Development of a snapback method of single-strand conformation polymorphism analysis for genotyping Golden Retrievers for the X-linked muscular dystrophy allele.

OBJECTIVE: To develop a snapback method of single-strand conformation polymorphism (SSCP) analysis for genotyping Golden Retrievers for the X-linked muscular dystrophy allele. ANIMALS: 20 Golden Retriever puppies from a colony with X-linked muscular dystrophy. PROCEDURE: DNA spanning the canine dystrophin mutation was amplified by means of a polymerase chain reaction (PCR), using a primer modified to have an additional sequence at the 5' terminus. The primer was designed so that 1 terminus of the single-stranded PCR product could anneal to the normal sequence flanking the region of the mutation in the allele but not in the mutant allele. True disease status of the dogs was determined by means of a PCR and restriction digest protocol. RESULTS: Snapback SSCP analysis allowed for accurate and unambiguous genotyping of unaffected, carrier, and affected dogs, whereas conventional SSCP analysis, using the unmodified primer, did not. Creatine kinase activities measured within 24 hours after birth were not consistent with genotype. CONCLUSION AND CLINICAL RELEVANCE: Snapback SSCP analysis provided a simple, fast, and accurate method for genotyping Golden Retrievers for the mutation known to cause X-linked muscular dystrophy.     

The electroretinographic phenotype of dogs with Golden Retriever muscular dystrophy

Objective To characterize the flash electroretinogram (ERG) in the Golden Retriever muscular dystrophy (GRMD) dog and to compare the results with those from a control group of Golden Retrievers. To investigate whether similar abnormalities of the ERG as those found in a majority of human patients with Duchenne muscular dystrophy (DMD) are also observed in the GRMD dog, the canine model for DMD. Animals Five GRMD dogs and five age‐matched clinically normal Golden Retrievers. Procedure An ophthalmic examination was carried out prior to performing electroretinography under general anesthesia. Rod, combined rod–cone and oscillatory potentials responses were recorded after dark adaptation. Responses to 30‐Hz‐flicker were recorded after light adaptation. The ERG responses of the GRMD dogs were compared with those of the control dogs by use of a Wilcoxon signed rank test. Results GRMD dogs had significantly reduced a and b‐wave amplitudes after dim white flash stimuli (rod response) and reduced a‐wave amplitude after bright white flash stimuli (rod–cone response). Conclusion and clinical relevance The ERG abnormalities observed in the GRMD dog suggest a dysfunction in the rod signaling pathway. These ERG alterations are different from those observed in human patients with DMD.     

Use of amnion and placenta in neonatal screening for canine GM1-gangliosidosis and the risk of diagnostic misclassifications

BACKGROUND: A closed breeding colony of Shiba dogs with GM1-gangliosidosis is maintained at Hokkaido University (Sapporo, Japan). Neonatal genotyping is essential to control the breeding colony genetically as an animal model for the human disease. OBJECTIVES: The purpose of the present study was to determine the utility of amnion and placenta in the neonatal screening or diagnosis for canine GM1-gangliosidosis. METHODS: Twenty neonatal Shiba dogs of a pedigree with GM1- gangliosidosis were differentiated into 3 genotypes--normal, heterozygous, and affected dogs--by using a previously reported DNA mutation assay. Acid beta-galactosidase activity was measured in amnion and placenta and compared among the 3 genotypes. RESULTS: The level of beta-galactosidase activity in the amnion of affected dogs was negligible and <2% of the mean activity in normal dogs; there was no significant difference among the 3 genotypes. In placenta, beta-galactosidase activity was significantly different among all the genotypes; however, there was wide overlap in enzyme activity between normal and heterozygous dogs. The level of activity in affected dogs was relatively high and >10% of the mean activity in normal dogs. The DNA mutation assay gave correct information about genotype with genomic DNA extracted from amnion but ambiguous information with DNA from placenta. CONCLUSIONS: Amnion and placenta were not useful as enzyme sources in neonatal screening in canine GM1-gangliosidosis because of the risk of misdiagnosis. DNA from amnion is applicable as a template for genotyping, whereas placenta should not be used because canine placenta contains maternal cells.     

