Atsutoshi Kuwano

Prevalence of superficial digital flexor tendonitis and suspensory desmitis in Japanese Thoroughbred flat racehorses in 1999

REASONS FOR PERFORMING STUDY Overstrain injuries to the superficial digital flexor tendon (SDFT) and suspensory ligament (SI) are among the most common musculoskeletal injuries which contribute to the considerable wastage of racing Thoroughbreds. Many epidemiological studies have demonstrated the prevalence of and risk factors for tendon injury when racing but have not included those injuries sustained during training. However, since tendon injury during training is seen commonly in clinical practice, it is appropriate to determine the overall prevalence of tendon injury sustained during both training and racing. OBJECTIVE To determine the prevalence of overstrain injury to the SDFT and SL during training and racing among Thoroughbred flat racehorses in Japan in 1999. METHODS A retrospective study was performed using a sample population of 10,262 Thoroughbred racehorses. The medical information database of Thoroughbred racehorses registered by the Japan Racing Association (JRA) in 1999 was analysed for SDFT and SL overstrain injury diagnosed by a veterinarian employed by JRA during training and racing. Jump racehorses were excluded from this study. RESULTS The prevalence of forelimb SDFT tendonitis and SL desmitis was 11.1% (1130 cases) and 3.61% (370 cases) of the population, respectively. In the hindlimb, there were 0.06% (6 cases) and 0.14% (14 cases), respectively. Risks of SDF tendonitis in the forelimb in 3-year-olds or older horses were significantly higher than in 2-year-olds. In contrast, the risk of SL desmitis in the forelimb at age 3 and 4 years was 2.23 and 2.11 times higher, respectively, than in 2-year-olds, but this increased to 5.07 times in those age > or = 5 years. Entire males were at greater risk in comparison to females and geldings. CONCLUSIONS The results suggest that the prevalence of SDF tendonitis and SL desmitis in the forelimb was associated with the horses age and sex. The prevalence of SL desmitis increased further with age compared with SDF tendonitis, possibly reflecting a more rapid accumulation of degeneration in this structure. POTENTIAL RELEVANCE The age-related risk demonstrated in this study provides further support that overstrain injuries are associated with accumulated degeneration. These data provide a valuable resource for further research into the aetiology of tendon injury in the racehorse.

The lamellar wedge.

In horses with chronic laminitis, an abnormal horn structure called the lamellar wedge develops within the lamellar region of the foot. This pathologic structure adversely affects normal foot function, and influences return to previous performance levels. Understanding the pathologic process that leads to the development of this structure is essential for correct supportive foot management of the horse with chronic laminitis. The ability to prevent or reduce the formation of the lamellar wedge may eventually lead to better outcomes in cases of laminitis.

TGF-β alters collagen XII and XIV mRNA levels in cultured equine tenocytes

Abstract The effects of TGF-β1, β2 and β3 (TGF-β) on levels of mRNA corresponding to the α chains of type XII and type XIV collagens in equine tenocyte cultures were assessed using the ribonuclease protection assay (RPA). The level of α1(XII) mRNA in untreated monolayer cultures was approximately 50- to 100-fold greater than α1(XIV) mRNA level. Addition of TGF-β resulted in an increase in the amount of α1(XII) present and a decrease of α1(XIV) mRNA in a dose-dependent manner. Specifically, the level of α1(XII) mRNA was doubled, but α1(XIV) was decreased to 30% of control by the addition of 2 ng/ml of TGF-β1 to the cultures. These effects were completely abrogated by neutralizing antibody specific for TGF-β. In addition, electron microscopy demonstrated that bundled collagen fibers were formed in the intercellular spaces of multilayered tenocytes which had been cultured for 2 weeks in the presence of exogenous TGF-β1 together with ascorbic acid phosphate. These results suggest that type XII and/or type XIV collagens modulate the structure of ECM formed by tenocytes in culture.

Muscle fiber population and biochemical properties of whole body muscles in thoroughbred horses.

