Bovine tuberculosis in African buffaloes : observations regarding Mycobacterium bovis shedding into water and exposure to environmental mycobacteria

Includes bibliographyBackground: African buffaloes are the maintenance host for Mycobacterium bovis in the endemically infected Kruger National Park (KNP). The infection is primarily spread between buffaloes via the respiratory route, but it is not known whether shedding of M. bovis in nasal and oral excretions may lead to contamination of ground and surface water and facilitate the transmission to other animal species. A study to investigate the possibility of water contamination with M. bovis was conducted in association with a BCG vaccination trial in African buffalo. Groups of vaccinated and nonvaccinated buffaloes were kept together with known infected in-contact buffalo cows to allow natural M. bovis transmission under semi-free ranging conditions. In the absence of horizontal transmission vaccinated and control buffaloes were experimentally challenged with M. bovis. Hence, all study buffaloes in the vaccination trial could be considered potential shedders and provided a suitable setting for investigating questions relating to the tenacity of M. bovis shed in water. Results: Serial water samples were collected from the drinking troughs of the buffaloes once per season over an eleven-month period and cultured for presence of mycobacteria. All water samples were found to be negative for M. bovis, but 16 non-tuberculous Mycobacterium spp. isolates were cultured. The non-tuberculous Mycobacterium species were further characterised using 5'-16S rDNA PCR-sequencing, resulting in the identification of M. terrae, M. vaccae (or vanbaalenii), M. engbaekii, M. thermoresistibile as well as at least two species which have not yet been classified. Conclusion: The absence of detectable levels of Mycobacterium bovis in the trough water suggests that diseased buffalo do not commonly shed the organism in high quantities in nasal and oral discharges. Surface water may therefore not be likely to play an important role in the transmission of bovine tuberculosis from buffalo living in free-ranging ecosystems. The study buffalo were, however, frequently exposed to different species of non-tuberculous, environmental mycobacteria, with an unknown effect on the buffaloes' immune response to mycobacteria.Peer Reviewe

Mycosin-1, a subtilisin-like serine protease of Mycobacterium tuberculosis, is cell wall-associated and expressed during infection of macrophages

BACKGROUND: Exported proteases are commonly associated with virulence in bacterial pathogens, yet there is a paucity of information regarding their role in Mycobacterium tuberculosis. There are five genes (mycP1-5) present within the genome of Mycobacterium tuberculosis H37Rv that encode a family of secreted, subtilisin-like serine proteases (the mycosins). The gene mycP1 (encoding mycosin-1) was found to be situated 3700 bp (four ORF's) from the RD1 deletion region in the genome of the attenuated vaccine strain M. bovis BCG (bacille de Calmette et Guérin) and was selected for further analyses due to the absence of expression in this organism. RESULTS: Full-length, 50 kDa mycosin-1 was observed in M. tuberculosis cellular lysates, whereas lower-molecular-weight species were detected in culture filtrates. A similar lower-molecular-weight species was also observed during growth in macrophages. Mycosin-1 was localized to the membrane and cell wall fractions in M. tuberculosis by Western blotting, and to the cell envelope by electron microscopy. Furthermore, M. tuberculosis culture filtrates were shown to contain a proteolytic activity inhibited by mixed serine/cysteine protease inhibitors and activated by Ca(2+), features typical of the subtilisins. CONCLUSIONS: Mycosin-1 is an extracellular protein that is membrane- and cell wall-associated, and is shed into the culture supernatant. The protein is expressed after infection of macrophages and is subjected to proteolytic processing. Although proteolytically active mycosin-1 could not be generated recombinantly, serine protease activity containing features typical of the subtilisins was detected in M. tuberculosis culture filtrates

Evolution and expansion of the Mycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions

