High Prevalence of Granulocytic Ehrlichiae and Borrelia burgdorferi Sensu Lato in Ixodes ricinus Ticks from Bulgaria

Bulgarian Ixodes ricinus ticks were examined for Ehrlichia and Borrelia coinfection: 34 and 32% of adult ticks and at least 2 and 10% of nymphs were positive for these infections, respectively. Coinfections and dual or triple Borrelia infections were frequent, although Ehrlichia phagocytophila heterogeneity was minimal. Multiple tick-borne bacteria coexist in I. ricinus ticks in southeastern Europe.     

Genetic Heterogeneity of Borrelia burgdorferi Sensu Lato in Ixodes ricinus Ticks Collected in Belgium

Borrelia burgdorferi sensu lato (s.l.), the etiological agent of Lyme disease, is transmitted by the bite of Ixodes ricinus. Four hundred eighty-nine ticks, collected in four locations of a region of southern Belgium where Lyme disease is endemic, were examined for the presence of the spirochete. In a PCR test with primers that recognize a chromosomal gene of all strains, 23% of the ticks were found to be infected. The species B. burgdorferi s.l. comprises at least three pathogenic genomospecies, B. burgdorferi sensu stricto (s.s.), Borrelia garinii, and Borrelia afzelii, which could be distinguished in PCR tests with species-specific primers that correspond to distinct plasmid sequences. B. garinii was most prevalent (53% of infected ticks), followed by B. burgdorferi s.s. (38%) and B. afzelii (9%). Of the infected ticks, 40% were infected with a single species, 40% were infected with two species, and 5% were infected with all three species. For 15% of the ticks, the infecting species could not be identified. No difference in rates of prevalence was observed among the four locations, which had similar ground covers, even though they belonged to distinct biogeographic regions. A greater heterogeneity of spirochetal DNA in ticks than in cultured reference DNA was suggested by a comparison of the results of PCRs with two different sets of species-specific primer sequences.     

Detection and Identification of Ehrlichia, Borrelia burgdorferi Sensu Lato, and Bartonella Species in Dutch Ixodes ricinus Ticks

A sensitive and specific PCR hybridization assay was developed for the simultaneous detection and identification of Ehrlichia and Borrelia burgdorferi sensu lato. In separate assays the 16S rRNA gene of Ehrlichia species and the 23S-5S rRNA spacer region of B. burgdorferi sensu lato were amplified and labeled by PCR. These PCR products were used in a reverse line blot hybridization assay in which oligonucleotide probes are covalently linked to a membrane in parallel lines. Hybridization of the samples with the oligonucleotide probes on this membrane enabled the simultaneous detection and identification of Ehrlichia, B. burgdorferi, and Bartonella species in 40 different samples. The application of the assay to DNA extracts from 121 Ixodes ricinus ticks collected from roe deer demonstrated that 45% of these ticks carried Ehrlichia DNA. More than half of these positive ticks carried species with 16S rRNA gene sequences closely related to those of E. phagocytophila and the human granulocytic ehrlichiosis agent. The majority of the other positive ticks were infected with a newly identified Ehrlichia-like species. In addition, 13% of the ticks were infected with one or more B. burgdorferi genospecies. In more than 70% of the ticks 16S rRNA gene sequences for Bartonella species or other species closely related to Bartonella were found. In five of the ticks both Ehrlichia and B. burgdorferi species were detected.     

Detection and Typing of Borrelia burgdorferi Sensu Lato in Ixodes ricinus Ticks Attached to Human Skin by PCR

Live Ixodes ricinus ticks attached to humans residing in Germany were examined for borreliae by dark-field microscopy and PCR. Borrelia species were identified by 16S rRNA sequence analysis, which showed the presence of several species, some not yet defined, and a high prevalence of multiply infected ticks.     

