By Russ Daly, Kelly Schmit, Travis Clement and Matt Dammen, South Dakota State University Veterinary and Biomedical Sciences Department
When sorting out the causes of respiratory signs in pig groups, veterinarians and animal caretakers can often differentiate between illness caused by influenza A virus and that resulting from porcine respiratory and reproductive syndrome virus. Clinical signs, incidence rates and rapidity of onset can be clues to the causes. There can be overlap, however — some acute respiratory signs can result from infections with either virus. Quick and accurate agent detection through laboratory testing comes into play.
Over the past decade, surveillance and diagnosis of respiratory agents have been revolutionized by the use of non-invasive techniques such as oral fluids sampling. This method, validated over the years and now widely accepted, collects viruses such as PRRSV or IAV shed by animals that chew on the rope.
While oral fluids have revolutionized sample collection, polymerase chain reaction technology has revolutionized agent detection in such samples. The speed, accuracy and sensitivity of PCR technology is unparalleled. It’s given swine veterinarians and producers the ability to confidently and quickly respond to new disease threats in the animals they care for. This testing method is particularly valuable in swine medicine compared to other disciplines: perhaps no other domestic animal species has been faced with so many emerging disease threats over the past couple decades (e.g., PRRS, porcine epidemic diarrhea virus, porcine circovirus).
In a typical PCR test, samples submitted to diagnostic labs are processed such that nucleic acid (DNA or RNA) is extracted from the various viruses and bacteria present in the particular sample. Then, primers and bases specific to the tested-for germ’s nucleic acid are added. Repeated cycles of heating and cooling result in an exponential amplification of the target nucleic acid. In “real-time” PCR tests, an indicator dye fluoresces in the midst of this reaction when sufficient nucleic acid has built up in the test chamber. This signifies a positive test: the presence of the target nucleic acid in the sample.
Most PCR tests run at veterinary diagnostic labs detect nucleic acid of a single pathogen. The section of nucleic acid targeted by the test must be one that’s “conserved” (the same) across multiple strains of a pathogen. On the other hand, PCR tests can be designed to target a very specific strain or portion of the germ.
Multiplex PCR tests utilize the same pig sample, but can detect more than one germ simultaneously. Developing these tests is not as simple as throwing two “single-plex” tests together. The reagents and time and temperature conditions for the test for agent A might be different than those for agent B. Tweaks and adjustments need to be made by the test developers so they work together. Current examples of multiplex PCR tests currently run at the South Dakota State University Animal Disease Research and Diagnostic Laboratory include porcine epidemic diarrhea virus/transmissible gastroenteritis/deltacoronavirus, and the multiplex PRRS PCR that detects both North American and European strains.
A new multiplex PCR test coming online at the SDSU Animal Disease Research and Diagnostic Laboratory will greatly help veterinarians and producers sort out clinical signs in growing pigs and other groups. For the first time, the influenza A PCR and PRRSV PCR tests are being combined in one multiplex procedure, developed in part by Tetracore. One PCR test on an oral fluids sample (or nasal swabs or tissues) will now be able to indicate the presence or absence of either virus (blood and serum samples will still be tested with individual single PCRs).
The multiplex test will be particularly valuable in light of the end of USDA support for IAV testing in swine populations. Its price is the same as a current single-plex test for either virus, making for more cost-effective diagnostics, whether for routine surveillance or for disease diagnostic investigations.
The development of tests such as the multiplex influenza-PRRS PCR demonstrates the responsiveness of today’s veterinary diagnostic laboratories to the needs of swine veterinarians and producers.