Hybridization studies to modify the host preference of Anopheles gambiae.

A strategy to decrease the vector competence of Anopheles gambiae sensu stricto (Diptera: Culicidae), the most efficient malaria vector in Africa, may consist of exploiting the genes involved in zoophily. Crossing and backcrossing experiments were performed between An. gambiae s.s. and the zoophilic sibling species Anopheles quadriannulatus. Mosquito strains were tested in a dual-choice olfactometer to investigate their responses to cow odour. Totals of 12% of An. gambiae s.s. and 59% of An. quadriannulatus selected the port with the cow odour. Crosses and backcrosses did not show a significant preference for the cow-baited port. The results indicated that anthropophilic behaviour in An. gambiae s.s. is a dominant or partially dominant trait, which, in conjunction with the unstable zoophilic behaviour observed in An. quadriannulatus, poses a serious obstacle to plans to decrease vector competence by modifying the anthropophilic trait.

Insecticide-treated bed-nets for malaria mosquito control.

Pyrethroid-treated bed-nets act against late-night biting mosquitoes, like traps baited by the body odor of the occupant. The personal protective effect of treated nets is considerable, even if they are torn. However, some biting of the occupants does occur, as shown by matching microsatellite alleles in mosquito blood meals to those of net occupants. When whole communities were provided with treated nets, ovarian age grading showed that mosquito survival was reduced, and so was the number of sporozoite-positive mosquitoes in malarious communities. Thus, a high percentage of coverage of all members of malaria-endemic communities is considered to be the most effective way of providing protection for highly malaria-vulnerable children and pregnant women. Teams distributing nets or retreating them free of charge show high productivity, and we consider this the most cost-effective way to proceed. There is evidence for reduced anti-malaria antibody levels in children in communities where treated nets have long been used. However, overall benefits in reduced anemia and mortality are sustained. A high frequency of the kdr resistance gene has not prevented pyrethroid-treated nets from functioning, but it is important to develop alternative fabric treatments in case stronger forms of resistance emerge.

Masked, controlled study to investigate the efficacy of a Staphylococcus intermedius autogenous bacterin for the control of canine idiopathic recurrent superficial pyoderma.

A masked, controlled study was designed to investigate the clinical efficacy of a staphylococcal autogenous bacterin for the control of canine idiopathic recurrent pyoderma (IRP). Ten dogs with at least three prior episodes of recurrent superficial pyoderma were recruited. All were screened and found to be free of ectoparasitic and fungal disease and failed to respond favourably to a dietary trial. Those exhibiting signs of pruritus responded completely to antibacterial therapy. Haematological and biochemical parameters were generally unremarkable and all dogs were euthyroid. Staphylococcus intermedius cultures from lesions were used to produce an autogenous bacterin for each animal. A numerical 'lesion score' was allocated and dogs were randomly divided into two groups of five (groups 1 and 2). Both groups received a 4-week course of antibiotic; group 1 also received concurrent s/c injections of bacterin, which continued until week 10. Group 2 received no additional therapy. All dogs were re-examined and rescored at weeks 5 and 10 and repeat blood samples were submitted at week 10 to screen for adverse effects. Comparison of scores at week 0 and week 5 (Mann-Whitney U-test) revealed no significant differences between the groups. At week 10, group 2 (control group) individual lesion scores were significantly higher compared with the group receiving bacterin (P < 0.05) and there was a significantly greater increase in the sum of the individual lesion scores for group 2 compared with group 1, from week 5 to week 10 (P < 0.05). No adverse reactions to bacterin therapy were detected. These results suggest that autogenous bacterins may provide an alternative, safe, effective method for the control of canine IRP. Further studies using larger groups of dogs and for a longer follow-up period are now warranted.

Comparison of coverage with insecticide-treated nets in a Tanzanian town and villages where nets and insecticide are either marketed or provided free of charge.

