REGULAR ARTICLE Genotypic characterization by spoligotyping and VNTR typing of Mycobacterium bovis and Mycobacterium caprae isolates from cattle of Tunisia Hela Lamine-Khemiri & Remigio Martínez & Waldo Luis García-Jiménez & Jose Manuel Benítez-Medina & Maria Cortés & Inés Hurtado & Mohammed Salah Abassi & Imed Khazri & Mohammed Benzarti & Javier Hermoso-de-Mendoza Accepted: 30 September 2013 # Springer Science+Business Media Dordrecht 2013 Abstract This work is an approach to the molecular epide- miology of Mycobacterium tuberculosis complex (MTBC) bovine infections in Tunisia. A total of 35 MTBC isolates from both lateral retropharyngeal lymph node samples of cattle slaughtered in different Tunisian regions were geno- typed by spoligotyping and variable number tandem repeat typing (VNTR)-typing. Spoligotyping allowed to identify two profiles not previously registered, namely SB2024, a Mycobacterium caprae isolate from Nabeul Region (North East Tunisia), the first description of this species in the coun- try, and SB2025 (Mycobacterium bovis ) from Sfax Region (Southern Tunisia). A second M . caprae isolate with a spoligotyping profile previously described in Europe main- land, SB0418, was also isolated from a bovine of Sfax region. Both isolates suggest the possibility of a widespread distribu- tion of this species in the country. The predominant spoligotype was SB0120, present in all Tunisian regions se- lected for the study but Nabeul. Molecular typing also allowed to describe a mixed infection caused by two different M . bovis isolates (SB0120 and SB0848) in the same animal. VNTR typing was highly discriminant by testing a panel of six loci. Loci QUB3232 and QUB11b were the most discriminant, whereas ETR-D and QUB11a had the lower diversity index. The value of allelic diversity can significantly vary among countries; thus, it is important to standardize a panel of loci for future inter-laboratory comparisons. Although VNTR typing proved to be useful for an efficient discrimination among MTBC isolates, especially in combination with spoligotyping, further studies are needed in order to assess the genetic diver- sity of the MTBC in Tunisia. Keywords Bovine tuberculosis . Mycobacterium tuberculosis complex (MTBC) . Mycobacterium bovis . Mycobacterium caprae . Spoligotyping . VNTR Introduction Bovine tuberculosis (bTB) is a major worldwide animal health problem, which causes huge losses in almost all countries, affecting animal production and national and international trade of livestock, especially cattle. The main agent of this disease is Mycobacterium bovis (M . bovis ) and, to a lesser extent, by Mycobacterium caprae (M . caprae ), both mem- bers of the Mycobacterium tuberculosis complex (MTBC). As a classical member of this complex, M . bovis is zoonotic and both the animal and human health concerns, together with the terrible impact of bTB in the developing world, are the reasons for the enormous effort to eradicate the disease since decades (Wedlock et al. 2002). Tunisia is an endemic country for tuberculosis (TB) with an incidence of 22.4 per 100,000 person-years in 2010 (Direction des Soins de Santé de Base 2011). In the last years, there was an increase in the incidence of lymph node tuberculosis from 2.3 to 4 per 100,000 person-years between 1993 and 2005 in this H. Lamine-Khemiri : M. S. Abassi : I. Khazri Laboratory of Animal Health, Institute of Veterinary Research of Tunisia, Tunis, Tunisia R. Martínez : W. L. García-Jiménez : J. M. Benítez-Medina : M. Cortés : I. Hurtado : J. Hermoso-de-Mendoza (*) Red de Grupos de Investigación Recursos Faunísticos, Facultad de Veterinaria, Universidad de Extremadura, Avenida de la Universidad s/n, 10003 Cáceres, Spain e-mail: jhermoso@unex.es M. Benzarti Laboratories of Pathology and Infectious Diseases, National School of Veterinary Medicine of Sidi Thabet, Sidi Thabet, Tunisia Trop Anim Health Prod DOI 10.1007/s11250-013-0488-y