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Tawee Saiwichai - One of the best experts on this subject based on the ideXlab platform.
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artesunate tafenoquine combination therapy promotes clearance and abrogates transmission of the avian malaria parasite Plasmodium gallinaceum
Veterinary Parasitology, 2017Co-Authors: Suchada Tasai, Morakot Kaewthamasorn, Tawee Saiwichai, Prayute Buddhirakkul, Sonthaya Tiawsirisup, Sirintip Chaichalotornkul, Sittiporn PattaradilokratAbstract:Clinical manifestations of malaria infection in vertebrate hosts arise from the multiplication of the asexual stage parasites in the blood, while the gametocytes are responsible for the transmission of the disease. Antimalarial drugs that target the blood stage parasites and transmissible gametocytes are rare, but are essentially needed for the effective control of malaria and for limiting the spread of resistance. Artemisinin and its derivatives are the current first-line antimalarials that are effective against the blood stage parasites and gametocytes, but resistance to artemisinin has now emerged and spread in various malaria endemic areas. Therefore, a novel antimalarial drug, or a new drug combination, is critically needed to overcome this problem. The objectives of this study were to evaluate the efficacy of a relatively new antimalarial compound, tafenoquine (TQ), and a combination of TQ and a low dose of artesunate (ATN) on the in vivo blood stage multiplication, gametocyte development and transmission of the avian malaria parasite Plasmodium gallinaceum to the vector Aedes aegypti. The results showed that a 5-d treatment with TQ alone was unable to clear the blood stage parasites, but was capable of reducing the mortality rate, while TQ monotherapy at a high dose of 30mg/kg was highly effective against the gametocytes and completely blocked the transmission of P. gallinaceum. In addition, the combination therapy of TQ+ATN completely cleared P. gallinaceum blood stages and sped up the gametocyte clearance from chickens, suggesting the synergistic effect of the two drugs. In conclusion, TQ is demonstrated to be effective for limiting avian malaria transmission and may be used in combination with a low dose of ATN for safe and effective treatment.
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Molecular detection of the avian malaria parasite Plasmodium gallinaceum in Thailand.
Veterinary Parasitology, 2015Co-Authors: Sittiporn Pattaradilokrat, Wisawa Tiyamanee, Phumin Simpalipan, Morakot Kaewthamasorn, Tawee Saiwichai, Jian Li, Pongchai HarnyuttanakornAbstract:Abstract Avian malaria is one of the most common veterinary problems in Southeast Asia. The standard molecular method for detection of the avian malaria parasite involves the phenol–chloroform extraction of parasite genomic (g)DNA followed by the amplification of parasite gDNA using polymerase chain reaction (PCR). However, the phenol–chloroform extraction method is time-consuming and requires large amounts of samples and toxic organic solvents, thereby limiting its applications for parasite detection in the field. This study aimed to compare the performance of chelex-100 resin and phenol/chloroform extraction methods for the extraction of Plasmodium gallinaceum gDNA from whole avian blood that had been dried on filter papers (a common field sampling method). The specificity and sensitivity of PCR assays for P. gallinaceum cytochrome B (cytb) and cytochrome oxidase subunit I (coxI) gene fragments (544 and 588 bp, respectively) were determined, and found to be more sensitive with gDNA extracted by the chelex-100 resin method than with the phenol/chloroform method. These PCR assays were also performed to detect P. gallinaceum in 29 blood samples dried on filter papers from domestic chickens in a malaria endemic area, where the reliable identification of seven field isolates of P. gallinaceum was obtained with an accuracy of 100%. The analysis of cytb and coxI gene nucleotide sequences revealed the existence of at least two genetically distinct populations of P. gallinaceum in Thailand, both of which differed from the reference strain 8A of P. gallinaceum. In conclusion, the chelex-100 resin extraction method is a simple and sensitive method for isolating gDNA from whole avian blood dried on filter paper. Genomic DNA extracted by the chelex method could subsequently be applied for the PCR-based detection of P. gallinaceum and DNA sequencing. Our PCR assays provide a reliable diagnostic tool for molecular epidemiological studies of P. gallinaceum infections in domestic chickens and wild birds.
