Antigen proteins specific to mycobacteria

More than 10 mycobacterial proteins (MPB or MPT) have been isolated from the nutrient fluids after cultivation of Mycobacterium bovis BCG (Japanese substrain) or M. tuberculosis H37Rv on Sauton medium. The genes for 4 antigens, MPB70, MPB64, alpha-antigen (MPB59), and MPB57, were cloned so far and the complete nucleotide and amino acid sequences were determined. MPB70 is a quite unique protein in 3 points; a highly species-specific antigen for M. bovis, a large amount of secretion more than 10% of the total protein amount in the culture fluid when the cells are actively growing, and a composition with hydrophobic amino acids in a high rate. MPB64 was also a unique antigen specific to M. bovis and M. tuberculosis with a strong reactivity in delayed-type hypersensitivity. MPB59, corresponds to alpha-antigen, was a protein of a group of several similar configurations and was commonly found in the mycobacterial species. These 3 antigens are synthesized in the cells binding with each signal peptide which is characteristic of secreted proteins. A heat stable protein, MPB57, a dominant component of PPD, corresponded to BCG-a or 10kD protein and is supposed to be one of the heat shock proteins.     

MPB70, a unique antigen of Mycobacterium bovis BCG

MPB70 is a constituent of Mycobacterium bovis BCG, which accounts for about 10% of the total protein content in the nutrient fluid after culture on Sauton medium. Previous studies of cell-mediated immune reactions have shown that MPB70 is a highly BCG-specific antigen occurring in markedly different concentrations in different substrains of BCG. The present studies were based on the reactivity of MPB70 with antibodies. The precipitate caused by MPB70 was identified in the crossed immunoelectrophoresis pattern of sonicates and concentrated culture fluids of BCG Tokyo. The antigen was quantified by radioimmunoassay inhibition tests on culture fluids standardized on the basis of identical total protein content. It was present in high concentrations in BCG substrains Tokyo, Moreau, Russia, and Sweden, and in M. bovis strain Ravenel. It was also detected in BCG substrains Glaxo, Copenhagen, Tice, and Pasteur, and in M. tuberculosis H37Rv, but in the latter 4 in only 1% or less of the concentration in BCG Tokyo. Six different species of slow- and rapid-growing mycobacteria showed no evidence of cross-reactivity, but a cross-reacting antigen was demonstrated in Nocardia asteroides. The antibody response after immunization of rabbits with BCG Tokyo and BCG Copenhagen was very similar in a polyvalent assay for antibodies reacting with various components of BCG. By contrast, there was a striking difference in anti-MPB70 antibodies, which were rapidly formed in large amounts after immunization with BCG Tokyo and only in very small amounts after immunization with BCG Copenhagen.     

The invasion of Mycobacterium tuberculosis into non-phagocytic cells

To explore the ability of tubercle bacilli to invade and survive within non-phagocytic cells, we used in this study a human fibroblast cell line, WI-38, derived from normal embryonic lung and a human epithelial cell line, SQ-5, derived from lung squamous cell carcinoma. Live M. tuberculosis Erdman and M. tuberculosis H37Rv invaded WI-38 cells more efficiently than live M. tuberculosis H37Ra, M. bovis Ravenel, M. bovis BCG Tokyo and M. bovis BCG Pasteur. The capability of tubercle bacilli to invade WI-38 cells was Erdman > or = H37Rv > BCG Pasteur [symbol: see text] M. bovis Ravenel [symbol: see text] BCG Tokyo > H37Ra. A similar invasive ability was observed using SQ-5 cells. In contrast with live bacilli, heat-killed bacilli failed to invade WI-38 cells, whereas they were detected within SQ-5 cells. These results and incorporation of latex beads suggest that SQ-5 cells, but not WI-38 cells, possess phagocytic activity. H37Rv multiplied most actively within WI-38 cells when compared to H37Ra and BCG Tokyo, suggesting that the ability to invade and survive within non-phagocytic cells reflects the more active invasion of virulent M. tuberculosis than avirulent M. tuberculosis. The assay system used in this study may help us to clarify the virulence of tubercle bacilli in vitro.     

