Research Review

2024 CEH Research Review

With the help of our donors, the UC Davis Center for Equine Health has worked to fund research that advances veterinary medicine and provides a knowledge base, establishing several focused research initiatives to concentrate resources, expertise, cutting-edge technology and state-of-the-art equipment in various areas of scientific research over the past 45 years.

*Download the 2024 Research Review (pdf).

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Faculty Research Projects

Drug Therapies

• Morphine concentrations in horses following oral administration: a new investigation of an old drug (#21-E)

• Investigator:
  Heather Knych, DVM, PhD, DACVCP

  Morphine is an effective pain reliever used commonly in human and small animal medicine. In some species, pain control effects of morphine have been attributed, at least in part, to one of the metabolites called M6G. In a crossover design, eight horses were given a single intravenous (IV) dose of 0.2 mg/kg morphine or oral doses of 0.2, 0.6, and 0.8 mg/kg of morphine. Levels of morphine and metabolites were determined. Physiologic and behavioral outcomes, including the number of steps taken, changes in heart rate, and gastrointestinal sounds were assessed. Oral administration of morphine resulted in higher concentrations of morphine metabolites, including M6G (maximum concentration: 11.6-37.8 ng/mL (0.6 mg/kg); 15.8-42.6 ng/mL (0.8 mg/kg)), as compared to IV. Oral absorption was moderately low (bioavailability was 36.5%, 27.6% and 28.0% for 0.2, 0.6 and 0.8 mg/kg, respectively). Behavioral and physiologic changes were noted in all groups but were less pronounced with oral administration compared to IV administration. These results are encouraging for further study, specifically of pain-relieving effects of morphine following oral administration.

  How does this research benefit horses?

  The study sought to investigate the behavior of a potent pain reliever when administered orally to horses as an important first step in assessing the utility of this method of administration for pain control, with the ultimate goal of adding to currently limited options for pain relief in horses.

  This research was reported in J Vet Pharmacol Therap 2023; 46(4), 238-249.

Genetics

• Confirming a genetic mutation for inherited hypocalcemia in Thoroughbred foals (#19-16)

• Investigators:
  Carrie J. Finno, DVM, PhD, DACVIM (LAIM)
  Gary Magdesian, DVM, DACVIM (LAIM), DACVECC, DACVCP
  Mustafa Khoka, PhD
  Andrew D. Miller, DVM, DAVP

  A syndrome of low blood calcium concentrations, leading to stiffness, weakness, and death, has been described in Thoroughbreds foals since 1997. With support through the Center for Equine Health in 2017, we identified a possible genetic mutation for this inherited disease. This grant confirmed the genetic mutation for this disease by testing two additional Thoroughbred foals and providing functional support for the genetic mutation in the gene RAPGEF5. This led to a DNA test that is now available through the UC Davis Veterinary Genetics Laboratory.

  How does this research benefit horses?

  Confirming the genetic mutation for inherited hypocalcemia of Thoroughbred foals has allowed DNA testing to screen for potential carriers and avoid breeding of carriers to carriers. Additionally, the implication of RAPGEF5 in a disease that leads to widespread hypocalcemia in the horse has strong implications across species.

  This research was reported in PLOS Genetics 2020; 16(9):e1009028. The genetic test is available at the UC Davis Veterinary Genetics Laboratory.

• Validating a biomarker test for equine neuroaxonal dystrophy/degenerative myeloencephalopathy (#19-17)

• Investigators:
  Carrie J. Finno, DVM, PhD, DACVIM (LAIM)

  Equine neuroaxonal dystrophy/degenerative myeloencephalopathy (eNAD/ EDM) is one of the top three causes of spinal ataxia in horses and is characterized by incoordination that typically develops in young foals 6-12 months of age. Currently, there is no diagnostic test available for eNAD/EDM other than an examination following euthanasia. In 2017, a Center for Equine Health funded resident project by Dr. Lisa Edwards supported the pilot investigation evaluating a biomarker of axon damage called phosphorylated neurofilament heavy subunit (pNfH) in horses with eNAD/EDM. We expanded upon this pilot study by sampling additional eNAD/EDM affected and unaffected horses in this current grant. We found that blood pNfH concentrations >1 ng/mL were significantly associated with eNAD/EDM (P =0.01), with only 12% sensitivity but 99% specificity. Spinal fluid pNfH concentrations >3 ng/mL were significantly associated with cervical vertebral compressive myelopathy (i..e “Wobbler’s disease; P = 0.0002), with 50% sensitivity and 86% specificity. This test became commercially available through the UC Davis Clinical Pathology Laboratory.

  How does this research benefit horses?

  A supportive diagnostic test for eNAD/EDM has provided veterinarians the necessary information to make informed decisions about horses with neurologic disease. As there is currently no effective therapy for eNAD/EDM, if this disease is diagnosed, an owner can make an informed decision to either euthanize or maintain the horse as a non-riding and non-breeding horse.

  This research was reported in Equine Vet J 2022; 54(2):290-298.

• Validating a genetic test for a bleeding disorder in Thoroughbred horses (#19-18)

• Investigators:
  Carrie J. Finno, DVM, PhD, DACVIM (LAIM)
  Fern Tablin, VMD, PhD

  Atypical equine thrombasthenia (AET) is a clotting disease that appears to be inherited in the Thoroughbred (TB) breed. Horses with AET have abnormal bleeding after injury. AET may also lead to bleeding from the airway, which is also the hallmark sign of exercise-induced pulmonary hemorrhage (EIPH). However, EIPH is only associated with bleeding from the airway post-exercise and affected horses do not demonstrate other problems with clotting. There is currently no way to distinguish between AET and EIPH in TBs without extensive specialized laboratory testing. Therefore, there is no way to predict if a nosebleed is due to EIPH or to the more serious clotting abnormalities of AET. The development of a genetic test for AET would allow for rapid diagnosis and breeding management. In this study, we defined the effects of a previously identified strong candidate missense mutation in suppressor/enhancer of lin-12-like (SEL1L) for AET. We confirmed that this is the most likely genetic candidate for this disease in Thoroughbred horses.

  How does this research benefit horses?

  On one breeding farm, AET was estimated to affect one in every 150 Thoroughbreds, causing bleeding from the airway with exercise and prolonged bleeding after injury. AET may also negatively affect a horse’s racing career due to repeated bleeding episodes. With the high prevalence of this disorder, a genetic test is required for accurate and prompt diagnosis. This genetic test is now part of a bleeding panel genetic screen that is available at UC Davis Veterinary Genetics Laboratory.

  This research was reported in Equine Vet J 2021;53(1):30-37.

• Validating a genetic susceptibility to melanoma in graying Connemara ponies (#19-19)

• Investigator:
  Carrie J. Finno, DVM, PhD, DACVIM (LAIM)
  Alain Theon, DVM, PhD, DACVR-RO

  Melanoma in graying horses is a hereditary disease with a complex mode of inheritance. Other than the graying mutation itself, no additional genetic associations have been identified for the risk of melanoma in graying horses. With support from the Center for Equine Health in 2017, we sequenced the entire genome of eight early onset (i.e. by 6 years of age) graying Connemara ponies with melanoma; six late onset (i.e. > 15 years of age) graying Connemara ponies with melanoma; and four graying Connemara ponies with no melanoma identified by 15 years of age. When comparing the early onset cases to either the unaffected cohort alone or the unaffected cohort plus the late onset cohort, a large region on chromosome 1 within a known tumor-suppressor gene (CAPN9) was identified. Because mutations of tumor-suppressor encoding-genes are common genetic abnormalities associated with inherited cancers, we hypothesized that this genetic region in CAP9N is associated with increased melanoma risk within graying Connemara ponies. In this study, we were unable to validate the association of the CAPN9 genetic region to melanoma susceptibility in graying Connemara ponies. Therefore, this genetic region does not appear to be associated with melanoma in graying Connemara ponies.

  How does this research benefit horses?

  Horses with graying hair coat have an 80% risk of developing melanoma. Graying ponies may develop melanoma late in life that is usually associated with a slow evolution and less likely to be life threatening. However, graying ponies that develop melanoma early in life are at high risk of dying of the disease because of faster evolution and earlier metastatic spread and represent a significant commercial loss for the industry. A susceptibility test for equine melanoma in graying horses would enable veterinarians to identify graying horses at high risk of early onset disease and monitor these individuals closely for tumor development.

• Identifying a genetic mutation for a new neurologic disease in Quarter Horse foals (#20-1)

• Investigator:
  Carrie J. Finno, DVM, PhD, DACVIM (LAIM)
  Kevin D. Woolard, DVM, PhD, DACVP

  In 2020, we identified six related Quarter horse foals that developed a severe neurologic disease between 1 and 4 weeks of age. These foals became uncoordinated and demonstrated severe weakness of their hindlimbs, culminating in the inability to stand within 3 to 7 days. Due to the poor prognosis, the foals were euthanized. Investigation after death identified lesions within the spinal cord. Based on pedigree analysis, the disease appeared to be inherited as an autosomal recessive trait. We performed whole-genome sequencing of four affected foals and their parents to identify the causative genetic variant. This study led to the identification of a genetic region on chromosome 11 that contained the genetic cause for this disease.

  How does this research benefit horses?

  Identifying a genetic mutation for this new neurologic disease in Quarter horse foals allows for DNA testing to screen for potential carriers and avoid breeding carriers to other carriers.

