Feline Spongy Encephalopathy With a Mutation in the ASPA Gene.

Canavan disease is an autosomal recessive leukodystrophy caused by mutations in the gene encoding aspartoacylase (ASPA), which hydrolyses N-acetylaspartate (NAA) to acetate and aspartate. A similar feline neurodegenerative disease associated with a mutation in the ASPA gene is reported herein. Comprehensive clinical, genetic, and pathological analyses were performed on 4 affected cats. Gait disturbance and head tremors initially appeared at 1 to 19 months of age. These cats eventually exhibited dysstasia and seizures and died at 7 to 53 months of age. Magnetic resonance imaging of the brain revealed diffuse symmetrical intensity change of the cerebral cortex, brainstem, and cerebellum. Gas chromatography-mass spectrometry analysis of urine showed significant excretion of NAA. Genetic analysis of the 4 affected cats identified a missense mutation (c.859G>C) in exon 6 of the ASPA gene, which was not detected in 4 neurologically intact cats examined as controls. Postmortem analysis revealed vacuolar changes predominantly distributed in the gray matter of the cerebrum and brain stem as well as in the cerebellar Purkinje cell layer. Immunohistochemically, these vacuoles were surrounded by neurofilaments and sometimes contained MBP- and Olig2-positive cells. Ultrastructurally, a large number of intracytoplasmic vacuoles containing mitochondria and electron-dense granules were detected in the cerebral cortex. All 4 cats were diagnosed as spongy encephalopathy with a mutation in the ASPA gene, a syndrome analogous to human Canavan disease. The histopathological findings suggest that feline ASPA deficiency induces intracytoplasmic edema in neurons and oligodendrocytes, resulting in spongy degeneration of the central nervous system.

Rapid multiple immunofluorescent staining for the simultaneous detection of cytokeratin and vimentin in the cytology of canine tumors.

BACKGROUND: Immunocytochemistry (ICC) is utilized as an advanced technique in veterinary cytology. In tumor diagnosis, cytokeratin and vimentin are markers used to distinguish the origin of tumor cells. Standard enzyme-based ICC has limitations in clinical use; and therefore, more convenient and reliable methods are needed. OBJECTIVES: The purpose of this study was to develop a rapid multiple immunofluorescent (RMIF) detection method for dual cytokeratin and vimentin staining on cytology slides in dogs. METHODS: Air-dried smear samples from solid tumors and sediments of pleural effusions were prepared from dogs (n = 14) that were admitted to the Veterinary Teaching Hospital, Kagoshima University, Japan. Mouse monoclonal anti-human cytokeratin (AE1/AE3) and rabbit monoclonal anti-human vimentin (SP20) antibodies were used as primary antibodies, followed by staining with Alexa Fluor-conjugated secondary antibodies. Staining using the RMIF method was compared with enzyme-based ICC staining. RESULTS: Rapid multiple immunofluorescent immunostaining was clear and specific in the evaluated smears, whereas the enzyme-based ICC showed nonspecific signals. By using the RMIF staining method, epithelial cells, mesenchymal cells, and mesothelial cells could be classified on a single smear of a pleural effusion. In smears of lymph nodes with epithelial tumor metastases, the RMIF method successfully detected metastatic epithelial tumor cells. CONCLUSIONS: The RMIF method might be a useful tool for diagnostic cytology in veterinary medicine.

Paraffin immunofluorescence for detection of immune complexes in renal biopsies: an efficient salvage technique for diagnosis of glomerulonephritis in dogs.