Diagnostic potential of natriuretic peptides in the occult phase of golden retriever muscular dystrophy cardiomyopathy

The objective of the study was to determine whether the plasma concentrations of atrial and brain natriuretic peptides (ANP and BNP, respectively) could be reliable markers of cardiac alterations during occult cardiomyopathy in Golden Retriever Muscular Dystrophy (GRMD). Fifty Golden Retrievers without any clinical or radiographic sign of heart disease were included in this study (21 GRMD dogs and 29 controls). Controls and GRMD dogs were divided into 2 subgroups according to age (< and > or =12 months old, respectively). All dogs underwent echocardiography and determination of BNP and ANP plasma concentrations by radioimmunoassay. No ventricular dilatation or dysfunction was observed in either control or GRMD dogs. ANP plasma concentration did not differ significantly between controls and GRMD dogs (mean +/- SD = 72 +/- 49 versus 58 +/- 23 pg/mL, respectively, P = .21). This finding was confirmed in both subgroups of dogs (ie, those < and > or =12 months old). In contrast, BNP plasma concentrations were significantly higher in GRMD dogs than in controls (mean +/- SD = 117 +/- 92 versus 46 +/- 22 pg/mL, respectively, P < .05). In dogs > or =12 months old, sensitivity and specificity of BNP for identifying GRMD with a cutoff of 65 pg/mL were 78 and 86%, respectively. For the same cutoff value, sensitivity dropped to 42%, whereas specificity reached 100% in dogs <12 months old. In conclusion, BNP may be a useful biochemical marker of asymptomatic cardiomyopathy. However, this peptide does not allow very early detection because its optimal discriminatory power was observed in adult dogs (ie, > or =12 months of age).     

Tissue Doppler assessment of diastolic and systolic alterations of radial and longitudinal left ventricular motions in Golden Retrievers during the preclinical phase of cardiomyopathy associated with muscular dystrophy

OBJECTIVE: To quantify radial and longitudinal left ventricular free wall (LVFW) velocities in dogs during the preclinical phase of Golden Retriever muscular dystrophy (GRMD)-associated cardiomyopathy by use of tissue Doppler imaging (TDI). ANIMALS: 9 dogs with GRMD and 6 healthy control dogs. PROCEDURE: All dogs (< 3 years old) were examined via conventional echocardiography and 2-dimensional color TDI. Myocardial velocities in the LVFW were recorded from right parasternal ventricular short-axis (radial motion) and left apical 4-chamber (longitudinal motion) views. Cardiac assessments via TDI included maximal systolic and early and late diastolic LVFW velocities in the endocardial and epicardial layers (for radial motion) and in the basal and apical segments (for longitudinal motion) (for longitudinal motion), RESULTS:-No notable ventricular dilatation or alteration of inotropism was detected in dogs with GRMD via conventional echocardiography. Compared with healthy dogs, endocardial velocities were significantly decreased in dogs with GRMD, resulting in marked decreases in radial myocardial velocity gradients during systole and early and late diastole. Similarly, basal and apical velocities were significantly decreased in systole and the former also in early diastole, resulting in significant decreases in the 2 corresponding longitudinal myocardial velocity gradients. The radial epicardial and longitudinal late diastolic velocities were comparable in the 2 groups. CONCLUSION AND CLINICAL RELEVANCE: Results indicated that GRMD-associated cardiomyopathy in dogs is associated with early marked dysfunction of both radial and longitudinal LVFW motions. These combined regional myocardial abnormalities might be useful criteria for detection of dilated cardiomyopathy at the preclinical stage of the disease in dogs.     

Identification of a 6 base pair insertion in West Highland White Terriers with erythrocyte pyruvate kinase deficiency.

OBJECTIVE: To define the disease-causing mutation in West Highland White Terriers (WHWT) with erythrocyte pyruvate kinase (R-PK) deficiency and to design a genetic test capable of recognizing affected (homozygous) and carrier (heterozygous) dogs. ANIMALS: 3 anemic WHWT littermates and 1 unaffected littermate; 16 dogs from the same kennel, including 4 unrelated, phenotypically normal dogs (control dogs), and 12 for which PK activity was not known; 2 PK-deficient Basenjis; 2 PK-deficient Beagles; 4 unaffected English Springer Spaniels; and 1 mixed-breed dog. PROCEDURES: cDNA was cloned and sequenced, and cDNA sequences were compared with the published sequence for canine R-PK cDNA to identify the putative disease-causing mutation. Genomic DNA spanning the affected region was cloned and sequenced to verify the mutation. Subsequently, polymerase chain reaction primers were designed to amplify the section of the gene containing the mutation from DNA in blood or buccal swab samples. Gel electrophoresis allowed assignment of genotypes on the basis of allele separation. RESULTS: 4 single base polymorphisms attributable to sequencing errors in the published sequence were identified, along with a 6 base pair (bp) insertion in exon 10 that was recognized as a putative disease-causing mutation. An identical insertion was found in genomic DNA. Amplification of genomic DNA yielded a 117 bp product for genotypically normal dogs and a 123 bp product for WHWT homozygous for PK deficiency. Carriers had 1 copy of each allele and variable heteroduplex structures. CONCLUSIONS AND CLINICAL RELEVANCE: A 6 bp insertion in the C domain of R-PK was identified in WHWT with PK deficiency. Affected and carrier dogs could be distinguished with a genetic test.     

