250 research outputs found
Microbial symbiosis and the control of vector-borne pathogens in tsetse flies, human lice, and triatomine bugs
No full textSymbiosis is a widespread biological phenomenon, and is particularly common in arthropods. Bloodsucking insects are among the organisms that rely on beneficial bacterial symbionts to complement their unbalanced diet. This review is focused on describing symbiosis, and possible strategies for the symbiont-based control of insects and insect-borne diseases, in three bloodsucking insects of medical importance: the flies of the genus Glossina, the lice of the genus Pediculus, and triatomine bugs of the subfamily Triatominae. Glossina flies are vector of Trypanosoma brucei, the causative agent of sleeping sickness and other pathologies. They are also associated with two distinct bacterial symbionts, the primary symbiont Wigglesworthia spp., and the secondary, culturable symbiont Sodalis glossinidius. The primary symbiont of human lice, Riesia pediculicola, has been shown to be fundamental for the host, due to its capacity to synthesize B-group vitamins. An antisymbiotic approach, with antibiotic treatment targeted on the lice symbionts, could represent an alternative strategy to control these ectoparasites. In the case of triatominae bugs, the genetic modification of their symbiotic Rhodococcus bacteria, for production of anti- Trypanosoma molecules, is an example of paratransgenesis, i.e. the use of symbiotic microorganism engineered in order to reduce the vector competence of the insect host
Evaluation of the in vitro expression of ATP binding-cassette (ABC) proteins in an Ixodes ricinus cell line exposed to ivermectin.
No full textBACKGROUND: Ticks are among the most important vectors of pathogens causing human and animal disease. Acaricides are used to control tick infestation, although there are increasing reports of resistance. Recently, over-expression of ATP-binding cassette (ABC) transporter proteins (P-glycoproteins, PgP) has been implicated in resistance to the acaricide ivermectin in the ticks Rhipicephalus (Boophilus) microplus and Rhipicephalus sanguineus sensu lato. Ixodid tick cell lines have been used to investigate drug resistance mechanisms. The aim of the present study was to evaluate expression of several PgPs in the Ixodes ricinus-derived cell line IRE/CTVM19 and to determine modulation of expression following treatment with ivermectin.
FINDINGS: IRE/CTVM19 cells were treated with different concentrations of ivermectin (0, 11, 22 or 33 μM) and incubated for 10 days. Evaluation of viability and relative expression of ABCB1, ABCB6, ABCB8 and ABCB10 genes were carried out at day 10 post treatment. Cell viability ranged between 84 % and 92 % with no significant differences between untreated and treated cells. qRT-PCR showed that ABC pump expression was not significantly modulated by ivermectin treatment. Expression of the ABCB8 PgP subfamily revealed a biphasic trend, based on the ivermectin concentration. ABCB6 and ABCB10 gene expression was not modulated by ivermectin treatment and ABCB1 expression was not detected.
CONCLUSIONS: This is the first report of PgP expression in an I. ricinus-derived tick cell line. Development of an in vitro model for the study of acaricide resistance mechanisms would greatly facilitate screening for drug resistance in ticks
Tick-Box for 3′-End Formation of Mitochondrial Transcripts in Ixodida, Basal Chelicerates and Drosophila
No full textAccording to the tRNA punctuation model, the mitochondrial genome (mtDNA) of mammals and arthropods is transcribed as large polycistronic precursors that are maturated by endonucleolytic cleavage at tRNA borders and RNA polyadenylation. Starting from the newly sequenced mtDNA of Ixodes ricinus and using a combination of mitogenomics and transcriptional analyses, we found that in all currently-sequenced tick lineages (Prostriata, Metastriata and Argasidae) the 3′-end of the polyadenylated nad1 and rrnL transcripts does not follow the tRNA punctuation model and is located upstream of a degenerate 17-bp DNA motif. A slightly different motif is also present downstream the 3′-end of nad1 transcripts in the primitive chelicerate Limulus polyphemus and in Drosophila species, indicating the ancient origin and the evolutionary conservation of this motif in arthropods. The transcriptional analyses suggest that this motif directs the 3′-end formation of the nad1/rrnL mature RNAs, likely working as a transcription termination signal or a processing signal of precursor transcripts. Moreover, as most regulatory elements, this motif is characterized by a taxon-specific evolution. Although this signal is not exclusive of ticks, making a play on words it has been named "Tick-Box", since it is a check mark that has to be verified for the 3′-end formation of some mt transcripts, and its consensus sequence has been here carefully characterized in ticks. Indeed, in the whole mtDNA of all ticks, the Tick-Box is always present downstream of nad1 and rrnL, mainly in non-coding regions (NCRs) and occasionally within trnL(CUN). However, some metastriates present a third Tick-Box at an intriguing site - inside the small NCR located at one end of a 3.4 kb translocated region, the other end of which exhibits the nad1 Tick-Box - hinting that this motif could have been involved in metastriate gene order rearrangement
