SNPs rs471462296, rs456245081 and rs438495570 in the 5'UTR region of DGAT1 gene in Nelore / SNPs rs471462296, rs456245081 e rs438495570 na região 5'UTR do gene DGAT1 em Nelore

O objetivo desta pesquisa foi avaliar os SNPs rs471462296, rs456245081 e rs438495570 do gene DGAT1 em bovinos Nelore. Foram analisados 109 bovinos. A extração do DNA genômico foi realizada do sangue dos animais, usando-se o kit Ilustra Blood Genomic Prep Mini Spin® (GE Healthcare, UK). A concentração e o grau de pureza do DNA foram determinados por meio de espectrofotômetro (Nanodrop - Thermo Fisher Scientifc, USA). A genotipagem dos SNPs ocorreu mediante o emprego do ensaio Taqman® (Applied Biosystems, USA). Na análise genômica, não foram encontradas alterações nas frequências alélicas e genotípicas (P≥0,05) para os SNPs testados. Dessa forma, a região 5'UTR analisada apresentou-se monomórfica e a variação de SNPs não foi observada, o que limita seu uso como marcadores moleculares para o gene DGAT1 em Nelore.(AU)

Assessment of BCL2/J(H) translocation in healthy individuals exposed to low-level radiation of 137CsCl in Goiânia, Goiás, Brazil.

Healthy radio-exposed individuals who received low levels of Cesium-137 radiation during the accident that occurred in Goiânia in 1987, their families and controls were tested for the detection of t(14;18)-rearranged B cells in peripheral blood by using a highly sensitive, real-time quantitative PCR method. The chromosomal translocation t(14;18)(q32;q21) is characteristic of follicular lymphoma and is a frequent abnormality observed in other types of non-Hodgkin's lymphoma. This translocation leads to constitutive activation of the BCL2 oncogene by the enhancers of the immunoglobulin heavy-chain locus. In healthy individuals, the same translocation may also be found in a small fraction of peripheral blood lymphocytes, and positive cells might serve as an indicator for environmental exposure to carcinogens and possibly correlate with the cumulative risk of developing t(14;18)- positive non-Hodgkin's lymphoma. Twenty healthy radio-exposed individuals, 10 relatives and 10 non-exposed healthy individuals were tested for the detection of this translocation. Only 1 non-exposed individual was positive for the chromosomal translocation, and healthy radio-exposed individuals presented lower levels of cells bearing the BCL2/J(H) rearrangement when compared to the levels of the patients with follicular lymphoma before treatment. However, evaluation of more cells would be required to confirm the total absence of circulating cells bearing BCL2/J(H) rearrangement.

Cattle fetal sex determination by polymerase chain reaction using DNA isolated from maternal plasma.

The objective of this study was to evaluate the use of polymerase chain reaction analysis (PCR) of fetal cells/DNA in the maternal plasma of pregnant cows to determine the sex of the fetus. Plasma was harvested from 35 cows of mixed genotype at different stages of pregnancy ranging from 5 to 35 weeks. A male calf and a heifer calf provided the control samples. Fetal sex was determined by amplification of Y-specific sequences. For the 35 cows, the fetal sex predicted by this technique was in accordance with the sex of the calf at birth in 88.6% of cases. The agreement between predicted and observed fetal sex was less for cows with a gestational length of 35-48 days (63.6%). Regression analysis showed that there was a strong relationship between the probability of correctly predicting fetal sex and the stage of gestation. It was estimated that the test performed at 43.8 days post fertilization would have 95% accuracy, increasing to 99% accuracy for testing at 48.4 days and 99.9% accuracy for tests at 55.0 days or later. It was concluded that PCR analysis of fetal cells in maternal plasma can be used to predict successfully the sex of the fetus in cattle.

Cytogenetic damage in the buccal epithelium of Brazilian aviators occupationally exposed to agrochemicals.