BACKGROUND: Renal biopsy is an essential tool for the diagnosis of proteinuric kidney diseases in dogs, and evaluation of immune complexes (IC) by immunofluorescence (IF) of frozen sections (IF-F) is required for the diagnosis of IC-mediated glomerulonephritis (ICGN). However, the use of frozen sections from renal biopsies can have limitations. The aim of this study was to develop a reliable IF method using formalin-fixed and paraffin-embedded (FFPE) sections to detect ICs in dog ICGN. METHODS: Renal biopsy specimens were obtained from dogs with protein-losing nephropathies. FFPE sections were prepared, and eight antigen retrieval pretreatment protocols were performed: digestion with trypsin, microwave (MW) heating in citrate buffer (MW-CB; pH 6.0), MW heating in Tris-EDTA buffer (MW-TEB; pH 9.0), as well as combinations of the above, and a non-treated control. RESULTS: A combination of trypsin for 30 min (Try-30) and MW-TEB; pH 9.0 was the most effective antigen retrieval pretreatment, with clear positive signals for IgG, IgA, IgM, and C3 detected by IF-FFPE. Granular signals, an important diagnostic indicator of ICGN, were clearly observed by both IF-F and IF-FFPE after combined pretreatment with Try-30 and MW-TEB, and IgG, IgA, IgM, and C3 signals were almost completely matched in all samples by IF-F and IF-FFPE. CONCLUSION: IF-FFPE with Try-30 and MW-TEB pretreatment is a valuable technique for the diagnosis of renal diseases in dogs. This method could be an efficient tool when standard IF-F cannot be used, or does not provide useful results due to lack of glomeruli in the specimens for IF-F.

Acquired Fanconi syndrome in two dogs following long-term consumption of pet jerky treats in Japan: case report.

Renal Fanconi syndrome has recently been associated with the ingestion of pet jerky treats from China in mostly small breed dogs in North America, Australia and Europe. We report here about two dogs with Fanconi syndrome following pet jerky treats exposure in Japan. A mixed-breed dog and a French bulldog showed weight loss, polyuria and polydipsia. For years, the owners had been feeding large quantities of pet jerky treats containing chicken prepared in China. Diagnostics revealed glycosuria without hyperglycemia, severe aminoaciduria, and in one case also ketonuria, hypokalemia and metabolic acidosis. A diagnosis of Fanconi syndrome associated with long-term consumption of Chinese pet jerky treats was made. Both dogs recovered fully following withdrawal of the pet jerky treats and supportive care. Fanconi syndrome of dogs in association with the consumption of pet jerky treats of Chinese origin can cause a broad proximal tubular defect with glycosuria and generalized amino aciduria, and should be also considered in Asia. Jerky treats associated Fanconi syndrome can be completely reversible following withdrawal of the treats and supportive care to correct the metabolic abnormalities.

Rapid immunocytochemistry for the detection of cytokeratin and vimentin: assessment of its diagnostic value in neoplastic diseases of dogs.

BACKGROUND: Immunocytochemistry (ICC) is an advanced diagnostic technique used in the field of veterinary cytology. We recently developed a rapid ICC method for the detection of cytokeratin and vimentin in dogs, which helps to determine whether tumor cells are of epithelial or nonepithelial origin. However, the diagnostic value of this rapid ICC method in neoplastic diseases of dogs has not been assessed yet. OBJECTIVES: The aim of the present study was to assess the diagnostic accuracy of rapid ICC compared to standard immunohistochemistry (IHC). METHODS: Air-dried smear samples and formalin-fixed paraffin sections were prepared from tumors excised from dogs (n = 30). Immunosignals for cytokeratin and vimentin were detected in smear samples by rapid ICC, and in paraffin sections by standard IHC. Signals in smear samples detected by rapid ICC were compared with positive staining in paraffin sections detected by standard IHC and analyzed for statistical significance (kappa statistic). RESULTS: Rapid ICC detected specific immunosignals in 25/30 cases (83.3%), and nonspecific signals were detected in 5/30 cases. Statistical analysis revealed fair agreement in epithelial tumors (n = 16) with cytokeratin (κ = 0.236) and vimentin (κ = 0.294). In nonepithelial tumors (n = 14), almost perfect agreement was demonstrated with cytokeratin (κ = 0.857) and vimentin (κ = 0.857). CONCLUSIONS: The rapid ICC method can be a useful tool for the diagnostic cytology of neoplastic tissues in dogs.

Degenerative myelopathy in the Collie breed: a retrospective immunohistochemical analysis of superoxide dismutase 1 in an affected Rough Collie, and a molecular epidemiological survey of the SOD1: c.118G>A mutation in Japan.

