Haemolytic anaemia and exercise intolerance due to phosphofructokinase deficiency in related springer spaniels

Phosphofructokinase (PFK) deficiency is an autosomal recessive inherited disorder in dogs causing haemolytic crises and exertional myopathy. The clinical signs may be confused with those of recurrent immune-mediated haemolytic anaemia. The deficiency has been commonly observed in field trial (working) English springer spaniels (ESSPs), but also in the conformation line of ESSPs in the USA over the past two decades. This report documents the first family of ESSPs found with PFK deficiency in Europe. Two related adult ESSPs in Denmark had intermittent signs of pigmenturia after exercise (hunting) and had evidence of a regenerative haemolytic anaemia. Based upon DNA sequencing data, both dogs had the previously described nonsense point mutation in the muscle-type PFK gene (delta2228G-->A). Study of 17 related family members using a simple and accurate PFK-DNA test revealed one additional PFK-deficient dog (with minor exercise intolerance), nine carriers and seven normal (or 'clear') ESSPs. Recently, the authors have also identified PFK carriers and affected ESSPs in the UK. Screening for PFK deficiency is recommended for ESSPs with suspicious clinical signs and before using any for field trials or breeding in order to prevent the further spread of this hereditary disorder.     

A comparative histochemical and morphometric study of canine skeletal muscle.

The purpose of this study was to determine whether there were differences in skeletal muscle properties in the hindlimb muscles of different types of dogs. Muscle samples were obtained from the gracilis, sartorius cranial head, sartorius caudal head and tibialis anterior muscles of mixed-breed and hound-type dogs and Beagles. Fiber type, fiber size and capillary morphometry determinations of each muscle from each dog were made from sections stained for myofibrillar ATPase activity. Individual animals were bilaterally symmetric for all measured variables. Fiber type, fiber size and capillary geometry varied between dogs of a given type and muscles within a given dog. There were no differences between dog types for fiber type or fiber size; significant variation in log(muscle)/log(body) mass ratios between dog types was observed for all muscles. The results indicate that for a given muscle, significant variation can occur in skeletal muscle characteristics between different types of dogs and that these differences can be independent of differences in exercise history.     

Bilateral Iliopsoas Muscle Contracture and Spinous Process Impingement in a German Shepherd Dog

Objective— To report diagnosis and treatment of bilateral iliopsoas muscle contracture in a dog with spinous process impingement. Study design— Case report. Animals— German Shepherd dog. Methods— A dog with chronic progressive lameness, flexion contracture of the coxofemoral joints, severe pain, and decreased femoral reflexes had severe spondylosis bridging the vertebral bodies from L1 to L4 and enlarged dorsal spinous processes from T8 to L6 with impingement and bony proliferation. Ultrasonographic and magnetic resonance imaging (MRI) findings were consistent with fibrosis, mineralization, and atrophy of the iliopsoas muscles bilaterally which was treated by staged tenectomy of the insertions of the iliopsoas muscles. Results— Because of severe perivascular fibrosis, the femoral vessels required ligation. Bilateral iliopsoas muscle tenectomy improved gait and provided pain relief. Histologic findings were consistent with fibrotic myopathy. Conclusions— Slow progression of severe clinical signs observed bilaterally in this dog differs from previous reports of iliopsoas myopathy. Findings were similar to the fibrotic myopathy of the gracilis or semitendinosus muscles described in dogs. Clinical Relevance— Iliopsoas muscle abnormalities should be considered in dogs with limited hip extension and pain. MRI is useful for diagnosing muscle fibrosis. Iliopsoas tenectomy may improve clinical function in dogs with fibrotic myopathy.     

Hemolysis caused by phosphofructokinase deficiency in English springer spaniels: seven cases (1983-1986)

Seven English Springer Spaniels (6 adult males and 1 female) with chronic hemolysis and sporadic intravascular hemolytic crises were determined to have a deficiency in erythrocyte phosphofructokinase (PFK) activity, a key regulatory enzyme of anaerobic glycolysis. Intermittent severe pigmenturia concomitant with weakness, lethargy, and anorexia were the major clinical signs and commonly were related to exercise or other stressful situations that caused panting or barking (hyperventilation). Pale or icteric mucous membranes, fever, mild hepatosplenomegaly, and muscle wasting sometimes were evident. Results of routine laboratory testing indicated a persistent marked bilirubinuria and reticulocytosis with normal PCV, to severe anemia and intermittent hemoglobinuria and hyperkalemia. Erythrocyte PFK activities were severely reduced to 8% to 22% of values for control dogs. The block of glycolysis at the PFK step caused a markedly diminished erythrocyte 2,3-diphosphoglycerate content, resulting in an increased hemoglobin-oxygen affinity and compensatory accelerated erythrocyte production. Phosphofructokinase-deficient erythrocytes had increased alkaline fragility in vitro and in vivo. Hemolytic crises were induced in vivo by hyperventilation that caused transient, mild alkalemia. Studies of family members of a PFK-deficient dog suggested an autosomal recessive mode of inheritance. Carrier dogs with half-normal erythrocyte PFK activities appeared clinically normal.     

