Research Review

2020 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 2020 Research Review here.

Faculty Research Projects

Drug Therapies

  • Investigation of vitamin E metabolism in horses and the effect of supplementation
  • Investigators:
    Birgit Puschner, DVM, PhD, DABVT
    Carrie J. Finno, DVM, PhD, DACVIM
    Xiaopeng Chen, PhD

    Vitamin E (vitE) is one of the most important fat-soluble vitamins and horses obtain most of their vitE from fresh pasture. Understanding the metabolic pathways of vitE is essential to define optimal dietary vitE intake in horses. Additionally, the cause for equine neuroaxonal dystrophy (eNAD) remains unknown but may be due to altered vitE metabolism. We were able to develop reliable assays to detect concentrations of vitE isoforms (α-tocopherol, γ-tocopherol, α-tocotrienol, γ-tocotrienol) and metabolites (α –CMBHC, α –CEHC and γ-CEHC) in serum and urine from healthy horses with adequate access to fresh pasture. Once we established reference ranges, we assayed them in a group of healthy horses and eNAD-affected horses before and after administration of 5000 IU of α-tocopherol orally. We found that horses with eNAD metabolized α-tocopherol more rapidly than unaffected horses. These results support the current hypothesis that the etiology of eNAD involves a genetic defect leading to abnormal metabolism of vitamin E.

    How does this research benefit horses?
    We have developed an assay to measure concentrations of vitE isoforms and metabolites in equine serum and urine samples. Preliminary results from this study indicate that this could potentially serve as an antemortem diagnostic test for eNAD; however, we will first validate these findings in a larger study population.

    This research is in press with the Journal of Veterinary Diagnostic Investigation.

Genetics

  • Investigating a genetic risk factor for eye and skin cancer in horses
  • Investigators:
    Rebecca R. Bellone PhD
    Mary Lassaline DVM, PhD, DACVO
    Christopher M Reilly MAS, DVM, DACVP
    Tammy Miller Michau DVM, MS, MSpVM, DACVO
    Jiayin Liu
    Savanna Vig DVM
    Moriel Singer-Berk BS

     

    Squamous cell carcinoma (SCC) is the most common cancer of eyes and the second most common tumor of the horse overall. A change in the DNA that also leads to a change in the gene product (missense mutation) in damage-specific DNA-binding protein 2 (DDB2, c.1012 C>T, p.Thr338Met) was previously found to be strongly associated with SCC in Haflingers. This study determined that this same mutation explains 76% of all ocular cases of the Haflinger and Belgian breeds. This variant also explained ocular SCC in the only reported case from the Rocky Mountain Horse breed. This variant was not associated with ocular SCC in the Arabian, Appaloosa, or Percheron breeds, or with oral or urogenital SCC of any breed investigated (P>0.05). Finally, functional analysis using recombinant DDB2 protein further supports that this variant (p.Thr338Met) is a causal genetic risk factor, as the protein with methionine at amino acid 338 is unable to bind to UV damaged DNA.

    How does this research benefit horses?
    This study confirmed that the DDB2 mutation is a risk factor specifically for ocular SCC in multiple breeds of horses (Haflinger, Belgian, and Rocky Mountain Horses, but not Appaloosa and Arabian). This further supports the use of this mutation as a breed specific DNA test to inform mating decisions and management practices, thus lowering the incidence of the disease, enabling earlier detection and better prognosis. minutes.

    This research was reported in Animal Genetics 2018 Oct; 49(5):457-460, Veterinary Ophthalmology 2019 Mar; 22(2):201-205, the International Journal of Genomics 2019 Sept; 3610965, and the Equine Veterinary Journal 2020 Jan;52(1):34-40.

  • Investigating genetic risk loci for ocular squamous cell carcinoma in horses
  • Investigators:
    Rebecca R. Bellone, PhD
    Mary E. Lassaline, DVM, PhD
    Kelly E. Knickelbein, VMD

    Squamous cell carcinoma (SCC) is the most common cancer of the equine eye and the second most common tumor of the horse overall. SCC frequently originates on the third eyelid or the limbus (where the clear cornea meets the white sclera) and can grow quickly to invade the eye and adjacent structures, leading to vision loss and destruction of the eye. A mutation in a DNA ultraviolet radiation repair enzyme (DDB2) was previously identified as associated with increased risk for ocular SCC development in the Haflinger and Belgian breeds, though not all affected horses could be explained by this mutation. The objective of this study was to identify additional DNA mutations for association with ocular SCC in Haflinger and Belgian horses. Investigating the previously associated locus did not identify another variant more strongly associated with ocular SCC in Haflingers or Belgians than the one identified previously in DDB2. When taking the DDB2 risk variant genotype into account, statistical analysis supported a variant located on horse chromosome 6 as a second risk factor for ocular SCC in both Belgian and Haflinger horses (P=2.75 X10-4). This variant is a missense variant in a gene involved in the same UV damage DNA repair process as DDB2.

    How does this research benefit horses?
    This work has advanced our understanding of the genetic risk for ocular squamous cell carcinoma in horses. These data further support the use of genetic testing for the DDB2 variant in Haflinger and Belgian breeds. Testing for this variant can inform management and breeding decisions. This study also identified an additional genetic risk factor that could be utilized in DNA testing to develop the most robust ocular SCC risk assessment in these two breeds. Functional studies are needed to confirm the role of this variant in cancer.

    This research was reported in the International Journal of Genomics 2019 Sept; 3610965.

  • Identification of the mutations resulting in blood group A factors to prevent or plan for neonatal isoerythrolysis
  • Investigators:
    Robert A. Grahn, PhD
    M. Cecilia Torres Penedo, PhD

    Blood group factor incompatibilities between mares and foals require immediate postpartum care. If untreated, they can result in the death of the foal as antibodies in the mare’s colostrum attach to the foal’s red blood cells, causing them to be destroyed. This research evaluated one genomic location with a blood factor (Aa) known to result in neonatal isoerythrolysis (NI). Twenty-one horses were genotyped across the genomic region associated with blood factor A, including obligate blood group factor Aa homozygous horses. These data did not identify a chromosomal segment segregating with the NI associated blood group factor Aa. These data prompted the complete genome sequencing of eight horses with known blood types. Five were type Aa, three were not Aa. The horse reference genome was included in the analysis and is also not type Aa. Again, no causal variants were identified in the region containing the A blood group. Evaluation of the remainder of the genome did not reveal any significant genetic variants in genes associated with cell surface proteins and carbohydrates. It is likely that the surface protein modification resulting in the Aa blood factor is caused by a gene at another location within the genome, but additional samples are required to refine the number of possible causal variants.

    How does this research benefit horses?
    The goal of this project was to identify causative mutations for the horse blood group A factors. As the majority of NI cases involve blood factor Aa incompatibilities, knowledge of stallion and mare genotypes at the A system has the potential to avoid mating, or minimally, anticipate and prepare for postpartum care required for NI foal survival. Identification of DNA variants associated with blood group factors involved in NI would also eliminate the current dependency on antibody reagents for serological typing. Production of reagents has virtually ceased worldwide, and future typing is limited to available stocks. The genome sequence data generated in this study has formed the basis of continued research efforts to identify the causal mutation as additional horses with known blood types are sequenced.

  • Finding the genetic mutation for equine neuroaxonal dystrophy
  • Investigator:
    Carrie J. Finno, DVM, PhD, DACVIM

    Equine neuroaxonal dystrophy (eNAD) is a devastating neurological condition that develops during the first year of life in genetically predisposed foals maintained on a vitamin E deficient diet. Affected horses suffer from incoordination, preventing their use as riding animals. Our previous study identified possible genetic regions of interest associated with eNAD. We then used whole-genome sequencing to find possible underlying mutations in these regions in a small number of eNAD-affected and unaffected horses. This study sought to validate these potential mutations in additional horses, particularly in the candidate gene, CYP4F2, which encodes the protein responsible for metabolizing vitamin E. Forty variants identified as possible candidates for eNAD were excluded. While no structural variants or segregating SNPs associated with eNAD/EDM were identified in CYP4F2, the gene demonstrated significantly increased expression (~1.63x, P=0.02) in liver from eNAD-affected horses.

    How does this research benefit horses?
    We have identified increased expression of CYP4F2, which encodes the major protein responsible for vitamin E metabolism in the liver, in eNAD-affected horses. In 2016, with CEH support, we had identified altered metabolism of α-tocopherol, the major isoform of vitamin E, in eNAD-affected horses. Therefore, increased metabolism of α-tocopherol, with upregulation of CYP4F2, is strongly associated with the eNAD phenotype. This supports the current recommendations for high dose vitamin E supplementation in genetically susceptible herds and further refines the search for an underlying genetic variant.

    This research was reported in Genes 2020 Jan 10;11(1):82.

  • Whole-genome sequencing to identify inherited genetic alterations associated with melanoma in graying Connemara ponies
  • Investigators:
    Alain P. Theon, DVM, MS, PhD
    Carrie J. Finno, DVM, PhD, DACVIM

    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. This study was designed to identify new actionable genetic targets for prevention and development of targeted treatment by comparing the whole genomes of graying Connemara Ponies with melanoma and without melanoma. When comparing the early onset cases to either the unaffected cohort alone or the unaffected cohort plus the late onset cohort, a large associated region on chromosome 1 was identified. This region contained a tumor-suppressing gene that is abnormal in affected ponies when compared to unaffected ponies. This is a significant finding because the presence of abnormal tumor suppressor genes is commonly found in all cancer cells. Because mutations of tumor-suppressor encoding genes are common genetic abnormalities associated with inherited cancers, the presence of the abnormal gene we identified is consistent with our hypothesis that early severe onset of melanoma in graying Connemara ponies is associated with a key genetic alteration.

    How does this research benefit horses?
    Horses with greying 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, greying ponies that develop melanoma early in life are at high risk of dying of disease due to faster evolution and earlier metastatic spread. This represents 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. This genetic region will be further validated, with the intent of providing a genetic test in the future for increased melanoma susceptibility in gray horses and ponies.

  • Finding the genetic mutation for juvenile idiopathic epilepsy in Arabian horses
  • Investigators:
    Monica Aleman, MVZ, PhD, DACVIM
    Carrie J. Finno, DVM, PhD, DACVIM

    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. The disorder is inherited, and our previous study 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. This study sought to validate these potential mutations in a large number of horses. Of the 27 variants genotyped across the ~5 Mb region on chromosome 1 (chr1), only two were associated with the JIE phenotype in the full subset of horses. These variants, in addition to the original GWAS variants, further narrowed the associated region to a 250 kb region on chr1. This associated region only included one annotated gene in the horse, mannose-binding lectin 2 (MBL2), a gene solely expressed in the liver and involved in the innate immune defense. However, none of the variants were located within this gene. There was no differential expression of hepatic MBL2 in n=1 JIE-affected Arabian vs. n=5 unaffected horses. MBL2 is not expressed in brain tissue from any species. Additionally, no associated variants were identified in the two flanking candidate genes, PCDH15 and ZWINT.

