Fulvio Esposito

Bacteria of the genus Asaia stably associate with Anopheles stephensi, an Asian malarial mosquito vector

Here, we show that an α-proteobacterium of the genus Asaia is stably associated with larvae and adults of Anopheles stephensi, an important mosquito vector of Plasmodium vivax, a main malaria agent in Asia. Asaia bacteria dominate mosquito-associated microbiota, as shown by 16S rRNA gene abundance, quantitative PCR, transmission electron microscopy and in situ-hybridization of 16S rRNA genes. In adult mosquitoes, Asaia sp. is present in high population density in the female gut and in the male reproductive tract. Asaia sp. from An. stephensi has been cultured in cell-free media and then transformed with foreign DNA. A green fluorescent protein-tagged Asaia sp. strain effectively lodged in the female gut and salivary glands, sites that are crucial for Plasmodium sp. development and transmission. The larval gut and the male reproductive system were also colonized by the transformed Asaia sp. strain. As an efficient inducible colonizer of mosquitoes that transmit Plasmodium sp., Asaia sp. may be a candidate for malaria control.

A community-based programme to provide prompt and adequate treatment of presumptive malaria in children

A community-based programme to ensure prompt and adequate treatment of presumptive episodes of clinical malaria in children has been established in a rural province of Burkina Faso. The implementation strategy was based on training a core group of mothers in every village and supplying community health workers with essential antimalarial drugs specially packed in age-specific bags containing a full course of treatment. Drugs were sold under a cost-recovery scheme. The programme was run in 1994 by the national malaria control centre (CNLP), and in 1995 it was developed to the provincial health team (PHT). Knowledge and awareness of malaria increased with the intervention. Drug consumption by age group was compatible with the distribution of disease, and no major problem of misuse emerged. The actual implementation costs of the intervention were US

An estimation of Toxocara canis prevalence in dogs, environmental egg contamination and risk of human infection in the Marche region of Italy.

0.06 per child living in the province. An evaluation of the impact of the intervention on the severity of malaria, using routine data from the health information system and taking as an indicator the proportion of malaria cases which were recorded as severe in health centres, was performed. In 1994, when the intervention was implemented on a provincial scale by CNLP, this proportion was lower than the average of the 4 preceding years (3.7% vs. 4.9%). In 1995, when the programme was implemented by the PHT, the proportion of severe cases was lower in health centres achieving a programme coverage of > or = 50% in their catchment area compared with the others (4.2% vs. 6.1%). Our experience shows that a low-cost, community-based intervention aimed at providing children with prompt and adequate treatment of presumptive episodes of clinical malaria is feasible, and suggests that it may lead to a reduction in the morbidity from severe malaria.

Prevalence and levels of antibodies to the circumsporozoite protein of plasmodium falciparum in an endemic area and their relationship to resistance against malaria infection

The human risk of infection with larvae of Toxocara canis was estimated in people from the Marche region of Italy. This region includes both urban and rural areas and its inhabitants frequently keep dogs for company, hunting, as guardians or shepherds. T. canis infection was diagnosed in 33.6% out of 295 dogs examined. Nearly half of the dogs (48.4%) living in rural areas were found T. canis positive, compared to about one-quarter of the dogs (26.2%) from urban areas. Analysis by provenance and role revealed the highest infection rate in rural hunting dogs (64.7%) and the lowest in urban companion dogs (22.1%). According to questionnaire data, the peridomestic environment, i.e. gardens and dog pens, is the most important defecation site in both rural and urban areas. Since over 40% of the dogs who defecate in dog pens are infected and 24% of urban and 47% of rural dogs who leave their droppings in the house surroundings harbour the parasite, it is clear that these environments may constitute sites of zoonotic risk. Our analysis of soil samples from 60 farms confirmed the high contamination level, revealing positive soil samples in more than half of the farms. Substantial egg contamination was also found in urban areas, as 3/6 parks examined were Toxocara spp. positive. Finally, our serological findings indicate that human infection actually occurs in the area: 7 out of 428 adults examined (1.6%) had very high levels of antibodies to T. canis antigen, suggesting a previous contact with the Larva migrans of the nematode.

Paternal transmission of symbiotic bacteria in malaria vectors.

A study on malaria transmission, prevalence of infection and anti-sporozoite antibodies was carried out in Burkina Faso (West Africa). The prevalence and the levels of antibodies to (NANP)3 were found to be related to the entomological sporozoite inoculation rates measured at the same time in a defined area. The major inducer of anti-(NANP)3 antibody production under field conditions is sporozoite inoculation by infected mosquitoes. Levels of antibodies to (NANP)3 vary considerably with age and transmission season. High levels of anti-(NANP)3 antibodies raised under field conditions might offer protection against small inocula of sporozoites.

