The wild hidden face of Lyme borreliosis in Europe

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1 Microbes and Infection, 2, 2000, Éditions scientifiques et médicales Elsevier SAS. All rights reserved S /REV Review The wild hidden face of Lyme borreliosis in Europe Pierre-François Humair, Lise Gern * Institut de Zoologie, Département de Parasitologie, Rue Emile-Argand 11, Case postale 2, 2007 Neuchâtel 7, Switzerland ABSTRACT Lyme borreliosis is a zoonosis affecting humans in the Northern hemisphere. The pathogen, Borrelia burgdorferi sensu lato (sl), persists in endemic areas through a maintenance cycle involving ticks and wild animals. The description of different genospecies associated with Lyme borreliosis in Europe has generated the question concerning the maintenance of these pathogens in nature: how do closely related bacterial species like B. burgdorferi sl circulate between one main tick species, Ixodes ricinus, and several vertebrate host species? Recent studies have provided evidence that specific associations exist in some areas between Borrelia species and vertebrate hosts. The present paper based on this recent knowledge discusses various aspects of the ecology of the disease in Western Europe, in particular the maintenance and dispersal of the pathogens, and brings up some interesting questions Éditions scientifiques et médicales Elsevier SAS Lyme borreliosis / ecology / ticks / Borrelia burgdorferi 1. Introduction Lyme borreliosis is a tickborne disease affecting humans in the Northern hemisphere, but Lyme borreliosis is above all a zoonosis, since it mainly infects a wide range of wild vertebrates and is only accidentally transmissible to man. Three protagonists are implicated in this vectorborne disease: the pathogen, Borrelia burgdorferi sensu lato (sl), the tick vector and the reservoir host. The pathogen persists through a maintenance cycle involving competent tick vectors and vertebrate reservoirs. Humans accidentally enter this cycle and represent dead-end hosts for the pathogen, as do vertebrates incompetent as reservoirs and ticks incompetent as vectors, since all are unable to transmit B. burgdorferi sl further. The tickborne zoonosis occurs in particular habitats named enzootic areas or natural foci where both competent vectors and reservoirs are present. The populations of vectors and reservoirs are both regulated by ecological factors which influence the ecology of the pathogen. The modalities that regulate the maintenance cycle (e.g., climate, phytocoenosis, zoocoenosis, vector and host diversity and density, frequency of tick-host contact, vector and host infection rates, etc.) may vary geographically and temporally and therefore influence the epidemiology of the disease in these foci. The study of the ecology of Lyme borreliosis, comprising pathogen-vector-reservoir interactions, is important for * Correspondence and reprints Microbes and Infection understanding the epidemiology of the disease in the human population. In Europe, the main vector of B. burgdorferi sl is the tick, Ixodes ricinus, and mammals such as small rodents (Apodemus, Clethrionomys, Microtus), rats, squirrels, dormice, shrews, hedgehogs and hares, and birds such as groundforaging passerines and pheasants are all known reservoirs. Five Borrelia genospecies associated with B. burgdorferi sl have been described up to now in questing I. ricinus ticks: B. burgdorferi sensu stricto (ss) [1], B. garinii [2], B. afzelii [3], B. lusitaniae [4] and B. valaisiana [5]. The description of these different genospecies generated new questions notably concerning the ecological aspects of the disease. Some recent studies provided strong evidence that specific associations exist in some areas between Borrelia species and reservoir hosts, particularly between B. afzelii and Apodemus mice and Clethrionomys voles [6, 7], between B. burgdorferi ss, B. afzelii and red squirrels [8], and between B. garinii, B. valaisiana and birds such as Turdus passerines and pheasants [9, 10]. This phenomenon implies that B. burgdorferi sl species are maintained in some areas in Europe through distinct maintenance cycles occurring simultaneously and involving the same tick vector but different reservoir species. The present review, based on the current knowledge, discusses various aspects of the ecology of B. burgdorferi sl in Western Europe, with the example of the situation in Switzerland. This paper deals in particular with the maintenance and the dispersal of the pathogens. The mainte- 915

