Discovery of diurnal resting sites of phlebotomine sand flies in a village in southern Egypt.

In an attempt to find diurnal resting sites of adult phlebotomine sand flies, potential phlebotomine adult habitats were aspirated in the village of Bahrif in Aswan, Egypt. During this survey, sand flies were aspirated from low (30-45 cm high) irregular piles of mud bricks found under high date palm canopies between the village and the Nile River. There were 5 males and 7 females of Phlebotomus papatasi and 3 males of Sergentomyia schwetzi. Six of the 7 aspirated females were engorged with blood. A total of 78 sand flies was captured on 3 glue boards placed overnight on the ground next to the mud bricks. Attempts to aspirate sand flies from adjacent walls and plants were unsuccessful. The identification of diurnal resting sites in less structured habitats may ultimately lead to more effective adult sand fly control.

Efficacy and duration of three residual insecticides on cotton duck and vinyl tent surfaces for control of the sand fly Phlebotomus papatasi (Diptera: Psychodidae).

This study evaluated the toxicity and duration of 3 residual insecticides against the Old World sand fly, Phlebotomus papatasi, an important vector of cutaneous leishmaniasis, on 2 types of tent material used by the US military in Afghanistan and the Middle East. Vinyl and cotton duck tent surfaces were treated at maximum labeled rates of lambda-cyhalothrin (Demand CS, Zeneca Inc, Wilmington, DE), bifenthrin (Talstar P Professional, FMC Corporation, Philadelphia, PA) and permethrin (Insect Repellent, Clothing Application, 40%), then subsequently stored in indoor, shaded spaces at room temperature (60%-70% relative humidity (RH), 22°C-25°C), and under sunlight and ambient air temperatures outdoors (20%-30% RH, 29°C-44°C). Insecticide susceptible colony flies (F110) obtained from the insectary of US Naval Medical Research Unit No. 3, Cairo, Egypt, were exposed to treated tent surfaces for 30-minute periods twice monthly for up to 5 months, then once monthly thereafter, using the World Health Organization cone assay. Lambda-cyhalothrin treated cotton duck tent material stored indoors killed P. papatasi for 8 months, while the complementary sun-exposed cotton duck material killed adult flies for 1 month before the efficacy dropped to less than 80%. Sand fly mortality on permethrin- and bifenthrin-treated cotton duck decreased below 80% after 2 weeks exposure to sunlight. Shade-stored permethrin and bifenthrin cotton duck material killed more than 80% of test flies through 5 months before mortality rates decreased substantially. Vinyl tent material provided limited control (less than 50% mortality) for less than 1 month with all treatment and storage regimes. Lambda-cyhalothrin-treated cotton duck tent material provided the longest control and produced the highest overall mortalities (100% for 8 months (shaded), more than 90% for 1 month (sunlight-exposed)) of both cotton duck and vinyl tents.

Experimental acquisition, development, and transmission of Leishmania tropica by Phlebotomus duboscqi.

We report experimental infection and transmission of Leishmania tropica (Wright), by the blood-feeding sand fly Phlebotomus duboscqi (Neveu-Lemaire). Groups of laboratory-reared female sand flies that fed "naturally" on L. tropica-infected hamsters, or artificially, via membrane feeding device, on a suspension of L. tropica amastigotes, were dissected at progressive time points post-feeding. Acquisition, retention and development of L. tropica through procyclic, nectomonad, and leptomonad stages to the infective metacyclic promastigote stage, and anterior progression of the parasites from abdominal midgut bloodmeal to the thoracic midgut were demonstrated in both groups. Membrane feeding on the concentrated amastigote suspension led to metacyclic promastigote infections in 60% of sand flies, whereas only 3% of P. duboscqi that fed naturally on an infected hamster developed metacyclics. Sand flies from both groups re-fed on naïve hamsters, but despite infections in 25-50% of membrane-fed and 2-3.5% of naturally fed flies, no skin lesions developed in the hamsters. After four months of observation these animals were euthanized and necropsied. Screening of the organs and tissue by polymerase chain reaction (PCR) that targeted the small subunit RNA gene, amplified generic Leishmania DNA from liver, spleen, bone marrow, and blood, but only from hamsters bitten by membrane-infected P. duboscqi. These results are notable in demonstrating the ability of P. duboscqi, originating from Kenya, to acquire, retain, develop, and transmit a Turkish strain of L. tropica originally isolated from a human case of cutaneous leishmaniasis. This marks the first demonstration of complete development and transmission of L. tropica by a member of the Phlebotomus subgenus of sand flies.

Effects of ivermectin on blood-feeding Phlebotomus papatasi, and the promastigote stage of Leishmania major.

