Biological cost of tolerance to heavy metals in the mosquito Anopheles gambiae.

The global rate of heavy metal pollution is rapidly increasing in various habitats. Anopheles malaria vector species (Diptera: Culicidae) appear to tolerate many aquatic habitats with metal pollutants, despite their normal proclivity for 'clean' water (i.e. low levels of organic matter). Investigations were conducted to establish whether there are biological costs for tolerance to heavy metals in Anopheles gambiae Giles sensu stricto and to assess the potential impact of heavy metal pollution on mosquito ecology. Anopheles gambiae s.s. were selected for cadmium, copper or lead tolerance through chronic exposure of immature stages to solutions of the metals for three successive generations. Biological costs were assessed in the fourth generation by horizontal life table analysis. Tolerance in larvae to cadmium (as cadmium chloride, CdCl(2)), copper [as copper II nitrate hydrate, Cu(NO(3))(2) 2.5 H(2)O] and lead [as lead II nitrate, Pb(NO(3))(2)], monitored by changes in LC(50) concentrations of the metals, changed from 6.07 microg/L, 12.42 microg/L and 493.32 microg/L to 4.45 microg/L, 25.02 microg/L and 516.69 microg/L, respectively, after three generations of exposure. The metal-selected strains had a significantly lower magnitude of egg viability, larval and pupal survivorship, adult emergence, fecundity and net reproductive rate than the control strain. The population doubling times were significantly longer and the instantaneous birth rates lower in most metal-selected strains relative to the control strain. Our results suggest that although An. gambiae s.s. displays the potential to develop tolerance to heavy metals, particularly copper, this may occur at a significant biological cost, which can adversely affect its ecological fitness.

Discriminative feeding behaviour of Anopheles gambiae s.s. on endemic plants in western Kenya.

Anopheles gambiae Giles s.s. (Diptera: Culicidae) is known to feed on plant sugars, but this is the first experimental study to consider whether it discriminates between plant species. Thirteen perennial plant species were selected on the basis of their local availability within the vicinity of human dwellings and larval habitats of An. gambiae s.s. in western Kenya. Groups of 100 or 200 mosquitoes were released into cages either with a cutting of one plant type at a time (single-plant assay) or with cuttings of all 13 plants simultaneously (choice assay), respectively, and left overnight. In the choice assay, direct observations of the percentages of mosquitoes perching or feeding on each plant were recorded over four 1-h periods each night. For both types of assay, mosquitoes were recaptured and the percentage that had fed on plants was assessed by testing them individually for the presence of fructose. To identify which plants the choice-assay mosquitoes had fed on, gas chromatography (GC) profiles of samples of mosquito homogenates were compared with GC profiles of extracts from relevant parts of each plant. Four of the plants that were observed to have been fed on most frequently in the choice assay (Parthenium hysterophorus L., Tecoma stans L., Ricinus communis L., and Senna didymobotrya Fresen) were also shown to have been ingested most often by mosquitoes in both types of assay, suggesting that An. gambiae is differentially responsive to this range of plants, regardless of whether the plants were presented singly or mixed together. Significantly more females than males fed on plants, with the exception of P. hysterophorus L., one of the plants most frequently fed on. For most plant species (ten of 13), GC profiles indicated that An. gambiae obtained sugars primarily from flowers. The exceptions were P. hysterophorus L., Lantana camara L. and R. communis L., on which An. gambiae fed more often from leaves and stems than from flowers.

