Development and use of innovative tools in medical entomology

Les arthropodes hématophages assurant la transmission biologique active des nombreux pathogènes d’un animal à un autre, dont l’homme. La lutte anti-vectorielle et la surveillance épidémiologique des vecteurs sont essentielles pour la lutte et le contrôle des maladies vectorielles.L’identification correcte des arthropodes, la détermination de l’origine du repas sanguin ainsi que des pathogènes associés sont des étapes cruciales dans la lutte contre les vecteurs. Nos travaux ont ainsi porté sur une combinaison des méthodes (morphologie, moléculaire et MALDI-TOF MS) pour l’identification, la détermination de repas de sang et des pathogènes associés avec les hôtes et leurs arthropodes. La première partie de notre travail, était utilisation d’outil MALDI-TOF MS pour identifier des tiques collectées au Mali et conservées dans de l’éthanol 70%. Les tiques étaient aussi testées par la biologie moléculaire pour rechercher les microorganismes. Nous avons également identifiés par MALDI-TOF MS des moustiques collectés au Tchad et conservés avec du silicagel. L’origine animale du repas sanguin de ces moustiques a également pu être étudiée par cet outil. La seconde partie nous a permis de détecter par la biologie moléculaire des microorganimes dans des animaux domestiques et sauvages et leurs ectoparasites en Algérie et au Mali. Nos travaux nous ont donc permis de confirmer l’intérêt du MALDI-TOF MS en entomologie médicale, avec pour la première fois son application sur des moustiques collectés sur le terrain au sud et conservé avec du siligalel, et de compléter le répertoire des agents pathogènes associés aux arthropodes au Mali et en Algérie.Blood-sucking arthropods ensure the active biological transmission of many pathogens from one animal to another, including humans. Vector control and epidemiological surveillance of vectors are essential for the control and monitoring of vector-borne diseases.The correct identification of arthropods, the determination of the origin of the blood meal and the associated pathogens are crucial steps in vector control. Our work thus focused on a combination of methods (morphology, molecular and MALDI-TOF MS) for the identification, determination of blood meals and pathogens associated with hosts and their arthropods. The first part of our work was to use the MALDI-TOF MS tool to identify ticks collected in Mali and stored in 70% ethanol. Ticks were also tested by molecular biology to look for microorganisms. We also identified by MALDI-TOF MS mosquitoes collected in Chad and preserved with silicagel. The animal origin of the blood meal of these mosquitoes could also be studied by this tool. The second part allowed us to detect microorganisms in domestic and wild animals and their ectoparasites in Algeria and Mali through molecular biology. Our work therefore enabled us to confirm the interest of MALDI-TOF MS in medical entomology, with for the first time its application on mosquitoes collected in the field in the south and preserved with siligalel, and to complete the directory of pathogens associated with arthropods in Mali and Algeria

Use of MALDI-TOF MS for the Identification of Chad Mosquitoes and the Origin of Their Blood Meal

International audienceMatrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a clinical microbiology tool for the systematic identification of microorganisms. It has recently been presented as an innovative tool for the rapid and accurate identification of mosquitoes and their blood meal. To evaluate the capacity of this tool to identify mosquitoes collected in a tropical environment and preserved with silica gel, we analyzed 188 mosquitoes of different species collected in Chad, which were preserved with silica gel for 2 months. The MALDI-TOF MS analysis correctly identified 96% of the mosquitoes and 37.5% of their blood meals. Using MALDI-TOF MS and molecular biology, eight mosquito species were identified, including Anopheles gambiae s.l., Anopheles rufipes, Culex quinquefasciatus, Culex neavei, Culex pipiens, Culex perexiguus, Culex rima, and Culex watti. Blood meal identification revealed that mosquitoes fed mainly on humans, birds, and cows. Matrix-assisted desorption/ionization time-of-flight mass spectrometry appears to be a promising, fast, and reliable tool to identify mosquitoes and the origin of their blood meal for samples stored with silica gel

Tick-Borne Diseases of Humans and Animals in West Africa

International audienceTicks are a significant group of arthropod vectors that transmit a large variety of pathogens responsible for human and animal diseases worldwide. Ticks are the second biggest transmitters of vector-borne diseases, behind mosquitoes. However, in West Africa, there is often only limited knowledge of tick-borne diseases. With the scarcity of appropriate diagnostic services, the prevalence of tick-borne diseases is generally underestimated in humans. In this review, we provide an update on tick-borne pathogens reported in people, animals and ticks in West Africa by microscopic, immunological and molecular methods. A systematic search was conducted in PubMed and Google Scholar. The selection criteria included all studies conducted in West Africa reporting the presence of Rickettsia, Borrelia, Anaplasma, Ehrlichia, Bartonella, Coxiella burnetii, Theileria, Babesia, Hepatozoon and Crimean–Congo haemorrhagic fever viruses in humans, animals or ticks. Our intention is to raise awareness of tick-borne diseases amongst human and animal health workers in West Africa, and also physicians working with tourists who have travelled to the region

