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1.  Detection and prevalence of Anaplasma phagocytophilum and Rickettsia helvetica in Ixodes ricinus ticks in seven study areas in Sweden 
Parasites & Vectors  2010;3:66.
Tick-borne Anaplasma phagocytophilum and Rickettsia spp. are considered to be emerging human pathogens, but only limited data are available on their occurrence in Sweden. Two real-time PCR assays followed by nested PCR and sequence analysis were carried out to investigate the prevalence of A. phagocytophilum and spotted fever rickettsiae in ticks from seven areas in Sweden.
In 139 pooled samples, representing a total of 1245 Ixodes ricinus ticks (204 larvae, 963 nymphs, 38 males, 40 females), the overall positive mean infection prevalence was 1.3-15.0% for A. phagocytophilum and 1.5-17.3% for R. helvetica. A. phagocytophilum was only detected in nymphs (1.7-19.4%), whereas R. helvetica was detected in all three tick stages. Support for vertical and transstadial transmission was only obtained for R. helvetica. Both agents showed similar infection rates across study areas, although infection rates were greater in coastal areas.
The results show that both pathogens occurred in all seven locations, indicating that they are prevalent in Sweden and should be considered etiological agents in patients recently bitten by ticks.
PMCID: PMC2923137  PMID: 20684755
2.  Dissemination of Spotted Fever Rickettsia Agents in Europe by Migrating Birds 
PLoS ONE  2010;5(1):e8572.
Migratory birds are known to play a role as long-distance vectors for many microorganisms. To investigate whether this is true of rickettsial agents as well, we characterized tick infestation and gathered ticks from 13,260 migratory passerine birds in Sweden. A total of 1127 Ixodes spp. ticks were removed from these birds and the extracted DNA from 957 of them was available for analyses. The DNA was assayed for detection of Rickettsia spp. using real-time PCR, followed by DNA sequencing for species identification. Rickettsia spp. organisms were detected in 108 (11.3%) of the ticks. Rickettsia helvetica, a spotted fever rickettsia associated with human infections, was predominant among the PCR-positive samples. In 9 (0.8%) of the ticks, the partial sequences of 17kDa and ompB genes showed the greatest similarity to Rickettsia monacensis, an etiologic agent of Mediterranean spotted fever-like illness, previously described in southern Europe as well as to the Rickettsia sp.IrITA3 strain. For 15 (1.4%) of the ticks, the 17kDa, ompB, gltA and ompA genes showed the greatest similarity to Rickettsia sp. strain Davousti, Rickettsia japonica and Rickettsia heilongjiangensis, all closely phylogenetically related, the former previously found in Amblyomma tholloni ticks in Africa and previously not detected in Ixodes spp. ticks. The infestation prevalence of ticks infected with rickettsial organisms was four times higher among ground foraging birds than among other bird species, but the two groups were equally competent in transmitting Rickettsia species. The birds did not seem to serve as reservoir hosts for Rickettsia spp., but in one case it seems likely that the bird was rickettsiemic and that the ticks had acquired the bacteria from the blood of the bird. In conclusion, migratory passerine birds host epidemiologically important vector ticks and Rickettsia species and contribute to the geographic distribution of spotted fever rickettsial agents and their diseases.
PMCID: PMC2797135  PMID: 20052286
3.  Ixodes ricinus ticks are reservoir hosts for Rickettsia helvetica and potentially carry flea-borne Rickettsia species 
Parasites & Vectors  2009;2:41.
Hard ticks have been identified as important vectors of rickettsiae causing the spotted fever syndrome. Tick-borne rickettsiae are considered to be emerging, but only limited data are available about their presence in Western Europe, their natural life cycle and their reservoir hosts. Ixodes ricinus, the most prevalent tick species, were collected and tested from different vegetation types and from potential reservoir hosts. In one biotope area, the annual and seasonal variability of rickettsiae infections of the different tick stages were determined for 9 years.
The DNA of the human pathogen R. conorii as well as R. helvetica, R. sp. IRS and R. bellii-like were found. Unexpectedly, the DNA of the highly pathogenic R. typhi and R. prowazekii and 4 other uncharacterized Rickettsia spp. related to the typhus group were also detected in I. ricinus. The presence of R. helvetica in fleas isolated from small rodents supported our hypothesis that cross-infection can occur under natural conditions, since R. typhi/prowazekii and R. helvetica as well as their vectors share rodents as reservoir hosts. In one biotope, the infection rate with R. helvetica was ~66% for 9 years, and was comparable between larvae, nymphs, and adults. Larvae caught by flagging generally have not yet taken a blood meal from a vertebrate host. The simplest explanation for the comparable prevalence of R. helvetica between the defined tick stages is, that R. helvetica is vertically transmitted through the next generation with high efficiency. The DNA of R. helvetica was also present in whole blood from mice, deer and wild boar.
Besides R. helvetica, unexpected rickettsiae are found in I. ricinus ticks. We propose that I. ricinus is a major reservoir host for R. helvetica, and that vertebrate hosts play important roles in the further geographical dispersion of rickettsiae.
