The Scoop From the Schools is a blog that brings news and information to those in the veterinary industry information from vet schools and equine research facilities. This month we feature news from Oklahoma State University, the University of Saskatchewan and the University of Pennsylvania.
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Donations Sought for Valuable EPM Research
Equine protozoal myeloencephalitis (EPM) has plagued the industry for years now and currently holds the title for “most frequently diagnosed neurologic disease of horses.” An estimated 1% of horses have EPM in the United States. That said, the seroprevalence of Sarcocystis neurona and Neospora hughesi are about 90% and 3-10%, respectively. Clearly not all of these horses with circulating antibodies against S. neurona and N. hughesi are infected and have EPM, but which ones will develop disease?
That is one question that Martin Furr, DVM, DACVIM, PhD, MA Ed, seeks to answer. Furr, head of the Department of Physiological Sciences at Oklahoma State University’s Center for Veterinary Health Sciences, said, “I am conducting a study to determine the nature of the immune response to S. neurona and attempting to understand why one horse gets the disease while other horses can resolve the infection without developing any clinical signs.”
In addition, Furr’s research will take a closer look at diagnostic testing—a persistent thorn in the sides of equine internists.
“The overall purpose of my research is to improve the diagnostic accuracy and usefulness of the current diagnostic testing,” said Furr. “While the current standard-of-care diagnostic test (spinal fluid antibody ratio) is highly accurate, there is a question that the site of sample collection (atlanto-occipital vs. lumbosacral) may influence the interpretation of the test result. The research will answer this question and help make the diagnostic test even more accurate.”
In order to conduct this research, Furr requires horses with clinically confirmed EPM caused by either S. neurona or N. Hughesi. CSF will be collected from the AO and LS sites, as well as blood for antibody determination. Finally, a post-mortem will be performed to confirm EPM and rule out other conditions.
For more information on how to make a donation, please contact Furr at firstname.lastname@example.org.
Treating Joint Infections: When to Say Whoa
Septic arthritis in adult horses occurs most frequently as a consequence of joint injections or direct trauma. Aggressively treating joint infections effectively clears infections in most cases (85-90%), but how long should these infections be treated?
Researchers from the University of Saskatchewan in Canada ventured to answer this key question in a series of experiments using standard laboratory diagnostics, as well as serum amyloid A (SAA).
Horses were divided into three groups, and their middle carpal joints were injected with 1) Escherichia coli to induce septic arthritis, 2) lipopolysaccharide for inciting synovitis, or 3) saline as a negative control.
Following standard protocols for managing joint infections (lavage, regional limb perfusion, administration of antibiotics), synovial fluid samples were collected and analyzed for evidence of infection.
The team, led by Drs Joe Bracamonte and Elemir Simko from the Western College of Veterinary Medicine, found that elimination of infection occurred by Day 4 post-infection in most horses included in the study. Bacterial culture, polymerase chain reaction and total nucleated cell counts proved useful in determining infection resolution; however, total protein and percent neutrophils were not.
The data also showed that serial measurements of the acute phase protein SAA in either serum or synovial might be useful for practitioners when assessing eradication of septic arthritis. SAA levels increased rapidly in horses injected with E. coli but not in the synovitis or control groups, peaking on Days 3 and 4. SAA slowly declined thereafter, but remained above baseline until Days 9-10.
Together, these assays will help determine when the “horse is clean.”
According to the research team, these data are important considering that current treatment plans remain largely empirical, as does estimating duration of therapy.
“This leads to an unnecessarily extended application of local, regional and systemic antimicrobial treatment. Such as extended treatment can lead to serious side effects in the treated horse, as well as to development of bacterial resistance to antibiotics,” wrote the investigators.
Putting a Halt on Gene Doping
We can all appreciate the importance of drug testing to abrogate drug abuse in competitive horses. One of the latest tactics to plague the industry involves gene doping—the administration of transgenes that are transcribed and translated into functional, performance-enhancing proteins. Unlike illegally administered medications, performance-enhancing proteins cannot be distinguished from endogenous proteins, making it extremely difficult—but not impossible—to detect.
When used appropriately, gene therapy holds great promise for the treatment of an array of conditions in humans. Many products are already available commercially and thousands more are in various phases of clinical trials. Gendicine, for example, reportedly treats head and neck squamous cell carcinoma. This gene therapy product is an adenovirus vector carrying the p53 tumor-suppressor gene. Gendicine enters tumor cells via receptor-mediated endocytosis and over-expresses genes coding the p53 protein. The mechanisms of p53 action appear to involve stimulating the apoptotic pathway in tumor cells and decreasing the expression of multi-drug resistance, vascular endothelial growth factor and matrix metalloproteinase-2 genes, and blocking transcriptional survival signals.
When used for doping purposes, horses are administered viral vectors encoding performance-enhancing genes. While those proteins themselves cannot be used to identify horses subjected to gene doping, researchers from the University of Pennsylvania recently devised an alternate means of detecting this illicit practice using polymerase chain reaction (PCR) to detect the viral vector.
The research was led by Mary A. Robinson, MA, VMD, PhD, an assistant professor of veterinary pharmacology and director of the Equine Pharmacology Laboratory. Robinson and her team used a recombinant adeno-associated viral vector (rAAV) carrying a transgene for the anti-inflammatory cytokine interleukin-10 (IL-10). That rAAV-IL10 or saline (for a negative control) was injected intra-articularly in 12 horses, and plasma and synovial fluid samples were collected intermittently for 84 days after administration.
To detect the viral vector, primers (small segments of DNA) were designed to recognize a specific region of the rAAV genome. Using these primers, blood and synovial samples collected from the horses were tested to determine the presence of the rAAV.
As the researchers hoped, rAAV was indeed detected in both plasma and synovial fluid. In some horses, the viral vector could be detected in the synovial fluid up to 84 days after administration, and up to 28 days in blood samples.
Robinson explained that this study used primers specific to rAAV-IL10 as an example of how PCR can “diagnose” gene doping.
“This study provided proof of principle that we could detect the intra-articular administration of this product in a blood sample for up to 28 days,” she said.
According to the research team, “This study is the first to validate that quantitative real time PCR can be used to systemically detect the local administration of a gene therapy product to horses.”
This data were presented in the study, “Detection of intra-articular gene therapy in horses using quantitative real time PCR in synovial fluid and plasma,” published in the June 2021 edition of Drug Testing and Analysis. Robinson and colleagues have continued their research and anticipate their new study to be published soon.