MOLECULAR CHARACTERIZATION OF MYCOBACTERIUM AVIUM SUBSP. AVIUM FROM ANIMALS IN CROATIA USING IS901 RFLP AND MIRU-VNTR TYPING

Mycobacterium (M.) avium subsp. avium, the causative agent of avian tuberculosis, primarily affects the birds but may often be isolated from granulomatous lesions in pigs and occasionally from cattle and other animals. In this study, a total of nine M. avium subsp. avium isolates collected between 2001 and 2006 from poultry (n=4), wild boars (n=2), pigs (n=2) and cattle (n=1) were investigated by IS907 restriction fragment length polymorphism (RFLP) analysis using two restriction endonucleases (PvuII and PstI) and by mycobacterial interspersed repetitive units - variable-number tandem repeat (MIRU-VNTR) typing. Digestion with the restriction endonuclease PvuII resulted in three RFLP types F, Q and M. Digestion with PstI was successfully accomplished in eight isolates demonstrating four RFLP types A29, A31, A32 and A33, of which the last three have not been described before. Combination of PvuII and PstI restriction patterns revealed four RFLP types F-A29, F-A31, F-A32 and M-A33, respectively. No epizootiological connection was found among the isolates expressing the predominant RFLP type F-A29, which was discovered in pig, wild boar and poultry. MIRU-VNTR typing resulted in four MIRU-VNTR types; among them, two were regarded as new. The most frequent type 34131127 was detected in four isolates from wild boars, pig and poultry. The combination of both typing methods revealed seven distinct RFLP/MIRU-VNTR genotypes; among them, six were unique. This work represents the first genotyping research of M. avium subsp. avium strains isolated from different animal species in Croatia. Notwithstanding the small number of investigated isolates, the results indicate a relatively high genetic diversity of M. avium subsp. avium in animals and suggest a combination of RFLP and MIRU-VNTR typing as a suitable approach to genotyping of M. avium subsp. avium isolates.