Johannes H. van der Kolk

The role of electromyography in clinical diagnosis of neuromuscular locomotor problems in the horse.

REASONS FOR PERFORMING STUDY Systematically performed EMG needle examination of muscles provides essential information about the functional aspects of the motor unit. However, clinical studies in which information is given on the diagnostic and discriminative values of electromyography (EMG) in the horse are scarce. OBJECTIVES To determine to what extent inclusion of EMG analysis in clinical examination contributes to determination of type and localisation of abnormality. METHODS EMG analysis, complete clinical examination and diagnosis of 108 horses (mean +/- s.d. age 75 +/- 3.8 years; bodyweight 548 +/- 86 kg; height 1.67 +/- 0.07 m) were performed, and results without and with EMG analysis compared. RESULTS Without EMG, myopathy and neuropathy were diagnosed in 20 and 58 horses, respectively, and with EMG in 17 and 82 horses. EMG changed localisation in myopathy and neuropathy in 12 and 37% of cases, respectively. Lesions in the C1-T2, T2-L3 and L3-S3 segments were, respectively, diagnosed without EMG in 7, 11 and 30%, and with EMG in 27, 7 and 17% of cases. Where no clinical diagnosis could be made prior to EMG, many patients appeared to be suffering from localised cervical lesions (29%) or generalised neuropathy (54%). CONCLUSIONS AND POTENTIAL RELEVANCE The assistance of EMG in discriminating between normal, neuropathy and myopathy, and in locating pathology, contributes to diagnosis of neuromuscular problems.

Development and evaluation of real-time PCR assays for the quantitative detection of Babesia caballi and Theileria equi infections in horses from South Africa

A quantitative real-time polymerase chain reaction (qPCR) assay using a TaqMan minor groove binder (MGB) probe was developed for the detection of Babesia caballi infection in equids from South Africa. Nine previously published sequences of the V4 hypervariable region of the B. caballi 18S rRNA gene were used to design primers and probes to target unique, conserved regions. The B. caballi TaqMan MGB qPCR assay was shown to be efficient and specific. The detection limit, defined as the concentration at which 95% of positive samples can be detected, was determined to be 0.000114% parasitized erythrocytes (PE). We further evaluated a previously reported Theileria equi-specific qPCR assay and showed that it was able to detect the 12 T. equi 18S rRNA sequence variants previously identified in South Africa. Both qPCR assays were tested on samples from two ponies experimentally infected with either T. equi or B. caballi. The qPCR assays were more sensitive than the indirect fluorescent antibody test (IFAT) and the reverse-line blot (RLB) during the early onset of the disease. The assays were subsequently tested on field samples collected from 41 horses, resident on three stud farms in the Northern Cape Province, South Africa. The IFAT detected circulating T. equi and B. caballi antibody in, respectively, 83% and 70% of the samples. The RLB detected T. equi parasite DNA in 73% of the samples, but none of the samples were positive for B. caballi, although 19 T. equi-positive samples also hybridized to the Babesia genus-specific probe. This could indicate a mixed T. equi and B. caballi infection in these samples, with either the B. caballi parasitaemia at a level below the detection limit of the B. caballi RLB probe, or the occurrence of a novel Babesia genotype or species. In contrast, the qPCR assays correlated fairly well with the IFAT. The B. caballi TaqMan MGB qPCR assay was able to detect B. caballi parasite DNA in 78% of the samples. The T. equi-specific qPCR assay could positively detect T. equi DNA in 80% of the samples. These results suggest that the qPCR assays are more sensitive than the RLB assay for the detection of T. equi and B. caballi infections in field samples.

Quantitative motor unit action potential analysis of skeletal muscles in the Warmblood horse

Motor unit action potential (MUP) analysis in human medicine is a valuable and important diagnostic technique enabling discrimination between myogenic and neurogenic problems. This study establishes normative data in subclavian, triceps and lateral vastus muscles for clinical application of MUP analysis in the Warmblood horse, and examines whether muscle differences are present. Electromyographic (EMG) needle examination and MUP analysis were performed of the triceps, lateral vastus and subclavian muscles in 7 awake, nonsedated, Warmblood horses age 4-10 years. The amplitude, duration, number of phases and turns were calculated from the recorded superimposed MUPs together with intramuscular and rectal temperatures. No significant differences were found in duration of insertional activity between the 3 muscles. The mean +/- s.d. duration of the insertional activity was 526 +/- 1483 ms. The MUP amplitude of all 3 muscles differed significantly, with the highest amplitude (427 +/- 3.20 microV) in the triceps and the lowest (220 +/- 2.08 microV) in the subclavian muscle. The number of turns of the lateral vastus (3.0 +/- 1.22) was significantly higher than that of the triceps muscle (2.7 +/- 1.51). No differences were found in MUP duration (5.9-6.4 ms).

