Lorraine Michelet

High-throughput screening of tick-borne pathogens in Europe

Due to increased travel, climatic, and environmental changes, the incidence of tick-borne disease in both humans and animals is increasing throughout Europe. Therefore, extended surveillance tools are desirable. To accurately screen tick-borne pathogens (TBPs), a large scale epidemiological study was conducted on 7050 Ixodes ricinus nymphs collected from France, Denmark, and the Netherlands using a powerful new high-throughput approach. This advanced methodology permitted the simultaneous detection of 25 bacterial, and 12 parasitic species (including; Borrelia, Anaplasma, Ehrlichia, Rickettsia, Bartonella, Candidatus Neoehrlichia, Coxiella, Francisella, Babesia, and Theileria genus) across 94 samples. We successfully determined the prevalence of expected (Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Rickettsia helvetica, Candidatus Neoehrlichia mikurensis, Babesia divergens, Babesia venatorum), unexpected (Borrelia miyamotoi), and rare (Bartonella henselae) pathogens in the three European countries. Moreover we detected Borrelia spielmanii, Borrelia miyamotoi, Babesia divergens, and Babesia venatorum for the first time in Danish ticks. This surveillance method represents a major improvement in epidemiological studies, able to facilitate comprehensive testing of TBPs, and which can also be customized to monitor emerging diseases.

Co-infection of Ticks: The Rule Rather Than the Exception

Introduction Ticks are the most common arthropod vectors of both human and animal diseases in Europe, and the Ixodes ricinus tick species is able to transmit a large number of bacteria, viruses and parasites. Ticks may also be co-infected with several pathogens, with a subsequent high likelihood of co-transmission to humans or animals. However few data exist regarding co-infection prevalences, and these studies only focus on certain well-known pathogens. In addition to pathogens, ticks also carry symbionts that may play important roles in tick biology, and could interfere with pathogen maintenance and transmission. In this study we evaluated the prevalence of 38 pathogens and four symbionts and their co-infection levels as well as possible interactions between pathogens, or between pathogens and symbionts. Methodology/principal findings A total of 267 Ixodes ricinus female specimens were collected in the French Ardennes and analyzed by high-throughput real-time PCR for the presence of 37 pathogens (bacteria and parasites), by rRT-PCR to detect the presence of Tick-Borne encephalitis virus (TBEV) and by nested PCR to detect four symbionts. Possible multipartite interactions between pathogens, or between pathogens and symbionts were statistically evaluated. Among the infected ticks, 45% were co-infected, and carried up to five different pathogens. When adding symbiont prevalences, all ticks were infected by at least one microorganism, and up to eight microorganisms were identified in the same tick. When considering possible interactions between pathogens, the results suggested a strong association between Borrelia garinii and B. afzelii, whereas there were no significant interactions between symbionts and pathogens. Conclusion/significance Our study reveals high pathogen co-infection rates in ticks, raising questions about possible co-transmission of these agents to humans or animals, and their consequences to human and animal health. We also demonstrated high prevalence rates of symbionts co-existing with pathogens, opening new avenues of enquiry regarding their effects on pathogen transmission and vector competence.

Multiple detection of pathogens in ticks: development of a high throughput real time PCR chip used as a new epidemiologic investigative tool

Worldwide, ticks transmit more pathogens than other arthropods. Around 60 bacteria, 30 parasites and 100 viruses have been registered as tick-borne pathogens; a third of these pathogens are responsible for zoonoses. Usually, detection of tick-borne pathogens depends on the tick species collected: assays are performed for a restricted number of pathogens that are known to be transmitted by a particular tick species collected at a particular site. To better understand the epidemiology of tick-borne pathogens, it will be important to detect for each sample (one tick or one pool of ticks) most of the diseases they potentially transmit, regardless of the tick species. The aim is therefore to develop a new epidemiologic investigative tool which could detect high number of tick-borne pathogens by real time PCR. We developed a chip (BioMark™ dynamic arrays, Fluidigm Corporation) targeting pathogens of worldwide distribution transmitted by ticks. The designed epidemiologic arrays may detect 48 pathogens in 48 samples corresponding to 2304 qPCR reactions on the same time. Specific primers and probe have been designed for each pathogen and their specificity have been tested in silico with Blast. To begin, we targeted: (i) 37 pathogens whose Francisella tularensis, Coxiella burnetii, Neoehrlichia mikurensis, 5 species of Anaplasma, 3 species of Ehrlichia, 8 species of Borrelia, 2 species of Bartonella, 4 species of Rickettsia, 10 species of Babesia and 2 species of Theileria, (ii) 5 species of ticks whose 3 species of Ixodes and 2 species of Dermacentor. Sensitivity of primers and probe has been tested on a dilution range of reference DNAs of the targeted pathogens on a Lightcycler 480. Specificity then has been tested on a Biomark™dynamic array. The chip was secondly evaluated on field samples corresponding to 47 pools of 25 nymphs collected in two sites in France, the Netherlands and Denmark (corresponding to 7050 nymphs in total). We succesfully detected and determined the prevalence of Anaplasma phagocytophilum, Neoehrlichia mikurensis, Rickettsia helvetica, Bartonella henselae, five different genospecies of Borrelia burgdorferi s.l., the recently identified pathogen Borrelia miyamotoi, and two parasite species Babesia divergens and Babesia venatorum. This fast and low-cost tool allows comprehensive testing of tick-borne pathogens and can be customized to fit regional demands or to accommodate new or emerging pathogens. The tool represents a major improvement for surveillance and future epidemiological studies.

