A Missense Mutation in the SERPINH1 Gene in Dachshunds with Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is a hereditary disease occurring in humans and dogs. It is characterized by extremely fragile bones and teeth. Most human and some canine OI cases are caused by mutations in the COL1A1 and COL1A2 genes encoding the subunits of collagen I. Recently, mutations in the CRTAP and LEPRE1 genes were found to cause some rare forms of human OI. Many OI cases exist where the causative mutation has not yet been found. We investigated Dachshunds with an autosomal recessive form of OI. Genotyping only five affected dogs on the 50 k canine SNP chip allowed us to localize the causative mutation to a 5.82 Mb interval on chromosome 21 by homozygosity mapping. Haplotype analysis of five additional carriers narrowed the interval further down to 4.74 Mb. The SERPINH1 gene is located within this interval and encodes an essential chaperone involved in the correct folding of the collagen triple helix. Therefore, we considered SERPINH1 a positional and functional candidate gene and performed mutation analysis in affected and control Dachshunds. A missense mutation (c.977C>T, p.L326P) located in an evolutionary conserved domain was perfectly associated with the OI phenotype. We thus have identified a candidate causative mutation for OI in Dachshunds and identified a fifth OI gene.

Evaluation of endosonography as a new diagnostic tool for feline pancreatitis

The purpose of this study was to evaluate endosonography (EUS) as a potential diagnostic tool for feline pancreatitis. Eleven healthy cats and six cats diagnosed with pancreatitis based on an increased serum feline pancreatic lipase immunoreactivity (fPLI) concentration were included. Transabdominal ultrasound (AUS) and EUS were performed in all cats. The widths of both pancreatic limbs and echogenicity and homogenicity were assessed by AUS and EUS. Finally, findings from both modalities were subjectively compared. In the healthy cats, the right pancreatic limb was significantly smaller on EUS compared to AUS. Also, subjectively, general visualization of the normal pancreas was superior with EUS and, the pancreatic margins and parenchyma could be resolved better with EUS in all sick patients. In this study, EUS findings did not alter the diagnosis in six cats with pancreatitis when compared to AUS. However, EUS may be useful in cases where AUS fails due to obesity, hyperechoic mesentery, or excessive intestinal gas.

A frameshift mutation in the cubilin gene (CUBN) in Beagles with Imerslund-Gräsbeck syndrome (selective cobalamin malabsorption).

Mammals are unable to synthesize cobalamin or vitamin B12 and rely on the uptake of dietary cobalamin. The cubam receptor expressed on the intestinal endothelium is required for the uptake of cobalamin from the gut. Cubam is composed of two protein subunits, amnionless and cubilin, which are encoded by the AMN and CUBN genes respectively. Loss-of-function mutations in either the AMN or the CUBN gene lead to hereditary selective cobalamin malabsorption or Imerslund-Gräsbeck syndrome (IGS). We investigated Beagles with IGS and resequenced the whole genome of one affected Beagle at 15× coverage. The analysis of the AMN and CUBN candidate genes revealed a homozygous deletion of a single cytosine in exon 8 of the CUBN gene (c.786delC). This deletion leads to a frameshift and early premature stop codon (p.Asp262Glufs*47) and is, thus, predicted to represent a complete loss-of-function allele. We tested three IGS-affected and 89 control Beagles and found perfect association between the IGS phenotype and the CUBN:c.786delC variant. Given the known role of cubilin in cobalamin transport, which has been firmly established in humans and dogs, our data strongly suggest that the CUBN:c.786delC variant is causing IGS in the investigated Beagles.

A frameshift mutation in the cubilin gene (CUBN) in Border Collies with Imerslund-Gräsbeck syndrome (selective cobalamin malabsorption).

Imerslund-Gräsbeck syndrome (IGS) or selective cobalamin malabsorption has been described in humans and dogs. IGS occurs in Border Collies and is inherited as a monogenic autosomal recessive trait in this breed. Using 7 IGS cases and 7 non-affected controls we mapped the causative mutation by genome-wide association and homozygosity mapping to a 3.53 Mb interval on chromosome 2. We re-sequenced the genome of one affected dog at ∼10× coverage and detected 17 non-synonymous variants in the critical interval. Two of these non-synonymous variants were in the cubilin gene (CUBN), which is known to play an essential role in cobalamin uptake from the ileum. We tested these two CUBN variants for association with IGS in larger cohorts of dogs and found that only one of them was perfectly associated with the phenotype. This variant, a single base pair deletion (c.8392delC), is predicted to cause a frameshift and premature stop codon in the CUBN gene. The resulting mutant open reading frame is 821 codons shorter than the wildtype open reading frame (p.Q2798Rfs*3). Interestingly, we observed an additional nonsense mutation in the MRC1 gene encoding the mannose receptor, C type 1, which was in perfect linkage disequilibrium with the CUBN frameshift mutation. Based on our genetic data and the known role of CUBN for cobalamin uptake we conclude that the identified CUBN frameshift mutation is most likely causative for IGS in Border Collies.

