Research Review

2022 CEH Research Review

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

*Download a pdf of the 2022 Research Review here.

Cover story: A Pioneering Approach to Equine Health - The Pioneer 100 Horse Health Project aims to lay the foundation for tailoring veterinary medical approaches to each horse’s specific needs through precision medicine.

Faculty Research Projects

Drug Therapies

• Implications of cytochrome P450 2D50 polymorphisms on drug clearance (#18-05)
• Investigator:
  Heather Knych, DVM, PhD, DACVCP
  
  Considering the large degree of individual variability between horses, establishing an appropriate treatment regimen for a drug can provide a challenge to veterinarians.  Variability in the activity of drug metabolizing enzymes attributable to genetic mutations is one documented reason for these differences in other species.  The goal of this study was to begin to assess whether mutations in drug metabolizing enzyme genes can impact drug behavior in horses. Codeine was chosen as the test drug and was administered orally to 12 research horses and blood samples collected.  Drug concentrations were measured and used to select times for collection of blood samples in a larger population of horses.  Following administration of codeine to this larger group of horses, drug concentrations were measured in blood and the relative amount of metabolism of codeine determined.  Based on the relative metabolism of codeine to metabolite, horses were categorized as low, normal and rapid metabolizers. A subset of horses was selected for whole genome sequencing and investigation of potential genetic mutations in drug metabolizing enzyme genes.  Codeine was well tolerated following oral administration and was rapidly converted to five different metabolites.  The range of codeine metabolism determined in this study identified a strong candidate gene for the metabolic enzyme, CYP2D82 in the horse and warrants further investigation of the implications of this finding on the clinical effect of drugs that are metabolized by this enzyme in horses.
  
  How does this research benefit horses?
  Data gathered in this study provides some insight into why differing responses are observed between horses when drugs that are metabolized by the CYP2D enzyme are administered.  Information from this study could potentially be used to develop a simple test, similar to that available for humans, which may aid in the development of individualized therapeutic regimens (“personalized medicine”) for each horse under care.
   

  This research was reported in Veterinary Anaesthesia and Analgesia 2020;47:694-704 and BMC Veterinary Research 2021;17(77).

• Pharmacokinetics and physiologic effects of meperidine in horses (#18-11)
• Investigator:
  Heather Knych, DVM, PhD, DACVCP
  
  Pain management is an important component of patient care. In horses, there are a limited number of analgesic drugs that have been fully characterized.  The goal of the current study was to begin to investigate the opioid drug, meperidine, which is similar in its actions to morphine, as a potential analgesic in horses.  Eight horses, on four different occasions, received a single intravenous administration of meperidine (0.5, 1 or 2 mg/kg) or saline.  Blood samples were collected up to 96 hours following drug administration, drug concentrations measured, and pharmacokinetic parameters determined.  Pre- and post-drug related behavior, locomotor activity, heart rate and gastrointestinal function were recorded.  Response to noxious stimuli was evaluated by determining the response to application of heat. Meperidine was rapidly cleared following IV administration of all three doses, however, adverse effects including excitation, increases in heart rate and the development of hives were noted with higher doses.  While meperidine did prevent horses from responding to the application of heat, the response was short-lived (up to 45 minutes).  Results of this study do not support routine clinical use of meperidine at a dose of 1 mg/kg IV.  Administration of a dose of 0.5 mg/kg decreased the response to the application of heat but the effect was short-lived, and the associated adverse effects suggest its use as a sole agent at this dose should be cautiously considered.
  
  How does this research benefit horses?
  A limited number of analgesics have been variably characterized in horses.  This study was designed to begin to assess the potential of the opioid, meperidine, as an additional option for pain relief in the horse.
   

  This research was reported in BMC Veterinary Research 2020;16(368).

• Characterization of a novel opioid analgesic in horses (#19-04)
• Investigator:
  Heather Knych, DVM, PhD, DACVCP
  
  Morphine is a potent pain-relieving drug. However, reports of adverse effects at clinical doses limits its use in horses.  In other species, pain-relieving and adverse effects have been attributed, at least partly, to two distinct metabolites of morphine (M3G and M6G).  The goal of this study was to describe how the horse processes the presumed pain-relieving metabolite (M6G) and determine its effects following intravenous administration.  Seven horses received a single intravenous administration of saline, 0.5 mg/kg morphine sulfate and 0.01 mg/kg M6G with a 2-week drug free period between doses.  Blood samples were collected up to 96-hours for determination of drug concentrations.  Drug related behavior and physiologic responses were recorded.  Response to noxious stimuli was evaluated by determining response to the application of heat at several times post administration.  M6G was cleared more rapidly than morphine.  Morphine administration resulted in signs of excitation, whereas M6G appeared to cause sedative like effects.  The response to a heat stimulus was significantly reduced until 4 hours post morphine administration and 1-hour post M6G administration.  A more favorable safety profile compared to morphine, coupled with favorable effects on response to a noxious stimuli (heat), are encouraging for further study of the effects of higher doses of M6G in the horse.
  
  How does this research benefit horses?
  Results of this study provide data to support further study of the morphine metabolite, M6G, as a pain medication for horses, devoid of the adverse effects associated with morphine administration.
   

  This research was reported in Veterinary Anaesthesia and Analgesia 2022;49(6):634-644.

Genetics

• Validating a diagnostic test for equine neuroaxonal dystrophy (#18-02)
• Investigators:
  Carrie J. Finno, DVM, PhD, DACVIM (LAIM)
  Birgit Puschner, DVM, PhD, DABVT

  During the first year of life, certain foals may develop an inherited neurologic disorder known as equine neuroaxonal dystrophy (eNAD). Until recently, the only way to definitively diagnose eNAD was on postmortem examination. Our previously funded studies from the Center for Equine Health allowed us to evaluate how vitamin E is metabolized in affected horses while investigating possible genetic regions associated with eNAD.  Excitingly, we have recently discovered that eNAD-affected horses metabolize vitamin E, specifically α-tocopherol, at a faster rate than age-matched unaffected horses. The next step is to perform this test in a larger number of horses, thereby validating the test as a way to diagnose eNAD. We hypothesized that eNAD-affected horses metabolize vitamin E, specifically α-tocopherol, at a faster rate than unaffected horses. We discovered that, similar to ataxia with vitamin E deficiency in humans, eNAD-affected horses metabolize α-tocopherol, but not λ-tocopherol, at a faster rate than unaffected horses.

  How does this research benefit horses?
  Abnormal α-tocopherol metabolism has been identified in ataxia with vitamin E deficiency in humans. Similar to that disease, horses with eNAD demonstrate a higher rate of metabolism of α-tocopherol than healthy horses. This further strengthens the link between eNAD and vitamin E deficiency and may provide an antemortem diagnostic assay.

  This research was reported in the Journal of Veterinary Internal Medicine 2021;35(5): 2473-2485.

