Sarah I Bonnet
email: sarah.bonnet@inrae.fr

Clémence Nadal
email: clemence.nadal@anses.fr

Estrella Montero

Jeremy Gray

Cheryl Ann Lobo

Luis Miguel González

Experimental Infection of Ticks: An Essential Tool for the Analysis of Babesia Species Biology and Transmission

Keywords: ticks, Babesia sp., biological cycle, experimental transmission, experimental models

Babesiosis is one of the most important tick-borne diseases in veterinary health, impacting mainly cattle, equidae, and canidae, and limiting the development of livestock industries worldwide. In humans, babesiosis is considered to be an emerging disease mostly due to Babesia divergens in Europe and Babesia microti in America. Despite this importance, our knowledge of Babesia sp. transmission by ticks is incomplete. The complexity of vectorial systems involving the vector, vertebrate host, and pathogen, as well as the complex feeding biology of ticks, may be part of the reason for the existing gaps in our knowledge. Indeed, this complexity renders the implementation of experimental systems that are as close as possible to natural conditions and allowing the study of tick-host-parasite interactions, quite difficult. However, it is unlikely that the development of more effective and sustainable control measures against babesiosis will emerge unless significant progress can be made in understanding this tripartite relationship. The various methods used to date to achieve tick transmission of Babesia spp. of medical and veterinary importance under experimental conditions are reviewed and discussed here.

Introduction

Babesiosis remains prevalent worldwide and represents an important threat for both humans and animals [1,[START_REF] Lobo | Human babesiosis: Recent advances and future challenges[END_REF]. The disease, impacting mainly cattle, sheep, goat, equidae, canidaecanidae, and accidentally humans, is caused by apicomplexan parasites belonging to the Babesia genus that exclusively infect erythrocytes of their vertebrate hosts [START_REF] Chauvin | Babesia and its hosts: Adaptation to long-lasting interactions as a way to achieve efficient transmission[END_REF]. To date, more than 100 Babesia species have been identified [1]. Babesia spp. are transmitted by hard ticks-occasionally by blood transfusion-and require both a competent vertebrate and invertebrate host to maintain the transmission cycle [START_REF] Chauvin | Babesia and its hosts: Adaptation to long-lasting interactions as a way to achieve efficient transmission[END_REF].

Human babesiosis is caused by Babesia microti, a Babesia crassa-like pathogen, Babesia divergens, Babesia duncani, and Babesia venatorum, as well as other parasites closely genetically related to these pathogens, such as B. divergens-like, B. duncani-like, and B. microti-like [START_REF] Krause | Human babesiosis[END_REF]. Infections in otherwise healthy individuals is usually mild to moderate and most cases of severe disease occur in immunocompromised individuals. B. microti is endemic in the northeastern and upper midwestern regions of the United States, while B. duncani is present on the west coast of the country [START_REF] Krause | Human babesiosis[END_REF]. In Europe, most of the human cases are due to B. divergens, whereas in Asia they are due to B. venatorum, B. crassa-like, and B. microti [START_REF] Krause | Human babesiosis[END_REF]. In cattle, Babesia spp. have a significant worldwide economic, social, and epidemiological impact and include, among the most important species, B. bovis, B. bigemina, Babesia major, and B. divergens [START_REF] Bock | Babesiosis of cattle[END_REF]. B. bovis and B. bigemina are present in many countries in Africa, Asia, Pathogens 2021, 10, 1403 2 of 14 Australia, Central and South America, and Southern Europe between 40 • N and 32 • S. B. major is present in Europe, Northwest Africa and Asia, and B. divergens is present in northern Europe [START_REF] Bock | Babesiosis of cattle[END_REF]. Ovine babesiosis due to Babesia ovis and Babesia motasi is considered as the most critical blood-borne parasitic disease of small ruminants in tropical and nontropical regions (occurring in South-eastern Europe, North Africa, and Asia) [START_REF] Yeruham | Some epizootiological and clinical aspects of ovine babesiosis caused by Babesia ovis-A review[END_REF]. In equids, Babesia caballi is (with Theileria equi and Theileria haneyi) the agent of equine piroplasmosis known to be endemic in several countries of Africa, Asia, the Americas, and mainly in the Mediterranean basin for Europe [START_REF] Tirosh-Levy | Twenty Years of Equine Piroplasmosis Research: Global Distribution, Molecular Diagnosis, and Phylogeny[END_REF][START_REF] Nadal | Eco-epidemiology of equine piroplasmosis and its associated tick vectors in Europe: A systematic literature review and a meta-analysis of prevalence[END_REF][START_REF] Knowles | Discovery of a novel species, Theileria haneyi n. sp., infective to equids, highlights exceptional genomic diversity within the genus Theileria: Implications for apicomplexan parasite surveillance[END_REF]. The disease represents a significant animal health issue and causes notable economic losses for the equine industry. Finally, babesiosis is one of the most important globally extended and quickly spreading tick-borne diseases in dogs worldwide. Babesia canis is the main cause of canine babesiosis in Europe and is only sporadically found around the world, whereas Babesia gibsoni, the most prevalent species, and Babesia vogeli have a global distribution. Babesia rossi, one of the most pathogenic species, is endemic in southern Africa [START_REF] Bilić | Canine Babesiosis: Where Do We Stand?[END_REF].

The current approaches available for babesiosis control have many important limitations, including increased resistance to acaricides by ticks, as well as the numerous drawbacks of these acaricides and of the current vaccines and babesicidal drugs (e.g., efficacy, toxicity, environmental effects) [START_REF] Suarez | Emerging perspectives in the research of bovine babesiosis and anaplasmosis[END_REF]. The development of improved control measures against babesiosis is limited by the numerous and significant gaps in our understanding of the biology of Babesia spp., especially regarding molecular interaction between parasites, vectors, and vertebrate hosts, as well as the factors that may influence both the development and the transmission of the parasite [START_REF] Rodriguez-Morales | Research on Babesia: A bibliometric assessment of a neglected tick-borne parasite[END_REF]. To fill these gaps, it is essential to be able to reproduce the life cycle of Babesia species in controlled experimental conditions, including transmission by ticks [START_REF] Bonnet | Laboratory artificial infection of hard ticks: A tool for the analysis of tick-borne pathogen transmission[END_REF][START_REF] De La Fuente | Tick-Pathogen Interactions and Vector Competence: Identification of Molecular Drivers for Tick-Borne Diseases[END_REF]. In addition, the validation of new methods of interruption of the cycle requires that it can be first carried out entirely under such conditions. Finally, it is important to mention that despite the promise of in vitro culture systems [START_REF] Muller | In vitro culture systems for the study of apicomplexan parasites in farm animals[END_REF], maintenance by in vitro culture [START_REF] Sondgeroth | Tick passage results in enhanced attenuation of Babesia bovis[END_REF] or needle-passage in the vertebrate host [START_REF] Stewart | Differences in the life cycles between a vaccine strain and an unmodified strain of Babesia bovis (Babes, 1889) in the tick Boophilus microplus (Canestrini)[END_REF] in the absence of tick passage may generate significant changes in the parasite population, potentially creating a bias in research results.

Several laboratory studies that aimed to understand babesiosis pathogenesis have focused their interest on infecting laboratory animals through artificial parasite injection [START_REF] Zaugg | Experimental infections of Babesia bigemina in American bison[END_REF][START_REF] Chauvin | Sheep as a new experimental host for Babesia divergens[END_REF]. Regarding tick-parasite interaction, the great majority of studies carried out concern mainly epidemiological studies focusing on the detection of parasites in ticks collected into the field [START_REF] Martinez-Garcia | Challenges in Tick-Borne Pathogen Detection: The Case for Babesia spp. Identification in the Tick Vector[END_REF]. Some in vitro studies have also been performed in order to understand the interactions between parasites and the cells of their vertebrate [START_REF] Sun | The invasion process of bovine erythrocyte by Babesia divergens: Knowledge from an in vitro assay[END_REF][START_REF] Spencer | In vitro host erythrocyte specificity and differential morphology of Babesia divergens and a zoonotic Babesia sp. from eastern cottontail rabbits (Sylvilagus floridanus)[END_REF][START_REF] O'connor | Cytoadherence of Babesia bovis-infected erythrocytes to bovine brain capillary endothelial cells provides an in vitro model for sequestration[END_REF] or invertebrate hosts [START_REF] Maeda | Initial development of Babesia ovata in the tick midgut[END_REF][START_REF] Bhat | The invasion and growth of Babesia bovis in tick tissue culture[END_REF][START_REF] Ribeiro | Babesia bigemina: In vitro multiplication of sporokinetes in Ixodes scapularis (IDE8) cells[END_REF][START_REF] De Rezende | In vitro cultivation and cryopreservation of Babesia bigemina sporokinetes in hemocytes of Rhipicephalus microplus[END_REF]. However, and certainly because of the difficulties inherent to the studied model-including Babesia spp. culture, tick colony maintenance, and animal models-relatively few studies have been based on the establishment, under experimental conditions, of complete parasite transmission cycles from one vertebrate host to another via the tick bite. Thanks to the experimental models developed for that purpose, these few studies have nevertheless made significant advances in (1) the definition/confirmation of the vector competence of various tick species; (2) the understanding of the modalities of parasite acquisition and transmission by ticks; (3) the discovery of the molecular interactions between the parasite and its invertebrate hosts; (4) the evaluation of some control methods. The aim of this review is to summarize studies that include both tick infection on Babesia-infected animals and Babesia infection of ticks through artificial systems, and to comment on the major results they achieved.