    How does this research benefit horses?
    We have confirmed and further narrowed the region of association for JIE onto chr1 and excluded the only protein-coding gene within this region. Next steps include using data generated by the functional annotation of the equine genome project to search for possible mutations that alter gene regulation. The ultimate goal is to develop a genetic test for JIE that breeders and veterinarians can use to prevent future cases.

    This research was reported in the Journal of Veterinary Internal Medicine 2018 Jan-Feb; 32(1):465-468.

  • Investigating the genetic cause of persistent hypocalcemia in Thoroughbred horses
  • Investigators:
    K. Gary Magdesian, DVM, DACVIM, ACVECC, ACVCP
    Carrie J. Finno, DVM, PhD, DACVIM

    A syndrome of idiopathic hypocalcemia has been described in Thoroughbreds foals since 1997 and appears to be inherited. All identified cases have been fatal, and a genetic test is necessary to prevent future cases. This study looked for genetic mutations in five genes that have been associated with inherited hypocalcemia in humans: calcium-sensing receptor (CASR), G protein subunit alpha 11 (GNA11), parathyroid hormone (PTH), glial cells missing transcription factor 2 (GCM2) and transient receptor potential channel melastatin 6 (TRPM6). Pedigree analysis revealed an autosomal mode of inheritance for idiopathic hypocalcemia in Thoroughbred foals. After evaluating whole-genome data and performing subsequent analysis, no variants were associated with the phenotype in the horses tested. Therefore, these five candidate genes are unlikely to be implicated in functional hypoparathyroidism of Thoroughbred foals.

    How does this research benefit horses?
    Idiopathic hypocalcemia in Thoroughbred foals has a 100% mortality rate. Discovery of a genetic mutation associated with this disease would allow for testing of breeding horses for the mutation, with subsequent avoidance of mating of carriers to prevent any future affected foals. As we have excluded the most likely candidate genes, the next step is to investigate the entire genome to attempt to identify a likely causal mutation.

Immunology

  • Vaccination of horses against rabies: Do we really need to revaccinate every year?
  • Investigators:
    W. David Wilson, DVMS, MS
    Johanna L. Watson, DVM, PhD
    Judy E. Edman, BS

    Rabies is an AAEP-designated core disease against which all horses in North America should be vaccinated. The recommended revaccination interval for horses is 1 year, whereas the same rabies vaccines are typically administered at 3-year intervals in dogs and cats. The aim of this study was to determine how long “protective” titers persist after booster vaccination of previously primed horses to determine whether an extended revaccination interval is feasible. In phase 1, 48 horses with an undocumented vaccination history were vaccinated against rabies. Blood samples were collected prior to vaccination, 3 to 7 weeks later, and at 6-month intervals for 2 to 3 years. Serum rabies virus– neutralizing antibody (RVNA) values were measured using the RFFI test. A RVNA value of ≥ 0.5 U/mL was used to define a predicted protective immune response. A protective RVNA value (≥ 0.5 U/mL) was maintained for 2 to 3 years in all horses inferred to have been previously vaccinated. In phase 2, sampling continued on 11 of the horses for as long as they remained available. All 11 maintained “protective” antibody levels at their last sampling time, 9.5 to 13 years after receiving their last rabies booster.

    The prolonged persistence of protective rabies antibody titers after revaccination of previously primed horses strongly suggests that an extended revaccination interval of 3 years or more is justified, particularly for horses that react adversely to rabies vaccines. In these horses, strategic revaccination based on measured serum RVNA titers is rational.

    How does this research benefit horses?
    Designation by the AAEP of rabies as a core vaccine for annual administration to horses has resulted in a substantial increase in the number of horses that receive this vaccine. The unintended negative impacts have been an increased expense to horse owners and an increase in the number of horses that experience adverse reactions to rabies vaccines. By showing that continual protection can be accomplished by administering rabies “boosters” at an interval of more than one year, this study identifies options for mitigating these unintended impacts, providing clear benefits to horse owners and to horses, particularly those that react adversely to rabies vaccines.

    This research was reported in the Journal of the American Veterinary Medical Association 2016 Aug; 249(4): 411-8.

  • Serological response to equine influenza virus following boost vaccination of adult horses using different commercially available killed vaccines
  • Genetic Investigation of Juvenile Idiopathic Epilepsy in Arabian Foals

    Investigators:

    Nicola Pusterla, DVM, PhD, DACVIM, DAVDC-Equine
    W. David Wilson, BVMS, MS, DACVIM
    Bruno Karam, DVM

    The best achieved protection against equine influenza virus (EIV) is based on AAEP vaccine recommendations that horses ages 4-6 months should be initially prime vaccinated with a series of 3 doses given 4-6 weeks apart, followed by boost vaccination every 6 to 12 months. Most veterinarians and owners use EIV vaccines interchangeably, despite differences in EIV strains, viral mass and adjuvant in commercially available vaccines. Our hypothesis was that a switch in EIV vaccine manufacturer requires a 2-dose vaccine series to trigger an antibody response to EIV that is similar, if not superior, to what a boost vaccination with the vaccine from the original manufacturer would induce.

    We enrolled 65 healthy, adult horses previously vaccinated bi-annually against EIV using the Fluvac Innovator vaccine (Zoetis). The horses were randomly assigned to one of three groups: 1- and 2-dose series with Fluvac Innovator, 1- and 2-dose series with Prestige II (Merck Animal Health) and 1- and 2-dose series with Vetera EIV (Boehringer Ingelheim). Five horses served as environmental sentinels. Whole blood was collected on the first day of vaccine administration and at 30, 60, 90 and 180 days. Serum samples were tested for antibody titers to EIV by hemagglutination inhibition assay against a contemporary EIV Clade I (Kentucky 2014) and Clade II (Richmond 2007) Florida sublineage strains. The geometric mean of the titers was compared amongst the EIV vaccine groups.

    For all vaccine groups, there was a significant difference between antibody responses pre- and post-initial vaccine administration. There was no statistical difference after day 30 between any booster and single dose vaccine groups.

    How does this research benefit horses?
    Equine influenza is one of the most devastating respiratory viruses. This study shows that, in previously immunized horses, a switch in vaccine manufacturer does not require a booster series in order to achieve superior antibody responses to Florida sublineage clade 1 and 2 EIV. This information will be relevant to educate veterinarians and owners on EIV vaccine protocols.

Medicine and Infectious Disease

  • Using microbiota analyses to understand and track a foal’s gastrointestinal health
  • Investigators:
    Michael J. Mienaltowski, DVM, PhD
    Elizabeth A. Maga, PhD
    K. Gary Magdesian, DVM, DACVIM, DACVECC, DACVCP
    Ubaldo De La Torre

    Foals need properly established gut microbiota to develop correctly and be healthy in their first years of life. Microorganisms associated with contact with the mare and consumption of colostrum and milk are the first to colonize a foal’s GI system. As the foal’s diet shifts from milk to solid food, so do the bacterial populations of the gut microbiota, enabling the foal to better obtain energy and nutrients from new feed sources. Disruptions to the microbiota, such as those that occur with diarrhea, can negatively affect foal health.

    Fecal samples from foals were analyzed to detect differences in gut establishment by age, changes in diet, and diarrhea status. Data showed that the composition of bacterial populations in foals followed an age-dependent pattern linked to changes in diet. Differences in microbial population patterns between healthy foals and those with diarrhea, as well as differences associated with management styles at individual facilities, were also observed.

    Samples from foals during the first week of life showed a high abundance of Proteobacteria. From day seven through weaning, the study population shifted to an abundance of Firmicutes, the most abundant bacterial phylum seen in horses overall. As foals in the study were progressively exposed to solid foods, the abundance of Bacteroidetes also began to rise. Samples from foals with diarrhea showed an increased abundance of Enterobacteriaceae, a family of bacteria that includes Salmonella and E. coli.

    How does this research benefit horses?
    Data from this study contributes to an improved understanding of the microbiota present in growing neonates and the role of the mare’s milk in microbiota establishment. This could be used as a simple diagnostic tool utilizing fecal bacteria to provide veterinarians with information about foals’ GI bacterial populations as they grow. A future goal would be for veterinarians to be able to suggest formulations of probiotics or screen for optimal gut microorganisms from healthy animals for transfer to sick animals. Further research could enable veterinarians to visit a farm that has horses with diarrhea and not just treat the horses, but combine genomics and epidemiology to troubleshoot a management issue on the farm to eliminate diarrhea, thus saving time, money, and improving equine welfare.

    This research was reported in PLoS One 2019 April; 14(4): e0216211.

  • Investigating the epidemiology of equine influenza virus in the USA from 2006 to 2016
  • Investigators:
    Beatriz Martinez Lopez, DVM, MPVM, PhD
    Nicola Pusterla, DVM, Ph.D., DACVIM
    Samantha Barnum, M.S.
    Kyuyoung Lee, DVM, MPVM

    Equine influenza virus (EIV) is a highly contagious respiratory virus responsible for outbreaks of equine influenza (EI) worldwide. Today, only EIV of the Clade I and Clade II Florida sub-lineage circulate amongst equids. Since the virus continuously evolves and alters its morphology to escape the immune system of the host, it is important to determine how the structure of proteins responsible for the induction of a solid immunity change over time to best evaluate vaccine effectiveness and provide recommendations to ensure that contemporary vaccines contain epidemiologically relevant EIV strains.

    This study investigated temporal and spatial molecular characteristics of H3N8 EIV isolates collected from horses in the USA from 2006 to 2016. Phylogenetic analysis showed that all current EIVs H3N8 were connected by two main trunk lineages, Florida Clade I and Florida Clade II. EIVs in Florida Clade I have endemically spread through North and South America, with a few spillovers into Japan, South Korea, Malaysia and Sweden. EIVs strains in Florida Clade II have mainly circulated among European countries and expanded to China, Mongolia and India. Among the study samples, 57 of the 58 EIVs were sublineages of Florida Clade I. A sample from a horse imported from Germany and quarantined in 2012 was a Florida Clade II virus.

