Relationships of a novel Lyme disease spirochete, Borrelia spielmani sp. nov., with its hosts in Central Europe

To determine whether the pathogenic variant of Lyme disease spirochetes, isolate A14S, is perpetuated in a particular reservoir-vector relationship, we screened vector ticks in various Central European sites for a related spirochete and determined its host association. A14S-like spirochetes infect numerous questing ticks in the Petite Camargue Alsacienne (PC). They frequently infect dormice, but no mice or voles. Garden dormice appear to be better reservoir hosts for A14S-like spirochetes than for Borrelia afzelii, because these spirochetes are retained longer and infect ticks more readily. Spirochetes associated with garden dormice in the PC site form a homologous entity with those isolated from a human patient in The Netherlands. Its unique biological relationship together with previous genetic characterization justifies designating this dormouse-associated genospecies as a distinct entity. Garden dormice serve as the main reservoir hosts of a novel genospecies, Borrelia spielmani sp. nov., one of several that cause Lyme disease in people.     

Seasonal Prevalence of Lyme Disease Spirochetes in a Heterothermic Mammal,the Edible Dormouse (Glis glis)

In Europe, dormice serve as competent reservoir hosts for particular genospecies of the tick-borne agent of Lyme disease (LD) and seem to support them more efficiently than do mice or voles. The longevity of edible dormice (Glis glis) and their attractiveness for ticks may result in a predominance of LD spirochetes in ticks questing in dormouse habitats. To investigate the role of edible dormice in the transmission cycle of LD spirochetes, we sampled skin tissue from the ear pinnae of dormice inhabiting five different study sites in south western Germany. Of 501 edible dormice, 12.6% harbored DNA of LD spirochetes. Edible dormice were infected most frequently with the pathogenic LD spirochete Borrelia afzelii. The DNA of B. garinii and B. bavariensis was detected in ca. 0.5% of the examined individuals. No spirochetal DNA was detectable in the skin of edible dormice until July, 6 weeks after they generally start to emerge from their obligate hibernation. Thereafter, the prevalence of spirochetal DNA in edible dormice increased during the remaining period of their 4 to 5 months of activity, reaching nearly 40% in September. Males were more than four times more likely to harbor LD spirochetes than females, and yearlings were almost twice more likely to be infected than adults. The seasonality of the prevalence of LD spirochetes in edible dormice was pronounced and may affect their role as a reservoir host in respect to other hosts.     

Characteristics of Garden Dormice That Contribute to Their Capacity as Reservoirs for Lyme Disease Spirochetes

To describe the contribution of garden dormice to the epizootiology of Lyme disease, we compared their reservoir capacity for these pathogens to that of other sympatric hosts. Garden dormice are trapped most abundantly during early spring and again during midsummer, when their offspring forage. They are closely associated with moist forests. Garden dormice serve as hosts to nymphal ticks far more frequently than do other small mammals. Spirochetal infection is most prevalent in dormice, and many more larval ticks acquire infection in the course of feeding on these than on other rodents in the study site. Mature dormice appear to contribute more infections to the vector population than juveniles do. Replete larval ticks generally detach while their dormouse hosts remain within their nests. The population of garden dormice contributes five- to sevenfold more infections to the vector population than the mouse population does. Their competence, nymphal feeding density, and preference for a tick-permissive habitat combine to favor garden dormice over other putative reservoir hosts of Lyme disease spirochetes.     

First report of Borrelia burgdorferi sensu lato in two threatened carnivores: the Marbled polecat, Vormela peregusna and the European mink

ABSTRACT: BACKGROUND: Lyme disease is a widespread cosmopolitan zoonosis caused by species belonging to the genus Borrelia. It is transmitted from animal reservoir hosts to humans through hard - ticks of genus Ixodes which are vectors of the disease. CASE PRESENTATION: Borrelia burgdorferi sensu lato infection was identified in a marbled polecat, Vormela peregusna, and two European minks, Mustela lutreola, from Romania, by PCR. RFLP revealed the presence of a single genospecies, Borrelia burgdorferi sensu stricto. CONCLUSIONS: This is the first report of the Lyme disease spirochetes in the two mentioned hosts.     

