Sporozoite-infected Mosquitoes Research Articles

Vectorial drivers of malaria transmission in Jabi Tehnan district, Amhara Regional State, Ethiopia

Among the factors affecting the effectiveness of malaria control is poor knowledge of the entomologic drivers of the disease. We investigated anopheline populations as part of a baseline study to implement house screening of windows and doors as a supplementary malaria control tool towards elimination in Jabi Tehnan district, Amhara Regional State of Ethiopia. The samples were surveyed monthly using CDC light traps between June 2020 and May 2021. Mosquito trap density (< 3 mosquitoes/trap) was low, however, with a high overall Plasmodium sporozoite rate (9%; indoor = 4.3%, outdoor = 13.1%) comprising P. falciparum (88.9%) and P. vivax (11.1%). Anopheles gambiae s.l., mostly An. arabiensis, comprised > 80% of total anopheline captures and contributed ~ 42% of Plasmodium-infected mosquitoes. On the other hand, morphologically scored Anopheles funestus s.l., constituting about 6% of anopheline collections, accounted for 50% of sporozoite-infected mosquitoes. Most of the infected An. funestus s.l. specimens (86.7%) were grouped with previously unknown or undescribed Anopheles species previously implicated as a cryptic malaria vector in the western Kenyan highlands, confirming its wider geographic distribution in eastern Africa. Other species with Plasmodium infection included An. longipalpis C, An. theileri, An. demillioni, and An. nili. Cumulatively, 77.8% of the infected mosquitoes occurred outdoors. These results suggest efficient malaria parasite transmission despite the low vector densities, which has implications for effective endpoint indicators to monitor malaria control progress. Additionally, the largely outdoor infection and discovery of previously unknown and cryptic vectors suggest an increased risk of residual malaria transmission and, thus, a constraint on effective malaria prevention and control.

Malaria hotspots explained from the perspective of ecological theory underlying insect foraging

Hotspots constitute the major reservoir for residual malaria transmission, with higher malaria incidence than neighbouring areas, and therefore, have the potential to form the cornerstone for successful intervention strategies. Detection of malaria hotspots is hampered by their heterogenous spatial distribution, and the laborious nature and low sensitivity of the current methods used to assess transmission intensity. We adopt ecological theory underlying foraging in herbivorous insects to vector mosquito host seeking and modelling of fine-scale landscape features at the village level. The overall effect of environmental variables on the density of indoor mosquitoes, sporozoite infected mosquitoes, and malaria incidence, was determined using generalized linear models. Spatial analyses were used to identify hotspots for malaria incidence, as well as malaria vector density and associated sporozoite prevalence. We identify household occupancy and location as the main predictors of vector density, entomological inoculation rate and malaria incidence. We propose that the use of conventional vector control and malaria interventions, integrated with their intensified application targeting predicted hotspots, can be used to reduce malaria incidence in endemic and residual malaria settings.

Mosquito Exposure and Malaria Morbidity: A Microlevel Analysis of Household Mosquito Populations and Malaria in a Population-Based Longitudinal Cohort in Western Kenya.

Malaria morbidity is highly overdispersed in the population. Fine-scale differences in mosquito exposure may partially explain this heterogeneity in individual malaria outcomes. In 38 households we explored the effect of household-level mosquito exposure and individual insecticide-treated net (ITN) use on relative risk (RR) of confirmed malaria. We conducted monthly active surveillance (n = 254; 2624 person-months) and weekly mosquito collection (2092 household-days of collection), and used molecular techniques to confirm human blood feeding and exposure to infectious mosquitoes. Of 1494 female Anopheles (89.8% Anopheles gambiae sensu lato), 88.3% were fed, 51.9% had a human blood meal, and 9.2% were sporozoite infected. In total, 168 laboratory-confirmed malaria episodes were reported (incidence rate 0.064 episodes per person-month at risk; 95% confidence interval [CI], .055-.074). Malaria risk was directly associated with exposure to sporozoite-infected mosquitoes (RR, 1.24; 95% CI, 1.11-1.38). No direct effect was measured between ITN use and malaria morbidity; however, ITN use did moderate the effect of mosquito exposure on morbidity. Malaria risk increases linearly with vector density and feeding success for persons with low ITN use. In contrast, malaria risk among high ITN users is consistently low and insensitive to variation in mosquito exposure.

