Borrelia burgdorferi infection in Ixodes ricinus from habitats in Denmark

A total of 2647 ticks of the genus Ixodes was sampled by flagging the vegetation in thirty-one sites in eastern Jutland, Denmark. All ticks were identified as Ixodes ricinus Linnaeus. A total of 317 ticks (202 nymphs and 115 adults) from three different sites were examined for the spirochaete Borrelia burgdorferi Johnson et al. by indirect fluorescent antibody staining; the frequency of infection varied from 7% to 22%. It is concluded that I. ricinus, known to be the most common tick in Denmark, is the vector largely responsible for the transmission of B. burgdorferi in this country.     

Problems of isolating Borrelia burgdorferi from ticks collected in United Kingdom foci of Lyme disease

Abstract. Many isolates of Borrelia burgdorferi have been obtained from ticks and vertebrate tissues collected in North America and continental Europe but only one established culture of United Kingdom Borrelia burgdorferi has been recorded. In this paper we report the isolation of B.burgdorferi from one of 108 tick pools representing 733 ticks and eighty-four tissue samples from twenty-six rodents collected in the U.K., and the subsequent failure to establish the isolate (from ticks collected in Fordingbridge) in culture. In contrast, using identical techniques and culture medium, B.burgdorferi was isolated from one of seven tick pools collected in Switzerland, and from a single pool of ticks collected in Slovakia, and both isolates were successfully passaged. Analysis of questing I.ricinus collected from Fordingbridge by direct immunofluorescence showed 6/32 (19%) of adults and 8/108 (7%) of nymphs were positive for B. burgdorferi , although only one nymph contained ≥ 1000 spirochaetes. To examine further the problem of isolating U.K. B.burgdorferi , twelve Ixodes ricinus tick samples from Fordingbridge, a recognized focus of Lyme disease, were subjected to isolation and culturing techniques, and the procedures monitored by use of the polymerase chain reaction (PCR). Whereas 11/12 samples were PCR positive after 2 weeks in culture, only one was PCR positive after 4 weeks. Motile spirochaetes were not visible by dark-field microscopy in any of the cultures. The results indicate that the standard BSK II medium routinely used to isolate and culture B. burgdorferi does not readily support the replication of the Borrelia species endemic to the U.K.     

Transmission cycles of Borrelia burgdorferi and B. bissettii in relation to habitat type in northwestern California

This study was undertaken to determine which rodent species serve as primary reservoirs for the Lyme disease spirochete Borrelia burgdorferi in commonly occurring woodland types in inland areas of northwestern California, and to examine whether chaparral or grassland serve as source habitats for dispersal of B. burgdorferi‐ or B. bissettii‐infected rodents into adjacent woodlands. The western gray squirrel (Sciurus griseus) was commonly infected with B. burgdorferi in oak woodlands, whereas examination of 30 dusky‐footed woodrats (Neotoma fuscipes) and 280 Peromyscus spp. mice from 13 widely‐spaced Mendocino County woodlands during 2002 and 2003 yielded only one infected woodrat and one infected deer mouse (P. maniculatus). These data suggest that western gray squirrels account for the majority of production by rodents of fed Ixodes pacificus larvae infected with B. burgdorferi in the woodlands sampled. Infections with B. burgdorferi also were rare in woodrats (0/47, 0/3) and mice (3/66, 1/6) captured in chaparral and grassland, respectively, and therefore these habitats are unlikely sources for dispersal of this spirochete into adjacent woodlands. On the other hand, B. bissettii was commonly detected in both woodrats (22/47) and mice (15/66) in chaparral. We conclude that the data from this and previous studies in northwestern California are suggestive of a pattern where inland oak‐woodland habitats harbor a B. burgdorferi transmission cycle driven primarily by I. pacificus and western gray squirrels, whereas chaparral habitats contain a B. bissettii transmission cycle perpetuated largely by I. spinipalpis, woodrats, and Peromyscus mice. The dominant role of western gray squirrels as reservoirs of B. burgdorferi in certain woodlands offers intriguing opportunities for preventing Lyme disease by targeting these animals by means of either host‐targeted acaricides or oral vaccination against B. burgdorferi.     

