Spoligotype diversity of Mycobacterium bovis strains isolated in France from 1979 to 2000

Abstract

The molecular fingerprints of 1,349 isolates of Mycobacterium bovis received between 1979 and August 2000 at Agence Française de Sécurité Sanitaire des Aliments (Afssa) have been obtained by spoligotyping. The majority of the isolates (1,266) were obtained from cattle living in France. An apparently high level of heterogeneity was observed between isolates. One hundred sixty-one spoligotypes were observed in total, of which 153 were from French isolates. The two predominant spoligotypes, designated BCG-like and GB54, accounted for 26 and 12% of the isolates, respectively. In addition, 84% of the spoligotypes were found fewer than 10 times. Analysis of the results by clustering and parsimony-based algorithms revealed that the majority of the spoligotypes were closely related. The predominant spoligotype was identical to that of the vaccine strain Mycobacterium bovis BCG, which was isolated in France at the end of the 19th century. Some spoligotypes were closely associated with restricted geographical areas. Interestingly, some spoligotypes, which were frequently observed in France, were also observed in neighboring countries. Conversely, few spoligotypes were common to France and England, and those that were shared were observed at very different frequencies. This last point illustrates the potential role for an international data bank, which could help trace the spread of M. bovis across national borders.

FIG. 1

Geographical distribution of the French isolates and spoligotypes. (Left) Number of isolates and number of spoligotypes per department. For each department, the shade of grey indicates the number of isolates for which a spoligotype was identified; the number on the map indicates the number of spoligotypes found in that region. (Right) Dominant spoligotype per department. “None” corresponds to departments where two or more spoligotypes were codominant (i.e., were represented by the same number of isolates).

FIG. 2

Spoligotype phenogram obtained from 1,349 M. bovis isolates and corresponding patterns. For each spoligotype, the spacer deletions are indicated. Because all spoligotypes share the BCG-like spoligotype deletions (spacers 3, 9, 16, 39 to 43), these deletions are not included in each spoligotype description. Therefore, for example, the AN5 description (5, 11–12, 22) indicates the deletions of spacers 3, 5, 9, 11 to 12, 16, 22, and 39 to 43. The phenogram was generated using the UPGMA clustering method, with the between-spoligotypes distances being Dice coefficients. The scale indicates dissimilarity proportions. Due to the length of the phylogram, it has been fractionated into two parts, the upper right part representing the continuation of the lower left part.

FIG. 3

Spoligotype phylogram obtained from 1,349 M. bovis isolates. Due to the length of the phylogram, it has been fractionated into two parts, the upper right part representing the continuation of the lower left part. For each spoligotype, the spacers' deletions can be found in the phenogram (Fig. 2). The phylogram was generated using PAUP*.

FIG. 4

F009 and F015 groups. Trees for the F009 group (A1) and the F015 group (A2) were obtained using cluster analysis as done for Fig. 2. (B1 and B2) Trees were obtained using PAUP*. (B1) The tree for the F009 group was done as the one for Fig. 3. (B2) F015 group. The proposed evolutionary tree was derived from the phylogram (Fig. 3) and from geographical distribution. The parental spoligotype is indicated in plain letters (excluding typical M. bovis deletions); successive deletions are indicated in italics. (C1 and C2) Geographical distributions of the isolates of the F009 group (C1) and the F015 group (C2) are shown. Each dot corresponds to one isolate randomly positioned inside the department from which the isolate originated.