Molecular typing of Mycobacterium bovis isolates from south-east Brazil by spoligotyping and RFLP.

The identification of 163 strains of Mycobacterium bovis by polymerase chain reaction (PCR) and microbiological tests was carried out on 252 tuberculous-like lesions (TLLs) collected from slaughtered cattle in south-east Brazil. This study compared the usefulness of three genotyping techniques, IS6110-restriction fragment length polymorphism (RFLP), polymorphic guanine-cytosine-rich sequence (PGRS)-RFLP and direct repeat (DR)-spoligotyping, as applied to M. bovis isolates. Based on IS6110-RFLP genotyping we selected a group of 23 isolates containing more than one IS6110 copy, along with 16 samples containing one IS6110 copy from different geographical areas, evenly distributed among dairy (eight) and beef cattle (eight). These selected isolates were analysed by PGRS-RFLP and DR-spoligotyping genotyping. Dairy cattle (17%) display a higher frequency of multiple IS6110 copies than beef cattle (10%). A comparison between the genotype data obtained fails to show a correlation between the main clusters found by the three techniques. However, the clustering of each genotyping procedure revealed that the majority of strains are closely related. The RFLP-PGRS patterns showed a sizable group (20.5%) containing a 5.5 kb fragment and the predominant spoligotype is similar to that from the BCG vaccine strain. Unexpectedly, four strains (2.4%) showed drug resistance to 0.2 microg/ml isoniazid and 20 microg/ml ethionamide, but none of them was resistant to rifampicin or other antibiotics tested.

A pncA polymorphism to differentiate between Mycobacterium bovis and Mycobacterium tuberculosis.

The pyrazinamidase gene coding for the enzyme that activates the bactericidal drug pyrazinamide contains a polymorphic site that is preserved in Mycobacterium bovis. We synthesized two sets of primers, one encompassing a 180 bp fragment, and the second spanning a 726 bp fragment including the full pncA gene. Following PCR of Mycobacterium tuberculosis and M. bovis samples, it is possible to discriminate by this polymorphism between these species by digestion with Eco065 I. Digestion of the 180 bp fragment results in two fragments of 101 and 79 bp, specific for M. tuberculosis. Alternatively, digestion of the 726 bp fragment yields three fragments of 452, 165 and 109 bp for M. tuberculosis, but only two fragments of 561 and 165 bp for M. bovis.

BCG lymphadenopathy detected in a BCG-vaccinated infant

Large-scale vaccination with BCG, the live attenuated strain of Mycobacterium bovis, is being adopted around the world, although sporadic complications have occurred after the procedure. Lymphadenopathy is not uncommon especially in babies under one year (0.73 percent of vaccinated infants), but the swelling subsides within 2 months in most cases, with no medical or surgical treatment. Brazil adopted BCG vaccination program earlier in the seventies and by 1995 more than 96 percent of the infant population received this immunization. We report here the occurrence of lymphadenopathy in a two-year-old child vaccinated with the Brazilian BCG strain. The diagnosis was made using a lymph node biopsy and intestinal aspirates that yielded a positive mycobacterial culture. The isolate was resistant to isoniazid, rifampicin, pyrazinamide and thiophen-2-carbonic acid hydrazide, sensitive to streptomycin, ethambutol, and p-nitrobenzoic acid, and reacted positively to cyclo-serine and negatively to niacin. The pncA gene involved in bacterial activation of pyrazinamide contains in M. bovis a point mutation that renders pyrazinamidase unable to catalyze drug activation. Therefore, this polymorphism is a good option for developing methods to differentiate M. bovis and M. tuberculosis. Taking advantage of this difference we further analyzed the isolates by single-stranded conformation polymorphism electrophoresis of DNA following PCR of the pncA gene. The isolate identity was confirmed by RFLP electrophoretic analysis of the amplified fragment following Eco065I digestion, which selectively cleaves M. tuberculosis DNA. From this result it is proposed that RFLP of pncA gene represents an alternative for differential diagnosis of M. bovis.

