Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR.

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Thin layer microcolony culture associated with PCR for early identification of Mycobacterium bovis.

The initial growth of mycobacteria from 49 samples of cattle and buffalo organs collected in commercial slaughterhouses was compared between modified Middlebrook 7H11 thin layer microcolony culture and Stonebrink medium used in the isolation of Mycobacterium bovis. Aliquots were decontaminated by Petroff's method, processed and cultured in both media. The identity of the acid-fast bacilli stained by Ziehl-Neelsen was confirmed by PCR. Optical microscopy showed that results of the early observation of Mycobacterium bovis colonies in thin layer culture were similar to those obtained in macroscopic observation of the colonies in Stonebrink medium. However, early observation of the colonies enabled early confirmation by PCR, given the shorter time to the visualization of colonies when thin layer culture was used (between the 12(nd) and 25(th) day of culture).

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Detection of Mycobacterium bovis in bovine and bubaline tissues using nested-PCR for TbD1.

In the present study, a nested-PCR system, targeting the TbD1 region, involving the performance of conventional PCR followed by real-time PCR, was developed to detect Mycobacterium bovis in bovine/bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. In terms of analytical sensitivity, the DNA of M. bovis AN5 was detected up to 1.56 ng with conventional PCR, 97.6 pg with real-time PCR, and 1.53 pg with nested-PCR in the reaction mixture. The nested-PCR exhibited 100% analytical specificity for M. bovis when tested with the DNA of reference strains of environmental mycobacteria and closely-related Actinomycetales. A clinical sensitivity value of 76.0% was detected with tissue samples from animals that exhibited positive results in the comparative intradermal tuberculin test (CITT), as well as from those with lesions compatible with tuberculosis (LCT) that rendered positive cultures. A clinical specificity value of 100% was detected with tissue samples from animals with CITT- results, with no visible lesions (NVL) and negative cultures. No significant differences were found between the nested-PCR and culture in terms of detecting CITT+ animals with LCT or with NVL. No significant differences were recorded in the detection of CITT- animals with NVL. However, nested-PCR detected a significantly higher number of positive animals than the culture in the group of animals exhibiting LCT with no previous records of CITT. The use of the nested-PCR assay to detect M. bovis in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Padronização do teste imunoalérgico aplicado ao diagnóstico da tuberculose e micobacterioses em suínos (Sus scrofa) experimentalmente sensibilizados com suspensões oleosas de Mycobacterium bovis ou M. avium inativados / Standardization of the immunoallergic skin test applied for the diagnosis of tuberculosis and mycobacterioses in swine (Sus scrofa) experimentally sensitized with oil suspensions of inactivated Mycobacterium bovis or M. avium

Foi investigado o valor diagnóstico da resposta alérgica cutânea em leitões experimentalmente sensibilizados, pela via intramuscular, com suspensões oleosas de Mycobacterium bovis ou M. avium inativados pelo calor.Foram utilizados 91 animais, divididos em quatro grupos: grupos A e B, cada um com 25 indivíduos, grupos C e D com 21 e 20 indivíduos respectivamente, balanceando-se as características de raça, linhagem, faixa etária e sexo. Aos 30 dias de idade, todos os animais foram submetidos a uma triagem com a aplicação de tuberculina PPD bovina, pela via intradérmica na base da orelha e não houve qualquer tipo de reação. Decorridos 60 dias do teste tuberculínico de triagem, o grupo A recebeu injeção intramuscular de 0,5 mL de uma suspensão oleosa de M. avium estirpe D4; o grupo B recebeu 0,5 mL de uma suspensão oleosa de M. bovis estirpe AN5; o grupo C (controle I), recebeu 0,5 mL do adjuvante oleoso; e o grupo D (controle II), recebeu 0,5 mL de solução fisiológica. Após 30 dias da sensibilização foi realizada a prova de tuberculinização comparativa com reação medida pela variação da espessura da pele com cutímetro de mola às 0h, 24h, 48h e 72h, após a aplicação das tuberculinas. No teste comparativo, lido às 48 ou 72 horas, a reação foi considerada negativa quando a diferença das reações entre o PPD bovino e o PPD aviário foi menor que 6,7 mm; suspeito ou inconclusivo quando a diferença se situou na faixa de 6,7 a 7,5 mm; e positiva de acordo com o tipo de PPD, considerando-se tuberculose para PPD M. bovis e micobacteriose para PPD M. avium, quando a diferença da reação foi superior a 7,5 mm.

