Effects of a P-class CpG-ODN administered by intramuscular injection on plasma cytokines and on white blood cells of healthy horses.

Cytosine-phosphate-guanosine (CpG-ODN) has been described as a potent immunostimulatory agent in different species. No study reported the effect of a P-class CpG when administered systemically in healthy horses. The aim of this study was to evaluate the tolerance and the effect of an intramuscularly administered P-class CpG-ODN on hematology and on plasma cytokines (IFN-α, IL-10, TNF-α, IFN-γ) in 8 healthy horses. Intra-muscular CpG-ODN or placebo (PBS) was administered twice at a 7 days-interval. Groups were inversed after 2 months of washout period. A physical examination, complete blood count (CBC) and plasma cytokine measurements were performed from 2 days before injection up to 21 days after injection. P-class CpG-ODN injection was well tolerated with minor side effects. After the first injection a significant transient drop in circulating total leukocytes, lymphocytes and an increase in monocytes were observed. A transient drop in eosinophils was also noted after each CpG injection. P-class CpG-ODN at a dose of 5 mg did not create major side effects in 7 horses, one horse showed transient pyrexia. A redistribution of white blood cells was observed in horses receiving CpG, but no change in plasma cytokines was observed at the indicated dose, route of administration and sampling times.

Characterization of collagen fibrils after equine suspensory ligament injury: an ultrastructural and biochemical approach.

Suspensory ligament (SL) injuries are an important cause of lameness in horses. The mechanical properties of connective tissue in normal and pathological ligaments are mainly related to fibril morphology, as well as collagen content and types. The purpose of this study was to evaluate, using biochemical and ultrastructural approaches, the alterations in collagen fibrils after injury. Eight Warmblood horses with visible signs of injury in only one forelimb SL were selected and specimens were examined by transmission electron microscope (TEM). Collagen types I, III and V were purified by differential salt precipitation after collagen extraction with acetic acid containing pepsin. TEM revealed abnormal organization as well as alterations in the diameter and shape of fibrils after SL injury. The bands corresponding to types I, III and V collagen were assessed by densitometry after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis indicated that the proportions of type III and type V collagen were higher (P < 0.001) in damaged tissues compared with normal tissues with a mean increase of 20.9% and 17.3%, respectively. Concurrently, a decrease (P < 0.001) in type I collagen within damaged tissues was recorded with a mean decrease of 15.2%. These alterations could be the hallmark of a decrease in the tissue quality and mechanical properties of the ligament. The findings provide new insight for subsequent research on tissue regeneration that may lead to the development of future treatment strategies for SL injury.

Comparison of superficial digital flexor tendon loading on asphalt and sand in horses at the walk and trot.

The incidence of superficial digital flexor tendon (SDFT) injuries is one of the highest of all equine musculoskeletal conditions. Horses with SDFT injuries commonly show no improvement of lameness on soft ground, unlike those suffering from distal bone or joint lesions. The aim of this study was to compare the SDFT loading in five horses at the walk and trot on asphalt and sand using a non-invasive ultrasonic tendon force measurement device. Three horses were equipped with the ultrasonic device, whereas the other two horses were equipped with the ultrasonic device and a dynamometric horseshoe (DHS); the DHS was used to calibrate the measured values of tendon speed of sound (SOS) converted to tendon force, while a previously established ground reaction force pattern was used to calibrate SOS measurements for the other three horses. Although the horses tended to be slower on S, maximal tendon force was higher on sand than on asphalt at the trot (+6%); there was no significant difference between the two surfaces at the walk. The duration of tendon loading was longer on S (+5%) and the area under the tendon force-time curve was larger on S (+10%) at both walk and trot. SDFT loading is significantly affected by the ground surface and the observed increase in SDFT loading on sand compared with asphalt is consistent with clinical observations in horses with SDFT injuries.

Comparative kinematic analysis of the leading and trailing forelimbs of horses cantering on a turf and a synthetic surface.

