First detection of Jingmen tick virus in Corsica, France and development of a real time detection system for multiple tick-associated jingmenviruses.

Jingmen tick virus (JMTV) is a recently discovered segmented RNA virus, closely related to flaviviruses. It was identified for the first time in 2014, in China and subsequently in Brazil. Following this discovery, JMTV-related sequences have been identified in arthropods, vertebrates (including humans), plants, fungus and environmental samples from Asia, America, Africa, Europe and Oceania. Several studies suggest an association between these segmented flavi-like viruses, termed jingmenviruses, and febrile illness in humans. The development of rapid diagnostic assays for these viruses is therefore crucial to be prepared for a potential epidemic, for the early detection of these viruses via vector surveillance or hospital diagnosis. In this study, we designed a RT-qPCR assay to detect tick-associated jingmenviruses, validated it and tested its range and limit of detection with six tick-associated jingmenviruses using in vitro transcripts. Then we screened ticks collected in Corsica (France) from different livestock species, in order to determine the distribution of these viruses on the island. In total, 6,269 ticks from eight species were collected from 763 cattle, 538 horses, 106 sheep and 218 wild boars and grouped in 1,715 pools. We report the first detection of JMTV in Corsica, in Rhipicephalus bursa, Hyalomma marginatum and R. sanguineus ticks collected from cattle and sheep. The highest prevalence was found in the Rhipicephalus genus. The complete genome of a Corsican JMTV was obtained from a pool of Rhipicephalus bursa ticks and shares between 94.7% and 95.1% nucleotide identity with a JMTV sequence corresponding to a human patient in Kosovo and groups phylogenetically with European JMTV strains. These results show that a Mediterranean island such as Corsica could act as a sentinel zone for future epidemics.

Rickettsial pathogens drive microbiota assembly in Hyalomma marginatum and Rhipicephalus bursa ticks.

Most tick-borne pathogens (TBPs) are secondarily acquired by ticks during feeding on infected hosts, which imposes 'priority effect' constraints, as arrival order influences the establishment of new species in a microbial community. Here we tested whether once acquired, TBPs contribute to bacterial microbiota functioning by increasing community stability. For this, we used Hyalomma marginatum and Rhipicephalus bursa ticks collected from cattle in different locations of Corsica and combined 16S rRNA amplicon sequencing and co-occurrence network analysis, with high-throughput pathogen detection, and in silico removal of nodes to test for impact of rickettsial pathogens on network properties. Despite its low centrality, Rickettsia showed preferential connections in the networks, notably with a keystone taxon in H. marginatum, suggesting facilitation of Rickettsia colonisation by the keystone taxon. In addition, conserved patterns of community assembly in both tick species were affected by Rickettsia removal, suggesting that privileged connections of Rickettsia in the networks make this taxon a driver of community assembly. However, Rickettsia removal had minor impact on the conserved 'core bacterial microbiota' of H. marginatum and R. bursa. Interestingly, networks of the two tick species with Rickettsia have similar node centrality distribution, a property that is lost after Rickettsia removal, suggesting that this taxon drives specific hierarchical interactions between bacterial microbes in the microbiota. The study indicates that tick-borne Rickettsia play a significant role in the tick bacterial microbiota, despite their low centrality. These bacteria are influential and contribute to the conservation of the 'core bacterial microbiota' while also promoting community stability.

Lack of Evidence for Crimean-Congo Hemorrhagic Fever Virus in Ticks Collected from Animals, Corsica, France.

In Corsica, France, 9.1% of livestock serum samples collected during 2014-2016 were found to have antibodies against Crimean-Congo hemorrhagic fever virus (CCHFV), an emerging tickborne zoonotic disease. We tested 8,051 ticks for CCHFV RNA and Nairovirus RNA. The results indicate that Corsica is not a hotspot for CCHFV.

Molecular detection of parapoxvirus in Ixodidae ticks collected from cattle in Corsica, France.