• Discovering a genetic mutation for elevated gamma-flutamyltransferase (GGT) concentrations in Thoroughbred racehorses (#18-09)

• Investigators:
  Carrie J. Finno, DVM, PhD, DACVIM (LAIM)
  K. Gary Magdesian, DVM, DACVIM (LAIM), DACVECC, DACVCP

  Elevations in serum gamma-glutamyltransferase (GGT) activity (i.e. “high GGT syndrome”) have been reported in Thoroughbred racehorses and associated with poor racing performance. The reasons for these increases in GGT are not known. Our proposal aimed to investigate an underlying genetic cause for isolated elevations in serum GGT concentrations in Thoroughbred racehorses using a combined approach of a genome-wide association study and whole-genome sequencing. We identified a region of suggestive genetic association on chromosome 5 for high GGT syndrome in Thoroughbred racehorses. Associated genetic markers in this region surround one candidate gene, cluster of differentiation 1a (CD1A1), a transmembrane gene related to the major histocompatibility complex. This candidate gene requires further evaluation in a larger cohort of high GGT horses.

  How does this research benefit horses?
  Abnormally high serum gamma-glutamyltransferase (GGT) is observed in Thoroughbred (TB) racing horses, particularly among those with a poor racing performance. In this study, we identified a possible underlying genetic region of association for high GGT syndrome in racing TBs.

  This research was reported in the Journal of Veterinary Internal Medicine 2022;36(6):2203-2212.

• Finding the genetic mutation for inherited epilepsy in Arabian foals (#18-10)

• Investigators:
  Carrie J. Finno, DVM, PhD, DACVIM (LAIM)
  Monica Aleman, MVZ, PhD, DACVIM (LAIM, Neurology)

  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. In children with similar benign familial neonatal convulsions, genetic mutations have been identified in the potassium voltage-gated channel genes, KCNQ2 and KCNQ3. In the horse, JIE is inherited and our previously funded studies from the Center for Equine Health allowed us to identify a genetic region of interest associated with JIE.  We then used whole-genome sequencing to find possible underlying mutations in this region in two JIE-affected horses. The region identified did not contain any strong candidate genes. However, sequence commonalities were identified to a region with a strong candidate gene, KCNQ1. We hypothesized that, either due to errors in the reference genome or structural rearrangements in JIE-affected foals, the underlying mutation for JIE resides in KCNQ1. This genetic mutation may result in a different proportion of the two RNA products produced.

  Long-range sequencing was performed using Nanopore technology in one affected and one unaffected foal with the associated haplotypes on chromosome 1. These sequences were then assembled into contiguous regions (i.e. “contigs”) that surrounded the region of interest. The homozygous contig for the JIE-affected foal correlated with the contig for the unaffected foal, suggesting no large homozygous structural variations. Direct genotyping of four putative genetic markers in KCNQ1 that were identified in the n=2 short-read whole-genome sequenced JIE foals were genotyped in a subset of affected (n=3) and unaffected (n=6) foals via Sanger Sequencing and no association was identified. Therefore, a structural rearrangement between chromosomes 1 and 12, in the region of KCNQ1, was excluded.

  How does this research benefit horses? 
  Epilepsy can result in loss of animals and a major financial burden due to elevated costs of antiepileptic treatment and hospitalization. Therefore, the identification of genetic variants will aid in strategic breeding and avoid the perpetuation of genetic mutations that affect the overall health and well-being of the Arabian breed.

  This research was reported in the Journal of Veterinary Internal Medicine 2022;36(6):2203-2212.

• Genetic investigation of equine recurrent uveitis in the Knabstrupper breed (#18-16)

• Investigator:
  Rebecca Bellone, PhD
  Lynne Sandmeyer, DVM, DVSc, DACVO
  Nicole Kingsley, PhD

  Equine recurrent uveitis (ERU) is the leading cause of blindness in horses. It has been documented in several breeds with the leopard complex spotting patterns (Appaloosa, Knabstrupper, Pony of the Americas). This investigation characterized prevalence, clinical features, and risk factors in the Knabstrupper breed. By evaluating horses in Denmark, Sweden, and the USA, it was determined that uveitis in the Knabstrupper is clinically very similar to a subset of ERU, insidious uveitis, in the Appaloosa. Risk for this eye disease increased with age as did having two copies (homozygous) of the genetic variant that causes the leopard complex spotting patterns (LP).  Therefore, genotyping for LP can assist with clinical and breeding decisions related to insidious uveitis in this breed. This analysis also determined that additional genetic loci likely contribute to disease risk in this breed and further genomic investigations are warranted.

  How does this research benefit horses?
  Equine recurrent uveitis is a complex disorder with several different etiologies suspected, but little is known about the mechanisms underlying this blinding disease within and across equine breeds. From this research, we have a better understanding of the clinical signs of ERU, specifically in the Knabstrupper.  Additionally, this research identified two significant risk factors (age and LP genotype). Based on the findings of this study, more frequent clinical evaluation are recommended for Knabstrupper horses between the ages of 11-20 years old with the LP/LP genotype to identify and manage early signs of disease.

  This research was published ahead of print in the Veterinary Journal 2022.

Medicine and Infectious Disease

• Identification of vaccine candidates for the neurological disease equine protozoal myeloencephalitis (#18-03)

• Investigators:
  Tatiana Paredes-Santos, PhD
  Jeroen P.J. Saeij, PhD

  Equine protozoal myeloencephalitis (EPM) is a debilitating neurologic disease that is associated with infection with the protozoan parasite Sarcocystis neurona. There are currently no vaccines available against S. neurona. For rational vaccine design, a better understanding of the basic biology of S. neurona is needed. For example, it is currently unclear how S. neurona modulates the host cells it infects to eventually cause pathology. We hypothesize that a better understanding of S. neurona-host interactions will allow the future rational design of vaccines or better therapies. A vaccine against S. neurona would reduce the prevalence of EPM. The goal of this study was to determine if the Toxoplasma secreted proteins that we know are important for Toxoplasma pathogenesis and that are conserved in S. neurona are also important for S. neurona pathogenesis.

  We identified 13 Toxoplasma proteins that are conserved in S. neurona and are known to be involved in crucial Toxoplasma-host interactions. We used the latest genetic techniques to manipulate the genome of S. neurona in a way that would have allowed us to visualize the localization of these conserved proteins. We also used genetic techniques to delete each of these genes individually in S. neurona. Unfortunately, we were unable to manipulate the genome of S. neurona. This shows that we will first need to optimize the technologies that we have previously established for Toxoplasma for S. neurona.

  How does this research benefit horses? 
  We want to determine which proteins S. neurona uses to acquire host nutrients or modulate the host cell. This will advance our understanding of S. neurona-host interactions. To date, no secreted proteins from S. neurona have been characterized. A better understanding of the basic biology of S. neurona will unveil new drug targets and vaccine candidates against EPM.

• Prevention of viral respiratory infections in weanling foals by using an intra-nasal equine influenza vaccine (#18-06)

• Investigators:
  Nicola Pusterla, DVM, PhD, DACVIM, DAVDC-Equine
  Jeanne Bowers, DVM
  Samantha Barnum, MS
  Virginia Hernandez
  Rebeca Scalco

  During stressful periods, such as weaning, foals can be more susceptible to viral respiratory infections, including equine herpes viruses. We hypothesized that an intra-nasal equine influenza vaccine, administered prior to weaning, would decrease the incidence of viral respiratory infections. Twenty healthy weaning foals received a single dose of FluAvert (equine influenza virus vaccine) 10 days prior to being weaned. An additional 20 healthy foals served as unvaccinated controls. Nasal secretions were collected prior to the vaccine administration, on the day of weaning, and weekly thereafter for 6 weeks. The nasal secretions were tested by PCR for herpes viruses. Physical parameters were collected daily for the entire study period. Molecular and physical parameters were then compared between the groups. The use of FluAvert was associated with a better clinical outcome in vaccinates. However, the EIV vaccine was unable to influence selected hematological parameters and viral kinetics of herpesviruses. The clinical benefit observed in vaccinates may explain the overall perception that the use of the modified live EIV vaccine induces cross-protection against respiratory agents.

