Differentiation of clinical Mycobacterium tuberculosis complex isolates by their GyrB polymorphism

Purpose: To evaluate the reliability of the gyrB PCR-RFLP technique in differentiating clinical Mycobacterium tuberculosis complex isolates. Materials and Methods: A primer pair MTUB-f and MTUB-r for M. tuberculosis complex (MTBC) was used to differentiate 79 mycobacterial isolates by specific amplification of the 1,020-bp fragment of the gyrB gene (gyrB-PCR1). The MTBC isolates were further differentiated using a set of specific primers MTUB-756-Gf and MTUB-1450-Cr that allowed selective amplification of the gyrB fragment specific for M. tuberculosis (gyrB-PCR2). The DNA polymorphisms in the 1,020-bp gyrB fragment for 7 M. tuberculosis strains confirmed by PCR as well as 2 reference strains; M. tuberculosis H37Rv and M. bovis BCG were analyzed with the restriction enzyme Rsa1. Results: Seventy-seven (97.5%) isolates were positive for gyrB-PCR1 and thus identified as members of M. tuberculosis complex (MTBC) and two (2.6%) isolates were negative and identified as Mycobacteria other than tuberculosis (MOTT). All the M. tuberculosis isolates showed the typical M. tuberculosis specific Rsa1 RFLP patterns (100, 360, 560-bp) while 360 and 480-bp fragments were generated from M. bovis BCG. Conclusion: The gyrB PCR-RFLP using the endonuclease Rsa1 can be used to differentiate M. tuberculosis from M. bovis in clinical isolates.     

A first insight into the genotypic diversity of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> from Rwanda

Background

Mycobacterium tuberculosis complex (MTC) is the causative agent of tuberculosis (TB). Globally, increasing evidence shows that in M. tuberculosis, transmission varies from strain to strain and that different strains exhibit a range of geographical and host specificities, pathogenicity, and drug susceptibility. Therefore rapid and accurate differentiation of the members of MTC is critical in guiding treatment and public health decisions. We carried out a study at different health units and the National Reference Laboratory in Rwanda identify Mycobacterium tuberculosis complex species prevalent in TB patients in Rwanda. We further characterized the isolates using spoligotyping in order to gain an insight into the strain diversity of drug resistant and susceptible isolates of M. tuberculosis in this setting.

Methods

A total of 151 isolates from culture positive sputum samples were harvested, heat killed at 80°C for two hours, and then shipped to Makerere University College of Health Sciences, Uganda, for speciation and typing. Species identification was achieved by regions of difference (RD) analysis, while Spoligotyping was done to identify strain types.

Results

Region of difference analysis identified all the 151 isolates as M. tuberculosis. Spoligotyping revealed predominance of the T2 family (58.3%, 88/151), with SIT 52 being the most prevalent strain (31.8%, 48/151). Among the 151 isolates, 64 (42.4%) were multidrug resistant (MDR) with 3 cases on mono-resistance. Of 94 retreatment cases, 48 (51.1%) were MDR and of 46 newly presenting cases 14 (30.4%) were MDR. There was a significant difference (p=0.01) in anti-TB drug resistance between new and retreatment cases in the sample. However, there was no significant relationship between HIV serostatus and the two major strain types SIT 52 (p =0.15and SIT 152 (p = 0.41).

Conclusion

Mycobacterium tuberculosis is the most prevalent species of Mycobacterium tuberculosis complex in Rwanda, and SIT 52 (T2) the predominant strain. There is significantly more MDR in the retreatment cases but no significant difference was observed by HIV status in relation to any spoligotypes.

