The first successful report of the in vitro life cycle of Chinese Leishmania: the in vitro conversion of Leishmania amastigotes has been raised to 94% by testing 216 culture medium compound

Abstract

Chinese Leishmania isolate MHOM/CN/90/SC10H2 (L. H2), which was obtained from the spinal cords of patients from the Sichuan province of China, is an uncharacterized, pathogenic species closely related to Leishmania tarentolae. The in vitro transformation rate of L. H2 promastigotes into amastigotes has not been studied. This study is the first to successfully define the in vitro life cycle of L. H2 by investigating the percent conversion of L.H2 promastigotes to amastigotes in vitro under 216 different culture conditions. The highest proportion of L. H2 amastigotes observed (94%) was significantly higher than that previously reported. After conversion, the axenic amastigotes remained viable as verified by the levels of stage-specific genes (Gp46, A2 and β-tubulin) detected by RT-PCR. Meanwhile, morphological and protein characterizations of these axenic amastigotes were carried out in order to confirm the successful conversion. Specific antibodies were only able to detect 46 kDa, 52 kDa and 75 kDa proteins in samples isolated from axenic amastigotes. Afterward, these converted axenic amastigotes were transformed into the promastigote form by altering the culture condition. These converted axenic promastigotes still have the ability to infect macrophages, and their morphology changed back to the amastigote form following infection. These findings will assist further investigations into the biological characteristics of the host-parasite relationship and the process of pathogenesis.

Molecular Detection of Suspected Leishmania Isolates Using Polymerase Chain Reaction

“…Cultivation of isolate: Procyclic promastigotes were cultivated in M199 medium supplemented with 10% HIFBS and 1% streptomycin/penicillin (Pen/Strep) then incubated at 26°C for 3 days for obtaining massive culture [14]. DNA extraction: DNA extraction from healthy cultures of the parasite was done by using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's instructions.…”

Molecular Typing of Two Suspected Cutaneous Leishmaniasis Isolates in Baghdad

“…The samples were previously isolated from skin ulcers of two patients attended AL-Karama Teaching hospitals in Baghdad and confirmed as cutaneous leishmaniasis by clinical presentation according to the dermatologist. Procyclic promastigotes were cultured in M199 medium supplemented with 10% heated inactivated foetal bovine serum and 1% Penicillin/Streptomycin then incubated at 26°C for 3 days to maintain logphase harvest culture (24,25).…”

Axenic amastigote cultivation and in vitro development of Leishmania orientalis

“…In immunochemistry analyses, recognition by amastigote-specific monoclonal antibodies (mAbs) and differential expression of stage-specific antigens and cysteine proteinase-specific antigens have been reported. Genes encoding beta-tubulin and p100/11E or stage-specific genes such as Gp46, A2 and β-tubulin, have been used to characterize molecular properties (Rainey et al 1991;Li et al 2017).…”