Genetic characterization of the human relapsing fever spirochete Borrelia miyamotoi in vectors and animal reservoirs of Lyme disease spirochetes in France

Background In France as elsewhere in Europe the most prevalent TBD in humans is Lyme borreliosis, caused by different bacterial species belonging to Borrelia burgdorferi sensu lato complex and transmitted by the most important tick species in France, Ixodes ricinus. However, the diagnosis of Lyme disease is not always confirmed and unexplained syndromes occurring after tick bites have become an important issue. Recently, B. miyamotoi belonging to the relapsing fever group and transmitted by the same Ixodes species has been involved in human disease in Russia, the USA and the Netherlands. In the present study, we investigate the presence of B. miyamotoi along with other Lyme Borreliosis spirochetes, in ticks and possible animal reservoirs collected in France. Methods We analyzed 268 ticks (Ixodes ricinus) and 72 bank voles (Myodes glareolus) collected and trapped in France for the presence of DNA from B. miyamotoi as well as from Lyme spirochetes using q-PCR and specific primers and probes. We then compared the French genotypes with those found in other European countries. Results We found that 3% of ticks and 5.55% of bank voles were found infected by the same B. miyamotoi genotype, while co-infection with other Lyme spirochetes (B. garinii) was identified in 12% of B. miyamotoi infected ticks. Sequencing showed that ticks and rodents carried the same genotype as those recently characterized in a sick person in the Netherlands. Conclusions The genotype of B. miyamotoi circulating in ticks and bank voles in France is identical to those already described in ticks from Western Europe and to the genotype isolated from a sick person in The Netherlands. This results suggests that even though no human cases have been reported in France, surveillance has to be improved. Moreover, we showed that ticks could simultaneously carry B. miyamotoi and Lyme disease spirochetes, increasing the problem of co-infection in humans.


Background
Human pathogenic Borrelia species are comprised of two main groups of spirochetes: the Lyme borreliosis group which cause human Lyme diseases, are widespread throughout Europe, Asia and North America and are transmitted between vertebrates by hard ticks (genus Ixodes) [1]; and the group causing severe relapsing fever in humans and which are transmitted by both, soft and hard ticks [2]. Borrelia miyamotoi, belonging to the relapsing fever group, is transmitted by the same Ixodes species that also transmits Borrelia species from the Lyme Borreliosis group. B. miyamotoi was isolated for the first time in Japan in 1995 from I. persulcatus ticks as well as from the blood of Apodemus argentus mice [3,4], and over recent decades, it has also been detected in Ixodes ticks in Europe, Russia and the USA [5][6][7]. The first human cases of B. miyamotoi infection were reported in Russia in 2011 [8], and more recently, human infections have been described in the USA and the Netherlands [9][10][11][12].
In Europe, Lyme borreliosis is the most common tickborne disease and is caused by different bacterial species belonging to the Borrelia burgdorferi sensu lato complex. They are transmitted by the tick species which has the most impact on European public health, Ixodes ricinus, and which are hosted by many animal species, like rodents, deer, birds and lizards. In France, over 10,000 new cases of Lyme borreliosis are estimated to occur each year. When there has been a history of tick bites, Lyme disease is commonly assumed, but in some cases there is an absence of diagnostic confirmation. Besides Lyme borreliosis typical cases, diagnosed in its first stage by the presence of an erythema migrans following tick bites and in its later phases by serological tests, a number of patients complain of polymorphic and unspecific clinical symptoms (asthenia, fever, myalgia, etc…), for which the diagnosis is not straightforward [13]. In recent years, unexplained syndromes occurring after tick bite has thus become a very important issue, grown considerably with alternate interpretations of the Lyme disease serology, which has led to considerable unrest between formal institutions of infectious disease and patient associations. The proportion of patients bitten by ticks with unexplained syndromes compared with patients who contracted Lyme disease is difficult to know precisely although it is estimated that 50% of fever after tick bites are of unknown etiological origin [14]. Unexplained syndromes, which have occurred following tick bites have thus become a very important issue in recent years.
In order to better characterize those tick-borne pathogens that are likely to be involved in unexplained tickborne diseases, we recently generated a global picture of their tick-borne bacteria by analyzing whole transcriptomes of I. ricinus ticks. Surprisingly, we identified the unanticipated B. miyamotoi in I. ricinus collected in France [15]. In the present study, our aim was to investigate the presence and prevalence of B. miyamotoi, along with Lyme borreliosis spirochetes, in both their vectors, i.e., I. ricinus, and their possible animal reservoirs (bank voles, Myodes glareolus). We have established that I. ricinus and bank voles are infected with B. miyamotoi along with Lyme disease spirochetes, that the same B. miyamotoi genotype circulate in both I. ricinus as well as in the bank voles and that this genotype is perfectly identical to those described in Western Europe, including those infecting humans [12].