  This research was reported in J Vet Intern Med 2024;38(3):1808-1814.

• Investigation of a candidate gene for equine neuroaxonal dystrophy (#20-2)

• Investigator:
  Carrie J. Finno, DVM, PhD, DACVIM (LAIM)

  Equine neuroaxonal dystrophy (eNAD) is an inherited neurologic disease in horses. Both a genetic susceptibility and a vitamin E deficiency during the first year of life are required for a foal to develop eNAD. Through previously funded studies from the Center for Equine Health, we localized the genomic region of interest to chromosome 7 (chr7), which contained 147 candidate genes. We subsequently refined the region to 46 prioritized genes and excluded one of these, ATCAY. Based on exciting new data evaluating the non-antioxidant role of vitamin E, one of the 46 remaining genes, acyl-coA synthetase bubblegum family member 2 (ACSBG2), was a very strong candidate. In this study, we tested an additional cohort of eNAD-affected and unaffected horses for variants in this gene. The possible association of this genetic region with eNAD was excluded in this larger population of horses. We also completed a retrospective study showing that eNAD was the second most common cause of spinal ataxia in horses seen at UC Davis. Additionally, we demonstrated that horses with eNAD metabolize vitamin E faster than unaffected horses.

  How does this research benefit horses?

  While we have demonstrated success in identifying a biomarker (pNfH) for eNAD, overall sensitivity is quite low. Therefore, a genetic test is still required to definitively identify eNAD-affected horses. Susceptible foals could be supplemented with a high dose of vitamin E at birth to prevent disease development.

  This research was reported in J Am Vet Med Assoc 2021;258(12):1386-1393; J Vet Intern Med 2021;35(5):2473-2485; J Vet Diagn Invest 2021;33(3):506-515; Drug Test Anal 2021;13(6):1158-1168.

• Confirming genetic markers for juvenile idiopathic epilepsy in Arabian horses (#20-6)

• Investigator:
  Carrie J. Finno, DVM, PhD, DACVIM (LAIM)< br> Monica Aleman, MVZ, PhD, DACVIM (LAIM and Neurology)< br> Tatiana Vinardell, DVM, PhD

  Juvenile idiopathic epilepsy (JIE) is a disorder of Egyptian Arabian foals that causes seizures and has potential life-threatening complications, including head injury and aspiration pneumonia. Currently, there is no genetic test available for JIE. Through support from previous grants funded by the Center for Equine Health, we successfully mapped the genetic location for the JIE mutation to chromosome 1. As new cases of JIE are exceedingly rare at UC Davis, we have been unable to validate these genetic markers in another set of cases and controls. However, we developed an exciting collaboration with Dr. Tatiana Vinardell at the Equine Veterinary Medical Center in Qatar to obtain samples from additional JIE-affected cases and controls. With these samples, we were able to exclude that the candidate region on chromosome 1 is associated with JIE.

  How does this research benefit horses?

  Epilepsy can result in loss of animals and is a major financial burden due to costs of antiepileptic treatment. Identification of genetic variants will aid in strategic breeding to improve the overall health of the Arabian breed. Additionally, our findings may have strong implications for human health.

• Investigating the genetic connection between coat color and eye diseases in the horse (#20-18)

• Investigator:
  Rebecca Bellone, PhD
  Callum Donnelly, BVetBiol/BVetSc, DACT, DACVIM 
  Kelly Knickelbein, VMD, DACVO
  Felipe Avila, PhD 
  Sara Thomasy, DVM, PhD, DACVO

  Horses have been selected for coat color since their domestication. The variety of colors and patterns is of great interest to the industry, but genetic components have only been identified for some of the variations. We extensively characterized coat color, white patterning, and countershading in a cohort of 99 horses from 17 different breeds at two time points (winter and spring). Additionally, we performed chemical analysis on fifteen horses to determine specific melanin pigment type and ratio in different areas of the body. Combined with the Pioneer 100 Horse Health Project whole genome sequencing data, an association analysis was performed that allowed us to rule out candidate causal variants for some of the coat color phenotypes observed in this cohort. These data together provide the basis for future studies to expand our knowledge of pigmentation biology in the horse. Previous research has documented a link between some coat colors and ocular disorders. Therefore, we also evaluated the eyes of 41 horses by performing complete ocular exams and dark and light adapted electroretinography (ERGs). We genotyped these horses for the genetic variant (CSNB2), found previously to cause night blindness (inability to see in dark conditions) in one Tennessee Walking Horse. While there was no link between CSNB2 and coat color, these data confirmed CSNB2 as causal for night blindness across three different breeds (Tennessee Walking Horse, Standardbred, and Missouri Fox Trotter) and identified CSNB2 in six other breeds, suggesting night blindness is underdiagnosed in the horse.

  How does this research benefit horses?

  This work expanded the knowledge of the genetics of night blindness in the horse, with CSNB2 only the second reported variant to be confirmed to cause night blindness. This work supports the use of genetic testing for CSNB2 to avoid producing animals that cannot see at night, particularly in those breeds noted to have the variant.

  This research was reported in Vet Ophthalmol 2024;27(3):248-255.

• The genetics of equine neuroaxonal (eNAD) dystrophy in Gypsy Vanner Horses (#22-B)

• Investigator:
  Carrie J. Finno, DVM, PhD, DACVIM (LAIM)

  Equine neuroaxonal dystrophy (eNAD) is an inherited neurologic disease in horses. Both a genetic susceptibility and a vitamin E deficiency during the first year of life are required for a foal to develop eNAD. Our previous work, funded through the Center for Equine Health, has extensively studied this disease in Quarter Horses (QHs). In eNAD-affected QHs, we have demonstrated increased vitamin E metabolism, identified the phosphorylated neurofilament heavy chain (pNfH) biomarker that, when elevated in serum, is highly specific (but not sensitive) for eNAD, and identified candidate genomic regions of interest. Despite years of effort, we have been unable to develop a genetic test for eNAD in the QH. However, we have recently identified a cohort of Gypsy Vanner Horses in the United States with confirmed eNAD. With only 2,200 Gypsy Vanner horses currently residing in the US, this limited gene pool may provide the necessary samples to uncover the genetic cause for eNAD. In this study, we clinically defined eNAD in the Gypsy Vanner horse and determined genomic regions of interest that overlapped with those in the QH.

  How does this research benefit horses?

  While we have demonstrated success in identifying a biomarker (pNfH) for eNAD in the QH, overall sensitivity is quite low. Therefore, alternate biomarkers or a genetic test is still required to definitively identify eNAD-affected horses. Susceptible foals could be supplemented with a high dose of vitamin E at birth to prevent disease development.

  This research was reported in J Vet Intern Med 2024;38(3):1792-1798.

Mathematical Models

• Integrating artificial intelligence into equine diagnostics (#21-Y)

• Investigators:
  Stefan Keller, DVM, Dr Med Vet, PhD
  Krystle Reagan, DVM, PhD, DACVIM (SAIM)
  Titus Brown, PhD
  Chris Brandt, DVM, MS
  Allison Zwingenberger, DVM, MS, DACVR, DECVRI

  Artificial intelligence (AI) has the potential to positively impact patient care; however, the UC Davis veterinary hospital has been lacking the computational infrastructure to apply AI algorithms to patient data in real time. This project created ANNA, an animal health analytics platform that can visualize laboratory data and apply AI algorithms to patient data. The development of ANNA was supported by joint funding from the Center for Equine Health and the Center for Companion Animal Health, and its vision is to provide data analytics services for multiple species. ANNA is a cloud-based application that can be accessed through a patient’s electronic medical record or directly via URL from anywhere with an internet connection. ANNA provides an intuitive overview of laboratory procedures performed on a patient including automated grading of abnormalities of routine blood parameters and data trends over time. We have demonstrated ANNA’s functionality to provide AI analyses in real-time by implementing AI classifiers for the detection of Addison’s disease and Leptospirosis in dogs. In addition to viewing AI analysis results by visiting the ANNA website, ANNA can display classifier results directly in the electronic medical records system. Our lab is working on an AI classifier to interpret routine blood work, which will be deployed on ANNA when ready.

  How does this research benefit horses? 

  Creating the computational infrastructure to facilitate the interpretation of test results by artificial intelligence will further elevate the level of medical care delivered to equine patients at UC Davis and will provide critical training for the concept of AI support for clinical decision making to the next generation of veterinarians.

Medicine and Infectious Disease

• Investigating Effective Antibiotic Treatments for Equine Corynebacterium pseudotuberculosis Infections Using Longitudinal Comparative Population Genomics (#20-5)

• Investigators:
  Bart Weimer, PhD
  Vasco Ariston De Carvalho Azvedo, DVM, PhD
  Barbara A. Byrne, DVM, PhD, DACVIM (LAIM), DACVMBM
  Cory L. Schlesener
  Bihua C. Huang
  Ashleigh Flores, MS
  Rodrigo Profeta

  The pathogenic bacteria species Corynebacterium pseudotuberculosis (C. ptb.) is an understudied organism that persistently infects horses and livestock. We performed whole genome DNA sequencing of over 400 bacterial isolates in a C. ptb. collection that had been amassed at UC Davis since 1996 to perform the largest population genomics analysis performed on this pathogen. Genomebased population structure shows two groups corresponding to known subgroup designations equis and ovis, respectively associated with infecting horses and sheep/goats. Surprisingly, there is little genomic diversity within each subgroup, even across years and geography (sampled from USA, mostly California); greater diversity has been seen in other parts of the world with less sampling. With the lack of diversity, no clear associations could be made to the presence of genes or major gene variants to antimicrobial resistance, body location of infection, or opposite host-subgroup infections. Genomic distinction between subgroups is clear, with shared genes diverging in DNA sequence and each having an exclusive set of ~100 genes. The most notable difference, as previously discovered, is that subgroup equis has a set of nar genes for nitrate/nitrite reduction, enabling high-energy yield metabolism without oxygen as in some deep abscess infections. We confirm that, in our large sample population, the full set of nar genes are present in all subgroup equis samples and that this is part of the equis exclusive set of genes, further supporting it as a marker for diagnostics. Although no antibiotic-specific gene associations were found, we can also confirm there are multiple drug efflux pump systems found in all our samples, each able to act on multiple antibiotics.