Phenotypic diagnosis of dwarfism in six Friesian horses

An extreme form of abnormal development, dwarfism, is common in man and some animals, but has not been officially reported in horses. Within the Friesian horse breed, congenital dwarfism has been recognised for many years, but no detailed report exists on its phenotype. The most salient feature of the dwarf syndrome is the physeal growth retardation in both limbs and ribs. Affected animals have approximately 25% shorter fore- and hindlimbs and approximately 50% reduced bodyweight. Postnatal growth is still possible in these animals, albeit at a slower rate: the head and back grow faster than the limbs and ribs leading to the characteristic disproportional growth disturbance. Thus, adult dwarfs exhibit a normal, but a relatively larger head conformation, a broader chest with narrowing at the costochondral junction, a disproportionally long back, abnormally short limbs, hyperextension of the fetlocks and narrow long-toed hooves. Furthermore, a dysplastic metaphysis of the distal metacarpus and metatarsus is radiographically evident. Microscopic analysis of the growth plates at the costochondral junction shows an irregular transition from cartilage to bone, and thickening and disturbed formation of chondrocyte columns, which is similar to findings in osteochondrodysplasia.

A missense mutation in the skeletal muscle chloride channel 1 (CLCN1) as candidate causal mutation for congenital myotonia in a New Forest pony

A 7-month-old New Forest foal presented for episodes of recumbency and stiffness with myotonic discharges on electromyography. The observed phenotype resembled congenital myotonia caused by CLCN1 mutations in goats and humans. Mutation of the CLCN1 gene was considered as possible cause and mutation analysis was performed. The affected foal was homozygous for a missense mutation (c.1775A>C, p.D592A) located in a well conserved domain of the CLCN1 gene. The mutation showed a recessive mode of inheritance within the reported pony family. Therefore, this CLCN1 polymorphism is considered to be a possible cause of congenital myotonia.

Idiopathic muscular hypertrophy of the oesophagus in the horse: a retrospective study of 31 cases.

REASONS FOR PERFORMING STUDY To present the first report of a case series concerning equine idiopathic muscular hypertrophy of the oesophagus (IMHO). OBJECTIVES To investigate the clinical and pathological features of the disorder. METHODS The medical records of 31 horses suffering from the disorder were reviewed retrospectively. In all these animals the diagnosis was confirmed at post mortem examination. RESULTS The median age of the affected horses was 12.5 +/- 5.6 years (range 1-26) without sex or breed predilection. Only 2 out of 31 horses showed clinical signs associated with oesophageal dysfunction, indicating that the muscular hypertrophy was rather a coincidental post mortem finding. Histology revealed thickening of the distal portion of the oesophagus mainly involving the circular layer of the tunica muscularis without fibrosis or inflammation. In 8 cases, the disorder was seen in concurrence with idiopathic hypertrophy of the tunica muscularis of various other parts of the gastrointestinal tract. CONCLUSIONS In the majority of patients, IMHO was a coincidental finding at post mortem examination usually confined to the smooth, circular muscle layer of the tunica muscularis externa. POTENTIAL RELEVANCE Further research is necessary to study the precise effect of IMHO on oesophageal function.

Differential expression of equine muscle biopsy proteins during normal training and intensified training in young standardbred horses using proteomics technology

The major aim of the present study was to investigate the proteome of standardbred horses at different stages of training and intensified training. We searched for biomarkers using small skeletal muscle biopsies of live animals. 2D gel electrophoresis and mass spectrometry were successfully applied to investigate training-induced differential expression of equine muscle biopsy proteins. Despite the poor resolution of the equine genome and proteome, we were able to identify the proteins of 20 differential spots representing 16 different proteins. Evaluation of those proteins complies with adaptation of the skeletal muscle after normal training involving structural changes towards a higher oxidative capacity, an increased capacity to take up long-chain fatty acids, and to store energy in the form of glycogen. Intensified training leads to additional changed spots. Alpha-1-antitrypsin was found increased after intensified training but not after normal training. This protein may thus be considered as a marker for overtraining in horses and also linked to overtraining in human athletes.

Over)training effects on quantitative electromyography and muscle enzyme activities in standardbred horses.