Reservoir competence of various rodents for the lyme disease Spirochete Borrelia spielmanii

To determine whether the pathogenic Lyme disease spirochete Borrelia spielmanii is adapted exclusively to garden dormice, we compared the reservoir competence of various rodent species for this spirochete, including sympatric and peridomestic rodents. The different kinds of rodents varied in their attractiveness to nymphal ticks and their level of susceptibility to tick-borne B. spielmanii infection, but only the edible dormouse appeared to be refractory. Although hazel dormice and Norway rats became infectious to ticks somewhat later than did garden dormice, they remained infectious for a longer period of time. During the course of a tick season, garden and hazel dormice contributed theoretically more than twice as many B. spielmanii-infected ticks than the somewhat less susceptible Norway rats and wood or yellow-necked mice. Hazel dormice appeared to be extraordinarily competent as reservoir hosts for B. spielmanii. Because peridomestic rodents proved to be reservoir competent, urban foci of transmission of B. spielmanii may affect the health of townspeople.     

Perpetuation of the Lyme Disease Spirochete Borrelia lusitaniae by Lizards

To determine whether the Lyme disease spirochete Borrelia lusitaniae is associated with lizards, we compared the prevalence and genospecies of spirochetes present in rodent- and lizard-associated ticks at a site where this spirochete frequently infects questing ticks. Whereas questing nymphal Ixodes ricinus ticks were infected mainly by Borrelia afzelii, one-half of the infected adult ticks harbored B. lusitaniae at our study site. Lyme disease spirochetes were more prevalent in sand lizards (Lacerta agilis) and common wall lizards (Podarcis muralis) than in small rodents. Although subadult ticks feeding on rodents acquired mainly B. afzelii, subadult ticks feeding on lizards became infected by B. lusitaniae. Genetic analysis confirmed that the spirochetes isolated from ticks feeding on lizards are members of the B. lusitaniae genospecies and resemble type strain PotiB2. At our central European study site, lizards, which were previously considered zooprophylactic for the agent of Lyme disease, appear to perpetuate B. lusitaniae.     

Perpetuation of the Lyme disease spirochete Borrelia lusitaniae by lizards

To determine whether the Lyme disease spirochete Borrelia lusitaniae is associated with lizards, we compared the prevalence and genospecies of spirochetes present in rodent- and lizard-associated ticks at a site where this spirochete frequently infects questing ticks. Whereas questing nymphal Ixodes ricinus ticks were infected mainly by Borrelia afzelii, one-half of the infected adult ticks harbored B. lusitaniae at our study site. Lyme disease spirochetes were more prevalent in sand lizards (Lacerta agilis) and common wall lizards (Podarcis muralis) than in small rodents. Although subadult ticks feeding on rodents acquired mainly B. afzelii, subadult ticks feeding on lizards became infected by B. lusitaniae. Genetic analysis confirmed that the spirochetes isolated from ticks feeding on lizards are members of the B. lusitaniae genospecies and resemble type strain PotiB2. At our central European study site, lizards, which were previously considered zooprophylactic for the agent of Lyme disease, appear to perpetuate B. lusitaniae.     

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.     

Introduced Siberian chipmunks (Tamias sibiricus barberi) harbor more-diverse Borrelia burgdorferi sensu lato genospecies than native bank voles (Myodes glareolus)

Little attention has been given in scientific literature to how introduced species may act as a new host for native infectious agents and modify the epidemiology of a disease. In this study, we investigated whether an introduced species, the Siberian chipmunk (Tamias sibiricus barberi), was a potentially new reservoir host for Borrelia burgdorferi sensu lato, the causative agent of Lyme disease. First, we ascertained whether chipmunks were infected by all of the B. burgdorferi sensu lato genospecies associated with rodents and available in their source of infection, questing nymphs. Second, we determined whether the prevalence and diversity of B. burgdorferi sensu lato in chipmunks were similar to those of a native reservoir rodent, the bank vole (Myodes glareolus). Our research took place between 2006 and 2008 in a suburban French forest, where we trapped 335 chipmunks and 671 voles and collected 743 nymphs of ticks that were questing for hosts by dragging on the vegetation. We assayed for B. burgdorferi sensu lato with ear biopsy specimens taken from the rodents and in nymphs using PCR and restriction fragment length polymorphism (RFLP). Chipmunks were infected by the three Borrelia genospecies that were present in questing nymphs and that infect rodents (B. burgdorferi sensu stricto, B. afzelii, and B. garinii). In contrast, voles hosted only B. afzelii. Furthermore, chipmunks were more infected (35%) than voles (16%). These results may be explained by the higher exposure of chipmunks, because they harbor more ticks, or by their higher tolerance of other B. burgdorferi sensu lato genospecies than of B. afzelii. If chipmunks are competent reservoir hosts for B. burgdorferi sensu lato, they may spill back B. burgdorferi sensu lato to native communities and eventually may increase the risk of Lyme disease transmission to humans.     

