Exploring new thermal fog and ultra-low volume technologies to improve indoor control of the dengue vector, Aedes aegypti (Diptera: Culicidae).

Control of the mosquito vector, Aedes aegypti (L.), inside human habitations must be performed quickly and efficiently to reduce the risk of transmission during dengue outbreaks. As part of abroad study to assess the efficacy of dengue vector control tools for the U.S. Military, two pesticide delivery systems (ultra-low volume [ULV] and thermal fog) were evaluated for their ability to provide immediate control of Ae. aegypti mosquitoes with a contact insecticide inside simulated urban structures. An insect growth regulator was also applied to determine how well each sprayer delivered lethal doses of active ingredient to indoor water containers for pupal control. Mortality of caged Ae. aegypti, pesticide droplet size, and droplet deposition were recorded after applications. In addition, larval and pupal mortality was measured from treated water samples for 4 wk after the applications. The ULV and the thermal fogger performed equally well in delivering lethal doses of adulticide throughout the structures. The ULV resulted in greater larval mortality and adult emergence inhibition in the water containers for a longer period than the thermal fogger. Therefore, the ULV technology is expected to be a better tool for sustained vector suppression when combined with an effective insect growth regulator. However, during a dengue outbreak, either delivery system should provide an immediate knockdown of vector populations that may lower the risk of infection and allow other suppression strategies to be implemented.

Evaluation of imidacloprid-treated traps as an attract and kill system for filth flies during contingency operations.

Two field trials were conducted to evaluate if filth fly trap efficacy was increased by augmentation with an insecticide application to the trap's exterior. Four Fly Terminator Pro traps (Farnam Companies, Inc, Phoenix, AZ) baited with Terminator Fly Attractant (in water) were suspended on polyvinyl chloride pipe framing at a municipal waste transfer site in Clay County, Florida. The outer surfaces of 2 traps were treated with Maxforce Fly Spot Bait (Bayer Environmental Science, Research Triangle Park, NC) (10% imidacloprid) to compare kill rates between treated and untreated traps. Kill consisted of total flies collected from inside traps and from mesh nets suspended beneath all traps, both treated and untreated. Each of 2 treated and untreated traps was rotated through 4 trap sites every 24 hrs. In order to evaluate operational utility and conservation of supplies during remote contingency operations, fly attractant remained in traps for the duration of the first trial but was changed daily during the second trial (following manufacturer's recommendations). In addition, ½ strength Terminator Fly Attractant was used during the first trial and traps were set at full strength during the second trial. Flies collected within the traps and in mesh netting were counted and identified. Three species, Musca domestica (L.), Chrysomya megacephala (F.), and Lucilia cuprina (Wiedemann), comprised the majority of samples in both trials. The net samples recovered more flies when the outer surface was treated with imidacloprid, however, treated traps collected fewer flies inside the trap than did untreated traps for both trials. No significant statistical advantage was found in treating Fly Terminator Pro trap exteriors with Maxforce Fly Spot Bait. However, reducing manufacturer's recommended strength of Terminator Fly Attractant showed similar results to traps set at full strength. Treating the outer surfaces may improve kill of fly species that do not enter the trap. Terminator Fly Attractant was also found to be more effective if traps were not changed daily and left to hold dead flies for longer periods.

Virus isolations and high population density implicate Culex antennatus (Becker) (Diptera: Culicidae) as a vector of Rift Valley Fever virus during an outbreak in the Nile Delta of Egypt.

In June, 2003, Egypt's hospital-based electronic disease surveillance system began to record increased cases of acute febrile illness from governorates in the Nile Delta. In response to a request for assistance from the Egyptian Ministry of Health and the World Health Organization (WHO), the U.S. Naval Medical Research Unit No. 3 (NAMRU-3) provided assistance in identifying the cause and extent of this outbreak. Testing of human clinical samples (n=375) from nine governorates in Egypt identified 29 cases of RVF viremia that spanned the period of June to October, and a particular focus of disease in Kafr el Sheikh governorate (7.7% RVF infection rate). Veterinary samples (n=101) collected during this time in Kafr el Sheikh and screened by immunoassay for RVFV-specific IgM identified probable recent infections in cattle (10.4%) and sheep (5%). Entomologic investigations that focused in rural, rice growing villages in the Sidi Salim District of Kafr el Sheikh during August-September, 2003, collected, identified, and tested host-seeking female mosquitoes for the presence of pathogenic viruses. Three isolates of RVF virus (RVFV) were obtained from 297 tested pools of female mosquitoes and all three RVFV isolates came from Cx. antennatus (Becker). While Cx. pipiens has been considered the primary vector of RVF virus in Egypt and is often the most common man-biting species found, Cx. antennatus was the dominant species captured at the 2003 outbreak location in Kafr el Sheikh governorate. This is the first time that Cx. antennatus has been found naturally infected with RVFV in Egypt.