Genetic screening of the canine zinc finger protein multitype 2 (cZFPM2) gene in dogs with tetralogy of Fallot (TOF)

A recent genetic study in humans where the ZFPM2 gene was screened for mutations revealed a heterozygous mutation that was found to contribute to several sporadic cases of tetralogy of Fallot (TOF). We hypothesized that this would also hold true for a study group of dogs with TOF. We isolated the full-length cDNA of the canine ZFPM2 and aligned the deduced amino acid sequence against sequences from other species. Our analysis indicated that the canine ZFPM2 was phylogenetically much closer to the human ZFPM2 compared with the mouse or rat sequences. Further gene expression analysis also failed to identify differential gene expression between affected and unaffected dogs. We screened the cZFPM2 for mutations in a group of dogs with TOF but found no significant base changes in either the splice sites or the coding regions.     

Physical therapy assessment tools to evaluate disease progression and phenotype variability in Golden Retriever muscular dystrophy

Dogs suffering from Golden Retriever muscular dystrophy (GRMD) present symptoms that are similar to human patients with Duchenne muscular dystrophy (DMD). Phenotypic variability is common in both cases and correlates with disease progression and response to therapy. Physical therapy assessment tools were used to study disease progression and assess phenotypic variability in dogs with GRMD. At 5 (T0), 9 (T1), 13 (T2) and 17 (T3) months of age, the physical features, joint ranges of motion (ROM), limb and thorax circumferences, weight and creatine kinase (CK) levels were assessed in 11 dogs with GRMD. Alterations of physical features were higher at 13 months, and different disease progression rates were observed. Passive ROM decreased until 1 year old, which was followed by a decline of elbow and tarsal ROM. Limb and thorax circumferences, which were corrected for body weight, decreased significantly between T0 and T3. These measurements can be used to evaluate disease progression in dogs with GRMD and to help discover new therapies for DMD patients.     

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.     

The ABCB1-1Δ mutation is not responsible for subchronic neurotoxicity seen in dogs of non-collie breeds following macrocyclic lactone treatment for generalized demodicosis

P-glycoprotein (P-gp), encoded by the multiple drug resistance gene ABCB1 (also known as MDR1 ), is an integral component of the blood brain barrier crucial in limiting drug uptake into the central nervous system. Altered expression or function of P-gp, as seen in dogs of the collie lineage homozygous for the nt228(del4) mutation of the ABCB1 gene ( ABCB1-1Δ ), can result in potentially fatal neurotoxicosis, especially following administration of systemic macrocyclic lactones (SML). Occasionally, dogs from unrelated breeds develop subchronic signs of neurotoxicity when receiving SML to treat generalized demodicosis. It is possible that these dogs are heterozygous carriers of the ABCB1-1Δ mutation, resulting in decreased P-gp activity and central neurotoxicosis. Cheek swabs were collected from 28 dogs with generalized demodicosis that had shown subchronic signs of neurotoxicity following daily oral administration of ivermectin or other SML. Ten of these animals received concurrent systemic treatment with other confirmed or putative P-gp substrates. After DNA extraction, the relevant portion of the ABCB1 gene was amplified by polymerase chain reaction, and sequenced. Twenty-seven dogs were homozygous normal while one dog was heterozygous for the ABCB1-1Δ mutation. Therefore, with the exception of one dog, the observed neurotoxicity could not be attributed to the ABCB1-1Δ mutation. Possible explanations for the adverse reactions observed include pharmacological interactions (administration of SML with other P-gp substrates or inhibitors), excessively high doses, polymorphisms in P-gp expression, uncharacterized mutations in the ABCB1 gene or in another gene, or phenomena unrelated to the SML–P-gp interaction.     