General Description of the Babesia Life Cycle

Babesia spp. are transmitted by hard tick (ixodid) vectors. The tick vectors and reservoir hosts differ depending on Babesia sp. and geographical location considered [1]. Several tick species have been mentioned in the literature as vectors of Babesia sp., but, as shown in Table 1, vector competence through experimental transmission has not been validated for all of them. For those mentioned here as "suspected vectors" without realization of the complete transmission cycle under experimental conditions, their involvement is mostly based on epidemiological evidence (e.g., correlation of tick species presence with disease occurrence). Each of the three active stages of hard ticks (larva, nymph, and adult) takes a single blood meal from a vertebrate host in order to mature to the next stage or lay eggs for the female. Most of the tick species listed as confirmed or suspected vectors of some Babesia species are three-hosts ticks-meaning that they take their three blood meals on three different hosts. Some of them, however, have a two-host cycle such as some Hyalomma spp., Rhipicephalus evertsi, and Rhipicephalus bursa, whereas Dermacentor nitens, Rhipicephalus annulatus, Rhipicephalus microplus, and Rhipicephalus decoloratus are one-host ticks that use the same individual host animal for all active tick stages. The Babesia life cycle includes both asexual multiplication in the erythrocytes of the vertebrate host and sexual reproduction in the tick vector [START_REF] Chauvin | Babesia and its hosts: Adaptation to long-lasting interactions as a way to achieve efficient transmission[END_REF][START_REF] Mehlhorn | The piroplasms: Life cycle and sexual stages[END_REF]. The general life cycle of the Babesia species is summarized in Figure 1 for Babesia sensu stricto (s.s.) species. Indeed, it is necessary to specify here that, quite recently, molecular phylogeny studies using the 18S rRNA gene have led to the division of Babesia species into two large groups: Babesia s.s. and Babesia sensu lato (s.l.). Species belonging to the latter group, such as B. microti, are not capable of transovarial transmission within the tick but only a transstadial mode of transmission [START_REF] Gray | Babesia microti[END_REF][START_REF] Jalovecka | Babesia Life Cycle-When Phylogeny Meets Biology[END_REF]. Vertebrate hosts are infected by the injection of sporozoites present in tick saliva during the tick bite. Each sporozoite penetrates the cell membrane of an erythrocyte with the aid of a specialized apical complex. Once inside, the parasite produces two merozoites by a process of merogony. Merozoites are then intermittently released following erythrocyte lysis to infect new erythrocytes. The parasite may then persist asymptomatically within its host for several years or lead to acute disease. When they are ingested by the tick during the blood meal, some parasites present in infected erythrocytes (pre-gametocytes) undergo further development in the passage from host blood to the midgut of the tick vector to evolve into gametocytes. The sexual reproduction between gametocytes takes place in the tick gut and leads to a zygote that penetrates the gut epithelium, where further multiplication occurs, with development to motile and haploid kinetes that escape into the tick hemolymph. The kinetes then infect a variety of tick cell types and tissues, including the ovary in the female tick-for Babesia s.s. species-and the tick salivary glands, where successive cycles of asexual multiplication take place. In this last organ, sporozoite development usually only begins when the infected tick attaches to the vertebrate host. The ticks thus transmit the sporozoites to a new host during a new blood meal of the next life-stage for the ticks with several hosts or of the next generation after transovarian transmission for the one-host ticks.

The Babesia life cycle includes both asexual multiplication in the erythrocytes of the vertebrate host and sexual reproduction in the tick vector [START_REF] Chauvin | Babesia and its hosts: Adaptation to long-lasting interactions as a way to achieve efficient transmission[END_REF][START_REF] Mehlhorn | The piroplasms: Life cycle and sexual stages[END_REF]. The general life cycle of the Babesia species is summarized in Figure 1 for Babesia sensu stricto (s.s.) species. Indeed, it is necessary to specify here that, quite recently, molecular phylogeny studies using the 18S rRNA gene have led to the division of Babesia species into two large groups: Babesia s.s. and Babesia sensu lato (s.l.). Species belonging to the latter group, such as B. microti, are not capable of transovarial transmission within the tick but only a transstadial mode of transmission [START_REF] Gray | Babesia microti[END_REF][START_REF] Jalovecka | Babesia Life Cycle-When Phylogeny Meets Biology[END_REF]. Vertebrate hosts are infected by the injection of sporozoites present in tick saliva during the tick bite. Each sporozoite penetrates the cell membrane of an erythrocyte with the aid of a specialized apical complex. Once inside, the parasite produces two merozoites by a process of merogony. Merozoites are then intermittently released following erythrocyte lysis to infect new erythrocytes. The parasite may then persist asymptomatically within its host for several years or lead to acute disease. When they are ingested by the tick during the blood meal, some parasites present in infected erythrocytes (pre-gametocytes) undergo further development in the passage from host blood to the midgut of the tick vector to evolve into gametocytes. The sexual reproduction between gametocytes takes place in the tick gut and leads to a zygote that penetrates the gut epithelium, where further multiplication occurs, with development to motile and haploid kinetes that escape into the tick hemolymph. The kinetes then infect a variety of tick cell types and tissues, including the ovary in the female tick-for Babesia s.s. species-and the tick salivary glands, where successive cycles of asexual multiplication take place. In this last organ, sporozoite development usually only begins when the infected tick attaches to the vertebrate host. The ticks thus transmit the sporozoites to a new host during a new blood meal of the next life-stage for the ticks with several hosts or of the next generation after transovarian transmission for the one-host ticks. Vertebrate hosts are infected following the bite of an infected hard tick, through the invasion of the host erythrocytes by sporozoites excreted in the tick saliva. Inside the erythrocyte, sporozoites develop into trophozoites that undergo an asexual multiplication called merogony, ending in the formation of either merozoites that can infect other erythrocytes, or of gametocytes, which eventually develop into gametes. Infected erythrocytes are taken up by the tick during its blood meal, but only the gametocytes survive, and they then undergo further development, changing into gametes in the tick midgut. Then, sexual multiplication-gamogony-takes place with the fusion of two gametes to form a motile zygote that enters the midgut epithelial cells to develop into motile kinetes through meiotic division. Kinetes disseminate to tick tissues, including ovarian and salivary gland cells. The invasion of tick ovaries results in transovarial transmission while those of salivary glands leads to transmission to the vertebrate host through injection of sporozoites with the saliva. Babesia microti-like species, which belong to Babesia spp. s.l. species, only invade the salivary glands, not the ovary. The schematic representation was made using the software biorender.com.

Experimental Models of Babesia Species-Transmission by Ticks

Tick Infestations on Babesia spp Infected Animals

The first experiments to transmit Babesia spp. naturally were carried out by applying ticks suspected of being vectors on infected animals. Due to the huge economic importance of bovine babesiosis, these studies on vector competence for Babesia sp. were first conducted on species that infect cattle. Indeed, in 1893, two American researchers, Smith and Kilborne-the first authors to demonstrate the transmission of a disease organism from an arthropod to a mammalian host-showed the vector competence of R. annulatus for B. bigemina by placing ticks infected on animal onto naïve cattle that developed the associated disease [START_REF] Smith | Investigations into the nature, causation and prevention of Southern cattle fever[END_REF]. Thereafter, different species of animals were used depending on the species of Babesia studied. Over time, the animal models and methodology used were refined to optimize the infection of both animals and ticks, and to comply with health and safety rules, and animal accommodation and tick containment methods during the tick feeding process have been the subject of several tests and evolutions (see examples in Figure 2A-F). In most cases, for cattle, animals were housed in individual, tick-proof pens surrounded by moats with or without detergent or insecticide [START_REF] Callow | Transmission experiments using Babesia bigemina, Theileria mutans, Borrelia sp. and the cattle tick, Boophilus microplus[END_REF][START_REF] Morzaria | Babesia bigemina in Kenya: Experimental transmission by Boophilus decoloratus and the production of tick-derived stabilates[END_REF]. Concerning the tick containment methods, in 1961, Callow and Hoyte used a hessian rug to protect the larvae until adult repletion to demonstrate the transmission of B. bigemina to cattle by R. microplus [START_REF] Callow | Transmission experiments using Babesia bigemina, Theileria mutans, Borrelia sp. and the cattle tick, Boophilus microplus[END_REF]. Ticks were either allowed to spread at will over the animal, or were confined to one site by releasing them under a fabric patch (nylon or organdie), which was glued along its edges to the flank of the bovine. A few years later, for the demonstration of the vector competence of R. decoloratus for B. bigemina in Kenya, ticks were fed on cattle until adult repletion by sprinkling larvae on the backs of the animals [START_REF] Morzaria | Babesia bigemina in Kenya: Experimental transmission by Boophilus decoloratus and the production of tick-derived stabilates[END_REF]. For the first experimental transmission of B. divergens by Ixodes ricinus achieved by Joyner et al in 1963, the ticks were contained in ear bags [START_REF] Joyner | The Experimental Transmission of Babesia divergens by Ixodes ricinus[END_REF], a method also used later for sheep [START_REF] Razmi | Transovarial Transmission of Babesia ovis by Rhipicephalus sanguineus and Hyalomma marginatum[END_REF]. Regarding rodents, several laboratory studies involving complete transmission cycles of B. microti to the vertebrate host through the tick bite were performed [START_REF] Dunn | Borrelia burgdorferi promotes the establishment of Babesia microti in the northeastern United States[END_REF][START_REF] Randolph | The effect of Babesia microti on feeding and survival in its tick vector, Ixodes trianguliceps[END_REF][START_REF] Ruebush | Tick transmission of Babesia microti to rhesus monkeys (Macaca mulatta)[END_REF][START_REF] Gray | Transmission studies of Babesia microti in Ixodes ricinus ticks and gerbils[END_REF][START_REF] Walter | A study on the transmission (transstadial, transovarial) of Babesia microti, strain "Hannover i", in its tick vector, Ixodes ricinus (author's transl)[END_REF][START_REF] Li | Experimental transmission of Babesia microti by Rhipicephalus haemaphysaloides[END_REF]. In most instances, rodents were maintained over trays of water from which detached engorged ticks-applied by brush to animals-were harvested, whereas in some cases, ticks were contained in plastic capsules attached with different adhesives. For horses, the first studies aimed to validate the vector competence of D. nitens for B. caballi used larvae applied by brush on the animal [START_REF] Roby | The Hereditary Transmission of Babesia Caballi in the Tropical Horse Tick, Dermacentor Nitens Neumann[END_REF], whereas in subsequent studies, tick feedings were accomplished by placing the larvae under a cloth patch glued to the back of the host [START_REF] Schwint | Transmission of Babesia caballi by Dermacentor nitens (Acari: Ixodidae) is restricted to one generation in the absence of alimentary reinfection on a susceptible equine host[END_REF].

