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.

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title: Biology of ticks
abstract: Part IV: Ecology, Behavior, and Host-Parasite Interactions.  Ecology of Non-Nidicolous Ticks.  Ecology of Nidiculous Ticks.  Acquired Immunity to Ticks. Part V: Tick-Borne and Tick-Caused Diseases.  Tick-Borne Protozoan Diseases. 27: Tick-Borne Arboviral Diseases.  Tick-Borne Rickettsioses. 29: Tick-Borne Bacteral Diseases. 30: Tick Paralysis and other Tick-Borne Toxicoses.  Control of Ticks and Tick-Borne Diseases.  Control of Ticks.

title: Public health significance of urban pests
abstract: of Urban Pests P ublic H ealth S ignicance of U ran P ests Xavier Bonnefoy Helge Kampen Kevin Sweeney The second half of the 20th century and the beginning of the 21st century have witnessed important changes in ecology, climate and human behaviour that favour the development of urban pests. Most alarmingly, urban planners are faced now with the dramatic expansion of urban sprawl, where the suburbs of our cities are growing into the natural habitats of ticks, rodents and other pests. Also, many city managers now erroneously assume that pest-borne diseases are relics that belong to the past.

title: Impacts of biodiversity on the emergence and transmission of infectious diseases
abstract: Current unprecedented declines in biodiversity reduce the ability of ecological communities to provide many fundamental ecosystem services. Here we evaluate evidence that reduced biodiversity affects the transmission of infectious diseases of humans, other animals and plants. In principle, loss of biodiversity could either increase or decrease disease transmission. However, mounting evidence indicates that biodiversity loss frequently increases disease transmission. In contrast, areas of naturally high biodiversity may serve as a source pool for new pathogens. Overall, despite many remaining questions, current evidence indicates that preserving intact ecosystems and their endemic biodiversity should generally reduce the prevalence of infectious diseases.

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title: Ecology of zoonoses: natural and unnatural histories
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title: Effects of environmental change on zoonotic disease risk: an ecological primer.
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title: Tick-borne disease systems emerge from the shadows: the beauty lies in molecular detail, the message in epidemiology
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title: Tick pests and vectors (Acari: Ixodoidea) in European towns: Introduction, persistence and management.
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title: Urbanization as a major cause of biotic homogenization
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title: Urban biodiversity: patterns and mechanisms
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title: The ecology of tick-borne diseases.
abstract: Zoonotic diseases are major causes of infection related morbidity and mortality worldwide. Of the various arthropods capable of transmitting pathogens that cause such diseases to humans, ticks, which are vectors of more kinds of pathogens than any other group of invertebrate, have become an increasing focus of attention. This is particularly the case in the temperate northern hemisphere where they are a significant vector of human disease. Here, we provide an overview of the complex ecological systems defining the various epidemiological cycles of tick-borne diseases. We highlight the abiotic and biotic factors influencing the establishment and persistence of tick populations and their associated pathogens. Furthermore, we emphasize the dynamic nature of such systems, especially when they are under the influence of both small and large-scale anthropogenic changes to the environment. Although a great deal of work has been done on ticks and the diseases which they transmit, the very dynamism of the system means that new factors are continually arising which shift the epidemiological pattern within specific areas. We therefore consider that more detailed, long-term (i.e. at least 10 years), multidisciplinary studies need to be carried out to define why and how these pattern shifts take place and to determine their public health significance.

title: Ticks and the city: ectoparasites of the Northern white-breasted hedgehog (Erinaceus roumanicus) in an urban park.
abstract: The European hedgehog (Erinaceus europaeus) is known to host several ectoparasites and also tick-borne pathogens, but there is scant information on its eastern relative, the Northern white-breasted hedgehog (Erinaceus roumanicus). We have studied an urban population of E. roumanicus in a city park of central Budapest, Hungary, for 2 years to investigate their tick and flea species. A total of 5063 ticks and 818 fleas were collected from 247 hedgehogs (including 46 recaptures). Ectoparasite prevalence and intensity differed significantly (p<0.001) between the 2 study years attributable to the enhanced tick removal rate due to anaesthesia used in the second year. The most common tick species was Ixodes ricinus (93.7%) followed by unidentified Ixodes larvae (5%). Only 57 hedgehog ticks (I. hexagonus) were removed from 22 hedgehogs. One I. acuminatus and one Hyalomma marginatum nymph were also collected. Mean intensity of tick infestation was 26.5 (range: 0-155 ticks/host) and mean intensity of flea infestation was 6.6 (range: 0-78 fleas/host). Most fleas (99.4%) collected were hedgehog fleas (Archaeopsylla erinacei), dog fleas (Ctenocephalides canis) were found on 2 hedgehogs. Hyalomma marginatum has previously not been found in Hungary, and I. acuminatus was only reported sporadically before. The large number of ectoparasites and the 2 imported tick species may thus survive in close proximity to humans if hedgehogs are present. This calls attention to the risk of possible tick-borne human infections that urban hedgehogs can pose.