    Data showed 11 fixed amino acid substitutions in the HA gene of current EIVs H3N8 Florida Clade I in the US compared to the OIE-recommended EIV Florida Clade I vaccine strain (Ohio/03) (HA1 subunit: 8 amino acids. HA2 subunit: 3 amino acids) and high selection pressure in the receptor binding site in HA1 subunits.

    How does this research benefit horses?
    Equine influenza is one of the most devastating respiratory viruses. This unique data set and analysis allow us to better understand why EIV cases in the US are becoming more prevalent today, even with the vaccine program combined with the implementation of quarantine measures for imported equids. Our study shows that introduction of novel strains of EIV or EIV clades 2 have not been observed in the US. However, continuous accumulation of fixed amino acid substitutions in the HA protein against the most recent EIV vaccine strains and their circulation could influence the effectiveness of commercially available EIV vaccines in the US. Updating EIV vaccine strains would help to improve the vaccine program against current EIVs in the US.

  • Investigation of the use of two different swabs for the detection of microorganisms in nasal secretions of healthy horses
  • Investigators:
    Nicola Pusterla, DVM, Ph.D., DACVIM
    Samantha Barnum, MS

    Quantitative PCR (qPCR) is the diagnostic tool of choice for detection of respiratory microorganisms based on high sensitivity and specificity, quick turnaround- time, cost-effectiveness and panel testing. Given the clinical relevance of respiratory pathogens, it is important to use the best collection system that provides the highest detection yield in nasal secretions. While standard rayon-tipped swabs are routinely used for the collection of nasal secretions in horses, flocked swabs have demonstrated superior performance for the detection of human respiratory, vaginal and gastrointestinal pathogens.

    The study objective was to determine if there is a difference in performance between flocked swabs and standard rayon swabs in detecting nasal carriage of ubiquitous viruses and bacteria among healthy adult horses. Thirty-one horses were swabbed with a flocked swab and rayon swab, processed for nucleic acid purification and assayed for target genes including the eukaryotic equine glyceraldehyde-3-phosphate dehydrogenase (eGAPDH) gene, the glycoprotein B (gB) gene of EHV-2 and EHV-5, the universal bacterial 16S rRNA gene and the ITS gene of Streptococcus equi subspecies zooepidemicus.

    Rayon swabs yielded significantly higher DNA and RNA concentrations than flocked swabs. There were no significant differences between the two swab types for the target genes. All swabs had detectable eGAPDH at the genomic DNA and complimentary DNA level and bacterial 16S rRNA genes. Seven and 14 horses tested positive in both swabs for EHV-2 and EHV-5, respectively. An additional 11 horses tested positive only in one of the two swabs for EHV-2 or EHV-5. Five horses tested positive in both swabs for S. zooepidemicus, while an additional nine horses only tested positive in one of the two swabs.

    How does this research benefit horses?
    Due to the economic impact of contagious respiratory pathogens, it is relevant that viruses and bacteria be detected with the most accurate and reliable protocol. Flocked swabs have been shown to be more accurate in the detection of human respiratory microorganisms. However, in this study, sampling with flocked swabs did not statistically improve the detection of selected target viruses and bacteria by qPCR. Some variability in the molecular detection of viruses and bacteria was observed between the two types of swabs collected from the same horse. The differences in rate of detection of microorganisms by qPCR between right and left nasal passages from the same horse highlight the importance of proper sampling and the need to standardize sampling procedures.

  • Investigating the genetic makeup of equine herpesvirus type 5 in healthy and sick horses
  • Investigators:
    Nicola Pusterla, DVM, Ph.D., DACVIM
    Emir Hodzic, DVM, MSci, PhD

    Equine herpesvirus-5 (EHV-5) is widespread in horse populations and is optimally adapted to its host, meaning that significant clinical expression of infection is rare. However, growing evidence supports the involvement of EHV- 5 with upper airway infections and the more devastating equine multinodular pulmonary fibrosis (EMPF), a sporadic, progressive and fibrosing interstitial lung disease of adult horses. The objective of this study was to determine the genetic diversity of EHV-5 in biological samples of healthy horses, horses with upper airway disease and horses with EMPF.

    Biological samples (nasal secretions, whole blood, bronchoalveolar lavage fluid and lung biopsies) were taken from 5 healthy horses, 5 horses with upper airway infection and 5 horses with EMPF. The samples were processed for next generation sequencing targeting the glycoprotein B (gB) gene of EHV-5. Genetic diversity of EHV-5 was determined for each sample type and compared amongst the various horse groups.

    Healthy horses and horses with upper airway infection displayed a greater genetic diversity compared to horses with EMPF. Horses with EMPF had significantly less diversity in EHV-5 partial gB gene sequences (median 1) compared to healthy horses (median 8) and horses with upper airway infection (median 9) in nasal secretions. Healthy horses and horses with respiratory signs tested negative for EHV-5 in whole blood, bronchoalveolar lavage fluid and lung biopsies. However, individual horses with EMPF displayed similar EHV-5 strains independent of the biological sample type.

    How does this research benefit horses?
    EHV-5 is a poorly characterized and fairly enigmatic gamma-herpesvirus. It is important to understand the viral dynamics of infections for EHV-5 in order to characterize the pathophysiology of this virus and develop better preventive and therapeutic protocols. Similar to human herpesvirus-4, the genetic diversity of EHV-5 appears to contribute to the clinical manifestation. While healthy horses and horses with respiratory signs are infected with multiple strains of EHV-5, cases of EMPF are generally infected with only one strain.

  • Investigation of the cellular response to Sarcocystis neurona in blood and cerebrospinal fluid of horses with and without equine protozoal myeloencephalitis
  • Investigators:
    Nicola Pusterla, DVM, PhD, DACVIM, DAVDCE
    Patricia Conrad, DVM, PhD
    Samantha Mapes, MS
    Andrea Packham, MS

    Equine protozoal myeloencephalitis (EPM) is a commonly diagnosed, economically costly neurologic condition, attributed to infection of the central nervous system with Sarcocystis neurona and/or Neospora hughesi. Laboratory diagnosis relies on the detection of antibodies specific to S. neurona and/or N. hughesi in serum and/or cerebrospinal fluid (CSF). Interferon-γ (INF-γ) is a cytokine involved in the host immune defense. This study aimed to evaluate the INF-γ response in horses with EPM.

    The INF-γ gene expression in blood and CSF from horses with laboratory confirmation of EPM (serum/CSF ratio ≤ 64; n=18) was compared to the IFN-γ response of horses with no evidence of EPM infection (serum/CSF ratio > 64; n=24). INF-γ gene expression against the positive mitogen control and S. neurona was measured in almost all blood samples from horses with and without evidence of S. neurona infection. Due to the low number of nucleated cells in the CSF samples, only 3 and 5 CSF samples from horses with and without evidence of S. neurona infection, respectively, had detectable INF-γ gene expression poststimulation with the positive mitogen. The upregulation of INF-γ gene expression in blood from horses with and without EPM following stimulation with S. neurona was similar and ranged from 2.4-19.3 (median 7.3) and 3.0 to 22.4 (median 8.3), respectively. Despite the small number of CSF samples with detectable INF-γ gene expression following stimulation with the positive mitogen and S. neurona, there was no upregulation of INF-γ gene expression against S. neurona in five horses without EPM, while three horses with suspected EPM showed a 1.3-4.1 upregulation of INF-γ gene expression against S. neurona.

    How does this research benefit horses?
    This study explored an untouched diagnostic field of EPM, which is the cellular response to S. neurona of mononuclear cells in blood and CSF from horses with and without EPM. While accurate diagnostic modalities are urgently needed in order to prevent overdiagnosing and underdiagnosing relevant infectious diseases, measuring cellular responses in the CSF of horses with EPM failed to consistently detect an upregulation of INF-γ gene expression secondary to S. neurona.

  • Development of a test that can identify the specific strain of multiple parasites thought to be associated with the neurological disease equine protozoal myeloencephalitis
  • Investigators:
    Jeroen P. Saeij, MS, PhD
    Nicola Pusterla, DVM, PhD, DACVIM, DAVDCE
    Patricia Conrad, DVM, PhD
    Monica Aleman, DVM, PhD

    Equine protozoal myeloencephalitis (EPM) is a debilitating neurologic disease associated with Sarcocystis neurona, and Neospora hughesi infection. Although many horses have antibodies against S. neurona (seroposivity), few develop EPM. Evidence suggests that co-infection with similar protozoan parasites, such as Toxoplasma gondii, is associated with a higher risk for developing EPM.

    Diagnosis of EPM is challenging and relies on an indirect immunofluorescence antibody test (IFAT) to detect antibodies to the parasites. This project sought to develop an indirect ELISA test using immunogenic and polymorphic peptides that would distinguish antibodies in horse sera against N. hughesi, N. caninum, S. neurona, S. fayeri, and different Toxoplasma strains.

    Serotyping is based on the exclusive reaction against the peptide from the genotype of the infecting parasite, but not against the corresponding peptide of other parasites. Seventeen new candidate peptides from dense granule (GRA) secreted proteins, rhoptry (ROP) and surface related antigens (SRSs) proteins were selected, synthesized and tested on a panel of horse serum including EPM cases, healthy animals, and experimentally infected animals. High background and cross-reactivity were noted, even in the negative samples, likely due to the “sticky” nature of horse serum, making analysis difficult. Further testing was performed with the best serum samples that had antibodies against one or more of the parasites. Using a standard indirect ELISA protocol, the majority of the peptides did not react, except a few from the GRA6 protein. This could indicate that their antigenicity is not enough or that the technical conditions for the ELISA need to be improved.

    How does this research benefit horses?
    Many horses are infected with Sarcocystis neurona, but only a small subset develop EPM. The ability to determine which parasite species/strains are present in ill horses is necessary to correlate infection specifics with disease outcomes and develop effective treatments. The information obtained from this study can be used to design serological tests to be able to identify the protozoan parasite/s that commonly infect horses and establish an etiological relation between EPM clinical signs and specific parasites.

Orthopedics and Lameness

  • Determining the mechanical properties of different arena surface types
  • Investigators:
    Susan Stover, DVM, PhD, DACVS
    Christina Rohlf, BS
    Tanya Garcia-Nolen, MS
    Shrinivasa Upadhyaya, PhD
    Sarah le Jeune, DVM, DACVS,CVA, DACVSMR, Cert Vet Chiro
    Sara Thomasy, DVM, Ph.D., DACVO

    This project was motivated by the need to understand the mechanical properties of dirt and synthetic arena surfaces in order to characterize the risk of injury to equine athletes performing on these surfaces. Previous studies within the laboratory indicated that synthetic surfaces exhibit a wide range of mechanical properties depending on factors such as surface preparation or composition, which may result in some synthetic surfaces having the same, or greater, injury risk than dirt surfaces. Although this laboratory has already developed validated equipment to test vertical impact via the arena impact device (AID), no validated equipment has been developed to measure shear surface properties. This study aimed to develop this shear testing equipment and quantify both shear and vertical impact on a wide range of dirt and synthetic arena surfaces.