BACKGROUND: There is much emphasis on social marketing as a means of scaling up coverage with insecticide-treated nets and the question has arisen whether nets provided free-of-charge will be looked after by householders. METHODS: Over several years questionnaires and surveys of usage and condition of nets were carried out throughout a town and 15 villages in north-east Tanzania, where nets and insecticide have to be purchased and in 24 other villages where over 15000 nets had been donated and annual re-treatment is provided free-of-charge. RESULTS: There was very high population coverage in the town but, in the villages where nets have to be purchased, only 9.3% of people used nets which were intact and/or had been insecticide-treated and could, therefore, provide protection. However, where nets had been provided free, over 90% of the nets were still present and were brought for re-treatment several years later. CONCLUSION: In this part of Tanzania, social marketing has performed well in a town but very poorly in villages. However, the study showed that people look after and bring for re-treatment nets which had been provided free-of-charge.

Tests of Olyset nets by bioassay and in experimental huts.

BACKGROUND: Olyset nets are a type of long-lasting insecticidal net made of polyethylene fibre with permethrin incorporated into it and with a 4 mm mesh size. BIOASSAYS: Olyset netting was wrapped around a wire frame and the mosquitoes were released inside for bioassays. There was significantly faster knockdown and higher percent mortality than bioassays with the netting attached to a WHO cone with a piece of cardboard on the other side of the net to prevent escapes through the large mesh. It is suggested that with the latter method some mosquitoes place their tarsae through the mesh on to the cardboard, thus avoiding insecticide contact. TRIALS IN EXPERIMENTAL HUTS: Four mm mesh nets were compared with conventional 1.5 mm mesh nets treated with permethrin. In further trials in huts Olyset nets which were either unwashed or five times washed, with or without subsequent heating, and a Olyset net which had been in domestic use for four years or a new Olyset net were compared with a net treated with bifenthrin. RESULTS & CONCLUSION: In all cases Anopheles biting on sleepers under the nets was reduced and Anopheles mortality was increased by the use of the insecticidal nets. No significant impact of washing or heating was detected and an Olyset net was as good as new after four years use, but did not cause as much mosquito mortality as bifenthrin treated nets.

Optimal life stage for radiation sterilization of Anopheles males and their fitness for release.

Mating by male Anopheles mosquitoes (Diptera: Culicidae) was evaluated in the laboratory to assess fitness effects of radio-sterilization applied during different life stages of the malaria vectors An. stephensi Liston and An. gambiae Giles sensu stricto. After reproductive sterilization by gamma-irradiation (120 Gy), equal proportions of sterile and fertile (unirradiated) male adults were released into cages with virgin females and allowed to compete for matings. Radio-sterilization was applied when the males were pupae aged 0-7 h or 24-32 h, or adults aged <24 h or 24-55 h. After being radio-sterilized in the adult stage, males of both species competed effectively with unirradiated males, whereas those sterilized in the pupal stage obtained significantly fewer matings than unirradiated males from the same cohort. There was no evidence of females obtaining multiple inseminations. These findings emphasize the need to radio-sterilize males as adults in order to minimize the fitness cost. Such males may be intended for sterile insect technique population suppression or for trial releases of transgenic anophelines.

Multi-country field trials comparing wash-resistance of PermaNet and conventional insecticide-treated nets against anopheline and culicine mosquitoes.