BACKGROUND: The PE and PPE multigene families of Mycobacterium tuberculosis comprise about 10% of the coding potential of the genome. The function of the proteins encoded by these large gene families remains unknown, although they have been proposed to be involved in antigenic variation and disease pathogenesis. Interestingly, some members of the PE and PPE families are associated with the ESAT-6 (esx) gene cluster regions, which are regions of immunopathogenic importance, and encode a system dedicated to the secretion of members of the potent T-cell antigen ESAT-6 family. This study investigates the duplication characteristics of the PE and PPE gene families and their association with the ESAT-6 gene clusters, using a combination of phylogenetic analyses, DNA hybridization, and comparative genomics, in order to gain insight into their evolutionary history and distribution in the genus Mycobacterium. RESULTS: The results showed that the expansion of the PE and PPE gene families is linked to the duplications of the ESAT-6 gene clusters, and that members situated in and associated with the clusters represent the most ancestral copies of the two gene families. Furthermore, the emergence of the repeat protein PGRS and MPTR subfamilies is a recent evolutionary event, occurring at defined branching points in the evolution of the genus Mycobacterium. These gene subfamilies are thus present in multiple copies only in the members of the M. tuberculosis complex and close relatives. The study provides a complete analysis of all the PE and PPE genes found in the sequenced genomes of members of the genus Mycobacterium such as M. smegmatis, M. avium paratuberculosis, M. leprae, M. ulcerans, and M. tuberculosis. CONCLUSION: This work provides insight into the evolutionary history for the PE and PPE gene families of the mycobacteria, linking the expansion of these families to the duplications of the ESAT-6 (esx) gene cluster regions, and showing that they are composed of subgroups with distinct evolutionary (and possibly functional) differences

Independent large scale duplications in multiple M. tuberculosis lineages overlapping the same genomic region

Mycobacterium tuberculosis, the causative agent of most human tuberculosis, infects one third of the world's population and kills an estimated 1.7 million people a year. With the world-wide emergence of drug resistance, and the finding of more functional genetic diversity than previously expected, there is a renewed interest in understanding the forces driving genome evolution of this important pathogen. Genetic diversity in M. tuberculosis is dominated by single nucleotide polymorphisms and small scale gene deletion, with little or no evidence for large scale genome rearrangements seen in other bacteria. Recently, a single report described a large scale genome duplication that was suggested to be specific to the Beijing lineage. We report here multiple independent large-scale duplications of the same genomic region of M. tuberculosis detected through whole-genome sequencing. The duplications occur in strains belonging to both M. tuberculosis lineage 2 and 4, and are thus not limited to Beijing strains. The duplications occur in both drug-resistant and drug susceptible strains. The duplicated regions also have substantially different boundaries in different strains, indicating different originating duplication events. We further identify a smaller segmental duplication of a different genomic region of a lab strain of H37Rv. The presence of multiple independent duplications of the same genomic region suggests either instability in this region, a selective advantage conferred by the duplication, or both. The identified duplications suggest that large-scale gene duplication may be more common in M. tuberculosis than previously considere

Detection of natural infection with Mycobacterium intracellulare in healthy wild-caught Chacma baboons (Papio ursinus) by ESAT-6 and CFP-10 IFN-γ ELISPOT tests following a tuberculosis outbreak

<p>Abstract</p> <p>Background</p> <p>Both tuberculous and non-tuberculous mycobacteria can cause infection in nonhuman primates (NHP), indicating the existence of potential zoonotic transmission between these animals and visitors to zoos or animal handlers in primate facilities. Screening of mycobacterial infections in NHP is traditionally done by tuberculin skin test (TST), which is unable to distinguish between pathogenic and non-pathogenic mycobacterial infections. In this study, we investigated the use of ESAT-6 and CFP-10 for detection of mycobacterial infections in a wild-caught baboon colony after one baboon died of tuberculosis (TB).</p> <p>Methods</p> <p>Peripheral blood lymphocytes for interferon-gamma enzyme-linked immunospot assay (IFN-γ ELISPOT) assay were obtained from TST positive baboons and those in contact with tuberculous baboons before being euthanased, autopsied and lung tissues taken for histology and mycobacterial culture.</p> <p>Results</p> <p>Both ESAT-6 and CFP-10 IFN-γ ELISPOT assays were able to detect early <it>M. tuberculosis </it>but also <it>M. intracellulare </it>infection. Although this indicates potential cross-reactivity with <it>M. intracellulare </it>antigens, the method was able to distinguish <it>M. bovis </it>BCG vaccination from <it>M. tuberculosis </it>infection. This assay performed better than the TST, which failed to detect one <it>M. tuberculosis </it>and two early <it>M. intracellulare </it>infections.</p> <p>Conclusion</p> <p>These results suggest that the IFN-γ ELISPOT assay could improve the detection of <it>M tuberculosis </it>infections when screening NHP. There is some doubt, however, concerning specificity, as the assay scored positive three animals infected with <it>M. intracellulare</it>.</p