  How does this research benefit horses?

  Infections by bacteria species Corynebacterium pseudotuberculosis cause “pigeon fever” in horses. Without a commercially available vaccine, isolation of infected animals and insect control remain the primary measures of disease prevention. Whole genome sequencing and bioinformatics innovations allow population comparative genomics that provide innovative solutions to identify new drug and vaccine targets. The results of this study provide a population scale approach to explore genome-based associations to improve approaches to diagnose, treat, and control this organism in animals. 

• Evaluation of the SarcoFluor antibody test for the detection of antibodies to Sarcocystis neurona, agent of equine protozoal myeloencephalitis, in blood and cerebrospinal fluid (#20-9)

• Investigators:
  Pranav Pandit, MPVM, PhD
  Patricia Conrad, DVM, PhD
  Woutrina Smith, DVM, MPVM, PhD
  Monica Aleman, MVZ, PhD, DACVIM (LAIM and Neurology)
  Nicola Pusterla, DVM, PhD, DACVIM, DAVDC-Equine

  Equine protozoal myeloencephalitis (EPM) is a challenging disease to diagnose in horses with neurological signs. To optimize contemporary diagnostic testing, the SarcoFluor antibody test for Sarcocystis neurona was reevaluated using 172 horses diagnosed with EPM and other neurological diseases. When differentiating between EPM and other neurological diseases, the combination of serum and CSF SarcoFluor testing added more information to the model accuracy than either test alone. Utilization of antibody titers against S. neurona in serum and CSF result in a high probability in support of EPM in the clinical setting.

  How does this research benefit horses?

  EPM is a diagnosis of exclusion and relies on the presence of neurological signs, ruling out other neurologic disorders, and the detection of antibodies to S. neurona in serum and CSF using quantitative immunodiagnostic tests. Accurate diagnostic modalities are needed to prevent over diagnosing or underdiagnosing relevant infectious diseases. The present reevaluation of the SarcoFluor using contemporary samples showed that a diagnosis of EPM can be accurately supported through the antibody testing against S. neurona in both serum and CSF. The use of serum to CSF antibody titer ratio against S. neurona was not superior to the combination of serum and CSF in support of EPM.

  This research was reported in Vet Parasitol 2024;:330:110219.

• Investigation of the role of horses in the COVID-19 pandemic (#20-12)

• Investigators:
  Emir Hodzic, DVM, PhD
  Nicola Pusterla, DVM, PhD, DACVIM, DAVDC-Equine

  Various domestic animals (cats and dogs) and large captive cats (lions, tigers) have been shown to become infected with SARS-CoV-2, often via direct or indirect interaction with clinically or asymptomatically infected humans. The role of companion animals such as cats and dogs are relevant considering the large number of such animals living in close contact with human beings and the many opportunities to acquire, amplify and potentially transmit SARS-CoV-2. While horses have been shown to have the angiotensin converting enzyme II (ACE2) receptor able to bind with the S protein of SARS-CoV-2, there is no available clinical or epidemiological data supporting the role of equids as a reservoir of infection contributing to human-to-human disease, infectivity and community spread. The present study investigated the status of healthy and sick horses regarding SARS-CoV-2. The investigators validated a commercial COVID-19 spike protein ELISA to determine the presence of specific SARS-CoV-2 antibodies in horses at a race venue that was closed after jockeys and track workers tested positive for SARS-CoV-2. The study results showed that amongst 587 racing Thoroughbreds, 35 (5.9%) horses had detectable antibodies to SARS-CoV-2.

  How does this research benefit horses?

  The research focus of the COVID-19 pandemic has understandably been on human health. However, little is known about the role of animals in this pandemic, and we are faced with many urgent questions that can only be answered through investigative studies and surveillance. While horses are apparently susceptible to SARS-CoV-2 and are likely to become infected through spillover from COVID-19 individuals, horses are unlikely to contribute to the spread of SARS-CoV-2. It is, however, important to continue to monitor possible human-to-horse transmission, especially with the emergence of highly transmissible SARS-CoV-2 variants, and to recommend that COVID-19 individuals avoid close contact with companion animals (dogs, cats, ferrets, horses).

  This research was reported in Animals (Basel) 2022;12(5):614.

• Investigation of newly discovered viruses in the nasal secretions of healthy and sick horses (#20-17)

• Investigators:
  Emir Hodzic, DVM, PhD
  Nicola Pusterla, DVM, PhD, DACVIM, DAVDC-Equine

  Three novel equine parvoviruses have recently been identified through metagenomics in the plasma and respiratory swabs of 13 horses with unexplained respiratory signs. These viruses are equine parvovirus-hepatitis (EqPV-H), equine parvovirus-CSF (EqPV-CSF) and equine copiparvovirus (Eqcopivirus). No large case-control study has been performed to test for statistically significant associations between the genetically characterized equine viruses and respiratory signs. Nasal fluid samples and blood from 667 equids with acute onset of fever and respiratory signs submitted to a diagnostic laboratory were analyzed for the presence of common equine respiratory pathogens (equine influenza virus, equine herpesvirus-1/-4, equine rhinitis A and B virus, S. equi subspecies equi) as well as EqPV-H, EqPV-CSF and Eqcopivirus by qPCR. An additional 87 clinically healthy horses served as controls. One hundred and seventeen sick horses tested qPCR-positive for at least one of the three parvoviruses. Co-infections with common respiratory pathogens and parvoviruses were seen in 39 sick equids. All 87 clinically healthy horses tested qPCR-negative for all tested common respiratory pathogens and 10 healthy horses tested qPCR-positive for one of the equine parvoviruses. When the frequency of detection for EqPV-H, EqPVCSF and Eqcopivirus of equids with respiratory signs was compared to that of clinically healthy horses, the difference was not statistically significant (p > 0.05), suggesting that the three recently identified equine parvoviruses do not contribute to the clinical picture of equids with respiratory disease.

  How does this research benefit horses?

  The relative neglect in the investigation of less characterized infectious respiratory pathogens may be related to the dominant position of EIV and EHV-1/-4 as causes of acute upper airway infection in horses, but is also related to a lack of sensitive, widely available and adopted diagnostic tests for less characterized respiratory viral pathogens. The past three decades have seen almost no discovery and characterization of new equine respiratory pathogens, compared to the human medical field. The investigation of less common pathogens and their possible interaction in co-infection is essential to improve the wellbeing of horses. It is imperative to gain a better epidemiological understanding of recently discovered equine respiratory viruses and to determine their possible association with respiratory disease. While EqPV-H, EqPV-CSF and Eqcopivirus can be found predominantly in the blood of equids with acute onset of fever and respiratory signs, it does not appear that these three newly identified parvoviruses contribute to the clinical picture of equids with respiratory disease.

  This research was reported in Animals (Basel) 2021; 19;11(10):3006.

• Is equine oral cancer associated with a virus? (#20-20)

• Investigators:
  Brian Murphy, DVM, PhD

  Squamous cell carcinoma (SCC) is one of the most common cancers of the equine oral cavity. Unfortunately, current treatment options for oral SCC are limited, often resulting in a poor prognosis and euthanasia. The cause of this important oral tumor in horses remains unknown. Research has shown that a cancer-associated virus is present in approximately half of the SCC lesions in the equine stomach. Closely related viruses are responsible for cervical cancer in women and are associated with cancer of the oral cavity in people. We suspected that the equine version of this virus, Equus caballus papillomavirus 2 (EcPV2), is associated with equine oral SCC. We performed polymerase chain reaction (PCR) and in-situ hybridization (ISH) for EcPV2 on 31 formalin-fixed paraffin-embedded equine oral SCCs (lingual, gingival, palate) and 10 equine nonSCC oral samples. PCR for EcPV2 was positive in 10/31 (32%) oral SCCs while all non-SCC oral samples were negative. Intense hybridization signals for EcPV2 nucleic acid were detected by ISH within neoplastic epithelial cells in 8/31 (26%) oral SCCs but not in the adjacent normal oral mucosa. No hybridization signals were detected within control samples. This study helps establish a link between EcPV2 and oral carcinoma, the most common oral tumor in horses.

  How does this research benefit horses?

  This study helps establish a link between equine papillomavirus and oral carcinoma, the most common oral tumor in horses. Once causality is understood, improved methods of detection, diagnosis and treatment will follow.

  This research was reported in J Comp Pathol 2023;205:1-6.