Das intrakranielle Meningeom: Befunde, Therapie und Ergebnisse bei neun Katzen und einem Hund

Nine cats and one dog with intracranial meningioma, which underwent surgery at the Department of Veterinary Surgery, University of Munich, Germany were evaluated retrospectively. One cat died shortly after surgery whereas the remaining 9 animals survived between 8 to 43 months (mean, 21.9 months) after surgery. Relapse was seen in 2 cats shortly after surgery and these animals were re-operated. After surgery, computed tomography was performed to ascertain that the tumour was completely removed. Pre-operative symptoms disappeared rapidly after surgery, except central blindness which persisted. Initial clinical observations and results of in vitro studies using feline meningioma cells indicated that hydroxy-urea can prolong survival time of affected animals.

Electrodiagnostic evaluation in feline hypertrophic muscular dystrophy

Standard needle electromyography (EMG) of 56 muscles and nerve conduction velocities (NCV) of the ulnar and common peroneal nerves were investigated in each of six cats affected with hypertrophic feline muscular dystrophy, 10 related heterozygote carriers and 10 normal cats. The EMG findings were considered normal in carrier and control cats, and consisted of 33% normal readings, 22% myotonic discharges, 18% fibrillation potentials, 11% prolonged insertional potentials, 10% complex repetitive discharges and 6% positive sharp waves in affected cats. Muscles of the proximal limbs were most frequently affected. No differences in NCV were found between the three cat groups. It was concluded that dystrophin-deficient dystrophic cats have widespread and frequent EMG changes, predominantly myotonic discharges and fibrillation potentials, which are most pronounced in the proximal appendicular muscles.

Looking the cow in the eye: deletion in the NID1 gene is associated with recessive inherited cataract in Romagnola cattle

Cataract is a known condition leading to opacification of the eye lens causing partial or total blindness. Mutations are known to cause autosomal dominant or recessive inherited forms of cataracts in humans, mice, rats, guinea pigs and dogs. The use of large-sized animal models instead of those using mice for the study of this condition has been discussed due to the small size of rodent lenses. Four juvenile-onset cases of bilateral incomplete immature nuclear cataract were recently observed in Romagnola cattle. Pedigree analysis suggested a monogenic autosomal recessive inheritance. In addition to the cataract, one of the cases displayed abnormal head movements. Genome-wide association and homozygosity mapping and subsequent whole genome sequencing of a single case identified two perfectly associated sequence variants in a critical interval of 7.2 Mb on cattle chromosome 28: a missense point mutation located in an uncharacterized locus and an 855 bp deletion across the exon 19/intron 19 border of the bovine nidogen 1 (NID1) gene (c.3579_3604+829del). RT-PCR showed that NID1 is expressed in bovine lenses while the transcript of the second locus was absent. The NID1 deletion leads to the skipping of exon 19 during transcription and is therefore predicted to cause a frameshift and premature stop codon (p.1164fs27X). The truncated protein lacks a C-terminal domain essential for binding with matrix assembly complexes. Nidogen 1 deficient mice show neurological abnormalities and highly irregular crystal lens alterations. This study adds NID1 to the list of candidate genes for inherited cataract in humans and is the first report of a naturally occurring mutation leading to non-syndromic catarct in cattle provides a potential large animal model for human cataract.

The dog as a genetic model for immunoglobulin A (IgA) deficiency: identification of several breeds with low serum IgA concentrations

Immunoglobulin A (IgA) serves as the basis of the secretory immune system by protecting the lining of mucosal sites from pathogens. In both humans and dogs, IgA deficiency (IgAD) is associated with recurrent infections of mucosal sites and immune-mediated diseases. Low concentrations of serum IgA have previously been reported to occur in a number of dog breeds but no generally accepted cut-off value has been established for canine IgAD. The current study represents the largest screening to date of IgA in dogs in terms of both number of dogs (n = 1267) and number of breeds studied (n = 22). Serum IgA concentrations were quantified by using capture ELISA and were found to vary widely between breeds. We also found IgA to be positively correlated with age (p < 0.0001). Apart from the two breeds previously reported as predisposed to low IgA (Shar-Pei and German shepherd), we identified six additional breeds in which ≥10% of all tested dogs had very low (<0.07 g/l) IgA concentrations (Hovawart, Norwegian elkhound, Nova Scotia duck tolling retriever, Bullterrier, Golden retriever and Labrador retriever). In addition, we discovered low IgA concentrations to be significantly associated with canine atopic dermatitis (CAD, p < 0.0001) and pancreatic acinar atrophy (PAA, p = 0.04) in German shepherds.

