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Browsing by Author "Larsen, Michelle H."

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    Draft Genome Sequence of the Mycobacterium tuberculosis Complex Pathogen M. mungi, Identified in a Banded Mongoose (Mungos mungo) in Northern Botswana
    Alexander, Kathleen A.; Larsen, Michelle H.; Robbe-Austerman, Suelee; Stuber, Tod P.; Camp, Patrick M. (American Society for Microbiology, 2016)
    Mycobacterium mungi, a member of the Mycobacterium tuberculosis complex, has emerged in wild banded mongoose in northern Botswana (1). Molecular examinations place the organism in wildlife-associated lineage six of the M. tuberculosis complex, which includes M. suricattae, infecting meerkats (Suricata suricatta) (2), and the dassie bacillus, infecting rock hyraxes (Procavia capensis) (3), with one reported case of infection in West Africa in a chimpanzee (Pan troglodytes) caused by a member of this complex (chimpanzee bacillus) (4). The pathogen is primarily transmitted between mongoose through an environmental pathway where infected secretions used in olfactory communication behaviors expose and invade the mongoose host through abrasions or injuries to the skin and/or nasal planum (5). In northern Botswana, banded mongoose have been the subject of intensive study since the pathogen emerged in 1999 (IACUC 13-164-FIW and Botswana Ministry of Environment, Wildlife and Tourism EWT 8/36/4 XXVI [24]). In 2013, postmortem samples were obtained opportunistically from a severely infected male mongoose. Because this Mycobacterium tuberculosis complex (MTBC) pathogen has not been successfully cultivated in vitro, wholegenome shotgun sequencing was performed directly from affected tissue. Several tissues from this mongoose were screened with a real-time IS6110 MTBC PCR (6), and a small portion (3 g) of the severely diseased liver with the lowest threshold cycle (CT) value (13.8) was homogenized thoroughly using a gentleMACSMtube (Milteny Biotec, San Diego, CA, USA). After hominization, 200 mg was placed in a 2.0-ml O-ring sealed microcentrifuge tube with a mixture of 1.0- and 0.1-mm glass beads, along with 400 µl of 1x Tris-EDTA buffer, and heated at 100°C for 30 min. Bead disruption was performed on a Mini-Beadbeater-96 (BioSpec Products, Bartlesville, OK, USA) for 2 min, the supernatant was purified with phenol-chloroform-isoamyl alcohol, and the aqueous layer was further purified through a spin column (Zymo Research, Irvine, CA, USA). Libraries were prepared using the Nextera XT kit and sequenced on a MiSeq for 2 x 250 paired-end reads. The total number of reads obtained was 1,324,968, with 1,208,783 (91.2%) reads mapping to M. tuberculosis H37Rv (GenBank accession no. NC_000962.3), for an estimated average depth of coverage of 120X. De novo alignments were performed using SeqMan NGen (DNAStar Lasergene, USA), and the reads were reduced to 130 contigs, for an estimated genome size of 4.4 Mb. We report here the first draft genome of the only knownMTBC species that has not been successfully cultured in vitro. Comparing this sequence to publicly available genomes, M. mungi has diverged by at least 623 single-nucleotide polymorphisms (SNPs) since sharing a common ancestor with M. suricattae (7).
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    Emerging Tuberculosis Pathogen Hijacks Social Communication Behavior in the Group-Living Banded Mongoose (Mungos mungo)
    Alexander, Kathleen A.; Sanderson, Claire E.; Larsen, Michelle H.; Robbe-Austerman, Suelee; Williams, Mark C.; Palmer, Mitchell V. (American Society for Microbiology, 2016-05)
    An emerging Mycobacterium tuberculosis complex (MTC) pathogen, M. mungi, infects wild banded mongooses (Mungos mungo) in Northern Botswana, causing significant mortality. This MTC pathogen did not appear to be transmitted through a primary aerosol or oral route. We utilized histopathology, spoligotyping, mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR), quantitative PCR (qPCR), and molecular markers (regions of difference [RDs] from various MTC members, including region of difference 1 [RD1] from M. bovis BCG [RD1(BCG)], M. microti [RD1(mic)], and M. pinnipedii [RD1(seal)], genes Rv1510 [RD4], Rv1970 [RD7], Rv3877/8 [RD1], and Rv3120 [RD12], insertion element IS1561, the 16S RNA gene, and gene Rv0577 [cfp32]), including the newly characterized mongoose-specific deletion in RD1 (RD1(mon)), in order to demonstrate the presence of M. mungi DNA in infected mongooses and investigate pathogen invasion and exposure mechanisms. M. mungi DNA was identified in 29% of nasal planum samples (n = 52), 56% of nasal rinses and swabs (n = 9), 53% of oral swabs (n = 19), 22% of urine samples (n = 23), 33% of anal gland tissue (n = 18), and 39% of anal gland secretions (n = 44). The occurrence of extremely low cycle threshold values obtained with qPCR in anal gland and nasal planum samples indicates that high levels of M. mungi can be found in these tissue types. Histological data were consistent with these results, suggesting that pathogen invasion occurs through breaks in the nasal planum and/or skin of the mongoose host, which are in frequent contact with anal gland secretions and urine during olfactory communication behavior. Lesions in the lung, when present, occurred only with disseminated disease. No environmental sources of M. mungi DNA could be found. We report primary environmental transmission of an MTC pathogen that occurs in association with social communication behavior. IMPORTANCE Organisms causing infectious disease evolve modes of transmission that exploit environmental and host conditions favoring pathogen spread and persistence. We report a novel mode of environmental infectious disease transmission that occurs in association with olfactory secretions (e.g., urine and anal gland secretions), allowing pathogen exposure to occur within and between social groups through intricate social communication behaviors of the banded mongoose host. The presence of M. mungi in these environmentally deposited secretions would effectively circumvent natural social barriers (e.g., territoriality), facilitating between-group pathogen transmission in the absence of direct physical contact, a rare occurrence in this highly territorial species. This work identifies an important potential mechanism of pathogen transmission of epidemiological significance in social species. We also provide evidence of a novel mechanism of pathogen transmission for the MTC complex, where pathogen movement in the environment and host exposure dynamics are driven by social behavior.