Etude des cycles épidémiologiques d'Anaplasma phagocytophilum en France : apport des approches de caractérisation génétique

A. phagocytophilum, a tick-borne bacterium, is responsible of the granulocytic anaplasmosis, an emerging disease that infects a large range of mammals including humans. Currently, the description of the epidemiological cycles of this bacterium is incomplete. The objective of this thesis was to characterize the genetic diversity of A. phagocytophilum in different host species to determine those involved in the same epidemiological cycle. First, we characterized the genetic diversity of A. phagocytophilum in sick domestic animals with a MLSA. We identified three groups of genotypes infecting cattle, including one group shared with horses and dogs, and another shared with roe deer. Then, we investigated what species of ticks can transmit the bacteria, and what wild mammals could be reservoirs. In Camargue, a genotype with high zoonotic potential was identified in five species of ticks of the genus Rhipicephalus, Dermacentor and Hyalomma. The prevalence in French rodents suggests that they may be reservoir hosts, but the presence of genotypes infecting cattle in rodents must be checked. Finally, comparing the bacterial genotypes in ticks and roe deer by 454 sequencing, showed that the contribution of the roe deer to tick infection was low in the site of “Vallons de Gascogne”. The absence of infected rodents in this location suggests that other reservoir mammals are present. This study demonstrates the complexity of the A. phagocytophilum cycle and the contribution of molecular tools.A. phagocytophilum, une bactérie transmise par les tiques, est responsable de l’anaplasmose granulocytaire, une maladie émergente qui infecte une large gamme de mammifères dont l’homme. Actuellement, la description des cycles épidémiologiques de cette bactérie est incomplète. L’objectif de cette thèse est de caractériser la diversité génétique d’A. phagocytophilum chez différentes espèces d’hôtes, afin de déterminer quelles espèces participent au même cycle épidémiologique. D’abord, nous avons caractérisé la diversité génétique d’A. phagocytophilum chez les animaux domestiques malades à l’aide d’une MLSA. Nous avons identifié trois groupes de génotypes infectant les bovins, dont un groupe est partagé avec les chevaux et les chiens, et un avec les chevreuils. Ensuite, nous avons recherché quelles espèces de tiques pouvaient transmettre la bactérie, et quels pouvaient être les réservoirs parmi les mammifères sauvages. En Camargue, un génotype au fort potentiel zoonotique a été identifié chez cinq espèces de tiques du genre Rhipicephalus, Dermacentor et Hyalomma. La prévalence chez des rongeurs suggère qu’ils peuvent être réservoirs, mais la présence de génotypes infectant les bovins chez les mulots est à vérifier. Enfin, la comparaison des génotypes obtenus chez les tiques et les chevreuils par séquençage 454, a montré que la contribution des chevreuils à l’infection des tiques était faible sur le site des Vallons de Gascogne. L’absence de rongeurs infectés sur ce site suggère que d’autres mammifères réservoirs sont présents. Cette étude montre la complexité des cycles d’A. phagocytophilum et l’intérêt des outils moléculaires

Genetic diversity of Anaplasma Phagocytophilum, the causative agent of granulocytic anaplasmosis, implications for epidemiology and control in france