PMCID: PMC2743653  PMID: 19732416
4.  Co-infection of Borrelia burgdorferi sensu lato and Rickettsia species in ticks and in an erythema migrans patient 
Parasites & Vectors  2013;6:347.
Lyme borreliosis is the most prevalent tick-borne disease in Europe. Ixodes ricinus also carries other pathogenic bacteria, but corresponding human diseases are rarely reported. Here, we compared the exposure to Rickettsia helvetica and Rickettsia monacensis with that to Lyme borreliosis spirochetes. We assumed that their exposure corresponds to their infection rate in questing I. ricinus.
Three Rickettsia species were detected in ticks with a total prevalence of 7.9%, of which the majority was R. helvetica (78%) and R. monacensis (21%). From the same geographic area, skin biopsies of erythema migrans patients were investigated for possible co-infections with Rickettsia spp.. Forty-seven out of 67 skin biopsies were PCR positive for Borrelia burgdorferi s.l. and one sample was positive for R. monacensis. The Borrelia genospecies from the R. monacensis positive patient was identified as Borrelia afzelii. The patient did not show any symptoms associated with rickettsiosis.
Co-infections of I. ricinus with Rickettsia spp. and B. burgdorferi s.l. were as high as expected from the individual prevalence of both pathogens. Co-infection rate in erythema migrans patients corresponded well with tick infection rates. To our knowledge, this is the first reported co-infection of B. afzelii and R. monacensis.
PMCID: PMC3878868  PMID: 24326096
Borrelia burgdorferi; Rickettsia monacensis; Rickettsia helvetica; Erythema migrans; Co-infection
5.  SNP-based real-time pyrosequencing as a sensitive and specific tool for identification and differentiation of Rickettsia species in Ixodes ricinus ticks 
BMC Infectious Diseases  2012;12:261.
Rickettsioses are caused by pathogenic species of the genus Rickettsia and play an important role as emerging diseases. The bacteria are transmitted to mammal hosts including humans by arthropod vectors. Since detection, especially in tick vectors, is usually based on PCR with genus-specific primers to include different occurring Rickettsia species, subsequent species identification is mainly achieved by Sanger sequencing. In the present study a real-time pyrosequencing approach was established with the objective to differentiate between species occurring in German Ixodes ticks, which are R. helvetica, R. monacensis, R. massiliae, and R. felis. Tick material from a quantitative real-time PCR (qPCR) based study on Rickettsia-infections in I. ricinus allowed direct comparison of both sequencing techniques, Sanger and real-time pyrosequencing.
A sequence stretch of rickettsial citrate synthase (gltA) gene was identified to contain divergent single nucleotide polymorphism (SNP) sites suitable for Rickettsia species differentiation. Positive control plasmids inserting the respective target sequence of each Rickettsia species of interest were constructed for initial establishment of the real-time pyrosequencing approach using Qiagen’s PSQ 96MA Pyrosequencing System operating in a 96-well format. The approach included an initial amplification reaction followed by the actual pyrosequencing, which is traceable by pyrograms in real-time. Afterwards, real-time pyrosequencing was applied to 263 Ixodes tick samples already detected Rickettsia-positive in previous qPCR experiments.
Establishment of real-time pyrosequencing using positive control plasmids resulted in accurate detection of all SNPs in all included Rickettsia species. The method was then applied to 263 Rickettsia-positive Ixodes ricinus samples, of which 153 (58.2%) could be identified for their species (151 R. helvetica and 2 R. monacensis) by previous custom Sanger sequencing. Real-time pyrosequencing identified all Sanger-determined ticks as well as 35 previously undifferentiated ticks resulting in a total number of 188 (71.5%) identified samples. Pyrosequencing sensitivity was found to be strongly dependent on gltA copy numbers in the reaction setup. Whereas less than 101 copies in the initial amplification reaction resulted in identification of 15.1% of the samples only, the percentage increased to 54.2% at 101-102 copies, to 95.6% at >102-103 copies and reached 100% samples identified for their Rickettsia species if more than 103 copies were present in the template.
The established real-time pyrosequencing approach represents a reliable method for detection and differentiation of Rickettsia spp. present in I. ricinus diagnostic material and prevalence studies. Furthermore, the method proved to be faster, more cost-effective as well as more sensitive than custom Sanger sequencing with simultaneous high specificity.
PMCID: PMC3532327  PMID: 23075233
Rickettsia helvetica; Rickettsia monacensis; Rickettsia massiliae; Rickettsia felis; Ixodes ricinus; Diagnostic; Sequencing
6.  Spotted Fever Group Rickettsiae in Ticks, Morocco  
Emerging Infectious Diseases  2008;14(7):1067-1073.
Identified rickettsiae were 4 pathogens, 2 suspected pathogens, and 1 incompletely described species.