  How does this research benefit horses? 
  Due to the economic impact of contagious respiratory pathogens, it is relevant that horses be protected against respiratory viruses, especially during stressful time such as the weaning period. While today’s vaccines offer a suboptimal protection against respiratory viruses, the use of local immunomodulators aiming at improving mucosal immunity could enhance anti-viral protection, therefore contributing to the overall health and wellbeing of horses.

  This research was reported in the Canadian Veterinary Journal 2020;61:517-520.

• Improving the molecular diagnostic field of Clostridium perfringens in foals with diarrhea (#18-15)

• Investigators:
  Nicola Pusterla, DVM, PhD, DACVIM, DAVDC-Equine
  K. Gary Magdesian, DVM, DACVIM, DACVECC, DACVCP
  Nathan Slovis, DVM, DACVIM, CHT

  Infections of Clostridium perfringens in foals are often severe and highly contagious. Thus, there is a need to improve the molecular diagnostic testing that is currently available in order to obtain rapid and accurate results. Feces from 50 healthy foals and 50 foals with diarrhea were collected for this study at two veterinary hospitals. The age of the study foals ranged from 1 week to 16 weeks. The samples were processed for nucleic acid extraction and tested for a panel of five C. perfringens target genes (alpha, beta, C. perfringens enterotoxin, beta2 and netF). The samples were also tested by qPCR for established foal diarrhea pathogens including equine rotavirus, equine coronavirus, C. difficile toxin A and B, Salmonella enterica, Lawsonia intracellularis, Neorickettsia risticii and Rhodococcus equi in order to document mono- versus co-infection. The results showed that there was no difference in the frequency of C. perfringens type and virulence genes between healthy and sick foals. There was further no difference between the two groups in the frequency of shedding of C. difficile. Salmonella enterica, N. risticii, L. intracellularis, R. equi, and ECoV were not detected in any of the study foals. Equine rotavirus was the only enteric pathogen with a higher frequency of detection in foals with diarrhea (P < 0.05). The study results showed that the use of a C. perfringens panel, targeting various toxin genes, was unable to definitively determine the role of this enteric pathogen as a cause of diarrhea in foals.

  

  
  How does this research benefit horses? 
  Equine veterinarians recognize that diarrhea is extremely common in foals of all ages but the condition is often self-limiting and caused by infectious and non-infectious conditions. Sensitivity and specificity of diagnostic tests vary for different agents. This mainly depends on the methodology but also relative prevalence of detection of the pathogens in healthy foals. Because of the severity and contagious nature of C. perfringens infections, reliable and timely qPCR assays may help manage foals with diarrhea. Unfortunately, the present study results showed that the detection of various virulence genes of C. perfringens was unable to differentiate between healthy foals and foals with diarrhea. 

• Study the role of Toxoplasma gondii as a cause of neurologic disease in horses (#18-21)

• Investigators:
  Nicola Pusterla, DVM, PhD, DACVIM, DAVDC-Equine
  Patricia Conrad, DVM, PhD
  Woutrina Smith, DVM, MPVM
  Kaitlyn James
  Eva Tamez-Trevino
  Monica Aleman MVZ, PhD, DACVIM (LAIM)
  Pedro Bernardino, PhD

  Equine protozoal myeloencephalitis (EPM) is caused by two recognized protozoal apicomplexan parasites, Sarcocystis neurona and Neospora hughesi. In other animal species, Toxoplasma gondii has been shown to be another protozoal parasite capable of causing encephalomyelitis. Banked serum and cerebrospinal fluid (CSF) samples from 210 horses with neurological deficits were analyzed for the detection of T. gondii, S. neurona and N. hughesi antibodies by immunofluorescence antibody test (IFAT). Based on a serum/CSF ratio ≤ 64, horses were characterized either as T. gondii-suspect or as S. neurona/N. hughesi-suspect. Frequency of immunodiagnostic status was determined, as well as selected prevalence factors between T. gondii-suspect and S. neurona/N. hughesi-suspect EPM cases. Based on a serum/CSF ratio ≤ 64, 22 and 43 horses fit the case definition of T. gondii and S. neurona/N. hughesi EPM-suspect, respectively. No statistically significant differences were found for age, sex, breed other than Warmblood, use, and clinical signs other than lameness between T. gondii- and S. neurona/N. hughesi-suspect EPM cases.

  How does this research benefit horses?EPM is a common neurologic disease of equids, and many facets of its epidemiology are still considered missing links. This study strives to illuminate the role of Toxoplasma gondii as another cause of neurologic disease in horses. The results of this study showed that 10% of EPM-suspect horses had evidence of antibodies in the CSF to Toxoplasma gondii. Warmblood breed and lack of lameness were significantly associated with T. gondii-suspect horses when compared to horses infected with well-established apicomplexan protozoa (S. neurona and/or N. hughesi).

• Using culture followed by PCR to determine the infectious nature of Streptococcus equi subspecies equi, agent of strangles (#18-23) 

• Investigators:
  Nicola Pusterla, DVM, PhD, DACVIM, DAVDC-Equine
  Barbara Byrne, DVM, PhD, DACVIM (LAIM), DACVMBM
  Samantha Barnum, MS

  The diagnosis of strangles requires the detection of S. equi by microbiological culture, PCR, or both on samples from the upper respiratory tract. Recent studies have shown that PCR is more sensitive than bacterial culture, despite the fact that PCR is unable to determine viability of streptococcal organisms. The objective of the present study was to investigate a novel approach that combined a 24-hour selective culture step, followed by PCR, in order to characterize the viability of S. equi in biological samples. Nasal secretions collected from 42 horses with suspected strangles were tested by culture and by qPCR prior to and 24-hours following a culture step. Viable S. equi was determined based on the detection of S. equi via culture, the detection of mRNA transcripts for the SeM gene of S. equi by qPCR and/or an increase in absolute number of SeM target genes of S. equi between pre- and post-cultured samples. The overall agreement between culture alone and the three criteria to determine viability was 59%. The overall agreement for the detection of mRNA transcripts and increase in absolute target genes was 88% and 74%, respectively. The combination of mRNA transcripts and increase in absolute target genes was able to determine the viability status in all 42 samples.

  How does this research benefit horses?The increasing application of qPCR for the detection of S. equi in practice settings has presented new dilemmas with regard to how test results are interpreted and used by equine practitioners since routine qPCR assays are unable to differentiate between viable streptococcal organisms from nonviable cells or from free nucleic acids in biological samples. The study results showed that, in the absence of a culture-positive result for S. equi, the determination of viability was achieved by using molecular strategies applied to samples undergoing a 24-hour culture step. Determining molecular viability of S. equi is essential in order to implement biosecurity protocols and reduce risk of transmission.