Anaplasma phagocytophilum is a tick-borne bacterium and the etiologic agent of granulocytic anaplasmosis, an emerging disease that affects a wide range of mammals. In this paper, we present the recent knowledge gained from studies on the genetic diversity of this pathogen in France. Multilocus sequence analysis (MLSA) was used to characterize the genetic diversity of A. phagocytophilum in populations of French cattle, horses, dogs, and roe deer. MLSA was based on nine loci (ankA, msp4, groESL, typA, pled, gyrA, recG, polA, and an intergenic region). Phylogenic analysis revealed three genetic clusters of bacterial variants in domesticated animals. The two principal clusters included 98% of the bacterial genotypes found in cattle, which were only distantly related to those in roe deer. One cluster comprised only cattle genotypes, while the second contained genotypes from cattle, horses, and dogs. The third contained all roe deer genotypes and three cattle genotypes. These results suggest that roe deer do not contribute to the spread of A. phagocytophilum in cattle in France. A Multiple-Locus Variable number tandem repeat (VNTR) Analysis typing technique was developed for A. phagocytophilum. Five VNTRs were selected based on the HZ human-derived strain genome, and were tested on the Webster human-derived strain and on 123 DNA samples. This study confirmed that A. phagocytophilum from roe deer or domestic ruminants belong to two different clusters, while A. phagocytophilum from red deer and domestic ruminants locate within the same cluster, questioning the respective roles of roe vs red deer as reservoir hosts for domestic ruminant strains in Europe. The molecular techniques recently developed have great potential to provide detailed information on A. phagocytophilum isolates,improving both epidemiological and phylogenic investigations, thereby helping in the development of relevant prevention and control measures.A. phagocytophilum, bactérie transmise par les tiques, est responsable de l’anaplasmose granulocytaire, une maladie émergente qui infecte une large gamme de mammifères dont l’homme. L’objectif de cet article est de présenter les nouvelles connaissances acquises sur la diversité génétique d’A. phagocytophilum chez différentes espèces d’hôtes en France, afin de déterminer quelles espèces participent au même cycle épidémiologique. Une analyse par séquençage multi-locus (MLSA) a été effectuée dans des populations de bovins, chevaux, chiens et chevreuils. Trois groupes de génotypes infectant les bovins ont été identifiés. Les deux groupes principaux incluent 98% des génotypes bactériens trouvés chez les bovins et sont éloignés de ceux des chevreuils. Un cluster ne comprenait que les génotypes de bovins, tandis que le second génotype contenant des bovins comprenait également des chevaux et des chiens. Le troisième cluster contenait tous les génotypes de chevreuils et trois génotypes de bovins. Ces résultats suggèrent que les chevreuils ne contribuent pas à la propagation d’A. phagocytophilum chez les bovins en France. Puis, une technique MLVA (Multiple Loci VNTR Analysis) a été développée pour A. phagocytophilum. Cinq VNTR ont été sélectionnés sur la base du génome de la souche d’origine humaine HZ, et ont été testés sur 123 échantillons d’ADN provenant d’animaux domestiques ou sauvages. Cette étude a confirmé que les souches d’A. phagocytophilum retrouvées chez les chevreuils et les ruminants domestiques appartiennent à deux groupes différents, alors que les souches identifiées chez les cerfs et les ruminants domestiques sont localisées dans le même cluster. Ces résultats remettent en question les rôles respectifs des chevreuils et des cerfs comme hôtes réservoirs pour les souches d’A. phagocytophilum de ruminants domestiques en Europe. Ces techniques moléculaires ont un grand potentiel pour améliorer nos connaissances sur les cycles épidémiologiques d’A. phagocytophilum, contribuant ainsi à l’élaboration de mesures de prévention et de contrôle pertinents

Metronomic Four-Drug Regimen Has Anti-tumor Activity in Pediatric Low-Grade Glioma; The Results of a Phase II Clinical Trial

Background: Metronomic chemotherapy (MC) is defined as the frequent administration of chemotherapy at doses below the maximal tolerated dose and with no prolonged drug-free break. MC has shown its efficacy in adult tumor types such as breast and ovarian cancer and has to some extent been studied in pediatrics.Objective: To assess the anti-tumor activity and toxicity of a four-drug metronomic regimen in relapsing/refractory pediatric brain tumors (BT) with progression-free survival (PFS) after two cycles as primary endpoint.Methods: Patients ≥4 to 25 years of age were included with progressing BT. Treatment consisted of an 8-week cycle of celecoxib, vinblastine, and cyclophosphamide alternating with methotrexate. Kepner and Chang two-steps model was used with 10 patients in the first stage. If stabilization was observed in ≥2 patients, 8 additional patients were recruited. Assessment was according WHO criteria with central radiology review.Results: Twenty-nine patients (27 evaluable) were included in two groups: ependymoma (group 1, N = 8), and miscellaneous BT (group 2): 3 medulloblastoma (MB), 5 high grade glioma (HGG), 11 low grade glioma (LGG), 2 other BT. After first stage, recruitment for ependymoma was closed [one patient had stable disease (SD) for 4 months]. Cohort 2 was opened for second stage since 1 HGG and 3 LGG patients had SD after two cycles. Recruitment was limited to LGG for the second stage and 2 partial responses (PR), 6 SD and 2 progressive disease (PD) were observed after two cycles. Of these patients with LGG, median age was 10 years, nine patients received vinblastine previously. Median number of cycles was 6.8 (range: 1–12). Treatment was interrupted in five patients for grade 3/4 toxicity.Conclusion: This regimen is active in patients with LGG, even if patients had previously received vinblastine. Toxicity is acceptable.Trial Registration: This study was registered under clinicaltrials.gov – NCT01285817; EUDRACT nr: 2010-021792-81

