Leishmania tropica: The comparison of two frequently-used methods of parasite load assay in vaccinated mice

• Published in: Asian Pacific Journal of Tropical Biomedicine
• Main Authors: Fatemeh Nemati Zargaran, Mosayeb Rostamian, Alisha Akya, Hamid M Niknam
• Format: Article in Journal/Newspaper
• Language: English
• Published: Wolters Kluwer Medknow Publications 2020
• Subjects: leishmania tropica; vaccinated mice; limiting dilution assay; parasite load assay; real-time pcr; Arctic medicine. Tropical medicine; RC955-962; Biology (General); QH301-705.5; Arctic
• Online Access: https://doi.org/10.4103/2221-1691.283938; https://doaj.org/article/91f71744af4446c7b30c88c21606690d

Objective: To compare limiting dilution assay and real-time PCR methods in Leishmania tropica parasite load measurement in vaccinated mice. Methods: BALB/c mice were vaccinated by Leishmania tropica soluble Leishmania antigen or recombinant Leishmania tropica stress- inducibleprotein-1 with/without adjuvant. After three vaccinations, mice were challenged by Leishmania tropica promastigotes. Two months after challenge, the draining lymph nodes of mice footpad were removed and parasite load was assayed by limiting dilution assay and real-time PCR methods. Limiting dilution assay was done by diluting tissue samples to extinction in a biphasic medium. For real-time PCR, DNA of the lymph nodes was extracted, equal dilutions of each sample were prepared and hot-start real-time PCR was done using appropriate primers. The data of the two methods were compared by appropriate statistical methods. Results: Both methods were able to measure different levels of parasite load in vaccinated/unvaccinated mice. In addition, wherever parasite load of a group was estimated high (or low) by one method, the estimated parasite load by another method was the same, although statistically significant differences were found between some groups. Conclusions: Both methods lead to approximately similar results in terms of differentiating parasite load of the experimental groups. However, due to the lower errors and faster process, the real-time PCR method is preferred.