Tick infection rates with Borrelia Ixodes ricinus versus Haemaphysalis concinna and Dermacentor reticulatus in two locations in eastern Germany

Unfed nymphal Ixodes ricinus, Haemaphysalis concinna, and adult Dermacentor reticulatus were collected in two locations of Saxony in July and September 1991 by flagging. In July, the abundance of nymphal I. ricinus was about 2-3 times higher than that of nymphal H. concinna, a time of the year when nymphs of both species are reported to have a seasonal peak of activity. No D. reticulatus were flagged concurrently. In September, host-seeking activity of nymphal I. ricinus was again quite high as was that of adult D. reticulatus but only low numbers of nymphal H. concinna were collected. The flagged ticks were individually examined for Borrelia by an indirect immunofluorescence assay (I. ricinus: n = 414; H. concinna: n = 96; D. reticulatus: n = 116). The prevalence of Borrelia (probably B. burgdorferi) in I. ricinus varied from 12.1% to 21.0%. No borreliae were found in H. concinna. Of the examined D. reticulatus from one site (n = 97) 11.3% contained either B. burgdorferi or a related Borrelia. This may be the first finding of Borrelia in an Eurasian Dermacentor species.     

A murine IgG1 monoclonal antibody thatbinds specifically to outer surface protein A of Lyme disease spirochete Borrelia burgdorferi

Abstract A murine monoclonal antibody, designated MA-2G9, directed against outer surface protein A (OspA) of the Lyme disease spirochete, Borrelia burgdorferi , has been produced. Antibody MA-2G9, IgG1 subclass, was purified by affinity chromatography on protein G Sepharose column and used for purification of OspA antigen from Borrelia burgdorferi cell lysate. Epitope specificity was studied by Western immunoblotting, using several strains of B. burgdorferi and non-Lyme disease bacteria such as Treponema pallidum and B. hermsii . The MA-2G9 monoclonal antibody reacted specifically with recombinant OspA aas well as with native OspA in sonicated B. burgdorferi strains. No reaction was observed with T. pallidum, Escherichia coli, Staphylococcus aureus and B. hermsii lysates. The MA-2G9 antibody also recognized the denatured form of OspA indicating that it is directed against sequential epitope and not conformational epitope.     

Absence of spirochaetes (Borrelia burgdorferi) and piroplasms (Babesia microti) in deer ticks (Ixodes dammini) parasitized by chalcid wasps (Hunterellus hookeri)

An entomophagous wasp (Hunterellus hookeri Howard) parasitizes about a third of the host-seeking nymphal Ixodes dammini Spielman et al. ticks on Naushon Island in Massachusetts (U.S.A.) where the agents of Lyme disease (Borrelia burgdorferi Johnson et al.) and human babesiosis (Babesia microti Franca) are enzootic. Following blood-feeding, wasp-parasitized ticks are destroyed by the developing wasp. The prevalence of either human pathogen in host-seeking ticks collected in wasp-infested sites is nearly 40% lower than that found in other sites. Nymphal ticks, collected early in their season of activity, are more frequently parasitized by the wasp and less frequently by the Lyme disease spirochaete than those collected later in the summer. Spirochaetes never infected wasp-infected ticks, and few wasp-infected ticks were concurrently infected by the Babesia piroplasm. Taken together, these correlations indicate that the wasp may render the tick inhospitable to both pathogens. The presence of the wasp may have reduced risk of human infection on the island by either pathogen by as much as a third.     

Multiplex PCR as a tool for validating plasmid content of Borrelia burgdorferi

Borrelia burgdorferi has an unusual genomic structure containing 21 plasmids. These plasmids carry genes that are essential for infectivity and survival of the spirochetes in vivo. Several plasmids are lost during cultivation in vitro, which might lead to a heterogeneous population after multiple passages and loss of infectivity in laboratory animals. Herein, we present a simple and inexpensive multiplex PCR method that detects the complete plasmid profile of B. burgdorferi B31 in just two PCR tubes.     

Adaptation of Borrelia burgdorferi in the tick and the mammalian host

Borrelia burgdorferi, the causative agent of Lyme disease, shows a great ability to adapt to different environments, including the arthropod vector, and the mammalian host. The success of these microorganisms to survive in nature and complete their enzootic cycle depends on the regulation of genes that are essential to their survival in the different environments. This review describes the current knowledge of gene expression by B. burgdorferi in the tick and the mammalian host. The functions of the differentially regulated gene products as well as the factors that influence their expression are discussed. A thorough understanding of the changes in gene expression and the function of the differentially expressed antigens during the life cycle of the spirochete will allow a better control of this prevalent infection and the design of new, second generation vaccines to prevent infection with the spirochete.     

