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1.  Prevalence of Neoehrlichia mikurensis in ticks and rodents from North-west Europe 
Parasites & Vectors  2012;5:74.
Background
Neoehrlichia mikurensis s an emerging and vector-borne zoonosis: The first human disease cases were reported in 2010. Limited information is available about the prevalence and distribution of Neoehrlichia mikurensis in Europe, its natural life cycle and reservoir hosts. An Ehrlichia-like schotti variant has been described in questing Ixodes ricinus ticks, which could be identical to Neoehrlichia mikurensis.
Methods
Three genetic markers, 16S rDNA, gltA and GroEL, of Ehrlichia schotti-positive tick lysates were amplified, sequenced and compared to sequences from Neoehrlichia mikurensis. Based on these DNA sequences, a multiplex real-time PCR was developed to specifically detect Neoehrlichia mikurensis in combination with Anaplasma phagocytophilum in tick lysates. Various tick species from different life-stages, particularly Ixodes ricinus nymphs, were collected from the vegetation or wildlife. Tick lysates and DNA derived from organs of wild rodents were tested by PCR-based methods for the presence of Neoehrlichia mikurensis. Prevalence of Neoehrlichia mikurensis was calculated together with confidence intervals using Fisher's exact test.
Results
The three genetic markers of Ehrlichia schotti-positive field isolates were similar or identical to Neoehrlichia mikurensis. Neoehrlichia mikurensis was found to be ubiquitously spread in the Netherlands and Belgium, but was not detected in the 401 tick samples from the UK. Neoehrlichia mikurensis was found in nymphs and adult Ixodes ricinus ticks, but neither in their larvae, nor in any other tick species tested. Neoehrlichia mikurensis was detected in diverse organs of some rodent species. Engorging ticks from red deer, European mouflon, wild boar and sheep were found positive for Neoehrlichia mikurensis.
Conclusions
Ehrlichia schotti is similar, if not identical, to Neoehrlichia mikurensis. Neoehrlichia mikurensis is present in questing Ixodes ricinus ticks throughout the Netherlands and Belgium. We propose that Ixodes ricinus can transstadially, but not transovarially, transmit this microorganism, and that different rodent species may act as reservoir hosts. These data further imply that wildlife and humans are frequently exposed to Neoehrlichia mikurensis-infected ticks through tick bites. Future studies should aim to investigate to what extent Neoehrlichia mikurensis poses a risk to public health.
doi:10.1186/1756-3305-5-74
PMCID: PMC3395572  PMID: 22515314
Vector-borne disease; Emerging zoonoses; Candidatus N. mikurensis; I. ricinus; Anaplasma phagocytophylum
2.  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
3.  Molecular Detection and Characterization of Tick-borne Pathogens in Dogs and Ticks from Nigeria 
Background
Only limited information is currently available on the prevalence of vector borne and zoonotic pathogens in dogs and ticks in Nigeria. The aim of this study was to use molecular techniques to detect and characterize vector borne pathogens in dogs and ticks from Nigeria.
Methodology/Principal Findings
Blood samples and ticks (Rhipicephalus sanguineus, Rhipicephalus turanicus and Heamaphysalis leachi) collected from 181 dogs from Nigeria were molecularly screened for human and animal vector-borne pathogens by PCR and sequencing. DNA of Hepatozoon canis (41.4%), Ehrlichia canis (12.7%), Rickettsia spp. (8.8%), Babesia rossi (6.6%), Anaplasma platys (6.6%), Babesia vogeli (0.6%) and Theileria sp. (0.6%) was detected in the blood samples. DNA of E. canis (23.7%), H. canis (21.1%), Rickettsia spp. (10.5%), Candidatus Neoehrlichia mikurensis (5.3%) and A. platys (1.9%) was detected in 258 ticks collected from 42 of the 181 dogs. Co- infections with two pathogens were present in 37% of the dogs examined and one dog was co-infected with 3 pathogens. DNA of Rickettsia conorii israelensis was detected in one dog and Rhipicephalus sanguineus tick. DNA of another human pathogen, Candidatus N. mikurensis was detected in Rhipicephalus sanguineus and Heamaphysalis leachi ticks, and is the first description of Candidatus N. mikurensis in Africa. The Theileria sp. DNA detected in a local dog in this study had 98% sequence identity to Theileria ovis from sheep.
Conclusions/Significance
The results of this study indicate that human and animal pathogens are abundant in dogs and their ticks in Nigeria and portray the potential high risk of human exposure to infection with these agents.
