Risk indicators for the tick Ixodes ricinus and Borrelia burgdorferi sensu lato in Sweden

The distributional area of the tick Ixodes ricinus (L.), the primary European vector to humans of Lyme borreliosis spirochaetes (Borrelia burgdorferi sensu lato) and tick‐borne encephalitis virus, appears to be increasing in Sweden. It is therefore important to determine which environmental factors are most useful to assess risk of human exposure to this tick and its associated pathogens. The geographical distribution of I. ricinus in Sweden was analysed with respect to vegetation zones and climate. The northern limit of I. ricinus and B. burgdorferi s.l. in Sweden corresponds roughly to the northern limit of the southern boreal vegetation zone, and is characterized climatically by snow cover for a mean duration of 150 days and a vegetation period averaging 170 days. The zoogeographical distribution of I. ricinus in Sweden can be classified as southerly–central, with the centre of the distribution south of the Limes Norrlandicus. Ixodes ricinus nymphs from 13 localities in different parts of Sweden were examined for the presence of B. burgdorferi s.l. and found to be infected with Borrelia afzelii and Borrelia garinii. Tick sampling localities were characterized on the basis of the density of Borrelia‐infected I. ricinus nymphs, presence of specific mammals, dominant vegetation and climate. Densities of I. ricinus nymphs and Borrelia‐infected nymphs were significantly correlated, and nymphal density can thus serve as a general indicator of risk for exposure to Lyme borreliosis spirochaetes. Analysis of data from this and other studies suggests that high densities of Borrelia‐infected nymphs typically occur in coastal, broadleaf vegetation and in mixed deciduous/spruce vegetation in southern Sweden. Ixodes ricinus populations consistently infected with B. burgdorferi s.l. can occur in: (a) biotopes with shrews, rodents, hares and birds; (b) biotopes with shrews, rodents, hares, deer and birds, and (c) island locations where the varying hare (Lepus timidus) is the only mammalian tick host.     

Rapid identification of Borrelia burgdorferi sensu lato by PCR-restriction fragment length polymorphism analysis of groEL gene

Thirty-one reference strains and 23 Korean isolates of the genus Borrelia were identified through the PCR-RFLP analysis using the groEL gene. This will be useful for the rapid differentiation of B. burgdorferi sensu lato and complements one of the 5S-23S intergenic spacers.     

Borrelia burgdorferi sensu lato and co‐infections with Anaplasma phagocytophilum and Rickettsia spp. in Ixodes ricinus in Hamburg,Germany

To obtain initial data on Borrelia burgdorferi sensu lato (Spirochaetales: Spirochaetaceae) in Ixodes ricinus (Ixodida: Ixodidae) ticks in Hamburg, Germany, 1400 questing ticks were collected by flagging at 10 different public recreation areas in 2011 and analysed using probe‐based quantitative real‐time polymerase chain reaction. The overall rate of infection with B. burgdorferi s.l. was 34.1%; 30.0% of adults were infected (36.7% of females and 26.0% of males), as were 34.5% of nymphs. Significant differences in tick infection rates were observed between the spring and summer/autumn months, as well as among sampling locations. Borrelia genospecies identification by reverse line blotting was successful in 43.6% of positive tick samples. The most frequent genospecies was Borrelia garinii/Borrelia bavariensis, followed by Borrelia afzelii, Borrelia valaisiana, B. burgdorferi sensu stricto, Borrelia spielmanii, Borrelia bissettii and Borrelia lusitaniae. Based on previously published data, co‐infection of Borrelia and Rickettsiales spp. was determined in 25.8% of ticks. Overall, 22.9% of ticks were co‐infected with Rickettsia spp. (Rickettsiales: Rickettsiaceae), 1.7% with Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), and 1.2% with both pathogens. Study results show a high prevalence of Borrelia‐positive ticks in recreation areas in the northern German city of Hamburg and the potential health risk to humans in these areas should not be underestimated.     

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 .     