Coinfection with Borrelia burgdorferi sensu stricto and Borrelia garinii alters the course of murine Lyme borreliosis

Ixodes ricinus ticks and mice can be infected with both Borrelia burgdorferi sensu stricto and Borrelia garinii. The effect of coinfection with these two Borrelia species on the development of murine Lyme borreliosis is unknown. Therefore, we investigated whether coinfection with the nonarthritogenic B. garinii strain PBi and the arthritogenic B. burgdorferi sensu stricto strain B31 alters murine Lyme borreliosis. Mice simultaneously infected with PBi and B31 showed significantly more paw swelling and arthritis, long-standing spirochetemia, and higher numbers of B31 spirochetes than did mice infected with B31 alone. However, the number of PBi spirochetes was significantly lower in coinfected mice than in mice infected with PBi alone. In conclusion, simultaneous infection with B. garinii and B. burgdorferi sensu stricto results in more severe Lyme borreliosis. Moreover, we suggest that competition of the two Borrelia species within the reservoir host could have led to preferential maintenance, and a rising prevalence, of B. burgdorferi sensu stricto in European I. ricinus populations.     

Detection of Lyme disease spirochete,Borrelia burgdorferi sensu lato,including three novel genotypes in ticks (Acari: Ixodidae) collected from songbirds (Passeriformes) across Canada

Lyme disease is reported across Canada, but pinpointing the source of infection has been problematic. In this three‐year, bird‐tick‐pathogen study (2004–2006), 366 ticks representing 12 species were collected from 151 songbirds (31 passerine species/subspecies) at 16 locations Canada‐wide. Of the 167 ticks/pools tested, 19 (11.4%) were infected with Borrelia burgdorferi sensu lato (s.l.). Sequencing of the rrf‐rrl intergenic spacer gene revealed four Borrelia genotypes: B. burgdorferi sensu stricto (s.s.) and three novel genotypes (BC genotype 1, BC genotype 2, BC genotype 3). All four genotypes were detected in spirochete‐infected Ixodes auritulus (females, nymphs, larvae) suggesting this tick species is a vector for B. burgdorferi s.l. We provide first‐time records for: ticks in the Yukon (north of 60° latitude), northernmost collection of Amblyomma americanum in North America, and Amblyomma imitator in Canada. First reports of bird‐derived ticks infected with B. burgdorferi s.l. include: live culture of spirochetes from Ixodes pacificus (nymph) plus detection in I. auritulus nymphs, Ixodes scapularis in New Brunswick, and an I. scapularis larva in Canada. We provide the first account of B. burgdorferi s. l. in an Ixodes muris tick collected from a songbird anywhere. Congruent with previous data for the American Robin, we suggest that the Common Yellowthroat, Golden‐crowned Sparrow, Song Sparrow, and Swainson's Thrush are reservoir‐competent hosts. Song Sparrows, the predominant hosts, were parasitized by I. auritulus harboring all four Borrelia genotypes. Our results show that songbirds import B. burgdorferi s.l.‐infected ticks into Canada. Bird‐feeding I. scapularis subadults were infected with Lyme spirochetes during both spring and fall migration in eastern Canada. Because songbirds disperse millions of infected ticks across Canada, people and domestic animals contract Lyme disease outside of the known and expected range.     

Nucleotide sequence and analysis of the gene in Borrelia burgdorferi encoding the immunogenic P39 antigen

Abstract The P39 antigen is a specific, highly conserved, and immunogenic protein of Lyne disease spirochetes, Borrelia burgdorferi sensu lato. The nucleotide sequence of the gene encoding this protein was determined and found to be the first of two tandemly arranged open reading frames located on the spirochete's chromosome. These two open reading frames were designated bmpA for the gene encoding P39 and bmpB for the gene encoding the putative protein ORF2 encoded by the second open reading frame. The nucleic acid sequence identity for the two open reading frames was 62% while their deduced amino acid sequences were 52% identical. Comparison to sequence data bases demonstrated that the deduced amino acid sequences of both P39 and ORF2 were homologous to TmpC, a putative outer or cytoplasmic membrane lipoprotein of the syphilis spirochete, Treponema pallidum .     