Insecticide-treated bednets (ITNs) are commonly used as a means of personal protection from malaria transmission by anopheline mosquitoes (Diptera: Culicidae). Long-lasting insecticidal nets (LLINs) have special treatments intended to remain effective after many washes. The present trials assessed the efficacy and wash-resistance of several production batches of PermaNet (polyester net coated with polymer resin containing pyrethroid insecticide deltamethrin 55 mg ai/m2) against malaria vectors in Pakistan, Iran and Tanzania compared to ITNs conventionally treated with alphacypermethrin 15 or 20 mg ai/m2, or deltamethrin 25 or 50 mg ai/m2. Insecticidal efficacy of the nets before and after repeated washing (using W.H.O. recommended and traditional local washing procedures) was monitored through contact bioassays with Anopheles and by experimental hut and outdoor platform tests. Local washing regimes gradually reduced the insecticidal efficacy of conventionally treated nets, but they were not exhausted, even after 21 washes. Using a more rigorous laboratory washing method, insecticide was more readily stripped from conventionally treated nets. PermaNet retained high efficacy after 21 washes, giving more than 97% mortality of Anopheles in contact bioassays with 3-min exposure. Using the more sensitive bioassay criterion of 'median time to knockdown', PermaNet showed no loss of insecticidal activity against Anopheles after washing repeatedly in 2 out of 6 trials; whereas in a further three trials knockdown activity of PermaNet and conventional ITNs declined at comparable rates. Higher mortality levels of Anopheles in contact bioassays did not always translate to superiority in experimental hut or enclosed platform trials. In only one of four comparative field trials did PermaNet out-perform conventional ITNs after washing: this was in the trial of PermaNet 2.0--the product with improved quality assurance. Because PermaNet and conventionally treated nets were both quite tolerant of local washing procedures, it is important in field trials to compare LLINs with conventional ITNs washed an equivalent number of times. Our comparison of PermaNet 2.0 against conventionally treated deltamethrin nets (CTDN) in Pakistan demonstrated superior performance of the LLIN after 20 washes in phase I and phase II bioassays, and this was corroborated by chemical assays of residual deltamethrin. Although PermaNet 2.0 has received WHOPES interim recommendation for malaria control purposes, its performance should be monitored in everyday use throughout its lifespan in various cultural settings to assess its durability and long-term effectiveness for malaria prevention and control. As many millions of conventionally treated nets are already in routine use, and these will require regular re-treatment, programme strategies should be careful to preserve the effectiveness of ITNS before and after establishing the reliability of LLINs in long-term use.

Investigation of negative cross-resistance as a resistance-management tool for insecticide-treated nets.

Resistance management for insecticide-treated nets (ITNs) remains a challenge. Options are limited, because a safe and highly active insecticide with a persistence of several months is required. These criteria have only been met by pyrethroids, although organophosphates (OPs) and carbamates have been considered as alternatives for impregnation of eave curtains. It has been observed that some pyrethroid-resistant mosquito strains show increased OP susceptibility over pyrethroid-susceptible strains (i.e., negative cross-resistance). The current study investigated whether this phenomenon applies to a range of mosquito species and strains, because a mixture or rotation strategy for resistance management could then be envisaged. Adult female mosquitoes from laboratory strains of Anopheles stephensi Liston, Anopheles gambiae Giles, and Culex quinquefasciatus Say were tested in World Health Organization susceptibility test kits. For An. stephensi, the highly pyrethroid-resistant DUB 234 strain showed the same level of resistance to malathion as the pyrethroid-susceptible DUB S. The malathion-resistant ST MAL strain was as susceptible to pyrethroids as the insecticide-susceptible BEECH. For An. gambiae, the malathion tolerance of the previously pyrethroid-resistant RSP strain was significantly higher than that of the insecticide-susceptible KWA. For Cx. quinquefasciatus, selection of the QUINQ strain with permethrin abolished preexisting resistance to the OP malathion as pyrethroid resistance increased, rendering the strain more susceptible to malathion than PEL SS. Some indication of negative cross-resistance to malathion was found for the permethrin-resistant MUHEZA strain. The occurrence of negative cross-resistance seems dependent on the history of insecticide selection and is not generally applicable. Resistance management for ITNs will need to use mechanisms other than negative cross-resistance to be effective.

Experimental hut comparisons of nets treated with carbamate or pyrethroid insecticides, washed or unwashed, against pyrethroid-resistant mosquitoes.