     

RD7 genotyping of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> strains isolated from patients with lung tuberculosis in various areas of Kazakhstan

A three-primer PCR assay has been designed for detecting possible deletions in the RD7 chromosomal region of the Mycobacterium tuberculosis complex. The assay gives rise to amplicons of different sizes depending on the presence or absence of deletions. The PCR assay was applied to 176 isolates from lung tuberculosis cases collected in various areas of Kazakhstan in the summer of 2004. Prior to assay, the isolates were characterized by culture and biochemical tests. The RD7 genotyping showed neither polymorphism nor deletions in the RD7 genome region. Some strains were additionally characterized by a PCR-RFLP analysis of the gyrB and hsp64 genes. The RFLP patterns corresponded to M. tuberculosis. The results of the study were consistent with certain previous studies, which indicates the population stability of RD7 in M. tuberculosis strains. Species identification of the isolates showed that M. tuberculosis sensu stricto was the principal causative agent of human lung tuberculosis in Kazakhstan.     

A Simple and Efficient Multiplex PCR Assay for the Identification of Mycobacterium Genus and Mycobacterium tuberculosis Complex to the Species Level

Purpose

The Mycobacterium tuberculosis complex comprises M. tuberculosis, M. bovis, M. bovis bacillus Calmette-Guérin (BCG) and M. africanum, and causes tuberculosis in humans and animals. Identification of Mycobacterium spp. and M. tuberculosis complex to the species level is important for practical use in microbiological laboratories, in addition to optimal treatment and public health.

Materials and Methods

A novel multiplex PCR assay targeting a conserved rpoB sequence in Mycobacteria spp., as well as regions of difference (RD) 1 and RD8, was developed and evaluated using 37 reference strains and 178 clinical isolates.

Results

All mycobacterial strains produced a 518-bp product (rpoB), while other bacteria produced no product. Virulent M. tuberculosis complex strains, M. tuberculosis, M. bovis and M. africanum, produced a 254-bp product (RD1), while M. bovis BCG, M. microti and nontuberculous mycobacteria produced no RD1 region product. Additionally, M. tuberculosis and M. africanum produced a 150-bp product (RD8), while M. bovis and M. bovis BCG produced a 360-bp product (deleted form of RD8). M. microti and nontuberculous mycobacteria produced no RD8 region product. This assay identified all Mycobacterium spp. and all M. tuberculosis complex strains to the species level.

Conclusion

The multiplex PCR assay of the present study could be implemented as a routine test in microbiology laboratories, and may contribute to more effective treatment and surveillance of tuberculosis stemming from the M. tuberculosis complex.     

Lateral flow assay for rapid differentiation of Mycobacterium tuberculosis complex and 97 species of mycobacteria other than tuberculosis grown in Löwenstein-Jensen and TK-SLC medium

Background: Mycobacterial antigen MPB64 is a secretory protein specific for Mycobacterium tuberculosis complex. A lateral flow immunochromatographic assay (ICA) is a method used for the rapid differentiation of M. tuberculosis complex. Aim: We aimed to evaluate the performance of ICA in rapid differentiation of M. tuberculosis complex from 97 Mycobacterium species other than tuberculosis (MOTT), which are grown in Löwenstein-Jensen and TK-selective (SLC) medium. Materials and Methods: The study was performed in our laboratory between January 2009 and January 2010. A total of 394 isolates consisting of reference strains of 34 M. tuberculosis from World Health Organization (WHO) collection, 97 different MOTT bacilli, 7 Mycobacterium bovis BCG substrains and total 256 clinical Mycobacterium isolates were tested by ICA, which is based on anti-MPB64 monoclonal antibodies. All the strains were inoculated onto a TK-SLC (selective) medium and Löwenstein-Jensen medium. TK-SLC is a new rapid mycobacterial culture medium that indicates mycobacterial growth by colour change. Results: The growth of mycobacterial strains was observed in 10–12 days on TK-SLC medium. ICA test was performed in 15 minutes. All strains belonging to M. tuberculosis complex group were found positive and all MOTT species were found negative on ICA slides. The results were confirmed with nucleic acid amplification by polymerase chain reaction (PCR) using primers specific for M. tuberculosis complex. Conclusion: With the additive effect of growth on TK-SLC medium in 10–12 days, the mycobacterial antigen MPB64 is a very useful and specific tool in rapid differentiation of M. tuberculosis and MOTT grown in culture.     