Study on recombinant BCG

For the purpose to establish the system to express foreign antigen from Mycobacterium bovis BCG. We have cloned, sequenced and expressed genes for secreting proteins, alpha antigen, MPB64, MPB57 and MPB70 from M. bovis BCG. The upstreams and structural genes were characterized. The gene for alpha antigen of Mycobacterium kansasii was also characterized. The gene for alpha antigen of M. kansasii (k-alpha) was chosen for the further study at first. This gene was fused with shuttle plasmid PIJ666-PAL5000 obtained from T. Kisser and transfected to M. bovis BCG (Tokyo). Transformant was obtained by a selection with kanamycin. It was able to secrete k-alpha antigen. DNA-containing a B-cell epitope (Glu-12-Leu-Asp-Arg-Trp-Glu-Lys-Ile-19) of human immunodeficiency virus type 1 P17 gag was fused to this vector at C terminal of k-alpha. Using this vector, we have succeeded to express foreign antigen in M. bovis BCG. The products were analyzed in one or two dimensional electro-phoresis. The results thus obtained will be reported elsewhere.     

Increased protection against bovine tuberculosis in the brushtail possum (Trichosurus vulpecula) when BCG is administered with killed Mycobacterium vaccae

SETTING: The Australian brushtail possum is the major wildlife reservoir for Mycobacterium bovis infection in New Zealand. Development of an effective tuberculosis vaccine for possums will reduce the spread of infection to cattle and farmed deer. OBJECTIVES: To determine whether killed M. vaccae can improve the efficacy of vaccination with M. bovis bacillus Calmette Guerin (BCG) against bovine tuberculosis in the possum. DESIGN: Groups of possums (n=6-8) were vaccinated via intranasal and intraconjunctival routes with BCG alone or BCG in combination with heat-killed M. vaccae. Controls were non-vaccinated or vaccinated with heat-killed M. vaccae alone. After challenge with virulent M. bovis, protection was assessed by a reduction in loss of body weight and bacterial counts in lungs and spleens. Blood lymphocyte proliferative responses to M. bovis purified protein derivative were monitored throughout. RESULTS: The earliest lymphocyte responses following vaccination were from animals inoculated with BCG plus 100 microg heat-killed M. vaccae. Loss of body weight was significantly reduced in all BCG-vaccinated groups compared control groups. Spleen bacterial counts were significantly lower in animals vaccinated with M. vaccae plus BCG compared to the non-vaccinated group. Furthermore, vaccination with 100 microg M. vaccae plus BCG significantly reduced spleen bacterial counts compared to vaccination with BCG alone. CONCLUSION: The possum infection model is one of the first to show that novel vaccine strategies may offer better protection against tuberculosis than BCG alone.     

Vaccination with DNA vaccines encoding MPB70 or MPB83 or a MPB70 DNA prime-protein boost does not protect cattle against bovine tuberculosis

SETTING: Bovine tuberculosis is a problem in a number of countries and protection of cattle by vaccination could be an important control strategy. OBJECTIVES: To determine the ability of DNA vaccines, which express the mycobacterial antigens MPB83 and MPB70 and a DNA prime-protein boost strategy to stimulate immune responses in cattle and protect against bovine tuberculosis. DESIGN: Groups of cattle (n=10) were vaccinated with MPB83 DNA, MPB70 DNA, or MPB70 DNA followed by MPB70 protein or injected with BCG or control plasmid DNA. Animals were challenged intratracheally with virulent Mycobacterium bovis at 13 weeks and protection assessed 17 weeks later at postmortem. RESULTS: In contrast to the strong cellular immune responses induced by BCG, the DNA vaccines induced minimal interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) responses. Cattle primed with MPB70 DNA and boosted with MPB70 protein induced a strong antibody response and a weak IFN-gamma response. BCG gave significant reduction in four pathological parameters of disease while the DNA vaccines and MPB70 DNA/protein did not protect animals against challenge with M. bovis. Moreover, cattle vaccinated with MPB70 DNA/protein had a significantly higher proportion of animals with severe lung lesions (>100 lesions) than the MPB70 DNA alone or the control group. Increased bovine PPD-specific IL-4 mRNA expression in cattle, post-challenge, correlated with the presence of tuberculous lung lesions. CONCLUSION: Vaccination of calves with MPB70 or MPB83 DNA vaccines or with a more immunogenic MPB70 DNA prime-protein boost strategy did not induce protection against bovine tuberculosis.     

