Equine intervertebral disc disease with dorsal protrusion and spinal cord compression: A computed tomography, myelography, MRI, and histopathologic case study.

A 3-year-old American Saddlebred gelding presented for progressive tetraparesis, ataxia, and cervical hyperaesthesia. Radiographic myelography identified spinal cord compression at C6-7 in neutral, extended, and flexed positions and at C4-5 in the flexed position. CT myelography and postmortem MRI identified severe vertebral canal stenosis/compression at C6-7. MRI further identified severe intervertebral disc herniation at C6-7 with intramedullary changes. Disc protrusion was confirmed macroscopically at postmortem. Lesions consistent with compressive myelopathy were confirmed microscopically at C6-7. This is the first report of equine disc protrusion and myelocompression confirmed by multiple advanced imaging modalities and postmortem examination.

Infectious Causes of Equine Placentitis and Abortion.

A variety of infectious agents including viral, bacterial, and fungal organisms can cause equine abortion and placentitis. Knowledge of normal anatomy and the common pattern distribution of different infectious agents will assist the practitioner in evaluating the fetus and/or placenta, collecting appropriate samples for further testing, and in some cases, forming a presumptive diagnosis. In all cases, it is recommended to confirm the diagnosis with molecular, serologic, or microbiological testing. If a causative agent can be identified, then appropriate biosecurity and vaccination measures can be instituted on the farm.

Donor age effects on in vitro chondrogenic and osteogenic differentiation performance of equine bone marrow- and adipose tissue-derived mesenchymal stromal cells.

BACKGROUND: Bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stromal cells (MSCs) have shown potential as cell-based therapies for cartilage and bone injuries and are used increasingly in human and veterinary practice to facilitate the treatment of orthopedic conditions. However, human and rodent studies have documented a sharp decline in chondrogenic and osteogenic differentiation potential with increasing donor age, which may be problematic for the important demographic of older orthopedic patients. The aim of this study was to identify the effect of donor age on the chondrogenic and osteogenic differentiation performance of equine BM- and AT-MSCs in vitro. BM- and AT-MSCs and dermal fibroblasts (biological negative control) were harvested from horses in five different age groups (n = 4, N = 60); newborn (0 days), yearling (15-17 months), adult (5-8 years), middle-aged (12-18 years), and geriatric (≥ 22 years). Chondrogenic differentiation performance was assessed quantitatively by measuring pellet size, matrix proteoglycan levels, and gene expression of articular cartilage biomarkers. Osteogenic differentiation performance was assessed quantitatively by measuring alkaline phosphatase activity, calcium deposition, and gene expression of bone biomarkers. RESULTS: Chondrogenic and osteogenic differentiation performance of equine BM- and AT-MSCs declined with increasing donor age. BM-MSCs had a higher chondrogenic differentiation performance. AT-MSCs showed minimal chondrogenic differentiation performance in all age groups. For osteogenesis, alkaline phosphatase activity was also higher in BM-MSCs, but BM-MSCs calcium deposition was affected by donor age earlier than AT-MSCs. Chondrogenic and osteogenic differentiation performance of BM-MSCs exhibited a decline as early as between the newborn and yearling samples. Steady state levels of mRNA encoding growth factors, chondrogenic, and osteogenic biomarkers were lower with increasing donor age in both MSC types. CONCLUSIONS: The data showed that chondrogenic and osteogenic differentiation performance of equine BM-MSCs declined already in yearlings, and that AT-MSCs showed minimal chondrogenic potential, but were affected later by donor age with regards to osteogenesis (calcium deposition). The results highlight the importance of donor age considerations and MSC selection for cell-based treatment of orthopedic injuries and will help inform clinicians on when to implement or potentially cryopreserve cells. Moreover, the study provides molecular targets affected by donor age.

Pathologic Conditions of the Nervous System in Horses.

The variety of neurologic diseases which affect horses makes pathologic examination of the nervous system a complex and lengthy process. An understanding of the common causes of neurologic disease, antemortem neurolocalization, and supplementation of the necropsy examination with ancillary testing will help to diagnose a large number of causes of neurologic disease. A general understanding of neuropathology and collaborative relationship with your local pathologists will aid in the definitive diagnosis of neurologic diseases.

Determining an approximate minimum toxic dosage of diphacinone in horses and corresponding serum, blood, and liver diphacinone concentrations: a pilot study.

