Molecular genetic and expression analysis of alpha-tocopherol transfer protein mRNA in German shepherd dogs with degenerative myelopathy

Degenerative Myelopathy (DM) is a progressive neurological disorder of the spinal cord preferentially occurring in German shepherd dogs. The pathogenesis of the disease is unknown. However, there are indications that vitamin E deficiency may be involved in the pathogenesis of DM. Therefore, we analyzed the expression and the nucleotide sequence of the canine alpha-tocopherol transfer protein (alpha Ttp) of German shepherd dogs with DM in order to determine whether a deficiency or a defect of the alpha Ttp could be a primary factor in the pathogenesis of DM, as found in human patients with Ataxia with vitamin E deficiency (AVED). The cDNA of the coding region of the canine alpha Ttp-mRNA was generated from total liver RNA using RT-PCR and 5' RACE technique. We determined the sequence of 707 out of 834 base pairs or 84.8% of the canine alpha Ttp coding region. Sequence comparison of canine alpha Ttp between affected and control dogs revealed no differences in either nucleotide or predicted amino acid sequence. Using Northern blot analysis alpha Ttp-mRNA expression was solely found in the liver of the dogs, rats and humans, while various other organs showed no alpha Ttp-mRNA expression. No significant differences in expression levels of canine alpha Ttp mRNA were found between DM and control dogs. Our data suggest that the canine alpha Ttp gene is unlikely to be involved in the pathogenesis of DM in German shepherd dogs.     

A degenerative myelopathy in young German shepherd dogs

Two young male German shepherd dogs, six and seven months old, were evaluated for progressive hindlimb ataxia and paresis. Both dogs were found to be thin and poorly muscled on physical examination, and on neurological examination, the lesions were localised to the spinal cord between T2 and L4. Microscopical examination of the spinal cords demonstrated widespread axonal degeneration similar to that seen in aged German shepherd dogs with chronic degenerative radiculomyelopathy (CDRM), although the distribution of these changes was not identical to CDRM.     

Central nervous system pathology in 25 dogs with chronic degenerative radiculomyelopathy

The neuropathology of 20 German shepherd dogs and five German shepherd dog crosses with chronic degenerative radiculomyelopathy were analysed by conventional techniques, immunocytochemistry and electron microscopy. There were previously unrecognised changes in brain nuclei. In the spinal cord, both motor and sensory tracts were involved, principally in their more distal regions. Wallerian degeneration affected the corticorubrospinal pathways in the lateral columns and the ventral funiculi, predominantly in the caudal thoracic and lumbar segments, although more cranial involvement was also observed. The dorsal columns were affected in the caudal lumbar region and the cervical fasciculus gracilis. The regional distribution was variable between cases. Within the brain, abnormalities, including chromatolysis, gliosis and neuronal loss were observed in the red nucleus, lateral vestibular nucleus and, occasionally, in the dentate nucleus. The changes in brain nuclei were compared with those found in dogs at various times after a focal spinal injury. The neuronal changes in the brain may be related to the primary site of damage, and possible aetiological mechanisms are discussed.     

Haemophilia A in German shepherd dogs

Haemophilia A was diagnosed in 14 male German shepherd dogs. Factor VIII: coagulant (FVIII:C) activities ranged from 1.13% of a normal canine plasma pool. von Willebrand's factor antigen values were normal or increased in all 9 of these dogs which were tested. Twelve of these dogs had a common maternal grandsire. Five dogs had exhibited no tendency to bleed (when tested between 2 to 23 months of age). They were tested because of pedigree links with clinically affected animals. Common clinical signs in the latter dogs included: bleeding from the mouth, subcutaneous and intramuscular haematomas and lameness. Since these dogs usually had a mild to moderate deficiency of FVIII:C, they may survive to adulthood without exhibiting clinical signs severe enough to necessitate veterinary attention.     

Computed tomographic pelvimetry in German shepherd dogs

Pelvimetry was performed on a computed tomographic scanner using dorsal and lateral scout-view images of 10 adult German shepherd dogs. The vertical and transverse diameters of the pelvis were measured and the pelvic inlet and pelvic outlet areas were also calculated. No significant correlations between the pelvic measurements and body weight, age and sex were found. Although the conjugata vera, diameter verticalis, diameter sacralis, sagittal diameter and the distance between the two medial tubera ischiadica were longer in females, no significant statistical difference was found between male and female animals for all measurements. It is proposed that the computed tomographic pelvimetry should be preferred for the sensitive measurements when the normal standards are determined for different animal species and the cost is reduced to an acceptable level.     

