Incidence of polysaccharide storage myopathy: necropsy study of 225 horses.

Muscle samples were obtained at necropsy from 225 horses and ponies 1 year of age or older. Samples were processed in routine manner and were stained with hematoxylin and eosin and with periodic acid-Schiff for glycogen. Sections were examined for abnormal glycogen content and amylase-resistant complex polysaccharide and for chronic myopathic change (excessive fiber size variation, increase in number of internal nuclei). A total of 101 horses and ponies with lesions of polysaccharide storage myopathy were identified. Age of affected horses ranged from one to 30 years, with a mean of 14.7 years. Mean age of nonaffected horses was 12 years. Incidence of polysaccharide storage myopathy varied depending on breed; Thoroughbreds had the lowest (27%) and draft-related horses had the highest (86%) incidence. Chronic myopathic changes were more severe in polysaccharide storage myopathy-affected horses than in nonaffected horses. Results of this study indicate that polysaccharide storage myopathy is a common disorder of many breeds of horses and ponies.

Fatal paraquat poisoning in seven Portland, Oregon, dogs.

Paraquat is one of the few broad-spectrum herbicides available in the US; however, it is extremely toxic to companion animals when ingested. Despite its restricted use status, poisoning of dogs and cats remains relatively common. This clinical report documents a series of chronologically and geographically related cases of presumed malicious and fatal sub-acute paraquat poisoning in 7 dogs in Portland, OR. All animals developed acute gastrointestinal disturbance, renal compromise and insidiously progressive respiratory failure. Hyperlipasemia and moderate hypertension were notable featured in 5/7 cases. Trace levels of paraquat were demonstrated in the urine of 4/7 animals by gas-liquid chromatography/mass spectroscopy. Diagnosis in the remaining 3 cases was made through a combination of history or exposure, clinical signs and their progression, and pulmonary and renal histopathology.

Complex polysaccharide inclusions in skeletal muscle adjacent to sarcomas in two dogs.

Inclusions of periodic acid-Schiff-positive, amylase resistant material were found within skeletal muscle fibers adjacent to an osteosarcoma in the proximal femur of an 8-year-old intact female Cocker Spaniel dog (dog No. 1) and adjacent to a synovial cell sarcoma of the stifle joint in a 7-year-old spayed female Bouvier des Flandres dog (dog No. 2). Inclusions were pale blue-gray with hematoxylin and eosin stain and formed irregular inclusions, replacing up to approximately 80% of the fiber diameter. Inclusions from dog No. 2 were of non-membrane-bound granular to filamentous material that occasionally formed discrete, elongate electron-dense masses. The features of these inclusions were similar to those of materials previously described as complex polysaccharide, polyglucosan bodies, amylopectin, and Lafora bodies. Evidence for a generalized metabolic disorder was not found in these two dogs, suggesting that storage of complex polysaccharide can occur as a relatively nonspecific response to metabolic alterations in skeletal muscle in a variety of conditions.

Sarcocystis neurona-like encephalitis in a Canada lynx (Felis lynx canadensis).

A 13-yr-old female Canada lynx (Felis lynx canadensis) died after a short clinical illness, and necropsy revealed multifocal, nonsuppurative encephalitis with protozoal schizonts present in cerebral vascular endothelial cells. The schizonts stained immunohistochemically with antiserum to Sarcocystis neurona. This is the first report of Sarcocystis encephalitis in the Canada lynx.

Subacute necrotising encephalopathy in an Alaskan husky.

A 29-month-old female Alaskan husky was presented recumbent, tetraparetic and in a state of dementia, with blindness and cranial nerve deficits. The dog's progress was followed for over two months, as the signs resolved to an non-progressive mild hypermetria with slight proprioceptive ataxia, a diminished menace response and inability to prehend food. Magnetic resonance imaging (MRI) revealed bilateral cavitation extending from the thalamus to the medulla, with less pronounced degenerative lesions in the caudate nucleus, putamen and claustrum. Cerebrospinal fluid lactate and pyruvate concentrations were in their normal ranges. Necropsy and histological examination confirmed the MRI findings as well as neuronal degeneration of the cerebellar cortex in the vermis and degenerative changes in the neocortex at the depths of the cerebral sulci. In view of the similarity of lesions to subacute necrotising encephalomyelopathy, known as Leigh's disease in humans, a tentative diagnosis of a mitochondrial encephalopathy was made.