Development of an Unbiased Antigen-Mining Approach To Identify Novel Vaccine Antigens and Diagnostic Reagents for Bovine Tuberculosis

Previous experiments for the identification of novel diagnostic or vaccine candidates for bovine tuberculosis have followed a targeted approach, wherein specific groups of proteins suspected to contain likely candidates are prioritized for immunological assessment (for example, with in silico approaches). However, a disadvantage of this approach is that the sets of proteins analyzed are restricted by the initial selection criteria. In this paper, we describe a series of experiments to evaluate a nonbiased approach to antigen mining by utilizing a Gateway clone set for Mycobacterium tuberculosis, which constitutes a library of clones expressing 3,294 M. tuberculosis proteins. Although whole-blood culture experiments using Mycobacterium bovis-infected animals and M. bovis BCG-vaccinated controls did not reveal proteins capable of differential diagnosis, several novel immunogenic proteins were identified and prioritized for efficacy studies in a murine vaccination/challenge model. These results demonstrate that Rv3329-immunized mice had lower bacterial cell counts in their spleens following challenge with M. bovis. In conclusion, we demonstrate that this nonbiased approach to antigen mining is a useful tool for identifying and prioritizing novel proteins for further assessment as vaccine antigens.     

Novel multiplex real-time PCR diagnostic assay for identification and differentiation of Mycobacterium tuberculosis, Mycobacterium canettii, and Mycobacterium tuberculosis complex strains

Tuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosis complex (MTC). Rapid detection of the MTC is necessary for the timely initiation of antibiotic treatment, while differentiation between members of the complex may be important to guide the appropriate antibiotic treatment and provide epidemiological information. In this study, a multiplex real-time PCR diagnostics assay using novel molecular targets was designed to identify the MTC while simultaneously differentiating between M. tuberculosis and M. canettii. The lepA gene was targeted for the detection of members of the MTC, the wbbl1 gene was used for the differentiation of M. tuberculosis and M. canettii from the remainder of the complex, and a unique region of the M. canettii genome, a possible novel region of difference (RD), was targeted for the specific identification of M. canettii. The multiplex real-time PCR assay was tested using 125 bacterial strains (64 MTC isolates, 44 nontuberculosis mycobacteria [NTM], and 17 other bacteria). The assay was determined to be 100% specific for the mycobacteria tested. Limits of detection of 2.2, 2.17, and 0.73 cell equivalents were determined for M. tuberculosis/M. canettii, the MTC, and M. canettii, respectively, using probit regression analysis. Further validation of this diagnostics assay, using clinical samples, should demonstrate its potential for the rapid, accurate, and sensitive diagnosis of TB caused by M. tuberculosis, M. canettii, and the other members of the MTC.     

Allele-Specific PCR Method Based on pncA and oxyR Sequences for Distinguishing Mycobacterium bovis from Mycobacterium tuberculosis: Intraspecific M. bovis pncA Sequence Polymorphism

An allele-specific amplification method based on two genetic polymorphisms to differentiate Mycobacterium tuberculosis from Mycobacterium bovis was tested. Based on the differences found at position 169 in the pncA genes from M. tuberculosis and M. bovis, a PCR system which was able to differentiate most of the 237 M. tuberculosis complex isolates tested in one of the two species was developed. All 121 M. tuberculosis strains showed the expected base (cytosine) at position 169. Most of the M. bovis isolates had a guanine at the cited position. Nevertheless, 18 of the 116 M. bovis isolates, all of them goat isolates, showed the pncA polymorphism specific to M. tuberculosis. These results suggest that goat M. bovis may be the nicotinamidase-missing link at the origin of the M. tuberculosis species. Based on the polymorphism found at position 285 in the oxyR gene, the same system was used to differentiate M. tuberculosis from M. bovis. In this case, DNAs from all 121 M. tuberculosis isolates had the expected base (guanine) at this position. In addition, all 116 M. bovis isolates, including those from goats, showed the identical polymorphism (adenine). The oxyR allele-specific amplification method can differentiate M. bovis from M. tuberculosis, is rapid (results can be obtained in less than 3 h), and is easy to perform.     