Prevalence of Borrelia burgdorferi and Granulocytic and Monocytic Ehrlichiae in Ixodes ricinus Ticks from Southern Germany

A total of 287 adult Ixodes ricinus ticks, collected in two regions of southern Germany (Frankonia and Baden-Württemberg) where Borrelia burgdorferi infections are known to be endemic, were examined for the presence of 16S ribosomal DNA specific for the Ehrlichia phagocytophila genogroup, E. chaffeensis, E. canis, and B. burgdorferi by nested PCR. Totals of 2.2% (6 of 275) and 21.8% (65 of 275) of the ticks were positive for the E. phagocytophila genogroup and B. burgdorferi, respectively. Two ticks (0.7%) were coinfected with both bacteria. Of 12 engorged I. ricinus ticks collected from two deer, 8 (67%) were positive for the E. phagocytophila genogroup and one (8%) was positive for B. burgdorferi. There was no evidence of infection with E. canis or E. chaffeensis in the investigated tick population. The nucleotide sequences of the 546-bp Ehrlichia PCR products differed at one or two positions from the original sequence of the human granulocytic ehrlichiosis (HGE) agent (S.-M. Chen, J. S. Dumler, J. S. Bakken, and D. H. Walker, J. Clin. Microbiol. 32:589-595, 1994). Three groups of sequence variants were detected; two of these were known to occur in other areas in Europe or the United States, whereas one has not been reported before. Thus, in the German I. ricinus tick population closely related granulocytic ehrlichiae are prevalent, which might represent variants of E. phagocytophila or the HGE agent.     

Molecular Evidence of Coinfection of Ticks with Borrelia burgdorferi Sensu Lato and the Human Granulocytic Ehrlichiosis Agent in Switzerland

Adult Ixodes ricinus ticks were collected in Switzerland and tested for the presence of coinfection with Borrelia burgdorferi sensu lato and the human granulocytic ehrlichiosis (HGE) agent by real-time PCR. Of 100 ticks, 49% were positive for B. burgdorferi and 2% were positive for the HGE agent. The two HGE agent-positive ticks were also found to be positive for B. burgdorferi.     

Coexistence of ehrlichiae of the phagocytophila group with Borrelia burgdorferi in Ixodes ricinus from Southern Germany

Human granulocytic ehrlichiosis (HGE) is an emerging infectious disease recognized in the Western hemisphere. HGE is well known to occur in North America, but records from outside the United States are sparse. The great majority of data from Europe are restricted to seroprevalence studies and molecular biological detection of granulocytic ehrlichiae (GE) in ticks and mammals, but include defined cases from Slovenia. They argue for the existence of this disease in many parts of Europe. In the present study, 510 Ixodes ricinus ticks collected in five different regions of Southern Germany were investigated for the presence of GE and Borrelia burgdorferi sensu lato using polymerase chain reaction. In all, 8 (1.6%) of the 492 ticks that could be evaluated (193 females, 208 males, and 91 nymphs) contained GE and 178 (36.2%) B. burgdorferi s.l.. Four of these ticks were infected with both pathogens. Interestingly, all ehrlichia-infected ticks were adults and all were collected in the English Garden, a recreational park area located in the city of Munich. Sequencing of the 16S rDNA (bp 1–1101) of four of the GE showed 100% sequence identity to each other and greater than 99.9% identity with the published sequence of the HGE agent. The four GE differed in respect to other hitherto described GE by a nucleotide exchange at position 336. These results show that GE that are closely related to the HGE agent are present in Southern Germany, and that coinfection with B. burgdorferi is common in GE-infected ticks. However, in contrast to B. burgdorferi which is endemic everywhere in Southern Germany, the distribution of GE seems to be focal. Received: 30 July 1999     

Specificity of Infection-Induced Immunity among Borrelia burgdorferi Sensu Lato Species

The specificity of infection-induced immunity in mice infected with cultured or host-adapted Borrelia burgdorferi sensu lato, the agent of Lyme disease, was examined. Sera obtained from mice following infection with high and low doses of cultured B. burgdorferi sensu stricto, transplantation of infected tissue (host-adapted spirochetes), or tick-borne inoculation all showed protective activity in passive immunization assays. Infection and disease were similar in mice infected with cultured spirochetes or by transplantation. Thus, the adaptive form of inoculated spirochetes did not influence the immune response during active infection. Mice infected with B. burgdorferi sensu stricto and then cured of infection with an antibiotic during early or late stages of infection were resistant to challenge with high doses of homologous cultured spirochetes for up to 1 year. In contrast, actively immune mice infected with different Borrelia species (B. burgdorferi sensu lato, B. burgdorferi sensu stricto cN40, Borrelia afzelii PKo, and Borrelia garinii PBi) and then treated with an antibiotic were resistant to challenge with cultured homologous but not heterologous spirochetes. Similar results were achieved for actively immune mice challenged by transplantation and by passive immunization with sera from mice infected with each of the Borrelia species and then challenged with cultured spirochetes. Arthritis and carditis in mice that had immunizing infections with B. afzelii and B. garinii and then challenged by transplantation with B. burgdorferi sensu stricto were equivalent in prevalence and severity to those in nonimmune recipient mice. These results indicate that protective immunity and disease-modulating immunity that develop during active infection are universal among species related to B. burgdorferi sensu lato but are species specific.     