Echocardiographic indices in normal German shepherd dogs

Sixty clinically normal German shepherd dogs, 31 males and 29 females, ranging in age from 1 and 5 years and with a body weight ranging from 22 to 37.2 kg, were examined by the two-dimensional mode, M-mode, and Doppler echocardiography. In Doppler mode, the mitral valve flows were obtained, where the aim was to determine the velocity peaks and ratios of the E and A waves and the mitral E wave deceleration time. The velocity peaks were obtained for the tricuspid, pulmonary and aortic valves. On the left ventricular outflow tract flow, the time velocity integral and aortic cross-sectional area was used to calculate the cardiac output. A statistically significant correlation with the body weight was found for the systolic left atrial and diastolic aortic diameter on two-dimensional mode. On M-mode, there was a significant correlation between the body weight and the systolic left atrium and diastolic aortic dimension, systolic and diastolic left ventricular, septal and posterior wall dimensions. Doppler echocardiography showed that there was no significant correlation between the body weight and the mitral, tricuspid, pulmonary and aortic valves flows. These results demonstrate that it is important to know the normal echocardiographic values for German shepherd dogs because there are some characteristics peculiar to this particular breed. The data obtained is expected to be helpful for studies on small animal cardiology.     

Medullary trichomalacia in 6 German shepherd dogs

Medullary trichomalacia is the name proposed for a hair shaft abnormality that was recognized in 6 German shepherd dogs. Affected dogs had multifocal areas of broken hairs, especially on the dorsolateral trunk. Microscopic examination of hair shafts revealed focal areas of loss of architecture, swelling, and apparent softening of the medulla, followed by longitudinal (length-wise) splitting and breakage of the hair shaft. No cause could be found. Affected dogs were otherwise healthy, and apparent spontaneous recovery was the usual outcome. Relapses may occur.     

Serum auto antibodies and clinical/pathological features in German shepherd dogs with a lupuslike syndrome

This study presents 8 dogs of German Shepherd breed (6 males, 2 females, 2-5 years of age at onset of the disease) with a lupus like syndrome characterized by febrile polyarthritis, wasting, nephropathy, cutaneous lesions and high positive titres of ANA (antinuclear antibodies) of speckled type. The serum autoantibodies were further characterized by double immunodiffusion against ENA (extractable nuclear antigen), ELISA for Histone antibodies (Histon fraction H-24A and H-3S), indirect IF on rat-liver sections, non treated and RNase/DNase digested sections for DNP/RNP antibodies, and smears of a hemoflagellate C. luciliae for antibodies vs doubbel strained DNA, (dsDNA). Thus, the high ANA titres in these dogs represent varying types of autoantibodies against nucleoproteins of both DNA and RNA nature, associated histone antigens and non-histone antibodies (RNA and Sm) as well. Rheumatoid Factor titres in serum from these dogs were low or negative. Immunoglobulin deposits at dermo-epidermal junctions were demonstrated in some of the dogs with hyperkeratotic skin lesions. High concentration of serum-IgG was a constant finding in combination with anemia and in most cases leukopenia probably related to the chronic inflammatory process in these animals. Autoimmune hemolytic anemia (AIHA) or thrombocytopenia was not detected in these dogs.     

Transient erythropoietic protoporphyria associated with chronic hepatitis and cirrhosis in a cohort of German shepherd dogs

Over the course of one year, slight jaundice and ascites suggestive of chronic liver disease occurred in 17 German shepherd dogs from one breeding colony. Blood analyses, performed twice with a six-month interval, revealed elevated serum activities of liver enzymes in 13 dogs. In addition, four young adult German shepherd dogs that showed severe ascites, slight jaundice and increased serum liver enzyme activities were referred for further evaluation. Because of their poor prognosis these four dogs were euthanased. There were no signs of photosensitivity. Postmortem examinations revealed macronodular darkened livers, which were characterised histopathologically by cirrhosis associated with aggregates of brown pigments showing a striking orange birefringence in polarised light. Ultrastructurally, the crystalline pigments were typical of protoporphyrins. High-performance liquid chromatographic analysis of liver samples revealed very high levels of protoporphyrins (mean 9550 nmol/g wet liver, reference value 0.41 nmol/g wet liver) and low activities of ferrochelatase (mean 0.274 mmol/mg protein/hour, reference value 0.684 nmol/mg protein/hour). Twenty-six months after the onset of the hepatopathies, the clinical condition of the 13 surviving dogs had improved and their serum liver enzyme activities were normal. The clinical histories and pedigree analyses were not in concordance with an inherited form of protoporphyria. There was no known history of exposure to toxic substances or drugs. The findings are in accordance with a transient erythropoietic protoporphyria associated with hepatic complications, presumably caused by exposure to a porphyrinogenic, ferrochelatase-inhibitory substance of unknown origin.     