The identification of a pathogen or pathogen DNA alone-which is even less convinc ing-in an arthropod cannot be sufficient to prove its ability to transmit this pathogen. Indeed, demonstrations of parasite presence in unfed field ticks, in tick salivary glands, eggs, or unfed larvae, while more convincing than detection in ticks collected from animals, also require confirmation only provided by the validation of vector competence in a controlled experimental model. The best illustration of this corresponds to the following studies performed in Iran in order to identify the vector of B. ovis to sheep. The kinetes of B. ovis were observed in hemolymph and egg smears of Rhipicephalus sanguineus and Hyalomma marginatum field ticks collected from sheep infected with B. ovis [START_REF] Razmi | An epidemiological study on ovine babesiosis in the Mashhad suburb area, province of Khorasan, Iran[END_REF], so the vector competence of both tick species was further evaluated by placing pairs of adult ticks on sheep inoculated with B. ovis, but no transmission by any of the succeeding tick stages could be demonstrated, thus showing that these tick species are not vectors [START_REF] Razmi | Transovarial Transmission of Babesia ovis by Rhipicephalus sanguineus and Hyalomma marginatum[END_REF]. (G) (H) [START_REF] Bonnet | Infection of Siberian chipmunks (Tamias sibiricus barberi) with Borrelia sp. reveals a low reservoir competence under experimental conditions[END_REF], (B) mouse [START_REF] Almazan | Multiple Antigenic Peptide-Based Vaccines Targeting Ixodes ricinus Neuropeptides Induce a Specific Antibody Response but Do Not Impact Tick Infestation[END_REF], (C,D) sheep [START_REF] Almazan | Experimental Ixodes ricinus-Sheep Cycle of Anaplasma phagocytophilum NV2Os Propagated in Tick Cell Cultures[END_REF], (E,F) rabbit [START_REF] Almazan | A Versatile Model of Hard Tick Infestation on Laboratory Rabbits[END_REF], and (F,H) a membrane artificial feeding system [START_REF] Bonnet | Transstadial and transovarial persistence of Babesia divergens DNA in Ixodes ricinus ticks fed on infected blood in a new skin-feeding technique[END_REF].

(A) (B) (C) (D) (E) (F)
The identification of a pathogen or pathogen DNA alone-which is even less convincing-in an arthropod cannot be sufficient to prove its ability to transmit this pathogen. Indeed, demonstrations of parasite presence in unfed field ticks, in tick salivary glands, eggs, or unfed larvae, while more convincing than detection in ticks collected from animals, also require confirmation only provided by the validation of vector competence in a controlled experimental model. The best illustration of this corresponds to the following studies performed in Iran in order to identify the vector of B. ovis to sheep. The kinetes of B. ovis were observed in hemolymph and egg smears of Rhipicephalus sanguineus and Hyalomma marginatum field ticks collected from sheep infected with B. ovis [START_REF] Razmi | An epidemiological study on ovine babesiosis in the Mashhad suburb area, province of Khorasan, Iran[END_REF], so the vector competence of both tick species was further evaluated by placing pairs of adult ticks on sheep inoculated with B. ovis, but no transmission by any of the succeeding tick stages could be demonstrated, thus showing that these tick species are not vectors [START_REF] Razmi | Transovarial Transmission of Babesia ovis by Rhipicephalus sanguineus and Hyalomma marginatum[END_REF].

As recently reviewed by Gray and co-workers, the establishment of experimental models of pathogens transmission by ticks using live animals has led to significant advances in the understanding of transmission modalities and tick-parasite interactions [START_REF] Gray | Vectors of Babesiosis[END_REF]. For example, it allowed the demonstration that transovarial transmission does not occur in B. microti, leading to no longer classifying this parasite in Babesia spp. s.s. [START_REF] Gray | Transmission studies of Babesia microti in Ixodes ricinus ticks and gerbils[END_REF][START_REF] Walter | A study on the transmission (transstadial, transovarial) of Babesia microti, strain "Hannover i", in its tick vector, Ixodes ricinus (author's transl)[END_REF][START_REF] Li | Experimental transmission of Babesia microti by Rhipicephalus haemaphysaloides[END_REF], and that, in I. ricinus, the infection only survives one molt [START_REF] Gray | Transmission studies of Babesia microti in Ixodes ricinus ticks and gerbils[END_REF]. Laboratory models have also demonstrated that B. microti may promote its transmission in rodents by enhancing the feeding success and survival of its tick vector, Ixodes trianguliceps [START_REF] Randolph | The effect of Babesia microti on feeding and survival in its tick vector, Ixodes trianguliceps[END_REF]. Likewise, the establishment of the transmission of B. divergens by I. ricinus in a gerbil experimental model [START_REF] Bonnet | Transstadial and transovarial persistence of Babesia divergens DNA in Ixodes ricinus ticks fed on infected blood in a new skin-feeding technique[END_REF] provided proof of sexual development of Babesia through DNA measurements on the developmental stages of B. divergens in the blood of the vertebrate host and in the gut, hemolymph, and salivary glands of the tick vector [START_REF] Lewis | The transmission of a human strain of Babesia divergens by Ixodes ricinus ticks[END_REF][START_REF] Mackenstedt | Sexual cycle of Babesia divergens confirmed by DNA measurements[END_REF]. In addition, several studies have been performed in order to establish which tick life stages are able to acquire and/or to transmit the parasite. Most of them have concluded that only adult stages were able to acquire Babesia sp. s.s. from infected animals, while all succeeding stages (larvae, nymphs, and adults) were able to re-transmit the parasites to susceptible animals [START_REF] Joyner | The Experimental Transmission of Babesia divergens by Ixodes ricinus[END_REF][START_REF] Donnelly | Experiments on the transmission of Babesia divergens to cattle by the tick Ixodes ricinus[END_REF][START_REF] Callow | Transmission experiments using Babesia bigemina, Theileria mutans, Borrelia sp. and the cattle tick, Boophilus microplus[END_REF][START_REF] Callow | Babesia Bigemina in Ticks Grown on Non-Bovine Hosts and Its Transmission to These Hosts[END_REF][START_REF] Morzaria | Experimental transmission of Babesia major by Haemaphysalis punctata[END_REF]. However, Schwint and co-workers demonstrated that only the first of three subsequent generations from D. nitens females was able to transmit B. caballi to naïve horses, showing that the parasite is unable to persist in ticks without continuing alimentary infection of adult females [START_REF] Schwint | Transmission of Babesia caballi by Dermacentor nitens (Acari: Ixodidae) is restricted to one generation in the absence of alimentary reinfection on a susceptible equine host[END_REF], whereas other studies have also shown the acquisition of this parasite by nymphs of R. evertsi [START_REF] De Waal | The transstadial transmission of Babesia caballi by Rhipicephalus evertsi evertsi[END_REF].

Models of transmission of B. microti to rodents via ticks in the laboratory have also made it possible to carry out studies on the phenomena of co-infections. In fact, a lower transmission efficiency of B. microti than Borrelia burgdorferi to Ixodes dammini from both hamsters [START_REF] Piesman | Intensity and duration of Borrelia burgdorferi and Babesia microti infectivity in rodent hosts[END_REF] and white-footed mice [START_REF] Mather | Borrelia burgdorferi and Babesia microti: Efficiency of transmission from reservoirs to vector ticks (Ixodes dammini)[END_REF] has been demonstrated. In the meantime, it was (C,D) sheep [START_REF] Almazan | Experimental Ixodes ricinus-Sheep Cycle of Anaplasma phagocytophilum NV2Os Propagated in Tick Cell Cultures[END_REF], (E,F) rabbit [START_REF] Almazan | A Versatile Model of Hard Tick Infestation on Laboratory Rabbits[END_REF], and (G,H) a membrane artificial feeding system [START_REF] Bonnet | Transstadial and transovarial persistence of Babesia divergens DNA in Ixodes ricinus ticks fed on infected blood in a new skin-feeding technique[END_REF].

As recently reviewed by Gray and co-workers, the establishment of experimental models of pathogens transmission by ticks using live animals has led to significant advances in the understanding of transmission modalities and tick-parasite interactions [START_REF] Gray | Vectors of Babesiosis[END_REF]. For example, it allowed the demonstration that transovarial transmission does not occur in B. microti, leading to no longer classifying this parasite in Babesia spp. s.s. [START_REF] Gray | Transmission studies of Babesia microti in Ixodes ricinus ticks and gerbils[END_REF][START_REF] Walter | A study on the transmission (transstadial, transovarial) of Babesia microti, strain "Hannover i", in its tick vector, Ixodes ricinus (author's transl)[END_REF][START_REF] Li | Experimental transmission of Babesia microti by Rhipicephalus haemaphysaloides[END_REF], and that, in I. ricinus, the infection only survives one molt [START_REF] Gray | Transmission studies of Babesia microti in Ixodes ricinus ticks and gerbils[END_REF]. Laboratory models have also demonstrated that B. microti may promote its transmission in rodents by enhancing the feeding success and survival of its tick vector, Ixodes trianguliceps [START_REF] Randolph | The effect of Babesia microti on feeding and survival in its tick vector, Ixodes trianguliceps[END_REF]. Likewise, the establishment of the transmission of B. divergens by I. ricinus in a gerbil experimental model [START_REF] Bonnet | Transstadial and transovarial persistence of Babesia divergens DNA in Ixodes ricinus ticks fed on infected blood in a new skin-feeding technique[END_REF] provided proof of sexual development of Babesia through DNA measurements on the developmental stages of B. divergens in the blood of the vertebrate host and in the gut, hemolymph, and salivary glands of the tick vector [START_REF] Lewis | The transmission of a human strain of Babesia divergens by Ixodes ricinus ticks[END_REF][START_REF] Mackenstedt | Sexual cycle of Babesia divergens confirmed by DNA measurements[END_REF]. In addition, several studies have been performed in order to establish which tick life stages are able to acquire and/or to transmit the parasite. Most of them have concluded that only adult stages were able to acquire Babesia sp. s.s. from infected animals, while all succeeding stages (larvae, nymphs, and adults) were able to re-transmit the parasites to susceptible animals [START_REF] Joyner | The Experimental Transmission of Babesia divergens by Ixodes ricinus[END_REF][START_REF] Donnelly | Experiments on the transmission of Babesia divergens to cattle by the tick Ixodes ricinus[END_REF][START_REF] Callow | Transmission experiments using Babesia bigemina, Theileria mutans, Borrelia sp. and the cattle tick, Boophilus microplus[END_REF][START_REF] Callow | Babesia Bigemina in Ticks Grown on Non-Bovine Hosts and Its Transmission to These Hosts[END_REF][START_REF] Morzaria | Experimental transmission of Babesia major by Haemaphysalis punctata[END_REF]. However, Schwint and co-workers demonstrated that only the first of three subsequent generations from D. nitens females was able to transmit B. caballi to naïve horses, showing that the parasite is unable to persist in ticks without continuing alimentary infection of adult females [START_REF] Schwint | Transmission of Babesia caballi by Dermacentor nitens (Acari: Ixodidae) is restricted to one generation in the absence of alimentary reinfection on a susceptible equine host[END_REF], whereas other studies have also shown the acquisition of this parasite by nymphs of R. evertsi [START_REF] De Waal | The transstadial transmission of Babesia caballi by Rhipicephalus evertsi evertsi[END_REF].

