Field evaluation of deet and a piperidine repellent against Aedes communis (Diptera: Culicidae) and Simulium venustum (Diptera: Simuliidae) in the Adirondack Mountains of New York.

Repellent efficacy of N,N-diethyl-3-methyl-benzamide (deet), the piperidine, 1-[3-cyclohexen-1-ylcarbonyl]-2-methylpiperidine (AI3-37220), and a 1:1 ratio of deet + AI3-37220 were evaluated topically (0.25 mg/cm2 applied in ethanol solution) on human volunteers against the mosquito Aedes communis (DeGeer) and the black fly Simulium venustum Say. The average repellency of all three formulations was > 95% at 4 h. For both mosquitoes and black flies, deet alone provided < 90% protection at 6 h, whereas AI3-37220 provided > 95% protection. Although repellent treatments were not significantly different overall, the contrasts between AI3-3720 versus deet were significant at 6 and 8 h. The 95% confidence interval on percent repellency at 6 h ranged from 90.1 to 98.9% for AI3-37220 versus 64.3 to 82.2% for deet, and at 8 h ranged 76.1 to 88.5% for AI3-37220 versus 47.8 to 64.0% for deet. Similarly, the confidence interval for protection against black flies at 6 h by (AI3-37220 ranged from 86.3 to 99.5% and did not overlap with the confidence interval provided by deet alone (51.2 to 78.8%). There was no evidence of synergistic repellency from a combination of the two compounds; i.e., protection from combined compounds was no better than either repellent used alone.

Gender-related efficacy difference to an extended duration formulation of topical N,N-diethyl-m-toluamide (DEET).

A clinical trial (n = 120, 60 males and 60 females) was conducted to assess the efficacy of an extended duration tropical insect/arthropod repellent (EDTIAR) topical formulation of N,N-diethyl-m-toluamide (DEET). The amount of EDTIAR (mean +/- confidence interval), applied by participants in accordance with label directions, was not significantly different between females (3.66 +/- 0.32 mg/cm2) and males (3.45 +/- 0.33 mg/cm2). There also was no significant difference in the number of Anopheles stephensi mosquitoes biting the control arm of females or males at 0, 3, 6, 9, and 12 hr. While gender had no effect on feeding, the time of day did effect mosquito feeding with fewer mosquitoes feeding in the afternoon than in the morning or evening. The percent protective efficacy at 0, 3, 6, 9, and 12 hr was 100.0, 99.3, 92.8, 79.7 and 66.3 for females, and 100.0, 100.0, 97.6, 91.9, and 77.5 for males. These data are inconsistent with the EDTIAR label claim that the repellent provides 95% or greater protection against mosquitoes for 12 hr or more under normal use conditions. The results of a multivariate regression analysis indicated that 1) protection decreased linearly as time after application of repellent increased (P < 0.001), 2) individuals who applied higher doses of repellent were better protected against mosquito bites (P < 0.001), 3) females experienced significantly less protection over time than did males (P = 0.005), and 4) the estradiol concentration in the blood had no effect on efficacy of the repellent (P = 0.110).

Long-term efficacy and immune responses following immunization with the RTS,S malaria vaccine.

The malaria sporozoite vaccine candidate RTS,S, formulated with an oil-in-water emulsion plus the immunostimulants monophosphoryl lipid A and the saponin derivative QS21 (vaccine 3), recently showed superior efficacy over two other experimental formulations. Immunized volunteers were followed to determine the duration of protective immune responses. Antibody levels decreased to between one-third and one-half of peak values 6 months after the last dose of vaccine. T cell proliferation and interferon-gamma production in vitro were observed in response to RTS,S or hepatitis B surface antigen. Seven previously protected volunteers received sporozoite challenge, and 2 remained protected (1/1 for vaccine 1, 0/1 for vaccine 2, and 1/5 for vaccine 3). The prepatent period was 10.8 days for the control group and 13.2 days for the vaccinees (P < .01). Immune responses did not correlate with protection. Further optimization in vaccine composition and/or immunization schedule will be required to induce longer-lasting protective immunity.

Continuous in vitro propagation of the malaria parasite Plasmodium vivax.

The difficulty in controlling Plasmodium vivax, the most common cause of human malaria, has been complicated by growing drug resistance. We have established a method to cycle parasite generations in continuous culture using human blood cells. Chesson strain parasites were passaged from owl monkey erythrocytes to human reticulocytes in McCoy's 5A medium modified with L-glutamine with 25 mM Hepes buffer supplemented with 20% AB+ human serum. Reticulocytes were separated by differential centrifugation in homologous plasma from the peripheral blood of a hemochromatosis patient. Parasites were grown during each 48-hr cycle in a static candle jar environment until the beginning of schizogony, at about 36-40 hr, when reticulocytes were added and cultures transferred to a shaker for 10-12 hr. The addition of a concentration of 10% reticulocytes resulted in stabilizing parasite densities between 0.28 and 0.57 after cycle 3 and increasing the total number of parasites at least 2-fold with each generational cycle. Cultured parasites successfully infected an owl monkey. The morphology of cultured parasites was typical of P. vivax, with highly ameboid trophozoites evident; however, infected erythrocytes were enlarged and distorted on thin film preparations. The species identity of cultivated parasites was confirmed by analysis of the A and C 18S rRNA genes from genomic DNA and expression of only the A gene during erythrocytic asexual growth. The ability to culture P. vivax opens new opportunities to develop vaccines, test drugs, and clone parasites for genome sequencing.

