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Johne’s Disease is a debilitating chronic enteritis caused by Mycobacterium avium subsp. paratuberculosis (Map), and has great economic impact on affected dairy farms.1, 2 Infection occurs in young calves, by exposure to manure from mature Map-shedding cattle. To reduce infection status on farms, prevention plans are in order, because no treatment is available for Johne’s Disease. Prevention plans for Johne’s disease rely on age-dependent susceptibility, with a cut-off at the age of six months. This cut-off is based on one paper from 1975, where only 9 animals were included in the experiment3. In a clinical trial at the University of Calgary this age dependent susceptibility is being investigated with a larger number of animals: 34 male Holstein-Friesian calves are subject of a research to age- and dose dependent susceptibility to infection with Map. They will be infected with a virulent Map strain at 5 different ages (2 weeks, 3-6-9 and 12 months) with a high-(5 x 109 CFU) and a low inoculum dose (5 x 107 CFU). Besides this pathogenic mycobacterium, there are also environmental mycobacteria where young calves are exposed to. These mycobacteria can be found in a wide variety of environmental reservoirs, including natural and municipal water, soils, aerosols and protozoans.4 Because of ingestion and close contact with these reservoirs, environmental mycobacteria can also be found in animals and humans. Evidence exists for a cross-reaction in immunity between rapid growing environmental mycobacteria and other pathogenic mycobacterial species (tuberculosis and leprosy) in humans4. That’s why we are interested in the impact of environmental mycobacteria on the infection with Map in calves. There are different protocols known for isolating mycobacteria from water, soil or milk5, 6, but there is none for isolating mycobacteria from faeces. That was the aim of this study: to identify an appropriate protocol to isolate different mycobacteria from faeces. Mycobacterium smegmatis was used as a model for rapid growing mycobacteria. Unfortunately, no perfect protocol was found. An important conclusion of this research is that HPC (Hexadecyl Pyridinium Chloride), frequently used to isolate Map from faeces, is not appropriate to isolate environmental mycobacteria, because M. smegmatis does not survive concentrations higher than 0,1%, and other faecal bacteria do.