    The data showed that vertical ground reaction forces did not depend on surface type (dirt, synthetic) for freshly harrowed cushion. However, dirt surfaces exhibited higher vertical ground reaction forces (and may have a greater risk of injury) than synthetic surfaces for a previously trampled surface. Dirt surfaces exhibited higher surface compaction (cohesion) than synthetic surfaces and synthetic surfaces exhibited higher resistance to slide (angle of internal friction) than dirt surfaces. Synthetic surfaces exhibited higher cushion depth and moisture content than dirt surfaces, while dirt surfaces exhibited higher temperatures than synthetic surfaces. Correlations were found between surface management factors (cushion depth, moisture content, and temperature), shear, and vertical impact surface properties, indicating that surface mechanical properties can be adjusted by altering surface management protocols.

    How does this research benefit horses?
    This research shows that dirt and synthetic arena surfaces vary in their mechanical properties. However, knowing that an arena has a dirt or synthetic surface is not enough information to understand how the surface behaves. Water, harrowing, and temperature affect the behavior of surfaces. Consistent surface properties would allow adaptation of the equine musculoskeletal system to the behavior of the surface, which would reduce the risk of injury.

  • How does horse forelimb motion during a jump relate to arena surface properties?
  • Investigators:
    Susan Stover, DVM, PhD, DACVS
    Christina Rohlf, BS
    Tanya Garcia-Nolen, MS
    Sarah le Jeune, DVM, DACVS,CVA, DACVSMR, Cert Vet Chiro

    Show jumping horses commonly injure tendons and ligaments in the lower limb, especially the suspensory ligament and superficial and deep digital flexor tendons as the result of large, repetitive loads that stretch the tendons and ligaments. The amount of stretching (strain) is affected by the amount and rate of fetlock joint extension. As footing can play an important role in fetlock joint extension, this study quantified relationships between surface properties and fetlock motion of show jumping horses to identify surface property variables that reduce the risk of injury. Fetlock motion data was collected from four horses jumping three times over a 1.1 meter parallel oxer on each of 12 surfaces (five dirt and seven synthetic). Landing exhibited higher fetlock extension (4.8⁰), rate of extension (668⁰/s) and flexion (579⁰/s), indicating that landing may put more strain on soft tissues that support the fetlock than takeoff. Fetlock angle was also higher when the instrumented limb was the leading leg (4.6⁰), indicating that the leading leg may support more load and help stabilize the horse as the hindlimb lands. The risk of injury may be highest for the leading leg at jump landing.

    Rate of fetlock flexion may represent the rebound energy available to the limb after contacting the surface. This was significantly higher in the dirt surface for takeoff (668⁰/s) and landing (273⁰/s), possibly because less energy is lost to soil movement. Rate of fetlock extension represents the extension rate of tendons crossing the fetlock joint and is maximized when hoof slide is reduced. High rates of fetlock extension may also lead to increased risk of injury.

    How does this research benefit horses?
    Arena surface management is important. The ‘feel’ of an arena surface to the horse varies markedly with the depth of the cushion (harrowing) and water content (e.g., sprinkling). As soon as a freshly harrowed surface is impacted by hooves, the surface becomes harder.

  • Effect of arena surface properties on hindlimb fetlock angle in show jumping horses
  • Investigators:
    Susan Stover, DVM, PhD, DACVS
    Lyndsey Marsh

    The objective of this study was to determine if hindlimb fetlock motion during takeoff and landing from a jump differs between jumping on a dirt or a synthetic arena surface. There were no significant differences in fetlock motion between dirt and synthetic surfaces during take-off for the jump. The rate of fetlock extension was greater for dirt surfaces during landing after the jump. However, horse jumping style affected fetlock motion during take-off and landing. Leading limb and hoof position at surface contact reflect jumping style. Jumping style was relatively consistent within horses but varied among horses. When jumping style variability was considered, there were no statistical differences in hindlimb fetlock motion during takeoff and landing between jumping on a dirt or synthetic surface for the surfaces studied.

    How does this research benefit horses?
    Lower limb injuries are prevalent in show jumping horses. While this research has suggested that there is not a significant difference between dirt and synthetic footing in the surfaces studied for hind limb fetlock motion in show jumping horses during takeoff and landing, it did provide insight that horse jumping style may play a significant role in fetlock motion. This study provides a window of opportunity into another avenue for injury prevention – training to influence jumping style.

  • The effect of horseshoe length on hoof wall changes that could lead to abnormal hoof conformation associated with injuries
  • Investigators:
    Vanessa Dahl, MS
    Susan M. Stover, DVM, PhD, DACVS
    Tanya C. Garcia-Nolen, MS

    Abnormal hoof conformation is related to muscle pain and lower limb lameness in performance horses, and fetlock injuries in racehorses. While horseshoes protect the hoof from excessive wear, they restrict other normal hoof functions. Therefore, shoeing techniques have the potential to affect hoof growth and cause abnormal hoof conformations, which in turn puts affected horses at risk for lameness and injury. We hypothesized that horseshoes mismatched in length to hoof size adversely affect hoof wall flexibility and cause hoof wall distortions that promote lameness and injury.

    The effect of horseshoe length on hoof wall flexibility and distortion, and on fetlock extension (an index of fetlock injury), was investigated using cadaveric forelimbs from eight horses. Forelimbs were fitted with shoes of different lengths and loaded in a mechanical testing system to replicate maximum limb load at the canter. Each limb was tested 5 times to compare the following conditions: unshod, with a short shoe, full shoe, long shoe, and again unshod for comparison to initial conditions.

    How does this research benefit horses?
    The data showed that fetlock extension decreased with the long shoe. In addition, application of a horseshoe to the hoof increased hoof wall strains and changed the way the hoof wall deformed. Horseshoes that extend to the back of the hoof (as opposed to shorter shoes) should reduce the likelihood of fetlock and flexor tendon injuries. Development of less rigid horseshoes may assist in preventing the development of abnormal hoof conformations.

  • The effect of horseshoe length on hoof growth, hoof horn tubule orientation and hoof wall angles
  • Investigators:
    Vanessa Dahl, MS
    Susan M. Stover, DVM, PhD, DACVS
    Tanya C. Garcia-Nolen, MS
    Ellen R. Singer, BA, DVM, DVSc, DACVS, DECVS, MRCVS
    David A. Hawkins, PhD

    Underrun heel hoof conformation is associated with muscle pain and lower limb lameness in performance horses, and fetlock injuries in racehorses. While horseshoes protect the hoof from excessive wear, they restrict other normal hoof functions, potentially affecting hoof growth and causing underrun heel hoof conformation. We hypothesized that horseshoes short in length relative to hoof size adversely affect hoof wall growth and cause hoof wall distortions that promote underrun heel hoof conformation. The effect of horseshoe length on hoof wall growth and distortion was studied in 50 Thoroughbreds over six shoeing intervals, while shod with a normal length horseshoe for three intervals and a short length horseshoe for three intervals. Hoof wall angles, tubule orientation, and growth were measured before and after each shoeing interval.

    Trends of decreasing toe angle and decreasing heel angle were found with a shorter shoe but seemed more associated with the time period (first half or second half), with no consistent results. Distal toe angles decreased for the short shoe on the left limb but increased for the right limb. No changes were noted for the angles along the quarter marks; however, a decrease in angle for the short shoe was found for the heels marks. Short shoe had a decrease in heel hoof length, quarter hoof length, and toe hoof length when compared to the full-length treatment.

    Hoof conformation was not consistently different at the end of 18-week shoeing periods between horses shod with a short horseshoe compared with horses shod with a long horseshoe.

    How does this research benefit horses?
    Racehorses with a long toe – underrun heel hoof conformation are at higher than normal risk for obtaining an unrecoverable fetlock injury. This study looked at one possible explanation for developing this abnormal hoof conformation. The length of the horseshoe does not appear to predispose sedentary horses to developing a long toe – underrun heel hoof conformation. Other factors, including exercise with differences in horseshoe length, should be examined.

  • How do horseshoe traction features alter hoof grip on performance footings?
  • Investigators:
    Christina Rohlf
    Tara Doherty
    Susan M. 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.

    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 that directly affect the risk for leg injury in sport horses. Our 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.

  • Improving visualization and assessment of cartilage in the horse foot
  • Investigators:
    Derek D. Cissell, VMD, PhD, DACVR
    Britton Nixon,DVM
    Mathieu Spriet, DVM, MS, DACVR, DECVDI

    Magnetic resonance imaging (MRI) is the gold standard imaging modality for non-invasive evaluation of joint injuries and damage to cartilage in people and animals. Our prior experience suggested that properties of cartilage in the joints of the horse foot cause it to appear different from cartilage in human joints. This research measured the properties of cartilage in the horse foot that influence its appearance on MRI. We further sought to develop a novel MRI protocol to specifically visualize horse cartilage toward improved detection of cartilage damage. The properties of healthy cartilage in the horse foot differed significantly between different joint regions and also differed from reported values for healthy adult human cartilage. Based on our findings, we synthesized an artificial gel with MRI properties identical to horse cartilage. Using the gel, we tested twelve different MRI protocols and identified the best protocol for visualizing horse cartilage.

    How does this research benefit horses?
    This study lead to the first MRI protocols developed specifically for imaging of cartilage in the horse foot. Protocols customized for MRI of the horse foot will improve visualization of cartilage in the foot toward better detection of cartilage changes associated with injury.

  • Use of Positron Emission Tomography as a new imaging modality for laminitis
  • Investigators:
    Mathieu Spriet, DVM, MS, DACVR, DECVDI
    Pablo Espinosa, DVM
    Larry Galuppo, DVM, DACVS
    Gary Magdesian, DVM, DACVIM

    Laminitis is an extremely debilitating and often fatal disease in horses. Conventional imaging modalities cannot identify the early stages of the disease and provide only little information on the activity of the disease. We hypothesized that Positron Emission Tomography (PET), an advanced imaging modality recently applied to the horse at UC Davis, will identify early changes of laminitis and improve the assessment of the progression of the disease. In this study, we imaged seven normal horses and seven horses with laminitis with the PET scanner under general anesthesia. The results showed that the feet of all normal horses had the same appearance on PET whereas all feet with laminitis appeared different from normal feet. The most common abnormality in feet with active laminitis was evidence of changes in the front of the hoof wall, as expected. Feet with chronic laminitis showed a different appearance with normalization of the front of the hoof wall, but persistent changes in the coronary band, indicative of improper growth of the hoof.

