Parasitol Res (1993) 79:340 342 Parasitnlogy Research 9 Springer-Verlag1993 Induction of complement-sensitivity in Leishmania amazonensis metacycSc promastigotes by protease treatment but not by specific antibodies N.M. Soares 1, E.M. Carvalho 2, R.T. Pinho 3, L.C. Pontes de Carvalho 1 1Laborat6rioAvangadode SafidePfiblica,Centro de Pesquisas Gon~alo Moniz, FIOCRUZ, 41945 Salvador, Brazil 2 HospitalProfessorEdgard Santos, UniversidadeFederal da Bahia, 40000 Salvador, Brazil 3 Departamentode Imunologia,[nstituto Oswaldo Cruz, FIOCRUZ, 21040 Rio de Janeiro, Brazil Received: 17 August 1992 / Accepted: 21 December 1992 During its life cycle, the protozoan parasite Leishmania has to differentiate into forms specialized in infecting vertebrate or invertebrate animals (Adler 1964). In this respect, leishmanias are similar to Trypanosoma eruzi, another trypanosomatid protozoan pathogenic to hu- man beings (Brenner 1973). Thus, Leishmania promasti- gores and T. cruzi epimastigotes multiply in the digestive tract of Phlebotominae and Triatominae insects, respec- tively. After a few days, T. eruzi epimastigotes differenti- ate into a mammal-infective stage, the metacyclic trypo- mastigote (reTry), acquiring the capacity to resist com- plement-mediated lysis when in contact with normal mammal sera (Nogueira et al. 1975). In a similar way, Leishmania promastigotes in the logarithmic phase of growth (logPro) differentiate into infective metacyclic promastigotes (mPro), also acquiring resistance to com- plement-mediated lysis (reviewed in Sacks 1989). T. eruzi trypomastigotes, however, although resistant to complement-mediated lysis in normal sera, are readily lysed by antibodies (lyric antibodies) present in the sera of patients with Chagas' disease (Krettli et al. 1984). These antibodies have been associated both with infec- tion activity and with immunity (Krettli et al. 1984). On the basis of the many similarities between reTry and mPro, the possibility that L. amazonensis rePro might also be lysed by antibodies was investigated in the present study. Antibodies were obtained from Leish- mania- or T. cruzi-infected patients (these antibodies react with several Leishmania promastigote antigens; Reed et al. 1987; dos Santos et al. 1987) and from Le- ishmania-immunized rabbits (a species normally resistant to infection by Leishmania). Diseases were diagnosed by the presence of specific antibodies in the sera as de- tected in an indirect immunofluorescence assay (Badar6 et al. 1983) and by the direct demonstration of parasites at biopsy or xenodiagnosis. Leishmania-antibody titers (maximal serum dilutions leading to fluorescent para- sites) ranged from 1:64 to 1:800. The titers of 100 nor- mal control sera never exceeded 1:16. The sera from Correspondence to: N.M. Stares patients with Chagas' disease were shown to lyse T. cruzi mTry as described elsewhere (Krettli et al. 1984). Pro- mastigotes in the stationary phase of growth (sPro), a proportion of which are repro (Sacks 1989), were ob- tained from modified liver-infusion-tryptose (LIT; Difco Laboratories, Detroit, USA) cultures (Sadigursky and Brodskyn 1986) grown at 26~ C and supplemented with 5% fetal calf serum. In cultures starting with 106 para- sites/ml, a stationary phase of growth was reached after 6 days. The Leishmania isolate used was identified as L. amazonensis by a panel of species-specific monoclonal antibodies (Grimaldi et al. 1987). The assay for lytic antibodies was similar to that de- scribed for T. cruzi (Krettli et al. 1984) and relied on the determination of the percentage of motile parasites remaining after a 30-rain period of incubation at 37~ C with fresh sera. At least 100 parasites were counted in a hemocytometer for each serum sample tested. Each immune serum specimen was tested in parallel with a normal sample of serum of the same species (human or rabbit) and with a sample of bovine fetal serum (Sig- ma Chemical Co., St. Louis, USA) as controls. To con- firm that the parasites surviving the lyric process were indeed viable, on some occasions they were put back into fresh LIT medium, cultured for 48 h, and assessed for motility by light microscopy. No difference was ob- served in the viability of these parasites as compared with untreated promastigotes. In fact, the absence of flagellar motility has been shown to be a useful parame- ter to indicate the lethal effect of sera on Leishmania (Pearson and Steigbigel 1980). As expected, virtually 100% of the promastigotes in the logarithmic phase of growth were susceptible to lysis in the presence of normal human sera (the mean percent- age of parasites susceptible to lysis in 25 determinations with 17 sera was 99.4% _+0.9%), whereas a proportion (ranging from 7% to 19%) of the stationary-phase pro- mastigotes (sPro) was not affected by treatment with 7 fresh samples of normal human sera (Fig. 1). Contrasting with the lytic activity of sera from pa- tients with Chagas' disease against T. cruzi trypomasti-