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1.  Detection of tick-borne ‘Candidatus Neoehrlichia mikurensis’ and Anaplasma phagocytophilum in Spain in 2013 
Parasites & Vectors  2014;7:57.
Background
‘Candidatus Neoehrlichia mikurensis’ is a tick-borne bacteria implicated in human health. To date, ‘Ca. Neoehrlichia mikurensis’ has been described in different countries from Africa, Asia and Europe, but never in Spain. However, according to the epidemiological features of the main vector in Europe, Ixodes ricinus, its circulation in our country was suspected.
Methods
A total of 200 I. ricinus ticks collected in the North of Spain were analyzed. DNAs were extracted and used as templates for PCRs targeting fragment genes for Anaplasma/Ehrlichia detection. The amplified products were sequenced and analyzed.
Results
‘Ca. Neoehrlichia mikurensis’ was amplified in two specimens. Furthermore, Anaplasma phagocytophilum was detected in 61 samples analyzed.
Conclusions
The detection of ‘Ca. Neoehrlichia mikurensis’ in I. ricinus ticks from Spain indicates its circulation and the potential risk of contracting a human infection in this country.
doi:10.1186/1756-3305-7-57
PMCID: PMC3912351  PMID: 24484637
‘Candidatus Neoehrlichia mikurensis’; Anaplasma phagocytophilum; Ixodes ricinus; Spain
2.  Infections and Coinfections of Questing Ixodes ricinus Ticks by Emerging Zoonotic Pathogens in Western Switzerland 
Applied and Environmental Microbiology  2012;78(13):4606-4612.
In Europe, Ixodes ricinus is the vector of many pathogens of medical and veterinary relevance, among them Borrelia burgdorferi sensu lato and tick-borne encephalitis virus, which have been the subject of numerous investigations. Less is known about the occurrence of emerging tick-borne pathogens like Rickettsia spp., Babesia spp., “Candidatus Neoehrlichia mikurensis,” and Anaplasma phagocytophilum in questing ticks. In this study, questing nymph and adult I. ricinus ticks were collected at 11 sites located in Western Switzerland. A total of 1,476 ticks were analyzed individually for the simultaneous presence of B. burgdorferi sensu lato, Rickettsia spp., Babesia spp., “Candidatus Neoehrlichia mikurensis,” and A. phagocytophilum. B. burgdorferi sensu lato, Rickettsia spp., and “Candidatus Neoehrlichia mikurensis” were detected in ticks at all sites with global prevalences of 22.5%, 10.2%, and 6.4%, respectively. Babesia- and A. phagocytophilum-infected ticks showed a more restricted geographic distribution, and their prevalences were lower (1.9% and 1.5%, respectively). Species rarely reported in Switzerland, like Borrelia spielmanii, Borrelia lusitaniae, and Rickettsia monacensis, were identified. Infections with more than one pathogenic species, involving mostly Borrelia spp. and Rickettsia helvetica, were detected in 19.6% of infected ticks. Globally, 34.2% of ticks were infected with at least one pathogen. The diversity of tick-borne pathogens detected in I. ricinus in this study and the frequency of coinfections underline the need to take them seriously into consideration when evaluating the risks of infection following a tick bite.
doi:10.1128/AEM.07961-11
PMCID: PMC3370488  PMID: 22522688
3.  “Candidatus Neoehrlichia mikurensis,” Anaplasma phagocytophilum, and Lyme Disease Spirochetes in Questing European Vector Ticks and in Feeding Ticks Removed from People 
Journal of Clinical Microbiology  2012;50(3):943-947.
