Orientadora: Márcia Ferreira da Rosa Sobreira / Coorientador: Hélio José Montassier / Banca: Ketherson Rodrigues Silva / Banca: Ruben Pablo Schocken Iturrino / Banca: Marita Vedovelli Cardozo / Banca: Diane Meyre Rassi / Resumo: A cinomose canina é uma das enfermidades infectocontagiosas mais importantes que acomete os cães. É uma doença causada pelo vírus da Cinomose ("Canine Distemper Virus" - CDV), um Paramyxovirus, do gênero Morbillivirus, de ocorrência mundial, sem sazonalidade, sem predileção de sexo ou raça, apresenta maior incidência em animais jovens, podendo acometer todas as idades. No Brasil, pesquisas sobre o uso de técnicas que comparam os diferentes testes diagnósticos direto para o vírus da cinomose (CDV) associado a análise molecular e filogenética do mesmo ainda continuam escassas. Além disso, são poucos os estudos sobre epidemiologia molecular que relatam diferenças marcantes na análise filogenética do gene hemaglutinina (H) do CDV entre os isolados de campo e as cepas de referência do CDV, especialmente àquelas utilizadas na produção de vacinas. Dessa maneira, o presente trabalho tem como principal objetivo avaliar os principais métodos diagnósticos laboratoriais para identificação do CDV em cães com suspeita de cinomose associando à análise filogenética do gene H dos isolados de campo. O Ensaio imunocromatografico direto (IC), a RT-PCR e a Nested-PCR foram avaliados para detecção do CDV em diferentes amostras clínicas (urina, suabes retais e conjuntivais) de 62 animais suspeitos de cinomose, provenientes do atendimento ambulatorial do hospital veterinário da Unesp (Jaboticabal-SP) e de clínicas particulares da região. Após detecção viral as amostras foram submetidas a sequenciame... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Canine distemper is one of the most important infectious diseases that affects dogs. It is a Canine Distemper Virus (CDV), a Paramyxovirus, of the genus Morbillivirus, a global deforest, without seasonality, without predilection of sex or race, that is constituted by the majority of the animals, being able to affect all ages. In Brazil, research on the use of techniques that compare the different direct diagnostic tests for the distemper virus (CDV) associated with a molecular and phylogenetic analysis of the same are still scarce. In addition, there are some studies on molecular epidemiology that report differences in the phylogenetic analysis of the hemagglutinin (H) gene from CDV between the fields of action and as a reference for CDV, especially the practices used in the production of vaccines. Thus, the main objective of this study is to identify laboratory diagnoses for the identification of CDV in dogs with suspected pair association to the phylogenetic analysis of the H gene of the field isolates. The direct immunochromatographic (CI) assay, RT-PCR and nested-PCR were evaluated for the detection of CDV in different animal species (urine, swabs and conjunctival) of 62 suspected cases of distemper from the outpatient clinic of the Unesp (Jaboticabal-SP) and the private medical records of the region. After viral detection as sequential sequencing followed by phylogenetic analysis of the CDV H gene. A total of 42 animals were positive at least one of the experiment techni... (Complete abstract click electronic access below) / Doutor

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The Dalmatian is affected by deafness more than any other breed of domestic dog, with 30% of the United States population suffering from unilateral or bilateral deafness. The genetic origin of deafness in the Dalmatian is unknown. The objective of this work was to identify, using linkage analysis, any chromosomal region(s) in which the gene(s) responsible for deafness in the Dalmatian may be located. To achieve this objective it was necessary to 1) develop multiplexed microsatellite markers for an efficient whole genome scan, 2) assemble a multigenerational Dalmatian kindred segregating deafness, 3) estimate the heritability of deafness and perform complex segregation analysis, and 4) perform linkage analysis of deafness, and other phenotypic traits, in the Dalmatian kindred. A set of 172 microsatellite markers, termed Minimal Screening Set 1 (MSS1), was characterized, prior to this work, for whole genome scans of the domestic dog. 155 of the MSS1 markers were multiplexed into 48 multiplex sets. Amplification of the multiplex sets was achieved using a single thermal cycling program. The markers were labeled with fluorescent dyes and optimized for resolution on an ABI 310 Genetic Analyzer or ABI 377 Sequencer. A kindred of 266 Dalmatians was assembled, of which 199 had been diagnosed using the brainstem auditory evoked response to determine auditory status. Of these, 74.4% (N = 148) had normal hearing, 18.1% (N = 36) were unilaterally deaf, and 7.5% (N = 15) were bilaterally deaf. A heritability of 0.73 was estimated considering deafness a dichotomous trait and 0.75 as a trichotomous trait. Although deafness in the Dalmatian is clearly heritable, the evidence for the presence of a major gene affecting the disorder was not persuasive. Dalmatians (N = 117) from the assembled kindred were genotyped for the MSS1 markers (149 were polymorphic). Linkage analysis was performed for deafness, eye color, and spot color. The maximum LOD scores for deafness were found with markers Cos15 on CFA17 (LOD = 1.69) and FH2585 on CFA28 (LOD = 1.34). No significant linkage was found with eye color. Significant linkage for spot color was found with marker FH2319 (LOD = 9.7) on CFA11.

