Effects of combined drug treatments on Plasmodium falciparum : in vitro assays with doxycycline, ivermectin and efflux pump inhibitors

There is great concern regarding the rapid emergence and spread of drug-resistance in Plasmodium falciparum, the parasite responsible for the most severe form of human malaria. Parasite populations resistant to some or all the currently available antimalarial treatments are present in different world regions. Considering the need for novel and integrated approaches to control malaria, combinations of drugs were tested on P. falciparum. The primary focus was on doxycycline, an antibiotic that specifically targets the apicoplast of the parasite. In combination with doxycycline, three different drugs known to inhibit efflux pumps (verapamil, elacridar and ivermectin) were tested, with the assumption that they could increase the intracellular concentration of the antibiotic and consequently its efficacy against P. falciparum. We emphasize that elacridar is a third-generation ABC transporters inhibitor, never tested before on malaria parasites. In vitro experiments were performed on asexual stages of two strains of P. falciparum, chloroquine-sensitive (D10) and chloroquineresistant (W2). Incubation times on asynchronous or synchronous cultures were 72h or 96h, respectively. The antiplasmodial effect (i.e. the IC50) was determined by measuring the activity of the parasite lactate dehydrogenase, while the interaction between drugs was determined through combination index (CI) analyses. Elacridar achieved an IC50 concentration comparable to that of ivermectin, approx. 10-fold lower than that of verapamil, the other tested ABC transporter inhibitor. CI results showed synergistic effect of verapamil plus doxycycline, which is coherent with the starting hypothesis, i.e. that ABC transporters represent potential targets, worth of further investigations, towards the development of companion molecules useful to enhance the efficacy of antimalarial drugs. At the same time, the observed antagonistic effect of doxycycline in combination with ivermectin or elacridar highlighted the importance of drug testing, to avoid the de-facto generation of a sub-dosage, a condition that facilitates the development of drug resistance

Molecular screening for bacterial pathogens in ticks (Ixodes ricinus) collected on migratory birds captured in northern Italy

Migratory birds have an important role in transporting ticks and associated tick-borne pathogens over long distances. In this study, 2,793 migratory birds were captured by nets in a ringing station, located in northern Italy, and checked for the presence of ticks. Two-hundred and fifty-one ticks were identified as nymphs and larvae of Ixodes ricinus (Linnaeus, 1758) and they were PCR-screened for the presence of bacteria belonging to Borrelia burgdorferi sensu lato, Rickettsia spp., Francisella tularensis and Coxiella burnetii. Four species of Borrelia (B. garinii, B. afzelii, B. valaisiana and B. lusitaniae) and three species of Rickettsia (R. monacensis, R. helvetica and Candidatus Rickettsia mendelii) were detected in 74 (30%) and 25 (10%) respectively out of 251 ticks examined. Co-infection with Borrelia spp. and Rickettsia spp. in the same tick sample was encountered in 7 (7%) out of the 99 infected ticks. We report for the first time the presence of Candidatus Rickettsia mendelii in I. ricinus collected on birds in Italy. This study, besides confirming the role of birds in dispersal of I. ricinus, highlights an important route by which tick-borne pathogens might spread across different countries and from natural environments towards urbanised areas

SYMBIOTIC CHIMERAS: NOVEL IMMUNOLOGICAL PROPERTIES OF ASAIA SYMBIONTS ENGINEERED TO BE COVERED BY THE WOLBACHIA SURFACE PROTEIN

