Differential microbial clearance and immunoresponse of Balb/c (Nramp1 susceptible) and DBA2 (Nramp1 resistant) mice intracerebrally infected with Mycobacterium bovis BCG (BCG)

In mice, the gene encoding Nramp1 (natural resistance-associated protein 1) exists in two allelic forms, differing for a point mutation. According to Nramp1 genotype, extensive literature documents a clear-cut distinction of inbred strains in two non-overlapping groups that phenotypically express resistance (Nramp1r) and susceptibility (Nramp1s) to systemic infections. Here, we provide evidence that Nramp1r (DBA/2) and Nramp1s (Balb/c) mice differently handle intracerebral infection with Mycobacterium bovis BCG. Distinct trends of microbial clearance from the brain and also different patterns of local immune responses occur, thus arguing on the involvement of Nramp1 gene product on the accomplishment of cerebral anti-mycobacterial defenses.     

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.     

Study of the immunological profile towards Mycobacterium bovis antigens in naturally infected cattle

A number of studies have determined the contribution of Th1 and Th2 responses to the protective immunity and pathology of Mycobacterium bovis infection. However, much of that information is derived from experimentally infecting cattle with M. bovis and few data from naturally infected animals are available. The aim of this study was to characterize the immunological profile towards M. bovis antigens of naturally infected cattle by measurement of cytokine mRNA expression in PBMC, and to determine which lymphocyte subsets are involved in recall responses of PBMC from M. bovis infected cattle to M. bovis antigens. Consistent with data from cattle experimentally infected with M. bovis , naturally infected animals were found to display a Th1 cytokine profile in response to M. bovis PPDB stimulation. Production of IFN-γ mRNA by PBMC after PPDB stimulation statistically distinguishes between infected and healthy herds, suggesting that this molecule is usable as an M. bovis -infection marker. As happens in experimentally infected cows, CD4, CD8 and γδTCR cells from a herd naturally infected with M . bovis are the predominant T cell subsets expanded in response to PPDB.     

Characterisation of a novel repetitive DNA sequence from Mycobacterium bovis

We report characterisation of three copies of a novel repeat sequence isolated from a Mycobacterium bovis genomic library. The repeat occurs within open reading frames, potentially encoding a conserved tandem array of a pentapeptide sequence with the consensus X-Gly-Asn-X-Gly. The tandem array is present up to five times in M. bovis and it is proposed that they may occur in a family of genes expressing functionally related proteins. We postulate that these proteins may play a role in binding of M. bovis to host cell receptors.     

Tubercle bacilli generate a novel cell wall-associated pigment after long-term anaerobic culture

Many cases of tuberculosis result from reactivation of previously acquired latent infections. Models to study such persister forms often involve gradual depletion of oxygen during culture as poor aeration is a characteristic of non-progressive TB granulomas. Anaerobically cultured bacilli develop a thickened outer-most cell wall layer. Here, we analyzed this layer from anaerobically cultured Mycobacterium tuberculosis and Mycobacterium bovis BCG. By six weeks of anaerobiosis a pigment was detected at levels > 60-fold higher in anaerobic than aerobic bacilli. This pigment was responsible for the electron-dense appearance of the thickened cell wall layer and gave an electrospray mass spectrometry peak at 409 Da (M+Na)+ or (M+H)+. We termed this pigment APP1, anaerobically produced pigment 1, the first pigment identified in M. tuberculosis.     

牛分枝杆菌mpb64基因的克隆、鉴定及其表达

以牛型分枝杆菌基因组DNA为模板,PCR方法扩增mpb64基因,纯化PCR产物并与pDM18-T载体连接、转化,经酶切及核苷酸序列鉴定为正确后,酶切产物亚克隆到原核表达载体pET30a(+)的KpnI／EcoRI位点,构建重组表达质粒pET30a+-mpb64,转化到大肠杆菌DE3内,以IPTG进行诱导,终浓度为1mmol／L,诱导产物进行SDS-PAGE电泳。结果表明,PCR方法成功扩增出mpb64基因,核苷酸序列测定验证了其正确性,重组表达质粒表达的pET30a+-mpb64融合蛋白相对分子量为30.4kDa,与实测相符。牛分枝杆菌pET30a+-mpb64的成功表达为牛结核病的诊断及新型疫苗的研究奠定了基础。     

cAMP levels within Mycobacterium tuberculosis and Mycobacterium bovis BCG increase upon infection of macrophages