The frequency of micronuclei in both buccal cells and peripheral blood lymphocytes is extensively used as a biomarker of chromosomal damage and genome stability in human populations. We examined whether prolonged exposure to complex mixtures of pesticides leads to an increase in cytogenetic damage. The exposed group comprised 50 agricultural aviators, mainly from Central and Southeast regions of Brazil, who had inhaled agrochemicals for more than 10 years without personal protection equipment; the control group consisted of 17 men from the same regions, without indication of exposure to pesticides, There were three times higher frequencies of micronuclei (P < 0.05) and 2.5 times higher frequencies of binucleated cells in the aviators when compared to controls. However, cytotoxic alterations such as broken eggs and karyorrhexis did not present statistically significant differences between the exposed and control groups. Therefore, diverse agrochemicals used to combat pests in agriculture possess genotoxic effects in the oral mucosa of the agricultural pilots, as showed in this study.

Sensibilidade da PCR na amplificação do DNA bovino em diluição seriada e mistura de amostra macho e fêmea / Sensitivity of PCR to amplify sex-specific bovine DNA in diluted samples

A mixture of bovine DNA from a male and a female Jersey (Bos taurus taurus) bred in different proportions was used to determine the sensitivity of PCR to amplify and discriminate the bovine DNA samples. Samples were obtained from the peripheral blood of a bull and a heifer and DNA was isolated using a commercial kit for extraction and purification of nucleic acids. Two primers sets were designed to flank genomic regions: one autosomal and one Y-specific. DNA samples were diluted in water to a final concentration of 4x10-14 ng. The results showed positive amplification of the samples diluted to a concentration of 4x10-10ng and 4x10-4ng for the autosomal and Y-specific regions, respectively. PCR was able to discriminate the male DNA in a mixture of 99:1 (DNA ♀: DNA ♂) heifer to bull ratio. Therefore, the PCR was successful in amplifying the bovine genome in samples containing low concentrations of DNA. Thus, PCR can be used as a sensitive and efficient tool to determine the sex of the fetus in pregnant cows, helping to promote correct and efficient animal management, sex selection, and breeding in commercial herds.(AU)

Sensibilidade da PCR na amplificação do DNA bovino em diluição seriada e mistura de amostra macho e fêmea / Sensitivity of PCR to amplify sex-specific bovine DNA in diluted samples

A mixture of bovine DNA from a male and a female Jersey (Bos taurus taurus) bred in different proportions was used to determine the sensitivity of PCR to amplify and discriminate the bovine DNA samples. Samples were obtained from the peripheral blood of a bull and a heifer and DNA was isolated using a commercial kit for extraction and purification of nucleic acids. Two primers sets were designed to flank genomic regions: one autosomal and one Y-specific. DNA samples were diluted in water to a final concentration of 4x10-14 ng. The results showed positive amplification of the samples diluted to a concentration of 4x10-10ng and 4x10-4ng for the autosomal and Y-specific regions, respectively. PCR was able to discriminate the male DNA in a mixture of 99:1 (DNA ♀: DNA ♂) heifer to bull ratio. Therefore, the PCR was successful in amplifying the bovine genome in samples containing low concentrations of DNA. Thus, PCR can be used as a sensitive and efficient tool to determine the sex of the fetus in pregnant cows, helping to promote correct and efficient animal management, sex selection, and breeding in commercial herds.

Association between male infertility and androgen receptor mutations in Brazilian patients.

The androgen receptor is encoded by a single-copy gene located in the long arm of the X chromosome (Xq11-12); it consists of eight exons and encodes an intracellular transcription factor that belongs to the steroid/nuclear receptor superfamily. Disturbances in the function of the androgen receptor can lead to several forms of male pseudohermaphroditism, such as androgen insensitivity syndrome, which can lead to infertility. Infertility affects around 20% of couples, and in half of the cases it is a male problem. Seventy male patients with idiopathic infertility were selected; data were obtained on age, drinking and smoking habits, occupation, and family history. The mean age of the patients was 37 years old (standard deviation = 12.3); 44% were azoospermic, 33% were oligozoospermic and 24% did not have alterations in the spermogram. Our objective was to evaluate a possible association between male infertility and mutations in the androgen receptor gene based on the presence or absence of exons 1 and 4 of this gene. These two exons were tested by PCR, and their products were separated on 1.5% agarose gels. We found that azoospermic patients had higher mutation rates on exons 1 and 4 of the androgen receptor gene, when compared to other alterations that also lead to infertility, such as oligozoospermia and teratozoospermia. So, we conclude that patients who do not produce sperm have a higher number of mutations in the androgen receptor gene when compared to those who only have impaired sperm production. Based on molecular analysis, we found that there was no correlation between alterations in the spermogram and mutations on exons 1 and 4 of the androgen receptor gene and no association between alterations in the spermogram and alcohol drinking or smoking.