To estimate the likelihood of people coming into contact with the recently described tick-borne agent “Candidatus Neoehrlichia mikurensis,” we compared its prevalence to those of Lyme disease spirochetes and Anaplasma phagocytophilum in questing adult Ixodes ricinus ticks collected in various Central European sites and examined ticks, which had been removed from people, for the presence of these pathogens. Whereas spirochetes infected questing adult ticks most frequently (22.3%), fewer than a third as many ticks were infected by “Ca. Neoehrlichia mikurensis” (6.2%), and about a sixth harbored A. phagocytophilum (3.9%). On average, every twelfth encounter of a person with an I. ricinus tick (8.1%) may bear the risk of acquiring “Ca. Neoehrlichia mikurensis.” Although a fifth of the people (20%) had removed at least one tick infected by “Ca. Neoehrlichia mikurensis,” none displayed symptoms described for this pathogen, suggesting that its transmission may not be immediate and/or that immunocompetent individuals may not be affected. Because immunosuppressed patients may be at a particular risk of developing symptoms, it should be considered that “Ca. Neoehrlichia mikurensis” appears to be the second most common pathogen in I. ricinus ticks. In our survey, only Borrelia afzelii appears to infect Central European vector ticks more frequently.
doi:10.1128/JCM.05802-11
PMCID: PMC3295140  PMID: 22205824
4.  Candidatus Neoehrlichia mikurensis and its co-circulation with Anaplasma phagocytophilum in Ixodes ricinus ticks across ecologically different habitats of Central Europe 
Parasites & Vectors  2014;7:160.
Background
Candidatus Neoehrlichia mikurensis is a newly emerging tick-borne bacterium from the family Anaplasmataceae. Its presence in Ixodes ricinus ticks was reported from various European countries, however, it’s ecology and co-circulation with another member of the same family, Anaplasma phagocytophilum has not been rigorously studied yet.
Findings
Candidatus N. mikurensis was detected in all sampling sites. In total, 4.5% of ticks were positive including larvae. The highest positivity was detected in Austria with a prevalence of 23.5%. The probability of Candidatus N. mikurensis occurrence increased with the proportion of ticks infected with Anaplasma phagocytophilum.
Conclusion
A positive association between the occurrences of Candidatus N. mikurensis and A. phagocytophilum indicates that both bacteria share similar ecology for their natural foci in Central Europe.
doi:10.1186/1756-3305-7-160
PMCID: PMC3984398  PMID: 24693971
Candidatus Neoehrlichia mikurensis; Anaplasma phagocytophilum; Ixodes ricinus; Human granulocytic anaplasmosis; Neoehrlichiosis
5.  Ixodes ricinus abundance and its infection with the tick-borne pathogens in urban and suburban areas of Eastern Slovakia 
Parasites & Vectors  2013;6:238.
Background
Raising abundance of ticks and tick-borne diseases in Europe is the result of multiple factors including climate changes and human activities. Herein, we investigated the presence and seasonal activity of Ixodes ricinus ticks from 10 urban and suburban sites in two different geographical areas of southeastern and northeastern Slovakia during 2008–2010. Our aim was to study the abundance of ticks in correlation with the environmental factors and their infection with Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Neoehrlichia mikurensis.
Methods
Questing I. ricinus ticks were collected from ten urban and suburban sites in Eastern Slovakia. A total of 670 ticks were further analysed for the presence of B. burgdorferi s.l., A. phagocytophilum and N. mikurensis by molecular methods. Tick site and environmental relations were analysed using General Linear Models (LM). The differences between the number of Lyme borreliosis cases between the Košice and Bardejov regions during a ten-year period were tested by Wilcoxon matched pairs test.
Results
In total, 2921 (1913 nymphs, 1008 adults) I. ricinus ticks were collected from 10 study sites during the main questing season. Tick activity and relative abundance differed between locations and months. Temperature and humidity were the main factors affecting the tick abundance and questing activity. Out of 670 examined ticks, 10.15% were infected with spirochetes from B. burgdorferi s.l. complex (represented by B. afzelii, B. garinii, B.valaisiana and B. burgdorferi s.s.), 2.69% with the A. phagocytophilum and 2.39% with N. mikurensis. The number of Lyme borreliosis cases per 100,000 inhabitants in the Bardejov region was significantly higher than in the Košice region.