Models of transmission of B. microti to rodents via ticks in the laboratory have also made it possible to carry out studies on the phenomena of co-infections. In fact, a lower transmission efficiency of B. microti than Borrelia burgdorferi to Ixodes dammini from both hamsters [START_REF] Piesman | Intensity and duration of Borrelia burgdorferi and Babesia microti infectivity in rodent hosts[END_REF] and white-footed mice [START_REF] Mather | Borrelia burgdorferi and Babesia microti: Efficiency of transmission from reservoirs to vector ticks (Ixodes dammini)[END_REF] has been demonstrated. In the meantime, it was shown that ticks that fed on mice with these concurrent pathogen infections exhibited twice the incidence of B. burgdorferi infection compared with B. microti [START_REF] Mather | Borrelia burgdorferi and Babesia microti: Efficiency of transmission from reservoirs to vector ticks (Ixodes dammini)[END_REF]. Twentyfive years later, however, this laboratory model also showed an increase of the frequency of B. microti-infected Ixodes scapularis (formerly I. dammini) nymphs when they fed as larvae on white-footed mice coinfected with B. burgdorferi, as well as an increase of B. microti parasitemia in co-infected mice [START_REF] Dunn | Borrelia burgdorferi promotes the establishment of Babesia microti in the northeastern United States[END_REF]. This enhancement of B. microti establishment by B. burgdorferi has been attributed to an immunological conflict in the adaptive immune response of the vertebrate host against the two tick-borne pathogens [START_REF] Diuk-Wasser | Coinfection by Ixodes Tick-Borne Pathogens: Ecological, Epidemiological, and Clinical Consequences[END_REF].

The development of experimental models using animals also provided knowledge on the infection acquisition by the vertebrate host following a Babesia-infected tick bite. For example, by developing a laboratory model of B. bovis infection of calves through the bite of R. microplus-infected ticks, Smith and co-workers demonstrated, in 1978, that tick-induced infection was more severe than in calves infected with carrier blood, even when very low numbers of infected larvae were applied [START_REF] Smith | Bovine babesiosis: Severity and reproducibility of Babesia bovis infections induced by Boophilus microplus under laboratory conditions[END_REF]. They attributed this difference in virulence to the large number of infective doses injected by each infected tick but Salivary-Assisted Transmission of tick-borne pathogens (see review in [START_REF] Bonnet | Tick saliva and its role in pathogen transmission[END_REF]) probably also contributed to this observation.

Although, compared to other pathogens, few molecular studies have involved Babesia parasites [START_REF] Gray | Vectors of Babesiosis[END_REF][START_REF] Liu | Hard tick factors implicated in pathogen transmission[END_REF], experimental tick-transmission models of Babesia spp. using animals have also made it possible to identify molecules potentially involved in this transmission. Such studies are helping us to better understand the interactions involved and to identify potential targets for blocking parasite transmission. Thus, infection of H. longicornis on dogs infected with B. gibsoni has allowed to implicate a tick protein, longipain, in the transmission of the parasite by its vector [START_REF] Tsuji | A cysteine protease is critical for Babesia spp. transmission in Haemaphysalis ticks[END_REF], and to demonstrate that the vitellogenin receptor on the surface of tick oocytes is essential for its transovarial transmission [START_REF] Boldbaatar | Tick vitellogenin receptor reveals critical role in oocyte development and transovarial transmission of Babesia parasite[END_REF]. Experimental models of B. bovis infection of cattle by R. microplus has also been used to study the function of the tick protein Bm86 during B. bovis infection [START_REF] Bastos | The Rhipicephalus (Boophilus) microplus Bm86 gene plays a critical role in the fitness of ticks fed on cattle during acute Babesia bovis infection[END_REF]. In the same way, experimental tick infection models of both B. bigemina and B. bovis-infected cattle were used to perform functional genomics studies on R. annulatus and R. microplus genes that are differentially expressed in response to parasite infection [START_REF] Antunes | Functional genomics studies of Rhipicephalus (Boophilus) annulatus ticks in response to infection with the cattle protozoan parasite, Babesia bigemina[END_REF][START_REF] Heekin | The ovarian transcriptome of the cattle tick, Rhipicephalus (Boophilus) microplus, feeding upon a bovine host infected with Babesia bovis[END_REF].

Both the discovery and validation of methods to control the transmission of tickborne pathogens require experimental designs that include complete transmission cycles. Regarding the Babesia spp. that infect cattle, experimental tick infection models for B. bigemina-infected cattle were used to evaluate the efficiency of some vaccine candidates and drugs against parasite transmission by R. microplus [START_REF] Merino | Targeting the tick protective antigen subolesin reduces vector infestations and pathogen infection by Anaplasma marginale and Babesia bigemina[END_REF][START_REF] Waldron | Transmission of Babesia spp by the cattle tick (Boophilus microplus) to cattle treated with injectable or pour-on formulations of ivermectin and moxidectin[END_REF]. Rhipicephalus microplus experimentally infected with B. bovis were also used to demonstrate the inefficiency of the injectable and pour-on forms of both ivermectin and moxidectin to prevent parasite transmission by ticks [START_REF] Waldron | Transmission of Babesia spp by the cattle tick (Boophilus microplus) to cattle treated with injectable or pour-on formulations of ivermectin and moxidectin[END_REF]. In Argentina, Mangold and co-workers developed a laboratory model of B. bovis transmission to cattle by R. microplus in order to demonstrate the nontransmissibility to ticks of an attenuated vaccine strain of the parasite [START_REF] Mangold | Evaluation of the infectivity of a vaccinal and a pathogenic Babesia bovis strain from Argentina to Boophilus microplus[END_REF]. Several studies also involved experimental transmission of B. canis to dogs through the bite of infected D. reticulatus ticks in order to evaluate the usefulness of different acaricides to prevent parasite transmission [START_REF] Jongejan | The prevention of transmission of Babesia canis canis by Dermacentor reticulatus ticks to dogs using a novel combination of fipronil, amitraz and (S)-methoprene[END_REF][START_REF] Beugnet | The ability of an oral formulation of afoxolaner to block the transmission of Babesia canis by Dermacentor reticulatus ticks to dogs[END_REF][START_REF] Taenzler | Prevention of transmission of Babesia canis by Dermacentor reticulatus ticks to dogs after topical administration of fluralaner spot-on solution[END_REF][START_REF] Jongejan | A novel combination of fipronil and permethrin (Frontline Tri-Act(R)/Frontect(R)) reduces risk of transmission of Babesia canis by Dermacentor reticulatus and of Ehrlichia canis by Rhipicephalus sanguineus ticks to dogs[END_REF], whereas similar experiments were performed regarding B. canis transmission by R. sanguineus [START_REF] Fourie | The efficacy of collars impregnated with flumethrin and propoxur against experimental infestations of adult Rhipicephalus sanguineus on dogs[END_REF][START_REF] Estrada-Pena | Efficacy of a collar impregnated with amitraz and pyriproxyfen for prevention of experimental tick infestations by Rhipicephalus sanguineus, Ixodes ricinus, and Ixodes scapularis in dogs[END_REF], and B. rossi transmission by Haemaphysalis elliptica [START_REF] Beugnet | Prevention of the transmission of Babesia rossi by Haemaphysalis elliptica in dogs treated with Nexgard((R))[END_REF].

Tick Infection through Artificial Feeding Systems

The use of natural hosts for direct infection of ticks on infectious animals remains the best method to obtain conditions that are closest to the physiological reality of tick-borne pathogen transmission. However, firstly, ethical considerations lead us to limit the use of animals as much as possible. Secondly, in addition to the constraints associated with licensing the experiments and host specificity, obtaining the animals, keeping them in the laboratory, and handling them can be expensive and difficult or even impossible in the case of most species of wildlife. Finally, the difficulty of controlling parasitaemias of infected animals for the whole feeding period of the ticks is an important consideration. All these reasons have led to the development of artificial methods of tick infection in order to complete the lifecycle of tick-borne pathogens under laboratory conditions. Artificial feeding of ticks, mimicking the natural process, has been used for different purposes including tick rearing, the study of tick physiology and the effects of antibodies or drugs on tick physiology, functional genomic studies, and vaccine candidate discovery, as well as the analysis of tick-borne pathogen transmission (see review by Bonnet and Liu [START_REF] Bonnet | Laboratory artificial infection of hard ticks: A tool for the analysis of tick-borne pathogen transmission[END_REF]). Nevertheless, relatively few studies have involved Babesia parasites. One of the factors limiting these studies is undoubtedly the need for successful in vitro cultivation of the Babesia species of interest [START_REF] Muller | In vitro culture systems for the study of apicomplexan parasites in farm animals[END_REF].

The use of blood-filled capillary tubes placed over the mouthparts of ticks was first reported in 1938 by Gregson, who used this technique to collect saliva from D. andersoni [START_REF] Gregson | Notes on some phenomenal feeding of ticks[END_REF].

Since then, this technique has been used to infect ticks with several tick-borne pathogens, mainly bacteria (see review in [START_REF] Bonnet | Laboratory artificial infection of hard ticks: A tool for the analysis of tick-borne pathogen transmission[END_REF]). Although this infection process has the advantage of using the natural route of infection, i.e., the digestive tract of the tick, and allows control of the quantity of pathogen ingested, it is quite far from natural conditions because the tick absorbs a large amount of pathogen at once and this, regardless of the "true" full blood meal. The other drawback is that it requires the use of animals before or after infection to feed ticks to repletion. However, its use has led to some significant advances in the study of Babesia spp. transmission. In 1998, Inokuma and Kemp used the capillary feeding technique in order to infect R. microplus with B. bigemina [START_REF] Inokuma | Establishment of Boophilus microplus infected with Babesia bigemina by using in vitro tube feeding technique[END_REF]. Adult ticks were pre-fed on cattle and then B. bigemina-infected red blood cells were offered to the ticks using glass capillaries on a warm plate at 35 • C for 18 h in the dark. The authors subsequently demonstrated that ticks were able to acquire the parasite and to transmit it to their progeny. The capillary feeding technique has also been used by Antunes and co-workers in order to evaluate the impact of purified rabbit polyclonal antibodies on some R. microplus proteins associated with both tick physiology and tick infection by B. bigemina [START_REF] Antunes | Tick capillary feeding for the study of proteins involved in tick-pathogen interactions as potential antigens for the control of tick infestation and pathogen infection[END_REF]. While an effect on tick weight and oviposition was observed, no effect was observed on pathogen DNA levels. However, this work has made it possible to set up an alternative system to the use of animals both to test vaccine candidates and to obtain essential data on tick-pathogen interaction, as this could also be done later with another protein, the calreticulin, identified as being involved in B. bigemina infection in R. annulatus ticks [START_REF] Antunes | Artificial feeding of Rhipicephalus microplus female ticks with anti calreticulin serum do not influence tick and Babesia bigemina acquisition[END_REF].