Poisoning of nontarget species is a major concern with the use of anticoagulant rodenticides (ARs). At postmortem examination, differentiating toxicosis from incidental exposure is sometimes difficult. Clotting profiles cannot be performed on postmortem samples, and clinically significant serum, blood, and liver AR concentrations are not well-established in most species. We chose diphacinone for our study because, at the time, it was the publicly available AR most commonly detected in samples analyzed at the University of Kentucky Veterinary Diagnostic Laboratory. We determined an approximate minimum toxic dosage (MTD) of oral diphacinone in 3 horses and measured corresponding serum, blood, and liver diphacinone concentrations. Diphacinone was administered orally to healthy horses. Prothrombin time (PT), activated partial thromboplastin time (aPTT), and serum and blood diphacinone concentrations were measured daily. At the study endpoint, the horses were euthanized, and diphacinone concentration was measured in each liver lobe. The horse that received 0.2 mg/kg diphacinone developed prolonged (>1.5× baseline) PT and aPTT; the horse that received 0.1 mg/kg did not. This suggests an approximate oral MTD in horses of 0.2 mg/kg diphacinone. Median liver diphacinone concentration at this dosage was 1,780 (range: 1,590-2,000) ppb wet weight. Marginal (model-adjusted) mean diphacinone concentrations of liver lobes were not significantly different from one another (p = NS). Diphacinone was present in similar concentrations in both serum and blood at each time after administration, indicating that both matrices are suitable for detection of diphacinone exposure in horses.

Compression generated by cortical screws in an artificial bone model of an equine medial femoral condylar cyst.

OBJECTIVE: Determine compression generated by lag and neutral screws over 12 h using two bone analogs. STUDY DESIGN: Experimental study. SAMPLE POPULATION: Bone analogs were made of composite synthetic bone (CSB) or three-dimensional printed polylactic acid (PLA). Analogs had a 2 mm exterior shell with a 10 mm thick internal layer of open-cell material. METHODS: Bone analogs were opposed, making a 4-sided box with open ends. A central channel contained the sensor and the screws passed through it to engage both paired analogs. Four screw/analog conditions were tested: neutral and lag screw with bicortical engagement, neutral and lag screw with unicortical engagement. All screws were tightened to 2 Nm torque and compression values recorded at 0, 0.5, 1, 2, 6, and 12 h (six trials per condition). Medians were compared across groups for statistical significance. RESULTS: There was no difference in median compression between lag and neutral bicortical screws. For PLA, greater median compression was generated by neutral (median 437 N) and lag (median 379 N) bicortical screws compared to neutral unicortical screws (median 208 N, p < .001); lag bicortical screws generated greater median compression than lag unicortical screws (median 265 N, p = .012). For CSB, lag bicortical screws (median 293 N) generated greater median compression than neutral unicortical screws (median 228 N, p = .008). CONCLUSION: Lag and neutral screws generated similar compression. Bicortical screws had higher median compression than unicortical screws in bone analogs. CLINICAL SIGNIFICANCE: Neutral screws generate compression in cancellous bone analogs that can be increased with bicortical bone engagement.

Microstructural features of subchondral radiolucent lesions in the medial femoral condyle of juvenile Thoroughbreds: A microcomputed tomography and histological analysis.

BACKGROUND: The aetiology of equine medial femoral condyle (MFC) subchondral bone radiolucencies (SR) is unknown. OBJECTIVES: Characterise the microstructural structural features of MFC SR in juvenile Thoroughbreds with microcomputed tomography (µCT) and histology. STUDY DESIGN: Cross-sectional post-mortem study. METHODS: Distal femurs were collected at post-mortem. Conventional tomodensitometry was employed to scout for MFCs with and without SR lesions (SR+ and SR-, respectively). Group 1 were CT MFC SR+ and Group 2 age-matched SR- controls. Both underwent µCT and histological analysis. Group 3 CT MFC SR- foals, <6 months, were selected to search for chondronecrosis. Histological sections, processed from the lesion (Group 1) and a corresponding site in Groups 2 and 3, were assessed for chondronecrosis, fibrin, fibroplasia and osteochondral separation. Group 3 sections were surveyed for chondronecrosis alone. RESULTS: A total of 178 femurs from 89 Thoroughbreds were harvested. Of these horses 19.1% (95% CI: 10.9%-27.3%) were CT MFC SR+ (17/23; 7.46 ± 4.36 months) and met the inclusion criteria for Group 1. Group 2 included 30 CT MFC SR- specimens (5.00 ± 2.73 months) and Group 3 had 44 CT MFC SR- s (2.68 ± 1.74 months). SR were located axially in foals <7 months of age, and centrally thereafter. All SRs had areas of thickened cartilage on histology and separation at the osteochondral junction containing fibrin (acute event) and fibroplasia (chronicity) in 73.9% (17/23; 95% CI: 56%-91.9%). In Group 1 specimens, chondronecrosis was present in 82.6% (19/23; 95% CI: 67.1%-98.1%) but four MFC SR+ had no evidence of chondronecrosis. Chondronecrosis was not detected in the Group 3 foal MFCs. MAIN LIMITATIONS: No longitudinal follow-up. CONCLUSIONS: The absence of chondronecrosis, pathognomic of osteochondrosis, in four MFC SR+s and in all of the CT MFC SR- foals suggests that osteochondrosis is not the cause, or the only cause, of these lesions and favours trauma as an alternate aetiological hypothesis.

