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ABSTRACT: A critical stage in malaria transmission occurs in the Anopheles mosquito midgut, when the malaria parasite, Plasmodium, ingested with blood, first makes contact with the gut epithelial surface. To understand the response mechanisms within the midgut environment, including those influenced by resident microbiota against Plasmodium, we focus on a midgut bacteria species' intra-specific variation that confers diversity to the mosquito's competency for malaria transmission. Serratia marcescens isolated from either laboratory-reared mosquitoes or wild populations in Burkina Faso shows great phenotypic variation in its cellular and structural features. Importantly, this variation is directly correlated with its ability to inhibit Plasmodium development within the mosquito midgut. Furthermore, this anti-Plasmodium function conferred by Serratia marcescens requires increased expression of the flagellum biosynthetic pathway that is modulated by the motility master regulatory operon, flhDC. These findings point to new strategies for controlling malaria through genetic manipulation of midgut bacteria within the mosquito.
Scientific Reports 04/2013; 3:1641.
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ABSTRACT: BACKGROUND: Malaria is the most significant human parasitic disease, and yet understanding of the energy metabolism of the principle pathogen, Plasmodium falciparum, remains to be fully elucidated. Amino acids were shown to be essential nutritional requirements since early times and much of the current knowledge of Plasmodium energy metabolism is based on early biochemical work, performed using basic analytical techniques, carried out almost exclusively on human plasma with considerable inter-individual variability. METHODS: In order to further characterize the fate of amino acid metabolism in malaria parasite, multivariate analysis using statistical modelling of amino acid concentrations (aminogram) of plasma and liver were determined in host infected with rodent malaria parasite, Plasmodium yoelii.Results and conclusion: Comprehensive and statistical aminogram analysis revealed that P. yoelii infection caused drastic change of plasma and liver aminogram, and altered intra- and inter-correlation of amino acid concentration in plasma and liver. These findings of the interactions between amino acids and Plasmodium infection may provide insight to reveal the interaction between nutrients and parasites.
Malaria Journal 01/2013; 12(1):19. · 3.19 Impact Factor
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ABSTRACT: Upon microbial invasion the innate immune system of Drosophila melanogaster mounts a response that comes in two distinct but complimentary forms, humoral and cellular. A screen to find genes capable of conferring resistance to the Gram-positive Staphylococcus aureus upon ectopic expression in immune response tissues uncovered imd gene. This resistance was not dependent on cellular defenses but rather likely a result of upregulation of the humoral response through increased expression of antimicrobial peptides, including a Toll pathway reporter gene drosomycin. Taken together it appears that Imd pathway is capable of playing a role in resistance to the Gram-positive S. aureus, counter to notions of traditional roles of the Imd pathway thought largely to responsible for resistance to Gram-negative bacteria.
Biochemical and Biophysical Research Communications 12/2012; · 2.48 Impact Factor
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ABSTRACT: mRNA and protein expression profiles for three peroxiredoxins (TPx-1, TPx-2 and 1-Cys Prx) of liver stage Plasmodium berghei were examined through quantitative reverse transcription-PCR (RT-PCR) and indirect immunofluorescence microscopy assay (IFA). RT-PCR experiments revealed that mRNA expression for the TPx-1 was detected shortly after the sporozoite infection and kept expressed until the schizont stage. In contrast, the mRNA expression for 1-Cys Prx had begun increasing when the parasite developed into the schizont stage. Using the IFA, TPx-1 and 1-Cys Prx were detected in the cytosol. These finding suggested the developmental stage-specific expression of the cytosolic enzymes in the liver stage parasite. On the other hand, the mRNA expression for TPx-2 had begun increasing at the trophozoite stage and peaked at the schizont stage. In the IFA, TPx-2 was found localized in the mitochondria. The increase of TPx-2 might be explained by the exponential development of the parasite during the schizont stage requiring ATP production which may induce reactive oxygen species (ROS) in the mitochondria.