Vector-borne diseases represent one-sixth of all infectious diseases and cause more than 1,000,000 deaths every year. The World Health Organization (WHO) proposes the adoption of sustainable integrated vector management, which includes strategies such as environmental remediation, information and education of the population, but also the integration of classical chemicals (e.g. insecticides) with non-chemical control methods for the control of arthropod vectors. Among these strategies, the symbiotic control (SC), which exploits microorganism symbionts of the insects, is getting more and more attention as it is particularly promising for the control of vector-borne diseases. Particularly, in this thesis, I studied the potentiality of bacteria belonging to two genera: Asaia and Wolbachia. The former includes acetic acid bacteria, symbionts of many insects such as mosquitoes and recently proposed as good candidates for paratransgenesis-based control strategies. The latter includes symbionts of arthropods and nematodes, with the capability to stimulate innate immune responses in mosquitoes with a reduction of their vectorial capability. We thus engineered the bacterium Asaia to express the Wolbachia surface protein (WSP), a potential inductor of innate immunity, derived from Wolbachia infecting the nematode Dirofilaria immitis (AsaiaWSP). My project has two main aims: i) to test the capability of the bacterium AsaiaWSP to stimulate the immune system of mosquitoes and thus to interfere with D. immitis development after infection; ii) to verify if AsaiaWSP is able to induce a macrophage polarization during the immune response towards the M1/Th1 phenotype and if this polarization could determine an anti-Leishmania effect. The obtained results to achieve these aims have been summarized in two articles: Article 1 The first study, here presented, consolidates the previous evidence on the immune-stimulating property of WSP. In fact, WSP from Wolbachia of D. immitis was shown to stimulate the immune response in mosquitoes and mammals (humans, rodents, dogs) in vitro, but here for the first time an engineered bacterium expressing WSP was tested. AsaiaWSP activated the expression of immune genes coding for effector molecules in Ae. aegypti and An. stephensi mosquitoes. Once verified the stimulation of the immune system of the mosquitoes, the capability to inhibit the development of the nematode D. immitis, ethiological agent of dirofilariasis, in the vector Ae. aegypti was investigated. We obtained a first evidence of an inhibition of D. immitis larval development after the infection of mosquitoes with the engineered bacterium AsaiaWSP. Article 2 In the second paper, I focused my attention on leishmaniases, vector-borne diseases widespread in more than one hundred countries in tropical, sub-tropical and temperate zones and caused by the protozoan parasite Leishmania. I investigated the capability of the chimeric bacterium AsaiaWSP to polarize the immune response towards the M1/Th1 phenotype, which is protective for the host; in fact, macrophage polarization towards the M1/Th1 or M2/Th2 side is crucial for the outcome of the visceral leishmaniasis. The chimeric bacterium AsaiaWSP acted as a polarizing agent, stimulating the phagocytosis and inducing the release of M1/Th1 cytokines, ROS and the expression of iNOS. Then, AsaiaWSP determined an anti-leishmanial effect with a reduction of the number of intracellular parasites. In conclusion the modified bacterium Asaia, here proposed, appears as a promising candidate for paratransgenesis-based control strategies for the control of vector-borne diseases in general, and as a successful immunomodulator, which could be used in combination with the classical chemotherapeutic agents for the treatment of leishmaniases and other M1-impaired diseases

Immunological properties of engineered Asaia symbionts: implications for the control of mosquito-borne diseases

The genus Wolbachia collects intracellular bacteria, which commonly infect arthropods. It is well-known that Wolbachia is capable of inducing the production of reactive oxygen species and innate immune effectors in mosquitoes; this immune stimulation is likely involved in the reduction of arthropod vector capacity, toward a variety of pathogens. In fact, the presence of Wolbachia has been associated with the inhibition of virus and parasites development in arthropods. Among Wolbachia molecules, the Wolbachia surface protein (WSP) has been shown to induce immune responses in mosquito cell lines, and Th1 polarization in mammalian hosts, after stimulation of innate-immunity receptors. Due to the intrinsic features of Wolbachia bacterium e.g. not culturable in cell-free media, its use in vector-borne diseases control is limited; for this reason we realized a chimeric bacterium of the genus Asaia, symbiont of Diptera vectors, to express WSP protein. The immunological effects of this engineered bacterium have been tested evaluating the stimulation of the innate immune response in mosquito cell lines and in vivo in presence of Asaia producing WSP. Investigations on the immunological properties of WSP in mosquitoes could generate novel insights into the insect-bacterium relationships, thus providing novel ideas toward the development of tools for the control of mosquito-borne diseases

Mosquito Trilogy: Microbiota, Immunity and Pathogens, and Their Implications for the Control of Disease Transmission

In mosquitoes, the interaction between the gut microbiota, the immune system, and the pathogens that these insects transmit to humans and animals is regarded as a key component toward the development of control strategies, aimed at reducing the burden of severe diseases, such as malaria and dengue fever. Indeed, different microorganisms from the mosquito microbiota have been investigated for their ability to affect important traits of the biology of the host insect, related with its survival, development and reproduction. Furthermore, some microorganisms have been shown to modulate the immune response of mosquito females, significantly shaping their vector competence. Here, we will review current knowledge in this field, focusing on i) the complex interaction between the intestinal microbiota and mosquito females defenses, both in the gut and at humoral level; ii) how knowledge on these issues contributes to the development of novel and targeted strategies for the control of mosquito-borne diseases such as the use of paratransgenesis or taking advantage of the relationship between Wolbachia and mosquito hosts. We conclude by providing a brief overview of available knowledge on microbiota-immune system interplay in major insect vectors

