Estimation of the minimum number of Leishmania major amastigotes required for infecting Phlebotomus duboscqi (Diptera: Psychodidae).

BACKGROUND: Leishmaniasis is a vector-borne disease in which Leishmania parasites are transmitted by the bite of phlebotomine sand flies. Amastigotes are ingested by the sand fly vector with a blood meal taken from an infected host. This is followed by their differentiation into metacyclic promastigotes which are selectively released and permitted to migrate interiorly so as to make them available for transmission by bite. However, the actual number of amastigotes ingested by the sand fly in the blood meal is not known. OBJECTIVE: To investigate the minimum number of Leishmania major amastigotes required to cause an infection in Phlebotomus duboscqi following an infective blood meal. DESIGN: A laboratory based study. SETTING: Centre for Biotechnology Research and Development, Kenya Medical Research institute, Nairobi. RESULTS: Dissection of all fed sand flies at six days post-infective blood meal revealed that blood containing one amastigote per 0.3 microl in a total volume of 0.5 ml was able to cause an infection in the sand flies, but very few sand flies got infected (7.6% and 9.6% respectively). Concentrations of ten amastigotes per 0.3 microl in 0.5 ml gave infection rates of 35.4% and 26.3% respectively, suggesting that even when the concentration of amastigotes in a bloodmeal was high, not all sand flies feeding on it were able to pick up the parasites. CONCLUSIONS: These observations suggests that one amastigote is sufficient to cause an infection to a sand fly and as a result of multiplication in the gut and the existence of mechanisms that increase the number of infective bites delivered by a female sand fly they are able to sustain the transmission of leishmaniasis in an area.

Phlebotomus guggisbergi (Diptera: Psychodidae), a vector of Leishmania tropica in Kenya.

Sand flies were collected in light traps and on oiled papers at four active case sites of human cutaneous leishmaniasis due to Leishmania tropica at Muruku Sublocation, Laikipia District, Kenya. Nearly 5,200 females of five species, including Phlebotomus guggisbergi, were dissected and examined for flagellates. Of 3,867 P. guggisbergi females collected at a multiple case site, 168 (4.3%) harbored mature infections (to include metacyclic promastigotes) of flagellates morphologically identical to Leishmania, while all other flies were negative. Of the infected flies, 164 were collected in a cave near the patients' home, three from crevices on an escarpment immediately behind the house, and one from the bedroom of one of the patients. One hundred sixty-four of the isolates were successfully grown in Schneider's Drosophila medium and harvested for typing by cellulose-acetate electrophoresis. Isoenzyme profiles of the first 22 of these were compared with those of WHO reference strains and well characterized local strains using 12 enzyme loci. The isolates yielded isoenzyme migration patterns that were indistinguishable from those of two L. tropica reference strains and of six L. tropica patient isolates from the same locality. This is the first reported isolation of L. tropica from a sand fly in Kenya, the first reported isolation of Leishmania parasites from P. guggisbergi, and the first confirmed isolation of this Leishmania from a sand fly other than P. sergenti. The finding of such a large number of P. guggisbergi naturally harboring mature infections of L. tropica at an active case site of cutaneous leishmaniasis due to this agent strongly implicates this fly as a vector.

Experimental immunization against cutaneous leishmaniasis using Leishmania major subcellular fractions alone or in combination with Phlebotomus duboscqi gut antigens.

Leishmania major-derived flagella and nuclear fractions, and a combination of flagella and sand fly gut antigens were assessed for protection against L. major infection in BALB/c mice. Mice immunized with flagella antigen developed a severe infection while nuclear fraction-immunized animals were partially protected at the onset of infection from week 1 to 4 post challenge. A combination/cock tail of flagella and sand fly gut antigens protected animals at a later stage from week 10 to 14 post-infection. Surviving cocktail-immunized animals did not ulcerate, and parasites did not metastasize to the viscera. These results provide preliminary evidence of the potential of a cock tail antigen derived from Leishmania flagella and sand fly gut in the protection against cutaneous leishmaniasis caused by L. major.

Animal reservoirs of leishmaniasis in Marigat, Baringo District, Kenya.

A total of 1128 rodents belonging to seven genera were examined for leishmanial parasites over a period of sixteen months. Parasites were isolated from 36 (12.5%) Tatera robusta, 3 (0.5%) Arvicanthis niloticus, and 2 (0.8%) Mastomys natalensis. All isolates were characterised by isoenzyme analysis using nine enzymes. The enzymes examined were: malate dehydrogenase (MDH), phosphoglucomutase (PGM), glucose phosphate isomerase (GPI), isocitrate dehydrogenase (ICD), nucleoside hydrolase (NH), glucose 6-phosphate dehydrogenase (G6PD), mannose phosphate isomerase (MPI), malic enzyme (ME) and 6-phosphogluconate dehydrogenase (6PGD). The enzyme profiles from these isolates were compared with those from Leishmania reference strains and also with isolates of Leishmania major from man and sandfly, P. duboscqci from the same area. All the isolates except one from a Mastomys were identified as L. major. The isolate from Mastomys was trypanosome-like and remains unidentified. The results in this study show that Tatera robusta is the main reservoir of cutaneous leishmaniasis in Baringo District. None of the animals trapped were found infected with Leishmania donovani suggesting that rodents do not play a role in the transmission of visceral leishmaniasis in this area.