Parasitology International 12/2012; · 2.13 Impact Factor
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ABSTRACT: BACKGROUND: Malaria parasites actively proliferate in the body of their vertebrate and insect hosts, and are subjected to the toxic effects of reactive oxygen species. The antioxidant defenses of malaria parasites are considered to play essential roles in their survival and are thus considered promising targets for intervention. We sought to identify the cellular function of thioredoxin peroxidase-2 (TPx-2), which is expressed in the mitochondria, by disrupting the TPx-2 gene (pbtpx-2) of the rodent malaria parasite Plasmodium berghei. FINDINGS: In three independent experiments, two disruptant populations (TPx-2 KO) and three wild-type parasite populations with pyrimethamine resistance (dhfr-ts/mt at the DHFR-TS locus) and intact pbtpx-2 (TPx-2 WT) were obtained and cloned. Null expression of TPx-2 in the KO population was confirmed by RT-PCR and Western blot analyses. The TPx-2 KO parasite developed normally in mouse erythrocytes and multiplied at a rate similar to that of the TPx-2 WT parasite during the experimental period. The peak period of gametocytemia was delayed by 1 day in the TPx-2 KO compared with that of the TPx-2 WT and the parent parasite, however, the highest gametocyte number was comparable. The number of midgut oocysts in the TPx-2 KO at 14 days post feeding was comparable to that of the TPx-2 WT. CONCLUSIONS: The present finding suggests that mitochondrial Prx TPx-2 is not essential for asexual and the insect stage development of the malaria parasite.
Parasites & Vectors 11/2012; 5(1):252. · 2.94 Impact Factor
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ABSTRACT: Malaria control relies heavily on treated bed nets and indoor residual spraying with pyrethroid insecticides. Unfortunately, the resistance to pyrethroid insecticides, mainly due to the kdr mutation, is spreading in the main malaria vector Anopheles gambiae s.l., decreasing the insecticides' efficacy. To manage the insecticide resistance rapidly and flexibly, simple and effective tools for the early detection of resistant mosquitoes are needed. This study aimed to develop an allele-specific, loop-mediated, isothermal amplification (AS-LAMP) method to detect the West African-type kdr mutation (kdr-w; L1014F) in field-collected mosquitoes.
DNA fragments of the wild-type and the mutated kdr gene were used to select the primers and develop the method. The primers were designed with the mutation at the 5' end of the backward inner primer (BIP). The AS-LAMP method was compared to the AS-PCR method using the genomic DNA of 120 field-collected mosquitoes.
The AS-LAMP method could discriminate between the wild-type homozygote, the heterozygote, and the kdr-w homozygote within 75 min. The AS-LAMP method has the advantage of being faster and at least as sensitive and specific as the AS-PCR method.
The AS-LAMP method can be used to detect the kdr mutation for quick decision-making, even in less well-equipped laboratories.
Malaria Journal 07/2012; 11:227. · 3.19 Impact Factor
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ABSTRACT: T-2307, a novel arylamidine, has been shown to exhibit broad-spectrum antifungal activities against clinically significant pathogens. Here, we evaluated the in vitro and in vivo antimalarial activity of T-2307. The 50% inhibitory concentrations (IC₅₀s) of T-2307 against Plasmodium falciparum FCR-3 and K-1 strains were 0.47 and 0.17 μM, respectively. T-2307 at 2.5 to 10 mg/kg of body weight/day exhibited activity against blood stage and liver stage parasites in rodent malaria models. In conclusion, T-2307 exhibited in vitro and in vivo antimalarial activity.
Antimicrobial Agents and Chemotherapy 01/2012; 56(4):2191-3. · 4.84 Impact Factor
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ABSTRACT: There is a need for improving the method for counting oocysts of Plasmodium berghei in the midgut of Anopheles mosquitoes. The two methods currently used, the formalin fixation method and the mercurochrome staining method, have contradicting advantages and disadvantages. In the formalin fixation method, the specimen can be preserved but unstained oocysts were often indistinct from the insect tissue. While in the mercurochrome staining method, stained oocysts can be clearly distinguished from insect tissue but the specimen are not well preserved. These two methods were combined in this study to develop a new improved technique in counting the oocysts, in which the specimen can be both stained and preserved well. This technique was evaluated for its accuracy and suitability in observing the oocyst development.
In the improved technique, the parasite-infected midgut was first stained with mercurochrome, and then fixed with formalin. The specimens were finally observed using light microscopy. To evaluate the accuracy in the oocyst counting with the improved technique, mosquitoes were infected with the green fluorescent protein (GFP)-expressing parasite. Then, the midgut oocysts were counted using both the GFP marker and the improved technique. Results were then compared and showed that the improved technique retrieved 78%-123% (arithmetic mean = 97%) of the oocysts counted using the GFP marker. Furthermore, it was also possible to evaluate the oocyst development with a green filter using the light microscopy.
The improved technique for oocyst counting will be a useful tool for evaluating midgut oocyst numbers and determining the developmental stage of oocysts in parasite-infected mosquitoes.