Protective efficacy of BCG Tokyo 172 in the guinea pig model of pulmonary tuberculosis

BCG Tokyo 172 strain was examined for its protective efficacy against pulmonary tuberculosis in a guinea pig model. Guinea pigs were vaccinated with an intradermal injection of 10(3) CFU of BCG Tokyo 172 strain. BCG Copenhagen 1331 was employed as a control strain. Eight weeks after the vaccination, the animals were infected with about 10 CFU of M. tuberculosis H37Rv by a respiratory route in an aerosol chamber. Five weeks after infection, the animals were euthanized and their spleens, lungs and livers were obtained for enumeration of M. tuberculosis H37Rv and histopathological examinations. The mean log10 CFU of M. tuberculosis H37Rv recovered from right lower lung lobes of guinea pigs vaccinated with BCG Tokyo 172 (frozen), BCG Tokyo 172 (freeze-dried), BCG Copenhagen 1331 (freeze-dried) and placebo were 4.72, 4.23, 4.35 and 5.76, respectively. The mean log10 CFU of the bacteria recovered from spleens were 2.11, 1.51, 1.37 and 5.90, respectively. There was a significant difference in bacterial recovery from both lung and spleen between the vaccinated and the non-vaccinated groups. No significant difference was seen among the groups vaccinated with different strains of BCG in any organ. The lungs exhibited just small granulomatous nodules and the spleens showed no granulomatous nodules in the BCG-vaccinated guinea pigs. On the other hand, the lungs and spleens from non-vaccinated guinea pigs showed much larger granulomatous nodules with central necrosis. These histopathological difference between the vaccinated and the non-vaccinated guinea pigs was consistent with the difference of bacterial growth between them. The results of this study have clearly indicated that BCG Tokyo 172 strain possesses a significant protective efficacy against M. tuberculosis as well as BCG Copenhagen 1331 strain. These results have also shown that the respiratory infection model in guinea pigs is very useful to evaluate efficacy of vaccines against pulmonary tuberculosis.     

Comparison of IFN-gamma responses to mycobacterial antigens as markers of response to BCG vaccination

An increase in interferon-gamma (IFN-gamma) production to Mycobacterium tuberculosis purified protein derivative (Mtb PPD), as measured in the cultured diluted whole blood assay, is one indicator of a protective immune response to BCG vaccine. We have explored the potential for this assay to be improved by measuring IFN-gamma responses to more defined antigens of M. tuberculosis (short-term and mid-term culture filtrates, ESAT-6, 38 kDa), Mycobacterium bovis (MPB70), M. bovis BCG (Antigen 85) and Mycobacterium leprae (35 kDa), in UK teenagers before and 1 year after BCG vaccination (or no vaccination as controls). There was a significant increase in response to the culture filtrates post-vaccination, but this was no greater than that to Mtb PPD. Many teenagers responded to the purified antigens, in particular to Antigen 85, prior to vaccination, and BCG vaccination could only augment this pre-existing response to a limited extent; prior exposure to environmental mycobacteria can thus induce cross-reactive responses to antigens which complicate interpretation of in vitro assays of vaccine response. In contrast, ESAT-6 was recognised by only one teenager prior to vaccination, and, as expected, responses were not boosted by BCG. We therefore conclude that Mtb PPD is the antigen preparation of choice for assessing the immunogenicity of BCG vaccination.     

Cloning and expression of recombinant MPB70 protein antigen from Mycobacterium bovis BCG for diagnosis of tuberculosis

