Leishmania tropica and L. major are etiologic agents of human cutaneous leishmaniasis. Delayed type hypersensitivity (DTH) is an immunologic response that has been frequently used as a correlate for protection against or sensitization to leishmania antigen. In BALB/c mice, L. tropica infection results in non-ulcerating disease, whereas L. major infection results in destructive lesions. In order to clarify the immunologic mechanisms of these 2 different outcomes, we compared the ability of these 2 leishmania species in induction of DTH response in this murine model. BALB/c mice were infected with L. major or L. tropica, and disease evolution and DTH responses were determined. The results show that the primary L. major infection can exacerbate the secondary L. major infection and is associated with DTH response. Higher doses of the primary L. major infection result in more disease exacerbation of the secondary L. major infection as well as higher DTH response. L. tropica infection induces lower DTH responses than L. major. We have previously reported that the primary L. tropica infection induces partial protection against the secondary L. major infection in BALB/c mice. Induction of lower DTH response by L. tropica suggests that the protection induced against L. major by prior L. tropica infection may be due to suppression of DTH response.
Animals ; Disease Models, Animal ; Ear/pathology ; Female ; Foot/pathology ; *Hypersensitivity, Delayed ; Leishmania major/*immunology ; Leishmania tropica/*immunology ; Leishmaniasis, Cutaneous/*immunology/*parasitology/pathology ; Mice ; Mice, Inbred BALB C

Leishmania tropica is one of the causative agents of leishmaniasis in humans. Routes of infection have been reported to be an important variable for some species of Leishmania parasites. The role of this variable is not clear for L. tropica infection. The aim of this study was to explore the effects of route of L. tropica infection on the disease outcome and immunologic parameters in BALB/c mice. Two routes were used; subcutaneous in the footpad and intradermal in the ear. Mice were challenged by Leishmani major, after establishment of the L. tropica infection, to evaluate the level of protective immunity. Immune responses were assayed at week 1 and week 4 after challenge. The subcutaneous route in the footpad in comparison to the intradermal route in the ear induced significantly more protective immunity against L. major challenge, including higher delayed-type hypersensitivity responses, more rapid lesion resolution, lower parasite loads, and lower levels of IL-10. Our data showed that the route of infection in BALB/c model of L. tropica infection is an important variable and should be considered in developing an appropriate experimental model for L. tropica infections.
Animals ; Disease Models, Animal ; Female ; Leishmania major/*immunology ; Leishmania tropica/*immunology/*pathogenicity ; Leishmaniasis/*immunology/parasitology/*pathology ; Mice ; Mice, Inbred BALB C ; Treatment Outcome

Experimental murine models with high, intermediate and low levels of genetically based susceptibility to Leishmania major infection reproduce almost entire spectrum of clinical manifestations of the human disease. There are increasing non-comparative studies on immune responses against isolated antigens of L. major in different murine strains. The aim of the present study was to find out whether there is an antigen that can induce protective immune response in resistant and susceptible murine strains. To do that, crude antigenic extract of procyclic and metacyclic promastigotes of L. major was prepared and subjected to SDS-PAGE electrophoresis. Western-blotting was used to search for antigen(s) capable of raising high antibody level of IgG2a versus IgG1 in the sera of both infected resistant and susceptible strains. Two novel antigens from metacyclic promastigotes of L. major (140 and 152 kDa) were potentially able to induce specific dominant IgG2a responses in BALB/c and C57BL/6 mice. The 2 antigens also reacted with IgG antibody of cutaneous leishmaniasis patients. We confirm that 140 and 152 kDa proteins of L. major promastigotes are inducing IgG production in mice and humans.
Protozoan Proteins/immunology/*isolation & purification ; Mice, Inbred C57BL ; Mice, Inbred BALB C ; Mice ; Life Cycle Stages/immunology ; Leishmaniasis, Cutaneous/immunology ; Leishmania major/*immunology ; Immunoglobulin G/*biosynthesis/blood ; Humans ; Female ; Blotting, Western/methods ; Antigens, Protozoan/immunology/*isolation & purification ; Animals

The identification and characterization of antigens that elicit human T cell responses is an important step toward understanding of Leishmania major infection and ultimately in the development of a vaccine. Micropreparative SDS-PAGE followed by electrotransfer to a PVDF membrane and elution of proteins from the PVDF, was used to separate 2 novel proteins from L. major promastigotes, which can induce antibodies of the IgG2a isotype in mice and also are recognized by antisera of recovered human cutaneous leishmaniasis subjects. Fractionation of the crude extract of L. major revealed that all detectable proteins of interest were present within the soluble Leishmania antigens (SLA). Quantitation of these proteins showed that their expression in promastigotes is relatively very low. Considering the molecular weight, immunoreactivity, chromatographic and electrophoretic behavior in reducing and non-reducing conditions, these proteins are probably 2 isoforms of a single protein. A digest of these proteins was resolved on Tricine-SDS-PAGE and immunoreactive fragments were identified by human sera. Two immunoreactive fragments (36.4 and 34.8 kDa) were only generated by endoproteinase Glu-C treatment. These immunoreactive fragments or their parent molecules may be ideal candidates for incorporation in a cocktail vaccine against cutaneous leishmaniasis.
Animals ; Antigens, Protozoan/*chemistry/*isolation & purification ; Blotting, Western ; Chromatography, High Pressure Liquid ; Electrophoresis, Polyacrylamide Gel/methods ; Humans ; Leishmania major/growth & development/*immunology ; Protein Isoforms/chemistry/isolation & purification

