Emerg Infect DisEIDEmerging Infectious Diseases1080-60401080-6059Centers for Disease Control and Prevention19046525263461007-113510.3201/eid1412.071135Dispatch Mycobacterium bovis Strains Causing Smear-Positive Human Tuberculosis, Southwest IrelandM. bovis and Human TB, Southwest IrelandOjoOlabisiSheehanStellaCorcoranG. DanielOkkerMelissaGoverKarenNikolayevskyVladyslavBrownTimothyDaleJamesGordonStephen V.1DrobniewskiFrancisPrenticeMichael B.University College Cork, Cork, Ireland (O. Ojo, M.B. Prentice)Cork University Hospital, Cork (S. Sheehan, G.D. Corcoran, M.B. Prentice)Veterinary Laboratories Agency Weybridge, Surrey, UK (M. Okker, K. Gover, J. Dale, S.V. Gordon)Health Protection Agency Barts and the London School of Medicine, London, UK (V. Nikolayevsky, T. Brown, F. Drobniewski)Current affiliation: University College Dublin, Dublin, Ireland.Address for correspondence: Michael B. Prentice, Department of Microbiology, University College Cork, Cork, Ireland; email: m.prentice@ucc.ie122008141219311934

Mycobacterium bovis caused 3% of human tuberculosis cases in southwest Ireland during 1998–2006. Of 11 M. bovis strains genotyped, 9 belonged to common animal spoligotypes. Seven strains were from sputum and potential sources of human-centered disease transmission. Ten-locus variable-number tandem repeat typing gave unique strain profiles and would detect disease outbreaks.

Keywords: Mycobacterium bovistuberculosismolecular epidemiologyrespiratory tract infectionsdispatch

Bovine tuberculosis occurs worldwide (13). It is caused by Mycobacterium bovis, a cattle-adapted member of the M. tuberculosis complex. M. bovis has the broadest host range of pathogenic mycobacteria, infecting domestic and wild mammals, and is classified as a Hazard Group 3 infectious agent (1). Human infection follows ingestion of unpasteurized milk or inhalation of droplet nuclei (1). In many countries, the risk for M. bovis infection in humans has been reduced by a test-and-slaughter program in which infected cattle are identified and culled. This program has eradicated M. bovis cattle infection from 11 states of the European Union (3). However, the Republic of Ireland and its neighbor the United Kingdom have failed to eradicate bovine tuberculosis (1,4). In the 1980s, 4%–6% of all cases of laboratory-confirmed tuberculosis in southwest Ireland were caused by M. bovis (5). Our study results suggest that this remains a problem in Ireland.

Molecular typing systems for pathogenic mycobacteria are important for epidemiologic control because they enable case-linking and outbreak tracing (6). We report a molecular epidemiology study that used spoligotyping, mycobacterial interspersed repetitive units–variable-number tandem repeat (MIRU-VNTR) typing, and region of difference (RD) typing of M. bovis strains isolated from human residents of Ireland.

The Study

During 1998–2006, the microbiology laboratory at Cork University Hospital obtained M. tuberculosis complex isolates from 501 patients (equivalent to 68.5% of notified cases) residing in southwest Ireland (counties Cork and Kerry); 15 were M. bovis isolates (3%). Eleven of these M. bovis strains were available for testing. Seven additional isolates obtained over this period (from inoculation abscesses) were identified as M. bovis BCG and not analyzed. Strains were identified as M. tuberculosis complex by using Accuprobe (Gene-Probe, San Diego, CA, USA) and as potential M. bovis strains by pyruvate dependence. Definitive identification was performed at the Mycobacterial Reference Unit in London and was based on absence of niacin production and nitrate reductase activity, thiophen-2-carboxylic acid hydrazide negativity, and pyrazinamide resistance (1). DNA extraction was performed as described (2). DNA controls were M. tuberculosis H37Rv and M. bovis AF2122/97. A 6-locus panel VNTR (exact tandem repeat [ETR]-A to ETR-F) (7) was used initially, then a 10-locus VNTR panel was used (Table 1) (8). Spoligotyping was as described by Kamerbeek (9). RD analysis was conducted by the method of Brosch et al. (10) for RD1, RD4, RD9, RD10, RD12, RDpan, RD17, N-RD17, and N-RD25. This study was reviewed and approved by the Clinical Research Ethics Committee of the Cork Teaching Hospitals (Ref ECM 3).