Estimation of the minimum number of leishmania major amastigotes required for infecting phlebotomus duboscqi (diptera: psychodidae)

Background. Leishmaniasis is a vector-borne disease in which Leishmania parasites are transmittedby the bite of phlebotomine sand flies. Amastigotes are ingested by the sand fly vector with ablood meal taken from an infected host. This is followed by their differentiation into metacyclicpromastigotes which are selectively released and permitted to migrate interiorly so as to makethem available for transmission by bite. However, the actual number of amastigotes ingested bythe sand fly in the blood meal is not known.Objective: Toinvestigate the minimum number of Leishmania major amastigotes required to causean infection in Phlebotomus duboscqi following an infective blood meal.Design: A laboratory based study.Setting: Centre for Biotechnology Research and Development, Kenya Medical Research institute, Nairobi.Results: Dissection of all fed sand flies at six days post-infective blood meal revealed that bloodcontaining one amastigote per 0.3µl in a total volume of 0.5ml was able to cause an infection in thesand flies, but very few sand flies got infected (7.6% and 9.6% respectively). Concentrations of tenamastigotes per 0.3µl in 0.5ml gave infection rates of 35.4% and 26.3% respectively, suggesting thateven when the concentration of amastigotes in a bloodmeal was high, not all sand flies feeding onit were able to pick up the parasites.Conclusions:These observations suggests that one amastigote is sufficient to cause an infection toa sand fly and as a result of multiplication in the gut and the existence of mechanisms that increasethe number of infective bites delivered by a female sand fly they are able to sustain the transmissionof leishmaniasis in an area

Development and survival of Anopheles gambiae eggs in drying soil: influence of the rate of drying, egg age, and soil type.

Little is known about the contribution made by the egg stage of African malaria vectors to the rapid rise in adult populations following the onset of seasonal rains. To examine this issue, we evaluated the viability of Anopheles gambiae eggs in drying soil in the laboratory. Survival data were collected from field-caught mosquitoes kept in sandy loam soil and laboratory-reared colonies kept in sandy loam soil and black cotton soil. Under high, medium, and low soil-moisture regimes, egg viability declined sharply with increased duration of drying. Eggs remained viable in drying sandy loam soil for 1, 5, and 10 days, but not after 15 or 20 days. The most dramatic decline in hatching success occurred between drying days 1 (78-83% hatch) and 5 (20-23% hatch). In contrast, eggs reared in high-moisture black cotton soil remained viable for up to 15 days. Furthermore, after 5 drying days, high-, medium-, and low-moisture soils averaged 59, 47, and 31% hatching success, respectively. We recovered unhatched eggs from sandy loam soils to examine the developmental status of the embryos. A majority of the unhatched eggs that were recovered from days 15 and 20 in sandy loam soils contained fully developed late-stage embryos. Thus, unhatched eggs completed embryonic development but probably died before receiving an appropriate hatching stimulus. Our results suggest that the absolute moisture content of the soil does not alone determine hatching success of anopheline eggs. Rather, soil moisture, together with the rate of drying, physiological factors associated with the age of the egg, and the type of soil in which the egg rests likely influence survival.

Feeding and survival of the malaria vector Anopheles gambiae on plants growing in Kenya.

The propensity of the malaria vector mosquito Anopheles gambiae Giles (Diptera: Culicidae) to ingest sugars from various plants, and subsequent survival rates, were assessed with laboratory-reared males and females offered eight species of plants commonly cultivated and/or growing wild in western Kenya. In cages (no-choice bioassay), mosquitoes given the opportunity to feed on castorbean (Ricinus communis L.) had the longest survival times (mean and median survival time of 6.99 +/- 0.23 and 5.67 +/- 0.17 days, respectively), comparable to mosquitoes given 6% glucose (mean and median survival time of 8.70 +/- 0.23 and 6.67 +/- 0.33 days, respectively). Survival rates of An. gambiae were low on the other plants, comparable to mosquitoes given only water. Three plants: sweet potato (Ipomoea batatas L.), wild sage (Lantana camara L.) and castorbean provided levels of sugar ingestion by both sexes of An. gambiae detectable using the cold anthrone method, showing a positive correlation between median survival and sugar consumption (Spearman rank correlation coefficient = 0.905, P < 0.0001). Equal numbers of males and females were released in an enclosed semi-field screenhouse system containing a range of local plants, but no host for blood, and allowed to feed ad libitum: 6.7 +/- 0.5% (11/64) of those recaptured were found to contain detectable fructose (all females). Common plants are clearly a viable source of nutrition for adult female An. gambiae, as well as males, and may constitute and important resource for this important malaria vector.

