Researchers discover protein that could improve strangles testing in horses

 Strangles is one of the most common infectious diseases of horses worldwide, but accurately determining whether a horse has been exposed to the bacterium that causes it can be challenging.

Researchers at the Texas A&M College of Veterinary Medicine and Biomedical Sciences (VMBS) have identified a protein that could help change that.

In a new study, the researchers found that a protein they called P92 could be used to more accurately detect antibodies against Streptococcus equi subspecies equi (SEE), the bacterium that causes strangles. The discovery could improve existing blood-based diagnostic testing and serve as the foundation for new tools that make detecting exposure to the bacterium easier.

“Having fewer false-positive and false-negative results will improve diagnostic accuracy when trying to assess exposure to SEE,” said Dr. Noah Cohen, a University Distinguished Professor, Regents Professor and the Glen Blodgett Chair of Equine Studies in the VMBS’ Department of Large Animal Clinical Sciences. “No test is perfect, but the more accurate the test, the better the clinical decisions that veterinarians can make.”

A common and highly contagious disease

Strangles is an ancient disease of horses that occurs around the world. It typically causes nasal discharge and swelling of the lymph nodes beneath the jaw and at the back of the throat.

In severe cases, the swelling can interfere with a horse’s ability to breathe — which is how the disease earned its name. The infection also can spread to other parts of the body, such as the brain and abdominal lymph nodes, or lead to potentially life-threatening immune-mediated complications. Those internal infections can be difficult to diagnose because samples cannot always be readily collected to confirm the presence of SEE.

Serological testing offers another way to assess exposure to SEE by looking for antibodies against the bacterium in serum, the liquid portion of blood that remains after clotting. Serology can be particularly useful when collecting a sample directly from the site of infection is difficult, such as when the bacterium has spread to an internal organ. It can also help veterinarians determine which horses may have been exposed during an outbreak or assess horses that may be at risk for immune-mediated complications following exposure or vaccination.

Researchers are also interested in whether serology could eventually help identify horses that may carry SEE without showing signs of disease.

“Evidence exists that horses can carry SEE without showing clinical signs,” Cohen said. “Finding such horses is likely important in controlling persistence and spread of strangles.”

Searching for a better test

The existing serological test that is most commonly used in the United States detects antibodies against a bacterial protein known as SeM. Although useful, the test has limitations that can result in false-positive or false-negative results.

One challenge is that the SeM protein is also found in Streptococcus equi subspecies zooepidemicus (SEZ), a close relative of SEE that is commonly found in the upper respiratory tract of healthy horses. As a result, antibodies produced following exposure to SEZ may cause a positive result even when a horse has not been exposed to SEE. Some strains of SEE also have mutations in the SeM protein that could lead to a negative test result in an infected horse.

To find a better target, Cohen and his colleagues compared strains of SEE and SEZ collected from horses in Texas, searching for genetic differences that could help them identify proteins unique enough to distinguish the two bacteria.

The researchers then collaborated with Dr. Philip Felgner’s laboratory at the University of California, Irvine, which used the candidate proteins to create a microarray — a tool that allowed the team to test how antibodies from different groups of horses responded to many proteins at once.

Using the microarray, the team narrowed the search to dozens of potential proteins and tested how antibodies from horses with strangles, horses with respiratory infections caused by SEZ, and horses with no known history of strangles responded to each. The protein P92 stood out for its ability to distinguish horses with strangles from the other groups.

“We believed and hoped we would identify a target that would be more accurate than the M protein, but in my experience, many, if not most, research efforts fail and success is rare,” Cohen said. “It was a happy surprise when our belief and hopes were realized.”

The researchers used P92 to develop a new serological test that detects antibodies against P92 and found that it was more accurate than the existing SeM-based test. Importantly, it was better able to distinguish strangles from exposure to SEZ, which can naturally live in the respiratory tract of healthy horses.  

The P92 test also relies on only one protein, which Cohen said could make it technically easier and less expensive than another strangles test developed in the United Kingdom that uses two protein targets and is not widely available in the U.S.

While the results are promising, Cohen said more research is needed to confirm P92’s accuracy in broader populations of horses. The technology has since been licensed by Southwest Veterinary Diagnostics, opening the door for the discovery to be translated into clinical use. In addition to a serological test that could be used by veterinary diagnostic laboratories, the antibody-based test could potentially be adapted into a point-of-care format for use stall-side.

Detecting SEE stall side

Because SEE is highly contagious, rapidly identifying infected horses is important not only for treating an individual horse but also for preventing the bacterium from spreading to others.

Horses showing classic signs of strangles can typically be diagnosed using PCR testing or bacterial culture, but those methods require samples to be sent to a diagnostic laboratory for processing, which can take days to provide an answer. Cohen’s laboratory previously developed a PCR test that simultaneously detects SEE and SEZ, which collaborators at the Texas A&M Veterinary Medical Diagnostic Laboratory adapted for diagnostic use.

To enable more rapid detection, Southwest Veterinary Diagnostics is now developing a rapid lateral-flow test — similar to an at-home COVID-19 test — designed to detect P92 itself as an indicator of SEE infection. This differs from the serological test described in the study, which detects a horse’s antibodies against P92. The lateral-flow test instead detects P92 as an indicator of the bacterium itself.

Cohen’s laboratory is collaborating with Southwest Veterinary Diagnostics to evaluate the analytical sensitivity and accuracy of the lateral-flow test, with the goal of eventually providing veterinarians and horse owners with a rapid point-of-care, or stall-side, method for determining whether a horse has strangles.

For Cohen, the effort also connects to an experience his family had about 15 years ago, when one of his daughter’s horses developed strangles after returning home from a show.

“My wife asked why there wasn’t a test like the one available at a physician’s office to diagnose strangles at the point of care or stall-side,” Cohen said.

That question stayed with Cohen, and the identification of P92 could help make that kind of diagnostic tool possible.

P92 may also have potential beyond diagnostics. Cohen is collaborating with colleagues at Baylor College of Medicine and seeking funding to investigate mRNA encoding P92, along with other proteins, as a potential component of a new strangles vaccine.

For Cohen, both possibilities point toward the larger goal of giving veterinarians better tools to control a disease that has affected horses for centuries.

“I hope they will recognize that researchers at our college are doing work to improve diagnostic tests and develop vaccines to help improve control and prevention of what is one of the most common infectious diseases of horses in the U.S. and abroad,” Cohen said.

By Camryn Haines, Texas A&M University College of Veterinary Medicine and Biomedical Sciences