Adenosine 3',5'-cyclic monophosphate (cAMP)-mediated signal transduction is common in both prokaryotes and eukaryotes, and several bacterial pathogens modulate cAMP signaling pathways of their mammalian hosts during infection. In this study, cAMP levels associated with Mycobacterium tuberculosis and Mycobacterium bovis BCG were measured during macrophage infection. cAMP levels within both bacteria increased c . 50-fold during infection of J774.16 macrophages, relative to the cAMP levels within bacteria incubated in tissue culture media alone. cAMP levels also increased within the macrophage cytoplasm upon uptake of live, but not dead, mycobacteria. The presence of albumin in the absence of oleic acid significantly decreased cAMP secretion and production by both M. tuberculosis and M. bovis BCG. These results suggest that cAMP signaling plays a role in the interaction of tuberculosis-complex mycobacteria with macrophages during infection, and that albumin may be a physiological indicator differentiating host environments during infection.     

Mycobacterium tuberculosis mammalian cell entry operon (mce) homologs in Mycobacterium other than tuberculosis (MOTT)

The cloned mammalian cell entry gene mce1a from Mycobacterium tuberculosis confers to non-pathogenic Escherichia coli the ability to invade and survive inside macrophages and HeLa cells. The aim of this work was to search for and characterize homologs of the four M. tuberculosis mammalian cell entry operons (mce1, mce2, mce3 and mce4) in mycobacteria other than tuberculosis (MOTT). The dot-blot and polymerase chain reaction (PCR) experiments performed on 24 clinical isolates representing 20 different mycobacterial species indicated that the mce operons were widely distributed throughout the genus Mycobacterium. BLAST search results showed the presence of mce1, mce2 and mce4 homologs in Mycobacterium bovis, Mycobacterium avium and Mycobacterium smegmatis. A homologous region for the mce3 operon was also found in M. avium and M. smegmatis. DNA and protein alignments were done to compare the M. tuberculosis mce operons and the deduced M. bovis, M. avium, and M. smegmatis homologs. The deduced proteins of M. bovis mce1, mce2 and mce4 operons had 99.6-100% homology with the respective M. tuberculosis mce proteins (MTmce). The similarity between M. avium mce proteins and the individual M. tuberculosis homologs ranged from 56.2 to 85.5%. The alignment results between M. smegmatis mce proteins and the respective MTmce proteins ranged from 58.5% to 68.5%. Primer sets were designed from the M. tuberculosis mce4a gene for amplification of 379-bp fragments. Amplification was successful in 14 strains representing 11 different mycobacterial species. The PCR fragments were sequenced from 10 strains representing eight species. Alignment of the sequenced PCR products showed that mce4a homologs are highly conserved in the genus Mycobacterium. In conclusions, the four mce operons in different mycobacterial species are generally organized in the same manner. The phylogenetic tree comparing the different mce operons showed that the mce1 operon was closely related to the mce2 operon and mce3 diverged from the other operons. The wide distribution of the mce operons in pathogenic and non-pathogenic mycobacteria implicates that the presence of these putative virulence genes is not an indicator for the pathogenicity of the bacilli. Instead, the pathogenicity of these factors might be determined by their expression.     

Characterization of PPD protein antigens in whole cell lysates of Mycobacterium bovis BCG

The low molecular mass protein antigens in PPD from M. bovis BCG were chemically oligomerized using sulfosuccinimidyl-4-(p-maleimidophenyl)-butyrate (S-SMPB) as a crosslinking agent. Protein oligomers with molecular mass over 90 kDa were obtained and used for the preparation of hyperimmune polyclonal rabbit antiserum. Using this antiserum four protein bands with molecular mass 120, 90, 75 and 65 kDA were detected in immunoblotting analysis of sonic extract from M. bovis BCG separated in SDS-polyacrylamide gel. We suggest that these immunoreactive proteins in the sonic extract represent the native forms of the heat stable low molecular mass protein antigens in PPD.     

