Screening of metal ion chelators against Leishmania donovani-infected Syrian hamsters.

Leishmania donovani-infected Syrian hamsters were treated intraperitoneally with 0.23 mmoles/kg/day of EDTA, EGTA, HEEDTA and 100 mg/kg/day of Pentostam R. The control group received 0.1 ml of phosphate buffered saline. After 30 days of treatment, the animals were sacrificed. Of the Pentostam-treated animals, 5 out 6 had negative spleen cultures, while all the chelator and PBS-treated ones yielded parasites. While all the Pentostam-treated animals had negative bone marrow cultures, only 1 out of 6 HEEDTA-treated hamsters yielded parasites. Spleen, liver and bone marrow parasite- loads calculated from chelator-treated animals were consistently significantly higher than for Pentostam-treated animals. These results suggest that although metal ion chelators have some antileishmanial potential, their in vivo activity against L. donovani is low compared to Pentostam.

Comparison of Giemsa and acridine orange stains for the diagnosis of Leishmania donovani in biopsies of infected hamsters.

Identical impression smears of spleen, liver and bone marrow biopsy materials from Leishmania donovani-infected hamsters were stained using either acridine orange or Giemsa. Spleen parasite-loads calculated from the two stains for identical biopsy material were significantly different from each other. However, liver and bone marrow parasite- loads calculated from either Giemsa-stained or acridine orange-stained biopsies were not significantly different from each other. This study has shown that acridine orange, which is a quick and simple technique, has great potential in the diagnosis of kala-azar when liver and bone marrow biopsies are used.

Pristane (2,6,10,14-Tetramethyl-pentadecane) inhibits disease progression in Leishmania-infected Balb/c mice.

The course of Leishmania infection in pristane-primed BALB/c mice infected with either Leishmania major or Leishmania donovani was examined. Pristane-primed L. donovani infected mice had spleen parasite-loads that were 13 times less than controls. Likewise pristane-primed L. major infected animals had significantly smaller footpad lesion areas than controls. Pristane-primed mice had an atypical haematology compared to controls. To the best of our knowledge, this is the first report to demonstrate that pristane inhibits progression of disease in Leishmania-infected BALB/c mice.

Ethyleneglycol-Bis-((-aminoethyl ether) N,N,N(1),N(1), -Tetraacetic acid (EGTA) inhibits Leishmania donovani in vitro.

Exposure of Leishmania donovani culture promastigotes to ethyleneglycol-bis-((-aminoethyl ether) N,N,N(1),N(1),-tetraacetic acid (EGTA) concentrations of between 0.2 to 1.6 mg/ml significantly inhibited their growth, though the different concentrations did not significantly differ between themselves on their effect on promastigotes in cell free media. EGTA concentrations of between 0.05 and 0.1 mg/ml were non-toxic to mouse peritoneal macrophages in vitro. Treatment of L. donovani-infected macrophages with EGTA at concentrations of 0.05, 0.1 and 0.2 mg/ml contributed significantly to a decline in amastigote parasite-loads in the macrophages. The higher the chelator concentration within the acceptable toxic levels for macrophages, the greater was the rate at which parasites were cleared from the macrophages. It is speculated that EGTA chelates manganese from phosphoenol pyruvate (PEP) carboxykinase, a TCA-rate limiting enzyme in the metabolism of Leishmania parasites. A manganese-complex is also probably used by these parasites as a defense mechanism against oxygen-derived radicals. Chelation of manganese would destabilise PEP carboxykinase, and therefore severely interfere with the parasite metabolism. All these factors would render the Leishmania parasite susceptible to digestion in the lysosomal vacuoles of the macrophage, hence the observed significant reduction in parasite-loads of L. donovani-infected EGTA-treated macrophages in vitro. These results suggest an exciting future potential use of chelators in the experimental chemotherapy of visceral leishmaniasis.

Establishment of an appropriate inoculum dose of Leishmania donovani promastigotes required to establish a visceral infection in laboratory animal rodent models.

Syrian hamsters and BALB/c mice were inoculated intraperitoneally with various doses of stationary phase Leishmania donovani promastigotes derived from primary, secondary and tertiary cultures. Axenic derived amastigotes from a tertiary culture and mass-culture derived promastigotes from primary, secondary, and tertiary cultures were also used. Animals were sacrificed after 30 days incubation period and parasite-loads quantified from Giemsa stained spleen smears. A primary inoculum dose of 1 x10(8) was found to be the most appropriate in effecting a visceral infection. This parasite dose resulted in a spleen parasite-load of 10-20 amastigotes per field of microscope view at x1,000 magnification. Those involved in candidate vaccine molecules or experimental drugs against kala-azar will find these results useful.