The CFP10/ESAT6 complex of Mycobacterium tuberculosis may function as a regulator of macrophage cell death at different stages of tuberculosis infection

Tuberculosis is a human disease caused by infection with Mycobacterium tuberculosis. M. tuberculosis (Mtb) is a facultative intracellular pathogen. The alveolar macrophages provide a critical niche for the intracellular pathogen. It has been shown that virulent strains mycobacteria (Mtb-H37Rv, Mycobacterium bovis) induce less apoptosis in host macrophage than avirulent mycobacteria strains (Bacillus Calmette-Guérin, H37Ra). Comparative genomics analysis has revealed that the region of difference (RD1) of M. tuberculosis is absent from all strains of Bacillus Calmette-Guérin (BCG). On the contrary, it presents in all virulent strains of M. tuberculosis and M. bovis. The culture filtrate protein 10 (CFP10) and early secretory antigenic target protein 6 (ESAT6) are encoded by RD1 genes Rv3874 and Rv3875, respectively. Recent studies indicated that the CFP10 and ESAT6 played an important role in M. tuberculosis virulence. It has been shown that incorporation of the RD1 region into BCG to restore the expression of CFP10 and ESAT6 leads to increasing the virulence and immunogenicity of bacterium. On the contrary, deletion of the genes encoding CFP10 and ESAT6 from the virulent M. bovis strain results in attenuation of virulence. Meanwhile, several studies showed that CFP10 and ESAT6 could inhibit and/or promote the production of tumor necrosis factor α (TNF-α) from macrophages. Furthermore, TNF-α can induce apoptosis and necrosis of infected macrophages in tuberculosis. Considering above results, we hypothesize that the CFP10 and ESAT6 may be involved in the virulence of Mycobacterium through modulating macrophage cell death.     

Frequency of <Emphasis Type="Italic">Mycobacterium bovis</Emphasis> as an etiologic agent in extrapulmonary tuberculosis in HIV-positive and -negative Mexican patients

Mycobacterium bovis can be an important etiological agent for extrapulmonary (EP) manifestations of tuberculosis, especially in HIV-infected persons. From January 2000 to December 2003, M. bovis as a cause of EP tuberculosis was investigated at the Pneumonology Service, Hospital General de Mexico, Mexico City. Eighty HIV-positive (HIV+) patients and 83 HIV-negative (HIV−) with EP involvement (ganglionar, genitourinary, meningeal, cutaneous, peritoneal, and pericardial) were analyzed using clinical, immunological, bacteriological, histopathological, and molecular biology methods. Mycobacterium species were identified by hsp65-RFLP analysis and species of M. tuberculosis complex isolates by spoligotyping. M. bovis was present in 6 HIV− cases (7.2%; 3 with lymphadenitis and 3 genitourinary) vs 11 in HIV+ cases (13.75%; 7 with lymphadenitis, 3 genitourinary, and 1 meningeal). Favorable response to retroviral and specific M. bovis chemotherapy was observed. Spoligotyping showed a unique profile in each isolate, 16 belonging to BOV1 lineage and 1 to BOV2 lineage. M. bovis is an significant re-emerging cause of EPTB in Mexico. Consumption of unpasteurized dairy products is the most likely source of transmission. Successful treatment depends on the adequate and opportune identification of the agent responsible.     

Use of Mycobacterium tuberculosis Complex-Specific Antigen Cocktails for a Skin Test Specific for Tuberculosis