    How does this research benefit horses?
    PET has the ability to distinguish different stages of the disease, which will help understand the progression of the disease and the response to different treatments. This will be extremely beneficial for development and assessment of new treatments for laminitis.

  • Stem cell tracking in the equine distal limb using Positron Emission Tomography
  • Investigators:
    Mathieu Spriet, DVM, MS, DACVR, DECVDI

    Assessing the fate of stem cells after their administration to a patient is critical to optimize treatments. Imaging techniques previously used in the horse for cell visualization provided only crude information regarding the localization of the cells. We hypothesized that Positron Emission Tomography (PET) will provide more accurate information regarding cell localization in the horse foot. The objectives of this study were to assess the distribution of stem cells in the horse limb after injection and assess localization of stem cells in the foot of horses with tendon injuries. Four research horses, including two with tendon lesions, were injected in an artery of the limb, under general anesthesia, with stem cells labelled with a PET radiotracer. PET images of the limb were obtained at the time of injection and up to 2 hours later. A CT scan was also performed, and the PET and CT images were combined to assess the distribution of the stem cells.

    How does this research benefit horses?
    PET confirmed that the majority of the injected stem cells remained in the limb. Stem cells were identified at the site of the tendon injuries. Stem cells had a tendency to accumulate at the tendon injury. This study confirmed that the injection of stem cell in an artery of the limb leads to accumulation of stem cells at tendon injury. This is further evidence that this injection technique can be used to treat tendon injuries, which are a common occurrence in sport horses.

  • 18F-Fluoride Positron Emission Tomography for detection of active osseous lesions in the equine distal limb.
  • Investigator:
    Mathieu Spriet, DVM, MS, DACVR, DECVDI

    Diagnostic imaging has made tremendous progress in identifying skeletal injuries in the horse over the past 15 years. However, early or subtle bone injuries remain difficult to detect. In addition, the significance of abnormalities identified with conventional imaging techniques is sometimes unknown. We hypothesized that Positron Emission Tomography (PET), an imaging technique only recently available to horses, will identify injuries not recognized using other imaging modalities and provide information regarding the significance of injuries. The objectives of this study were to validate the new imaging technique in research horses and apply it to clinical cases for which conventional imaging did not provide sufficient information to diagnose the cause of the lameness.

    The results showed that PET imaging was well tolerated by all horses and provided high quality images. PET identified injuries not recognized with other imaging modalities. These include early injury in the fetlocks of racehorses (in particular in the sesamoid bones), early injury to the bone of joints including the hock, the fetlock, the pastern and coffin joints, early injury to the navicular bone and injuries at the attachment of the suspensory ligament on the cannon bone and of small ligaments in the foot. PET also helped distinguish between active and inactive injuries, which was particularly helpful in the hock.

    How does this research benefit horses?
    PET proved to be a valuable addition to other existing imaging techniques to assess injuries of bones in horses. This is of particular interest in racehorses, where PET can identify early bone changes before they progress to catastrophic injuries. PET is also very useful for the assessment of lameness in sport horses in the foot, fetlock and hock.

  • Development of a new technique for imaging bone and soft tissue of the horse limb
  • Investigator:
    Mathieu Spriet, DVM, MS, DACVR, DECVDI
    Pablo Espinosa, DVM
    Scott Katzman, DVM, DACVS
    Larry Galuppo, DVM, DACVS

    Positron Emission Tomography (PET) is an imaging modality that has recently become available to the horse. PET consists in administering a small amount of a special marker that will accumulate at the site of injuries and be detected by the scanner. There are different markers for soft tissue or bone injuries. So far, our team has validated the use of these markers used individually. However, in many horses, soft tissue and bone lesions coexist and it is important to recognize both. The goal of this study was to see if the markers could be combined within one scan. Six horses with soft tissue and bone injuries in the foot were scanned with the two tracers independently, and then combined. Overall, the scan with the combined tracers detected the majority of the injuries identified with the individual scans. The main exception was when a soft tissue injury was in close proximity to a bone injury and could be hidden by the bone injury. Almost all bone injuries could be recognized when the markers were combined. The combination of the tracers is advantageous as it decreases the amount of time a horse needs to be anesthetized to collect the information on both soft tissue and bone injuries. Although it is important to keep in mind the limitation identified, this study validated this technique for use in horses with lameness.

    How does this research benefit horses?
    This study confirmed that combining two PET tracers in one scan could provide the information previously collected under two different scans. This technique is now commonly used at the UC Davis veterinary hospital, leading to better identification of causes of lameness in horses and helping to select treatment to improve healing and recovery.

  • Establishing the relationships between training and racing programs and bone damage and bone loss for the future prediction of fetlock fracture risk in Thoroughbred racehorses.
  • Investigators:
    Susan M. Stover, DVM, PhD, DACVS
    David P. Fyhrie, MS, PhD
    Tanya Garcia-Nolen, MS
    Sarah K. Schaffer, BS

    Bone fractures result in removal of Thoroughbred racehorses from training and racing. When fractures are catastrophic, they often result in horse death and sometimes jockey injury. During racing, the fetlock sustains the highest loads of any part of the limb and often extends beyond the joint’s physiological range of motion, making it extremely susceptible to injury. The most common fetlock injury is fracture of the proximal sesamoid bone (PSB). Evidence indicates PSB fractures in racehorses are repetitive overuse injuries. We used morphological microcomputed tomography (uCT) and histological samples to determine if grossly observed changes in the bone tissue of PSB fractures are correlated with the horse’s training history.

    Data showed that focal lesions exist in bones from the PSBs of racehorses euthanized due to unilateral biaxial PSB fracture. These focal lesions are not present in racehorses that died from other musculoskeletal injuries. There is a correlation between uCT morphological parameters and the horse’s exercise history.

    How does this research benefit horses?
    There are significant benefits from knowing the conditions that predispose racehorses to proximal sesamoid bone fracture, the most common unrecoverable fetlock injury. The study demonstrated that PSB fracture is the acute manifestation of a process that develops over time. It also documented the location, shape, and size of the preexisting abnormality that predisposes horses to catastrophic PSB fracture. There are significant differences in how horses are trained and raced that separate horses that fracture PSBs from horses that do not fracture these bones. The data collected will be used to validate a finite element model that predicts internal changes in bone density and microdamage beneath the subchondral surface of PSBs in response to horse-specific training histories.

  • How are bones that fracture different from bones that don’t fracture in racehorses with fetlock breakdown?
  • Investigators:
    Sarah Shaffer, B.S.
    Susan M. Stover, DVM, PhD, DACVS
    David P. Fyhrie, PhD

    Identification of abnormalities in the fetlock bones of racehorses that died because of a fetlock breakdown can be used to understand why some horses have a fetlock breakdown. The proximal sesamoid bones (PSBs) are bones within the fetlock whose fracture cause 45-50% of racehorse fatalities due to injury.

    Microscopic examination of fractured and non-fractured PSBs from racehorses revealed clusters of cracks that were most numerous in fractured bones, present but less numerous in non-fractured bones from racehorses that had another fetlock bone fractured, and rare in racehorses that died for reasons unrelated to fetlock breakdown. Cracks were in a consistent location in the bones. These cracks represent pre-existing microscopic damage that weaken bones and may predispose to bone fracture. There was also evidence of bone resorption to remove the microscopic cracks, which could further weaken bones until new bone has time to replace the damaged bone.

    How does this research benefit horses?
    Fracture of the proximal sesamoid bones (PSB) is the most common fracture in Thoroughbred racehorses, causing approximately half of fetlock related fatalities. This study demonstrated that there are changes leading up to fracture that can be used to identify horses at risk for fracture – before they fracture. High-risk horses could be treated until the abnormalities heal, and then return to racing. Additionally, knowledge of the presence and consistent locations of these abnormalities can be used with advanced imaging, like PET scans, to detect horses at risk for fracture. Further, data from this study will be used to identify training and racing schedules that put a horse at high risk for fetlock breakdown.

    This research was reported in the Equine Veterinary Journal 2020 May; 13291.

  • Determining the mean peak concentration of amikacin sulfate in the coffin joint during regional limb perfusion
  • Investigators:
    Isabelle Kilcoyne MVB, DACVS
    Jorge Nieto MVZ, PhD, DACVS, ACVSMR
    Julie Dechant DVM, DACVS, DACVECC
    Heather Kynch DVM, PhD,DACVCP

    Synovial sepsis of equine distal limb joints and traumatic wounds are a commonly encountered problem in equine practice. Regional limb perfusion allows the delivery of a high concentration of antibiotic to the affected region. Standardization and reduction of the tourniquet time required to perform these perfusions would allow treatment of these conditions in a more efficient manner.

    The purpose of the study was to determine the most appropriate length of time for tourniquet application during distal limb perfusion with antibiotics in order to reduce pain and collateral side effects, as well as to increase the efficiency of this procedure. Seven healthy horses underwent intravenous regional limb perfusion (IVRLP) using standing sedation with 2g amikacin sulfate diluted to 60mls using 0.9% saline in the cephalic vein of a front limb using a pneumatic tourniquet placed 10 cm proximal to accessory carpal bone. Synovial fluid was collected from the coffin joint at 5, 10, 15, 20 and 30 minutes after IVRLP. The concentration of amikacin within the joint was measured at each time point. The median peak concentration (Cmax) of amikacin and the time to median peak concentration (Tmax) within the DIP joint was determined.

    Results showed that the median peak amikacin concentration for the distal interphalangeal joint (DIP) joint was 550 (range 37-2167) μg/mL. The median time to peak concentration for the DIP joint was 15 minutes.

    How does this research benefit horses?
    Traumatic wounds in the limbs of horses are common and frequently involve synovial structures that can affect the life and performance career of the animal. Distal limb perfusion with antibiotics is a simple procedure, which allows local administration of effective levels with minimal systemic effects. This study demonstrated that tourniquet application of 15 minutes is sufficient for completion of intravenous regional limb perfusion when trying to achieve adequate synovial levels of amikacin in the coffin joint.

    This research was reported in the American Journal of Veterinary Research 2018 Mar; 79(3): 282-286.