Canine model and genomic structural organization of glycogen storage disease type Ia (GSD Ia)

A canine model of glycogen storage disease Ia (GSD Ia), similar clinically, biochemically, and pathologically to the human disease, was established by crossbreeding Maltese and Beagle dogs carrying a mutated, defective glucose-6-phosphatase (G-6-Pase) gene. Ten puppies were born in three litters from these crossbreedings. Six were homozygous for the previously described M121I GSD Ia mutation. Of these six affecteds, two were stillborn, and one died at 2, 32, and 60 days of life, respectively (puppies A, B, C, D, E), while one is alive at age 15 months (puppy F). Affected puppies exhibited tremors, weakness, and neurologic signs when hypoglycemic. They had postnatal growth retardation and progressive hepatomegaly. Biochemical abnormalities included fasting hypoglycemia, hyperlactacidemia, hypercholesterolemia, hypertriglyceridemia, and hyperuricemia. Microscopic examination of tissues from affected puppies showed diffuse, marked hepatocellular vacuolation, with distended clear hepatocytes and central to marginally located rounded nuclei. In the kidneys of puppies D and E, there was segmental glomerular sclerosis and vacuolation of proximal convoluted tubular epithelium. Biochemical analysis revealed increased liver glycogen content and isolated markedly reduced G-6-Pase enzyme activity in liver and kidney. The canine G-6-Pase gene was characterized by screening a canine genomic library. It spans approximately 11.8 kb and consists of five exons with >90% amino acid sequence homology to the derived human sequence. The first 1.5 kb of the 5' region was sequenced and contains several putative response element motifs homologous to the human 5' region. Establishment of this canine colony of GSD Ia that closely resembles human disease and isolation of the canine genomic gene provides an excellent model for studying pathophysiology and long-term complications and an opportunity to develop novel therapeutic approaches such as drug and gene therapy.     

Association of p53 Gene Mutation with the Prognosis of Canine Lymphoid Tumors

Introduction:  Many dogs with lymphoid tumors develop resistance to chemotherapy. As a mechanism of drug resistance in canine lymphoma, ATP‐dependent drug efflux by P‐glycoprotein was reported, however, inhibition of apoptosis mediated by P53 inactivation has not been investigated. In this study, we investigated the relationship between p53 gene mutation and clinical drug resistance in canine lymphoid tumors.
Methods:  Tumor specimens were obtained from 44 dogs with lymphoid tumors. Mutations of p53 gene at exon 4–8 of these tumor tissues were examined by PCR‐SSCP (single strand conformational polymorphism) analysis, followed by nucleotide sequencing of the abnormal bands. The cases were treated with UW‐Madison protocol, and its response was evaluated by the tumor size or the number of peripheral leukemic cells.
Results:  Of the 44 dogs, 15 dogs (34%) had p53 mutation, whereas 29 dogs (66%) were devoid of p53 mutation, before or during the chemotherapeutic protocol. Rate of good response (CR and PR) to chemotherapy was significantly lower in the dogs with p53 mutation (20%) than those without p53 mutation (55%)(p = 0.022). Median overall survival duration after examination of p53 mutation was significantly shorter in dogs with p53 mutation (101days) than those without p53 mutation (223days)(p = 0.008).
Conclusions:  Lymphoid tumors with p53 mutations were shown to have worse prognosis than those without p53 mutation.     

Comparison of polymerase chain reaction-restriction fragment length polymorphism assay and enzyme assay for diagnosis of G(M1)-gangliosidosis in Shiba dogs.

In the present study, diagnostic methods for canine G(M1)-gangliosidosis were examined by comparing a DNA mutation assay with an enzyme assay. Sixty-two Shiba dogs of a pedigree with G(M1)-gangliosidosis were differentiated into 3 genotypes, i.e., normal, heterozygous, and homozygous affected dogs, using a DNA mutation assay, which consists of polymerase chain reaction amplification and the determination of restriction fragment length polymorphisms. The beta-galactosidase activity in leukocytes, umbilical cords, and plasma was measured using 4-methylumbelliferyl beta-D-galactoside and p-nitrophenyl beta-D-galactoside as artificial substrates and compared among the 3 genotypes. The results showed that it was possible to identify homozygous dogs with the enzyme assay using leukocytes and umbilical cords. When using leukocytes, heterozygous carriers could be differentiated from normal dogs in many cases. However, the use of the DNA mutation assay is essential for a complete determination of heterozygous carriers because of the overlap in the distribution of enzyme activity between these 2 groups. When umbilical cords were used, heterozygous carriers could not be differentiated from normal dogs because of no significant difference in enzyme activity between these 2 groups. The beta-galactosidase activity in plasma was not applicable to the diagnosis and genotyping of G(M1)-gangliosidosis in Shiba dogs.     