Too intensive training may lead to overreaching or overtraining. To study whether quantitative needle electromyography (QEMG) is more sensitive to detect training (mal)adaptation than muscle enzyme activities, 12 standardbred geldings trained for 32 wk in age-, breed-, and sex-matched fixed pairs. After a habituation and normal training (NT) phase (phases 1 and 2, 4 and 18 wk, respectively), with increasing intensity and duration and frequency of training sessions, an intensified training (IT) group (phase 3, 6 wk) and a control group (which continued training as in the last week of phase 2) were formed. Thereafter, all horses entered a reduced training phase (phase 4, 4 wk). One hour before a standardized exercise test (SET; treadmill), QEMG analysis and biochemical enzyme activity were performed in muscle or in biopsies from vastus lateralis and pectoralis descendens muscle in order to identify causes of changes in exercise performance and eventual (mal)adaptation in skeletal muscle. NT resulted in a significant adaptation of QEMG parameters, whereas in muscle biopsies hexokinase activity was significantly decreased. Compared with NT controls, IT induced a stronger adaptation (e.g., higher amplitude, shorter duration, and fewer turns) in QEMG variables resembling potentially synchronization of individual motor unit fiber action potentials. Despite a 19% decrease in performance of the SET after IT, enzyme activities of 3-hydroxyacyl dehydrogenase and citrate synthase displayed similar increases in control and IT animals. We conclude that 1) QEMG analysis is a more sensitive tool to monitor training adaptation than muscle enzyme activities but does not discriminate between overreaching and normal training adaptations at this training level and 2) the decreased performance as noted in this study after IT originates most likely from a central (brain) rather than peripheral level.

The role of quantitative electromyography (EMG) in horses suspected of acute and chronic grass sickness

REASONS FOR PERFORMING THE STUDY Clinical evidence of motor neuron involvement in equine grass sickness (EGS) has not been reported. HYPOTHESIS Quantitative electromyography (EMG) analysis can elucidate subtle changes of the lower motor neuron system present in horses with EGS, performed ante mortem. METHODS Fourteen horses diagnosed clinically with acute, subacute or chronic EGS were examined and quantitative EMG performed. Previously published data on healthy horses and horses with proven lower motor neuron disease (LMND) were used as controls. In 8 horses post mortem examination was performed, and in 7 muscle biopsies of the lateral vastus muscle underwent histopathology and morphometry. RESULTS Clinical electrophysiological evidence of neuropathy was present in 12 horses. Analysis of data from the first 4 horses resulted in 95% confidence intervals (CI) of nontransformed data for motor unit action potential (MUP) duration in subclavian, triceps and lateral vastus muscle of 11.0-13.7, 14.8-20.3 and 12.2-17.2 msecs, respectively, and for MUP amplitude 291-453, 1026-1892 and 957-1736 microV, respectively. For number of phases the 95% CI was 3.6-4.4, 2.9-3.6 and 2.9-3.4, respectively, and for number of turns 5.0-6.5, 4.3-5.3 and 3.7-4.6, respectively. No changes in duration of insertional activity were measured. Pathological spontaneous activity was observed in all horses. EGS as evidenced by degenerative changes in the autonomic ganglia in combination with minor degenerative changes of the spinal lower motor neurons was observed on post mortem examination in all 8 available autopsies. In muscle biopsies of 4 out of 7 horses changes consistent with slight neurogenic atrophy were found. CONCLUSIONS AND POTENTIAL RELEVANCE EMG results demonstrated the presence of a neuropathy of skeletal muscles in all horses suspected to have EGS. The combination of clinical and electrophysiological evidence may aid differential diagnosis of neurogenic disease in cases of weight loss and colic.

Effect of Clostridium botulinum toxin type A injections into the deep digital flexor muscle on the range of motion of the metacarpus and carpus, and the force distribution underneath the hooves, of sound horses at the walk

In the treatment of laminitis, reducing deep digital flexor muscle (DDFM) activity might diminish its pull on the distal phalanx, thereby preventing displacement and providing pain relief. Injection of Clostridium botulinum toxin type A into the DDFM of horses is potentially therapeutic. However, the effects of C. botulinum toxin type A on the gait characteristics of sound horses at the walk are not known. The aim of this study was to test if a reduced DDFM activity would lead to (1) alterations of the sagittal range of motion of the metacarpus (SROM) and range of motion of the carpal joint (CROM); (2) changes in the force distribution underneath the hoof (toe vs. heel region: balance index); and (3) changes in the force distribution between the treated and untreated limb (symmetry index). The DDFMs of the left forelimbs of seven sound Royal Dutch Sport Horses were injected with 200 IU C. botulinum toxin type A using electromyography and ultrasound guidance. Measurements using an inertial sensor system and dynamically calibrated pressure plate were performed before and after injections. The SROM and CROM of the treated limb were significantly increased after C. botulinum toxin type A injections. No significant changes were detected in the balance index or in the symmetry index, indicating that no lameness was induced. C. botulinum toxin type A injections into the DDFM of sound horses do not appear to result in substantial gait alterations at the walk.