Avian malaria alters the dynamics of blood feeding in Culex pipiens mosquitoes

BackgroundSome Plasmodium species have the ability to modify the behaviour of their mosquito vectors. This is thought to be an adaptive strategy that maximizes the parasite’s transmission.MethodsThe effect of Plasmodium relictum infections on the blood feeding behaviour of Culex pipiens quinquefasciatus mosquitoes was monitored.ResultsPlasmodium infections did not alter the proportion of blood fed mosquitoes but they did affect the dynamics and the size of the blood meal. Sporozoite-infected mosquitoes completed their blood meal 1.3 times later than uninfected mosquitoes and ended up with smaller blood meals.ConclusionThe potential adaptive nature of this manipulation of mosquito behaviour is discussed in the light of previous studies on other malaria models.

Humans frequently exposed to a range of non-human primate malaria parasite species through the bites of Anopheles dirus mosquitoes in South-central Vietnam.

BackgroundRecent studies have described natural human infections of the non-human primate parasites Plasmodium knowlesi and Plasmodium cynomolgi. In Southeast Asia, mosquitoes of the Anopheles leucosphyrus group bite both humans and monkeys in the forest and thus offer a possible route for Plasmodium species to bridge the species barrier. In this study we analysed the species composition of malarial sporozoites infecting the salivary glands of Anopheles dirus in order to determine their potential role as bridge vectors of Plasmodium parasites from monkeys to humans.MethodsMosquitoes were collected in the forest and forest fringe area of Khanh Phu commune by human-baited landing collection. Anopheles species were determined on the basis of morphologic features. Sporozoite-infected salivary glands were applied to filter paper and dried in an ambient atmosphere, before storage in closed vials at 4–6 °C. Detection and identification of Plasmodium species in salivary glands were carried out by nested-PCR of the small subunit ribosomal RNA gene.ResultsSix species of Plasmodium parasites were detected by PCR, of which P. vivax was the most common, followed by P. knowlesi, P. inui, P. cynomolgi, P. coatneyi and P. falciparum. Twenty-six of the 79 sporozoite infected mosquitoes showed multiple infections, most of which were a combination of P. vivax with one or more of the non-human primate Plasmodium species.ConclusionsThese results suggest that humans overnighting in this forest are frequently inoculated with both human and non-human primate malaria parasites, leading to a situation conducive for the emergence of novel zoonotic malaria.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0995-y) contains supplementary material, which is available to authorized users.

Development of cultured Plasmodium falciparum blood-stage malaria cell banks for early phase in vivo clinical trial assessment of anti-malaria drugs and vaccines.

BackgroundThe ability to undertake controlled human malaria infection (CHMI) studies for preliminary evaluation of malaria vaccine candidates and anti-malaria drug efficacy has been limited by the need for access to sporozoite infected mosquitoes, aseptic, purified, cryopreserved sporozoites or blood-stage malaria parasites derived ex vivo from malaria infected individuals. Three different strategies are described for the manufacture of clinical grade cultured malaria cell banks suitable for use in CHMI studies.MethodsGood Manufacturing Practices (GMP)-grade Plasmodium falciparum NF54, clinically isolated 3D7, and research-grade P. falciparum 7G8 blood-stage malaria parasites were cultured separately in GMP-compliant facilities using screened blood components and then cryopreserved to produce three P. falciparum blood-stage malaria cell banks. These cell banks were evaluated according to specific criteria (parasitaemia, identity, viability, sterility, presence of endotoxin, presence of mycoplasma or other viral agents and in vitro anti-malarial drug sensitivity of the cell bank malaria parasites) to ensure they met the criteria to permit product release according to GMP requirements.ResultsThe P. falciparum NF54, 3D7 and 7G8 cell banks consisted of >78% ring stage parasites with a ring stage parasitaemia of >1.4%. Parasites were viable in vitro following thawing. The cell banks were free from contamination with bacteria, mycoplasma and a broad panel of viruses. The P. falciparum NF54, 3D7 and 7G8 parasites exhibited differential anti-malarial drug susceptibilities. The P. falciparum NF54 and 3D7 parasites were susceptible to all anti-malaria compounds tested, whereas the P. falciparum 7G8 parasites were resistant/had decreased susceptibility to four compounds. Following testing, all defined release criteria were met and the P. falciparum cell banks were deemed suitable for release. Ethical approval has been obtained for administration to human volunteers.ConclusionsThe production of cultured P. falciparum blood-stage malaria cell banks represents a suitable approach for the generation of material suitable for CHMI studies. A key feature of this culture-based approach is the ability to take research-grade material through to a product suitable for administration in clinical trials.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0663-x) contains supplementary material, which is available to authorized users.

