A. Hanafi

Rift Valley Fever Virus Epidemic in Kenya, 2006/2007: The Entomologic Investigations

In December 2006, Rift Valley fever (RVF) was diagnosed in humans in Garissa Hospital, Kenya and an outbreak reported affecting 11 districts. Entomologic surveillance was performed in four districts to determine the epidemic/epizootic vectors of RVF virus (RVFV). Approximately 297,000 mosquitoes were collected, 164,626 identified to species, 72,058 sorted into 3,003 pools and tested for RVFV by reverse transcription-polymerase chain reaction. Seventy-seven pools representing 10 species tested positive for RVFV, including Aedes mcintoshi/circumluteolus (26 pools), Aedes ochraceus (23 pools), Mansonia uniformis (15 pools); Culex poicilipes, Culex bitaeniorhynchus (3 pools each); Anopheles squamosus, Mansonia africana (2 pools each); Culex quinquefasciatus, Culex univittatus, Aedes pembaensis (1 pool each). Positive Ae. pembaensis, Cx. univittatus, and Cx. bitaeniorhynchus was a first time observation. Species composition, densities, and infection varied among districts supporting hypothesis that different mosquito species serve as epizootic/epidemic vectors of RVFV in diverse ecologies, creating a complex epidemiologic pattern in East Africa.

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.

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, Egypts 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.

Use of Bioassay and Microplate Assay to Detect and Measure Insecticide Resistance in Field Populations of Culex pipiens from Filariasis Endemic Areas of Egypt

ABSTRACT 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.

Efficacy of Commercial Mosquito Traps in Capturing Phlebotomine Sand Flies (Diptera: Psychodidae) in Egypt

ABSTRACT Four types of commercial mosquito control traps, the Mosquito Magnet Pro (MMP), the Sentinel 360 (S360), the BG-Sentinel (BGS), and the Mega-Catch Ultra (MCU), were compared with a standard Centers for Disease Control and Prevention (CDC) light trap for efficacy in collecting phlebotomine sand flies (Diptera: Psychodidae) in a small farming village in the Nile River Valley 10 km north of Aswan, Egypt. Each trap was baited with either carbon dioxide (CO2) from combustion of butane gas (MMP), dry ice (CDC and BGS traps), light (MCU and S360), or dry ice and light (CDC). Traps were rotated through five sites in a 5 × 5 Latin square design, repeated four times during the height of the sand fly season (June, August, and September 2007) at a site where 94% of sand flies in past collections were Phlebotomus papatasi (Scopoli). A total of 6,440 sand flies was collected, of which 6,037 (93.7%) were P. papatasi. Of the CO2-baited traps, the BGS trap collected twice as many P. papatasi as the MMP and CDC light traps, and at least three times more P. papatasi than the light-only MCU and S360 traps (P < 0.05). Mean numbers (±SE) of P. papatasi captured per trap night were as follows: BGS 142.1 (±45.8) > MMP 56.8 (±9.0) > CDC 52.3 (±6.1) > MCU 38.2 (±6.4) > S360 12.6 (±1.8). Results indicate that several types of commercial traps are suitable substitutes for the CDC light trap in sand fly surveillance programs.

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 IVMs 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.

Profiling of Human Acquired Immunity Against the Salivary Proteins of Phlebotomus papatasi Reveals Clusters of Differential Immunoreactivity

Phlebotomus papatasi sand flies are among the primary vectors of Leishmania major parasites from Morocco to the Indian subcontinent and from southern Europe to central and eastern Africa. Antibody-based immunity to sand fly salivary gland proteins in human populations remains a complex contextual problem that is not yet fully understood. We profiled the immunoreactivities of plasma antibodies to sand fly salivary gland sonicates (SGSs) from 229 human blood donors residing in different regions of sand fly endemicity throughout Jordan and Egypt as well as 69 US military personnel, who were differentially exposed to P. papatasi bites and L. major infections in Iraq. Compared with plasma from control region donors, antibodies were significantly immunoreactive to five salivary proteins (12, 26, 30, 38, and 44 kDa) among Jordanian and Egyptian donors, with immunoglobulin G4 being the dominant anti-SGS isotype. US personnel were significantly immunoreactive to only two salivary proteins (38 and 14 kDa). Using k-means clustering, donors were segregated into four clusters distinguished by unique immunoreactivity profiles to varying combinations of the significantly immunogenic salivary proteins. SGS-induced cellular proliferation was diminished among donors residing in sand fly-endemic regions. These data provide a clearer picture of human immune responses to sand fly vector salivary constituents.