The presence of Borrelia valaisiana-related genospecies in ticks and a rodent in Taiwan

A field survey was conducted to investigate the presence of Borrelia burgdorferi sensu lato (s.l.) in six counties of Taiwan. Spirochetes were successfully isolated from one rodent ear sample out of 485 rodent ears and 53 live, fed tick (Ixodes granulatus) samples. The spirochetes were confirmed to be B. burgdorferi s.l. by real-time PCR. In addition, 23 of 113 tick samples were tested positive for Borrelia DNA according to real-time PCR. The Borrelia isolate from the rodent and the 23 Borrelia DNA samples from the ticks were identified as B. valaisiana-related genospecies by phylogenetic analysis based on flagellin gene sequences. These findings suggest that the Borrelia valaisiana-related strains are maintained in a zoonotic cycle between tick vectors and reservoir hosts in Taiwan.     

Diversity of European Lyme Disease Spirochetes at the Southern Margin of Their Range

We determined whether the genospecies diversity of Lyme disease spirochetes in vector ticks questing on a subtropical island is as broad as that in Central Europe. Although spirochetes infected <1% of the ticks sampled on Madeira Island, these infections included all three genospecies implicated in human disease. Therefore, spirochetal diversity is as great at the southern margin as it is in the center of this pathogen’s range.     

Failure of Ixodes Ticks To Inherit Borrelia afzelii Infection

To define conditions promoting inherited infection by Lyme disease spirochetes in Ixodes ticks, we variously infected ticks with Borrelia afzelii and examined their progenies by dark-field microscopy, immunofluorescence, PCR, and serial passage. No episode of inherited infection was evident, regardless of instar or gender infected or frequency of exposure. We suggest that these spirochetes rarely, if ever, are inherited by vector ticks.     

Habitat-Specific Diversity of Borrelia burgdorferi Sensu Lato in Europe, Exemplified by Data from Latvia

The distribution of Borrelia burgdorferi sensu lato genospecies in questing Ixodes ricinus ticks from ecologically distinct habitats in Latvia was analyzed. A significant variation in the frequency of the genospecies across sites was observed, pointing to the importance of the host community in the ecology of Lyme borreliosis.     

Species Co-Occurrence Patterns among Lyme Borreliosis Pathogens in the Tick Vector Ixodes ricinus

Mixed infections have important consequences for the ecology and evolution of host-parasite interactions. In vector-borne diseases, interactions between pathogens occur in both the vertebrate host and the arthropod vector. Spirochete bacteria belonging to the Borrelia burgdorferi sensu lato genospecies complex are transmitted by Ixodes ticks and cause Lyme borreliosis in humans. In Europe, there is a high diversity of Borrelia pathogens, and the main tick vector, Ixodes ricinus, is often infected with multiple Borrelia genospecies. In the present study, we characterized the pairwise interactions between five B. burgdorferi sensu lato genospecies in a large data set of I. ricinus ticks collected from the same field site in Switzerland. We measured two types of pairwise interactions: (i) co-occurrence, whether double infections occurred more or less often than expected, and (ii) spirochete load additivity, whether the total spirochete load in double infections was greater or less than the sum of the single infections. Mixed infections of Borrelia genospecies specialized on different vertebrate reservoir hosts occurred less frequently than expected (negative co-occurrence) and had joint spirochete loads that were lower than the additive expectation (inhibition). In contrast, mixed infections of genospecies that share the same reservoir hosts were more common than expected (positive co-occurrence) and had joint spirochete loads that were similar to or greater than the additive expectation (facilitation). Our study suggests that the vertebrate host plays an important role in structuring the community of B. burgdorferi sensu lato genospecies inside the tick vector.     