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.     

Hereditary phosphofructokinase deficiency in wachtelhunds

Hereditary phosphofructokinase (PFK) deficiency was diagnosed in two Wachtelhund dogs and suspected in three related Wachtelhund dogs with exercise intolerance, hemolytic anemia, and pigmenturia. Severe, persistent reticulocytosis in light of only mild anemia together with hemoglobinuria after strenuous exercise suggested PFK deficiency. Low erythrocyte PFK activity together with low 2,3-diphosphoglycerate concentrations and a high hemoglobin-oxygen affinity confirmed the diagnosis. The PFK deficiency is due to a single missense mutation in the muscle-type PFK M-PFK gene in English springer and American cocker spaniels, whippets, and mixed-breed dogs; however, these PFK-deficient Wachtelhunds do not have the same PFK mutation.     

Dysphagia as the primary clinical abnormality in two dogs with inflammatory myopathy

Two adult Boxers were evaluated because of chronic dysphagia of several years' duration. Serum creatine kinase activity was high in both dogs, but other hematologic or serum biochemical abnormalities were not detected. Esophagraphy revealed abnormalities of the cricopharyngeal phase of swallowing in both dogs, and electromyography of the pharyngeal and laryngeal muscles revealed complex repetitive discharges, positive sharp waves, and fibrillation potentials characteristic of primary myopathy or neuropathy. Because of the severity of their condition, both dogs were euthanatized. Histologically, mixed-cell infiltrates were seen in sections of the masseter and thyropharyngeal muscles. Results of indirect immunofluorescence staining for proteins associated with dystrophic myopathy were unremarkable, except for decreased staining for integrin alpha7. A diagnosis of chronic inflammatory myopathy was made. The clinical importance of reduced staining for integrin alpha7 could not be determined but was considered to be a result of the myopathy.     

Masticatory and skeletal muscle myositis in canine leishmaniasis (Leishmania infantum)

Twenty-four dogs with a parasitologically and serologically established diagnosis of leishmaniasis were studied to investigate the atrophy of the masticatory muscles which commonly occurs in this disease, and to compare the lesions in the masticatory muscles with those in the cranial tibial muscles. The 24 animals were divided into three groups of eight, group A dogs with no muscular atrophy, group B dogs with different degrees of atrophy in the masticatory and skeletal muscles, and group C dogs with similar degrees of atrophy in the masticatory and skeletal muscles. Increased activities of creatine phosphokinase and lactate dehydrogenase were recorded in only some of the dogs in groups B and C, but there were no significant differences between the mean activities in the three groups. Electromyographic changes indicating myopathy and involving both the temporalis and cranial tibial muscles, were observed in two of the dogs in group A, seven of those in group B, and in all the dogs in group C. Muscle histopathology revealed a variable degree of muscle fibre necrosis and atrophy, mononuclear infiltrates and neutrophilic vasculitis in all the dogs except two in group A. Leishmanial amastigotes were found within macrophages and myofibres in 16 of the dogs, some in each group. IgG immune complexes were detected in muscle samples, and circulating antibodies against myofibres were detected in serum samples from all the 24 dogs.     

Myotonic dystrophy-like disease in a dog

A mature female Rhodesian Ridgeback was determined to have a progressive, degenerative myopathy associated with myotonia, dysphagia, and marked muscle wasting. Clinical findings revealed a diffuse muscular disease with percussion dimpling, dysphagia, and creatine kinase elevation. A paroxysmal atrial tachycardia was found. Electromyography revealed a diffuse myopathy with high-frequency bizarre waves, myotonic discharges especially in the masticatory, laryngeal, and pharyngeal muscles. A few positive sharp waves were found in some of the appendicular muscles. Histopathologic and histochemical stains on skeletal muscle biopsy specimens demonstrated moderate fiber-size variation, myofiber architectural changes, muscle-fiber splitting, focal necrosis and phagocytosis, high percentage of internal nuclei, and atrophy of type-2 muscle fibers. A review of myotonic myopathies in the dog is presented. The clinical, electrophysiologic, and histochemical findings are similar to those for myotonic muscular dystrophy in man.     

