Culture and molecular identification of microorganisms from Digital Dermatitis lesions in dairy cattle: Leptospira, an unexpected finding / Cultivo e identificação molecular de microrganismos isolados de lesões de dermatite digital em bovinos leiteiros: Leptospira, um achado inesperado

Bovine digital dermatitis (BDD) is an infectious and contagious disease characterized by ulcerative and proliferative lesions affecting the skin on the bulbs of the heel or the interdigital cleft in dairy cattle, often associated with lameness. Evidences on the etiology of BDD indicate that it is multifactorial, involving environmental factors and multiple bacterial colonization. We isolated and identified microorganisms from BDD biopsy samples obtained from five Holstein Friesian and two Jersey cows by cultivation and molecular identification of bacterial isolates using 16S rRNA gene sequence analysis. We identified six bacterial species: Spirochetes as Treponema pedis and Leptospira broomi/L. fainei, L. licerasiae/L. wolffii; Corynebacterium appendicis, Cupriavidus gilardii and Enterococcus casseliflavus/E. gallinarum. It was quite surprising to have isolated and identified Leptospira species in three out of seven cultures, from different individual cows and two different farms. The species identified belong to the intermediate pathogenic clade, which is a group found to cause human and animal disease. Our findings indicate the need to further investigate the association of Leptospira of intermediate pathogenicity with BDD lesions and whether its presence would have any veterinary and medical significance both in Leptospirosis and with the pathogenesis of BDD lesions, especially in tropical countries.(AU)

Dermatite digital bovina (DDB) é uma doença infecciosa, contagiosa, caracterizada por lesões ulcerativas e proliferativas da região dos talões e/ou do espaço interdigital, frequentemente associada com claudicação. Evidências indicam que a etiologia da DDB é multifatorial, envolvendo fatores ambientais e colonização polimicrobiana. Relata-se aqui o isolamento e a identificação bacteriana em amostras de biópsias em lesões de DDB, obtidas de cinco vacas da raça Holandesa e duas da raça Jersey, por meio de cultivo e identificação molecular de isolados, com base na análise de sequências de genes 16S rRNA. São identificadas seis espécies bacterianas: as espiroquetas Treponema pedis e Leptospira broomi/L. fainei, L. licerasiae/L. wolffii; Corynebacterium appendicis, Cupriavidus gilardii e Enterococcus casseliflavus/E. gallinarum. O isolamento e a identificação de espécies de Leptospira surpreenderam, destacando-se sua presença em três dos sete cultivos obtidos em diferentes vacas, de duas fazendas distintas. As espécies identificadas pertencem ao grupo tipificado como de patogenicidade intermediária, causador de doenças em animais e no homem. Os resultados apresentados indicam a necessidade de maiores investigações sobre a associação entre Leptospira de patogenicidade intermediária e a patogênese das lesões DDB, investigando-se sua presença e significado nas medicinas veterinária e humana, especialmente em países tropicais.(AU)

A new tetra-primer ARMS-PCR for genotyping bovine kappa-casein polymorphisms.

Kappa-casein (κ-casein) is one of the most abundant milk proteins. Its main function is to avoid the aggregation of casein micelles, keeping them, and therefore calcium phosphate, in pockets in solution. In bovines, a κ-casein functional polymorphism has been associated with fat, calcium, and protein milk contents and faster curd contraction in cheese production. Quicker curd contraction reduces the loss of milk solids, enhancing cheese yield. This polymorphism induces a double amino acid substitution (Thr136Ile and Ala148Asp). The polymorphism is normally detected by PCR-RFLP, which is a laborious method. An interesting methodological alternative is the tetra-primer amplification refractory mutation system PCR (tetra-primer ARMS-PCR). A tetra-primer ARMS-PCR for the detection of this κ-casein polymorphism has been described. However, specificity was not achieved, probably due to problems with primer design. We developed a new tetra-primer ARMS-PCR for the detection of the κ-casein polymorphism. This new method was validated in a double-blind test, by comparison with the results obtained for 50 Guzerá bulls formerly genotyped by PCR-RFLP. This new method achieved 100% sensitivity and specificity. We conclude that this method is a useful, cost-efficient alternative for the detection of functional κ-casein polymorphisms.

