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.

FIG. 1.

Schematic illustrations of the hemoflagellate stages discussed in this review. Morphological distinctions include the length of the flagellum and its location, and the position of the kinetoplast with respect to the nucleus of the cell. (A) Promastigote stage of Leishmania spp. found within the insect vector; (B) epimastigote stage typical of Trypanosoma spp. in the insect vector; (C) trypomastigote stage typical of the bloodstream form of Trypanosoma spp.; (D) intracellular amastigote stage of T. cruzi and Leishmania spp. Abbreviations: N, nucleus; K, kinetoplast; F, flagellum. Adapted from reference with permission of the publisher.

FIG. 2.

(A) Epimastigote stage of T. cruzi from culture, the stage normally seen in the insect vector. Flagella can be seen arising from the slender organisms. The dark-staining body within the cell is the nucleus. Magnification, ×1,000. (B) Promastigote stage of L. donovani from culture, the stage normally seen in the insect vector. The organisms are clustered in the forms of rosettes, held together by their intertwined flagella. Magnification, ×800.

FIG. 3.

Generalized scheme for isolation of representatives of T. cruzi and the African trypanosome complex from blood and other tissues of a vertebrate host. Samples are taken from blood as well as lymphoid tissue and transferred to NNN diphasic blood agar medium. Once established in an enriched medium, trypanosomes can be inoculated into animals or transferred into semidefined or defined culture medium. Isolates behave differently from one another, and it may not be possible to establish all isolates in defined media. The crude media are a good backup in case the organism will not grow under more defined circumstances.