Parasites & Vectors 06/2011; 4:118. · 2.94 Impact Factor
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ABSTRACT: The infection by the malaria parasite of its mammalian host is initiated by the asexual reproduction of the parasite within the host hepatocyte. Before the reproduction, the elongated sporozoites undergo a depolarizing morphogenesis to the spherical exo-erythrocytic form (EEF). This change can be induced in vitro by shifting the environmental conditions, in the absence of host hepatocytes. Using rodent malaria parasites expressing a FRET-based calcium sensor, YC3.60, we observed that the intracellular calcium increased at the center of the bulbous structure during sporozoite transformation. Modulators of intracellular calcium signaling (A23187 and W-7) accelerated the sporozoite-rounding process. These data suggest that calcium signaling regulates the morphological development of the malaria parasite sporozoite to the EEF, and support a fundamental role for calcium as a universal transducer of external stimuli in the parasitic life cycle.
Experimental Parasitology 02/2011; 128(2):176-80. · 2.12 Impact Factor
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ABSTRACT: The proboscis is an essential head appendage in insects that processes gustatory code during food intake, particularly useful considering that blood-sucking arthropods routinely reach vessels under the host skin using this proboscis as a probe.
Here, using an automated device able to quantify CO(2)-activated thermo (35°C)-sensing behavior of the malaria vector Anopheles stephensi, we uncovered that the protruding proboscis of mosquitoes contributes unexpectedly to host identification from a distance. Ablation experiments indicated that not only antennae and maxillary palps, but also proboscis were required for the identification of pseudo-thermo targets. Furthermore, the function of the proboscis during this behavior can be segregated from CO(2) detection required to evoke mosquito activation, suggesting that the proboscis of mosquitoes divide the proboscis into a "thermo-antenna" in addition to a "thermo-probe".
Our findings support an emerging view with a possible role of proboscis as important equipment during host-seeking, and give us an insight into how these appendages likely evolved from a common origin in order to function as antenna organs.
Parasites & Vectors 01/2011; 4:10. · 2.94 Impact Factor
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Kosuke Nakamura,
Sunao Fujioka, Shinya Fukumoto,
Noboru Inoue,
Kimitoshi Sakamoto,
Haruyuki Hirata,
Yasutoshi Kido,
Yoshisada Yabu,
Takashi Suzuki,
Yoh-ichi Watanabe,
Hiroyuki Saimoto,
Hiroshi Akiyama,
Kiyoshi Kita
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ABSTRACT: Trypanosoma brucei rhodesiense and T. b. gambiense are known causes of human African trypanosomiasis (HAT), or "sleeping sickness," which is deadly if untreated. We previously reported that a specific inhibitor of trypanosome alternative oxidase (TAO), ascofuranone, quickly kills African trypanosomes in vitro and cures mice infected with another subspecies, non-human infective T. b. brucei, in in vivo trials. As an essential factor for trypanosome survival, TAO is a promising drug target due to the absence of alternative oxidases in the mammalian host. This study found TAO expression in HAT-causing trypanosomes; its amino acid sequence was identical to that in non-human infective T. b. brucei. The biochemical understanding of the TAO including its 3 dimensional structure and inhibitory compounds against TAO could therefore be applied to all three T. brucei subspecies in search of a cure for HAT. Our in vitro study using T. b. rhodesiense confirmed the effectiveness of ascofuranone (IC(50) value: 1 nM) to eliminate trypanosomes in human infective strain cultures.
Parasitology International 12/2010; 59(4):560-4. · 2.13 Impact Factor
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ABSTRACT: Malaria parasites undergo two rounding-up transformations in their life cycle: the ookinete-to-oocyst transformation in the mosquito midgut, and the sporozoite-to-EEF (exo-erythrocytic form) differentiation in the host hepatocyte. Both events are characterized by the loss of polarity, implying that cytoskeletal reorganization is involved. In other eukaryotes, regulation of the actin skeleton is fundamental to subcellular remodeling. Although filamentous actin is well known to be involved in the motility of apicomplexan parasites, its participation in their morphological regulation is still largely unexplored. Here we describe the fundamental role of Actin depolymerization factor 2 (ADF2), a vector-stage-specific ADF isoform, in morphological changes accompanying the parasite life cycle. Among protozoan parasites, Plasmodium is unique in having two actin and two ADF genes. Disruption of the ADF2 gene in the rodent malaria parasite P. berghei had no effect on ookinete development or its subsequent invasion of the midgut. However, both the ookinete-to-oocyst and sporozoite-to-EEF transformations showed significant defects. These results indicated that Plasmodium ADF2 plays a pivotal role in transformation in the malaria parasite life cycle.