  • Unraveling the effect(s) of osteoporosis on bones and joints of the neck of horses suffering with neck stiffness and pain
  • Investigators:
    Susan M. Stover, DVM, PhD, DACVS
    Mathieu Spriet, DVM, MS, DACVR, DECVDI
    Sheley Nola
    Brian Murphy, DVM, PhD, DACVP
    Neil Willits, PhD

    Neck stiffness and pain are common in horses that have osteoporosis and lung disease caused by breathing dust with toxic silicate particles. Affected horses may be unable to reach food on the ground and lose weight as a result. Increased understanding of the changes in the bones of the neck may lead to earlier diagnosis of horses with lung-associated osteoporosis and better management and preventive strategies to improve quality of life of affected horses.

    We hypothesized that neck pain and stiffness in horses affected with osteoporosis can be attributed to bone degeneration and arthritis in the bones and joints of the neck. The necks of horses that died due to osteoporosis and of horses that died for reasons unrelated to osteoporosis were examined using computed tomography (3-dimensional radiographs). Abnormalities of the bones and joints were compared between affected and unaffected horses.

    How does this research benefit horses?
    The reason for neck stiffness and pain in horses suffering from osteoporosis is not well understood, and thus, cannot be appropriately managed. Our results showed that silicate associated osteoporosis (osteoporosis due to inhalation of a toxic substance) affected bones of the neck, with the neck bones close to the chest being more severely affected. Degeneration of the bones also caused collapse of the intervertebral disks. Severely affected horses had marked neck stiffness and pain. Knowledge of these neck abnormalities in the neck allows for 1) early radiographic diagnosis of horses with osteoporosis, 2) early treatment of affected horses with medication, 3) appropriate management of affected horses (e.g., elevation of feed and water troughs), and 4) prevention of diagnosis of incorrect causes (e.g., wobbler) of neck pain in affected horses.

Regenerative Medicine

  • Moon blindness: Defining immune cells and how stem cells may help decrease inflammation associated with moon blindness
  • Investigators:
    Dori L. Borjesson, DVM, PhD, DACVP
    Mary A. Lassaline, DVM, PhDDACVO, MA
    Rebecca R. Bellone, PhD
    Naomi J. Walker, BS
    Seldy G. Nelson, BS

    Equine recurrent uveitis (ERU), or moon blindness, is the most common cause of equine blindness. It is caused by T lymphocyte-driven inflammation in the eyes (flares), and there is currently no cure. Mesenchymal stem cells (MSCs) are known to decrease T lymphocyte inflammation and may be an effective therapy for ERU. We hypothesized that horses with ERU have a specific “blood lymphocyte profile” that differs from unaffected horses, and that MSCs can switch specific lymphocytes from an inflammatory to a regulatory state.

    Data showed that normal horses and horses with ERU had similar percentages of T and B lymphocyte subsets in blood. However in horses with ERU a subset of these T lymphocytes were shifted towards a pro-inflammatory response, they secreted significantly more interferon gamma (IFNγ) than T lymphocytes from normal horses. Many ERU horses also had T lymphocytes did not secrete as much of an anti-inflammatory cytokine, IL-10, as did normal horses. Lymphocytes from ERU horses also had high expression of a receptor that helps them move from blood to enter lymph nodes. This receptor means that the lymphocytes have been “primed” by previous exposure to an antigen. This is a common finding in animals with immune-mediated disease. These findings confirm that horses with ERU have a pattern of activated, pro-inflammatory lymphocytes that could serve as biomarkers of disease.

    Expression of Foxp3 on CD8 T cells may be increased in ERU diseased horses. ERU horses tended to have increased expression of Foxp3 on CD8+ T lymphocytes compared to the control horses. There was no difference in Foxp3+ expression on CD4+ T lymphocytes between ERU horses and control horses.

    How does this research benefit horses?
    ERU is a devastating immune-mediated disease that requires long-term medical management and can result in blindness. These initial data highlighted that CD4 T cells were the most interesting subset of cells altered by ERU. Overall, MSCs were seen to decrease the CD4+ T cell activation phenotype including, most notably, the ability to decrease CD4+ T cell IFNγ concentration, which is elevated in ERU horses. Stem cell treatment continues to be a promising indicator for treatment of CD4+ T cell mediated diseases in horses. This study also provides critical data in horses to compare to data in cats, dogs and humans, where MSC modulation of T cell subsets, including CD8+ cells, is increasingly recognized, and inform human clinical trials for patients with autoimmune uveitis for which the horse is an excellent model of disease. A deeper understanding of how MSCs work in the context of ERU will lead to a personalized medical approach to therapy.

Reproduction

  • Variation in sperm mitochondrial function varies with stallion age and cryopreservation success and provides rationale for novel treatment and prevention strategies for male subfertility.
  • Investigators:
    Stuart Meyers, DVM, PhD
    Gino Cortopassi, PhD
    Evelyn Bulkeley, BS

    Mitochondrial dysfunction has been implicated as a major factor in aging and age-related diseases in numerous species and tissue types. The objective of this study was to evaluate the relationship between stallion age, sperm quality, and mitochondrial function for age-related patterns of dysfunction in fresh stallion semen. This was accomplished by employing an array of mitochondrial oxidative phosphorylation inhibitors and uncouplers of mitochondrial oxygen consumption (MITOX) in fresh semen from Quarter Horse stallions. Results indicated a significant negative correlation between stallion age and both MITOX and spare respiratory capacity (SRC). No significant correlations were found between age and ejaculate volume, concentration, viability, sperm deformity index, percent morphologically normal sperm, or motility parameters, but significant, although weak, positive correlations were observed between Compα-t and stallion age, SRC, and sperm deformity index. Significant positive correlations were found between MITOX and both total and progressive motility. Increasing age resulted in a significant decrease in MITOX, while increases in progressive motility resulted in a significant increase in MITOX. A negative interaction of effects was observed between age and mitochondrial uncoupling, with each year increase in age resulting in a 6.2% reduction in SRC. Interestingly, a significant negative relationship was found between age and progressive motility during ETC inhibition. In the presence of ETC inhibition, every year increase beyond the age of 11 resulted in a 9.8% decrease in progressive motility.

    How does this research benefit horses?
    These results confirm that aging is accompanied by decreased sperm mitochondrial function and indicate an age-related increase in dependence on mitochondrial oxidative function for progressive motility maintenance, strongly implicating mitochondrial dysfunction in the known age-related decrease in stallion fertility and sperm quality. There are detectable differences in mitochondrial efficiency that vary with stallion. Despite a decline in motility over time, oxygen consumption continues to increase since sperm are still viable and respiring even though they are becoming less motile. When we can powerfully predict the relationship of mitochondrial function to sperm function, effects from aging, and stallion fertility, we will be able to provide rational design of semen or male treatment to optimize stallion utilization. This could enable drug, stem cell, or immunologic-based therapies that may prevent or reverse oxidative injury, or other mechanisms that affect male fertility. Ultimately, this could allow more stallions to remain commercially viable for additional breeding seasons and this, in turn, will allow greater participation of stallions from various breeds in expanding international breeding programs.

  • Use of non-invasive embryo imaging to detect early embryonic developmental changes that predict the likelihood of later embryonic survival
  • Investigators:
    > Bruce W. Christensen, DVM, MS, DACT
    Stuart A. Meyers, DVM, PhD, DACT
    Ghislaine A. Dujovne, DVM, MS, DACT

    Early embryonic death is poorly understood in horse breeding. Higher rates of early embryonic death are noted when breeding older mares and performing advanced reproductive techniques like intracytoplasmic sperm injection (ICSI). Finding changes in early embryonic development that correlate with high or low embryo survivability would help target future research into causes of early embryonic death, and help with synchronizing recipient mares for ICSI procedures. We hypothesized that equine embryos have landmarks of developmental competence that are based on cell cycle and mitotic behavior, making possible the predictability of cultured embryos to progress to transferrable embryos.

    We have established one pregnancy from an ICSI-derived embryo and successfully performed oocyte recovery a total of 52 times while successfully recovering immature oocytes that have been in vitro matured (IVM) and cultured prior to ICSI. In our initial experiments, 52% of IVM oocytes extruded a polar body and were injected with sperm collected the morning of ICSI, 30 hours post- TVA. A very low (<5%) blastocyst rate was observed. A second experiment was then performed using improved media. Culture conditions demonstrated marked improvement in maturation oocytes aspirated from mares and in successful ICSI, with a near 30% blastocyst rate, which is the industry standard. This demonstrated a significantly higher level of blastocyst development reflected by decreased timing from oocyte aspiration to in vitro culture, and from removal of any blood from oocyte culture medium. The results show a marked improvement in successful embryo development.

    Further improvements in in vitro maturation, including follicular fluid and recombinant hormones, resulted in increased oocyte quality from aspirations and resulted in a blastocyst rate of 20%. One blastocyst from this cohort was transferred to a synchronized recipient mare and resulted in a positive pregnancy diagnosis, the first “all UC Davis” ICSI pregnancy.

    How does this research benefit horses?

    Predicting how many post-ICSI embryos are going to mature out of a cohort, and when they will mature, would be a tremendous help in synchronizing recipient mares for fresh embryo transfers. Abnormalities associated with later pregnancy losses could be detected and choosing not to transfer those embryos could save time and money. Identifying stages in embryonic development when problems first occur will help focus future research into the causes of and possible solutions for preventing early embryonic death.

Surgery/Anesthesiology

  • Pilot study: evaluating dosage and safety of a new pain drug in horses
  • Investigators:
    Robert Brosnan, DVM, PhD, DACVAA
    Claudia Sonder, DVM

    Pain in horses is commonly managed with anti-inflammatory and opioid drugs. However, these drugs are not always effective and can produce undesirable side effects. We hypothesized that a new volatile analgesic discovered at UC Davis might exhibit analgesic effects in horses. This study examined dose ranges and safety of the analgesic by evaluating behavioral and biochemical effects of escalating doses in horses with spontaneously occurring orthopedic pain and lameness that was not fully ameliorated by conventional therapy.

    Administration of this new volatile analgesic discovered at UC Davis produced improvement of pain for up to two hours for most doses studied. Horses also took significantly more steps in the four hours after drug administration than in the four hours prior to drug administration. It is possible that the drug allowed horses to be more active because it helped alleviate their pain. Additionally, we were able to collect expired breath samples from horses and measure drug concentrations in the lungs and extrapolate drug concentrations that were present in the blood. Adverse effects were deemed minimal as, except at the two highest drug doses administered, no horse exhibited evidence of ataxia or sedation. At all doses, horses had no statistical changes in complete blood counts (CBC) parameters or chemistry panels that were repeated over the course of 2 days. A future modification of this technique could allow veterinarians to measure the realtime concentration of this volatile analgesic in horses to help better direct drug therapy and dosing.

    How does this research benefit horses?