• Frequency of detection of a novel EHV-1 genotype (H752) in nasal secretions from horses with acute onset of respiratory disease (#21-B)

• Investigators:
  Nicola Pusterla, DVM, PhD, DACVIM, DAVDC-Equine

  Since the full genome sequencing of two prototype EHV-1 strains, Ab4 (neuropathogenic) and V592 (non-neuropathogenic), most diagnostic laboratories in the USA rely on allelic discrimination qPCR assays to detect EHV1. Unfortunately, such a molecular approach may lead to missed EHV-1 cases as exemplified by recent outbreaks from Europe and the USA. With the discovery of a new H752 genotype of EHV-1 and the validation of a SNP discriminatory assay, every EHV-1 infection can be successfully attributed to one of three genotypes. A total of 297 EHV-1 qPCR-positive swabs collected from 2019 to 2022 from horses with respiratory disease (EHV-1), neurological disease (equine herpesvirus-1 myeloencephalopathy (EHM)) and abortion were tested for the three different EHV-1 genotypes (N752, D752 and H752) using qPCR allelic discrimination assays. All submissions originated from the USA and included 257 EHV-1 cases, 35 EHM cases and 5 cases of abortion. EHV1 qPCR-positive cases were predominantly seen during winter and spring. N752 was the predominant genotype detected in EHV-1 cases (87.5%), EHM cases (74.3%) and abortions (80%). D752 was detected less frequently in EHV-1 cases (9.3%) and EHM cases (25.7%), while H752 was only detected in EHV-1 cases (3.1%).

  How does this research benefit horses?

  While the N752 genotype has remained the predominant genotype affecting horses with respiratory disease and abortion, it has also become a leading genotype in cases of EHM, when compared to historical data. The new H752 genotype, first reported in the United States in 2021, has remained confined to a cluster of geographically and temporally related outbreaks and the data showed no emerging spread of H752 since it was first reported. While the monitoring of EHV-1 genotypes is important from a diagnostic and epidemiological standpoint, it may also help establish medical interventions and preventive protocols to reduce the risk of severe complications associated with EHV-1 infection.

  This research was reported in J Equine Vet Sci 2023;123:104244.

• Investigation of the antibody response and antigen detection against EHV-1 following the intranasal administration of two commercially available EHV-1 vaccines (#21-C)

• Investigators:
  Nicola Pusterla, DVM, PhD, DACVIM, DAVDC-Equine

  Two EHV-1 vaccines are routinely used intranasally, especially in high-risk horses and outbreak situations. One of these vaccines is a killed-adjuvanted EHV-1 vaccine (Calvenza-EHV, Boehringer Ingelheim Animal Health), labeled for intramuscular and intranasal administration. The second vaccine is a modifiedlive EHV-1 vaccine (Rhinomune, Boehringer Ingelheim Animal Health), labeled for intramuscular administration. For this study, the investigators enrolled 30 healthy adult horses with no recent EHV-1 vaccination. The horses were randomly allocated to one of three groups: (i) Rhinomune intranasal vaccine group, (ii) Calvenza-EHV intranasal vaccine group, and (iii) Control group. Prior to vaccine administration and daily thereafter for 7 days, nasal secretions were collected for the detection of EHV-1 via qPCR. Further, whole blood was collected on the day of vaccine administration and 30 days later for antibody detection using an EHV-1 specific ELISA. Vaccine-derived EHV-1 was only detected in the two EHV-1 vaccine groups with 9/10 horses in the Rhinomune group and 8/10 horses in the Calvenza group testing qPCRpositive for EHV-1 for 1 to 4 days. Total Ig and isotype-specific IgG4/7 against EHV-1 measured pre- and 30-days post-vaccination were not different amongst the three study groups.

  How does this research benefit horses?

  The main benefit of this study was to determine how long vaccine-derived EHV-1 could be detected in nasal secretions following intranasal administration. This is relevant information as vaccine-derived antigen and naturally occurring EHV-1 are not routinely differentiated using molecular diagnostic tools. Vaccine derived EHV-1 could be detected in most of the intranasally vaccinated horses up to 4-days post-vaccine administration, potentially affecting diagnostic interpretation of EHV-1 during outbreak situations. A second benefit of this study was to determine if intranasally administered EHV-1 vaccines could induce a measurable antibody response to EHV-1. Unfortunately, the intranasally administered commercial EHV-1 vaccines did not elicit a systemic immune response. Therefore, this vaccine route seems suboptimal and should not be used to vaccinate adult horses during and outside an EHV-1 outbreak.

  This research was reported in J Equine Vet Sci 2023;122:104229 and Viruses 2024;16(7):1070.

• Investigation of neurological outbreaks of herpesvirus-1 (#21-I)

• Investigators:
  Nicola Pusterla, DVM, PhD, DACVIM, DAVDC-Equine

  Recent outbreaks of equine herpesvirus-1 myeloencephalopathy (EHM) around the world have highlighted the complexity of controlling the spread of EHV-1 and determining the infection state of exposed horses. Further, every well-investigated outbreak has added valuable information regarding the virus, the affected population, the clinical presentation, and the efficacy of specific preventive and therapeutic strategies. However, most EHM outbreaks remain poorly investigated mainly because of the laboratory costs associated with horse testing. The present study aimed at investigating a contemporary outbreak of EHM caused by the novel genotype H752. The morbidity of the EHV-1 outbreak was 84% with 26 clinically infected horses displaying fever, and less frequently anorexia and swelling of the hind limbs. Four horses showed mild transient neurological deficits. Clinically diseased horses experienced high viral load of EHV-1 in blood and/or nasal secretions via qPCR, while subclinically infected horses had detectable EHV-1 mainly in nasal secretions. Most infected horses showed a rise in antibody titers to EHV-1 during the outbreak. All 31 horses were treated with valacyclovir, while clinically infected horses further received flunixin meglumine and sodium heparin.

  How does this research benefit horses?

  This study aimed at investigating an unusual contemporary outbreak of EHM by supporting laboratory analysis (molecular and serological testing) and by collecting demographic, clinical and laboratory information. This investigation highlights various relevant aspects of an EHV-1 outbreak caused by a new H752 genotype: (i) importance of early detection of EHV-1 infection; (ii) diagnostic challenge to assess H752 genotype; (iii) apparent benefit of valacyclovir use in the early stage of the outbreak; and (iv) weekly testing of blood and nasal secretions by qPCR to monitor individual infection status and lift quarantine.

  This research was reported in Pathogens 2021;10(6):747.

• Full genome sequencing of EHV-1 strains associated with recent outbreaks (#21-U)

• Investigators:
  Nicola Pusterla, DVM, PhD, DACVIM, DAVDC-Equine
  Daniel Rejmanek
  Beate Crossley, DVM, MPVM, PhD
  Beatriz Martinez Lopez, DVM, MPVM, PhD
  Shadira Gordon, DVM, MPVM

  Current research suggests a multifaceted interplay between viral genetics, host characteristics, and environmental factors in the development of equine herpesvirus-1 myeloencephalopathy (EHM). While specific gene deletions and mutations have been identified, their association with disease severity remains unclear. Additionally, the influence of host characteristics such as age, breed, and vaccination status on EHM development requires further elucidation. Understanding these factors is crucial for developing targeted interventions and preventive strategies. A total of 22 isolated EHV-1 positive samples detected by PCR performed on nasopharyngeal swabs and blood samples were obtained from the California Department of Food and Agriculture (CDFA) during outbreaks from 2022-2023. The isolated samples were characterized by allelicdiscrimination analysis for seven single-nucleotide polymorphisms (SNPs) within the open reading frames (ORFs) 30, 33, and 72, which are genes known to have the greatest level of divergence among EHV-1 strains. EHV-1 DNA was sequenced in collaboration with colleagues from the California Animal Health & Food Safety Laboratory (CAHFS). Various SNPs were identified among the different EHV-1 isolates and associated with disease form (respiratory versus EHM). Further, direct or indirect relationships between the various outbreaks could be established by homology of the various SNPs. The established genomic patterns of the three targeted EHV-1 genes were associated with disease outcome and allowed us to establish relationships between the various outbreaks.

  How does this research benefit horses?

  One key aspect of this research lies in its potential to advance ongoing genomic surveillance efforts. This not only aids in the early detection of EHM outbreaks but also enables the identification of emerging trends, novel genetic variants, and potential outbreak clusters. The identification of genetic markers and future analysis of transmission networks can help develop risk assessment tools tailored to specific events and equine populations. In addition to enhancing disease surveillance and risk management, this research also has significant implications for vaccination protocols. After understanding the specific genetic markers and host characteristics associated with EHM susceptibility, vaccination strategies can be tailored to individual horses or vulnerable populations.

Orthopedics and Lameness

• Mitotherapy for equine joint disease: evaluating mitochondria’s potential to treat joint inflammation and prevent osteoarthritis (#19-02)

• Investigators:
  Jennifer Cassano, DVM, PhD
  Krzysztof Marycz, PhD
  Natalia Vapniarsky-Arzi, DVM, PhD, DACVP

  Inflammation of the lining of the joint (synovitis) is a key factor in the development of osteoarthritis, which contributes to 60% of lameness in horses. In our study, a mild synovitis was induced in the left and right knee joints of 6 horses by injecting lipopolysaccharide (bacterial cell wall component). Mitochondria isolated from each horse’s own blood was injected into the right joint. The horses tolerated this well, with no signs of inflammation. There was no change in general physical exam parameters after injecting mitochondria or the control. Total white blood cell counts from peripheral blood remained stable throughout the study. Synovial fluid cell counts increased following the induction of synovitis with lipopolysaccharide, indicating the induced synovitis model was working as expected. There was significant variation in numerous cytokines over time, with inflammatory markers peaking at 8 hours. Gene expression analysis of synovial fluid cells and microRNA library preparation are in progress. If a biomarker is found in response to mitochondria treatment, the next step will be to evaluate the benefit of mitochondria in naturally occurring osteoarthritis.

