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Morphology and Life Cycle

Leishmaniaspp. 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 Leishmaniathe life cycle stage in the vertebrate is the amastigote and in the

insect, the promastigote. Leishmania exist in two basic body forms: the amastigote, theintracellular 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 25

oC 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.

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Amastigotes in a phagocytic cell by TEM

Life Cycle

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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.

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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 sandflys 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

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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.

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Thanks to K.P. Chang for these images.

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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 todifferentiate 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.

Clickhere to see several streaming videos ofLeishmania tarentolae cells in culture under the phase contrast

microscope.

Study questions:

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?

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