Influence of Arg72Pro polymorphisms of TP53 on the response of buccal cells to radiotherapy.

Polymorphisms in the TP53 gene codon 72 (Arg72Pro) influence apoptosis induction and DNA damage repair. We evaluated how variants of protein p53 (p53Arg and p53Pro) affect cell death and DNA damage repair by analyzing the frequencies of karyorrhexis and micronuclei. There were significant differences in the frequency of karyorrhexis between the three p53 genotypes (Arg/Arg, Arg/Pro, and Pro/Pro), between samples taken before and after radiotherapy, and between patients and controls. The frequency of micronucleated cells increased significantly after radiotherapy. There were no significant differences in the micronucleus frequency in healthy tissues of these patients compared to controls, or in the comparisons between the three genotypes. We conclude that Arg72Pro polymorphism influences cell apoptotic capacity. This is the first study investigating karyorrhexis and micronuclei, as indicators of apoptosis after radiotherapy, and how these indicators are influenced by the TP53 polymorphism Arg72Pro.

Variations in genotype-phenotype correlations in phenylketonuria patients.

Phenylalanine hydroxylase deficiency is a trait inherited in an autosomal recessive pattern; the associated phenotype varies considerably. This variation is mainly due to the considerable allelic heterogeneity in the phenylalanine hydroxylase enzyme locus. We examined the genotype-phenotype correlation in 54 phenylketonuria (PKU) patients from Minas Gerais, Brazil. Two systems were used. The first was a phenotype prediction system based on arbitrary values (AV) attributed to each mutation and the second was a correlation analysis. An AV was assigned to each mutation: AV = 1 for classical PKU mutation; AV = 2 for moderate PKU mutation; AV = 4 for mild PKU mutation, and AV = 8 for non-PKU hyperphenylalaninemia mutation. The observed phenotype for AV analysis was the clinical diagnosis established by the overloading phenylalanine test. Among the 51 PKU patients that we analyzed based on this trait, in 51% the predicted phenotype did not match the observed phenotype; the highest degree of concordance was found in patients with null/null genotypes. The genotype was observed to be a good predictor of the clinical course of the patients and significant correlations were found between phenylalanine values at first interview and predicted residual activity, genotype and arbitrary value sum.

Detecção de QTL em dados de famílias estruturadas como as de um núcleo MOET por meio do método da regressão / QTL detection in MOET nucleus family structure-like data using the regression method

O objetivo deste trabalho foi avaliar a eficiência do método de regressão em detectar QTL com base na utilização de dados da estrutura de família (irmãos completos e meios-irmãos), como aqueles gerados em um núcleo MOET. Foram simulados dados fenotípicos e genotípicos em uma estrutura de núcleo MOET fechado de seleção. Três arquivos foram analisados, contendo: a) informações conjuntas de irmãos completos e meios-irmãos; b) apenas informações de irmãos completos e c) apenas informações de meios-irmãos. Verificou-se que o método da regressão, para dados discretos ou contínuos, foi capaz de detectar associações entre marcador e QTL em níveis bastante expressivos de significância (P<0.001 e P<0,0001), quando se utilizou o arquivo que continha informações conjuntas de irmãos completos e meios-irmãos. Os resultados indicaram a possibilidade de utilização dessa metodologia para estudos de detecção/validação de QTL em rebanhos ou núcleos de seleção que utilizam MOET.

The objective of this study was to evaluate the efficiency of the regression method to detect QTL using data from full and half-sib families, like those generated in a MOET nucleus. For this study, genotypic and phenotypic data were simulated in a structure of a closed selection MOET nucleus. Three files were analyzed containing: a) the joint information of full and half sibs; b) only full sibs data; and c) only half sibs data. The method of regression, for continuous or discrete data, was able to detect associations of marker and QTL in very expressive levels of significance (P<0.001 P<0.0001), when the file containing the joint information of full and half sisters was used. The results indicated the possibility of using this methodology for studies of QTL detection / validation in MOET nucleus or herds under selection.

Technical note: A new and cost-effective method for detection of the bovine acyl-CoA:diacylglycerol acyltransferase 1 K232A polymorphism in cattle.

