Epiploic foramen entrapment (EFE) is a common cause of small intestinal colic in horses and may lead to intestinal strangulation. Strangulating intestinal obstruction impairs the gastrointestinal outflow and can lead to secondary gastric rupture and endotoxemia. Clostridioides difficile can cause enterotyphlocolitis with colic in horses of all ages, and the process is commonly referred to as C. difficile-associated disease (CDAD). Here we report the results of the postmortem examination of a 7-y-old Thoroughbred racehorse with concurrent CDAD, EFE, and gastric rupture that was euthanized following a history of colic over several days. A segment of distal jejunum and proximal ileum had passed through the epiploic foramen, and the intestinal wall was thickened and dark-red. The remaining small intestinal loops were distended and filled with blood-tinged contents. Peritonitis had resulted from escape of gastric contents into the abdominal cavity through a tear in the major curvature of the stomach. Histologically, the incarcerated segment had acute transmural hemorrhage with congestion and mucosal necrosis; neutrophilic infiltrates with fibrin thrombi were in the mucosa of the non-incarcerated small intestinal segments. C. difficile toxins were detected in the small intestinal contents, and C. difficile was isolated from the small intestine, colon, and cecum.

BACKGROUND: Lidocaine is a local anesthetic that is sometimes administered in combination with epinephrine. The addition of epinephrine increases the time lidocaine remains at the site of administration, thus prolonging the duration of effect. Due to their potential to prevent the visual detection of lameness, the administration of local anesthetics is strictly regulated in performance and racehorses. Recent reports of positive regulatory findings for lidocaine in racehorses suggests a better understanding of the behavior of this drug is warranted. The objective of the current study was to describe serum and urine concentrations and the pharmacokinetics of lidocaine and its primary metabolites following administration in combination with epinephrine, as a palmar digital nerve block in horses. Twelve horses received a single administration of 1 mL of 2% lidocaine HCl (20 mg/horse) with epinephrine 1:100,000, over the palmar digital nerve. Blood samples were collected up to 30 h and urine samples up to 48 h post administration. Lidocaine and metabolite concentrations were determined by liquid chromatography- mass spectrometry and pharmacokinetic (non-compartmental and compartmental) analysis was performed. RESULTS: Serum concentrations of lidocaine and 3-hydroxylidocaine were above the LOQ of the assay at 30 h post administration and monoethylglycinexylidide (MEGX) and glycinexylidide (GX) were below detectable levels by 24 and 48 h, respectively. In urine, lidocaine, MEGX and GX were all non-detectable by 48 h post administration while 3-hydroxylidocaine was above LOQ at 48 h post administration. The time of maximal concentration for lidocaine was 0.26 h (median) and the terminal half-life was 3.78 h (mean). The rate of absorption (Ka) was 1.92 1/h and the rate of elimination (Kel) was 2.21 1/h. CONCLUSIONS: Compared to previous reports, the terminal half-life and subsequent detection time observed following administration of lidocaine in combination with epinephrine is prolonged. This is likely due to a decrease in systemic uptake of lidocaine because of epinephrine induced vasoconstriction. Results of the current study suggest it is prudent to use an extended withdrawal time when administering local anesthetics in combination with epinephrine to performance horses.

More and more studies are reporting on the natural transmission of SARS-CoV-2 between humans with COVID-19 and their companion animals (dogs and cats). While horses are apparently susceptible to SARS-CoV-2 infection based on the homology between the human and the equine ACE-2 receptor, no clinical or subclinical infection has yet been reported in the equine species. To investigate the possible clinical role of SARS-CoV-2 in equids, nasal secretions from 667 horses with acute onset of fever and respiratory signs were tested for the presence of SARS-CoV-2 by qPCR. The samples were collected from January to December of 2020 and submitted to a commercial molecular diagnostic laboratory for the detection of common respiratory pathogens (equine influenza virus, equine herpesvirus-1/-4, equine rhinitis A and B virus, Streptococcus equi subspecies equi). An additional 633 serum samples were tested for antibodies to SARS-CoV-2 using an ELISA targeting the receptor-binding domain of the spike protein. The serum samples were collected from a cohort of 587 healthy racing Thoroughbreds in California after track personnel tested qPCR-positive for SARS-CoV-2. While 241/667 (36%) equids with fever and respiratory signs tested qPCR-positive for at least one of the common respiratory pathogens, not a single horse tested qPCR-positive for SARS-CoV-2. Amongst the racing Thoroughbreds, 35/587 (5.9%) horses had detectable antibodies to SARS-CoV-2. Similar to dogs and cats, horses do not seem to develop clinical SARS-CoV-2 infection. However, horses can act as incidental hosts and experience silent infection following spillover from humans with COVID-19. SARS-CoV-2-infected humans should avoid close contact with equids during the time of their illness.