Sensitive Detection of Borrelia burgdorferi Sensu Lato DNA and Differentiation of Borrelia Species by LightCycler PCR

In order to differentiate species within the Borrelia burgdorferi sensu lato complex, LightCyler PCR and melting-curve analysis of the amplicons of two genes with intraspecies variability, the p66 gene and the recA gene, were performed. It was demonstrated that nested LightCycler PCR amplification of p66 is more sensitive in the detection of borrelia DNA than amplification of the recA gene. B. burgdorferi sensu stricto could be differentiated from Borrelia garinii and Borrelia afzelii by melting-curve analysis of the p66 gene amplicon. B. garinii could be differentiated from B. afzelii and B. burgdorferi sensu stricto by melting-curve analysis of the recA gene amplicon. Therefore, the PCRs complement each other in subtyping different Borrelia species, and combined LightCycler PCR and melting-curve analysis of both target genes is a rapid method to distinguish the three species of B. burgdorferi sensu lato.     

Swiss Army Survey in Switzerland to Determine the Prevalence of Francisella tularensis, Members of the Ehrlichia phagocytophila Genogroup, Borrelia burgdorferi Sensu Lato, and Tick-Borne Encephalitis Virus in Ticks

 A total of 6071 Ixodes ricinus ticks were collected on Swiss Army training grounds in five regions of Switzerland. The aim of the survey was to assess the prevalence of ticks infected with the human pathogens Francisella tularensis, members of the Ehrlichia phagocytophila genogroup, Borrelia burgdorferi sensu lato, and the European tick-borne encephalitis virus. TaqMan PCR (PE Biosystems, USA) and TaqMan RT-PCR (PE Biosystems) analyses were performed on DNA and RNA extracted from pools of ten ticks grouped by gender. Here, for the first time, it is shown that ticks may harbor Francisella tularensis in Switzerland, at a rate of 0.12%. Furthermore, 26.54% of the ticks investigated harbored Borrelia burgdorferi sensu lato, 1.18% harbored members of the Ehrlichia phagocytophila genogroup, and 0.32% harbored the European tick-borne encephalitis virus. A new instrumentation was applied in this study to carry out and analyze more than 2300 PCR reactions in only 5 days. Furthermore, the results reveal that people working in outdoor areas, including army personnel on certain training grounds contaminated with ticks containing tick-borne pathogens, are at risk for different tick-borne diseases.     

Specificities and Sensitivities of Four Monoclonal Antibodies for Typing of Borrelia burgdorferi Sensu Lato Isolates

Borrelia burgdorferi, the agent of Lyme borreliosis, is genetically more heterogeneous than previously thought. In Europe five genospecies have been described from the original B. burgdorferi sensu lato (sl): B. burgdorferi sensu stricto (ss), B. garinii, B. afzelii, B. lusitaniae, and B. valaisiana. In the United States, B. burgdorferi ss as well as B. bissettii in California and B. andersonii on the East Coast were differentiated. In Asia, B. japonica has been identified along, with B. garinii, B. afzelii, and B. valaisiana. In order to evaluate sensitivity and specificity of four species-specific monoclonal antibodies, we analyzed 210 B. burgdorferi sl isolates belonging to eight genospecies by immunoblot and confirmed genospecies by restriction fragment length polymorphism (RFLP) of rrf (5S)-rrl (23S) intergenic spacer amplicon. Monoclonal antibody H3TS had 100% sensitivity for 55 B. burgdorferi ss isolates but showed reactivity with all four isolates belonging to B. bissetii. Monoclonal antibody I 17.3 showed 100% specificity and sensitivity for 45 B. afzelii isolates. Monoclonal antibody D6 was 100% specific for B. garinii but missed 1 of 64 isolates (98.5% sensitivity). Monoclonal antibody A116k was 100% specific for B. valaisiana but was unreactive with 4 of 24 isolates (83.5% sensitivity). Genetic analysis correlated well with results of reactivity and confirmed efficacy of the phenotypic typing of these antibodies. Some isolates showed atypical RFLP. Therefore, both phenotypic and genotypic analyses are needed to characterize new Borrelia isolates.     

