Introduction

Guillaume-Benjamin-Amand Duchenne described the first human case of Duchenne muscular dystrophy (DMD) in 1861 in his book Paraplégie hypertrophique de l’enfance de cause cérébrale.¹ In 1987, the causative mutation was identified in the gene encoding dystrophin (DYS) located on the X chromosome.² In modern times, DMD is the most common cause of muscular dystrophy, affecting 1 in 5000 live human male births.^3,4 X-linked muscular dystrophy (XLMD) is now a well-recognized condition in veterinary medicine, with 20 different DYS gene variants affecting 16 different dog breeds.⁵ To the authors’ knowledge, XLMD has not been described in the Australian cattle dog.

Case Report

A 9 mo old intact male Australian cattle dog was evaluated at the Veterinary Neurological Center for slowly progressive muscle weakness and dysphagia beginning at 4 mo of age. The dog was rescued at 5 mo of age, and prior history is unknown. On examination by the referring veterinarian, the dog was noted to have difficulty with food prehension and had a right pelvic limb lameness. Radiographs of the lumbar spine, pelvis, and right stifle were unremarkable. Treatment with carprofen (5 mg/kg q 24 hr for 2 wk) and gabapentin (10 mg/kg q 12 hr for 3 mo) did not result in significant improvement. Dysphagia progressed to dropping food and water. On further examination, the dog was reluctant to walk and unable to fully open the jaw with jaw pain. A complete blood count (CBC) and serum chemistries revealed lymphocytosis (9.21 K/µL, reference 1.05–5.10 K/µL) and elevated alanine transaminase (ALT; 582 IU/L, reference 8–75 IU/L) activity. Carprofen was discontinued and prednisone (4 mg/kg q 24 hr) and hydrocodone/acetaminophen (0.6/20 mg/kg q 8 hr) were initiated. Doxycycline was also initiated at an unknown dose.

Owing to the lack of improvement, the dog was referred to an internal medicine specialist. Cytology from a right carpal joint tap was normal. A serum 2M antibody titer for masticatory myositis was negative. A CBC and a serum chemistry analysis were performed but did not include creatine kinase (CK) or aspartate aminotransferase activity. The chemistry revealed a markedly elevated ALT activity (620 U/L, reference 8–75 U/L). Esophagoscopy showed severe swelling and stiffness at the base of the tongue and superficial ulcers of the esophagus. Cytology of the tongue showed rare mesenchymal cells and a few muscle aggregates without infectious organisms. A computed tomography scan of the skull and pelvis showed subjective contrast enhancement of the muscles of mastication and epaxial muscles consistent with possible inflammation or muscle necrosis. A minimally compressive lumbosacral disk protrusion was noted. Prednisone was tapered and omeprazole (1 mg/kg q 12 hr) and amoxicillin/clavulanic acid (20 mg/kg q 12 hr) were started.

Owing to lack of improvement, the dog was then referred to the Veterinary Neurological Center. On examination, significant swelling at the base of the tongue was noted. The dog was reluctant to open the jaw, and a stiff, stilted gait was noted in all limbs. Proprioception was intact in all limbs and segmental reflexes were normal. The distal appendicular muscles of the thoracic and pelvic limbs appeared hypertrophied. An awake electromyogram was performed and showed extensive complex repetitive discharges with a dive bomber sound in all appendicular muscles. Serum antibody titers for Toxoplasma, Neospora, and coccidioidomycosis were negative. An antinuclear antibody test was also negative. A CBC and serum chemistries including CK activity were performed. Marked elevation of ALT (507 IU/L; reference 12–118 IU/L), aspartate aminotransferase (1075 IU/L; reference 15–66 IU/L), and CK (77,807 IU/L; reference 59–895 IU/L) activities were noted. A presumptive diagnosis of a form of muscular dystrophy was made based on the clinical examinations, age of onset, progression of disease, and markedly elevated CK activity. Genetic testing for the DMD mutation identified in golden retrievers⁶ and for the CLN5 mutation causing neuronal ceroid lipofuscinosis identified in border collies⁷ and Australian cattle dogs⁸ was clear for the variants. The latter testing was performed to exclude genetic variants known in the breed. Genetic testing for the SOD1 mutation causing degenerative myelopathy was performed and the dog tested at risk for degenerative myelopathy. This positive result was thought to be unrelated to the current clinical signs. The condition did not improve with supportive treatment and the dog was euthanized because of the presumptive diagnosis and continued progression of clinical signs.

A necropsy was performed, and muscle specimens were collected from the diaphragm, appendicular and facial muscles, tongue, and esophagus. Histopathology revealed variability in myofiber size, with multifocal clusters of degenerating fibers (myonecrosis), regenerating fibers, and scattered calcific deposits in all muscles examined (Figure 1). Degenerating fibers were at varying stages within different muscles indicating polyphasic changes. Inflammation was minimal and there was no evidence of infection. The pathologic changes were consistent with a dystrophic phenotype.