    This pilot study has provided a dose range for a novel analgesic that does not cause sedation or toxicity in horses, but which may be associated with clinically relevant efficacy. These results support the need to conduct a larger, blinded, controlled analgesia study in horses that includes the highest no-observed-adverse-effect level dose of this new drug. Improved pain management will improve welfare of horses and potentially reduce the need for euthanasia of horses. New analgesics may also alleviate systemic complications that are associated with pain, such as decreased GI mobility, gastric ulceration, kidney insult, decreased wound healing strength, and possibly development of secondary laminitis.

Resident Grants

Genetics

  • Investigation of a region on horse chromosome 19 as the cause for bilateral corneal stromal loss in Friesian horses
  • Investigators:
    Kelly E. Knickelbein, VMD
    Rebecca Bellone, PhD
    Mary E. Lassaline, DVM, PhD

    Bilateral Corneal Stromal Loss (BCSL), a disease resulting in progressive thinning of a small region of the cornea, is implicated as a genetic disorder in Friesian horses. Bilaterally symmetric thinning of the cornea can progress to permanent ocular damage and blindness if globe rupture occurs, which can be career or life ending.

    A region on chromosome 19 previously identified as associated with BCSL was evaluated for differences in the DNA sequence between affected and unaffected horses. Fifty single nucleotide variants were identified in the region of interest. While none were perfectly concordant with the disease, the most associated variant was a missense variant in the mucin 4 (MUC4) gene (c.778G>C, p.Val260Leu, P=1.04x10-4). This variant was present in 10 of 22 cases and found in only 4 of 54 controls. Mucin 4 has been linked to a corneal disease in humans. Additionally, two deletions (a 242 base pair deletion in NECTIN3 and an 809 base pair intergenic deletion) and one insertion (a 241 base pair insertion in LSAMP) were identified and genotyped in the same horses. None of these structural variants were perfectly concordant with disease phenotype, and only the intronic insertion in LSAMP was statistically associated with disease status (P=0.019). As such, these structural variants are not likely to be the cause of BCSL. Given the lack of identification of a variant perfectly concordant with the disease phenotype, it is likely that this is a complex disease with multiple genetic variants potentially contributing to the disease process.

    How does this research benefit horses?

    Bilateral Corneal Stromal Loss in Friesian horses is a disease that typically requires emergency surgery to prevent rupture of the globe. If the disease progresses to globe rupture, loss of use of the horse is likely, making this an expensive and potentially devastating disease for horse owners. This work elucidated details about the inherited component of BCSL in Friesian horses and identified 53 variants for further investigation. The most associated variants are being further scrutinized for use in genetic testing for marker assisted selection and identification of at-risk horses for clinical evaluation.

Immunology

  • Investigation into the effect of common anti-inflammatory medications on the acute inflammatory marker serum amyloid A
  • Investigators:
    Callum G Donnelly, BVBiol, BVSc (Hons I), DACT, DACVIM
    Nicola Pusterla, DVM, PhD, DACVIM, DAVDC
    Sharon J Spier, DVM, PhD, DACVIM

    Serum amyloid A (SAA), an acute phase protein, is a widely accepted monitoring tool for acute infectious or inflammatory diseases in horses, especially for sport horses traveling long distances for competition. These horses are also likely to be concurrently and/or chronically treated with anti-inflammatory drugs (NSAIDs) and antihistamines. The effect of NSAIDs and antihistamines administration on the acute inflammatory response in horses as measured by SAA is unknown and may reduce the diagnostic sensitivity of this early inflammatory marker.

    Thirty horses of known Corynebacterium pseudotuberculosis exposure status (by titer) were pre-treated with flunixin meglumine (n=6), firocoxib (n=6) or diphenhydramine (n=6) prior to receiving a strong inflammatory stimulus – Corynebacterium pseudotuberculosis bacterin/toxoid vaccine. Two control groups included horses receiving the vaccine (and no drugs) (n=6) and those receiving no vaccine and no drugs (n=6). Horses were monitored daily over 14 days for the development of side effects Blood samples were collected every 24 hours for the first seven days following vaccination and then every 48 hours until day 14 post vaccination. SAA concentrations were determined using a commercial stall-side lateral flow immunoassay. Vaccination was repeated 30 days following the initial administration. Horses followed the same protocol of pre-treatment, observation and sampling as before.

    Administration of flunixin meglumine, firocoxib or diphenhydramine did not reduce the acute inflammatory response as monitored by SAA. Booster vaccination resulted in a more pronounced inflammatory response compared to the initial vaccination, regardless of concurrent treatment.

    How does this research benefit horses?

    Co-administration of NSAIDs or antihistamines at the time of vaccination does not result in a reduction in SAA and therefore does not appear to modify the acute inflammatory process induced by a strong antigenic vaccine.

    This research is under review at the Equine Veterinary Journal.

Medicine and Infectious Disease

  • A new tool in the diagnosis of equine neurodegenerative diseases
  • Investigators:
    Lisa Edwards, DVM, DACVIM
    Carrie J Finno, DVM, PhD, DACVIM

    Diagnosis of neurologic disease in horses is challenging; investigation involves multiple diagnostics in addition to thorough clinical examination. For diseases such as equine neuroaxonal dystrophy (eNAD) and equine degenerative myeloencephalopathy (EDM), there is no definitive antemortem diagnostic available. Neurofilaments, structural proteins unique to the neuron, have been studied as biomarkers of neurologic disease in multiple species and show promising diagnostic utility.

    Phosphorylated heavy protein (pNfH) has unique properties that make it useful as a biomarker of neurologic disease. We hypothesized that horses with neurologic disease (eNAD/EDM or cervical compressive vertebral myelopathy (CVCM)) have higher pNfH levels in blood and cerebrospinal fluid (CSF) compared to normal horses, with eNAD/EDM affected horses having the highest pNfH levels. We hypothesized that blood and CSF pNfH levels in normal horses would be < 2 ng/ml.

    Blood and CSF pNfH levels in non-neurologic horses and neurologic horses diagnosed with eNAD/EDM or CVCM were determined using a species-validated enzyme linked immunosorbent assay (ELISA) kit.Blood and CSF pNfH was < 1.2 ng/ml in neurologically normal horses. Horses with blood pNfH of 1–2 ng/ml were 8.8 times more likely to have eNAD/EDM at necropsy. Horses with blood pNfH of > 2 ng/ml were 4.4 times more likely to have eNAD/EDM at necropsy. Horses with CSF pNfH > 2 ng/ml were 2.35 times more likely to have eNAD/EDM at necropsy. Serum appears to be more predictive of eNAD/EDM than CSF and may differentiate eNAD and CVCM affected horses.

    How does this research benefit horses?

    Currently, there is no diagnostic test available for eNAD/EDM other than necropsy. Even at necropsy, the disease may go unrecognized due to the subtlety of the microscopic changes that occur in the nervous system. Measurement of blood pNfH can support the clinical diagnosis of eNAD/EDM without the need for euthanasia and necropsy evaluation. Elevated CSF pNfH levels can also document injury to the spinal cord, as seen in CVCM, and reduce the need for expensive and invasive diagnostics. Lastly, identification of eNAD/EDM affected horses via blood pNfH testing can facilitate selective breeding and targeted vitamin E supplementation in susceptible pregnant broodmares and foals. It is important to note that while increased pNfH levels provide useful prognostic information, normal blood or CSF pNfH does not rule out neurologic disease. This test is now available at UC Davis.

    This research has been submitted to the Equine Veterinary Journal.

  • Comparison of white and red blood cell counts in Warmblood and Thoroughbred horses
  • Investigators:
    Emily A. Schaefer, VMD, DACVIM
    K. (Gary) Magdesian DVM, DACVIM, DACVECC, DACVCP, CVA
    Judy Edman

    Complete blood counts (CBCs) are routinely performed in adult horses to evaluate for clinical or subclinical inflammation and infection based on equine reference intervals. Clinically, we have noted that certain breeds of horses have CBC values that fall outside of established reference intervals and could be miscategorized as unhealthy based on these differences.

    We hypothesized that warmbloods will have lower values for red cell parameters and white blood cell counts than Thoroughbreds and other breeds 3-19 years of age. We collected whole blood (10mL) from healthy horses and performed Complete Blood Counts at the hematology lab at the UC Davis veterinary hospital. The horses were grouped by breed and CBC parameters compared between groups.

    Warmblood horses had statistically significantly fewer total white cells compared to Thoroughbreds and other breeds. Warmbloods had statistically significantly fewer lymphocytes, a particular type of white blood cell. In fact, 26.5% of warmbloods had lymphocyte counts that were below the previously established lower reference interval. Warmbloods had statistically significantly lower red blood cell counts than Thoroughbreds. There were no statistically significant differences between breeds in eosinophil, basophil, or monocyte counts, nor fibrinogen concentration.

    How does this research benefit horses?

    It has been previously documented that certain breeds of horses have differing numbers of certain types of blood cells. Thoroughbreds, for example, have higher red blood cell counts than many other breeds, a necessity for highly aerobic exercise such as racing as red blood cells carry oxygen throughout the body. This study documented for the first time that normal horses within a generalized subset of breeds (warmbloods) often have lower white blood cell counts than Thoroughbreds and other breeds. This is relevant in a clinical setting because it may alter treatment decisions when evaluating a warmblood horse with “low” white cell count and avoid unnecessary costs to client and delays in elective treatments for other conditions.

  • Seasonal variation of endogenous adrenocorticotropic hormone in healthy donkeys in Northern California
  • Investigators:
    Sarah Schale, DVM
    Erin Goodrich, DVM, DACVPM
    Philip Kass, DVM, MPVM, MS, PhD
    Emily Berryhill, DVM, DACVIM

    Pituitary pars intermedia dysfunction (PPID, or Equine Cushing’s disease) is the most common endocrinopathy of aging equids and predisposes affected horses to significant health problems, including laminitis. A common test to diagnose PPID is assessing blood levels of adrenocorticotropic hormone (ACTH), which increases in horses with PPID. Seasonal variation of ACTH has been validated in normal horses to enable accurate year-round testing; however, these values have not been established in donkeys.

    This study measured plasma ACTH concentrations in healthy, nongeriatric donkeys in northern California on a monthly basis to establish seasonal variation of ACTH. The hypotheses were that ACTH concentrations would be higher in donkeys than in horses in all seasons, and that similarly to horses, ACTH would further increase in the fall.

    Twenty-five donkeys were recruited based on their age (median age 6 years, range 2 to 13 years) and appearance of good health based on physical examinations and complete blood counts. They were kept at the same property under the same management practices. Venipuncture was performed on a monthly basis from March 2019 through February 2020, and a validated ACTH assay was run on all samples. Months were grouped into seasons: spring (March through May), summer (June through August), fall (September through November) and winter (December through February).