Conclusions
Our data indicate that the risk of infection with tick-borne pathogens in Eastern Slovakia is common since 15.2% of ticks were infected at least with one of the tested microorganisms. Even though the abundance of ticks was affected by the microclimatic conditions and the prevalence of pathogens differed between the habitats, the infection risk for humans is also affected by human activities leading to an increased contact with infected ticks.
doi:10.1186/1756-3305-6-238
PMCID: PMC3751762  PMID: 23952975
Ixodes ricinus; Borrelia burgdorferi sensu lato; Anaplasma phagocytophilum; Neoehrlichia mikurensis; PCR-RFLP; Lyme borreliosis; Anaplasmosis
6.  A Novel High-Resolution Melt PCR Assay Discriminates Anaplasma phagocytophilum and “Candidatus Neoehrlichia mikurensis” 
Journal of Clinical Microbiology  2013;51(6):1958-1961.
“Candidatus Neoehrlichia mikurensis” (Anaplasmataceae) is an emerging pathogen transmitted by Ixodes ticks. Conventional PCR and the newly developed high-resolution melt PCR were used to detect and discriminate “Candidatus Neoehrlichia mikurensis” and Anaplasma phagocytophilum. Both bacterial species were frequently found in Ixodes ricinus and Ixodes hexagonus but virtually absent from Dermacentor reticulatus. In rodents, “Candidatus N. mikurensis” was significantly more prevalent than A. phagocytophilum, whereas in cats, only A. phagocytophilum was found.
doi:10.1128/JCM.00284-13
PMCID: PMC3716091  PMID: 23576542
7.  First evidence of Candidatus Neoehrlichia mikurensis in Hungary 
Parasites & Vectors  2013;6:267.
Altogether 2004 Ixodes ricinus ticks, from 37 places in Hungary, were analysed in pools with a recently developed multiplex real-time PCR for the presence of Candidatus Neoehrlichia mikurensis and for other representatives of the genus. Ca. Neoehrlichia mikurensis was identified in nine sampling sites, indicating three separated endemic regions along the borders of Hungary. In addition, results of samples from seven places (except for the western part of the country) were positive in the genus-specific (Ca. Neoehrlichia sp.) PCR, but were negative for Ca. Neoehrlichia mikurensis.
doi:10.1186/1756-3305-6-267
PMCID: PMC3849741  PMID: 24341500
Tick-borne diseases; Zoonosis; Epidemiology
8.  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.
doi:10.3389/fcimb.2013.00036
PMCID: PMC3726834  PMID: 23908971
vector-borne disease; Borrelia burgdorferi; Candidatus Neoehrlichia mikurensis; Rickettsia helvetica; Rickettsia conorii; Anaplasma phagocytophilum; Babesia; Ixodes ricinus
9.  Occurrence and identification of risk areas of Ixodes ricinus-borne pathogens: a cost-effectiveness analysis in north-eastern Italy 
Parasites & Vectors  2012;5:61.
Background
Ixodes ricinus, a competent vector of several pathogens, is the tick species most frequently reported to bite humans in Europe. The majority of human cases of Lyme borreliosis (LB) and tick-borne encephalitis (TBE) occur in the north-eastern region of Italy. The aims of this study were to detect the occurrence of endemic and emergent pathogens in north-eastern Italy using adult tick screening, and to identify areas at risk of pathogen transmission. Based on our results, different strategies for tick collection and pathogen screening and their relative costs were evaluated and discussed.
Methods
From 2006 to 2008 adult ticks were collected in 31 sites and molecularly screened for the detection of pathogens previously reported in the same area (i.e., LB agents, TBE virus, Anaplasma phagocytophilum, Rickettsia spp., Babesia spp., "Candidatus Neoehrlichia mikurensis"). Based on the results of this survey, three sampling strategies were evaluated a-posteriori, and the impact of each strategy on the final results and the overall cost reductions were analyzed. The strategies were as follows: tick collection throughout the year and testing of female ticks only (strategy A); collection from April to June and testing of all adult ticks (strategy B); collection from April to June and testing of female ticks only (strategy C).