  This research was reported in the Journal of Equine Veterinary Science 2021;97:103328.

• Develop PCR assays able to differentiate between field and vaccine strains of Streptococcus equi subspecies equi, agent of strangles (#19-14)

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

  The highly contagious, host-adapted bacterium, Streptococcus equi subspecies equi (S. equi), is the etiologic agent of equine strangles. S. equi primarily targets the lymphoid tissues of the upper respiratory tract of equids, which leads to acute swelling and abscess formation of mandibular and retropharyngeal lymph nodes. Killed or modified-live (ML) S. equi vaccines are commonly used in the prevention of strangles. However, the ML S. equi vaccine strain has retained some virulence and has been associated with clinical disease post-vaccination. Because of the strong difference in contagiousness between vaccine (not contagious) and field (highly contagious) S. equi isolates, it is important to quickly differentiate between the two strain types. Field and vaccine strains associated infections can be differentiated using subtyping methods. These methods are costly and very time-consuming.

  In order to further characterize a S. equi positive sample, our laboratory has successfully developed two real-time qPCR assays able to differentiate between field and vaccine S. equi strains. One hundred and seventy-one field samples from horses, which tested qPCR-positive for S. equi, were used for the validation of the differentiating assays. Twenty-two and 147 samples were consistent with the vaccine and field S. equi strains, respectively. One additional samples tested positive for both strains. All horses with detectable S. equi vaccine strain had a history of been recently vaccinated with the ML S. equi vaccine or in close contact with a recently vaccinated horse.

  How does this research benefit horses?
  The ML S. equi vaccine is commonly used on endemic farms and during outbreaks of strangles. Since the ML vaccine strain of S. equi has retained some virulence and can cause mild disease, it is imperative to characterize a S. equi strain to determine its origin. The established and validated S. equi PCR assays, able to differentiate between field and vaccine S. equi strains, are available for the fine-tuning of molecular diagnostics in special conditions when a recently vaccinated horse develops strangles. Determining the S. equi strain type in a clinically infected horse is important to institute proper biosecurity protocols and characterize the source of the infection.

• Investigation of the genetic make-up of equine coronavirus from foals and adult horses (#19-13)

• Investigators:
  Nicola Pusterla, DVM, PhD, DACVIM, DAVDC-Equine
  Beatriz Martinez Lopez, DVM, MPVM, PhD

  Equine coronavirus (ECoV) is a newly recognized enteric virus of adult horses that has been associated with fever, lethargy and anorexia, as well as colic and diarrhea. Since 2010, clinical ECoV infections have predominantly been reported in adult horses. Data from our laboratory showed that the age distribution of confirmed ECoV infections was 20.5% in foals (age 0-6 months), 25.3% in horses aged 6 months to 5 years and 54.2% in horses older than 5 years. Usually, foals with clinical gastrointestinal disease are only positive for ECoV if other co-infections are present (e.g. rotavirus or Clostridium perfringens) with the prevalence of ECoV being similar between healthy and clinically affected foals. Adult horses with clinical signs experience a mono-infection with ECoV. Disease expression, as observed between foals and adult horses, may relate to various factors originating from the host or the virus.

  In an attempt to determine if viral genetic factors are responsible for the observed age-dependent disease expression, our laboratory performed genetic sequencing of 40 individual ECoV strains from 9 young and 31 adult horses. Sequence comparison of the nucleocapsid (N) gene amongst the 40 strains of ECoV showed very high level of homology, ranging between 97.9 to 99.0%. While geographic clusters of ECoV were determined, there was no marked difference of the N gene between strains originating from foals and from adult horses. The study results showed that age-dependent susceptibility to ECoV infection is not mediated by genetic differences of the N gene of ECoV and is more likely to depend on host factors.

  How does this research benefit horses?
  ECoV has major clinical implications for the equine industry since it causes outbreaks with morbidity rates ranging from 15 to 85%. As with many viral infections, disease expression is often a multifactorial process, depending on host, viral and environmental factors. Determining risk factors is essential in order to establish proper preventive measures. While the study results showed that there are no genetic differences in the virus strong enough to account for the age-dependent disease expression of ECoV, it remains prudent to separate young from adult horses, as foals have been shown to be subclinically infected and potentially act as a source of infection for older animals.

Orthopedics and Lameness

• Bolstering tendon repair with mesenchymal stem cells that secrete biglycan (#18-01)

• Investigators:
  Michael Mienaltowski, DVM, PhD
  Elizabeth Maga, PhD

  Mesenchymal stem cells (MSCs) have been used in regenerative strategies to improve healing of tendon; they have been shown to have immunomodulatory effects and to serve as resources for trophic factors to bolster tissue healing. In order to study the effects of these cells using a lab-based assay, optimization of the current cell culture model is required. We hypothesized that transfection of expression vectors expressing small leucine-rich repeat proteoglycans (SLRP) like biglycan and decorin would bolster tenogenic properties of cells. However, after transfection with these plasmids, we found that adipose-derived MSCs did not express any higher levels of SLRPs than controls. Instead, the MSCs seemed to be stressed by the introduction of the plasmids. Because adipose-derived MSCs are injected into tendons alone, we recognize that they will interact with tendon proper and peritenon cells. Thus, we also examined the relationships between these tendon cells and the adipose-derived MSCs through co-culture of tendon cells with adipose-derived MSCs. In co-culture, all three cell types – tendon proper cells, peritenon cells, and adipose-derived MSCs – exhibited gene expression profiles considered favorable for a tenogenic phenotype. That is, it appears that these cell types all secrete factors that promote tendon-forming features. Tenogenic transcription factors and extracellular matrix markers were most affected. In this study, we determined that application of expression vectors for SLRPs require further optimization and consideration of stresses to MSCs. Furthermore, adipose-derived MSCs, tendon proper cells and peritenon cells all interact in a manner favorable for tendon formation in vitro. It is likely that these positive interactions occur in the tissue as well.

  How does this research benefit horses?
  Our findings highlighted the fragile nature of adipose-derived MSCs when it comes to cell manipulation – particularly the introduction of expression vectors to promote the secretion of extracellular matrix (tendon structural and regulatory proteins) – ahead of stem cell injection. Moreover, the co-culture research confirms utility of adipose-derived MSCs for bolstering tendon-forming expression profiles. This study helped to optimize our current in vitro model, which can now be used to systematically investigate the role of MSCs on tendon repair.

• How do horseshoe traction features alter hoof grip on performance footings? (#18-07)

• Investigators:
  Christina Rohlf, PhD
  Tara Doherty, DVM
  Susan Stover, DVM, PhD, DACVS

  The grip of performance surfaces is a risk factor for lower leg injuries of sport horses by affecting the extent of hoof slide and stability of the leg. Horseshoe traction features may modify surface interactions. Surface grip was measured with eight paired cadaver hooves on a dirt surface (sand) and a synthetic surface (sand with fiber). Hooves were shod with positive (low toe grab), neutral (flat), and negative (sliding plate) traction characteristics. Unshod hooves served as a control. Surface material had a greater effect than horseshoe traction characteristics on surface grip, with the synthetic surface exhibiting significantly higher grip than the dirt surface. Traction characteristics altered the shear force most notably on the synthetic surface, with sliding plates exhibiting lower grip than the unshod hoof. However, traction characteristics did not affect the grip on the dirt surface. These results indicate that the addition of fiber alone to surface material can significantly alter the grip at the hoof-surface interface, while horseshoe traction features have a lesser effect on grip properties.