2 Review Humair and Gern Figure 1. Ixodes ricinus: larval and female stages on the left and nymphal and male stages on the right. Source: Institut de Zoologie, Neuchâtel, Switzerland. 1 small square = 1mm 2. nance cycle of B. burgdorferi sl in nature is basically composed of four steps: (1) Borrelia occurs in the tick vectors, (2) is transmitted from the tick to the host, (3) occurs in the hosts, and (4) is transmitted from the host to the tick. 2. Borrelia in ticks I. ricinus ticks (figure 1) acquire B. burgdorferi sl infection during an infective meal on a reservoir host. The transstadial transmission (or passage) consisting of the persistence of spirochetes in the tick during its moult to the next instar is essential for the maintenance of spirochetes in natural foci [11]. In unfed ticks the spirochetes remain generally in the midgut, but systemic (generalized) infections affecting different organs may occur in 12 to 56% of infected ticks [12, 13]. In a female tick, the systemic infection may reach the ovaries and the developing embryos, leading to the transovarial transmission of spirochetes from female ticks to their larval progeny [11], but this is a rare event in I. ricinus (about 1% of females transmit) [11]. It is not known at present if the transstadial and transovarial transmission rates, or the development of a systemic infection in the tick, are influenced by the Borrelia genospecies. In Europe, B. burgdorferi ss, B. afzelii and B. garinii are present in various foci. Few isolates of the newly described genospecies, B. valaisiana and B. lusitaniae [4, 5] have been obtained at present. Further investigations should be done to elucidate whether these two genospecies are really less prevalent in the tick population or whether they multiply at a low rate and are usually undetectable by cultivation. In fact, very few isolates of B. valaisiana have been obtained from ticks up to now, except perhaps in the Netherlands [5] and Switzerland [9]. In contrast, DNA of B. valaisiana has been amplified from numerous ticks from Ireland [14]. This suggests that B. valaisiana may grow inadequately in BSK medium or is more prone to spirochetal decrease during tick moult, which makes isolation more laborious. A very clustered geographical distribution could also explain the low number of isolates of some genospecies. Very few isolates of B. lusitaniae, for example, have been reported, and only from particular areas in Europe (for a review see [15]). Preliminary observations on the occurrence of Borrelia genospecies related to tick stages show that B. afzelii tends to be more frequent in questing nymphs, whereas B. garinii and B. burgdorferi ss are more frequently found in questing adults (Gern et al., unpublished). This predominance is directly related to trophic preferences of feeding ticks of the previous stages, and studies on reservoir hosts and their associated Borrelia species support this observation [6 10, 16]. Concerning the ecology of the vector, questing I. ricinus ticks generally occur from February to November as, for example, on the Swiss Plateau [17, 18]. In general the number of questing ticks is high in spring and decreases during the remaining activity season. On the Swiss Plateau, the seasonal pattern is generally monomodal with pseudobimodal (monomodal pattern with summer depression) pattern during some years [17, 18]. At present, there is no evidence of a seasonal pattern of ticks infected by any genospecies of B. burgdorferi sl. 916 Microbes and Infection

3 The wild hidden face of Lyme borreliosis in Europe Review The spatial distribution of questing I. ricinus ticks is defined by vertical and horizontal components. In Switzerland, the vertical distribution of questing ticks on the vegetation is related to tick stages: larvae quest in the leaf litter and very near the ground, nymphs quest on the low vegetation at an average height of 10 cm, whereas adults quest on the vegetation at a height ranging between 10 and 50 cm or even higher [17]. The horizontal distribution of questing ticks is determined by the drop-off sites of engorged ticks of the previous stages, since I. ricinus show little horizontal movement (up to 1 m for adults) [17]. Larvae are present in the close vicinity of the site of the egg laying by the females and clearly show a clustered distribution [19], they may spread horizontally in a circle of cm in diameter around the site of egg laying [17]. Little is known about the distribution of nymphs and adults, but a study on a small scale (3m 2 ) has shown that the distribution of nymphs and adults is neither regular nor random but aggregated [17]. Mosaic distribution of ticks infected by B. burgdorferi sl has recently been observed [20]. However, the microgeographical distribution of infected ticks in a focus has never been studied in relation to the infection by the different genospecies of B. burgdorferi sl. However, in view of the preliminary observations on the occurrence of Borrelia genospecies related to tick stages (Gern et al., unpublished), we can hypothesize that adult ticks mostly infected by B. garinii and B. burgdorferi ss would be found higher in the vegetation than nymphs mostly infected by B. afzelii. Moreover, considering the horizontal distributions of the different tick stages, the occurrence of microfoci (clusters) of ticks infected by one species or another can be suspected. Anyway, the study of the spatial distribution of tick stages related to their infection should give some interesting answers. 