MAGNETIC RESONANCE IMAGING IN THE DIAGNOSIS OF CANINE INFLAMMATORY MYOPATHIES IN THREE DOGS

In humans affected with inflammatory myopathies, regions of altered signal intensity are found on magnetic resonance (MR) images of affected muscles. Although electromyography (EMG) is more practical for muscle disease evaluation, and a muscle biopsy is the only manner in which a definitive diagnosis can be made, MR imaging has proven useful if a specific anatomic localization is difficult to achieve. Three dogs with focal inflammatory myopathy diagnosed with the assistance of MR imaging are discussed and the findings are compared with those found in humans. MR images of the affected muscles in each dog were characterized by diffuse and poorly marginated abnormal signal on T1- and T2-weighted images. Marked enhancement was noted in these muscles after contrast medium administration. An inflammatory myopathy was confirmed histologically in all three dogs. A good association existed between the MR images and muscle inflammation identified histopathologically. MR imaging may be a useful adjunctive procedure for canine inflammatory myopathies.     

Epidermal growth factor and active caspase-3 expression in the levator ani muscle of dogs with and without perineal hernia

Objectives : To perform a histological and immunohistochemical study of epidermal growth factor, transforming growth factor‐alpha and their receptor, as well as the apoptotic signal active caspase‐3 in the levator ani muscle of dogs with and without perineal hernia. Methods : Biopsy specimens of the levator ani muscle were obtained from 25 dogs with perineal hernia and 4 non‐affected dogs and were processed for Masson and immunohistochemical staining. Results : The affected dogs exhibited myopathological features, internalised nuclei, destruction and abnormal size of muscle fibres, which were replaced by collagen. The immunohistochemical study revealed active caspase‐3, epidermal growth factor, transforming growth factor‐alpha and epidermal growth factor receptor in the levator ani. Compared to the healthy muscle, transforming growth factor‐alpha staining intensity was lower in the affected muscle, whereas epidermal growth factor receptor and active caspase‐3 staining were higher. Clinical Significance : Pelvic diaphragm muscle weakening is the leading cause of perineal hernia in the dog. Survival and death signals expressed in these muscles may contribute to the pathogenesis of this disease. This study reports epidermal growth factor, transforming growth factor‐alpha and epidermal growth factor receptor immunohistochemical expression in the skeletal muscle and suggests that perineal hernia in the dog is accompanied by levator ani muscle atrophy, increased expression of epidermal growth factor receptor, caspase‐3 activation, and decreased expression of transforming growth factor‐alpha.     

Inflammatory myopathies.

Inflammatory myopathies are the result of infiltration of inflammatory cells into striated muscle, with or without an association with an underlying cause. Two broad classifications are IIMs and secondary inflammatory myopathies associated with other diseases. Standard diagnostic criteria for inflammatory myopathy include the presence of weakness or loss of specific muscle group function, an increase in CK, EMG changes associated with muscle membrane instability, and histologic evidence of inflammation. Not all these criteria, however, must be present. Fresh-frozen biopsy from two proximal muscles is recommended for biopsy confirmation. IIM can either focally affect head or neck muscles or be more diffuse. MMM is an immune-mediated disease characterized by a humoral antibody produced against the unique type IIM and type I variant mvofibers of masticatory muscles of dogs, which causes inflammation and loss of function of the muscles of mastication. Idiopathic polymyositis can affect focal muscle groups (extraocular, laryngeal) or present as multifocal or diffuse involvement of skeletal muscle in the cat and dog. Familial canine DM is an inflammatory disease of the striated muscle, skin, and vasculature in young Collies, Shetland Sheepdogs (Shelties), and, rarely, Collie-crossbred dogs. Immunosuppressive therapy is the key to successful treatment. Protozoal parasitic myopathies are the most common cause of clinically relevant secondary inflammatory myopathies. The degree of systemic involvement is often the limiting factor to successful treatment. Early recognition of the clinical signs for proper diagnostic testing and institution of appropriate therapy can result in a rewarding outcome in treating inflammatory myopathies in the cat and dog.     