Plasmodium falciparum malaria disease manifestations in humans and transmission to Anopheles gambiae: a field study in Western Kenya.

Transmission of the malaria parasite Plasmodium is influenced by many different host, vector and parasite factors. Here we conducted a field study at Mbita, an area of endemic malaria in Western Kenya, to test whether parasite transmission to mosquitoes is influenced by the severity of malaria infection in its human host at the time when gametocytes, the transmission forms, are present in the peripheral blood. We examined the infectivity of 81 Plasmodium falciparum gametocyte carriers to mosquitoes. Of these, 21 were patients with fever and other malaria-related symptoms, and 60 were recruited among apparently healthy volunteers. Laboratory-reared Anopheles gambiae s.s. (local strain) were experimentally infected with blood from these gametocyte carriers by membrane-feeding. The severity of the clinical symptoms was greater in febrile patients. These symptomatic patients had higher asexual parasitaemia and lower gametocyte densities (P = 0.05) than healthy volunteers. Ookinete development occurred in only 6 out of the 21 symptomatic patients, of which only 33.3% successfully yielded oocysts. The oocyst prevalence was only 0.6% in the 546 mosquitoes that were fed on blood from this symptomatic group, with mean oocyst intensity of 0.2 (range 0-2) oocysts per mosquito. In contrast, a higher proportion (76.7%) of healthy gametocyte carriers yielded ookinetes, generating an oocyst rate of 12% in the 1332 mosquitoes that fed on them (mean intensity of 6.3, range: 1-105 oocysts per mosquito). Statistical analysis indicated that the increased infectivity of asymptomatic gametocyte carriers was not simply due to their greater gametocyte abundance, but also to the higher level of infectivity of their gametocytes, possibly due to lower parasite mortality within mosquitoes fed on blood from healthy hosts. These results suggest that blood factors and/or conditions correlated with illness reduce P. falciparum gametocyte infectivity.

Efficacy of Bacillus thuringiensis israelensis, Bacillus sphaericus and temephos for managing Anopheles larvae in Eritrea.

We evaluated the larvicidal activity of the granular formulation of Bacillus thuringiensis israelensis (Bti) serotype H-14 (Vectobac G, 200 ITU/mg) and Bacillus sphaericus (Bsph) serotype H5a5b (Vectolex CG, 670 Bs ITU/mg) against Anopheles arabiensis and other mosquitoes in breeding habitats in 3 sites, Gash-Barka, Anseba, and Debub zones, in Eritrea. The primary objective was to determine the optimal application rate and duration of effect for Bti and Bsph in representative larval habitats as compared with the organophosphate temephos. The biolarvicides were tested at 100% (high) and 50% (low) of the maximum recommended application rate. Temephos was applied at a rate of 100 ml/ha. At least 4 replicate experiments with Vectobac G (5.6 and 11.2 kg/ha), Vectolex CG (11.2 and 22.4 kg/ha) were conducted in each study site. All 3 larvicides caused significant mortality of the main malaria vector species, An. arabiensis, and other mosquito species (Anopheles cinereus, Anopheles pretoriensis, Culex quinquefasciatus). The larvicidal activity for Bti and Bsph was variable depending upon breeding habitat, mosquito species, and general ecology of the area. Both biopesticides had a similar duration of activity (2-3 wk) and were generally as effective as temephos for these time periods. In some cases, the high and low application rates for Bti and Bsph produced equivalent control over 2-3 wk. The 2 Bacillus biopesticides were less effective in habitats with high algal content and in fast flowing streams primarily because of the inability to penetrate algal mats and dilution effect, respectively. The results show that application of the 2 biolarvicides bimonthly to streambed pools, rain pools, and similar habitats would maintain control of the anopheline mosquito population.

Treatment failure of pyrimethamine-sulphadoxine and induction of Plasmodium falciparum gametocytaemia in children in western Kenya.

