Technical note: molecular typing of Corynebacterium bovis isolates by pulsed-field gel electrophoresis

A typing method for Corynebacterium bovis based on pulsed-field gel electrophoresis (PFGE) is described and applied to type 162 strains isolated from mastitic milk from 57 Friesian dairy herds in northern Spain. Isolates were identified to the species level using the API-Coryne system and a polymerase chain reaction-restriction fragment length polymorphism of the 16S rDNA amplicon. Cultures in logarithmic phase were harvested, mixed with agarose, and treated with lisozyme and proteinase K to release intact genomic DNA that was digested with XbaI and submitted to PFGE. Seven different pulsotypes were identified; none of them were similar to that of the reference strain (ATCC 7715). The most frequent pulsotype was pattern I (51.9%) followed by pattern II (29.6%). The majority of the herds showed infection with one single pulsotype, but 12 herds yielded 2 pulsotypes, and one herd had 3 pulsotypes. No relationship was found between the geographical location of the herd and the pulsotype. This is the first description of a typing method for C. bovis; the technique might prove useful for epidemiological studies on this species.