Identification of ticks from an old collection by MALDI-TOF MS

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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

Using MALDI-TOF MS to Identify Mosquitoes from Senegal and the Origin of Their Blood Meals

International audienceMosquitoes are arthropods that represent a real public health problem in Africa. Morphology and molecular biology techniques are usually used to identify different mosquito species. In recent years, an innovative tool, matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), has been used to identify many arthropods quickly and at low cost, where equipment is available. We evaluated the ability of MALDI-TOF MS to identify mosquitoes collected in Senegal and stored for several months in silica gel, and to determine the origin of their blood meal. A total of 582 mosquitoes were collected and analysed. We obtained 329/582 (56.52%) MALDI-TOF MS good-quality spectra from mosquito legs and 123/157 (78.34%) good-quality spectra from engorged abdomens. We updated our home-made MALDI-TOF MS arthropod spectra database by adding 23 spectra of five mosquito species from Senegal that had been identified morphologically and molecularly. These included legs from Anopheles gambiae, Anopheles arabiensis, Anopheles cf. rivulorum, Culex nebulosus, Anopheles funestus, and three spectra from abdomens engorged with human blood. Having updated the database, all mosquitoes tested by MALDI-TOF MS were identified with scores greater than or equal to 1.7 as An. gambiae (n = 64), Anopheles coluzzii (n = 12), An. arabiensis (n = 1), An. funestus (n = 7), An. cf rivulorum (n = 1), Lutzia tigripes (n = 3), Cx. nebulosus (n = 211), Culex quinquefasciatus (n = 2), Culex duttoni (n = 1), Culex perfescus (n = 1), Culex tritaeniorhynchus (n = 1), and Aedes aegypti (n = 2). Blood meal identification by MALDI-TOF MS revealed that mosquitoes had fed on the blood of humans (n = 97), cows (n = 6), dogs (n = 2), goats (n = 1), sheep (n = 1), and bats (n = 1). Mixed meals were also detected. These results confirm that MALDI-TOF MS is a promising technique for identifying mosquitoes and the origin of their blood meal

Detection of Tick-Borne Pathogens in Ticks from Cattle in Western Highlands of Cameroon

International audienceThis study aimed to detect and identify microorganisms in ticks collected in the Western Highlands of Cameroon. Quantitative real-time and standard PCR assays, coupled with sequencing, were used. A total of 944 ticks collected from cattle in five distinct sites in Cameroon were selected for the analyses. They belonged to five genera (Amblyomma, Hyalomma, Rhipicephalus, Haemaphysalis, and Ixodes) and twelve species. Real-time PCR revealed that 23% (n = 218) of the ticks were positive for Rickettsia spp., 15% (n = 141) for bacteria of the Anaplasmataceae family, 3% (n = 29) for Piroplasmida, 0.5% (n = 5) for Coxiella burnetii, 0.4% (n = 4) for Borrelia spp., and 0.2% (n = 2) for Bartonella spp. The co-infection rate (3.4%, n = 32) involved mainly Rickettsia spp. and Anaplasmataceae. Of the Rickettsia spp. positive ticks, the targeted PCR and sequencing yielded Rickettsia africae (78.9%), Rickettsia aeschlimannii (6.4%), Rickettsia massiliae (7.8%), Candidatus Rickettsia barbariae (0.9%), and Rickettsia sp. (0.9%). Anaplasmataceae included Anaplasma marginale (4.3%), Anaplasma platys (1.4%), Anaplasma centrale (0.7%), Ehrlichia ruminantium (0.7%), Wolbachia sp., Candidatus Ehrlichia rustica (13.5%), Candidatus Ehrlichia urmitei (7%), and an uncultured Ehrlichia sp. (4.2%). Borrelia theileri was identified in one Rhipicephalus microplus tick. Unfortunately, Piroplasmida could not be identified to the species level. This study demonstrates that in Cameroon, ticks harbour a wide variety of microorganisms and present a risk of zoonotic diseases.</jats:p

Molecular identification and evaluation of Coxiella-like endosymbionts genetic diversity carried by cattle ticks in Algeria