In a search for developing new skin test reagents, MPB70 protein antigen was a candidate antigen for the Diagnosis of bovine tuberculosis. First M. bovis BCG genomic DNA was extracted purified and the mpb70 gene was amplified by PCR. The gene was then ligated to an expression vector, PQE. After transformation of the expression E. coil M15 host strain with the PQE plasmid, the expression was induced using 10 mM of IPTG. Two bands were seen in the SDS-PAGE analysis the 44 and 50 KDa represents the dimmers of the nonglycosylated and glycosylated form of the reMPB70 antigen. The His-tagged reMPB70 antigen was then purified by metal affinity chromatography using Ni-NTA agarose. Protein refolding was done by the use of the co solvent Polyethylene glycol MW 3000. The diagnostic potential of the re-MPB70 was evaluated using sera from experimentally sensitized guinea pigs with different strains of mycobacteria (M. bovis BCG, M. tuberculosis, M. kansasii and M. intracellular) using ELISA test. The results indicated the efficiency of MPB70 but not bovine PPD to discriminate between M. bovis sensitized guinea pigs and those sensitized with other mycobacterial strains at serum dilution of 1150. In a field trials to using reMPB70 antigen for the serodiagnosis of bovine tuberculosis using ELISA test. Fifty serum samples from tuberculin +ve and 6 from tuberculin -ve cattle were used as well as 10 tuberculin +ve buffaloes. All +ve animals were confirmed to be M. bovis infected by P/M analysis, bacteriological examination. ELISA results revealed that reMPB70 could recognize the tuberculin +ve infected animals at serum dilution of 1/50 and that it could diagnose tuberculosis in cattle as well as buffaloes.     

MPB64分泌蛋白检测鉴定结核分枝杆菌复合群的应用研究

目的 对应用MPB64分泌蛋白检测鉴定结核分枝杆菌复合群进行方法学评价.方法 对临床分离菌株,取BacT/ALERT 3D系统的液体培养液或改良罗氏固体培养基分离培养菌落室温静置约2 h、浊度为1麦氏单位的生理盐水菌悬液0.1 ml为检测样本,应用胶体金标记抗MPB64单克隆抗体采用免疫层析色谱法进行MPB64分泌蛋白检测,而后与菌种鉴定结果相比较.对参考菌株,则同时采用两种样本进行检测.结果 对23株分枝杆菌参考菌株、267株临床分离菌株进行了检测.①对于参考菌株的两种样本,MPB64检测结果均为:结核分枝杆菌、牛分枝杆菌、田鼠分枝杆菌菌株阳性,其他20株非结核分枝杆菌为阴性.分群鉴定准确度为100%.②在111株BacT/ALERT 3D系统分离培养临床株中,55株为结核分枝杆菌,其中51株MPB64检测阳性;56株为非结核分枝杆菌,其中54株MPB64检测阴性.分群鉴定准确度为94.59%.③在156株改良罗氏培养基分离培养临床菌株中,121株为结核分枝杆菌,其中115株MPB64检测阳性;35株为非结核分枝杆菌,MPB64检测全部阴性.分群鉴定准确度为96.15%.④对于所有研究样本,应用MPB64分泌蛋白检测鉴定结核分枝杆菌复合群的敏感度、特异度与准确度分别为94.41%、98.20%和95.86%.结论 应用MPB64分泌蛋白检测鉴定结核分枝杆菌复合群是一种准确、快速、简便而又不需要任何仪器设备的实用型方法,值得临床推广应用.     

结核分枝杆菌MPT64蛋白适配子的筛选与鉴定

目的 利用指数富集配体系统进化(SELEX)技术筛选能与结核分枝杆菌分泌蛋白MPT64特异性结合的寡核苷酸适配子,寻找早期诊断结核病的方法.方法 体外合成随机单链DNA(ssDNA)文库,以MPT64蛋白为靶物质,采取SELEX技术进行12轮筛选,将适配子库克隆、测序后,用DNAMAN软件对其结构进行分析,经生物素-链亲和素-辣根过氧化物酶显色系统测定亲和力,并利用捕获适配子与检测适配子组成的"三明治"夹心法对获得的适配子进行初步验证.将13个非结核分枝杆菌菌株和BCG菌株作为阴性组计为0,将结核分枝杆菌和牛结核分枝杆菌组作为阳性组计为1,采用MedCalc软件分析受试者操作特征曲线,确定最佳阳性判定值,并构建散点图.结果 经12轮筛选后,随机挑选15个适配子与MPT64蛋白的亲和性进行分析,吸光度值为0.492～1.243,73.3％的适配子的吸光度值在1.0以上;二级结构分析显示,适配子与MPT64蛋白亲和性的基础主要是大口袋茎环结构,口袋与环之间的茎桥含有不同数量的GC碱基对;"三明治"夹心法对阴性组[非结核分枝杆菌标准菌株及卡介苗(BCG)株]和阳性组(结核分枝杆菌实验室H37Rv株及临床株、牛结核分枝杆菌标准株)共47个菌株培养上清的检测结果显示,在临界(cut-off)值为0.61时,H37Rv、牛结核分枝杆菌组为阳性,BCG株为阴性;阴性组标本的阴性检出率为85.7％,阳性组标本的阳性检出率为87.9％,表现出一定的检测价值.结论 已初步筛选到与MPT64蛋白具有高亲和性的DNA适配子.     