Study of epidemiological cycles of Anaplasma phagocytophilum in France : contribution of characterization by genetic approaches

A. phagocytophilum, une bactérie transmise par les tiques, est responsable de l’anaplasmose granulocytaire, une maladie émergente qui infecte une large gamme de mammifères dont l’homme. Actuellement, la description des cycles épidémiologiques de cette bactérie est incomplète. L’objectif de cette thèse est de caractériser la diversité génétique d’A. phagocytophilum chez différentes espèces d’hôtes, afin de déterminer quelles espèces participent au même cycle épidémiologique. D’abord, nous avons caractérisé la diversité génétique d’A. phagocytophilum chez les animaux domestiques malades à l’aide d’une MLSA. Nous avons identifié trois groupes de génotypes infectant les bovins, dont un groupe est partagé avec les chevaux et les chiens, et un avec les chevreuils. Ensuite, nous avons recherché quelles espèces de tiques pouvaient transmettre la bactérie, et quels pouvaient être les réservoirs parmi les mammifères sauvages. En Camargue, un génotype au fort potentiel zoonotique a été identifié chez cinq espèces de tiques du genre Rhipicephalus, Dermacentor et Hyalomma. La prévalence chez des rongeurs suggère qu’ils peuvent être réservoirs, mais la présence de génotypes infectant les bovins chez les mulots est à vérifier. Enfin, la comparaison des génotypes obtenus chez les tiques et les chevreuils par séquençage 454, a montré que la contribution des chevreuils à l’infection des tiques était faible sur le site des Vallons de Gascogne. L’absence de rongeurs infectés sur ce site suggère que d’autres mammifères réservoirs sont présents. Cette étude montre la complexité des cycles d’A. phagocytophilum et l’intérêt des outils moléculaires.A. phagocytophilum, a tick-borne bacterium, is responsible of the granulocytic anaplasmosis, an emerging disease that infects a large range of mammals including humans. Currently, the description of the epidemiological cycles of this bacterium is incomplete. The objective of this thesis was to characterize the genetic diversity of A. phagocytophilum in different host species to determine those involved in the same epidemiological cycle. First, we characterized the genetic diversity of A. phagocytophilum in sick domestic animals with a MLSA. We identified three groups of genotypes infecting cattle, including one group shared with horses and dogs, and another shared with roe deer. Then, we investigated what species of ticks can transmit the bacteria, and what wild mammals could be reservoirs. In Camargue, a genotype with high zoonotic potential was identified in five species of ticks of the genus Rhipicephalus, Dermacentor and Hyalomma. The prevalence in French rodents suggests that they may be reservoir hosts, but the presence of genotypes infecting cattle in rodents must be checked. Finally, comparing the bacterial genotypes in ticks and roe deer by 454 sequencing, showed that the contribution of the roe deer to tick infection was low in the site of “Vallons de Gascogne”. The absence of infected rodents in this location suggests that other reservoir mammals are present. This study demonstrates the complexity of the A. phagocytophilum cycle and the contribution of molecular tools

Raw sequencing data of Anaplasma phagocytophilum loci (ankA, msp4, groEL) obtained from 454 and parameter files to clean these data using MOTHUR.

Lien zenodo pour télécharger les données de la recherche : https://www.zenodo.org/record/375497 Année de la première version : 2017Lien zenodo pour télécharger les données de la recherche : https://www.zenodo.org/record/375497il s'agit d'un type de produit dont les métadonnées ne correspondent pas aux métadonnées attendues dans les autres types de produit : SOFTWARERaw sequencing data of [i]Anaplasma phagocytophilum[/i] loci (ankA, msp4, groEL) obtained from 454 and parameter files to clean these data using MOTHUR

Raw sequencing data of Anaplamsa phagocytophilum loci (ankA, msp4, groEL) obtained from 454 and parameter files to clean these data using MOTHUR

<p>A compressed archive including: i) raw sequences in ssf file; ii) Mothur oligo files to sort out sequences among loci and individual samples.</p

Single Genotype of Anaplasma phagocytophilum Identified from Ticks, Camargue, France

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Evaluation of the Pathogenicity and the Escape from Vaccine Protection of a New Antigenic Variant Derived from the European Human-Like Reassortant Swine H1N2 Influenza Virus