Osteoclast density is not increased in bone adjacent to radiolucencies (cysts) in juvenile equine medial femoral condyles.

BACKGROUND: There is a knowledge gap about how equine MFC subchondral radiolucencies (SR) arise and evolve. Osteoclasts are believed to have a role but have not been studied in situ. OBJECTIVES: To measure and compare osteoclast density and the percentage of chondroclasts in healthy and MFC SR specimens from juvenile Thoroughbreds. STUDY DESIGN: Cadaveric study. METHODS: Medial femoral condyles (MFC) from a tissue bank of equine stifles were studied. Inclusion criteria were MFCs (≤8 months old) with a computed tomography SR lesion and histological focal failure of endochondral ossification (L group). Contralateral, lesion-free, MFCs were a control group (CC). Osteochondral slabs were cut through the lesion (L), a healthy site immediately caudal to the lesion, (internal control; IC) and the contralateral, site-matched controls (CC). Histological sections were immunostained with Cathepsin K for osteoclast counting. Osteoclasts in contact with the growth cartilage (chondroclasts) were also counted. The sections were segmented into regions of interest (ROI) at different depths in the subchondral bone: ROI1 (0-1 mm), ROI2 (1-3 mm) and ROI3 (3-6 mm). Osteoclasts were counted and the bone area was measured in each ROI to calculate their density. Chondroclasts were counted in ROI1 . RESULTS: Sections were studied from L and IC (n = 6) and CC sites (n = 5). Osteoclast density was significantly higher in ROI1 when compared with ROI3 in all groups. Although higher osteoclast density was measured in ROI1 in the L group, no significant differences were detected when compared with control ROIs. The proportion of chondroclasts in ROI1 was lower in the L sections when compared with controls but no significant differences were detected. MAIN LIMITATIONS: Limited sample size. CONCLUSIONS: Osteoclasts are important actors in MFC subchondral bone development, digesting both growth cartilage (chondroclasts) and bone, but the pathophysiology of early MFC SRs cannot be explained solely by an increased osteoclast presence in the subchondral bone.

Detection and residence time of bisphosphonates in bone of horses.

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.

Chondrogenic differentiation potential of adult and fetal equine cell types.

OBJECTIVE: To determine the chondrogenic potential of cells derived from interzone tissue, the normal progenitor of articular cartilage during fetal development, compared to that of adult bone marrow-derived and adipose-derived mesenchymal cell isolates. The objective of this study was to compare the chondrogenic potential of fetal musculoskeletal progenitor cells to adult cell types, which are currently used therapeutically to facilitate joint cartilage repair in equine clinical practice. The hypothesis tested was that cells derived from interzone tissue have a chondrogenic potential that exceeds that of adult bone marrow-derived and adipose-derived mesenchymal cell isolates. STUDY DESIGN: In vitro study. ANIMALS: Six young adult horses (15-17 months of age) and 6 equine fetuses aged 45-46 days of gestation. METHODS: Three-dimensional pellet cultures were established under chondrogenic conditions with fresh, primary cells isolated from adult (articular cartilage, bone marrow, adipose, dermis) and fetal (interzone, skeletal anlagen cartilage, dermis) tissues. Cellular morphology, pellet architecture, and proteoglycan synthesis were assessed in the pellet cultures. Steady state levels of ACAN (aggrecan core protein), COL2A1 (collagen type II), and COL1A1 (collagen type I) messenger RNA (mRNA) were compared among these cell types as pellet cultures and monolayer cultures. RESULTS: Adult articular chondrocytes, fetal interzone cells, and fetal anlage cells generated the largest pellets under these chondrogenic culture conditions. Pellets derived from adult articular chondrocytes and fetal anlage cells had the highest scores on a neocartilage grading scale. Fetal anlage and adult articular chondrocyte pellets had low steady-state levels of COL1A mRNA but high COL2A1 expression. Anlage chondrocyte pellets also had the highest expression of ACAN. CONCLUSION: Adult articular chondrocytes, fetal interzone cells, and fetal anlage chondrocytes exhibited the highest chondrogenic potential. In this study, adult adipose-derived cells exhibited very limited chondrogenesis, and bone marrow-derived cells had limited and variable chondrogenic potential. CLINICAL SIGNIFICANCE: Additional investigation of the high chondrogenic potential of fetal interzone cells and anlage chondrocytes to advance cell-based therapies in diarthrodial joints is warranted.