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.

Evaluation of commercial and field-expedient baited traps for house flies, Musca domestica L. (Diptera: Muscidae).

A comparison of nine commercial baited fly traps on Florida dairy farms demonstrated that Terminator traps collected significantly more (13,323/trap) house flies (Musca domestica L.) than the others tested. Final Flight, Fly Magnet, and FliesBeGone traps collected intermediate numbers of flies (834-2,166), and relatively few were caught with ISCA, Advantage, Fermone Big Boy, Squeeze & Snap, or OakStump traps (<300). Terminator traps collected about twice as many flies (799.8/trap) as FliesBeGone traps (343.8) when each trap was baited with its respective attractant, but when the attractants were switched between the two trap types, collections were significantly lower (77-108) than was observed with traps baited with their respective attractant. Solutions of molasses were significantly more attractive to house flies than honey, maple syrup, or jaggery (date palm sugar). Field-expedient traps constructed from discarded PET water bottles were much less effective than commercial traps, but painting the tops of such traps with black spray paint resulted in a six-fold increase in trap capture.

Laboratory demonstration of the acquisition and development of leishmania major in the sand fly Phlebotomus kazeruni (Diptera: Psychodidae).

Phlebotumus kazeruni, a blood-feeding, xerophilic sand fly species found broadly throughout North Africa and Western Asia, is a suspected vector of cutaneous leishmaniasis (CL). Following successful laboratory colonization of this species, we employed the murine (BALB/c) infection model to determine whether our Sinai strain of P. kazeruni was able to successfully acquire, develop, and transmit a Sinai strain of Leishmania major. Groups of female sand flies were fed 1) by membrane, hamster blood containing culture-produced L. major promastigotes, 2) by membrane, hamster blood containing a suspension of L. major tissue amastigotes, and 3) directly upon L. major lesions in BALB/c mice. Samples of blood-fed sand flies from each group were dissected on selected days post-feeding and examined by light microscope for acquired and developing Leishmania infections. Female P. kazeruni acquired viable parasites by the three feeding methods. Development of ingested parasites to infective-stage metacyclic forms was observed and seen to progress from midgut to the mouthparts. Promastigote infection rates were 20% in flies fed directly upon infected mice, 35% in those fed amastigotes via membrane, and 100% in flies fed culture promastigotes via membrane. Direct blood fee-ding upon BALB/c mice was more avid (P < 0.001) among previously blood-fed flies, possibly indicative of selection and colony adaptation to murine blood-feeding. Although we failed to demonstrate clear transmission of infective-stage L. major promastigotes by feeding infected flies upon a susceptible murine host, and producing lesions in the animal, the progressive development of L. major from amastigote to metacyclic-stage promastigotes, and movement of the parasites from sand fly midgut to its mouthparts, provides evidence that P. kazeruni could serve as a vector for this parasite.

Rickettsial pathogens in the tropical rat mite Ornithonyssus bacoti (Acari: Macronyssidae) from Egyptian rats (Rattus spp.).

We collected and tested 616 tropical rat mites (Ornithonyssus bacoti (Hirst)) from rats (Rattus norvegicus (Berkenhout) and R. rattus (Linnaeus)) throughout 14 governorates in Egypt and tested DNA extracts from pools of these mites for Bartonella spp., Coxiella burnetii, and Rickettsia spp. by PCR amplification and sequencing. Three different mite-associated bacterial agents, including one Bartonella and two Rickettsia spp., were detected in eight pools of mites. Further research could demonstrate the vector potential of mites and pathogenicity of these agents to humans or animals.

Population survey of Egyptian arthropods for rickettsial agents.

Between June 2002 and July 2003, 987 fleas, representing four species, and 1019 ticks, representing one argasid and eight ixodid species, were collected from Egyptian animals. These arthropods were tested for rickettsial agents using polymerase chain reaction. DNAs from Anaplasma and Ehrlichia spp. were detected in 13 ticks. Previously undescribed Bartonella spp. were detected in 21 fleas. Coxiella burnetii was detected in two fleas and 20 ticks. Rickettsia typhi was detected in 27 fleas from 10 cities. Spotted fever group rickettsiae were detected in both fleas and ticks and included Rickettsia aeschlimanii and an unnamed Rickettsia sp.

