Innate Cellular Immune Responses in Aedes caspius (Diptera: Culicidae) Mosquitoes.

Mosquitoes transmit a variety of pathogens that have devastating consequences for global public and veterinary health. Despite their capacity to serve as vectors, these insects have a robust capacity to respond to invading organisms with strong cellular and humoral immune responses. In Egypt, Aedes caspius (Pallas, 1771) has been suspected to act as a bridge vector of Rift Valley Fever virus between animals and humans. Microscopic analysis of Ae. caspius hemolymph revealed the presence of phagocytic cells called granulocytes. We further evaluated cellular immune responses produced by Ae. caspius as a result of exposure to a Gram-negative, and Gram-positive bacterium, and to latex beads. After challenge, a rapid and strong phagocytic response against either a natural or synthetic invader was evident. Hemocyte integrity in bacteria-inoculated mosquitoes was not morphologically affected. The number of circulating granulocytes decreased with age, reducing the overall phagocytic capacity of mosquitoes over time. The magnitude and speed of the phagocytic response suggested that granulocytes act as an important force in the battle against foreign invaders, as has been characterized in other important mosquito vector species.

Filariasis elimination in Egypt: impact of low microfilaraemics as sources of infection for mosquitoes.

The elimination strategy for lymphatic filariasis aims at reducing blood microfilaraemia to levels at which vector transmission cannot be sustained. We aimed to determine whether patients with pre-treatment low or ultra-low microfilaria (MF) counts could be a reservoir of infection after mass drug administration (MDA) with a combined regimen. Laboratory-reared mosquitoes were fed on 30 volunteers after 2 rounds of MDA. Microfilaria uptake, infectivity rates and number of Wuchereria bancrofti L3 per mosquito were assessed. One year after MDA-1, 6 subjects transmitted MF, but up to 9 months after MDA-2 transmission failed. Six months after MDA-2 > 90% had clear MF smears and either failed to transmit MF or transmitted MF that did not develop to L3. We conclude that the transmission cycle is seriously weakened after MDA-2.

Filariasis elimination in Egypt: impact of low microfilaraemics as sources of infection for mosquitoes

The elimination strategy for lymphatic filariasis aims at reducing blood microfilaraemia to levels at which vector transmission cannot be sustained. We aimed to determine whether patients with pre-treatment low or ultra-low microfilaria [MF] counts could be a reservoir of infection after mass drug administration [MDA] with a combined regimen. Laboratory-reared mosquitoes were fed on 30 volunteers after 2 rounds of MDA. Microfilaria uptake, infectivity rates and number of Wuchereria bancrofti L3 per mosquito were assessed. One year after MDA-1, 6 subjects transmitted MF, but up to 9 months after MDA-2 transmission failed. Six months after MDA-2 > 90% had clear MF smears and either failed to transmit MF or transmitted MF that did not develop to L3. We conclude that the transmission cycle is seriously weakened after MDA-2

Short report: A practical technique for the cryopreservation of Dirofilaria immitis, Brugia malayi, and Wuchereria bancrofti microfilariae.

A technique to cryopreserve microfilariae has been developed. This method was used to cryopreserve microfilariae of Dirofilaria immitis, Brugia malayi, and Wuchereria bancrofti at a controlled rate of 1 degree C/min by use of a freezing tank. Microfilariae of each of these species retained their ability to infect susceptible mosquito species and develop to the infective stage after cryopreservation. The method presented here is quickly and easily carried out with inexpensive equipment.

Detection of Wuchereria bancrofti in mosquitoes by the polymerase chain reaction: a potentially useful tool for large-scale control programmes.

Focally endemic bancroftian filariasis is targeted for elimination in the Nile delta of Egypt. Improved methods are needed for identifying endemic villages to be included in the control programme and for monitoring its success. We have evaluated the performance of a polymerase chain reaction (PCR) assay in estimating Wuchereria bancrofti infection in pools of Culex pipiens (1-25 females) from 2 adjacent villages with high (El Qolzom, 10.8%) and low (Kafr Shorafa, 2.1%) prevalence rates of human filariasis. This assay detects a repeated sequence in W. bancrofti deoxyribonucleic acid (DNA). Mosquitoes resting within houses were captured by aspiration and pooled by house. Houses were classified as positive or negative for human filarial infection based on night blood examinations of residents. The assay detected parasite DNA in mosquitoes from 60% of 25 infected houses and 24% of 25 uninfected houses. PCR processing of mosquitoes caught within houses of unknown filariasis infection status (44 in El Qolzom, 37 in Kafr Shorafa) identified 31.8% and 8.1% of houses, respectively, as containing infected mosquitoes. These results support the validity of the PCR assay for evaluating filarial prevalence in different villages. C. pipiens collected outdoors in dry ice-baited traps and tested by PCR (266 in Qolzom, 82 in Kafr Shorafa) did not contain parasite DNA. Pools of female mosquitoes (296 in Qolzom, 240 in Kafr Shorafa) captured in oviposition traps were also negative. We concluded that the PCR based assay is a powerful epidemiological tool that can be used for evaluating W. bancrofti infection in villages in the Nile delta and for monitoring the application of control programmes in filariasis endemic areas.