Exclusion of DMRT1 as a candidate gene for canine SRY-negative XX sex reversal

DMRT1, which encodes a zinc finger-like DNA binding motif, is a well-conserved gene that is involved in testis differentiation in a variety of mammalian and non-mammalian vertebrates. The objective of this study was to determine whether a DMRT1 microsatellite marker allele is associated with the affected phenotype in a pedigree of canine SRY-negative XX sex reversal generated from an American Cocker spaniel founder. Ten affected dogs and their parents and grandparents were genotyped. Four alleles at this locus and five different genotypes were found in this pedigree. All affected dogs inherited this trait from the foundation sire of this colony. Thus, the disease-causing mutation should be identical by descent in all affected dogs. Six affected dogs were found to have genotypes at this locus that were different from those of the founder sire. These results indicate that DMRT1 is an unlikely candidate gene for SRY-negative XX sex reversal in this model.     

Estimated frequency of the canine hyperuricosuria mutation in different dog breeds

Background: Hyperuricosuria is a condition that predisposes dogs to urate urolithiasis. A mutation that causes canine hyperuricosuria was previously identified in 3 unrelated dog breeds. The occurrence of the mutation in additional breeds was not determined. Hypothesis/Objectives: Identify additional breeds that have the hyperuricosuria mutation and estimate the mutant allele frequency in those breeds. Animals: Three thousand five hundred and thirty dogs from 127 different breeds were screened for the hyperuricosuria mutation. Methods: DNA samples were genotyped by pyrosequencing and allele‐specific polymerase chain reaction methods. Results: Mutant allele frequencies that range from 0.001 to 0.15 were identified in the American Staffordshire Terrier, Australian Shepherd, German Shepherd Dog, Giant Schnauzer, Parson (Jack) Russell Terrier, Labrador Retriever, Large Munsterlander, Pomeranian, South African Boerboel, and Weimaraner breeds. Conclusions and Clinical Importance: The hyperuricosuria mutation has been identified in several unrelated dog breeds. The mutant allele frequencies vary among breeds and can be used to determine an appropriate breeding plan for each breed. A DNA test is available and may be used by breeders to decrease the mutant allele frequency in breeds that carry the mutation. In addition, veterinarians may use the test as a diagnostic tool to identify the cause of urate urolithiasis.     

Evaluation of the phospholamban gene in purebred large-breed dogs with dilated cardiomyopathy

OBJECTIVE: To evaluate the role of the phospholamban gene in purebred large-breed dogs with dilated cardiomyopathy (DCM). ANIMALS: 6 dogs with DCM, including 2 Doberman Pinschers, 2 Newfoundlands, and 2 Great Danes. PROCEDURE: All dogs had clinical signs of congestive heart failure, and a diagnosis of DCM was made on the basis of echocardiographic findings. Blood samples were collected from each dog, and genomic DNA was isolated by a salt extraction method. Specific oligonucleotides were designed to amplify the promoter, exon 1, the 5'-part of exon 2 including the complete coding region, and part of intron 1 of the canine phospholamban gene via polymerase chain reaction procedures. These regions were screened for mutations in DNA obtained from the 6 dogs with DCM. RESULTS: No mutations were identified in the promoter, 5' untranslated region, part of intron 1, part of the 3' untranslated region, and the complete coding region of the phospholamban gene in dogs with DCM. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that mutations in the phospholamban gene are not a frequent cause of DCM in Doberman Pinschers, Newfoundlands, and Great Danes.     