Genome-Wide Analyses Suggest Mechanisms Involving Early B-Cell Development in Canine IgA Deficiency.

Immunoglobulin A deficiency (IgAD) is the most common primary immune deficiency disorder in both humans and dogs, characterized by recurrent mucosal tract infections and a predisposition for allergic and other immune mediated diseases. In several dog breeds, low IgA levels have been observed at a high frequency and with a clinical resemblance to human IgAD. In this study, we used genome-wide association studies (GWAS) to identify genomic regions associated with low IgA levels in dogs as a comparative model for human IgAD. We used a novel percentile groups-approach to establish breed-specific cut-offs and to perform analyses in a close to continuous manner. GWAS performed in four breeds prone to low IgA levels (German shepherd, Golden retriever, Labrador retriever and Shar-Pei) identified 35 genomic loci suggestively associated (p <0.0005) to IgA levels. In German shepherd, three genomic regions (candidate genes include KIRREL3 and SERPINA9) were genome-wide significantly associated (p <0.0002) with IgA levels. A ~20kb long haplotype on CFA28, significantly associated (p = 0.0005) to IgA levels in Shar-Pei, was positioned within the first intron of the gene SLIT1. Both KIRREL3 and SLIT1 are highly expressed in the central nervous system and in bone marrow and are potentially important during B-cell development. SERPINA9 expression is restricted to B-cells and peaks at the time-point when B-cells proliferate into antibody-producing plasma cells. The suggestively associated regions were enriched for genes in Gene Ontology gene sets involving inflammation and early immune cell development.

A Multi-Breed Genome-Wide Association Analysis for Canine Hypothyroidism Identifies a Shared Major Risk Locus on CFA12.

Hypothyroidism is a complex clinical condition found in both humans and dogs, thought to be caused by a combination of genetic and environmental factors. In this study we present a multi-breed analysis of predisposing genetic risk factors for hypothyroidism in dogs using three high-risk breeds-the Gordon Setter, Hovawart and the Rhodesian Ridgeback. Using a genome-wide association approach and meta-analysis, we identified a major hypothyroidism risk locus shared by these breeds on chromosome 12 (p = 2.1x10-11). Further characterisation of the candidate region revealed a shared ~167 kb risk haplotype (4,915,018-5,081,823 bp), tagged by two SNPs in almost complete linkage disequilibrium. This breed-shared risk haplotype includes three genes (LHFPL5, SRPK1 and SLC26A8) and does not extend to the dog leukocyte antigen (DLA) class II gene cluster located in the vicinity. These three genes have not been identified as candidate genes for hypothyroid disease previously, but have functions that could potentially contribute to the development of the disease. Our results implicate the potential involvement of novel genes and pathways for the development of canine hypothyroidism, raising new possibilities for screening, breeding programmes and treatments in dogs. This study may also contribute to our understanding of the genetic etiology of human hypothyroid disease, which is one of the most common endocrine disorders in humans.

Spatial Distribution of Stem Cell-Like Keratinocytes in Dissected Compound Hair Follicles of the Dog

Hair cycle disturbances are common in dogs and comparable to some alopecic disorders in humans. A normal hair cycle is maintained by follicular stem cells which are predominately found in an area known as the bulge. Due to similar morphological characteristics of the bulge area in humans and dogs, the shared particularity of compound hair follicles as well as similarities in follicular biomarker expression, the dog is a promising model to study human hair cycle and stem cell disorders. To gain insight into the spatial distribution of follicular keratinocytes with stem cell potential in canine compound follicles, we microdissected hair follicles in anagen and telogen from skin samples of freshly euthanized dogs. The keratinocytes isolated from different locations were investigated for their colony forming efficiency, growth and differentiation potential as well as clonal growth. Our results indicate that i) compound and single hair follicles exhibit a comparable spatial distribution pattern with respect to cells with high growth potential and stem cell-like characteristics, ii) the lower isthmus (comprising the bulge) harbors most cells with high growth potential in both, the anagen and the telogen hair cycle stage, iii) unlike in other species, colonies with highest growth potential are rather small with an irregular perimeter and iv) the keratinocytes derived from the bulbar region exhibit characteristics of actively dividing transit amplifying cells. Our results now provide the basis to conduct comparative studies of normal dogs and those with hair cycle disorders with the possibility to extend relevant findings to human patients.