Use of bioassay and microplate assay to detect and measure insecticide resistance in field populations of Culex pipiens from filariasis endemic areas of Egypt.

Insecticide and resistance bioassays and microplate assays were performed on Culex pipiens mosquitoes to determine the level and mechanisms of resistance. Culex pipiens larvae were collected from three filariasis-endemic areas of Egypt and reared to adults for subsequent production and testing of F1 generation larvae and adults. Bioassays were performed using World Health Organization (WHO) methods with the diagnostic doses of 6 organophosphate insecticides for larvae and 1 organochlorine (OC), 4 pyrethroid, 2 organophosphate, and 2 carbamate insecticides for adults. Microplate assays were performed to measure levels of beta esterase, acetylcholinesterase, insensitive acetylcholinesterase, oxidases, and glutathione-S-transferase enzymes. Larval bioassay results showed clear indications of resistance to organophosphate insecticides. Adult bioassays also showed widespread, significant resistance to many insecticides from all four classes, including the OC, DDT. The Qalubiya larval population was susceptible only to malathion, whereas Sharkiya larvae were susceptible to malathion, temephos, and chlorpyrifos. On the other hand, larval specimens from Assiut were resistant to all insecticides tested. Larval bioassay results were supported by those of microplate assays in showing elevated levels of glutathione S-transferase in populations from all three areas. In general, microplate results confirmed patterns of resistance observed using bioassays, and mechanisms of resistance were evident for all three areas sampled. Mechanisms of resistance are discussed in relation to microplate and bioassay results for the areas sampled and pesticides used.

Rickettsial agents in Egyptian ticks collected from domestic animals.

To assess the presence of rickettsial pathogens in ticks from Egypt, we collected ticks from domestic and peridomestic animals between June 2002 and July 2003. DNA extracts from 1019 ticks were tested, using PCR and sequencing, for Anaplasma spp., Bartonella spp., Coxiella burnetii, Ehrlichia spp., and Rickettsia spp. Ticks included: 29 Argas persicus, 10 Hyalomma anatolicum anatolicum, 55 Hyalomma anatolicum excavatum, 174 Hyalomma dromedarii, 2 Hyalomma impeltatum, 3 Hyalomma marginatum rufipes, 55 unidentified nymphal Hyalomma, 625 Rhipicephalus (Boophilus) annulatus, 49 Rhipicephalus sanguineus, and 17 Rhipicephalus turanicus. Ticks were collected predominantly (>80%) from buffalo, cattle, and camels, with smaller numbers from chicken and rabbit sheds, sheep, foxes, a domestic dog, a hedgehog, and a black rat. We detected Anaplasma marginale, Coxiella burnetii, Rickettsia aeschlimannii, and four novel genotypes similar to: "Anaplasma platys," Ehrlichia canis, Ehrlichia spp. reported from Asian ticks, and a Rickettsiales endosymbiont of Ixodes ricinus.

Surveillance of Egyptian fleas for agents of public health significance: Anaplasma, Bartonella, Coxiella, Ehrlichia, Rickettsia, and Yersinia pestis.

Serologic surveys in Egypt have documented human and animal exposure to vector-borne bacterial pathogens, but the presence and distribution of these agents in arthropods has not been determined. Between July 2002 and July 2003, fleas were collected from 221 mammals trapped in 17 cities throughout Egypt. A total of 987 fleas were collected, representing four species (Ctenocephalides felis, Echidnophaga gallinacea, Leptopsylla segnis, and Xenopsylla cheopis); 899 of these fleas were X. cheopis from rats (Rattus spp.). Fleas were tested for DNA from Anaplasma spp., Bartonella spp., Coxiella burnetii, Ehrlichia spp., Rickettsia spp., and Yersinia pestis. Rickettsia typhi, the agent of murine typhus, was detected in X. cheopis and L. segnis from rats from nine cities. A spotted-fever group Rickettsia sp. similar to "RF2125" was detected in E. gallinacea, and two unidentified spotted fever group Rickettsia were detected in two X. cheopis. Novel Bartonella genotypes were detected in X. cheopis and L. segnis from three cities. Coxiella burnetii was detected in two fleas. Anaplasma, Ehrlichia, and Y. pestis were not detected.