MicroRNAs (miRNAs) play a vital role in differentiation, proliferation and tumorigenesis by binding to messenger RNAs (mRNA) and inhibiting translation. To initiate an investigation into the identification of miRNAs in the domestic dog, an emerging model for human disease, a comparison of the human and canine genetic databases was conducted. The bioinformatics work revealed significant conservation of miRNA genes between the two species. Proof of principle experiments, including serial dilutions and sequencing, were performed to verify that primers made to amplify human mature miRNAs can be used to amplify canine miRNAs, providing that the mature sequences are conserved. TaqMan® Real-time RT-PCR, a sensitive and specific method, was used to isolate the first miRNA mature products from canine tissues. The expression levels of miR-17-3p, miR-17-5p, miR-18, miR-19a, miR-19b, miR-20, and miR-92 were evaluated in five canine tissues (heart, lung, brain, kidney, and liver). Because miRNAs have been found to act as both tumor suppressors and oncogenes in several different cancers, expression patterns of ten miRNAs (miR-15a, miR-16, miR-17-5p, miR-21, miR-29b, miR-125b, miR-145, miR-155, miR-181b, let-7f) known to be associated with human breast cancer were compared between malignant canine mammary tumors (n=6) and normal canine mammary tissue (n=10). Resulting data revealed miR-29b and miR-21 to have a statistically significant (p<0.05) up-regulation in cancerous samples. Overall expression patterns showed nine of the ten miRNAs follow the same pattern of expression in the domestic dog as the human, while the miR-145 expression does not show a difference between the normal and cancerous samples.

The domestic dog, Canis lupus familiaris, is a unique model system for the dissection of hereditary diseases. Selective breeding practices have created more than 300 distinct breeds of dogs, born from a desire to create specific physical and behavioral characteristics. Breeds represent closed breeding populations and the extensive records maintained for members of each breed (e.g., multi-generational pedigrees, veterinary medical records) present an incredible tool for genetic research. Two closed populations were used in the work presented here: a colony of mixed-breed dogs segregating resistance and sensitivity to cholesterol feeding, and a purebred pet population of Havanese experiencing a high frequency of developmental abnormalities. Estimates of heritability were calculated for each disease to evaluate the degree of phenotypic variation attributable to genetics among dogs in the populations used. A heritability of 0.55 (± 0.16) was identified for cholesterol resistance and sensitivity in the mixed-breed colony. The small sample size prevented the use of complex segregation analyses to examine mode of transmission. A heritability of 0.36 (± 0.26) was calculated for the composite phenotype in the Havanese, encompassing the spectrum of abnormalities in the breed. Polygenic inheritance was identified for the composite phenotype, but the action of a major gene was identified by complex segregation analyses in the Havanese. Complex diseases preclude the use of a candidate gene approach, owing to the multitude of genes involved in the disease process. Whole genome screens provide a practical approach to the identification of chromosomal region(s) associated with a disease phenotype by narrowing the search for candidate gene(s). The Minimal Screening Set – 2 (MSS-2) was used in the present studies to evaluate the segregation of microsatellite markers in pedigrees for both the mixed-breed colony and the Havanese. No significant LOD scores were identified, though suggestive LOD scores were obtained in both analyses. A canine-specific oligonucleotide microarray was used to create gene expression profiles for developmental abnormalities in the Havanese and for cholesterol sensitivity in the mixed-breed colony dogs. Distinct expression profiles were generated for each group, and several genes of interest were identified as being both differentially expressed (>±2-fold change) and statistically significant (p-value<0.05).