Heterogeneity of each MIRU-VNTR locus of <italic>Mycobacterium bovis</italic> isolates, southwest Ireland, 1998–2006*
VNTR locusIsolateNo. tandem repeat copies
HGDIAllelic
variants
01234567891011
1895QUB18950012800000000.473
2163aQUB11a0002000000450.693
2163bQUB11b0102800000000.473
2165ETR-A0000120800000.473
2461ETR-B0001180100000.494
2687MIRU240650000000000.552
2996MIRU260001172000000.604
3232QUB32321000000071110.625
3336QUB33360016300100000.674
4052QUB260021800000000.473

*MIRU, mycobacterial interspersed repetitive units; VNTR, variable-number tandem repeats; HGDI, Hunter Gaston Discriminatory Index; QUB, Queen’s University Belfast; ETR, exact tandem repeat.

Spoligotype signatures were recorded as binary numbers (1 and 0 denoting presence and absence of oligonucleotide spacer, respectively). Signatures were matched against the M. bovis spoligotype database (www.mbovis.org) and the global spoligotype database, SpolDB4. Unmatched spoligotypes were sent to the M. bovis spoligotype database curator for an authoritative name assignment (SB number). The discriminatory power of VNTR and spoligotyping was calculated by using the Hunter Gaston Discriminatory Index. Two or more spoligotype or VNTR patterns with 100% identity were considered a cluster.

Eight spoligotypes (Table 2) were identified among 11 isolates. Two clusters were identified: 3 strains of SB0140 (ST683) and 2 strains corresponding to SB0139 (ST680) (Table 2). SB0140 (also known as spoligotype A1 or ST1), is the most common spoligotype in animals in Ireland and the United Kingdom (1,11) (Table 2). SB0139 was previously detected as an isolate from a cow in Northern Ireland in 2000 (SpolDB4; R. Skuce, pers. comm.). Other spoligotypes we identified that had been previously reported in isolates from animals in Ireland were SB0130 (ST691), also described as D1 or ST7; SB0142 (ST679), also described as A2 or ST2; and SB1047 (not found in SpolDB4). Three strains were not found in either of the databases. These strains were assigned spoligotypes SB1185, SB1186, and SB1187 through the M. bovis spoligotype database. SB1186 and SB1187 differed from the ubiquitous SB140 by absence of spacer 35 and spacers 35 and 37, respectively. Previously reported SB0139, SB0142, and SB1047 differed from SB0140 by absence of spacers 33, 33–34, and 35–36, respectively. SB1185 differed from the established animal strain SB0130 by the absence of spacers 20–21.

Molecular characteristics of strains and clinical and demographic parameters of 11 patients from whom <italic>Mycobacterium bovis</italic> was isolated, southwest Ireland, 1998–2006*
Study no.SpoligotypeSpolDB4†Mbovis. org‡Other name§VNTR pattern¶Patient data
Sex/ age, ySample siteSputum smear
HB002■■□■■□■□□□□□■■■□■■■■■■■■■■■■■■■■■■■■■■□□□□□ST683SB0140A14-11-4-7-5-
1-5-8-3-2F/81Sputum+
HB008■■□■■□■□□□□□■■■□■■■■■■■■■■■■■■■■■■■■■■□□□□□ST683SB0140A13-10-4-5-5-
1-5-8-3-4F/86Sputum
HB010■■□■■□■□□□□□■■■□■■■■■■■■■■■■■■■■■■■■■■□□□□□ST683SB0140A14-3-4-7-5-
1-5-8-3-4F/33Sputum+
HB011■■□■■□■□□□□□■■■□■■■■■■■■■■■■■■■■□■■■■■□□□□□ST680SB0139NA4-3-4-7-3-
1-5-8-3-4F/29Sputum+
HB012■■□■■□■□□□□□■■■□■■■■■■■■■■■■■■■■□■■■■■□□□□□ST680SB0139NA4-10-4-7-4-
1-5-8-3-4M/62Sputum+
HB006■■□■■□■□□□□□■■■□■■■■■■■■■■■■■■■■■■□■■■□□□□□NDSB1186NA4-11-4-5-5-
2-5-9-4-4M/60UrineNA
HB007■■□■■□■□□□□□■■■□■■■■■■■■■■■■■■■■■■□□■■□□□□□NDSB1047NA4-10-3-7-7-
2-4-x-4-2F/74Neck abscessNA
HB003■■□■■□■□□□□□■■■□■■■■■■■■■■■■■■■■□□■■■■□□□□□ST679SB0142D13-11-4-7-5-
1-6-8-3-4M/37Spinal disc aspirateNA
HB009■■□■■□■□□□□□■■■□■■■■■■■■■■■■■■■■■■□■□■□□□□□NDSB1187NA4-11-4-7-5-
2-5-10-4-4F/80Sputum+
HB001■■□■■■■■□■□■■■■□■■■■■■■■■■■■■■■■■■■■■■□□□□□ST691SB130A22-11-3-7-5-
2-3-8-2-3M/16Sputum+
HB005■■□■■■■■□■□■■■■□■■■□□■■■■■■■■■■■■■■■■■□□□□□NDSB1185NA4-10-1-4-5-
2-6-11-7-4M/68Testis biopsy sampleNA

