103 Axenic cultivation and partial characterization of Leishmania braziliensis amastigote-like stages J. M. F. BALANCO, E. M. F. PRAL, S.  SILVA, A. T. BIJOVSKY, R. A. MORTARA and S. C. ALFIERI*  Departamento de Parasitologia, Instituto de Cie  ncias Biome  dicas, Universidade de Sa  o Paulo, Av. Prof. Lineu Prestes 1374, CEP 05508-900, Sa  o Paulo, S.P., Brasil  Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina and Centro de Microscopia Eletro  nica, Universidade Federal de Sa  o Paulo, R. Botucatu 862, CEP 04062-040, Sa  o Paulo, S.P., Brasil (Received 14 May 1997 ; revised 12 September 1997 ; accepted 12 September 1997)  Leishmania braziliensis strain M2903 was adapted for growth and serially maintained as amastigotes at 34 C in modified UM-54 medium, with growth curves exhibiting typical log and stationary phases. In late passages, amastigote growth took place in the absence of supplementary haemin and was unaffected when the initial medium pH was adjusted between 54 and 63. In contrast to promastigotes, which were elongated and exhibited very long free flagella endowed with the paraflagellar rod (PFR), axenic amastigotes were rounded to ovoid and displayed a short flagellum restricted to the pocket area. The absence of PFR in axenic amastigotes was confirmed in Western blots and confocal immunofluorescence microscopy, by lack of reactivity with mAb 1B10. The antibody, which specifically labelled the paraflagellar structure, recognized a 7072 kDa doublet in Trypanosoma cruzi epimastigotes and two 7074 kDa related proteins in L. braziliensis promastigotes. Surface I-labelling experiments identified promastigote-specific components ( 100, 74, 4547 and 28 kDa) and at least 1, a 76 kDa polypeptide was specific for the amastigote stage. While axenic amastigotes were agglutinated by both peanut (PNA) and Lens culinaris (LCA) agglutinins, respectively at 50 and 125 μgml, promastigotes were not agglutinated by PNA and agglutinated in the presence of LCA at concentrations of 100 μgml and higher. Axenic amastigotes infected rat bone marrow-derived macrophages and were avidly taken up by J774 cells, from which numerous organisms, able to proliferate at 34 C in UM-54 medium, could be recovered 48 h later. Key words : Protozoa, Leishmania braziliensis, amastigote-like forms, axenic amastigotes.  Trypanosomatid flagellates of the genus Leishmania exist as promastigotes within the alimentary tract of blood-feeding Phlebotomine sandflies, and as in- tracellular amastigotes within phagolysosomes of mammalian macrophages (see Molyneaux & Killick- Kendrick, 1987). Promastigotes can be easily main- tained at temperatures below 28 C in axenic, commercially available growth media (Chang, Nacy & Pearson, 1986) but amastigotes have to be isolated from either animal lesions or macrophage cultures. These procedures do not assure that preparations are free from host-derived contaminants, such as organ- elles and macromolecules. Indeed, studies with L. mexicana have clearly demonstrated the presence of host immunoglobulins at the surface of lesion amastigotes (Peters et al. 1995). Contamination of parasite preparations can be avoided by using amastigotes from axenic cultures. * Corresponding author : Departamento de Parasitologia, Instituto de Cie  ncias Biome  dicas, Universidade de Sa  o Paulo, Av. Prof. Lineu Prestes 1374, CEP 05508-900, Sa  o Paulo, SP, Brasil. Tel : 55 11 818 7263, Fax : 55 11 818 7417. E-mail : salfieribiomed.icb2.usp.br Different Leishmania strains have been adapted for growth as amastigotes at temperatures above 28 C in cell-free media (reviewed by Pan et al. 1993; Hodgkinson et al. 1996). Despite the different conditions that have been used, in all instances the differentiation of promastigotes into amastigotes was achieved by increasing the temperature andor dropping the medium pH. As reviewed elsewhere (Zilberstein & Shapira, 1994), alterations of both temperature and pH trigger changes in gene ex- pression, leading to the appearance of new stages. Axenic amastigotes have been shown to be morpho- logically and biochemically similar to lesion-derived parasites (Pan, 1984; Pan & Pan, 1986; Eperon & McMahon-Pratt, 1989 a ; Rainey et al. 1991 ; Bates et al. 1992; Pan et al. 1993; Pral et al. 1993), infective to animals and macrophage cultures (Pan & Honigberg, 1985; Pan & Pan, 1986; Eperon & McMahon-Pratt, 1989 a ; Pan et al. 1993), and to display amastigote-specific surface antigens (Pan & McMahon-Pratt, 1988 ; Eperon & McMahon-Pratt, 1989 b ; Pan et al. 1993 ; Hodgkinson et al. 1996). Axenic amastigote cultures so far described have been established and serially maintained under conditions that differ as to the composition and pH Parasitology (1998), 116, 103–113. Printed in the United Kingdom  1998 Cambridge University Press