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

Bovine granulocytic anaplasmosis in France : characterization of first available bovine Anaplasma phagocytophilum genome and epidemiologic study of ruminants strains by MLVA

Anaplasma phagocytophilum est une alpha-protéobactérie, parasite intracellulaire stricte, à localisation intra-granulocytaire et principalement vectorisée par des tiques du genre Ixodes. Elle est notamment l'agent de l'anaplasmose granulocytaire bovine, ou Tick-borne fever, une maladie provoquant d'importantes pertes économiques chez les bovins en Europe. Cette bactérie, peut également infecter un large spectre d'hôtes, tels que des ruminants sauvages ou des rongeurs. Cependant, l'épidémiologie de l'infection par A. phagocytophilum est encore mal connue. Le(s) réservoir(s) des souches de ruminants domestiques en Europe n'est/ne sont notamment pas identifié(s) à l'heure actuelle. Il est donc nécessaire d'approfondir nos connaissances dans ce domaine, notamment afin de lutter plus efficacement contre l'atteinte des bovins. L'objectif de cette thèse était de caractériser la diversité génétique des variants d'A. phagocytophilum circulant chez les ruminants en France. Pour cela, nous avons, dans un premier temps, étudié la circulation des souches d'A. phagocytophilum au sein des ruminants sauvages et domestiques en développant et en utilisant une technique MLVA (Multiple-Locus Variable-number tandem repeat Analysis). Cela nous a permis de suspecter fortement l'existence d'au moins deux cycles épidémiologiques impliquant des variants d'A. phagocytophilum présents chez les ruminants en France. Le premier pourrait impliquer le cerf comme espèce réservoir et les ruminants domestiques (entre autres) comme espèces sensibles, alors que le second impliquerait le chevreuil comme réservoir, sans impact significatif chez les bovins. Dans un deuxième temps, après avoir développé une technique de séquence capture pour A. phagocytophilum, nous avons séquencé et caractérisé le premier génome de cette bactérie issu d'un bovin. La comparaison de ce génome aux neuf autres génomes actuellement disponibles a permis d'identifier quatre protéines uniquement présentes chez la souche bovine, et neuf uniquement chez les deux souches de ruminants domestiques étudiés, ce qui amène à envisager leur implication potentielle dans le tropisme d'hôte de ces souchesAnaplasma phagocytophilum is an obligate intracellular alpha-proteobacterium mainly transmitted by Ixodes ticks. In domestic ruminants, it is the causative agent of tick-borne fever, which causes significant economic losses in Europe. It also infects a large range of hosts, including wild ruminants and rodents. The epidemiology of A. phagocytophilum is not yet fully understood. For example, the reservoir host(s) for European domestic ruminant strains has/have not been identified to date, which doesn't facilitate control of cattle infection. Our objective was to explore the genetic diversity of A. phagocytophilum obtained from ruminants in France. For this purpose, we first studied the circulation of this pathogen in domestic and wild ruminants, using a new MLVA technique. Our results potentially reveal the existence of at least two different epidemiological transmission cycles of A. phagocytophilum. The first cycle may involve red deer as reservoir hosts, and possibly domestic ruminants, as either accidental or longer-term hosts, whereas the second might involve roe deer. In a second study, we have sequenced and characterized the genome of A. phagocytophilum obtained from a cow. Following comparison with nine available genomes, we identified four genes specific to the A. phagocytophilum bovine genome, and nine common to both genomes from domestic ruminants (i.e. a cow and a sheep). These genes could be involved in host tropism of ruminant strain

Comparative genomics of first available bovine Anaplasma phagocytophilum genome obtained with targeted sequence capture.

Background: [br/] Anaplasma phagocytophilum is a zoonotic and obligate intracellular bacterium transmitted by ticks. In domestic ruminants, it is the causative agent of tick-borne fever, which causes significant economic losses in Europe. As A. phagocytophilum is difficult to isolate and cultivate, only nine genome sequences have been published to date, none of which originate from a bovine strain. Our goals were to; 1/ develop a sequencing methodology which efficiently circumvents the difficulties associated with A. phagocytophilum isolation and culture; 2/ describe the first genome of a bovine strain; and 3/ compare it with available genomes, in order to both explore key genomic features at the species level, and to identify candidate genes that could be specific to bovine strains.[br/] [br/] Results:[br/] DNA was extracted from a bovine blood sample infected by A. phagocytophilum. Following a whole genome capture approach, A. phagocytophilum DNA was enriched 197-fold in the sample and then sequenced using Illumina technology. In total, 58.9% of obtained reads corresponded to the A. phagocytophilum genome, covering 85.3% of the HZ genome. Then by performing comparisons with nine previously-sequenced A. phagocytophilum genomes, we determined the core genome of these ten strains. Following analysis, 1281 coding DNA sequences, including 1001 complete sequences, were detected in the A. phagocytophilum bovine genome, of which four appeared to be unique to the bovine isolate. These four coding DNA sequences coded for "hypothetical proteins of unknown function” and require further analysis. We also identified nine proteins common to both European domestic ruminants tested.[br/] [br/] Conclusion:[br/] Using a whole genome capture approach, we have sequenced the first A. phagocytophilum genome isolated from a cow. To the best of our knowledge, this is the first time that this method has been used to selectively enrich pathogenic bacterial DNA from samples also containing host DNA. The four proteins unique to the A. phagocytophilum bovine genome could be involved in host tropism, therefore their functions need to be explored

