Iowa State University (ISU) scientists have developed and launched the first official test to detect antibodies against porcine reproductive and respiratory syndrome (PRRS) virus in swine oral fluids.

ISU’s diagnostic laboratory is also organizing an oral fluids working group to pursue development of other antibody-based and polymerase chain reaction (PCR)–based assays for use in the detection of a variety of swine diseases, such as swine influenza virus, as well as assays to detect and monitor antimicrobials. A $250,000 grant from the USDA National Institute for Food in Agriculture will support those efforts.

 

Test Development

ISU Veterinary Diagnostic Laboratory researchers developed methods to adapt the IDEXX PRRS antibody enzyme-linked immunosorbent assay (ELISA) to the oral fluid matrix, according to Rodger Main, DVM, director of the ISU diagnostic lab. The oral fluid test is interpreted exactly as the current PRRS serum ELISA: sample-to-positive (S:P) ratios equal to or greater than 0.4 are considered positive for PRRS, adds ISU’s Jeff Zimmerman, DVM, diagnostic lab scientist. An S:P ratio is a measure of the amount of antibody in a sample.

Significantly, the new test has been shown to be 95% sensitive and close to 100% specific for PRRS at the 0.4 cutoff, Zimmerman adds. The test detects maternal antibody, as well as antibody produced in response to PRRS virus infection.

So far the test is only available through ISU, but the university is working with seven other U.S. diagnostic labs to assist them in implementing the PRRS oral fluids ELISA.

Zimmerman explains important distinctions in PRRS assays. “This is the first oral fluid antibody-based assay for PRRS; we have been using PCR-based assays for detecting PRRS in oral fluids for some time. PCRs look for virus genetic material, but ELISAs look for antibodies. PCR can tell you what is happening right now — is the pig excreting virus? But antibodies give you a historical perspective — has the pig ever seen PRRS virus?”

PCR tests for PRRS are valuable in that they provide quick antigen (substance capable of stimulating an immune response) detection in the case of an acute infection, adds Main. “Antibodies persist, so an antibody test can tell you if the pig has seen the virus even if it has cleared the virus from its bloodstream. Because antibodies will still be present, the test can help determine whether the pig (or the population) is positive or negative for PRRS.”

 

Testing Improvements

Zimmerman stresses that the oral fluid antibody ELISA is a modification of the new generation IDEXX PRRS X3 ELISA that Apisit Kittawornrat, DVM, John Johnson, DVM, and he adapted to oral fluid samples.

Johnson, section serology leader, says the recently introduced PRRS X3 ELISA provided a “tremendous improvement in specificity and reduces the number of false positives compared to the previous kit.” With the old kit, singleton positives used to crop up when trying to move a group of negative pigs into a negative population. The result was often “spending a lot of money chasing something you didn’t think was there and many times wasn’t there,” he says.

This improvement in the PRRS ELISA, plus the fact that oral fluid samples are so easy to collect, provides a major advancement in the frequency of herd monitoring.

“The message is that instead of trying to monitor every quarter or every six weeks, you can do it every week or two with easy-to-collect (oral) samples and (get) really informative test results,” Zimmerman points out.

Main notes lab assays to improve testing results for PRRS are changing at a rapid pace. “There isn’t one of the core assays that we are using for PRRS that is the same as the ones we were using in March 2010,” he says.

 

Power of Oral Fluid

Zimmerman says their research indicates that in a 20-minute period, about 70% of the pigs in a pen of 25 will make contact with a rope and leave a fluid sample. “You gain tremendous statistical power through oral fluid collection because of the greater number of pigs being sampled for each assay conducted,” Johnson explains.

Routine surveillance usually involves taking 30 serum samples once a month. “Instead, we can collect six oral fluid samples in a building every week, effectively sampling 80% of the pigs and generating real-time test results, but only running 24 ELISAs per month,” he says.

Swine veterinarians no longer have to catch and bleed 2,000 pigs in a day. Producers can hang ropes and do their own sample collection, alleviating biosecurity concerns about veterinarians walking through multiple buildings. The ease of sampling means that for about the same dollars spent for serum sampling, producers can sample a lot more animals, providing a huge economic advantage, Main says.

Currently, producer monitoring of the health status of pig populations using oral fluids has been almost exclusively focused on PCR, while the antibody-based assays used today have required serum. The new antibody-based PRRS X3 Oral Fluid ELISA will provide a new, cost-effective diagnostic tool to assist in objectively measuring the PRRS-status of swine herds. Main believes it will also be helpful in the regional PRRS control projects.

 

Beyond Pathogen Identification

Hans Coetzee, DVM, a new addition to the faculty at the diagnostic lab, and Locke Karriker, DVM, are heading up efforts to take oral fluid collection technology beyond pathogen identification and onto antimicrobial issues.

“Our lab is interested in measuring drug concentrations in materials of animal origin, including tissues and oral fluids,” Coetzee says. “The latter would be a convenient sample that producers can collect from live animals to detect whether they have had exposure to a drug (e.g. antibiotic).”

The ability to detect antibiotic concentrations in oral fluids will enable the swine industry to respond to challenges related to antimicrobial use. This approach has three distinct advantages for producers:

• Determining whether animals have received antibiotics. Antimicrobial concentrations in oral fluids may be used as a surrogate marker to determine if pigs have been exposed to a medicated feed.

• Determining pen-level drug exposure would allow producers to determine if animals are being treated appropriately. “If it turns out that the pigs weren’t receiving sufficient antimicrobials through medication in feed, we could then make the decision to inject that pen of pigs with an antibiotic to ensure they are getting enough of that drug,” Coetzee says.

• By comparing drug depletion rates in oral fluids with other tissues, producers could make informed decisions as to whether their pigs can be marketed and establish appropriate withdrawal periods.

Coetzee hopes the industry will support research on the correlation between oral fluid concentrations and tissue concentrations for antibiotics to determine whether adequate withdrawal times have been established.

 “I think the consumer wants to see that we are responsible stewards of antimicrobial use. I don’t think they necessarily want our pigs to go without antimicrobials because of the animal welfare consequences,” he adds.

Main stresses the Cyclone Analytical Detection Services lab at the Iowa State Veterinary Diagnostic Laboratory will be one of a very few laboratories in the United States conducting applied food-animal pharmacologic research and delivering producer-focused pharmacologic detection services.

The discovery of oral fluids as an advancement in PRRS assays doesn’t spell the end of serum use in swine diagnostics, Main clarifies. The PRRS oral fluid antibody test provides an easy, sensitive means of monitoring swine populations. Particularly in cases where there are sick pigs, producers and their veterinarians may choose to use the diagnostic procedures and techniques that have been developed and proven over the decades, including serum-based tests.

A nine-minute video provides basic details about oral fluid collection, developed by staff at Iowa State University, in partnership with USDA’s Animal and Plant Health Inspection Service (www.aphis.usda.gov), the American Association of Swine Veterinarians (515-465-5255), the National Pork Board (www.pork.org or 800-456-7675) and the Center for Food Security and Public Health (www.cfsph.iastate.edu or 515-294-7189).

Additional information about oral fluids testing can be obtained by contacting Johnson at 515-294-1950 or visiting www.vdpam.iastate.edu.  

For more information see "Tips and Protocols for Oral Fluid Sampling for PRRS Virus."