REASONS FOR PERFORMING STUDY: The relationship between track surface properties and limb kinematics is poorly understood. Hoof orientation within the track surface has never been quantified under training conditions. Previously described kinematic and dynamic differences between leading and trailing forelimbs at the canter poorly correlate with epidemiological data regarding injuries. OBJECTIVES: To compare joint kinematics and hoof orientation in the leading and trailing forelimbs of horses cantering on turf and on a synthetic surface. STUDY DESIGN: Noninvasive experimental study. METHODS: The right forelimb of 5 horses was equipped with markers facing the main joints while markers and a dynamometric horseshoe were placed on the hoof. The horses were filmed with 2 high-speed cameras (1000 Hz) while cantering (30 km/h). Recordings were repeated at each lead and alternated on turf and on a synthetic surface. Joint angles and angles of the hoof and limb to the track were measured from the 2-dimensional coordinates of the markers. RESULTS: Elbow, carpus and fetlock were more maximally flexed during swing and had a larger range of motion throughout the stride in the leading forelimb. Maximal carpal extension during stance was also larger on this limb, which had a more toe-up orientation. Comparing surfaces, the limb was more oblique at landing, the range of motion of the hoof into the surface was larger, most kinematic events were delayed and fetlock and carpus extension velocities were smaller on the synthetic surface. CONCLUSIONS: The differences between limbs were more prominent than those between surfaces and the more toe-up orientation on the hoof of the leading forelimb suggests a different loading of that limb's joints and tendons. Differences between limbs may be important in the interpretation of lead changes in lame horses. While the synthetic surface appears to be less strenuous for the joints in the forelimbs, it was associated with changes in timing of the kinematic events of the stride.

Longitudinal development of equine forelimb conformation from birth to weaning in three different horse breeds.

There is limited published data on conformational changes in the forelimbs of growing foals. This study was designed to describe the changes in conformation of the carpus and distal forelimb from birth to weaning in foals of three different breeds. Evaluation of the conformation of the carpus, fetlock, pastern and foot was carried out in 134 Thoroughbreds, 162 French Trotters and 98 Selle Français (French Warmblood) within 1 month of age and then at approximately 2 month intervals until weaning at approximately 6 months of age. The prevalence of limb deviations decreased from birth to weaning. Angular limb deformities were the predominant conditions in the first month (63.6% of all observed limb deviations) and flexural limb deformities were the most common abnormalities at weaning. The most frequent congenital abnormalities were carpal valgus (42.1% of the foals), fetlock valgus (31.2%), over-at-the-knee (30.8%) and dropped fetlocks (13.0%), with French Trotters and Thoroughbred foals being more affected than Selle Français foals. During the study period, the carpal and fetlock conformation became less valgus. The predominant abnormalities at weaning were fetlock valgus (19.1%), club feet (13.0%) and fetlock varus (11.2%). These observations show that carpal, fetlock and foot conformations changed substantially from birth and weaning. There were significant conformational differences between the three breeds, especially in the first months of life. An understanding of the peculiarities of specific breeds may be useful for the evaluation of individual foal conformation.

Evolution of radiological findings detected in the limbs of 321 young horses between the ages of 6 and 18 months.

The objective of this study was to evaluate the spontaneous evolution of radiological findings (RF) indicative of juvenile osteochondral conditions (JOCC) in a field study. A radiographic survey was performed at the age of 6 and 18months in 321 young horses from three breeds (French Trotter Standardbred, Selle-Français Warmblood and Thoroughbred). Each RF identified at 6months was re-evaluated at 18months, and classified as (1) disappeared, (2) improved, (3) stable, (4) deteriorated or (5) new when it was not identified at 6months. Only 32.3% of all RF identified at the age of 6months were stable, while at 18months 46.6% had disappeared and 38.7% were new radiological abnormalities. Evolution of RF varied according to the joint involved and the type of lesion. In the stifle, 85.7% of the RF of osteochondral fragmentation (OCF) of the lateral ridge of the femoral trochlea regressed (disappeared or improved). In the hock, 53.1% of the RF of OCD of the intermediate ridge of the tibial cochlea were stable. In the fetlock, dorsal and plantar OCF were frequently observed as new radiological abnormalities at 18months (51% of the dorsal RF and 45.8% of the plantar). As many lesions develop or change after 6months, a definitive radiographic assessment of the JOCC status is more reliable at 18months. Knowing the spontaneous healing capacity of some lesions is useful in helping to decide the appropriate management.