The tuberculin skin test currently used to diagnose infection with Mycobacterium tuberculosis has poor diagnostic value, especially in geographic areas where the prevalence of tuberculosis is low or where the environmental burden of saprophytic, nontuberculous mycobacteria is high. Inaccuracy of the tuberculin skin test often reflects a low diagnostic specificity due to the presence in tuberculin of antigens shared by many mycobacterial species. Thus, a skin test specific for tuberculosis requires the development of new tuberculins consisting of antigens specific to M. tuberculosis. We have formulated cocktails of two to eight antigens of M. tuberculosis purified from recombinant Escherichia coli. Multiantigen cocktails were evaluated by skin testing guinea pigs sensitized with M. bovis BCG. Reactivity of multiantigen cocktails was greater than that of any single antigen. Cocktail activity increased with the number of antigens in the cocktail even when the same amount of total protein was used for cocktails and for each single antigen. A cocktail of four purified antigens specific for the M. tuberculosis complex elicited skin test responses only in BCG-immunized guinea pigs, not in control animals immunized with M. avium. These findings open the way to designing a multiantigen formulation for a skin test specific for tuberculosis.     

M. bovis and M. caprae infections in Aquitaine: A clinico-epidemiologic study of 15 patients

The agent of bovine tuberculosis, Mycobacterium bovis, is a zoonosis which can be transmitted to human beings. In France, the prevalence of tuberculosis due to M. bovis has drastically decreased, both for animals and humans, since public health measures were introduced to prevent its transmission. However, a new outbreak of the disease is noted among cattle in several French areas and more particularly in Aquitaine. In 2008, 40% of bovine tuberculosis French cases provided from Aquitaine. From November 2004 to October 2008, 15 cases were registered at Bordeaux's academic hospital (CHU). Thirteen of them were due to M. bovis and two to Mycobacterium caprae. It represents 2.9% of tuberculosis due to tuberculosis complex. An analysis of the 15 patients’ medical files showed that it occurred either to old people who reactivated a former infection, or to younger ones who were born in countries with a strong M. bovis endemic disease. Extrapulmonary forms and especially ganglionics ones are the most frequent. M. caprae seems to be an emergent species among animal mycobacteries transmissible to human being. An epidemiological monitoring seems to be necessary to establish a relation between the regional outbreak of bovine tuberculosis and human tuberculosis.     

Delayed-Type Hypersensitivity Responses to ESAT-6 and MPT64 from Mycobacterium tuberculosis in the Guinea Pig

Two antigens from Mycobacterium tuberculosis, ESAT-6 and MPT64, elicited delayed-type hypersensitivity (DTH) skin responses in outbred guinea pigs infected with M. tuberculosis by the aerosol and intravenous routes but not those sensitized with M. bovis BCG or M. avium. The DTH epitope of ESAT-6 was mapped to the C terminus. Nonresponders to the individual antigens were found, but all animals responded to a combination of ESAT-6 and MPT64 or their respective minimal target peptides. Correspondingly, these molecules could form the basis of a new skin test for tuberculosis.     

Evaluation of the Speed-oligo Direct Mycobacterium tuberculosis Assay for Molecular Detection of Mycobacteria in Clinical Respiratory Specimens

We present the first evaluation of a novel molecular assay, the Speed-oligo Direct Mycobacterium tuberculosis (SO-DMT) assay, which is based on PCR combined with a dipstick for the detection of mycobacteria and the specific identification of M. tuberculosis complex (MTC) in respiratory specimens. A blind evaluation was carried out in two stages: first, under experimental conditions on convenience samples comprising 20 negative specimens, 44 smear- and culture-positive respiratory specimens, and 11 sputa inoculated with various mycobacterium-related organisms; and second, in the routine workflow of 566 fresh respiratory specimens (4.9% acid-fast bacillus [AFB] smear positives, 7.6% MTC positives, and 1.8% nontuberculous mycobacteria [NTM] culture positives) from two Mycobacterium laboratories. SO-DMT assay showed no reactivity in any of the mycobacterium-free specimens or in those with mycobacterium-related organisms. Compared to culture, the sensitivity in the selected smear-positive specimens was 0.91 (0.92 for MTC and 0.90 for NTM), and there was no molecular detection of NTM in a tuberculosis case or vice versa. With respect to culture and clinical data, the sensitivity, specificity, and positive and negative predictive values for the SO-DMT system in routine specimens were 0.76 (0.93 in smear positives [1.0 for MTC and 0.5 for NTM] and 0.56 in smear negatives [0.68 for MTC and 0.16 for NTM]), 0.99, 0.85 (1.00 in smear positives and 0.68 in smear negatives), and 0.97, respectively. Molecular misidentification of NTM cases occurred when testing 2 gastric aspirates from two children with clinically but not microbiologically confirmed lung tuberculosis. The SO-DMT assay appears to be a fast and easy alternative for detecting mycobacteria and differentiating MTC from NTM in smear-positive respiratory specimens.     