Immunologic study of systemic aspergillosis in German shepherd dogs

Data are presented from a series of eight cases of disseminated canine aspergillosis (A. terreus) in German Shepherd dogs referred to Murdoch University Veterinary Hospital. Immunoglobulin determination revealed depression of serum IgA (cases 1 and 5) and IgM (case 2) levels and elevated levels of IgG in all cases. Total complement activity (CH50) and complement components tests, (C3, C4) were present in normal amounts in all cases. Using agar gel diffusion, serum antibody to A. terreus was found in only one case and aspergillus antigenaemia in two of the remainder. Lectin transformation of lymphocytes in two dogs was found to be depressed relative to normal controls in case 1 and initially in case 2. Two dogs failed to respond to the intradermal injection of A. terreus antigen.     

Spontaneous laryngeal paralysis in four white-coated German shepherd dogs

Four cases of spontaneous laryngeal paralysis in juvenile white-coated German shepherd dogs are described. The presenting signs were typical for laryngeal paralysis, with stridor present in all four cases. Laryngoscopy revealed bilateral laryngeal paralysis in three cases, and unilateral paralysis in one. Concurrent megaoesophagus was also identified in one dog. All dogs underwent surgical treatment for laryngeal paralysis. Euthanasia was performed in one case due to intractable regurgitation and aspiration pneumonia. A possible association with white coat colour is discussed.     

Immunohistochemical evidence for immunoglobulin and complement deposition in spinal cord lesions in degenerative myelopathy in German shepherd dogs.

The purpose of this study was to examine the distribution of immunoglobulin and complement component C3 in spinal cord tissues of dogs with degenerative myelopathy. Sections of formalin-fixed paraffin-embedded spinal cord from five German Shepherd dogs with clinical and histological features consistent with degenerative myelopathy (DM) and one normal dog were tested immunohistochemically for deposition of immunoglobulin G (IgG) and the third component of complement (C3). In all dogs there was staining associated with large and small blood vessels. In addition, in the dogs with DM there was focal staining for IgG and C3 in spinal nerve tracts characteristically affected in DM. Deposition of IgG and C3 was found in histological lesions, and in addition, in other areas independent of visible lesions, suggesting that IgG and C3 deposition may precede histological evidence of spinal cord damage. These findings suggest a role for immune-mediated destruction of the spinal cord which may contribute to the pathogenesis of DM in German Shepherd dogs.     

Age-associated changes in the immune system of German shepherd dogs

In order to look into the ageing of the canine immune system we investigated age-related changes and associated gender-related differences in parameters of innate and acquired immunity in German Shepherd dogs. We obtained the following findings: white blood cell counts, peripheral blood lymphocytes, lymphocyte proliferative activity and interleukin-2 (IL-2) serum concentrations were significantly lower in the group of old animals, whereas the concentrations of gamma-globulins and the functional activity of the complement system were significantly higher in the elderly. Phagocytic and bactericidal activity of polymorphonuclear cells, as well as their 'killing function,' the serum cytokine-like activities of tumour necrosis factor-alpha and the plasma concentrations of immunoglobulin G, as well as of alpha- and beta-globulins, were not significantly affected by age, whereas natural killer-cell activity and the serum cytokine-like activities of IL-1 were significantly higher only in the group of female old animals. With regard to gender-related differences, lymphocyte proliferative activities as well as plasma concentrations of alpha-globulin were significantly higher in the group of female animals, whereas the absolute numbers of segmented neutrophils were significantly lower. Species analogies with regard to ageing as presumed to exist between man and laboratory rodents also seem to be applicable to the dog. The observed age-related changes in the canine immune system are probably among the main causes for the multimorbidity of old age, affecting life expectancy and mortality in the dog and should be recognized and considered by the attending veterinarian.