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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 Firouz    , Zahra Sadat Mousavi Shafi , Flora Forouzesh , Akram Abouie Mehrizi , Jafar Joorbandi Sani , Hemn Yousefi , Sakineh Pirahmadi1 |
| 1- , s.pirahmadi2020@googlemail.com |
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Abstract: (39 Views) |
Background and Aim: Malaria is a prevalent infectious disease caused by parasites of the genus Plasmodium, transmitted to vertebrate hosts through 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 sporozoites infection in Anopheles stephensi.
Materials and Methods: Female BALB/c mice infected with Plasmodium berghei were used for blood feeding of Anopheles stephensi. Then 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 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 mosquitoes 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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Type of Study: Original Research |
Subject:
Parasitology and Mycology
Received: 2025/02/22 | Accepted: 2026/07/18
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