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effects of artesunate treatment on Plasmodium gallinaceum transmission in the vectors aedes aegypti and culex quinquefasciatus
Veterinary Parasitology, 2015Co-Authors: Mintra Pruckngern, Sittiporn Pattaradilokrat, Pongchai Harnyuttanakorn, Prayute Buddhirakkul, Kamlang Chumpolbanchorn, Suntorn Pimnon, Somphong Narkpinit, Tawee SaiwichaiAbstract:In the absence of vaccines, chemotherapy is an effective and economical way for controlling malaria. Development of anti-malarial drugs that target pathogenic blood stage parasites and gametocytes is preferable for the treatment as it can alleviate the host's morbidity and mortality and block transmission of the Plasmodium parasite. Recently, our laboratory has developed an in vivo transmission blocking assay that involves administration of 7 consecutive daily doses of a test compound into domestic chickens (Gallus gallus domesticus) infected with the avian malaria parasite Plasmodium gallinaceum with 10% parasitaemia and 1% gametocytaemia. To compromise the cost and time for artesunate (ATN) treatment, this study aimed to investigate effects of a 5-day consecutive administration of 10 milligrams per kilogram (mg/kg) ATN on P. gallinaceum infection in chickens and transmission to two natural vectors, Aedes aegypti and Culex quinquefasciatus. Our study showed that the treatment with 10 mg/kg ATN for 7 days, but not 5 days, completely eliminated blood stage infections, prevented recrudescence and blocked gametocyte production and transmission of P. gallinaceum to its vectors, thereby confirming the potent schizontocidal and gametocytocidal activities of ATN. This regimen should be further evaluated in field trials.
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refractoriness of the natural malaria vector culex quinquefasciatus to Plasmodium gallinaceum
The Journal of Tropical Medicine and Parasitology, 2014Co-Authors: Mintra Pruckngern, Sittiporn Pattaradilokrat, Pongchai Harnyuttanakorn, Prayute Buddhirakkul, Kamlang Chumpolbanchorn, Suntorn Pimnon, Tawee SaiwichaiAbstract:Development of effective strategies to control malaria transmission is an important issue in malaria research. This study aimed to investigate refractoriness of laboratory and field strains of the malaria vector Culex quinquefasciatus to the avian malaria parasite Plasmodium gallinaceum , a model of the human malaria disease. Transmission potential was determined by feeding batches of Cx. quinquefasciatus on P. gallinaceum infected chickens with 10% and 30% parasitemia. The mosquitoes were examined for percent infectivity and numbers of oocysts. Our study showed that when the mosquitoes were fed on the infected chickens with high parasitemia, the laboratory isolate of Cx. quinquefasciatus was more susceptible to P. gallinaceum transmission and produced significantly higher oocyst numbers than the field isolate. There were, however, no differences in term of the transmission potential and infectivity of the malaria parasite when the mosquitoes were allowed to feed on the P. gallinaceum -infected chickens with low parasitemia. In conclusion, our study demonstrated the differences in refractoriness of the natural vector Cx. quinquefasciatus to the transmission of the avian malaria P. gallinaceum , implying the existence of the natural pathogen resistance mechanisms in the Culex mosquito.
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in vivo transmission blocking activities of artesunate on the avian malaria parasite Plasmodium gallinaceum
Veterinary Parasitology, 2013Co-Authors: Rapeeporn Kumnuan, Sittiporn Pattaradilokrat, Pongchai Harnyuttanakorn, Kamlang Chumpolbanchorn, Suntorn Pimnon, Somphong Narkpinit, Tawee SaiwichaiAbstract:Infection and transmission of the avian malaria parasite Plasmodium gallinaceum in domestic chickens is associated with high economic burden and presents a major challenge to poultry industry in South East Asia. Development of drugs targeting both asexual blood stage parasites and sexual stages of the avian malarias will be beneficial for malaria treatment and eradication. However, current drugs recommended for treatment of the avian malaria parasites target specifically the asexual blood stage parasites, but have little or no impact to the gametocytes, the major target for development of transmission-blocking strategies. In the present work, we established a simple procedure to evaluate gametocytocidal and transmission blocking activities in a P. gallinaceum-avian model. The assays involved administration of seven consecutive daily doses of test compounds into P. gallinaceum-infected chickens with 10% parasitaemia and 1% gametocytaemia. Our studies indicated that intramuscular injection with seven daily low doses (the minimum effective dose of 10mg/kg) of artesunate blocked the gametocyte production and transmission to the mosquito vector Aedes aegypti. This assay can be further applicable for testing new compounds against P. gallinaceum and for other parasitic protozoa infecting birds.