Identification of parasitic communities within European ticks using next-generation sequencing.

Background Risk assessment of tick-borne and zoonotic disease emergence necessitates sound knowledge of the particular microorganisms circulating within the communities of these major vectors. Assessment of pathogens carried by wild ticks must be performed without a priori, to allow for the detection of new or unexpected agents. Methodology/Principal Findings We evaluated the potential of Next-Generation Sequencing techniques (NGS) to produce an inventory of parasites carried by questing ticks. Sequences corresponding to parasites from two distinct genera were recovered in Ixodes ricinus ticks collected in Eastern France: Babesia spp. and Theileria spp. Four Babesia species were identified, three of which were zoonotic: B. divergens, Babesia sp. EU1 and B. microti; and one which infects cattle, B. major. This is the first time that these last two species have been identified in France. This approach also identified new sequences corresponding to as-yet unknown organisms similar to tropical Theileria species. Conclusions/Significance Our findings demonstrate the capability of NGS to produce an inventory of live tick-borne parasites, which could potentially be transmitted by the ticks, and uncovers unexpected parasites in Western Europe.

Discriminating Francisella tularensis and Francisella-like endosymbionts in Dermacentor reticulatus ticks: evaluation of current molecular techniques.

Francisella tularensis, the causative agent of tularemia, is commonly transmitted by ticks. To ensure accurate F. tularensis reporting rates in epidemiological surveys, specific discrimination between F. tularensis and Francisella-like tick endosymbionts (FLEs) is absolutely critical. Four molecular available techniques capable of distinguishing Francisella spp. were compared here for the first time in French Dermacentor reticulatus ticks in order to estimate their specificity as well as their ease and speed of use. Results showed that tul4 and fopA real-time PCR assays can easily and effectively discriminate between F. tularensis and FLEs in D. reticulatus. In addition, a high prevalence of FLEs in D. reticulatus collected in France was reported by the use of fopA real-time PCR assay (79%). Finally, phylogenetic analysis showed that FLEs isolated from D. reticulatus correspond to a well-defined group compared to FLEs originating from various tick species.

To be or not to be co-infected

Ticks can transmit a large spectrum of pathogens including bacteria, viruses and parasites with a significant number of these pathogens being agents of emerging infectious diseases. In Europe, the most prevalent tick-borne disease is Lyme Borreliosis, caused by the bacteria Borrelia burgdorferi s.l. In most cases, Lyme Borreliosis is well diagnosed. However, beside these typical cases, patients bitten by ticks can be infected by many other pathogens (bacteria: Anaplasma spp., Bartonella spp., Rickettsia spp.; parasites: Babesia spp., Theileria spp.; and arboviruses: TBEV) that are more difficult to diagnose. Moreover, co-infections between several of these pathogens might also occur. Clinical surveys show that patients coinfected by several tick-borne pathogens present more severe symptoms and a longer duration of illness than those infected by a single pathogen. The overall objective of our study was to evaluate tick-borne pathogen coinfection in ticks and the consequence of those coinfections for human health. Using the high throughput real-time PCR chip, we detected the DNA of the 37 major tick-borne pathogens in a cohort of questing adult of Ixodes ricinus ticks collected in Ardennes (France). We identified that 60% of ticks were infected by at least one pathogen and half of the infected ticks were coinfected. We then evaluated the risk for simultaneous infection of those pathogens to humans by detecting the DNA of the most prevalent tick-borne pathogens in the blood of patients bitten by ticks. Our data illustrated the importance of coinfection, and highlighted the necessity to evaluate coinfection in the context of tick-borne diseases.

