67% respectively while the combined prevalence rate was 52.33%. The epidemiological association of TTSuV1 and 2 with the common etiological agents of the porcine respiratory disease complex (PRDC) namely porcine PRRSV, PCV2, Mycoplasma hyopneumoniae and swine influenza virus (SIV) was estimated in lung tissue derived from 45 pigs showing clinical signs of PRDC. Notably, 86.67% of the PRDC-suspect samples were positive for TTSuV1 in comparison to the baseline population prevalence rate of 47.34%. However, the prevalence of TTSuV2 (26.67%) was not significantly different. TTSuV1 was detected in 80.00%, 81.81%, 75.00% and 77.78% of the PRRSV, SIV, M. hyopneumoniae and PCV2 positive PRDC-suspect samples respectively. Our results indicate that TTSuV1 is strongly associated with clinical PRDC and support the hypothesis that TTSuVs might function as co-factors in PRDC. Further studies to define their possible role in the pathogenesis of swine respiratory diseases are warranted.

Comparison of RNA extraction and real-time reverse transcription polymerase chain reaction methods for the detection of Porcine reproductive and respiratory syndrome virus in porcine oral fluid specimens.

The objective of the current study was to evaluate various RNA extraction and polymerase chain reaction (PCR) protocols for the detection of Porcine reproductive and respiratory syndrome virus (PRRSV) in porcine oral fluids. Extraction protocols were selected based on ease of use and compatibility with high-throughput, automated systems. The results showed marked differences among extraction protocols, PCR protocols, and combinations thereof in detecting PRRSV in the oral fluid matrix. An important finding was that PCR reactions were partially inhibited by unknown factors in the oral fluid matrix and that inhibition was reduced by use of a higher concentration of PCR enzymes. The results suggest that further optimization of PCR assays for porcine oral fluids is needed and that laboratories should not assume that methods optimized for detection of virus in serum will perform equally with porcine oral fluids.

Porcine reproductive and respiratory syndrome virus (PRRSV) in serum and oral fluid samples from individual boars: will oral fluid replace serum for PRRSV surveillance?

The purpose of this study was to determine whether oral fluid samples could be used to monitor individually-housed adult boars for porcine reproductive and respiratory syndrome virus (PRRSV) infection. In 3 trials, 24 boars were intramuscularly (IM) inoculated with a modified-live PRRSV (MLV) vaccine (Trial 1), a Type 1 PRRSV isolate (Trial 2), or a Type 2 isolate (Trial 3). Oral fluid samples were collected daily and serum samples were collected twice weekly. Following the completion of the study, samples were randomized and blind-tested for PRRSV by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR). PRRSV was detected in oral fluids at DPI 1 and all oral fluid specimens were PRRSV qRT-PCR positive at DPI 4. Although PRRSV was detected in both serum and oral fluid specimens through DPI 21, a comparison of matched samples from individual boars showed that oral fluid was equal to serum for the detection of PRRSV at DPI 7 and more likely to be positive than serum on DPI 14 and 21. Overall, oral fluid was superior to serum for the detection of PRRSV using PCR over the 21-day observation period in this study. The results of this experiment suggest that individually-penned oral fluid sampling could be an efficient, cost-effective approach to PRRSV surveillance in boar studs and other swine populations.

The effect of vaccination against Porcine reproductive and respiratory syndrome virus (PRRSV) on the Porcine circovirus-2 (PCV-2) load in porcine circovirus associated disease (PCVAD) affected pigs.

A diagnostic project was initiated across the United States in 2006 to improve the understanding of porcine circovirus associated diseases (PCVAD) as well as to identify co-factors in PCVAD-affected farms. A Porcine circovirus-2 (PCV-2) DNA real-time polymerase chain reaction quantitation (qPCR) was established according to a published method and sera from a total of 23pig farms across the United States were examined for viral loads for PCV-2 and analyzed for any possible effects of Porcine reproductive and respiratory syndrome virus (PRRSV) vaccination on this parameter. Vaccination against PRRS resulted in significantly lower viral loads for PCV-2 in animals 13 wk or older compared with nonvaccinated animals, but vaccination of pigs against PRRS had no effect on qPCR results for PCV-2 in 4- to 12-week-old pigs. Interestingly, PRRS vaccinates had significantly lower viral loads when peak wasting disease was observed in the herds. The qPCR method for PCV-2 proved to be an important tool for help in the antemortem diagnosis of PCVAD as well as in the monitoring of infection dynamics.

Diagnostic performance of assays for the detection of anti-Porcine reproductive and respiratory syndrome virus antibodies in serum and muscle transudate ("meat juice") based on samples collected under experimental conditions.

Three assays were evaluated for their ability to detect antibodies against Porcine reproductive and respiratory syndrome virus (PRRSV) in porcine muscle transudate ("meat juice") samples. Samples were derived from 91 pigs inoculated with PRRSV isolate VR-2332 and 46 age-matched controls. Serum and muscle (Musculus longissimus dorsi) samples were collected from randomly selected animals euthanized at approximately 14-day intervals from 28 to 202 days postinoculation. Serum samples were assayed at a dilution of 140, and muscle transudate samples were assayed at 5 dilutions (12, 15, 110, 120, 140) using a commercial PRRSV antibody enzyme-linked immunosorbent assay (ELISA). In addition, muscle transudate samples were tested using an indirect fluorescent antibody test (IFAT) at 5 dilutions (12, 15, 110, 120, 140). Attempts to assay muscle transudate samples for neutralizing antibodies using a modified fluorescent focus neutralization assay were unsuccessful. Receiver operator characteristic (ROC) curve analyses were used to estimate cutoff thresholds and the associated diagnostic sensitivities and specificities for ELISA and IFAT at each dilution. For ELISA, muscle transudate samples at the ROC-optimized cutoffs were >95% sensitive and 100% specific at each dilution. At a cutoff dilution of > or =15, the IFAT diagnostic sensitivity and specificity of muscle transudate was estimated at 63.3% and 100%, respectively. These findings validated the use of muscle transudate samples in PRRSV surveillance programs based on ELISA antibody testing.

Simultaneous detection of North American and European porcine reproductive and respiratory syndrome virus using real-time quantitative reverse transcriptase-PCR.

Porcine reproductive and respiratory syndrome (PRRS) is 1 of the most economically important diseases of swine. Detection of the etiologic agent, PRRS virus (PRRSV), represents a diagnostic challenge due to the heterogeneity of field isolates as well as the propensity for swine to develop persistent infection in which virus is difficult to detect. Recently European (EU) lineage PRRSV isolates, which are genetically divergent from North American (NA) isolates, have been introduced into NA swine further complicating efforts to diagnose this disease. In this study, real-time (TaqMan) reverse transcriptase (RT)-PCR assays were developed for multiplex detection, differentiation, and quantification of NA and EU PRRSV field isolates. Oligonucleotide primers and dual-labeled probes were selected from conserved regions of open-reading frame 7 and the 3'-untranslated region. The real-time RT-PCR assays described for the NA or EU genotype of PRRSV detected viral RNA from 83/83 strains (74 NA; 9 EU) previously isolated by cell culture between 1992 and 2003. The analytical sensitivity of both assays was consistently found to be less than a single TCID50, which corresponded to 5-10 RNA molecules, and was not significantly reduced when the reactions were performed in a multiplex format. When performing multiplex reactions, sensitive detection was possible even when 1 viral RNA concentration was up to 5,000-fold higher than the second. The diagnostic sensitivity and specificity of the multiplex reaction was found to be at a minimum equivalent to that of both nested RT-PCR and virus isolation.