Sub-Saharan Africa faces increasing levels of resistance of Plasmodium falciparum parasites to the first-line drug pyrimethamine-sulphadoxine (SP). Successful treatment with SP is reported to induce gametocytes and drug resistance may further increase gametocytaemia after treatment. Treatment success, gametocyte prevalence and gametocyte density were determined in 224 asymptomatic children in western Kenya on day 7 after treatment with SP. Treatment failure (R2 or R3 resistance) was observed in 22% of the children. The relative risk to show gametocytes on day 7 after treatment in children with treatment failure was 4.1 (95% CI 1.4-11.6) times higher compared to children with a sensitive infection, after adjustment for age and trophozoite density at the start of treatment. In addition, the gametocyte density was also higher upon SP treatment failure. These findings are reason for concern, as the increased gametocyte prevalence and density after SP treatment failure may increase the spread of SP-resistant strains in the population.

Infectivity of Plasmodium falciparum gametocytes in patients attending rural health centres in western Kenya.

BACKGROUND: Experimentally studying the transmission of the malaria parasite and its regulating factors requires availability of human blood donors carrying infectious gametocytes. The difficulty of identifying gametocyte carriers from the community is often limited due to financial and human resources constraints. The available alternative is rural health centres where malaria patients go for treatment. In this study, the potential of recruiting volunteers and acquiring infectious blood for experimental infections from rural health centers in malaria endemic area was examined through routine patient diagnosis. OBJECTIVE: To examine the patients presenting at rural health centers for the potential to carry sexual stage malaria parasite and test their infectivity to Anopheles gambiae mosquitoes. SETTING: Mbita Health Centre, Mbita Town Ship, Suba District, western Kenya. METHODOLOGY: Routine survey of all patients attending Mbita Health Centre with suspected malaria. Patients were examined for Plasmodium falciparum trophozoites and gametocytes. Gametocyte-positive volunteers were recruited for their potential to infect Anopheles mosquitoes via membrane feeding. RESULTS: Three thousand nine hundred and eighty seven patients were screened between May 2000 and April 2001. Plasmodium falciparum was the predominant parasite species and P. malariae being the only minor species, accounting for 0.9% of malaria cases. Clinical malaria varied with age and prevailed throughout the year with a slight seasonality. Gametocyte prevalence was low (0.9-6.6%), and gametocyte densities were generally very low with a geometric mean of 39 gametocytes per microl blood. Children aged > 5 years constituted 67% of all gametocyte carriers. Only 22 volunteers with mean gametocytes density of 39.62 per microl blood (range: 16-112) were recruited for study of parasite infectiousness to laboratory-reared mosquitoes. Only two patients infected 1% of 1099 mosquitoes with one or two oocysts. CONCLUSION: The low gametocyte densities or other possible host and vector related factors regulating infectivity of gametocyte carriers to mosquitoes may have caused the poor infections of mosquitoes. This study indicates that rural health centers in malaria-endemic areas may not be suitable for recruiting infectious gametocyte donors for studies of vector competence. They are suitable for passive clinical case surveillance and for evaluation of the effects of control measures.

Behavioural determinants of gene flow in malaria vector populations: Anopheles gambiae males select large females as mates.

BACKGROUND: Plasmodium-refractory mosquitoes are being rapidly developed for malaria control but will only succeed if they can successfully compete for mates when released into the wild. Pre-copulatory behavioural traits maintain genetic population structure in wild mosquito populations and mating barriers have foiled previous attempts to control malaria vectors through sterile male release. METHODS: Varying numbers of virgin male and female Anopheles gambiae Giles, from two strains of different innate sizes, were allowed to mate under standardized conditions in laboratory cages, following which, the insemination status, oviposition success and egg batch size of each female was assessed. The influence of male and female numbers, strain combination and female size were determined using logistic regression, correlation analysis and a simple mechanistic model of male competition for females. RESULTS: Male An. gambiae select females on the basis of size because of much greater fecundity among large females. Even under conditions where large numbers of males must compete for a smaller number of females, the largest females are more likely to become inseminated, to successfully oviposit and to produce large egg batches. CONCLUSIONS: Sexual selection, on the basis of size, could either promote or limit the spread of malaria-refractory genes into wild populations and needs to be considered in the continued development and eventual release of transgenic vectors. Fundamental studies of behavioural ecology in malaria vectors such as An. gambiae can have important implications for malaria control and should be prioritised for more extensive investigation in the future.