A total of 370 ticks, encompassing 7 species from 4 genera, were collected during 2002–2006 from domestic animals and vegetation in the Taza region of northeastern Morocco. Rickettsial DNA was identified in 101 ticks (27%) by sequencing PCR products of fragments of the citrate synthase and outer membrane protein genes of Rickettsia spp. Seven rickettsiae of the spotted fever group were identified, including 4 pathogens: R. aeschlimannii in Hyalomma marginatum marginatum, R. massiliae in Rhipicephalus sanguineus, R. slovaca in Dermacentor marginatus, and R. monacensis in Ixodes ricinus. Two suspected pathogens were also detected (R. raoultii in D. marginatus and R. helvetica in I. ricinus). An incompletely described Rickettsia sp. was detected in Haemaphysalis spp. ticks.
PMCID: PMC2600325  PMID: 18598627
Ticks; Morocco; rickettsia; spotted fever; research
7.  Genetic Identification of Rickettsiae Isolated from Ticks in Japan 
Journal of Clinical Microbiology  2002;40(6):2176-2181.
Following the description in Japan of Japanese spotted fever, caused by Rickettsia japonica, a search for the vector of this disease led to the isolation of several rickettsiae from various tick species. Sixty-three rickettsial isolates were obtained from six different tick species, and six type strains were described by PCR and monoclonal antibody testing. We identified these six strains by amplification and sequencing of the genes encoding 16S rRNA and citrate synthase. We confirmed that the isolates from Dermacentor taiwanensis and Haemaphysalis flava ticks were R. japonica isolates. In Ixodes ovatus, Ixodes persulcatus, and Ixodes monospinosus, we identified a Rickettsia identical or closely related to Rickettsia helvetica, a species that is pathogenic for humans and that to date has only been found in Europe. Finally, we identified a new genotype of unknown pathogenicity, genotype AT, that was isolated from Amblyomma testudinarium ticks and that is closely related to a Slovakian genotype obtained from Ixodes ricinus ticks.
PMCID: PMC130657  PMID: 12037083
8.  Life cycle, growth characteristics and host cell response of Rickettsia helvetica in a Vero cell line 
Experimental & Applied Acarology  2011;56(2):179-187.
Rickettsia helvetica, a spotted fever rickettsia and emerging pathogen with Ixodes ricinus ticks as the main vector, is an agent of human disease and may cause febrile illness as well as meningitis. In three parallel series the isolated standard type of R. helvetica, obtained from a PCR-positive I. ricinus tick, was high-passaged and propagated in a Vero cell line. By using quantitative real-time PCR, the generation time from inoculation to stationary phase of growth was calculated to 20–22 h. In the static cultivation system the stationary phase was observed from the seventh day after inoculation, and there was no observed degradation of R. helvetica DNA during the 14 days studied. Microscopy showed that the organisms invaded the host cells rapidly and were primarily found free in the cytoplasm and only occasionally located in the nucleus. Four days after inoculation some of the host cells were broken and many indifferent stages of cytoplasmic organic decomposition were seen. However the R. helvetica organism did not show any morphologic alterations and the number of organisms was stable after the replication peak which may indicate that R. helvetica is adapted to growth in a Vero cell line and/or that the phase of degradation occurs later than the 14 days studied. The findings differ from what has been reported for other rickettsiae of the spotted fever group and may be of importance for invasiveness and virulence of R. helvetica.
PMCID: PMC3253991  PMID: 22116301
Rickettsia helvetica; qPCR; Vero cells; Life cycle; Ultrastructure
9.  Characterization of a spotted fever group Rickettsia from Ixodes ricinus ticks in Sweden. 
Journal of Clinical Microbiology  1997;35(1):243-247.
A spotted fever group rickettsia isolated from the common tick, Ixodes ricinus, was genetically characterized by PCR and genomic sequencing. This study was performed with nymphal and adult ticks collected in southern and central Sweden. I. ricinus is the only North European tick species of medical importance which is regularly collected from humans. No species of the genus Rickettsia has previously been found in Scandinavian ticks, nor has any case of domestic rickettsial infection in humans or animals been reported. According to the nucleotide sequencing, the present Rickettsia sp. belongs to the spotted fever group of rickettsiae. Ticks are the most common arthropod reservoirs and vectors of the rickettsiae of this group. Among 748 ticks investigated, 13 (1.7%) were positive for a Rickettsia sp. Borrelia burgdorferi was detected in 52 (7%) of the ticks, a prevalence similar to or somewhat lower than that previously been recorded in other Swedish studies. There was no evidence of ehrlichial or chlamydial DNA in these ticks. The Rickettsia sp. was further characterized by 16S ribosomal DNA (rDNA) sequencing and restriction fragment length polymorphism (RFLP). The 16S rDNA sequencing resulted in a sequence identical to that described for Rickettsia helvetica, but the pattern obtained with RFLP of the citrate synthetase gene diverged from previously known patterns. The rickettsial agent of one tick which was positive by PCR was confirmed by transmission electron microscopy. The morphology of this rickettsia was similar to that of the spotted fever and typhus group rickettsiae. This represents the first documented isolate of a Rickettsia sp. from Swedish ticks.