  
  

  
  How does this research benefit horses?
  The choice of surface material and design of horseshoe traction features to improve grip have previously been driven by subjective opinions. This study scientifically quantified the effects of surface material and horseshoe traction features on performance factors, which directly affect the risk for leg injury in sport horses. Results of this study will inform horseshoe practices and guide the installation of surface materials which enhance the welfare and safety of performance horses.

• PET scans improve the detection of injuries in the horse foot when compared with MRI (#18-13)

• Investigators:
  Mathieu Spriet, DVM, MS, DECVDI, DACVR-EDI
  Jannah Pye, DVM
  Larry Galuppo, DVM, DACVS-LA
  Scott Katzman, DVM, DACVS-LA

  MRI is the primary reference imaging technique used to investigate injuries responsible for foot pain in horses. However, MRI cannot explain the origin of the pain in some horses. Positron emission tomography (PET) is a new imaging modality that provides information about injuries by using a small amount of a radioactive dye. PET is able to recognize very early injuries and distinguish between active injuries and old scars. In this study, 12 horses that recently had MRI for investigation of foot pain were also imaged with PET. PET and MRI identified the same injuries in the majority of cases but there were a few situations were PET provided more information than MRI. These included:

  -      MRI typically only detects large bone bruises of the coffin bone, while PET is excellent at demonstrating even small areas with abnormal bone,

  -      The attachments of ligaments on bones can be challenging to evaluate by MRI. PET was very helpful at demonstrating abnormal marker accumulation, either in the bone or the soft tissue part of the ligament.

  -      Early arthritis is associated with small bone spurs that are challenging to recognize on MRI. As these developing spurs are actively growing, they display good PET marker accumulation, helping with their identification.

  -      MRI is excellent at identifying tendon injuries, but can be limited in its ability to distinguish active injuries from scar tissue. The soft tissue PET marker is excellent at distinguishing between the two, as it only accumulates within active injuries.

  

  Figure 1. Images of the left hind suspensory origin (top 3 rows, A-I) and proximal aspect of the body of the suspensory (bottom 3 rows, J-R) of a 12-year-old Warmblood. A dual tracer 18F-NaF and 18F-FDG PET scan was performed. 18F-NaF images (C, L) were obtained first. 18F-FDG was them injected during the same anesthesia and combined tracer images (F, O) were obtained. The 18F-NaF images were subtracted from the combined tracer images to obtain 18F-FDG-like images (I, R). The left column displays CT images (bone window (A, J) and soft tissue window (D,G,M,P), the central column shows PET/CT fused images. Lateral is to the left. There was marked focal increased 18F-NaF uptake (long arrow) at the lateral plantar proximal aspect of the third metatarsal bone, visible in both 18F-NaF (B, C) and combined 18F-NaF / 18F-FDG (E,F) images, at the attachment of the suspensory ligament, with associated contour irregularity on the CT, indicative of active enthesopathy. There was no abnormal 18F-FDG uptake at the suspensory origin. The focal uptake adjacent to the second metatarsal bone (arrowhead) was vascular uptake. The bone uptake was not apparent on the subtracted images (H,I). There was moderate increased 18F-FDG uptake at the lateral aspect of the body of the suspensory ligament (short arrow), associated with loss of normal architecture of the suspensory ligament on CT (M,P) appreciated on both the combined 18F-NaF / 18F-FDG (N,O) and the subtracted (Q,R) images. This indicated an active suspensory body desmitis. The arrowheads indicate incidental vascular uptake.
  
  How does this research benefit horses?
  This study confirmed that PET is an excellent tool for imaging the horse foot to explain the cause of the lameness and plan appropriately for treatment. Depending on the type of injury expected, PET can be used in addition to MRI, but in some cases,  PET can be used prior to or instead of doing an MRI. The development of equine PET has added a very powerful tool to help figure out foot lameness in horses.
  
  This research was reported in the American Journal of Veterinary Research 2022;83(7): ajvr.22.03.0051.

• PET scan improves the assessment of the hock and suspensory apparatus in lame horses (#18-22)

• Investigators:
  Mathieu Spriet, DVM, MS, DECVDI, DACVR-EDI
  Stefanie Arndt, DVM
  Sabrina Wilson, DVM
  Larry Galuppo, DVM, DACVS-LA
  Scott Katzman, DVM, DACVS-LA
  Marcos Perez Nogues, DVM, DACVS-LA

  The hock and suspensory apparatus are two common sites of injuries responsible for hind limb lameness in horses. Distinguishing which of the two areas is responsible for the lameness can be challenging due to their proximity. These areas are commonly imaged with X-rays and ultrasound, but there is sometimes limited association between the imaging findings and the clinical signs. Mild changes are common both with X-rays and ultrasound and may or may not be the source of pain. Positron emission tomography (PET) is a newly available imaging modality in horses that has the ability to detect subtle injuries and also distinguish between active and inactive injuries when changes are seen with other modalities. This study was the first to investigate the value of PET to assess both the bone and soft tissue changes in horses with pain localizing to the distal hock / suspensory area. The study confirmed that, in this population, changes could be seen in several regions. It was possible to distinguish horses with abnormalities in their hock joints, horses with abnormality in the attachment of the suspensory ligament on the cannon bone, and horses with changes in the suspensory ligament itself. There was high agreement between the three different observers assessing the images.

  How does this research benefit horses?
  Dual tracer PET of the equine distal hock and suspensory  apparatus is now used on regular bases to evaluate horses with pain originating from this region. The results of PET are very helpful for treatment and rehabilitation planning.

• Evaluating the effect of footing properties on tendon and ligament strains in show jumping horses (#19-07)

• Investigators:
  Christina Rohlf, PhD
  David Hawkins, PhD
  Tanya C. Garcia-Nolen, MS
  Susan M. Stover, DVM, PhD, DACVS-LA

  Show jumping horses commonly injure tendons and ligaments in the lower limb, especially the suspensory ligament and superficial and deep digital flexor tendons. Due to limb anatomy, the amount of tendon and ligament stretching (strain) is affected by the amount of fetlock, pastern, and coffin joint extension during exercise. This study used high speed video to track fetlock, pastern, and coffin joint movement of four horses jumping a 1.1 meter parallel oxer on 12 different surfaces in northern California (5 dirt, 7 synthetic). The horses averaged 9.8 years of age and included 1 mare and 3 geldings. Average takeoff velocity was 5.97 m/s, with an average jump height of 1.27 m at the girth. Our findings showed that extension of all three of these joints was significantly greater at landing than takeoff. Additionally, fetlock and coffin joint extension were greater on dirt than synthetic surfaces, while pastern extension was greater on synthetic than dirt surfaces. The reported surface effects on coffin and pastern extension were greater on the leading limb compared to the trailing limb.