Transmission blocking vaccine studies in leishmaniasis: II. Effect of immunisation using Leishmania major derived 63 kilodalton glycoprotein, lipophosphoglycan and whole parasite antigens on the course of L. major infection in BALB/c mice.

BACKGROUND: Safe, effective and inexpensive vaccines may be the most practical tool for control of any form of leishmaniasis. Leishmaniasis produces a state of pre-immunition which is the underlying mechanism for prolonged immunity to re-infection. Low doses of parasites has been shown to be able to induce protection in mice. It is not known, however, how immune sera from a susceptible host immunised with Leishmania-derived antigens when taken in by the sandfly affects the development and the subsequent transmission of the parasite to naive hosts. OBJECTIVE: To monitor the course of disease in BALB/c mice following challenge using L. major infected P. duboscqi which had previously fed on immunised mice. METHODS: BALB/c mice were immunised adequately with Leishmania major-derived antigens namely, crude whole parasite (WPA), recombinant 63 kilodalton glycoprotein (rgp63), lipophosphoglycan (LPG) and a cocktail composed of rgp63 plus LPG antigens. Laboratory reared Phlebotomus duboscqi sandflies, the natural vector for L. major were later allowed to feed on immunised animals, interrupted and allowed to continue feeding on infected animals for an equal amount of time until they became fully engorged. The sandflies were maintained on apples as a carbohydrate source in an insectary maintained at a temperature of 25 degrees C and 80% relative humidity. On the seventh day these sandflies were used to infect naive BALB/c mice and the course of infection followed for a period of at least three months. RESULTS: Mice infected using sandflies which had previously fed on WPA or rgp63-immunized mice showed disease exacerbation as the infection progressed, whereas those infected using sandflies which had previously fed on LPG-immunised mice had the least lesion sizes compared to control mice infected using sandflies which had fed on saline immunised mice (p < 0.05). CONCLUSIONS: Results from this study indicate that the course of L. major infection in BALB/c mice was dependent on the infective dose of parasites transmitted by the sandflies. Results from this study suggests that sub-infective doses of the parasite from sandflies previously fed on animals immunised with Leishmania-derived antigens needs to be evaluated for their potential in vaccine development against Leishmania infections.

Transmission blocking vaccine studies in leishmaniasis: I. Lipophosphoglycan is a promising transmission blocking vaccine molecule against cutaneous leishmaniasis.

BACKGROUND: New strategies for control of leishmaniasis is needed as chemotherapy using antimonial drugs is prolonged, expensive, associated with side effects and relapses. Vector control has limitations and a vaccine which may be the best approach is not available. OBJECTIVES: To assess the level of inhibition of promastigote development and gut morphology in infected Phlebotomus duboscqi sandflies fed on different groups of BALB/c mice immunised with rgp63, lipophosglycan (LPG) or their cocktail and whole parasite antigens prepared from L. major culture-derived promastigotes. METHODS: BALB/c mice were immunised adequately with Leishmania major-derived antigens namely, crude whole parasite (WPA), recombinant 63 kilodalton glycoprotein (rgp63), LPG and a cocktail composed of rgp63 plus LPG antigens. Laboratory reared Phlebotomus duboscqi sandflies, the natural vector for L. major were later allowed to feed on immunised animals, interrupted and allowed to continue feeding on infected animals for an equal amount of time until they became fully engorged. The sandflies were maintained on apples as a carbohydrate source in an insectary maintained at a temperature of 25 degrees C and 80% relative humidity. Some of the sandflies were dissected on days 2, 4 and 6 after feeding and observed using the light and the transmission electron microscopy for any changes in their gut morphology. The remaining sandflies were all dissected on the sixth day post-feeding and examined for procyclics, nectomonads, haptomonads and metacyclic promastigote forms of Leishmania. RESULTS: Sandflies which had previously fed on WPA, LPG plus rgp63 cocktail and LPG-immunised mice showed the lowest infection rates compared to control sandflies fed on saline immunised mice (p < 0.05). A significant number of procyclic promastigotes, the first developmental form of the parasite in culture as well as in the sandfly was observed in sandflies which fed on LPG-immunised mice (p < 0.05). The dominant parasite form in sandflies which fed on rgp63 or LPG-immunised mice was the nectomonad form but very few of the infective metacyclic forms (p < 0.05). Control sandflies fed on saline immunised or infected mice alone displayed a normal pattern of parasite development up to the metacyclic stage. Studies showed that two possible mechanisms through which immune sera from immunised mice may cause inhibition of parasite development is by exflagellation of nectomonad forms and degeneration of the sandfly midgut epithelium as revealed by light and electron microscopy studies respectively. CONCLUSIONS: This study has shown that immune-mediated transmission blocking may be applied to Leishmania infections. Based on observation of the procyclic promastigotes, the dominance of the nectomonad forms, low infectivity rates in sandflies fed on LPG-immunised mice, we concluded that LPG stands out to be a promising transmission blocking vaccine candidate in leishmaniasis.