PMCID: PMC229547  PMID: 8968916
10.  Rickettsia Species in Ticks Removed from Humans in Istanbul, Turkey 
Vector Borne and Zoonotic Diseases  2012;12(11):938-941.
A total of 167 ticks collected from humans in Istanbul (Turkey) in 2006 were screened for Rickettsia species, and nested PCRs targeting gltA and ompA rickettsial fragment genes were carried out. Rickettsia monacensis (51), R. aeschlimannii (8), R. conorii subsp. conorii (3), R. helvetica (2), R. raoultii (1), R. africae (1), R. felis (1), and other Rickettsia spp. (2), were detected. To our knowledge, these Rickettsia species (except R. conorii) had never been reported in ticks removed from humans in Turkey. The presence of R. africae also had not been previously described, either in Hyalomma ticks or in any European tick species. In addition, R. aeschlimannii and R. felis had not been found associated with Rhipicephalus bursa specimens. The presence of human pathogenic Rickettsia in ticks removed from humans provides information about the risk of tick-borne rickettsioses in Turkey.
PMCID: PMC3491622  PMID: 22925016
Rickettsia species; Turkey; Ticks from humans.
11.  Rickettsia helvetica in Ixodes ricinus Ticks in Sweden 
Journal of Clinical Microbiology  1999;37(2):400-403.
In the present study further characterization of the amplified sequence of the citrate synthase gene of the spotted fever group Rickettsia isolated from Ixodes ricinus ticks in Sweden showed that it has 100% homology with the deposited sequence of the citrate synthase gene of Rickettsia helvetica. The restriction fragment length polymorphism (RFLP) pattern of an amplified 382-bp product of the citrate synthase sequence, defined by primers RpCS877 and RpCS1258, yielded fragments for our isolate that could be visualized as a double band that migrated at approximately 44 bp, another double band at 85 bp, and a single band at nearly 120 bp after digestion with the restriction enzyme AluI. When calculating a theoretical PCR-RFLP pattern of the sequence of the citrate synthase gene of R. helvetica from the known positions where the AluI enzyme cuts, we arrived at the same pattern that was obtained for our isolate, a pattern distinctly different from the previously published PCR-RFLP pattern for R. helvetica. Investigation of 125 living I. ricinus ticks showed a higher prevalence of rickettsial DNA in these ticks than we had found in an earlier study. Rickettsial DNA was detected by amplification of the 16S rRNA gene, for which a seminested primer system consisting of two oligonucleotide primer pairs was used. Of the 125 ticks, some were pooled, giving a total of 82 tick samples, of which 20 were found to be positive for the rickettsial DNA gene investigated. When considering the fact that some of the positive samples were pooled, the minimum possible prevalence in these ticks was 20 of 125 (16%) and the maximum possible prevalence was 46 of 125 (36.8%). These prevalence estimates conform to those of other studies of spotted fever group rickettsiae in hard ticks in Europe.
PMCID: PMC84320  PMID: 9889227
12.  Exotic Rickettsiae in Ixodes ricinus: fact or artifact? 
Parasites & Vectors  2010;3:54.
Several pathogenic Rickettsia species can be transmitted via Ixodes ricinus ticks to humans and animals. Surveys of I. ricinus for the presence of Rickettsiae using part of its 16S rRNA gene yield a plethora of new and different Rickettsia sequences. Interpreting these data is sometimes difficult and presenting these findings as new or potentially pathogenic Rickettsiae should be done with caution: a recent report suggested presence of a known human pathogen, R. australis, in questing I. ricinus ticks in Europe. A refined analysis of these results revealed that R. helvetica was most likely to be misinterpreted as R. australis. Evidence in the literature is accumulating that rickettsial DNA sequences found in tick lysates can also be derived from other sources than viable, pathogenic Rickettsiae. For example, from endosymbionts, environmental contamination or even horizontal gene transfer.
PMCID: PMC2904298  PMID: 20569494
13.  Rickettsia felis from Cat Fleas: Isolation and Culture in a Tick-Derived Cell Line 
Rickettsia felis, the etiologic agent of spotted fever, is maintained in cat fleas by vertical transmission and resembles other tick-borne spotted fever group rickettsiae. In the present study, we utilized an Ixodes scapularis-derived tick cell line, ISE6, to achieve isolation and propagation of R. felis. A cytopathic effect of increased vacuolization was commonly observed in R. felis-infected cells, while lysis of host cells was not evident despite large numbers of rickettsiae. Electron microscopy identified rickettsia-like organisms in ISE6 cells, and sequence analyses of portions of the citrate synthase (gltA), 16S rRNA, Rickettsia genus-specific 17-kDa antigen, and spotted fever group-specific outer membrane protein A (ompA) genes and, notably, R. felis conjugative plasmids indicate that this cultivatable strain (LSU) was R. felis. Establishment of R. felis (LSU) in a tick-derived cell line provides an alternative and promising system for the expansion of studies investigating the interactions between R. felis and arthropod hosts.