Characterization of Borrelia burgdorferi isolated from different organs of Ixodes ricinus Ticks collected in nature

Borrelia burgdorferi was isolated from 22 out of 133 adult Ixodes ricinus ticks collected from vegetation at two sites in Switzerland. From 17 ticks, spirochetes could be isolated from more than one organ. When the different isolates obtained from one tick were compared by SDS-PAGE analysis, differences in the protein profiles were observed in 8 cases. The isolates were further compared by immunological methods using mono- and polyclonal antibodies. Differences were observed in the proteins of 31–35 kDA and 18–25 kDa. Genetic divergence among isolates was evaluated by use of a B. burgdorferi specific gene probe for ospA. Correlation could be observed between immunological differences in OspA defined by monoclonal antibody LA31 and genetic variation of ospA as judged by restriction fragment length polymorphism (RFLP). Our findings indicate that systemic infection in unfed I. ricinus adults, as reflected by isolation of B. burgdorferi from multiple organs of one tick, is more frequent (8/22, 36%) than previously described (5%). Moreover, the presence of different B. burgdorferi phenotypes/genotypes in one tick is described for the first time. The findings may have bearings (i) on the time of tick attachment required for spirochete transmission since borreliae are already present in the salivary glands of systemically infected ticks at the beginning of the blood meal and (ii) perhaps also on the diversity of B. burgdorferi phenotypes inoculated by these ticks.     

Prevalence of Borrelia burgdorferi in Ixodes ricinus ticks in urban recreational areas of Helsinki

Lyme borreliosis, an infection caused by the tick-borne spirochete Borrelia burgdorferi, is a major health problem for populations in areas of endemicity in the Northern Hemisphere. In the present study we assessed the density of ticks and the prevalence of B. burgdorferi sensu lato among ticks in popular urban recreational areas of Helsinki, Finland. Altogether 1,688 Ixodes ricinus ticks were collected from five areas located within 5 km of the downtown section of Helsinki, and 726 of them (303 nymphs, 189 females, and 234 males) were randomly chosen for laboratory analysis. The midguts of the ticks were divided into three pieces, one for dark-field microscopy, one for cultivation in BSK-II medium, and one for PCR analysis. Ticks were found in all the study areas; their densities varied from 1 to 36 per 100 m along which a cloth was dragged. The rate of tick infection with B. burgdorferi sensu lato varied from 19 to 55%, with the average being 32%. Borellia afzelii was the most predominant genospecies in all the areas, and no B. burgdorferi sensu stricto isolates were detected. Only two ticks were concurrently infected with both B. afzelii and Borrelia garinii. Dark-field microscopy gave more positive results for B. burgdorferi than did cultivation or PCR analysis. However, the agreement between all three methods was fairly good. We conclude that Lyme borreliosis can be contracted even in urban environments not populated with large mammals like deer or elk. The disease should be taken into account in the differential diagnosis of certain symptoms of patients from these areas, and the use of measures to improve the awareness of the general population and health care officials of the risk of contracting the disease is warranted.     

Prevalence rates of Borrelia burgdorferi sensu lato in host-seeking Ixodes ricinus ticks in Europe

Borrelia burgdorferi sensu lato spirochetes have been found in all examined Ixodes ricinus (L.) populations in Europe. The overall mean proportions of unfed I. ricinus infected with B. burgdorferi s.l. were 1.9% (range 0–11%), 10.8% (2–43%) and 17.4% (3–58%) for larvae (n = 5699), nymphs (n = 48 804) and adults (n = 41 666), respectively. However, the results varied according to the method used. Cultivation in BSK medium is the least sensitive technique (an average of 11% adult ticks found infected), whereas polymerase chain reaction detecting spirochetal DNA is probably the most sensitive method (29% adults found infected). Microscopic methods (dark field, phase contrast, direct or indirect fluorescence) are generally comparable to each other (17–20% adults found infected) and should be regarded as standard procedures because they also make possible a quantitative estimation of spirochetes in the vector. Some technical problems of these methods are discussed. Received: 18 August 1997 / Accepted: 12 September 1997     