Both infected and uninfected mosquitoes are attracted toward malaria infected birds

BackgroundThe biting behaviour of mosquitoes is crucial for the transmission of malaria parasites. This study focuses on the feeding behaviour of Culex pipiens mosquitoes with regard to the infection status by the avian malaria parasite Plasmodium relictum (lineage SGS1).MethodsUninfected and sporozoite-infected mosquitoes were provided with a choice between an uninfected bird and a bird undergoing a chronic P. relictum infection. Mosquito choice is assessed by microsatellite typing of the ingested blood.ResultsChronically infected birds are more attractive to mosquitoes. This choice is not altered by the infection status of the mosquitoes: both infected and uninfected mosquitoes have similar host choice behaviours and are more attracted towards infected birds.ConclusionsThese results support some, but not all predictions derived from the hypothesis that malaria parasites can manipulate the behaviour of their mosquito vectors to enhance their transmission. The possible mechanisms driving this manipulation, the evolutionary dynamics leading to the modification of the biting behaviour of mosquitoes by Plasmodium sp. as well as the implications for malaria epidemiology are discussed.

Synchronous peaks in trap catches of malaria-infected mosquito species at Daeseongdong, a border village between North and South Korea

Malaria continues to be a major health threat near the Demilitarized Zone (DMZ) that separates North and South Korea. Adult mosquitoes were collected from 20 July through 21 October, 2010 at Daeseongdong, a small village within the DMZ. Molecular techniques were used to identify Anopheles to species and for detection of Plasmodium vivax sporozoites in their head and thorax. Trap catches showed concordant peaks of Anopheles belenrae and An. kleini early in the study period and concordant peaks of An. pullus and An. sinensis later in the season. Three well defined peaks of the 107 sporozoite positive mosquitoes were observed: 34.6% were An. kleini, 23.4% were An. belenrae, 21.5% were An. sinensis, 19.6% were An. pullus, and 0.9% were An. lesteri. Estimation of the extrinsic incubation period from daily temperatures did not help identify preceding biting peaks of An. pullus and An. sinensis, when infection should have been acquired. We explore possible reasons for the sudden appearance and disappearance of sporozoite-infected mosquitoes, including the influx of infected mosquitoes from adjoining areas, and weather patterns. Regular surveillance for infected mosquitoes near border areas of the Republic of Korea may provide advance warning of increased malaria risk potential.

Intradermal immunization of mice with radiation-attenuated sporozoites of Plasmodium yoelii induces effective protective immunity

BackgroundIntravenous injection of mice with attenuated Plasmodium berghei sporozoites induces sterile immunity to challenge with viable sporozoites. Non-intravenous routes have been reported to yield poor immunity. Because intravenous immunization has been considered to be unacceptable for large scale vaccination of humans, assessment was made of the results of intradermal immunization of mice with Plasmodium yoelii, a rodent malaria parasite whose infectivity resembles that of human malaria.MethodsMice were immunized with two injections of isolated, radiation-attenuated P. yoelii sporozoites, either by intravenous (IV) or intradermal (ID) inoculation. In an attempt to enhance protective immunogenicity of ID-injections, one group of experimental mice received topical application of an adjuvant, Imiquimod, while another group had their injections accompanied by local "tape-stripping" of the skin, a procedure known to disrupt the stratum corneum and activate local immunocytes. Challenge of immunized and non-immunized control mice was by bite of sporozoite-infected mosquitoes. Degree of protection among the various groups of mice was determined by microscopic examination of stained blood smears. Statistical significance of protection was determined by a one-way ANOVA followed by Tukey's post hoc test.ResultsTwo intravenous immunizations produced 94% protection to mosquito bite challenge; intradermal immunization produced 78% protection, while intradermal immunization accompanied by "tape-stripping" produced 94% protection. There were no statistically significant differences in degree of protective immunity between immunizations done by intravenous versus intradermal injection.ConclusionsThe use of a sub-microlitre syringe for intradermal injections yielded excellent protective immunity. ID-immunization with large numbers of radiation-attenuated P. yoelii sporozoites led to levels of protective immunity comparable to those achieved by IV-immunization. It remains to be determined whether an adjuvant treatment can be found to substantially reduce the numbers of attenuated sporozoites required to achieve a strong protective immunity with as few doses as possible for possible extension to immunization of humans.