Author Summary
In Nigeria, dogs are not only kept as pets, but are also used for hunting as well as a source of animal protein among some ethnic groups. Ticks are known to infest dogs and serve as vectors for some pathogens of zoonotic and veterinary importance. There is limited information on the prevalence and distribution of vector borne pathogens in dogs and ticks in Nigeria. The aim of the study was to detect and characterize vector borne pathogens in dogs and ticks from Nigeria using molecular methods. The results of this study showed a high estimate of vector borne pathogens in Nigerian dogs (77.3%) and ticks (63.3%). DNA of Candidatus N. mikurensis, an emerging pathogen of humans was detected in Rhipicephalus sanguineus and Heamaphysalis leachi ticks. Another human pathogen, Rickettsia conorii israelensis the causative agent of Mediterranean spotted fever was detected in Rhipicephalus sanguineus ticks. This is the first description of Candidatus N. mikurensis in Africa and Rickettsia conorii israelensis in Nigeria. These results indicate that the use of molecular techniques for the diagnosis of emerging infections in developing countries is of utmost importance in assisting physicians and veterinarians in making accurate diagnoses and providing the appropriate treatment for their patients.
doi:10.1371/journal.pntd.0002108
PMCID: PMC3591325  PMID: 23505591
4.  Infection Dynamics of the Tick-Borne Pathogen “Candidatus Neoehrlichia mikurensis” and Coinfections with Borrelia afzelii in Bank Voles in Southern Sweden 
The tick-borne bacterium “Candidatus Neoehrlichia mikurensis” has recently been recognized as a human pathogen. Together with Borrelia afzelii, it is one of the most common pathogens found in the tick Ixodes ricinus. Here, we compared the epidemiologies of “Ca. Neoehrlichia mikurensis” and B. afzelii by longitudinal sampling from May to September in one of their most abundant vertebrate hosts, the bank vole (Myodes glareolus), using real-time PCR for detection and quantification. The prevalences of “Ca. Neoehrlichia mikurensis” and B. afzelii were determined to be 19% (50/261) and 22% (56/261), respectively. The prevalence of “Ca. Neoehrlichia mikurensis” increased significantly during the sampling season. The clearance rate of “Ca. Neoehrlichia mikurensis” was significantly higher than that of B. afzelii. We found a high frequency of double infections; 46% of all samples infected with “Ca. Neoehrlichia mikurensis” also had a coinfection with B. afzelii. The frequency of coinfections was significantly higher than expected from the prevalence of each pathogen. The high level of coinfections can be caused by interactions between the pathogens or might reflect variation in general susceptibility among voles.
doi:10.1128/AEM.03469-13
PMCID: PMC3957614  PMID: 24375128
5.  “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
6.  Ticks infesting humans in Italy and associated pathogens 
Parasites & Vectors  2014;7:328.
Background
Ticks may transmit a large variety of pathogens, which cause illnesses in animals and humans, commonly referred to as to tick-borne diseases (TBDs). The incidence of human TBDs in Italy is underestimated because of poor surveillance and the scant amount of studies available.
Methods
Samples (n = 561) were collected from humans in four main geographical areas of Italy (i.e., northwestern, northeastern, southern Italy, and Sicily), which represent a variety of environments. After being morphologically identified, ticks were molecularly tested with selected protocols for the presence of pathogens of the genera Rickettsia, Babesia, Theileria, Candidatus Neoehrlichia mikurensis, Borrelia and Anaplasma.
Results
Ticks belonged to 16 species of the genera Argas, Dermacentor, Haemaphysalis, Hyalomma, Ixodes and Rhipicephalus, with Ixodes ricinus (59.5%) being the species most frequently retrieved, followed by Rhipicephalus sanguineus sensu lato (21.4%). Nymphs were the life stage most frequently retrieved (41%), followed by adult females (34.6%). The overall positivity to any pathogen detected was 18%. Detected microorganisms were Rickettsia spp. (17.0%), Anaplasma phagocytophilum (0.8%), Borrelia afzelii (0.5%), Borrelia valaisiana (0.3%), C. N. mikurensis (0.5%) and Babesia venatorum (0.6%).