Lyme Disease Borrelia spp. in Ticks and Rodents from Northwestern China

In May 1999, field surveys of Lyme disease spirochetes were conducted around the Tianshan Mountains in Xinjiang Uygur Autonomous Region in northwestern People's Republic of China. Ixodes persulcatus ticks were obtained in a Tianchi Lake valley with primary forest, while the tick fauna was poor in the semidesert or at higher altitudes in this region. Species identities were confirmed by molecular analysis in which an internal transcribed spacer sequence was used. Of 55 adult ticks, 22 (40%) were positive for spirochetes as determined by Barbour-Stoenner-Kelly culture passages. In addition, some rodents, including Apodemus uralensis (5 of 14 animals) and Cricetulus longicaudatus (the only animal examined), and some immature stages of I. persulcatus (4 of 11 ticks) that had fed on A. uralensis were positive for spirochetes. Based on 5S-23S rRNA intergenic spacer restriction fragment length polymorphism analysis and reactivity with monoclonal antibodies, 35 cultures (including double isolation cultures) were identified as Borrelia garinii (20 isolates, including 9 Eurasian pattern B isolates and 11 Asian pattern C isolates), Borrelia afzelii (10 pattern D isolates), and mixed cultures (5 cultures, including isolates that produced B. garinii patterns B and C plus B. afzelii pattern D). These findings revealed that Lyme disease pathogens are distributed in the mountainous areas in northwestern China even though it is an arid region, and they also confirmed the specific relationship between I. persulcatus and genetic patterns of Borrelia spp. on the Asian continent.     

Lyme disease Borrelia spp. in ticks and rodents from northwestern China.

In May 1999, field surveys of Lyme disease spirochetes were conducted around the Tianshan Mountains in Xinjiang Uygur Autonomous Region in northwestern People's Republic of China. Ixodes persulcatus ticks were obtained in a Tianchi Lake valley with primary forest, while the tick fauna was poor in the semidesert or at higher altitudes in this region. Species identities were confirmed by molecular analysis in which an internal transcribed spacer sequence was used. Of 55 adult ticks, 22 (40%) were positive for spirochetes as determined by Barbour-Stoenner-Kelly culture passages. In addition, some rodents, including Apodemus uralensis (5 of 14 animals) and Cricetulus longicaudatus (the only animal examined), and some immature stages of I. persulcatus (4 of 11 ticks) that had fed on A. uralensis were positive for spirochetes. Based on 5S-23S rRNA intergenic spacer restriction fragment length polymorphism analysis and reactivity with monoclonal antibodies, 35 cultures (including double isolation cultures) were identified as Borrelia garinii (20 isolates, including 9 Eurasian pattern B isolates and 11 Asian pattern C isolates), Borrelia afzelii (10 pattern D isolates), and mixed cultures (5 cultures, including isolates that produced B. garinii patterns B and C plus B. afzelii pattern D). These findings revealed that Lyme disease pathogens are distributed in the mountainous areas in northwestern China even though it is an arid region, and they also confirmed the specific relationship between I. persulcatus and genetic patterns of Borrelia spp. on the Asian continent.     

Genetic Variability within Borrelia burgdorferi Sensu Lato Genospecies Established by PCR-Single-Strand Conformation Polymorphism Analysis of the rrfA-rrlB Intergenic Spacer in Ixodes ricinus Ticks from the Czech Republic

In Europe the Borrelia burgdorferi sensu lato complex is represented by five distinct genospecies: Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana, and Borrelia lusitaniae. These taxonomic entities are known to differ in their specific associations with vertebrate hosts and to provoke distinct clinical manifestations in human patients. However, exceptions to these rules have often been observed, indicating that strains belonging to a single genospecies may be more heterogeneous than expected. It is, therefore, important to develop alternative identification tools which are able to distinguish Borrelia strains not only at the specific level but also at the intraspecific level. DNA from a sample of 370 Ixodes ricinus ticks collected in the Czech Republic was analyzed by PCR for the presence of a ~230-bp fragment of the rrfA-rrlB intergenic spacer of Borrelia spp. A total of 20.5% of the ticks were found to be positive. The infecting genospecies were identified by analyzing the amplified products by the restriction fragment length polymorphism (RFLP) method with restriction enzyme MseI and by single-strand conformation polymorphism (SSCP) analysis. The two methods were compared, and PCR-SSCP analysis appeared to be a valuable tool for rapid identification of spirochetes at the intraspecific level, particularly when large samples are examined. Furthermore, by using PCR-SSCP analysis we identified a previously unknown Borrelia genotype, genotype I-77, which would have gone unnoticed if RFLP analysis alone had been used.     