The diagnostic value of the cutaneous allergic response to tuberculin in piglets experimentally sensitized intramuscularly with the oily suspensions of heat inactivated M. bovis or M. avium was investigated. Ninety-one animals were used and divided into four groups: groups A and B were formed each with 25 individuals, and groups C and D, with 21 and 20 individuals, respectively, balancing the characteristics of race, ancestry, age and sex. At the age of 30 days, all the animals were submitted to the screening test with the use of M. bovis PPD, by the intradermal route at the base of the ear and no reaction was detected. Sixty days after the screening tuberculin test, animals of the group A were injected intramuscularly with 0.5 mL of oily suspension of M. avium D4 strain; animals of the group B received 0.5 mL of an oily suspension of M. bovis, AN5 strain; group C (control I) received 0.5 mL of an oily adjuvant; and the individuals of the group D (control II) received 0.5 mL of saline solution. Following 30 days of sensitization, comparative skin reactions were measured by the variation in skin thickness with a caliper at 0h, 24h, 48h an 72h after applications of tuberculins. In the comparative test measured at 48 or 72h, the reaction was considered negative when the difference of the reactions between bovine PPD and avian PPD was less than 6.7 mm; suspected or inconclusive, when the difference stood in the range of 6.7 to 7.5 mm; and positive according to the type of PPD, considering tuberculosis the M. bovis PPD and mycobacteriosis the M. avium PPD, when the difference of the reaction was greater than 7.5 mm.

Thin layer microcolony culture associated with PCR for early identification of Mycobacterium bovis

The initial growth of mycobacteria from 49 samples of cattle and buffalo organs collected in commercial slaughterhouses was compared between modified Middlebrook 7H11 thin layer microcolony culture and Stonebrink medium used in the isolation of Mycobacterium bovis. Aliquots were decontaminated by Petroff's method, processed and cultured in both media. The identity of the acid-fast bacilli stained by Ziehl-Neelsen was confirmed by PCR. Optical microscopy showed that results of the early observation of Mycobacterium bovis colonies in thin layer culture were similar to those obtained in macroscopic observation of the colonies in Stonebrink medium. However, early observation of the colonies enabled early confirmation by PCR, given the shorter time to the visualization of colonies when thin layer culture was used (between the 12nd and 25th day of culture).

Evaluation of polymerase chain reaction (PCR) for identifying Mycobacterium bovis isolates from the modified Middlebrook 7H11 agar thin layer technique

The associated use of the modified Middlebrook 7H11 agar thin layer technique and the Polymerase Chain Reaction (PCR) assay enabled to perform the early identification of microcolonies of Mycobacterium bovis from 12th to 25th day of culture. In order to reduce the time for performing the Mycobacterium bovis identification, the combined use of these two techniques was evaluated by analyzing the microcolonies of mycobacteria at the 8th day after culturing. Until the last day of analysis, all of the PCR-positive samples already showed the microcolonies. Therefore, the early diagnosis of bovine tuberculosis is feasible, without an apparent macroscopic colonies growth.

Comparative study on PCR and microbiological culture performance for identifying Mycobacterium tuberculosis complex and Mycobacterium bovis specie in bovine samples

The present study aimed at evaluating the concordance between PCR and microbiological culture techniques for analysing organs samples from cattle with suspected lesions of tuberculosis. Fifty-twosamples collected from slaughter houses were analyzed by microbiological culture, and the extracted DNA was amplified by PCR using NZ1 and NZ2 primers. These primers identify the mycobacteria belongingto M. tuberculosis complex, and the primers pair pncA differentiate the M . bovis from M. tuberculosis species. The colonies isolated from 30 samples were suspended, and the extracted DNA was amplifiedby PCR using the same primer pairs. Although the agreement has been considered weak (k = 0.175) between microbiological culture and PCR performed directly in clinical samples using NZ1 and NZ2 primers, the two pairs of primers could amplify the target genes when 100% of the extracted DNA from 30 isolated colonies were used. Thus, PCR employing pncA primer pair enabled to identify M.bovis in the isolated colonies at a short time when compared with the biochemical assays. The concomitant use of PCR and bacteriologic culture techniques hastens the confirmation of detected agent, which is essential inconducting the epidemiological studies and in taking preventive control measures.

Comparação entre a técnica de cultivo em camada delgada de ágar Middlebrook 7H11 e meio de Stonebrink para isolamento de Mycobacterium Bovis em amostras de campo / Microcolony detection of Mycobacterium Bovis in Middlebrook 7H11 thin layer culture