The surveillance of swine influenza A viruses in France revealed the emergence of an antigenic variant following deletions and mutations that are fixed in the HA-encoding gene of the European human-like reassortant swine H1N2 lineage. In this study, we compared the outcomes of the parental (H1huN2) and variant (H1huN2&Delta;14&ndash;147) virus infections in experimentally-inoculated piglets. Moreover, we assessed and compared the protection that was conferred by an inactivated vaccine currently licensed in Europe. Three groups of five unvaccinated or vaccinated piglets were inoculated with H1huN2 or H1huN2&Delta;14&ndash;147 or mock-inoculated, respectively. In unvaccinated piglets, the variant strain induced greater clinical signs than the parental virus, in relation to a higher inflammatory response that involves TNF-&alpha; production and a huge afflux of granulocytes into the lung. However, both infections led to similar levels of virus excretion and adaptive (humoral and cellular) immune responses in blood. The vaccinated animals were clinically protected from both infectious challenges and did not exhibit any inflammatory responses, regardless the inoculated virus. However, whereas vaccination prevented virus shedding in H1huN2-infected animals, it did not completely inhibit the multiplication of the variant strain, since live virus particles were detected in nasal secretions that were taken from H1huN2&Delta;14&ndash;147-inoculated vaccinated piglets. This difference in the level of vaccine protection was probably related to the poorer ability of the post-vaccine antibodies to neutralize the variant virus than the parental virus, even though post-vaccine cellular immunity appeared to be equally effective against both viruses. These results suggest that vaccine antigens would potentially need to be updated if this variant becomes established in Europe

Ecological factors influencing small mammal infection by <i>Anaplasma phagocytophilum</i> and <i>Borrelia burgdorferi</i> s.l. in agricultural and forest landscapes

International audienceSmall mammals are key components of numerous tickborne disease systems, as hosts for immature ticks and pathogen reservoirs. To study the factors influencing tick-borne infection in small mammals, we trapped small mammals and collected questing ticks in spring and autumn in 2012 and 2013 at 24 sites in a 10 3 15 km rural landscapes (Brittany, France). Tissue samples were screened by real-time PCR for Anaplasmaphagocytophilum and Borrelia burgdorferi sensu lato. Of the two dominant small mammal species captured, bank voles (Myodes glareolus) had higher prevalence than wood mice (Apodemus sylvaticus) for both infections, presumably because of specific differences in immunological defenses. Prevalence of infections was higher in 2013 than in 2012, likely because small mammals were fivefold less abundant in 2013, favouring tick aggregation. Bacterial prevalence, which was higher in autumn, was not associated to questing Ixodes ricinus nymph abundance which was six times higher in spring, but rather to the structure of the small mammal community. These findings suggest the involvement of endophilic tick species, I. trianguliceps and/or I. acuminatus, in bacterial transmission. Our study highlights that the entire community of hostsand vectors, and their interactions, should be considered to fully understand the epidemiology of vectorborne diseases

Relationships between landscape structure and the prevalence of two tick-borne infectious agents, Anaplasma Phagocytophilum and Borrelia burgdorferi sensu lato, in small mammal communities

International audienceContext - By modifying ecosystems, land cover changes influence the emergence, the spread and the incidence of vector-borne diseases.Objective - This study aimed at identifying associations between landscape structure and the prevalence of two tick-borne infectious agents, Anaplasma phagocytophilum and Borrelia burgdorferi s.l., in small mammal communities.Methods - Small mammals were sampled in 24 sites along a gradient of woodland fragmentation and hedgerow network density, and screened for infectious agents with real-time PCR techniques. For each site, structural variables (composition and configuration) of the surrounding landscape at various scales (0–500 m) and variables of wooded habitats connectivity based on graph theory and least cost path distances for the two dominant species, bank voles (Myodes glareolus) and wood mice (Apodemus sylvaticus), were computed.Results - The A. phagocytophilum prevalence increased with wooded habitats cover (0–500 m), likely through host population size, and increased slightly with bank vole abundance, which has a higher reservoir competence than wood mouse. The B. burgdorferi s.l. prevalence increased with wooded ecotones only at local scales (50–100 m). Wooded habitats connectivity measures did not improve models built with simple land cover variables. A more marked spatial pattern was observed for the prevalence of A. phagocytophilum than B.burgdorferi s.l.Conclusions - This study highlights the interest of considering together the ecology of infectious agents (e.g. host specificity) and the host species community ecology to better understand the influence of the landscape structure on the spatial distribution of vector-borne infectious agents