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Abstract. 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 3Ј-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 TCID 50 , 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.

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Abstract. 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.

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Abstract. 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 ,14-day intervals from 28 to 202 days postinoculation. Serum samples were assayed at a dilution of 1:40, and muscle transudate samples were assayed at 5 dilutions (1:2, 1:5, 1:10, 1:20, 1:40) 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 (1:2, 1:5, 1:10, 1:20, 1:40). 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 $1:5, 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.

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