Detection and typing of Borrelia burgdorferi sensu lato genospecies in Ixodes persulcatus ticks in West Siberia, Russia

The prevalence of Borrelia burgdorferi sensu lato (s.l.) genospecies in West Siberia as well as in many other regions of Russia remains insufficiently investigated. In the present study a total of 151 adult female ticks Ixodes persulcatus Schulze, collected at three localities in eastern regions of West Siberia, where Lyme disease is endemic, were examined for the presence of the spirochete B. burgdorferi s.l. by polymerase chain reaction targeting the 23S-5S rRNA intergenic spacer regions. Spirochetal DNA was detected in on average 15.2+/-3.0% of the ticks examined. The infection rate of adult ticks with B. burgdorferi s.l. at various localities ranged from 8.6+/-3.4% to 29.0+/-7.6%, being greatest in the northernmost site studied and decreasing southwards. The restriction patterns obtained after MseI digestion of the 23S-5S rRNA intergenic spacer amplicons assigned 23 DNA samples to the following genomic groups: 19 to B. garinii (12 to group NT29 and seven to group 20047(T)), three to B. afzelii, and one to mixed B. afzelii and B. garinii NT29. We have not detected other genospecies, which were found in ticks in Europe, the Russian Far East and Japan. Thus, the ticks examined were associated only with two genospecies of Borrelia burgdorferi s.l. pathogenic to humans (B. garinii and B. afzelii), and B. garinii was the major genospecies infecting adult I. persulcatus in eastern regions of West Siberia.     

Sequence diversity of a gene encoding a putative primary sigma factor among Borrelia burgdorferi sensu lato strains

Twenty-six strains of Borrelia burgdorferi sensu lato were subjected to polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis for assessing the sequence divergence of rpoD gene encoding the primary sigma factor. Four and five RFLP patterns were observed from two fragments of rpoD gene. Sequence analysis of a subgenic fragment covering region 1 through 4 from 13 strains of Borrelia burgdorferi s. l. revealed that 21 of 450 deduced amino acid residues were diverged. These results indicate that the sequence heterogeneity of rpoD is present in different strains of Borrelia burgdorferi s. l., and agreed well with the current classification of genospecies.     

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.     

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.     

An alternative real-time PCR method for the detection of thermotolerant Bacillus sensu lato contaminants in naturally-contaminated gelatine

Aims:  Comparison of an internally-controlled real-time PCR assay with the current plate-based assay for the detection of Bacillus sensu lato contaminants in gelatine.
Methods and Results:  A comprehensive TaqMan^® probe was designed allowing the real-time PCR assay to be fully inclusive for the gelatine-contaminating Bacillus s.l. species. An internal amplification control was implemented at 500 copies per reaction without impact on target detection. Specific and selective detection of target cells was achieved with a quick and simple DNA preparation procedure. No significant difference (Kappa value = 0·94) was observed between the performance of the real-time PCR and the current plate-based method on naturally contaminated gelatines ( n  = 162). Relative accuracy, relative sensitivity and relative specificity were 97·5%.
Conclusions:  The real-time PCR assay is an adequate alternative of the current plate-based assay.
Significance and Impact of the Study:  The real-time PCR assay decreased the time between sample collection and result from 2 days to 2 h. The gelatine-producing industry can ensure gelatine quality in a much faster way.     

Detection of Borrelia burgdorferi and Anaplasma phagocytophilum in the black‐legged tick,Ixodes scapularis,within southwestern Pennsylvania

Prevalence studies of Borrelia burgdorferi and Anaplasma phagocytophilum have been rare for ticks from southwestern Pennsylvania. We collected 325 Ixodes scapularis ticks between 2011 and 2012 from four counties in southwestern Pennsylvania. We tested for the presence of Borrelia burgdorferi and Anaplasma phagocytophilum using PCR. Of the ticks collected from Pennsylvania, B. burgdorferi (causative agent of Lyme disease) was present in 114/325 (35%) and Anaplasma phagocytophilum (causative agent of Human Granulocytic Anaplasmosis) was present in 48/325 (15%) as determined by PCR analysis.     