Immune Responses in Cattle Inoculated with Mycobacterium bovis,Mycobacterium tuberculosis,or Mycobacterium kansasii

Cattle were inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii to compare the antigen-specific immune responses to various patterns of mycobacterial disease. Disease expression ranged from colonization with associated pathology (M. bovis infection) and colonization without pathology (M. tuberculosis infection) to no colonization or pathology (M. kansasii infection). Delayed-type hypersensitivity and gamma interferon responses were elicited by each mycobacterial inoculation; however, the responses by the M. bovis- and M. tuberculosis-inoculated animals exceeded those of the M. kansasii-inoculated animals. Specific antibody responses were detected in all M. tuberculosis- and M. bovis-inoculated cattle 3 weeks after inoculation. From 6 to 16 weeks after M. tuberculosis inoculation, the antibody responses waned, whereas the responses persisted with M. bovis infection. With M. kansasii inoculation, initial early antibody responses waned by 10 weeks after inoculation and then increased 2 weeks after the injection of purified protein derivative for the skin test at 18 weeks after challenge. These findings indicate that antibody responses are associated with the antigen burden rather than the pathology, cellular immune responses to tuberculin correlate with infection but not necessarily with the pathology or bacterial burden, and exposure to mycobacterial antigens may elicit an antibody response in a presensitized animal.Tuberculosis (TB) in humans and animals may result from exposure to bacilli within the Mycobacterium tuberculosis complex (i.e., M. tuberculosis, M. bovis, M. africanum, M. pinnipedi, M. microti, M. caprae, or M. canetti [8]). Despite their ∼99.95% sequence identity (12), M. bovis and M. tuberculosis exhibit distinct differences in virulence and host adaptation. Compared to M. tuberculosis, experimental M. bovis infection of mice or rabbits results in a more severe pathology and shorter mean survival times (9, 17, 18). Mycobacterium tuberculosis is primarily a human pathogen that demonstrates a high level of attenuation in cattle (as reviewed by Francis in 1947 [10]), whereas M. bovis has a wider host range and affects many domesticated and free-ranging mammals as well as humans. Prior to the initiation of control and eradication campaigns in the early to mid-1900s, M. bovis infection accounted for up to 30% of human tuberculosis cases, with M. bovis being transmitted to humans primarily by the consumption of unpasteurized dairy products and contact with infected livestock. Control efforts, including slaughter surveillance and test/cull campaigns, have dramatically reduced the prevalence of M. bovis infection in domestic cattle herds, thereby reducing the spread of M. bovis to humans. However, in developing countries, M. bovis infection of humans persists as a serious and relatively common zoonosis (16).Although Mycobacterium kansasii is not a member of the M. tuberculosis complex, it may cause disease in otherwise healthy humans, albeit infrequently, that is clinically indistinguishable from M. tuberculosis infection (1, 3). As with humans, M. kansasii infection of cattle is uncommon; however, it is occasionally associated with granulomatous lesions within lymph nodes and the respiratory tract of cattle (B. Harris, unpublished observations). Of particular relevance for the diagnosis of tuberculosis, M. kansasii infection/sensitization may elicit responses to antigens generally considered to be tuberculosis specific, such as ESAT-6, CFP-10, and MPB83 (2, 30, 35).With experimental M. bovis infection of cattle, the levels of MPB83-specific antibody correlate with disease severity, bacterial burden, and specific cell-mediated immune responses (15, 33). With this particular scenario, disease severity (i.e., pathology) and bacterial burden are intimately linked; thus, it is difficult to define a potential correlation of a particular immune response to either readout independently. Prior studies have demonstrated that virulent and attenuated strains of M. bovis induce similar delayed-type hypersensitivity responses in cattle; however, only the virulent M. bovis strain induces a persistent gamma interferon (IFN-γ), interleukin-2 (IL-2), and antibody response (34). The objective of the present study was to compare mycobacterium-specific immune responses to the patterns of mycobacterial disease expression in which the mycobacterial burden is uncoupled from pathological changes. Disease expression patterns included persistent colonization with an associated pathology (i.e., M. bovis infection), colonization without an associated pathology (i.e., M. tuberculosis infection), and no colonization or pathology (i.e., M. kansasii infection). Antigen-specific immune responses were evaluated for their correlation to manifestations of disease expression.     