Prevalence and distribution of non-tuberculous mycobacteria (NTM) in cattle, African buffaloes (Syncerus caffer) and their environments in South Africa

It has been hypothesized that a variety of non-tuberculous mycobacteria (NTM) species to which livestock and wildlife species are naturally exposed induce broadly cross-reactive anti-mycobacterial immune responses which interfere with current standard diagnostic assays. Non-tuberculous mycobacteria have also been implicated in Mycobacterium bovis-specific immune responsiveness, hence potentially the development of tuberculosis. Cattle and African buffaloes are both maintenance hosts of bovine tuberculosis (BTB) in South Africa, yet the effective diagnosis and control in these species may be hampered by adverse effects of NTM. As part of an investigation of the role of NTM in the immune responsiveness of cattle and African buffaloes to NTM, we conducted a countrywide survey to establish the prevalent NTM species and their distribution in the natural environments of these animals. A total of 1123 samples (water, soil, nasal and pharyngeal swabs) were collected for mycobacterium isolation. In addition, NTM isolated from tissue samples between 1991 and 2011 were included in the analysis. Mycobacteria were isolated from 56% of the samples from the countrywide survey. A total of 420 NTM isolates from soil, water, animal tissues and animal-derived swab samples were genotyped with the following results: 302 belonged to 40 known NTM species, 79 were found to be closely related to 23 known NTM species, and 38 isolates were found to be potentially novel species that are not currently listed in the RIDOM and NCBI BLAST databases. The four NTM species or closely related groups most frequently isolated in this survey included Mycobacterium terrae (11.2% of isolates), a group of mycobacteria closely related to Mycobacterium moriokaense (referred to as M. moriokaense-like) (8.1% of isolates), Mycobacterium nonchromogenicum (7.4% of isolates) and Mycobacterium vaccae/M. vanbaalenii (5.2% of isolates). The phylogenetic analysis of the M. moriokaense-like isolates, based on the 16S rRNA sequences, revealed at least eight clusters, possibly associated with eight different NTM species. Our findings provide account of NTM species diversity and associated prevalences in cattle and African buffaloes and their environments in South Africa. The identification of the most prevalent NTM species in this study will allow for a targeted investigation of their effects on host immune responses.WOTRO Integrated Programme 2008http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1865-1682hb2014ab201

Assessing the progress of Mycobacterium tuberculosis H37Rv structural genomics

Tuberculosis threatens human health nowhere more than in developing countries with large malnourished and/or immune-compromised (e.g. HIV infected) populations. The etiological agent, Mycobacterium tuberculosis (Mtb), is highly infectious and current interventions demonstrate limited ability to control the epidemic in particular of drug resistant Mtb strains. New drugs and vaccines are thus urgently required. Structural biologists are critical to the TB research community. By identifying potential drug targets and solving their three dimensional structures they open new avenues of identifying potential inhibitors complementing the screening of novel compounds and the investigation of Mtb's molecular physiology by pharmaceutical companies and academic researchers. Much effort has gone into structurally elucidating the Mtb proteome though much remains to be done with progress primarily limited by technological constraints. We review the currently available data for Mtb H37Rv to extract the lessons they have taught us.http://intl.elsevierhealth.com/journals/tubehb201