Plasmodium yoelii-Infected A. stephensi Inefficiently Transmit Malaria Compared to Intravenous Route

It was recently reported that when mosquitoes infected with P. berghei sporozoites feed on mice, they deposit approximately 100–300 sporozoites in the dermis. When we inoculate P. yoelii (Py) sporozoites intravenously (IV) into BALB/c mice, the 50% infectious dose (ID50) is often less than 3 sporozoites, indicating that essentially all Py sporozoites in salivary glands are infectious. Thus, it should only take the bite of one infected mosquito to infect 100% of mice. In human subjects, it takes the bite of at least 5 P. falciparum-infected mosquitoes to achieve 100% blood stage infection. Exposure to 1–2 infected mosquitoes only leads to blood stage infection in approximately 50% of subjects. If mosquitoes carrying Py sporozoites inoculate 100–300 sporozoites per bite, and 1 to 2 mosquito bites achieve 50% blood stage infection rates, then this would suggest that the majority of sporozoites inoculated by mosquitoes into the dermis are not responsible for a productive infection, or that a significant number of sporozoite-infected mosquitoes do not inoculate any sporozoites. The objective of this study was to determine if this is the case. We therefore studied the infectivity to mice of the bites of 1, 2, 4, or 5–8 Py-infected mosquitoes. The bite of one Py sporozoite-infected mosquito caused blood stage infection in 41.4% (12/29) of mice, two bites infected 66.7% (22/33), four bites infected 75% (18/24), and five to eight bites infected 100% (21/21). These findings demonstrate that inoculation of sporozoites by mosquito bite is much less efficient than IV inoculation of Py sporozoites by needle and syringe. Such data may have implications for determining the best route and dose of administration to humans of our attenuated P. falciparum sporozoite vaccine, the scientific basis of which is immunity by bites from irradiated infected mosquitoes, and suggest that the challenge is to develop a method of administration that approximates IV inoculation, not one that mimics mosquito bite.

Plasmodium falciparum: Effect of radiation on levels of gene transcripts in sporozoites

Humans immunized by the bites of irradiated Plasmodium falciparum (Pf) sporozoite-infected mosquitoes are protected against malaria. Radiation attenuates the sporozoites preventing them from fully developing and replicating in hepatocytes, but the effects of radiation on gene expression in sporozoites are unknown. We used RT-PCR (35 cycles of PCR followed by densitometry) to assess the expression of ten genes in Pf sporozoites, and in sporozoites irradiated with 15,000 cGy. Irradiation reduced expression substantially (>60%) of two DNA repair genes; moderately (30–60%) of PfUIS3, the Pf orthologue of PbUIS3, a gene up-regulated in Plasmodium berghei sporozoites and of a third DNA repair gene; and minimally (<30%) of the Pf18S ribosomal RNA, PfCSP, PfSSP2/TRAP, and PfCELTOS genes. Irradiation increased expression of PfSPATR minimally. PfLSA1 RNA was not detectable in sporozoites. These results establish that radiation of sporozoites affects gene expression levels and provide the foundation for studies to identify specific genes involved in attenuation and protective immunity.

