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Due to advances in laboratory technologies, multiple methods are commercially available throughout the world for diagnosing the presence of PRRSV. This article will focus on selected antigen and antibody detection methods based on their application by practicing veterinarians to field cases in the US.
Antigen tests
Virus isolation
PRRSV can be cultured in a limited number and type of cell lines, including porcine alveolar macrophages and highly permissive clones of MA-104 cells. PRRSV varies in its ability to replicate in different cell lines; therefore, the use of porcine alveolar macrophages in conjunction with MA-104 cells enhances the sensitivity of virus isolation assays. While serum, buffy coat, lung, alveolar washing, lymph nodes, and tonsils are the specimens of choice for virus isolation, the efficacy of virus detection in semen via cell culture methods is limited due to the cytotoxicity of semen. The presence of PRRSV in cell culture is confirmed by observation of cytopathic effect (CPE). CPE is usually observed within 2 to 6 days. Because virus isolation require the presence of viable virus in a sample, the sensitivity of the test can be influenced by sample-handling procedures, including the temperature in which samples have been stored, and the interval of time following collection of the samples.
Blood sampling is an effective means to assess patterns of PRRSV transmission in swine populations
Polymerase chain reaction (PCR)
The polymerase chain reaction (PCR) is an in-vitro enzymatic method which allows the exponential amplification of a specific nucleic acid sequence through repeated cycles at different temperatures. Reverse transcription-PCR (RT-PCR) has been applied to detect PRRSV RNA from several field samples, including serum, tissues, and semen. It has been considered to be a highly sensitive and rapid method to detect PRRSV nucleic acid, providing results within 24 to 48 hours. The use of PCR has improved the detection of PRRSV for boar semen. Nested RT-PCR has been described to be an extremely sensitive technique for the detection of low concentrations of PRRSV in boar semen. The minimum number of PRRS virions per unit volume detected by nested RT-PCR was 10 to 30 infectious particles/ml and the concentration of PRRSV per unit volume was 10-2 to 102 TCID50/ml. Despite a high degree of sensitivity and specificity, increased false positive results due to carry-over contamination of amplicons are major drawbacks of the nested RT-PCR assay.
In the US, the TaqManTM PCR assay is widely regarded as an excellent assay for the detection of PRRSV RNA. Based on the ORF 6, region, the sensitivity of TaqManTM PCR has been shown to be similar to that of nested RT-PCR (10-2 to 10-1 TCID50/ml). The TaqManTM PCR assay eliminates the need for analyzing PCR products on agarose gels. The samples are read by an automated fluorescent reader, using a closed-tube system to hold samples, which prevents carry-over contamination, resulting in more rapid and higher throughout assays.
Litters of infected pigs provide excellent samples for identification of PRRSV
Nucleic acid Sequencing
Recent advances in molecular diagnostics has allowed for the characterization of the open reading frame (ORF) 5 regions by molecular sequencing. The ORF 5 encodes for the virus envelope protein, is the most highly variable portion of the virus genome and is frequently prone to mutation. Therefore, it is useful for characterizing genetically distinct isolates of PRRSV. The higher the percentage of homology among isolates, the genetically closer the isolates are considered to be related. Sequencing has allow veterinarians to conduct epidemiological investigations of acute epizootics of PRRS, as well as to better understand the epidemiology of PRRS within chronically infected farms having a history of recurrent reproductive failure secondary to PRRS.
Antibody tests
PRRSV antibodies can be detected by enzyme linked immunosorbent assay (ELISA) as well as other serological tests, including immunoperoxidase monolayer assay (IPMA), indirect fluorescent antibody test (IFA), and serum neutralization test (SN). However, in the US, the primary antibody test applied to pig populations is the ELISA.
At this time, the IDEXX ELISA is widely applied to diagnose the presence of PRRSV antibodies following PRRSV infection in many countries, including the United States, Canada, Asia and Latin America. The IDEXX ELISA has been reported to perform at high levels of both sensitivity (100%) and specificity (99.5%), and automated aspects of this assay allow for the rapid test of large scale number of samples, providing results within 24 hours. Similar to tests with a high degree of sensitivity, the IDEXX ELISA can produce false positive reactions under field conditions. Therefore, it is important to note that the IDEXX ELISA should be applied to detect PRRSV antibodies as a population test at the herd level, rather than at the individual animal level.
The presence of PRRSV antibodies detected by the IDEXX ELISA is determined by measuring the sample to positive ratio (s/p ratio) which is corrected for non-specificity. A s/p ratio of 0.4 or greater is considered positive for PRRSV infection. The ELISA detects PRRSV antibodies (IgG) on 7 to 14-day after infection, reaches maximal titers by 30 to 50 days, and gradually decline to low or undetectable levels by 4 to 6 months post-infection. The PRRSV vaccination status, age, and regional prevalence of PRRSV must be considered when interpreting serologic test results because the ELISA and any other serological tests do not distinguish PRRSV antibodies of vaccinated pigs from those of naturally infected pigs. Furthermore, young pigs may possess passive antibodies from their mothers for up to 3 to 4 week of age. Finally, the PRRSV antibodies that are detected by the ELISA are only indicative of exposure to PRRSV, not protection against the disease of PRRS.