Canine degenerative myelopathy (DM) is an adult-onset, progressive neurodegenerative disease that occurs in multiple dog breeds. A DM-associated mutation of the canine superoxide dismutase 1 (SOD1) gene, designated as c.118G>A (p.E40K), has been implicated as one of pathogenetic determinants of the disease in many breeds, but it remains to be determined whether the c.118G>A mutation is responsible for development or progression of DM in Collies. Previously, a Rough Collie was diagnosed clinically and histopathologically as having DM in Japan, suggesting the possibility that the Collie breed may be predisposed to DM due to the high frequency of c.118G>A in Japan. In this study, accumulation and aggregate formation of SOD1 protein were retrospectively demonstrated in the spinal cord of the DM-affected dog by immunohistochemical analysis. Furthermore, a molecular epidemiological survey revealed a high carrier rate (27.6%) and mutant allele frequency (0.138) of c.118G>A in a population of Collies in Japan, suggesting that the Collie breed may be predisposed to DM associated with c.118G>A, and the prevention of DM in Collies in Japan should be addressed through epidemiological and genetic testing strategies.

Localization of a mutant SOD1 protein in E40K-heterozygous dogs: Implications for non-cell-autonomous pathogenesis of degenerative myelopathy.

Canine degenerative myelopathy (DM) is a fatal neurodegenerative disorder. Dogs with typical clinical signs carry homozygous mutations in the superoxide dismutase 1 (SOD1) gene; therefore, DM is regarded as a naturally-occurring model of amyotrophic lateral sclerosis (ALS). Despite the presence of a toxic mutant protein, E40K-SOD1 heterozygotes rarely develop clinical signs. Therefore, E40K-heterozygotes may provide insights into the subclinical and early phase of mutant SOD1-related pathology. In order to identify the distribution of mutant SOD1 in the spinal cords of E40K-heterozygotes, we developed a monoclonal antibody 16G9 that reacts to the mutant E40K-SOD1 protein. We found that the spinal cords of E40K-heterozygotes display white matter degeneration, the severity of which was markedly less than that in E40K-homozygotes. In E40K-heterozygotes, 16G9-reactive SOD1 accumulated predominantly in reactive astrocytes, while spinal neurons remained almost completely free of this form of SOD1 proteins. In contrast, all symptomatic E40K-homozygotes contained 16G9-reactive SOD1 in their spinal neurons and reactive astrocytes. These results suggest that mutant SOD1 proteins accumulate in reactive astrocytes during the early phase of DM pathology, which may contribute to subclinical neurodegeneration. The early involvement of reactive astrocytes in the pathogenesis of DM is strongly suspected and warrants further investigations in the context of non-cell autonomous neuronal death, as proposed for ALS.

Low expression of cyclooxygenase-2 in chronic kidney disease in young dogs.

Chronic kidney disease (CKD) often results in end-stage renal failure in young dogs; however, the pathogenesis of this disease is not established. This study investigated renal expression of cyclooxygenase (COX)-1 and COX-2 proteins in three dogs with chronic kidney disease by immunohistochemistry. Histopathology showed asynchronous differentiation of renal tissues, including immature glomeruli. COX-1 signals were not detected in diseased or normal kidneys. COX-2 signals were low or undetectable in diseased kidneys, while normal kidneys showed clear positive signals in the macula densa (MD). Quantitative scores of COX-2 in diseased kidneys were significantly lower than those in normal kidneys. These findings demonstrate low renal COX-2 expression in CKD in young dogs, but whether this is correlated with disease pathogenesis remains unclear.

Recovery with a regular dose of antibiotics from bacillary hemoglobinuria in a Holstein cow.

One Holstein cow housed with 21 other cows exhibited clinical signs of pyrexia, anorexia and diarrhea along with severe hemoglobinuria. Hematological and biochemical analyses conducted before and after antibiotic therapy indicated severe hemolytic anemia and disruption of hepatic function. A general improvement in conditions was observed after an 11-day program of treatment comprising a regular dose of antibiotics and prescribed supportive therapies. A tentative diagnosis of bacillary hemoglobinuria was made based on the clinical and clinico-pathologic features on day 7. A molecular diagnosis was made by a PCR amplification of the flagellin gene of Clostridium haemolyticum using DNA extracted from the whole blood. The cow was diagnosed with the first recorded occurrence of bacillary hemoglobinuria of Holstein cattle in Japan.

