GENETIC-DIFFERENCES BETWEEN NITRATE-NEGATIVE AND NITRATE-POSITIVE C-PSEUDOTUBERCULOSIS STRAINS USING RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISMS

Corynebacterium pseudotuberculosis has been classified into two biotyp es according to ability to breakdown nitrate (Biberstein et al., 1971) . Restriction enzyme analysis (REA) has shown to reflect this differen tiation, but numerous bands generated by this technique make interpret ation difficult (Songer et al., 1988), Restriction fragment length pol ymorphism's (RFLP's) has become an accepted genetic tool and was used in this study to determine if differences in nitrate reduction and oth er phenotypic characteristics could be identified genetically. Thirtee n C. pseudotuberculosis isolates from four species of domestic animals from different parts of the world were investigated for phenotypic an d genetic differences. Three closely related bacteria, Corynebacterium ulcerans, Actinomyces pyogenes (previously C, pyogenes), and Rhodococ cus equi (previously C. equi) were included in the study to determine if the RFLP bands were unique to C. pseudotuberculosis. All C. pseudot uberculosis isolates were positive for urease production. Some differe nces in maltose and sucrose fermentation ability and nitrate reduction were recorded. Genetic differences were identified between the nitrat e-positive group and the nitrate-negative group using non-radioactive ribosomal RNA (rRNA) probes Southern blotted to restriction digests of ApaI, PstI, and Sst I. A small number of bands were seen, with distin ct differences between the nitrate-positive and the nitrate-negative s trains. No genetic variations were seen between strains which reflecte d differences in carbohydrate fermentation. Strains isolated from diff erent animal species and from different parts of the world could not b e differentiated genetically using these three restriction enzymes.