3. Transmission of borrelia from ticks to hosts The transmission of B. burgdorferi sl from ticks to hosts is trivially the result of the encounter of an infected starving tick with a vertebrate host. Once this tick attaches to the host, spirochetes which are present in the tick midgut migrate through the midgut wall and hemocoel, reach the salivary glands and are inoculated with the tick saliva into the host 2 3 days after attachment [21]. Successful transmission of spirochetes may occur even 17 or 29 h after I. ricinus attachment [22]. The presence of spirochetes in salivary glands of host-seeking ticks (systemically infected) could explain this reduction of time before transmission, as suggested by Lebet et al. [12] and Leuba-Garcia et al. [13]. The infected tick-host encounter depends on the frequency of contacts between the vector and the host species and on the infection prevalence of the vector. Considering the high prevalence of infection in nymphs compared to the very low prevalence in larvae, nymphal ticks were thought to be crucial in the transmission of Borrelia infection to hosts. This is certainly the case for hosts such as passerine birds, squirrels, and hares, which are frequently infested by I. ricinus nymphs. But this is less obvious for Microbes and Infection hosts such as small mammals which are less frequently exposed to nymphs. In this case, two routes of infection can be considered: small mammals may become infected either through transovarially infected larvae or transstadially infected nymphs. Mathematical models show that, although the infection rate in larvae is low [11], the greater abundance of larvae in the biotope compared with the abundance of nymphs and adults and the high larval infestation on small mammals enhance the importance of larvae in the transmission of spirochetes to these hosts [23]. On the other hand, although I. ricinus nymphs are not abundant on small mammals, they are not so uncommon and could also contribute to the infection of rodents: ten to 31% of these hosts are infested by nymphs [24, 25] and nymphs are on average about 20 times as infected as larvae. Thus, what are the respective chances for a small mammal to be infected with either transovarially infected larvae or infected nymphs? Only 1% of the I. ricinus egg batches are infected by B. burgdorferi sl [11]. Yet, in an infected egg batch, % of eggs and 47 97% of larvae are infected [11]. On the other hand, on average 30% of nymphs are infected by B. burgdorferi sl [26], and the abundance of nymphs in a biotope is higher ten times as abundant as females, according to Randolph and Craine [23] than that of egg batches, which depends on the abundance of females. Consequently, considering these parameters the probability for small mammals to encounter infected nymphs seems to be higher than that to encounter clustered larvae issued from an infected egg batch. Apparently, small mammals may acquire the infection from I. ricinus larvae or nymphs, but the role of larvae or nymphs might vary geographically and temporally. Knowledge on the seasonal pattern of ticks attached on hosts is rather limited. The seasonal occurrence of I. ricinus attached on hosts varies among tick stages and host species. In Switzerland, for example, I. ricinus larvae on rodents peaked in May-June and in August-September with a summer depression in July [16, 24], whereas the number of larvae per bird is almost constant throughout the season with a slight increase in August [27]. Onthe other hand, I. ricinus nymphs are not abundant on rodents and can be found without real peak in May, July, August and October [16, 24], whereas nymphs on birds are abundant and peaked in (April)-May [27]. If we consider that the I. ricinus nymph is the infecting stage for either host species, the seasonal patterns of attached ticks on hosts are important to ensure a maintenance cycle as efficient as possible: nymphs should peak before larvae. This seasonal pattern occurs clearly in the case of birds [27] and apparently also in the case of rodents: nymphs are present on rodents during and/or before the two peaks of larvae. 4. Borrelia in hosts Once in the host skin, spirochetes may remain at the inoculation site for a few days and finally disseminate [28]. Little is known about the development of B. burgdorferi sl in the host. Borrelia has been isolated and/or detected in various organs or tissues such as skin, blood, joints, 917

4 Review Humair and Gern internal organs (spleen, heart, liver, urinary bladder, kidneys, nervous system) in various vertebrate hosts [29 33]. In humans the different genospecies tend to cause distinct clinical manifestations affecting different systems: B. burgdorferi ss tends to be present in joint fluid and cause arthritis, B. afzelii is present in skin and induces cutaneous manifestations and B. garinii is present in cerebrospinal fluid and is responsible for neurologic troubles [34 36].In a reservoir host the skin surface frequently exposed to tick bites is an essential interface of epidemiological importance, since this site is the entry point for spirochetes into the host and especially the exit point out of the host, allowing the tick infection and the cycle to continue. Few authors have studied the occurrence of Borrelia species in European vertebrate hosts. The difficulty of isolating spirochetes from hosts has been a constraint for a long time. BSK medium optimized by Sinsky and Piesman [30] has greatly increased the isolation rate from host tissues. The association observed between small mammals and B. afzelii [16] is in accordance with the identity of the first European rodent isolate obtained