Results
Eleven pathogens were detected in 77 out of 193 ticks collected in 14 sites. The most common microorganisms detected were Borrelia burgdorferi sensu lato (17.6%), Rickettsia helvetica (13.1%), and "Ca. N. mikurensis" (10.5%). Within the B. burgdorferi complex, four genotypes (i.e., B. valaisiana, B. garinii, B. afzelii, and B. burgdorferi sensu stricto) were found. Less prevalent pathogens included R. monacensis (3.7%), TBE virus (2.1%), A. phagocytophilum (1.5%), Bartonella spp. (1%), and Babesia EU1 (0.5%). Co-infections by more than one pathogen were diagnosed in 22% of infected ticks. The prevalences of infection assessed using the three alternative strategies were in accordance with the initial results, with 13, 11, and 10 out of 14 sites showing occurrence of at least one pathogen, respectively. The strategies A, B, and C proposed herein would allow to reduce the original costs of sampling and laboratory analyses by one third, half, and two thirds, respectively. Strategy B was demonstrated to represent the most cost-effective choice, offering a substantial reduction of costs, as well as reliable results.
Conclusions
Monitoring of tick-borne diseases is expensive, particularly in areas where several zoonotic pathogens co-occur. Cost-effectiveness studies can support the choice of the best monitoring strategy, which should take into account the ecology of the area under investigation, as well as the available budget.
doi:10.1186/1756-3305-5-61
PMCID: PMC3337281  PMID: 22452970
Ixodes ricinus; tick-borne diseases; surveillance; economic evaluation; Italy.
10.  Candidatus Neoehrlichia mikurensis in rodents in an area with sympatric existence of the hard ticks Ixodes ricinus and Dermacentor reticulatus, Germany 
Parasites & Vectors  2012;5:285.
Background
Candidatus Neoehrlichia mikurensis (CNM) has been described in the hard tick Ixodes ricinus and rodents as well as in some severe cases of human disease. The aims of this study were to identify DNA of CNM in small mammals, the ticks parasitizing them and questing ticks in areas with sympatric existence of Ixodes ricinus and Dermacentor reticulatus in Germany.
Methods
Blood, transudate and organ samples (spleen, kidney, liver, skin) of 91 small mammals and host-attached ticks from altogether 50 small mammals as well as questing I. ricinus ticks (n=782) were screened with a real-time PCR for DNA of CNM.
Results
52.7% of the small mammals were positive for CNM-DNA. The majority of the infected animals were yellow-necked mice (Apodemus flavicollis) and bank voles (Myodes glareolus). Small mammals with tick infestation were more often infected with CNM than small mammals without ticks. Compared with the prevalence of ~25% in the questing I. ricinus ticks, twice the prevalence in the rodents provides evidence for their role as reservoir hosts for CNM.
Conclusion
The high prevalence of this pathogen in the investigated areas in both rodents and ticks points towards the need for more specific investigation on its role as a human pathogen.
doi:10.1186/1756-3305-5-285
PMCID: PMC3533915  PMID: 23216786
Candidatus Neoehrlichia mikurensis; Bank vole; Yellow-necked mouse; Ixodes ricinus; Dermacentor reticulatus; Recreational area; Host survey; Vector-host relation
11.  Close Geographic Association of Human Neoehrlichiosis and Tick Populations Carrying “Candidatus Neoehrlichia mikurensis” in Eastern Switzerland 
Journal of Clinical Microbiology  2013;51(1):169-176.
Neoehrlichiosis caused by “Candidatus Neoehrlichia mikurensis” is an emerging zoonotic disease. In total, six patients have been described in Europe, with the first case detected in 2007. In addition, seven patients from China were described in a report published in October 2012. In 2009, we diagnosed the first human case of “Ca. Neoehrlichia mikurensis” infection in the Zurich area (Switzerland). Here, we report two additional human cases from the same region, which were identified by broad-range 16S rRNA gene PCR. Both patients were immunocompromised and presented with similar clinical syndromes, including fever, malaise, and weight loss. A diagnostic multiplex real-time PCR was developed for specific detection of “Ca. Neoehrlichia mikurensis” infections. The assay is based on the signature sequence of a 280-bp fragment of the “Ca. Neoehrlichia mikurensis” 16S rRNA gene and incorporates a “Ca. Neoehrlichia mikurensis” species, a “Ca. Neoehrlichia” genus, and an Anaplasmataceae family probe for simultaneous screening. The analytical sensitivity was determined to be below five copies of the “Ca. Neoehrlichia mikurensis” 16S rRNA gene. Our results show that the assay is suitable for the direct detection of “Ca. Neoehrlichia mikurensis” DNA in clinical samples from, for example, blood and bone marrow. In addition, it allows for monitoring treatment response during antibiotic therapy. Using the same assay, DNA extracts from 1,916 ticks collected in four forests in close proximity to the patients' residences (<3 km) were screened. At all sampling sites, the minimal prevalence of “Ca. Neoehrlichia mikurensis” was between 3.5 to 8% in pools of either nymphs, males, or females, showing a strong geographic association between the three patients and the assumed vector.