Hereditary polioencephalomyelopathy of the Australian cattle dog.

A vacuolar degeneration affecting primarily the gray matter in the central nervous system (CNS) of young Australian Cattle Dogs is described. An initial presentation of seizures was followed by a progressive spastic tetraparesis. Grossly evident bilateral and symmetrical foci of malacia were in the nuclei of the cerebellum and brain stem and the gray matter of the spinal cord. Microscopically, vacuolation of glial cells, dilation of the myelin sheaths and reactive astrocytosis characterized mild CNS changes. More advanced lesions displayed progressive dissolution of the neuropil, prominent vacuolation of reactive astrocytes, numerous glial fibrillary acidic protein-positive coiled astrocytic processes, neuronal vacuolation and loss with relative sparing of large neurons. Ultrastructurally marked mitochondrial accumulation and swelling were seen in astrocytes. In the appendicular muscles, changes interpreted as long-term denervation atrophy accompanied by widespread expression of the neonatal isoform of myosin were observed. The character of the neurological sings, the nature and the distribution of the lesions within the neuroaxis have not been reported in domestic animals. An inherited biochemical defect, possibly mitochondrial, is proposed as the cause. Selected conditions with a bilateral and symmetrical distribution affecting the gray matter of domestic animals are summarized.

Severe polysaccharide storage myopathy in Belgian and Percheron draught horses.

A severe myopathy leading to death or euthanasia was identified in 4 Belgian and 4 Percheron draught horses age 2-21 years. Clinical signs ranged from overt weakness and muscle atrophy in 2 horses age 2 and 3 years, to recumbency with inability to rise in 6 horses age 4-21 years. In 5 horses there was mild to severe increases in muscle enzyme levels. Clinical diagnoses included equine motor neuron disease (2 horses), post anaesthetic myopathy (2 horses), exertional myopathy (2 horses), myopathy due to unknown (one horse), and equine protozoal myelitis (one horse). Characteristic histopathology of muscle from affected horses was the presence of excessive complex polysaccharide and/or glycogen, revealed by periodic acid-Schiff staining in all cases and by electron microscopy in one case. Evaluation of frozen section histochemistry performed on 2 cases indicated that affected fibres were Type 2 glycolytic fibres. Subsarcolemmal and intracytoplasmic vacuoles were most prominent in 3 horses age 2-4 years, and excessive glycogen, with little or no complex polysaccharide, was the primary compound stored in affected muscle in these young horses. Myopathic changes, including fibre size variation, fibre hypertrophy, internal nuclei, and interstitial fat infiltration, were most prominent in 5 horses age 6-21 years, and the accumulation of complex polysaccharide appeared to increase with age. Mild to moderate segmental myofibre necrosis was present in all cases.

Mutation segregation and rapid carrier detection of X-linked muscular dystrophy in dogs.

OBJECTIVES: To use exon 7-specific genomic polymerase chain reaction (PCR) products to identify the genotypes of normal, affected, and carrier female dogs in pedigrees segregating Golden Retriever muscular dystrophy (GRMD), and to confirm the concordant segregation of the mutation in all carrier and affected dogs presently available. DESIGN: The GRMD mutation is found in the consensus splice acceptor site in intron 6 of the canine dystrophin gene. PCR cycle-sequencing and restriction fragment length polymorphism/PCR were used for determination of the pattern of segregation of the point mutation which causes GRMD. ANIMALS: Normal, clinically affected, and obligate carrier dogs in pedigrees of GRMD. PROCEDURE: DNA from blood was amplified, using PCR and primers that bracket all of exon 7 of the canine dystrophin gene as well as 100 base pairs of intron on either side. PCR products were either cycle-sequenced directly or submitted to a second round of PCR, using 1 of the original primers coupled with a mutagenic restriction fragment length polymorphism-primer, which thus creates an artificial restriction site. Digestion with Stu I detected the normal allele. To detect the affected allele, Sau96 I was used to digest the 310-base pair exon 7 genomic fragment directly. CONCLUSIONS: Simple, clear diagnosis of carrier status was possible using these methods. This mutation is passed through all carrier and affected dogs in both United States GRMD colonies and the colony in Australia. CLINICAL RELEVANCE: Rapid, accurate diagnosis of carrier and affected dogs will enhance study of this homologue of Duchenne muscular dystrophy.

Stable fetal cardiomyocyte grafts in the hearts of dystrophic mice and dogs.