title: Urban zoonoses caused by Bartonella, Coxiella, Ehrlichia, and Rickettsia species.
abstract: The last half of the 20th Century witnessed an increase in the occurrence and recognition of urban zoonoses caused by members of the genera Bartonella, Coxiella, Ehrlichia, and Rickettsia, all traditionally considered to be members of the family Rickettsiaceae. In recent years, new human pathogens (Bartonella elizabethae, Bartonella henselae, and Rickettsia felis) have been recognized in urban environments. Other newly recognized pathogens (Ehrlichia chaffeensis and Ehrlichia phagocytophila in the United States) have sylvan zoonotic cycles but are present in urban areas because their vertebrate hosts and associated ectoparasitic arthropod vectors are able to survive in cities. Still other agents, which were primarily of historical importance (Bartonella quintana) or have not traditionally been associated with urban environments (Rickettsia rickettsii), have been recognized as causes of human disease in urban areas. Some diseases that have traditionally been associated with urban environments, such as rickettsialpox (caused by Rickettsia akari) and murine typhus (caused by Rickettsia typhi), still occur in large cities at low or undetermined frequencies and often go undetected, despite the availability of effective measures to diagnose and control them. In addition, alternate transmission cycles have been discovered for Coxiella burnetii, Rickettsia prowazekii, and R. typhi that differ substantially from their established, classic cycles, indicating that the epidemiology of these agents is more complex than originally thought and may be changing. Factors leading to an increase in the incidence of illnesses caused by these bacteria in urban areas include societal changes as well as intrinsic components of the natural history of these organisms that favor their survival in cities. Transovarial and transstadial transmission of many of the agents in their arthropod hosts contributes to the highly focal nature of many of the diseases they cause by allowing the pathogens to persist in areas during adverse times when vertebrate amplifying hosts may be scarce or absent. Domesticated animals (primarily cats, dogs, and livestock) or commensal rodents [primarily Norway rats (Rattus norvegicus) and house mice (Mus musculus)] can serve as vertebrate amplifying hosts and bring these agents and their ectoparasitic arthropod vectors into direct association with humans and help maintain transmission cycles in densely populated urban areas. The reasons for the increase in these urban zoonoses are complex. Increasing population density worldwide, shifts in populations from rural areas to cities, increased domestic and international mobility, an increase in homelessness, the decline of inner-city neighborhoods, and an increase in the population of immunosuppressed individuals all contribute to the emergence and recognition of human diseases caused by these groups of agents. Due to the focal nature of infections in urban areas, control or prevention of these diseases is possible. Increased physician awareness and public health surveillance support will be required to detect and treat existing urban infections caused by these agents, to determine the disease burden caused by them, to design and implement control programs to combat and prevent their spread, and to recognize emerging or resurging infections caused by members of these genera as they occur.

title: A clear and present danger: tick-borne diseases in Europe
abstract: Ticks can transmit a variety of viruses, bacteria or parasites that can cause serious infections or conditions in humans and animals. While tick-borne diseases are becoming an increasing and serious problem in Europe, tick-borne diseases are also responsible for major depressions in livestock production and mortality in sub-Saharan Africa, Latin America and Asia. This review will focus on the most important circulating tick-transmitted pathogens in Europe (Borrelia spp., Anaplasma phagocytophilum, Babesia spp., tick-borne encephalitis virus, Rickettsia spp. and Crimean-Congo hemorrhagic fever virus).

title: Ticks and Tick-borne Diseases: Geographical Distribution and Control Strategies in the Euro-Asia Region
abstract: 1. Emerging (re-emerging) tick-borne infections and dissemination of ticks 2. Generic morphological and geographic distribution of tick species that are involved transmission of animal pathogens including zoonoses 3. Factors influencing the spread and distribution of ticks 4.Tick-borne animal infections (including zoonoses) in Europe and the Mediterranean basin 5. Maps of reported occurrence of tick-borne pathogens 6. Maps of reported occurrence of ticks 7. Surveillance and control measures Appendix 1 - Tables of geographic data of tick-borne pathogens Appendix.2 - List (by ID number) of the references from which geographic data was extracted Appendix 3 - List of references that could not be retrieved on time for the data extraction Appendix 4 - Keys for genera and species of ticks in the Mediterranean basin.