*VNTR, variable number tandem repeats; +, smear positive by auramine and Ziehl-Neelsen stains; −, smear negative by auramine and Ziehl-Neelsen stains; NA, not applicable; ND, not described; x, not determined.
†Spoligotype designation as described in the SpolDB4 database (www.pasteur-guadeloupe.fr/tb).
‡Spoligotype assignment as described in the Mbovis.org database (www.mbovis.org).
§Spoligotype assignment (11).
¶VNTR profile based on a 10-loci scheme for M. bovis strains from Ireland in this order: QUB1895, QUB11a, QUB11b, ETR-A, ETR-B, MIRU 24, MIRU 26, QUB 3232, QUB 3336, and QUB26 (8).

Six-locus VNTR (ETR-A–ETR-F) showed little variation. The predominant profile (5 strains) was 7-5-5-4*-3-3.1. All strains except CUH-HB005 had ETR A–F profiles, matching spoligotype SB0140 strains isolated from cattle in England and Wales (12). A 10-locus expanded panel derived from loci recently described for Irish zoonotic M. bovis strains (8) improved strain discrimination by producing different profiles for every isolate (Table 1). Hence, VNTR typing was able to split the clustered spoligotypes into individual profiles. Analysis of regions of difference confirmed that none of the strains were derived from M. bovis BCG.

Conclusions

A recent outbreak report describing sputum-positive M. bovis disease transmitted by person-to-person contact (6) underlines the need for precise genetic markers of M. bovis to aid epidemiologic traceback. We studied strains of M. bovis isolated from humans in the Republic of Ireland, and we have defined an optimal set of markers using a combination of spoligotyping and VNTR. We detected a group of isolates (Table 2) of spoligotype SB0140, which is predominant in animal strains of M. bovis reported from the Republic of Ireland (51.8% of isolates) (11) and the United Kingdom (1). It forms the single largest group of M. bovis strains isolated from humans in the United Kingdom (30%) (1). This group was not reported in a recent series of M. bovis isolates from humans in Italy (13) or France (14). Indeed, none of the spoligotypes in our survey and these reports overlap. Predominant strains by spoligotype in animals and those infecting humans in the same country are known to be linked (1,13,14). We found 3 novel spoligotypes similar to SB0140 in a small group of patients, showing that a wider variety of strains infect humans than animals, as described in similar studies (1,2,13).

In our study, 81% of patients infected were >30 years of age (Table 2), comparable with findings of a previous survey of the southwest Ireland population (5). Primary infection of this group is likely to have been several decades before diagnosis, and our isolates probably represent reactivation of disease acquired earlier in life, effectively a record of past prevalence in animals. Spoligotype SB0140 strains were isolated from 2 patients who were >80 years of age, showing that the current predominance of SB0140 in animals (11) is therefore of long duration in Ireland, potentially going back 8 decades.

Molecular typing by insertion sequence (IS) 6110–based restriction fragment length polymorphism (RFLP) is inadequate for M. bovis because of low copy numbers of IS6110. A combination of MIRU-VNTR and spoligotyping gives better discrimination than the RFLP method (15). Traditional 6-locus VNTR (ETR-A–ETR-F) has been described for typing of M. tuberculosis complex strains (7) including M. bovis (2), but an expanded panel with an additional 10 loci applied to our strains greatly improved discrimination and enabled individual identification of each isolate.

Seven of our 11 isolates were from sputum, and 6 were detected on direct smear with potential for transmission. A VNTR-typing scheme based on the loci established on M. bovis isolates from animals (8) would detect M. bovis clusters derived from foodborne outbreaks or horizontal transmission of disease between humans in Ireland. Our study provides ways to markedly improve the ability to identify and contact-trace future clusters of M. bovis infection in humans.

