Infection, Genetics and Evolution 12 (2012) 877–882

Contents lists available at SciVerse ScienceDirect

Infection, Genetics and Evolution

journal homepage: www.elsevier .com/locate /meegid

A database for animal tuberculosis (mycoDB.es) within the contextof the Spanish national programme for eradication of bovine tuberculosis

Sabrina Rodriguez-Campos a,b,1, Sergio González a,1, Lucía de Juan a,b, Beatriz Romero a, Javier Bezos a,Carmen Casal a, Julio Álvarez c, Isabel G. Fernández-de-Mera a,b, Elena Castellanos a, Ana Mateos a,b,José L. Sáez-Llorente d, Lucas Domínguez a,b, Alicia Aranaz a,b,⇑, The Spanish Network on SurveillanceMonitoring of Animal Tuberculosis 2

a Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense de Madrid, 28040 Madrid, Spainb Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spainc Instituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM-JCCM), Ronda de Toledo s/n, 13071 Ciudad Real, Spaind Subdirección General de Sanidad de la Producción Primaria, Dirección General de Recursos Agrícolas y Ganaderos, Ministerio de Medio Ambiente, y Medio Rural y Marino, 28071Madrid, Spain

a r t i c l e i n f o

Article history:Available online 19 October 2011

Keywords:Animal tuberculosisEpidemiologyMolecular typingDatabase

1567-1348/$ - see front matter � 2011 Elsevier B.V. Adoi:10.1016/j.meegid.2011.10.008

⇑ Corresponding author at: Departamento de SanidE-mail address: alaranaz@vet.ucm.es (A. Aranaz).

1 These authors contributed equally to this work.2 Members of the Spanish Network on Surveillance an

staff of Government and Regional and Research LaborN. Abacens, I. Belanche, J. Gracia, S. Izquierdo, J.M. MaFernández, M.G. Gradillas, M. Gutiérrez, E. Sola (CantabrJ.A. Viñuelas (Castilla La Mancha), J.A. Anguiano, I. BurónRomero (Castilla y León), J. Gou (Cataluña), J.R. Puy (EusBaleares), J.M. Cámara, J. Carpintero, C. Delso, R. Díaz, E.Fernández (Navarra), F.J. Puértolas, J.F. Soldevilla (La RioFaculties of Veterinary Sciences [S. Lavin, G. MentaberrMendoza, A. Parra, (Universidad de Extremadura), E.F.Mazzucchelli, C. Novoa, X. Pickering, M. Pizarro, G. SantQuesada (Universidad de Las Palmas de Gran Canaria), MVidal (CRESA), J. Garrido, R. Juste (NEIKER), M. Galka, CAsturias)];I. Carpio (Unión de Criadores del Toro de LidiaJ.L. del Pozo (Madrid)];M.D. E. Gómez-Mampaso (H. RaFernández (A.D.S. Cabra del Guadarrama, Madrid), C. GilP.J. Mora (Ciudad Real), J.M. Amigo, N. Castro, V. ColladoSantos (Toledo),J. Fonbellida (Zamora), J. Rodríguez (Lainformation.

a b s t r a c t

Spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) analysis are the international standard techniques for molecular typing of members of theMycobacterium tuberculosis complex. To enable the exploitation of molecular typing data for epidemiolog-ical purposes, the creation of large databases is indispensable. Here we describe mycoDB.es, a databasefor animal tuberculosis which forms part of the Spanish national programme for eradication of bovinetuberculosis. This database has been created as an epidemiological tool at national level and contains spo-ligotype patterns of 17,273 isolates clustered in 401 different spoligotypes of Mycobacterium bovis,Mycobacterium caprae and M. tuberculosis. The database offers an overview of the present spoligotypes,to a lower extent also of MIRU-VNTR types, affected animal species and furthermore of the spatial distri-bution of these genotypes.

� 2011 Elsevier B.V. All rights reserved.