The most prevalent causes of death in racehorses are musculoskeletal injuries, causing ~83% of deaths within the racing industry in California and elsewhere. The vast majority of these injuries have preexisting lesions that predispose to fatal injury. A 4-y-old Thoroughbred colt suffered an acute suspensory apparatus failure, including biaxial proximal sesamoid bone fractures of the right front fetlock, causing loss of support of the fetlock joint and consequent fall with fractures of the cervical and sacral spine. Cervical fracture caused spinal cord damage that resulted in sudden death. A preexisting lesion in the medial proximal sesamoid bone likely predisposed to complete fracture of this bone and fetlock breakdown. Interestingly, a comparable osteopenic lesion was present in the intact medial proximal sesamoid bone of the left forelimb, which is consistent with bilateral repetitive overuse injury in racehorses. The morphologic features of the cervical and sacral spine fractures were compatible with acute injury; no evidence of preexisting lesions was seen. Most likely, these acute vertebral fractures occurred as a result of the horse falling. This case emphasizes the importance of performing a detailed autopsy in horses that suffer an appendicular musculoskeletal injury, particularly in fatal cases when the horse dies following a leg injury.

The in vivo metabolism and pharmacokinetics of flunixin meglumine and phenylbutazone have been extensively characterized; however, there are no published reports describing the in vitro metabolism, specifically the enzymes responsible for the biotransformation of these compounds in horses. Due to their widespread use and, therefore, increased potential for drug-drug interactions and widespread differences in drug disposition, this study aims to build on the limited current knowledge regarding P450-mediated metabolism in horses. Drugs were incubated with equine liver microsomes and a panel of recombinant equine P450s. Incubation of phenylbutazone in microsomes generated oxyphenbutazone and gamma-hydroxy phenylbutazone. Microsomal incubations with flunixin meglumine generated 5-OH flunixin, with a kinetic profile suggestive of substrate inhibition. In recombinant P450 assays, equine CYP3A97 was the only enzyme capable of generating oxyphenbutazone while several members of the equine CYP3A family and CYP1A1 were capable of catalyzing the biotransformation of flunixin to 5-OH flunixin. Flunixin meglumine metabolism by CYP1A1 and CYP3A93 showed a profile characteristic of biphasic kinetics, suggesting two substrate binding sites. The current study identifies specific enzymes responsible for the metabolism of two NSAIDs in horses and provides the basis for future study of drug-drug interactions and identification of reasons for varying pharmacokinetics between horses.

Methylphenidate is a powerful central nervous system stimulant with a high potential for abuse in horse racing. The detection of methylphenidate use is of interest to horse racing authorities for both prior to and during competition. The use of hair as an alternative sampling matrix for equine anti-doping has increased as the number of detectable compounds has expanded. Our laboratory developed a liquid chromatography-high-resolution mass spectrometry method to detect the presence of methylphenidate in submitted samples. Briefly, hair was decontaminated, cut, and pulverized prior to liquid-liquid extraction in basic conditions before introduction to the LC-MS system. Instrumental analysis was conducted using a Thermo Q Exactive mass spectrometer using parallel reaction monitoring using a stepped collision energy to obtain sufficient product ions for qualitative identification. The method was validated and limits of quantitation, linearity, matrix effects, recovery, accuracy, and precision were determined. The method has been applied to confirm the presence of methylphenidate in official samples submitted by racing authorities.

Recombinant human erythropoietin (rHuEPO) is a well-known performance enhancing drug in human athletes, and there is anecdotal evidence of it being used in horse racing for the same purpose. rHuEPO, like endogenous EPO, increases arterial oxygen content and thus aerobic power. Micro-doping, or injecting smaller doses over a longer period of time, has become an important concern in both human and equine athletics since it is more difficult to detect. Horses offer an additional challenge of a contractile spleen, thus large changes in the red blood cell mass occur naturally. To address the challenge of detecting rHuEPO doping in horse racing, we determined the transcriptomic effects of rHuEPO micro-dosing over seven weeks in exercised Thoroughbreds. RNA-sequencing of peripheral blood mononuclear cells isolated at several time points throughout the study identified three transcripts (C13H16orf54, PUM2 and CHTOP) that were significantly (PFDR < 0.05) different between the treatment groups across two or three time point comparisons. PUM2 and CHTOP play a role in erythropoiesis while not much is known about C13H16orf54, but it is primarily expressed in whole blood. However, gene expression differences were not large enough to detect via RT-qPCR, thereby precluding their utility as biomarkers of micro-doping.

