Besnoitia besnoiti: Long term cultivation in tick cell lines

Abstract

The life cycle of Besnoitia besnoiti Marotel (1912) and its transmission in nature are unclarified, though cats were shown to be the final hosts (Petesheva et al. 1974). The besnoitiosis disease of cattle can be induced mechanically by blood transfusion (Pols 1960) or by bloodsucking flies shortly after they have fed on an infected animal (Bigalke 1968). The causative agent has been cultivated successfully in some mammalian cell cultures and with less success in others (Bigalke 1962; Bigalke et al. 1974; Neuman 1974; Shkap et al., 1987). No attempts to grow Besnoitia sp. in invertebrate cells have been reported as yet. Ticks were suggested as possible vectors of B. besnoiti (Pols 1960). B. besnoiti tachyzoites (BI strain from Israel), grown in Vero cells, were injected intraperitoneally at 2 x 10’ into gerbils. After clinical signs appeared, the peritoneum was washed with culture medium (MEM, Eagle’s supplemented with 10% calf serum and 0.3% tryptose phosphate broth). The washings were centrifuged (4OOg), and the parasites were resuspended in the medium. Two tick cell lines, RML-15 and LSTH-RA-243, were maintained as described elsewhere (Samish et al. 1985). Tick cells (8 x 106) were seeded in three replications in tissue culture flasks (25 cm2 growth area) and, l-2 hr later, 16 x 106 parasites were added, adjusted to 10 ml. The cultures were incubated at 28 or 36 C and checked daily for cytopathic effects. The time interval needed for the destruction of the cell monolayer by the parasites as well as the number of extracellular parasites at the time of subculturing were observed. Whenever it was visually estimated that 80% of the monolayer became destroyed, the cells were suspended and half of the media, including free parasites and tick cells, and transferred to new flasks, seeded previously as described above. A sample was stained with 0.5% Trypan blue and the parasites were counted in a hematocytometer. Giemsa stain smears and immunofluorescence antibody tests (Shkap et al., 1987) were done. For infectivity tests tick cell cultures, inoculated previously with B. besnoiti, were placed on Vero cells and when zoites were seen, they were harvested by centrifugation and injected intraperitoneally into two or three gerbils. In addition, the previously inoculated tick cells were injected directly intraperitoneally into two or three gerbils. Three gerbils serving as positive control groups were injected with 2 x 10’ parasites harvested from infected Vero cells and three additional control gerbils received uninfected tick cells. Six weeks after the first infection, the survivors were challenged with 2 x 10’ parasites harvested from infected Vero cells. Whenever the inoculated gerbils showed clinical signs, they were sacrificed and washings of the peritoneal cavity were examined microscopically. At 36 C, the destruction of the RA-243 and RML-15 cell monolayer started l-2 days after seeding. Therefore, subculturing was needed within 3-4 days after the start of the experiment. At 28 C, the parasites required 45 days to destroy 80% of the RA-243 monolayer and 67 days to destroy the RML-15 cells derived from the first seeding. Afterward, subculturing at 28 and 36 C was quite similar throughout the experiment, and no “adaptation” period was needed for RA-243, while RML-15 had to be subcultured at weekly intervals. The number of extracellular parasites in three RA-243 cultures incubated at 36 C decreased during the first 5 days after inoculation to nearly half and continued to decrease for up to 15-35 days. Subsequently, the amount of free parasites became stable within a certain range but began to increase rapidly to a peak of 3.1-4.0 x 106/ml about 2 months after seeding, reaching up to four times more extracellular parasites per day. After this stage, the number of free parasites started to decrease, disappearing in the course of 7 months after the first seeding. When B. besnoiti was seeded on RML-15 cells, the amount of extracellular parasites at 36 C decreased gradually, and they became invisible after 7-9 subcul-