Fatores de virulência em linhagens de Escherichia coli isoladas de mastite bovina / Virulence factors in Escherichia coli strains isolated from bovine mastitis

Avaliou-se a ocorrência de fatores de virulência e do sorotipo O157:H7 em 120 linhagens de Escherichia coli, isoladas de 80 casos de mastite clínica bovina e 40 de mastite subclínica. Verificou-se alfa-hemolisina em oito (6,7%) linhagens, isoladas de cinco casos de mastite clínica e três de mastite subclínica e em nenhuma das estirpes detectou-se enteroemolisina. A presença de sideróforos foi encontrada em 11 (9,2%) linhagens, sete de mastite clínica e quatro de subclínica. Em duas (1,7%) estirpes isoladas de mastite subclínica, identificou-se enterotoxina STa. Observou-se efeito citopático em células vero compatível com a produção de verotoxina-VT em cinco (4,2%) linhagens, duas de mastite clínica e três subclínicas. Em uma (0,8%) linhagem isolada de mastite clínica, detectou-se efeito citopático compatível com o fator necrosante citotóxico. Nenhuma estirpe apresentou-se sorbitol-negativa no MacConkey-sorbitol, tampouco aglutinou com o sorotipo O157:H7. Os antimicrobianos mais efetivos foram polimixina B (97,5%) e norfloxacina (95,8%). Observou-se multi-resistência a dois ou mais antimicrobianos em 24 (20%) estirpes, principalmente com o uso de ampicilina e ceftiofur.(AU)

The occurrence of different virulence factors and O157:H7 serotype investigation in 120 Escherichia coli strains isolated from clinical (80 cases) and subclinical (40 cases) bovine mastitis was evaluated. Alpha-haemolysin was detected in 8 (6.7%) strains (5 clinical and 3 subclinical cases). None strain showed enterohaemolysin production. E. coli growth under iron restriction conditions (siderophores production) was observed in 11 (9.2%) strains (7 clinical and 4 subclinical cases). STa enterotoxin was detected in 2 (1.7%) strains from subclinical cases. Cytotoxic effect in vero cells compatible with verotoxin-VT production was observed in 5 (4.2%) strains (2 clinical and 3 subclinical cases). One strain (0.8%) isolated from clinical mastitis showed cytophatic effect in vero cells compatible with cytotoxic-necrotic-factor production. None strain showed non-sorbitol fermenting colonies using sorbitol MacConkey agar and agglutination with specific anti-sera for E. coli O157:H7 serotype investigation. Polymixin B (97.5%) and norfloxacin (95.8%) were the most effective drugs. Multiple-drug resistance for 2 or more antimicrobials was observed in 24 (20.0%) strains, mainly with use of ampicillin and ceftiofur.(AU)

Fatores de virulência em linhagens de Escherichia coli isoladas de mastite bovina / Virulence factors in Escherichia coli strains isolated from bovine mastitis

Avaliou-se a ocorrência de fatores de virulência e do sorotipo O157:H7 em 120 linhagens de Escherichia coli, isoladas de 80 casos de mastite clínica bovina e 40 de mastite subclínica. Verificou-se alfa-hemolisina em oito (6,7 por cento) linhagens, isoladas de cinco casos de mastite clínica e três de mastite subclínica e em nenhuma das estirpes detectou-se enteroemolisina. A presença de sideróforos foi encontrada em 11 (9,2 por cento) linhagens, sete de mastite clínica e quatro de subclínica. Em duas (1,7 por cento) estirpes isoladas de mastite subclínica, identificou-se enterotoxina STa. Observou-se efeito citopático em células vero compatível com a produção de verotoxina-VT em cinco (4,2 por cento) linhagens, duas de mastite clínica e três subclínicas. Em uma (0,8 por cento) linhagem isolada de mastite clínica, detectou-se efeito citopático compatível com o fator necrosante citotóxico. Nenhuma estirpe apresentou-se sorbitol-negativa no MacConkey-sorbitol, tampouco aglutinou com o sorotipo O157:H7. Os antimicrobianos mais efetivos foram polimixina B (97,5 por cento) e norfloxacina (95,8 por cento). Observou-se multi-resistência a dois ou mais antimicrobianos em 24 (20 por cento) estirpes, principalmente com o uso de ampicilina e ceftiofur.