Malaria Plasmodium agent induces alteration in the head proteome of their Anopheles mosquito host

Despite increasing evidence of behavioural manipulation of their vectors by pathogens, the underlying mechanisms causing infected vectors to act in ways that benefit pathogen transmission remain enigmatic in most cases. Here, 2-D DIGE coupled with MS were employed to analyse and compare the head proteome of mosquitoes (Anopheles gambiae sensu stricto (Giles)) infected with the malarial parasite (Plasmodium berghei) with that of uninfected mosquitoes. This approach detected altered levels of 12 protein spots in the head of mosquitoes infected with sporozoites. These proteins were subsequently identified using MS and functionally classified as belonging to metabolic, synaptic, molecular chaperone, signalling, and cytoskeletal groups. Our results indicate an altered energy metabolism in the head of sporozoite-infected mosquitoes. Some of the up-/down-regulated proteins identified, such as synapse-associated protein, 14-3-3 protein and calmodulin, have previously been shown to play critical roles in the CNS of both invertebrates and vertebrates. Furthermore, a heat shock response (HSP 20) and a variation of cytoarchitecture (tropomyosins) have been shown. Discovery of these proteins sheds light on potential molecular mechanisms that underlie behavioural modifications and offers new insights into the study of intimate interactions between Plasmodium and its Anopheles vector.

RE-INGESTION OF PLASMODIUM BERGHEI SPOROZOITES AFTER DELIVERY INTO THE HOST BY MOSQUITOES

Malaria-infected mosquitoes feeding on a mammalian host inject sporozoites into the skin to induce a malaria infection. The numbers of sporozoites ultimately able to reach the liver may be important determinants of the characteristics of the ensuing blood infection. Because feeding mosquitoes not only inject sporozoites into the host but concomitantly ingest blood to obtain their bloodmeal, some sporozoites are re-ingested by the feeding mosquito. We studied transmission of fluorescent Plasmodium berghei sporozoites injected into mice by Anopheles stephensi mosquitoes and found that the numbers of sporozoites re-ingested by mosquitoes are comparable to numbers previously reported to be delivered directly into mice. Thus, re-ingestion of sporozoites likely plays a significant role in transmission dynamics of malaria by mosquitoes, and may account for the failure of some sporozoite-infected mosquitoes to induce a blood infection.

Plasmodium falciparum sporozoites increase feeding-associated mortality of their mosquito hosts Anopheles gambiae s.l.

There is some evidence that pathology induced by heavy malaria infections (many oocysts) increases mortality of infected mosquitoes. However, there is little or no published evidence that documented changes in feeding behaviour associated with malaria infection also contribute to higher mortality of infected mosquitoes relative to uninfected individuals. We show here for the first time that, in a natural situation, infection by the sporozoites of the malaria parasite Plasmodium falciparum significantly reduced survival of blood-feeding Anopheles gambiae, the major vector of malaria in sub-Saharan Africa. To estimate feeding-associated mortality of infected mosquitoes, we compared the percentage of sporozoite infection in host-seeking mosquitoes caught before and after feeding. The infection rate was 12% for mosquitoes caught during the night as they were entering a tent to feed; however, only 7.5% of the surviving members of the same cohort caught after they had had the opportunity to feed were infected. Thus, Plasmodium falciparum sporozoites increased the probability of dying during the night-time feeding period by 37.5%. The increase in mortality was probably due to decreased efficiency in obtaining blood and by increased feeding activity of the sporozoite-infected mosquitoes that elicited a greater degree of defensive behaviour of hosts under attack.

Vector competence of Aedes hendersoni (Diptera: Culicidae) for La Crosse virus: lack of impaired function in virus-infected salivary glands and enhanced virus transmission by sporozoite-infected mosquitoes.

Previous research has shown Aedes hendersoni Cockerell to be an incompetent vector of La Crosse (LAC) virus because of a salivary gland escape (SGE) barrier; that is, the salivary glands are infected but the mosquito fails to transmit the virus orally. Intradermal probing behavior and ability to locate blood were studied in infected mosquitoes as indicators of salivary gland impairment to determine if the SGE barrier was due to virus-induced pathology of the salivary glands. No evidence of salivary gland impairment as a result of virus infection was detected in infected Ae. hendersoni. This was also true for Aedes triseriatus (Say), a competent vector of LAC virus, which was used as a control. However, coinfection of Ae. hendersoni with Plasmodium gallinaceum and LAC virus dramatically increased virus transmission (72 versus 8%), whereas transmission by coinfected Ae. triseriatus was not significantly affected. Possible causes for the SGE barrier in Ae. hendersoni are discussed.

