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Three methods for initiating in vitro cultures of Theileria annulata from infected cattle were compared: 1) separation of buffy coat cells by centrifugation in a plastic syringe; 2) separation of lymphocytes by a commercial Ficoll-Paque gradient and 3) trypsinization of liver biopsy material. Successful cultures were obtained in 26 out of 48 trials (54 p. 100) by method 1, in 28 out of 56 trials (50 p. 100) by method 2 and 17 out of 21 trials (81 p. 100) by method 3. Once established, cultures, initiated by any method can be subcultivated for years.

Soluble antigens from culture-grown Besnoitia besnoiti endozoites were identified following their partial purification by affinity chromatography. A specific eluate obtained after affinity chromatography on a column to which antibodies from serum of a naturally infected cow were bound exhibited seven polypeptide bands on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Five bands were observed in the eluate from an immunoadsorbent to which antibodies from an experimentally infected calf were coupled. Eluted antigens were reactive in the enzyme-linked immunosorbent assay (ELISA). Reactivity of electrophoretically resolved antigens with sera of endozoite-inoculated cattle and sera from field cases of besnoitiosis were studied using the Western immunoblotting technique.

Soluble antigens from Besnoitia besnoiti cell culture-grown endozoites, obtained either by hypotonic lysis or by freeze-thawing and ultrasonication (FTS) of the organisms, were detected by the agar gel immunodiffusion method. Each antigenic preparation yielded 1-4 precipitin lines when reacted with the corresponding rabbit hyperimmune serum, while no reaction was observed with Besnoitia-positive sera from naturally infected cattle. Soluble exoantigens released by viable Besnoitia endozoites into the supernatant of infected cell cultures formed two precipitin lines with rabbit anti-FTS hyperimmune serum and appeared as positively charged protein in immunoelectrophoresis. The precipitin lines observed were parasite specific since no reaction occurred with either lysates of normal Vero cells or with supernatants from non-infected cell cultures.

Cultivation of Besnoitia besnoiti on five mammalian cell types showed that Vero (green monkey kidney), L929 (mouse fibroblasts) and BEK (bovine embryo kidney) cells were highly susceptible and were almost destroyed after 5 days of incubation, while MDBK (Madin-Darby bovine kidney) and BL (Theileria annulata-infected bovine lymphoid) cells were less affected. Intraperitoneal infection with culture-derived endozoites was fatal for gerbils (Meriones tristrami) and sand rats (Psammomys obesus). Rabbits showed fever, conjunctivitis and orchitis, but survived the infection. Mice, rats, guinea pigs, hamsters and Microtus guentheri did not show signs of illness, but developed specific antibodies to B. besnoiti. B. besnoiti subcultivated for 2 years in BL cells remained virulent for gerbils.

The use of the ELISA method for the detection of antibodies to B. besnoiti in cattle is described and compared to the IFAT technique. One hundred and twenty-one sera were examined, of which 61 were sera of calves experimentally infected with B. besnoiti, 52 sera from field animals and eight were sera with high titres of antibodies to other parasites. The specificity of both assays correlates but ELISA seemed to be more sensitive. The ELISA technique provides a rapid and reliable method for the screening of B. besnoiti infection in cattle.

Specific precipitable antibodies of both IgG and IgM classes were detected in sera of cattle naturally infected with B. besnoiti. The amount of specific antibodies of the IgG class precipitated by soluble antigen was in the range of 17-50 micrograms/ml serum while that of the IgM class ranged between 5 and 24 micrograms/ml serum. Specific antibodies precipitated by live B. besnoiti parasites were in amounts of 10 to 22 micrograms/ml serum for IgG and 4 to 26 micrograms/ml serum for IgM. Different ratios of IgG/IgM were obtained by the two methods of precipitation. This might indicate that antibodies to B. besnoiti of the IgM class can be precipitated and detected in sera of naturally infected cattle in similar amounts either by live parasites or by solubilized antigen, whereas antibodies of the IgG class can be preferentially detected when solubilized antigen is used for precipitation.

Studies the ultrastructure of the tissue cysts and bradyzoites of the Besnoitia besnoiti, an economically important tissue cyst-forming apicomplexan of cattle in Africa and Israel. Removal of enigmatic bodies as a generic character of the bradyzoites of Besnoitia species; Analysis of pertinent topics and relevant issues; Implications on eukaryotic microbiology.