Visceral leishmaniasis (VL) or kala-azar mainly affects children in endemic areas. This study was conducted to determine the seroprevalence of VL using direct agglutination test (DAT) in children living in rural districts of Alborz Province located 30 km from Tehran capital city of Iran. Multi-stage cluster random sampling was applied. Blood samples were randomly collected from 1,007 children under 10 years of age in the clusters. A total of 37 (3.7%) of the studied population showed anti-Leishmania infantum antibodies with titers of > or =1:800. There was a significant association between positive sera and various parts of the rural areas of Alborz Province (P<0.002). Two children with anti-Leishmania infantum antibodies titers of > or =1:3,200 indicated kala-azar clinical features and treated with anti-leishmaniasis drugs in pediatric hospital. The findings of this study indicated that Leishmania infection is prevalent in rural areas of Alborz Province. Therefore, it is necessary to increase the awareness and alertness among physicians and public health managers, particularly in high-risk rural areas of the province in Iran.
Antibodies, Protozoan/blood ; Child ; Child, Preschool ; Female ; Health Policy ; Humans ; Iran/epidemiology ; Leishmania infantum/immunology/isolation & purification/physiology ; Leishmaniasis, Visceral/blood/*epidemiology/parasitology ; Male ; *Rural Health ; Seroepidemiologic Studies

The protective effect of the Synadenium carinatum latex lectin (ScLL), and the possibility of using it as an adjuvant in murine model of vaccination against American cutaneous leishmaniasis, were evaluated. BALB/c mice were immunized with the lectin ScLL (10, 50, 100 microgram/animal) separately or in association with the soluble Leishmania amazonensis antigen (SLA). After a challenge infection with 10(6) promastigotes, the injury progression was monitored weekly by measuring the footpad swelling for 10 weeks. ScLL appeared to be capable of conferring partial protection to the animals, being most evident when ScLL was used in concentrations of 50 and 100 microgram/animal. Also the parasite load in the interior of macrophages showed significant reduction (61.7%) when compared to the control group. With regard to the cellular response, ScLL 50 and 100 microgram/animal stimulated the delayed-type hypersensitivity (DTH) reaction significantly (P < 0.05) higher than SLA or SLA plus ScLL 10 weeks after the challenge infection. The detection of high levels of IgG2a and the expression of mRNA cytokines, such as IFN-gamma, IL-12, and TNF-alpha (Th1 profiles), corroborated the protective role of this lectin against cutaneous leishmaniasis. This is the first report of the ScLL effect on leishmaniasis and shows a promising role for ScLL to be explored in other experimental models for treatment of leishmaniasis.
*Adjuvants, Immunologic ; Animals ; Antibodies, Protozoan/immunology ; Antibody Formation ; Antigens, Protozoan/immunology ; Cytokines/genetics/immunology ; Euphorbiaceae/*chemistry ; Hypersensitivity, Delayed/immunology ; Immunization ; Immunoglobulin G/immunology ; Latex/chemistry ; Leishmania/immunology ; Leishmaniasis, Cutaneous/*immunology/pathology ; Mice ; Mice, Inbred BALB C ; Nitric Oxide Synthase Type II/genetics/immunology ; Plant Lectins/*immunology/isolation & purification ; Protozoan Vaccines/immunology/pharmacology ; Skin/pathology

The beta-glucans derived from yeast cell walls have been reported for having many immunomodulatory activities in vivo and in vitro. In this study, Aureobasidium-derived soluble branched (1,3-1,6) beta-glucan (Sophy beta-glucan) was checked for natural killer (NK) activity and for the production of IFN-gamma and IL-4 in Leishmania amazonensis infection. The main experiment was performed with a group of female C57BL/6 and BALB/c mice, orally supplemented with 5% of Sophy beta-glucan and infected with promastogotes of L. amazonensis (1 x 10(7)) into the footpad. Increase in the footpad thickness with time was observed in BALB/c mice in spite of the oral Sophy beta-glucan supplement, but it was less in C57BL/6 mice. The difference in overall mean footpad thickness between 'infection only' versus 'infection + glucan' groups was statistically significant (P < 0.001). High NK activity in C57BL/6 than BALB/c mice was observed in 'glucan only' group compared to the control group and also in 'infection + glucan' group compared to 'infection only' group. The difference in the NK activity among these groups was significant (P < 0.05). The IFN-gamma level increased at weeks 7 and 8 post-infection in C57BL/6 mice and was significantly high in 'infection + glucan' group compared to the 'infection only' group (P < 0.05). IL-4 levels did not increase up to detectable levels throughout the study. The results led a conclusion that Sophy beta-glucan enhances NK activity and cellular immunity in L. amazonensis-infected mice.
Administration, Oral ; Animals ; Ascomycota/*chemistry ; Cytotoxicity Tests, Immunologic ; Female ; Foot/pathology ; Glucans/administration & dosage/*isolation & purification/pharmacology/*therapeutic use ; Immunologic Factors/administration & dosage/*isolation & purification/pharmacology/*therapeutic use ; Interferon-gamma/biosynthesis ; Interleukin-4/biosynthesis ; Killer Cells, Natural/drug effects/*immunology ; Leishmania mexicana/*immunology ; Leishmaniasis, Cutaneous/*drug therapy/immunology/pathology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Severity of Illness Index ; Time Factors