In situ detection of GM1 and GM2 gangliosides using immunohistochemical and immunofluorescent techniques for auxiliary diagnosis of canine and feline gangliosidoses.

BACKGROUND: GM1 and GM2 gangliosidoses are progressive neurodegenerative lysosomal storage diseases resulting from the excessive accumulation of GM1 and GM2 gangliosides in the lysosomes, respectively. The diagnosis of gangliosidosis is carried out based on comprehensive findings using various types of specimens for histological, ultrastructural, biochemical and genetic analyses. Therefore, the partial absence or lack of specimens might have resulted in many undiagnosed cases. The aim of the present study was to establish immunohistochemical and immunofluorescent techniques for the auxiliary diagnosis of canine and feline gangliosidoses, using paraffin-embedded brain specimens stored for a long period. RESULTS: Using hematoxylin and eosin staining, cytoplasmic accumulation of pale to eosinophilic granular materials in swollen neurons was observed in animals previously diagnosed with GM1 or GM2 gangliosidosis. The immunohistochemical and immunofluorescent techniques developed in this study clearly demonstrated the accumulated material to be either GM1 or GM2 ganglioside. CONCLUSIONS: Immunohistochemical and immunofluorescent techniques using stored paraffin-embedded brain specimens are useful for the retrospective diagnosis of GM1 and GM2 gangliosidoses in dogs and cats.

Canine IgA nephropathy: a case report.

Immunoglobulin (Ig) A nephropathy is a rare form of canine glomerular disease. This report describes a case of canine IgA nephropathy showing characteristics typical of human IgA nephropathy. An 8-year-old, spayed female Miniature Dachshund showed persistent severe proteinuria without azotemia. She was receiving long-term glucocorticoid therapy due to chronic gastritis and an intra-abdominal suture granuloma. A renal biopsy demonstrated mesangial proliferative glomerulonephritis with predominantly mesangial IgA deposition and electron-dense deposits in the paramesangium. These findings closely resembled those of human IgA nephropathy. Glucocorticoid treatment was discontinued, and the angiotensin-converting enzyme inhibitor enalapril was administrated as an antiproteinuric agent. The proteinuria subsequently went into remission, and the patient has maintained good condition without recurrence.

Real-time PCR genotyping assay for canine progressive rod-cone degeneration and mutant allele frequency in Toy Poodles, Chihuahuas and Miniature Dachshunds in Japan.

Canine progressive rod-cone degeneration (PRCD) is a middle- to late-onset, autosomal recessive, inherited retinal disorder caused by a substitution (c.5G>A) in the canine PRCD gene that has been identified in 29 or more purebred dogs. In the present study, a TaqMan probe-based real-time PCR assay was developed and evaluated for rapid genotyping and large-scale screening of the mutation. Furthermore, a genotyping survey was carried out in a population of the three most popular breeds in Japan (Toy Poodles, Chihuahuas and Miniature Dachshunds) to determine the current mutant allele frequency. The assay separated all the genotypes of canine PRCD rapidly, indicating its suitability for large-scale surveys. The results of the survey showed that the mutant allele frequency in Toy Poodles was high enough (approximately 0.09) to allow the establishment of measures for the prevention and control of this disorder in breeding kennels. The mutant allele was detected in Chihuahuas for the first time, but the frequency was lower (approximately 0.02) than that in Toy Poodles. The mutant allele was not detected in Miniature Dachshunds. This assay will allow the selective breeding of dogs from the two most popular breeds (Toy Poodle and Chihuahua) in Japan and effective prevention or control of the disorder.

GM1 gangliosidosis in a Japanese domestic cat: a new variant identified in Hokkaido, Japan.