Development of a multiple locus VNTR analysis for <em>Anaplasma phagocytophilum</em>

National audienc

Diversité génétique d’<em>Anaplama phagocytophilum</em>, agent de l’anaplasmose granulocytaire, - Conséquences épidémiologiques et implications pour le contrôle en France -

National audienc

Diversité génétique d’<em>Anaplama phagocytophilum</em>, agent de l’anaplasmose granulocytaire, - Conséquences épidémiologiques et implications pour le contrôle en France -

National audienc

Multiple-locus variable-number tandem repeat analysis potentially reveals the existence of two groups of Anaplasma phagocytophilum circulating in cattle in France with different wild reservoirs

Abstract Background Anaplasma phagocytophilum is the causative agent of tick-borne fever, a disease with high economic impact for domestic ruminants in Europe. Epidemiological cycles of this species are complex, and involve different ecotypes circulating in various host species. To date, these epidemiological cycles are poorly understood, especially in Europe, as European reservoir hosts (i.e. vertebrate hosts enabling long-term maintenance of the bacterium in the ecosystem), of the bacterium have not yet been clearly identified. In this study, our objective was to explore the presence, the prevalence, and the genetic diversity of A. phagocytophilum in wild animals, in order to better understand their implications as reservoir hosts of this pathogen. Methods The spleens of 101 wild animals were collected from central France and tested for the presence of A. phagocytophilum DNA by msp2 qPCR. Positive samples were then typed by multi-locus variable-number tandem repeat (VNTR) analysis (MLVA), and compared to 179 previously typed A. phagocytophilum samples. Results Anaplasma phagocytophilum DNA was detected in 82/101 (81.2%) animals including 48/49 red deer (98%), 20/21 roe deer (95.2%), 13/29 wild boars (44.8%), and 1/1 red fox. MLVA enabled the discrimination of two A. phagocytophilum groups: group A contained the majority of A. phagocytophilum from red deer and two thirds of those from cattle, while group B included a human strain and variants from diverse animal species, i.e. sheep, dogs, a horse, the majority of variants from roe deer, and the remaining variants from cattle and red deer. Conclusions Our results suggest that red deer and roe deer are promising A. phagocytophilum reservoir host candidates. Moreover, we also showed that A. phagocytophilum potentially circulates in at least two epidemiological cycles in French cattle. The first cycle may involve red deer as reservoir hosts and cattle as accidental hosts for Group A strains, whereas the second cycle could involve roe deer as reservoir hosts and at least domestic ruminants, dogs, horses, and humans as accidental hosts for Group B strains

One particular <i>Anaplasma phagocytophilum</i> ecotype infects cattle in the Camargue, France

Abstract Background Anaplasma phagocytophilum is a zoonotic tick-borne pathogen responsible for granulocytic anaplasmosis, a mild to a severe febrile disease that affects man and several animal species, including cows and horses. In Europe, I. ricinus is the only proven vector for this pathogen, but studies suggest that other tick genera and species could be involved in its transmission. Our objective was to assess the presence and genetic diversity of A. phagocytophilum in domestic animals and different tick species from the Camargue region, located in the south of France. Methods A total of 140 ticks and blood samples from 998 cattle and 337 horses were collected in Camargue and tested for the presence of A. phagocytophilum DNA by msp2 quantitative real-time PCR. Molecular typing with four markers was performed on positive samples. Results Anaplasma phagocytophilum DNA was detected in 6/993 (0.6%) cows, 1/20 (5%) Haemaphysalis punctata, 1/57 (1.75%) Rhipicephalus pusillus, and was absent in horses (0%). All cattle A. phagocytophilum presented a profile identical to an A. phagocytophilum variant previously detected in Dermacentor marginatus, Hyalomma marginatum, and Rhipicephalus spp. in Camargue. Conclusions Our results demonstrate that one particular A. phagocytophilum variant infects cattle in Camargue, where I. ricinus is supposed to be rare or even absent. Dermacentor marginatus, Rhipicephalus spp. and Hyalomma spp., and possibly other tick species could be involved in the transmission of this variant in this region

Development of new markers for the characterization of <em>Anaplasma phagocytophilum</em> by MLST

National audienc

Three clusters of <em>Anaplasma phagocytophilum</em> isolates from clinical cases of French domestic animals revealed by multi-locus sequence analysis

International audienc