Prevalence of Borrelia burgdorferi, Bartonella spp., Babesia microti, and Anaplasma phagocytophila in Ixodes scapularis ticks collected in Northern New Jersey

PCR analysis of Ixodes scapularis ticks collected in New Jersey identified infections with Borrelia burgdorferi (33.6%), Babesia microti (8.4%), Anaplasma phagocytophila (1.9%), and Bartonella spp. (34.5%). The I. scapularis tick is a potential pathogen vector that can cause coinfection and contribute to the variety of clinical responses noted in some tick-borne disease patients.     

Delineation of a New Species of the Borrelia burgdorferi Sensu Lato Complex,Borrelia americana sp. nov.

Analysis of borrelia isolates collected from ticks, birds, and rodents from the southeastern United States revealed the presence of well-established populations of Borrelia burgdorferi sensu stricto, Borrelia bissettii, Borrelia carolinensis, and Borrelia sp. nov. Multilocus sequence analysis of five genomic loci from seven samples representing Borrelia sp. nov. isolated from nymphal Ixodes minor collected in South Carolina showed their close relatedness to California strains known as genomospecies 1 and separation from any other known species of the B. burgdorferi sensu lato complex. One nucleotide difference in the size of the 5S-23S intergenic spacer region, one substitution in 16S rRNA gene signature nucleotides, and silent nucleotide substitutions in sequences of the gene encoding flagellin and the gene p66 clearly separate Borrelia sp. nov. isolates from South Carolina into two subgroups. The sequences of isolates of each subgroup share the same restriction fragment length polymorphism patterns of the 5S-23S intergenic spacer region and contain unique signature nucleotides in the 16S rRNA gene. We propose that seven Borrelia sp. nov. isolates from South Carolina and two California isolates designated as genomospecies 1 comprise a single species, which we name Borrelia americana sp. nov. The currently recognized geographic distribution of B. americana is South Carolina and California. All strains are associated with Ixodes pacificus or Ixodes minor and their rodent and bird hosts.Spirochetes of the Borrelia burgdorferi sensu lato complex parasitize vertebrates and are transmitted by hard-bodied ticks (Ixodidae) throughout the temperate zones of the northern hemisphere (48). Four species of Ixodes, Ixodes scapularis, Ixodes pacificus, Ixodes ricinus, and Ixodes persulcatus, account for the majority of Ixodes species-vectored human disease. Certain Ixodes ticks are host specific, whereas others are not. Those with nonspecific feeding habits, (e.g., I. scapularis, I. pacificus, I. ricinus, and I. persulcatus) not only feed on species that are competent reservoirs for multiple tick-borne pathogens but also readily bite humans. The list of potential reservoir hosts is great and variable and includes species in classes Mammalia, Aves, and Reptilia (9, 15). The worldwide distribution of B. burgdorferi sensu lato may be caused by long-distance dispersal of infected birds that serve as hosts for ticks (11, 46, 53).The blacklegged tick, I. scapularis, is the main vector of B. burgdorferi sensu lato for humans in the eastern half of the United States, both in the northeastern and southern parts, while I. pacificus is the main vector in the far-western part of the United States. Other species of Ixodes known to be naturally infected and to transmit B. burgdorferi among wildlife include Ixodes spinipalpis (formerly Ixodes neotomae) (38), Ixodes jellisoni (25), and Ixodes angustus (47) in the western United States and Ixodes dentatus, Ixodes affinis, and Ixodes minor in the eastern part of the country (39). The non-human-biting tick I. spinipalpis serves as a maintenance vector, and I. pacificus serves as the “bridge” vector for humans. In some areas of the southeastern United States, I. minor (which usually does not bite humans) appears to be more important as a maintenance vector in the enzootic cycle of B. burgdorferi sensu lato than the “bridge” vector I. scapularis, which feeds on nonhuman species and humans. The role of I. minor and I. affinis and several species of birds in the enzootic cycle of B. burgdorferi in the southern United States is currently under investigation (J. H. Oliver, Jr., unpublished data).Data generated during the last decade demand reevaluation of the previously held concepts about Lyme borreliosis in the United States, particularly in the western and southern United States. Recent results confirm the presence of well-established populations of B. burgdorferi sensu stricto, Borrelia andersonii, Borrelia bissettii, and the recently described Borrelia carolinensis in the southern part of the country (29-32, 39-45, 58). Now we present data that support delineation of another species from the B. burgdorferi sensu lato complex, Borrelia americana sp. nov. B. americana includes American strains isolated in California from 1989 to 1991 that were designated as genomospecies 1 in 2007 and South Carolina strains isolated from 1994 to 1995 and identified as Borrelia sp. nov. in 2007. The enzootiology of Lyme disease in California differs fundamentally from that reported in the northeastern United States but is quite similar to the enzootiology of Lyme borreliosis in the southeastern region of the United States. The explanation for the presence of identical strains of the same species, B. americana sp. nov., on opposite sides of the United States (California and South Carolina) is an enigma worthy of further investigation.     