Conclusions
Results indicate that people living in the Italian peninsula are at risk of being bitten by different tick species, which may transmit a plethora of TBD causing pathogens and that co-infections may also occur.
doi:10.1186/1756-3305-7-328
PMCID: PMC4223688  PMID: 25023709
Ticks; Pathogens; Humans; Tick-borne diseases; Italy; Distribution
7.  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
8.  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
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.  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
11.  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
12.  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
13.  High-throughput screening of tick-borne pathogens in Europe 
Due to increased travel, climatic, and environmental changes, the incidence of tick-borne disease in both humans and animals is increasing throughout Europe. Therefore, extended surveillance tools are desirable. To accurately screen tick-borne pathogens (TBPs), a large scale epidemiological study was conducted on 7050 Ixodes ricinus nymphs collected from France, Denmark, and the Netherlands using a powerful new high-throughput approach. This advanced methodology permitted the simultaneous detection of 25 bacterial, and 12 parasitic species (including; Borrelia, Anaplasma, Ehrlichia, Rickettsia, Bartonella, Candidatus Neoehrlichia, Coxiella, Francisella, Babesia, and Theileria genus) across 94 samples. We successfully determined the prevalence of expected (Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Rickettsia helvetica, Candidatus Neoehrlichia mikurensis, Babesia divergens, Babesia venatorum), unexpected (Borrelia miyamotoi), and rare (Bartonella henselae) pathogens in the three European countries. Moreover we detected Borrelia spielmanii, Borrelia miyamotoi, Babesia divergens, and Babesia venatorum for the first time in Danish ticks. This surveillance method represents a major improvement in epidemiological studies, able to facilitate comprehensive testing of TBPs, and which can also be customized to monitor emerging diseases.
doi:10.3389/fcimb.2014.00103
PMCID: PMC4114295  PMID: 25120960
tick borne diseases; molecular epidemiology; surveillance; Europe; microfluidic analyses
14.  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
15.  Candidatus Neoehrlichia mikurensis and Anaplasma phagocytophilum: prevalences and investigations on a new transmission path in small mammals and ixodid ticks 
Parasites & Vectors  2014;7(1):563.
Background
Small mammals are crucial for the life history of ixodid ticks, but their role and importance in the transmission cycle of tick-borne pathogens is mostly unknown. Candidatus Neoehrlichia mikurensis (CNM) and Anaplasma phagocytophilum are both tick-borne pathogens, and rodents are discussed to serve as main reservoir hosts for CNM but not for the latter especially in Germany. Analysing the prevalence of both pathogens in small mammals and their ticks in endemic regions may help to elucidate possible transmission paths in small mammal populations and between small mammals and ticks.
Methods
In 2012 and 2013, small mammals were trapped at three different sites in Germany. DNA was extracted from different small mammal tissues, from rodent neonates, foetuses and from questing and attached ticks. DNA samples were tested for CNM and A. phagocytophilum by real-time PCR. Samples positive for A. phagocytophilum were further characterized at the 16S rRNA gene locus.
Results
CNM was detected in 28.6% of small mammals and in 2.2% of questing and 3.8% of attached ticks. Altogether 33 positive ticks were attached to 17 different hosts, while positive ticks per host ranged between one and seven. The prevalences for this pathogen differed significantly within small mammal populations comparing sites (χ2: 13.3987; p: 0.0004) and between sexes. Male rodents had an approximately two times higher chance of infection than females (OR: 1.9652; 95% CI: 1.32-2.92). The prevalence for CNM was 31.8% (95% CI: 22-44) in rodent foetuses and neonates (23 of 67) from positive dams, and 60% (95% CI: 35.7-80.25) of positive gravid or recently parturient rodents (9 out of 15) had at least one positive foetus or neonate. Anaplasma phagocytophilum was detected at a low percentage in rodents (0-5.6%) and host-attached ticks (0.5-2.9%) with no significant differences between rodent species. However, attached nymphs were significantly more often infected than attached larvae (χ2: 25.091; p: <0.0001).
Conclusion
This study suggests that CNM is mainly a rodent-associated pathogen and provides evidence for a potential transplacental transmission in rodents. In contrast, most of the rodent species captured likely represent only accidental hosts for A. phagocytophilum at the investigated sites.
doi:10.1186/s13071-014-0563-x
PMCID: PMC4264555  PMID: 25465390
Candidatus Neoehrlichia mikurensis; Anaplasma phagocytophilum; Ixodes ricinus; Emerging pathogen; Small mammals; Tick-borne Pathogen
16.  Asymptomatic “Candidatus Neoehrlichia mikurensis” Infections in Immunocompetent Humans 
Journal of Clinical Microbiology  2014;52(8):3072-3074.