This report documents the formation of stable fetal cardiomyocyte grafts in the myocardium of dystrophic dogs. Preliminary experiments established that the dystrophin gene product could be used to follow the fate of engrafted cardiomyocytes in dystrophic mdx mice. Importantly, ultrastructural analyses revealed the presence of intercalated discs consisting of fascia adherens, desmosomes, and gap junctions at the donor-host cardiomyocyte border. To determine if isolated cardiomyocytes could form stable intracardiac grafts in a larger species, preparations of dissociated fetal canine cardiomyocytes were delivered into the hearts of adult CXMD (canine X-linked muscular dystrophy) dogs. CXMD dogs, like Duchenne muscular dystrophy patients and mdx mice, fail to express dystrophin in both cardiac and skeletal muscle. Engrafted fetal cardiomyocytes, identified by virtue of dystrophin immunoreactivity, were observed to be tightly juxtaposed with host cardiomyocytes as long as 10 wk after engraftment, the latest date analyzed. Confocal laser scanning microscopy revealed the presence of connexin43, a major constituent of the gap junction, at the donor-host cardiomyocyte border. The presence of intracardiac grafts was not associated with arrhythmogenesis in the CXMD model. These results indicate that fetal cardiomyocyte grafting can successfully augment cardiomyocyte number in larger animals.

Septic cholangiohepatitis and cholangiocarcinoma in a horse.

Septic cholangiohepatitis was diagnosed in an 11-year-old Warmblood gelding with a history of intermittent colic and fever. Klebsiella pneumoniae, susceptible to gentamicin, was cultured from the biopsy specimen. However, treatment with gentamicin was unsuccessful, and histologic examination and bacteriologic culture of a biopsy specimen obtained 3 weeks later revealed progression of the hepatic inflammation and yielded growth of gentamicin-resistant K pneumoniae. At this time, several discrete hyperechoic structures, suggestive of biliary calculi, were seen ultrasonographically. A change in antibiotic treatment was associated with gradual resolution of clinical signs. Five months after initial examination, the horse had a sudden onset of severe right forelimb lameness. The horse responded to treatment with antibiotics and phenylbutazone, but lameness and fever that was unresponsive to treatment recurred 7 months later, and the horse was euthanatized. Necropsy revealed nodules throughout the liver and a mass associated with the right metacarpophalangeal joint. Histologic and immunohistochemical examination revealed carcinomatous infiltration of the liver and metacarpophalangeal joint. The tumor was probably of biliary origin. Carcinoma should be considered in cases of septic cholangiohepatitis unresponsive to antibiotic treatment.

Reduction of the transient outward potassium current in canine X-linked muscular dystrophy.

BACKGROUND: The xmd dog develops a cardiomyopathy similar to that seen in Duchenne muscular dystrophy patients. In both the canine and human diseases, ECG abnormalities may precede the development of overt cardiac pathological lesions. The purpose of this study was to determine whether specific cellular electrical abnormalities occur in dystrophic ventricular tissue. METHODS AND RESULTS: Action potentials were recorded in epicardial tissue strips obtained from normal and xmd dogs. Phase 1 amplitude was increased from 86.8 +/- 2.7 mV in normal dogs to 94.3 +/- 1.8 mV in xmd dogs (mean +/- SEM; P < .05). The 4-aminopyridine-sensitive transient outward potassium current (Ito), as recorded in isolated epicardial myocytes using the whole-cell patch-clamp technique, was reduced in xmd dogs compared with age-matched normal dogs. Cell capacitance also was reduced significantly in xmd compared with normal cells, as was the current density (3.6 +/- 0.3 versus 5.4 +/- 0.8 pA/pF, respectively). No differences were observed in the time constants of current decay or in the kinetics of recovery from inactivation between groups. The slope factor (k) of steady-state inactivation was significantly greater in xmd compared with normal cells (7.2 +/- 0.9 versus 5.4 +/- 0.5, respectively), whereas the V1/2 of inactivation did not differ (-38.2 +/- 2.4 versus -36.8 +/- 1.6 mV, respectively). CONCLUSIONS: These data indicate that the magnitude of Ito is reduced in dystrophic epicardial myocytes, resulting in an increase in phase 1 amplitude. The reduction of Ito may alter the balance of inward and outward currents in dystrophic myocardium and thereby contribute to the development of cardiac pathology.

Deletion of the dystrophin muscle promoter in feline muscular dystrophy.