Mosquito-Bacteria Symbiosis: The Case of Anopheles gambiae and Asaia

Summary Bacteria of the genus Asaia are associated with different species of malaria vectors and are located in the midgut, salivary glands and reproductive organs of female and male mosquitoes. Based on current evidence, the spreading of these bacteria in mosquito populations occurs through different mechanisms: co-feeding, sexual mating, and maternal transmission [1,2]. Even though paternal transmission of insect symbionts to progeny is not commonplace, the presence of Asaia in the male reproductive organs makes this additional transmission route worth being investigated. Here, we show that male-borne Asaia are transferred to females during the mating of Anopheles stephensi mosquitoes. Subsequently, the bacteria acquired by the female are vertically transmitted to the progeny. It would thus be possible to use male mosquitoes, which do not bite, to spread Asaia strains interfering with malaria transmission.

First detection of spotted fever group rickettsiae in Ixodes ricinus from Italy

The symbiotic relationship between Asaia, an α-proteobacterium belonging to the family Acetobacteriaceae, and mosquitoes has been studied mainly in the Asian malaria vector Anopheles stephensi. Thus, we have investigated the nature of the association between Asaia and the major Afro-tropical malaria vector Anopheles gambiae. We have isolated Asaia from different wild and laboratory reared colonies of A. gambiae, and it was detected by PCR in all the developmental stages of the mosquito and in all the specimens analyzed. Additionally, we have shown that it localizes in the midgut, salivary glands and reproductive organs. Using recombinant strains of Asaia expressing fluorescent proteins, we have demonstrated the ability of the bacterium to colonize A. gambiae mosquitoes with a pattern similar to that described for A. stephensi. Finally, fluorescent in situ hybridization on the reproductive tract of females of A. gambiae showed a concentration of Asaia at the very periphery of the eggs, suggesting that transmission of Asaia from mother to offspring is likely mediated by a mechanism of egg-smearing. We suggest that Asaia has potential for use in the paratransgenic control of malaria transmitted by A. gambiae.

Different mosquito species host Wickerhamomyces anomalus (Pichia anomala): perspectives on vector-borne diseases symbiotic control

Ixodes ricinus from Italy were examined for the first time to detect whether rickettsiae were present. Using molecular methods, we detected three different spotted fever group rickettsiae, including Rickettsia helvetica. Our results raise the possibility that bacteria other than R. conorii are involved in rickettsial diseases in Italy.

Synthesis and immunological characterization of 104-mer and 102-mer peptides corresponding to the N- and C-terminal regions of the Plasmodium falciparum CS protein

The genetic manipulation of the microbial community associated with hematophagus insects is particularly relevant for public health applications. Within mosquito populations, this relationship has been overlooked until recently. New advances in molecular biotechnology propose the genetic manipulation of mosquito symbionts to prevent the transmission of pathogens to humans by interfering with the obligatory life cycle stages within the insect through the use of effector molecules. This approach, defined as ‘paratransgenesis’, has opened the way for the investigation and characterization of microbes residing in the mosquito body, particularly those localised within the gut. Some interesting bacteria have been identified as candidates for genetic modification, however, endosymbiotic yeasts remain largely unexplored with little information on the symbiotic relationships to date. Here we review the recent report of symbiotic relationship between Wickerhamomyces anomalus (Pichia anomala) and several mosquito vector species as promising methods to implement control of mosquito-borne diseases.

The yeast Wickerhamomyces anomalus (Pichia anomala) inhabits the midgut and reproductive system of the Asian malaria vector Anopheles stephensi

We investigated the immunogenicity and the conformational properties of the non-repetitive sequences of the Plasmodium falciparum circumsporozoite (CS) protein. Two polypeptides of 104 and 102 amino acids long, covering, respectively, the N- and C-terminal regions of the CS protein, were synthesized using solid phase Fmoc chemistry. The crude polypeptides were purified by a combination of size exclusion chromatography and RP-HPLC. Sera of mice immunized with the free polypeptides emulsified in incomplete Freunds adjuvant strongly reacted with the synthetic polypeptides as well as with native CS protein as judged by ELISA and IFAT assays. Most importantly, these antisera inhibited the sporozoite invasion of hepatoma cells. In addition, sera derived from donors living in a malaria endemic area recognized the CS 104- and 102-mers. Conformational studies of the CS polypeptides were also performed by circular dichroism spectroscopy showing the presence of a weakly ordered structure that can be increased by addition of trifluoroethanol. The obtained results indicate that the synthetic CS polypeptides and the natural CS protein share some common antigenic determinants and probably have similar conformation. The approach used in this study might be useful for the development of a synthetic malaria vaccine.