Study of restriction fragment length polymorphism analysis and spoligotyping for epidemiological investigation of Mycobacterium bovis infection

Abstract

Restriction fragment length polymorphism (RFLP) analysis with probes derived from the insertion element IS6110, the direct repeat sequence, and the polymorphic GC-rich sequence (PGRS) and a PCR-based typing method called spacer oligonucleotide typing (spoligotyping) were used to strain type Mycobacterium bovis isolates from the Republic of Ireland. Results were assessed for 452 isolates which were obtained from 233 cattle, 173 badgers, 33 deer, 7 pigs, 5 sheep, and 1 goat. Eighty-five strains were identified by RFLP analysis, and 20 strains were identified by spoligotyping. Twenty percent of the isolates were the most prevalent RFLP type, while 52% of the isolates were the most prevalent spoligotype. Both the prevalent RFLP type and the prevalent spoligotype were identified in isolates from all animal species tested and had a wide geographic distribution. Isolates of some RFLP types and some spoligotypes were clustered in regions consisting of groups of adjoining counties. The PGRS probe gave better differentiation of strains than the IS6110 or DR probes. The majority of isolates from all species carried a single IS6110 copy. In four RFLP types IS6110 polymorphism was associated with deletion of fragments equivalent in size to one or two direct variable repeat sequences. The same range and geographic distribution of strains were found for the majority of isolates from cattle, badgers, and deer. This suggests that transmission of infection between these species is a factor in the epidemiology of M. bovis infection in Ireland.

FIG. 1

Correlation between IS6110 and DR types associated with deletion of fragments corresponding in size to one or two DVRs. IS6110 type A1 was the most prevalent IS6110 type. The larger fragments of IS6110 patterns A2 and A3 were approximately 70 and 140 bp, respectively, less than the 3.6-kb fragment of IS6110 pattern A1. The smaller fragments of IS6110 patterns B2 and B1 were approximately 70 and 140 bp, respectively, less than the 1.9-kb fragment of IS6110 pattern A1. DR type A was the most prevalent DR type. The largest fragments of DR patterns B and G were approximately 70 and 140 bp, respectively, less than the largest fragment of DR pattern A. The second largest fragments of DR patterns K and C were approximately 70 and 140 bp, respectively, less than the second largest fragment of DR pattern A. There was a correlation between IS6110 type A2 and DR type B, between IS6110 type A3 and DR type G, between IS6110 type B2 and DR type K, and between IS6110 type B1 and DR type C.

FIG. 2

Schematic representation of 20 spoligotype patterns identified among 452 M. bovis isolates. The 43 spacer oligonucleotides are numbered as described by Kamerbeek et al. (11).