The impact of environmental and entomological factors on intervillage filarial focality in the Nile Delta.

In the Nile Delta of Egypt, levels of W. bancrofti infection in humans vary among nearby villages. Ecological and entomological factors that might explain variability between adjacent villages, El Qolzom (QOL) and Kafr Shorafa (KSH) with respective 10.8% and 2.1% microfilaria (MF) prevalence were examined. The epidemiological study covered 127 and 79 houses scattered in QOL and KSH, respectively, and described 25 items relating to housing characters, socio-economic state and human activities. It revealed that QOL is more rural than KSH, and therefore would be more favorable to the vector mosquito population and hence, filarial parasite transmission in QOL. Weekly records (N = 81 and 62 for QOL and KSH, respectively) of ambient temperature, relative humidity and wind speed taken at sunset, over 3 months during summer, revealed no significant variation between villages. Those measured at sunrise revealed significant, although inconsistent, differences at a particular month, but no difference over the whole period. Whether climatological conditions could have influenced mosquito bionomics in the study villages is questionable. Abundance of female Cx. pipiens collected weekly by standard sampling methods using 247 and 240 dry ice-baited CDC trap-nights in QOL and KSH, respectively, oral aspiration from within 346 and 304 respective house-nights, and 65 and 40 respective ovitrap-nights, did not vary significantly over the whole study period. Daily survival and survival to infectivity rates of wild-caught mosquitoes were based on parity and were generally more elevated in QOL than KSH. Monthly records of abundance and survival seemed to favor filaria transmission by mosquitoes in QOL. Autogeny amounted to 6.5 and 20% for QOL and KSH, respectively. Experimental infection of Cx. pipiens from the study villages with W. bancrofti revealed that QOL females were 3.3 times more efficient vectors than KSH ones, mainly because QOL mosquitoes survived longer. The ultimate outcome of observed entomological factors might explain its preponderance in QOL.

Host feeding of mosquitoes (Diptera: Culicidae) associated with the recurrence of Rift Valley fever in Egypt.

In 1993, Rift Valley fever (RVF) virus reappeared in Egypt. We determined the prevalence and feeding patterns of mosquitoes in 5 villages where the virus was active. Of 10 species recovered, Aedes caspius (Pallas), Culex pipiens L., Cx. antennatus (Becker), and Cx. perexiguus Theobald constituted 99% of > 35,000 mosquitoes captured in dry ice-baited CDC light traps. Ae. caspius was most prevalent, except at Nag' El Hagar where it was replaced by Cx. perexiguus. Cx. pipiens ranked 2nd, except at Nag' El Ghuneimiya, where it was replaced by Cx. antennatus. Most blood meals analyzed by an enzyme-linked immunosorbent assay reacted to > or = 1 antiserum. Cx. pipiens was mainly anthropophagic, and therefore may have been the main vector of RVF virus among humans. Ae. caspius feeds were chiefly from humans, bovines, and equines. Cx. antennatus and Cx. perexiguus fed generally on bovines. Mixed blood meals from humans and RVF virus susceptible animals were identified in the predominant mosquitoes. Prevalence and host selection, as well as predicted probability for a blood meal being interrupted, indicated that Ae. caspius may have served as a bridge vector between humans and bovines in 4 of the villages. Cx. perexiguus may have played this role at Nag' El Hagar. Because potential vectors are abundant, susceptible domestic animals are associated closely with humans, and surveillance of imported livestock is not systematic, we conclude that RVF virus sporadically will recur in Egypt.

Filariasis transmission potential of mosquitoes to humans of different age groups.

