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

Abstract

Two cloned lines of Leishmania major promastigotes, one attenuated (CO1H) and one virulent (CO1R), differing in molecular karyotype and expression of the major surface glycoprotein (gp63), were mixed to produce two heterogeneous populations: MP-1 (100 CO1R promastigotes/CO1H promastigote); and MP-2 (10000 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 electrophoresis so that the molecular karyotypes and gp63-expression characteristics of the promastigote populations could be determined.

In spite of the initial predominance of the virulent CO1R, the attenuated CO1H always outgrew it. The patterns of growth of pure cultures of CO1H or CO1R did not fully explain this observation. When grown alone, CO1H acidified the culture medium much more and much faster than CO1R, low pH values eventually inhibiting multiplication. Decreasing the medium's initial glucose concentration or increasing its initial pH prolonged the growth phase of CO1H, probably by slowing its acidification of the medium to inhibitory values. It is possible that, in mixed populations, the CO1R promastigotes help to buffer the medium, permitting faster and longer multiplication of the CO1H promastigotes than occurs when they are cultured alone. CO1H promastigotes may also inhibit CO1R multiplication; CO1H promastigotes in mid-logarithmic phase entered stationary phase within a day of being transferred into cell-free supernatants from 3-day-old CO1R cultures.