Identification of arthropods and associated pathogens by MALDI-TOF MS and study of the relationship between arthropods and bacteria

Ce travail est composé de 3 parties. La première est une étude épidémiologique avec la détection moléculaire des spécimens appartenant à six espèces d’Argasidés collectées en Algérie et identifiées morphologiquement et par biologie moléculaire. Nous avons pu détecter Borrelia hispanica dans des Ornithodoros occidentalis et Borrelia cf turicatae dans des Carios Carpensis. Dans des Argas persicus nous avons pu identifier un nouveau génotype de Bartonella spp ainsi qu’un génotype appartenant à une nouvelle espèce dans la famille des Anaplasmataceae. Dans la 2e partie, nous avons évalué la capacité vectorielle des punaises de lit à transmettre Borrelia recurrentis, l’agent de la fièvre récurrente. Pour ce fait, nous avons utilisé un modèle expérimental d’infection artificielle de Cimex lectularius par B. recurrentis pour ensuite détecter la présence de la bactérie dans les fèces. Nous avons utilisé quatre approches : la détection par qPCR, la culture à partir des fèces, la FDA (Fluorescein Diacetate) et l’inoculation des fèces aux souris. Nous avons également utilisé l’Immunofluorescence pour localiser la bactérie dans le corps de la punaise. Nous avons constaté que les punaises de lit acquièrent la bactérie et excrètent des microorganismes vivants dans les fèces. Elles peuvent être considérées comme vecteur potentiel de Borrelia recurrentis. La troisième partie s’intéresse à l’évaluation de la capacité du MALDI-TOF MS à identifier les puces, les punaises et les pathogènes associés.This work focuses on three main parts, a first part presents an epidemiological study of bacteria associated with soft ticks in Algeria, or we identified morphologically and confirmed by molecular biology six species of Argasidae. In addition, looking further we could detect Borrelia hispanica in Ornithodoros occidentalis and Borrelia cf turicatae in Carios Carpensis. On the other hand, in Argas persicus a new genotype of Bartonella spp has been identified as well as a new species of Anaplasmatacea bacteria.A second part evaluates the vectorial capacity of bed bugs to transmit Borrelia recurrentis, the agent of the relapsing fever. For this reason an experimental model of artificial infection of Cimex lectularius by Borrelia recurrentis has been developed, to study the presence of bacteria in feces. In this model, four approaches were used: qPCR, fece’s culture, FDA (Fluorescein Diacetate) and fece’s inoculation to mice. Immunofluorescence was also used to detect the location of the bacteria in the body of the bed bug. We confirmed that bed bugs acquire the bacteria and excrete live microorganisms in the feces. They can be considered as potential vector of Borrelia recurrentis.The third part is an assessment of the capacity of MALDI-TOF MS to identified fleas, bed bugs and associated pathogens. This innovative tool, which has revolutionized medical entomology and has shown its efficiency to identify several species of arthropods, has also been able to distinguish between infected and uninfected fleas and bugs, and even distinguish between fleas and bugs infected by the same species of bacteria

Detection of Bartonella spp. in Cimex lectularius by MALDI-TOF MS

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Fleas and flea-borne diseases of North Africa

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Detection of Rickettsia raoultii in Dermacentor reticulatus and Haemaphysalis inermis ticks in Slovakia

International audienceTicks are vector arthropods responsible for the transmission of several pathogenic agents that affect both human and animal health worldwide. In this study our objective was to analyse, using molecular tools, the bacterial community of Dermacentor reticulatus and Haemaphysalis inermis ticks collected in south-eastern Slovakia. Using real-time PCR, we identified the presence of Rickettsia spp. DNA at levels of 14/59 (23.72 %) and 29/173 (16.76 %) in D. reticulatus and H. inermis , respectively. In addition, using standard PCR and sequencing, we identified the presence of Rickettsia raoultii DNA in 13 ticks belonging to the two investigated species. Rickettsia raoultii blast results revealed an average identification percentage of 99.62 %. Following the results of this molecular study there is a possibility that D. reticulatus and H. inermis play a potential role in the transmission of R. raoultii . To prove the possibility of validity of this hypothesis, we suggest performing experimental models in future studies. Our results can serve as preliminary data for future transmission models

Testing the Competence of Cimex lectularius Bed Bugs for the Transmission of Borrelia recurrentis, the Agent of Relapsing Fever

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MALDI-TOF MS identification of Cimex lectularius and Cimex hemipterus bedbugs