O presente trabalho teve por objetivo avaliar a eficiência da técnica de cultivo em camada delgada de ágar Middlebrook 7H11 (TL7H11) no isolamento de Mycobacterium bovis de lesões sugestivas de tuberculose em bovinos, comparando seus resultados com os métodos tradicionais de cultivo. Numa primeira fase foram utilizadas estirpes padrão de M. bovis AN5 e M. tuberculosis H37Rv mantidas em laboratório, para comparação de desempenho entre o cultivo em TL7H11 e nos meios de Stonebrik e Petragnani. Ambas estirpes apresentaram crescimento visível em TL7H11 no terceiro dia de cultivo enquanto que nos meios de Stonebrink e Petragnani só houve crescimento a partir do 14° dia. Aos 13 dias de cultivo em TL7H11 foi possível diferenciar as duas estirpes pelas características morfológicas das colônias. Numa segunda fase, 62 amostras de campo foram cultivadas em TL7H11 e Stonebrink para isolamento de M. bovis. As amostras isoladas foram detectadas pelo TL7H11 até os 21 dias de cultivo contra nenhum crescimento dos tubos de Stonebrink. O tempo médio de crescimento no TL7H11 foi de 19,0 dias contra 49,0 dias do meio de Stonebrink (p = 0,014).

The aim of this article is to evaluate the efficiency of the cultivation technique in thin layer of Middlebrook 7H11 (TL7H11) for isolating Mycobactererium bovis from suggestive lesions of tuberculosis in cattle and to compare the results with traditional methods of cultivation. At the first step it was used M. bovis AN5 and M. tubercuolosis H37Rv standard strain. The both performance were compared between the cultivation in TL7H11 and in the Stonebrink and Petragnani media. The strains presented visible growing in TL7H11 at the third day of cultivation, while the Stonebrink and Petragnani there were growing just at the14° day. At the 13 day of cultivation it was possible to differentiate both strains by their colony morphological characteristics. The second step was to cultivate 62 clinicals samples in TL7H11 and Stonebrink for tentative isolation of M. bovis. The isolated samples were detected in TL7H11 until 21 days of cultivation where as none samples were grown in Stonebrink tubes. The median time of growing in TL7H11 was 19,0 days against 49,0 days of Stonebrink (p=0,014).

Isolation and identification of mycobacteria from livestock specimens and milk obtained in Brazil.

The prevalence of Mycobacterium bovis and other mycobacterial species in livestock specimens and milk was evaluated. An emphasis was placed upon the distribution of these organisms in milk that is readily available to the public that was either untreated, pasteurized, or treated using ultra high temperature. Twenty-two pathologic specimens from livestock (bovine, swine and bubaline) in five Brazilian states and 128 bovine milk samples from retail markets in the State of S o Paulo were examined for mycobacteria. Identification was made by classical biochemical tests, thin layer chromatography of mycolic acids and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Mycobacteria were isolated from 15 (68.2%) caseous lesions and from 23 (18%) milk samples. Eleven isolates were identified as M. bovis, and the remaining 27 nontuberculous mycobacterial isolates were represented by five species and six unidentified rapidly growing mycobacterial strains. The data demonstrate that animal products in Brazil are frequent reservoirs of mycobacteria and may pose a risk to the public.

Isolation and identification of mycobacteria from livestock specimens and milk obtained in Brazil

The prevalence of Mycobacterium bovis and other mycobacterial species in livestock specimens and milk was evaluated. An emphasis was placed upon the distribution of these organisms in milk that is readily available to the public that was either untreated, pasteurized, or treated using ultra high temperature. Twenty-two pathologic specimens from livestock (bovine, swine and bubaline) in five Brazilian states and 128 bovine milk samples from retail markets in the State of São Paulo were examined for mycobacteria. Identification was made by classical biochemical tests, thin layer chromatography of mycolic acids and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Mycobacteria were isolated from 15 (68.2 percent) caseous lesions and from 23 (18 percent) milk samples. Eleven isolates were identified as M. bovis, and the remaining 27 nontuberculous mycobacterial isolates were represented by five species and six unidentified rapidly growing mycobacterial strains. The data demonstrate that animal products in Brazil are frequent reservoirs of mycobacteria and may pose a risk to the public

Virulent Mycobacterium fortuitum restricts NO production by a gamma interferon-activated J774 cell line and phagosome-lysosome fusion.

The virulence of different isolates of Mycobacterium has been associated with two morphologically distinguishable colonial variants: opaque (SmOp) and transparent (SmTr). In this report we used an in vitro assay to compare macrophage (Mphi) responses to SmOp and SmTr Mycobacterium fortuitum variants, taking advantage of the fact that these variants were derived from the same isolate. Cells preactivated or not with gamma interferon (IFN-gamma) were infected with SmOp or SmTr M. fortuitum. We showed that SmOp and SmTr induced different levels of nitric oxide (NO) production by IFN-gamma-stimulated Mphi. Indeed, the amount of IFN-gamma-induced NO production by J774 cells was 4.8 to 9.0 times higher by SmOp (23.1 to 37.7 micro M) compared to SmTr infection (3.9 to 4.8 micro M) (P = 0.0332), indicating that virulent SmTr bacilli restricted NO production. In addition, IFN-gamma-induced NO production by Mphi was higher when correlated with reduction of only avirulent SmOp bacillus viability. SNAP (S-nitroso-N-acetyl-DL-penicillamine)-induced NO production did not modify SmTr viability, indicating its resistance to nitrogen radicals. Electron microscopy studies were performed to evaluate the capacity of phagosomes to fuse with lysosomes labeled with bovine serum albumin-colloidal gold particles. By 24 h postinfection, 69% more phagosome-containing SmOp variant had fused with lysosomes compared to the SmTr-induced phagosomes. In conclusion, these data indicate that virulent SmTr bacilli may escape host defense by restricting IFN-gamma-induced NO production, resisting nitrogen toxic radicals, and limiting phagosome fusion with lysosomes.