Antoniana U. Krettli - One of the best experts on this subject based on the ideXlab platform.
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virus expressed recombinant single chain antibody blocks sporozoite infection of salivary glands in Plasmodium gallinaceum infected aedes aegypti
American Journal of Tropical Medicine and Hygiene, 2000Co-Authors: M De L Capurro, Antoniana U. Krettli, Brenda T. Beerntsen, Judy Coleman, Kevin M Myles, Ken E Olson, E Rocha, Anthony A. JamesAbstract:Transgenic mosquitoes resistant to malaria parasites are being developed to test the hypothesis that they may be used to control disease transmission. We have developed an effector portion of an antiparasite gene that can be used to test malaria resistance in transgenic mosquitoes. Mouse monoclonal antibodies that recognize the circumsporozoite protein of Plasmodium gallinaceum can block sporozoite invasion of Aedes aegypti salivary glands. An anti-circumsporozoite monoclonal antibody, N2H6D5, whose corresponding heavy- and light-chain gene variable regions were engineered as a single-chain antibody construct, binds to P. gallinaceum sporozoites and prevents infection of Ae. aegypti salivary glands when expressed from a Sindbis virus. Mean intensities of sporozoite infections of salivary glands in mosquitoes expressing N2scFv were reduced as much as 99.9% when compared to controls.
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antisporozoite antibodies with protective and nonprotective activities in vitro and in vivo correlations using Plasmodium gallinaceum an avian model
Journal of Eukaryotic Microbiology, 1995Co-Authors: Adelina Diaz De Ramirez, Eliana M. M. Rocha, Antoniana U. KrettliAbstract:ABSTRACT. A correlation was observed between in vivo and in vitro activity of six monoclonal antibodies (mAb) against the major circumsporozoite protein of the avian malaria Plasmodium gallinaceum as follows. (1) Two mAb were protective, totally abrogating sporozoite infectivity to chicks, its natural host, in vivo; they caused 100% inhibition of sporozoite invasion (ISI) in vitro to SL-29 chicken fibroblasts and intense ISI to cultured chicken macrophages, as well as inhibited the exoerythrocytic development of sporozoites taken up by macrophages, the initial cell host of P. gallinaceum sporozoites. (2) Two mAb were partially protective in that they reduced sporozoite infectivity to chicks, caused partial ISI to SL-29 and macrophage cells and partial inhibition to the exoerythrocytic development of sporozoites in macrophages in vitro. (3) Two mAb were totally inactive in vivo although they both bound to the sporozoite antigens as detected by indirect immunofluorescence, western blot, and ELISA; they both failed to induce ISI or inhibit the exoerythrocytic development in macrophages. The possible participation of macrophages as the initial cell type involved in sporozoite destruction in the presence of anti-circumsporozoite antibodies is discussed.
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exoerythrocytic development of Plasmodium gallinaceum sporozoites in a chicken fibroblast cell line and inhibition of the cell invasion by specific anti sporozoite monoclonal antibodies
Journal of Eukaryotic Microbiology, 1993Co-Authors: Eliana M. M. Rocha, Michael R. Hollingdale, Robert W Gwadz, Antoniana U. KrettliAbstract:Cultivation of the Plasmodium gallinaceum exoerythrocytic forms from sporozoites was attempted in three different cell lines: HEPG2-A16 (from a human hepatoma), VERO (monkey kidney epithelial cells) and SL-29 (chicken embryo fibroblast cells). The sporozoites invaded all three cells types but their development into exoerythrocytic forms occurred only in the SL-29 cells. In the presence of specific monoclonal antibodies against the major circumsporozoite protein, there were varying degrees of inhibition of parasite invasion of the SL-29 cells. Of seven monoclonal antibodies tested, two completely inhibited cell invasion at high concentrations and caused intense inhibition at concentrations as low as 2.5 micrograms/ml, four caused intense inhibition at these various concentrations, and one had no effect on sporozoite invasion.
