Cultivation of Clinically Significant Hemoflagellates

Abstract

The hemoflagellates, Trypanosoma spp. and Leishmania spp., are causal agents of a number of parasitic diseases having a major impact on humans and domestic animals over vast areas of the globe. Among the diseases are some of the most pernicious and deadly of human afflictions: African sleeping sickness, Chagas' disease, kala-azar, and Oriental sore. The organisms have complex, pleomorphic life cycles typically involving a vertebrate and an invertebrate host, the latter serving as a vector. In the vertebrate host, they are primarily blood and tissue parasites. In their transition from one host to another, the hemoflagellates undergo morphological, physiological, and biochemical changes that facilitate their growth and subsequent transmission. A major goal in the study of the hemoflagellates has been the cultivation in vitro of both vertebrate and invertebrate stages of the organisms. The first types of media used in their cultivation, and still useful for establishment of cultures, were undefined and contained a complex of ingredients. These gave way to semidefined formulations which included tissue culture media as a base and, as a next step, addition of tissue culture cells as a feeder layer to promote parasite growth. More recently developed media are completely defined, having replaced the feeder cells with various supplements. Serum, a sometimes-variable component of the media, can be replaced by various serum substitutes. This review focuses on the hemoflagellates that infect humans, describing stages in the development of media leading to the fully defined formulations that are now available for the cultivation of many of these organisms

Trypanosoma rhipicephalis sp. nov. (Protozoa: Kinetoplastida) isolated from Rhipicephalus microplus (Acari: Ixodidae) ticks in Rio de Janeiro, Brazil

“…The great diversity of culture media may, for example, provide nutritional requirement information for these parasites (Schuster and Sullivan, 2002). Knowledge of the replication stage of trypanosomes is important when studying new species.…”

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

“…Folic acid has been implicated in tymidine and methionine synthesis and in the interconversion of serine into glycine by Leishmania (Ouellete et al 2002). It was also showed that absence of folic acid inhibited L. (L.) donovani growth (Schuster & Sullivan 2002). Therefore, we favor the depletion of an essential component as a more likely explanation.…”

“…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 (Schuster & Sullivan 2002). L. (V.) braziliensis promastigotes show low growth rates in in vitro cell culture, when they are compared with other Leishmania genus species (Armstrong & Paterson 1994).…”

LB broth-lyophilized Rabbit serum (LLR) as a new and suitable culture medium for cultivation of promastigotes of Leishmania major

“…FCS is highly expensive, and reliable supply is very difficult to obtain, especially in developing countries (Newman 2003) and using it in cell culture is problematic for several reasons as complexity, highly variable, and difficult to characterizing (Armstrong and Patterson 1994). Several attempts have been made to replace FCS in leishmania culture media with different kinds of sera, bovine serum albumin, a mixture of purine bases, vitamins, large concentrations of certain amino acids, hormones, hemin, hemoglobulin, human and animal urine and, more recently chicken serum (Trager 1953;Herman 1966;Ghoshal et al 1986;Ali et al 1998;Merlen et al 1999;Shamsuzzaman et al 1999;Pal and Joshi-Purandare 2001;Schuster et al 2002;Nasiri et al 2013a, b;Nasiri 2013) that the later introduced an alternative low-cost serum that can be used in culture medium for primary isolation, routine cultivation and mass cultivation of Leishmania parasites (Nasiri et al 2011(Nasiri et al , 2013a.…”