The occurrence of different virulence factors and O157:H7 serotype investigation in 120 Escherichia coli strains isolated from clinical (80 cases) and subclinical (40 cases) bovine mastitis was evaluated. Alpha-haemolysin was detected in 8 (6.7 percent) strains (5 clinical and 3 subclinical cases). None strain showed enterohaemolysin production. E. coli growth under iron restriction conditions (siderophores production) was observed in 11 (9.2 percent) strains (7 clinical and 4 subclinical cases). STa enterotoxin was detected in 2 (1.7 percent) strains from subclinical cases. Cytotoxic effect in vero cells compatible with verotoxin-VT production was observed in 5 (4.2 percent) strains (2 clinical and 3 subclinical cases). One strain (0.8 percent) isolated from clinical mastitis showed cytophatic effect in vero cells compatible with cytotoxic-necrotic-factor production. None strain showed non-sorbitol fermenting colonies using sorbitol MacConkey agar and agglutination with specific anti-sera for E. coli O157:H7 serotype investigation. Polymixin B (97.5 percent) and norfloxacin (95.8 percent) were the most effective drugs. Multiple-drug resistance for 2 or more antimicrobials was observed in 24 (20.0 percent) strains, mainly with use of ampicillin and ceftiofur.

Evaluation of the microbiological status of milk and various structures in mammary glands from naturally infected dairy cows.

A knowledge of the microbiological status of milk and of the different structures in the mammary glands has great importance in elucidating the pathogenesis of mammary gland infections. The objective of this study was to evaluate the microbiological status of various structures in the mammary glands from naturally infected dairy cows following slaughter. A total of 94 samples of milk, 184 samples of mammary parenchyma, 168 samples of gland cisterns, and 168 samples of teat cisterns were collected for microbiological examination. Microorganisms were detected in 59.9% of all samples, 67.0% of the milk samples, 70.1% of the mammary parenchymas, 55.9% of the gland cisterns and 48.8% of the teat cistern samples. When all samples were considered, coagulase-negative Staphylococcus were the most prevalent (35.7%) followed by coagulase-positive Staphylococcus (12.2%), Corynebacterium bovis (2.4%), Prototheca sp. (1.9%), and Streptococcus dysgalactiae (1.5%). There was a significantly higher occurrence of microorganisms in the milk and mammary parenchyma compared to the gland cisterns and teat cisterns.

Fator necrosante citotóxico em Escherichia coli isolada de mastite clínica bovina / Cytotoxic necrotizing factor in Escherichia coli strain isolated from clinical bovine mastitis

This report describes the production of cytotoxic necrotizing factor (CNF) by an Escherichia coli strain isolated from clinical bovine mastitis with clinical signs of toxemia The animal had hemorrhages and necrosis of the mammary glands, and died within 24 hours after the onset of clinical signs. In addition to CNF identification, alpha-haemolysin and siderophores production were also characterized in this strain. This report reinforce the association of CNF and alpha-haemolysin production in E. coli virulence associated with clinical cases of severe bovine mastitis

Aetiology and histopathology of bovine mastitis of espontaneous occurrence.