International audienceCoxiella-like bacteria are a large group of yet-to-isolate and characterize bacteria phylogenetically close to the agent of Q fever, Coxiella burnetii, and often associated with ixodid ticks worldwide. This study was designed to assess the presence of Coxiella-like endosymbionts (CLE) in ticks and to describe their genetic diversity in different tick species infesting cattle in Algeria. A total of 765 ticks were collected from three locations. The screening of 20 % of sampled ticks (147/765) exhibited the presence of Coxiella-like in 51.7 % (76/147). The sequencing of partial 16S rRNA and the GroEl genes showed an identity higher than 98 % with different Coxiella-like endosymbionts. The phylogenetic analysis based on the 16S rRNA gene showed the positions of identified Coxiella bacteria. Eleven of the 13 sequences from Rhipicephalus, Dermacentor and Hyalomma ticks were grouped in a distinct Glade, the other two each represent an independent Glade. This study reported that CLE are prevalent in cattle ticks. Most of the identified Coxiella-like bacteria, from different species of ticks found on cattle, were identical. This may mean that, unlike the currently accepted paradigm, Coxiella-like bacteria are not only tick host-associated, but rather can be transmitted from one tick species to another via the vertebrate host

Microorganisms associated with hedgehog arthropods

Abstract Hedgehogs are small synanthropic mammals that live in rural areas as well as in urban and suburban areas. They can be reservoirs of several microorganisms, including certain pathogenic agents that cause human and animal public health issues. Hedgehogs are often parasitized by blood-sucking arthropods, mainly hard ticks and fleas, which in turn can also carry various vector-born microorganisms of zoonotic importance. Many biotic factors, such as urbanization and agricultural mechanization, have resulted in the destruction of the hedgehog’s natural habitats, leading these animals to take refuge near human dwellings, seeking food and shelter in parks and gardens and exposing humans to zoonotic agents that can be transmitted either directly by them or indirectly by their ectoparasites. In this review, we focus on the microorganisms detected in arthropods sampled from hedgehogs worldwide. Several microorganisms have been reported in ticks collected from these animals, including various Borrelia spp., Anaplasma spp., Ehrlichia spp., and Rickettsia spp. species as well as Coxiella burnetii and Leptospira spp. As for fleas, C. burnetii, Rickettsia spp., Wolbachia spp., Mycobacterium spp. and various Bartonella species have been reported. The detection of these microorganisms in arthropods does not necessarily mean that they can be transmitted to humans and animals. While the vector capacity and competence of fleas and ticks for some of these microorganisms has been proven, in other cases the microorganisms may have simply been ingested with blood taken from an infected host. Further investigations are needed to clarify this issue. As hedgehogs are protected animals, handling them is highly regulated, making it difficult to conduct epidemiological studies on them. Their ectoparasites represent a very interesting source of information on microorganisms circulating in populations of these animals, especially vector-born ones. Graphical abstrac

Detection of Tick-Borne Pathogens in Ticks from Cattle in Western Highlands of Cameroon

This study aimed to detect and identify microorganisms in ticks collected in the Western Highlands of Cameroon. Quantitative real-time and standard PCR assays, coupled with sequencing, were used. A total of 944 ticks collected from cattle in five distinct sites in Cameroon were selected for the analyses. They belonged to five genera (Amblyomma, Hyalomma, Rhipicephalus, Haemaphysalis, and Ixodes) and twelve species. Real-time PCR revealed that 23% (n = 218) of the ticks were positive for Rickettsia spp., 15% (n = 141) for bacteria of the Anaplasmataceae family, 3% (n = 29) for Piroplasmida, 0.5% (n = 5) for Coxiella burnetii, 0.4% (n = 4) for Borrelia spp., and 0.2% (n = 2) for Bartonella spp. The co-infection rate (3.4%, n = 32) involved mainly Rickettsia spp. and Anaplasmataceae. Of the Rickettsia spp. positive ticks, the targeted PCR and sequencing yielded Rickettsia africae (78.9%), Rickettsia aeschlimannii (6.4%), Rickettsia massiliae (7.8%), Candidatus Rickettsia barbariae (0.9%), and Rickettsia sp. (0.9%). Anaplasmataceae included Anaplasma marginale (4.3%), Anaplasma platys (1.4%), Anaplasma centrale (0.7%), Ehrlichia ruminantium (0.7%), Wolbachia sp., Candidatus Ehrlichia rustica (13.5%), Candidatus Ehrlichia urmitei (7%), and an uncultured Ehrlichia sp. (4.2%). Borrelia theileri was identified in one Rhipicephalus microplus tick. Unfortunately, Piroplasmida could not be identified to the species level. This study demonstrates that in Cameroon, ticks harbour a wide variety of microorganisms and present a risk of zoonotic diseases