Depletion of total salivary gland protein in blood-fed Anopheles mosquitoes.

Reduction in total salivary gland protein from four anopheline vectors of human malaria, Anopheles stephensi Liston, An. albimanus Wiedmann, An. gambiae Giles, and An. freeborni Aitken, was quantified after mosquitoes blood-fed to repletion on human volunteers, hamsters or through a Baudruche artificial membrane. Total salivary gland protein from pools of six unfed mosquitoes ranged from 4.33 to 7.91 micrograms/ml. The difference between the total protein of glands from unfed and blood-fed mosquitoes for all species ranged from 1.77 to 3.12 (micrograms/ml for six pooled salivary glands. Total salivary gland protein for mosquitoes blood-fed to repletion was significantly less than that of unfed controls from the same cohort. Reduction in total salivary gland protein for An. freeborni and An stephensi blood fed to repletion on human volunteers, hamsters, and a Baudruche membrane ranged from 24 to 46%, from 43 to 56%, and from 24 to 51%, respectively. An. stephensi mosquitoes were allowed to blood feed on humans for 0 (unfed), 0.5-, 1.0-, 2.0-min time periods or to repletion (> 2-5 min). As feeding time increased, there was a significant decrease in total amount of protein in the salivary glands. This decrease was proportional over time, indicating that salivation occurred continuously from the beginning (probing) of blood feeding to withdrawal of the mosquito mouthparts at repletion. These data indicate that during blood feeding there difference between species in the salivary gland output measured as amount of protein depleted from the salivary glands and that depletion of salivary protein from the glands occurred continuously as mosquitoes fed to repletion.

Immunoenzymatic labeling of multiple plasmodial salivary gland sporozoites in a single test.

A direct, double- and triple-staining immunoenzymatic method detected and differentiated sporozoites by color in Anopheles stephensi salivary glands and in mixed sporozoite slide preparations. A double-staining method used beta-galactosidase- and alkaline phosphatase-labeled monoclonal antibodies to the circumsporozoite (CS) proteins of Plasmodium berghei and P. falciparum in mosquito salivary glands. The CS proteins were distinguished clearly by the blue-green and red substrate products of beta-galactosidase and alkaline phosphatase, respectively. A triple-staining method differentiated by color among a mixture of P. falciparum and two strains of P. vivax sporozoites. Monoclonal antibodies to the CS proteins conjugated to beta-galactosidase (P. falciparum), alkaline phosphatase (P. vivax variant), and horseradish peroxidase (P. vivax predominant) readily color differentiated sporozoites by the blue-green, purple-blue, and orange-brown substrate products, respectively. This assay may have potential use in malaria transmission studies, genetic crosses of variant strains of plasmodia to determine assortment of CS antigen alleles, and as a technique to determine the fate of the CS antigen in infected mosquitoes.

Plasmodium falciparum and P. berghei: detection of sporozoites and the circumsporozoite proteins in the saliva of Anopheles stephensi mosquitoes.

Sporozoites and free circumsporozoite (CS) protein were stained immunoenzymatically in 1-min saliva samples collected from Anopheles stephensi mosquitoes infected with either Plasmodium berghei or P. falciparum. The number of sporozoites in 1-min saliva-streak samples significantly increased as the salivary gland index rose from 3+ to 4+. For P. berghei-infected mosquitoes from which saliva had been collected before 30 days postfeed, the median sporozoite counts for 3+ and 4+ gland indexes were 4.5 and 116, respectively. For P. falciparum-infected mosquitoes, the median counts obtained in two experiments were 4.5 and 14.5 (3+) and 97 and 107 (4+), respectively. The frequency of sporozoite detection in the saliva of mosquitoes containing < 100 salivary-gland sporozoites was low (0.1), whereas that in the saliva of mosquitoes with > 100 sporozoites was high (0.96). In highly infected 4+ P. berghei-infected mosquitoes from which saliva had been collected after 30 days postinfection, both the volume of saliva collected and the median number of sporozoites recovered decreased significantly.

The distribution of circumsporozoite protein (CS) in Anopheles stephensi mosquitoes infected with Plasmodium falciparum malaria.

We monitored the distribution of Plasmodium falciparum circumsporozoite protein (CS) in Anopheles stephensi using an immunohistochemical method. An alkaline phosphatase-labeled monoclonal antibody, specific for the CS protein of P. falciparum, was incubated with tissue sections from infected and non-infected mosquitoes. Sections were stained for phosphatase activity using a new fuchsin/naphthol AS-BI phosphate capture system. Distribution of the CS protein in mosquitoes was dependent on the time after post-infective blood meal. CS protein was first detected in immature oocysts on the mosquito midgut. As oocysts differentiated to mature sporoblasts, detectable CS protein increased. Between 11-16 days post infective blood meal, CS protein was detected on the surface of sporozoites that were released into the hemolymph from oocysts. Although sporozoites were found throughout the hemocoel, they were most frequently associated with the salivary glands and flight muscle. Once in the salivary glands, sporozoites massed into bundles. The amount of CS protein associated with bundles of sporozoites was highly variable.