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Plasmodium gallinaceum antibodies to circumsporozoite protein prevent sporozoites from invading the salivary glands of aedes aegypti
Experimental Parasitology, 1992Co-Authors: Alon Warburg, Antoniana U. Krettli, M G Touray, Louis H. MillerAbstract:A circumsporozoite protein-specific monoclonal antibody (N2H6D5) was injected into malaria-infected mosquitoes to determine its effect on the sporogonic cycle. After injection of antibody into mosquitoes (100 ng each), positive immunofluorescence (measured on air-dried sporozoites) reactions in hemolymph extracts were observed at a dilution of 1:1000. At 72 hr postinjection the levels dropped to 1:10. Sporozoites coinjected with antibody did not invade the salivary glands. In naturally infected mosquitoes, sporozoites were released over a period of 3 to 4 days. Therefore, mosquitoes were injected twice. The first injection was a day before the beginning of sporozoite release and the second, 2 days later. Sporozoite invasion of the salivary glands was assessed 3 days after the second injection, by microscopic examination of dissected glands. At this stage, all oocysts had completed maturation and released the sporozoites. Salivary gland infections were totally prevented in mosquitoes given two injections of 100 ng N2H6D5. Hence, sustained presence of anti-circumsporozoite antibodies in the hemolymph can render female Aedes aegypti refractory to Plasmodium gallinaceum.
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in vitro development of exoerythrocytic forms of Plasmodium gallinaceum sporozoites in avian macrophages
Journal of Eukaryotic Microbiology, 1991Co-Authors: Adelina D Ramirez, Eliana M. M. Rocha, Antoniana U. KrettliAbstract:Exoerythrocytic forms of Plasmodium gallinaceum were cultured in vitro using salivary gland sporozoites extracted from experimentally infected Aedes fluviatilis mosquitoes. The host cells were macrophage precursors from chicken bone marrow. At various times after introduction of sporozoites, the cultures were stained by Giemsa or by immunofluorescence assay (IFA) using anti-sporozoite-specific monoclonal antibodies (MAb). The time to complete parasite development in vitro was 50-70 h. By 70 h, ruptured segmenters and free merozoites were visible within the cells. Inoculation of normal chickens with infected cultures induced parasitemia after a pre-patent period of 10-11 days. In vitro young exoerythrocytic forms, late schizonts that include the matured segmenters, and free merozoites shared common antigens with the sporozoites as revealed by IFA using anti-sporozoite-specific MAbs. Our data indicate that macrophages support development of P. gallinaceum sporozoites and that the circumsporozoite proteins are present until the end of the primary exoerythrocytic schizogony.
Clive J Ellory - One of the best experts on this subject based on the ideXlab platform.
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two functionally distinct organic osmolyte pathways in Plasmodium gallinaceum infected chicken red blood cells
Biochimica et Biophysica Acta, 2002Co-Authors: Henry M Staines, Stephane Egee, Franck Lapaix, Edmund M Godfrey, Serge Thomas, Clive J ElloryAbstract:AbstractRed cells infected with the human malaria parasite Plasmodium falciparum have an increased permeability to a range ofsmall, structurally unrelated solutes via a malaria-induced pathway. We report here a similar pathway present in parasitisedred cells from chickens infected with the avian malaria parasite, Plasmodium gallinaceum. Parasitised cells showed a markedincrease in the rate of influx of sorbitol (76-fold) and, to a lesser degree, taurine (3-fold) when compared with red cells fromuninfected chickens. Pharmacological data suggest that both sorbitol and taurine are transported via a single malaria-induced pathway, which is sensitive to inhibition by 5-nitro-2-(3-phenylpropylamino)benzoic acid (IC 50 V7 WM). Themalaria-induced pathway differed in its inhibition by a range of anion channel inhibitors when compared to theendogenous, volume-activated osmolyte pathway of chicken red cells. There were also differences in the selectivity ofsorbitol and taurine by the two permeation routes. The data presented here are consistent with the presence of two distinctorganic solute pathways in infected chicken red cells. The first is an endogenous volume-activated pathway, which is notactivated by the parasite and the second is a malaria-induced pathway, similar to those that are induced by other types ofmalaria in other host species. s 2002 Elsevier Science B.V. All rights reserved.