The LACK gene from Leishmania, an analogue of the receptor of activated protein kinase C, was discovered recently. In this study, the LACK gene of Leishmania donovani was obtained from the recombinant plasmid T-LACK by PCR. The gene was cloned into eukaryotic expressed plasmid pcDNA3.1(+) to construct recombinant plasmid. This recombinant plasmid then was transfected into the eukaryotic cell COS-7, and the expression of LACK gene in eukaryotic cell was detected by RT-PCR and immunofluorescent staining. Both RT-PCR and immunofluorescent staining of recombinant plasmid transfected COS-7 showed positive reaction, thus indicating that the recombinant plasmid pcDNA3-LACK can express LACK protein in euka ryotic cell COS-7.
Animals ; Antigens, Protozoan ; biosynthesis ; genetics ; immunology ; COS Cells ; Cloning, Molecular ; DNA, Recombinant ; biosynthesis ; genetics ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; Leishmania donovani ; Plasmids ; genetics ; Protozoan Proteins ; biosynthesis ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Vaccines, DNA

A 12-year-old spayed female mixed-bred dog presented with nasal bleeding of 2 days duration and a skin nodule in the left flank. No abnormalities were found in coagulation profiles and blood pressure. Cytological evaluation of the nodule revealed numerous characteristic round organisms having a nucleus and a bar within macrophages and in the background, consistent with leishmaniasis. In vitro culture was unsuccessful but PCR of the nodular aspirate identified the organisms as Leishmania infantum, and the final diagnosis was canine leishmaniasis. No history of travel to endemic countries was noted. Because the dog had received a blood transfusion 2 years before the illness, serological screening tests were performed in all donor dogs of the commercial blood bank using the commercial Leishmania ELISA test kit, and there were no positive results. Additional 113 dogs with hyperglobulinemia from Seoul were also screened with the same kits but no positive results were obtained. To the best of the author's knowledge this is the first autochthonous case of canine leishmaniasis in Korea.
Animals ; Base Sequence ; DNA, Protozoan/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Dog Diseases/*diagnosis/epidemiology ; Dogs ; Enzyme-Linked Immunosorbent Assay/veterinary ; Female ; Giant Cells/pathology ; Leishmania infantum/genetics/immunology/*isolation & purification ; Leishmaniasis, Visceral/diagnosis/*veterinary ; Molecular Sequence Data ; Polymerase Chain Reaction/veterinary ; Protozoan Proteins/genetics ; Republic of Korea ; Sequence Analysis, DNA/veterinary ; Serologic Tests/veterinary

This study investigated whether trinitroglycerine (TNG) as nitric oxide (NO) releasing agent had anti-leishmanial effects and mediated pathology in BALB/c mice infected with Leishmania major. Cutaneous leishmaniasis (CL), a zoonotic infection caused by leishmania protozoa is still one of the health problems in the world and in Iran. NO is involved in host immune responses against intracellular L. major, and leishmania killing by macrophages is mediated by this substance. Moreover, application of CL treatment with NO-donors has been recently indicated. In our study, TNG was used for its ability to increase NO and to modify CL infection in mice, in order to evaluate NO effects on lesion size and formation, parasite proliferation inside macrophages, amastigote visceralization in target organs, and NO induction in plasma and organ suspensions. Data obtained in this study indicated that TNG increased plasma and liver-NO, reduced lesion sizes, removed amastigotes from lesions, livers, spleens, and lymph nodes, declined proliferation of amastigotes, hepatomegaly, and increased survival rate. However, TNG reduced spleen-NO and had no significant effects on spelenomegaly. The results show that TNG therapy reduced leishmaniasis and pathology in association with raised NO levels. TNG had some antiparasitic activity by reduction of positive smears from lesions, livers, spleens, and lymph nodes, which could emphasize the role of TNG to inhibit visceralization of L. major in target organs.
Animal Structures/parasitology ; Animals ; Antiprotozoal Agents/chemistry/*therapeutic use ; Female ; Leishmania major/*drug effects/immunology ; Leishmaniasis, Cutaneous ; Macrophages/parasitology ; Mice ; Mice, Inbred BALB C ; Nitric Oxide/blood/metabolism/*pharmacology ; Nitroglycerin/*analogs & derivatives/*therapeutic use ; Severity of Illness Index ; Skin/pathology ; Survival Analysis