Louse-borne bacterial pathogens in lice (Phthiraptera) of rodents and cattle from Egypt.

We collected 1,023 lice, representing 5 species, from rats and domestic cattle throughout 13 governorates in Egypt and tested these lice for Anaplasma marginale, Bartonella spp., Brucella spp., Borrelia recurrentis, Coxiella burnetii, Francisella tularensis, and Rickettsia spp. by PCR amplification and sequencing. Five different louse-borne bacterial agents were detected in lice from rodents or cattle, including "Bartonella rattimassiliensis", "B. phoceensis", and Bartonella sp. near Bartonella tribocorum, Coxiella burnetii, and Rickettsia typhi. More lice from governorates bordering the Mediterranean and Red Seas contained pathogens. Our data indicate that lice of urban and domestic animals harbor pathogenic or potentially pathogenic bacterial agents throughout Egypt.

Detection of malaria sporozoites by standard ELISA and VecTestTM dipstick assay in field-collected anopheline mosquitoes from a malaria endemic site in Ghana.

We compared the VecTestTM dipstick assay for detection of Plasmodium sporozoites in Anopheles vectors of malaria with standard circumsporozoite (CS) microplate ELISA for detection of Plasmodium falciparum circumsporozoite protein (PfCSP) in Anopheles mosquitoes. Mosquitoes were collected from a malaria endemic site (Kassena Nankana district) in northern Ghana. Of 2620 randomly sampled mosquitoes tested, the standard CS-ELISA gave a sporozoite rate of 10.8% compared with 11.2% by VecTestTM, which was not statistically different (P = 0.66). Visual reading of the CS-ELISA results gave a sporozoite rate of 13.4%, which was higher than the other tests (P > 0.05). To allow a more objective evaluation of the sensitivity of the dipstick, an additional 136 known CS-ELISA-positive specimens were analysed. The prevalence of the test (including the additional samples) was 14.6% and 14.7% for CS-ELISA and dipstick, respectively (P > 0.05). The estimated prevalence by visual assessment of the CS-ELISA results was 17.5%. The relative specificity and sensitivity of the VecTestTM dipstick and visually read ELISA were estimated based on the CS-ELISA as a gold standard. The specificities of the dipstick and visual ELISA were high, 98.0% and 96.6%, respectively. However, the sensitivities of the two assays were 88.8% for VecTest and 100% for visual ELISA (P < 0.01). Concordance between VecTest and CS-ELISA was good (kappa = 0.86). Similarly, there was a good concordance between the dipstick and the visually read ELISA (kappa = 0.88). Extrapolating from PfCSP controls (titrated quantities of P. falciparum sporozoites), mean sporozoite loads of CS-ELISA-positive An. gambiae (286 +/- 28.05) and An. funestus (236 +/- 19.32) were determined (P = 0.146). The visual dipstick grades showed high correlation with sporozoite load. The more intense the dipstick colour, the higher the mean sporozoite load (+ = 108, ++ = 207, +++ = 290, r = 0.99, r2 = 1). The VecTest dipstick offers practical advantages for field workers needing rapid and accurate means of detection of sporozoites in mosquitoes.

Behavioral responses and mortality in German cockroaches (Blattodea: Blattellidae) after exposure to dishwashing liquid.

Behavioral responses and mortality of the German cockroach, Blattella germanica (L.), after exposure to solutions of common household dishwashing liquid diluted in tap water without other known insecticidal active ingredients are described. Soap solutions of 0.05, 0.1, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0% were tested on first and second instars, fifth and sixth instars, and adults. Cockroaches were treated individually or in groups. Behavioral observations after treatment included an immediate knockdown period, an awakening and a struggle period, an unresponsive period, and either death or recovery between 30 min and 18 h after treatment. Probit analysis gave an excellent fit of unresponsive rates 18 h after treatment with the various soap concentrations, and LD50 and LD99 values were calculated to be 0.4 and 3.0% soap, respectively. Adult females were the most difficult to kill, but at soap concentrations of 1% or higher, 95% or greater unresponsiveness and death occurred. Soap concentrations of 3% or greater resulted in 100% unresponsiveness after 3 min, and eventually resulted in 100% mortality within 72 h for all adults and nymphs treated. Observations on the mode and site of action for soap are consistent with the blockage of spiracles and/or tracheae that results in asphyxiation and death. Future investigations into the efficacy of soaps against other arthropod groups are warranted and may be beneficial in certain situations.