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<i>Leishmania major-phlebotomus duboscqi</i> interactions: inhibition of anti-LPG antibodies and characterisation of two proteins with shared epitopes
Abstract
Objectives: To assess the effect of monoclonal antibodies (MABS) raised against L. major derived LPG on L. major development in vitro and in its natural vector P. duboscqi. Also determine whether LPG molecule and the sand fly the gut Iysates have sharedepitopes.
Design: A laboratory based study.
Setting: Colony bred P. duboscqi sand flies and all other experiments were done under laboratory conditions.
Methods: Laboratory reared sand flies were allowed to feed beneath a blood filled membrane feeder containing 1 x106 amastigotes in 20µl mixed with 0.5 ml of defibrinated rabbit blood with a 1:100 dilution of anti-LPG MABS. Control blood contained a similar number of amastigotes but no MABS. At least five female previously fed sand flies were later dissected on days two, four, and six post-feeding and examined for promastigote forms and parasite loads in the sand fly mid gut. In vitro, the same number of amastigotes in 100µl complete Schneider's Drosophila medium was mixed in a 96 well plate with either 100µl of 1: 100 anti-LPG MABS, 1:1000 anti LPG MABS or undiluted sera from L. major infected mice. The control well contained a similar number of amastigotes but no antibodies added. Following an overnight incubation in a CO2 incubator at 37ºC and growth at 26ºC, parasites were assessed at 3, 6 and 24 hour intervals for changes in their developmental forms.
Results: 1:100 dilution of anti-LPG MABS when mixed with amastigotes were effective in reducing L. major development at the early log phase or procyclic stage both in vitro and within the sand fly (p<0.05). The control cultures or sand flies that fed on amastigotes alone and no MABS supported full parasite development up to the metacyclic stage. Results also showed that flies, which had fed on MABS, showed low parasitemia levels of 2+, compared to a high density of 4+ for their controls (p<0.5).
Conclusions: These findings showed that anti-LPG MABS were effective in reducing sand fly infections. This study also showed that P. duboscqi gut lysates and proteins present in L. major-derived LPG share two common proteins of molecular weights 105kDa and 106kDa. Further analysis of these individual proteins from the gut should be studied with a view of determining their vaccine potential.
East African Medical Journal Vol. 81 No. 2 February 2004: 97-103
Design: A laboratory based study.
Setting: Colony bred P. duboscqi sand flies and all other experiments were done under laboratory conditions.
Methods: Laboratory reared sand flies were allowed to feed beneath a blood filled membrane feeder containing 1 x106 amastigotes in 20µl mixed with 0.5 ml of defibrinated rabbit blood with a 1:100 dilution of anti-LPG MABS. Control blood contained a similar number of amastigotes but no MABS. At least five female previously fed sand flies were later dissected on days two, four, and six post-feeding and examined for promastigote forms and parasite loads in the sand fly mid gut. In vitro, the same number of amastigotes in 100µl complete Schneider's Drosophila medium was mixed in a 96 well plate with either 100µl of 1: 100 anti-LPG MABS, 1:1000 anti LPG MABS or undiluted sera from L. major infected mice. The control well contained a similar number of amastigotes but no antibodies added. Following an overnight incubation in a CO2 incubator at 37ºC and growth at 26ºC, parasites were assessed at 3, 6 and 24 hour intervals for changes in their developmental forms.
Results: 1:100 dilution of anti-LPG MABS when mixed with amastigotes were effective in reducing L. major development at the early log phase or procyclic stage both in vitro and within the sand fly (p<0.05). The control cultures or sand flies that fed on amastigotes alone and no MABS supported full parasite development up to the metacyclic stage. Results also showed that flies, which had fed on MABS, showed low parasitemia levels of 2+, compared to a high density of 4+ for their controls (p<0.5).
Conclusions: These findings showed that anti-LPG MABS were effective in reducing sand fly infections. This study also showed that P. duboscqi gut lysates and proteins present in L. major-derived LPG share two common proteins of molecular weights 105kDa and 106kDa. Further analysis of these individual proteins from the gut should be studied with a view of determining their vaccine potential.
East African Medical Journal Vol. 81 No. 2 February 2004: 97-103
