An eradication programme against paratuberculosis implemented in this institute over ten years has shown that this infection is spread particularly among young animals. To gain a better understanding of the pathogenesis of this diseases, we have started to investigate the effect of the environment (barns and pasture), low vertebrates and invertebrates (worms, diptera and beetles) on disease transmission. From 1993-1998, fourteen ruminant herds (cattle, wild goats and moufflons) were examined. Samples included: 725 from the environment, 34 organs from low vertebrates and 1700 from invertebrates (38 worms, 17 beetles and 1645 diptera). Strains of M.avium. subsp. paratuberculosis were isolated from environmental samples originating from infected herds with a prevalence of clinical paratuberculosis >3%. Isolated strains were always of the same DNA type. However, one cattle herd was infected with strains of various DNA types: 55.8% type A-C10, 21.1% type B-C1, 15.4% type B-C9, 5.8% type B-C10 and 1.9% type B-C14. The environment in this cattle herd was contaminated by the most common DNA types: 69.2% type A-C10, 7.7% type B-C1 and 23.1% type B-C9. 17 mice were trapped from a pastoral area, from which two strains of M.a.paratuberculosis were isolated. However, we were unable to determine their DNA fingerprint type. In addition, identification of M.a.paratuberculosis in samples collected from invertebrates was unsuccessful. Dungworms, flies (at different stages of life-cycle) and beetles were either artificially infected with M.a.paratuberculosis from intestines of sick cows or with faeces from cattle and goats. After 4-6 weeks, M.a.paratuberculosis could be isolated from these faeces. In addition, M.paratuberculosis (with the same DNA type) could be detected for up to 12 months after the infection in faeces of more than 300 hamsters, whilst in liver and spleen, survival was even longer (24 months). Our research was partially supported by the Ministry of Agriculture (grant no. EP0960006087) and the Ministry of Health of the Czech Republic (grant no. 4211-3/97). Permanent address of Robin du Maine - Hogeschool van Utrecht, Netherlands.