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Although apicomplexan parasites of the group Piroplasmida represent commonly identified global risks to both animals and humans, detailed knowledge of their life cycles is surprisingly limited. Such a discrepancy results from incomplete literature reports, nomenclature disunity and recently, from large numbers of newly described species. This review intends to collate and summarize current knowledge with respect to piroplasm phylogeny. Moreover, it provides a comprehensive view of developmental events of , and representative species, focusing on uniform consensus of three consecutive phases: (i) schizogony and merogony, asexual multiplication in blood cells of the vertebrate host; (ii) gamogony, sexual reproduction inside the tick midgut, later followed by invasion of kinetes into the tick internal tissues; and (iii) sporogony, asexual proliferation in tick salivary glands resulting in the formation of sporozoites. However, many fundamental differences in this general consensus occur and this review identifies variables that should be analyzed prior to further development of specific anti-piroplasm strategies, including the attractive targeting of life cycle stages of or tick vectors.

Cervids are known to be reservoirs of zoonotic bacteria transmitted by ticks. This study aimed to identify the Anaplasma species carried by captive red deer and swamp deer in a wild fauna reserve in France. Blood from 59 red deer and 7 swamp deer was collected and analyzed over a period of two years. A semi-nested PCR targeting the 23S rRNA was performed to detect and characterize Anaplasma spp. and determine the presence of zoonotic species. Anaplasma phagocytophilum was identified in 14/59 red deer (23.7%) but it was not identified in any of the swamp deer (7 animals). Three sequences could not be assigned to any particular species based on the 23S rRNA sequences. Complementary nested PCR targeting 16S rRNA, gltA and groEL genes and sequencing analysis then identified these sequences as a recently reported zoonotic species, Anaplasma capra; this species was found in 2 red deer (Cervus elaphus) and 1 swamp deer (Rucervus duvaucelii). This is the first report of the tick-borne zoonotic bacterium A. capra in France, a species otherwise described only in China, Japan, Malaysia and South Korea in goats, sheep, deer, cattle and Japanese serows (Capricornis crispus). While this bacterium may have been introduced into the reserve by infected imported animals, its local epidemiological cycle via tick transmission seems possible as locally born deer were found infected. Diagnostic methods, especially molecular ones, should take into account the potential infection of animals and humans with this species.

The vector competence of blood-feeding arthropods is influenced by the interaction between pathogens and the immune system of the vector. The Toll and IMD (immune deficiency) signaling pathways play a key role in the regulation of innate immunity in both the Drosophila model and blood-feeding insects. However, in ticks (chelicerates), immune determination for pathogen acquisition and transmission has not yet been fully explored. Here, we have mapped homologs of insect Toll and IMD pathways in the European tick Ixodes ricinus , an important vector of human and animal diseases. We show that most genes of the Toll pathway are well conserved, whereas the IMD pathway has been greatly reduced. We therefore investigated the functions of the individual components of the tick Toll pathway and found that, unlike in Drosophila , it was specifically activated by Gram-negative bacteria. The activation of pathway induced the expression of defensin ( defIR ), the first identified downstream effector gene of the tick Toll pathway. Borrelia , an atypical bacterium and causative agent of Lyme borreliosis, bypassed Toll-mediated recognition in I . ricinus and also resisted systemic effector molecules when the Toll pathway was activated by silencing its repressor cactus via RNA interference. Babesia , an apicomplexan parasite, also avoided Toll-mediated recognition. Strikingly, unlike Borrelia , the number of Babesia parasites reaching the salivary glands during tick infection was significantly reduced by knocking down cactus . The simultaneous silencing of cactus and dorsal resulted in greater infections and underscored the importance of tick immunity in regulating parasite infections in these important disease vectors.

Background Hyalomma marginatum is a hard tick vector of various pathogens, including Crimean-Congo Hemorrhagic fever virus, recently detected in French specimens. This species has a wide distribution from North Africa to Eastern Europe and has only recently been considered established in Southern France. These changes in species distribution led us to explore the genetic structure of tick populations in the Mediterranean basin and attempt to infer the origin of French populations. Methods We used two mitochondrial markers (12S rRNA and Cytochrome Oxidase 1) and genotyped ticks from nine Mediterranean countries. We compared genetic indices and haplotypic composition between these countries and the various French geographical populations. Results Across all countries, we showed significant genetic differentiation, with a certain proximity between neighboring countries. We found very different genetic compositions among the French geographic populations: some exhibited signs of recent expansion, while others suggested the presence of ancient populations. Conclusions It is possible that small populations of H. marginatum were already present in France and are now more abundant. This recent change in population structure could be owing to increased human activity and climate change. These factors, combined with a potentially high level of phenotypic plasticity, could facilitate H. marginatum conquest of more northerly latitudes in France and other European countries. Graphical Abstract

In Europe, Babesia divergens is responsible for most of the severe cases of human babesiosis. In the present study, we describe a case of babesiosis in a splenectomized patient in France and report a detailed molecular characterization of the etiological agent, named Babesia sp. FR1, as well as of closely related Babesia divergens, Babesia capreoli and Babesia sp. MO1-like parasites. The analysis of the conserved 18S rRNA gene was supplemented with the analysis of more discriminant markers involved in the red blood cell invasion process: rap-1a (rhoptry-associated-protein 1) and ama-1 (apical-membrane-antigen 1). The rap-1a and ama-1 phylogenetic analyses were congruent, placing Babesia sp. FR1, the new European etiological agent, in the American cluster of Babesia sp. MO1-like parasites. Based on two additional markers, our analysis confirms the clear separation of B. divergens and B. capreoli. Babesia sp. MO1-like parasites should also be considered as a separate species, with the rabbit as its natural host, differing from those of B. divergens (cattle) and B. capreoli (roe deer). The natural host of Babesia sp. FR1 remains to be discovered.