Myopathy associated with hyperadrenocorticism in the dog

Naturally occurring or iatrogenic hyperadrenocorticism was associated with myopathy in six dogs. One dog had muscle weakness and muscle atrophy but normal electromyographic findings. Five dogs had muscle stiffness, proximal appendicular muscle enlargement, and myotonic discharges on electromyography. Histologic, electron microscopic, and histochemical findings in the musculature of dogs that were examined were characteristic of noninflammatory degenerative myopathy. Clinical signs of the myopathy improved to varying degrees in five dogs that were treated for the hyperadrenocorticism.     

Amylopectinosis in fetal and neonatal Quarter Horses

Three Quarter Horses, a stillborn filly (horse No. 1), a female fetus aborted at approximately 6 months of gestation (horse No. 2), and a 1-month-old colt that had been weak at birth (horse No. 3), had myopathy characterized histologically by large spherical or ovoid inclusions in skeletal and cardiac myofibers. Smaller inclusions were also found in brain and spinal cord and in some cells of all other tissues examined. These inclusions were basophilic, red-purple after staining with periodic acid-Schiff (both before and after digestion with diastase), and moderately dark blue after staining with toluidine blue. The inclusions did not react when stained with Congo red. Staining with iodine ranged from pale blue to black. Their ultrastructural appearance varied from amorphous to somewhat filamentous. On the basis of staining characteristics and diastase resistance, we concluded that these inclusions contained amylopectin. A distinctly different kind of inclusion material was also present in skeletal muscle and tongue of horse Nos. 1 and 3. These inclusions were crystalline with a sharply defined ultrastructural periodicity. The crystals were eosinophilic and very dark blue when stained with toluidine blue but did not stain with iodine. Crystals sometimes occurred freely within the myofibers but more often were encased by deposits of amylopectin. This combination of histologic and ultrastructural features characterizes a previously unreported storage disease in fetal and neonatal Quarter Horses, with findings similar to those of glycogen storage disease type IV. We speculate that a severe inherited loss of glycogen brancher enzyme activity may be responsible for these findings. The relation of amylopectinosis to the death of the foals is unknown.     

Hemolysis,myopathy, and cardiac disease associated with hereditary phosphofructokinase deficiency in two Whippets

Abstract: Two male castrated Whippet littermates were presented at 1 year of age for pallor, tachycardia, systolic heart murmur, dark yellow to orange feces, intermittent lethargy, pigmenturia, and muscle shivering or cramping after exercise. Persistent macrocytic hypochromic anemia with marked reticulocytosis and metarubricytosis was found when CBC results were compared with reference values for Whippets. Increased serum creatine kinase activity and hyperkalemia also were sometimes present over the 4‐year period of evaluation. Progressively increasing serum concentrations of N‐terminal prohormone brain natriuretic peptide suggested cardiac disease. Erythrocytes from the whippets were less osmotically fragile but more alkaline fragile than those from control dogs. Erythrocyte phosphofructokinase (PFK) activities and 2,3‐diphosphoglycerate concentrations were decreased. Restriction enzyme‐based DNA test screening and DNA sequencing revealed the same mutation in the muscle‐PFK gene of the Whippets as seen in English Springer Spaniel dogs with PFK deficiency. This is the first report of PFK deficiency in Whippet dogs. In addition to causing hemolysis and exertional myopathy, heart disease may be a prominent clinical component of PFK deficiency in this breed and has not been previously recognized in PFK‐deficient English Springer Spaniels.     

Increased Serum Alanine Aminotransferase Activity Associated With Muscle Necrosis in the Dog

Serum activity of alanine aminotransferase (ALT) was consistently increased in dogs with canine X-linked muscular dystrophy (CXMD), a primary myopathy characterized by profound and on-going skeletal muscle necrosis. In order to determine whether the ALT was of liver origin, serum activity of creatine kinase (CK), aspartate aminotransferase (AST), ALT, and sorbitol dehydrogenase (SDH) obtained from dystrophic dogs was compared with enzyme activity present in clinically normal dogs. In dystrophic dogs at all ages tested, serum activity of CK, AST, and ALT was increased, and significant increases were present in dogs four weeks or older. In contrast, SDH activity in dystrophic dogs was not statistically different from values in clinically normal dogs. Ultrastructural examination of liver tissue revealed no evidence of hepatic degeneration in dystrophic dogs. It was concluded that increased serum activity of ALT in the dog may be associated with severe skeletal muscle degeneration, without concurrent hepatocellular necrosis.     

