A total of 1 750 strains of Mycobacterium avium subsp. paratuberculosis isolated from cattle, sheep, goats, wild ruminants (moufflon, deer etc.), non-vertebrates, small vertebrates (cat, mouse, rabbit etc.), external environment, milk from cattle, moufflon, deer, fallow deer and other animals and Crohn's patients were examined by the use of standardised RFLP method with two restriction endonucleases (RE) PstI and BstEII, and standard IS900 probe prepared by PCR a non-radioactive labelled. Strains were obtained from Europe (15 countries), USA (13 states), Canada, Africa (2 countries), Australia, and New Zealand. After digestion by RE PstI, 15 RFLP types (A-O) were detected; whereas digestion by RE BstEII resulted in 34 RFLP types (C1-C29, I1-I2 and S1-S3). A combination of both BstEII and PstI RFLP results revealed o total of 40 RFLP types. We have analysed the similarity of certain BstEII RFLP types and noticed that some RFLP profiles might have been a mixture of several different strains. Such isolates were subcultivated and progeny from single bacterial colony was analysed. By this way one "new" BstEII RFLP type was identified as mixture of two "known" RFLP types. For more detailed differentiation of PstI RFLP types three different hybridisation probes were developed. As RE PstI digests inside of the element IS900. Due to this fact one probe was designed so that it covered both part of the digested fragments (total probe) and two half-probe hybridised to different halves of IS900. A combination of hybridisation by these three probes enabled to identify new RFLP subtypes inside original types. RFLP typing was used for the epidemilogical studies in animal and human populations, in the environment and for the study of localisation of the causal agent in the host organism's tissue.

Supported by the grants FAIR6-CT98-4373 and QLK2-CT-2000-00928 (Brussels, EC).