Evaluation of canine heat shock transcription factor 4 (HSF4) as a candidate gene for primary cataracts in the Dachshund and the Entlebucher Mountain dog

OBJECTIVE: Testing of the cataract-causing insertion/deletion mutation in the canine HSF4 gene for its linkage and association with primary cataracts (CAT) in Dachshunds and Entlebucher Mountain dogs. MATERIALS: Exon 9 with flanking intronic regions of the canine HSF4 gene was sequenced in 24 Dachshunds and 20 Entlebucher Mountain dogs. The HSF4 cDNA sequence of lens tissue was analyzed in a CAT-unaffected mixed-breed dog and in three CAT-affected dogs of different breeds, including a Wire-haired Dachshund, a Dachshund-mix and a German Shepherd dog. RESULTS: In all dogs investigated here, the previously reported CAT-causing mutation did not exist. We found a single nucleotide polymorphism (SNP) in intron 9, which was neither associated nor linked with the CAT phenotype in the two dog breeds. CONCLUSION: The CAT phenotype in the two dog breeds investigated here was not caused by the same mutation found to be associated with early-onset CAT in the Staffordshire Bull Terrier and Boston Terrier. The intronic SNP may be useful to test HSF4 for linkage with CAT in further dog breeds.     

Sequencing of the Von Hippel-Lindau Gene in Canine Renal Carcinoma

Background: Similarities in human and canine renal cell carcinoma (RCC) epidemiology and biologic behavior suggest that molecular mechanisms of tumorigenesis may be similar in both species. Approximately 75% of RCC in people are of the clear cell subtype, up to 85% of which are associated with mutation of the von Hippel-Lindau ( VHL ) gene. The canine VHL coding deoxyribonucleic acid (DNA) shares 90% identity with the human VHL gene.
Objective: To determine whether or not RCC in dogs are associated with VHL mutations, and if so determine the prevalence, type, and location of these mutations.
Animals: Thirteen dogs with RCC, 2 dogs with primary renal sarcomas, and 10 dogs without neoplastic kidney disease.
Methods: DNA was extracted from paraffin-embedded RCC tissue; DNA extracts from paraffin-embedded and snap-frozen nonneoplastic canine kidneys and canine whole blood were used as negative controls. Polymerase chain reaction and sequencing of the 3 VHL exons was performed, and results compared with the accessioned canine sequence.
Results: All VHL exons were amplified from 9 of 13 canine RCC samples, both renal sarcomas, 8 of 10 nonneoplastic kidney samples, and canine whole blood; only exon 2 could be amplified from 2 RCC samples. Mutations were not identified in any exons. A maximal prevalence of 33.6% for VHL mutations in canine RCC was determined.
Conclusion and Clinical Importance: Although similarities between canine and human RCC merit further investigation of the dog as a model for some subtypes of renal tumors, the lower prevalence of VHL mutations suggests that oncogenesis in these 2 species differs.     

The T963C mutation of TP53 gene does not participate in the clonal origin of canine TVT

In dogs, the canine transmissible venereal tumor (CTVT) is the only neoplasm which is not produced by neoplastic transformation of normal cells; the tumor is transmitted from the affected dog to healthy dogs by implantation of one or various clones of cancer cells. Thus, the CTVT of dogs analyzed in various countries reveals similar genetic characteristics and consequently CTVT is considered to have a clonal origin.The CTVTs obtained from dogs in Korea showed the T963C mutation on TP53 gene; this mutation was thought to be a molecular alteration which participates in the origin of the ancestral clone, CTVT. Nonetheless, this supposed mutation has not been identified in other studies which were carried out for the purpose of clarifying the clonal origin of CTVT. Thus we have considered it important to identify the role of the T963C mutation of the TP53 gene in the clonal origin of CTVT in dogs.Consequently the region which includes the mutation of the TP53 gene in twenty samples of CTVT obtained from various canine breeds was PCR amplified and afterwards its sequence of nucleotides was determined. We conclude that this mutation did not participate in the clonal origin of the tumor, but was acquired at a later stage.     

Disseminated aspergillosis in dogs

Clinical and pathological findings are reported from a series of 12 cases of disseminated aspergillosis (A. terreus) in 11 German Shepherd dogs and one Dalmatian referred to Murdoch University Veterinary Hospital (MUVH) over the period 1980 to 1984. A preliminary study of humoral and cell mediated immune components and complement levels revealed no consistent abnormality in 9 dogs tested apart from raised IgG levels. Serum IgA levels were depressed in 30% of cases. Serial data from one extensively monitored case is presented. The unusual epidemiological and pathogenetic features of the disease are discussed.