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malaria parasite Plasmodium gallinaceum up regulates host red blood cell channels
FEBS Letters, 2001Co-Authors: Serge L Y Thomas, Stephane Egee, Franck Lapaix, Lars Kaestner, Henry M Staines, Clive J ElloryAbstract:The properties of the malaria parasite-induced permeability pathways in the host red blood cell have been a major area of interest particularly in the context of whether the pathways are host- or parasite-derived. In the present study, the whole-cell configuration of the patch-clamp technique has been used to show that, compared with normal cells, chicken red blood cells infected by Plasmodium gallinaceum exhibited a 5–40-fold larger membrane conductance, which could be further increased up to 100-fold by raising intracellular Ca2+ levels. The increased conductance was not due to pathways with novel electrophysiological properties. Rather, the parasite increased the activity of endogenous 24 pS stretch-activated non-selective cationic (NSC) and 62 pS calcium-activated NSC channels, and, in some cases, of endogenous 255 pS anionic channels.
Pongchai Harnyuttanakorn - One of the best experts on this subject based on the ideXlab platform.
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Molecular detection of the avian malaria parasite Plasmodium gallinaceum in Thailand.
Veterinary Parasitology, 2015Co-Authors: Sittiporn Pattaradilokrat, Wisawa Tiyamanee, Phumin Simpalipan, Morakot Kaewthamasorn, Tawee Saiwichai, Jian Li, Pongchai HarnyuttanakornAbstract:Abstract Avian malaria is one of the most common veterinary problems in Southeast Asia. The standard molecular method for detection of the avian malaria parasite involves the phenol–chloroform extraction of parasite genomic (g)DNA followed by the amplification of parasite gDNA using polymerase chain reaction (PCR). However, the phenol–chloroform extraction method is time-consuming and requires large amounts of samples and toxic organic solvents, thereby limiting its applications for parasite detection in the field. This study aimed to compare the performance of chelex-100 resin and phenol/chloroform extraction methods for the extraction of Plasmodium gallinaceum gDNA from whole avian blood that had been dried on filter papers (a common field sampling method). The specificity and sensitivity of PCR assays for P. gallinaceum cytochrome B (cytb) and cytochrome oxidase subunit I (coxI) gene fragments (544 and 588 bp, respectively) were determined, and found to be more sensitive with gDNA extracted by the chelex-100 resin method than with the phenol/chloroform method. These PCR assays were also performed to detect P. gallinaceum in 29 blood samples dried on filter papers from domestic chickens in a malaria endemic area, where the reliable identification of seven field isolates of P. gallinaceum was obtained with an accuracy of 100%. The analysis of cytb and coxI gene nucleotide sequences revealed the existence of at least two genetically distinct populations of P. gallinaceum in Thailand, both of which differed from the reference strain 8A of P. gallinaceum. In conclusion, the chelex-100 resin extraction method is a simple and sensitive method for isolating gDNA from whole avian blood dried on filter paper. Genomic DNA extracted by the chelex method could subsequently be applied for the PCR-based detection of P. gallinaceum and DNA sequencing. Our PCR assays provide a reliable diagnostic tool for molecular epidemiological studies of P. gallinaceum infections in domestic chickens and wild birds.