1. Introduction Mycobacterium bovis and Mycobacterium caprae. Both pathogens

The Mycobacterium tuberculosis complex (MTBC) includes themain aetiological agents of bovine and caprine tuberculosis,

ll rights reserved.

ad Animal, Facultad de Veterinaria

d Monitoring of Animal Tuberculosatories of Autonomous Communitilo (Aragón), M.F. Copano, E. Fernánia), V. Alcaide, J. Alia, J. Alonso, M.R., J. Cermeño, C. Domínguez, F. Fernákadi), E. Dorado, C. Sanz (ExtremadFernández, C. Fernández-Zapata, M.ja), C. Caballero, M. Lázaro (Valenciae (Universidad Autónoma de BarcelRodríguez-Ferri, O. González-Llamazurde, I. Simarro (Universidad Comp.V. Latre (Universidad de Zaragoza),. Sánchez, (P.N. Doñana), J. de la F);veterinary inspectors at abattoirsmón y Cajal, Madrid) and R. Borrás, F. Moneo-López, I. Larrauri (Albacet, J.L. Cumbreño, J.M. Finat, M.P. Herboratorios Syva), among many othe

can cause disease in a wide range of animal species and also in hu-mans. Since implementation of molecular fingerprinting of MTBCisolates (van Embden et al., 1993), several techniques have contrib-

, Universidad Complutense de Madrid, 28040 Madrid, Spain. Tel.: +34 913944006.

is are F. Garrido (Laboratorio Central de Sanidad Animal de Santa Fé, Granada, MARM),es [C. Fornell, J.M. Gómez, A. Jiménez, I. Muñoz, J.A. Téllez, E.J. Villalba (Andalucía),dez, I. Merediz (Asturias), P. Peláez, C. Pieltain, V. Vigo (Canarias), C. Fernández, F.M.Bermúdez, C. Fernández, P. García, E. Grande, F. Plaza, M.L. Rando, C. Rojas, A. Sánchez,ndez, A. Grau, S. Marques, O. Martín, C. Martínez, O. Mínguez, F. Moreno, F. Reviriego, I.ura), C. Calvo, D. Fernández, J.E. Mourelo (Galicia), C. Aguilo, M.J. Portau, C. Vidal (IslasGarcía, E. Pages, J.J. Urquía (Madrid), J. Pastor, C. Rivas (Murcia), J, Eguiluz, F. Eslava, C.)];A. Jacoste, M. Moreno (Patrimonio Nacional);academic and research members from

ona), I. García-Bocanegra, A. Perea (Universidad de Córdoba), A. García, J. Hermoso deares (Universidad de León), J. Blanco, M. Castaño, A.A. Díez-Guerrier, J.V. González, F.lutense de Madrid), A. Contreras, J. Sánchez (Universidad de Murcia), A. Fernández, O.];colleagues from research centers on Animal Health [M. Domingo, B. Pérez, S. López, D.uente, C. Gortázar, J. Vicente (IREC-CSIC), A. Espí, J.M. Prieto and A. Balseiro (SERIDA,[A.J. Domínguez, M. Fernández, J.M. Rubio (Ciudad Real), M. García, J. Guedeja, F. Osuna,(Facultad de Medicina, Valencia);and veterinary practitioners [P. Díez de Tejada, J.M.e), J. Cermeño, D. Martín (Badajoz), J.L. García (Burgos), A. Rodríguez, E. Sainz (Cáceres),ranz, E. Legaz, L.M. Portas, J. Rodríguez, L. Sánchez, J.M. Sebastián, T. Yuste (Madrid), A.rs, which have made this study possible by submitting samples and epidemiological

878 S. Rodriguez-Campos et al. / Infection, Genetics and Evolution 12 (2012) 877–882

uted to a more refined understanding of infectious disease epidemi-ology (Durr et al., 2000; Moonan et al., 2009). The most widely usedmolecular typing methods are nowadays spoligotyping (Kamerbeeket al., 1997) and MIRU-VNTR analysis (Frothingham and Meeker-O’Connell, 1998; Supply et al., 2000).