The efficacy against mosquitoes (Diptera: Culicidae) of a bednet treated with carbamate insecticide [carbosulfan capsule suspension (CS) 200 mg/m(2)] was compared with four types of pyrethroid-treated nets in veranda-trap huts at Yaokoffikro near Bouaké, Côte d'Ivoire, where the malaria vector Anopheles gambiae Giles carries the kdr gene (conferring pyrethroid resistance) at high frequency and Culex quinquefasciatus Say is also pyrethroid resistant. Pyrethroids compared were lambdacyhalothrin CS 18 mg/m(2), alphacypermethrin water dispersible granules (WG) 20 mg/m(2), deltamethrin 50 mg/m(2) (Permanet) and permethrin emulsifiable concentrate (EC) 500 mg/m(2). Insecticidal power and personal protection from mosquito bites were assessed before and after the nets were used for 8 months and hand washed five times in cold soapy water. Before washing, all treatments except permethrin significantly reduced blood-feeding and all had significant insecticidal activity against An. gambiae. The carbosulfan net gave significantly higher killing of An. gambiae than all pyrethroid treatments except the Permanet. Against Culex spp., carbosulfan was more insecticidal and gave a significantly better protective effect than any of the pyrethroid treatments. After washing, treated nets retained various degrees of efficacy against both mosquito genera - but least for the carbosulfan net. Washed nets with three types of pyrethroid treatment (alphacypermethrin, lambdacyhalothrin, permethrin) gave significantly higher mortality rates of Culex than in huts with the same pyrethroid-treated nets before washing. After five washes, the Permanet, which is sold as a long-lasting insecticidal product, performed no better than the other nets in our experimental conditions.

Chronic illness as a result of low-level exposure to synthetic pyrethroid insecticides: a review of the debate.

Synthetic pyrethroid insecticides are widely used in public health and agriculture throughout the world and generally considered to be the safest class of insecticides available. In contrast to this belief stands a recent debate in Germany, around possible chronic effects of exposure to low levels of pyrethroid residues, particularly in the household. The debate is reviewed with a view to its potential implications on the use of synthetic pyrethroids on insecticide-treated nets (ITNs) for malaria control. Media coverage, rather than conclusive evidence from carefully planned scientific investigations, seems the underlying factor in the debate. However, chronic effects can presently not be excluded with certainty, as relevant toxicological data do not exist in the open scientific literature. Properly designed neuro-behavioural studies on groups with long-term exposure to low doses of synthetic pyrethroids should be conducted. This will require establishment of a working definition of "case" and "exposure". Meanwhile pyrethroids should continue to be used for public health interventions that contribute substantially to morbidity and mortality reduction, such as ITNs for malaria control.

Vectors and malaria transmission in a gem mining area in Sri Lanka.

Anopheline vectors of malaria were studied in the Kaluganga gem mining area in the dry zone of central Sri Lanka. Adult mosquitoes were collected using cattle-baited huts and nets, pyrethrum spray sheets, window exit traps and light traps and partial and full-night human landing collections over a period of 17 mo. The collections produced a total of 13,591 anophelines belonging to 14 species. Using ELISA for circumsporozoite proteins of Plasmodium vivax and P. falciparum, Anopheles culicifacies, An. subpictus and An. varuna were incriminated as malaria vectors. For these species, estimated entomological inoculation rates were 5.9, 5.7, and 6.7 infective bites per person per year, respectively. An. culicifacies and An. subpictus show endophagic behavior, while An. varuna were exophagic. Mosquito larval surveys indicated that the gem pits, when filled with water, contributed 60% of the larvae of the three vector species. Parasitological data were collected by two mobile malaria clinics and by hospitals. The incidence rates of fever per 1,000 person-years with P. vivax and P. falciparum were 122.8 and 26.1 respectively, with a significantly higher rate in males over 15 years of age than in females of that age. Changes in the environment due to gem mining may have caused the emergence of An. subpictus and An. varuna as significant malaria vectors. The conventional view of An. culicifacies being the main vector of malaria in Sri Lanka needs to be reconsidered when planning the vector control programs in this study area. Due to migration of malaria patients, there is a likelihood the disease could be spread to other parts of the island. Therefore, action should be taken to eliminate these pits as breeding sites.

Control of vectors and incidence of malaria in an irrigated settlement scheme in Sri Lanka by using the insect growth regulator pyriproxyfen.