in Sweden [37]. An intimate association between birds and B. garinii has also been reported in Europe [9, 38 40]. Recently, B. valaisiana in addition to B. garinii was described in birds [9, 10] and their transmission from infected passerines to uninfected I. ricinus ticks was demonstrated [9]. Moreover, the predominance of B. afzelii and B. burgdorferi ss in squirrels was reported [8]. Various methods may be used to assess the infection status of a host. Isolation by BSK cultivation of tissue samples and DNA detection in host tissues after PCR amplification are two of these methods. The isolation has the advantage of obtaining live spirochetes, demonstrating that the infection is current, but has the disadvantage of being a fastidious technique and not so sensitive. In contrast, PCR amplification has the advantage of being very sensitive, allowing detection of 1 10 spirochetes but has the disadvantage of detecting DNA from both live and dead organisms, demonstrating a current or resolving infection. To obtain a better assessment of the infection status of hosts, both methods can be used concomittantly [8, 9, 16]. For the isolation, skin surfaces exposed to tick bites (ear for rodents, chin for birds) were removed by skin biopsy, inoculated in culture medium and characterized by PCR/ RFLP. Hosts analysed this way clearly showed differential infection: B. afzelii in small rodents, B. garinii and B. valaisiana in birds and B. burgdorferi ss and B. afzelii in squirrels. These associations observed between the host groups and the genospecies present an exclusive pattern, since no other genospecies has been isolated. A PCR/RFLP screening performed on BSK medium containing skin biopsies confirmed results obtained from isolation except in two cases where B. burgdorferi ss DNA was detected in a sample from a vole [16] and B. garinii DNA was detected in a sample from a red squirrel [8]. In these two cases, only DNA of borrelia was amplified and no live borrelia has ever been isolated, suggesting that these genospecies may occur at least at the site of inoculation in inadequate hosts in which they were maintained with difficulty. The specific maintenance cycles might be explained by the specific effect of the complement present in the host serum on each genospecies of B. burgdorferi sl. A recent study showed that the pattern of serum sensitivity of different Borrelia genospecies matched the reservoir status of various vertebrate species for B. burgdorferi sl: B. afzelii, for example, was resistant to rodent sera, whereas B. garinii was readily lysed by rodent sera [41]. Thus, the complement would have an effect not only in the vertebrate hosts, which will not accept all Borrelia species, but possibly also in the tick by the loss of spirochetes in ticks fed on inadequate hosts. If a strong association has been described between specific hosts and Borrelia species in Switzerland and in the UK, nonspecific maintenance cycles involving small mammals and various Borrelia species have been described mainly in Russia and eastern parts of Europe, where different Borrelia species were demonstrated in internal organs of small rodents using BSK cultivation and/or PCR amplification [42 44]. The presence of different subtypes of B. garinii in these areas as well as the occurrence of different reservoir and vector species may explain the different situations. Hedgehogs are also involved in a nonspecific maintenance cycle in Switzerland, since they were found infected by various genospecies [45]. This suggests that nonspecific maintenance cycles involving host species and various Borrelia species may occur in addition to specific maintenance cycles involving host species and particular Borrelia species. Little is known about the duration of Borrelia infection in vertebrate hosts. Reservoirs are defined as hosts maintaining the Borrelia infection for a long period of time even during nontransmission periods. At present, only wild small rodents were shown to remain infected during winter, outside of the tick activity period until the following spring [16]. Hosts incompetent as reservoirs would fight the infection through their immune system to avoid the establishment of infection. However, the skin of such hosts can constitute an interface for the transmission of spirochetes among cofeeding ticks attached on these hosts. Cofeeding transmission allows transmission of spirochetes from infected to uninfected ticks feeding together on a host presenting no generalized infection [28] as observed in sheep and deer [46, 47]. Although vertebrate hosts may present a systemic (generalized) infection affecting different organs, the spirochetal infection has never been detected in ovaries, and transplacental transmission has been excluded in the case of Peromyscus leucopus in North America [48]. To our knowledge, this way of transmission has never been studied in Europe, but this route does not seem to be very likely, since young wild rodents, for example, are rarely found infected [49]. Ticks are absolutely needed for the maintenance cycle to continue. 5. Transmission of borrelia from hosts to ticks The presence of borrelia in a host does not mean that this host is infective to ticks and acts as a reservoir. The success of the transmission of spirochetes from host to 918 Microbes and Infection

