Entamoeba histolytica: concurrent irreversible loss of infectivity-pathogenicity and encystment potential after prolonged maintenance in axenic culture in vitro

The NIH-200 strain of Entamoeba histolytica became avirulent after more than 2 yr maintenance in axenic culture in vitro. In an attempt to restore virulence to the amoeba, it was transferred to Locke's egg rice-flour medium with various combinations of the following bacteria: Bacteroides sp., Clostridium perfringens, Escherichia coli, and Streptococcus faecalis. Similar cultures were established with a mixed bacterial flora (comprising many unknown species), with and without rice flour, and an attempt was made to induce encystation. Subsequent inoculation of amoebae from the various amoeba-bacteria cultures into the cecum of germfree and exgermfree guinea pigs harboring the same bacteria, as the culture-produced inoculum did not in any instance produce amoebic lesions or prolonged amoebic infections of the enteric lumen. All attempts to induce encystation were unsuccessful; the amoeba had lost its encystment potential, and this was believed to be intimately related to the irreversible loss of virulence.