Genus Leishmania

The genus Leishmania: its two morphological forms and how they differ from Trypanosoma, how the parasite gets inside macrophages, its vectors and its reservoir hosts, how the species are told apart in the laboratory, the life cycle of Leishmania donovani and the visceral leishmaniasis (kala-azar) it causes, what the parasite looks like under the light and electron microscope, and the other species that infect humans, Leishmania tropica and Leishmania brasiliensis, with the cutaneous and mucocutaneous diseases they produce.

Leishmania is a kinetoplastid, like Trypanosoma, but it uses only two of the four kinetoplastid forms.

These parasites have two forms:

• amastigote (intracellular) in the vertebrate host;
• promastigote (extracellular) in the vector.

The promastigotes that penetrate the bite are allowed to be phagocytosed by macrophages and dendritic cells in the dermis, after which they lose their flagella and become amastigotes. The word "allowed" is deliberate: the parasite does not resist phagocytosis, it uses it as its way into the cell.

Unlike T. cruzi, Leishmania, once an amastigote, remains in this form while in the vertebrate host, and forms a flagellum only if taken up by the vector. T. cruzi alternates between amastigote and trypomastigote inside the mammal; Leishmania does not alternate at all.

More than 20 species of the genus Leishmania can infect humans. The disease is called leishmaniasis.

• Predators of the Canidae family, as well as rodents, are also hosts. These animals are a natural reservoir of Leishmania. Dogs are drawn in the life cycle beside the human for exactly this reason.
• Leishmaniasis in Europe, Asia and Africa is transmitted by female sandflies of the genus Phlebotomus, and in North and South America by related dipteran insects.
• The vector is Phlebotomus papatasii (sand fly) - only the females, because only they suck blood.
• Meiosis, production of gametes, and gamete fusion occur in the insect, which therefore is the definitive host.
• The different species of Leishmania do not differ morphologically. Molecular biology methods and monoclonal antibodies are used for diagnosis. This is the practical point: you cannot name the species down a microscope, so the species is decided by the clinical picture, the geography, and the laboratory.

The figure of the infected skin shows the phlebotomine sandfly biting, the promastigote entering the dermis, the macrophage taking it up, the amastigotes multiplying inside it, and lymphocytes gathering around, with the parasites also drawn inside a blood vessel.

a) In the elongated promastigote form of the parasite, the flagellum extends freely from the anterior end (no undulating membrane), so it appears longer than in Trypanosoma. The kinetoplast is also at the anterior end.

b) The amastigotes are rounded, without a flagellum and much smaller than promastigotes because they are intracellular.

The drawing of the two forms labels, in both of them, the flagellum, the flagellar pocket, the mitochondria, the kinetoplast, the Golgi, the multi-vesicular bodies, the nucleus and the endoplasmic reticulum - the same organelles are present in each, but the amastigote's flagellum is reduced to a stub inside the flagellar pocket.

• Promastigote (extracellular) form, seen in the insect. While trypomastigotes curl like little worms because the undulating membrane transmits the movements of the flagellum to the entire cell, promastigotes have a straight cell body.
• Electron microscopy of an amastigote (intracellular) form in a mammalian cell. An arrow points to the kinetoplast, above which is the basal body of the flagellum on the left and the nucleus on the right.
• In a stained smear, two large cells are macrophages, in which a large number of amastigote Leishmania are visible. Note the characteristic "double dot" pattern - a large dot (nucleus) next to a small dot (kinetoplast).

The "double dot" is the practical recognition sign of Leishmania amastigotes in a smear, and it is worth remembering that the small dot is mitochondrial DNA.

It causes visceral leishmaniasis (kala-azar).

After the bite, the parasites migrate to the internal organs, mainly to the liver, spleen, and bone marrow. The main symptoms are fever, weight loss, anaemia, and enlarged (due to inflammation) spleen and liver. The photograph in the lecture shows a child with the grossly distended abdomen that the enlarged liver and spleen produce.

If left untreated, the disease almost always results in the death of the host.

On the world map, visceral leishmaniasis is distributed across South America, much of Africa, the Mediterranean, the Middle East, Central and South Asia, that is, across the range of the sandfly vectors.

The cycle has a mammalian half and a sandfly half.

Mammalian stages:

• The sandfly takes a blood meal and injects the promastigote stage into the skin.
• Promastigotes are phagocytised by macrophages.
• Promastigotes transform into amastigotes inside macrophages.
• Amastigotes multiply in cells (including macrophages) of various tissues.

Sandfly stages:

• The sandfly takes a blood meal and ingests macrophages infected with amastigotes.
• Ingestion of the parasitised cell.
• Amastigotes transform into the promastigote stage in the midgut.
• Amastigotes divide in the midgut and migrate to the proboscis, from where the next bite delivers them.

A dog is drawn beside the human in the cycle, as a reservoir host.

Leishmania tropica

• In Africa, Europe, and Asia.
• It causes cutaneous leishmaniasis - ulcers on the skin of the face and limbs around the bite site.
• The most "harmless" leishmaniasis - even without treatment, it heals in about a year, although a scar may remain.

Leishmania brasiliensis

• In Central and South America.
• Causes mucocutaneous (American) leishmaniasis with skin ulcers similar to the cutaneous form or deeper, but if the bite is on the face, the parasite gradually invades and destroys the oral and nasal mucosa.
• Late untreated stages are characterised by the so-called "tapir nose" - the nasal septum is missing.

Note that the lecture writes the species as Leishmania brasiliensis; it is also met as L. braziliensis, and the exam paper follows the spelling used here.

• *Leishmania* is a kinetoplastid with only two forms: the promastigote, extracellular, in the vector, and the amastigote, intracellular, in the vertebrate host.
• The promastigote is taken up by macrophages and dendritic cells in the dermis, loses its flagellum and becomes an amastigote, and stays an amastigote for as long as it is in the vertebrate - unlike T. cruzi, which alternates.
• The vector is the female sandfly: *Phlebotomus* (Ph. papatasii) in Europe, Asia and Africa, related dipterans in the Americas. Only females suck blood. Meiosis, gametogenesis and gamete fusion occur in the insect.
• Canidae and rodents are the natural reservoir.
• More than 20 species infect humans, and they are morphologically identical: molecular methods and monoclonal antibodies are needed to identify them.
• The amastigote in a smear shows the "double dot": the large dot is the nucleus, the small dot the kinetoplast.
• *L. donovani* causes visceral leishmaniasis (kala-azar) in liver, spleen and bone marrow, with fever, weight loss, anaemia and hepatosplenomegaly, and is almost always fatal untreated.
• *L. tropica* causes cutaneous leishmaniasis which heals in about a year; *L. brasiliensis* causes mucocutaneous leishmaniasis which can destroy the nasal septum ("tapir nose").