Genetic parameters of juvenile osteochondral conditions (JOCC) in French Trotters.

Juvenile osteochondral conditions (JOCC) have been defined as lesions resulting from biomechanical influences (compressive, tensional or shear forces) on the developing and growing musculoskeletal system. They include different types of osteochondrosis, osteochondral fragmentation of the articular surface or of the periarticular margins, juvenile subchondral bone cysts, osteochondral collapse, avulsion fractures of epiphyseal (or metaphyseal) ossifying bone and 'physitis'. The aim of this study was to estimate heritability of JOCC in a sample of 2106 French Trotters from four different sources, comprising representative samples of the Trotter population, as well as material from auctions. Horses were aged 6-24months and were either not yet in training or just beginning training. Radiographs were taken of fore and hind feet, including proximal interphalangeal (pastern) joints, metacarpophalangeal and metatarsophalangeal (fetlock) joints, tarsocrural (hock) joints, carpi and femoropatellar (stifle) joints. The threshold model used included sex, age, region and month of birth, sampling group and sire (n=159) with all inter-sire relationships. The main results were a moderate heritability for findings in the hind fetlock (0.29) and the hock (0.19). There was a weak genetic correlation between findings in fetlocks and hocks (0.26). Higher heritability was found for findings in the hock (0.37 for findings in the distal row and 0.49 for the proximal row of tarsal bones) in that part of the data (699 horses) in which it was possible to integrate the grade, bilateral occurrence or not, and distal or proximal location of the lesions. It is possible to use these genetic parameters in breeding selection with more efficiency when detailed phenotypes are considered.

Radiographic findings of juvenile osteochondral conditions detected in 392 foals using a field radiographic protocol.

Radiographic examination in young horses is commonly used to detect juvenile osteochondral conditions (JOCC). The aim of this study is to present the radiographic findings (RF) detected at the age of 6months in the joints of 392 foals from three breeds: French Trotter Standardbreds (FT), Selle-Français Warmbloods (SF) and Thoroughbreds (TB). Radiographic examination included lateromedial views of the fore and hind digits, tarsi and stifles, and dorsopalmar projections of both carpi. In all areas RF indicative of JOCC were classified according to a four-grade severity scoring system. Distribution and severity of the RF varied according to breed with the SF most affected. The most frequently affected site was the hind fetlock (28.3% of foals). The second most affected site was the fore fetlock in TB and SF, while the carpus and proximal tarsus were the second and third affected sites in FT. The radiographic protocol and grading system used can serve as a basis for field research and further studies on the evolution of RF, risk factors for JOCC or the correlation between RF and the subsequent athletic career of horses.

Comparison between standard ultrasonography, angle contrast ultrasonography, and magnetic resonance imaging characteristics of the normal equine proximal suspensory ligament.

Previous studies have proposed that standard ultrasonography may not adequately represent the pertinent anatomic characteristics of the equine proximal suspensory ligament. The purpose of the study was to compare the use of standard ultrasonography, angle contrast ultrasonography, MRI, and histology for identification of the anatomic characteristics of the normal equine suspensory ligament in the forelimb. Horses free from forelimb lameness with no palpable abnormalities in the region of the suspensory ligament were included in the study. The proximal suspensory ligaments in 20 forelimbs were examined using the standard ultrasound technique, angle contrast ultrasound technique, and MRI, followed by histologic evaluation. Total transverse (cross-sectional) area of the proximal suspensory ligament was estimated using the standard ultrasound and the angle contrast ultrasound techniques, MRI, and histologic sections for the following parameters: total area of the ligament, ligament fibers, muscle, and fat. The proximal suspensory ligament lobe size and tissue distribution were compared and subjectively graded (0-4) for asymmetry. Subjectively, angle contrast ultrasound technique improved differentiation of fibers from the remaining tissue types and allowed identification of the peripheral ligament margin. There was no significant difference in asymmetry scores between modalities. The asymmetry scores of the right and left forelimbs were significantly different with both ultrasound and MRI, based on the level of measurement. The angle contrast ultrasound technique has limitations compared to MRI. However, it provides additional diagnostic information that is not available with the standard ultrasound technique.