Suggested citation for this article: Ojo O, Sheehan S, Corcoran GD, Okker M, Gover K, Nikolayevsky V, et al. Mycobacterium bovis strains causing smear-positive human tuberculosis, southwest Ireland. Emerg Infect Dis [serial on the Internet]. 2008 Dec [date cited]. Available from http://www.cdc.gov/EID/content/14/12/1931.htm

Acknowledgment

We thank Margaret O’Sullivan for details of the numbers of cases of tuberculosis notified in Cork and Kerry Counties.

O.O. was funded by a Department of Microbiology Studentship, a University College Cork Non–European Union Fee Waiver scholarship, and a Society for Applied Microbiology Anniversary Fellowship.

Mr Ojo was formerly associated with the College of Medicine, University of Ibadan, Ibadan, Nigeria. He is currently a doctoral candidate at the Microbiology Department, University College Cork, Cork, Ireland. His research focuses on molecular epidemiology and phylogenetic analysis of M. tuberculosis complex local clinical isolates.

Referencesde la Rua-Domenech R Human Mycobacterium bovis infection in the United Kingdom: incidence, risks, control measures and review of the zoonotic aspects of bovine tuberculosis.Tuberculosis (Edinb) 2006;86:77109 10.1016/j.tube.2005.05.00216257579Cadmus S, Palmer S, Okker M, Dale J, Gover K, Smith N, Molecular analysis of human and bovine tubercle bacilli from a local setting in Nigeria.J Clin Microbiol 2006;44:2934 10.1128/JCM.44.1.29-34.200616390943Reviriego Gordejo FJ, Vermeersch JP Towards eradication of bovine tuberculosis in the European Union.Vet Microbiol 2006;112:1019 10.1016/j.vetmic.2005.11.03416388921More SJ, Good M The tuberculosis eradication programme in Ireland: a review of scientific and policy advances since 1988.Vet Microbiol 2006;112:23951 10.1016/j.vetmic.2005.11.02216337345Cotter TP, Sheehan S, Cryan B, O’Shaughnessy E, Cummins H, Bredin CP Tuberculosis due to Mycobacterium bovis in humans in the south-west region of Ireland: is there a relationship with infection prevalence in cattle?Tuber Lung Dis 1996;77:5458 10.1016/S0962-8479(96)90053-29039448Evans JT, Smith EG, Banerjee A, Smith RM, Dale J, Innes JA, Cluster of human tuberculosis caused by Mycobacterium bovis: evidence for person-to-person transmission in the UK.Lancet 2007;369:12706 10.1016/S0140-6736(07)60598-417434402Frothingham R, Meeker-O’Connell WA Genetic diversity in the Mycobacterium tuberculosis complex based on variable numbers of tandem DNA repeats.Microbiology 1998;144:1189969611793Roring S, Scott AN, Glyn Hewinson R, Neill SD, Skuce RA Evaluation of variable number tandem repeat (VNTR) loci in molecular typing of Mycobacterium bovis isolates from Ireland.Vet Microbiol 2004;101:6573 10.1016/j.vetmic.2004.02.01315201034Kamerbeek J, Schouls L, Kolk A, van Agterveld M, van Soolingen D, Kuijper S, Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology.J Clin Microbiol 1997;35:907149157152Brosch R, Gordon SV, Marmiesse M, Brodin P, Buchrieser C, Eiglmeier K, A new evolutionary scenario for the Mycobacterium tuberculosis complex.Proc Natl Acad Sci U S A 2002;99:36849 10.1073/pnas.05254829911891304Costello E, O’Grady D, Flynn O, O’Brien R, Rogers M, Quigley F, Study of restriction fragment length polymorphism analysis and spoligotyping for epidemiological investigation of Mycobacterium bovis infection.J Clin Microbiol 1999;37:32172210488180Smith NH, Dale J, Inwald J, Palmer S, Gordon SV, Hewinson RG, The population structure of Mycobacterium bovis in Great Britain: clonal expansion.Proc Natl Acad Sci U S A 2003;100:152715 10.1073/pnas.203655410014657373Lari N, Rindi L, Bonanni D, Tortoli E, Garzelli C Molecular analysis of clinical isolates of Mycobacterium bovis recovered from humans in Italy.J Clin Microbiol 2006;44:421821 10.1128/JCM.01216-0616988015Mignard S, Pichat C, Carret G Mycobacterium bovis infection, Lyon, France.Emerg Infect Dis 2006;12:1431317073096Supply P, Allix C, Lesjean S, Cardoso-Oelemann M, Rusch-Gerdes S, Willery E, Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis.J Clin Microbiol 2006;44:4498510 10.1128/JCM.01392-0617005759