The membrane feeding technique-consisting of feeding ticks on blood or culture media through a membrane-was first developed by Pierce and Pierce in 1956 in order to feed R. microplus using embryonated hen eggs [START_REF] Pierce | A note on the cultivation of Boophilus microplus (Canestrini, 1887) (Ixodidae: Acarina) on the embryonated hen egg[END_REF]. Since then, several membranes of animal or artificial origin have been used both to feed and to infect ticks with different pathogens (see review in [START_REF] Bonnet | Laboratory artificial infection of hard ticks: A tool for the analysis of tick-borne pathogen transmission[END_REF]). In this case, as the pathogen is mixed in blood and absorbed throughout the blood meal via the digestive tract, the method mimics the natural conditions of tick infection more closely than other methods. Its main disadvantages, however, are the need to regularly change the blood used, and the need to use antibiotics and antifungals to avoid contamination. It is also necessary to test pathogen viability under these feeding conditions at regular intervals. In the case of artificial membranes such as silicone ones, feeding systems also need olfactory stimuli for attachment and feeding. Regarding their use to infect ticks with Babesia spp., and due to red blood cell sedimentation, it is important to note that this requires the placing of blood above the membrane to produce a continuous gravitational pressure, ensuring tick absorption of the intraerythrocytic parasites. In 2007, Bonnet and co-workers developed a skin-feeding technique using the skins of both gerbils (for larvae and nymphs) and rabbits (for nymphs and adults) to infect I. ricinus with B. divergens without the need for additional stimuli (Figure 2G,H) [START_REF] Bonnet | Transstadial and transovarial persistence of Babesia divergens DNA in Ixodes ricinus ticks fed on infected blood in a new skin-feeding technique[END_REF]. To our knowledge, this is the only membrane-feeding technique that has been used to date to infect ticks with Babesia spp. All tick instars were allowed to acquire the parasite from infected red blood cells maintained at 37 • C in a glass feeder through the animal skin until repletion. Contrary to what was previously observed [START_REF] Joyner | The Experimental Transmission of Babesia divergens by Ixodes ricinus[END_REF][START_REF] Donnelly | Experiments on the transmission of Babesia divergens to cattle by the tick Ixodes ricinus[END_REF], this system showed that in addition to adult females, I. ricinus larvae and nymphs can also acquire B. divergens infections, which persists transtadially in the subsequent nymphal and adult stages (as determined by the detection of DNA in their salivary glands). Babesia divergens DNA was also detected in eggs and larvae produced by females that had fed on parasitized blood, demonstrating the transovarial transmission of the parasite. Later, the use of this artificial tick infection system also allowed the discovery of molecular markers for B. divergens sexual stages [START_REF] Becker | Identification of three CCp genes in Babesia divergens: Novel markers for sexual stages parasites[END_REF][START_REF] Becker | Validation of BdCCp2 as a marker for Babesia divergens sexual stages in ticks[END_REF]. Lastly, the same membrane feeding technique allowed the validation of the vector competence of I. ricinus for Babesia (EU1) venatorum [START_REF] Bonnet | Experimental in vitro transmission of Babesia sp. (EU1) by Ixodes ricinus[END_REF].

Finally, in order to understand tick-Babesia interactions and to follow parasite development in the vector, Maeda and co-workers used a "semi-artificial" mouse skin membrane feeding technique to infect H. longicornis with B. ovata [START_REF] Maeda | Establishment of a novel tick-Babesia experimental infection model[END_REF]. In this case, female adult ticks were first allowed to feed on the shaved back of mice, and after 4 to 5 days, a section of the mouse skin with the ticks attached was removed immediately after euthanasia, and set up in artificial feeding units. The ticks were then fed on a mix of media and B. ovata-infected red blood cells through the piece of mouse skin. This technique was then used to demonstrate the transovarial persistence of B. ovata DNA in H. longicornis [START_REF] Umemiya-Shirafuji | Transovarial persistence of Babesia ovata DNA in a hard tick, Haemaphysalis longicornis, in a semi-artificial mouse skin membrane feeding system[END_REF]. Thus, the mixing of animal use and membrane feeding makes it possible to control the parasitemia of the meal offered to ticks, but does not prevent the use of live animals.

Tick Infection through Injection

Although this method is more distant from physiological reality than the ones previously detailed, some studies have also performed tick infections by injecting the pathogen through the cuticle of the tick. In addition to requiring live animals to feed ticks after the infection, this invasive method also has the disadvantage of a low survival rate of ticks after injection [START_REF] Rechav | Comparison of methods for introducing and producing artificial infection of ixodid ticks (Acari: Ixodidae) with Ehrlichia chaffeensis[END_REF]. Nevertheless, its use by some authors has led to important results concerning Babesia spp. In 2018, Antunes and co-workers used a R. bursa-B. ovis-sheep infection model to characterize tick salivary gland genes that were differentially expressed in response to blood feeding and B. ovis infection [START_REF] Antunes | Rhipicephalus bursa Sialotranscriptomic Response to Blood Feeding and Babesia ovis Infection: Identification of Candidate Protective Antigens[END_REF]. In that experiment, female ticks were inoculated with B. ovis in the trochanter-coxae articulation and allowed to feed on rabbits. Vector competence was then confirmed by feeding B. ovis-infected ticks on a naïve lamb. This study allowed both increased understanding of the role of tick salivary gland genes in Babesia infections and identification of potential candidate vaccine antigens for innovative control strategies.

Conclusions

It is likely that vector competence for Babesia spp. has yet to be determined in some tick species. Furthermore, our understanding of the life cycles of Babesia spp. is still incomplete, especially regarding the intimate mechanisms of molecular dialogue between the parasite and its vertebrate and invertebrate hosts. Field experiments are less easy to control than those under laboratory conditions, and the gaps in our knowledge can probably only be filled by experimentally reproducing the transmission cycle as closely as possible to reality and by involving all three actors of the vectorial system. Indeed, this review has shown that the use of experimental systems for tick-borne pathogen infections that permit the complete transmission cycles of Babesia parasites has led to major scientific advances in the study of these pathogens. Despite these successes, efforts are still needed to standardize and simplify laboratory protocols to improve our ability to exploit tick artificial infection systems. It is hoped that, in the future, such models of artificial infection will be further developed in order to acquire new knowledge and develop new control strategies while avoiding the use of animals.

Figure 1 .

 1 Figure 1. Life cycle of Babesia spp. sensu stricto. Vertebrate hosts are infected following the bite of an infected hard tick, through the invasion of the host erythrocytes by sporozoites excreted in the tick saliva. Inside the erythrocyte, sporozoites develop into trophozoites that undergo an asexual multiplication called merogony, ending in the formation of either merozoites that can infect other erythrocytes, or of gametocytes, which eventually develop into gametes. Infected erythrocytes

Figure 1 .

 1 Figure 1. Life cycle of Babesia spp. sensu stricto. Vertebrate hosts are infected following the bite of an infected hard tick, through the invasion of the host erythrocytes by sporozoites excreted in the tick saliva. Inside the erythrocyte, sporozoites develop into trophozoites that undergo an asexual multiplication called merogony, ending in the formation of either merozoites that can infect other erythrocytes, or of gametocytes, which eventually develop into gametes. Infected erythrocytes are taken up by the tick during its blood meal, but only the gametocytes survive, and they then undergo further development, changing into gametes in the tick midgut. Then, sexual multiplication-gamogony-takes place with the fusion

Figure 2 .

 2 Figure 2. Cont.

Figure 2 .

 2 Figure 2. Experimental feeding of Ixodes ricinus ticks on (A) Siberian chipmunks (Tamias sibiricus barberi)[START_REF] Bonnet | Infection of Siberian chipmunks (Tamias sibiricus barberi) with Borrelia sp. reveals a low reservoir competence under experimental conditions[END_REF], (B) mouse[START_REF] Almazan | Multiple Antigenic Peptide-Based Vaccines Targeting Ixodes ricinus Neuropeptides Induce a Specific Antibody Response but Do Not Impact Tick Infestation[END_REF], (C,D) sheep[START_REF] Almazan | Experimental Ixodes ricinus-Sheep Cycle of Anaplasma phagocytophilum NV2Os Propagated in Tick Cell Cultures[END_REF], (E,F) rabbit[START_REF] Almazan | A Versatile Model of Hard Tick Infestation on Laboratory Rabbits[END_REF], and (F,H) a membrane artificial feeding system[START_REF] Bonnet | Transstadial and transovarial persistence of Babesia divergens DNA in Ixodes ricinus ticks fed on infected blood in a new skin-feeding technique[END_REF].

Figure 2 .

 2 Figure 2. Experimental feeding of Ixodes ricinus ticks on (A) Siberian chipmunks (Tamias sibiricus barberi) [65], (B) mouse [66],(C,D) sheep[START_REF] Almazan | Experimental Ixodes ricinus-Sheep Cycle of Anaplasma phagocytophilum NV2Os Propagated in Tick Cell Cultures[END_REF], (E,F) rabbit[START_REF] Almazan | A Versatile Model of Hard Tick Infestation on Laboratory Rabbits[END_REF], and (G,H) a membrane artificial feeding system[START_REF] Bonnet | Transstadial and transovarial persistence of Babesia divergens DNA in Ixodes ricinus ticks fed on infected blood in a new skin-feeding technique[END_REF].

Table 1 .