Anopheline mosquito survival strategies during the dry period in western Kenya.

The dry season survival mechanism of Anopheles gambiae Giles is one of the most vexing deficiencies in our understanding of the biology of the major malaria vectors. In this study, we examined the dynamics of anopheline adult mosquitoes, their larval habitats, and egg survival potential during the dry season in the basin region of Lake Victoria, western Kenya. Through field surveys, we demonstrated two survival strategies of An. gambiae sensu stricto during the dry season: continuous reproduction throughout the year and embryo dormancy in moist soil for at least several days. We further demonstrated that An. gambiae shows a strong preference for moist soil as an oviposition substrate rather than dry soil substrate under the insectary conditions. The observation that anopheline eggs remain a dormant stage to resist desiccation clearly contrasts the conventional wisdom that anopheline eggs hatch shortly after they are laid. Our results from western Kenya are consistent with the suggestion that anopheline mosquitoes do not necessarily suffer a severe population bottleneck during the dry season and thus maintain a large effective population size.

Short report: entomologic inoculation rates and Plasmodium falciparum malaria prevalence in Africa.

Epidemiologic patterns of malaria infection are governed by environmental parameters that regulate vector populations of Anopheles mosquitoes. The intensity of malaria parasite transmission is normally expressed as the entomologic inoculation rate (EIR), the product of the vector biting rate times the proportion of mosquitoes infected with sporozoite-stage malaria parasites. Malaria transmission intensity in Africa is highly variable with annual EIRs ranging from < 1 to > 1,000 infective bites per person per year. Malaria control programs often seek to reduce morbidity and mortality due to malaria by reducing or eliminating malaria parasite transmission by mosquitoes. This report evaluates data from 31 sites throughout Africa to establish fundamental relationships between annual EIRs and the prevalence of Plasmodium falciparum malaria infection. The majority of sites fitted a linear relationship (r2 = 0.71) between malaria prevalence and the logarithm of the annual EIR. Some sites with EIRs < 5 infective bites per year had levels of P. falciparum prevalence exceeding 40%. When transmission exceeded 15 infective bites per year, there were no sites with prevalence rates < 50%. Annual EIRs of 200 or greater were consistently associated with prevalence rates > 80%. The basic relationship between EIR and P. falciparum prevalence, which likely holds in east and west Africa, and across different ecologic zones, shows convincingly that substantial reductions in malaria prevalence are likely to be achieved only when EIRs are reduced to levels less than 1 infective bite per person per year. The analysis also highlights that the EIR is a more direct measure of transmission intensity than traditional measures of malaria prevalence or hospital-based measures of infection or disease incidence. As such, malaria field programs need to consider both entomologic and clinical assessments of the efficacy of transmission control measures.

