Leishmania (Viannia) braziliensis growth in vitro culture relies more on folic acid availability than Leihsmania (Leishmania) amazonensis

Abstract

We compared the in vitro growth of promastigotes from two Leishmania species in TC-100 and Schneider media. Leishmania (Leishmania) Leishmania are parasitic protozoa that infect many mammals including man. Despite the epidemiological impact of Leishmania (Viannia) braziliensis in the New World (OPS 1996), it has been studied less than other species, principally due to the difficulties of keeping this parasite in axenic conditions. These difficulties were used in the past to differentiate this Leishmania species and were related to media composition differences (Walton et al. 1977, Shaw & Laison 1981. Biphasic media with solid phases enriched with variety of components have been used to keep Leishmania, but harvesting large numbers of parasites is difficult, thus liquid media is preferred for promastigote cultivation ( MATERIALS AND METHODSParasites were cultured as previously described (Forero et al. 1999). L. (V.) braziliensis (HOM/BR/75M2903) and L. (L.) amazonensis (FLA/BR/67/PH8) isolates, kindly donated by Dr Nancy Gore Saravia (Cideim, Cali, Colombia) were used in the present study. Promastigotes, at an initial concentration of 1 x 10 6 , were cultured at 24 o C in 25 cm 2 flasks (Costar) in TC-100 or Schneider media (Invitrogen) supplemented with 10% FBS (Hyclone). Cultures were allowed to reach their metacyclic phase, determined by the stationary behavior of parasite number and rosette formation. Samples of parasites at different time points were counted in a Neubauer hematocytometer in a solution of 5% Giemsa, 2% formaldehyde in 0.14 M NaCl, under light microscopy. Data were analyzed with the Student's t test and p values < 0.05 were considered significant. RESULTSL. (L.) amazonensis replication rates were similar in the two culture media during the period studied, and maximum replication rates occurred within 48 h (Fig. 1A). L. (V.) braziliensis replication rates doubled during the first 48 h and no differences were found between the two culture media (p = 0.4). However, between 72 to 96 h postseeding, the replication rates of L. (V.) braziliensis promastigotes tripled in TC-100 but only doubled in Schneider. This difference was significant (p = 0.02; Fig. 1B). Although L. (V.) braziliensis took 96 h to reach its optimal replication rate compared to L. (L.) amazonensis, parasite numbers were similar. The percentage of metacyclics increased over the period studied for both species, and achieved their maximum value during the stationary phase. To further investigate the impact of particular nutritional factors on the recorded differences, folic acid was added to Schneider media. L. (V.) braziliensis replication rates increased and the maximum replication time was reduced to 48 h post-seeding, suggesting that the limiting nutritional factor was folic acid (Fig. 1C). DISCUSSIONWe found no differences between replication rates of L. (L.) amazonensis promastigotes in the two media studied. In contrast L. (V.) braziliensis promastigote replication rates were significantly higher in TC-100 compared to Schn...

Separation of Leishmania-infected macrophages by step-SPLITT fractionation

“…Promastigotes, at an initial concentration of 1 × 10 6 , were cultured at 24 • C in 25 cm 2 flasks in TC-100 or Schneider's medium (Invitrogen) supplemented with 10% FBS [9]. Promastigotes were allowed to reach their stationary stage (stationary growth, rosette formation and good infectivity) and either diluted to maintain the culture or harvested for infection.…”

Hiperadrenocorticismo Canino E Felinorevisão De Literatura

“…O meio de Schneider tem sido empregado em cultura de formas promastigotas de Leishmania, há décadas (HENDRICKS; WOOD; HAJDUK, 1978;HOCKMEYER et al, 1981;NEAL, 1984;NIÑO;CAMACHO, 2005;MADEIRA et al, 2006;SANTARÉM et al, 2014;SANTOS et al, 2018). O SFB é uma das suplementações mais comumente utilizada em diversos meios de cultura de Leishmania com concentrações de 10 a 30% (HENDRICKS;WOOD;HAJDUK, 1978;NEAL, 1984;HOWARD et al, 1991;NIÑO;CAMACHO, 2005;MADEIRA et al, 2006;SANTARÉM et al, 2014;SANTOS et al, 2018). Contudo, o SFB tem sido criticado devido ao alto custo, à sua composição variável, às implicações éticas e à contraindicação de seu uso em processos de desenvolvimento para produtos de uso humano.…”

“…gêneros Leishmania e Trypanosoma (HENDRICKS; WOOD; HAJDUK, 1978) e, desde então, tem sido usado por alguns pesquisadores (HENDRICKS;WOOD;HAJDUK, 1978;HOCKMEYER et al, 1981;NEAL, 1984;NIÑO;CAMACHO, 2005;MADEIRA et al, 2006;SANTARÉM et al, 2014;SANTOS et al, 2018).…”

Achados Radiográficos Torácicos De Cães E Gatos Atendidos Em Uma Clínica Veterinária Na Cidade De Teresina – Pi

“…O meio de Schneider tem sido empregado em cultura de formas promastigotas de Leishmania, há décadas (HENDRICKS; WOOD; HAJDUK, 1978;HOCKMEYER et al, 1981;NEAL, 1984;NIÑO;CAMACHO, 2005;MADEIRA et al, 2006;SANTARÉM et al, 2014;SANTOS et al, 2018). O SFB é uma das suplementações mais comumente utilizada em diversos meios de cultura de Leishmania com concentrações de 10 a 30% (HENDRICKS; WOOD;HAJDUK, 1978;NEAL, 1984;HOWARD et al, 1991;NIÑO;CAMACHO, 2005;MADEIRA et al, 2006;SANTARÉM et al, 2014;SANTOS et al, 2018). Contudo, o SFB tem sido criticado devido ao alto custo, à sua composição variável, às implicações éticas e à contraindicação de seu uso em processos de desenvolvimento para produtos de uso humano.…”