 1 Major Babesia species infecting humans, dogs, cattle, sheep, goat, and equids; their suspected or confirmed main vector and vertebrate hosts in the field; and the realization of the whole transmission cycle under experimental conditions.

Babesia spp. Suspected or Confirmed Main Vectors Main Vertebrate Hosts Realization of the Complete Transmission Cycle Under Experimental Conditions

		Ixodes persulcatus		ND
		Ixodes ovatus		ND
		Ixodes scapularis		[28]
	Babesia microti	Ixodes trianguliceps Ixodes dammini	Human, rodent	[29] [30]
		Ixodes ricinus		[31,32]
		Rhipicephalus haemaphysaloides		[33]
		Haemaphysalis longicornis		[34]
	Babesia divergens	Ixodes ricinus	Human, cattle	[35-39]
	Babesia venatorum	Ixodes ricinus	Human, roe deer	[40]
	Babesia duncani	Dermacentor albipictus	Human, mule deer	ND
		Rhipicephalus microplus		[41]
	Babesia bovis	Rhipicephalus annulatus	Cattle, buffalo	ND
		Rhipicephalus geigyi		ND
		Rhipicephalus annulatus		[42]
		Rhipicephalus microplus		[43,44]
	Babesia bigemina	Rhipicephalus decoloratus	Cattle, buffalo	[45-47]
		Rhipicephalus geigyi		ND
		Rhipicephalus evertsi		ND
	Babesia major	Haemaphysalis punctata	Cattle	[48,49]
	Babesia ovata	Haemaphysalis longicornis	Cattle	[50]
	Babesia orientalis	Rhipicephalus haemaphysaloides	Water buffalo	ND
		Dermacentor nitens		[51,52]
	Babesia caballi	Dermacentor sp. Hyalomma sp.	Horse, Donkey, Mule	ND ND
		Rhipicephalus evertsi		[53]
	Babesia ovis	Rhipicephalus bursa	Sheep and Goat	[54]
	Babesia motasi	Rhipicephalus bursa Haemaphysalis punctata	Sheep and Goat	ND ND
		Dermacentor reticulatus		[55-59]
	Babesia canis	Haemaphysalis spp.	Dog	ND
		Hyalomma spp.		ND
	Babesia gibsoni	Haemaphysalis sp. Rhipicephalus sanguineus	Dog	ND ND
	Babesia vogeli	Rhipicephalus sanguineus	Dog	ND
	Babesia rossi	Haemaphysalis elliptica Rhipicephalus sanguineus	Dog	[60] ND

ND: no identified data.

Funding:

The PhD of CN is funded by the Institut Français du Cheval et de l'Equitation (IFCE) and by the Fonds Eperon.

Conflicts of Interest:

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.


Laura Rouch
email: laura.rouch.pro@gmail.com

  Trajectoires de diversification de l'offre touristique dans les territoires de moyenne montagne : réseaux d'acteurs et dynamiques apprenantes Principaux résultats de thèse Les territoires de moyenne montagne structurés par le tourisme des sports d'hiver sont impactés directement par divers changements globaux (climatiques, évolution des clientèles, etc.) qui questionnent la continuité de l'économie neige Objectifs de la recherche : -Donner un référentiel d'analyse de la diversification de l'offre touristique sur le temps long et sur des critères d'adaptation, pas seulement sur des critères économiques -Comprendre le rôle de la diversification de l'offre touristique dans la transformation des territoires de moyenne montagne -Identifier les capacités adaptatives, en termes d'organisation d'acteurs et d'apprentissages, qui découlent de la diversification Une méthode qualitative … Basée sur 65 Entretiens semi-directifs, des documents historiques, des recensements des acteurs de la diversification, une enquête en ligne, de l'observation participante Quelles capacités adaptatives de ces territoires ?Quel rôle de la diversification de l'offre touristique ?Taille du domaine skiable selon le moment de puissance (pers*km/h)

  La diversification s'établit le long de trajectoires de différentes natures, qui peuvent coexister sur un même espace

	1
	Massif Sancy					Haut-Chablais
	1830		Ski	Déploiement d'activités culturelles, sportives, etc.	2020	1930 Ski	1970 Festival film Avoriaz	VTT	2020	À l'échelle des
										stations de ski
	Thermalisme intégré 1 trajectoire de diversification liée à la Organisation intercommunale autour du tourisme Le massif du Sancy comme périmètre cohérent de diversification construction d'un territoire touristique		Valorisation des spécificités locales Déploiement d'activités culturelles et sportives en périphérie des	internationales À l'échelle du Haut-Chablais
									stations
									2 trajectoires de diversification qui coexistent
									sur le périmètre du Haut-Chablais
	2	Des trajectoires de diversification qui transforment les territoires via l'établissement de
		systèmes touristiques plus diversifiés
	Des systèmes	Un STD structuré sur le Massif du Sancy	Un STD en cours de construction sur le Haut-Chablais,
	touristiques							structuré autour des domaines skiables avec des
	diversifiés (STD)						tentatives de transformation vers un autre modèle
	organisés autour:						touristique
	D'activités		à l'année				été-hiver
	D'un périmètre	À l'échelle du massif du Sancy, avec un		2 sous-espaces de diversification aux caractéristiques
	spatial		fort sentiment d'appartenance			territoriales distinctes
	D'acteurs		Office de Tourisme Intercommunal leader	Ø leader et une multiplicité d'organismes touristiques
				dans la diversification + polarise les		qui se partagent difficilement les compétences tourisme
				relations entre acteurs parties prenantes
	3	Ces transformations peuvent se caractériser par des apprentissages et des collaborations
		entre acteurs, déterminants de capacités adaptatives renforcées sur les territoires
	Capacités des acteurs à collaborer et	Outils et témoins d'apprentissages
	à apprendre ensemble
	partagée du vision mémoire et Création Définition du rôle d'acteurs leaders	citoyennes locales Dynamiques	adaptative Gouvernance Interaction entre acteurs et apprentissages comme garantie d'un territoire apprenant sur le long terme	et savoirs informations Accès aux	stratégies prospective, Formations Évaluations,	sur le Massif du Sancy Actions engagées, en phase de réflexion ou fonctionnements existants observés le long des trajectoires de diversification : sur le Haut-Chablais
	territoire	Plateformes
			et lieux
	Collaborations autour d'expérimenta	d'échanges		Ingénierie touristique et territoriale
	-tions

Un cadre qui permet d'identifier les marges de manoeuvre en termes d'adaptabilité des territoires


Brigitte Juanals
email: brigitte.juanals@univ-amu.fr

Jean-Luc Minel
email: jean-luc.minel@u-paris10.fr

Analyzing Museums and Key Influential Users on Twitter during the European Night of Museums 2016

Keywords: circulation of information, influence, instrumented methodology, social network, Twitter, European Night of Museums

In this paper, we first present a representation of message flows and their lexical and topic contents on Twitter, then an instrumented methodology to describe and analyze these flows and their distribution among the various stakeholders. The aim is to explore the engagement and interactions between different types of stakeholders and to identify key influential users. We apply our methodology and tools to the 12th edition of the cultural event "European Night of Museums" (NDM16).

Introduction

It is a fact that in France and in Europe, over the last decade, temporary cultural events have multiplied, in the form of public events or festivals -such as the 'Journées Européennes du Patrimoine', 'la Nuit des Musées', 'La Nuit de l'Archéologie', 'Passeurs dimages'. These events have now become recurrent, generating initiatives that contribute to cultural outreach in society as well as to the development of cultural tourism. Lastly, the 'eventualization' of culture, following the explosion of temporary exhibitions, has amplified a shift of patrimonial institutions to the logic of working like streaming media.

We consider that these profound ongoing changes in the museum context [START_REF] Juanals | La construction dun espace patrimonial partagé dans le web de données ouvert ; entre interopérabilité et stratégies institutionnelles Communication[END_REF] are a necessary framework to understand the relationship between museums and digital media and more specifically the presence of museums on social networks. Therefore, we do not attempt to assess the 'impact' of these digital media on museum practices, but rather to apprehend the place and the role they play in the evolution of these practices. In this perspective, this article focuses on the representation and analysis of message flows on Twitter during a cultural event and on the instrumented method designed to analyze these flows. The purpose is twofold: first, to devise a representation of message flows and their contents on this platform; second, to develop an instrumented methodology to describe and analyze these flows and their distribution among stakeholders in order to reflect the engagement and interactions between different types of stakeholders.

Our study is based on the 12th edition of the cultural event 'European Night of Museums' (NDM16). This event took place in the heritage institutions that were partners in the event and was extended to digital media on the website dedicated to the operation as well as on social networks (Twitter, Facebook, Instagram). The outline of this paper is the following. First, in Section 2, we will present the specificities and the contribution of our approach and our methodology to collect and analyze flows of tweets. In Section 3, the results from quantitative and lexical analysis are discussed. Finally, we conclude in Section 4.

Related Work and Contribution

The concept of participatory museums proposed by Nina Simon in her book [START_REF] Simon | The Participatory Museum[END_REF] departs from the vision of museology; her proposal is built on the articulation between the audiences who contribute to the activities of the institution and the museums that supervise encourage and evaluate collaborative projects and co-creation activities with visitors. In this section, we begin with a review of the literature in order to present and situate the contribution of our approach, and then to present the interdisciplinary approach and methodology we developed to conduct the analysis of messages.

Related Work

A number of scientific studies have investigated how museums use social media, focusing on the communication and mediation policies and practices of museums and their uses of platforms (including Facebook, Twitter) for various purposes [3] [4]. The aim is to move from a vertical logic of diffusion to the establishment of closer relationships based on audience interaction and participation. Nowadays, museums and cultural institutions use social media as a means to communicate and promote their cultural activities, as well as to interact and engage with their visitors, the main use of social media by museums remaining information and promotion of activities such as exhibition openings or events [START_REF] Fletcher | Current social media uses and evaluations in American museums[END_REF]. Some museums choose to adopt social media for audience development and engagement in order to maintain their position of cultural and educational leaders. A qualitative study on the ways that museums use Twitter in this perspective shows that they choose to link resources, engage the public with new social media tools and favor a two-way form of communication [START_REF] Osterman | Museums and Twitter: An Exploratory Qualitative Study of How Museums Use Twitter for Au-dience Development and Engagement[END_REF]. In their commitment to a digital policy of mediation of cultural resources, institutional communication strategies and marketing strategies are also a common concern. Indeed, the image of the institution is affected by its capacity to ensure visibility on media considered as innovative in their modes of public involvement and in the sharing of their cultural resources.