In Europe, human infections with “Candidatus Neoehrlichia mikurensis” have mainly been restricted to immunocompromised patients. We report here the first cases of asymptomatic “Ca. Neoehrlichia mikurensis” infection in immunocompetent humans (5/316 [1.6%] were infected). Due to the potential threats of infections with “Ca. Neoehrlichia mikurensis” in healthy persons to the safety of the blood supply, further study of this phenomenon is required.
doi:10.1128/JCM.00741-14
PMCID: PMC4136151  PMID: 24899023
17.  First Case of Human “Candidatus Neoehrlichia mikurensis” Infection in a Febrile Patient with Chronic Lymphocytic Leukemia ▿  
Journal of Clinical Microbiology  2010;48(5):1956-1959.
An immunocompromised patient presented with febrile episodes, an erysipelas-like rash, and thromboembolic complications. Amplification of 16S rRNA gene sequences from blood and sequence analysis revealed “Candidatus Neoehrlichia mikurensis.” We report the first case of human disease caused by “Ca. Neoehrlichia mikurensis.”
doi:10.1128/JCM.02423-09
PMCID: PMC2863919  PMID: 20220155
18.  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
19.  Birds as potential reservoirs of tick-borne pathogens: first evidence of bacteraemia with Rickettsia helvetica 
Parasites & Vectors  2014;7:128.
Background
Birds have long been known as carriers of ticks, but data from the literature are lacking on their role as a reservoir in the epidemiology of certain tick-borne disease-causing agents. Therefore, the aim of this study was to evaluate the presence of three emerging, zoonotic tick-borne pathogens in blood samples and ticks of birds and to assess the impact of feeding location preference and migration distance of bird species on their tick infestation.
Methods
Blood samples and ticks of birds were analysed with TaqMan real-time PCRs and conventional PCR followed by sequencing.
Results
During the spring and autumn bird migrations, 128 blood samples and 140 ticks (Ixodes ricinus, Haemaphysalis concinna and a Hyalomma specimen) were collected from birds belonging to 16 species. The prevalence of tick infestation and the presence of tick species were related to the feeding and migration habits of avian hosts. Birds were shown to be bacteraemic with Rickettsia helvetica and Anaplasma phagocytophilum, but not with Candidatus Neoehrlichia mikurensis. The prevalence of rickettsiae was high (51.4%) in ticks, suggesting that some of them may have acquired their infection from their avian host.
Conclusion
Based on the present results birds are potential reservoirs of both I. ricinus transmitted zoonotic pathogens, R. helvetica and A. phagocytophilum, but their epidemiological role appears to be less important concerning the latter, at least in Central Europe.
doi:10.1186/1756-3305-7-128
PMCID: PMC3976504  PMID: 24679245
Ground feeding birds; Migratory birds; Ticks; Rickettsia helvetica; Anaplasma phagocytophilum; Candidatus Neoehrlichia mikurensis
20.  “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
21.  Characterization of ‘Candidatus Neoehrlichia lotoris’ (family Anaplasmataceae) from raccoons (Procyon lotor) 
Recently, a novel ehrlichial organism was isolated from a raccoon (Procyon lotor) and the isolate (RAC413) was infectious to two naïve raccoons but not laboratory mice, rats or rabbits. In this study, amplification and sequencing of four gene targets (16S rRNA gene, groESL, gltA and rpoB) confirmed that the novel ehrlichial organism was a member of the family Anaplasmataceae and was most closely related to, but distinct from, ‘Candidatus Neoehrlichia mikurensis’ TK4456R and IS58. RAC413 shared the highest sequence similarity with members of the genus Ehrlichia (94.2–95.1, 80.9–83.1, 67.9–71.9 and 39.9–40.7 % similarity for the 16S rRNA gene, groESL, gltA and rpoB, respectively). No sequence variation in three sequences (16S rRNA gene, groESL and gltA) was observed between the RAC413 isolate and five additional sequences amplified from blood of naturally infected raccoons from several geographically isolated populations in the south-eastern USA. Serum samples from four experimentally infected raccoons did not react to Ehrlichia canis, Ehrlichia chaffeensis, Anaplasma marginale or Anaplasma phagocytophilum antigens in an immunofluorescence assay or an Ehrlichia ewingii peptide in an ELISA format. On the basis of the distinctive molecular and serological characteristics and apparent host specificity of this ehrlichial organism, it is proposed that this organism be designated ‘Candidatus Neoehrlichia lotoris’ (reference strain RAC413R).