Age-specific levels of Wuchereria bancrofti infection determined by microfilaremia in filarious 10 to 60 years old males from endemic village revealed that microfilaria intensity was higher in younger age, presumably because of increased vector biting rate of younger individuals. Out of 1334 Cx. pipiens captured while attempting to bite 51 child-adult bait pairs, 54.0% selected a host in the 10-15 age group. Biting preference towards children was more evaluated (79.2%) later than earlier in the night. Frequency distribution of age-specific biting collections indicated that 62.7% recorded exceeding numbers of bites on children, 21.6% adult biting, and 15.7% equal biting of one of the baits in the pair. Furthermore, 21.6% of the children were exposed to nightly bites ranging from 21 to 64 and only 7.8% of the adults experienced from 21 to 85 bites. Mosquito biting intensity averaged 14.2 and 11.6 bites/night for children and adults respectively. Children experienced 22.4% more bites per night than adults. Thus, children are more exposed to filaria infection and seem to represent an important source of infection. It is concluded that attempts at widespread control of filariasis in the densely populated Nile Delta, where youngsters may account for an immense proportion of the local population, should concentrate on immatures.

A polymerase chain reaction-based assay for detection of Wuchereria bancrofti in human blood and Culex pipiens.

Human blood samples and indoor-resting Culex pipiens were collected in 33 randomly selected houses from different sectors of a village in the Nile Delta of Egypt which was endemic for Wuchereria bancrofti. Blood was also collected from subjects with no history of living in filarial endemic areas. Human blood samples were divided and assessed by both membrane filtration and polymerase chain reaction (PCR). Similarly, mosquito samples were assessed by both dissection and PCR. Blood pools representing each household were tested by PCR. If a pool gave a positive result, then individual blood specimens were also tested by PCR. Of the 33 houses tested, both membrane filtration and blood pools assayed by PCR identified 14 (42.4%) 'infected houses'. PCR detected parasite deoxyribonucleic acid (DNA) in blood pools from an additional 3 households that gave negative results by membrane filtration. Of 178 endemic blood samples tested by membrane filtration, 22 (12.3%) had microfilariae and all were individually positive by PCR. Although microfilaria counts were lower in blood collected during the day than in night-collected blood, the PCR results were consistent, regardless of time of collection. All non-endemic blood samples were negative by PCR. Among the 33 houses rested, mosquito pools assayed by PCR identified 17 (51.5%) as 'infected households'. Of these, 8 houses (47%) contained at least one microfilaraemic resident. One 'infected household' was identified by mosquito dissection. We concluded that PCR is a powerful epidemiological tool for screening villages for the prevalence of W. bancrofti. PCR detection of W. bancrofti DNA in blood-fed mosquitoes could be used initially to locate endemic areas with transmission of bancroftian filariasis. PCR detection of W. bancrofti DNA in blood collected during the day could then be used to assess W. bancrofti infection rates.

Uptake and development of Wuchereria bancrofti in Culex pipiens L. and Aedes caspius pallas.

Studies were conducted to compare early phenomena associated with W. bancrofti infection and further development in the filaria vector Culex pipiens and the refractory mosquito Aedes caspius. Ingestion rates evaluated immediately after simultaneous feeding on an infected human were 69.1% for 43 Cx. pipiens and 35.7% for 28 Ae. caspius. The observed number of mf ingested by either mosquito did not vary significantly (2.7 +/- 1.4, and 2.3 +/- 0.9, respectively) and, based on the size of the blood meal ingested (2.8 and 2.3 microliters respectively), a two-fold mf concentration factor was recorded for both species. Blood ingested by Cx. pipiens (N = 16) and Ae. caspius (N = 10) clotted within 120 and 90 min post-feeding respectively. The time difference observed, however, did not affect significantly the rates of migration into the hemocele (56 and 67% respectively). Comparison of initial infection rates with those obtained after the extrinsic incubation period of the parasite was completed, indicated that the proportion of infected Cx. pipiens was reduced by 3.9% and that of Ae. caspius by 30.0%. Furthermore, the observed infectivity ratio of 265 Cx. pipiens that had an infective blood meal was 0.74 and only 0.009 for 70 Ae. caspius. It is concluded that refractoriness of Ae. caspius to W. bancrofti is expressed through the feeding mechanism itself, by severely limiting the mf ingestion rate, and through physiological processes that inhibited the development of ingested worms.

Morphological keys for the separation of the Rhipicephalus sanguineus group of ticks (Acarina:Ixodidae) in Egypt.

Rhipicephalus sanguineus sensu stricto is the taxonomic baseline for the R. sanguineus group of ticks which consists of four species in Egypt. Because of the potential confusion in the differentiation of species within the group, particularly in the Middle East, there is an urgent need for the development of new diagnostic keys. Keys, based on morphological characters, are provided herein for the various stages of R. sanguineus ticks in Egypt. An additional plan provides a useful tool for distinguishing most field collected specimens.

