Inhibition of Plasmodium falciparum proliferation in vitro by double-stranded RNA nanoparticle against malaria topoisomerase II.

The need to develop new effective antimalarial agents is urgent due to the rapid emergence of drug resistance to all current drugs by the most virulent human malaria parasite, Plasmodium falciparum. A promising avenue is in the development of antimalarials based on RNA interference targeting expression of malaria parasite vital genes, viz. DNA topoisomerase II gene (PfTOP2). Biodegradable chitosan nanoparticle system has proven to be effective in delivering DNA and small double-stranded interfering RNA to target cells. We have employed a long double-stranded (dsRNA) targeting the coding region of PfTOP2 that is complexed with chitosan nanoparticles in order to interfere with the cognate mRNA expression and examined its effect on P. falciparum growth in culture. Exposure of ring stage-infected erythrocytes to 10 µg/ml PfTOP2 chitosan/dsRNA nanoparticles for 48 h resulted in 71% growth inhibition as determined by [(3)H] hypoxanthine incorporation and microscopic assays, compared with 41% inhibition using an equivalent amount of free PfTOP2 dsRNA or 12% with unrelated chitosan/dsRNA nanoparticles. This inhibition was shown to occur during maturation of trophozoite to schizont stages. RT-PCR analysis indicated 56% and 38% decrease in PfTOP2 transcript levels in P. falciparum trophozoites treated with PfTOP2 dsRNA nanoparticles and free PfTOP2 dsRNA respectively. These results suggest that chitosan-based nanoparticles might be a useful tool for delivering dsRNA into malaria parasites.

Chemically induced behavioral responses in Anopheles minimus and Anopheles harrisoni in Thailand.

Behavioral responses of female mosquitoes representing two species in the Minimus Complex exposed to an operational field dose of bifenthrin or DEET (N,N-diethyl-m-toluamide) were described using an excito-repellency test system. Two test populations of An. minimus, one from the field (Tak Province, western Thailand), the other from a long-established laboratory colony, and Anopheles harrisoni collected from Kanchanaburi Province, western Thailand, were used. Results showed that all test populations rapidly escaped after direct contact with surfaces treated with either bifenthrin or DEET compared to match-paired untreated controls. Greater escape response by exposed females to bifenthrin and DEET were observed in the An. minimus colony compared to the two field populations. Field-collected An. minimus demonstrated a more rapid escape response to DEET than to bifenthrin, whereas An. harrisoni showed a converse response. Although fewer females escaped from test chambers without direct contact with treated surfaces compared to contact tests, the spatial repellency response was significantly pronounced in all test populations compared to match-paired controls (P < 0.05). DEET was found to perform as both a contact stimulant and moderate spatial repellent.

Human-landing patterns of Anopheles dirus sensu lato (Diptera: Culicidae) in experimental huts treated with DDT or deltamethrin.

Anopheles dirus females landing on humans inside experimental huts treated with residual applications of DDT or deltamethrin were observed during the wet season in Pu Teuy Village, Kanchanaburi Province, western Thailand. Two identical experimental huts were constructed in the fashion of typical local rural Thai homes. Pretreatment (baseline) human-landing collections (HLC) in both huts showed an early evening peak of activity between 1900 and 2000 h with no significant difference in numbers of mosquitoes captured between huts over a period of 30 collection nights. During posttreatment HLC, female mosquitoes continued to show greater landing activity inside huts fitted with insecticide-treated panels during the first half of the evening compared with the second half. A greater number (proportion) of An. dirus females landed on humans in the hut treated with deltamethrin compared with DDT. Comparing pre- and posttreatment HLC, the DDT-treated hut showed a 79.4% decline in attempted blood feeding, whereas exposure to deltamethrin resulted in a 56.3% human-landing reduction. An odds ratio was performed to demonstrate the relative probability (risk) of mosquitoes entering and attempting to blood feed in the two treated huts compared with untreated control huts. Mosquitoes were approximately 0.47 times less likely to land on humans inside a DDT-treated hut compared with the deltamethrin-treated hut. Although both chemicals exerted strong excitatory responses, DDT appears to have a more pronounced and significant (P = 0.002) effect on behavior than deltamethrin, resulting in greater movement away from the insecticide source and thus potential reduction of blood-feeding activity.

Feeding response of Aedes aegypti and Anopheles dirus (Diptera: Culicidae) using out-of-date human blood in a membrane feeding apparatus.

The colonization of Aedes aegypti and Anopheles dirus was performed using out-of-date human blood from a blood bank as a nutritional supply dispensed from a common artificial feeder. Preserved human blood was collected and used for feeding on days 5, 15, and 25 after date of expiration and dispensed from a common artificial feeder to rear the mosquitoes. Ae. aegypti had a feeding rate of 78.7, 62, and 18% at the respective intervals while An. dirus had a rate of 80, 56.8, and 7.3% on the same respective days. Direct feeding on live hamsters resulted in a rate of 96 and 90% for Ae. aegypti and An. dirus, respectively. Although egg production rates decreased from the day 5 feeding to the day 25 feeding, all of the developmental stages resulting from An. dirus fed at day 5 and 15 showed insignificant differences when compared with direct feeding on the blood of a hamster.

