During mosquito transmission, malaria ookinetes must cross a chitin-containing structure known as the peritrophic matrix (PM), which surrounds the infected blood meal in the mosquito midgut. In turn, ookinetes produce multiple chitinase activities presumably aimed at disrupting this physical barrier to allow ookinete invasion of the midgut epithelium. Plasmodium chitinase activities are demonstrated targets for human and avian malaria transmission blockade with the chitinase inhibitor allosamidin. Here, we identify and characterize the first chitinase gene of a rodent malaria parasite, Plasmodium berghei. We show that the gene, named PbCHT1, is a structural ortholog of PgCHT1 of the avian malaria parasite Plasmodium gallinaceum and a paralog of PfCHT1 of the human malaria parasite Plasmodium falciparum. Targeted disruption of PbCHT1 reduced parasite infectivity in Anopheles stephensi mosquitoes by up to 90%. Reductions in infectivity were also observed in ookinete feeds-an artificial situation where midgut invasion occurs before PM formationsuggesting that PbCHT1 plays a role other than PM disruption. PbCHT1 null mutants had no residual ookinete-derived chitinase activity in vitro, suggesting that P. berghei ookinetes express only one chitinase gene. Moreover, PbCHT1 activity appeared insensitive to allosamidin inhibition, an observation that raises questions about the use of allosamidin and components like it as potential malaria transmission-blocking drugs. Taken together, these findings suggest a fundamental divergence among rodent, avian, and human malaria parasite chitinases, with implications for the evolution of Plasmodium-mosquito interactions.After ingestion of infectious Plasmodium gametocytes by the mosquito, motile ookinetes develop in the midgut lumen and traverse the chitin-containing peritrophic matrix (PM), the microvillus-associated network, and the midgut epithelium to form sporozoite-producing oocysts on the hemocoel side of the midgut (11,18). After the demonstration that ookinetes secrete multiple chitinase activities (6), two distinct Plasmodium chitinase genes were isolated. The first was isolated from the human malaria parasite Plasmodium falciparum (PfCHT1) (14), while the second was found in the avian malaria parasite Plasmodium gallinaceum (PgCHT1) (15). The primary structures of these two chitinase genes are markedly different: PgCHT1 encodes putative amino-terminal proenzyme and carboxy-terminal chitin-binding domains, which are both absent in PfCHT1. P. gallinaceum secretes a second chitinase activity provisionally named PgCHT2, believed to be orthologous to that encoded by PfCHT1 based on its molecular mass and physiological properties (pH optimum and sensitivity to the chitinase inhibitor allosamidin), and it may have additional chitinase activities (15).The Streptomyces-produced molecule allosamidin is a 622-dalton pseudo-oligosaccharide that inhibits Plasmodium chitinase activities in vitro (10,14,15). Moreover, the presence of allosamidin in an infected blood meal inhibited ooc...

BackgroundThe major malaria vector in Sri Lanka is reported to be Anopheles culicifacies with Anopheles subpictus, Anopheles annularis, and Anopheles varuna considered as potential vectors. The occurrence of Anopheles stephensi, which is the key vector of urban malaria in India and the Middle East, had never been reported from Sri Lanka.MethodsA series of entomological investigations were carried out by the Anti Malaria Campaign, Ministry of Health, Sri Lanka during December 2016 to April 2017 in two localities of the Mannar District in the Northern Province of the country. Adult mosquito collections were done through indoor and outdoor resting collections, animal and human biting collections and emergence traps. Potential mosquito breeding sites were investigated through larval surveys. The larvae and adults of An. stephensi were initially identified using morphological keys, and subsequently confirmed by sequencing the barcode region of the cytochrome c oxidase I (COI) gene.ResultsThis is the first report of the presence of An. stephensi in the island of Mannar in the Northern Province of Sri Lanka. Anopheles stephensi (36.65%) was the most abundant anopheline species in the larval habitats in Mannar. It was found breeding together with An. culicifacies (20.7%), An. subpictus (13.5%) and An. varuna (28.13%). Anopheles stephensi was found to be abundantly breeding in built wells used for domestic purposes. Adult females of An. stephensi were observed in emergence trap collections (93.9%), human landing catches all night (79.2%), pyrethrum spray sheet collections (38.6%), outdoor collections (8.3%), donkey-baited trap collections (14.3), and cattle-baited net trap collections (0.7%).ConclusionsSri Lanka was certified as malaria-free by the WHO in September 2016, however, this new finding may pose a serious challenge to the efforts of the Ministry of Health to prevent the re-introduction of malaria transmission in the country, considering the role that An. stephensi could play in urban and high vulnerability areas of Sri Lanka.

