New vectors in northern Sarawak, Malaysian Borneo, for the zoonotic malaria parasite, Plasmodium knowlesi

Abstract

Background Plasmodium knowlesi is a significant cause of human malaria in Sarawak, Malaysian Borneo. Only one study has been previously undertaken in Sarawak to identify vectors of P. knowlesi, where Anopheles latens was incriminated as the vector in Kapit, central Sarawak. A study was therefore undertaken to identify malaria vectors in a different location in Sarawak. Methods Mosquitoes found landing on humans and resting on leaves over a 5-day period at two sites in the Lawas District of northern Sarawak were collected and identified. DNA samples extracted from salivary glands of Anopheles mosquitoes were subjected to nested PCR malaria-detection assays. The small subunit ribosomal RNA (SSU rRNA) gene of Plasmodium was sequenced, and the internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the mosquitoes were sequenced from the Plasmodium-positive samples for phylogenetic analysis. Results Totals of 65 anophelines and 127 culicines were collected. By PCR, 6 An. balabacensis and 5 An. donaldi were found to have single P. knowlesi infections while 3 other An. balabacensis had either single, double or triple infections with P. inui, P. fieldi, P. cynomolgi and P. knowlesi. Phylogenetic analysis of the Plasmodium SSU rRNA gene confirmed 3 An. donaldi and 3 An. balabacensis with single P. knowlesi infections, while 3 other An. balabacensis had two or more Plasmodium species of P. inui, P. knowlesi, P. cynomolgi and some species of Plasmodium that could not be conclusively identified. Phylogenies inferred from the ITS2 and/or cox1 sequences of An. balabacensis and An. donaldi indicate that they are genetically indistinguishable from An. balabacensis and An. donaldi, respectively, found in Sabah, Malaysian Borneo. Conclusions Previously An. latens was identified as the vector for P. knowlesi in Kapit, central Sarawak, Malaysian Borneo, and now An. balabacensis and An. donaldi have been incriminated as vectors for zoonotic malaria in Lawas, northern Sarawak.

New vectors that are early feeders for Plasmodium knowlesi and other simian malaria parasites in Sarawak, Malaysian Borneo

“…With regards to the detection of Plasmodium , observations including a higher sporozoite rate in vectors compared to other studies 8 , 9 , 11 , 13 – 15 ; discrepancy between PCR and sequencing results, and the presence of Plasmodium species which could not be detected with the primers specific for the five simian malaria parasites that are reported in the current study, are consistent with the observations from our previous study in Lawas, Sarawak 18 . We have suggested that the high sporozoite rates observed could be attributed to a higher sensitivity of the sampling and detection methods since we used all the salivary glands for DNA extraction and we detected Plasmodium with molecular methods.…”

“…Some of the sequences generated in our study did not fall into any particular clade following phylogenetic analysis and the reasons for this could be either that the SSUrRNA gene is not a suitable marker for identification of species of Plasmodium or that there are species of Plasmodium circulating among wildlife in Betong and Lawas that have not previously been characterised 18 . It would be necessary to sequence additional genes in order to confirm that there are novel species of Plasmodium carried by these mosquitoes as was done recently with non-nuclear genes 5 .…”

“…dirus in Vietnam) and An. donaldi (Malaysia) have previously been incriminated in the transmission of P. knowlesi and other simian malaria parasites in Southeast Asia 8 , 9 , 11 – 16 , 18 , 49 . In all the studies where peak biting times were described, An.…”

“…leucosphyrus s.l., and more recently An. donaldi 11 , 18 , as vectors of knowlesi malaria, it is possible that other vectors could have been discovered if collections were also conducted during the 12-h period between dawn to dusk. With the incrimination of An.…”

“…Genomic DNA of the dried salivary glands was extracted using DNeasy Blood and Tissue Kit (QIAGEN, Germany) according to the manufacturer's protocol and stored at 4 °C until required. Nested PCR assays to detect Plasmodium DNA were initially undertaken and Plasmodium -positive samples were then subjected to PCR assays with primers specific for P. coatneyi, P. cynomolgi, P. fieldi, P. inui and P. knowlesi as described previously 18 .…”