Effect of Prior Exposure to Noninfected Ticks on Susceptibility of Mice to Lyme Disease Spirochetes

To determine whether prior exposure to Nearctic Ixodes vector ticks protects native reservoir mice from tick-borne infection by Lyme disease spirochetes, we compared their infectivities for white-footed mice and laboratory mice that had been repeatedly infested by noninfected deer ticks. Nymphal ticks readily engorged on tick-exposed laboratory mice, but their feeding success on white-footed mice progressively declined. Tick-borne spirochetes readily infected previously tick-infested mice. Thus, prior infestation by Nearctic ticks does not protect sympatric reservoir mice or Palearctic laboratory mice from infection by sympatric tick-borne spirochetes.     

Distribution of cp32 Prophages among Lyme Disease-Causing Spirochetes and Natural Diversity of Their Lipoprotein-Encoding erp Loci

Lyme disease spirochetes possess complex genomes, consisting of a main chromosome and 20 or more smaller replicons. Among those small DNAs are the cp32 elements, a family of prophages that replicate as circular episomes. All complete cp32s contain an erp locus, which encodes surface-exposed proteins. Sequences were compared for all 193 erp alleles carried by 22 different strains of Lyme disease-causing spirochete to investigate their natural diversity and evolutionary histories. These included multiple isolates from a focus where Lyme disease is endemic in the northeastern United States and isolates from across North America and Europe. Bacteria were derived from diseased humans and from vector ticks and included members of 5 different Borrelia genospecies. All erp operon 5′-noncoding regions were found to be highly conserved, as were the initial 70 to 80 bp of all erp open reading frames, traits indicative of a common evolutionary origin. However, the majority of the protein-coding regions are highly diverse, due to numerous intra- and intergenic recombination events. Most erp alleles are chimeras derived from sequences of closely related and distantly related erp sequences and from unknown origins. Since known functions of Erp surface proteins involve interactions with various host tissue components, this diversity may reflect both their multiple functions and the abilities of Lyme disease-causing spirochetes to successfully infect a wide variety of vertebrate host species.     

Why Lyme disease is common in the northern US,but rare in the south: The roles of host choice,host-seeking behavior,and tick density

Lyme disease is common in the northeastern United States, but rare in the southeast, even though the tick vector is found in both regions. Infection prevalence of Lyme spirochetes in host-seeking ticks, an important component to the risk of Lyme disease, is also high in the northeast and northern midwest, but declines sharply in the south. As ticks must acquire Lyme spirochetes from infected vertebrate hosts, the role of wildlife species composition on Lyme disease risk has been a topic of lively academic discussion. We compared tick–vertebrate host interactions using standardized sampling methods among 8 sites scattered throughout the eastern US. Geographical trends in diversity of tick hosts are gradual and do not match the sharp decline in prevalence at southern sites, but tick–host associations show a clear shift from mammals in the north to reptiles in the south. Tick infection prevalence declines north to south largely because of high tick infestation of efficient spirochete reservoir hosts (rodents and shrews) in the north but not in the south. Minimal infestation of small mammals in the south results from strong selective attachment to lizards such as skinks (which are inefficient reservoirs for Lyme spirochetes) in the southern states. Selective host choice, along with latitudinal differences in tick host-seeking behavior and variations in tick densities, explains the geographic pattern of Lyme disease in the eastern US.

Lyme disease is common in the northeastern United States, but rare in the southeast, even though the tick vector is found in both regions. This study shows that this is largely because the tick vectors attach abundantly to rodents (which are good hosts for the Lyme bacteria) in the north, and to lizards (which are relatively poor hosts for Lyme bacteria) in the south.