Determinants of Adherence to a Gluten-Free Diet in Children with Celiac Disease and the Influence of the Methods Used to Assess It
No full textLifelong adherence to a gluten-free diet (GFD) is the cornerstone of management of celiac disease (CD), but adhering to a GFD can be hard. Although several factors are positively associated with adherence of pediatric CD patients to a GFD, it is unknown whether these are influenced by variability caused by the specific tool used to assess adherence to a GFD. Here, we aimed to evaluate how individual patient characteristics and dietary counselling by a trained dietitian influence adherence to a GFD in children with CD, as assessed by two validated questionnaires: the Biagi questionnaire and the Leffler short questionnaire adapted for pediatric patients. Some 139 children and adolescents were recruited in a cross-sectional, multicenter study. Concordance between the two questionnaires in defining adherence was fair (weighted Cohen's kappa coefficient 0.39, 95%CI 0.19-0.60). Upon regression analysis, having a cohabiting family member with CD, being of Italian origin, and receiving specialized dietary counselling during follow-up were found to positively influence stricter adherence to a GFD for children with CD. Neither questionnaire detected a significant relationship between adherence to a GFD and the presence of symptoms after gluten ingestion. This study provides important new data on the factors influencing GFD adherence in the pediatric population, and highlights the importance of dietician input and overcoming language and cultural barriers when educating patients
Engineering of the yeast Wickerhampmyces anomalus, symbiont of mosquito species relevant to public health, for paratransgenic control strategies.
No full textEngineering of the yeast Wickerhampmyces anomalus, symbiont of mosquito species relevant to public health, for paratransgenic control strategies
Presence of wolbachia in three hymenopteran species : diprion pini (Hymenoptera: Diprionidae), neodiprion sertifer (Hymenoptera: Diprionidae), and dahlbominus fuscipennis (Hymenoptera: Eulophidae)
No full textSawflies are important pests of various plant species. Diprion pini (L.) and Neodiprion sertifer (Geoffroy) (Hymenoptera: Diprionidae) are two of the most important sawfly pests in Italy, and both species are parasitized by the hymenopteran parasitoid Dahlbominus fuscipennis (Zetterstedt). Bacterial endosymbionts are currently studied for their high potential in strategies of biocontrol in a number of insect species. In this study, we investigated the presence of symbiotic bacteria (Wolbachia and Cardinium) in the three species of hymenoptera mentioned earlier, both in wild and laboratory populations. Although all samples were negative for the presence of Cardinium, 100% prevalence for Wolbachia was detected, as all examined individuals resulted to be PCR positive. Furthermore, 16S rDNA and ftsZ gene sequencing indicated that all individuals from the three hymenopteran species are infected by a single Wolbachia strain. Additionally, we report the presence of gynandromorphic individuals in D. pini, both in wild and laboratory-reared populations. Heat treatments on D. pini colonies removed the Wolbachia symbionts, but they also prevented the development of adults
Gene silencing through RNAi and antisense Vivo-Morpholino increases the efficacy of pyrethroids on larvae of Anopheles stephensi
No full textBACKGROUND:
Insecticides are still at the core of insect pest and vector control programmes. Several lines of evidence indicate that ABC transporters are involved in detoxification processes against insecticides, including permethrin and other pyrethroids. In particular, the ABCG4 gene, a member of the G subfamily, has consistently been shown to be up-regulated in response to insecticide treatments in the mosquito malaria vector Anopheles stephensi (both adults and larvae).
METHODS:
To verify the actual involvement of this transmembrane protein in the detoxification process of permethrin, bioassays on larvae of An. stephensi, combining the insecticide with a siRNA, specifically designed for the inhibition of ABCG4 gene expression were performed. Administration to larvae of the same siRNA, labeled with a fluorescent molecule, was effected to investigate the systemic distribution of the inhibitory RNA into the larval bodies. Based on siRNA results, similar experiments using antisense Vivo-Morpholinos (Vivo-MOs) were effected. These molecules, compared to siRNA, are expected to guarantee a higher stability in environmental conditions and in the insect gut, and present thus a higher potential for future in-field applications.
RESULTS:
Bioassays using two different concentrations of siRNA, associated with permethrin, led to an increase of larval mortality, compared with results with permethrin alone. These outcomes confirm that ABCG4 transporter plays a role in the detoxification process against the selected insecticide. Moreover, after fluorescent labelling, it was shown the systemic dissemination of siRNA in different body districts of An. stephensi larvae, which suggest a potential systemic effect of the molecule. At the same time, results of Vivo-MO experiments were congruent with those obtained using siRNA, thus confirming the potential of ABCG4 inhibition as a strategy to increase permethrin susceptibility in mosquitoes. For the first time, Vivo-MOs were administered in water to larvae, with evidence for a biological effect.