PMCID: PMC1538700  PMID: 16885313
14.  Absence of Rickettsia rickettsii and Occurrence of Other Spotted Fever Group Rickettsiae in Ticks from Tennessee 
Rocky Mountain spotted fever (RMSF) is the most common tick-borne illness in Tennessee. Little is known about the occurrence of R. rickettsii, the causative agent, in ticks in Tennessee. To better understand the prevalence and distribution of rickettsial agents in ticks, we tested 1,265 Amblyomma, Dermacentor, and Ixodes adult and nymphal ticks. Additionally, we tested 231 Amblyomma americanum larvae. Ticks were collected from 49 counties from humans, wild animals, domestic canines, and flannel drags. Spotted fever group rickettsiae (SFGR) DNA was detected by polymerase chain reaction (PCR) in 32% of adult and nymphal ticks. A total minimum infection rate of 85.63 was found in larval pools tested. Three rickettsial species, Rickettsia montana, Rickettsia amblyommii, and Rickettsia cooleyi were identified by molecular analysis. Rickettsia rickettsii was not detected. This study suggests that some RMSF cases reported in Tennessee may be caused by cross-reactivity with other SFGR antigenically related to R. rickettsii.
PMCID: PMC2929065  PMID: 20810834
15.  Spotted fever group rickettsiae in Dermacentor reticulatus and Haemaphysalis punctata ticks in the UK 
Parasites & Vectors  2013;6:212.
Spotted fever group (SFG) rickettsiae have recently been identified for the first time in UK ticks. This included the findings of Rickettsia helvetica in Ixodes ricinus and Rickettsia raoultii in Dermacentor reticulatus. This paper further investigates the occurrence of SFG rickettsiae in additional geographically distinct populations of D. reticulatus, and for the first time, investigates the occurrence of SFG rickettsiae in UK populations of Haemaphysalis punctata ticks.
Questing D. reticulatus and H. punctata were collected at a number of sites in England and Wales. DNA from questing ticks was extracted by alkaline lysis and detection of rickettsiae DNA was performed, in addition to detection of A. phagocytophilum, N. mikurensis, C. burnetii and B. burgdorferi sensu lato.
This paper builds on previous findings to include the detection of spotted fever Rickettsia which showed the highest homology to Rickettsia massiliae in Haemaphysalis punctata, as well as R. helvetica in D. reticulatus. The occurrence of SFG rickettsiae in D. reticulatus in the UK appears to be confined only to Welsh and Essex populations, with no evidence so far from Devon. Similarly, the occurrence of SFG rickettsiae in H. punctata appears confined to one of two farms known to be infested with this tick in North Kent, with no evidence so far from the Sussex populations. Anaplasma phagocytophilum, Neoehrlichia mikurensis, Coxiella burnetii and Borrelia burgdorferi sensu lato DNA was not detected in any of the ticks.
These two tick species are highly restricted in their distribution in England and Wales, but where they do occur they can be abundant. Following detection of these SFG rickettsiae in additional UK tick species, as well as I. ricinus, research should now be directed towards clarifying firstly the geographic distribution of SFG rickettsiae in UK ticks, and secondly to assess the prevalence rates in ticks, wild and domesticated animals and humans to identify the drivers for disease transmission and their public health significance.
PMCID: PMC3725166  PMID: 23870197
Haemaphysalis; Rickettsia massiliae; UK; Dermacentor; Rickettsiae; Ticks
16.  Detection of “Candidatus Rickettsia sp. strain Argentina”and Rickettsia bellii in Amblyomma ticks (Acari: Ixodidae) from Northern Argentina 
Experimental & applied acarology  2010;52(1):93-100.
Ixodid ticks were collected from vegetation and from humans, wild and domestic mammals in a rural area in the semi-arid Argentine Chaco in late spring 2006 to evaluate their potential role as vectors of Spotted Fever Group (SFG) rickettsiae. A total of 233 adult ticks, identified as Amblyomma parvum, Amblyomma tigrinum and Amblyomma pseudoconcolor, was examined for Rickettsia spp. We identified an SFG rickettsia of unknown pathogenicity, “Candidatus Rickettsia sp. strain Argentina”, in A. parvum and A. pseudoconcolor by PCR assays targeting gltA, ompA, ompB and 17-kDa outer membrane antigen rickettsial genes. Rickettsia bellii was detected in a host-seeking male of A. tigrinum. Amblyomma parvum is widespread in the study area and is a potential threat to human health.
PMCID: PMC3072707  PMID: 20186466
Amblyomma spp.; Candidatus Rickettsia sp. strain Argentina; Rickettsia bellii; Argentina; Spotted fever; Tick-borne disease
17.  Hemocytic rickettsia-like organisms in ticks: serologic reactivity with antisera to Ehrlichiae and detection of DNA of agent of human granulocytic ehrlichiosis by PCR. 