Ivermectin (IVM) is a chemically modified macrocyclic lactone of Streptomyces avermitilis that acts as a potent neurotoxin against many nematodes and arthropods. Little is known of IVM's effect against either blood-feeding Phlebotomus sand flies, or the infective promastigote stage of Leishmania transmitted by these flies. We injected hamsters subcutaneously with two standard IVM treatments (200 and 400 µg/kg body weight) and allowed cohorts of Leishmania major-infected Phlebotomus papatasi to blood-feed on these animals at various posttreatment time points (4 h, 1, 2, 6, and 10 days). Infected and uninfected sand flies that bit treated and untreated hamsters served as controls. Serum levels of IVM in low- and high-dose-treated hamsters were determined at the five time points. Sand fly mortality following blood feeding was recorded at 24-h intervals and, in relation to IVM treatment, was time and dose dependent. Mortality was most rapid and greatest among infected flies that fed nearest to time of dosing. Mean survival of infected sand flies after feeding on untreated hamsters was 11.5 days, whereas that of infected sand flies that fed 4 h, 1 day, or 2 days posttreatment on high-dose-treated hamsters (400 µg/kg) was 1.6, 2.1, and 2.7 days, respectively. Infected and uninfected sand flies that blood fed 6 days following low-dose IVM treatment (200 µg/kg) still experienced significantly greater mortality (p < 0.02) than controls. Promastigotes dissected out of surviving flies that fed on IVM-treated hamsters showed typical motility and survival. Moreover, 21.7% of IVM-treated hamsters developed lesions after being fed upon by infected sand flies. L. major promastigotes appeared to be tolerant to ng/mL blood levels of IVM that caused significant mortality for up to 10 days posttreatment in blood-feeding P. papatasi.

Operational vector-borne disease surveillance and control: closing the capabilities gap through research at overseas military laboratories.

Malaria, dengue fever, chikungunya virus, leishmaniasis, and a myriad of other vector-borne diseases pose significant threats to the warfighter and to the overall combat effectiveness of units. Military preventive medicine (PM) assets must accurately evaluate the vector-borne disease threat and then implement and/or advise the commander on countermeasures to reduce a particular threat. The success of these measures is contingent upon the biology of the disease vector and on the tools or methods used to conduct vector/pathogen surveillance and vector control. There is a significant gap between the tools available and those required for operational PM assets to provide real-time, effective surveillance and control. A network of US Army and US Navy overseas laboratories is focused on closing the current capabilities gap. Their mission is to develop and field test tools and methods to enhance the combatant commander's ability to identify and mitigate the threat posed by these vector-borne diseases.

First report of Leishmania tropica from a classical focus of L. major in North-Sinai, Egypt.

Cutaneous leishmaniasis (CL) is prevalent in the Egyptian Sinai Peninsula and previous research has consistently documented the etiologic agent to be Leishmania major. We report the first isolation of Leishmania tropica from human cases of CL in a Northern Sinai community bordering Palestine. Parasite culturing, real-time polymerase chain reaction (PCR), gene sequencing, and restriction fragment length polymorphism (RFLP) analyses indicate CL cases in this community were caused by either L. major or L. tropica (three cases each). Two wild-caught rodents (Gerbillus pyramidum floweri) were infected with L. tropica. Phlebotomus papatasi sand flies were found harboring L. major, however only non-infected individuals of Phlebotomus sergenti, a vector for L. tropica, were caught. Patients with L. tropica had not traveled from the region in over a year, suggesting these cases are autochthonous. This scenario is consistent with an incursion of L. tropica from bordering countries and raises concerns about expansion of this parasite further into Egypt.

Fractionation of organ extracts of Culex pipiens mosquitoes for the isolation of active components that impair biological parameters of the insect.

The previous phase of the present study revealed that when crude extracts of Culex pipiens midgut, ovaries, and salivary glands are injected into New Zealand White (NZW) rabbits (Oryctolagus cuniculus), rabbits immunized with midgut extract exert the greatest negative impact on adult Cx. pipiens survival and fecundity. This study was conducted to further our understanding of the immunogenic nature of the aforementioned antigenic preparations, thus providing data for the ultimate goal of developing a vaccine against the numerous Cx. pipiens-vectored diseases that affect human populations throughout the world. Extracts of Cx. pipiens midgut, ovaries, and salivary glands were fractionated using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The high (> 80.0 to >106.0 kDa) and low (< 18.5 kDa) molecular weight (MW) fractions of midgut extract, high MW fractions (75.0 - <106.0 kDa) of salivary gland extract, and low MW fraction (27.5 kDa) of ovary extract were excised, and used to immunize rabbits. Following immunization, anti-sera from all immunized rabbits were assayed for antibody response using Enzyme-Linked Immunosorbent Assay (ELISA), Enzyme Immunoblot transfer (EIB), and Indirect Fluorescent Antibody Techniques (IFAT). These assays resulted in both high and low MW fractions of midgut extract, with special reference to the midgut extract low MW fraction (18.0 kDa), eliciting the strongest humoral responses in immunized hosts. When Cx. pipiens were fed on rabbits immunized with the low MW fractions of midgut extract, the fecundity and survival rates were significantly less than those of mosquitoes fed on rabbits immunized with the high MW fractions of midgut extract and control rabbits (P < 0.001). It is concluded that, the low MW fraction of midgut extract is highly immunogenic, and the antibody response of immunized rabbits contributes to a significant disturbance in the life cycle of Cx. pipiens and their progeny. This impairment of feeding behavior and reproduction, in turn, could interfere with pathogen transmission.