The first database for molecular typing results, created by theInstitute Pasteur Guadeloupe focused on human tuberculosis andincluded information on spoligotyping results of a limited numberof M. tuberculosis isolates from few geographical settings (Solaet al., 1999). By systematically collecting published spoligotypesa better representation of the worldwide M. tuberculosis diversitywas achieved (Sola et al., 2001; Filliol et al., 2003). The latest up-date of this database, SpolDB4 (Brudey et al., 2006), provides ahigher resolution image of the worldwide M. tuberculosis genomediversity and enables large-scale studies of epidemiology and pop-ulation genetics, including spoligotyping and MIRU-VNTR typingresults, in total 39,609 entries from 121 countries (http://www.pasteur-guadeloupe.fr/tb/bd_myco.html, consulted on 10thApril 2011). MIRU-VNTRplus (http://www.miru-vntrplus.org/MIRU/index.faces) is a new database created with a similar pur-pose (Allix-Béguec et al., 2008; Weniger et al., 2010). Apart fromthe spoligotyping pattern and a 24-locus MIRU-VNTR profile itadditionally includes information on single-nucleotide- andlarge-sequence-polymorphisms leading to optimal phylogeneticidentification. Both databases are freely accessible, but unfortu-nately their use for bovine TB epidemiology is limited due to alow number of animal isolates. In response to the need of an inter-national nomenclature for spoligotypes of animal origin, in 2003the M. bovis spoligotype database was created (Smith and Upton,in press, this issue). The implementation of a common nomencla-ture has provided an easy way to compare spoligotyping resultsand hence is of great value for the scientific community.

Herein we describe a national database for spoligotyping and, toa lower extent, MIRU-VNTR typing data of animal tuberculosiswhich has been created as a tool for molecular epidemiology with-in the Spanish national eradication programme for bovinetuberculosis.

2. Material and methods

2.1. Informatics

The mycoDB database is accessible via a XHTML/CSS web appli-cation implemented in php language running in an Apache WebServer (The Apache Software Foundation, MD) under Linux (The Li-nux Kernel Organization Inc., CA). The mycoDB database makes useof AJAX technologies (Asynchronous JavaScript and XML, OpenAjaxAlliance) and JavaScript must be activated in the browser.

On the main page, a brief description of the techniques is pro-vided and the user manual of the main functionalities can bedownloaded. Four possible queries are available: spoligotypesearch, MIRU-VNTR type search, isolate search and isolate maps.Each search summarizes the results in a box and displays the re-trieved data in a map showing their geographical distribution.Moreover, the detailed data are shown in a series of tables, namelyisolate table, MIRU-VNTR table, region table, animal table, spoligotable and data table. The query result pages use ALOV Map JavaApplet (TimeMap Project, University of Sydney) (Johnson, 2004),a web-based geographic viewer where maps are described byxml files, and Java software (Oracle Corporation, CA) is necessaryfor correct display. The smallest geographical unit considered inthe database and therefore shown in the maps is the municipality(basic local entity of the Spanish government). This geographicalinformation system (GIS) allows the visualisation of data in amap with variable size detail.

Access to the webpage is restricted to Official Veterinary Ser-vices and Regional Laboratories involved in the national bovinetuberculosis eradication programme. For demonstration purposesa demo version of mycoDB which contains simulated informationis available at http://www.mycodb.es; any resemblance with real-ity is purely coincidental.

2.2. Data collection

The 17,273 isolates included in the database were collectedfrom 1996 until 2011 in the national territory. The control of bo-vine tuberculosis is defined by the Spanish Programme on Eradica-tion on Bovine Tuberculosis which stipulates the inclusion ofspoligotyping results in the national database since 2008. The Re-gional Laboratories collaborate at different stages in the tuberculo-sis analysis, so that at the Centro de Vigilancia Sanitaria Veterinaria(VISAVET) different types of samples can be received: tissue sam-ples for culture (to date 37% of the samples), mycobacterial cul-tures (15%), DNA (47%) or spoligotyping data (1%). Monthlyupdates are performed to actualise mycoDB with the latest results.

At present, data include M. bovis (n = 15,898), M. caprae(n = 1369) and M. tuberculosis (n = 6) isolates from cattle (Bostaurus, n = 14,723), domestic goats (Capra aegagrus hircus, n = 1078),domestic pigs (Sus scrofa domestica, n = 179), sheep (Ovis aries, n = 15),alpacas (Lama pacos, n = 3), wild boar (Sus scrofa, n = 618), red deer (Cer-vus elaphus, n = 282), fallow deer (Dama dama, n = 348), Iberian lynxes(Lynx pardinus, n = 5), foxes (Vulpes vulpes, n = 4), chamois (Rupicaprarupicapra, n = 2), badgers (Meles meles, n = 9), cats (Felis silvestris catus,n = 3), a dog (Canis lupus familiaris, n = 1), a yellow-crowned parrot(Amazona ochrocephala, n = 1), a mouflon (Ovis musimon, n = 1, zoo ani-mal), and a peccary (Tayassusu sp. n = 1, zoo animal).