BackgroundAnaplasma phagocytophilum is a tick-transmitted Gram-negative obligate intracellular bacterium able to infect a wide variety of wild and domestic animals worldwide. Based on the genetic diversity observed with different molecular markers, several host-specific lineages have been identified. Roe deer is one of the most important reservoirs of this bacterium and hosts different genetic groups sometimes found on domestic animals. We therefore developed an ankA cluster-specific nested PCR (nPCR) to evaluate the prevalence of the three different ankA genetic groups described in roe deer (clusters II, III and IV) at three locations in France and the level of co-infections.ResultsThe specificity of the three nPCRs was assessed by partially sequencing 35 amplicons of ankA genes obtained from the different nested PCRs. All three genetic lineages were detected in roe deer from all three geographical locations. Of the infected deer population, 60.7% were co-infected by two or three different genetic variants. Co-infections varied from 42.9 to 70.6% of the infected population depending on the local infection prevalences (from 33.3 to 73.9%). All types of mixed infections occurred, suggesting the absence of a strict variant exclusion by another variant.ConclusionsMixed infections by two or three genetic variants of A. phagocytopilum are a common feature in roe deer. Genetic variants (cluster IV) also found in domestic ruminants (cattle and sheep) were present in all the roe deer populations analyzed, suggesting a shared epidemiological cycle

Although Babesia divergens is the the principal confirmed zoonotic Babesia sp. in Europe, there are gaps in our knowledge of its biology and transmission by the tick Ixodes ricinus. In order to reproduce the part of the parasite cycle that occurs in the vector, an in vitro animal skin feeding technique on blood containing in vitro cultivated B. divergens was developed. Parasite DNA was detected in all samples of salivary glands of nymphs and adults that had fed on parasitized blood as larvae and nymphs, respectively, indicating acquisition as well as a transtadial persistence of B. divergens. PCR performed on eggs and larvae produced by females that had fed on parasitized blood demonstrated the existence of a transovarial transmission of the parasite. Gorging B. divergens infected larvae on non-infected gerbils showed persistance of the parasite over moulting into the resulting nymphs. These results indicate that the parasitic stages infective for the vector (i.e. the sexual stages) can be produced in vitro. To our knowledge, this is the first report of artificial feeding of I. ricinus via membrane as well as in vitro transmission of B. divergens to its vector. The opportunities offered by the use of such a transmission model of a pathogen by I. ricinus are discussed.

Background Babesia divergens is the most common blood parasite in Europe causing babesiosis, a tick-borne malaria-like disease. Despite an increasing focus on B. divergens , especially regarding veterinary and human medicine, the sexual development of Babesia is poorly understood. Development of Babesia sexual stages in the host blood (gametocytes) plays a decisive role in parasite acquisition by the tick vector. However, the exact mechanism of gametocytogenesis is still unexplained. Methods Babesia divergens gametocytes are characterized by expression of bdccp1 , bdccp2 and bdccp3 genes. Using previously described sequences of bdccp1 , bdccp2 and bdccp3 , we have established a quantitative real-time PCR (qRT-PCR) assay for detection and assessment of the efficiency of B. divergens gametocytes production in bovine blood. We analysed fluctuations in expression of bdccp genes during cultivation in vitro, as well as in cultures treated with different drugs and stimuli. Results We demonstrated that all B. divergens clonal lines tested, originally derived from naturally infected cows, exhibited sexual stages. Furthermore, sexual commitment was stimulated during continuous growth of the cultures, by addition of specific stress-inducing drugs or by alternating cultivation conditions. Expression of bdccp genes was greatly reduced or even lost after long-term cultivation, suggesting possible problems in the artificial infections of ticks in feeding assays in vitro. Conclusions Our research provides insight into sexual development of B. divergens and may facilitate the development of transmission models in vitro, enabling a more detailed understanding of Babesia -tick interactions.

Hyalomma marginatum is an invasive tick species reported in regions bordering the Mediterranean Sea. It has recently become established in mainland France in 2016. H. marginatum hosts a diverse range of human and animal pathogens such as Rickettsia aeschlimannii, Theileria equi, Anaplasma phagocytophilum, Anaplasma marginale, Ehrlichia minasensis and West Nile virus. The objective of this study was to characterize the current spatial distribution of H. marginatum-borne pathogens in the region Occitanie, southern France and the influence of other factors (tick sex, engorgement status). A large-scale tick collection program was organized in the region Occitanie to answer these questions. A total of 510 ticks from 27 sites of four departments were analysed for pathogens presences and loads using both a high-throughput microfluidic real time PCR system and quantitative PCR. In the analysed ticks, we identified the tick-borne pathogens and their prevalence: Theileria equi (9.2%), Theileria orientalis (0.2%), Anaplasma phagocytophilum (1.6%), Anaplasma marginale (0.8%) and Rickettsia aeschlimannii (87.3%). Interestingly, we highlighted a spatial clustered distribution for the pathogen R. aeschlimannii between two geographically isolated areas with infection rates and bacterial loads significantly lower in Herault/Gard departments (infection rate 78.6% in average) compared to Aude/Pyrenees-Orientales (infection rate 92.3% in average). R. aeschlimannii infection rates varied from one site to another, ranging from 29% to 100%, highlighting the importance of the sampling effort to better characterize the circulation of pathogens. Such high infection rate (87.3% in average) and the effective maternal transmission of R. aeschlimannii suggest a role as a tick symbiont in H. marginatum. On the other hand, currently identified as a human pathogen, its pathogenic status in humans is questioned given the low number of human cases. Further studies are thus needed to understand both the status and the role of R. aeschlimannii in H. marginatum ticks.Competing Interest StatementThe authors have declared no competing interest.