Real-time PCR undergoes real world testing

Have you ever watched dollar signs disappear before your eyes? That’s what an owner of a cow-calf operation sees when his pregnancy rate decreases and too few calves are born.

Poor fertility can often be the result of challenges with nutrition or bull management. Some infectious agents can also cause sudden drops in reproductive performance.

One of these agents is a bacteria called Campylobacter fetus subspecies venerealis (Cfv), known as “vibrio.” This organism hides in the bull’s prepuce (skin that connects the sheath to the penis).

A bull can sometimes carry the infection indefinitely without giving any sign that they are infected, but problems begin when he introduces the bacteria into the cow at breeding. The Cfv bacterium causes inflammation in the uterus and interferes with the development of the fetus — often causing fetal loss.

“This infection is often missed until the producer sees a lot of cows cycling at the end of breeding season or the veterinarian finds too many open cows in the fall,” says Dr. Cheryl Waldner, a large animal veterinarian at the Western College of Veterinary Medicine (WCVM) who examines the causes of reproductive failure in beef cattle herds.

“In a few of the cases we investigated, the problem wasn’t recognized until calving season when a large number of cows failed to calve.”

Because it is often difficult to determine the cause of reproductive problems, a group of researchers at the University of Saskatchewan, including Waldner, are working together to develop and evaluate new diagnostic tests.

The standard method of detecting Cfv was to culture the bacteria and then use a technique called conventional PCR (cPCR) on the colonies that most look like Cfv. Using cPCR, laboratory staff can see if the DNA from the culture matches known samples of Cfv.

As Cfv organisms are very sensitive to temperature changes and can be lost due to overgrowth of other bacteria, culture only works when the samples from the bull are handled carefully and arrive at the lab very quickly.

In Western Canada, this may not be possible due to the large distances between ranches, veterinary clinics and labs. Extreme temperature conditions at collection and during transport can also make successful culture impossible.

“We needed a more practical test that would work for producers and veterinarians under field conditions,” Waldner says.

A more convenient test that works directly on the bull sample and uses real-time PCR (or qPCR) was recently developed. Dr. Alvaro Garcia Guerra, a recent Master of Science program graduate at the WCVM, evaluated this new approach. His goal was to determine the accuracy of the qPCR for detecting Cfv: how good was the test at finding infected bulls?

Garcia Guerra found that the direct qPCR was better at detecting Cfv in samples that were stored at the correct temperature and tested within expected transport times.

“This was exciting news,” says Waldner. “However, we still have extremes in weather during sample collection and often have delays during transport. We needed to know how far we could push this new test and still get good results.”

Cue my summer job where my goal was to determine if time and temperature do indeed change the sensitivity of the direct qPCR. We compared samples managed under ideal conditions (4 C for two hours before testing) to samples that encountered typical transport times (48 hours) as well as extended transport times (96 hours).

We also had a group of samples that were allowed to get too warm for each of the three time periods (30 C) and samples that were frozen (-20 C) for each time period before testing.

But why is all of this important?  It may seem trivial to have samples held at temperatures for certain amounts of time just to see if you can still find Cfv, but the results may help to solve a real-life issue.

Producers, veterinarians and laboratories are interested in this project. Laboratories want to know so they can determine whether it’s worth going ahead with testing on samples that arrive late or aren’t stored in ideal temperatures. Veterinarians want to know so they can transport the samples they collect properly and efficiently.

But most of all producers want to know. They require the correct information as quickly as possible with no uncertainties involved to make good management decisions, minimize their losses and to decrease the chance that they will have to watch those dollar signs disappear.

Nicole Macdonald of Kelowna, B.C., is a third-year veterinary student whose 2013 summer research position was supported by the Merial Veterinary Scholars program and the WCVM. Visit WCVM Today to read more WCVM-related animal health news.