2.3. Molecular typing

Spoligotyping is performed according to Kamerbeek et al.(1997). Assignation of M. tuberculosis complex organisms to M. bo-vis, M. caprae or M. tuberculosis species is based on recognition ofspecific spoligotyping signatures plus additional identification ofabsence/presence of region of difference (RD)4 and RD9 (Mostowyet al., 2002; Brosch et al., 2002) for confirmation of the pre-assigned types.

MIRU-VNTR typing is carried out as per Frothingham et al.(1998) and Supply (2006). The loci used for MIRU-VNTR typingare the six loci recommended by VENoMYC (Supply, 2006)[VNTR2165 (ETR-A), VNTR2461 (ETR-B), VNTR580 (ETR-D, MIRU4),VNTR2163a (QUB11a), VNTR2163b (QUB11b) and VNTR3232(QUB3232)] plus additional three loci [VNTR3192 (ETR-E, MIRU31),VNTR2996 (MIRU26), VNTR4052 (QUB26)] (Supply et al., 2001;Skuce et al., 2002).

3. Results

3.1. Database organization

The database, mycoDB, is operating since 2007 and is availablein Spanish and English. Its use is intuitive, providing necessaryinformation on spoligotyping and MIRU-VNTR analysis on the wel-come page. A large amount of results included in this database hasbeen previously published and information of these publications isat hand on the publicly accessible demo version. The database al-lows users to recall data through four different types of request,namely spoligotype search, MIRU-VNTR type search, isolate searchand isolate maps.

Fig. 1. Maps obtained with spoligotype search showing differential distribution of spoligotypes in Spain. Map of the most frequent spoligotype SB0121 in comparison with amap of a geographically localised profile, SB1230, in Doñana National Park.

Fig. 2. Maps of the presence of Mycobacterium bovis in cattle and in different wildlife species in Spain. These have been obtained performing the isolate search selecting thedifferent animal species. (A) Cattle; (B) Wild boar; (C) Red deer/fallow deer; (D) other species (badger, Iberian lynx, fox and chamois).

S. Rodriguez-Campos et al. / Infection, Genetics and Evolution 12 (2012) 877–882 879

880 S. Rodriguez-Campos et al. / Infection, Genetics and Evolution 12 (2012) 877–882

3.2. Spoligotype search

The spoligotype search offers the possibility to explore a dis-tinct spoligotype pattern using the international nomenclature(SB or SIT number) or the internal nomenclature for spoligotypes.The search also offers a conversion tool from the binary code of aspoligotype pattern to the official or internal name and vice versa.This query provides an overview of the geographical distribution ofa spoligotype and of the animal species from which it has been iso-lated. To date, the isolates cluster in 401 different spoligotype pat-terns, 379 of which correspond to M. bovis strains, 17 to M. capraeand five to M. tuberculosis (Supplementary Table 1). The highestspoligotype diversity of M. bovis was found in cattle (350 spoligo-type patterns out of 13166 isolates) followed by wild boar (43types out of 450 isolates). As an example Fig. 1 shows maps ofthe distribution of the most frequent spoligotype in Spain,SB0121 (Rodríguez et al., 2010), and a geographically localised pro-file, SB1230, in Doñana National Park (Romero et al., 2008).

3.3. MIRU-VNTR type search

MIRU-VNTR type search retrieves every isolate with the re-quested type [entered either as number of repeats for each locusor as composite (numbers separated by hyphens)]. The MIRU-VNTRtype can include up to nine loci, if the result for a locus is unknown itappears as ‘‘?’’ and if the amplification failed as ‘‘F’’. Information in-cludes the M. tuberculosis complex organism, the spoligotype, plussame information as above. Currently, 389 M. bovis isolates weresubtyped into 157 different MIRU-VNTR types and 21 M. caprae in15 MIRU-VNTR types (Supplementary Table 2).

3.4. Isolate search

Isolate search allows for the localisation of isolates combiningthe above mentioned queries of typing patterns with independentor combined selection criteria such as year of isolation (from 1996to 2011) or periods of time, geographical spread [AutonomousCommunity (n = 17), province (n = 50), municipality (n = 8810)],and animal species (n = 18). As before, the information is shownin a table and displayed in a map. This query is useful for epidemi-ological studies since it enables the visualisation of transmissionbetween different regions or between wildlife and livestock;Fig. 2 shows the presence of M. bovis in different wildlife speciesin Spain.