Infectious bovine mastitis is responsible for serious economic losses in dairy cattle breeding. Information about the damages caused by micro-organisms is of great interest and importance. The purpose of the present study was to report on the microbiological and histopathological aspects of the mammary parenchymas of slaughtered dairy cows. A total of 184 mammary glands were examined and samples of mammary parenchyma were collected for microbiological and histopathological examinations. Micro-organisms were isolated from 69.6% samples; 23 (12.5%) of the 184 samples did not show histological changes; inflammatory response was observed in 56 (30.4%) samples; inflammatory response and repair were present in 82 (44.6%) samples; repair process was verified in 23 (12.5%) of the mammary glands. Coagulase-negative Staphylococci were observed in 53.8% of the samples followed by coagulase-positive Staphylococci (7.6%), Prototheca sp. (2.2%) and Streptococcus dysgalactiae (1.6%). Coagulase-negative Staphylococci and coagulase-positive Staphylococci were associated mainly to chronic inflammatory response and chronic inflammatory response and repair. Samples from which no micro-organisms were isolated (n = 56) had no histological changes in 82.6% of the cases. These results were higher (P < 0.05) when compared to the samples with micro-organisms and without histological changes (17.4%).

Susceptibility and features of the ultrastructure of Prototheca zopfii following exposure to copper sulphate, silver nitrate and chlorexidine.

One of the most important forms of the occurrence of protothecosis is bovine mastitis. Studies on the "in vivo" and "in vitro" susceptibility to antimicrobials have shown that the microorganism is resistant to most of them. Looking for alternative treatments this study aimed to study the susceptibility to copper sulphate (which has an important algicide effect) and silver nitrate (used in dairy cattle breeding for the cauterization of mammary glands) and also to chlorexidine (an important post-dipping anti-septic used in dairy practice), and the effect of these antimicrobials in the ultrastructure of Prototheca zopfii before and after the exposure to these drugs. The "in vitro" susceptibility tests to chlorexidine, silver nitrate and copper sulphate of the strains of Prototheca zopfii for the determination of their minimal microbicidal concentrations (MMC), were performed using the tube dilution method in Sabouraud dextrose broth and evaluation of colony growth after plating in Sabouraud dextrose agar. The MMCs of chlorexidine, copper sulphate and silver nitrate of the 50 strains tested were 0.01%, 0.1% and 0.3%, respectively. The tubes containing the material used in the antimicrobial susceptibility tests were prepared for the examination in an electron microscope. The untreated controls of P. zopfii showed a similar ultrastructural appearance with the typical characteristics of the microorganism. Cells exposed to silver nitrate showed changes suggesting thickness of the cell wall. Cells exposed to chlorexidine showed changes suggesting degradation of intra-cellular organelles present in the cytoplasm. P. zopfii treated with copper sulphate showed changes suggesting fibrilation of inner layer of cell wall.

Fator necrosante citotóxico em Escherichia coli isolada de mastite clínica bovina

This report describes the production of cytotoxic necrotizing factor (CNF) by an Escherichia coli strain isolated from clinical bovine mastitis with clinical signs of toxemia The animal had hemorrhages and necrosis of the mammary glands, and died within 24 hours after the onset of clinical signs. In addition to CNF identification, alpha-haemolysin and siderophores production were also characterized in this strain. This report reinforce the association of CNF and alpha-haemolysin production in E. coli virulence associated with clinical cases of severe bovine mastitis.

Bacterial septicemia in water snakes (Helicops modestus) in Brazil

Relata-se infecção septicêmica em cobras d’água (Helicops modestus) causada por Proteus vulgaris. Os animais iniciavam o processo apresentando abscessos puntiformes de coloração esbranquiçada na pele e após rápida evolução, morriam (100% de mortalidade). Descreve-se, ainda, a sensibilidade do P. vulgaris isolado dos animais a drogas antibacterianas.

Bacterial septicemia in water snakes (Helicops modestus) in Brazil

Relata-se infecção septicêmica em cobras dágua (Helicops modestus) causada por Proteus vulgaris. Os animais iniciavam o processo apresentando abscessos puntiformes de coloração esbranquiçada na pele e após rápida evolução, morriam (100% de mortalidade). Descreve-se, ainda, a sensibilidade do P. vulgaris isolado dos animais a drogas antibacterianas.

Antimicrobial susceptibility of Staphylococcus spp. isolated from mammary parenchymas of slaughtered dairy cows.