ネズミマラリア Plasmodium yoelii nigeriensis に感染した Anopheles stephensi の吸血行動の変動

Using a cage-test method, reduction and restoration of behavioral activity of Plasmodium-infected Anopheles stephensi was demonstrated. Reduction in the ability to pass through a net barrier and the rate of blood-feeding during the period oocysts are present was notable. However, net passage and bait-feeding activities in sporozoite-infected mosquitoes following disappearance of the oocyst were entirely restored.

Plasmodium falciparum: Sporozoite boosting of immunity due to a T-cell epitope on a sporozoite vaccine

The impact of a malaria sporozoite vaccine may be enhanced if protective immunity elicited by the vaccine is boosted by natural exposure to sporozoites. For this to occur, a helper T lymphocyte epitope present on the vaccine must be shared by sporozoites. These studies show that T cells from mice immunized with R32tet 32, the Plasmodium falciparum sporozoite vaccine candidate, recognize an epitope of ⩽7 amino acids derived from the circumsporozoite protein repeat region of R32tet 32, as well as an epitope on the tet 32 fusion protein tail of R32tet 32. Exposure of R32tet 32 immunized animals to P. falciparum sporozoites elicits a significant secondary antibody response which suggests that humans who are immunized and respond to this vaccine may be boosted by field exposure to sporozoite infected mosquitoes.

SAFETY AND EFFICACY OF A RECOMBINANT DNA PLASMODIUM FALCIPARUM SPOROZOITE VACCINE

A recombinant DNA Plasmodium falciparum sporozoite vaccine produced in Escherichia coli (FSV-1) was tested in doses of 10 μg to 800 μg protein in fifteen volunteers. No serious adverse reactions occurred. Antibodies that reacted with P falciparumsporozoite antigens by enzyme-linked immunoassay developed in twelve of the volunteers. The highest antibody titres induced were similar to those resulting from lifelong natural exposure to sporozoite-infected mosquitoes. Postimmunisation serum samples from a majority of volunteers mediated the circumsporozoite (CS) precipitation reaction and inhibited sporozoite invasion of hepatoma cells in vitro. Serum from the three volunteers who received 800 μg doses reacted with the surface of sporozoites in an immunofluorescence assay. Six immunised volunteers receiving a fourth dose of FSV-1 and two non-immunised controls were challenged by bites of mosquitoes infected from cultured P falciparumgametocytes. Parasitaemia did not develop in the volunteer with the highest titre of CS antibodies, and parasitaemia was delayed in two other immunised volunteers. This study confirms that human beings can be protected by CS protein subunit vaccines and provides a framework for the further development and testing of more immunogenic sporozoite vaccines.

Increased biting rate and reduced fertility in sporozoite-infected mosquitoes.

Because salivary function and blood location are impaired in sporozoite-infected mosquitoes, we determined whether such pathology also could lead to an increased biting rate. For 5 days, we compared relative daily biting rates of Plasmodium gallinaceum sporozoite-infected mosquitoes (Aedes aegypti) and noninfected mosquitoes with an olfactometer. Mosquitoes then were exposed for 5 min to an anesthetized guinea pig. Infected mosquitoes exhibited a significant increase in olfactometer response which was also reflected in a decreased egg output. We conclude that if duration of contact with a host is limited, then infected mosquitoes may make more attempts at probing before being successful, and thus enhance transmission.

Increased intradermal probing time in sporozoite-infected mosquitoes.

Because malaria sporozoites destroy segments of the salivary glands of vector mosquitoes, we determined whether salivary function is impaired. Such pathology would result in a prolonged intradermal probing phase of feeding behavior, because the role of saliva is to help locate blood vessels. Indeed, non-infected Aedes aegypti mosquitoes probed for a shorter period than did either sporozoite-infected or saliva-deprived mosquitoes. Salivary apyrase activity is reduced to a third following maturation of sporozoites. Apyrase activity, normally, is confined to those regions invaded by sporozoites. Sporozoite-infected and non-infected mosquitoes produced equal volumes of saliva. We conclude that sporozoite infection impairs the vector's ability to locate blood vessels by affecting the quality of salivary product, thereby increasing potentially infective host contacts.