  How does this research benefit horses?

  Synovitis and osteoarthritis are common clinical problems in horses and exert a significant economic burden on the equine industry. In this study, we expanded our knowledge of a commonly used equine model for synovitis and evaluated a novel therapy using mitochondria, which has the potential to restore synovial cells’ anti-oxidative defense and reduce their pro-inflammatory activity, halting the progression of synovitis to osteoarthritis.

• Promoting tendon formation through the inhibition of glycolysis (#19-10)

• Investigators:
  Michael Mienaltowski, DVM, PhD

  Previous studies demonstrated that slowing down glycolysis reduced scar formation and inappropriate cartilage formation for progenitor cells from injured human tendons. In this study, we used 2-Deoxy-D-glucose to inhibit glycolysis in tendon proper and peritenon progenitors grown in three-dimensional tendon constructs – an in vitro tendon formation model. Glycolysis inhibition was shown early on to promote transcription factors that encourage tendon differentiation. Glycolysis inhibition also slowed down collagen synthesis in equine superficial digital flexor tendon progenitor cells, while promoting the expression of other collagen regulatory molecules that work to better organize the collagen in tendon. From this in vitro study, it seems that modulation of glycolysis could ultimately lead to improved tendon repair in horses, suggesting potential for glycolysis inhibition as a strategy to aid in tendon repair. We are working on the development of further mechanistic follow-up studies to optimize concentrations and timing of the use of this inhibitor to improve outcomes for the cellular responses.

  How does this research benefit horses?

  The study demonstrated the promise of using the 2-deoxy-d-glucose sugar molecule to reduce imperfections like scar formation within the culture model. Further optimization is required, but the goal is to ultimately consider how conservative use of such an affordable sugar could be used to promote tendon repair.

• Which equine muscles are important for jumping? (#20-3)

• Investigators:
  Christina Rohlf, PhD
  David Hawkins, PhD
  Tanya Garcia, MS
  Susan Stover, DVM, PhD, DACVS

  Show jumping horses commonly injure tendons and ligaments in the lower limb. To identify factors associated with the risk of injury, it is important to know how jumping loads are distributed between muscles and tendons. Although muscle forces cannot be measured directly in jumping horses, computer modeling techniques can be used to determine muscle activity. This requires two inputs: associated lower limb motions and ground reaction forces (GRFs) for the same jumping trial. Lower limb motions were collected from 4 horses during trotting and jumping on 12 different arena surfaces (5 dirt, 7 synthetic) using marker-based video capture. Ground reaction forces were collected indirectly with a surface tester. We chose to indirectly measure GRFs, because direct measures of GRF may affect natural movement by imposing additional weight on the hoof (force sensing horseshoe) or by altering the surface (force plate). GRFs as measured by the surface tester were fit to an established surface mathematical model and extrapolated to hoof GRFs using measurements of hoof displacement and velocity. However, the magnitude of GRFs was 2-3x higher than the 7-9kN expected at jump landing when using this method. Our findings suggest that future work into the development of a direct method of GRF measurement that does not alter natural locomotion is a necessary step toward building a computer model to evaluate equine muscle activity.

  How does this research benefit horses?

  These results suggest a possible direction of further research to understand equine biomechanics during show jumping and continue the development of a dynamic computer model. Recent developments such as pressure sensitive fabrics and microscale sensors show promise for the development of a novel force sensitive device attached to the hoof.

  This research was reported in Animals 2023;13(13):2122.

• Supplementing vitamin C to promote tendon formation (#20-7)

• Investigators:
  Michael Mienaltowski, DVM, PhD

  Horse tendinopathy greatly impacts the well-being and performance of horses of all breeds and uses. There is great value in the development of novel therapeutic strategies to improve repair responses for tendon injuries. In this study, we examined the impacts of vitamin C supplementation on cells collected from within the superficial digital flexor tendon and those along the outside of the tendon of horses - both of which are essential to proper tendon function. We found that vitamin C had several roles in promoting tendon formation, including bolstering anti-inflammatory proteins while inhibiting pro-inflammatory proteins, preventing tendon calcification (which structurally weakens tendon), and promoting matrix assembly. In some instances, there were increases in collagen synthesized, which is helpful for forming new strong tendon. Finally, vitamin C could protect tendon cells from cell death caused by corticosteroid exposure. These findings offer promising results that indicate that vitamin C should be further considered in therapeutic strategies. Follow-up studies will focus on understanding the role of vitamin C in protecting tendon cells against adverse effect of other drugs used with tendinopathy and in how best to deliver vitamin C locally to affected sites.

  How does this research benefit horses?

  Our studies demonstrate the potential for supplemental vitamin C to promote tendon formation in tendon proper and peritenon cells. By working from multiple approaches, this affordable biomolecule could potentially be added to injury sites or sites of tendinopathy for a positive outcome. While this is an in vitro study, the promising results will lead our group to design studies in which vitamin C could be added to tendinopathy treatments.

• PET imaging of the racehorse fetlock after surgical repair of fracture (#20-10)

• Investigators:
  Mathieu Spriet, DVM, MS, DACVR, DECVDI, DACVR-EDI
  Thomas Bergstrom, DVM
  Ryan Carpenter, DVM, MS, DACVS-LA

  Fractures of the cannon bone are common in racehorses, but novel imaging may help prevent them by detecting early signs. Fourteen horses that had sustained a surgically repaired fracture of the cannon bone were imaged with Positron Emission Tomography (PET) to understand the changes that led to the fracture and to see how fractures heal over time. Both the fractured limb and the other limb were imaged up to 3 times - immediately after fracture repair, 3 months later and 5 months later. The scans demonstrated that there were associated findings that preceded fractures based on imaging of the non-fractured limbs. The fractured limbs demonstrated a specific pattern with evidence of altered stress on the limb, with more stress on the outside than the inside, which is likely to contribute to the fracture. PET also confirmed healing of most of the fracture by 3 months; however, a few fractures had evidence of delayed healing on PET scans, leading to delay or inability to resume racing. Complications from the fracture with development of degenerative changes could also be detected with PET scans.

  How does this research benefit horses?

  This study confirmed the value of PET to recognize horses at risk for fracture and potentially prevent the fractures by modifying the training and racing program. PET was also useful post fracture repair to guide rehabilitation and predict ability to resume racing.

  This research was reported in Vet Surg 2024;53(1):131-142.

• Calibrating a model of racehorse proximal sesamoid bone stress fractures for injury prevention (#21-L)

• Investigators:
  Sarah K. Shaffer, PhD
  Susan Stover, DVM, PhD, DACVS
  David P. Fyhrie, PhD

  Fracture of the proximal sesamoid bones (PSBs) is the most common fracture in Thoroughbred racehorses. PSB fracture is related to the presence of focal subchondral bone lesions (regions of abnormally porous, damaged bone beneath the articular surface). Importantly, lesion severity is associated with training intensity. However, it is difficult to predict how training causes lesion formation because bone responds to both mechanical loading and damage. The goal of this study was to perform an initial calibration of a compartment model of bone turnover.

  First, we derived steady-state rate constants for a compartment model of bone’s tissue turnover cycle using data from fractured and non-fractured racehorse proximal sesamoid bones (Figure 1). Model rate constants were determined in two regions: the lesion and a control region. Second, these rate constants were related to high-speed training history.

  Steady-state rate constants were determined using bone volume fraction, tissue mineral density, and microdamage area fraction measurements. We were unable to collect data on newly formed bone due to the prevalence of damaged tissue at the lesion site. To solve the model at steady state, we assumed a fixed primary mineralization rate (k3) and derived a relationship for tissue mineral density (TMD) based on literature. In horses, k3 is unknown; however, in other species, there is limited evidence it is affected by mechanical loading (unlike the other model rate constants). Our results indicate the choice of k3 did not affect the relationships observed between other rate constants and exercise.

  The derived undamaged bone resorption rate, damage formation rate, and osteoid formation rate had significant robust regression relationships to exercise intensity (rate) variables, layup (time out of exercise), and exercise 2-10 months before death. The direction of these relationships varied between the stress reaction and non-damaged regions, reflecting that the biological response to damage differs from the response to changes in non-damaging exercise.

  How does this research benefit horses?

  We are developing a computer model to determine how existing and novel horse training programs influence lesion formation and, therefore, PSB fracture risk. This study provided an initial calibration of a compartment model of bone turnover in racehorses that can help us make these predictions. The compartment model allows observable measures in bone (e.g., damaged mineralized bone) to be related to exercise history. To link this model with a computer program capable of predicting lesion formation under different training programs, we first needed to establish if and how model rate constants could be related to exercise. Under steady-state conditions (e.g., a habitual training program), observed relationships between model rate constants and exercise differed based on if they were found in a damaged or control region. These relationships are consistent with bone biology. In the future, these relationships could be used to dynamically drive the rate constants with strain (or another exercise-related parameter) in a finite element model capable of predicting lesion formation under virtual training conditions.

  This research was reported in Sci Rep 2023;13:205.

