Lymphocytes of dogs immunised with purified excreted-secreted antigens of Leishmania infantum co-incubated with Leishmania infected macrophages produce IFN gamma resulting in nitric oxide-mediated amastigote apoptosis.

The role of nitric oxide (NO) in the anti-leishmanial activity has been confirmed both in vitro and in vivo. Recently, we demonstrated that NO-mediated apoptosis-like amastigote death pathway is an important and highly regulated mechanism used for the clearance of Leishmania within infected murine macrophages stimulated to produce NO endogenously. To further characterize these important effector mechanisms in dog, a natural host-reservoir of L. infantum/L. chagasi, we have developed an ex vivo infection model of canine macrophages. Exposure of L. infantum-infected macrophages to autologous peripheral lymphocytes derived from dogs immunised with purified excreted-secreted antigens of L. infantum promastigotes (LiESAp) formulated with muramyl dipeptide (MDP) as adjuvant resulted in a significant leishmanicidal effect due to interferon (IFN)-gamma dependent macrophage activation. Concomitant accumulation of NO(3)(-)/NO(2)(-) in supernatants of co-cultured cells and in situ staining of parasites with terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and YOPRO-1 showed that NO-mediated apoptosis of intracellular L. infantum amastigotes is occurring in canine macrophages as previously observed in mouse models. Monitoring these parameters in dogs after immunisation and before experimental challenge can represent a useful and easy way to rapidly evaluate vaccine candidates against canine visceral leishmaniasis.

Experimental studies on the evolution of antimony-resistant phenotype during the in vitro life cycle of Leishmania infantum: implications for the spread of chemoresistance in endemic areas.

Pentavalent antimonial unresponsiveness is an emerging problem in endemic areas and information on factors which could modulate the transmission of drug-resistant phenotypes and parasites during life cycle are warranted. Using axenic amastigotes resistant to potassium antimonyl tartrate (Sb(III)) we investigated the modulation of antimonyl resistance during the in vitro life cycle. We assessed: (i) the stability of the drug-resistant phenotype during the in vitro life cycle; (ii) the transmission of drug-resistant clones when mixed with a wild-type clone at different susceptible/chemoresistant ratios (50/50,90/10,10/90) after one or two in vitro life cycles. We demonstrate that: (i) mutants which were 12,28,35 and 44 fold more resistant to Sb(III)-antimonial than their parental wild-type, were Glucantime Sb(V)-resistant when growing in THP-1 cells; (ii) the drug-resistant phenotype was partially retained during long-term in vitro culture (3 months) in drug free medium; (iii) the antimonyl-resistant phenotype was retained after one or more in vitro life cycles. However, when drug-resistant parasites were mixed with susceptible, mutants could not be detected in the resulting population, after one or two in vitro life cycles, whatever the initial wild-type/chemoresistant ratio. These results could be explained by the lower capacity of drug-resistant amastigotes to undergo the amastigote-promastigote differentiation process, leading probably to their sequential elimination during life cycle. Taken together, these observations demonstrate that different factors could modulate the transmission of Leishmania drug resistance during the parasite's life cycle.

Axenically grown amastigotes of Leishmania infantum used as an in vitro model to investigate the pentavalent antimony mode of action.

The mechanism(s) of activity of pentavalent antimony [Sb(V)] is poorly understood. In a recent study, we have shown that potassium antimonyl tartrate, a trivalent antimonial [Sb(III)], was substantially more potent than Sb(V) against both promastigotes and axenically grown amastigotes of three Leishmania species, supporting the idea of an in vivo metabolic conversion of Sb(V) into Sb(III). We report that amastigotes of Leishmania infantum cultured under axenic conditions were poorly susceptible to meglumine [Glucantime; an Sb(V)], unlike those growing inside THP-1 cells (50% inhibitory concentrations [IC50s], about 1.8 mg/ml and 22 microg/ml, respectively). In order to define more precisely the mode of action of Sb(V) agents in vivo, we first induced in vitro Sb(III) resistance by direct drug pressure on axenically grown amastigotes of L. infantum. Then we determined the susceptibilities of both extracellular and intracellular chemoresistant amastigotes to the Sb(V)-containing drugs meglumine and sodium stibogluconate plus m-chlorocresol (Pentostam). The chemoresistant amastigotes LdiR2, LdiR10, and LdiR20 were 14, 26, and 32 times more resistant to Sb(III), respectively, than the wild-type one (LdiWT). In accordance with the hypothesis described above, we found that intracellular chemoresistant amastigotes were resistant to meglumine [Sb(V)] in proportion to the initial level of Sb(III)-induced resistance. By contrast, Sb(III)-resistant cells were very susceptible to sodium stibogluconate. This lack of cross-resistance is probably due to the presence in this reagent of m-chlorocresol, which we found to be more toxic than Sb(III) to L. infantum amastigotes (IC50s, of 0.54 and 1.32 microg/ml, respectively). Collectively, these results were consistent with the hypothesis of an intramacrophagic metabolic conversion of Sb(V) into trivalent compounds, which in turn became readily toxic to the Leishmania amastigote stage.

Lactate dehydrogenase as a marker of Plasmodium infection in malaria vector Anopheles.

Lactate dehydrogenase (Ldh) electrophoresis showed the presence of Plasmodium yoelii yoelii in Anopheles stephensi and An. gambiae. The Ldh appeared as an additional band (pLdh) whose activity was more intense with 3-acetyl pyridine adenine dinucleotide as coenzyme than with beta nicotin-amide adenine dinucleotide. Several allelic forms occurred both in the vector and the host. The isoelectric point of Ldh, similar in the vector and host, differed from those of Ldh from mosquito and mouse. The presence of pLdh was detected from the 2nd to the 28th day of infection. The pLdh appeared to be proportional to the number of sporozoites present in infected salivary glands. However, pLdh was not found in salivary glands or midguts, but it was detected in the rest of the corresponding mosquito. The origin and use of pLdh as a marker of Plasmodium in its vector is discussed.

[Control by traps of Glossina fuscipes fuscipes for protecting livestock in Central African Republic. IV. Entomological, parasitological and zootechnical impact]. / La lutte par piégeage contre Glossina fuscipes fuscipes pour la protection de l'élevage en République centrafricaine. IV. Impact entomologique, parasitologique et zootechnique.

Neighteen Mbororo zebu herds were monitored to evaluate the impact of a trapping campaign against Glossina fuscipes fuscipes restricted to the watering places. This programme is part of the strategy of an integrated campaign against bovine trypanosomoses in the Central African Republic. Trapping reduces the densities of G. f. fuscipes and causes trypanosome prevalence to fall. These effects are shown by the improved hematocrit values and the reduced number of trypanocidal treatments administered. The impact on productivity is more difficult to assess over a short period; nevertheless, it seems clear from the parameters calculated.