doi:10.1128/JCM.01955-12
PMCID: PMC3536216  PMID: 23115262
12.  Detection of “Candidatus Neoehrlichia mikurensis” in Two Patients with Severe Febrile Illnesses: Evidence for a European Sequence Variant▿  
Journal of Clinical Microbiology  2010;48(7):2630-2635.
Recently, a new genus of Anaplasmataceae termed “Candidatus Neoehrlichia” was discovered in ticks and rodents. Here, we report on two patients who suffered from febrile bacteremia due to “Candidatus Neoehrlichia mikurensis” associated with thrombotic or hemorrhagic events. 16S rRNA and groEL gene sequencing provided evidence of three groups of sequence variants.
doi:10.1128/JCM.00588-10
PMCID: PMC2897504  PMID: 20519481
13.  “Candidatus Neoehrlichia mikurensis” Infection in a Dog from Germany▿ 
Journal of Clinical Microbiology  2011;49(5):2059-2062.
“Candidatus Neoehrlichia mikurensis” is a new intracellular pathogen associated with human infection and death. “Candidatus Neoehrlichia mikurensis” infection in a chronically neutropenic dog from Germany was confirmed by DNA sequencing. The same organism was previously described from ticks and two sick human beings from Germany.
doi:10.1128/JCM.02327-10
PMCID: PMC3122698  PMID: 21367991
14.  Septicemia Caused by Tick-borne Bacterial Pathogen Candidatus Neoehrlichia mikurensis 
Emerging Infectious Diseases  2010;16(7):1127-1129.
We have repeatedly detected Candidatus Neoehrlichia mikurensis, a bacterium first described in Rattus norvegicus rats and Ixodes ovatus ticks in Japan in 2004 in the blood of a 61-year-old man with signs of septicemia by 16S rRNA and groEL gene PCR. After 6 weeks of therapy with doxycycline and rifampin, the patient recovered.
doi:10.3201/eid1607.091907
PMCID: PMC3358111  PMID: 20587186
Candidatus Neoehrlichia mikurensis; septicemia; human infection; 16S rRNA gene PCR; therapy; tick-borne pathogen; bacteria; dispatch
15.  Anaplasma phagocytophilum in ticks in Slovenia 
Parasites & Vectors  2010;3:102.
Ticks act as vectors of many pathogens of domestic animals and humans. Anaplasma phagocytophilum in Europe is transmitted by the ixodid tick vector Ixodes ricinus. A. phagocytophilum causes a disease with diverse clinical signs in various hosts. A great genetic diversity of the groESL operon of A. phagocytophilum has been found in ticks elsewhere. In Slovenia, the variety of the groESL operon was conducted only on deer samples. In this study, the prevalence of infected ticks was estimated and the diversity of A. phagocytophilum was evaluated. On 8 locations in Slovenia, 1924 and 5049 (6973) I. ricinus ticks were collected from vegetation in the years 2005 and 2006, respectively. All three feeding stages of the tick's life cycle were examined. The prevalence of ticks infected with A. phagocytophilum in the year 2005 and in the year 2006 was 0.31% and 0.63%, respectively, and it did not differ considerably between locations. The similarity among the sequences of groESL ranged from 95.6% to 99.8%. They clustered in two genetic lineages along with A. phagocytophilum from Slovenian deer. One sequence formed a separate cluster. According to our study, the prevalence of A. phagocytophilum in ticks is comparable to the findings in other studies in Europe, and it does not vary considerably between locations and tick stages. According to groESL operon analysis, two genetic lineages have been confirmed and one proposed. Further studies on other genes would be useful to obtain more information on genetic diversity of A. phagocytophilum in ticks in Slovenia.