牛分枝杆菌单基因及双价融合基因DNA疫苗免疫效果的研究

利用PCR技术和SOE技术扩增牛分枝杆菌ag85b、esat-6、hsp65、mpb64基因和ag85b-esat-6、hsp65-esat-6和mpb64-esat-6融合基因,连接真核表达载体pCDNA3.1(+),构建重组质粒pCA、pCE6、pCH、pCM、pCAE、pCHE和pCME。转染SP2/0细胞,检测目的基因的表达。以各重组质粒和pCDNA3.1(+)及PBS免疫BALB/c小鼠后检测血清特异性抗体水平、脾淋巴细胞增殖情况和IFN~γ分泌情况。结果表明,七种重组质粒免疫后小鼠血清抗体水平持续上升,与 pCDNA3.1(+)对照组和PBS对照组相比差异显著 （P<0.05）,其中pCA组血清抗体水平明显高于其他六种DNA疫苗免疫组 （P<0.05）;三免两周后,融合基因免疫组的刺激值（SI值）与单基因免疫组相比差异显著（P<0.05）,其中以pCME组的SI值最高;PPD刺激后融合基因DNA疫苗免疫组小鼠脾细胞分泌的IFN~γ高于单基因DNA疫苗组（P<0.05）,而两对照组则未检测到IFN~γ的产生。本试验成功构建了牛分枝杆菌ag85b、esat-6、hsp65、mpb64单基因和ag85b-esat-6、hsp65-esat-6、mpb64-esat-6双价融合基因DNA疫苗,从而为牛结核病新型疫苗的研制奠定了基础。     

Paleopathological and biomolecular study of tuberculosis in a medieval skeletal collection from England

Nine human skeletons of medieval date from a rural English burial site show signs of skeletal tuberculosis. They were subject to polymerase chain reaction (PCR) assays aimed at detecting traces of DNA from infecting mycobacteria, with the purpose both of confirming the paleopathological diagnosis of tuberculosis and determining in individual cases whether disease was due to M. tuberculosis or M. bovis. In all nine cases, evidence for M. tuberculosis complex DNA was found, and in all instances it appeared that disease was due to M. tuberculosis rather than M. bovis. The significance of the findings for understanding tuberculous infection in rural agrarian communities in medieval England is discussed.     

宠物结核病的危害及其防治

随着生活水平的提高,宠物的数量和种类增长迅速。结核病是目前病死人数最多的人兽共患病,犬、猫和鸟类都能感染发病,对人类,尤其是免疫抑制人群威胁很大。监测和防治宠物结核病对人类结核根除计划具有重要意义。     

Comparison of decontamination methods for primary isolation of Mycobacterium bovis in paucibacillary bovine tissues

Aims: To compare three decontamination methods applied to paucibacillary samples for primary isolation of Mycobacterium bovis from suspect lesions. Tuberculosis caused by Myco. bovis is an important infectious disease of cattle in Brazil and also has zoonotic potential. Although a national campaign based on testing and slaughtering cattle has achieved good results, there is a strong need to develop better diagnostic methods to identify cattle with recent infections harbouring few bacilli. Methods and Results: A dairy herd (274 adult crossbred cows) located in the state of Rio de Janeiro was tested for tuberculosis with both single intradermal tuberculin test and comparative intradermal tuberculin test. Reactive cows (n = 27, 9·8%) were slaughtered and suspect lesions were collected (one sample per cow). Samples considered paucibacillary (based on microscopy) were decontaminated with 0·75% hexadecylpyridinium chloride (HPC), 4% sodium hydroxide (Petroff) or 6% sulphuric acid. Using these methods, 10, five and six, respectively, of the 27 samples yielded positive cultures. Overall, Myco. bovis was isolated from 14 of 24 cows. Although the HPC method resulted in isolation of more Myco. bovis strains than either Petroff or sulphuric acid methods (P = 0·015), it did not result in the recovery of Myco. bovis from all samples. However, using both HPC and 6% sulphuric acid methods for decontamination was possible to identify 13 of 14 (92·9%) of infected cows. Conclusions: At least two methods should be used concurrently for primary isolation of Myco. bovis from bovine tissues, particularly for paucibacillary samples. Significance and Impact of the Study: Detection of low numbers of Myco. bovis in tissue is an important goal in optimizing the detection of bovine tuberculosis and should assist in identification of infected cattle, in particular, those with few Myco. bovis bacilli. This was apparently the first study comparing three decontamination methods for the detection of Myco. bovis in paucibacillary samples from naturally infected cattle.     

环介导等温可视扩增检测牛分枝杆菌方法的建立

目的:建立一种快速简便检测牛分枝杆菌的方法。方法:根据已发表的牛分枝杆菌特殊基因序列,设计并合成6对特异扩增牛分枝杆菌特异性基因片段的引物,通过条件优化,建立针对牛分枝杆菌的环介导等温扩增(LAMP)检测法,测定其特异性和敏感性,并对采集的牛临床样品的DNA分别进行检测。结果:采用该法只检出牛分枝杆菌,检测的最低拷贝数为1×102拷贝/μL。结论:建立的LAMP方法简便、快速、特异性高,可用于临床上牛分枝杆菌的快速检测。