  

   

  

  
  
  How does this research benefit horses?
  These results help contribute to the scientific development of a set of standards for arena surface properties, designed to minimize tendon and ligament injuries of jumping horses. These standards would guide the construction and management of arena surfaces to reduce the risk of injury for horses that train and compete on such surfaces.
  
  This research was reported in The Veterinary Journal 2023;291:105930.

• Assessment of bone growth in horse fetuses and newborn foals (#19-23)

• Investigators:
  Mathieu Spriet, DVM, MS, DECVDI, DACVR-EDI
  Catherin Renaudin, DVM, DECAR

  Equine fetal growth is routinely assessed by ultrasonography using fetal growth measurements. Femur length and eye volume can predict gestational age in Quarter Horses up to 6 months of age with an accuracy of within 2 weeks. As pregnancy advances, age predictions are not as precise. This study aimed to evaluate a new fetal growth parameter: the first phalange (P1). The length of P1 was measured and P1 bone maturation was described by recording the time of appearance and closure of P1 secondary ossification centers. Correlation was assessed between ultrasonographic findings observed within the last 2 weeks of gestation and radiographic findings obtained at birth was evaluated. Data was obtained from 10 healthy Quarter Horse mares with known gestation dates that underwent transrectal ultrasound every 2 weeks from 9 months of gestation until parturition. Within 48 hours of birth, radiographs of each foal’ 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 (Figure 3). The proximal and distal ossification centers both appeared between 277 and 303 days of gestation (2 weeks). The proximal ossification center did not close as opposed to the distal one that closed between 306 and 333 days of gestation (2 weeks). All ultrasonographic findings were confirmed on radiographs, with the exception of the length of P1. As P1 becomes too long to be measured close to parturition, the latest ultrasonographic measurement is therefore underestimated.
   

  

   

  

   

  

  
  How does this research benefit horses?
  We confirm that P1 length can be used as a novel fetal growth parameter. In addition to the other growth parameters already known, P1 length will improve the prediction of the 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 Quarter Horses that may be used in the future in the decision-making process of inducing parturition in the mare.  It may help also identify risk of foal health issues at birth, such as prematurity or dysmaturity due to abnormal development.

Reproduction

• Ovarian hormones in follicular fluid (FF) are predictive markers of oocyte quality and maturation rates in mares (#18-12)

• Investigators:
  Stuart Meyers, DVM, PhD, DACT
  Alan Conley, BVSc, MS, PhD, FRCVS, DACT
  Alejandro de la Fuente, DVM, MS

  The follicular fluid of antral follicles is part of the environment where an oocyte acquires its competence to become a healthy embryo. Molecules secreted by the mural granulosa cells (GC) and cumulus cells (CC) surrounding the oocyte are part of the signaling mechanism between the oocyte and its environment that control the development and maturation of the oocyte before ovulation. Anti-Müllerian hormone (AMH), Inhibin-B (Inh-B), and Inhibin-A (Inh-A) are important hormones secreted by GC and CC that exert effects not only in the follicle. However, their concentration in the follicular fluid of horses has not been characterized. In our study, we determined the concentration of AMH, Inh-B, and Inh-A in follicles ranging from 5-40 mm in size. We found that AMH presents a peak concentration in 15 mm follicles. Inhibin-B increases its concentration as the follicle grows. However, it presents a small decrease after the follicle reaches a size of 30mm. Inhibin-A concentration on the other hand, increases as the follicle grows without showing a decrease. Additionally, from the whole pool of follicles aspirated, size 10, 15, and 20 mm were the more abundant and we obtained a better oocyte recovery rate in follicles of size 15mm.

  How does this research benefit horses?
  The goal of this study is to understand the hormonal regulation of follicle growth, to improve oocyte quality from growing follicles, and to increase fecundity of mares by improving embryo production. ICSI is an IVF technique that is becoming an important part of reproductive success in valuable horses. Understanding the factors controlling oocyte maturation will result in improved embryo development, thereby increasing pregnancy rates and production of healthy foals.

• DNA sequencing of equine embryos for detection of chromosomal errors (#18-14)

• Investigators:
  Stuart Meyers, DVM, PhD, DACT
  Ghislaine A. Dujovne, DVM, MS, DACT
  S Chavez
  Alejandro de la Fuente, DVM, MS

  Early embryonic death in horses is costly and poorly understood, with higher loss rates in older mares and with advanced reproductive techniques. Detecting abnormalities in early development associated with later pregnancy loss would guide embryo selection and direct future research on causes of early embryonic death. Noninvasive imaging to precisely determine the duration of vital early embryonic events has allowed us to create the first predictive model of equine embryo development (Meyers et al, 2019; Table 1). Time-lapse imaging was used to develop a predictive model of success to blastocyst stage by identifying precise timing of cellular divisions and aberrations of embryo development. Chromosomal abnormalities were assessed by DNA sequencing in dividing embryos and indicate that cell division timing influences reproductive successes of horse embryos. DNA sequencing of embryos surviving to blastocysts has generated a complex of genes that we are currently studying that will be used to identify genes associated with developmental success, and targets for future therapeutic options. In the figures below, we show that equine embryos display varying characteristics that indicate abnormal cell divisions and fragmented DNA and chromosomes are associated with embryo failure and associated infertility.

  After generating embryos in vitro using ICSI, noninvasive imaging key cell division times were marked and noted as seen in Table 1. Mitotic parameters were retrospectively analyzed in embryos that succeeded or fail to reach blastocyst stage to identify events in equine embryonic development that are predictive of early embryonic failure and death. 
   

  

  
  
  Using fluorescence staining, we have been able to identify precisely which cells in the developing embryo shows abnormal chromosomes and mitotic structures. In Figure 1, we have shown that normal- appearing embryo cells display very abnormal chromosomes and spindle structures when special fluorescence stains are used. The asterisks in Fig 1b below shows a very erratic pattern of chromosome defects with multiple unaligned (blue) chromosomes.
   

  

  
  
  Figure 2, below, is a comparison of the chromosomal status of embryos between 2-12 cell stages using single-cell sequencing. 11/16 embryos include at least 1 blastomere with chaotic aneuploidy, which is defined as having 5 or more chromosome losses and gains. Additionally, in 9/16 embryos, segmental aneuploidy was detected where segmental deletions, duplications, and amplifications had occurred.
   

  

  
  How does this research benefit horses?

  Assisted reproductive techniques (ART) in the horse industry are growing in clinical use and importance in breeding valuable mares. While the proportions of embryos that survive to blastocyst remains low, the significant cost of embryonic loss can be minimized by transferring high quality embryos of predicted success. The development of a set of markers of embryonic success will allow a deep understanding of equine embryo development, improve preimplantation genetic diagnostics, and result in birth of healthy foals.