Journal of Clinical Microbiology  1995;33(10):2710-2714.
Ixodid ticks were collected from Connecticut, Massachusetts, Missouri, Pennsylvania, Rhode Island, and British Columbia (Canada) during 1991 to 1994 to determine the prevalence of infection with hemocytic (blood cell), rickettsia-like organisms. Hemolymph obtained from these ticks was analyzed by direct and indirect fluorescent antibody (FA) staining methods with dog, horse, or human sera containing antibodies to Ehrlichia canis, Ehrlichia equi, or Rickettsia rickettsii. Of the 693 nymphal and adult Amblyomma americanum, Dermacentor variabilis, Ixodes scapularis, and Ixodes pacificus ticks tested with dog anti-E. canis antiserum, 209 (32.5%) contained hemocytic bacteria. The prevalence of infected ticks varied greatly with species and locale. In parallel tests of duplicate hemolymph preparations from adult I. scapularis ticks, the hemocytic organisms reacted positively with E. canis and/or E. equi antisera, including sera from persons who had granulocytic ehrlichiosis. In separate PCR analyses, DNA of the agent of human granulocytic ehrlichiosis was detected in 59 (50.0%) of 118 adult and in 1 of 2 nymphal I. scapularis ticks tested from Connecticut. There was no evidence of Ehrlichia chaffeensis DNA in these ticks. In indirect FA tests of hemolymph for spotted fever group rickettsiae, the overall prevalence of infection was less than 4%. Specificity tests of antigens and antisera used in these studies revealed no cross-reactivity between E. canis and E. equi or between any of the ehrlichial reagents and those of R. rickettsii. The geographic distribution of hemocytic microorganisms with shared antigens to Ehrlichia species or spotted fever group rickettsiae is widespread.
PMCID: PMC228561  PMID: 8567911
18.  Comparison of Real-Time Quantitative PCR and Culture for the Diagnosis of Emerging Rickettsioses 
Isolation of Rickettsia species from skin biopsies may be replaced by PCR. We evaluated culture sensitivity compared to PCR based on sampling delay and previous antibiotic treatment.
Methodology/Principal Findings
Skin biopsies and ticks from patients with suspected Rickettsia infection were screened for Rickettsia spp. using qPCR, and positive results were amplified and sequenced for the gltA and ompA genes. Immunofluorescence for spotted fever group rickettsial antigens was done for 79 patients. All skin biopsies and only ticks that tested positive using qPCR were cultured in human embryonic lung (HEL) fibroblasts using the centrifugation-shell vial technique. Patients and ticks were classified as definitely having rickettsioses if there was direct evidence of infection with a Rickettsia sp. using culture or molecular assays or in patients if serology was positive. Data on previous antibiotic treatments were obtained for patients with rickettsiosis. Rickettsia spp. infection was diagnosed in 47 out of 145 patients (32%), 41 by PCR and 12 by culture, whereas 3 isolates were obtained from PCR negative biopsies. For 3 of the patients serology was positive although PCR and culture were negative. Rickettsia africae was the most common detected species (n = 25, [17.2%]) and isolated bacterium (n = 5, [3.4%]). The probability of isolating Rickettsia spp. was 12 times higher in untreated patients and 5.4 times higher in patients from our hometown. Rickettsia spp. was amplified in 24 out of 95 ticks (25%) and we isolated 7 R. slovaca and 1 R. raoultii from Dermacentor marginatus.
We found a positive correlation between the bacteria copies and the isolation success in skin biopsies and ticks. Culture remains critical for strain analysis but is less sensitive than serology and PCR for the diagnosis of a Rickettsia infection.
Author Summary
Diagnosis of Rickettsia infection would benefit by use of the more rapid and sensitive method of quantitative real-time PCR than the time-intensive and less sensitive method of culturing Rickettsia species from skin biopsies. We evaluated culture sensitivity compared to PCR according to sampling delay and previous antibiotic treatment. We found that skin biopsies can be positive even when molecular tests were negative, and a negative result using molecular assays did not exclude the diagnosis of Rickettsia spp. infection. Rickettsia africae was the most common species in skin biopsies and R. slovaca was most common in ticks. We found a positive correlation between the number of bacteria copies and the isolation success in skin biopsies and ticks. The probability of isolating Rickettsia spp. was higher in untreated patients and in patients from our hometown. To increase the sensitivity of culture, skin biopsies should be sampled before treatment early in the course of the disease and should be inoculated as soon as possible.
PMCID: PMC3295807  PMID: 22413026
19.  Absence of zoonotic Bartonella species in questing ticks: First detection of Bartonella clarridgeiae and Rickettsia felis in cat fleas in the Netherlands 
Parasites & Vectors  2011;4:61.