Nitric oxide plays a key role in adaptive control of locomotion in cat

Diverse roles in cellular functions have been ascribed to nitric oxide (NO), and its involvement in induction of long-term depression in cerebellar Purkinje cells has been demonstrated. Manipulations of NO concentration or its synthesis in cerebellar tissues therefore provide a means for investigating roles of NO in cerebellar functions at both cellular and behavioral levels. We tested adaptive control of locomotion to perturbation in cats, and found that this form of motor learning was abolished by application of either an inhibitor of NO synthase or a scavenger of NO to the cerebellar cortical locomotion area. This finding supports the view that NO in the cerebellum plays a key role in motor learning.

Estudo retrospectivo das afecções orais em 754 felinos domésticos (Felis catus) atendidos no Laboratório de Odontologia Comparada da Universidade de São Paulo

Objetivou-se fazer um estudo retrospectivo avaliando quais as afecções da cavidade oral foram mais frequentes nos gatos domésticos atendidos no Laboratório de Odontologia Comparada da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, relatando estatisticamente a prevalência das afecções da cavidade oral de gatos, enfatizando se há correlação entre elas e com características como raça, sexo, faixa etária e estado reprodutivo. Os dados analisados dos 754 prontuários foram raça, idade, sexo, estado reprodutivo, diagnóstico, tratamento e, no caso de neoplasia, sua localização e diagnóstico histopatológico. As principais doenças diagnosticadas foram doença periodontal, fratura dentária, gengivoestomatite crônica felina, lesão de reabsorção dentária felina, neoplasia oral e traumatismo do sistema estomatognático (luxação de articulação temporomandibular, fenda palatina, fratura de processo coronoide, fratura de zigomático, disjunção de sínfise, fratura de maxila e mandíbula). A idade dos animais variou de menos de um ano a 20 anos, sendo que, os animais tinham, em média 7,2 anos (desvio padrão = 4,9) e a faixa etária mais frequente foi de um a cinco anos. Os gatos sem raça definida (66,5%), siameses (19,0%) e persas (10,2%) totalizaram 95,7% de todos os felinos atendidos no LOC. A doença periodontal foi a afecção mais frequente e esteve presente em 38,3% da população estudada. A fratura dentária, segunda mais frequente, esteve presente em 27,2% dos animais. Houve associação estatisticamente significativa (p=0,026) entre fratura dentária e faixa etária, já que a proporção de animais entre um e cinco anos de idade com fratura foi maior do que a das outras faixas etárias. A lesão de reabsorção dentária felina (LRDF) esteve presente em 19,6% dos gatos estudados, sendo a terceira afecção mais prevalente dentre as pesquisadas. Esta lesão foi mais frequente em gatos com idade entre 11 e 15 anos e houve associação estatisticamente significativa entre a LRDF e a doença periodontal e entre LRDF e gengivite. A prevalência de gengivoestomatite crônica felina foi de 15,7% entre os felinos pesquisados e a proporção de animais com idades entre seis e dez anos com esta doença foi maior do que em outras faixas etárias. As neoplasias estavam presentes em 9,8% dos gatos, sendo que em 46 dos 72 animais que apresentaram alguma neoplasia tinham mais de dez anos de idade. O carcinoma de células escamosas foi o neoplasma mais comum, correspondendo a 63,2% das neoformações que foram submetidas ao exame histopatológico. As fraturas ósseas do sistema estomatognático corresponderam a 19,3% dos atendimentos, sendo a sínfise mentoniana e o corpo da mandíbula os locais mais comuns de fraturas. Concluiu-se que: existe grande variedade de afecções que acometem a cavidade oral de gatos, sendo a doença periodontal, fratura dentária, lesão de reabsorção dentária, gengivite, gengivoestomatite crônica, neoplasias orais e fraturas dos ossos do sistema estomatognático as mais prevalentes delas; é de extrema importância que as anotações nas fichas de atendimento sejam feitas da maneira mais completa possível, para que informações não sejam perdidas

A influência do cão na expressividade emocional de crianças com transtorno do espectro do autismo