Selective delipidation of Mycobacterium bovis BCG enables direct pulmonary vaccination and enhances protection against Mycobacterium tuberculosis

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), is the leading killer due to an infectious organism. Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine approved against TB, however, its efficacy against pulmonary TB is poor. While BCG is currently inoculated intradermally, the natural route of M.tb infection is through the lung. Excessive lung pathology caused by pulmonary inoculation of BCG has prevented the use of this immunization route. Here, we show that selective chemical treatment of BCG with petroleum ether removes inflammatory lipids from the bacterial surface while keeping BCG viable. Pulmonary vaccination using this modified BCG attenuated inflammatory responses, prevented immunopathology of the lung, and significantly increased protection against M.tb infection in mice. We further directly linked IL-17A as the responsible contributor of improved immunity against M.tb infection. These results provide evidence that selective removal of cytotoxic lipids from the BCG surface attenuates inflammation and offers a safer and superior vaccine against TB causing less damage post-infectious challenge with M.tb.     

Gamma Interferon Responses Induced by a Panel of Recombinant and Purified Mycobacterial Antigens in Healthy, Non-Mycobacterium bovis BCG-Vaccinated Malawian Young Adults

We have previously shown that young adults living in a rural area of northern Malawi showed greater gamma interferon (IFN-γ) responses to purified protein derivatives (PPD) prepared from environmental mycobacteria than to PPD from Mycobacterium tuberculosis. In order to define the mycobacterial species to which individuals living in a rural African population have been exposed and sensitized, we tested T-cell recognition of recombinant and purified antigens from M. tuberculosis (38 kDa, MPT64, and ESAT-6), M. bovis (MPB70), M. bovis BCG (Ag85), and M. leprae (65 kDa, 35 kDa, and 18 kDa) in >600 non-M. bovis BCG-vaccinated young adults in the Karonga District of northern Malawi. IFN-γ was measured by enzyme-linked immunosorbent assay (ELISA) in day 6 supernatants of diluted whole-blood cultures. The recombinant M. leprae 35-kDa and 18-kDa and purified native M. bovis BCG Ag85 antigens induced the highest percentages of responders, though both leprosy and bovine tuberculosis are now rare in this population. The M. tuberculosis antigens ESAT-6 and MPT64 and the M. bovis antigen MPB70 induced the lowest percentages of responders. One of the subjects subsequently developed extrapulmonary tuberculosis; this individual had a 15-mm-diameter reaction to the Mantoux test and responded to M. tuberculosis PPD, Ag85, MPT64, and ESAT-6 but not to any of the leprosy antigens. We conclude that in this rural African population, exposure to M. tuberculosis or M. bovis is much less frequent than exposure to environmental mycobacteria such as M. avium, which have antigens homologous to the M. leprae 35-kDa and 18-kDa antigens. M. tuberculosis ESAT-6 showed the strongest association with the size of the Mantoux skin test induration, suggesting that among the three M. tuberculosis antigens tested it provided the best indication of exposure to, or infection with, M. tuberculosis.     