    Median ACTH concentration was 19.0 pg/mL in spring, 49.8 pg/mL in summer, 83.9 pg/mL in fall, and 12.6 pg/mL in winter. Each season’s median ACTH concentration was significantly different from each other. Median ACTH concentrations fell within horse reference ranges in winter and spring months (< 35 pg/mL).

    How does this research benefit horses?

    Based on the results in this group of donkeys, the best time to assess ACTH concentrations in donkeys is during the winter and spring months (December through May). This will avoid the large increases in ACTH concentrations that may occur in the summer and fall months. For winter and spring months it appears that horse reference ranges may be used. These findings will aid equine veterinarians in appropriate management of aging donkeys.

Orthopedics

  • Comparison of two advanced imaging modalities (PET/CT and MRI) for diagnosis of lameness localized to the foot in horses.
  • Investigators:
    Jannah Pye, BVSc
    Mathieu Spriet, DVM, MS, DACVR, DECVDI

    Computed tomography (CT) and magnetic resonance imaging (MRI) have considerably improved our ability to identify lameness-causing lesions in the equine distal limb, but the significance of some findings remains uncertain. Our group has demonstrated the value of Positron Emission Tomography (PET) in equine patients to assess bone and soft tissue lesions associated with lameness, but these should be compared to changes seen on MRI to better characterize the clinical relevance of such changes.

    We hypothesized that dual PET/CT would allow assessment of both bone and soft tissue lesions detected on MRI in a clinical population of lame horses and detect lesions not visible on standing low field MRI. This study aimed to compare dual tracer PET/CT findings with MRI findings from horses that have lameness localized to the distal limb and correlate imaging results with lameness exam findings and outcome following treatment of identified lesions.

    Horses (n = 8) that underwent both dual tracer PET/CT and MRI within the same month for evaluation of lameness localized to the distal limb by diagnostic anesthesia (palmar digital or abaxial sesamoid nerve block) were selected. The PET/CT findings were compared with the MRI findings and correlated with the patient’s history, clinical findings, and outcome.

    Results showed that PET/CT identified abnormalities not seen with MRI include deep digital flexor tendinopathy, desmitis of the chondrosesamoidean ligament and desmitis of the collateral ligament of the navicular bone. PET/CT also helped differentiate between “active” and “inactive” lesions in a horse with both navicular bone and deep digital flexor abnormalities seen on MRI.

    How does this research benefit horses?
    PET/CT technique has the potential to improve detection of early or subtle injuries and help distinguish active from non-active lesions. With this modality now available at UC Davis, we anticipate that PET/CT will be a useful tool to help explain clinical findings and improve outcomes for horses with lameness localized to the foot.

Surgery/Anesthesiology

  • Can anesthesia be induced by intravenous etomidate in horses?
  • Investigators:
    Juhana M Honkavaara, DVM, PhD
    Robert J Brosnan, DVM, PhD, DACVAA

    Fewer anesthetic induction drug techniques exist for horses than for all other domestic species. The few available drug options can sometimes produce severe, undesirable cardiopulmonary effects in very ill horses. Etomidate is an injectable general anesthetic commonly used in humans and small animals that does not cause significant cardiovascular or respiratory depression at clinical doses. However, etomidate efficacy as a general anesthetic has never been tested in horses.

    We hypothesized that etomidate can be used to induce general anesthesia in healthy horses. Sedated horses were administered etomidate in a stepwise manner based on the responses observed in the previous study horse (“Upand- Down” method) to determine the median effective dose (i.e. the dose that will immobilize 50% of the study population) for general anesthesia. Clinically relevant physiologic responses (heart rate, respiratory rate, oxygen saturation, venous carbon dioxide), anesthetic depth signs (eye position, palpebral reflex, pupil size, limb or head motor activity), and induction and recovery quality were also be assessed.

    Etomidate 0.5mg/kg administered to sedated horses was sufficient to produce recumbency and unconsciousness and allow intubation. However, induction quality in most horses was poor due to brief trembling and galloping behavior after anesthetic induction. Quality of anesthetic induction was not improved by higher etomidate doses or by addition of a benzodiazepine muscle relaxant. Although recoveries from etomidate alone were excellent, etomidate is an unsuitable anesthetic induction agent in horses.

    How does this research benefit horses?

    Cardiovascular and respiratory depression can increase perianesthetic morbidity and mortality in sick horses (e.g., ill colic patients). General anesthesia that can be induced with minimal cardiopulmonary effect may provide a safer alternative to current agents in debilitated horses. However, based on the results of this study, etomidate is not suitable for general anesthesia in horses.

  • The efficacy of a 0.2% polyhexamethylene biguanide-impregnated gauze dressing against common orthopedic bacteria found in horses.
  • Investigators:
    Charlene V. Noll BA, BE, MSME, DVM
    Isabelle Kilcoyne, MVB, DACVS
    Jorge E. Nieto, MVZ, PhD, DACVS, DACVSMR
    Barbara A. Byrne DVM PhD DACVIM, DACVM

    Traumatic wounds of distal limb joints are commonly encountered problems in equine practice, and frequently involve synovial structures. Such injuries can be career limiting or life threatening. Distal limb wounds in horses heal more slowly than wounds on other parts of the body because of a comparatively decreased blood supply, greater mobility over joints, and predisposition for bacterial contamination because of proximity to the ground. Common isolates from equine wounds include Staphylococcus spp. in addition to Enterobacteriaceae, Streptococcal spp., Pseudomonas spp., and anaerobes.

    We hypothesized there would be significantly less bacterial growth of certain species of bacteria using the PHMB-impregnated gauze compared to the control gauze. Cultures of wounds, draining tracts and incisional infections were submitted for aerobic culture. Eleven aerobic bacteria were identified and banked at -20°C. Squares of PHMB-impregnated and non-impregnated control gauze were placed on Muller-Hinton agar plates inoculated with commonly isolated bacterial species (n=11). Growth under each gauze was assessed qualitatively after a 24-hour incubation period. Zones of inhibition were measured to a standardized scale, using image-processing software. A numerical scale was used to record level of inhibition of bacterial growth.

    PHMB-impregnated gauze provided greater inhibition of growth of 4/6 Gram-positive species and 4/5 Gram-negative species on inoculated plates compared with control gauze. Growth inhibition (%) using the 0.2% PMHB-impregnated gauze for Staphylococcal spp. (n=4) ranged from 33-83.1% and for Escherichia coli spp. (n=4) ranged from 6.5-37% compared to 0% using the control gauze. There was no inhibition of growth of Pseudomonas aeruginosa or Enterococcus spp.

    How does this research benefit horses?

    Use of a 0.2% polyhexamethylene biguanide (PHMB)-impregnated dressing resulted in variable growth inhibition of different Staphylococcal spp. and Escherichia coli spp, bacteria commonly encountered when treating orthopedic conditions such as infections postoperatively and wounds in horses. These dressings may be useful for reducing contamination of underlying wounds by common equine bacterial pathogens in clinical practice.

  • Arthroscopic surgery of the caudal cervical facets using a needle arthroscope
  • Investigators:
    Marcos Perez Nogues, LV, MSc
    Betsy Vaughan, DVM, DACVSMR
    Kathryn L. Phillips, DVM, DACVR
    Larry D. Galuppo, DVM, DACVS

    Caudal cervical osteoarthritis is a commonly found pathology that has a significant career impact in horses. The progression of the osteoarthritis in the lower neck can lead to neck pain, lameness, or subsequent spinal cord compression and ataxia, and can ultimately lead to retirement. Currently, the imaging modalities available usually have low yield correlating clinical signs with the severity of the lesions, and advance imaging is sometimes impossible due to disparity of the equipment and horse´s size. In this study, we performed needle arthroscopy of the caudal cervical facet joint in healthy horses. The goal was to refine this technique in order to diagnose, prognosticate, and better guide treatment for osteoarthritic lesions, osteochondral (OCD) lesions and flush septic processes in these joints.

    How does this research benefit horses?

    The study results showed that needle arthroscopic surgery of the caudal cervical facet joints can be done safely and successfully with the horses standing just under sedation. This minimal invasive surgery will be useful in the future for the diagnosis of neck pathology, could be used in future studies to discover how osteoarthritis impinge the spinal cord developing ataxia, and could be the start point of the developing of future treatments for neck osteoarthritis and other neck pathologies.

    This research was reported in Veterinary Surgery 2020 Apr;49(3):463-471.

  • Determining whether or not the tarsocrural joint communicates with the talocalcaneal joint in the equine tarsus.
  • Investigators:

    Thomas Cullen BVMS
    Katrijn Dow Whisenant DVM
    Derek D. Cissell, VMD, Ph.D., DACVR
    Larry D Galuppo, DVM, DACVS

    As the hock consists of four joints, communication between joints is an important aspect to consider when performing diagnostic or therapeutic joint injections. To date, the communication of the distal hock joints has been well investigated, but potential communication of the proximal joints remains unclear. If communication of the proximal joints exists, joint injection approaches could be altered to make injections more feasible in the field.

    We hypothesized that injection into the front of the tarsocrural joint would result in diffusion to the talocalcaneal joint in the back of the hock. CT was performed on twelve paired limbs from horses euthanized for reasons unrelated to disease of the tarsus. The tarsocrural joints were injected with a mixture of iodinated contrast and methylene blue stain via a routine dorsomedial approach. One joint was injected with 60 mls (high volume) and the other was injected with 7 mls (low volume). Repeat CT following injection and flexion of the tarsus was performed. The tarsi were subsequently dissected to assess the methylene blue staining of the talocalcaneal joint cartilage.

    With CT, contrast was noted to fill the tarsal sinus and surround the facets of the talocalcaneal joint in all 12 limbs. Discernable contrast between the articular surfaces of the talocalcaneal facets was only seen in 3/12 limbs. Methylene blue staining of all talocalcaneal facets was present in 12/12 limbs. For the high-volume limbs, the stain uptake was subjectively increased when compared with the low-volume injectate. The medial talocalcaneal facet had consistently less stain uptake than the lateral facets.

    How does this research benefit horses?

    Methylene blue stain confirmed tarsocrural-talocalcaneal joint communication in all cases and was far superior to contrast-enhanced CT for this determination. Clinically, the majority of talocalcaneal joint disease is concentrated at the medial facet of the joint. Knowing that, in healthy limbs, there is limited communication with the tarsocrural joint into the medial facet decreases the likelihood that intraarticular treatments injected into the tarsocrural joint will be efficacious in clinical cases.