O Transtorno do Espectro do Autismo (TEA) inclui um conjunto de sintomas, tais como dificuldade para sustentar contato visual direto e comprometimento da linguagem. Apesar da Terapia Assistida Por Animais (TAA) com cães ser considerada uma modalidade terapêutica eficaz para promover o desenvolvimento de pessoas com TEA, ainda não são se sabe quais características dos cães possibilitam alcançar sucesso na terapia. Esta análise quantitativa tem como objetivo verificar o impacto de abordagens laterais e frontais de cães e humanos nas expressões emocionais de alegria e rejeição de crianças com TEA. Através da análise de vídeos de TAA, foram mensuradas duração e frequência das abordagens laterais e frontais de cães e humanos dirigidas às crianças para comparar possíveis diferenças entre ambos e também para verificar se a abordagem escolhida afetava o tipo de expressão emocional exibida pela criança. Os participantes deste projeto foram 11 crianças, 8 do sexo masculino e 3 do sexo feminino, entre 5 e 11 anos. Seis crianças foram atendidas por uma psicóloga, uma condutora e um Border Collie. O segundo grupo era composto pela mesma psicóloga, uma condutora e uma Golden Retriever. Escalas de avaliação foram aplicadas para confirmar o diagnóstico de TEA. Os cães foram previamente avaliados e treinados por uma instituição que atua na área de TAA. Cinco minutos de 8 sessões foram analisadas: um bloco de seis sessões com o cão, uma sessão anterior e uma sessão posterior a este bloco. Para verificar possíveis diferenças temperamentais entre cães, o C-barq (Canine Behavioral Assesment & Research Questionnaire) foi aplicado para analisar o temperamento de ambos. Embora esta análise tenha demonstrado diferenças em relação às categorias busca de atenção e nível de energia dos cães, não foram verificadas diferenças estatísticas entre os cães, em relação às variáveis analisadas neste estudo. Na comparação entre cães e humanos, os cães foram mais efetivos para conseguir expressões de alegria independentemente do tipo de abordagem escolhida. Comparando-se o tempo de abordagem de cães e humanos até obterem expressão emocional das crianças, observou-se uma importante diferença estatística. Os resultados sugerem que os cães exibiram menor latência que humanos para todas expressões emocionais analisadas: alegria (2= 7,312, p=0,007), de rejeição (2= 11,277, p-0,001) e neutras (2=9,097, p=0,043). Além disso, os resultados sugerem que, no contexto da TAA, não há relação entre abordagem lateral ou frontal e expressões de alegria, rejeição ou neutras de crianças com TEA. As expressões de alegria foram mais frequentes diante das abordagens laterais dos cães do que das abordagens frontais, no entanto não foi verificada significância estatística. Em relação aos humanos também não foi verificada preferência por uma abordagem especifica. Assim, os resultados sugerem que a latência para a exibição de uma expressão emocional das crianças depende mais de quem aborda do que do posicionamento lateral ou frontal quando a abordagem é realizada

Neospora caninum calcium-dependent protein kinase 1 is an effective drug target for neosporosis therapy.

Despite the enormous economic importance of Neospora caninum related veterinary diseases, the number of effective therapeutic agents is relatively small. Development of new therapeutic strategies to combat the economic impact of neosporosis remains an important scientific endeavor. This study demonstrates molecular, structural and phenotypic evidence that N. caninum calcium-dependent protein kinase 1 (NcCDPK1) is a promising molecular target for neosporosis drug development. Recombinant NcCDPK1 was expressed, purified and screened against a select group of bumped kinase inhibitors (BKIs) previously shown to have low IC50s against Toxoplasma gondii CDPK1 and T. gondii tachyzoites. NcCDPK1 was inhibited by low concentrations of BKIs. The three-dimensional structure of NcCDPK1 in complex with BKIs was studied crystallographically. The BKI-NcCDPK1 structures demonstrated the structural basis for potency and selectivity. Calcium-dependent conformational changes in solution as characterized by small-angle X-ray scattering are consistent with previous structures in low Calcium-state but different in the Calcium-bound active state than predicted by X-ray crystallography. BKIs effectively inhibited N. caninum tachyzoite proliferation in vitro. Electron microscopic analysis of N. caninum cells revealed ultra-structural changes in the presence of BKI compound 1294. BKI compound 1294 interfered with an early step in Neospora tachyzoite host cell invasion and egress. Prolonged incubation in the presence of 1294 interfered produced observable interference with viability and replication. Oral dosing of BKI compound 1294 at 50 mg/kg for 5 days in established murine neosporosis resulted in a 10-fold reduced cerebral parasite burden compared to untreated control. Further experiments are needed to determine the PK, optimal dosage, and duration for effective treatment in cattle and dogs, but these data demonstrate proof-of-concept for BKIs, and 1294 specifically, for therapy of bovine and canine neosporosis.