An evaluation of pyriproxyfen as a larval control agent with the aim of reducing malaria vector populations and incidence of malaria was conducted in 12 villages in an irrigated settlement scheme in the dry zone of central Sri Lanka. In these villages, there are many pools in the beds of rivers, streams, and irrigation ditches during the dry season of the year. These are the major breeding places of the malaria vectors Anopheles culicifacies and An. subpictus. Collections of adult mosquitoes were carried out by using standard methods and parasitological data were collected by daily malaria clinics set up for the project and through the 2 government hospitals. All villages in the study area were under residual house spraying with lambdacyhalothrin water-dispersible powder. Using the 1st year's baseline data collection, the villages were stratified into 6 villages with high malaria incidence and 6 villages with low incidence. Within each group, 3 villages were randomly assigned for larval control by treating all the pools in the beds of rivers, streams, and irrigation ditches and agricultural wells with a granular formulation of the insect growth regulator pyriproxyfen at the rate of 0.01 mg active ingredient/liter. The field bioassays indicated that a single treatment of pyriproxyfen effectively inhibited the emergence of adult mosquitoes in the riverbed pools for a period of 190 days. The treatment caused significant reduction of the adult populations of An. culicifacies (78%) and An. subpictus (72%). Similarly, incidence of malaria was reduced in the treatment villages by about 70% (95% confidence interval 58-78%) compared with the controls. The conclusion is made that pyriproxyfen can be a very effective means of malaria control if all possible vector breeding places in the area can be located.

Horizontal and vertical dispersal of dengue vector mosquitoes, Aedes aegypti and Aedes albopictus, in Singapore.

To study the dispersal of dengue vector mosquitoes in Singapore, females of Aedes aegypti (L.) and Aedes albopictus (Skuse) (Diptera: Culicidae) were fed blood containing rubidium (Rb), which was detectable in their eggs by means of Graphite Furnace Atomic Absorption Spectrophotometry (GFAAS). Laboratory calibration of the Rb reading, for a range of egg numbers from Rb-fed females, indicated a reasonably linear relationship and an unequivocal distinction between results with zero and one marked egg. Rb-marked female Aedes mosquitoes aged 3-5 days were released in semi-rural and urbanized parts of Singapore, with an array of ovitraps extending to a radius of 320 m from the release point. Subsequently, Rb-marked Aedes eggs were detected throughout the array, with similar distributions on each of the 4 days after release. More Rb was detected nearer the release point. However, when correction was made for the greater areas of zones further from the release point (and therefore presumably existence of more alternative oviposition sites), there were no significant differences in the numbers of marked eggs per ovitrap in the zones nearer or further from the release points. It is concluded that females of both these Aedes (Stegomyia) species could disperse easily and quickly throughout areas of radius 320 m in search of oviposition sites. This contrasts with the general belief that Ae. aegypti seldom flies more than 50 m and that control operations can safely be based on such an assumption. Releases on level 12 of a 21-storey apartment block, with ovitraps on each storey, showed similar easy and rapid dispersal to the top and bottom of the block.

Comparative evaluation of carbosulfan- and permethrin-impregnated curtains for preventing house-entry by the malaria vector Anopheles gambiae in Burkina Faso.

Pyrethroid-impregnated bednets and curtains are widely employed to reduce the risk of malaria transmission, but pyrethroid-resistance is becoming more prevalent among malaria vector Anopheles mosquitoes (Diptera: Culicidae). As an alternative treatment for curtains, we assessed carbosulfan (a carbamate insecticide) in comparison with permethrin as the standard pyrethroid, against endophilic female mosquitoes of the Anopheles gambiae Giles complex in a village near Ouagadougou, Burkina Faso. The main criterion evaluated was the impact of curtains (hung inside windows, eaves and doorways) on the number of An. gambiae s.l. females active indoors at night. Light-traps were operated overnight (21.00-06.00 hours beside occupied untreated bednets) to sample mosquitoes in houses fitted with net curtains treated with carbosulfan 0.2 g ai/m2 or permethrin 1 g ai/m2 or untreated, compared with houses without curtains. The treated and untreated curtains significantly reduced the numbers of mosquitoes collected indoors, compared with houses without curtains. Carbosulfan-treated curtains had a highly significantly greater effect than permethrin-treated or untreated curtains, the scale of the difference being estimated as three-fold. However, there was no significant difference between the impact of untreated and permethrin-treated curtains on densities of An. gambiae s.l. trapped indoors. Samples of the An. gambiae complex comprised An. arabiensis Patton and both the S- and M-forms of An. gambiae Giles s.s. Susceptibility tests revealed some resistance to DDT and low frequencies of permethrin-resistance, insufficient to explain the poor performance of permethrin on curtains. Among survivors from the diagnostic dosage of permethrin were some specimens of all three members of the An. gambiae complex, but the kdr resistance mechanism was detected only in the S-form of An. gambiae s.s. Questions arising for further investigation include clarification of resistance mechanisms in, and foraging behaviour of, each member of the An. gambiae complex in this situation and the need to decide whether carbosulfan-treated curtains are acceptably safe for use to reduce risks of malaria transmission.

