Development and use of innovative tools in medical entomology and malacology : molecular and protein approach

Cette thèse se divise en deux grands axes. Le premier concerne l’utilisation du MALDI-TOF MS en malacologie en mettant au point un protocole adapté à l’identification des différentes espèces de mollusques y compris les hôtes intermédiaires des schistosomes et le deuxième est consacré à d’autres axes en entomologie médicale. En collaboration avec l’équipe de parasitologie de Dakar, nous avons mis au point un protocole MALDI-TOF MS qui permet l'identification rapide des mollusques d'eau douce congelés et un autre protocole adapté aux mollusques conservés dans l'éthanol 70%. Dans un deuxième temps, nous avons rapporté pour la première fois en France un cas d’infestation humaine par les punaises d’hirondelles (Cimex hirundinis). Nous avons, par la suite, utilisé le MALDI-TOF MS pour identifier cette espèce et évaluer l’efficacité de l’approche pour distinguer les nymphes des adultes. Également, nous avons détecté pour la première fois la nouvelle souche de Wolbachia (Wolbachia massiliensis) infectant les Cimex hirundinis. Nous avons aussi mené une étude sur les tiques de dromadaires du sud-est d’Algérie afin d’éprouver une nouvelle fois la capacité du MALDI-TOF MS pour l'identification des tiques, d’incrémenter la base de données de notre laboratoire par des spectres des tiques conservées dans l’éthanol 70% et accroitre notre connaissance sur les microorganismes associés à ces tiques.Nos travaux, présentés dans cette thèse, mettent en évidence l’importance de l’utilisation du MALDI-TOF MS en entomologie médicale et en malacologie en l’occurrence dans le cadre de la surveillance épidémiologique.This thesis is divided into two main axes. The first axis concerns the use of MALDI-TOF MS in malacology and the second axis concerns the use of this tool in medical entomology. Therefore, the first objective of this thesis was to standardize a protocol for the identification of different species of freshwater snails including the intermediate hosts of schistosomes. The second objective was to use this high-throughput approach to increment our MS database with spectra of hematophagous arthropods. In the first project, we optimized an MS protocol for the rapid and correct identification of frozen and ethanol-preserved freshwater snails without requiring any malacological expertise. In the second step of the thesis, we reported for the first time in France a case of human infestation by swallow bugs (Cimex hirundinis). Moreover, we used MALDI-TOF MS to assess its ability to identify this species and compared it with laboratory-reared bed bugs. The effectiveness of the approach to distinguish nymphs from adults was evaluated as well. We also detected the novel strain of Wolbachia (Wolbachia massiliensis) infecting Cimex hirundinis. Our particular interest in Cimicids led us to write a literature review on Cimicidae of medical and veterinary importance.Finally, we conducted a study on dromedary ticks from South-Eastern Algeria in order to enrich our MS database with spectra of ticks preserved in 70% ethanol and detect their associated microorganisms using molecular tools

Cimicids of Medical and Veterinary Importance

International audienceMembers of the Cimicidae family are significant pests for mammals and birds, and they have attracted medical and veterinary interest. A number of recent studies have investigated bed bugs, due to their dramatic resurgence all over the world. Indeed, bed bugs are of significant public health and socioeconomic importance since they lead to financial burdens and dermatological complications and may have mental and psychological consequences. It is important to note that certain cimicids with a preference for specific hosts (birds and bats) use humans as an alternative host, and some cimicids have been reported to willingly feed on human blood. In addition, members of the Cimicidae family can lead to economic burdens and certain species are the vectors for pathogens responsible for diseases. Therefore, in this review, we aim to provide an update on the species within the Cimicidae family that have varying medical and veterinary impacts, including their distribution and their associated microorganisms. Various microbes have been documented in bed bugs and certain important pathogens have been experimentally documented to be passively transmitted by bed bugs, although no conclusive evidence has yet associated them with epidemiological outbreaks. Additionally, among the studied cimicids (bat bugs, chicken bugs, and swallow bugs), only the American swallow bug has been considered to be a vector of several arboviruses, although there is no proven evidence of transmission to humans or animals. Further studies are needed to elucidate the reason that certain species in the Cimicidae family cannot be biologically involved in transmission to humans or animals. Additional investigations are also required to better understand the roles of Cimicidae family members in the transmission of human pathogens in the field

Molecular and MALDI-TOF MS identification of swallow bugs Cimex hirundinis (Heteroptera: Cimicidae) and endosymbionts in France