A review of terminology for equine juvenile osteochondral conditions (JOCC) based on anatomical and functional considerations.

This manuscript describes a new classification of the various joint-related lesions that can be seen in the young, growing horse based on their anatomical and functional aetiopathogenesis. Juvenile osteochondral conditions (JOCC) is a term that brings together specific disorders according to their location in the joint and their biomechanical origin. When a biomechanical insult affects the process of endochondral ossification different types of osteochondrosis (OC) lesions may occur, including osteochondral fragmentation of the articular surface or of the periarticular margins, or the formation of juvenile subchondral bone cysts. In severe cases, osteochondral collapse of the articular surface or the epiphysis or even an entire small bone may occur. Tension on ligament attachments may cause avulsion fractures of epiphyseal (or metaphyseal) ossifying bone, which are classified as JOCC, but do not result from a disturbance of the process of endochondral ossification and are not therefore classified as a form of OC. The same applies to 'physitis' which can result from damage to the physeal growth plate.

Morphometric analyses of the body and the branches of the normal third interosseous muscle (suspensory ligament) in Standardbreds.

The third interosseous muscle (suspensory ligament, TIOM) is composed of connective tissue (CT) with a variable proportion of muscle (MT) and adipose tissue (AT). The aim of our study is to quantify the CT, MT and AT within the body and the branches of right thoracic and pelvic limbs TIOM in sound horses to determine whether there are differences in CT, MT and AT between age, sex, limbs and levels. Right limbs from 11 sound horses were collected. Samples from 6 levels of the TIOM were embedded in paraffin or in Tissue-Tek(®) . Most of the paraffin sections were shredded. Using the cryosection, some artefacts appeared. Cryoprotection was carried out, which produced the best results. Hematoxylin-phloxine-saffron and Hematoxylin-eosin gave a good contrast of colours between the tissues observed allowing the use of an image analysis programme to calculate percentage of each tissue within the TIOM. The percentage of MT and AT decreased significantly (P < 0.0001), whereas the percentage of CT increased significantly (P < 0.0001) with age and when descending from the proximal to the distal level of the TIOM. The percentage of MT was significantly higher (P < 0.0001) in females than males, while the percentage of CT was significantly higher (P < 0.0001) in males than females. The percentage of AT was significantly higher (P = 0.0278) in pelvic limbs than in thoracic limbs. These results confirm the variation in tissue composition within the TIOM of sound horses.

Genome-wide association studies for osteochondrosis in French Trotter horses.

A genome-wide association study for osteochondrosis (OC) in French Trotter horses was carried out to detect QTL using genotype data from the Illumina EquineSNP50 BeadChip assay. Analysis data came from 161 sire families of French Trotter horses with 525 progeny and family sizes ranging from 1 to 20. Genotypes were available for progeny (n = 525) and sires with at least 2 progeny (n = 98). Radiographic data were obtained from progeny using at least 10 views to reveal OC. All radiographic findings were described by at least 2 veterinary experts in equine orthopedics, and severity indices (scores) were assigned based on the size and location of the lesion. Traits used were a global score, the sum of all severity scores lesions (GM, quantitative measurement), and the presence or absence of OC on the fetlock (FM), hock (HM), and other sites (other). Data were analyzed using 2 mixed models including fixed effects, polygenic effects, and SNP or haplotype cluster effects. By combining results with both methods at moderate evidence of association threshold P < 5 × 10(-5), this genome-wide association study displayed 1 region for GM on the Equus caballus chromosome (ECA) 13, 2 for HM on ECA 3 and 14, and 1 for other on ECA 15. One region on ECA 3 for HM represented the most significant hit (P = 3 × 10(-6)). By comparing QTL between traits at a decreased threshold (P < 5 × 10(-4)), the 4 QTL detected for GM were associated to a QTL detected for FM or HM but never both. Another interesting result was that no QTL were found in common between HM and FM.