Host-parasite relationships of Wuchereria bancrofti and mosquito hosts, Culex pipiens L. and Aedes caspius pallas.

We compared the defense mechanisms directed against Wuchereria bancrofti by a filaria susceptible mosquito, Culex pipiens, and a refractory one, Aedes caspius. The reciprocal deleterious effects of both the mosquito host and its parasite are reported. Anatomical and histological examinations of mosquitoes revealed minor differences in the general structure of the foregut of either species. The cibarial pump of Ae. caspius, however, lacked a cibarial armature and damage inflicted to some of the microfilariae (mf) ingested by this mosquito was not mechanical. In contrast, the cibarial armature of Cx. pipiens consisted of 24 delicate teeth which did not seem to affect ingested mf. The peritrophic membrane (pm) did not constitute a significant barrier to mf migrating through the midgut wall of either mosquito. Indeed, mf forced their way out into the hemocoel, immediately after they have been ingested, and up to 60 min post-feeding, when the pm was still thin and soft. It hardened 16 h post-feeding. Traversing mf perforated the pm and displaced the basement membrane. Within the thorax of Cx. pipiens, the parasite completed its development synchronously. In contrast, mf ingested by Ae. caspius did not develop beyond the sausage stage, and many of these larvae were vacuolated. In both mosquitoes, parasitized thoracic muscle fibers were tunnelled and, only in Ae. caspius, their nuclei were pyknotic. No worm encapsulation or melanization was observed in either mosquito. It is concluded that defense lines to invading parasites in Cx. pipiens are insignificant, and that in Ae. caspius, the main barrier to parasite development resides within the thoracic muscles. In the absence of cellular evidence, it is suggested that such a barrier is physiological in nature.

Identification of endemic foci of filariasis by examination of mosquitoes for microfilariae.

Studies were conducted in the Nile Delta of Egypt to determine the feasibility of detecting Wuchereria bancrofti microfilariae (Mf) in mosquitoes as a primary surveillance method for the identification of filariasis-endemic villages. Initial experimental studies evaluated the ingestion, survival, and migration rates of W. bancrofti Mf in Culex pipiens and Culex antennatus after mosquitoes were fed on infected volunteers. In 2 villages, 1,684 bloodfed mosquitoes were dissected during the night immediately after collections inside houses. In the village of Kafr Tahoria, Mf were found in 27 of 519 Cx. pipiens and in one of 8 Anopheles pharoensis. In Tahoria, Mf were detected in 7 of 799 Cx. pipiens and in one of 302 Cx. antennatus. Identifying filariasis-endemic villages based on the detection of Mf in mosquitoes may be a useful strategy for epidemiologic studies or for filariasis control programs.

Host-feeding patterns of Culex pipiens and Cx. antennatus (Diptera: Culicidae) from a village in Sharqiya Governorate, Egypt.

Host-selection patterns of mosquitoes were determined over a 1-yr period at Abu Heif, a village in Sharqiya Governorate, Egypt, that has had a history of Rift Valley fever virus (RVFV) transmission. Culex pipiens L. and Cx. antennatus Becker were the most common mosquito species collected, and 8,252 blood meals from both species were analyzed by a precipitin technique. Host availability was estimated by a monthly census of human and animal populations. Both mosquito species exhibited opportunistic endophagic behavior. In bedrooms, 79% of Cx. pipiens fed on humans, compared with 53% of the Cx. antennatus. In animal sheds, 35% of Cx. pipiens and 68% of Cx. antennatus fed on sheep or goats. Cx. pipiens was primarily anthropophilic (forage ratio = 2.7) whereas Cx. antennatus was mainly an ovine feeder (forage ratio = 2.4). These findings indicated that both species probably were involved in the transmission of RVFV in Sharqiya Governorate during the epidemics of 1977 and 1978, Cx. pipiens being mainly responsible for transmission to humans, and Cx. antennatus for transmission among domestic animals. The persistent custom of keeping sheep and goats inside human dwellings, combined with the opportunistic host selection by the local mosquitoes, continues to make this area receptive to RVFV transmission.

Preimaginal vitellogenic development and repeated autogeny in Culex pusillus (Diptera: Culicidae) from Egypt.