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effects of artesunate treatment on Plasmodium gallinaceum transmission in the vectors aedes aegypti and culex quinquefasciatus
Veterinary Parasitology, 2015Co-Authors: Mintra Pruckngern, Sittiporn Pattaradilokrat, Pongchai Harnyuttanakorn, Prayute Buddhirakkul, Kamlang Chumpolbanchorn, Suntorn Pimnon, Somphong Narkpinit, Tawee SaiwichaiAbstract:In the absence of vaccines, chemotherapy is an effective and economical way for controlling malaria. Development of anti-malarial drugs that target pathogenic blood stage parasites and gametocytes is preferable for the treatment as it can alleviate the host's morbidity and mortality and block transmission of the Plasmodium parasite. Recently, our laboratory has developed an in vivo transmission blocking assay that involves administration of 7 consecutive daily doses of a test compound into domestic chickens (Gallus gallus domesticus) infected with the avian malaria parasite Plasmodium gallinaceum with 10% parasitaemia and 1% gametocytaemia. To compromise the cost and time for artesunate (ATN) treatment, this study aimed to investigate effects of a 5-day consecutive administration of 10 milligrams per kilogram (mg/kg) ATN on P. gallinaceum infection in chickens and transmission to two natural vectors, Aedes aegypti and Culex quinquefasciatus. Our study showed that the treatment with 10 mg/kg ATN for 7 days, but not 5 days, completely eliminated blood stage infections, prevented recrudescence and blocked gametocyte production and transmission of P. gallinaceum to its vectors, thereby confirming the potent schizontocidal and gametocytocidal activities of ATN. This regimen should be further evaluated in field trials.
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refractoriness of the natural malaria vector culex quinquefasciatus to Plasmodium gallinaceum
The Journal of Tropical Medicine and Parasitology, 2014Co-Authors: Mintra Pruckngern, Sittiporn Pattaradilokrat, Pongchai Harnyuttanakorn, Prayute Buddhirakkul, Kamlang Chumpolbanchorn, Suntorn Pimnon, Tawee SaiwichaiAbstract:Development of effective strategies to control malaria transmission is an important issue in malaria research. This study aimed to investigate refractoriness of laboratory and field strains of the malaria vector Culex quinquefasciatus to the avian malaria parasite Plasmodium gallinaceum , a model of the human malaria disease. Transmission potential was determined by feeding batches of Cx. quinquefasciatus on P. gallinaceum infected chickens with 10% and 30% parasitemia. The mosquitoes were examined for percent infectivity and numbers of oocysts. Our study showed that when the mosquitoes were fed on the infected chickens with high parasitemia, the laboratory isolate of Cx. quinquefasciatus was more susceptible to P. gallinaceum transmission and produced significantly higher oocyst numbers than the field isolate. There were, however, no differences in term of the transmission potential and infectivity of the malaria parasite when the mosquitoes were allowed to feed on the P. gallinaceum -infected chickens with low parasitemia. In conclusion, our study demonstrated the differences in refractoriness of the natural vector Cx. quinquefasciatus to the transmission of the avian malaria P. gallinaceum , implying the existence of the natural pathogen resistance mechanisms in the Culex mosquito.
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in vivo transmission blocking activities of artesunate on the avian malaria parasite Plasmodium gallinaceum
Veterinary Parasitology, 2013Co-Authors: Rapeeporn Kumnuan, Sittiporn Pattaradilokrat, Pongchai Harnyuttanakorn, Kamlang Chumpolbanchorn, Suntorn Pimnon, Somphong Narkpinit, Tawee SaiwichaiAbstract:Infection and transmission of the avian malaria parasite Plasmodium gallinaceum in domestic chickens is associated with high economic burden and presents a major challenge to poultry industry in South East Asia. Development of drugs targeting both asexual blood stage parasites and sexual stages of the avian malarias will be beneficial for malaria treatment and eradication. However, current drugs recommended for treatment of the avian malaria parasites target specifically the asexual blood stage parasites, but have little or no impact to the gametocytes, the major target for development of transmission-blocking strategies. In the present work, we established a simple procedure to evaluate gametocytocidal and transmission blocking activities in a P. gallinaceum-avian model. The assays involved administration of seven consecutive daily doses of test compounds into P. gallinaceum-infected chickens with 10% parasitaemia and 1% gametocytaemia. Our studies indicated that intramuscular injection with seven daily low doses (the minimum effective dose of 10mg/kg) of artesunate blocked the gametocyte production and transmission to the mosquito vector Aedes aegypti. This assay can be further applicable for testing new compounds against P. gallinaceum and for other parasitic protozoa infecting birds.