Borrelia Burgdorferi Sensu Lato in Ixodes Ricinus Ticks and Rodents in a Recreational Park in South-Western Ireland

Ixodes ricinus ticks infected with Borrelia burgdorferi sensu lato were numerous on the edges of paths and roads in a recreational park in south-western Ireland. The abundance of ticks at different sites was related to the presence of deer, but a negative relationship was shown between tick abundance and tick infection rates. This is thought to be due to the deposition of large numbers of uninfected ticks by deer, which are apparently not good reservoir hosts of B. burgdorferi s.l. Blood meal analysis only detected deer DNA in uninfected nymphs. Reservoir competent rodents, Apodemus sylvaticus and Clethrionomys glareolus, were abundant at all sites and a high proportion of captured specimens were infested with larval ticks. However, very few rodents were infected with B. burgdorferi s.l. and none of the unfed infected nymphs analysed for the identity of their larval blood meal had fed on rodents. The spirochaetes detected in I. ricinus in the study area may be poorly adapted to rodents or are not transmitted readily because of the absence of nymphal infestation. The majority of spirochaetes in these ticks were apparently acquired from non-rodent hosts, such as birds.     

Lyme disease in transgenic mice expressing the Borrelia burgdorferi flagellin epitope implicated in human neuroborreliosis

Because of an association of human neuroborreliosis with the development of an antibody response against an antigen in neural tissue that cross-reacts with an epitope on the flagellin protein of Borrelia burgdorferi, C3H transgenic mice were created that expressed the flagellin epitope (amino acids 213–224) as a fusion protein with myelin basic protein. The transgenic mice expressed the flagellin epitope selectively in myelinated regions of the nervous system. Both transgenic and non-transgenic mice developed an antibody response to the flagellin epitope during B. burgdorferi infection and both developed arthritis and carditis. However, no lesions were found in the central nervous system of either type of mouse for up to 8 weeks after infection. The data indicate that expression of the flagellin 213–24 epitope in mice does not result in neurologic disease, suggesting that B. burgdorferi flagellin antibodies may not be directly implicated in neuroborreliosis.     

Interstadial variation in the attachment sites of Ixodes ricinus ticks on sheep

The spatial aggregation of ticks feeding on vertebrate hosts has been recognized for some time but, for hosts supporting more than one stage of the tick, observations of interstadial variation in the site of attachment have not previously been quantified. This study showed that all three parasitic stages of Ixodes ricinus ticks feeding on sheep attach most commonly to the hair-covered areas of the head and limbs while few ticks attach to the fleeced region of the body. However, significant differences were observed in the site of attachment of the three feeding stages of the tick. Larvae attached to distal limbs and rostral areas of the head and adult females attached to the proximal areas of the limbs and around the neck and ears, while nymphs attached in locations between the larvae and adults. The importance of the spatial aggregation of the ticks and interstadial variation in their distribution on the host, for the transmission of tick-borne pathogens and the epidemiology of the diseases they cause, is discussed. © Rapid Science Ltd. 1998     

The spatial distribution of Borrelia burgdorferi-infected Ixodes ricinus in the Connemara region of County Galway,Ireland

Studies were carried out in the Connemara area of County Galway in the west of Ireland in order to determine the abundance and distribution of the tick, Ixodes ricinus and the prevalence of its infection with Borrelia burgdorferi. The tick was very abundant locally, in particular when associated with cattle, sheep and enclosed red deer. Large numbers of ticks not only occurred on the pastures, but also on adjacent roadside verges. No infections with B. burgdorferi could be demonstrated when nymphal ticks were sampled from central areas of the pastures, suggesting that livestock and red deer are probably not significant reservoirs of the spirochaete. Small numbers of infected nymphal and adult ticks were associated with hedges, dry stone walls, the margins of woodland adjoining infested pastures and in woodland from which livestock were excluded. Woodmice (Apodemus sylvaticus) were most numerous in such habitats and the majority were infected with B. burgdorferi.     

Dynamics of Borrelia burgdorferi infection in nymphal Ixodes ricinus ticks during feeding

We report the sequential developmental events of Borrelia burgdorferi in histological sections of Ixodes ricinus nymphs before, during and after feeding. During the blood meal a decrease of approximately 50% in the number of infected ticks was recorded (eight out of 76, 11%) in comparison with the infection rate of unfed ticks (12 out of 56, 21%). Spirochetes were detected in tick salivary glands only after 2 days of attachment. From day 3 until drop-off, the number of infected ticks increased to 31% (15 out of 49). A quadratic logistic regression analysis showed that the variation in the number of infected ticks was significant, but only during the blood meal. The drop in the percentage of infected ticks during the first hours following attachment to the host is explained by our observation of spirochetes in the faeces of the ticks. The increase in the infection rate of replete ticks may be due to an uptake of spirochetes from the host skin at the feeding site.     