Vector-related case-control study of severe malaria in Kilifi District, Kenya.

A case-control study examined vector-related and environmental parameters associated with severe malaria in Kilifi District along the coast of Kenya. Over an 11-month period, 119 children identified with severe malaria infections at the Kilifi District Hospital were matched by age with control children who reported to the outpatient clinic with nonsevere infections. Intensive mosquito sampling was done in each of the case-control houses over a four-day period, beginning within a week of index case admission. A total of 109 environmental, demographic, behavioral, and animal husbandry variables were characterized for each household. Vector species (Anopheles gambiae s.l. and An. funestus) were detected in 40.1% and 36.1% of case and control houses, respectively. The relative abundance of vectors in individual houses was stable over the two-week resampling periods (r = 0.9). Both the overall abundance of anopheline mosquitoes (odds ratio [OR] = 1.5) and P. falciparum sporozoite rates (OR = 1.5) were not significantly different between case and control houses. In a matched analysis, 11 of 109 house variables associated significantly with severe malaria were also associated with vector abundance, as determined by chi-square linear trend analysis. Under conditions of year-round, low-level transmission on the coast of Kenya, the risk of severe disease in children is multifactorial and not governed strictly by transmission intensity or environmental heterogeneity affecting vector abundance and distributions. This suggests that current interventions that appear to be achievable only in areas where transmission is already low to moderate should be appropriate. However, such interventions should be monitored so that inappropriate and possibly disastrous control activities can be avoided in Africa.

Phlebotomine sand fly (Diptera: Psychodidae) seasonal distribution and infection rates in a defined focus of Leishmania tropica.

A two-year study was conducted of phlebotomine sand fly fauna in a defined focus of Leishmania tropica. A total of 17,947 sand flies representing 10 species were collected from the location. Phlebotomus guggisbergi, a vector of L. tropica in Kenya, was the most prevalent species through the entire period, representing about 80% of the total catch. There was marked seasonal fluctuation in the populations of the three most common species, with highest population levels reached in December and lowest levels reached in July and August. Leishmania-like infections were encountered in 489 P. guggisbergi. No flagellate infections were observed in any other species of sand fly. Although infected P. guggisbergi were collected during each month of the year, the percent parous infected flies was highest (27.5%) during the November through January time period. These data show that the greatest risk of transmission to humans at this focus occurs during December, when the vector is prevalent and infections are common.

Evaluation of a standardized direct agglutination test (DAT) for the diagnosis of visceral leishmaniasis in Kenya.

A prototype test kit being developed, by the World Health Organization (WHO), for the diagnosis of visceral leishmaniasis (VL) was evaluated in the Baringo district of Rift Valley province in Kenya. The screening of approximately 10,000 individuals for the signs of VL produced 305 suspected cases. These cases and 304 controls matched for sex and age (+/- 2 years) were then tested with the kit, which is based on a direct agglutination test (DAT). The evaluation was a three-stage process. The first stage, the field screening, involved screening filter-paper samples of dried blood from the suspects and controls at a DAT titre of 1:500. The second stage, the laboratory titration, involved screening of the same individuals by testing freshly eluted filter-paper samples at 1:500 to 1:2000 dilution. In the third stage, the full-scale titration, all samples that had been positive at 1:2000 were titrated at 1:500-1:512,000. All the suspects giving DAT titres of 1:2000 or higher were considered positive for VL. This diagnosis was checked, whenever possible, by the examination of smears and/or cultures of splenic aspirates for leishmanial parasites. Those found to be parasitologically positive were put on a standard treatment regime of 20 mg sodium stibogluconate (Pentostam)/kg.day. Although 42 (13.8%) of the 305 clinical suspects investigated were DAT-positive (at 1:2000), it was only possible to take splenic aspirates from 32. Four (12.5%) of these 32 were apparently false-positives by DAT, as no parasites could be detected in their splenic aspirates. The others provided positive smears and cultures (27 cases) or a negative smear but a positive culture (one case). It was possible to re-examine two of the four serologically positive but parasitologically negative VL suspects at a 3-month follow-up: neither had a palpable spleen, one had seroconverted and the other had much lower DAT titre (1:32,000) than when investigated previously (1:128,000). All the parasitologically confirmed cases remained DAT-positive (1:2000) at this follow-up. The low cut-off titre (1:2000) and the simple procedure should make the kit suitable for use by health workers at all levels of primary-health care, including those with limited training and skills, for screening rural communities at risk of VL.