5 The wild hidden face of Lyme borreliosis in Europe Review ticks demonstrates the infectivity of the host and its role as reservoir in a focus of Lyme borreliosis. Tick xenodiagnosis is the best way to assess the reservoir status of a vertebrate species. Naive ticks derived from a laboratory colony free of infection are allowed to engorge on tested hosts and are analyzed after the moult to detect the pathogen. This method is easily applicable to animals which can be kept in captivity such as small rodents. Using xenodiagnosis, various mammal and bird species were found to be competent reservoirs of B. burgdorferi sl in enzootic areas in Europe (for a review see [50]). Further examinations of xenodiagnostic ticks allow researchers to identify the Borrelia species which are transmitted from the vertebrate to the ticks. It was reported that only B. afzelii in Switzerland and B. burgdorferi ss in the UK were transmitted from rodents to feeding ticks [7, 10, 16]. Humair et al. [9] clearly demonstrated that blackbirds (Turdus merula) are reservoirs and that they only transmit B. valaisiana and B. garinii to feeding ticks, and Kurtenbach et al. [10] showed the reservoir role of pheasants for these genospecies. However, xenodiagnosis is not always possible for other animals. The alternative method is to detect the infection in the field-derived ticks, especially larvae, which feed on the hosts and to compare the prevalence of infection in the feeding ticks and in the questing ticks in the same habitat. A higher prevalence of infection in the host-feeding ticks compared to the prevalence in the questing ticks suggests that the transmission occurs between hosts and ticks. Yet, it is difficult to distinguish between cofeeding transmission and transmission from reservoirs to naive ticks. However, this method was used with various vertebrates [50]. In addition, the Borrelia genospecies can be determined in feeding ticks using identification methods. It was shown, for example, that spirochetal transmission occurred from red squirrels (Sciurus europaeus) to field-derived feeding ticks and that these ticks were mainly infected by two genospecies, B. burgdorferi ss and B. afzelii [8]. In studies investigating Borrelia infection in vertebrate hosts and in feeding ticks, the Borrelia genospecies present in the hosts were also predominant in the feeding ticks, showing that a complete correlation occurs between host infection and host infectivity [6 9, 16]. Each group of hosts contributes in its own way to the maintenance of the different Borrelia genospecies in the ticks in the focus. In the foci where Borrelia-host associations occur, the variation in the distribution of Borrelia genospecies in tick populations would be correlated to the occurrence and the density of the different reservoir species. The explanation of the occurrence of distinct associations between Borrelia species and vertebrate hosts must probably be searched for in the past. We hypothesize that the different genospecies currently known and possibly those not yet identified come from a common ancestor that has evolved differently and has adapted to various environments. In its maintenance cycle, borrelia is linked with tick and host environments. As the main vectors (I. ricinus complex) are tick species showing no particular parasitic specificity towards hosts, the ancestor of Borreliae had to adapt to many different vertebrate hosts, such as mammalian, avian and reptilian species. However, the trend of a differential parasitic specificity related to tick Microbes and Infection stages (larvae mainly on small mammals, nymphs mainly on birds and medium-sized mammals) could have helped in driving the diversity in Borrelia species. B. garinii is the species which has the most opportunity to be dispersed in completely different habitats via infected ticks carried by migratory birds, meaning that it has to adapt to different conditions. The highest diversity observed among B. garinii isolates [51] may be the results of the adaptation of this species to different environmental conditions (other vertebrate hosts and tick species). The existence of Borrelia-host associations generates the question about the destiny of Borrelia species transmitted to inadequate hosts. Apparently, the maintenance in unsuitable hosts and the transmission to feeding ticks are not ensured. To this purpose, the observation of a specific association between host and borrelia allows us to understand why blackbirds were revealed incompetent as reservoirs [52]. In the experiment, blackbirds were infected by ticks previously fed on infected rodents. In view of the specific association occurring between rodents and B. afzelii, ticks would have harbored B. afzelii infection and transmitted it to blackbirds. Considering the specific association existing between birds and B. garinii / B. valaisiana, blackbirds were unable to maintain and transmit B. afzelii spirochetes to further feeding ticks. This actually demonstrates that birds are inadequate hosts for maintaining B. afzelii. Similarly, it is almost impossible to obtain B. garinii-infected ticks from mice experimentally infected by this genospecies (Gern, personal observation). This demonstrates the importance of different reservoir species in the distinct maintenance cycles of B. burgdorferi sl in nature and suggests that inadequate host species may have a zooprophylactic role towards some Borrelia species, as observed in lizards in California [53]. Finally, the key question is 'what is a reservoir?'