Villaespesa Cantalapiedra [START_REF] Villaespesa | Measuring Social Media Success: The value of the Balanced Scorecard as a tool for evaluation and strategic management in museums PhD[END_REF] carried out fieldwork including a series of interviews with museum professionals which showed that the term engagement, 'can be interpreted in a variety of ways (. . . ): From fostering inspiration and creativity in the user, originating a change of behavior, increasing the user's knowledge, receiving interaction from the user in the form of a like or a comment,' [START_REF] Villaespesa | Measuring Social Media Success: The value of the Balanced Scorecard as a tool for evaluation and strategic management in museums PhD[END_REF]. In particular, when Langa [START_REF] Langa | Does Twitter Help Museums Engage with Visitors?[END_REF] studied the building of a relationship that forty-eight museums engaged on Twitter with online users, she showed that its primary use was as a marketing tool (public relations, events announcements, fact of the day, etc.) and that it led to a lesser engagement and a low audience participation. However, when museums led a more engaging and interactive communication (including games, live tweeting events and co-curating projects), this greater engagement was related to a higher level of participation [START_REF] Langa | Does Twitter Help Museums Engage with Visitors?[END_REF]. In this study, it therefore appears that the kinds of communication museums are conducting with online users have a meaningful influence on user participation.

Studying the Relationships between Stakeholders

So far, scientific studies on the forms of digital communication engaged in by cultural institutions and audiences have analyzed the practices of institutions and audiences as well as their uses of digital platforms. Our contribution aims to design, in an inter-institutional cultural space, a model for the circulation of message flows on a social network platform, taking the case of Twitter. We chose an inter-institution space that corresponds both to a social network platform widely used by institutions and to a growing communication situation in the cultural field at the present time. This choice led us to focus on the category of cultural event programmed on a national scale. This enables us to explore how information circulates and is exchanged as well as the communication relations established between the different stakeholders present in an inter-institution space. It also avoids making any presuppositions about the various categories of stakeholders taking part in cultural events, as well as their statuses and roles. We focus particularly on the configurations of the authority and legitimacy relations that institutions maintain with the public through the processes of communication and mediation that they implement. Such an approach emphasizes the socio-political aspects of museums' editorial practices, in particular the relations of legitimacy between different categories of actors. Based on the analysis of message flows on the scale of the cultural event studied, we attempt to answer questions concerning the communicative practices of the stakeholders, such as for example, what is the current strategic usage of social media conducted by different types of stakeholders (not only museums or cultural institutions) during cultural events? We expect marketing and promotional messages to be present but we also inquire into initiatives fostering audience participation, providing cultural contents and favoring interaction with users. We will specifically examine the communication policies of cultural institutions during a cultural event on Twitter in order to assess whether they are part of their mission to democratize culture for a wide audience on this platform or whether it is rather a marketing campaign to promote a place or an event. More broadly, do stakeholders focus on marketing, advertising, education, cultural information or institutional communication?

An Instrumented Methodology

In order to build a representation of the modes of circulation of message flows, we developed an instrumented and interdisciplinary methodology that calls on Communication Sciences, Natural Language Processing (NLP) and Computer Science. Our methodology is based on statistical analyses of all the Twitter messages sent during the period of a cultural event as well as a content analysis of the messages. As mentioned in [START_REF] Foucault | Automatic Classification of Tweets for Analyzing Communication Behavior Museums Proceedings of LREC[END_REF], tweet analysis has led to a large number of studies in many do-mains such as ideology prediction in Information Sciences [START_REF] Djemili | What does Twitter have to say about ideology? In NLP 4 CMC: Natural Language Processing for Computer-Mediated Communication[END_REF], spam detection [START_REF] Yamasaki | A Trust Rating Method for Information Providers over the Social Web Service: A Pragmatic Protocol for Trust among Information Explorers, and Provider in Formation[END_REF], natural disaster anticipation in Emergency [START_REF] Sakaki | Tweet Analysis for Real-Time Event Detection and Earthquake, Reporting System Development[END_REF] and tracking epidemic [START_REF] Missler | Tracking Dengue Epidmeics using Twitter Content Classification and Topic Modelling[END_REF] while work in Social Sciences and Digital Humanities has developed tweet classifications [START_REF] Shiri | Twitter Content Categorisation: A Public Library Perspective[END_REF]. However, few studies aim at classifying tweets according to communication classes. They mostly rely on small reference sets analyzed by experts in Information Communication (InfoCom) rather than by Twitter users. An exception worth mentioning is the work presented in Lovejoy and Saxton [START_REF] Lovejoy | Information, Community, and Action: How Nonprofit Organizations Use Social Media[END_REF] in which the authors (Twitter users) analyze the global behavior of nonprofit organizations on Twitter based on three communication classes: Information, Community and Action classes. Recently, several studies on tweet classification have been carried out in NLP [START_REF] Missler | Tracking Dengue Epidmeics using Twitter Content Classification and Topic Modelling[END_REF][START_REF] Kothari | Detecting Comments on News Articles in Microblogs[END_REF][START_REF]Proceedings of the 2nd Workshop on Natural Language Pro-cessing for Social Media[END_REF] but to the best of our knowledge, only [START_REF] Courtin | A Tool-Based Methodology to Analyze Social Network Interactions in Cultural Fields: The Use Case "MuseumWeek[END_REF] has classified cultural institutional tweets in communication categories based on NLP techniques. The advantage of NLP approaches is that they can automatically classify large corpora of tweets. The most commonly used models are supervised learning, Support Vector Machine (SVM) and Naive Bayes (NB) [START_REF] Missler | Tracking Dengue Epidmeics using Twitter Content Classification and Topic Modelling[END_REF][START_REF] Sriram | Short Text Classification in Twitter to Improve Information Filtering In[END_REF][START_REF] Kouloumpis | Twitter Sentiment Analysis: The Good the Bad and the OMG![END_REF][START_REF] Malandrakis | SAIL: Sentiment Analysis using Semantic Similarity and Contrast Features[END_REF]. In supervised learning, features are extracted from tweets and metadata and then vectorized as training examples to build models. Our analysis focuses on Twitter messages (called tweets) sent by accounts of cultural institutions and by other institutional or non-institutional stakeholders who participated in a cultural event. A first step was to build a terminology to describe the objects studied according to three dimensions: the message, the stakeholders, and the forms of stakeholder participation. Concerning messages, we will call a message sent by a twitter account an "original tweet" and an original message sent by an account different from the issuing account a "retweet". The current Twitter API gives access to the original tweet (and its sending account) of a retweet. The generic term tweet includes original tweet and retweet. Regarding stakeholder qualification, we distinguished Twitter accounts, accounts managed by institutions (called organizational account, OA), and accounts managed by individuals (called private account, PA). This distinction is based both on the official list of museums in France provided by the French open data website, and the description on the Twitter account provided by the Twitter API. The analysis of the description field is necessary because non-museum institutions such as the City of Paris (@Paris) participated in the Night of Museums. Analysis of the flows during the MuseumWeek 2014 and 2015, European Night of Museums 2016, Europeans Days Heritage 2016 events, led us to identify six attributes used to qualify accounts according to their modes of participation and one computed score was associated at each attribute (cf. Table 1). We used the terms participant, producer, relayed, relaying, mentioned and passing. The attribute "participant" was assigned to an account if it produced at least one original tweet or retweet during of of the the two temporal periods of the event (before and during the event). The attribute "producer" was assigned to an account if it produced at least one original tweet. The attribute "relayed" was assigned to an account if at least one of its tweets was retweeted. The "relaying" attribute was assigned to an account if the account retweeted at least one tweet. The "mentioned" attribute was assigned to an account if its Twitter account name was mentioned at least once in a tweet. The attribute "passing" was assigned to an account if it was both relayed and relaying. Our hypothesis is that an account with a high passing score is a key influential user (see section 3.2). Note that these attributes were calculated irrespective of the number of followers. From these six attributes, it is possible, on the one hand, to compare the behavior of several accounts and, on the other hand, to calculate an overall score to classify them (see 3.2). Both quantitative and lexical analyses were carried out. Quantitative analyses focused on the observation of flows and aimed to identify accounts that contributed to the circulation of information during the cultural event. For each attribute (see above) a ranking of the accounts was computed, keeping only the first 10 or 15 accounts in this ranking. This made it possible to order the accounts that produced the largest number of original tweets, which were the most retweeted, and so on. In order to rank passing accounts, we computed for each account a score that is the product between the number of accounts that this account retweeted and the number of accounts that retweeted it. The value of this index is not significant in itself; it simply provides a means of comparing accounts. The lexical analysis focused on the contents of the messages. Lexical analysis of tweets was carried out as well as an automatic classification of tweets, in three stages. First, a team composed of two linguists and 10 community managers of cultural institutions designed a model, that is to say, determined the classes in which to categorize the tweets, and the features used to assign a tweet to a class. Four classes were identified (Encouraging participation, Interacting with the community, Promoting the event and informing about it, Sharing experience). The features selected were semio-linguistic (mostly lexical, but also including punctuation marks, emoticons), tweet-specific features (for example, the presence / absence of hashtags in tweets) as well as metadata such as the identity of the account. In the second stage, a classifier was built based on a corpus of sample tweets drawn from two cultural events, MuseumWeek2014 and MuseumWeek 2015 [START_REF] Courtin | A Tool-Based Methodology to Analyze Social Network Interactions in Cultural Fields: The Use Case "MuseumWeek[END_REF]. In our case, these examples take the form of 1000 tweets annotated by hand by cultural experts according to the categories defined in the previous step. The classifier is based on the Naive Bayes and SVM models, with unanimous vote. This means that both models had to predict the same category for a tweet; otherwise, the tweet was not classified. In a third stage, the classifier was applied to the corpus of tweets to categorize all the tweets. Results of the thorough evaluation of the quality of the classifier carried out on the Mu-seumWeek 2015 campaign are detailed in [START_REF] Foucault | Automatic Classification of Tweets for Analyzing Communication Behavior Museums Proceedings of LREC[END_REF]; the F-measure F 0.5 coefficient is 0.696.

Applying our methodology at the European Night of Museums (#NDM16) on Twitter

The analyses of the European Night of Museums (NDM16) were carried in the structuring content of the organizational and media framework in order to understand what happened during this cultural event, going beyond the display of quantitative data communicated at the time of its closure, i.e. 3000 events organized in France and in Europe, more than 2 million visitors who participated in the European Night of Museums in France.