Detection, molecular characterization and phylogenetic analysis of G3P[12] and G14P[12] equine rotavirus strains co-circulating in central Kentucky.

Equine rotavirus A (ERVA) is the leading cause of diarrhea in neonatal foals and a major health problem to the equine breeding industry worldwide. The G3P[12] and G14P[12] ERVA genotypes are the most prevalent in foals with diarrhea. Control and prevention strategies include vaccination of pregnant mares with an inactivated vaccine containing a prototype ERVA G3P[12] strain with limited and controversial field efficacy. Here, we performed the molecular characterization of ERVA strains circulating in central Kentucky using fecal samples collected during the 2017 foaling season. The data indicated for the first time that the G14P[12] genotype is predominant in this region in contrast to a previous serotyping study where only G3 genotype strains were reported. Overall, analysis of antigenic sites in the VP7 protein demonstrated the presence of several amino acid substitutions in the epitopes exposed on the surface including a non-conserved N-linked glycosylation site (D123N) in G14P[12] strains, while changes in antigenic sites of VP8* were minor. Also, we report the successful isolation of three ERVA G14P[12] strains which presented a high identity with other G14 strains from around the world. These may constitute ideal reference strains to comparatively study the molecular biology of G3 and G14 strains and perform vaccine efficacy studies following heterologous challenge in the future.

Common lesions of the distal end of the third metacarpal/metatarsal bone in racehorse catastrophic breakdown injuries.

Equine catastrophic skeletal breakdown injury is a serious issue within the racing industry, given the impact on equine and human health. The metacarpo- and metatarso-phalangeal (fetlock) joints are common sites of catastrophic injury. However, lesions involving articular cartilage, subchondral bone, and synovium are commonly identified within the fetlock of the contralateral limb; hence, it is imperative that lesions in both limbs are evaluated and characterized during postmortem examination. Bone and articular cartilage changes typically occur in specific locations, related to cyclic fetlock load and overextension during high-speed exercise. Associations between preexisting degenerative fetlock lesions and catastrophic injury are a focus of continued research. These lesions often occur because of adaptive failure related to cumulative damage. Further investigation of these lesions is imperative to determine their impact on equine performance or injury. Ultimately, consistent documentation of catastrophic versus non-catastrophic osteochondral lesions provided by pathologists, in the context of training history, diagnostic imaging, and the presence or absence of catastrophic injury, will contribute to further understanding of skeletal responses associated with catastrophic failure.

A diagnostic pathologist's guide to carpal disease in racehorses.

As a pathologist, postmortem examination of the equine carpus can be daunting. The anatomy is complex and oftentimes, small or subtle lesions have significant impact on lameness and secondary lesions such as catastrophic musculoskeletal fractures and other injuries. In performance horses, particularly racehorses, the carpus is a common site of injury and source of lameness. Given the predisposition of racehorses to developing carpal disease, familiarity with clinically relevant anatomy and common developmental, degenerative, traumatic, and inflammatory processes are imperative for thorough postmortem examination. Our aim is (1) to provide a concise summary of clinically relevant anatomy and function that serves as a guide for postmortem evaluation of the equine carpus, and (2) to review common carpal injuries and diseases in actively training, racing, or retired racehorses, including developmental lesions (incomplete ossification, osteochondromata), infectious and inflammatory lesions (septic arthritis and tenosynovitis), and degenerative and traumatic lesions (degenerative and traumatic osteoarthritis, osteochondral fragmentation, and polyostotic catastrophic "breakdown" fractures). Representative gross and histologic images are presented along with corresponding antemortem and postmortem diagnostic images, and a review of current scientific literature pertaining to the pathogenesis of these equine carpal lesions.