Comparison of three carbon dioxide sources on phlebotomine sand fly capture in Egypt.

ABSTRACT Lighted Centers for Disease Control and Prevention (CDC) light traps were baited with carbon dioxide (CO2) produced from three different sources to compare the efficacy of each in collecting phlebotomine sand flies in Bahrif village, Aswan Governorate, Egypt. Treatments consisted of compressed CO2 gas released at a rate of 250 ml/min, 1.5 kg of dry ice (replaced daily) sublimating from an insulated plastic container, CO2 gas produced from a prototype FASTGAS (FG) CO2 generator system (APTIV Inc., Portland, OR), and a CDC light trap without a CO2 source. Carbon dioxide was released above each treatment traps catch opening. Traps were placed in a 4 × 4 Latin square designed study with three replications completed after four consecutive nights in August 2007. During the study, 1,842 phlebotomine sand flies were collected from two genera and five species. Traps collected 1,739 (94.4%) Phlebotomus papatasi (Scopoli), 19 (1.0%) Phlebotomus sergenti, 64 (3.5%) Sergentomyia schwetzi, 16 (0.9%) Sergentomyia palestinensis, and four (0.2%) Sergentomyia tiberiadis. Overall treatment results were dry ice (541) > FG (504) > compressed gas (454) > no CO2 (343). Total catches of P. papatasi were not significantly different between treatments, although CO2-baited traps collected 23–34% more sand flies than the unbaited (control) trap. Results indicate that the traps baited with a prototype CO2 generator were as attractive as traps supplied with CO2 sources traditionally used in sand fly surveillance efforts. Field-deployable CO2 generators are particularly advantageous in remote areas where dry ice or compressed gas is difficult to obtain.

Expression plasticity of Phlebotomus papatasi salivary gland genes in distinct ecotopes through the sand fly season

BackgroundSand fly saliva can drive the outcome of Leishmania infection in animal models, and salivary components have been postulated as vaccine candidates against leishmaniasis. In the sand fly Phlebotomus papatasi, natural sugar-sources modulate the activity of proteins involved in meal digestion, and possibly influence vectorial capacity. However, only a handful of studies have assessed the variability of salivary components in sand flies, focusing on the effects of environmental factors in natural habitats. In order to better understand such interactions, we compared the expression profiles of nine P. papatasi salivary gland genes of specimens inhabiting different ecological habitats in Egypt and Jordan and throughout the sand fly season in each habitat.ResultsThe majority of investigated genes were up-regulated in specimens from Swaymeh late in the season, when the availability of sugar sources is reduced due to water deprivation. On the other hand, these genes were not up-regulated in specimens collected from Aswan, an irrigated area less susceptible to drought effects.ConclusionExpression plasticity of genes involved with vectorial capacity in disease vectors may play an important epidemiological role in the establishment of diseases in natural habitats.

Phlebotomus papatasi SP15: mRNA expression variability and amino acid sequence polymorphisms of field populations

BackgroundThe Phlebotomus papatasi salivary protein PpSP15 was shown to protect mice against Leishmania major, suggesting that incorporation of salivary molecules in multi-component vaccines may be a viable strategy for anti-Leishmania vaccines.MethodsHere, we investigated PpSP15 predicted amino acid sequence variability and mRNA profile of P. papatasi field populations from the Middle East. In addition, predicted MHC class II T-cell epitopes were obtained and compared to areas of amino acid sequence variability within the secreted protein.ResultsThe analysis of PpSP15 expression from field populations revealed significant intra- and interpopulation variation.. In spite of the variability detected for P. papatasi populations, common epitopes for MHC class II binding are still present and may potentially be used to boost the response against Le. major infections.ConclusionsConserved epitopes of PpSP15 could potentially be used in the development of a salivary gland antigen-based vaccine.