A male Japanese domestic cat with retarded growth in Hokkaido, Japan, showed progressive motor dysfunction, such as ataxia starting at 3 months of age and tremors, visual disorder and seizure after 4 months of age. Finally, the cat died of neurological deterioration at 9 months of age. Approximately half of the peripheral blood lymphocytes had multiple abnormal vacuoles. Magnetic resonance imaging showed bisymmetrical hyperintensity in the white matter of the parietal and occipital lobes in the forebrain on T2-weighted and fluid-attenuated inversion recovery images, and mild encephalatrophy of the olfactory bulbs and temporal lobes. The activity of lysosomal acid ß-galactosidase in leukocytes was negligible, resulting in the biochemical diagnosis of GM1 gangliosidosis. Histologically, swollen neurons characterized by accumulation of pale, slightly granular cytoplasmic materials were observed throughout the central nervous system. Dysmyelination or demyelination and gemistocytic astrocytosis were observed in the white matter. Ultrastructually, membranous cytoplasmic bodies were detected in the lysosomes of neurons. However, genetic analysis did not identify the c.1448G>C mutation, which is the single known mutation of feline GM1 gangliosidosis, suggesting that the cat was affected with a new variant of the feline disease.

GM2 Gangliosidosis Variant 0 (Sandhoff Disease) in a Mixed-Breed Dog.

GM2 gangliosidosis variant 0 (Sandhoff disease, SD) is a fatal, progressive, neurodegenerative lysosomal storage disease caused by simultaneous deficiencies of acid ß-hexosaminidases A and B. Canine SD has so far been identified only in two purebreeds. In this article, we present the case of a 10 mo old, male dog of mixed breed that developed progressive neurological signs including ataxia, postural deficit, and visual deficits and finally died at the age of 21 mo. The dog was diagnosed with SD on the basis of the results of biochemical and histopathological analyses. This is the third report of canine SD and the first time it has been identified in a mixed breed.

Real-time PCR genotyping assay for feline erythrocyte pyruvate kinase deficiency and mutant allele frequency in purebred cats in Japan.

Erythrocyte pyruvate kinase (PK) deficiency is an inherited glycolytic erythroenzymopathy caused by mutations of the PKLR gene. A causative mutation of the feline PKLR gene was originally identified in Abyssinian and Somali cats in the U.S.A. In the present study, a TaqMan probe-based real-time PCR genotyping assay was developed and evaluated for rapid genotyping and large-scale screening for this mutation. Furthermore, a genotyping survey was carried out in a population of four popular purebred cats in Japan to determine the current mutant allele frequency. The assay clearly displayed all genotypes of feline PK deficiency, indicating its suitability for large-scale survey as well as diagnosis. The survey demonstrated that the mutant allele frequency in Abyssinian and Somali cats was high enough to warrant measures to control and prevent the disease. The mutant allele frequency was relatively low in Bengal and American Shorthair cats; however, the testing should still be carried out to prevent the spread of the disease. In addition, PK deficiency should always be considered in the differential diagnosis of anemia in purebred cats in Japan as well as worldwide.

Real-time PCR genotyping assay for GM2 gangliosidosis variant 0 in toy poodles and the mutant allele frequency in Japan.

GM2 gangliosidosis variant 0 (Sandhoff disease, SD) is a fatal, progressive neurodegenerative lysosomal storage disease caused by mutations of the HEXB gene. In canine SD, a pathogenic mutation (c.283delG) of the canine HEXB gene has been identified in toy poodles. In the present study, a TaqMan probe-based real-time PCR genotyping assay was developed and evaluated for rapid and large-scale genotyping and screening for this mutation. Furthermore, a genotyping survey was carried out in a population of toy poodles in Japan to determine the current mutant allele frequency. The real-time PCR assay clearly showed all genotypes of canine SD. The assay was suitable for large-scale survey as well as diagnosis, because of its high throughput and rapidity. The genotyping survey demonstrated a carrier frequency of 0.2%, suggesting that the current mutant allele frequency is low in Japan. However, there may be population stratification in different places, because of the founder effect by some carriers. Therefore, this new assay will be useful for the prevention and control of SD in toy poodles.

High frequency of a single nucleotide substitution (c.-6-180T>G) of the canine MDR1/ABCB1 gene associated with phenobarbital-resistant idiopathic epilepsy in Border Collie dogs.