IgM and IgG significant reactivity to Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii among Italian patients affected by Lyme arthritis or neuroborreliosis

Abstract This survey evaluates the specificity of band patterns in immunoblot of sera taken from clinically defined cases of Lyme arthritis and neuroborreliosis, towards three locally isolated strains of Borrelia burgdorferi , belonging to the three species: Borrelia sensu stricto, Borrelia garinii and Borrelia afzelii . To assess specificity, patient sera were statistically ( χ ², P ≤ 0.05) compared with blood donors sera samples. Both IgG and IgM antibodies were considered. The overall reactivity of the three Borrelia strains in IgG immunoblots indicated that ten protein bands were significant, with a different prevalence of some of them in the two groups of patient sera: bands at 60-58, 30–33, 36–37 and 28-27 kDa were markers for neuroborreliosis sera; proteins at 100-83, 72-70 and 18-17 kDa behaved like markers for Lyme arthritis. The IgM Immunoblots revealed significant bands at 100-83, 72-70, 51, 24-21 and 18-17 kDa only with neuroborreliosis sera. Though there were variable band reactivities in each strain, a correlation emerged between the three genospecies and the clinical symptoms: in fact B. afzelii and B. garinii were prevalent in Lyme arthritis sera, (IgG Immunoblots); B. garinii was associated to neuroborreliosis (IgG and IgM Immunoblots); B. sensu stricto was strongly reactive with neuroborreliosis in IgM immunoblots. These data indicate that the three locally isolated strains of Borrelia representing the three genospecies should be used together in immunoblot to detect antibodies elicited in neuroborreliosis and Lyme arthritis.     

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.     

Biopsy of canine ear tissue for detection of Borrelia burgdorferi following Ixodes dammini feeding

A punch biopsy method was developed to detect and cultivate Borrelia burgdorferi from canine ear tissue. The biopsy procedure was sensitive in that spirochetes were detected in 100% of the dogs upon which infected ticks had fed. B. burgdorferi could be cultured as early as 6 days after tick feeding at which time the organisms could not be concurrently recovered from peripheral blood. The ear punch biopsy provides a reliable method for sampling live canines and should prove useful for detecting early B. burgdorferi infection and for monitoring duration of non-disseminated infection. Additionally, the procedure will aid in the development of an animal model for evaluation of preventive and therapeutic treatment modalities.     

Organization of Ribosomal RNA Genes in Borrelia burgdorferi sensu lato Isolated from Ixodes ovatus in Japan

Borrelia burgdorferi sensu lato was obtained from adult ixodid ticks, Ixodes ovatus, collected in Nagano, Japan, and was named NT112. The genomic DNA was digested with enzymes, electrophoresed, blotted and hybridized with rRNA gene probes obtained from B. burgdorferi sensu stricto B31. The results showed that the borrelial chromosome contains a single rrs (16S rRNA gene) sequence and two copies of rrl/rrf (23S/5S rRNA genes) sequences. The rrl/rrf genes were tandemly repeated at intervals of 3.2 kb and were located separately from the rrs gene on the genome. Our findings indicate that the organization of rRNA genes in Borrelia from I. ovatus ticks is identical to that of B. burgdorferi sensu stricto.     

N-terminal amino acid sequence of the Borrelia burgdorferi flagellin

Abstract The 41 kDa flagellar protein of Borrelia burgdorferi appears to be an immunodominant antigen producing an early and strong response in most, if not all, individuals during infection in humans. It would represent a very good antigen for serodiagnosis of Lyme disease, if its crossreactivity with flagella of other bacteria was low. To gain information on this point we isolated the B. burgdorferi flagellin by preparative two-dimensional electrophoresis for N-terminal amino acid analysis. By comparing the N-terminal amino acid sequences of flagellar proteins from other eubacteria we found that the first six out of twenty nine amino acids were identical to the Treponema pallidum and Treponema phagedenis 'class B' flagellins. All 29 N-terminal residues exhibited a moderate inter-genus homology (44–55%), in contrast to the high degree (67–95%) of inter-species conservation of the treponemal 'class B' flagellar N-terminal sequences. There was little similarity to other flagellins except the B. subtilis flagellar protein.