TOC Summary: Forests harboring these mosquitoes may be a reservoir for transmission of P. knowlesi.
Summary During the last decade, major progress in malaria control has been achieved in Vietnam, Laos and Cambodia. However, malaria is still a potentially fatal disease in some hilly‐forested areas and continues to be endemic in a few coastal foci. To estimate the risk that stems from the major vectors after a decade of intensive malaria control, an entomological study based on human landing collections was conducted between April 1998 and November 2000 in six study villages (four in Vietnam, one in Cambodia and one in Laos) located in different physio‐geographical areas. Five villages were selected in places where new cases of malaria still occurred. In the sixth village, in the northern hilly area of Vietnam, no case of malaria was detected during the past 3 years. In three study villages of the hilly forested areas of Cambodia and central Vietnam, Anopheles dirus A still played an important role in malaria transmission and maintain perennial transmission inside the villages despite its low density. Anopheles minimus A was found in all study villages except in the southern coastal village of Vietnam. Its role in malaria transmission, however, varied between localities and surveys. In one study village of central Vietnam it was almost absent (one specimen collected over 480 man nights), and in another village sporozoite positive specimens (2.8%) were only observed during the first two surveys whereas this species disappeared from the collections from November 1998 onwards (six surveys: 360 man nights). In the northern study site An. minimus A and C were found in all collections, but no local malaria transmission occurred. However, the constant presence of these two species associated with a high longevity (parous rate up around 80% and 65%, respectively), suggests that transmission can occur at almost any time if parasite reservoirs are reintroduced in the area. The proper management of malaria cases and population movement is, therefore, important to prevent outbreaks and the reintroduction of malaria in northern Vietnam. In the study site of the Mekong delta, An. sundaicus occurred at high densities (up to 190 bites/man/night). The recent changes in land use from rice cultivation to shrimp farming probably explains the increase of this brackish water breeding species during the study period. However, none of the 11 002 specimens was positive for Plasmodium circumsporozoite protein (CSP). The relative low survival rate as estimated by the parous rate (around 47 %) may reflect its low vectorial status that could explain the very low malaria incidence (1.9 case/100 persons/year) in this study site. A calculated sporozoite rate of maximum 1/300 000 is enough to explain this low malaria incidence. Despite the successes in malaria control, the vector An. dirus A continues to play an important role in malaria transmission, whereas An. minimus A showed temporal and spatial variation in its role as vector. The role of An. sundaicus as vector could not be confirmed because of the low incidence in the coastal study villa...
BackgroundRecent studies have described natural human infections of the non-human primate parasites Plasmodium knowlesi and Plasmodium cynomolgi. In Southeast Asia, mosquitoes of the Anopheles leucosphyrus group bite both humans and monkeys in the forest and thus offer a possible route for Plasmodium species to bridge the species barrier. In this study we analysed the species composition of malarial sporozoites infecting the salivary glands of Anopheles dirus in order to determine their potential role as bridge vectors of Plasmodium parasites from monkeys to humans.MethodsMosquitoes were collected in the forest and forest fringe area of Khanh Phu commune by human-baited landing collection. Anopheles species were determined on the basis of morphologic features. Sporozoite-infected salivary glands were applied to filter paper and dried in an ambient atmosphere, before storage in closed vials at 4–6 °C. Detection and identification of Plasmodium species in salivary glands were carried out by nested-PCR of the small subunit ribosomal RNA gene.ResultsSix species of Plasmodium parasites were detected by PCR, of which P. vivax was the most common, followed by P. knowlesi, P. inui, P. cynomolgi, P. coatneyi and P. falciparum. Twenty-six of the 79 sporozoite infected mosquitoes showed multiple infections, most of which were a combination of P. vivax with one or more of the non-human primate Plasmodium species.ConclusionsThese results suggest that humans overnighting in this forest are frequently inoculated with both human and non-human primate malaria parasites, leading to a situation conducive for the emergence of novel zoonotic malaria.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0995-y) contains supplementary material, which is available to authorized users.
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 ...
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