Although typically thought of as a beloved companion or indispensable aide, the domestic dog (Canis lupus familiaris) has emerged as an excellent model for the study of human hereditary diseases. Many hereditary diseases of the dog have nearly identical clinical presentations as those of the human and are, most often, caused by mutations in the same genes. One such disease is hereditary nephropathy; an inherited glomerular disease in the domestic dog that is similar to Alport syndrome of the human. Both diseases are caused by mutations in the type IV collagens genes, and the disease has nearly identical pathology and clinical presentations in the dog and human. By studying this disease in the dog, our laboratory hopes to increase understanding of the disease so that information that can be applied to both the human and the dog. Reported here is 1) the development of a genomic based test to determine genotypes of mixed breed dogs in a colony presenting with X-linked hereditary nephropathy, 2) the determination of patterns of X-chromosome inactivation in normal dogs and dogs that are carriers of Xlinked hereditary nephropathy, 3) the design of a synthetic COL4A5 cDNA to be used for gene therapy treatment of dogs with X-linked hereditary nephropathy, 4) the investigation of type IV collagen gene expression changes in normal dogs and those affected with X-linked and autosomal recessive hereditary nephropathy, and 5) the discovery of the mutation causative for autosomal recessive hereditary nephropathy in the English Cocker Spaniel. Utilization of the colony of dogs affected with X-linked hereditary nephropathy (for which the causative mutation was previously identified) allowed for comparisons of type IV collagen gene expression to English Cocker Spaniels with autosomal recessive hereditary nephropathy. These data were critical to identification of the gene harboring the causative mutation for autosomal recessive hereditary nephropathy. Sequencing was performed to identify the mutation. With the ability to test for carriers of this disease, it is our hope that breeders will use it to to maintain the desired traits in the ECS while simultaneously eliminating the production of affected offspring.

The domestic dog, Canis lupus familiaris, is a unique model system for the dissection of hereditary diseases. Selective breeding practices have created more than 300 distinct breeds of dogs, born from a desire to create specific physical and behavioral characteristics. Breeds represent closed breeding populations and the extensive records maintained for members of each breed (e.g., multi-generational pedigrees, veterinary medical records) present an incredible tool for genetic research. Two closed populations were used in the work presented here: a colony of mixed-breed dogs segregating resistance and sensitivity to cholesterol feeding, and a purebred pet population of Havanese experiencing a high frequency of developmental abnormalities. Estimates of heritability were calculated for each disease to evaluate the degree of phenotypic variation attributable to genetics among dogs in the populations used. A heritability of 0.55 (± 0.16) was identified for cholesterol resistance and sensitivity in the mixed-breed colony. The small sample size prevented the use of complex segregation analyses to examine mode of transmission. A heritability of 0.36 (± 0.26) was calculated for the composite phenotype in the Havanese, encompassing the spectrum of abnormalities in the breed. Polygenic inheritance was identified for the composite phenotype, but the action of a major gene was identified by complex segregation analyses in the Havanese. Complex diseases preclude the use of a candidate gene approach, owing to the multitude of genes involved in the disease process. Whole genome screens provide a practical approach to the identification of chromosomal region(s) associated with a disease phenotype by narrowing the search for candidate gene(s). The Minimal Screening Set – 2 (MSS-2) was used in the present studies to evaluate the segregation of microsatellite markers in pedigrees for both the mixed-breed colony and the Havanese. No significant LOD scores were identified, though suggestive LOD scores were obtained in both analyses. A canine-specific oligonucleotide microarray was used to create gene expression profiles for developmental abnormalities in the Havanese and for cholesterol sensitivity in the mixed-breed colony dogs. Distinct expression profiles were generated for each group, and several genes of interest were identified as being both differentially expressed (>±2-fold change) and statistically significant (p-value<0.05).

The Dalmatian is affected by deafness more than any other breed of domestic dog, with 30% of the United States population suffering from unilateral or bilateral deafness. The genetic origin of deafness in the Dalmatian is unknown. The objective of this work was to identify, using linkage analysis, any chromosomal region(s) in which the gene(s) responsible for deafness in the Dalmatian may be located. To achieve this objective it was necessary to 1) develop multiplexed microsatellite markers for an efficient whole genome scan, 2) assemble a multigenerational Dalmatian kindred segregating deafness, 3) estimate the heritability of deafness and perform complex segregation analysis, and 4) perform linkage analysis of deafness, and other phenotypic traits, in the Dalmatian kindred. A set of 172 microsatellite markers, termed Minimal Screening Set 1 (MSS1), was characterized, prior to this work, for whole genome scans of the domestic dog. 155 of the MSS1 markers were multiplexed into 48 multiplex sets. Amplification of the multiplex sets was achieved using a single thermal cycling program. The markers were labeled with fluorescent dyes and optimized for resolution on an ABI 310 Genetic Analyzer or ABI 377 Sequencer. A kindred of 266 Dalmatians was assembled, of which 199 had been diagnosed using the brainstem auditory evoked response to determine auditory status. Of these, 74.4% (N = 148) had normal hearing, 18.1% (N = 36) were unilaterally deaf, and 7.5% (N = 15) were bilaterally deaf. A heritability of 0.73 was estimated considering deafness a dichotomous trait and 0.75 as a trichotomous trait. Although deafness in the Dalmatian is clearly heritable, the evidence for the presence of a major gene affecting the disorder was not persuasive. Dalmatians (N = 117) from the assembled kindred were genotyped for the MSS1 markers (149 were polymorphic). Linkage analysis was performed for deafness, eye color, and spot color. The maximum LOD scores for deafness were found with markers Cos15 on CFA17 (LOD = 1.69) and FH2585 on CFA28 (LOD = 1.34). No significant linkage was found with eye color. Significant linkage for spot color was found with marker FH2319 (LOD = 9.7) on CFA11.

