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Development of an experimental infection model of Anopheles stephensi with Plasmodium berghei and molecular detection of sporozoites using qPCR
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Zeinab Mohammadi Firouz1    , Zahra Sadat Mousavi Shafi1 , Flora Forouzesh2 , Akram Abouie Mehrizi3 , Jafar Joorbandi Sani3 , Hemn Yousefi3 , Sakineh Pirahmadi4 |
1- Department of Genetics, TeMS.C., Islamic Azad University, Tehran, Iran; Malaria and Vector Research Group (MVRG), Biotechnology Research Center (BRC), Pasteur Institute of Iran, Tehran, Iran
2- . Department of Genetics, TeMS.C., Islamic Azad University, Tehran, Iran
3- . Malaria and Vector Research Group (MVRG), Biotechnology Research Center (BRC), Pasteur Institute of Iran, Tehran, Iran
4- . Malaria and Vector Research Group (MVRG), Biotechnology Research Center (BRC), Pasteur Institute of Iran, Tehran, Iran , s.pirahmadi2020@googlemail.com |
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Abstract: (402 Views) |
Background and Aim: Malaria is a prevalent infectious disease caused by parasites of the genus Plasmodium and transmitted to vertebrate hosts by Anopheles vectors. This study focuses on developing an experimental model using Plasmodium berghei, BALB/c mice, and Anopheles stephensi, emphasizing the application of qPCR for sensitive detection of Plasmodium berghei sporozoite infection in Anopheles stephensi.
Materials and Methods: Female BALB/c mice infected with Plasmodium berghei were used to feed Anopheles stephensi. Subsequently, the mosquitoes were kept for 24 days at 19-21°C and 60%-80% relative humidity. Microscopic evaluation of mosquito salivary glands for sporozoites was conducted. To confirm infection, a qPCR assay targeting the Plasmodium berghei cytb gene was conducted. Finally, to assess the infectivity of sporozoites for the second host, the infected mosquitoes were used for blood feeding of BALB/c mice on day 21 after blood feeding.
Results: qPCR analysis confirmed sporozoites in 100% of the infected mosquitoes, with a mean Cq value of 18.84 ± 2.33. Standard curve analysis of the positive control sample showed an R² value of 0.98 and a slope of -3.38, with a Cq detection limit of 34 cycles. Additionally, the blood smears of mice after 5 days of mosquito blood feeding showed that all of the mice were infected with Plasmodium berghei, indicating 100% infectivity of sporozoites.
Conclusion: This study developed a qPCR analysis with specific amplification of the P. berghei cytb gene, demonstrating higher sensitivity compared to microscopic examination. The qPCR results showed a high infection frequency in the mosquitoes. This model can be instrumental in future research on transmission-blocking strategies or parasite-Anopheles interactions |
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| Keywords: Anopheles stephensi, Malaria, Plasmodium berghei, qPCR test, Sporozoite |
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Full-Text [PDF 858 kb]
(47 Downloads)
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Type of Study: Original Research |
Subject:
Parasitology and Mycology
Received: 2025/02/22 | Accepted: 2026/07/18 | Published: 2026/09/8
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