Identification and characterization of Leishmania amastigote and axenic form antigens.

This study was aimed to identify and characterize Leishmania amastigote, and axenic form antigens. Two in vitro techniques were used to change leishmania parasite isolates from promastigote form to amastigotes and amastigote like (axenic) forms. The main strategy relied upon in vitro infection of murine macrophages cell line J774 with leishmania promastigote, at 37°C with 5% CO2, while the second technique relied upon the culture of promastigote at 37°C with low pH (5.5), and 5-10% CO2. Proteins were extracted and fractionated utilizing 12% Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE). Antigens were recognized using both immune dot blot and western blot procedures. PCR was performed for recognition of leishmania parasites in infected J774 macrophages. L. major was quicker in infectivity of macrophages cell line than L. donovani. Shared proteins ranging from 26-116 kDa were identified by SDS PAGE in all stages. Immune Dot-blot method showed positive outcomes, while western blot identified an exceptional antigen band of 16 kDa in amastigote, this unique band could be of value in diagnosis and vaccination of leishmaniasis. PCR results confirmed presence of both isolates demonstrating that coinfection is conceivable, and no indications of hereditary recombination at kinetoplast DNA (kDNA) were identified in macrophages simultaneously infected by L. major and L. donovani.

Identification and characterization of Leishmania amastigote and axenic form antigens

@#This study was aimed to identify and characterize Leishmania amastigote, and axenic form antigens. Two in vitro techniques were used to change leishmania parasite isolates from promastigote form to amastigotes and amastigote like (axenic) forms. The main strategy relied upon in vitro infection of murine macrophages cell line J774 with leishmania promastigote, at 37°C with 5% CO2, while the second technique relied upon the culture of promastigote at 37°C with low pH (5.5), and 5-10% CO2. Proteins were extracted and fractionated utilizing 12% Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE). Antigens were recognized using both immune dot blot and western blot procedures. PCR was performed for recognition of leishmania parasites in infected J774 macrophages. L. major was quicker in infectivity of macrophages cell line than L. donovani. Shared proteins ranging from 26-116 kDa were identified by SDS PAGE in all stages. Immune Dot-blot method showed positive outcomes, while western blot identified an exceptional antigen band of 16 kDa in amastigote, this unique band could be of value in diagnosis and vaccination of leishmaniasis. PCR results confirmed presence of both isolates demonstrating that coinfection is conceivable, and no indications of hereditary recombination at kinetoplast DNA (kDNA) were identified in macrophages simultaneously infected by L. major and L. donovani.

Concomitant Infection with Leishmania donovani and L. major in Single Ulcers of Cutaneous Leishmaniasis Patients from Sudan.

In Sudan human leishmaniasis occurs in different clinical forms, that is, visceral (VL), cutaneous (CL), mucocutaneous (ML), and post-kala-azar dermal leishmaniasis (PKDL). Clinical samples from 69 Sudanese patients with different clinical manifestations were subjected to a PCR targeting the cytochrome oxidase II (COII) gene for Leishmania species identification. Mixed infections were suspected due to multiple overlapping peaks presented in some sequences of the COII amplicons. Cloning these amplicons and alignment of sequences from randomly selected clones confirmed the presence of two different Leishmania species, L. donovani and L. major, in three out of five CL patients. Findings were further confirmed by cloning the ITS gene. Regarding other samples no significant genetic variations were found in patients with VL (62 patients), PKDL (one patient), or ML (one patient). The sequences clustered in a single homogeneous group within L. donovani genetic group, with the exception of one sequence clustering with L. infantum genetic group. Findings of this study open discussion on the synergetic/antagonistic interaction between divergent Leishmania species both in mammalian and vector hosts, their clinical implications with respect to parasite fitness and response to treatment, and the route of transmission with respect to vector distribution and or adaptation.