This tool also performs queries regarding a mycobacterialspecies. At the moment, 1369 M. caprae isolates are registered,27.3% of which occurring in cattle and not necessarily linked to

Fig. 3. Distribution of Mycobacterium caprae in cattle and other species using the isolaretrieves 1369 isolates affecting seven animal species in a total of 197 municipalities.

the presence of goats (Fig. 3), a fact that underlines the observationof an increase of M. caprae in cattle in Spain (Rodríguez et al.,2011).

3.5. Isolate maps

Additionally, annual isolate maps that display the respectivepositive isolations centralised in the database are at the user’s dis-posal. These isolate maps can be used as a control for regional par-ticipation. Fig. 4 shows the isolate map for the year 2010 incomparison with the corresponding annual herd prevalence mapfor cattle.

4. Discussion

In this report we describe the development and the operation ofa database of genotyping of pathogens causing animal tuberculosis.Mycobacterium bovis, and to a lesser extent M. caprae, are success-ful animal pathogens in Spain; extensive human and economic ef-forts directed toward eradication achieved reduction of the herdprevalence from 2.11% to 1.51% in the last decade, costing over50 million € per year (unpublished data, MARM 2010).

This database has resulted from the commitment of numerouscontributors to the study of the epidemiology of animal tuberculo-sis. This is reflected on the annual isolate maps, which emphasisesthe progress in using and centralising molecular data made since1996 and the almost complete coverage of the national territory.Understanding the epidemiology of the infection should be a col-laborative task that benefits from the science-policy interface (TaskForce Bovine Tuberculosis Subgroup, 2006; Proceedings of the 4thInternational Conference on Mycobacterium bovis, 2006). It requiresthe collection of a large amount of information combining classicaland molecular data. Although a high number of entries difficult themanagement of data, the design of mycoDB intends to be intuitiveand user-friendly and offers the possibility of both large-scale andlocal enquiries. The exploitation of the database provides supportto the control programme. On the one hand, results have beenapplied to increase our knowledge of the demography of bothpathogens in a broad context (Rodríguez et al., 2010, 2011) andto corroborate transmission of M. bovis between wildlife anddomestic animals (Romero et al., 2008). On the other hand, itwould be useful to explain the relevance of small-scale transmis-sion at local or farm level, such as relative contribution of reactiva-tion of latent infection in the farm or exogenous infection throughcontact by trade or communal pastures. On long term basis, thiscould offer clues about the existence of isolates of atypicalbehaviour, for example emerging strains with increased clonal

te search tool by combining mycobacterial and animal species criteria. The search

Fig. 4. Comparison of the isolate map for the year 2010 shows that the collection of molecular typing information is consistent with the corresponding annual herdprevalence map for cattle.

S. Rodriguez-Campos et al. / Infection, Genetics and Evolution 12 (2012) 877–882 881

expansion, hypervariable isolates or isolates which may induce al-tered immune response that hinder diagnosis.

Although the database has been successful in many ways, we areaware that there are several possibilities for improvement. Thesearch based on the spoligotype search tool may in some situationspresent limitations, because the analysis is currently on perfectmatch basis. This could be misleading, because epidemiologicallyrelated, but not-matching isolates, for example due to deletion ofa DVR or block of DVRs or convergence due to homoplasy are notconsidered; in these cases, the combined interpretation of profilesalong with classical epidemiological information and the focused

additional use of MIRU-VNTR analysis are required to unravel thepotential link (Romero et al., 2006). However, the search for a cer-tain MIRU-VNTR type has only recently been implemented andtherefore contains less data. This search for spoligotype patternscould be modified to a less stringent criterion, in order to allowfor a degree of mutation in the profile to be compared.