Inherited myopathy of great Danes

A hereditary, non-inflammatory myopathy occurring in young great Danes with distinctive histological features in muscle biopsy specimens is reviewed. Onset of clinical signs is usually before one year of age and both sexes are affected. Clinical signs are characterised by exercise intolerance, muscle wasting, and an exercise-induced tremor. Although most affected dogs have a severe form of the disease, occasional dogs may have a less pronounced form and survive into adulthood with an acceptable quality of life. Litters containing affected puppies are born to clinically unaffected parents, and an autosomal recessive pattern of inheritance is likely. All recorded cases have had fawn or brindle coat coloration. Elevated serum creatinine kinase concentrations and spontaneous electrical activity in skeletal muscles are frequently found. While originally reported (Targett and others 1994) as a central core myopathy in this breed, the histochemical characteristics of the distinct cytoarchitectural structures differ from those of the well-characterised central core myopathy in human beings. In fact, these structures differ from any known myopathy in human beings and likely represents a unique non-inflammatory myopathy affecting dogs. Until this myopathy is characterised further, the name inherited myopathy in great Danes is suggested.     

Histopathological characterization of the skeletal myopathy in rasH2 mice carrying human prototype c-Ha-ras gene

A skeletal myopathy is found in approximately 100% of rasH2 mice. To confirm detailed features of the rasH2 skeletal myopathy, the biceps femoris, diaphragm, triceps brachii, gastrocnemial (types I and II fiber-mixed muscles) and soleus muscle (type I fiber-dominant muscle) obtained from male rasH2 and non-transgenic littermates aged 10-13 and 34 weeks were examined. Variations in the muscle fiber size, early-scattered degeneration/necrosis and regeneration of muscle fibers were detected in 10-13-week-old rasH2 mice. The severity of the above muscular lesions was more prominent in older rasH2 mice. These lesions were noted in the type II myofiber dominant muscles (biceps femoris, triceps brachii and gastrocnemial). NADH-TR stain clearly demonstrated a disorganized intermyofibrillar network and necrotic change in muscle fibers. No specific morphological changes, like rod structure or tubular aggregation seen in some types of myopathy, were noted in Gomori trichrome and NADH-TR stains in the rasH2 mouse like in many types of muscular dystrophy. Electronmicroscopically, occasional muscle fiber degeneration/regeneration, invaded phagocytic cells, indistinct Z-band suggesting excessive contraction and dilatation of the sarcoplasmic reticulum were observed. In summary, the skeletal myopathy occurring in rasH2 mice is consistent with muscular dystrophy characterized morphologically by progressive degeneration and regeneration of myofibers. The myopathy is confined to the type II myofiber predominant muscles and is not associated with any pathognomonic lesions. These characteristics will provide us with a useful model for research in muscular dystrophy of diverse myofibers.     

Pathogenesis, laboratory diagnosis, and clinical implications of erythrocyte enzyme deficiencies in dogs, cats, and horses

Deficiencies of enzymes involved in erythrocyte metabolism can have significant effects on erythrocyte function and survival. Animals with pyruvate kinase (PK) or phosphofructokinase (PFK) deficiencies have shortened erythrocyte life spans and regenerative anemia. PK-deficient dogs (but not PK-deficient cats) develop progressive myelofibrosis and osteosclerosis of bone marrow and hemochromatosis and cirrhosis of the liver. PFK-deficient dogs have sporadic episodes of hyperventilation-induced intravascular hemolysis and hemoglobinuria. Cytochrome b5 reductase (Cb5R) deficiency in dogs and cats results in persistent methemoglobinemia and cyanotic mucous membranes. Severe deficiency of glucose-6-phosphate dehydrogenase, the rate-controlling enzyme in the pentose phosphate pathway, resulted in anemia with eccentrocytosis in an American saddlebred colt. Horses with erythrocyte flavin adenine dinucleotide (FAD) deficiency have both eccentrocytosis (attributable to severe deficiency in glutathione reductase activity) and methemoglobinemia (attributable to Cb5R deficiency); the dual enzyme deficiency occurs because FAD is a required cofactor for both enzymes. Erythrocyte enzyme deficiencies do not usually shorten life expectancy, except for PK-deficient dogs and potentially PFK-deficient dogs during a hemolytic crisis. Although enzyme deficiencies are rare causes of anemia and methemoglobinemia, the ability to diagnose deficient animals allows for the possibility of eliminating these undesirable traits in future breeding. DNA-based assays are available for PK and PFK deficiencies; whereas, biochemical tests of enzyme activity are required for other deficiencies. Continued research is needed to document additional enzyme deficiencies that likely occur and to develop additional DNA-based assays to detect heterozygous animals.