Susceptibility of various mosquitoes of Thailand to nocturnal subperiodic Wuchereria bancrofti.

Ten different mosquito species representing five genera were allowed to feed on human blood containing microfilariae (5.5-6.5 mf/microl) of nocturnal subperiodic Wuchereria bancrofti from Kanchanaburi Province, Thailand. Aedes aegypti, Aedes albopictus, Aedes desmotes, Downsiomyia species (=Finlaya Niveus Group), Culex quinquefasciatus, Anopheles dirus A, An. maculatus, An. minimus, Armigeres subalbatus, and Mansonia uniformis were fed under laboratory conditions using an artificial membrane feeding procedure. All species had adequate feeding responses (68-87.3%) and survival (66.4-81.3%) 12-15 days post-feeding. Anopheles maculatus was the most susceptible (73.1%) for development of infective third-stage larvae (L3), followed by An. minimus (61.0%), Downsiomyia species (27.3%), Ae. desmotes (24.2%), An. dirus A (24.7%), Cx. quinquefasciatus (19.2%), Ma. uniformis (9.2%), and Ae. albopictus (0.01%). Aedes aegypti and Ar. subalbatus were found to be completely refractory. An. maculatus produced the greatest mean number of L3 per infective mosquito (5.6 L3) and Ae. albopictus the least (1 L3). The two Anopheles demonstrated significantly greater susceptibility (P<0.05%) to W. bancrofti than the two Aedes species regarded as natural vectors.

Behavioral responses of catnip (Nepeta cataria) by two species of mosquitoes, Aedes aegypti and Anopheles harrisoni, in Thailand.

An investigation of the biological effect of catnip oil (Nepeta cataria L.) on the behavioral response of field collected Aedes aegypti and Anopheles harrisoni was conducted using an automated excitorepellency test system. Aedes aegypti showed significantly higher escape rates from the contact chamber at 5% catnip oil compared to other concentrations (P < 0.05). With Anopheles harrisoni, a high escape response was seen at 2.5% catnip oil from the contact chamber, while in the noncontact chamber a higher escape response was observed at a concentration of 5%. Results showed that this compound exhibits both irritant and repellent actions.

Behavioral responses of malaria vectors, Anopheles minimus complex, to three classes of agrochemicals in Thailand.

Behavioral responses of two wild-caught populations of Anopheles minimus complex, species A and C, exposed to operational field doses of three commonly used agricultural insecticides, carbaryl (carbamate), malathion (organophosphate) and cypermethrin (pyrethroid), were characterized using an excito-repellency test system. Test populations were collected from different localities in Kanchanaburi Province, western Thailand. Both populations showed strong irritancy by quickly escaping test chambers after direct contact with individual surfaces treated with each insecticide compared with match-paired untreated controls. Noncontact repellency response to cypermethrin and carbaryl was significantly pronounced in both A and C populations, but comparatively weak when exposed to malathion. Noncontact repellency produced much weaker escape response in both populations, but in some species-chemical combinations, it remained significant compared with controls. We conclude that contact irritancy is a major behavioral response of both A and C when exposed directly to any of the three compounds, whereas only cypermethrin produced a significant repellency response in species A.

Comparison of blood feeding response and infection of Aedes aegypti to Wuchereria bancrofti using animal membranes and direct host contact.

Comparison of an artificial, whole-blood membrane feeding procedure was performed by feeding Aedes aegypti (Liverpool strain) on the blood of patients infected with Wuchereria bancrofti microfilariae with the use of 3 types of membranes produced from chicken and mouse skin and swine intestine. Direct feeding of Ae. aegypti on the skin of infected human patients served as control. For all 3 types of membranes, mosquito survival, infection, and number of infective-stage larvae per mosquito did not differ significantly from the control. However, the blood feeding response between swine intestine layer (32%) compared to chicken skin (75.3%), mouse skin (70%), and direct feeding (84%) differed significantly. The response in direct feeding method was significantly higher than those in all membranes tested (F = 18.89; df = 3; P < 0.05) Chicken skin preparation was shown to be the preferred membrane for blood feeding Ae. aegypti and experimental infection with W. bancrofti.

Inhibition of Plasmodium falciparum proliferation in vitro by antisense oligodeoxynucleotides against malarial topoisomerase II.

The development of new effective antimalarial agents is urgently needed due to the ineffectiveness of current drug regimes on the most virulent human malaria parasite Plasmodium falciparum. Antisense (AS) oligodeoxynucleotides (ODNs) have shown promise as chemotherapeutic agents. Phosphorothioate AS ODNs against different regions of P. falciparum topoisomerase II gene were investigated. Chloroquine- and pyrimethamine-resistant P. falciparum K1 strain was exposed to phosphorothioate AS ODNs for 48 h and growth was determined by flow cytometric assay or by microscopic assay. Exogenous delivery of phosphorothioate AS ODNs between 0.01 and 0.5 microM significantly inhibited parasite growth compared with sense sequence controls suggesting sequence specific inhibition. This inhibition was shown to occur during maturation stages, with optimal inhibition being detected after 36 h. These results should prove useful in future designs of novel antimalarial agents.