Spatial distribution and habitat characterization of anopheline mosquito larvae in Western Kenya.

Studies were conducted to characterize larval habitats of anopheline mosquitoes and to analyze spatial heterogeneity of mosquito species in the Suba District of western Kenya. A total of 128 aquatic habitats containing mosquito larvae were sampled, and 2,209 anopheline and 10,538 culicine larvae were collected. The habitats were characterized based on size, pH, distance to the nearest house and to the shore of Lake Victoria, coverage of canopy, surface debris, algae and emergent plants, turbidity, substrate, and habitat types. Microscopic identification of third- and fourth-instar anopheline larvae did not yield any Anopheles funestus or other anophelines. A total of 829 An. gambiae s.l. larvae from all habitats were analyzed further by rDNA-polymerase chain reaction to identify individual species within the An. gambiae species complex. Overall, An. arabiensis was the predominant species (63.4%), and An. gambiae was less common (31.4%). The species composition of An. gambiae s.l. varied significantly among the sampling sites throughout Suba District. The larval habitats in the southern area of the district had a higher proportion of An. gambiae than in the northern area. Multiple logistic analysis did not detect any significant association between the occurrence of anopheline larvae and habitat variables, and principal component analysis did not identify key environmental factors associated with the abundance of An. gambiae. However, significant spatial heterogeneity in the relative abundance of An. gambiae within the Suba district was detected. When the effect of larval habitat locality was considered in the analysis, we found that the distance to the nearest house and substrate type were significantly associated with the relative abundance of An. gambiae. Future studies integrating detailed water chemistry analysis, remote sensing technology, and the ecology of predators may be required to further elucidate the mechanisms underlying the observed spatial variation of anopheline larval distribution.

Relationship between parasite load and immune responses in early stages of Leishmania donovani infection in inbred BALB/c mice.

The aim of this study was to determine if a correlation exists between Leishmania donovani parasite load and the corresponding humoral and cellular mediated immune responses in the early stages of Leishmania donovani infection in inbred BALB/c mice. Five groups of ten BALB/c mice each were inoculated intraperitoneally with stationary phase metacyclic promastigotes of Leishmania donovani at doses 1 x 10(2), 1 x 10(4), 10(6) and 1 x 10(8) respectively per mouse. Group five mice were not manipulated in any way and were left to serve as control. At weekly intervals, for five weeks, the mice were assayed for cellular mediated immune responses to leishmania antigen by the delayed type hypersensitivity skin test (DTH) and humoral responses by the direct agglutination test (DAT) and the enzyme linked immunosorbent assay (ELISA). A correlation was established between parasite load and humoral responses as assayed by DAT and ELISA techniques. This study demonstrates that it is possible to diagnose visceral leishmaniasis in experimentally infected laboratory mice by DAT and ELISA. These techniques have the potential in screening large numbers of animals suspected to be reservoirs of visceral leishmaniasis by examining the peripheral blood taken from the tail of the animal.

Experimental infection of domestic sheep with culture-derived Leishmania donovani promastigotes.

Domestic sheep were intradermally inoculated with culture-derived stationary phase Leishmania donovani promastigotes. Sampling of site of inoculation, liver and spleen for 244 days showed that this parasite can stay alive in the skin for up to 28 days post-inoculation. Apart from pyrexia that was evident in all the animals for 42 days, no other symptoms of kala-azar were seen. No parasites were recovered from the visceral organs throughout the sampling period, suggesting that sheep are not susceptible to infection with L. donovani. It is therefore unlikely that sheep can be synanthropic reservoirs for this parasite.