Members of the M. tuberculosis complex are also serious humanpathogens. The mycoDB database can improve data exchange be-tween public health and veterinary services; therefore, it can helpto fully recognise the role as a zoonosis and therefore the value ofthe eradication programme in terms of human health. Since 1989

882 S. Rodriguez-Campos et al. / Infection, Genetics and Evolution 12 (2012) 877–882

the microbiological information system regarding cases of M. bovisand M. caprae affecting humans in Spain had been based on volun-tary weekly reporting by the laboratories; currently, notificationhas become compulsory. Comparison of patterns can include aswell the primary human pathogen M. tuberculosis. Isolations ofM. tuberculosis from samples of animal origin are rare findings(Prasad et al., 2005; Jenkins et al., 2011) though the first cases inSpain have recently been described (Romero et al., accepted).

Providing the GIS of the application with orthophotographs andparcel of land layers is one of the future objectives which will im-prove the visualisation of overlapping livestock and wildlife habi-tats and shared pastures.

In conclusion, the national database is being used as a valuabletool for animal tuberculosis epidemiology and forms one of thepillars of the current Spanish national eradication programme. Westrongly encourage the use of databases for centralisation of molec-ular typing results and emphasise the importance of a comprehen-sive collaboration between the veterinary and public health services.

Acknowledgements

The database on animal tuberculosis is financed by and in thepossession of the Spanish Ministry of the Environment, Rural andMarine Affairs (MARM). S. Rodriguez-Campos was financed byPhD studentship AP2006-01630 of the Spanish Ministry of Educa-tion. The continuous encouragement of L. Carbajo and B. Muñóz(MARM) is very much appreciated. We would like to acknowledgethe excellent technical assistance of C. Ancochea and E. Celeiro(Computer and Communication Unit, VISAVET Centre), F. Lozano,N. Moya, T. Alende, A. Gutiérrez, C. Viñolo, L. Guijarro, J. Gimenoand E. Mateos (Mycobacteria Unit, VISAVET Centre) and TragsatecS.A. (Tragsa Group, Madrid).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.meegid.2011.10.008.

References

Allix-Béguec, C., Harmsen, D., Weniger, T., Supply, P., Niemann, S., 2008. Evaluationand strategy for use of MIRU-VNTRplus, a multifunctional database for onlineanalysis of genotyping data and phylogenetic identification of Mycobacteriumtuberculosis complex isolates. J. Clin. Microbiol. 46, 2692–2699.

Brosch, R., Gordon, S.V., Marmiesse, M., Brodin, P., Buchrieser, C., Eiglmeier, K.,Garnier, T., Gutiérrez, C., Hewinson, G., Kremer, K., Parsons, L.M., Pym, A.S.,Samper, S., van Soolingen, D., Cole, S.T., 2002. A new evolutionary scenario forthe Mycobacterium tuberculosis complex. Proc. Natl. Acad. Sci. USA 99, 3684–3689.

Brudey, K., Driscoll, J.R., Rigouts, L., Prodinger, W.M., Gori, A., Al Hajoj, S.A., Allix, C.,Aristimuno, L., Arora, J., Baumanis, V., Binder, L., Cafrune, P., Cataldi, A., Cheong,S., Diel, R., Ellermeier, C., Evans, J.T., Fauville-Dufaux, M., Ferdinand, S., Garcia,d.V., Garzelli, C., Gazzola, L., Gomes, H.M., Gutierrez, M.C., Hawkey, P.M., vanHelden, P.D., Kadival, G.V., Kreiswirth, B.N., Kremer, K., Kubin, M., Kulkarni, S.P.,Liens, B., Lillebaek, T., Ho, M.L., Martin, C., Martin, C., Mokrousov, I., Narvskaia,O., Ngeow, Y.F., Naumann, L., Niemann, S., Parwati, I., Rahim, Z., Rasolofo-Razanamparany, V., Rasolonavalona, T., Rossetti, M.L., Rusch-Gerdes, S., Sajduda,A., Samper, S., Shemyakin, I.G., Singh, U.B., Somoskovi, A., Skuce, R.A., vanSoolingen, D., Streicher, E.M., Suffys, P.N., Tortoli, E., Tracevska, T., Vincent, V.,Victor, T.C., Warren, R.M., Yap, S.F., Zaman, K., Portaels, F., Rastogi, N., Sola, C.,2006. Mycobacterium tuberculosis complex genetic diversity: mining the fourthinternational spoligotyping database (SpolDB4) for classification, populationgenetics and epidemiology. BMC. Microbiol. 6, 23–25.

Durr, P.A., Clifton-Hadley, R.S., Hewinson, R.G., 2000. Molecular epidemiology ofbovine tuberculosis. II. Applications of genotyping. Rev. Sci. Tech. 19, 689–701.