The effect of mycobacterial virulence and viability on MAP kinase signalling and TNF alpha production by human monocytes

SETTING: The success of Mycobacterium tuberculosis as a human pathogen depends on its ability to tolerate and perhaps manipulate host defense mechanisms. OBJECTIVE: To determine the induction of tumour necrosis factor-alpha (TNF alpha), a central mediator of immunity, by human monocytes infected with virulent M. tuberculosis, M. leprae and attenuated M. bovis BCG. DESIGN: Mycobacteria-induced cellular activation pathways of TNF alpha production was investigated using an inhibitor of protein tyrosine kinase (PTKs) and an inhibitor of mitogen-activated protein (MAP) kinases. RESULTS: TNF alpha production was significantly lower during infection with virulent M. tuberculosis than with BCG and this differential response was independent of mycobacterial viability. TNF alpha production involved the PTK and MAP kinase pathways. Reduced TNF alpha induction by M. tuberculosis was associated with a reduction in the extent and duration of phosphorylation of extracellular-signal regulated kinases (ERK 1/2). Infection with M. leprae triggered low and transient ERK 1/2 activation as well as low TNF alpha production. CONCLUSION: Maintenance of the differential response in both live and heat-killed preparations suggests that the reduced TNF alpha response associated with virulent mycobacteria is due to differences in the presence of components capable of triggering host pattern recognition receptors, rather than events associated with phagosome trafficking or the active release of intracellular modulators.     

Tumor necrosis factor α is a determinant of pathogenesis and disease progression in mycobacterial infection in the central nervous system

The pathogenesis of tuberculous meningitis, a devastating complication of tuberculosis in man, is poorly understood. We previously reported that rabbits with experimental tuberculous meningitis were protected from death by a combination of antibiotics and thalidomide therapy. Survival was associated with inhibition of tumor necrosis factor alpha (TNF-alpha) production by thalidomide. To test whether cerebrospinal fluid (CSF) levels of TNF-alpha correlated with pathogenesis, the response of rabbits infected in the central nervous system (CNS) with various mycobacterial strains was studied. CNS infection with Mycobacterium bovis Ravenel, M. bovis bacillus Calmette-Guérin (BCG) Pasteur, and M. bovis BCG Montreal were compared. M. bovis Ravenel induced the highest levels of TNF-alpha in the CSF in association with high leukocytosis, protein accumulation, and severe meningeal inflammation. BCG Pasteur had intermediate effects, and BCG Montreal was the least virulent. In addition, M. bovis Ravenel numbers were highest in the brain and CSF and the bacilli also disseminated more efficiently to distant organs, compared with BCG Pasteur and BCG Montreal. In subsequent experiments, rabbits were infected with either recombinant M. bovis BCG Montreal (vector), or BCG Montreal expressing the murine gene for TNF-alpha (BCG mTNF-alpha). BCG Montreal was rendered virulent by the expression of murine TNF-alpha, as demonstrated by high CSF leukocytosis, high protein accumulation, severe meningeal inflammation, persistent bacillary load, and progressive clinical deterioration. Taken together, these results demonstrate that the level of TNF-alpha produced during mycobacterial CNS infection determines, at least in part, the extent of pathogenesis.     

Utility of PCR assays for rapid diagnosis of BCG infection in children.