Ticks and rodents
Ticks (268 questing Ixodes ricinus female ticks) and bank voles (72 Myodes glareolus) were collected and trapped in the French Ardennes, an endemic region for rodent-borne hantaviruses, along a transect line of about 80 km in the course of a study of the epidemiology of Puumala hantavirus [16]. Along this transect, we sampled six sites in forested areas and four sites in fragmented habitats (i.e. hedge network). Animals were killed by cervical dislocation. They were weighed, sexed and dissected. All collected ticks were washed and crushed as previously described [17].

DNA extraction
Voles genomic DNA was extracted from a piece of spleen using the DNeasy kit (Qiagen) according to manufacturer's instructions with a final elution volume of 100 μL. Tick genomic DNA was extracted using the Wizard genomic DNA purification kit (Promega) according to manufacturer's instructions with a final elution volume of 50 μL.  cycling conditions were as follows: 95°C for 5 min, 45 cycles at 95°C for 10s and 60°C for 15 s and then 40°C for 10 s.

Phylogenetic analysis of B. miyamotoi genotypes
Three genomic regions, including partial sequences of 16S rRNA (1256 bp), glpQ (379 bp) and p66 (532 bp) genes were amplified and sequenced as previously described [18] from B. miyamotoi positive-testing tick and rodent DNA extracts. Sequence alignment and phylogenetic analysis was performed using SeaView version 4.4.2 as previously described [19].

Ethical approval
Animals have been treated in accordance with the guidelines of the European Union legislation (Directive 86/609/EEC). The CBGP laboratory has received the approval (no. B 34-169-1) from the regional Head of Veterinary Service (Hérault, France), for the sampling and killing of rodents and the harvesting of their tissues. Dr Cosson has personally received the agreement "certificat d'autorisation d'expérimenter sur animaux vivants" (i.e. "certificate of authorization to experiment on live animals") (no. C34-105) by the French administration.

Results and discussion
Tick and bank vole DNA samples were assessed for the presence of B. miyamotoi via quantitative real-time PCR using primers and probes described in [15]. The prevalence of B. miyamotoi in ticks and bank voles was 3.0% (8/ 267 ticks) and 5.55% (4/72) respectively. The prevalence of B. miyamotoi in ticks was similar to that reported for I. ricinus collected from other European countries, which ranged from 0.4% in Estonia to 3.5% in Germany [5,18] , also with close proximity to human dwellings in some cases.
B. miyamotoi has previously been isolated from the blood of different rodent species such as the small Japanese field mouse (Apodemus argentus) from Japan [3], and the white-footed mouse (Peromyscus leucopus) from the USA [6], all of these species are likely potential B. miyamotoi host reservoirs. Our results support the importance of different rodent species in the circulation of B. miyamotoi, by adding the bank vole (Myodes glareolus) in the list of potential B. miyamotoi reservoirs in Europe.
In order to better characterize B. miyamotoi genotypes, three genomic regions, including partial sequences of 16S rRNA (1256 bp), glpQ (379 bp) and p66 (532 bp) genes were amplified and sequenced as previously described [18] from positive-testing tick and rodent DNA extracts. All three genes were amplified in all B. miyamotoi positive tick DNA samples, but the 16S RNA gene was amplified in four B. miyamotoi positive bank vole DNA samples, glpQ in only three, and the p66 gene was not amplified at all in bank vole DNA samples.
Nucleotide sequence similarity analysis of the three genomic regions (two for the bank vole samples) showed that all B. miyamotoi tick sequences were 100% identical to those amplified from bank voles (Figure 1), suggesting that the same genotype circulates between I. ricinus and bank voles. Phylogenetic analyses of these three genes were internally consistent with each other and gave strong support to the proposed genetic structuring of B. miyamotoi into three distinct geographic clades of Western Palearctic, Eastern Palearctic and Nearctic respectively (Figure 1). French sequences clustered to those amplified from I. ricinus in Switzerland, the Netherlands, Sweden, Estonia, Poland and the European part of Russia. They also clustered with the strain recently described from the patient in the Netherlands [12]. All of these sequences were clearly separated from a second group, which includes sequences from I. ricinus, I. persulcatus and human blood from Estonia, Russia and Japan. A third group includes sequences generated from ticks and human tissues from the USA. Finally, it is relevant to note that all three of these B. miyamotoi genetic groups include strains that are pathogenic for humans.
(See figure on previous page.) Figure 1 Phylogenetic trees of B. miyamotoi genotypes based on the partial sequences of A) 16S rRNA (GenBank accession numbers of the French data from KJ412189 to KJ412199), B) glpQ (GenBank accession numbers the French data from KJ425352 to KJ425363) and C) p66 genes (GenBank accession numbers the French data from KJ425364 to KJ425374). Phylogenetic analysis for each of the three genes was performed via the neighbor-joining method and a Kimura 2-parameter distance. Bootstrap analysis was performed on 1,000 replicates. Trees were rooted with Borrelia turicatae and Borrelia ionestari (not shown). Numbers beside branches indicate bootstrap values. Abbreviations: I. for Ixodes, and M. for Myodes. Scale bars indicate Jukes-Cantor evolutionary distances. Samples sequenced in the present study are marked with _*.