Awareness for flea- and tick-borne infections has grown in recent years and the range of microorganisms associated with these ectoparasites is rising. Bartonella henselae, the causative agent of Cat Scratch Disease, and other Bartonella species have been reported in fleas and ticks. The role of Ixodes ricinus ticks in the natural cycle of Bartonella spp. and the transmission of these bacteria to humans is unclear. Rickettsia spp. have also been reported from as well ticks as also from fleas. However, to date no flea-borne Rickettsia spp. were reported from the Netherlands. Here, the presence of Bartonellaceae and Rickettsiae in ectoparasites was investigated using molecular detection and identification on part of the gltA- and 16S rRNA-genes.
The zoonotic Bartonella clarridgeiae and Rickettsia felis were detected for the first time in Dutch cat fleas. B. henselae was found in cat fleas and B. schoenbuchensis in ticks and keds feeding on deer. Two Bartonella species, previously identified in rodents, were found in wild mice and their fleas. However, none of these microorganisms were found in 1719 questing Ixodes ricinus ticks. Notably, the gltA gene amplified from DNA lysates of approximately 10% of the questing nymph and adult ticks was similar to that of an uncultured Bartonella-related species found in other hard tick species. The gltA gene of this Bartonella-related species was also detected in questing larvae for which a 16S rRNA gene PCR also tested positive for "Candidatus Midichloria mitochondrii". The gltA-gene of the Bartonella-related species found in I. ricinus may therefore be from this endosymbiont.
We conclude that the risk of acquiring Cat Scratch Disease or a related bartonellosis from questing ticks in the Netherlands is negligible. On the other hand fleas and deer keds are probable vectors for associated Bartonella species between animals and might also transmit Bartonella spp. to humans.
PMCID: PMC3087693  PMID: 21501464
20.  Prevalence of Tick-Borne Pathogens in Ixodes ricinus and Dermacentor reticulatus Ticks from Different Geographical Locations in Belarus 
PLoS ONE  2013;8(1):e54476.
Worldwide, ticks are important vectors of human and animal pathogens. Besides Lyme Borreliosis, a variety of other bacterial and protozoal tick-borne infections are of medical interest in Europe. In this study, 553 questing and feeding Ixodes ricinus (n = 327) and Dermacentor reticulatus ticks (n = 226) were analysed by PCR for Borrelia, Rickettsia, Anaplasma, Coxiella, Francisella and Babesia species. Overall, the pathogen prevalence in ticks was 30.6% for I. ricinus and 45.6% for D. reticulatus. The majority of infections were caused by members of the spotted-fever group rickettsiae (24.4%), 9.4% of ticks were positive for Borrelia burgdorferi sensu lato, with Borrelia afzelii being the most frequently detected species (40.4%). Pathogens with low prevalence rates in ticks were Anaplasma phagocytophilum (2.2%), Coxiella burnetii (0.9%), Francisella tularensis subspecies (0.7%), Bartonella henselae (0.7%), Babesia microti (0.5%) and Babesia venatorum (0.4%). On a regional level, hotspots of pathogens were identified for A. phagocytophilum (12.5–17.2%), F. tularensis ssp. (5.5%) and C. burnetii (9.1%), suggesting established zoonotic cycles of these pathogens at least at these sites. Our survey revealed a high burden of tick-borne pathogens in questing and feeding I. ricinus and D. reticulatus ticks collected in different regions in Belarus, indicating a potential risk for humans and animals. Identified hotspots of infected ticks should be included in future surveillance studies, especially when F. tularensis ssp. and C. burnetii are involved.
PMCID: PMC3551763  PMID: 23349900
21.  Characterization of Spotted Fever Group Rickettsiae in Flea and Tick Specimens from Northern Peru 
Journal of Clinical Microbiology  2004;42(11):4961-4967.
Evidence of spotted fever group (SFG) rickettsiae was obtained from flea pools and individual ticks collected at three sites in northwestern Peru within the focus of an outbreak of febrile disease in humans attributed, in part, to SFG rickettsia infections. Molecular identification of the etiologic agents from these samples was determined after partial sequencing of the 17-kDa common antigen gene (htrA) as well as pairwise nucleotide sequence homology with one or more of the following genes: gltA, ompA, and ompB. Amplification and sequencing of portions of the htrA and ompA genes in pooled samples (2 of 59) taken from fleas identified the pathogen Rickettsia felis. Four tick samples yielded molecular evidence of SFG rickettsiae. Fragments of the ompA (540-bp) and ompB (2,484-bp) genes were amplified from a single Amblyomma maculatum tick (tick 124) and an Ixodes boliviensis tick (tick 163). The phylogenetic relationships between the rickettsiae in these samples and other rickettsiae were determined after comparison of their ompB sequences by the neighbor-joining method. The dendrograms generated showed that the isolates exhibited close homology (97%) to R. aeschlimannii and R. rhipicephali. Significant bootstrap values supported clustering adjacent to this nodule of the SFG rickettsiae. While the agents identified in the flea and tick samples have not been linked to human cases in the area, these results demonstrate for the first time that at least two SFG rickettsia agents were circulating in northern Peru at the time of the outbreak. Furthermore, molecular analysis of sequences derived from the two separate species of hard ticks identified a possibly novel member of the SFG rickettsiae.