We examine the muscle fiber population and metabolic properties of skeletal muscles from the whole body in Thoroughbred horses. Postmortem samples were taken from 46 sites in six Thoroughbred horses aged between 3 and 6 years. Fiber type population was determined on muscle fibers stained with monoclonal antibody to each myosin heavy chain isoform and metabolic enzyme activities were determined spectrophotometrically. Histochemical analysis demonstrated that most of the muscles had a high percentage of Type IIa fibers. In terms of the muscle characteristic in several parts of the horse body, the forelimb muscles had a higher percentage of Type IIa fiber and a significantly lower percentage of Type IIx fiber than the hindlimb muscles. The muscle fiber type populations in the thoracic and trunk portion were similar to those in the hindlimb portion. Biochemical analysis indicated high succinate dehydrogenase activity in respiratory‐related muscle and high phosphofructokinase activity in hindlimbs. We suggested that the higher percentage of Type IIa fibers in Thoroughbred racehorses is attributed to training effects. To consider further the physiological significance of each part of the body, data for the recruitment pattern of each muscle fiber type during exercise are needed. The muscle fiber properties in this study combined with the recruitment data would provide fundamental information for physiological and pathological studies in Thoroughbred horses. Anat Rec, 2009.

Anatomical location and culture of equine corneal epithelial stem cells

OBJECTIVE To identify morphologically the locations of equine corneal epithelial stem cells (CESCs) and to culture these cells. ANIMALS STUDIED We studied the eyes of 12 adult thoroughbred horses. PROCEDURES Eye tissues were immunostained for two positive stem cell markers (p63, CK14) and one negative marker (CK3) to identify the locations of CESCs, so we could compare their immunostaining patterns with those of human stem cells previously reported. We compared the proliferation rates and morphological features of epithelial cells isolated from the corneal limbus and central cornea. RESULTS Undifferentiated cells expressing the same immunostaining pattern as human CESCs were present in the equine corneal limbus. Cultured epithelial cells isolated from the limbus expressed the same immunostaining pattern that CESCs show histologically, but cells isolated from the central cornea did not proliferate and could not be evaluated. CONCLUSIONS Equine CESCs were localized in the epithelial basal layer of the corneal limbus, where melanocytes reside. They could be cultured without loss of their undifferentiated nature. When collecting such stem cells, it may be useful to harvest and culture corneal epithelial tissues in the limbus where melanocytes serve as an indicator of the collecting area.

Regulation of Tenomodulin Expression Via Wnt/β-catenin Signaling in Equine Bone Marrow-derived Mesenchymal Stem Cells.

ABSTRACT Tenomodulin has been recognized as a biomarker for tendon differentiation, and its gene expression is regulated by several transcription factors including Scleraxis and Mohawk. In this study, we found a novel regulatory mechanism of tenomodulin expression. Equine bone marrow-derived mesenchymal stem cells (BMSCs) in monolayer culture showed a low mRNA level of tenomodulin in comparison with the level in the tendon. When cultured in collagen gel containing a glycogen synthase kinase-3 (GSK-3) inhibitor (BIO), expression of tenomodulin in BMSCs increased up to the level in the tendon. Participation of GSK-3 in its gene expression was further demonstrated by a gene silencing experiment with small interference RNA corresponding to GSK-3, suggesting that Wnt/β-catenin signaling mediated expression of tenomodulin. These results were confirmed by nuclear translocation of β-catenin in BIO-treated BMSCs cultured in collagen gel. Under this culture condition, expression of tenomodulin-related transcription factors including Scleraxis and Mohawk was not affected, suggesting that Wnt/β-catenin signaling was independent from these transcription factors. Additionally, BIO strongly enhanced expression of type XIV collagen in collagen-embedded BMSCs up to the level in the tendon, and other tendon-related extracellular matrix components such as decorin and fibromodulin were also upregulated. Taken together, these results indicated that activation of Wnt/β-catenin signaling could induce differentiation of BMSCs into tenomodulin-expressing tendon cells in collagen gel.

Effects of bedding material on the lying behavior in stabled horses.

The objective of this study was to investigate the effect of straw, sawdust, coconut husk (husk), and coconut fiber (fiber) on the welfare of stable horses by observing their resting behavior. Twenty horses with ages ranging from 3 to 21 years were used at the Equine Research Institute of the Japan Racing Association, Utsunomiya, Japan. Five horses were allocated to each bedding condition. The behavior of each horse was recorded by video camera for 3 days and was continuously sampled from 17:00 to 05:00. The total duration, the number of bouts, and the mean and the maximum duration of bouts in standing rest, sternal lying, and lateral lying were calculated and analysed by the Kruskal-Wallis test and post hoc Steel-Dwass test. There was no difference in the standing rest and the sternal lying among beddings. Significant differences were observed in these values in the lateral lying among the different beddings (P<0.05). The values of the means of the total duration, the number of bouts, and the mean and the maximum duration of bout in the lateral lying were greater when husk was used as the bedding material than when sawdust were used (P<0.05). The results of the observations show that the new bedding materials would be as usable as straw. However, lateral lying was observed less frequently when sawdust were used as bedding; this indicates that use of sawdust as bedding material will decrease the welfare of stabled horses.