A new, quick, and inexpensive method for detecting the bovine acyl-CoA:diacylglycerol acyltransferase1 (DGAT1) polymorphism (K232A) through tetra-primer amplification refractory mutation system by PCR (ARMS-PCR) was developed in the present investigation. The DGAT1 gene was recently identified as underlying variation in milk production traits. To date, PCR techniques such as PCR-RFLP have been used for detecting the DGAT1 K232A polymorphism, despite being expensive and laborious. The method proposed here, a tetra-primer ARMS-PCR, showed 100% sensitivity and specificity when compared with PCR-RFLP results obtained in a sample of 80 animals tested in a double-blind system. Our results suggest that the use of tetra-primer ARMS-PCR for DGAT1 K232A polymorphism genotyping could greatly reduce costs providing information for both research purposes and for dairy cattle breeders who perform DGAT1 genotyping for gene-assisted selection.

Genetic diversity and population structure in Brazilian Mangalarga Marchador horses.

One hundred and fifteen unrelated Mangalarga Marchador horses were sampled from three geographically distinct regions of Minas Gerais State, Brazil (South, Southeast, and Northeast) and tested for 10 microsatellite loci. Genetic diversity and population structure parameters were estimated with ARLEQUIN 3.0, CERVUS 2.0, POPGENE 1.31, GENEPOP on the web, STRUCTURE 2.0, and SPAGEDI 1.2 software packages. Under Hardy-Weinberg assumptions, seven markers were at equilibrium (LEX014, LEX017, LEX019, SGCV23, TKY321, VHL20, and VIASH39), while two (ASB3 and LEX031) presented significant homozygote excess. Seventy-four alleles were identified in these nine markers, with a mean of 8.22 alleles. Mean heterozygosity was 0.637 and polymorphism information content was 0.662. Markers ASB3, LEX019, SGCV23, TKY321, and VHL20 were highly informative (PIC >0.7) and may be useful for eventual expansion of parentage test panels. The F(ST) value (0.0562) indicated relatively little geographical structure. However, based on a Bayesian-based cluster analysis under a three-cluster model, 94% of the 115 individuals were correctly assigned to the subpopulations from where they were sampled. Mean pairwise f was relatively high (0.11), and in spite of the efforts towards non-consanguineous sampling, 1% of the pairs of individuals shared over 50% of the alleles. These results strongly suggest that the population is genetically structured. Under a conservation genetics approach, two strategies are recommended: avoidance of crosses between highly endogamic individuals and stimulation of crosses between individuals from those regions for which low genetic flow was identified.

PKU in Minas Gerais State, Brazil: mutation analysis.

This work was undertaken in order to ascertain the PKU mutational spectrum in Minas Gerais, Brazil, the relative frequency of the mutations in the State and the origin of these mutations by haplotype determination. Minas Gerais is a trihybrid population formed by miscegenation from Europeans, Africans and Amerindians. All 13 exons of the PAH gene from 78 PKU patients were analyzed, including splicing sites and the promoter region. We identified 30 different mutations and 98% of the PAH alleles were established. A new mutation (Q267X) was identified as well. The most common mutations found were V388M (21.2), R261Q (16.0%), IVS10-11G>A (15.3%), I65T (5.8%), IVS2+5G>C (5.8%), R252W (5.1%), IVS2+5G>A (4.5%), P281L (3.8%) and L348V (3.2%). These nine mutations correspond to 80% of the PKU alleles in the state. Haplotypes were determined to characterize the origin of the PAH alleles. The majority of the mutations found, with respective haplotypes, are frequent in the Iberian Peninsula. However, there were some mutations that are rare in Europe and four previously unreported mutation-haplotype associations. I65T and Q267X were found in association with haplotype 38 and may be African in origin or the result of miscegenation in the Brazilian population.

Genetic polymorphism of the kappa-casein gene in Brazilian cattle.