Filliol, I., Driscoll, J.R., van, S.D., Kreiswirth, B.N., Kremer, K., Valetudie, G., Dang, D.A.,Barlow, R., Banerjee, D., Bifani, P.J., Brudey, K., Cataldi, A., Cooksey, R.C., Cousins,D.V., Dale, J.W., Dellagostin, O.A., Drobniewski, F., Engelmann, G., Ferdinand, S.,Gascoyne-Binzi, D., Gordon, M., Gutierrez, M.C., Haas, W.H., Heersma, H., Kassa-Kelembho, E., Ho, M.L., Makristathis, A., Mammina, C., Martin, G., Mostrom, P.,Mokrousov, I., Narbonne, V., Narvskaya, O., Nastasi, A., Niobe-Eyangoh, S.N.,Pape, J.W., Rasolofo-Razanamparany, V., Ridell, M., Rossetti, M.L., Stauffer, F.,Suffys, P.N., Takiff, H., Texier-Maugein, J., Vincent, V., De Waard, J.H., Sola, C.,

Rastogi, 2003. Snapshot of moving and expanding clones of Mycobacteriumtuberculosis and their global distribution assessed by spoligotyping in aninternational study. J. Clin. Microbiol. 41, 1963–1970.

Frothingham, R., Meeker-O’Connell, W.A., 1998. Genetic diversity in theMycobacterium tuberculosis complex based on variable numbers of tandemDNA repeats. Microbiology 144, 1189–1196.

Jenkins, A.O., Cadmus, S.I., Venter, E.H., Pourcel, C., Hauk, Y., Vergnaud, G., Godfroid,J., 2011. Molecular epidemiology of human and animal tuberculosis in Ibadan,Southwestern Nigeria. Vet. Microbiol. doi:10.1016/j.vetmic.2011.02.037.

Johnson, I., 2004. Putting Time on the Map. Using TimeMap™ for map animationand web delivery. GEO Informatics July/August, 26–29.

Kamerbeek, J., Schouls, L., Kolk, A., van Agterveld, M., van Soolingen, D., Kuijper, S.,Bunschoten, A., Molhuizen, H., Shaw, R., Goyal, M., van Embden, J., 1997.Simultaneous detection and strain differentiation of Mycobacterium tuberculosisfor diagnosis and epidemiology. J. Clin. Microbiol. 35, 907–914.

Moonan, P.K., Chatterjee, S.G., Lobue, P.A., 2009. The molecular epidemiology ofhuman and zoonotic Mycobacterium bovis: the intersection between veterinarymedicine and public health. Prev. Vet. Med. 88, 226–227.

Mostowy, S., Cousins, D., Brinkman, J., Aranaz, A., Behr, M.A., 2002. Genomicdeletions suggest a phylogeny for the Mycobacterium tuberculosis complex. J.Infect. Dis. 186, 74–80.

Prasad, H.K., Singhal, A., Mishra, A., Shah, N.P., Katoch, V.M., Thakral, S.S., Singh, D.V.,Chumber, S., Bal, S., Aggarwal, S., Padma, M.V., Kumar, S., Singh, M.K., Acharya,S.K., 2005. Bovine tuberculosis in India: potential basis for zoonosis.Tuberculosis (Edinb.) 85, 421–428.

Proceedings of the 4th International Conference on Mycobacterium bovis, Dublin,Ireland, 22–26 August 2005. 2006. Vet. Microbiol. 112, 89–391.

Rodríguez, S., Romero, B., Bezos, J., de Juan, L., Álvarez, J., Castellanos, E., Moya, N.,Lozano, F., González, S., Sáez-Llorente, J.L., Mateos, A., Domínguez, L., Aranaz, A.,2010. High spoligotype diversity within a Mycobacterium bovis population:clues to understanding the demography of the pathogen in Europe. Vet.Microbiol. 141, 89–95.

Rodríguez, S., Bezos, J., Romero, B., de Juan, L., Álvarez, J., Castellanos, E., Moya, N.,Lozano, F., Javed, M.T., Sáez-Llorente, J.L., Liébana, E., Mateos, A., Domínguez, L.,Aranaz, A., 2011. Mycobacterium caprae infection in livestock and wildlife.Spain. Emerg. Infect. Dis. 17, 532–535.