Autogenous ovarian development was investigated in pupal and adult stages of Culex pusillus Macquart from Egypt. Vitellogenesis was initiated synchronously in > 90% of 1-d-old pupae, and primary follicles of all pupae reached stages III and IV 1 d later. All adult females oviposited their initial autogenous egg batches within 1-4 d after emergence. Females were capable of ovipositing up to four successive autogenous egg batches. The expression of repeated autogeny was confirmed by the presence of an equivalent number of dilatations and the concurrent existence of mature primary follicles and maturing secondary follicles in the same ovarioles. In addition, the total fecundity of 33% of the females that oviposited repeatedly exceeded the ovariole number of the two ovaries. This species failed to ingest a blood meal when offered a variety of hosts. Therefore, Cx. pusillus from Egypt expressed autogenous ovarian development throughout life, a characteristic apparently unique among the genus Culex.

Absence of mating barriers between autogenous and anautogenous Culex pipiens L. in Egypt.

The existence of pre-mating and post-mating barriers between Culex pipiens L. populations and the possibility of interbreeding between autogenous and anautogenous mosquitoes were examined. Test mosquito populations were stenogamous with > 83% insemination rates in 20 x 20 x 20 cm cages, regardless of autogeny rates ranging from 10 to 91%. Bi-directional crosses between three geographical populations resulted in > 80% hatch rates, indicating full cytoplasmic compatibility. Both autogenous and anautogenous Cx. pipiens individuals were encountered in the progenies of autogenous or anautogenous female parents. Moreover, more than 75% of field caught females produced mixed progenies. thus, the authors suggest there is no limitation for hybridization between Cx. pipiens mosquitoes in Egypt due to the lack of premating and post-mating barriers.

Electrophoretic separation of two Aedes caspius Pallas forms from Egypt.

Isozyme banding patterns of the recently recognized autogenous stenogamous and anautogenous eurygamous sexually isolated Aedes caspius forms were investigated using horizontal starch gel electrophoresis of 5 enzymes. The esterase assay clearly separated in 2 forms, a faster band being characteristic of autogenous individuals. Other tested enzymes were encoded by 5 loci that were highly polymorphic. The "malic" enzyme locus was unambiguously diagnostic, specific, non-overlapping alleles at that locus were revealed in each form. Other enzyme loci screened exhibited at least 2 alleles common to both forms, allelic frequency differences at least loci could not separate the 2 forms except at the cathodal MDH-1 locus (malate dehydrogenase assay). Probability of correct diagnosis at the MDH-1 locus was as high as 93% so that it may be used as a confirmatory test.

Biochemical key to eight species of adult Egyptian mosquitoes.

1. An electrophoretic key is described, based on enzyme relative mobility, to distinguish eight species of Egyptian mosquito adults. 2. Using horizontal starch gel electrophoresis, five different enzyme assays unambiguously separated Culex pipiens L., Cx antennatus (Becker), Cx pusillus Macquart, Aedes caspius (Pallas), Culiseta longiareolata (Macquart), Uranotaenia unguiculata Edwards, Anopheles multicolor Cambouliu and An.pharoensis Theobald. 3. Diagnostic loci between species were: Ak-2, G6pd, alpha-Gpd, Gpi and 6-Pgd; the probability of correct diagnosis in each case was at least 0.989.

Genetic similarity among Egyptian populations of Culex pipiens (Diptera: Culicidae).

Geographic variation among Culex pipiens L. populations was studied along a north-south transect across Egypt. Horizontal starch gel electrophoresis of 10 enzyme loci permitted estimates of adult polymorphism for 10 demes of Cx. pipiens. Levels of genetic variability, as indicated by a mean of 1.25 alleles per locus, a mean expected heterozygosity of 0.023, and a proportion of 0.25 of the loci being polymorphic, indicated limited genetic heterogeneity within and among demes. Nei's estimates of genetic divergence indicated considerable genetic exchange among demes (average genetic distance, 0.013). Thus, Cx. pipiens in Egypt was not differentiated into genetically isolated demes but, rather, seemed to consist of a single panmictic population.

Tropical extension of temperature zone Culex pipiens throughout Egypt.

To explore the southern limit of the temperate zone form of the Culex pipiens complex of mosquitoes in North Africa, we compared the structure of the copulatory structures of such male mosquitoes sampled along the length of Egypt. Larval mosquitoes were collected in 11 sites distributed along the entire Egyptian span of the Nile Valley as well as in sites along the central Red Sea coast and in the western desert. Four of these sites were sampled repeatedly, at various times of the year. A DV/D ratio was calculated for each adult male mosquito. Although all DV/D values conformed to criteria normally attributed to the temperate zone form, even at the southernmost site (22 degrees N), values tended toward those of the tropical form in southern sites and during the summer months. We concluded that Cx. pipiens mosquitoes of the Middle East are predominantly Mediterranean in form due to an isolating effect of the Sahara Desert which separates these temperate zone insects from those of tropical Africa.