. From an ecological viewpoint, a reservoir is any animal capable of maintaining a pathogen for a long time and capable of transmitting the pathogen to competent vectors. From a biological viewpoint, the reservoir status is given to any animal capable of coexisting with a pathogen. This status is the fruit of a long evolution in which each organism adapts little by little to the other. At the very beginning, the inoculation of the pathogen into the host would have activated the development of specific and nonspecific immune responses against the invader. In response, the pathogen would have developed several mechanisms to overcome vertebrate host defenses such as antigenic variation, intracellular location, and would also have begun to take advantage of immunosuppressive properties of saliva of tick vectors [54] to settle in the host. Little by little, the host-pathogen relationship has evolved, leading to a physiological adaptation to each other and the coexistence of both organisms. The relationship is so old that the pathogen may persist in the host without causing any disease and the host becomes a reservoir. 6. Dispersal of borrelia Infective vertebrate species may contribute to the dispersal of infected ticks in their respective home range. Any 919

6 Review movements outside the home range offer additional possibilities of dispersal inside the focus or in close suitable habitats. Due to their great capacity of movement, birds are more likely to contribute to the local dispersal of infected ticks and migrant birds may contribute to longdistance dispersal of B. burgdorferi sl-infected ticks [39] and presumably of B. valaisiana and B. garinii spirochetes according to the Borrelia-host associations. The dispersal may occur along the migration routes northward/ southward in Europe and southward as far as Africa for some bird species. The successful establishment of infected tick in new locations along the migration route depends upon diverse conditions: the tick must engorge successfully to repletion, detach in a suitable habitat for survival during its moult and find an adequate host for the subsequent stage. Since the transovarial transmission of B. burgdorferi sl rarely occurs, suitable hosts are needed to ensure horizontal transmission. But given the association between Borrelia species and hosts, this last condition could be very restrictive for establishing new foci. Anyway, the presence of Borrelia-infected I. ricinus ticks on migrating birds collected in Sweden demonstrates that infected ticks are carried during bird migrations between different European foci [39]. Additionally, infective migrating birds may also transmit spirochetal infection to susceptible ticks encountered during the migration. In this case, based on recent studies [9, 10], only B. valaisiana and B. garinii would be dispersed in tick populations of new areas. In these sites, competent vectors as well as suitable hosts such as indigeneous birds are needed for these Borrelia species to become established. Interestingly, most borrelia found in Ireland were characterized as B. valaisiana and B. garinii [14], suggesting that birds are perhaps responsible for the presence of Lyme Borreliae on this island. The presence of B. garinii in Ixodes uriae ticks collected in various seabird colonies in both hemispheres indicates that an exchange of spirochetes and/or Borrelia-infected I. uriae occurs between colonies of the Northern hemisphere and those of the Southern hemisphere [38]. 7. Conclusion The present review shows that the puzzle of the ecology of Lyme borreliosis is still incomplete and that further investigations are needed to understand fully the wild hidden face of the disease. The relationships between Borrelia species associated with Lyme borreliosis and vertebrate hosts acting as reservoirs need to be investigated more extensively in Europe and elsewhere to understand more accurately the intimacy of such associations. Local considerations on the biology and the ecology of vectors and reservoirs may be some pieces of the puzzle. Phylogenetic studies of B. burgdorferi sl including biogeography and Borrelia-host-vector associations could answer the question about the evolution of this pathogen in its geographical distribution. Future knowledge in this field will demonstrate that each focus of Lyme borreliosis has its own transmission pattern because each focus has its own history. References Humair and Gern [1] Johnson R.C., Schmid G.P., Hyde F.W., Steigerwalt A.G., Brenner D.J., Borrelia burgdorferi sp. nov.: etiologic agent of Lyme disease, Int. J. Syst. Bacteriol. 34 (1984) [2] Baranton G., Postic D., Saint Girons I., Boerlin P., Piffaretti J.C., Assous M., Grimont P.A.D., Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis, Int. J. Syst. 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