The high prevalence of the great resistance to antimicrobials shown by the aetiological agents of infectious bovine mastitis, often leads to chronic recurrent mastitis, one of the main causes of early culling of dairy cows. The purpose of this study was to determine the in vitro susceptibility pattern of Staphylococcus spp. isolated from mammary parenchymas of slaughtered dairy cows, to different anti-microbials. A total of 45 Staphylococcus spp. strains [33 coagulase-negative Staphylococcus (CNS) and 12 Staphylococcus aureus (SA)] were used. These strains were tested with 12 different antimicrobials by the Kirby and Bauer standardized disc diffusion method; 84.44% and 86.66% of the 45 strains were resistant to ampicillin and penicillin, respectively. The highest sensitivity was to cephalothin (84.44%), gentamicin (80%) and to sulphazotrin (77.77%). CNS showed higher resistance (P < 0.05) than SA. The in vitro susceptibility pattern of Staphylococcus spp. strains isolated from mammary parenchymas studied in the present investigation was similar to that observed in recent studies of in vitro and in vivo susceptibility patterns of Staphylococcus spp. isolated from cases of bovine mastitis.

Evaluation of the susceptibility of Prototheca zopfii to milk pasteurization.

Protothecosis has been reported in humans (gastroenteritis, bursitis, etc.) and in many other animal species. Bovine mastitis represents the main form of occurrence of protothecosis in cattle. Milk as well as dairy products, when contaminated with Prototheca spp., represent a potential means of transmission of this zoonosis. The purpose of this study was to evaluate the susceptibility of forty Prototheca zopfii strains isolated from milk from intramammary infections in dairy cows and also from bulk milk tanks of dairy farms, to the different ratios of temperature/time employed in the thermal treatment of milk: 72-75 degrees C/15 seconds, 72-75 degrees C/20 seconds and 62-65 degrees C/30 minutes. The samples were subjected to these different temperature/time ratios. The evaluation of the thermal susceptibility of the P. zopfii strains showed that 34 strains were resistant in at least one of the tests. The results point out the need to consider the importance of mastitis caused by Prototheca spp. as representing a public health risk.

Infectious bovine mastitis caused by environmental organisms.

The purpose of this study was to determine the prevalence of environmental mastitis in dairy herds and identify the main environmental pathogens, and to evaluate the influence of season, housing and management. A total of 20,310 quarters of 5216 animals from 52 dairy herds in 32 counties was examined. Milk samples were aseptically collected for laboratory examination from mammary glands testing positive to any of the field tests. From these, 736 environmental infections were identified. The most frequently isolated environmental pathogens were algae of the genus Prototheca sp. (41.2%), Streptococcus uberis (21.1%), fungi (19.5%), enterobacteriacea (8.3%) and Nocardia sp. (6.6%). The occurrence of mastitis was not influenced by the herd size, use of dry cow therapy, or post milking teat dipping. A tendency for increased occurrence of environmental mastitis during the months of September to February (hot and wet weather) was observed, suggesting a seasonal influence.

Métodos para diagnóstico microbiológico de mastite clínica bovina / Microbiological methods for diagnosis of clinical bovine mastitis

O objetivo deste trabalho foi comparar três procedimentos de semeadura do leite para diagnóstico microbiológico da mastite bovina: "in natura", pós-congelamento de 12 horas a -20ºC e pós-incubaçäo a 37§C por 12 horas. Foram examinados 256 amostras de leite procedentes de 200 vacas com mastite clínica de cinco propriedades leiteiras. Os resultados bacteriológicos após os tratamentos descritos mostraram aumento dos microrganismos isolados. Observou-se aumento significativo do Staphylococcus sp nas amostras congeladas e incubadas, aumento da Klebsiella sp e do Pseudomonas sp apenas no pós-incubaçäo, e reduçäo do Corynebacterium bovis, nos isolamentos pós-incubaçäo. Näo se obervou diferença entre os tratamentos "in natura" e pós-congelamento. Pode-se concluir que a melhor conduta para diagnóstico da mastite seria a associaçäo destes procedimentos para se obter maior taxa de isolamento, permitindo estabelecer o diagnóstico e instituir a conduta terapêutica adequada