Molecular detective work in a University of Nebraska veterinary science laboratory has played an important role in the national pseudorabies eradication effort. Now, scientists are using the molecular diagnostic know-how they honed on pseudorabies to target a new, major swine disease.

Virologist Fernando Osorio, working in NU's Veterinary Diagnostic Center, was the first nationwide to use a powerfully sensitive molecular technique called Polymerase Chain Reaction or PCR for pseudorabies diagnosis. PCR rapidly detects pseudorabies even when the virus is latent and invisible in the animal's immune system. Over the years, Osorio's lab refined the test and became a leader in pseudorabies molecular diagnostics.

The Institute of Agriculture and Natural Resources team began PCR testing for pseudorabies in 1990, about a year after the nationwide eradication program was launched. PCR reduced from three weeks to two days the time required to determine whether a seemingly healthy pseudorabies suspect that showed positive in routine herd blood tests actually had the disease. Most single reactors ain blood tests are false positives. PCR results provide the ultimate answer.

"This is a very specific, very sophisticated test but each test we run is very significant because the fate of the whole herd, potentially thousands of animals, depends on the result," Osorio said. If itÍs positive, the herd is quarantined, infected animals can be sold only for slaughter and remaining animals must be disease-free before the quarantine is lifted.

PCR involves complex biotechnology techniques that essentially allow scientists to multiply genetic material so it's easier to see what's there. One molecule becomes a million in a few hours. Using PCR, IANR researchers can easily spot the tell-tale gene. If it's there, the pig was infected.

Osorio's lab soon began testing samples from around the nation, and other state and federal labs adopted Nebraska's test. The lab advises state and federal animal health officials on pseudorabies diagnosis and is a national reference center for the disease. Regulators consider Nebraska's PCR results an official verdict.

In many states, eradication efforts are winding down. Nebraska is in the surveillance phase, the fourth of five eradication stages, but the lab still tests samples from hot spots nationwide.

"When we developed this test for pseudorabies, PCR was brand new," Laboratory Supervisor Judi Galeota said. "Everything we went through trying to make it as good a test as possible applies to other diseases as well."

Know-how gained through intensive pseudorabies molecular diagnostics also advanced the team's research, Osorio said. Today, they're using this knowledge to diagnose and better understand Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), which is emerging as the world's leading infectious swine disease. Initially, PRRSV causes abortions and piglet deaths. Overall herd performance suffers if it becomes persistent.

"The momentum we have built with pseudorabies applies directly to PRRSV," Osorio said.

Scientists worldwide are scrambling to understand PRRSV. Diagnosing it is more difficult than pseudorabies, Osorio said, and vaccines confound diagnosis.

"The jury is still out on the efficacy and benefits of vaccinating against PRRSV," he said. Osorio's and several other labs showed that modified live-virus PRRSV vaccines may revert to virulence and cause disease in unvaccinated swine.

His team was the first to isolate and completely genetically sequence a U.S. PRRSV strain. Comparing the strain's genetic characteristics to other known PRRSV strains, including vaccines, they found this strain most likely came from a vaccine.

"We were able to determine that, at best, the vaccines were so-so," he said.

USDA's National Research Initiative competitive grant program, the National Pork Producers, Nebraska Pork Producers and USDA special grants helped fund Osorio's PCR work.

– Vicki Miller

IANR Virologist Fernando Osorio was the first to use Polymerase Chain Reaction or PCR to diagnose pseudorabies. As a leader in pseudorabies molecular diagnostics, his lab has assisted the national pseudorabies eradication campaign. Above: Osorio loads a pseudorabies sample into a DNA thermo cycler, which provides the precise time and temperature needed to rapidly copy specific sections of DNA using PCR.

Lab Supervisor Judi Galeota examines a sample of a pig's trigeminal ganglia. Scientists conduct PCR on this nerve tissue to detect pseudorabies even when it's latent. The team now is using its molecular diagnostic skills to target Porcine Reproductive and Respiratory Syndrome Virus.