We report Mycobacterium bovis BCG infection in two children vaccinated with BCG (Tokyo strain) on the first day of life. Their diagnoses were made by biopsy of skin lesions and pus from an anterior chest wall abscess, respectively, yielding a positive culture of mycobacteria fully susceptible to rifampicin, isoniazid and ethambutol, but resistant to pyrazinamide. M. bovis BCG was identified by a negative niacin test, absence of nitrate reductase and resistance to pyrazinamide and cycloserine. The diagnoses were further confirmed by a combination of an allele-specific polymerase chain reaction ated strain of Mycobacterium bovis, is the only available vaccine for the prevention of tuberculosis. Although complications are rare after BCG vaccination and the outcome is usually favourable, serious BCG infections can occur. We report two cases of M. bovis BCG infection in children, a 4-year-old immunocompetent girl and an 8-month-old immunodeficient boy. To our knowledge, this is the first report of BCG complications in children in which two recently developed polymerase chain reaction (PCR) based methods were used for rapid identification of M. bovis BCG infection. (PCR) and a multiplex PCR method. Based on the drug susceptibility results, treatment with rifampicin, isoniazid and ethambutol was instituted. One patient (Case 1) improved clinically and is well after treatment. However, the other patient with severe combined immunodeficiency died of disseminated BCG infection in spite of intensive anti-tuberculosis therapy. Although BCG is considered to be a safe vaccine, it should be kept in mind that complications related to BCG do occur.     

Genomics of Mycobacterium bovis.

The imminent completion of the genome sequence of Mycobacterium bovis will reveal the genetic blueprint for this most successful pathogen. Comparative analysis with the genome sequences of M. tuberculosis and M. bovis BCG promises to expose the genetic basis for the phenotypic differences between the tubercle bacilli, offering unparalleled insight into the virulence factors of the M. tuberculosis complex. Initial analysis of the sequence data has already revealed a novel deletion from M. bovis, as well as identifying variation in members of the PPE family of proteins. As the study of bacterial pathogenicity enters the postgenomic phase, the genome sequence of M. bovis promises to serve as a cornerstone of mycobacterial genetics.     

Inactivation of the Mycobacterium bovis homologue of the polymorphic RD1 gene Rv3879c (Mb3909c) does not affect virulence

The RD1 locus is deleted from all strains of Mycobacterium bovis BCG but present in virulent isolates of M. bovis and Mycobacterium tuberculosis. The RD1 gene Rv3879c encodes a proline- and alanine-rich protein that shows sequence polymorphism across members of the M. tuberculosis complex. The role of this protein in virulence was investigated by deleting the Rv3879c homologue from M. bovis (Mb3909c) and testing the virulence of the mutant in the guinea pig model. The M. bovis Delta Mb3909c mutant was not attenuated in the guinea pig model, showing that this gene does not encode a virulence factor and plays no role in the attenuation caused by loss of RD1.     

Evaluation of the Mycobacterium smegmatis and BCG models for the discovery of Mycobacterium tuberculosis inhibitors

The objective of this study was to measure the efficacy of Mycobacterium smegmatis as a surrogate in vitro model for the detection of compounds which are inhibitory to the growth of Mycobacterium tuberculosis. A chemical screen of the LOPAC library for anti-mycobacterial compounds was performed using M. smegmatis. Parallel screens were conducted with another tuberculosis model, Mycobacterium bovis BCG, and with M. tuberculosis under identical growth conditions and the inhibitors detected across the three species were compared. 50% of compounds that were detected as active against M.?tuberculosis were not detected using M. smegmatis compared to 21% of compounds using M. bovis BCG. To examine whether these findings were unique to LOPAC, screens were performed with the NIH Diversity Set and Spectrum Collection. An even higher proportion of M. tuberculosis inhibitors were not detected from the NIH Diversity Set and Spectrum Collection using M. smegmatis compared to M. bovis BCG. These data reveal that a significant proportion of M. tuberculosis inhibitors are missed in library screening with M.?smegmatis. The basis of the variation in the inhibitory profiles of M. smegmatis and M. tuberculosis has yet to be fully determined, however, our genomic comparisons indicate that approximately 30% of M.?tuberculosis proteins lack conserved orthologues in M. smegmatis compared to 3% being absent in M.?bovis BCG. In conclusion, although M. smegmatis offers some technical benefits such as a shorter generation time and negligible risk to laboratory workers, it is significantly less effective in the detection of anti-M. tuberculosis compounds relative to M. bovis BCG. This limitation needs to be taken into consideration when selecting an in vitro screening model for tuberculosis drug discovery.     

Vaccination of cattle against Mycobacterium bovis.