Infection with Mycobacterium microti in Animals in France

ABSTRACT We describe here 35 animal cases of tuberculosis due to Mycobacterium microti in France (2002–2014). Recently, molecular tools that overcome the difficulty of confirming infection by this potentially zoonotic agent have revealed an increasing number of cases, suggesting that its prevalence may have been underestimated.

Transmission differentials for multiple pathogens as inferred from their prevalence in larva, nymph and adult of Ixodes ricinus (Acari: Ixodidae)

Ixodes ricinus serves as vector for a range of microorganisms capable of causing clinical illness in humans. The microorganisms occur in the same vector populations and are generally affected by the same tick–host interactions. Still, the instars have different host preferences which should manifest in different transmission patterns for various microorganisms in the tick populations, i.e., most microorganisms increase in prevalence rate from larvae to nymphs because their reservoirs are among small mammals and birds that serve as blood hosts for larvae. Other microorganisms, like Anaplasma phagocytophilum, mainly increase in prevalence rates from nymphs to adults, because their reservoirs are larger ungulates that serve as primary blood hosts for nymphs and adults. We sampled a representative sample of ticks from 12 locations on Zealand and Funen, Denmark, and investigated the differences in prevalence rate of infection in larvae, nymphs and adults for multiple pathogens. Prevalence of infection for larvae, nymphs and adults, respectively, was: 0, 1.5 and 4.5% for Borrelia burgdorferi; 0, 4.2 and 3.9% for Borrelia garinii; 0, 6.6 and 6.1% for Borrelia afzelii; 0, 0 and 0.6% for Borrelia valaisiana; 0, 3.7 and 0.6% for Borrelia spielmanii; 0, 0.7 and 1.2% for Babesia divergens; 0, 0, 0.6% for Babesia venatorum; 0, 1.5 and 6.1% for A. phagocytophilum. The results were in general compatible with the hypothesis i.e., that differences in blood host for larvae and nymphs define differences in transmission of infectious agents, but other factors than differences in blood hosts between larvae and nymphs may also be important to consider.

Mycobacterium bovis Infection of Red Fox, France

Mycobacterium bovis infection in wild red foxes was found in southern France, where livestock and other wildlife species are infected. Foxes frequently interact with cattle but have been underestimated as a reservoir of M. bovis. Our results suggest a possible role of the red fox in the epidemiology of bovine tuberculosis.

Tick species, tick-borne pathogens and symbionts in an insular environment off the coast of Western France.

Insular environments provide ideal natural conditions to study disease ecology, especially emerging diseases, due to clear differentiation between local and long-distance transmission. Such environments are of particular interest regarding tick-borne pathogens (TBP), since animal exchange with the mainland (along with any ticks they carry) is limited, and because such locations could lie on migratory routes for birds carrying ticks. Therefore both tick species and TBP may display different prevalence than those observed on the continent. As such, an epidemiological survey was performed on Belle-Ile-en-Mer, an island off the coast of Western France, in order to estimate the prevalence of tick species and the microorganisms they carried. Three tick species, Dermacentor marginatus, D. reticulatus, and Haemaphysalis punctata were collected at five different sites in 2010 and 2011. All ticks were tested for pathogens and symbionts DNA by (i) PCR for Anaplasma spp., Borrelia spp., Rickettsia spp.; (ii) real-time PCR for Francisella tularensis, Francisella-like endosymbionts (FLE) and Coxiella spp. and (iii) PCR-RLB for Babesia-Theileria spp. Pathogen DNA detected in D. marginatus including Borrelia spp. (18%), Rickettsia spp. (13%) which was identified as R. slovaca, Babesia spp. (8%), and Theileria spp. (1%). Pathogens detected in D. reticulatus including Rickettsia spp. (31%) identified as R. raoulti, Francisella-like endosymbiont (86%), and Babesia spp (21%). Pathogens detected in H. punctata including Rickettsia spp. (1%) identified as R. aeschlimannii, FLE (0.4%), Babesia spp. (18%), and Theileria spp. (7%). Anaplasma spp., F. tularensis, or Coxiella spp. were not detected in any of the collected ticks. This study represents the first epidemiological survey of the insular Belle-Ile-en-Mer environment. It demonstrated the presence of expected pathogens, consistent with reports from island veterinarians or physicians, as well as unexpected pathogens, raising questions about their potential introduction through infected animals and/or their dispersion by migratory birds.