Biomarkers Help Crack the Code on Saving More Equine Lives

In both animals and humans, biomarkers can signal that something has gone awry. For clinicians, they are valuable tools – once they are identified.

In human and animal medicine, biomarkers are used in several ways, including to diagnose, predict, or monitor health issues. Human health care consumers are familiar with biomarkers as mundane as blood pressure to gauge heart health, or more sophisticated testing for BRCA1 or BRCA2 gene mutations to predict the likelihood of breast cancer.

At Penn Vet, David Levine, associate professor of clinical large animal surgery, uses biomarkers as effective detectors of inflammation – early indicators of infection that can then be stopped, avoiding complications and even saving equine lives.

Mary Robinson, associate professor of veterinary pharmacology, is director of Penn Vet’s Equine Pharmacology Laboratory, and acting director of the Pennsylvania Equine Toxicology and Research Laboratory (PETRL). She uses biomarkers to determine whether horses that race at Pennsylvania’s Standardbred tracks have been illegally doped by horse trainers attempting to gain an unethical advantage and potentially harming the horses.

At both of her labs, Robinson and her staff have studied biomarkers and continue to work to break new ground. Their work helps enforce the rules and regulations for horse racing, but for Robinson, there is an even greater issue at stake.

“For me as a veterinarian, the health and welfare of the horse come first,” Robinson said. “Everything that we’re doing is to protect them. Then the integrity piece naturally falls into place.”

Using a biomarker to improve equine surgery outcomes

For Levine, an equine surgeon, his go-to biomarker is serum amyloid A (SAA), an acute-phase protein produced by the liver that increases in response to inflammation, infection, and trauma.

“SAA is very sensitive to infection-related inflammation. It goes really high, especially for bacterial infection,” Levine said. “It also goes high, but a little lower, for viral infections.”

Levine has found that SAA detects infection-related inflammation earlier and more accurately than fibrinogen, a long-used acute phase protein biomarker. In addition, there are handheld devices that can easily and quickly measure SAA from a simple blood sample.

For a surgeon and his patients, early detection is crucial.

“When a horse gets an infection post-operatively, your success rate goes down substantially,” Levine said.

In Levine’s own research, comparing fibrinogen use before and after surgery with SAA, SAA proved to be the more effective biomarker for equine patient outcomes.

“It showed if you have an early detection, before even getting clinical signs of infection,” Levine said, “you could start treating the infection earlier and increase your success rate. Because when an equine patient gets an infection, it can often be endgame for the horse.”

Research recently submitted for publication by Levine and colleagues also found that SAA is a superior biomarker compared with fibrinogen. “It was much more accurate in detecting horses that had disease or didn’t have disease, so it’s a better pre-operative test,” the surgeon said.

Another significant use Levine has found for SAA is in what he calls “antimicrobial stewardship.”

“It has given a better assessment of whether my antibiotic is working, so I’m not just switching antibiotics all the time, and I’m able to shorten the amount of time I give antibiotics to animals,” Levine said. “This has really supported our stewardship around antibiotics.”

Scientists like Levine are also on the lookout for new biomarkers.

“We’ll test all these biomarkers and see if one jumps out. I don’t know if we’ll have another like SAA soon, but we weren’t expecting this one either,” Levine said. “As new biomarkers are found, we use them. The problem is, they have to be detectable and easy to test. If you have a biomarker that takes a week and a lab to test, it doesn’t really help.”

Fast and accurate SAA is one biomarker that is helping.

“It’s our number one tool to diagnose infection, post-operative. It’s our number one tool for early detection of infection, for sure,” Levine said. “And it’s our number one tool to monitor response to treatment.”

Biomarkers detect illegal and harmful doping practices in PA racehorses

Mary Robinson’s Penn Vet Equine Pharmacology Laboratory and PETRL can detect many of the substances that may be illegally administered to racehorses. But not all. For those that would elude conventional detection methods, biomarkers are called in.

Robinson gave a few common examples that can be hazardous to horses.

“Total carbon dioxide is something that we’ve been measuring for many, many years in racehorses to determine if the horse has been given what’s called a ‘milkshake.’ In layman’s terms, it’s a box of baking soda,” Robinson said.

Some trainers mistakenly believe it helps horses perform better by neutralizing the lactic acid horses produce when they exercise, she said. Horses given a “milkshake” are administered bicarbonate by a nasogastric tube that passes through the nose and down into the stomach.

“That can be a dangerous practice,” Robinson said.

Using an instrument called a Radiometer, the technician compares a horse’s blood gas values with the carbon dioxide levels that are normal according to published work predominantly by her Penn Vet mentor, the late Lawrence Soma, V’57, an emeritus professor of anesthesiology. They can then tell if the horse has been given an illegal ‘milkshake.’

Soma’s research also established the normal levels of testosterone in stallions, mares, and geldings. With those normalcy levels, Robinson’s PETRL staff are able to detect illegally administered testosterone using the gold standard for drug testing, a technology called liquid chromatography coupled with mass spectrometry.

Robinson said research at PETRL and the Equine Pharmacology Laboratory is using omics technologies to search for new biomarkers. The different branches of omics provide the opportunity to systematically search for the many different biological factors produced in a horse that may change due to illegal treatment with a drug. 

Transcriptomics, for example, measures RNA in a sample, Robinson said, while proteomics measures proteins and metabolomics measures metabolites.

The challenge for Robinson and her team is determining which factors are specific and sensitive enough to use as a biomarker.

Bethany Keen, a resident working with Robinson, is identifying metabolites of bisphosphonates, a class of drugs taken by older humans to build bones but that paradoxically has been associated with an increased risk of fracture.

“We are also worried about horses receiving bisphosphonates because they are very potent analgesics, which means they decrease pain,” Robinson said. “If a horse has some sort of bone injury, we don’t want them to not feel it. If they are working despite the injury because they can’t feel it, that creates a huge increased risk of having a catastrophic fracture.”

Biomarkers are enabling scientists like Robinson to detect substances they couldn’t have detected before.

“If we didn’t have biomarkers like the one for total carbon dioxide, we wouldn’t be able to tell if a horse was given a ‘milkshake’. There’s no way to test for that without using biomarkers,” she said. “Same for testosterone. There’s no way for us to know if a horse has been doped with testosterone without measuring testosterone and comparing it to the normal range.”

Biomarkers are by no means Robinson’s and her colleagues’ only tool, but they are an important one.

“We’ve got methods that test for hundreds of drugs, so we can test for a lot of the drugs themselves,” Robinson said. “The biomarkers give us a tool to be able to detect things that are traditionally difficult to find and not detectable. That’s where the biomarkers can really help us.”