Protection of cattle against bovine tuberculosis by vaccination could be an important control strategy in countries where there is persistence of Mycobacterium bovis infection in wildlife and in developing countries where it is not economical to implement a 'test and slaughter' control programme. Early field trials with Bacille Calmette Guerin (BCG) M. bovis vaccine in cattle produced disappointing results, with induction of tuberculin skin-test reactivity following vaccination and low levels of protection. However, recent studies using a low dose of BCG vaccine in cattle have produced more encouraging results and field trials should now be carried out in developing countries to determine whether this low dose BCG vaccination strategy will reduce the spread of infection. The options for new candidate tuberculosis vaccines have increased markedly in the last decade with the advent of new attenuated strains of M. bovis, and sub-unit protein and recombinant DNA vaccines. Some of these new types of vaccines have recently been tested in cattle. New attenuated M. bovis vaccines induced greater protection than BCG vaccine in cattle which had been sensitized to environmental mycobacteria prior to vaccination. In contrast, it has proved difficult to stimulate appropriate immune responses in cattle necessary for protection with sub-unit protein and recombinant DNA vaccines and better immunological adjuvants are required for these types of vaccines. Progress in the development of new tuberculosis vaccines has been very rapid in the past decade and the prospects for vaccination to control and eradicate bovine tuberculosis are encouraging.     

Activation of ERK1/2 and TNF-alpha production are mediated by calcium/calmodulin, and PKA signaling pathways during Mycobacterium bovis infection

Mycobacterium bovis bacillus Calmette-Guérin (BCG)-induced tumor necrosis factor (TNF)-alpha secretion via an extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase-dependent mechanism is an important host defence mechanism against Mycobacterium tuberculosis in human monocytes. We now define distinct signaling pathways that regulate induction of TNF-alpha and activation of ERK1/2 by intracellular signaling mechanisms during M. bovis infection. We determined that M. bovis BCG-induced ERK 1/2 activation occurs through a mechanism that requires intracellular calcium and likely involves a calmodulin-sensitive step. In contrast, M. bovis BCG can induce p38 mapk activation by a calcium (Ca2+)/calmodulin-independent mechanism. Interestingly, we present evidence that M. bovis BCG activates protein kinase A (PKA), since pretreatment of monocytes with H-89, a inhibitor of PKA activity, blocked the ability of M. bovis BCG to induce ERK1/2 activation. These results were further supported by the fact that treatment of cells with KT5720, another well-described inhibitor of PKA activity, significantly diminished the effect of M. bovis BCG on ERK1/2 activation. Furthermore, secretion of TNF-alpha in M. bovis-infected human monocytes was also dependent on Ca2+/calmodulin, and PKA pathways. Finally, addition of H-89 significantly diminished TNF-alpha mRNA expression in M. bovis-infected human monocytes. These results indicate that the Ca2+/calmodulin, and PKA pathways play important regulatory roles in monocyte signaling upon M. bovis infection.     

Clinical evaluation of the recombinant 38 kDa protein of Mycobacterium tuberculosis

The diagnosis of tuberculosis remains among public health concerns due to shortcomings of the purified protein derivative (PPD). Recombinant truncated 38 kDa protein (rTPA38) of Mycobacterium tuberculosis was evaluated to screen new tuberculosis-specific tuberculin. 539 patients, 1133 healthy controls, and 55 guinea pigs were recruited to assess their sensitivity and specificity to rTPA38; 221 healthy controls, with negative responses to rTPA38 and PPD, were vaccinated with M. bovis BCG to determine their cross-reactions with M. bovis BCG. The Mantoux technique was adopted to perform skin tests. No difference in the sensitivity of skin tests was detected between rTPA38 and PPD (78.2% vs 83.4%), but there was a significant difference in the specificity of skin tests between rTPA38 and PPD (75.2% vs 47.0%). Compared to PPD, rTPA38 elicited low positive responses for those recruitments vaccinated with M. bovis BCG. The rTPA38 had significant skin reactions in M. tuberculosis-sensitized guinea pigs, and the opposite was true for both M. fortuitum- and M. kansasii-sensitized guinea pigs. These findings indicate that rTPA38 may have potential as a tuberculosis-specific skin test antigen.