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To further characterize a specific form of muscular dystrophy, cryosections from the biceps femoris muscle and an archived control muscle were cut (8 μm) and stained for indirect immunofluorescence as previously described.⁹ Several monoclonal or polyclonal antibodies were used, including those against the rod (1:100, NCL-DYS1) and carboxy-terminus (1:100, NCL-DYS2) of dystrophin and against utrophin (1:20, NCL-DRP2), developmental myosin heavy chain (dMHC1:20, NCL-dMHC)), α-sarcoglycan ( 1:200, gift of Eva Engvall),¹⁰ β-dystroglycan (1:100, NCL, bDG), laminin α2 (gift of Eva Engvall, 4F11, direct apply),¹⁰ and collagen VI (gift of Eva Engvall, 3G7, direct apply)¹¹ (Figure 2). Staining was absent for both the rod-domain and the carboxy terminus of dystrophin and reduced for α-sarcoglycan and β-dystroglycan. Scattered groups of dMHC-positive regenerating fibers were identified. Utrophin antibody staining was increased on the muscle sarcolemma. The staining pattern for collagen VI was increased, indicative of fibrosis. Antibody staining for laminin α2 was similar to control tissue. Dystrophin-deficient muscular dystrophy with secondary reduction of dystrophin-associated proteins was confirmed based on immunofluorescence staining. Informed consent was obtained from the owners to publish this case report. The dog was clinically managed according to the contemporary standards of care as described in JAAHA instructions for authors.

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Discussion

This case report describes X-linked dystrophin-deficient muscular dystrophy in a juvenile male Australian cattle dog. To the authors’ knowledge, XLMD has not been described in the Australian cattle dog breed. Clinical, laboratory, and electrophysiological testing and histopathological and immunohistochemical changes were consistent with those described in the literature.^5,6 As in people, dystrophin-deficient muscular dystrophy is the most common form of muscular dystrophy described in dogs,⁵ and the list of breeds and number of DYS variants continues to grow. Since the recent report in 2023 confirming 16 different dog breeds with XLMD and describing 20 different variants,⁵ an additional article has been published describing a second DYS variant affecting a young male border collie.¹² In previous reports in other breeds, clinical signs are normally recognized between 8 and 10 wk of age and tend to progress over 3 to 6 mo. Clinical signs include weakness, exercise intolerance, stiff gait, difficult prehension, and inability to fully open the jaw. The affected animals also show progressive muscle atrophy of the limbs, head, and trunk. Some muscles may be hypertrophied such as the tongue, semitendinosus and semimembranosus muscles, or the cranial sartorius in golden retrievers.^13–15 Importantly, the CK activity is markedly and persistently elevated and can be detected at a few weeks of age. Clinical signs were first recognized in the dog of this report at around 4 mo of age and may not have been recognized earlier as the dog was a stray and may not have had a veterinary evaluation. The progression of clinical signs and histopathologic changes noted in the reported dog are consistent with XLMD reported in other breeds

Identification of a specific DYS variant in our case would be important for development of a diagnostic genetic test for suspected affected dogs of this breed and identification of carriers for breeding programs. However, identification of the variant requires sequencing of the DYS gene or whole-genome sequencing, which was not performed at the time of evaluation. In a clinical setting, identification of a gene variant responsible for a condition such as XLMD in a breed where this has not yet been confirmed can be problematic. Whole-genome sequencing is not readily available, can still be expensive, may require bioinformatic analysis, and may need several months to reach an answer. Owners often have limited information on the pedigree of their animal, which makes it difficult to follow genetic variants and determine patterns of inheritance. Breeders may not be cooperative and may sometimes import studs or bitches from other countries, which can make it difficult to obtain information on related animals. When a genetic neuromuscular disease is not known in a breed, a phenotypic diagnosis from muscle biopsies at the time of clinical evaluation or from collection of samples at necropsy can be achieved. DNA can be extracted from archived frozen muscle biopsy samples for future testing.

In this case, clinical signs progressed over several months and impaired quality of life, resulting in humane euthanasia. No improvement was noted despite attempted therapy. The phenotypic diagnosis explains the lack of improvement with therapy as no specific treatments or cures are available for any of the dystrophies. Unfortunately, a CK activity was not performed early in the course of diagnostic testing. The diagnosis of neuromuscular diseases can be long and difficult. When faced with an animal displaying signs of muscular weakness, a CK activity should be routinely added to the serum chemistry analysis to evaluate muscular damage.¹⁶ Elevated CK activity, particularly if persistently or markedly elevated as in this case, should prompt a veterinarian to perform muscle biopsies, thus allowing a pathogenic diagnosis and realistic treatment options, if any.

Conclusion

A congenital neuromuscular disease should be considered in young Australian cattle dogs with an early onset of fatigue, appendicular muscle atrophy or hypertrophy, and an enlarged tongue. A form of muscular dystrophy should be ruled out and muscle biopsies performed when the CK activity is markedly and persistently elevated. Identification of a genetic variant and development of diagnostic genetic tests are essential in aiding breeding programs.