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species specific nested pcr for detecting Plasmodium gallinaceum in fresh chicken blood
The Journal of Tropical Medicine and Parasitology, 2009Co-Authors: Tawee Saiwichai, Pongchai Harnyuttanakorn, Montree Maneepak, Pucharee Songprakhon, Suwannee NithiuthaiAbstract:Abstract Avian malaria, epidemic in Thailand’s chicken farms, is caused by Plasmodium gallinaceum. The standard method for detecting avian malaria is microscopic diagnosis, but parasites may go undetected in light infections. Therefore, the objective of this study was to detect P. gallinaceum DNA in infected fresh-blood samples by nested polymerase chain reaction (nested PCR). Primers were designed from small subunit ribosomal RNA (SSUrRNA) genes, with genus-specific primers for initial amplification, and species-specific primers for nested amplification. The PCR conditions were optimized using P. gallinaceum DNA as templates. The optimum conditions for the 1 st and 2 nd amplifications were different at the annealing stages, using 60 o C and 45 o C, respectively. The first amplification resulted in unobservable PCR products at 420 bp of the target fragment in all diluted blood samples. These products were continued in the 2 nd amplification, when the species-specific 120 bp-DNA fragment of P. gallinaceum was found. This nested PCR condition was sensitive to detecting 0.0000085% parasitemia or 0.2 infected red cells/μl of the lowest blood dilution in the study. The results suggest that this assay had high sensitivity and species-specificity using whole blood samples without DNA extraction. Keywords : Plasmodium gallinaceum, detection, nested PCR, whole blood
Mohammed Shahabuddin - One of the best experts on this subject based on the ideXlab platform.
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Plasmodium gallinaceum differential killing of some mosquito stages of the parasite by insect defensin
Experimental Parasitology, 1998Co-Authors: Mohammed Shahabuddin, Iesha Fields, Philippe Bulet, Jules A Hoffmann, Louis H. MillerAbstract:We examined several insect antimicrobial peptides to study their effect on Plasmodium gallinaceum zygotes, ookinetes, oocysts, and sporozoites. Only two insect defensins-Aeschna cyanea (dragon fly) and Phormia terranovae (flesh fly)-had a profound toxic effect on the oocysts in Aedes aegypti and on isolated sporozoites. The defensins affected the oocysts in a time-dependent manner. Injecting the peptide into the hemolymph 1 or 2 days after an infectious blood meal had no significant effect on prevalence of infection or relative oocyst density per mosquito. When injected 3 days after parasite ingestion, the relative oocyst density was significantly reduced. Injection on day 4 or later damaged the developing oocysts, although the oocysts density per mosquito was not significantly different when examined on day 8. The oocysts were swollen or had extensive internal vacuolization. The peptides had no detectable effect on the early stages of the parasite: the zygotes and ookinetes tested in vitro. Both the defensins were highly toxic to isolated sporozoites in vitro as indicated by disruption of the membrane permeability barrier, a change in morphology, and loss of motility. In contrast to the toxicity of cecropin and magainin for mosquitoes, defensin, at concentrations that kill parasites, is not toxic to mosquitoes, suggesting that defensin should be studied further as a potential molecule to block sporogonic development of Plasmodium.