BACKGROUND: Several viruses belonging to the family Poxviridae can cause infections in humans and animals. In Corsica, livestock farming (sheep, goats, pigs, and cattle) is mainly mixed, leading to important interactions between livestock, wildlife, and human populations. This could facilitate the circulation of zoonotic diseases, and makes Corsica a good example for studies of tick-borne diseases. OBJECTIVES: To gain understanding on the circulation of poxviruses in Corsica, we investigated their presence in tick species collected from cattle, sheep, horses, and wild boar, and characterized them through molecular techniques. METHODS: Ticks were tested using specific primers targeting conserved regions of sequences corresponding to two genera: parapoxvirus and orthopoxvirus. RESULTS: A total of 3555 ticks were collected from 1549 different animals (687 cattle, 538 horses, 106 sheep, and 218 wild boars). They were tested for the presence of parapoxvirus DNA on one hand and orthopoxvirus DNA on the other hand using Pangeneric real-time TaqMan assays. Orthopoxvirus DNA was detected in none of the 3555 ticks. Parapoxvirus DNA was detected in 6.6% (36/544) of ticks collected from 23 cows from 20 farms. The remaining 3011 ticks collected from horses, wild boars, and sheep were negative. The infection rate in cow ticks was 8.0% (12/148) in 2018 and 6.0% (24/396) in 2019 (p = 0.57). Parapoxvirus DNA was detected in 8.5% (5/59) of Hyalomma scupense pools, 8.2% (15/183) of Hyalomma marginatum pools, and 6.7% (16/240) of Rhipicephalus bursa pools (p = 0.73). We successfully amplified and sequenced 19.4% (7/36) of the positive samples which all corresponded to pseudocowpox virus. CONCLUSIONS: Obviously, further studies are needed to investigate the zoonotic potential of pseudocowpox virus and its importance for animals and public health.

Molecular screening of Anaplasmataceae in ticks collected from cattle in Corsica, France.

Bacteria belonging to the family Anaplasmataceae cause infections in humans and domestic animals. The consequences of infection can be significant economic losses for farmers. To better understand the epidemiology of tick-borne Anaplasmataceae in Corsica, we used molecular methods to detect and characterize Anaplasmataceae in ixodid ticks collected from cattle. Anaplasmataceae were detected by using a real-time polymerase chain reaction (PCR) targeting the 23S rRNA gene. Partial sequencing of rpoB and groEL allowed identifying species and conducting phylogenetic analyses. Infection rates were calculated using maximum likelihood estimation (MLE) with 95% confidence intervals (CIs). In total, 597 Rhipicephalus bursa, 216 Hyalomma marginatum, and seven Ixodes ricinus were collected from cattle during July-August 2017 and July-December 2018. Overall, Anaplasmataceae DNA was detected in 15 of 255 tick pools (MLE = 1.7%; 95% CI 0.9-2.7%). The molecular analysis revealed two species within the genus Anaplasma: A. marginale and A. phagocytophilum. We also detected bacteria within the genus Ehrlichia: we confirmed the detection of E. minasensis DNA in H. marginatum and R. bursa tick pools collected from cattle in Corsica and detected, for the first time to our knowledge, Candidatus E. urmitei in Corsican R. bursa ticks and a potential new species, Candidatus E. corsicanum. Further studies are needed to ascertain the pathogenesis and zoonotic potential of the strains and their importance for animals and public health.

Molecular Detection of Spotted-Fever Group Rickettsiae in Ticks Collected from Domestic and Wild Animals in Corsica, France.

