Electron Microscope Study of the Proliferative Form of Besnoitia jellisoni

Abstract

Electron microscope studies of the proliferative form of Besnoitia jellisoni confirm its close relationship to Toxoplasma gondii. Its pellicle consists of an outer and an inner membrane under which 22 longitudinal fibrils are found. A micropyle is formed by an invagination of the outer membrane. At the anterior end a polar ring surrounds the conoid. Club-like "toxonemes" extend posteriorly from within the conoid. A large vesicle with a tube connecting its lumen to the exterior through the conoid may represent a feeding mechanism. One or two mitochondria, rough endoplasmic reticulum, and free ribosomes are distributed throughout the cytoplasm. The Golgi zone is anterior to the nucleus. A large vacuole of unknown function is found near the Golgi zone. Endodyogeny, a division by internal budding, occurs producing two daughter cells within a parent cell. Stages possibly representing binary fission were also observed. Besnoitia jellisoni was first described by Frenkel (1953). It was recognized that the organism morphologically resembled Toxoplasma gondii. By using silver protein staining Goldman, Carver, and Sulzer (1957) observed similar internal structures and a similar method of reproduction in B. jellisoni and T. gondii. However, B. jellisoni differs from T. gondii in the type of cyst formed, in the pathology produced, and in serological reaction (Frenkel, 1953). Recently, Lunde and Jacobs (1965) demonstrated that although common antigens were present in the two parasites they were immunologically different. Since the fine structure of T. gondii has been described by several authors an electron microscope study of B. jellisoni was undertaken to compare its internal morphology with that reported for T. gondii. MATERIALS AND METHODS Proliferative forms of Besnoitia jellisoni were obtained by collecting peritoneal fluid from mice 4 days after inoculation. The peritoneal fluid was centrifuged and the supernatant discarded. The organisms were then fixed by adding 1% osmium tetroxide buffered at pH 7.4 with veronal acetate and sucrose (Caulfield, 1957) to the pellet which was subsequently divided into small pieces (less than 1.0 mm3). After fixation for 2 hr the specimens were rinsed in distilled water and dehydrated for 1 hr and 15 min with graded concentrations of ethyl alcohol. Fixation and dehydration were carried out in an ice bath. The specimens were then placed in propylene oxide followed by a 1:1 mixture of propylene oxide and Epon. The Epon consisted of 10 ml Epon Received for publication 6 December 1965. 812, 25 ml DDSA (dodecenyl succinic anhydride), and 0.8 ml DMP-30 (2,4,6-tri( dimethylaminomethyl)phenol) (Sporn, Wanko, and Dingman, 1962). After infiltration for 1 hr with the propylene oxide-Epon mixture the specimens were placed in Epon and then transferred to BEEM capsules and polymerized for approximately 16 hr at 60 C. Sections were cut with a diamond knife on an LKB Ultrotome, stained with lead (Karnovsky, 1961), and examined in an RCA EMU-3G electron microscope operating at 50 kv.