BCG lymphadenopathy detected in a BCG-vaccinated infant.

Large-scale vaccination with BCG, the live attenuated strain of Mycobacterium bovis, is being adopted around the world, although sporadic complications have occurred after the procedure. Lymphadenopathy is not uncommon especially in babies under one year (0.73% of vaccinated infants), but the swelling subsides within 2 months in most cases, with no medical or surgical treatment. Brazil adopted BCG vaccination program earlier in the seventies and by 1995 more than 96% of the infant population received this immunization. We report here the occurrence of lymphadenopathy in a two-year-old child vaccinated with the Brazilian BCG strain. The diagnosis was made using a lymph node biopsy and intestinal aspirates that yielded a positive mycobacterial culture. The isolate was resistant to isoniazid, rifampicin, pyrazinamide and thiophen-2-carbonic acid hydrazide, sensitive to streptomycin, ethambutol, and p-nitrobenzoic acid, and reacted positively to cyclo-serine and negatively to niacin. The pncA gene involved in bacterial activation of pyrazinamide contains in M. bovis a point mutation that renders pyrazinamidase unable to catalyze drug activation. Therefore, this polymorphism is a good option for developing methods to differentiate M. bovis and M. tuberculosis. Taking advantage of this difference we further analyzed the isolates by single-stranded conformation polymorphism electrophoresis of DNA following PCR of the pncA gene. The isolate identity was confirmed by RFLP electrophoretic analysis of the amplified fragment following Eco065I digestion, which selectively cleaves M. tuberculosis DNA. From this result it is proposed that RFLP of pncA gene represents an alternative for differential diagnosis of M. bovis.

Evaluation of the ratio of omega(6: omega3 fatty acids and vitamin E levels in the diet on the reproductive performance of cockerels.

Three hundred and twenty 30-week old White Leghorn cockerels were housed in individual cages and distributed in a completely randomized factorial design of 5 x 3, with five oil sources (sunflower, soybean, canola, linseed and fish/soybean) and three levels of antioxidant (30, 200 and 400 mg of vitamin E/kg). The aim of this study was to evaluate the effect of the ratio of omega6: omega3 fatty acids by the inclusion of different oil sources and of dietary supplementation with vitamin E on the reproductive performance of cockerels. The use of the fish/soybean combination determined the lowest total antioxidant status of the semen. However, the addition of vitamin E to the fish/soybean-oil-based diet resulted in a linear increase in semen volume, motility and sperm vigour in the 38th week and again in the 52nd week for motility and for sperm vigour and fertility rate in the periods from 50-53 and 41-53 weeks of age. The use of canola oil in the diet resulted in the highest fertility rate during 50-53 and 41-53 weeks of life. Animals receiving the soybean oil based diet showed the smallest fertility rate in the range from 50-53 weeks of age and concomitantly the highest level of cholesterol in the spermatozoa in the range from 47-51 weeks. An interaction between the vitamin E level and soybean oil was verified by the linear increase in motility and sperm vigour at 38 weeks of age. Later, the contrary was shown by a linear reduction in fertility in the periods from 44-46, 47-49 and 41-53 weeks of age. Cockerels that had been fed on the sunflower-oil-based diet showed the highest content of saturated fatty acids in the spermatozoa from 48-51 weeks. An interaction effect was observed between the vitamin E level and sunflower oil shown by a linear increase in the content of saturated fatty acids in the spermatozoa and a linear reduction in the C18: 1omega9, C18 :2 omega6 and PUFA (C18 : 2omega6 + C20 : 4omega6) contents in the spermatozoa at 48-51 weeks and in sperm volume at 52 weeks of age.

Mycobacterium bovis: polymerase chain reaction identification in bovine lymphonode biopsies and genotyping in isolates from Southeast Brazil by spolygotyping and restriction fragment length polymorphism.