Frequencies of kappa-casein gene alleles were determined in 1316 animals from the Brazilian Bos indicus genetic groups (Sindhi cows, Gyr sires, Gyr cows, Guzerat sires, Guzerat cows, Nellore sires, and Gyr x Holstein crossbreds) by means of polymerase chain reaction-restriction fragment length polymorphism analysis using two independent restriction nucleases (Hinf I and HaeIII). The genotyping of kappa-casein alleles (A and B) is of practical importance, since the B allele is found to correlate with commercially valuable parameters of cheese yielding efficiency. The frequencies of the B allele of kappa-casein among breeds ranged from 0.01 to 0.30. The Sindhi breed had the highest frequency for the B allele (0.30), while the frequencies of this allele in other breeds ranged from 0.01 to 0.18. A wide variation in the B allele frequency among B. indicus breeds was found suggesting that molecular selection for animals carrying the B allele could impact breeding programs for dairy production.

Frequency of FMR1 premutation in individuals with ataxia and/or tremor and/or parkinsonism.

A late onset neurological syndrome in carriers of premutation in FMR1 gene was recently described. The condition was named fragile-X-associated tremor/ataxia syndrome (FXTAS) and includes intentional tremor, cerebellar ataxia, parkinsonism, and cognitive deficit. We ascertained the contribution of FMR1 premutation to the phenotypes ataxia, tremor and/or parkinsonism. Sixty-six men over 45 years old presenting these symptoms, isolated or combined, were tested. Also, 74 normal men, randomly chosen in the population, formed the control group. In the patient group, no premutation carrier was found, which is in agreement with other observed frequencies reported elsewhere (0-5% variation). No significant differences were found when comparing gray zone allele frequencies among target and control groups. The FXTAS contribution in patients with phenotypic manifestations of FXTAS was 15/748 (2%). The presence of gray zone alleles is not correlated with FXTAS occurrence.

Frequency of FMR1 premutation in individuals with ataxia and/or tremor and/or parkinsonism

A late onset neurological syndrome in carriers of premutation in FMR1 gene was recently described. The condition was named fragile-X-associated tremor/ataxia syndrome (FXTAS) and includes intentional tremor, cerebellar ataxia, parkinsonism, and cognitive deficit. We ascertained the contribution of FMR1 premutation to the phenotypes ataxia, tremor and/or parkinsonism. Sixty-six men over 45 years old presenting these symptoms, isolated or combined, were tested. Also, 74 normal men, randomly chosen in the population, formed the control group. In the patient group, no premutation carrier was found, which is in agreement with other observed frequencies reported elsewhere (0-5% variation). No significant differences were found when comparing gray zone allele frequencies among target and control groups. The FXTAS contribution in patients with phenotypic manifestations of FXTAS was 15/748 (2%). The presence of gray zone alleles is not correlated with FXTAS occurrence.

PERMANENT GENETIC RESOURCES: Characterization of eight microsatellite loci in the woolly mouse opossum, Micoureus paraguayanus, isolated from Micoureus demerarae.

Eight novel microsatellite markers were isolated from the woolly mouse opossum from the Amazon Forest in Peru, Micoureus demerarae, using a partial genomic DNA library and an enrichment protocol. These loci were polymorphic in M. demerarae and Micoureus paraguayanus populations from the Atlantic Forest in Brazil with the number of alleles ranging from two to 23. Those eight loci plus another five already described for M. paraguayanus will allow for the evaluation of genetic diversity of populations from the 'Rio Doce' Park, one of the last Atlantic Forest fragments in Minas Gerais state, Brazil.

Logistic regression analysis of pregnancy rate following transfer of Bos indicus embryos into Bos indicus x Bos taurus heifers.

Factors affecting pregnancy rate of 5627 Zebu embryos in crossbred females with unknown proportions of Holstein and Zebu breeding were examined. After evaluation for developmental stage, quality, and viability, embryos were immediately transferred to recipients. Pregnancy diagnosis was conducted approximately 53 d after transfer; pregnancy rate was coded as a binomial event and analyzed using logistic regression models. Maximum likelihood methodology and the likelihood ratio statistic were used to estimate regression coefficients and test hypotheses. Explanatory variables were year of transfer (1992-1999), season of transfer (summer, autumn, winter and spring), breed of the embryo (Guzerat, Gyr or Nellore), stage of the embryo (morula, early blastocyst, blastocyst, expanded blastocyst, and hatching blastocyst), quality of the embryo (excellent, good or regular), and donor-recipient synchrony (estrus in the recipient occurred 2-3 d before, 1 d before, the day of, 1 d after, or 2-3 d after estrus in the donor). Average pregnancy rate was 63.7%. Pregnancy rates were not significantly affected by breed of embryo. The best multiple-logistic model to explain the pregnancy result included the effects of year and season of transfer, embryo stage and quality, and estrous synchrony between donor and recipient (P

DGAT1 K232A polymorphism in Brazilian cattle breeds.