Reproduction

• Evaluation of the use of different treatments to control uterine inflammation after breeding (#18-24)

• Investigators:
  Ghislaine A. Dujovne, DVM, MS, DACT
  Daniela Orellana-Guerrero, DVM, MS, DACT
  Eduardo Santos-Villanueva, DVM, DACT
  Elizabeth Scholtz, DVM, DACT
  Pouya Dini, DVM, PhD, PhD, DACT, DECAR

  Breeding induced endometritis is one of the leading causes of infertility. Several treatments are used and controlled studies are necessary to establish the best treatment for use in older mares and mares susceptible to persistent endometritis. In this study, mares who are susceptible to persistent breeding induced endometritis were treated with either systematic dexamethasone or intrauterine platelet rich plasma. While there was no difference in inflammation or pregnancy success with either treatment, an expanded understanding of how these treatments work was identified using a molecular technique (endometrial transcriptome).

  How does this research benefit horses?

  The information obtained in this study helps us understand the response of the uterus (endometrium) at the molecular level to the use of systemic dexamethasone and intrauterine platelet rich plasma to better understand their mechanism of action. This knowledge helps to develop and determine the most appropriate treatments for endometritis post-breeding.

• Understanding the interaction between the bacteria and placenta during equine nocardioform placentitis (#20-4)

• Investigators:
  Pouya Dini, DVM, PhD, PhD, DACT, DECAR
  Bart Weimer, PhD
  Margo Verstraete, DVM
  Machteld van Heule, DVM

  Nocardioform placentitis is a disease affecting pregnant horses, usually in the later stages of pregnancy. Several studies have reported nocardioform placentitis in various states and countries, dispelling the belief that this condition is limited to central Kentucky. This condition is associated with certain types of bacteria that have a characteristic appearance under the microscope. They are "filamentous" and "branching" and stain positively in a Gram stain, a common way to classify bacteria. The main types of bacteria connected with this disease are Crossiella equi, Amycolatopsis spp., and Streptomyces spp. Despite this, not all horses with the disease have these bacteria, and sometimes these bacteria are found in the placentas of healthy horses after they have given birth. To better understand this condition, we worked with veterinary clinics and practitioners to collect placental samples. We then examined the diversity of microbes present in the placenta of both healthy and disease-affected horses. Our analysis indicates that there could be a wider range of bacteria that might contribute to the disease than previously thought. These bacteria could lead to changes in the placenta's tissue structure that look like those caused by the traditionally recognized bacteria associated with nocardioform placentitis. This information led us to start investigating this disease based on the activity of the microbes.

  How does this research benefit horses?

  Our research demonstrated that the condition known as nocardioform placentitis is associated with a broader range of microbes than previously recognized. This variability may explain the inconsistent efficacy of treatments observed in clinical cases. Our findings underscore the complexity of nocardioform placentitis and emphasize the need to identify the specific underlying cause of the disease prior to initiating antibiotic therapy.

  This research was reported in Theriogenology 2023;206:60-70; J Vet Eq Sci 2023;125: 104789.

• Regulation of gene expression in the equine placenta; what makes the placenta function? (#20-13)

• Investigators:
  Pouya Dini, DVM, PhD, PhD, DACT, DECAR
  Carrie J. Finno, DVM, PhD, DACVIM (LAIM)
  Margo Verstraete, DVM
  Daniela Orellana-Guerrero, DVM, MS, DACT

  The placenta is a vital organ during pregnancy, impacting the long-term health of both the foal and the dam. The placenta works by changing its gene activity at different stages of pregnancy. Our study explored these changes by examining DNA methylation in horse placentas, which is a key process influencing gene activity. We analyzed placenta samples at four, six, and ten months of gestation to track how DNA methylation patterns evolve. We found that, as pregnancy progresses, the placenta's DNA becomes more methylated. By comparing gene activity data with our methylation findings, we pinpointed genes that were turned up or down in relation to methylation levels at various stages. Our results showed that these changes mainly control important functions like cell movement, blood vessel formation, and the processing of vital nutrients. Among the identified genes, Fibrillin 2 (FBN2) stood out as it is known to produce placensin (a hormone critical for glucose management during pregnancy) in humans. Since glucose is the primary energy source for the fetus, and changes in glucose and insulin resistance levels are documented in horses, our findings hint at FBN2's potential role in equine gluconeogenesis. Understanding the expression of FBN2 in the equine placenta and the levels of placensin in the mare's blood may shed light on glucose homeostasis in pregnant mares. Exploring this further could reveal how alterations in maternal glucose and insulin might affect fetal development—a phenomenon well documented in humans. This was the first detailed study of how DNA methylation in horse placentas changes throughout pregnancy, providing a basis for future research into how deviations from this pattern could affect pregnancy outcomes. 

  How does this research benefit horses?

  By understanding the normal methylation patterns during pregnancy, breeders can potentially identify deviations that may predict complications, allowing for early interventions.

  This research was reported in Int J Mol Sci 2023;24(8):7084 and J Eq Vet Sci 2023;125:104778.

• Gene expression of equine cumulus cells during oocyte maturation: in vitro and in vivo conditions (#20-14)

• Investigators:
  Stuart Meyers, DVM, PhD, DACT
  Pouya Dini, DVM, PhD, PhD, DACT, DECAR
  Ghislaine A. Dujovne, DVM, MS, DACT

  Assisted reproductive techniques (ART), including in vitro embryo production, have become important tools for the successful breeding of mares. These include normal mares and mares that cannot conceive by other natural means, or mating to deceased or unavailable stallions that possess a reduced cryopreserved semen bank or have inherent low sperm quality. Retrieval of immature oocytes represents an important tool in achieving this goal; however, in vitro matured oocytes have not been optimized, while in vivo matured oocytes have shown a better maturation rate and developmental competence. In this study, we determined differences in the gene expression of cumulus cells (cells that surround oocytes and play critical roles in oocyte maturation) of those cumulusoocyte complexes (COCs) matured in vivo and those matured in vitro using RNA sequencing which could reveal insights into oocyte maturation and fertility. We also aimed to relate these gene expression profiles to the cellular morphologies of the oocyte and the cumulus expansion rate in order to increase our understanding of equine oocyte maturation and improve the existing maturation conditions.

  How does this research benefit horses?

  In vitro production of embryos is an important and growing tool in the equine breeding industry and is crucial in mares that cannot conceive by other means. This study will help to understand the differences between in vivo and in vitro maturation processes and serve to optimize production of high-quality embryos.

  This research was reported in J Eq Vet Sci 2022;113:103963 and Reprod Fertil Devel 2023;35:242-243.

• Can cumulus cells during in vitro maturation predict successful fertilization and embryo development in horses? (#20-19)

• Investigators:
  Stuart Meyers, DVM, PhD, DACT
  Pouya Dini, DVM, PhD, PhD, DACT, DECAR
  Ghislaine A. Dujovne, DVM, MS, DACT

  Cumulus cells surround the oocyte and play an essential role in the processes of oocyte maturation and readiness for fertilization. This study aimed to test our ability to predict fertilization and embryo development before in vitro fertilization based on the quality of the cumulus cells. This is a continuation of our study to understand equine embryo development using a non-invasive timelapse imaging system. Previously, we proposed to track pronuclear formation to predict the first mitotic division in real-time. We were able to visualize some of the structural changes in the zygote prior to the first division, allowing us to predict fertilization outcome in the early stages of embryogenesis. In this study, our goal was to assess the oocyte milieu, including its surrounding cells, during maturation to understand its effect on post-fertilization events and embryo development. We performed RNA sequencing studies to examine immature and in vitro matured equine oocytes for gene expression and morphological changes that could be detected in our time-lapse imaging system during the maturation process of equine oocytes. 

  How does this research benefit horses?

  By developing a new understanding of the influence of cumulus cells on embryo development and quality, we can optimize production of high-quality embryos from valuable mares. Our findings will provide horse breeders and researchers with new methods for successful equine reproduction, with strong application to clinical management of mare subfertility.

  This research was reported in Int J Mol Sci 2023;24(18):13718.

• Assessment of bone growth in horse fetuses and newborn Warmblood foals (#20-21)

• Investigators:
  Catherine Renaudin, DVM, DECAR
  Mathieu Spriet, DVM, MS, DACVR, DECVDI, DACVR-EDI
  Fiona Wensley, BVMS, MRCVS
  Jessica Morgan, DVM, PhD, DACVSMR
  Jennifer Cassano, DVM, PhD

  Ultrasonographic evaluation of the first phalanx (P1) has been successfully evaluated in Quarter Horses (QH) and can be used to assess fetal growth and bone development late in gestation. This study aimed to evaluate the first phalanx (P1) in Warmbloods (WB). The length of P1 was measured. P1 bone maturation was described by recording the time of appearance and closure of P1 secondary ossification centers. Correlation between ultrasonographic findings was observed within the last 2 weeks of gestation and radiographic findings obtained at birth were evaluated. Data was obtained from 9 healthy Warmblood mares with known gestation dates that were ultrasounded transrectally every 2 weeks from 9 months of gestation until parturition. Within 48 hours of birth, radiographs of each foal’s lower limb were taken. We found that P1 could be imaged in most examinations and that P1 length was strongly correlated with days of gestation. The proximal and distal ossification centers both appeared between 268 and 298 days of gestation. The proximal ossification center did not close as opposed to the distal one that closed between 306 and 333 days of gestation. Ultrasound findings were confirmed on radiographs except for the length of P1. As P1 becomes too long to be measured close to parturition, the last ultrasonographic measurement is therefore underestimated. The results are very similar to those obtained in QHs.