CONCLUSIONS:
Targeting ABCG4 gene for silencing through both techniques resulted in an increased pyrethroid efficacy. These results open the way toward the possibility to exploit ABCG4 inhibition in the context of integrated programmes for the control An. stephensi mosquitoes and malaria transmission
Molecular and serological evidence for the circulation of the tick symbiont Midichloria (Rickettsiales: Midichloriaceae) in different mammalian species
No full textBackground: The Midichloriaceae is a novel family of the order Rickettsiales, that encompasses intracellular bacteria associated with hard ticks (Ixodidae) and other arthropods. The most intensively investigated member of this family is Midichloria mitochondrii, a symbiotic bacterium of the sheep tick Ixodes ricinus, characterized by the capacity of multiplying inside the mitochondria. A recent study suggested that these bacteria might be inoculated into the human host during the tick bite. The purpose of this study was to determine the potential infectivity of Midichloria bacteria for non-human animals exposed to the risk of tick bite. Methods: Blood from horses, cattle, sheep and dogs exposed to the risk of tick bite was included in this study. DNAs were extracted, and amplified using 16S ribosomal RNA primers conserved in the Midichloria genus. Furthermore, sera from dogs exposed to the risk of tick bite were analyzed in order to evaluate the presence of antibodies against the recombinant flagellar protein (rFliD) from M. mitochondrii using an ELISA test. Results: Here we present two lines of evidence that support the possibility that bacteria from the genus Midichloria are inoculated into vertebrate hosts during a tick bite: (i) a direct evidence, i. e. the detection of circulating DNA from bacteria related with M. mitochondrii, in the blood of vertebrates exposed to tick parasitism; (ii) a further indirect evidence, i. e. the presence of antibodies against an antigen from M. mitochondrii in dogs exposed to the risk of tick bite. It is interesting to note that variability was detected in the Midichloria gene sequences recovered from positive animals, and that some of these sequences were identical to those generated from tick-associated Midichloria. Conclusions: Based on the results, and on the overall information so far published on the genus Midichloria, we suggest that these bacteria are likely to represent a novel group of vector-borne agents, with the potential of infecting mammalian hosts. Whether inoculation of Midichloria bacteria could cause a true infection and pathological alteration in mammalian hosts is still to be determined. Surely, results emphasize the relevance of Midichloria bacteria in investigations on tick immunology and tick-bite markers
MONITORING OF LEISHMANIA TARENTOLAE AND LEISHMANIA INFANTUM IN SAND FLIES AND SYNANTHROPIC REPTILES OF NORTHERN ITALY: EMERGING EPIDEMIOLOGICAL SCENARIOS?
No full textVector-borne diseases represent one-sixth of all infectious diseases and cause over one million deaths annually. Insect vectors can transmit pathogenic viruses, bacteria, and parasites. Among parasitic infections, leishmaniases, caused by protozoan parasites of the genus Leishmania, rank second only to malaria in terms of mortality. These parasites are transmitted to vertebrate hosts through the bites of infected sand flies (Diptera: Psychodidae: Phlebotominae). In Italy, leishmaniasis is primarily caused by Leishmania infantum, which is responsible for both the cutaneous and visceral forms in humans, as well as for the canine form. Since the 1990s, leishmaniasis, caused by L. infantum, became endemic in northern Italy. This has been associated with the movement of infected dogs and the expansion of sand fly populations due to climate change. The main vectors of L. infantum in Italy are Phlebotomus perniciosus and Phlebotomus perfiliewi.
Another Leishmania species found in Italy is Leishmania tarentolae, which primarily infects reptiles and is transmitted by the sand fly Sergentomyia minuta. L. tarentolae is considered non-pathogenic to mammals and exposure to this parasite may elicit protective responses against pathogenic Leishmania infections. For this reason, screening for L. tarentolae is essential in leishmaniasis-endemic areas. However, little is known about L. tarentolae ecology and distribution in Italy, and S. minuta is often overlooked in Leishmania epidemiological studies. Despite this, S. minuta sand flies have been reported to feed on human blood and have tested positive for L. infantum DNA in molecular screenings. Synanthropic reptiles may also serve as reservoirs for Leishmania parasites that are pathogenic to humans. These reptiles are well-adapted to human environments and regularly interact with domestic animals, potentially exposing dogs and cats that prey on them to Leishmania parasites.
Considering this scenario, my PhD project had two main objectives:
i) To develop a highly sensitive and specific assay for the simultaneous and differential detection of DNA of the non-pathogenic L. tarentolae and the pathogenic L. infantum, in sand flies and dog blood;
ii) To conduct a screening of L. tarentolae and L. infantum in sand flies and synanthropic reptiles in northern Italy, in an area where leishmaniasis is an emerging disease.