Previous studies examining the canine intestinal microflora have focused on cultivation of bacteria from intestinal content. Recently, it has been recognized that the majority of bacteria cannot be identified using standard culture techniques. The aim of this study was to describe the composition and dynamics of the canine intestinal microflora using molecular methods based on identification of the 16S ribosomal DNA (16S rDNA) and to evaluate the clinical use of a 13C-glycocholic acid blood test (13CGCBT) as a serum marker for small intestinal bacterial biomass. Intestinal content was obtained from healthy dogs and the microflora was characterized in different compartments of each dog by denaturing gradient gel electrophoresis (DGGE) and comparative 16S rDNA analysis. A 13C-glycocholic acid blood test (13C-GCBT) was developed as a marker for small intestinal bacterial biomass and the influence of tylosin administration on the 13C-GCBT, serum concentrations of cobalamin, folate, and unconjugated cholic acid (SUCA) was evaluated. There was marked variation in DGGE profiles between individual dogs and also between different intestinal compartments within dogs. DGGE profiles from duodenal juice samples collected endoscopically at different time-points varied within individuals, possibly due to variations over time or a slight variation in sampling location. Direct sequencing revealed 106 individual 16S rDNA sequences. Forty-two sequences showed less than 98% similarity to described sequences in public databases and may constitute previously uncharacterized bacterial species. Serum folate concentrations, SUCA, and the cumulative percent dose/min of 13C administered as 13C-glycocholic acid (CUMPCD) increased significantly following tylosin administration (p<0.01). The results indicate that dogs have a complex intestinal microflora with marked differences between individual dogs. Different intestinal compartments appear to host a unique microflora and the assessment of a fecal sample does not yield accurate information about the composition of the microflora in proximal compartments of the gut. The intestine harbors many previously uncharacterized bacterial species. The clinical significance of these uncharacterized intestinal bacterial species needs to be further investigated in dogs with gastrointestinal disease. Increased serum folate, SUCA, and CUMPCD in the 13C-GCBT suggest that, in the dogs described here, tylosin administration increased the biomass of organisms carrying out these metabolic functions.

Although typically thought of as a beloved companion or indispensable aide, the domestic dog (Canis lupus familiaris) has emerged as an excellent model for the study of human hereditary diseases. Many hereditary diseases of the dog have nearly identical clinical presentations as those of the human and are, most often, caused by mutations in the same genes. One such disease is hereditary nephropathy; an inherited glomerular disease in the domestic dog that is similar to Alport syndrome of the human. Both diseases are caused by mutations in the type IV collagens genes, and the disease has nearly identical pathology and clinical presentations in the dog and human. By studying this disease in the dog, our laboratory hopes to increase understanding of the disease so that information that can be applied to both the human and the dog. Reported here is 1) the development of a genomic based test to determine genotypes of mixed breed dogs in a colony presenting with X-linked hereditary nephropathy, 2) the determination of patterns of X-chromosome inactivation in normal dogs and dogs that are carriers of X-linked hereditary nephropathy, 3) the design of a synthetic COL4A5 cDNA to be used for gene therapy treatment of dogs with X-linked hereditary nephropathy, 4) the investigation of type IV collagen gene expression changes in normal dogs and those affected with X-linked and autosomal recessive hereditary nephropathy, and 5) the discovery of the mutation causative for autosomal recessive hereditary nephropathy in the English Cocker Spaniel. Utilization of the colony of dogs affected with X-linked hereditary nephropathy (for which the causative mutation was previously identified) allowed for comparisons of type IV collagen gene expression to English Cocker Spaniels with autosomal recessive hereditary nephropathy. These data were critical to identification of the gene harboring the causative mutation for autosomal recessive hereditary nephropathy. Sequencing was performed to identify the mutation. With the ability to test for carriers of this disease, it is our hope that breeders will use it to to maintain the desired traits in the ECS while simultaneously eliminating the production of affected offspring.