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We have studied the transmission of visceral leishmaniasis by blood transfusion in the CB hamster model. Five normal CB hamsters (females, 2.5 months old) received a 0.1-ml blood transfusion from a donor that had been infected with 10(7) amastigotes of Leishmania donovani 90 days prior to the blood harvest. The development of the disease in transfused animals was monitored by the increase in anti-Leishmania serum antibodies, splenomegaly, and spleen and liver parasitic burdens. The transfused hamsters developed all the typical signs of the disease, i.e., ascites, cachexia and death. The scores of anti-Leishmania antibodies (1.345) and the level of parasite load (spleen Leishman Donovan units of Stauber (LDU) = 471, liver LDU = 378) in transfuse hamsters were similar to those observed in hamsters experimentally infected with 10(7) amastigotes (P > 0.05, Student t-test). Our results demonstrate that blood transfusion is an effective route for transmission of visceral leishmaniasis, and we point out that adequate precautions should be taken at blood banks in the regions where leishmaniasis is endemic.

An improved method for isolation of human and Rhesus monkey band-3 separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is described. Purified band-3 was obtained from human hemoglobin-free ghosts (Hb-free ghosts) after SDS-PAGE by chemical elution+sonication (CE+S). The section of the gel corresponding to the antigen was cut out, mechanically disrupted and incubated in 1% NaHCO3 containing 1% SDS, for 2 h, with shaking, at room temperature, followed by overnight incubation at 4 degrees C. The preparation was subsequently sonicated and clarified by centrifugation. Supernatants were dialyzed against distilled water, their protein contents were measured, and the presence of purified band-3 was demonstrated by SDS-PAGE. A calibration curve was developed for assay of CE+S material using densitometric evaluation of the protein profile on SDS-PAGE. An amount of 37.5 mg of Hb-free ghosts gave 3.15 mg of purified band-3 after CE+S, corresponding to an 8.4% yield. Rabbits were immunized with 50 micrograms CE+S antigen. Sera were collected and assayed by Western blot analysis against its proteolytic fragments, which were obtained from packed red blood cells by treatment with protease type VI from Streptomyces griseus (1 h at 37 degrees C), followed by extensive washing and hypotonic lysis. Specific antibodies recognized band-3 and its proteolytic fragments 60 and 63 kDa in human ghosts obtained from different blood donors, confirming the genetic polymorphism. Analogous serum obtained against the Rhesus monkey band-3 proteolytic fragment 63 kDa recognized the human antigen and its respective fragments. These results indicate the existence of similarities between these two species of band-3, suggesting the potential use of this technique in taxonomic and phylogenetic studies. Purification by CE+S is an efficient and rapid method for isolation of band-3 and its fragments with satisfactory yields and maintenance of both their immunogenic and antigenic properties.

The fucose-mannose ligand (FML) of Leishmania donovani is a complex glycoprotein fraction present in pro- and amastigotes, that interferes with parasite-macrophage interactions in vitro. In the present study, we have tested the potential immunoprotective effect of FML on L. donovani infection in inbred female BALB/c mice. The protection schemes included three weekly intraperitoneal administrations of FML, supplemented or not with saponin. Mice were challenged by intravenous injections of 2 x 10(7) amastigotes of Leishmania donovani (LD-1S/MHOM/SD/00-strain 1S) obtained from CB hamsters' infected spleens. After 15 days of infection, we monitored the splenocyte proliferative response to FML in vitro by ELISA for specific antibody response, and by parasite quantification as "Leishman-Donovan Units" in liver. A significant (P < 0.001) protective effect of FML with saponin, but not of FML or saponin alone, was shown by the reduction of parasite burden in liver and by the enhancement of splenocyte proliferation. The antibody response, very low at 15 days of infection in both untreated and control animals, showed a pronounced increase (P < 0.001) in animals sensitized with FML/saponin. Taken together, our results represent a 79.1 and 89.1% increase in specific proliferative and antibody responses, respectively, and an 84.4% protection in reduction of parasite liver burden. The protective potential was specifically due to FML (P < 0.001). Under the present conditions, no toxic or nonspecific effect could be attributed to saponin. A detailed study of the molecular events related to vaccination against murine visceral leishmaniasis with total and fractionated FML is currently underway.