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Plasmodium gallinaceum preferentially invades vesicular atpase expressing cells in aedes aegypti midgut
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Mohammed Shahabuddin, Paulo F P PimentaAbstract:Penetration of the mosquito midgut epithelium is obligatory for the further development of Plasmodium parasites. Therefore, blocking the parasite from invading the midgut wall disrupts the transmission of malaria. Despite such a pivotal role in malaria transmission, the cellular and molecular interactions that occur during the invasion are not understood. Here, we demonstrate that the ookinetes of Plasmodium gallinaceum, which is related closely to the human malaria parasite Plasmodium falciparum, selectively invade a cell type in the Aedes aegypti midgut. These cells, unlike the majority of the cells in the midgut, do not stain with a basophilic dye (toluidine blue) and are less osmiophilic. In addition, they contain minimal endoplasmic reticulum, lack secretory granules, and have few microvilli. Instead, these cells are highly vacuolated and express large amounts of vesicular ATPase. The enzyme is associated with the apical plasma membrane, cytoplasmic vesicles, and tubular extensions of the basal membrane of the invaded cells. The high cost of insecticide use in endemic areas and the emergence of drug resistant malaria parasites call for alternative approaches such as modifying the mosquito to block the transmission of malaria. One of the targets for such modification is the parasite receptor on midgut cells. A step toward the identification of this receptor is the realization that malaria parasites invade a special cell type in the mosquito midgut.
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Adhesion of Plasmodium gallinaceum ookinetes to the Aedes aegypti midgut: sites of parasite attachment and morphological changes in the ookinete.
The Journal of eukaryotic microbiology, 1998Co-Authors: Helge Zieler, Claude F. Garon, Elizabeth R. Fischer, Mohammed ShahabuddinAbstract:Plasmodium gallinaceum ookinetes adhered to Aedes aegypti midgut epithelia when purified ookinetes and isolated midguts were combined in vitro. Ookinetes preferentially bound to the microvillated luminal surface of the midgut, and they seemed to interact with three types of structures on the midgut surface. First, they adhered to and migrated through a network-like matrix, which we have termed microvilli-associated network, that covers the surface of the microvilli. This network forms on the luminal midgut surface in response to blood or protein meals. Second, the ookinetes bound directly to the microvilli on the surface of the midgut and were occasionally found immersed in the thick microvillar layer. Third, the ookinetes associated with accumulations of vesicular structures found interspersed between the microvillated cells of the midgut. The origin of these vesicular structures is unknown, but they correlated with the surface of midgut cells invaded by ookinetes as observed by TEM. After binding to the midgut, ookinetes underwent extensive morphological changes: they frequently developed one or more annular constrictions, and their surface roughened considerably, suggesting that midgut components remain bound to the parasite surface. Our observations suggest that, in a natural infection, the ookinete interacts in a sequential manner with specific components of the midgut surface. Initial binding to the midgut surface may activate the ookinete and cause morphological changes in preparation for invasion of the midgut cells.
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antibody mediated inhibition of aedes aegypti midgut trypsins blocks sporogonic development of Plasmodium gallinaceum
Infection and Immunity, 1996Co-Authors: Mohammed Shahabuddin, David C Kaslow, Francisco J A Lemos, Marcelo JacobslorenaAbstract:The peritrophic matrix (PM) that forms around a blood meal is a potential barrier of Plasmodium development in mosquitoes. Previously, we have shown that to traverse the PM, Plasmodium ookinetes secrete a prochitinase and that an inhibitor of chitinase blocks further parasite development. Here we report that it is the mosquito trypsin that activates the Plasmodium prochitinase. Trypsin was identified as the chitinase-activating enzyme by two criteria: (i) trypsin activity and activating activity comigrated on one-dimensional gels, and (ii) activating activity and penetration of the PM by Plasmodium parasites were both hindered by trypsin-specific inhibitors. Subsequently, we examined the effect of antitrypsin antibodies on the parasite life cycle. Antibodies prepared against a recombinant blackfly trypsin effectively and specifically inhibited mosquito trypsin activity. Moreover, when incorporated into an infective blood meal, the antitrypsin antibodies blocked infectivity of Aedes aegypti mosquitoes by Plasmodium gallinaceum. This block of infectivity could be reversed by exogenously provided chitinase, strongly suggesting that the antibodies act by inhibiting prochitinase activation and not on the parasite itself. This work led to the identification of a mosquito antigen, i.e., midgut trypsin, as a novel target for blocking malaria transmission.