doi:10.1186/1756-3305-3-102
PMCID: PMC2988007  PMID: 21050436
16.  Spotted fever group rickettsiae in Dermacentor reticulatus and Haemaphysalis punctata ticks in the UK 
Parasites & Vectors  2013;6:212.
Background
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1186/1756-3305-6-212
PMCID: PMC3725166  PMID: 23870197
Haemaphysalis; Rickettsia massiliae; UK; Dermacentor; Rickettsiae; Ticks
17.  Association of Borrelia garinii and B. valaisiana with Songbirds in Slovakia 
In Europe, 6 of the 11 genospecies of Borrelia burgdorferi sensu lato are prevalent in questing Ixodes ricinus ticks. In most parts of Central Europe, B. afzelii, B. garinii, and B. valaisiana are the most frequent species, whereas B. burgdorferi sensu stricto, B. bissettii, and B. lusitaniae are rare. Previously, it has been shown that B. afzelii is associated with European rodents. Therefore, the aim of this study was to identify reservoir hosts of B. garinii and B. valaisiana in Slovakia. Songbirds were captured in a woodland near Bratislava and investigated for engorged ticks. Questing I. ricinus ticks were collected in the same region. Both tick pools were analyzed for spirochete infections by PCR, followed by DNA-DNA hybridization and, for a subsample, by nucleotide sequencing. Three of the 17 captured songbird species were infested with spirochete-infected ticks. Spirochetes in ticks that had fed on birds were genotyped as B. garinii and B. valaisiana, whereas questing ticks were infected with B. afzelii, B. garinii, and B. valaisiana. Furthermore, identical ospA alleles of B. garinii were found in ticks that had fed on the birds and in questing ticks. The data show that songbirds are reservoir hosts of B. garinii and B. valaisiana but not of B. afzelii. This and previous studies confirm that B. burgdorferi sensu lato is host associated and that this bacterial species complex contains different ecotypes.
doi:10.1128/AEM.69.5.2825-2830.2003
PMCID: PMC154513  PMID: 12732554
18.  Genetic variants of Anaplasma phagocytophilum from 14 equine granulocytic anaplasmosis cases 
Parasites & Vectors  2011;4:161.
Background
Equine Granulocytic Anaplasmosis (EGA) is caused by Anaplasma phagocytophilum, a tick-transmitted, obligate intracellular bacterium. In Europe, it is transmitted by Ixodes ricinus. A large number of genetic variants of A. phagocytophilum circulate in nature and have been found in ticks and different animals. Attempts have been made to assign certain genetic variants to certain host species or pathologies, but have not been successful so far. The purpose of this study was to investigate the causing agent A. phagocytophilum of 14 cases of EGA in naturally infected horses with molecular methods on the basis of 4 partial genes (16S rRNA, groEL, msp2, and msp4).
Results
All DNA extracts of EDTA-blood samples of the horses gave bands of the correct nucleotide size in all four genotyping PCRs. Sequence analysis revealed 4 different variants in the partial 16S rRNA, groEL gene and msp2 genes, and 3 in the msp4 gene. One 16S rRNA gene variant involved in 11 of the 14 cases was identical to the "prototype" variant causing disease in humans in the amplified part [GenBank: U02521]. Phylogenetic analysis revealed as expected for the groEL gene that sequences from horses clustered separately from roe deer. Sequences of the partial msp2 gene from this study formed a separate cluster from ruminant variants in Europe and from all US variants.