PMCID: PMC525230  PMID: 15528680
22.  Spatiotemporal dynamics of emerging pathogens in questing Ixodes ricinus 
Ixodes ricinus transmits Borrelia burgdorferi sensu lato, the etiological agent of Lyme disease. Previous studies have also detected Rickettsia helvetica, Anaplasma phagocytophilum, Neoehrlichia mikurensis, and several Babesia species in questing ticks in The Netherlands. In this study, we assessed the acarological risk of exposure to several tick-borne pathogens (TBPs), in The Netherlands. Questing ticks were collected monthly between 2006 and 2010 at 21 sites and between 2000 and 2009 at one other site. Nymphs and adults were analysed individually for the presence of TBPs using an array-approach. Collated data of this and previous studies were used to generate, for each pathogen, a presence/absence map and to further analyse their spatiotemporal variation. R. helvetica (31.1%) and B. burgdorferi sensu lato (11.8%) had the highest overall prevalence and were detected in all areas. N. mikurensis (5.6%), A. phagocytophilum (0.8%), and Babesia spp. (1.7%) were detected in most, but not all areas. The prevalences of pathogens varied among the study areas from 0 to 64%, while the density of questing ticks varied from 1 to 179/100 m2. Overall, 37% of the ticks were infected with at least one pathogen and 6.3% with more than one pathogen. One-third of the Borrelia-positive ticks were infected with at least one other pathogen. Coinfection of B. afzelii with N. mikurensis and with Babesia spp. occurred significantly more often than single infections, indicating the existence of mutual reservoir hosts. Alternatively, coinfection of R. helvetica with either B. afzelii or N. mikurensis occurred significantly less frequent. The diversity of TBPs detected in I. ricinus in this study and the frequency of their coinfections with B. burgdorferi s.l., underline the need to consider them when evaluating the risks of infection and subsequently the risk of disease following a tick bite.
PMCID: PMC3726834  PMID: 23908971
vector-borne disease; Borrelia burgdorferi; Candidatus Neoehrlichia mikurensis; Rickettsia helvetica; Rickettsia conorii; Anaplasma phagocytophilum; Babesia; Ixodes ricinus
23.  Survey of Tickborne Infections in Denmark 
Emerging Infectious Diseases  2005;11(7):1055-1061.
We conducted a study of the distribution and prevalence of tickborne infections in Denmark by using roe deer as sentinels. Blood samples from 237 roe deer were collected during the 2002–2003 hunting season. Overall, 36.6% of deer were Borrelia seropositive, while 95.6% were Anaplasma phagocytophilum positive; all animals were negative for Bartonella quintana and B. henselae by indirect immunofluorescence assay. When a hemagglutination-inhibition test was used, 8.7% of deer were found positive for tickborne encephalitis (TBE)-complex virus. A total of 42.6% were found positive by polymerase chain reaction (PCR) for A. phagocytophilum with significant seasonal variation. All were PCR negative for Rickettsia helvetica. PCR and sequencing also showed a novel bacterium in roe deer previously only found in ticks. The study showed that the emerging pathogen A. phagocytophilum is widely distributed and that a marked shift has occurred in the distribution of TBE-complex virus in Denmark. This finding supports studies that predict alterations in distribution due to climatic changes.
PMCID: PMC3371797  PMID: 16022780
Borrelia; Anaplasma phagocytophilum; Bartonella; Rickettsia; Tickborne encephalitis viruses; climate; roe deer; Ixodes ricinus; serology; sentinel surveillance
24.  Spotted Fever Group Rickettsiae in Ticks, Germany 
Emerging Infectious Diseases  2011;17(5):890-892.
To explore increased risk for human Rickettsia spp. infection in Germany, we investigated recreational areas and renatured brown coal surface-mining sites (also used for recreation) for the presence of spotted fever group rickettsiae in ticks. R. raoultii (56.7%), R. slovaca (13.3%), and R. helvetica (>13.4%) were detected in the respective tick species.
PMCID: PMC3321775  PMID: 21529404
Rickettsia; vector-borne infections; bacteria; ticks Germany; dispatch
25.  Detection of Rickettsia massiliae in Rhipicephalus sanguineus from the Eastern United States 
We report the first evidence of Rickettsia massiliae in the brown dog tick, Rhipicephalus sanguineus, from the East Coast of the United States. As part of routine pathogen surveillance, DNA samples from ixodid ticks were tested for spotted fever group rickettsiae by nested PCR. A R. massiliae-positive tick was collected off a beagle mix recently rescued from North Carolina. Infection was confirmed by partial sequence analysis of the htrA, gltA, ompB, ompA, and sca4 genes, which had 100% identity to a R. massiliae isolate from Arizona.
PMCID: PMC3540894  PMID: 23199270
Rickettsia massiliae; Rhipicephalus sanguineus

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