Pyrethroid resistance/susceptibility and differential urban/rural distribution of Anopheles arabiensis and An. gambiae s.s. malaria vectors in Nigeria and Ghana.

Resistance to pyrethroid insecticides and DDT caused by the kdr gene in the malaria vector Anopheles gambiae Giles s.s. (Diptera: Culicidae) has been reported in several West African countries. To test for pyrethroid resistance in two more countries, we sampled populations of the An. gambiae complex from south-western Ghana and from urban and rural localities in Ogun State, south-west Nigeria. Adult mosquitoes, reared from field-collected larvae, were exposed to the WHO-recommended discriminating dosage of exposure for 1 h to DDT 4%, deltamethrin 0.05% or permethrin 0.75% and mortality was recorded 24 h post-exposure. Susceptibility of An. gambiae s.l. to DDT was 94-100% in Ghana and 72-100% in Nigeria, indicating low levels of DDT resistance. Deltamethrin gave the highest mortality rates: 97-100% in Ghana, 95-100% in Nigeria. Ghanaian samples of An. gambiae s.l. were fully susceptible to permethrin, whereas some resistance to permethrin was detected at 4/5 Nigerian localities (percentage mortalities 75, 82, 88, 90 and 100%), with survivors including both An. arabiensis Patton and An. gambiae s.s. identified by PCR assay. Even so, the mean knockdown time was not significantly different from a susceptible reference strain, indicating absence or low frequency of kdr-type resistance. Such low levels of pyrethroid resistance are unlikely to impair the effectiveness of pyrethroid-impregnated bednets against malaria transmission. Among Nigerian samples of An. gambiae s.l., the majority from two urban localities were identified as An. arabiensis, whereas the majority from rural localities were An. gambiae s.s. These findings are consistent with those of M. Coluzzi et al. (1979). Differences of ecological distribution between molecular forms of An. gambiae s.s. were also found, with rural samples almost exclusively of the S-form, whereas the M-form predominated in urban samples. It is suggested that 'urban island' populations of An. arabiensis and of An. gambiae s.s. M-form in the rainforest belt of West Africa might be appropriate targets for elimination of these malaria vectors by the sterile insect technique.

The pyrethroid knock-down resistance gene in the Anopheles gambiae complex in Mali and further indication of incipient speciation within An. gambiae s.s.

In Mali the Anopheles gambiae complex consists of An. arabiensis and Mopti, Savanna and Bamako chromosomal forms of An. gambiae s.s. Previous chromosomal data suggests a complete reproductive isolation among these forms. Sequence analysis of rDNA regions led to the characterization of two molecular forms of An. gambiae, named M-form and S-form, which in Mali correspond to Mopti and to Savanna/Bamako, respectively, while it has failed so far to show any molecular difference between Savanna and Bamako. The population structure of An. gambiae s.l. was analysed in three villages in the Bamako and Sikasso areas of Mali and the frequency of pyrethroid resistance of the knock-down resistance (kdr) type was calculated. The results show that the kdr allele is associated only with the Savanna form populations and absent in sympatric and synchronous populations of Bamako, Mopti and An. arabiensis. This is the first molecular indication of barriers to gene flow between the Bamako and Savanna chromosomal forms. Moreover, analyses of specimens collected in the Bamako area in 1987 show that the kdr allele was already present in the Savanna population at that time, and that the frequency of this allele has gradually increased since then.