Conclusions
The results show that more than one variant of A. phagocytophilum seems to be involved in EGA in Germany. The comparative genetic analysis of the variants involved points towards different natural cycles in the epidemiology of A. phagocytophilum, possibly involving different reservoir hosts or host adaptation, rather than a strict species separation.
doi:10.1186/1756-3305-4-161
PMCID: PMC3170280  PMID: 21843364
19.  Wide Distribution and Genetic Diversity of “Candidatus Neoehrlichia mikurensis” in Rodents from China 
“Candidatus Neoehrlichia mikurensis” was detected by PCR in 4.0% (34/841) of the rodents tested in this study. The 34 rodents represented nine species from seven regions of China. Phylogenetic analyses based on the partial groEL and nearly entire 16S rRNA gene sequences of the agent revealed genetic diversity, which was correlated with its geographic origins.
doi:10.1128/AEM.02917-12
PMCID: PMC3568564  PMID: 23183973
20.  A system to simultaneously detect tick-borne pathogens based on the variability of the 16S ribosomal genes 
Parasites & Vectors  2013;6:269.
Background
DNA microarrays can be used to quickly and sensitively identify several different pathogens in one step. Our previously developed DNA microarray, based on the detection of variable regions in the 16S rDNA gene (rrs), which are specific for each selected bacterial genus, allowed the concurrent detection of Borrelia spp., Anaplasma spp., Francisella spp., Rickettsia spp. and Coxiella spp.
Methods
In this study, we developed a comprehensive detection system consisting of a second generation DNA microarray and quantitative PCRs. New oligonucleotide capture probes specific for Borrelia burgdorferi s.l. genospecies and Candidatus Neoehrlichia mikurensis were included. This new DNA microarray system required substantial changes in solution composition, hybridization conditions and post-hybridization washes.
Results
This second generation chip displayed high specificity and sensitivity. The specificity of the capture probes was tested by hybridizing the DNA microarrays with Cy5-labeled, PCR-generated amplicons encoding the rrs genes of both target and non-target bacteria. The detection limit was determined to be 103 genome copies, which corresponds to 1–2 pg of DNA. A given sample was evaluated as positive if its mean fluorescence was at least 10% of the mean fluorescence of a positive control. Those samples with fluorescence close to the threshold were further analyzed using quantitative PCRs, developed to identify Francisella spp., Rickettsia spp. and Coxiella spp. Like the DNA microarray, the qPCRs were based on the genus specific variable regions of the rrs gene. No unspecific cross-reactions were detected. The detection limit for Francisella spp. was determined to be only 1 genome copy, for Coxiella spp. 10 copies, and for Rickettsia spp., 100 copies.
Conclusions
Our detection system offers a rapid method for the comprehensive identification of tick-borne bacteria, which is applicable to clinical samples. It can also be used to identify both pathogenic and endosymbiontic bacteria in ticks for eco-epidemiological studies, tick laboratory colony testing, and many other applications.
doi:10.1186/1756-3305-6-269
PMCID: PMC3850910  PMID: 24330462
Tick-borne bacteria; DNA microarray; Quantitative PCR
21.  Candidatus Neoehrlichia mikurensis in Bank Voles, France 
Emerging Infectious Diseases  2012;18(12):2063-2065.
To further assess the geographic occurrence, possible vectors, and prevalence of Candidatus Neoehrlichia mikurensis, we analyzed spleen tissues from 276 voles trapped close to human settlements in France; 5 were infected with the organism. Sequencing showed the isolates carried the same genotype as the bacteria that caused disease in humans and animals elsewhere in Europe.
doi:10.3201/eid1812.120846
PMCID: PMC3557860  PMID: 23171720
Candidatus Neoehrlichia mikurensis; rodents; bank vole; Myodes glareolus; France; zoonoses; wildlife; vector-borne infections; ticks; Ixodes ricinus; bacteria
22.  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.
doi:10.1371/journal.pone.0054476
PMCID: PMC3551763  PMID: 23349900
23.  Identity of Ehrlichial DNA Sequences Derived from Ixodes ricinus Ticks with Those Obtained from Patients with Human Granulocytic Ehrlichiosis in Slovenia 
Journal of Clinical Microbiology  1999;37(1):209-210.