International audienceAbstract Background The Cimicidae are obligatory blood-feeding ectoparasites of medical and veterinary importance. We aim in the current study to assess the ability of MALDI-TOF MS to identify Cimex hirundinis swallow bugs collected in house martin nests. Methods Swallow bugs were picked out from abandoned nests of house martin swallows and identified morphologically to the species level. The bugs were randomly selected, dissected and then subjected to MALDI-TOF MS and molecular analyses. Results A total of 65 adults and 50 nymphs were used in the attempt to determine whether this tool could identify the bug species and discriminate their developmental stages. Five adults and four nymphs of C. hirundinis specimens were molecularly identified to update our MS homemade arthropod database. BLAST analysis of COI gene sequences from these C. hirundinis revealed 98.66–99.12% identity with the corresponding sequences of C. hirundinis of the GenBank. The blind test against the database supplemented with MS reference spectra showed 100% (57/57) C. hirundinis adults and 100% (46/46) C. hirundinis nymphs were reliably identified and in agreement with morphological identification with logarithmic score values between 1.922 and 2.665. Ninety-nine percent of C. hirundinis specimens tested were positive for Wolbachia spp. The sequencing results revealed that they were identical to Wolbachia massiliensis , belonging to the new T-supergroup strain and previously isolated from C. hemipterus . Conclusions We report for the first time to our knowledge a case of human infestation by swallow bugs ( C. hirundinis ) in France. We also show the usefulness of MALDI-TOF MS in the rapid identification of C. hirundinis specimens and nymphs with minimal sample requirements. We phylogenetically characterized the novel Wolbachia strain ( W. massiliensis ) infecting C. hirundinis and compared it to other recognized Wolbachia clades. Graphical Abstrac

MALDI-TOF MS Identification of Dromedary Camel Ticks and Detection of Associated Microorganisms, Southern Algeria

International audienceThis study used MALDI-TOF MS and molecular tools to identify tick species infesting camels from Tamanrasset in southern Algeria and to investigate their associated microorganisms. Ninety-one adult ticks were collected from nine camels and were morphologically identified as Hyalomma spp., Hyalomma dromedarii, Hyalomma excavatum, Hyalomma impeltatum and Hyalomma anatolicum. Next, the legs of all ticks were subjected to MALDI-TOF MS, and 88/91 specimens provided good-quality MS spectra. Our homemade MALDI-TOF MS arthropod spectra database was then updated with the new MS spectra of 14 specimens of molecularly confirmed species in this study. The spectra of the remaining tick specimens not included in the MS database were queried against the upgraded database. All 74 specimens were correctly identified by MALDI-TOF MS, with logarithmic score values ranging from 1.701 to 2.507, with median and mean values of 2.199 and 2.172 ± 0.169, respectively. One H. impeltatum and one H. dromedarii (2/91; 2.20%) tested positive by qPCR for Coxiella burnetii, the agent of Q fever. We also report the first detection of an Anaplasma sp. close to A. platys in H. dromedarii in Algeria and a potentially new Ehrlichia sp. in H. impeltatum

MALDI-TOF MS Identification of Dromedary Camel Ticks and Detection of Associated Microorganisms, Southern Algeria

International audienceThis study used MALDI-TOF MS and molecular tools to identify tick species infesting camels from Tamanrasset in southern Algeria and to investigate their associated microorganisms. Ninety-one adult ticks were collected from nine camels and were morphologically identified as Hyalomma spp., Hyalomma dromedarii, Hyalomma excavatum, Hyalomma impeltatum and Hyalomma anatolicum. Next, the legs of all ticks were subjected to MALDI-TOF MS, and 88/91 specimens provided good-quality MS spectra. Our homemade MALDI-TOF MS arthropod spectra database was then updated with the new MS spectra of 14 specimens of molecularly confirmed species in this study. The spectra of the remaining tick specimens not included in the MS database were queried against the upgraded database. All 74 specimens were correctly identified by MALDI-TOF MS, with logarithmic score values ranging from 1.701 to 2.507, with median and mean values of 2.199 and 2.172 ± 0.169, respectively. One H. impeltatum and one H. dromedarii (2/91; 2.20%) tested positive by qPCR for Coxiella burnetii, the agent of Q fever. We also report the first detection of an Anaplasma sp. close to A. platys in H. dromedarii in Algeria and a potentially new Ehrlichia sp. in H. impeltatum

MALDI-TOF mass spectrometry for the identification of freshwater snails from Senegal, including intermediate hosts of schistosomes

International audienceFreshwater snails of the genera Biomphalaria , Bulinus , and Oncomelania are intermediate hosts of schistosomes that cause human schistosomiasis, one of the most significant infectious neglected diseases in the world. Identification of freshwater snails is usually based on morphology and potentially DNA-based methods, but these have many drawbacks that hamper their use. MALDI-TOF MS has revolutionised clinical microbiology and has emerged in the medical entomology field. This study aims to evaluate MALDI-TOF MS profiling for the identification of both frozen and ethanol-stored snail species using protein extracts from different body parts. A total of 530 field specimens belonging to nine species ( Biomphalaria pfeifferi , Bulinus forskalii , Bulinus senegalensis , Bulinus truncatus , Bulinus globosu s, Bellamya unicolor , Cleopatra bulimoides , Lymnaea natalensis , Melanoides tuberculata ) and 89 laboratory-reared specimens, including three species ( Bi . pfeifferi , Bu . forskalii , Bu . truncatus ) were used for this study. For frozen snails, the feet of 127 field and 74 laboratory-reared specimens were used to validate the optimised MALDI-TOF MS protocol. The spectral analysis yielded intra-species reproducibility and inter-species specificity which resulted in the correct identification of all the specimens in blind queries, with log-score values greater than 1.7. In a second step, we demonstrated that MALDI-TOF MS could also be used to identify ethanol-stored snails using proteins extracted from the foot using a specific database including a large number of ethanol preserved specimens. This study shows for the first time that MALDI-TOF MS is a reliable tool for the rapid identification of frozen and ethanol-stored freshwater snails without any malacological expertise