THEILERIA-ANNULATA - ALTERED GENE-EXPRESSION AND CLONAL SELECTION DURING CONTINUOUS IN-VITRO CULTURE

Kept in continuous in vitro culture, the protozoan parasite Theileria annulata gradually loses virulence when inoculated into cattle. These attenuated cell lines form the basis of the in vitro live vaccines whi ch have been used successfully to control tropical theileriosis in sev eral endemic regions. In the study reported here, events occurring dur ing in vitro culture of an Indian (Hisar) cell line, which may be asso ciated with the reduction in virulence, have been investigated. Hybrid ization with two polymorphic DNA probes following Southern blotting sh owed that selection of particular parasite genotypes occurs very rapid ly with culture; a novel hybridization pattern is observed with both p robes after 50-100 passages in vitro. In addition to this selection pr ocess, immunofluorescence studies using a monoclonal antibody which sp ecifically recognizes virulent T. annulata revealed alterations in ant ibody reactivity following in vitro culture. This loss of reactivity w as observed in three cloned cell lines derived from the early, virulen t Hisar line and implies that phenotypic changes resulting from altera tions to parasite gene expression are taking place during the attenuat ion process. When considered with the results from in vivo infections with serial passages of this cell line, it can be proposed that both a ltered gene expression and selection may be involved in the loss of pa thogenicity of T. annulata during continuous in vitro culture. (C) 199 6 Academic Press, Inc.