Biochemical and Biophysical Research Communications 07/2010; 397(4):668-72. · 2.48 Impact Factor
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ABSTRACT: Vector-borne diseases, such as malaria and lymphatic filariasis, are co-endemic in large parts of the world. To develop a multiplex amplification method for the simultaneous detection of multiple insect-borne infectious diseases, we used LAMP with fluorescently labeled primers to identify the SPECT2 gene of Plasmodium berghei and the cytochrome oxidase subunit I gene of Dirofilaria immitis in mosquitoes. This technique could detect as few as 100 P. berghei-infected red blood cell-equivalents or one D. immitis microfilaria. Moreover, individual species of parasites in mosquitoes could be identified when a mixture of fluorescently labeled primer sets was used. These findings suggest that the multiplex LAMP assay is sensitive and specific enough to identify parasite-bearing mosquitoes in areas where several diseases occur simultaneously. This procedure could increase the efficiency and effectiveness of arthropod-borne disease elimination programs.
Experimental Parasitology 06/2010; 125(2):179-83. · 2.12 Impact Factor
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ABSTRACT: We have developed two loop-mediated isothermal amplification (LAMP) assays for specific detection of Trypanosoma cruzi and Trypanosoma rangeli based on the 18S ribosomal RNA (rRNA) and the small nucleolar RNA (snoRNA) genes, respectively. The detection limit of the assays is 100 fg and 1 pg for T. cruzi and T. rangeli, respectively, with reactions conducted in 60 minutes. The two LAMP assays were used in detection of T. cruzi and T. rangeli infections in comparison with polymerase chain reaction (PCR) for DNA samples extracted from Rhodnius pallescens bugs collected from palm trees in Panama. Out of a total of 52 DNA samples from R. pallescens bugs 17 (33%) and 14 (27%) were T. cruzi-positive by LAMP and PCR, respectively, while, 7 (13%) and 4 (8%) were T. rangeli-positive by LAMP and PCR, respectively. Further evaluation of these LAMP assays is needed, especially with specimens collected from human patients as well as blood kept for transfusion purposes.
The American journal of tropical medicine and hygiene 05/2010; 82(5):855-60. · 2.59 Impact Factor
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Maria S Herbas,
Yoshiko Y Ueta,
Chie Ichikawa,
Mayumi Chiba,
Kana Ishibashi,
Mototada Shichiri, Shinya Fukumoto,
Naoaki Yokoyama,
Motohiro Takeya,
Xuenan Xuan,
Hiroyuki Arai,
Hiroshi Suzuki
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ABSTRACT: Various factors impact the severity of malaria, including the nutritional status of the host. Vitamin E, an intra and extracellular anti-oxidant, is one such nutrient whose absence was shown previously to negatively affect Plasmodium development. However, mechanisms of this Plasmodium inhibition, in addition to means by which to exploit this finding as a therapeutic strategy, remain unclear.
alpha-TTP knockout mice were infected with Plasmodium berghei NK65 or Plasmodium yoelii XL-17, parasitaemia, survival rate were monitored. In one part of the experiments mice were fed with a supplemented diet of vitamin E and then infected. In addition, parasite DNA damage was monitored by means of comet assay and 8-OHdG test. Moreover, infected mice were treated with chloroquine and parasitaemia and survival rate were monitored.
Inhibition of alpha-tocopherol transfer protein (alpha-TTP), a determinant of vitamin E concentration in circulation, confers resistance to malarial infection as a result of oxidative damage to the parasites. Furthermore, in combination with the anti-malarial drug chloroquine results were even more dramatic.
Considering that these knockout mice lack observable negative impacts typical of vitamin E deficiency, these results suggest that inhibition of alpha-TTP activity in the liver may be a useful strategy in the prevention and treatment of malaria infection. Moreover, a combined strategy of alpha-TTP inhibition and chloroquine treatment might be effective against drug resistant parasites.
Malaria Journal 01/2010; 9:101. · 3.19 Impact Factor
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ABSTRACT: In recent studies, heterologous prime-boost approaches, employing plasmid DNA and viral vector pathogen-delivering sequences, have been considered an effective protection strategy for intracellular parasite infections. Here, we evaluated the efficacy of such a strategy against the canine Babesia gibsoni infection. The DNA (pCAGGS-P29) and recombinant vaccinia virus (vvP29) both encoding the P29 of B. gibsoni were used in this study. The dogs were immunized 3 times with priming DNA and boosted once with recombinant virus. The dogs immunized with P29 developed a significant level of IgG2 antibody against P29. The response was strongly boosted by the inoculation of vvP29. The peripheral IFN-gamma responses of the dogs immunized with P29 were significantly higher than those of controls after the parasite inoculation. Moreover, the P29 immunized group showed a significantly low level of parasitemia. In conclusion, this study supports the efficacy of a prime-boost strategy for dogs against canine B. gibsoni infection.