The results of my PhD were presented in three scientific articles:
Article 1 - Research paper [submitted to Scientific Reports]
This study focused on developing a highly sensitive and specific assay for the simultaneous and differential detection of the DNA of the protozoan parasites L. tarentolae and L. infantum. Accurately detecting both species in sand flies and vertebrate hosts is crucial for understanding the epidemiology of the investigated area. Traditional diagnostic methods often suffer from limitations in sensitivity, specificity, and quantification, especially when dealing with complex biological samples that contain low concentrations of parasite DNA. To address these challenges, we used droplet digital PCR (ddPCR) to develop the assay. The method was validated using dog blood spiked with DNA from various isolates of both Leishmania species, as well as DNA extracted from wild-caught sand flies previously tested for Leishmania presence via qPCR. The method demonstrated high sensitivity, specificity, and no substantial cross-reactions.
Article 2 - Research paper [submitted to PLOS Neglected Tropical Diseases]
In this study, I investigated the biodiversity of sand flies and the prevalence of Leishmania spp. in sand flies and synanthropic reptiles in the Bergamo district, in an area where new cases of autochthonous canine leishmaniasis have recently been reported. The district is densely populated and includes numerous peri-urban areas where domestic, farm, and wild animals coexist. Few studies have focused on sand fly populations in the Bergamo district, and none have explored Leishmania prevalence in these vectors. I conducted a two-year survey of sand flies in peri-urban sites to better understand the area's sand fly biodiversity. Additionally, I performed a molecular screening for Leishmania spp. on female sand flies, as well as on the blood and faeces of synanthropic lizards, using conventional PCR and also the ddPCR method described in Article 1. Sand flies of the species P. perniciosus, Phlebotomus neglectus, and S. minuta were collected, and females of all three species tested positive for L. tarentolae and L. infantum DNA. Reptile samples also tested positive for both L. tarentolae and L. infantum DNA. This study was made possible thanks to the training I received from colleagues in the Parasitology and Micology laboratory of the University of Bari Aldo Moro.
Article 3 - Review paper [published in Parasites & Vectors]
I spent my PhD in the EntoPar laboratory, in a research group where one of the expertise is the study of immunological aspects of L. tarentolae and the biotechnological applications of L. tarentolae. I was therefore able to participate in the drafting of a review regarding L. tarentolae biotechnological applications, which presented also an overview of the biological and epidemiological aspects of the parasite. The review was published with the colleagues of the Parasitology and Micology laboratory of the University of Bari Aldo Moro.
In conclusion, the results obtained during my PhD work revealed that the Bergamo district, which can be considered a model area for Lombardy and northern Italy for the study of leishmaniasis in newly and/or recently endemic zones, is characterized by the presence of sand flies and the circulation of both L. tarentolae and L. infantum in vectors and in synanthropic reptiles. The findings from my PhD research contributed to the advancement of molecular screening methods for Leishmania and provided data on the prevalence and distribution of Leishmania parasites, particularly in less-studied vectors and hosts. In addition, my research highlighted the potential emergence of a new epidemiological scenario for leishmaniasis in the context of northern Italy. Moreover, the detection method I developed will contribute to realize screenings in areas where leishmaniasis is endemic and L. tarentolae and L. infantum occur in sympatry
A new strain of wolbachia in an alpine population of the viviparous oreina cacaliae (Coleoptera: Chrysomelidae)
No full textMicrobial symbionts played a central role in insect evolution.Oreinacacaliae(Schrank, 1785) (Coleoptera: Chrysomelidae) is a rare example of a viviparous insect, able to feed on toxic plants and sequester toxic compounds. In the current study, the microbiota associated withO.cacaliae was characterized using a culture-independent approach, targeting the 16S rRNA bacterial gene. The obtained 16S rRNA gene sequences were analyzed and identiÞed at different taxonomic levels. Wolbachiawas the dominant bacterium, both in male and female (100 and 91.9%, respectively) individuals; the detected Wolbachiawas described as a new sequence type based on multilocus sequence typing ( WolbachiaST375 Ocac_A_wVdO). After phylogenetic analyses, WolbachiaST375 Ocac_A_wVdO was attributed to the supergroup A. Immunoßuorescence assays and electron mi-croscopy conÞrmed the presence ofWolbachiawithin O.cacaliaeoocytes, conÞrming its transovarial transmission in this species. Representatives of six species of Oreina were tested for the presence of Wolbachiathrough speciÞc polymerase chain reaction, and a dendrogram was generated for these species based on coxI gene sequences. The Wolbachiaharbored by different species of Oreina were characterized by multilocus sequence typing. Five out of the six examined Oreina species were positive for Wolbachia, with four of these harboring the same sequence type
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