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Journal of Clinical Microbiology, March 2009, p. 636-644, Vol. 47, No. 3
0095-1137/09/$08.00+0     doi:10.1128/JCM.01192-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Molecular Typing of Mycobacterium bovis Strains Isolated in Italy from 2000 to 2006 and Evaluation of Variable-Number Tandem Repeats for Geographically Optimized Genotyping{triangledown}

M. Beatrice Boniotti,1* Maria Goria,2 Daniela Loda,1 Annalisa Garrone,2 Alessandro Benedetto,2 Alessandra Mondo,3 Ernesto Tisato,4 Mariagrazia Zanoni,1 Simona Zoppi,2 Alessandro Dondo,2 Silvia Tagliabue,1 Stefano Bonora,3 Giorgio Zanardi,1 and M. Lodovica Pacciarini1

Centro Nazionale di Referenza per la Tubercolosi Bovina, Istituto Zooprofilattico Sperimentale Lombardia e Emilia-Romagna, Via Bianchi 9, 25124 Brescia, Italy,1 Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Via Bologna 148, 10154 Turin, Italy,2 Department of Infectious Diseases, University of Torino, Corso Svizzera 164, 10149 Turin, Italy,3 Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell'Università 10, 35020 Legnaro (PD), Italy4

Received 24 June 2008/ Returned for modification 15 August 2008/ Accepted 7 January 2009

Spoligotyping and exact tandem repeat (ETR) analysis of Mycobacterium bovis and M. caprae isolated strains has been routinely carried out in Italy since 2000 to obtain a database of genetic profiles and support traditional epidemiological investigations. In this study, we characterized 1,503 M. bovis and 57 M. caprae isolates obtained from 2000 to 2006 in 747 cattle herds mainly located in northern Italy. We identified 81 spoligotypes and 113 ETR profiles, while the combination of spoligotyping/ETR analysis differentiated 228 genotypes, with genotypic diversity indices of 0.70 (spoligotyping), 0.94 (ETR-A to -E typing), and 0.97 (spoligotyping/ETR-A to -E typing), respectively. Despite the high degree of resolution obtained, the spoligotyping/ETR methods were not discriminative enough in the case of genotypes characterized by the combination of SB0120, the predominant spoligotype in Italy, with the most common ETR profiles. To obtain a more informative subset of typing loci, 24 mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) markers were evaluated by analyzing a panel of 100 epidemiologically unrelated SB0120 isolates. The panel was differentiated into 89 profiles with an overall genotypic diversity of 0.987 that could be also achieved by using a minimal group of 13 loci: ETR-A, -B, and -E; MIRU 26 and 40; and VNTR 2163a, 2163b, 3155, 1612, 4052, 1895, 3232, and 3336. The allelic diversity index and the stability of single loci was evaluated to provide the most discriminative genotyping method for locally prevalent strains.

* Corresponding author. Mailing address: Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Via Bianchi 9, 25124 Brescia, Italy. Phone: (39)0302290273. Fax: (39)0302290360. E-mail: mariabeatrice.boniotti{at}bs.izs.it

{triangledown} Published ahead of print on 14 January 2009.

Journal of Clinical Microbiology, March 2009, p. 636-644, Vol. 47, No. 3
0095-1137/09/$08.00+0     doi:10.1128/JCM.01192-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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