• Characterization of intracellular calcium stores in stallion sperm (#18-17)

• Investigators:
  Stuart Meyers, DVM, PhD, DACT
  Gino Cortopassi, PhD

  As the only method for long-term sperm storage, cryopreservation is widely used in the equine breeding industry, despite causing significant declines in fertility rates and sperm motility. Efforts to improve post-thaw motility are limited by a critical gap in knowledge of the regulatory mechanisms of motility. Intracellular calcium (Ca2+i) signaling is known to be important for motility maintenance and is significantly altered by cryopreservation in stallion sperm. We confirmed the presence of ER-like Ca2+I stores in fresh stallion sperm by demonstrating dose-dependent Ca2+i release to thapsigargin (sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor4; Tg) treatment. The approximately 3-fold elevation in Ca2+ suggests that the ER-like Ca2+i stores are separate from other, larger intracellular Ca2+ stores, which could represent the acrosome. Additional work is needed to determine the intracellular localization of these ER-like Ca2+, to investigate whether they are altered by cryopreservation, and their implications in sperm motility, mitochondrial function and fertility. The work has demonstrated that stallion sperm oxidative and mitochondrial functions are unique to the equine species among livestock species.

  How does this research benefit horses?

  In demonstrating that equine sperm are biochemically unique among livestock species, this project could significantly improve our understanding of how cryopreservation affects sperm physiology. The work also provides an opportunity to enable rational development of drug, stem cell, or immunologic-based therapies that may prevent or reverse oxidative injury to sperm, or by other mechanisms that affect male fertility. Producers desire stallions to remain commercially viable for additional breeding seasons and additional production of healthy foals. This project could allow greater participation of stallions from various breeds and expand domestic and international shipping of semen.
  
  This research was reported in Reproduction in Domestic Animals 2019;54(S3):22–28 and Clinical Theriogenology 2019;11(3):353-359.

• The role of critical factors in equine egg maturation using time-lapse microscopy (TLM) (#19-08)

• Investigators:
  Stuart Meyers, DVM, PhD, DACT
  Ghislaine A. Dujovne, DVM, MS, DACT
  Anna Denicol, DVM, MPVM, PhD

  During oocyte maturation, the cumulus cells go through a process of mucification, which enables the cumulus oophorous to expand. In vivo processes like ovulation of oocytes, sperm capacitation, and fertilization depend upon granulosa and cumulus cell expansion. During in vitro maturation of cumulus-oocyte complexes, cumulus expansion has been reported to be positively correlated with blastocyst rate in bovine oocytes, although never described for horses. However, the time needed for expansion is longer in the in vitro culture as compared to that of in vivo maturation. Moreover, the magnitude of expansion is greater in the latter. In this study, we matured individual horse cumulus-oocyte complexes in vitro using the Miri™ Time-Lapse microscopy system, which allowed us to measure the area of the cumulus every hour for a period of 24 hours for the first time. Then, cells were processed for RNA-sequencing to determine their gene expression. Our results indicate that the gene expression of cumulus cells from in vitro matured COCs present a low expression of genes related to expansion, extracellular matrix, collagen synthesis, and hyaluronic acid synthesis when compared to the gene expression of in vivo matured COCs. We are currently exploring all the genes that present different expressions and determine their correlation with the cumulus expansion rates.
   

  

   

  

  
  
  How does this research benefit horses?

  Reproductive success of many horses depends on Assisted Reproductive Technology (ART). This study will increase our understanding of factors regulating oocyte maturation. Potential benefits of this study would be an improvement of in vitro maturation of oocytes, improving embryo production, and ultimately more healthy foals that otherwise would not have been produced.
  
  This research is under review at Reproduction in Domestic Animals.

• Predicting the 1st cell division of equine embryos using time-lapse imaging (#19-09)

• Investigators:
  Stuart Meyers, DVM, PhD, DACT
  Candace Lyman, DVM, DACT
  Ghislaine A. Dujovne, DVM, MS, DACT
  Momoe Kato

  This is a continuation of our study to develop a predictive model of successful embryo selection using a non-invasive time-lapse imaging embryo culture system. Previously, we tracked and defined cell cycle parameters establishing key morphological events leading to a successful embryo. In this study, we focused exclusively on the earliest stages of embryo development, which occurs post-fertilization but before first cell division. We identified two important and crucial events in this project related to the timing of the first cell division after fertilization: 1. the extrusion of cytoplasmic debris (called “cytoplasmic extrusion, CE”) and 2. reorganization of the fertilized zygote’s chromosomes and other nuclear content. We can now describe both of these events for horses for the first time based on the critical hypothesis that equine embryos have detectable mitotic landmarks of developmental competence by assessing the association between embryo morphology, developmental times, blastomere symmetry, ploidy status, and gene expression. These findings will help us gain a better understanding of equine embryogenesis, further improving early predictions that may lead to successful pregnancy outcomes.
   

  

   

  

   

  

  
  
  How does this research benefit horses?

  Assisted reproductive technology (ART) provides the ability to transfer embryos to produce genetically valuable horses. Abnormalities in early embryo development are crucial to understanding the causes of early embryonic death. Non-invasive imaging allows us to accurately determine key morphological events and the first predictive model of equine embryo development.
  
  This research was reported in Reproduction, Fertility and Development 2019;31:1874-1884.

• Baseline systemic and abdominal parameters after transvaginal aspiration of oocytes in mares (#19-11)

• Investigators:
  Ghislaine A. Dujovne, DVM, MS, DACT
  Daniela Orellana-Guerrero, DVM, DACT

  Aspiration of oocytes (TVA) is critical for in vitro production of horse embryos. The procedure includes the aspiration of oocytes using a needle, which is guided by ultrasound from the vagina into the ovaries. Most mares recover uneventfully after the procedure, but others will develop complications including fever, bleeding and peritonitis. The goal of this study was to determine baseline information encompassing normal changes in blood parameters and changes in peritoneal fluid due to inflammation after the procedure. This study showed that all mares had some degree of inflammation in the peritoneal fluid 24 hrs after uneventful procedures with no changes observed in bloodwork at that stage.

  This information will help clinicians to more quickly determine when further treatment is required following complications post-TVA.

  How does this research benefit horses?

  This research provides clinically relevant information to evaluate optimal patient recovery after the procedure and be able to determine the plan of action in cases that have complications after aspiration of oocytes.
  
  This research was reported in Journal of Equine Veterinary Science 2022;114:103949.

Surgery/Anesthesiology

• Evaluation of a new pain reliever in horses with chronic lameness (#18-18)

• Investigators:
  Robert Brosnan, DVM, PhD, DACVAA
  Alessia Cenani, DrMedVet, MS, DACVAA
  Susan Stover, DVM, PhD, DACVS-LA
  Tanya Garcia-Nolen, MS
  Antonio Jose de Araujo Aguiar, DVM

  3,4,4,4,-tetrafluoro-3-(trifluoromethyl)-butan-1-ol (TFMB) is a novel analgesic discovered at the UC Davis School of Veterinary Medicine.  A pilot study (16-02) in lame horses identified a TFMB route and dose range that caused no observable adverse effects.  Drug administration was associated with improved lameness scores, improved pain scores, and increased step frequency, presumably due to less pain.  However, the efficacy of TFMB compared to a standard treatment (positive control) and a placebo (negative control) needed to be evaluated.

  The objective of this study was to measure subjective and objective measures of pain over time in horses with chronic orthopedic lameness following administration of TFMB, morphine (positive control), or saline (negative control) to determine if the pain-relieving effects of TFMB are as good, or better, and persists as long, or longer, than morphine.

  Horses with naturally occurring static and chronic orthopedic disease received each of three treatments (TFMB, morphine, and placebo) in different orders with a 1-week drug washout period between treatments.  Investigators unaware of the administered treatment performed analgesic assessments and orthopedic examinations over 24 hours to provide multidimensional comparisons of drug efficacy.  Horses also wore pedometers to count the number of steps taken after each treatment.

