EVIDENCE FROM GENOTYPIC AND PHENOTYPIC MARKERS THAT AN ATTENUATED LINE OUTGROWS A VIRULENT ONE IN A MIXED POPULATION OF LEISHMANIA-MAJOR PROMASTIGOTES CULTURED IN-VITRO

Two cloned lines of Leishmania major promastigotes, one attenuated (CO 1H) and one virulent (CO1R), differing in molecular karyotype and expr ession of the major surface glycoprotein (gp63), were mixed to produce two heterogeneous populations: MP-1 (100 COIR promastigotes/CO1H prom astigote); and MP-2 (10 000 CO1R promastigotes/CO1H promastigote). The mixed populations were cultured for 1 month in vitro in HO-MEM medium and sub-samples taken on days 4 and 30 were subjected to electrophore sis so that the molecular karyotypes and gp63-expression characteristi cs of the promastigote populations could be determined. In spite of th e initial predominance of the virulent CO1R, the attenuated CO1H alway s outgrew it. The patterns of growth of pure cultures of CO1H or CO1R did not fully explain this observation. When grown alone, CO1H acidifi ed the culture medium much more and much faster than CO1R, low pH valu es eventually inhibiting multiplication. Decreasing the medium's initi al glucose concentration or increasing its initial pH prolonged the gr owth phase of CO1H, probably by slowing its acidification of the mediu m to inhibitory values. It is possible that, in mixed populations, the CO1R promastigotes help to buffer the medium, permitting faster and l onger multiplication of the CO1H promastigotes than occurs when they a re cultured alone. CO1H promastigotes may also inhibit CO1R multiplica tion; CO1H promastigotes in mid-logarithmic phase entered stationary p hase within a day of being transferred into cell-free supernatants fro m 3-day-old CO1R cultures.