A single nucleotide substitution (c.-6-180T>G) associated with resistance to phenobarbital therapy has been found in the canine MDR1/ABCB1 gene in Border Collies with idiopathic epilepsy. In the present study, a PCR-restriction fragment length polymorphism assay was developed for genotyping this mutation, and a genotyping survey was carried out in a population of 472 Border Collies in Japan to determine the current allele frequency. The survey demonstrated the frequencies of the T/T wild type, T/G heterozygote, and G/G mutant homozygote to be 60.0%, 30.3%, and 9.8%, respectively, indicating that the frequency of the mutant G allele is extremely high (24.9%) in Border Collies. The results suggest that this high mutation frequency of the mutation is likely to cause a high prevalence of phenobarbital-resistant epilepsy in Border Collies.

Molecular epidemiology of canine GM1 gangliosidosis in the Shiba Inu breed in Japan: relationship between regional prevalence and carrier frequency.

BACKGROUND: Canine GM1 gangliosidosis is a fatal disease in the Shiba Inu breed, which is one of the most popular traditional breeds in Japan and is maintained as a standard breed in many countries. Therefore, it is important to control and reduce the prevalence of GM1 gangliosidosis for maintaining the quality of this breed and to ensure supply of healthy dogs to prospective breeders and owners. This molecular epidemiological survey was performed to formulate an effective strategy for the control and prevention of this disease. RESULTS: The survey was carried out among 590 clinically unaffected Shiba Inu dogs from the 8 districts of Japan, and a genotyping test was used to determine nation-wide and regional carrier frequencies. The number and native district of affected dogs identified in 16 years from 1997 to June 2013 were also surveyed retrospectively. Of the 590 dogs examined, 6 dogs (1.02%, 6/590) were carriers: 3 dogs (2.27%, 3/132) from the Kinki district and the other 3 dogs from the Hokkaido, Kanto, and Shikoku districts. The retrospective survey revealed 23 affected dogs, among which, 19 dogs (82.6%) were born within the last 7 years. Of the 23 affected dogs, 12 dogs (52.2%) were from the Kinki district. Pedigree analysis demonstrated that all the affected dogs and carriers with the pedigree information have a close blood relationship. CONCLUSIONS: Our results showed that the current carrier frequency for GM1 gangliosidosis is on the average 1.02% in Japan and rather high in the Kinki district, which may be related to the high prevalence observed over the past 16 years in this region. This observation suggests that carrier dogs are distributed all over Japan; however, kennels in the Kinki district may face an increased risk of GM1 gangliosidosis. Therefore, for effective control and prevention of this disease, it is necessary to examine as many breeding dogs as possible from all regions of Japan, especially from kennels located in areas with high prevalence and carrier frequency.

Genotyping assays for the canine degenerative myelopathy-associated c.118G>A (p.E40K) mutation of the SOD1 gene using conventional and real-time PCR methods: a high prevalence in the Pembroke Welsh Corgi breed in Japan.

Canine degenerative myelopathy is an adult-onset, progressive neurodegenerative disease that occurs in multiple dog breeds, particularly Pembroke Welsh Corgis. Recently, a degenerative myelopathy-associated mutation of the canine SOD1 gene was identified as c.118G>A (p.E40K). In the present study, genotyping assays using conventional and real-time PCR methods were developed, and a preliminary genotyping survey was performed on 122 randomly selected Pembroke Welsh Corgis without any degenerative myelopathy-related clinical signs to determine the current allele frequency in Japan. Both of the assays provided clear-cut genotyping. The survey demonstrated the frequencies of the G/G wild-type, G/A heterozygote and A/A homozygote to be 9.0, 42.6 and 48.4%, respectively, indicating that the prevalence of the mutant A allele (69.7%) in Pembroke Welsh Corgis is extremely high in Japan.

Real-time PCR genotyping assay for canine trapped neutrophil syndrome and high frequency of the mutant allele in Border collies.

Trapped neutrophil syndrome is an autosomal recessive inherited neutropenia in Border collies. The causative mutation is a 4base pair deletion in exon 19 of the canine VPS13B gene. In this study, a real-time PCR assay was developed and a genotyping survey was carried out in Border collies in Japan. The carrier frequency was 11.1%, suggesting that the mutant allele frequency is high enough to warrant measures to control and prevent the disease.