Recent reports identified DGAT1 (EC 2.3.1.20) harboring a lysine to alanine substitution (K232A) as a candidate gene with a strong effect on milk production traits. Our objective was to estimate the frequency of the DGAT1 K232A polymorphism in the main Zebu and Taurine breeds in Brazil as well as in Zebu x Taurine crossbreds as a potential QTL for marker-assisted selection. Samples of 331 animals from the main Brazilian breeds, Nellore, Guzerat, Red Sindhi, Gyr, Holstein, and Gyr x Holstein F1 were genotyped for DGAT1 K232A polymorphism (A and K alleles) using the PCR-RFLP technique. The highest frequency of the A allele was found in the Holstein sample (73%) followed by Gyr x Holstein F1 (39%). Gyr and Red Sindhi showed low frequencies of A alleles (4 and 2.5%, respectively). The A allele was not found in the Nellore and Guzerat samples. Our results could be used to guide association studies between this locus and milk traits in these breeds.

DGAT1 K232A polymorphism in Brazilian cattle breeds

Recent reports identified DGAT1 (EC 2.3.1.20) harboring a lysine to alanine substitution (K232A) as a candidate gene with a strong effect on milk production traits. Our objective was to estimate the frequency of the DGAT1 K232A polymorphism in the main Zebu and Taurine breeds in Brazil as well as in Zebu x Taurine crossbreds as a potential QTL for marker-assisted selection. Samples of 331 animals from the main Brazilian breeds, Nellore, Guzerat, Red Sindhi, Gyr, Holstein, and Gyr x Holstein F1 were genotyped for DGAT1 K232A polymorphism (A and K alleles) using the PCR-RFLP technique. The highest frequency of the A allele was found in the Holstein sample (73%) followed by Gyr x Holstein F1 (39%). Gyr and Red Sindhi showed low frequencies of A alleles (4 and 2.5%, respectively). The A allele was not found in the Nellore and Guzerat samples. Our results could be used to guide association studies between this locus and milk traits in these breeds.

Aspectos Genéticos do Alcoolismo / Genetic aspects of alcoholism

O termo alcoolismo refere-se aos sintomas que se desenvolvem a partir do consumo inadequado de álcool. Por ser o álcool a principal droga psicoativa utilizada no mundo, é de se esperar que o desenvolvimento da doença dependa da interaçäo entre fatores neurobiológicos e psicossociais. Diversos estudos têm mostrado que o alcoolismo é mais frequente nas famílias de alcoolitas do que na populaçäo em geral. Entretanto, além das influências genéticas, os fatores ambientais também säo responsáveis pela agregaçäo familiar observada, caracterizando, assim, uma herança multifatorial. A subdivisäo do alcoolismo nos tipos 1 e 2 tem permitido uma melhor compreensäo da doença do ponto de vista etiopatogênico e uma melhor abordagem da mesma. Apesar das diferenças metodológicas, a grande maioria dos estudos familiares sugere que os fatores säo muito importantes na determinaçäo do alcoolismo. Já foram identificados os seguintes fatores predisponentes ao alcoolismo: o alelo A1 do gene do receptor de dopammina DRD2, a reduçäo da atividade da MAO-B plaquetária, e a reduçäo da amplitude da onda P300 nos ERPs. Também evidenciou-se que uma anomalia da enzima transcetolase predispöe à Síndrome de Wernicke-Korsakoff. Além disto, foram identificados fatores protetores à doença, como a presença dos alelos ADH2*2 e ALDH2*2 dos genes das enzimas álcool desidrogenase, rspectivamente. Apesar da identificaçäo desses fatores associados ao alcoolismo, pouco ainda se sabe sobre o modo pelo qual eles interagem entre si e, portanto, muitos estudos ainda seräo necessários para um melhor esclarecimento do assunto