Porcine Reproductive and Respiratory Syndrome Virus Diagnostics
by Joseph F. Connor, DVM, MS

Porcine reproductive and respiratory syndrome virus (PRRSV) has arguably had the single greatest impact on the cost of production in the swine industry. Management of this disease and understanding the epidemiology has continued to drive development of various diagnostic techniques. It is important that veterinarians understand the advantages and disadvantages of the currently available technology associated with PRRSV diagnostic assays. Strategic use and interpretation of the available diagnostic tests provide the veterinarian with useful information in diagnosing, managing, eliminating and understanding the epidemiology, as well as in implementing biosecurity protocols. Diagnostic tests have various applications depending on the diagnostic goal and limitations of each test. This article reviews some of the common areas of use and tests for the veterinary practitioner.

Breeding-Herd Stability
Breeding-herd stability occurs when PRRSV is not circulating in the breeding herd. In general, the objective of this screening is to assess a recent PRRSV episode in the breeding herd and/or the effectiveness of the implemented control measures, which frequently include replacement animal acclimatization, mass vaccination, planned exposure, or herd closure following an outbreak. Samples tested with the enzyme-linked immunosorbent assay (ELISA) can be used as screens and over time the average S/P ratio and minimum/maximum will decline as the herd stabilizes. To determine if there is active circulation of PRRSV, at least one piglet per each weaned litter can be sampled at two-week intervals for two to four weeks, or multiple pigs sampled from low-viability pigs at birth. Samples can be pooled into sets of five and submitted for RT-nested PCR or real-time PCR. If all pools are negative, average ELISA S/P ratios are declining, and performance parameters have returned to target, sentinels can be introduced into the breeding herd and serum sampled biweekly for PRRSV ELISA. The sentinels should have nose-to-nose contact with the herd population. The last serum samples should be tested by RT-nested PCR or real-time PCR. As a final step in determining herd stability to PRRSV, tonsils and lymph nodes of cull animals can be collected and homogenates tested with RT-nested PCR or real-time PCR.

Replacement Animal Monitoring
Replacement animals purchased or produced internally should be tested with ELISA. The IDEXX HerdChek® PRRS 2XR Antibody Test Kit is the test of choice. The HerdChek PRRS 2XR Antibody Test Kit uses nucleocapsid proteins as the antigen. Antibodies against PRRSV that are used diagnostically to detect infected animals are directed against the nucleocapsid protein encoded by open reading frame 7. These are non-neutralizing antibodies. The sampling of population should be conducted statistically and consideration given for the small sample size. Since it requires 10 to 14 days for production of the non-neutralizing antibodies from the animals' initial contact with the PRRSV, consideration of this should be given on the time of the sampling post-delivery. If the biosecurity of the isolation facility is poor, using the RT-nested PCR in combination with the ELISA will detect a false-negative ELISA due to the interval from infection to antibody detection. Sampling should be conducted as close to the exit date from isolation as possible and, as a minimum, 14 days post-entry. Sample size needs to consider the duration of time from entry and probable exposure to PRRSV and the risk of exposure. For example, if sampling a population within two weeks post-delivery, the statistical sampling should be 95/1, which would be the probability of detecting one PRRSV-positive with a 1% prevalence, but if the sampling is conducted seven weeks post-delivery, the sampling may more appropriately be 95/10, which is the probability of detecting one PRRSV-positive with a 10% prevalence.

[For more information about sample size according with disease prevalence, review our July 2004 Newsletter.]

Acclimatizing Gilt Monitoring
Replacement animals are frequently acclimatized either via vaccination (BI-Vet Medica, Inc.) or natural exposure with a homologous strain. ELISA screening is the most commonly used test to determine the success of exposure. Sampling timing and sample size should be based on the method of acclimatization, length of acclimatization, and available time until entry into a sow herd. Herds should strive to acclimatize replacements at a young age, allowing recovery of 90 days or more before entry into the sow herd and ample time for diagnostic screening. Screening is also used to evaluate recovery from acclimatization. The same animals sampled in 30–60–day intervals using ELISA can be used to determine if the animals are recovering or if there has been exposure to additional PRRSV strains. The sampling should have a declining average S/P ratio and lower maximum and minimum S/P ratio if recovery is occurring without re-exposure. It is important to keep in mind that recovery is not the same as nonshedding and there is no correlation between the S/P ratio and the probability of PRRSV shedding. Frequently, veterinarians will use the RT-nested PCR at the end of acclimatization to determine if there is circulating virus. The RT-nested PCR is highly sensitive, but negative results do not mean that the animal will not shed PRRSV under certain conditions at herd entry.

Semen Monitoring
Real-time PCR is the best diagnostic test to ensure semen is free of PRRSV. Recently, several practitioners have begun collecting serum from boar studs during the collection process for submission for real-time PCR. Since PRRS virus is in the blood before it enters semen and there is less foreign material, testing blood/serum is more sensitive than semen. Since semen contains components that are toxic to cell cultures, determining the quantity of PRRSV in semen is difficult, but testing of blood/serum avoids this. This assay also has the advantage of having same-day turnaround, which allows sow herds to use the semen the same day.

When using the RT-nested PCR as an adjunct to ELISA to ensure replacement animals are PRRSV-naïve, the age of the pig related to the length of time of viral shedding will drive the statistical sample size. The use of RT-nested PCR can identify recent infection, contamination during transport, or both, depending on when sampling occurs. These diagnostics when combined with source herd history and diagnostics, will identify whether the animals were infected at delivery, in transport or in isolation through a lateral source.

False-Positives
Serological tests should be interpreted on a population basis and not on an individual animal basis. Due the high sensitivity of some of the serological tests for PRRSV, false-positives will occur. To confirm that a result is a true positive, request an alternative test such as immunofluorscence assay (IFA), virus neutralization (VN) test or immuno peroxidase monolayer assay (IPMA); and RT-nested PCR or real-time PCR under viremic conditions such as monitoring boars.

Summary

• ELISA and real-time PCR can be complementary tools for the monitoring and surveillance of PRRS-infected herds.
• ELISA is able to detect antibodies 16 days post-infection/vaccination and up to 140 days post-infection/vaccination.
• Real-time PCR can detect positive animals 2–3 days post-infection and up to 45 days post-infection.
• ELISA and real-time PCR can be the cornerstone for your control and elimination programs for PRRS virus.

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