Comparison of Commercial Real-Time Reverse Transcription-PCR Assays for Reliable, Early, and Rapid Detection of Heterologous Strains of Porcine Reproductive and Respiratory Syndrome Virus in Experimentally Infected or Noninfected Boars by Use of Different Sample Types

Abstract

The aims of this study were to compare three commercial porcine reproductive and respiratory syndrome virus (PRRSV) realtime reverse transcription-PCR (RT-PCR) assays for detection of genetically diverse PRRSV isolates in serum, semen, blood swabs, and oral fluids collected from experimentally infected boars and to evaluate the effects of sample pooling. Six groups of three boars negative for PRRSV were each inoculated with one of six PRRSV isolates (sharing 55 to 99% nucleotide sequence identity in ORF5). Samples were collected on days ؊2, P orcine reproductive and respiratory syndrome virus (PRRSV) continues to be the most important pathogen affecting pigs in North America (1). PRRSV is a small, enveloped, single-stranded positive-sense RNA virus of the family Arteriviridae and can be divided into two genotypes: type 1 (European type [EU]) and type 2 (North American type [NA]) (2). Although a variety of commercial vaccines are available on the global market, the virus remains difficult to control, and the demand for PRRSV-naïve replacement genetics and, with it, the need for highly sensitive and specific assays that can detect genetically diverse strains and provide information on the most appropriate samples for testing continue to grow.Presently, many boar studs in the United States are PRRSV negative and are routinely tested for PRRSV to ensure that PRRSV-free semen is used in breeding herds for artificial insemination (1). If previously negative boar studs become infected with PRRSV, it is critical to detect the virus as soon as possible so that any shipments of possible PRRSV-contaminated semen can be stopped.The reverse transcription-PCR (RT-PCR) method in the realtime format is one of the most commonly used techniques for detection of PRRSV RNA because of its sensitivity and specificity and relatively short test turnaround time. However, the high mutation rate, rapid evolution, and genetic variability of PRRSV strains complicate the development of long-term reliable diagnostic assays, and consequently cases of false-negative results with commercially available assays have been reported (3-5).Active PRRSV surveillance in boar studs relies mainly on collection and testing of serum, semen, blood swabs, and, more recently, oral fluids (6, 7). The choice of sample type should take into consideration the availability and ease of collection in addition to the sensitivity and specificity of the RT-PCR assay. Studies have shown that PRRSV RNA can be detected in boar serum, oral fluids, and blood swabs as early as 24 to 48 h postinfection and in semen samples as early as 48 to 120 h postinfection (6-9). Isolatespecific differences in the levels of PRRSV replication and shedding in the host have been reported (10), and as veterinarians and diagnosticians consider using alternative sampling methods, it is important to conduct an unbiased comparison of the ability of different commercial real-time RT-PCR tests to detect genetically diverse isolates of PRRSV in new and conventional sample types.The sample collection ...

Comparison of PRRSV Nucleic Acid and Antibody Detection in Pen-Based Oral Fluid and Individual Serum Samples in Three Different Age Categories of Post-Weaning Pigs from Endemically Infected Farms

“…The same reasoning could hold true for the two pens that were missed in the youngest age category (8-12w). The negative oral fluid result in the non-vaccinating farm (F1: 10w) at the moment that virus circulation was detected for the first time in serum could however potentially also be explained by our previous finding that oral fluid becomes later PRRSV positive than serum after initial infection [1]. Before concluding that PRRSV monitoring in oral fluid is less sensitive than monitoring in serum, it should also be noted that current monitoring and surveillance programs that are based on blood sampling usually not sample all animals in the pen but often only select and test 5 animals per age category.…”

“…Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of PRRS, a chronic, infectious and economically important disease of swine that is still difficult to control despite the availability of different types of vaccines [1]. PRRSV is differentiated into genetically distinct genotype 1 (European genotype) and genotype 2 (American genotype) strains.…”

“…Oral fluid collected at pen level namely possesses several benefits compared to blood sampling: it is a non-invasive collection technique; samples can be collected at group level, and time and money can be saved due to the ease of collection. Many different aspects related to oral fluid sampling and its use as a diagnostic matrix for PRRSV detection have been studied during the past decade: RNA extraction methods and PCR tests were compared and developed for PRRSV detection [1,9–10]; tests for PRRSV specific antibody detection of different isotypes were developed and compared [11–14]; issues related to collection material, sample collection protocol and sample storage were studied and optimized [10,14–17]; and comparison was made between oral fluid and serum diagnostics for PRRSV in individual pigs [18–23]. All these studies showed that oral fluid could be a promising matrix for PRRSV surveillance.…”

Diagnosis of the Lelystad strain of Porcine reproductive and respiratory syndrome virus infection in individually housed pigs: comparison between serum and oral fluid samples for viral nucleic acid and antibody detection

“…8 In Europe, PRRSV is endemic in nearly all countries with major pig production, whereas countries with smaller swine stocks such as Finland, Norway, Sweden, and Switzerland are considered PRRSV free. 18 PRRSV is differentiated into 2 genetically distinct genotypes: type 1, or European genotype, and type 2, or North American genotype.…”

Development and Validation of an Assay To Detect Porcine Reproductive and Respiratory Syndrome Virus-Specific Neutralizing Antibody Titers in Pig Oral Fluid Samples

“…In pigs, shedding of PRRSV (17,18) and the presence of virusspecific antibody in oral fluid samples have been reported (19). Recently, swine researchers have conducted pioneering studies to survey PRRSV and porcine circovirus 2 (PCV2) using oral fluid samples (19)(20)(21)(22)(23)(24).…”