Morphology and Life Cycle
Leishmania spp. are digenetic or heteroxenous parasites, whose life cycle involves two hosts, a vertebrate and an invertebrate, the sandfly. Hemoflagellates may have several morphological stages that differ from one another in the placement of the kinetoplast relative to the nucleus, as well as the location and origin of the flagellum. In Leishmania the life cycle stage in the vertebrate is the amastigote and in the insect, the promastigote. Leishmania exist in two basic body forms: the amastigote, the intracellular form in the vertebrate host, and the promastigote, the extracellular form in the sandfly (Phleobotomus spp. and Lutzomyia spp.) vector. Amastigotes are taken up from the blood of an infected host when the female sandfly bites, and in the sandfly gut they develop into promastigotes where they multiply by binary fission; promastigotes move anteriorly into the proboscis, and are introduced into the vertebrate host when the sandfly bites again. The promastigotes injected by the sandfly during feeding are phagocytized and develop into intracellular amastigotes.
The amastigote, literally “without a flagellum,” is the intracellular, non-motile form in the vertebrate host, and it divides by longitudinal binary fission at 37oC. Intracellular amastigotes are 3-6 um in length and 1.5-3.0 um in width. The amastigote is also called the Leishman-Donovan (LD) body. The amastigote is not really devoid of a flagellum, it is simply that the flagellum does not protrude beyond the body surface and by light microscopy cannot be seen.
The promastigote, literally the body form with “an anterior flagellum” is 15-30 um in body length and 5mm in width; it is extracellular, motile, and grows and divides by longitudinal binary fission at 27oC in the sandfly. Promastigotes can be grown in vitro at 25oC temperature on NNN medium, which has a solid phase of blood agar and a liquid phase containing a physiologic salt solution. Liquid media that support promastigote growth are also available. Amastigotes usually are grown inside tissue culture cells and can also be grown extracellularly at 37oC under special conditions.
Amastigotes in a phagocytic cell by TEM
Leishmania invade the phagocytic cells of the reticulo-endothelial system and macrophages, but are able to evade destruction in these phagocytic cells. Transmission is by the bite of female sandflies in the genera, Phleobotomus and Lutzomyia.
Sandflies are pool feeders and when infected macrophages are taken up by the blood-feeding sandfly, the amastigotes transform into promastigotes.
The promastigotes in the gut of the female sandfly attach to the midgut-hindgut epithelium where they divide by binary fission. Development in the sandfly takes 8-20 days after initial ingestion of blood, and after multiplication in the midgut-hindgut they move forward to the pharynx where they produce a partial or complete blockage of the sucking apparatus. (These are called metacyclic promastigote forms and the trigger for metacyclogenesis is a decline in tetrahydrobiopterin). When such a sandfly attempts to feed (one could say the ”fly clears its throat”) it regurgitates a bolus of metacyclic promastigotes and this is injected into the bite wound. There is no infection of the sandfly’s salivary glands as in the case of the african trypanosomes in the tsetse fly.
The promastigotes are phagocytized by macrophages or other “professional” phagocytes, and transform into intracellular amastigotes.
Although promastigotes specifically interact with the surface of the host cell they cannot actively penetrate the host cell (unlike T. cruzi) and thus infection is dependent on host cell phagocytosis.
Thanks to K.P. Chang for these images.
The Leishmania Life Cycle in vitro
For experimental purposes the ability to have parasites progress through the entire life cycle in axenic cultures (no other cells present) would be extremely helpful. The insect stage of Leishmania can easily be grown in axenic culture at 26-27oC, and as discussed in another chapter, metacyclic promastigote forms appear in late stationary phase in culture which are infective for the mammalian host. Continuous in vitro axenic culture of amastigote-like cells at 37oC has also been achieved. The problems have been that promastigotes rapidly lose virulence in culture and cultured amastigotes have not been widely used for genetic studies. A recent study by Goyard et al. (2003) describes a clonal line of L. donovani (Ld bob or Ldb) which retains the ability to differentiate repeatedly between the promastigote and amastigote stages in culture, both forms can be easily plated on agar and easily transfected with plasmids. This line was originally developed in the Dwyer laboratory (Joshi et al. 1993). Several stage specific markers such as LPG, sAP and the A2 protein family were expressed as expected. The axenic amastigotes are also as virulent as wt L. donovani cells.
Click here to see several streaming videos of Leishmania tarentolae cells in culture under the phase contrast microscope.
1. Can the entire life cycle of Leishmania sp. be studied in culture?
2. Why do you think that the parasites become promastigotes in the sandfly and why amastigotes in the vertebrate host?