The Sociocultural and Media Context

The European Night of Museums in 2016 had a European and international dimension thanks to the patronage of the Council of Europe, the French National Commission for UNESCO and the International Council of Museums (ICOM). It included both public and private partnerships. The event therefore had a political dimension of cultural outreach and democratization, an international dimension (although the event was mainly French) and a media dimension through the support of major media. The press release issued by the Ministry of Culture highlighted the characteristics underpinning the Night of Museums event -marketing, cultural mediation and pedagogy. They form the basis of the constituent dimensions of the event, which are reflected in its organizational structure and in the program of activities. The event was free of charge. NDM 2016 can be considered a hybrid cultural event in its purposes of marketing, cultural mediation and pedagogy. It welds together events communication and cultural marketing devices, an educational system and cultural mediation operations. The latter, supervised by the Ministry of Culture, gave rise to forms of engagement which associate the participating institutions with the public. We will now examine the forms of communication on the Twitter platform.

Key Influential Users

Data Acquisition. The data acquisition stage consisted in harvesting tweets with only one hashtag, the official event hashtag #NDM16. We developed a script Python, based on Twarc(http://github.com/docnow/twarc) module proposed by Ed Summers using the streaming option of Twitter Application Programming Interface (API) . However, Twitter data collection poses a number of challenges which introduce biases and problematic assumptions [START_REF] Boyd | Critical questions for big data: Provocations for a cultural, technological, and scholarly phenomenon[END_REF]. We followed best practice in terms of data collection and outline our data model and framework (see section 2.3). In this paper, we limit the analysis to tweets in French sent during the week preceding the event and the day of the event, that is to say from 14 May to 21 May 2016 midnight. Some flows were analyzed over the time period of the day of the event, from May 21 at 6pm to May 21 at midnight. The main figures are the following: 11 264 tweets of which 3 301 original tweets (29%), 7 963 retweets (71%) sent by 4 012 participants.

Table 2 shows some specific figures : more than half (56%) of organizational accounts and only 25% of private accounts produced original tweets. Participation in this event was largely limited to the action of retweeting (75% of tweets) the messages sent by the institutional partners. Passing Accounts . Table 3 shows, in descending order, the 10 passing accounts that had a significant score (see 2.3). These accounts that are both relayed and relaying played a central role in the flow of information on the platform. We have indicated for each of them the number of followers as they appeared on their Twitter account on May 28, 2016. A remarkable point is the non-correlation between the passing score and the number of followers. The Pearson-correlation computed on the first fifteen passing accounts (NuitdesMusées excluded) is equal to 0.15875, that is very low. Consequently, while the growth of followers of museums Twitter accounts is considered as a marketing target, the circulation of information during a cultural event is depending of key influential accounts which are not necessarily what is called 'big players' [START_REF] Espinos | Museums on social media : Analyzing growth through case studies[END_REF]. Some characteristics of these passing accounts must be pointed out. They are all organizational accounts with the exception of the account '@Mariette Escalier'. The owner of this private account is a professional in the field of cultural mediation. She holds the position of 'Mediation Coordinator' for the 'Quai in Toulouse', which is an institution that supports Scientific, Technical and Industrial Culture (CSTI), but she tweets on her own behalf. Ranked second after the organizational account of the event but with less than 2 000 followers, she can be considered as an key influential user. The first account, 'NuitdesMusées', is the institutional account dedicated to the event. It thus fulfills its role of dissemination account of NDM 2016, as does the account of the Ministry of Culture and Communication (@MinistèreCultureCom). Other accounts include museums located in Paris, as well as a regional museum (in Brittany) that reaches the same level of visibility as the Paris museums. In 5th place, the presence of the account of Alain Juppé, a French politician, mayor of the city of Bordeaux and former prime minister, shows that culture has become a political issue (he built an ambitious cultural policy program during his candidacy for the right-wing primary of the 2017 presidential elections) and plays an important role in marketing Cities and territories. The circulation of cultural information appears to be a dynamic process that goes beyond the cultural sphere and achieves wider political and social appropriation. Great diversity was observed in the communicative practices of museum accounts. Some museums relay exclusively organizational accounts, while others relay only private accounts. We also noted that the passing accounts during the event are not the same as the passing accounts of the period preceding the event. Only the 'NuitdesMusées' and 'MinistèreCultureCom" accounts are common to both these periods.

Lexical Analysis and Supervised Classification. The 10 most frequent terms in the text of the tweets (after deleting prepositions, conjunctions and terms related to the event) sent before the event are presented in table 4. Before the event, organizations used 'vous' (you) and 'votre' (your) to address the public and 'nous' (us) and 'notre' (our) to present their offer. They stressed that the event was free of charge 'gratuit' and the notion of discovery 'découvrir, découvrez' (to discover). During the event, organizational accounts used 'vous' and 'tous' (all), 'notre', an invitation to discover 'découvrez' emphasizing participation. They also reported on specific events such as 'concert'. The findings of the supervised classification of original tweets sent during the event (see section 2.3) are shown in table 5. It should be noted that some tweets have been categorized twice (that is why the sum of the total percentages is greater than 100%). This table shows that the organizational accounts tweets mainly (93%) serve to promote and inform about local events planned for the occasion. This result is consistent with the organizational framework of the cultural event, the success of which is partly linked to its attendance. There is also some interaction with the public but messages of engagement are very sparse. Comparing practices . In order to compare the 15 most active accounts, we performed a principal component analysis with 4 variables for each account: number of original tweets, mentioned score, relayed score and passing score (see section 2.3). We did not take into account the official account of the event (@Nu-itdesMusees) which due to its official position, presents specific characteristics. This analysis shows the positions occupied by the 15 most active stakeholders in the 2016 NDM (Figure 1). Among them, the Musée dOrsay is in a remarkable position, far on the right; it was mentioned and relayed many times because it organized an event (a jazz concert in the museum) which was very well at- tended. The success of this in situ event was relayed on Twitter which worked in this respect as a soundboard whereas the Louvre Museum, which organized conferences, was seldom mentioned or relayed despite its reputation. The Cluny Museum, the Guimet Museum and the CMN occupy positions that place them more as passing accounts. There are also several stakeholders who were not partners in the events and who took the opportunity of NDM 2016 to enhance their visibility in the cultural field. Similarly, the position of the private account Mariette Escalier (cultural mediator) occupies another remarkable position, in the left top corner; she is a passing account and she sent a lot of tweets, unlike most private accounts. Another private account, 'Scribe', in the center, occupies a position closer to that of the organizational accounts, that is to say, rather well retweeted. One can also see an activist account 'Musées Debout' (Standing Museums), a collective in line with the political movement 'Nuit Debout' (Standing Night), launched by a curator at the Louvre Museum with a program 'to put the museum at the heart of public debate, and public debate at the heart of the museum', whose tweets were quite well retweeted but he was not a passing account. The private account of the politician Alain Juppé (see 3.2.3) who sought to enhance his cultural program was largely relayed by the collective who supports him (fig. 2). France Culture, the only media, occupies an average position of relayed account.

The topological representation of the networks of the two most active accounts, one organizational, @Ministry of Culture and one private, @Mariette Escalier reveals very different forms of information flows. There is a spin-off in clusters for the organizational account of the Musé d'Orsay (Figure 2), in dark blue in the center (accounts are in blue and tweets are in green). This account sent information to other accounts which play the role of broadcaster towards the collectives to which they are connected. In contrast, the network of the private account of Mariette Escalier in dark blue in the center, shows a circulation of information that is more fragmented and more diffuse. The third picture shows clearly that the supporters of Alain Juppé (black label on the far right) network is not connected with others networks like for instance the NuitdesMusées (green label) network.

Conclusion

In this study, we described how to design an interdisciplinary and instrumented methodology in an inter-institutional cultural space. We proposed a model based on six attributes to qualify accounts according to their modes of participation and a machine learning classifier to categorize all the accounts. We applied the methodology and the tools to the cultural event European Night of Museums 2016. We pointed out that one private account played a major role in disseminating information and that culture has become a political issue in France. We performed a correspondence analysis that shows the specificity of this private account and the role of two museums in Paris, the Musée d'Orsay and the Centre Pompidou. We are working on the extension of our method in order to take into account the incremental analysis of graphs.

Fig. 1 .Fig. 2 .

 12 Fig. 1. Principal component analysis

Table 1 .

 1 Score calculation method

	Attribute	Method
	Participant	0 (no participation in the considered period of time) or 1
	Producer	Total amount of tweets and retweets
	Relayed	Total number of accounts which relayed his/her original tweets
	Relaying	Total number of accounts she/he relayed
	Mentioned	Total number of mention of the account in text tweets
	Passing	Product of relayed score by relaying score

Table 2 .

 2 Specific figures

	Type of account	Number Percentage of original tweets Percentage of retweets
	Institutional accounts 411	56%	44%
	Private accounts	3601	25%	75%

Table 3 .

 3 Passing accounts

	Name	Type of accounts retweeted	Retweeted by Followers
	NuitdesMusées	Mainly organizational	Mainly private	22 422
	MarietteEscalier	Organizational	Mainly private	1 842
	MuséeCluny	Half organizational, half private Mainly private	44 839
	MinistèreCultureCom	Organizational	Private	184 521
	AlainJuppé	Two Organizational	Private	352 837
	MuséeOrsay	MuséeOrangerie	Private	463 184
	LeCMN	Organizational	Mainly Private	27 856
	MusédesAugustins	Private	Mainly Private	4 774
	MuséGuimet	Half organizational, half private	Private	29 500
	MuséBretagne	Half private	Private	7 708

Table 4 .

 4 Most frequent terms

	Before the Event	Number	During the event	Number
	Vous (you)	160	Vous (you)	49
	On(one)	78	Tous(all)	28
	Votre (your)	78	Nous (we)	18
	Notre (our)	76	Découvrir (to discover)	18
	Merci (thanks)	55	Notre (our)	18
	Nos(ours)	53	Monde(world)	18
	Gratuit(free of charge)	45	Nos(ours)	18
	Découvrir (to discover)	40	Concert	18
	Venez(come)	40	Belle(Beautiful)	17
	Découvrez (discover)	40	Voir (to see)	15

Table 5 .

 5 Supervised classification

	Classes