Publications
66 results found
Bass C, Nikou D, Blagborough AM, et al., 2008, PCR-based detection of plasmodium in Anopheles mosquitoes: a comparison of a new high-throughput assay with existing methods, Malaria Journal, Vol: 7, ISSN: 1475-2875
Background: Detection of the four malaria-causing Plasmodium species (Plasmodium falciparum, Plasmodium vivax,Plasmodium ovale and Plasmodium malariae) within their mosquito hosts is an essential component of vectorcontrol programmes. Several PCR protocols have been developed for this purpose. Many of these methods, whilesensitive, require multiple PCR reactions to detect and discriminate all four Plasmodium species. In this study anew high-throughput assay was developed and compared with three previously described PCR techniques.Methods: A new assay based on TaqMan SNP genotyping was developed to detect all four Plasmodium speciesand discriminate P. falciparum from P. vivax, P. ovale and P. malariae. The sensitivity and the specificity of the newassay was compared to three alternative PCR approaches and to microscopic dissection of salivary glands in ablind trial of 96 single insect samples that included artificially infected Anopheles stephensi mosquitoes. Theperformance of the assays was then compared using more than 450 field-collected specimens that had beenstored on silica gel, in ethanol or in isopropanol.Results: The TaqMan assay was found to be highly specific when using Plasmodium genomic DNA as template.Tests of analytical sensitivity and the results of the blind trial showed the TaqMan assay to be the most sensitiveof the four methods followed by the 'gold standard' nested PCR approach and the results generated using thesetwo methods were in good concordance. The sensitivity of the other two methods and their agreement with thenested PCR and TaqMan approaches varied considerably. In trials using field collected specimens two of themethods (including the nested protocol) showed a high degree of non-specific amplification when using DNAderived from mosquitoes stored in ethanol or isopropanol. The TaqMan method appeared unaffected when usingthe same samples.Conclusion: This study describes a new high-throughput TaqMan assay that very effectively detects the
Habtewold T, Povelones M, Blagborough AM, et al., 2008, Transmission blocking immunity in the malaria non-vector mosquito Anopheles quadriannulatus species A, Plos Pathogens, Vol: 4, ISSN: 1553-7374
Despite being phylogenetically very close to Anopheles gambiae, the major mosquito vector of human malaria in Africa,Anopheles quadriannulatus is thought to be a non-vector. Understanding the difference between vector and non-vectormosquitoes can facilitate development of novel malaria control strategies. We demonstrate that An. quadriannulatus islargely resistant to infections by the human parasite Plasmodium falciparum, as well as by the rodent parasite Plasmodiumberghei. By using genetics and reverse genetics, we show that resistance is controlled by quantitative heritable traits andmanifested by lysis or melanization of ookinetes in the mosquito midgut, as well as by killing of parasites at subsequentstages of their development in the mosquito. Genes encoding two leucine-rich repeat proteins, LRIM1 and LRIM2, and thethioester-containing protein, TEP1, are identified as essential in these immune reactions. Their silencing completelyabolishes P. berghei melanization and dramatically increases the number of oocysts, thus transforming An. quadriannulatusinto a highly permissive parasite host. We hypothesize that the mosquito immune system is an important cause of naturalrefractoriness to malaria and that utilization of this innate capacity of mosquitoes could lead to new methods to controltransmission of the disease.
Liu Y, Tewari R, Ning J, et al., 2008, The conserved plant sterility gene <i>HAP2</i> functions after attachment of fusogenic membranes in <i>Chlamydomonas</i> and <i>Plasmodium</i> gametes, GENES & DEVELOPMENT, Vol: 22, Pages: 1051-1068, ISSN: 0890-9369
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- Citations: 222
Tewari R, Liu Y, Ning J, et al., 2008, A plant sterility gene has a conserved function in <i>Plasmodium berghei</i> gamete membrane fusion and is essential for mosquito transmission of malaria., 3rd Molecular Approaches to Malaria Meeting (MAM 2008), Publisher: PERGAMON-ELSEVIER SCIENCE LTD, Pages: S20-S20, ISSN: 0020-7519
Dittrich S, Mitchell SL, Blagborough AM, et al., 2007, An atypical orthologue of 6-pyruvoyltetrahydropterin synthase can provide the missing link in the folate biosynthesis pathway of malaria parasites, Molecular Microbiology, Vol: 67, Pages: 609-618, ISSN: 1365-2958
Folate metabolism in malaria parasites is a long-standing, clinical target for chemotherapy and prophylaxis. However, despite determination of the complete genome sequence of the lethal species Plasmodium falciparum, the pathway of de novo folate biosynthesis remains incomplete, as no candidate gene for dihydroneopterin aldolase (DHNA) could be identified. This enzyme catalyses the third step in the well-characterized pathway of plants, bacteria, and those eukaryotic microorganisms capable of synthesizing their own folate. Utilizing bioinformatics searches based on both primary and higher protein structures, together with biochemical assays, we demonstrate that P. falciparum cell extracts lack detectable DHNA activity, but that the parasite possesses an unusual orthologue of 6-pyruvoyltetrahydropterin synthase (PTPS), which simultaneously gives rise to two products in comparable amounts, the predominant of which is 6-hydroxymethyl-7,8-dihydropterin, the substrate for the fourth step in folate biosynthesis (catalysed by 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase; PPPK). This can provide a bypass for the missing DHNA activity and thus a means of completing the biosynthetic pathway from GTP to dihydrofolate. Supported by site-directed mutagenesis experiments, we ascribe the novel catalytic activity of the malarial PTPS to a Cys to Glu change at its active site relative to all previously characterized PTPS molecules, including that of the human host.
Ramjanee S, Robertson JS, Franke-Fayard B, et al., 2007, The use of transgenic <i>Plasmodium berghei</i> expressing the <i>Plasmodium vivax</i> antigen P25 to determine the transmission-blocking activity of sera from malaria vaccine trials, VACCINE, Vol: 25, Pages: 886-894, ISSN: 0264-410X
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- Citations: 43
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