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SUMMARY OF IN VITRO AND IN VIVO CYST FORMATION

The recent development of stage-specific antibodies makes it possible to study in vitro conversion of bradyzoites to tachyzoites and in vitro conversion of tachyzoites to bradyzoites. Soête et al. (156, 157) inoculated bradyzoites onto MRC-5 cell cultures and monitored the appearance of bradyzoite-specific (B+) or tachyzoite-specific (P30) antigens. At 15 h after inoculation, the organisms had both B+ and P30 antigens. Organisms began to divide at approximately 24 h p.i. and vacuoles containing two doubly labelled organisms were seen. By 48 h p.i. some organisms had lost B+ antigens and were considered to be tachyzoites. A quantitative analysis was not possible after 48 h p.i. because some parasites had ruptured and reinvaded other host cells. These authors concluded that not all bradyzoites transform into tachyzoites, since they found multiplying bradyzoites (156); these results were different from in vivo transformation of all bradyzoites into tachyzoites by 48 h p.i. as determined by the cat bioassay. The same authors also observed that cell cultures inoculated with tachyzoites from the peritoneal exudate of mice had completely lost tachyzoite-specific markers by 72 h p.i. Bohne et al. (9, 10) obtained similar results in cell cultures inoculated with bradyzoites. The bradyzoite-specific antigens declined from 100 to 15% from day 1 to 3 after inoculation of cultures. Conversely, the bradyzoite-specific antigens increased steadily from day 1 to 6 in cultures inoculated with tachyzoites.

By using stage-specific monoclonal antibodies, bradyzoite-specific antigens were detected in the brains of mice on day 9 (127). Mice were examined 6, 7, 9, 12, and 14 days after being fed 20 tissue cysts of the 76K strain. The brains of the mice were stained with tachyzoite-specific antibodies (P30) and bradyzoite-specific antibodies (P36). On days 6 and 7, only P30-specific organisms were present. On day 9, groups of parasites were labelled doubly with P30 and P36 antibodies. On days 12 and 14, the number of P30-positive organisms decreased whereas the number of P36-positive organisms increased.

BAG-5 develops early in vitro and is probably a small HSP-like molecule associated with development. Weiss et al. (179), using the BAG-5 bradyzoite-specific antibody, found that tissue cysts had formed by day 3 after inoculation of human foreskin fibroblasts with bradyzoites of the ME-49 strain; it appears that some bradyzoites formed tissue cysts directly without conversion to tachyzoites. Using a monoclonal antibody specific for the tissue cyst wall, Halonen et al. (87) found tissue cysts in human fetal neuronal culture beginning on day 2 after inoculation with the ME-49 strain. The development of bradyzoites in vitro occurs in a series of steps, whereas the development of mature tissue cysts is less frequent and requires a minimum of 6 days in culture. This is supported by studies of antigen expression as ascertained by the various bradyzoite-specific sera available. Most bradyzoite-specific monoclonal antibodies and recombinant polyclonal sera available are expressed after 1 day in culture under conditions which induce bradyzoite switching (9-11,155, 156). The late-appearing antigen is not seen until 5 to 6 days of culture. In addition, results of feeding experiments suggest that even at 6 days, not all of the observed tissue cyst-like structures are mature, since only a subset behave as tissue cysts in animal bioassays (88, 109). Thus, to some extent, the data on in vitro cystogenesis mimic the data seen in this study. Early on, one would expect tachyzoites that are in process of becoming mature bradyzoites to express BAG-5 antigen (day 1 of transition), but full biologic maturity would take several more days. It is clear from the above discussion that for in vitro work, markers of mature functional cysts are needed.



While these in vitro studies are helpful in elucidating stage conversion, the results do not completely agree with those of in vivo studies. The 15 h needed to convert bradyzoites to tachyzoites in cell culture agrees with the data obtained with mice, where functional tachyzoites were not found 18 h after bradyzoites were fed to mice (42). The 72 h needed for tachyzoites to completely lose their tachyzoite-specific markers also agrees with the 3 days needed to form biologically functional tissue cysts after inoculation of tachyzoites into mice (48). However, when bradyzoites were inoculated into mice by any route, the minimum period needed to form biologically functional tissue cysts was 6 days (48). Unlike in vitro studies, all bradyzoites had converted to biologically defined tachyzoites by 18 h p.i. (42). Therefore, biologic measurements should be examined in the context of parasitologic and host factors (74), and caution should be used in interpreting in vitro phenomena as having biological significance.


Date: 2016-01-03; view: 693


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