Experimental Parasitology 09/2009; 123(4):296-301. · 2.12 Impact Factor
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ABSTRACT: Hosts employ a combination of two distinct yet compatible strategies to defend themselves against parasites: resistance, the ability to limit parasite burden, and tolerance, the ability to limit damage caused by a given parasite burden. Animals typically exhibit considerable genetic variation in resistance to a variety of pathogens; however, little is known about whether animals can evolve tolerance. Using a bacterial infection model in Drosophila, we uncovered a p38 MAP kinase-mediated mechanism of tolerance to intracellular bacterial infection as measured by the extent to which the host's survival rate increased or was maintained despite increasing bacterial burden. This increased survival was conferred primarily by a tolerance strategy whereby p38-dependent phagocytic encapsulation of bacteria resulted in enlarged phagocytes that trap bacteria. These results suggest that phagocytic responses are not restricted to resistance mechanisms but can also be applied to tolerance strategies for intracellular encapsulation of pathogens during the invertebrate immune response.
Cell host & microbe 09/2009; 6(3):244-52. · 13.02 Impact Factor
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ABSTRACT: Despite recent advances in our understanding of the basic biology behind transmission of zoonotic infectious diseases harbored by arthropod vectors these diseases remain threatening public health concerns. For effective control of vector and treatment, precise sampling indicating the prevalence of such diseases is essential. With an aim to develop a quick and simple method to survey zoonotic pathogen-transmitting vectors, LAMP (loop-mediated isothermal amplification) was applied to the detection of filarial parasites using a filarial parasite-transmitting experimental model that included one of the mosquito vectors, Aedes aegypti, and the canine heartworm, Dirofilaria immitis.
LAMP reactions amplifying the cytochrome oxidase subunit I gene demonstrated high sensitivity when a single purified D. immitis microfilaria was detected. Importantly, the robustness of the LAMP reaction was revealed upon identification of an infected mosquito carrying just a single parasite, a level easily overlooked using conventional microscopic analysis. Furthermore, successful detection of D. immitis in wild-caught mosquitoes demonstrated its applicability to field surveys.
Due to its simplicity, sensitivity, and reliability, LAMP is suggested as an appropriate diagnostic method for routine diagnosis of mosquito vectors carrying filarial parasites. This method can be applied to the survey of not only canine filariasis but also lymphatic filariasis, another major public health problem. Therefore, this method offers great promise as a useful diagnostic method for filarial parasite detection in endemic filariasis regions.
Parasites & Vectors 04/2009; 2(1):15. · 2.94 Impact Factor
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ABSTRACT: Genetic variation in susceptibility to pathogens is a central concern both to medicine and agriculture and to the evolution of animals. Here, we have investigated the link between such natural genetic variation and the immune response in wild-type Drosophila melanogaster, a major model organism for immunological research. We found that within nine wild-type strains, different Drosophila genotypes show wide-ranging variation in their ability to survive infection from the pathogenic bacteria Listeria monocytogenes. Canton-S, a resistant strain, showed increased capacity to induce stronger innate immune activities (antimicrobial peptides (AMPs), phenol oxidase activity, and phagocytosis) compared to the susceptible strain (white) at early time points during bacterial infection. Moreover, PGRP-LE-induced innate immune activation immediately after infection greatly improves survival of the susceptible strain strongly suggesting a mechanism behind the natural genetic variation of these two strains. Taken together we provide the first experimental evidence to suggest that differences in innate immune activity at early time points during infection likely mediates infection susceptibility in Drosophila.
Biochemical and Biophysical Research Communications 01/2009; 379(1):6-10. · 2.48 Impact Factor
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ABSTRACT: Mosquitoes are critical vectors in many arboviral transmission cycles. Considering the increasing incidence of arboviral infections throughout the world, monitoring of vector populations for the presence of an arbovirus could be considered an important initial step of risk assessment to humans and animals. In response to this need, increased efforts to develop rapid and reliable diagnostic techniques have been undertaken; a single-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect virus in vector mosquitoes (Aedes aegypti) using the Flock House Virus (FHV) as a model. The robustness of the RT-LAMP reaction was revealed by its ability to detect FHV from an "all-in-one" template using whole mosquito bodies within 30min. Furthermore, RT-LAMP identified successfully a mosquito carrying just a single FHV particle, a level easily overlooked in conventional analysis such as plaque forming assays. These observations suggest that RT-LAMP is more reliable and useful for routine diagnosis of vector mosquitoes in regions where the prevalence of vector-borne diseases such as West Nile fever or dengue fever are common.
Journal of Virological Methods 12/2008; 156(1-2):32-6. · 2.01 Impact Factor