Differentiation between Mycobacterium bovis BCG-Vaccinated and M. bovis-Infected Cattle by Using Recombinant Mycobacterial Antigens

Tuberculosis continues to be a worldwide problem for both humans and animals. The development of tests to differentiate between infection with Mycobacterium tuberculosis or Mycobacterium bovis and vaccination with M. bovis BCG could greatly assist in the diagnosis of early infection as well as enhance the use of tuberculosis vaccines on a wider scale. Recombinant forms of four major secreted proteins of M. bovis—MPB59, MPB64, MPB70, and ESAT-6—were tested in a whole-blood gamma interferon (IFN-γ) assay for differentiation between cattle vaccinated with BCG and those experimentally infected with M. bovis. BCG vaccination induced minimal protection in the present study, with similar numbers of animals infected with M. bovis in BCG-vaccinated and nonvaccinated groups. Following vaccination with BCG, the animals produced moderate IFN-γ responses to bovine purified protein derivative (PPDB) but very weak responses to the recombinant antigens. Cattle from both the BCG-vaccinated and nonvaccinated groups which were M. bovis culture positive following challenge produced IFN-γ responses to PPDB and ESAT-6 which were significantly stronger than those observed in the corresponding M. bovis culture-negative animals. IFN-γ responses to MPB59, MPB64, and MPB70 were significantly weaker, and these antigens could not discriminate between vaccinated animals which develop disease and the culture-negative animals. The results of the study indicate that of the four antigens tested in the IFN-γ assay, only ESAT-6 would be suitable for differentiating BCG-vaccinated animals from those infected with bovine tuberculosis.     

Extrapulmonary Dissemination of Mycobacterium bovis but Not Mycobacterium tuberculosis in a Bronchoscopic Rabbit Model of Cavitary Tuberculosis

The rabbit model of tuberculosis is attractive because of its pathophysiologic resemblance to the disease in humans. Rabbits are naturally resistant to infection but may manifest cavitary lung lesions. We describe here a novel approach that utilizes presensitization and bronchoscopic inoculation to reliably produce cavities in the rabbit model. With a fixed inoculum of bacilli, we were able to reproducibly generate cavities by using Mycobacterium bovis Ravenel, M. bovis AF2122, M. bovis BCG, M. tuberculosis H37Rv, M. tuberculosis CDC1551, and the M. tuberculosis CDC1551 ΔsigC mutant. M. bovis infections generated cavitary CFU counts of 10⁶ to 10⁹ bacilli, while non-M. bovis species and BCG yielded CFU counts that ranged from 10⁴ to 10⁸ bacilli. Extrapulmonary dissemination was almost exclusively noted among rabbits infected with M. bovis Ravenel and AF2122. Though all of the species yielded secondary lesions at intrapulmonary sites, M. bovis infections led to the most apparent gross pathology. Using the M. tuberculosis icl and dosR gene expression patterns as molecular sentinels, we demonstrated that both the cavity wall and cavity lumen are microenvironments associated with hypoxia, upregulation of the bacterial dormancy program, and the use of host lipids for bacterial catabolism. This unique cavitary model provides a reliable animal model to study cavity pathogenesis and extrapulmonary dissemination.     

Performance of a Noninvasive Test for Detecting Mycobacterium bovis Shedding in European Badger (Meles meles) Populations

The incidence of Mycobacterium bovis, the causative agent of bovine tuberculosis, in cattle herds in the United Kingdom is increasing, resulting in substantial economic losses. The European badger (Meles meles) is implicated as a wildlife reservoir and is the subject of control measures aimed at reducing the incidence of infection in cattle populations. Understanding the epidemiology of M. bovis in badger populations is essential for directing control interventions and understanding disease spread; however, accurate diagnosis in live animals is challenging and currently uses invasive methods. Here we present a noninvasive diagnostic procedure and sampling regimen using field sampling of latrines and detection of M. bovis with quantitative PCR tests, the results of which strongly correlate with the results of immunoassays in the field at the social group level. This method allows M. bovis infections in badger populations to be monitored without trapping and provides additional information on the quantities of bacterial DNA shed. Therefore, our approach may provide valuable insights into the epidemiology of bovine tuberculosis in badger populations and inform disease control interventions.