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Detection of Bartonella spp. in fleas by MALDI-TOF MS

International audienceBackground Matrix-Assisted Laser Desorption/lonization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has recently emerged in the field of entomology as a promising method for the identification of arthropods and the detection of associated pathogens. Methodology/Principal findings An experimental model of Ctenocephalides felis (cat fleas) infected with Bartonella quintana and Bartonella henselae was developed to evaluate the efficacy of MALDI-TOF MS in distinguishing infected from uninfected fleas, and its ability to distinguish fleas infected with Bartonella quintana from fleas infected with Bartonella henselae. For B. quintana, two groups of fleas received three successive blood meals, infected or not. A total of 140 fleas (100 exposed fleas and 40 control fleas) were engorged on human blood, infected or uninfected with B. quintana. Regarding the second pathogen, two groups of fleas (200 exposed fleas and 40 control fleas) were fed in the same manner with human blood, infected or not with Bartonella henselae. Fleas were dissected longitudinally; one-half was used for assessment of B. quintana and B. henselae infectious status by real-time PCR, and the second half was subjected to MALDI-TOF MS analysis. Comparison of MS spectra from infected fleas and uninfected fleas revealed distinct MS profiles. Blind queries against our MALDI-TOF MS arthropod database, upgraded with reference spectra from B. quintana and B. henselae infected fleas but also non-infected fleas, provided the correct classification for 100% of the different categories of specimens tested on the first model of flea infection with Bartonella quintana. As for Bartonella henselae, 81% of exposed qPCR-positive fleas, 96% of exposed qPCR-negative fleas and 100% of control fleas were correctly identified on the second model of flea infection. MALDI-TOF MS successfully differentiated Bartonella spp.-infected and uninfected fleas and was also able to correctly differentiate fleas infected with Bartonella quintana and fleas infected with Bartonella henselae. MALDI-TOF MS correctly identified flea species as well as their infectious status, consistent with the results of real-time PCR. Conclusions/Significance MALDI-TOF is a promising tool for identification of the infection status of fleas infected with Bartonella spp., which allows new possibilities for fast and accurate diagnosis in medical entomology and vector surveillance

Detection of relapsing fever Borrelia spp., Bartonella spp. and Anaplasmataceae bacteria in argasid ticks in Algeria.

Argasid ticks (soft ticks) are blood-feeding arthropods that can parasitize rodents, birds, humans, livestock and companion animals. Ticks of the Ornithodoros genus are known to be vectors of relapsing fever borreliosis in humans. In Algeria, little is known about relapsing fever borreliosis and other bacterial pathogens transmitted by argasid ticks.Between May 2013 and October 2015, we investigated the presence of soft ticks in 20 rodent burrows, 10 yellow-legged gull (Larus michahellis) nests and animal shelters in six locations in two different bioclimatic zones in Algeria. Six species of argasid ticks were identified morphologically and through 16S rRNA gene sequencing. The presence and prevalence of Borrelia spp., Bartonella spp., Rickettsia spp. and Anaplasmataceae was assessed by qPCR template assays in each specimen. All qPCR-positive samples were confirmed by standard PCR, followed by sequencing the amplified fragments. Two Borrelia species were identified: Borrelia hispanica in Ornithodoros occidentalis in Mostaganem, and Borrelia cf. turicatae in Carios capensis in Algiers. One new Bartonella genotype and one new Anaplasmataceae genotype were also identified in Argas persicus.The present study highlights the presence of relapsing fever borreliosis agents, although this disease is rarely diagnosed in Algeria. Other bacteria of unknown pathogenicity detected in argasid ticks which may bite humans deserve further investigation

Detection of <i>Bartonella</i> spp. in fleas by MALDI-TOF MS - Fig 3

<p><b>(A, B, C and D)</b> Comparison of MALDI-TOF MS profiles of body half of <i>Ctenocephalides felis infected</i> or not by <i>Bartonella quintana</i> using ClinProTools 2.2 software. Red and green peaks indicated by arrows correspond to discriminating peaks of control and infected fleas respectively.</p

Reproducible and specific MALDI-TOF MS spectra of <i>Bartonella quintana</i> strain, body half of fresh <i>Ctenocephalides felis</i> infected with <i>Bartonella henselae</i>, exposed and control analyzed by Flex analysis 3.3 software.

<p>a.u., arbitrary units; m/z, mass-to-charge ratio.</p