We have characterized the mutation in a feline model of DMD that selectively eliminates expression of the muscle and Purkinje neuronal dystrophin isoforms. The cortical neuronal isoform was expressed at a detectable level in skeletal muscle in the absence of the muscle promoter and levels of PCR products representing cortical neuronal-type transcripts in dystrophic muscle were comparable to those of normal feline skeletal muscle. Although localized at the sarcolemma, cortical neuronal dystrophin apparently failed to protect skeletal muscle. Neuronal transcripts could not be amplified from feline heart, indicating that these promoters are not active in this tissue in the cat.

Expression of utrophin (dystrophin-related protein) during regeneration and maturation of skeletal muscle in canine X-linked muscular dystrophy.

The regulation of utrophin, the autosomal homologue of dystrophin, has been studied in the canine X-linked model of Duchenne muscular dystrophy. Dystrophic muscle has been shown to exhibit abnormal sarcolemmal expression of utrophin, in addition to the normal expression at the neuromuscular junction, in peripheral nerves, vascular tissues and regenerating fibres. To establish whether this abnormal presence of utrophin in dystrophic muscle is a consequence of continued expression following regeneration, or is attributable to a disease related up-regulation, the expression of utrophin was compared immunocytochemically with that of dystrophin, beta-spectrin and neonatal myosin in regenerating normal and dystrophic canine muscle, following necrosis induced by the injection of venom from the snake Notechis iscutatis. In normal regenerating muscle, sarcolemmal utrophin and dystrophin were detected concomitantly from 2-3 d post-injection, prior to the expression of beta-spectrin. Down-regulation of utrophin was apparent in some fibres from 7 d, and it was no longer present on the extra-junctional sarcolemma by 14 d. Neonatal myosin was still present in all fibres at this stage, but dystrophin and beta-spectrin had been fully restored. In dystrophic regenerating muscle, down-regulation of utrophin occurred from 7 d, although it persisted on some fibres until 28 d, longer than in normal muscle. At 42 d, however, utrophin in dystrophic muscle was only detected in a population of small fibres thought to represent a second cycle of regeneration, with no immunolabelling of mature fibres.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental regeneration in canine muscular dystrophy--2. Expression of myosin heavy chain isoforms.

The sequential expression of neonatal, fast and slow myosin heavy chain isoforms was examined during the regeneration of normal and dystrophic dog muscle from 1 to 56 days, following necrosis induced by the venom of Notechis scutatis, to assess the regenerative potential of dystrophic muscle. Regeneration was equally rapid in normal and dystrophic dogs but morphological and immunocytochemical abnormalities were more apparent in the dystrophic fibres. New myotubes were formed by 3 days, and by 4 days all myosin isoforms were expressed. Neonatal myosin persisted in normal dogs after morphological restoration of the muscle and after dystrophin and beta-spectrin expression had returned to normal. Neonatal myosin was considerably reduced by 21 days in normal dogs, but persisted beyond 28 days in dystrophic dogs, suggesting a delay in maturation. At 42 and 56 days, dystrophic dogs showed a population of small fibres expressing neonatal myosin, which may represent a second cycle of degeneration and regeneration. The reciprocal pattern of fast and slow myosin was not fully restored in normal or dystrophic regenerating muscle, and co-expression persisted. Thus, dystrophic muscle retains its potential to regenerate, but maturation is slower than normal. The persistent co-expression of isoforms has implications for the long-term function of fibres formed after myoblast therapy, but the results imply that a stable state can be achieved if dystrophin expression is restored by gene or myoblast transfer therapy.

Notechis scutatus venom increases the yield of proliferating muscle cells from biopsies of normal and dystrophic canine muscle--a possible source for myoblast transfer studies.

The injection of 1 micrograms Notechis scutatus (Australian tiger snake) venom (notexin) induces localized necrosis in the muscles of normal and dystrophic dogs. Biopsies taken from the muscles on the second day of postnecrotic regeneration provide about 8-16 x 10(6) cells capable of proliferation per g tissue, about 100 fold more than the untreated adult dog muscles. Muscle specific markers, such as the capacity of the cells to fuse, surface labelling with N-CAM antibodies (Leu-19 and 5.1.H11), and immunostaining with desmin, indicated that over 90% of the cultivated cells are indeed myogenic. The method is a safe and cost effective way to generate large amounts of proliferating muscle cells from biopsies of adult animals, which could provide a useful step in the therapeutic efforts in inherited muscle diseases by the implantation of normal myoblasts or genetically corrected myoblasts.