doi:10.1099/ijs.0.65836-0
PMCID: PMC4278589  PMID: 19060060
22.  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
23.  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
24.  Ixodes ricinus and Its Transmitted Pathogens in Urban and Peri-Urban Areas in Europe: New Hazards and Relevance for Public Health 
Tick-borne diseases represent major public and animal health issues worldwide. Ixodes ricinus, primarily associated with deciduous and mixed forests, is the principal vector of causative agents of viral, bacterial, and protozoan zoonotic diseases in Europe. Recently, abundant tick populations have been observed in European urban green areas, which are of public health relevance due to the exposure of humans and domesticated animals to potentially infected ticks. In urban habitats, small and medium-sized mammals, birds, companion animals (dogs and cats), and larger mammals (roe deer and wild boar) play a role in maintenance of tick populations and as reservoirs of tick-borne pathogens. Presence of ticks infected with tick-borne encephalitis virus and high prevalence of ticks infected with Borrelia burgdorferi s.l., causing Lyme borreliosis, have been reported from urbanized areas in Europe. Emerging pathogens, including bacteria of the order Rickettsiales (Anaplasma phagocytophilum, “Candidatus Neoehrlichia mikurensis,” Rickettsia helvetica, and R. monacensis), Borrelia miyamotoi, and protozoans (Babesia divergens, B. venatorum, and B. microti) have also been detected in urban tick populations. Understanding the ecology of ticks and their associations with hosts in a European urbanized environment is crucial to quantify parameters necessary for risk pre-assessment and identification of public health strategies for control and prevention of tick-borne diseases.
doi:10.3389/fpubh.2014.00251
PMCID: PMC4248671  PMID: 25520947
ticks; Ixodes ricinus; tick-borne pathogens; urban habitats; Europe
25.  Pathogens in ticks collected from dogs in Berlin/Brandenburg, Germany 
Parasites & Vectors  2014;7(1):535.
Background
Tick-borne diseases are a major health risk for humans and dogs. In addition to collection and analysis of questing ticks, analysis of host-associated ticks for the presence of pathogens is a valuable method to gain insight into transmission patterns of tick-borne diseases.
Methods
Ticks were collected from dogs living in the Berlin/Brandenburg area. The three tick species Ixodes ricinus, Ixodes hexagonus and Dermacentor reticulatus were examined for the presence of Babesia spp., Borrelia spp., Rickettsia spp. and Anaplasmataceae. Conventional PCR followed by sequencing was used for pathogen detection and characterization.
Results
Babesia spp. were found in 2.5% and 3% of I. ricinus and I. hexagonus, respectively. Sequencing revealed the presence of Babesia microti, Babesia capreoli and Babesia venatorum. D. reticulatus were free of Babesia canis. Rickettsia spp. were detected in 61% of I. ricinus, 44% of I. hexagonus and 39% of D. reticulatus. Specifically detected were Rickettsia raoulti in D. reticulatus and I. hexagonus, Rickettsia helvetica in I. ricinus and I. hexagonus and Rickettsia monacensis in I. hexagonus. Anaplasma phagocytophilum and Candidatus Neoehrlichia mikurensis have been reported previously in I. ricinus (6.5% and 4.3%, respectively) and I. hexagonus (3.9% and 5.9%). Borrelia spp. were found in 11.6% of I. ricinus and 11.2% of I. hexagonus. Subsequent genospecies analysis revealed Borrelia afzelii, Borrelia garinii, Borrelia burgdorferi sensu stricto and Borrelia miyamotoi. Simultanous presence of more than one pathogen was found in 20% of I. ricinus and in 59% of I. hexagonus whereas the total frequency of any pathogen was 65% in I. ricinus, 59% in I. hexagonus and 64% in D. reticulatus. Ticks in which A. phagocytophilum was detected had a significantly increased risk of also containing Rickettsia. Ticks harbouring a pathogen had significantly higher scutal indices than ticks without presence of any pathogen.
Conclusions
Frequencies of potential human or canine pathogens in ticks were considerable and DNA of all four groups of pathogens was detected. Differences in scutal indices might suggest that pathogens are frequently taken up by ticks when feeding on dogs in Berlin/Brandenburg.
Electronic supplementary material
The online version of this article (doi:10.1186/s13071-014-0535-1) contains supplementary material, which is available to authorized users.
doi:10.1186/s13071-014-0535-1
PMCID: PMC4262381  PMID: 25441762
Canine vector-borne diseases; Borrelia; Babesia; Rickettsia; Anaplasma; Candidatus neoehrlichia mikurensis

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