Anopheles dirus co-infection with human and monkey malaria parasites in Vietnam

Abstract

The feasibility of identifying parasite DNA and specific mRNAs from wild caught Anopheles dirus mosquitoes was assessed using dried mosquito salivary glands preserved on filter paper. We were able to detect Plasmodium falciparum, Plasmodium vivax This study also shows that the preservation of mosquito salivary glands on filter paper, and the down-stream extraction of parasite DNA and RNA from those, offers a powerful resource for molecular epidemiological studies on malaria. Keywords: Salivary glands, Plasmodium knowlesi, PCR, RT-PCRMalaria transmission occurs in the forested areas in the southern and central provinces of Vietnam (Erhart et al., 2004) and constitutes a considerable public health problem in these areas. In order to fully understand the complexities of malaria transmission, it is important to identify and characterize malaria parasites in mosquito vectors. Evidence for the generation of genetic diversity during sexual proliferation in mosquito vectors has previously been accumulated using laboratory isolates of genetically diverse parasites, as well as with samples from field settings (Babiker et al., 1994; Menegon et al., 2000 knowlesi, were spotted onto chromatography-grade filter papers (ET31CHR;Whatman, Maidstone, UK). Each blood-spotted filter paper was immediately air-dried and stored in a sealed plastic bag at room temperature until RT-PCR or PCR analysis was performed. To examine the detection limit for mosquito salivary gland-extracted parasite mRNA, salivary glands from laboratory reared Anopheles stephensi were prepared in the same way as were the wild-caughtAn. dirus. Salivary glands were dissected from laboratory colony reared An.stephensi more than 1 week after blood feeding on either mouse or human blood and dried on E31CHR filter paper at room temperature. Gametocyte suspensions were obtained from cultured 3D7-9A by pyrimethamine treatment at 10 -6 M from day 7 after thawing until harvest on day 12. Ten times serially diluted cultured gametocyte suspension was added to the spot on the filter paper where the salivary glands were attached. Extraction of RNA and reverse transcription was carried out as previously described (Maeno et al., 2003). Briefly, dried spotted filter paper was cut into small pieces and total RNA and genomic DNA (gDNA) was extracted with ISOGEN (Nippon gene, Tokyo, Japan) according to the manufacturer's instructions. The extracted total RNA was transcribed to synthesize cDNA which was subsequently subjected to PCR using specific The detection limit of parasite mRNA by RT-PCR was one gametocyte per salivary gland for pfg377 mRNA and 10 gametocytes per gland for pfs16 mRNA (Fig. 1A). We extracted both parasite gDNA and mRNA from the salivary glands of wild caught mosquitoes. Region 3 of pfg377 mRNA was detected in all of the three dried salivary gland samples and both the mRNA and gDNA of pfg377 were detected in two samples (Fig. 1B). The PCR products showed the same molecular size as the RT-PCR products by electrophoresis. No PCR product was observed by ...

Diversity of Anopheles mosquitoes in Binh Phuoc and Dak Nong Provinces of Vietnam and their relation to disease

“…Besides these primary vectors, published reports also indicate the possible role of other Anopheles species, including An. pampanai , in malaria transmission [3,5,7,23]. The P. vivax infection rate in Binh Phuoc Province (0.41%; 2/486) is higher than the infection rate in the Anopheles population of Khe Ngang in Quang Binh Province (0.24%; 9/3,770) [1] and in seven species of Anopheles from Vietnamese and Cambodian populations (0.08%; 13/16,160) assayed by Durnez et al [52].…”

“…Moreover, use of insecticides and prophylactic drugs has resulted in resistance in some Anopheles species [11-13] and malarial parasites [14,15]. Furthermore, the monkey malarial protozoan Plasmodium knowlesi [16-21] has been found in both humans [7,22] and Anopheles mosquitoes in Vietnam [7,23]. Marchand et al [7] reported co-infection of P. falciparum , P. vivax and P. knowlesi in an An.…”

Human Infections and Detection of Plasmodium knowlesi

“…None of these vectors of P. knowlesi were found to be infected with the human malaria parasites P. falciparum, P. vivax, P. malariae, and P. ovale during recent entomological surveys in Sarawak (82,83) and Peninsular Malaysia (44). Interestingly, at 4 collection sites in the forest and forest fringe areas near a village in south central Vietnam, A. dirus mosquitoes were found to harbor sporozoites of P. knowlesi alone and mixed with P. vivax or P. falciparum and also mixed with both P. falciparum and P. vivax (62,85). Since A. dirus is the only known vector for human malaria in this area (62), this raises the possibil- ity that human-to-human transmission of P. knowlesi could occur and may actually be occurring in this region in Vietnam.…”

Vector Biology and Malaria Transmission in Southeast Asia

“…All four human malaria parasites and P. knowlesi have been reported in Vietnam [181,182,183]. Reported malaria cases are mostly due to P. falciparum (66%), followed by P. vivax (34%), while P. malariae and P. ovale are seldom recorded [3].…”

“…Plasmodium knowlesi has been found in several Anopheles species, especially An. dirus considered as the main malaria vector in Vietnam [181,183].…”