Acta Protozool. (2005) 44: 113 - 121 Free-living Amoebae Serve as a Host for the Chlamydia-like Bacterium Simkania negevensis Rolf MICHEL 1 , Karl-Dieter MÜLLER 2 , Lothar ZÖLLER 1 , Julia WALOCHNIK 3 , Mathias HARTMANN 4 and Ernst-N. SCHMID 2 1 Central Institute of the Federal Armed Forces Medical Services, Koblenz, Germany; 2 Institut für Medizinische Mikrobiologie der Universität Essen, Essen, Germany; 3 Department of Medical Parasitology, Clinical Institute of Hygiene and Medical Microbi- ology, Vienna, Austria; 4 Institut für Medizinische Mikrobiologie des Klinikums, Jena, Germany Summary. Members of the novel family Parachlamydiaceae are commonly observed in free-living amoebae (FLA) as host cells. Therefore, we examined the potential of 14 different species of free-living amoebae to serve as hosts of the Chlamydia-like bacterium, Simkania negevensis, previously isolated as a contaminant from a cell culture in Israel (Kahane et al. 1993, 1995). The inoculum of the obligate intracellular agent was prepared from Buffalo Green Monkey (BGM) cells. The infection of Acanthamoeba strain HLA and of Naegleria clarki (N-DMLGo) revealed typical morphological stages of a Chlamydia-like life cycle, including the presence of elementary and reticulate bodies, as could be shown by electron microscopy. Subsequent infection studies with an Acanthamoeba-adapted Simkania isolate showed that also Balamuthia mandrillaris and one of two Hartmannella strains supported the growth of Simkania. Balamuthia can be considered as an experimental host for mass production of elementary bodies. This is based on the finding that the host amoebae expelled great numbers of bacteria leading to a long-term survival of the infected trophozoites. The observation that Simkania negevensis can survive and replicate within at least four of tested FLA species suggests that various free-living amoebae may serve as survival and multiplication vehicles supporting the spread of these pathogens in aquatic environments. The concept that Simkania may fall into the group of environmentally preadapted pathogens is discussed as well. Key words: Acanthamoeba, Balamuthia, Chlamydia, endoparasite, Hartmannella, host range, Neochlamydia, Simkania negevensis, ultrastructure, Waddlia. INTRODUCTION Chlamydiae are well known as important obligate intracellular pathogens. They are the causative agents of a variety of diseases in humans and animals, e.g. infections of the eye, as well as of the respiratory and genital tracts. The life cycle of chlamydiae is character- ized by the development of reticulate bodies (RBs) that divide intracellularly by binary fission and the more resistant elementary bodies (EBs) that are specialized for transmission to another cell of the same host and to a new host (Moulder 1984). Recent isolation of several novel Chlamydia-related bacteria from contaminated cell culture (Kahane et al. 1993, 1995), from an aborted bovine foetus (Dilbeck et al. 1990, Rurangirwa et al. 1999, Henning et al. 2002) Address for correspondence: Rolf Michel, Zentrales Institut-BW-Koblenz, c/o Rheinkaserne Geb. 5, Lab. Abt. I. (Mikrobiologie), Andernacher Str. 100, 56070 Koblenz; E-mail: rolf_michel@hotmail.com