  Lame horses treated with TFMB walked significantly more for the first hour after treatment than the same horses after either morphine or saline control.  However, by the 3-hour time point, step counts in TFMB horses returned to baseline whereas morphine-treated horse step counts were back to baseline by the 6-hour assessment time.  Other subjective analgesia assessments (lameness and pain scores) were not statistically different between control, morphine and TFMB treatments.

  How does this research benefit horses?

  TFMB provides effective analgesia in lame horses, with a duration of at least 1 hour but less than 3 hours for the dose and route studied.  It is possible that this duration of effect may be extended with repeated dosing, and follow-up studies measuring drug concentrations after single and multiple doses in horses are currently underway.  Nonetheless, even the shorter action of the single TFMB dose may be useful in acute pain scenarios, such as in horses recovering from anesthesia after surgery, where better analgesia may improve recovery quality and reduce perioperative injury risk.

Resident Grants

Medicine and Infectious Disease

• Comparing whether a horse's inflammatory response to West Nile virus vaccine is similar to their antibody-mediated response (#19-26)

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

  The purpose of this study was to establish if peak serum amyloid A (SAA) concentrations could be used to determine an appropriate immune response to a vaccine containing West Nile virus (WNV) antigen.

  A pilot study was performed using 20 clinically healthy horses to identify peak SAA concentration post vaccination with a commercial multivalent WNV vaccine. Blood was collected at baseline and for up to 7 days post vaccination. After this pilot study, an additional 40 horses underwent the study protocol with SAA measurements collected before and 72 hours after vaccination. Ninety percent of the population of horses had an increase in SAA in response to WNV vaccination, although no significant correlation was identified between SAA peak and antibody titer fold changes. The main conclusions were that SAA was an unreliable predictor of immune response to WNV and is therefore not useful as a point of care test 72 hours following vaccination to determine adequate antibody response.

  How does this research benefit horses?

  Antibody testing remains the gold standard method for determining vaccination response.

  This research was reported in the Journal of Equine Veterinary Science 2021;106:103755.

• Response of acute phase protein levels in healthy horses receiving injections with antimicrobials and anti-inflammatory drugs (#20-23)

• Investigators:
  Jurica Trsan, DVM
  Bridget Nottle, BVSc, DACVS
  Nicola Pusterla, DVM, PhD, DACVIM, DAVDC-Equine

  Serum amyloid A (SAA), an acute phase protein, is a monitoring tool for acute infectious or inflammatory diseases in horses. SAA is often used to monitor complications post-elective procedures. With many postoperative horses receiving injections of antimicrobials and anti-inflammatory drugs, the goal of this study was to determine the response of SAA to these injectable medications in healthy horses. Six healthy adult horses were used in crossover design study, including one control and three treatment groups. The control group received no treatment, the procaine penicillin G (PPG) group received PPG only via  intramuscular injection q12h for 72h, the flunixin meglumine group received flunixin meglumine only via intravenous injection q24h for 72h, and the combined treatment group received PPG (intramuscularly q12h for 72h) with flunixin meglumine (intravenously q24h for 72h). Whole blood was collected at 0, 24, 48, 72, 96 and 120 hours post-initial drug administration to measure SAA concentrations. The washout period between treatments was 30 days.

  Individual SAA values were within the reference range of ≤ 20 µg/mL for almost all horses in the control group. One control horse displayed a SAA value of 28 µg/mL at 72 hours. All horses from the PPG group showed normal SAA values throughout the study. Except for one horse (SAA of 24 µg/mL at 96 hours) from the flunixin meglumine group, all horses showed normal SAA values. For the PPG and flunixin meglumine group, 5 horses had SAA values within the reference range. One horse displayed SAA values from 32-45 µg/mL between 48 to 96 hours post-drug administration. There was no statistical significance in area under the SAA time curve amongst control and three treatment groups (P > 0.05).

  How does this research benefit horses?

  Local tissue injury caused by intramuscular PPG administration, intravenous flunixin meglumine or both does not elicit an inflammatory response that induces SAA values above the reference range in the majority of adult healthy horses. This information is important in order to determine that an increase in SAA is due to the infection and not medical treatment and/or the procedure.

Orthopedics

• A novel fiberglass hoof casting technique for treatment of equine coffin bone fractures (#20-24)

• Investigators:
  Shane Westman, SPD, GradDip ELR
  Thomas Cullen, BVMS, DACVS
  Thomas Bergstrom, DVM
  Lisa Edwards, DVm, DACVIM-LAIM
  Tanya Garcia-Nolen, MS
  Susan Stover, DVM, PhD, DACVS-LA

  Coffin bone fractures in horses can be difficult to manage. Our team performed a study, using cadaver specimens from horses euthanized for other reasons, to test a fiberglass casting technique for coffin bone fractures. Our goal was to determine the effects on the size of the fracture gap under loading of the limb.

  To perform this study, we first successfully used a novel methodology for creating minimally invasive coffin bone fractures in horse limbs while preserving all the collateral soft tissues. The limbs were then loaded using a biomechanical press and the effects on the fractures were assessed with radiographs. Our study showed that this fiberglass cast reduced the size of the fracture gap at the joint surface within the coffin joint and at other locations along the fracture when compared to non-casted feet.

  How does this research benefit horses?
  Our results suggest that this technique could provide a very useful conservative treatment for coffin bone fractures in horses, negating the need for complex surgery and general anesthesia in some instances.

Surgery

• Comparison of tourniquet number for maximizing antibiotic concentration in horses undergoing intravenous regional limb perfusion (#20-22)

• Investigators:
  Thomas C Bergstrom, DVM
  Isabelle Kilcoyne, MVB, DACVS
  K. Gary Magdesian, DVM, DACVIM (LAIM), DACVECC, DACVCP
  Jorge Nieto, MVZ, PhD, DACVS, DACVSMR

  Joint infections in horses are serious medical conditions that may result in damage to the joint leading to career limiting lameness or even euthanasia of the horse. Intravenous regional limb perfusion (IVRLP) delivers high concentrations of antibiotics to a region of a horse’s leg, including the associated joints, to treat infections. This procedure can be performed with one or two tourniquets to effectively isolate the area from the systemic circulation. In joints, such as the carpus (knee), there has been little research to determine if using a second tourniquet results in higher levels of antibiotics in the joint.

  Our study compared the effect of IVRLP performed with one or two tourniquets on the concentration of antibiotics in a joint in the carpus. The results of the study indicate that there is no significant difference in peak antibiotic concentration when performing an IVRLP with one or two tourniquets. Similarly, the number of tourniquet(s) did not affect the time to peak concentration of antibiotic in the carpus. This finding indicates that a single tourniquet is sufficient to maximize antibiotic concentration when performing IVRLP in the carpus.

  How does this research benefit horses?

  Data from this research will provide equine practitioners with information that will allow them to make informed decisions concerning how best to perform an IVRLP to deliver antibiotics to the equine carpus when treating orthopedic infections of that region.
  
  This research was reported in American Journal of Veterinary Research. 2022;83(4):364-370.