Romero, B., Aranaz, A., Juan, L., Álvarez, J., Bezos, J., Mateos, A., Gómez-Mampaso, E.,Domínguez, L., 2006. Molecular epidemiology of multidrug-resistantMycobacterium bovis isolates with the same spoligotyping profile as isolatesfrom animals. J. Clin. Microbiol. 44, 3405–3408.

Romero, B., Aranaz, A., Sandoval, A., Álvarez, J., de Juan, L., Bezos, J., Sánchez, C.,Galka, M., Fernández, P., Mateos, A., Domínguez, L., 2008. Persistence andmolecular evolution of Mycobacterium bovis population from cattle and wildlifein Doñana National Park revealed by genotype variation. Vet. Microbiol. 132,87–95.

Romero, B., Rodríguez, S., Bezos, J., Díaz, R., Copano, M.F., Merediz, I., Mínguez, O.,Marqués, S., Palacios, J.J., García de Viedma, D., Sáez, J.L., Mateos, A., Aranaz, A.,Domínguez, L., de Juan, L. Humans as the source of Mycobacterium tuberculosisinfection in cattle. Emerg. Infect. Dis. accepted.

Skuce, R.A., McCorry, T.P., McCarroll, J.F., Roring, S.M., Scott, A.N., Brittain, D.,Hughes, S.L., Hewinson, R.G., Neill, S.D., 2002. Discrimination of Mycobacteriumtuberculosis complex bacteria using novel VNTR-PCR targets. Microbiology 148,519–528.

Smith, N.H., Upton, P., in press. Naming spoligotype patterns for the RD9-deletedlineage of the Mycobacterium tuberculosis complex: www.Mbovis.org. Infect.Genet. Evol. This issue.

Sola, C., Devallois, A., Horgen, L., Maïsetti, J., Filliol, I., Legrand, E., Rastogi, N., 1999.Tuberculosis in the Caribbean: using spacer oligonucleotide typing tounderstand strain origin and transmission. Emerg. Infect. Dis. 5, 404–414.

Sola, C., Filliol, I., Gutiérrez, M.C., Mokrousov, I., Vincent, V., Rastogi, N., 2001.Spoligotype database of Mycobacterium tuberculosis: biogeographic distributionof shared types and epidemiologic and phylogenetic perspectives. Emerg. Infect.Dis. 7, 390–396.

Supply, P., Mazars, E., Lesjean, S., Vincent, V., Gicquel, B., Locht, C., 2000. Variablehuman minisatellite-like regions in the Mycobacterium tuberculosis genome.Mol. Microbiol. 36, 762–771.

Supply, P., Lesjean, S., Savine, E., Kremer, K., van Soolingen, D., Locht, C., 2001.Automated high-throughput genotyping for study of global epidemiology ofMycobacterium tuberculosis based on mycobacterial interspersed repetitiveunits. J. Clin. Microbiol. 39, 3563–3571.

Supply, P., 2006. Protocol and Guidelines for Multilocus Variable Number TandemRepeat Genotyping of M. bovis VENoMYC (Veterinary Network of LaboratoriesResearching into Improved Diagnosis and Epidemiology of MycobacterialDiseases) WP7 Workshop, October 19–22 2006, Toledo, Spain, pp.15–16. WP7Workshop VENoMYC Coordination Action EU SSPE-CT-2004-501903.

Task Force Bovine Tuberculosis Subgroup, Working Document on Eradication ofBovine Tuberculosis in the EU accepted by the Bovine tuberculosis subgroup ofthe Task Force on monitoring animal disease eradication, 2006. SANCO/10200/2006.

van Embden, J.D., Cave, M.D., Crawford, J.T., Dale, J.W., Eisenach, K.D., Gicquel, B.,Hermans, P., Martin, C., McAdam, R., Shinnick, T.M., 1993. Strain identificationof Mycobacterium tuberculosis by DNA fingerprinting: recommendations for astandardized methodology. J. Clin. Microbiol. 31, 406–409.

Weniger, T., Krawczyk, J., Supply, P., Niemann, S., Harmsen, D., 2010. MIRU-VNTRplus: a web tool for polyphasic genotyping of Mycobacterium tuberculosiscomplex bacteria. Nucleic Acids Res. 38, W326–W331.