To obtain a better understanding of the current magnitude of tick-borne rickettsioses in Corsica, we used molecular methods to characterize the occurrence of Rickettsia spp. in ixodid ticks collected from domestic and wild animals. The presence of Rickettsia spp. was evaluated using real-time polymerase chain reaction targeting the gltA gene and by sequencing of gltA and ompA partial genes for species identification and phylogenetic analysis. Infection rates were calculated as the maximum-likelihood estimation (MLE) with 95% confidence intervals (CI). In total, 1117 ticks belonging to four genera (Rhipicephalus, Hyalomma, Ixodes, and Dermacentor) were collected from cattle, sheep, wild boars, and companion animals during July-August 2017 and July 2018-January 2019. Overall, Rickettsia DNA was detected in 208 of 349 pools of ticks (MLE = 25.6%, 95% CI: 22.6-28.8%). The molecular analysis revealed five different rickettsial species of the spotted-fever group (SFG). We highlighted the exclusive detection of Candidatus Ri. barbariae in R. bursa and of Ri. aeschlimanii in H. marginatum. Rickettsia slovaca was detected in D. marginatus collected from wild boars. This study provides the first evidence of the presence of Ri. monacensis in I. ricinus ticks isolated from a dog in Corsica. In conclusion, our data revealed wide dispersal of SFG Rickettsiae and their arthropod hosts in Corsica, highlighting the need for surveillance of the risk of infection for people living and/or working close to infected or infested animals.

First molecular detection of Rickettsia africae in a tropical bont tick, Amblyomma variegatum, collected in Corsica, France.

Here we report the first detection of Amblyomma variegatum, a tick species of medical and veterinary importance, and the first molecular evidence of a pathogen, Rickettsia africae, both new to Corsica (France). In August 2018, an ixodid tick with an unusual morphology was removed from the ventral part of a cow's whole skin in a slaughterhouse located in the village of Ponte-Leccia (Haute-Corse). The tick was morphologically identified as an adult male of A. variegatum. This result was confirmed by 16S rDNA sequence analysis with a close relative being a sequence from Senegal showing 99% nucleotide identity. We tested the tick for Ehrlichia and Rickettsia. The tick was positive to Rickettsia and the corresponding sequence matched with R. africae. There is little or no risk of the introduction and establishment of a viable population of A. variegatum in Corsica by migrating birds. However, if it did, it could produce major economic losses for livestock production. Further studies and sustained surveillance are indicated, not only focusing on this species of tick and this rickettsia, but also on other microorganisms of veterinary and medical importance that might be transmitted in Corsica and other Mediterranean islands.

First detection of Ehrlichia minasensis in Hyalomma marginatum ticks collected from cattle in Corsica, France.

Ehrlichiosis are severe, feverish tick-borne illnesses caused by specific species within the genus Ehrlichia (Anaplasmataceae family). Recent data suggest that ruminants in Corsica area reservoir for several Anaplasmataceae species. The purpose of our study was to determine whether Ehrlichia species could be detected in ticks collected in Corsican ruminants by using molecular methods. Ticks were collected in northern Corsica: (i) in May 2016 from sheep bred in one farm located in a 5000-inhabitants village and (ii) from cattle in June and July 2016 in a slaughterhouse. There sheep and cattle whole skin was inspected and ticks were collected manually. A total of 647 ticks was collected in northern Corsica during this study: 556 (86%) belonged to the Rhipicephalus bursa species and 91 (14%) to Hyalomma marginatum. The 91 H. marginatum ticks were organized in 27 pools, of which one (3.7%) was found positive for the presence of E. minasensis; this pool consisted of six ticks collected from a cow bred and raised northwestern Corsica. Ehrlichial DNA was not detected in R. bursa ticks. The 16S rRNA and groEL gene sequences of Ehrlichia detected in the H. marginatum pool showed 100% (303/303 bp) and 99.8% (555/556) of nucleotide identity with E. minasensis, respectively. Phylogenetic analyses demonstrated the highest closeness with E. minasensis UFMG-EV genotype than to any other E. canis strains. To our knowledge, this is the first report of E. minasensis outside of Brazil, Ethiopia and Canada. This identification of E. minasensis in H. marginatum merits to be further investigated and pleads for translational studies addressing the potential impact of vector-borne diseases of human and veterinary impact through large-scale research and surveillance programmes in Corsica.