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Year : 2020  |  Volume : 10  |  Issue : 6  |  Page : 248-253

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

1 Infectious Diseases Research Center, Health Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran
2 Immunology Department, Pasteur Institute of Iran, 69 Pasteur Avenue, Tehran 13169-43551, Iran

Correspondence Address:
Hamid M Niknam
Immunology Department, Pasteur Institute of Iran, 69 Pasteur Avenue, Tehran 13169-43551
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Source of Support: This work was supported by Pasteur Institute of Iran (funding No: 754) and Kermanshah University of Medical Sciences (funding No: 980467), Conflict of Interest: None

DOI: 10.4103/2221-1691.283938

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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.

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