Background

Exercise induced cardiac fatigue (EICF) and cardiac dysrhythmias are well described conditions identified in high-level human athletes that increase in frequency with intensity and duration of exercise. Identification of these conditions requires an understanding of normal pre- and post-race cardiac assessment values. The objectives of this study were to (1) characterize selected indices of cardiac function, electrophysiologic parameters, and biochemical markers of heart dysfunction prior to and immediately after high level racing in Thoroughbred horses receiving furosemide; and (2) create pre- and post-race reference values in order to make recommendations on possible screening practices for this population in the future.

Results

Thirty Thoroughbred horses were enrolled in the study with an age range of 3-6 years. All horses received furosemide prior to racing. Physical exams, ECGs, and echocardiograms were performed prior to racing (T0) and within 30-60 min following the race (T1). Blood samples were obtained at T0, T1, 4 h post-race (T4) and 24 h after the race (T24). Electrolytes, hematocrit, cardiac troponin I, and partial pressure CO2 values were obtained at all time points. Heart rate was significantly increased post-race compared to baseline value with a median difference of 49 bpm, 95% CI [31,58],(P < 0.0001). No dysrhythmias were noted during ECG assessment. Following the race, an increase in number of horses demonstrating regurgitation through the aorta and AV valves was noted. Systolic function measured by fractional shortening increased significantly with a mean difference of 7.9%, 95% CI [4.8, 10.9], (P < 0.0001). Cardiac troponin I was not different at pre- and immediately post-race time points, but was significantly increased at T4 (P < 0.001). Troponin returned to baseline value by T24.

Conclusions

This study utilized a before and after study design where each horse served as its own control, as such the possible effect of regression to the mean cannot be ruled out. The reference intervals generated in this study may be used to identify selected echocardiographic and electrocardiographic abnormalities in racing horses receiving furosemide.

Bisphosphonates are potent anti-resorptive agents that have the potential to adversely affect bone healing in equine athletes, and normal bone adaption in young racehorses. A concern exists that bisphosphonate inhibition of normal bone metabolism could lead to increased bone fractures during high-intensity exercise. We found only a single report describing concentrations of tiludronate in the bone of horses, and no studies describing clodronate. Knowledge of the residence time in bone could allow for a better understanding of the long-term effects of these compounds. Our objectives were to develop a method for detection of bisphosphonates in bone and add to the limited information available regarding the disposition of these drugs in the bone of horses. Two horses received clodronate and 2 tiludronate disodium. Postmortem collection of bones and teeth occurred either 4 or 30 d post drug administration. Additionally, postmortem blood, synovial fluid, aqueous humor, and bone samples from racehorses with various histories of bisphosphonate administration were collected, and concentrations determined using the developed LC-MS/MS method. Bisphosphonates were detected in bones and teeth tested at 4 and 30 d. In a postmortem sample, clodronate was detected in bone from a horse with reported administration 18 mo prior; clodronate was not detected in other sample types collected from this horse. Bisphosphonates reside in bone for extended periods of time, which could lead to potential long-term effects, increasing the potential for bone fractures in young and/or athletic horses.

Five horses originating from 4 different California race tracks were submitted to the California Animal Health and Food Safety Laboratory for necropsy and diagnostic workup. The 5 horses had a history of sudden collapse and death during exercise. In all of them, massive hemoperitoneum and hemorrhages in other cavities or organs were observed. The liver from these 5 animals and from 27 horses that had been euthanized due to catastrophic leg injuries (controls) were subjected to a rodenticide anticoagulant screen. Traces of brodifacoum, diphacinone, or bromadiolone were detected in the 5 horses with massive bleeding (5/5), and no traces of rodenticides were detected in control horses (0/27). Other frequent causes of massive hemorrhages in horses were ruled out in 4 of the cases; one of the horses had a pelvic fracture. Although only traces of anticoagulants were found in the livers of these horses and the role of these substances in the massive bleeding remains uncertain, it is speculated that exercise-related increases in blood pressure may have reduced the threshold for toxicity of these anticoagulants.