Methicillin-resistant Staphylococcus aureus ulcerative keratitis in a Thoroughbred racehorse.

ABSTRACT We report the first case of methicillin-resistant Staphylococcus aureus (MRSA) keratitis in a racehorse. A 5-year-old mare developed punctate keratitis after racing. The corneal ulcer continued to expand despite ophthalmic antimicrobial therapy. On day 6, a conjunctival graft surgery was performed. The mare was euthanized, following colitis and laminitis development on day 10. MRSA was isolated from the corneal swab taken at the time of euthanasia. Immunohistochemical analysis demonstrated gram-positive and anti-S. aureus monoclonal antibody-positive cocci infiltration of the corneal stroma; and a diagnosis of MRSA ulcerative keratitis was made. An ophthalmic antimicrobial against the isolated MRSA did not improve the ocular lesion. The MRSA strain was found to be staphylococcal cassette chromosome mec type II, a strain frequently isolated from humans in Japan.

Meticillin-resistant Staphylococcus aureus colonisation and infection in Thoroughbred racehorses and veterinarians in Japan

Meticillin-resistant Staphylococcus aureus (MRSA) infections have been confirmed in hospitalised Thoroughbred racehorses at the hospitals of two training centres in Japan since 2009. To investigate the source of infection, the authors examined the rate of nasal MRSA colonisation in 600 healthy Thoroughbred racehorses, 53 veterinarians and 16 office staff at the racehorse hospitals of the two training centres. MRSA was not isolated from healthy Thoroughbred racehorses or hospital office staff. However, MRSA was isolated from 16 veterinarians (30.1 per cent), and the colonisation rate was significantly higher in veterinarians than in the office staff of the same hospitals. Also, 10 of the 16 MRSA strains (62.5 per cent) isolated from veterinarians were classified as type II by staphylococcal cassette chromosome mec (SCCmec) typing and ST5 by multilocus sequence typing. Pulsed-field gel electrophoresis analysis demonstrated that these 10 MRSA strains of SCCmec type II and ST5 were genetically identical or very similar to 9 MRSA strains isolated from infected horses hospitalised at these hospitals between 2009 and 2013. These results indicate that SCCmec type II and ST5 MRSA strains were probably transmitted between veterinarians and infected horses.

Immunohistochemical and ultrastructural detection of intestinal spirochetes in Thoroughbred horses

Studies of equine intestinal spirochetes have long focused on intestinal contents alone, but intestinal spirochetosis has been reported recently in a 21-month-old Thoroughbred colt in Japan. To define the clinical and pathological significances of intestinal spirochetosis in several horses, an epizootiologic survey with histologic, immunohistochemical, and ultrastructural methods was conducted for Brachyspira antigen–containing intestinal spirochetes in 12 diseased or injured Thoroughbred horses, aged from 35 days to 17 years. Brachyspira antigen–containing spirochetes were found in 7 of 12 horses (58.3%) and were more frequent in the cecum than in other parts of the bowel. It was not clear whether the infection was clinically related to diarrhea or dysentery, but histopathology revealed a close association between the bacterial infection and epithelial hyperplasia. Crypt epithelium consisted mainly of goblet cells and showed frequent mitosis throughout its length. Inflammatory cells and congestion were also present. There were numerous spirochetes in the crypts, and some invaded the cecal and colonic epithelia and underlying lamina propria. Ultrastructurally, the spirochetes were divided into 4 types. Three types were identified in degenerative epithelial cells or intracellularly. Brachyspira antigen–containing intestinal spirochetes invading the mucosa were capable of causing epithelial hyperplasia in the cecum and colon in the horses. The findings in this study will increase awareness of the importance of intestinal spirochetosis and may also be helpful for diagnosis and treatment of this condition.