Discrimination of members of the Mycobacterium avium complex by polymerase chain reaction

Mycobacterium avium complex (MAC) species cannot be discriminated by the usual methods of biochemical identification of mycobacteria. This study showed that amplification by PCR of DT1 and DT6, two single copy sequences identified in the genome of M. avium serotype 2, the insertion sequence IS1245, found to be consistently present in M. avium strains and the heat-shock protein gene hsp65, followed by restriction polymorphism analysis, are rapid and accurate tests for the differentiation of the species M. avium, M. intracellulare, and M. scrofulaceum.

M. bovis como causa de zoonose / Zoonosis caused by M. bovis

A tuberculose bovina, como a humana, volta a assumir grande importância em todo o mundo, especialmente nos países em desenvolvimento, alcançando índices acima de 1 por cento, acometendo aproximadamente 3,5 milhöes de animais só entre Brasil e Argentina. Crescentes säo as perdas econômicas com baixa produtividade do rebanho e condenaçäo de carcaças em matadouro. Näo se sabe em nosso meio a real importância do M. bovis como doença de caráter profissional ou sua transmissäo à populaçäo por meio do consumo de leite e seus derivados. Conceitos de epidemiologia, patogenia e diagnóstico säo revistos com a finalidade de atualizar os conhecimentos sobre o aspecto da tuberculose bovina como zoonose.

Evoluçäo das lesöes induzidas por micobactérias BCG e por H37RV em hamster (Mesocricetus auratus) / Evoution of the injuries induced by BCG mycobacterium and by H37RV in hamster (Mesocricetus auratus)

Foram investigados experimentalmente alguns aspectos da patogenia da tuberculose através da inoculaçao de uma cepa de micobactéria de virulência atenuada - BCG e outra cepa altamente virulenta - M. tuberculosis H37 Rv no tecido sub-epitelial do coxim plantar e no tecido sub-epitelial do terço distal da bolsa cervical de hamster, um sítio desprovido de drenagem linfática. A inoculaçao desses agentes determinou diferentes perfis de evoluçao das lesoes, sendo que as lesoes produzidas pelo BCG na pata apresentaram uma tendência de declínio do tamanho após 21 dias e as lesoes produzidas pelo H37 Rv mostraram uma evoluçao progressiva. O mesmo perfil foi observado nas lesoes induzidas na bolsa cervical por ambos os agentes. Contudo, diferentemente do BCG, o H37 Rv disseminou-se para os órgaos internos, causando extensas lesoes. A inoculaçao de BCG ou H37 Rv na pata provocou um aumento de 21 e 68 vezes, respectivamente, o peso do linfonodo satélite em relaçao ao controle, evidenciado a disseminaçao do bacilo por via linfática. Todavia, quando essas amostras foram inoculadas na bolsa, nao determinaram significativas mudanças de peso do linfonodo cervical, mas evidenciou-se a disseminaçao do bacilo virulento por via sangüínea, pelas lesoes produzidas no baço e pulmao.

Patogenia comparada das cepas de micobactérias BCG e H37Rv em hamster (Mesocrisetus auratus): influência das vias sanguínea e linfática / Comparative pathogenesis of mycobacterial strains BCG and H37Rv in hamster (Mesocrisetus auratus): influence of blood and lymphatic pathways

Some aspects of the pathogeny of tuberculosis was experimentally investigated by inoculating either an attenuated mycobacteria strain BCG, or a virulent strainM. tuberculosis H37Rv into the foot pad or the cheek pouch of hamster. The inoculation of these agents determined different patterns of lesion. The BCG-induced lesionsin the foot pad had a tendency to descrease in size. On the other hand, the lesions induced by H37Rv showed a progressive evolution. The same pattern of evolution was observed with the lesions induced in the cheek pouch. However, the H37Rv disseminated to internal organs and the BCG did not. When BCG or H37Rv were inoculated in the foot pad of animals, the popliteal lymph node showed respectively a 21 fold and 68 fold increase in weight