Adult Ixodes ricinus (Acari: Ixodidae) ticks collected near Ljubljana, Slovenia, were tested for the agent of human granulocytic ehrlichiosis (HGE) by using PCR assays based on the 16S rRNA gene. Three (3.2%) of 93 ticks were found to contain granulocytic ehrlichiae. Nucleotide sequences of portions of the bacterial groESL heat shock operon amplified from these ticks were identical or nearly (99.8%) identical to those previously determined for human patients with HGE from Slovenia, providing additional evidence that the ticks were infected with the HGE agent. This study identified I. ricinus as the likely vector for these ehrlichial pathogens of humans in this part of Europe.
PMCID: PMC84210  PMID: 9854093
24.  Ecological Factors Characterizing the Prevalence of Bacterial Tick-Borne Pathogens in Ixodes ricinus Ticks in Pastures and Woodlands ▿ §  
Applied and Environmental Microbiology  2010;76(13):4413-4420.
Ecological changes are recognized as an important driver behind the emergence of infectious diseases. The prevalence of infection in ticks depends upon ecological factors that are rarely taken into account simultaneously. Our objective was to investigate the influences of forest fragmentation, vegetation, adult tick hosts, and habitat on the infection prevalence of three tick-borne bacteria, Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, and Rickettsia sp. of the spotted fever group, in questing Ixodes ricinus ticks, taking into account tick characteristics. Samples of questing nymphs and adults were taken from 61 pastures and neighboring woodlands in central France. The ticks were tested by PCR of pools of nymphs and individual adults. The individual infection prevalence was modeled using multivariate regression. The highest infection prevalences were found in adult females collected in woodland sites for B. burgdorferi sensu lato and A. phagocytophilum (16.1% and 10.7%, respectively) and in pasture sites for Rickettsia sp. (8.7%). The infection prevalence in nymphs was lower than 6%. B. burgdorferi sensu lato was more prevalent in woodlands than in pastures. Forest fragmentation favored B. burgdorferi sensu lato and A. phagocytophilum prevalence in woodlands, and in pastures, the B. burgdorferi sensu lato prevalence was favored by shrubby vegetation. Both results are probably because large amounts of edges or shrubs increase the abundance of small vertebrates as reservoir hosts. The Rickettsia sp. prevalence was maximal on pasture with medium forest fragmentation. Female ticks were more infected by B. burgdorferi sensu lato than males and nymphs in woodland sites, which suggests an interaction between the ticks and the bacteria. This study confirms the complexity of the tick-borne pathogen ecology. The findings support the importance of small vertebrates as reservoir hosts and make a case for further studies in Europe on the link between the composition of the reservoir host community and the infection prevalence in ticks.
doi:10.1128/AEM.00610-10
PMCID: PMC2897445  PMID: 20453131
25.  Relapsing Fever–Like Spirochetes Infecting European Vector Tick of Lyme Disease Agent 
Emerging Infectious Diseases  2003;9(6):697-701.
To determine whether relapsing fever–like spirochetes associated with hard ticks may infect Ixodes ricinus ticks in central Europe, we screened questing ticks for 16S rDNA similar to that of Asian and American relapsing fever–like spirochetes. We compared the prevalence of these spirochetes to that of Lyme disease spirochetes transmitted by the same vector. Relapsing fever-like spirochetes infect 3.5% of questing vector ticks in our three central European sites near the Rhein Valley. These spirochetes differ genetically from their American and Asian analogs while being relatively homogeneous in the region we sampled. The Lyme disease genospecies most commonly detected in central Europe are distributed broadly, whereas those that are less frequently found appear to be place-specific. The absence of co-infected ticks suggests that relapsing fever–like and Lyme disease spirochetes may not share hosts. Exposure risk for relapsing fever–like spirochetes is similar to that of certain Lyme disease genospecies. Although many persons may be bitten by ticks infected by relapsing fever–like spirochetes, health implications remain unknown.
doi:10.3201/eid0906.020459
PMCID: PMC3000135  PMID: 12781009
spirochetes; relapsing fever; Lyme disease; hard ticks; genospecies; Ixodes ricinus; research

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