Canine X-linked muscular dystrophy as an animal model of Duchenne muscular dystrophy: a review.

Canine X-linked muscular dystrophy is a spontaneously occurring, progressive, degenerative myopathy of dogs that is clinically and pathologically similar to Duchenne muscular dystrophy in man. The molecular basis for the disease has been shown to be a lack of dystrophin, the protein product of the Duchenne muscular dystrophy gene. Breeding colonies of dystrophic dogs have been established. This report reviews the findings of genetic, clinical, pathologic, molecular biologic, and immunocytochemical studies of the canine model, and compares the features of the canine disease to those of Duchenne dystrophy in man.

Experimental regeneration in canine muscular dystrophy--1. Immunocytochemical evaluation of dystrophin and beta-spectrin expression.

The expression of dystrophin and beta-spectrin was examined from 1 to 56 days in regenerating muscle fibres in normal and dystrophic dogs, following necrosis induced by the venom of Notechis scutatis. Normal and dystrophic dog muscle regenerated at an equal rate and new myotubes were present in both at the periphery of necrotic fibres by 3 days. In normal dogs dystrophin was detected in the sarcoplasm of the regenerating fibres by 3 days and was localized to the plasma membrane by 4 days. The localization of dystrophin is independent of beta-spectrin and was detected before beta-spectrin, which was not observed until 5-6 days. Normal peripheral labelling of both was restored by 14 days in normal dogs. Normal beta-spectrin labelling of regenerating dystrophic fibres was also restored by 14 days and is not dependent on the presence of dystrophin in dystrophic dogs. A proportion of regenerating fibres in normal and dystrophic dogs showed weak immunolabelling of beta-spectrin prior to 14 days. This is a feature of immature muscle fibres. Antibodies to different domains of dystrophin bound to the periphery and sarcoplasm of regenerating fibres in dystrophic dogs, particularly during the first 7 days of regeneration, but the fluorescence was less intense than in normal dogs. Weak labelling with antibodies corresponding to the C-terminus of the rod domain of dystrophin persisted on dystrophic regenerating fibres up to 21 days. This may relate to developmental isoforms of dystrophin.

Duchenne's cardiomyopathy in a canine model: electrocardiographic and echocardiographic studies.

Thirteen dogs affected with X-linked Duchenne's muscular dystrophy and 11 female carrier dogs were studied by electrocardiography (ECG) and echocardiography. Twelve of the affected dogs were studied as immature animals and followed at 1 to 6 month intervals until they were 7 to 46 months of age. Compared with control dogs, affected dogs had significantly increased (p less than 0.02) Q/R ratios in ECG leads II, III, aVF, CV6LL (V2) and CV6LU (V4). Carrier dogs had significantly increased (p less than 0.02) Q/R ratios in leads V2 and V4. The Q/R ratio increased in three of six dogs followed up from age 6 months to greater than 2 years. The PR intervals were significantly shorter (p less than 0.02) in affected dogs. Ventricular arrhythmias were identified in four of six mature affected dogs. Two-dimensional echocardiography revealed distinctive hyperechoic lesions in 12 of the 13 affected dogs and in 6 of the 11 carrier dogs. Hyperechoic lesions corresponded to calcified myocardium and surrounding dense connective tissue. This study establishes the dog affected with Duchenne's muscular dystrophy as an animal model of Duchenne's cardiomyopathy and demonstrates that the heart in carrier dogs is affected by the dystrophic process.

In vitro characteristics of normal and dystrophic skeletal muscle from dogs.

Explants were prepared from skeletal muscle tissue from 5 nondystrophic pups and from 5 pups with X-linked muscular dystrophy; pups were 2 to 17 weeks old. A serial reexplant method resulted in optimal cell density with minimal fibroblast growth. Cultures were examined daily by use of phase-contrast microscopy; differentiated (post-fusion) cultures were examined by electron microscopy. Moderate nuclear pleomorphism and cell clustering were observed in cultures of normal and dystrophic muscle cells. Cultures were maintained to 27 days after plating. Minimal myofilament synthesis was observed in multinucleate cells from nondystrophic and dystrophic pups, but spontaneous contraction of myotubes was not observed during this period. Differences in growth, fusion, or differentiation of myogenic cells into multinucleate cells and myotubes were not found between dystrophic and normal muscle.