SUMMARYThe transmission-blocking monoclonal antibody 13.1, which recognizes the ookinete surface antigen Pbs21 ofPlasmodium berghei, and an IgG2a isotype control antibody 26.37 were purified by caprylic acid and ammonium sulphate precipitation. Fab fragments were prepared by papain digestion. IgG but not Fab from antibody 13.1 reduced ookinete formation byP. bergheiin culture by as much as 94% at a concentration of 100 μg/ml. There was little difference in antibody efficacy in the range 6·25–400 μg/ml in this assay. The parasite was most sensitive to antibody activity in the first 6–9 h of culture, i.e. the gamete/zygote and early retort stages. Peripheral blood leucocytes (PBL) were essential to achieve maximal inhibition by mAb 13.1 (activity was abrogated totally if PBL were removed). Together the data suggest that one of the mechanisms of action of this antibody is antibody-mediated PBL killing. Phagocytosis of parasites was noted in these experiments in all cultures. We have not attempted in this study to distinguish between Fc-mediated opsonization, as opposed to antibody-dependent cellular cytotoxicity.

The effects of purified monoclonal immunoglobulins from control, or transmission-blocking anti-Pbs21 antibodies, upon the infection of Anopheles stephensi by ookinetes of Plasmodium berghei are compared. Anti-Pbs21 antibody reduced mean intensity and prevalence of infection by 94.7 and 58.7% respectively if added to the infectious bloodfeed at a concentration of 100 micrograms/ml. Fab fragments were of similar efficacy. No transmission enhancement was detected with declining antibody concentrations. Addition to subsequent (second) feeds reduced mean oocyst intensity but not prevalence. The reduction in blockade declined from 41% at day 2, to 4% at day 8. Second bloodfeeds, containing control globulin taken 4 or 6 days (but not 2 days) after infection, increased sporozoite burden in the salivary glands. At all times anti-Pbs21 reduced sporozoite number in the thorax compared to time-matched controls, but again highest gland intensities were obtained when the second bloodfeed was given on day 4. We conclude that second bloodfeeds containing transmission-blocking antibody simultaneously serve two opposing roles, (1) inhibition of parasite development and (2) the supply of nutrients which permit more sporozoites to be produced by each oocyst.

The impact of immune sera, and peripheral blood cells (PBC) from mice immunized with Plasmodium berghei ookinetes; and of purified immunoglobulin or Fab fragments from anti-Pbs21 monoclonal antibody 13.1, upon establishment of oocyst infections in the mosquito was studied. Infections were initiated either from gametocyte-infected mice, or membrane feeders which contained either gametocytes or mature ookinetes. PBC from ookinete-immunized mice presented with non-immune serum failed to show any transmission-blocking activity. Anti-ookinete serum, intact anti-Pbs21 monoclonal antibody 13.1 or its Fab fragments, all inhibited oocyst formation significantly. When gametocyte-infected mice or gametocytes in membrane feeds were used, inhibition did not directly correlate with antibody concentration. In membrane feeders that contained ookinetes and antibody, concentration-dependent inhibition usually occurred. The efficacy of purified 13.1 IgG was dependent upon the ookinete concentration. The ookinete plasmalemma and cytoplasm were significantly disturbed after 12 h in bloodmeals that contained antibody 13.1, but not in the isotype controls. These changes may have caused the observed failure of the ookinete to migrate as rapidly as the controls from the destructive environment of the bloodmeal.