Common Ancestry of Borrelia burgdorferi Sensu Lato Strains from North America and Europe

Ten atypical European Borrelia burgdorferi sensu lato (Borrelia spp. ) strains were genetically characterized, and the diversity was compared to that encountered among related Borrelia spp. from North America. Phylogenetic analyses of a limited region of the genome and of the whole genome extend existing knowledge about borrelial diversity reported earlier in Europe and the United States. Our results accord with the evidence that North American and European strains may have a common ancestry.     

Characterization of Borrelia burgdorferi sensu lato strains isolated from Ixodes ricinus in Mecklenburg-Vorpommern,Germany

Epidemiological studies in Mecklenburg-Vorpommern have shown a high prevalence ofBorrelia burgdorferi-infected ticks. A total of 17B. burgdorferi sensu lato strains were isolated from ticks and investigated by Western blots (immunoblot) with eight monoclonal antibodies against different epitopes of the outer surface protein A (OspA). Except for one, all strains could be classified using this system. The majority of strains belonged to theB. garinii-associated OspA serotypes 3, 5 and 6. Three isolates were classified as OspA serotype 2 (B. afzelii).B. burgdorferi sensu stricto strains (Ospa serotype 1) as well asB. garinii-associated OspA serotype 4 were not present.     

Detection and identification of Anaplasma phagocytophilum, Borrelia burgdorferi, and Rickettsia helvetica in Danish Ixodes ricinus ticks

Borreliosis is an endemic infection in Denmark. Recent serosurveys have indicated that human anaplasmosis may be equally common. The aim of this study was to look for Anaplasma phagocytophilum and related pathogens in Ixodes ricinus ticks and estimate their prevalence, compared to Borrelia, using PCR. Ticks were collected from three locations in Denmark: Jutland, Funen, and Bornholm. Ticks from Jutland and Funen were analysed individually, ticks from Bornholm were analysed in pools of 20. A. phagocytophilum was found in ticks from all areas. A. phagocytophilum was found in 23.6% of ticks from Jutland and Funen, while 11% were positive for Borrelia burgdorferi. The Borrelia genotype B. afzelii was most prevalent, followed by B. valaisiana, B. burgdorferi s.s. and B. garinii.A. phagocytophilum was found in 14.5% of nymphs and 40.5% of adult ticks, while Borrelia was found in 13% of nymphs and 8% of adult ticks. The difference in prevalence between Anaplasma and Borrelia in adult ticks supports the idea that their maintenance cycles in nature may be different. Ticks were also infected with Rickettsia helvetica. Our study indicates that A. phagocytophilum prevalence in ticks in Denmark is as high as Borrelia prevalence and that human anaplasmosis may be unrecognized.     

Species-Specific Plasmid Sequences for PCR Identification of the Three Species of Borrelia burgdorferi Sensu Lato Involved in Lyme Disease

Species-specific sequences were shown to be carried by plasmids of the three main species of Borrelia burgdorferi sensu lato involved in Lyme disease. Libraries of the 16-, 33-, and 25-kb plasmids of B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii, respectively, were then built and used to isolate species-specific sequences. After sequencing of the cloned inserts, three sets of primers were designed. They were shown to determine species-specific PCR amplification products. The sensitivities of the PCR assay with these primers were 100 spirochetes for B. burgdorferi sensu stricto and 1,000 spirochetes for B. garinii and B. afzelii. The usefulness of these primers for the identification of species in biological samples (tick, serum, and cerebrospinal fluid samples) was ascertained.