A microsporidian parasite Enterocytozoon bieneusi is the most common microorganism recognized in AIDS patients, and slow scientific progress is attributed to our inability to propagate the parasite. We report upon the development of a system of propagation using the pig biliary system. The parasite spores were continuously detected in the bile samples post onset of spore shedding in the gall bladder, which suggests that this organism maintain persistent infection in the biliary system and that the hepatobiliary tree may represent a reservoir of infection. In conclusion the biliary tree is an adequate niche for the propagation of E. bieneusi. This work has also resulted in the development of a procedure of ultrasound-guided cholecystocentesis for aspirating biles. This is a simple and non-surgical procedure, and creates no signs of clinical complications in the livers and the gall bladders after dozens of separate attempts. Thus, this is a very useful and safe technique for the aspiration of bile from live animals.