Diagnosis of the Mycobacterium tuberculosis complex by direct PCR of mediastinal lymphnode DNA and microbiological tests were compared in cattle suspicious of bearing tuberculous-like lesions detected during slaughter. The PCR procedure applied on DNA samples (n=54) obtained by adding alpha -casein into the thiocyanate extraction mix was positive in 70% of the samples. PCR confirmed the identification of 23 samples (100%) that grew in culture, 9 samples (60%) that failed to grow in culture, plus 6 (37.5%) samples that resulted in growth of bacterial contaminants. Genotyping by IS6110-RFLP and DR-spoligotyping analysis of seven samples revealed the presence of several polimorphisms. Seven of the isolates contained multiple copies of IS6110, thus defining the existence of five singular genotypes.

Mycobacterium bovis: polymerase chain reaction identification in bovine Lymphonode biopsies and genotyping in isolates from Southeast Brazil by spolygotyping and restriction fragment length polymorphism

Diagnosis of the Mycobacterium tuberculosis complex by direct PCR of mediastinal lymphnode DNA and microbiological tests were compared in cattle suspicious of bearing tuberculous-like lesions detected during slaughter. The PCR procedure applied on DNA samples (n=54) obtained by adding alpha -casein into the thiocyanate extraction mix was positive in 70 percent of the samples. PCR confirmed the identification of 23 samples (100 percent) that grew in culture, 9 samples (60 percent) that failed to grow in culture, plus 6 (37.5 percent) samples that resulted in growth of bacterial contaminants. Genotyping by IS6110-RFLP and DR-spoligotyping analysis of seven samples revealed the presence of several polimorphisms. Seven of the isolates contained multiple copies of IS6110, thus defining the existence of five singular genotypes

Detection of Mycobacterium bovis in milk by polymerase chain reaction.

A simple method was developed for DNA extraction of Mycobacterium bovis in milk and further detection of the bacterium by polymerase chain reaction (PCR). Milk previously seeded with M. bovis was used as the starting material. The procedure involved overnight digestion of a milk sample with proteinase K at 56 degrees C and phenol extraction, followed by ethanol precipitation and PCR. The amplification pattern obtained was analyzed with primers BW8-BW9 which amplify a 248 bp in strains of M. bovis. By using the BW8-BW9 primers, 10(3) CFU were detected on silver-stained PAGE gels. The procedure was validated by PCR analysis of milk in tuberculin-positive animals. It is anticipated that this method can be used for routine diagnosis of M. bovis in milk samples.

Use of a cysteine proteinase from Carica candamarcensis as a protective agent during DNA extraction

We describe the use of a plant cysteine proteinase isolated from latex of Carica candamarcensis as a protective agent during isolation of bacterial DNA following growth in culture of these cells. Between 100 to 720 units of proteinase (1 µg = 6 units) afforded good DNA protection when incubated with various kinds of microorganisms. Agarose gel electrophoresis showed that the resulting DNA was similar in size to DNA preparations obtained by treatment with proteinase K. The viability of the resulting material was checked by PCR amplification using species-specific primers. After standing at room temperature (25oC) for 35 days, the enzyme lost 10 percent of its initial activity. The enzyme stability and good yield of DNA suggest the use of this proteinase as an alternative to proteinase K

Use of a cysteine proteinase from Carica candamarcensis as a protective agent during DNA extraction.

We describe the use of a plant cysteine proteinase isolated from latex of Carica candamarcensis as a protective agent during isolation of bacterial DNA following growth in culture of these cells. Between 100 to 720 units of proteinase (1 microgram = 6 units) afforded good DNA protection when incubated with various kinds of microorganisms. Agarose gel electrophoresis showed that the resulting DNA was similar in size to DNA preparations obtained by treatment with proteinase K. The viability of the resulting material was checked by PCR amplification using species-specific primers. After standing at room temperature (25 degrees C) for 35 days, the enzyme lost 10% of its initial activity. The enzyme stability and good yield of DNA suggest the use of this proteinase as an alternative to proteinase K.