  How does this research benefit horses?

  We confirm that P1 length can be used as a fetal growth parameter in Warmbloods. In addition to the other growth parameters already known, P1 length will improve prediction of unknown due dates in late pregnancy and the prediction of fetal growth when due date is known. The presence or absence of P1 ossification centers can serve as markers of bone maturation in Warmblood horses that may be used in the future in the decision-making process of inducing parturition in the mare. It may also help identify risk of foal health issues at birth, such as prematurity or dysmaturity, due to abnormal development. 

  This research was reported in J Eq Vet Sci 2023;125:104781.

• Equine cumulus cell culture: an in vitro model to study equine oocyte maturation (#21-O)

• Investigators:
  Stuart Meyers, DVM, PhD, DACT
  Pouya Dini, DVM, PhD, PhD, DACT, DECAR
  Alan Conley, BVSc, MS, PhD, FRCVS, DACT
  Janet Roser, MS, PhD

  In vitro maturation (IVM) of oocytes is an important step of in vitro embryo production (IVP), preparing oocytes for fertilization via intracytoplasmic sperm injection. However, IVM is a limiting step since only a few oocytes typically mature and are available for fertilization. Unfortunately, IVM studies that aim to optimize maturation culture conditions in the horse are limited by the low number of oocytes obtained due to the difficulties of retrieving cumulus oocyte complexes (COCs) from live mares. An important component of COCs that play a critical role during maturation is the cumulus cell layer surrounding the oocyte. These cells provide a niche with important molecules and metabolites that help the oocyte become an embryo. In this study, we aimed to establish and characterize a primary cell culture of equine cumulus cells (CC) obtained from COCs. Our goal is to utilize the CC culture as a valuable and useful model for IVM studies in equine oocytes. Based on our long-term cultures of immature and mature cumulus, it appears that they are unable to be cultured for longer than 2 months, which is logical since in vivo cumulus cells are typically only present short-term. Cumulus cells also die after the oocyte resumes meiosis, so it makes sense that cumulus cells cannot be successfully cultured for longer than 2 months. Morphologically, we observed subtle differences in immature and mature cumulus cells. The immature cells are more cuboidal with large cytoplasms, while the mature cells are thinner and more fibroblastic. However, as the immature cells are passaged, they become more fibroblastic in shape and look similar to the mature cells. Because of this, it is possible that the cumulus cells that have grown long term will display abnormal metabolism and behavior. As a result, we have elected to modify our methods. Studies are ongoing to further understand equine cumulus expansion.

  How does this research benefit horses?

  Availability of COCs is the limiting factor to meticulously study the effectiveness of equine oocyte IVM conditions and embryo development. Our goal is to establish a functional primary equine cumulus cell culture model for advanced studies of oocyte IVM and media optimization, thereby reducing the need to perform transvaginal aspiration of COCs.

  This research was reported in J Eq Vet Sci 2022;113:103963 and Int J Mol Sci 2023;24(18):13718.

• Selection and characterization of high-quality stallion sperm using microfluidics and sperm cell membrane charge (#21-P)

• Investigators:
  Pouya Dini, DVM, PhD, PhD, DACT, DECAR
  Stuart Meyers, DVM, PhD, DACT
  Morgan Orsolini, MS

  Intracytoplasmic sperm injection (ICSI) is currently the sole clinical technique for producing horse embryos in a lab setting. The success of this process depends heavily on the quality of the sperm used. In human fertility treatments, a property called zeta potential (ZP), which measures the electric charge on the sperm surface, has been linked to sperm health and the success of creating embryos. It was unknown whether this is also true for horse sperm. In this study, we measured the ZP of horse sperm to see if it relates to sperm quality. We also compared traditional sperm sorting methods to a new technique that uses a microfluidic chip. We looked at fresh and chilled sperm samples and tested their motility, viability, and normal shape both before and after sorting them with different methods. We found that horse sperm has a net negative ZP, much like human sperm, and this negative charge is associated with how well the sperm moves and survives. Interestingly, the microfluidic chip was especially good at selecting high-quality sperm without the damage that can be caused by other methods. This study shows that horse sperm has a net negative electric charge that could predict its quality and that the microfluidic chip is a promising new tool for selecting the best sperm for creating horse embryos.

  How does this research benefit horses?

  The optimization and application of microfluidic devices in the selection of equine sperm represent significant technological advancements in horse breeding. This innovation, which we finetuned for practical use in clinical settings, offers a superior method of sperm selection. Its increased adoption is a testament to its effectiveness, promising to enhance the success rates of in vitro embryo production.

  This research was reported in Theriogenol 2022;192:1-8, Animals 2021;11(11):3248 and 3319, J Eq Vet Sci 2022;113:103966.

• Mechanisms of placental blood vessel formation in cloned equine pregnancies (#21-R)

• Investigators:
  Pouya Dini, DVM, PhD, PhD, DACT, DECAR
  Stuart Meyers, DVM, PhD, DACT
  Margo Verstraete, DVM

  Somatic cell nuclear transfer (SCNT), or “cloning,” enables production of offspring from horses regardless of reproductive status, age, health status, as well as post-mortem. Despite significant technical progress, the overall success of horse cloning is still low and placental abnormalities have been associated with pregnancy failures and decreased survival rates of cloned foals. The most common placental abnormalities in cloned pregnancies include abnormal blood vessel formation, excessive edema, and excessive accumulation of fluid (hydrops). We previously identified a gene that is essential for normal placental blood vessel formation in horses and demonstrated that abnormal expression of this gene is associated with hydrops. The current study evaluated gene expression and DNA methylation of placental samples from cloned equine pregnancies with different outcomes. The results highlight the underlying molecular pathways associated with abnormal placental development in cloned equine fetuses. Moreover, the data demonstrated the disruption of angiogenesis in the cloned placenta, manifested as excessive edema. Similarly, in a previous study, we demonstrated the association between abnormal angiogenesis and placental edema and hydrallantois in equine pregnancy, highlighting the importance of adequate angiogenesis in placental function.

  How does this research benefit horses?

  This research sheds light on the probable causes of the high incidence of pregnancy abnormalities arising from equine somatic cell nuclear transfers, a method used in cloning. Through our studies, we have been able to pinpoint factors that could lead to these complications, providing a foundation for improving cloning techniques and outcomes in horses.

  This research was reported in J Eq Vet Sci 2022;113:103983.

• Equine placentitis: a new approach to understand an old problem (#21-S)

• Investigators:
  Pouya Dini, DVM, PhD, PhD, DACT, DECAR
  Bart Weimer, PhD
  Fabio Lima, DVM, PhD
  Machteld van Heule, DVM

  Ascending placental infections and subsequent placental inflammation (ascending placentitis) are among the most common problems of late gestation in mares. Although placentitis is one of the costliest diseases in the equine breeding industry, the exact cause of the disease is not fully understood, leading to inefficient preventive, diagnostic, and treatment protocols. Previously, we characterized the molecular changes in placental tissue at the final stage of this condition. In the current study, we delved into the specific small molecules and virulence factors (VFs) produced by the bacteria involved in this condition. We identified 20 genes linked to virulence that are active in diseased placental samples but not active in healthy placentas. This breakthrough paves the way for refining diagnostic methods and, ultimately, treatment strategies for placentitis. By applying this knowledge, we aim to decrease the rates of pregnancy loss and the economic impact associated with this disease in the equine industry. 

  How does this research benefit horses?

  This research will lead to identifying better diagnostic tools for equine placentitis. The resulting insights will be used to optimize diagnosis and treatment, and consequently lower pregnancy loss rates and financial loss in the equine breeding industry.

Sports Medicine

• How common are abnormal heart rhythms in horses, and which horses are at risk? (#20-25)

• Investigators:
  Jessica Morgan, DVM, PhD, DACVSMR
  Joshua Stern, DVM, PhD, DACVIM
  Ashley Hill, DVM, MPVM, PhD

  Electrical disturbances in the heart, known as cardiac arrythmias, are common in horses. While some of these electrical disturbances are considered normal, others are not. In some cases, abnormal electrical disturbances can be problematic and associated with decreased performance, collapse, or sudden death. More information is needed about frequency of arrhythmias in normal horses to be able to make recommendations regarding safety and risk of future cardiac events. We recorded electrical activity of the heart over a 24-hour period on 98 horses from a mixed breed population with a mean age of 12 years. Abnormal rhythms were present in 91.8% (90/98) of horses, with 22.4% (22/98) of those horses having more than one arrythmia per hour of recording. Of the horses that had arrhythmias, 94.4% (85/90) had supraventricular arrhythmias (premature beats that occur prior to the expected beat) and 25.5% (23/90) had ventricular arrhythmias (concerning electrical disturbances in the lower chambers of the heart). While preliminary analysis found no significant relationships between number of arrythmias and age, weight, troponin values, body condition score, or heart girth circumference, there was a trend for a greater number of arrythmias in horses with a higher body condition score. This data will be made available for future studies looking for risk factors for cardiac arrhythmias as more information about these horses is discovered through ongoing work in the Pioneer 100 Horse Health Project.

  How does this research benefit horses?

  Abnormal heart rhythms can be associated with poor performance, collapse, and sudden death. These events have catastrophic consequences for all participants in equine sport. By being able to prioritize horses for evaluation and improve our understanding of normal electrocardiogram findings, this study aims to improve identification of horses at risk of arrythmias, benefiting the safety of both horses and humans.