Characterization of fungal community of black soldier fly (Hermetia illucens) larvae reared for large-scale feed production

Population, urbanization and rising incomes are expected to double the demands for livestock products in the developing countries. FAO indicates insect-derived flour such innovative protein source and the black soldier fly (Hermetia illucens) as one of the most promising species of insect for a production of large-scale feed. H. illucens is a fly (Diptera) member of the Stratiomyidae family, which is native from America, but it is now widespread in tropical and warmer temperate regions. The aim of the present research was to study the intestinal fungal community of black soldier fly larvae fed on vegetable substrates and to characterize any toxin-producing fungi. The safety of using insects as feed ingredient is one of the priorities for both animals and humans as final consumers in the food chain; in fact the use of insects or insect-derived flour incorporated in the feed may be a risk of potential zoonoses, pathogens, toxins and contamination with heavy metals. The research has been focused on the isolation of yeasts and moulds from the intestinal content of larvae, fed on heat treated vegetable waste, and on the typing of isolates by ITS-RFLP and sequencing of the 26S rRNA D1/D2 domain. The isolated yeasts which, according to literature sources, were producers of toxins, were then used for inhibition assays on sensitive yeast strains to test their killer phenotype. Finally, the variability of the fungal community of each sample was also evaluated by metabarcoding using Next Generation Sequencing 454 technology of fungal ribosomal ITS region

The killer yeast Wickerhamomyces anomalus associated to the sand fly Phlebotomus perniciosus: possible implication against the pathogen Leishmania spp.

While arthropod-associated bacteria are the focus of several research programs aimed at developing strategies to control vector-borne diseases, such as malaria, dengue, and trypanosomiasis, arthropod-associated yeasts and their killer toxins have not yet been deeply investigated. I focused my work on the sand fly\ua0Phlebotomus perniciosus, the main vector of human and canine leishmaniasis in the western Mediterranean area, with the aim of investigating if a yeast associated with this arthropod could exert inhibitory/killing activity against the pathogen Leishmania spp. We isolated and phylogenetically characterized strains of the yeast\ua0species Wickerhamomyces anomalus, from laboratory-reared adults and larvae of P. perniciosus. The yeast W. anomalus is renowned for its potential in biocontrol and has been isolated from different substrates and habitats including several mosquito species, in witch it showed the ability to produce a killer toxin active against other yeast species. In the present research, the isolated yeast strains of the species W. anomalus were tested against sensitive yeast strains, demonstrating its killer phenotype. Finally, in order to explore the possibility that this yeast could exert inhibitory/killing activity against pathogens, I tested the in vitro activity of W. anomalus strains against Leishmania infantum. This study offers the basis for the development of a method for vector-borne disease control that is environment-friendly and safe for human health. The use of yeast symbionts with antimicrobial proprieties could be included in the integrated approach for the control of leishmaniases, a worldwide re-emerging public health problem

Silencing ABCG4 transporter gene to increase insecticide efficacy against mosquito larvae

According to WHO, more than half of the world population lives under the risk of contracting illnesses carried by mosquitoes, and 3.2 billion people are at risk of getting malaria. Vector control is one of the main components in vector-borne diseases control programs, but resistance insurgence is nowadays an emerging problem threatening global efforts. A novel strategy for pest management implies the use of RNA interference against various gene targets in different arthropod species. Here, I performed the gene silencing of an ATP-binding cassette (ABC) transporter, ATP-dependent efflux pumps located in the cellular membranes both in prokaryotes and eukaryotes. These proteins are associated with trans-membrane transport of several substrates, including toxic compounds. In previous studies we have demonstrated the involvement of ABCs in larvae of Anopheles stephensi in the defence against permethrin. In particular, three families of ABC transporters (ABC-B, ABC-C and ABC-G) are implied through up and down-regulation at different time points, with maximum up-regulation of the ABCG4 gene. In my study, I treated three instar larvae of An. stephensi with permethrin alone or in combination with siRNAs at different concentrations and I evaluated larval mortality and the relative expression ABCG4 gene at different time points. My preliminary results demonstrate that RNAi on ABCG4 is effective and dose-dependent and significantly decrease the relative expression of the gene after 48h. Mortality results higher at 48h at higher siRNA concentration. This data show that silencing of ABC transporters through RNAi could be an effective and reliable method for increase chemicals efficacy against mosquitoes, allowing reduction of insecticide dosage and environmental pollution