Animal and human antibodies reactive with the outer surface protein A and B of Borrelia burgdorferi are borreliacidal, in vitro, in the presence of complement

Abstract Polyspecific antibodies present in ascitic fluids of mice (pMIAFs) immunized with whole Borrelia burgdorferi cells exerted borreliacidal activity in vitro when tested with complement and homologous antigen but not with heterologous B. hermsii . Similarly, monospecific mouse antibodies obtained by immunizing mice with purified preparations of outer surface protein A and B of B. burgdorferi were borreliacidal. On the contrary, mouse monospecific antibodies raised against the 41-kDa flagellar protein of B. burgdorferi did not kill borreliae in the presence of complement. A complement-mediated, in vitro, borreliacidal activity was observed in human sera from patients with Lyme disease when antibodies against OspA and/or OspB were detectable in sera by the Western blotting technique. The in vitro borreliacidal activity of human sera was evident after 14 h incubation with live B. burgdorferi spirochaetes and complement, whereas antibodies present in mouse immune ascitic fluids killed borreliae after 1 h incubation.     

Antigens and glycoproteins of larvae, nymphs and adults of the tick Ixodes ricinus

Abstract Protein components of homogenates of unfed larvae and nymphs of Ixodes ricinus (L.), and of ovary, haemolymph, Malpighian tubules, rectal ampulla, fat body, integument, salivary glands and midgut of partially fed adult females were studied for their antigenicity and carbohydrate moieties using immunoblotting and lectin affinity blotting (LAB) techniques. Comparing the individual anti-larval, anti-nymphal and anti-adult immune sera for their capacity to recognize the specific and trans-stadially cross-reactive antigenic proteins, larval feeding induced the most effective humoral response. The majority of immunogens recognized by rabbit anti-tick immune sera are glycoproteins. Most of the glycosylated antigens were modified with N-type glycans; however, O-type glycans were also demonstrated in some antigens. The correlation of the type of glycosylation with antigenicity, and the sharing of common antigenic epitopes by various tissues, are discussed.     

Risk factors in habitats of the tick Ixodes ricinus influencing human exposure to Ehrlichia phagocytophila bacteria

Ixodes ricinus L. (Acari: Ixodida) were sampled during 1996-99 in southern Scotland, on vegetation using cloth drags, on humans by removal from clothing and on roe deer (Capreolus capreolus L.) by searching legs of culled deer. Developmental microclimate was recorded by automatic recorders and questing microclimate by portable instruments during tick collections. Ticks and deer were examined for infection with Ehrlichia phagocytophila bacteria (Rickettsiales) using microscopy and polymerase chain reaction. This pathogen causes tick-borne fever of sheep in Europe and human granulocytic ehrlichiosis in North America, but in Europe human clinical ehrlichiosis due to E. phagocytophila has not been recorded despite serological evidence of exposure. Among three types of habitat, coniferous woodland was most infested with questing ticks (560 ticks/km of drag; mean numbers collected on long trousers: 24.3 larvae, 13.5 nymphs and 0.8 adult ticks/km walked), deciduous woodland had slightly lower infestation (426 ticks/km drag) and upland sheep pasture had much lower infestation (220 ticks/km drag). Of the three main vegetation types, bracken was least infested (360 ticks/km drag), ericas most (430 ticks/km drag) and grassland had intermediate infestation density (413 ticks/km drag). Questing and developmental microclimates were poor predictors of exposure within these habitats, except lower infestation of pastures was attributed to greater illumination there. Collectors who walked a total of 300 km through all habitats (taking 360 h in all seasons), wearing cotton trousers hanging outside rubber boots, were bitten by only four nymphs and 11 larvae of I. ricinus (but no adult ticks). There was a negative correlation between densities of deer and ticks collected, although presence of deer remains a major indicator of exposure. The proportion of infected ticks was fairly uniform at four sites studied. Overall prevalence of E. phagocytophila in I. ricinus was 3.3% in nymphs (40/1203) but only approximately 1.5% in adults of both sexes (although males do not bite). It was estimated that nymphs of I. ricinus gave 4.4% probability of one infected bite/person/year (for occupational exposure during this research) due to presence in all seasons and habitats, their human biting rate of 0.011 nymphs/h or 0.013 nymphs/km and widespread infection with E. phagocytophila. The frequency distribution of intensity of infection in ticks was approximately normal (mean 98 morulae/nymph infected), thus there is a high risk of receiving a high dose from any one infected tick bite.