Light and transmission electron microscopic studies on the encystation of Histomonas meleagridis

Abstract

The study deals with the pleomorphic zooflagellate Histomonas meleagridis, which was cultivated under different stress conditions to induce a possible encystation. In the present paper, the morphological changes were analyzed by light and electron microscopy. The determination of the proliferation under different adverse conditions led to conclusions on the tenacity of the flagellate. H. meleagridis parasitizes in the intestinal tract of galliform birds and may cause enormous losses in poultry farming. For the development of new therapy approaches, clarification of the transmission pathways will be helpful. Different clonal cultures of H. meleagridis established by micromanipulation and exposed to media lacking different ingredients, inappropriate temperatures, and/or distinct reagents were investigated. Lowering of temperature was proven to have adverse effects on the survival of H. meleagridis. The flagellate could not survive in a frozen medium, and survival in a temperature of 4°C lasted no longer than 23 h. An addition of sodium chloride induced an increased proliferation; pH values between 2 and 8 set limits for the survival of the parasite in different ways. H. meleagridis was able to survive under high acidic conditions for only 1 h. The major amount of cells, which could be discovered in the controls, measured 8–12 μm appeared amoebic (stage 1) and were filled with enclosures of rice starch. A rounding of most cells was noted 4 h at 4°C after incubation in minimal essential medium in the absence of rice starch and fetal calf serum. A higher osmolarity of the medium, which was initiated by the addition of sodium chloride or magnesium chloride, did not induce an encystation process. After addition of hypochlorite base and cultivating at pH values between 7 and 8, spherical stages without a flagellum were formed (stage 2) measuring about 8–12 μm in diameter. Their interior consisted of a central and a peripheral region when studied by transmission electron microscopy. This aspect was due to the location of the glycogen granules. The central zone was described as totally filled with the carbohydrates, which made totally invisible the other organelles. The solidity of the amorphous layer below the cell membrane seemed to hinder the invasion of the glycogen granules. The amorphous layer below the cell membrane made it apparently possible that the cell might survive under adverse conditions—at least for a short time. This special structure might enable H. meleagridis to proceed a fast transmission and to infect many birds in a rather short time, which was shown in the past by several studies. Double-membraned cells, which were guessed to be cyst-like structures of the parasite, were also detected (stage 3). The size of these cells, however, was much smaller than that of the amoebic stages or the above-described spherical forms of H. meleagridis. Furthermore, the small cells were characterized by other granula structures. These findings might be interpreted that the small stages are possibly long-term (true) cysts and that the spherical stages with the amorphous layer beneath the cell membrane might be short-term cysts. Both, however, should be able to survive situations outside of a body and thus might be transmitted from feces to another animal.