  This research is under review at Eq Vet J.

• Effects of Lasix (furosemide) on the hormone system responsible for blood pressure regulation (#21-07)

• Investigators:
  Jessica Morgan, DVM, PhD, DACVSMR
  Marisa Ames, DVM, DACVIM
  Mallory Lehman, DVM

  Furosemide, a commonly used diuretic, activates the renin-angiotensinaldosterone system (RAAS) in non-equine species, but little is known about RAAS activation in horses. The objective of this study was to measure circulating angiotensin peptide (AP) concentrations in horses at baseline and in response to the administration of furosemide to improve our understanding of RAAS activation. A secondary aim of this study was to correlate the use of routine sample handling (the RAS-Fingerprint® analysis using equilibrium dialysis) to immediate protease-inhibited sample handling. Equilibrium dialysis results were analogous to immediate protease inhibition, supporting the use of this practical approach to RAAS analysis in the horse. Baseline levels of circulating APs were low in healthy horses compared to the levels described for other species. Only Angiotensin II was consistently detectable in healthy unstimulated horses. Furosemide produced an increase in hormones associated with both the classical and alternative RAAS pathways. Specifically, there was an increase in Angiotesin I, Angiotensin II, Angiotensin IV, Angiotensin 1-5 and aldosterone in furosemidetreated horses 4 hours after administration. This comprehensive RAAS data raises new questions about the evolutionary advantages of decreased RAAS activation in the horse and provides critical fundamental knowledge to study RAAS activation in cardiovascular and kidney disease.

  How does this research benefit horses?

  Horses have remarkably low circulating RAAS activity compared to other species. Furosemide, a common diuretic used in horses, activates RAAS in the horse at doses commonly used in clinical practice. This information provides critical baseline information to direct the future study of this hormone system as a biomarker for disease and a therapeutic pathway in disease treatment.

Surgery/Anesthesiology

• Morphine concentrations in the carpal joints after intravenous regional limb perfusion (#21-V)

• Investigators:
  Isabelle Kilcoyne, MVB, DACVS-LA
  Jorge Nieto, MVZ, PhD, DACVS, DACVSMR
  Bridget F. Nottle, BVSc, DACVS-LA
  Harriet Flynn, MVB, DACVAA
  Heather K. Knych, DVM, PhD, DACVCP

  Intravenous regional limb perfusion (IVRLP) is a commonly used technique to deliver high concentrations of antibiotics to the distal limb when treating wounds, septic arthritis, or osteomyelitis. These conditions can be very painful and co-administration of morphine, which is an opioid analgesic, may provide substantial pain relief for these conditions with fewer side effects than have been associated with systemic administration. The main objective of this study was to determine morphine concentrations in the carpal joint following IVRLP with morphine alone or in combination with amikacin (a commonly administered antibiotic). This was a randomized crossover study. Six horses underwent IVRLP with 0.1mg/kg morphine sulfate diluted to 60 mLs using 0.9% NaCl (M group) or combined with 2g amikacin and 0.9% NaCl (MA group) with a 2-week washout period between treatments. Synovial fluid was collected from the radiocarpal joint (RCJ) at different time points after IVRLP. The tourniquet was removed after the 30-minute sample was collected. Synovial concentrations of morphine and major metabolites were measured using liquid chromatography–tandem mass spectrometry. Amikacin concentrations were quantified by a fluorescence polarization immunoassay. Measurable concentrations of morphine were apparent in the RCJ of all horses. Peak concentrations of morphine in the M group were 4753.1 (2115.7-14,934.5) ng/mL and 4477 (3434.3-7363) ng/mL in the MA group (p=0.5). Median peak concentrations of synovial amikacin were 322.6 (157.5-1371.6 g/mL). IVRLP using morphine is a feasible technique and synovial morphine concentrations were measurable following IVRLP and were not affected when used concurrently with amikacin. Administration of morphine via IVRLP may be beneficial as an analgesic technique for orthopedic conditions of the distal limb while limiting potential serious systemic side effects.

  How does this research benefit horses?

  Administration of opioids via IVRLP has potential benefits as an analgesic and anti-inflammatory agent in horses with painful, traumatic conditions of the distal limb, while potentially limiting the negative gastrointestinal effects associated with systemic administration. Techniques to improve weight bearing in horses with severe orthopedic injuries is paramount to reduce the incidence of complications such as support-limb laminitis.

  This research was reported in Eq Vet J 2024 June 17 (online ahead of print).

Resident Grants

Medicine and Infectious Disease

• To investigate if vaccines are less effective in horses that receive immuno-suppressive drugs such as dexamethasone

• Investigators:
  Resident: Nicole Kreutzfeldt, DVM
  Co-investigators: Thomas M. Chambers, PhD
  Stephanie Reedy
  Kennedy Spahn
  Mentor: Nicola Pusterla, DVM, PhD, DACVIM, DAVDC-Equine

  Corticosteroids such as dexamethasone are commonly used to treat horses; however, the effects of these immuno-suppressive medications on the immune response to vaccination is not well understood. In this study, horses that received one or several doses of dexamethasone at the time of vaccination did not have lower antibody levels at 30 days after vaccination against equine influenza compared to horses that did not receive any dexamethasone. However, horses that received multiple doses of dexamethasone (i.e. 3 intravenous doses at 24-hour intervals) at the time of vaccination had lower antibody levels at 30 days after vaccination against equine herpes virus 1 compared to horses that did not receive any dexamethasone. Multiple doses of dexamethasone at the time of vaccination may dampen the immune response in horses depending on the type of vaccine used. It remains unclear if the lower levels of antibodies are still protective against disease.

  How does this research benefit horses?

  Veterinarians should be cautious when using multiple doses of dexamethasone to treat horses around the time of vaccination to prevent the risk of a potential vaccine failure and thereby increased risk of spread of infectious diseases in the horse population.

  This research was reported in J Vet Intern Med 2024;38(1):424-430.

Reproduction

• Effect of euthanasia solution on oocyte’s developmental competence and fertilization

• Investigators:
  Resident: Soledad Martin-Pelaez, DVM, DACT
  Co-investigator: Margo Verstraete, DVM
  Mentor: Pouya Dini, DVM, PhD, PhD, DACT, DECAR

  The collection of oocytes from mares’ ovaries before or immediately after euthanasia is the last resource for producing offspring from these animals. However, it was unclear how euthanasia solution affected the production of embryos. In this study, we tested the effect of pentobarbital, a euthanasia agent commonly used in horses, on the developmental competence of oocytes. We evaluated the presence of pentobarbital in follicular fluid after euthanasia, and observed that this drug reaches the oocyte's environment almost immediately. Then, we used a bovine in vitro embryo production model to evaluate the potential toxicity of this drug on embryo development. We found a lower maturation rate of oocytes, but with an equal embryo development capacity. Lastly, in a follow-up study on equine oocytes, we observed the same trends as in the bovine model. The results of our study shed light on an unknown area of clinical practice: do we need to remove ovaries before or after euthanasia if the goal is in vitro embryo production? According to our results, removing ovaries under anesthesia prior to euthanasia is the most desirable protocol, as we could obtain a higher total number of embryos. On the other hand, due to logistical considerations, this is not always possible in the day-to-day equine practice, so we as also demonstrated that production of embryos from oocytes that have been exposed to pentobarbital is possible.

  How does this research benefit horses?

  The production of offspring from critically ill and injured mares that must undergo euthanasia is a key resource to maintain and rescue valuable genetics. It is also an invaluable resource for owners that must make the hard decision to euthanize their mares and would like to have their progeny. Improving the techniques for post-mortem gamete retrieval and in vitro embryo production is key to ensuring this process is successful. The reproductive toxicity of euthanasia agents is an enigma that needs to be solved to increase the success rates. Here, we demonstrated the rapid presence of pentobarbital in the follicle after the administration of the drug and showed its effect on embryo production. Our research will aid owners and veterinarians in making informed decisions regarding euthanasia, while also encouraging the industry to explore post-mortem in vitro embryo production. Our study showed that, although it is less beneficial to remove the ovaries after euthanasia, the oocytes are still capable of producing embryos.

  This research was reported in Theriogenol 2023;205:1-8.

Surgery

• Comparison of durations of perfusate injection for delivery of antibiotics to horses undergoing regional perfusion

• Investigators:
  Resident: Laurel Saldinger, DVM
  Mentor: Isabelle Kilcoyne, MVB, DACVS-LA

  Joint infections in horses are a very serious medical condition that may lead to lameness, loss of performance, and even euthanasia. To aid in treatment of equine distal limb joint infection, regional administration of antimicrobials (intravenous regional limb perfusion) is commonly performed. Antimicrobials are infused into the leg via injection into the venous circulation distal to a tourniquet. In this study, two possible durations of time for perfusate instillation were evaluated to see if there was a change in joint or systemic concentrations.

  The results of this study found that faster administration of perfusate (given over 1 minute) did not result in a significant change in joint concentration of antibiotic when compared to slower administration (5 minutes). However, faster administration did increase the systemic concentration of antibiotic during the perfusion. This suggests that faster instillation of perfusate results in leakage from the tourniquet and supports the practice of slower perfusate instillation time in practice.

  How does this research benefit horses?

  Results from this research will help to provide equine clinicians with the best technique for achieving maximum concentration in the radiocarpal joint following intravenous regional limb perfusion with minimal systemic leakage. This will help to provide the best care for horses with orthopedic infections.