title: Driving forces for changes in geographical distribution of Ixodes ricinus ticks in Europe
abstract: Many factors are involved in determining the latitudinal and altitudinal spread of the important tick vector Ixodes ricinus (Acari: Ixodidae) in Europe, as well as in changes in the distribution within its prior endemic zones. This paper builds on published literature and unpublished expert opinion from the VBORNET network with the aim of reviewing the evidence for these changes in Europe and discusses the many climatic, ecological, landscape and anthropogenic drivers. These can be divided into those directly related to climatic change, contributing to an expansion in the tick’s geographic range at extremes of altitude in central Europe, and at extremes of latitude in Scandinavia; those related to changes in the distribution of tick hosts, particularly roe deer and other cervids; other ecological changes such as habitat connectivity and changes in land management; and finally, anthropogenically induced changes. These factors are strongly interlinked and often not well quantified. Although a change in climate plays an important role in certain geographic regions, for much of Europe it is non-climatic factors that are becoming increasingly important. How we manage habitats on a landscape scale, and the changes in the distribution and abundance of tick hosts are important considerations during our assessment and management of the public health risks associated with ticks and tick-borne disease issues in 21st century Europe. Better understanding and mapping of the spread of I. ricinus (and changes in its abundance) is, however, essential to assess the risk of the spread of infections transmitted by this vector species. Enhanced tick surveillance with harmonized approaches for comparison of data enabling the follow-up of trends at EU level will improve the messages on risk related to tick-borne diseases to policy makers, other stake holders and to the general public.

title: Biology of ticks
abstract: Part IV: Ecology, Behavior, and Host-Parasite Interactions.  Ecology of Non-Nidicolous Ticks.  Ecology of Nidiculous Ticks.  Acquired Immunity to Ticks. Part V: Tick-Borne and Tick-Caused Diseases.  Tick-Borne Protozoan Diseases. 27: Tick-Borne Arboviral Diseases.  Tick-Borne Rickettsioses. 29: Tick-Borne Bacteral Diseases. 30: Tick Paralysis and other Tick-Borne Toxicoses.  Control of Ticks and Tick-Borne Diseases.  Control of Ticks.

title: Monitoring human tick-borne disease risk and tick bite exposure in Europe: available tools and promising future methods.
abstract: Ticks are the main vector for infectious disease pathogens in both humans and animals, and tick-borne diseases are currently spreading throughout Europe. Various surveillance methods have been developed to estimate the burden and risk of tick-borne diseases and host exposure to tick bites. The ultimate aims of these approaches are to determine the risk level of a tick-borne disease in a given area, determine its health priority, identify the at-risk population and propose specific countermeasures or complementary studies as needed. The purpose of this review is to present the current methods for monitoring the circulation of tick-borne diseases and to highlight the use of salivary antigens as original and recently developed serological tools that could be useful for tick bite risk assessment and could improve the current surveillance methods.

title: Vertical distribution of the tick Ixodes ricinus and tick-borne pathogens in the northern Moravian mountains correlated with climate warming (Jeseníky Mts., Czech Republic).
abstract: A study of the vertical distribution of the common tick Ixodes ricinus and tick-borne pathogens--tick-borne encephalitis virus (TBEV) and genospecies of Borrelia burgdorferi s.l.--was performed in the highest part of the Jeseniky mountain area (the Hrubý Jesenik Mts. with the highest summit Praded, 1,491 m above see level). Altogether 1,253 specimens of all tick stages (607 larvae, 614 nymphs, 8 females and 24 males) were collected at the altitude 990-1,300 m above sea level on 12 collection sites by the flagging method. Altogether 1,207 ticks (8 females, 24 males, 568 nymphs and 607 larvae) were examined for the presence of tick-borne encephalitis virus and B. burgdorferi s.l. None of the samples contained TBEV, 35 samples (6% of adult ticks, 5% of nymphs, 0.7% of larvae) were positive for B. burgdorferi s.l. The most prevalent genospecies were B. afzelii (44%), B. garinii (28%), less frequent were B. burgdorferi sensu stricto (5%), B. valaisiana (3%). The rather large number of ticks (in absolute numbers as well as recounted to the index: average number of nymphs/worker/collection hour) and the presence of all developmental stages clearly demonstrate that there are viable local tick populations in all the sites, and that recorded ticks were not randomly individuals brought into higher altitudes by birds or game animals. The results are compared with the long-term (2002-2007) monitoring of the tick altitudinal distribution in the Krkonose Mts. and the conditions, which allow ticks to establish local populations up to the timberline in both mountain areas, are discussed. Simultaneously, changes in climatic conditions (especially the air temperature) monitored at 3 meteorological stations in the area of the Jeseníky Mts. were compared with the records from another 8 stations in other mountain areas in the Czech Republic. A very similar statistically significant trend of increasing mean air temperatures during the last three decades is found at all analyzed stations. The trend is most pronounced in the spring and summer months with the highest activity of I. ricinus ticks.

title: Multi-source analysis reveals latitudinal and altitudinal shifts in range of Ixodes ricinus at its northern distribution limit
abstract: BackgroundThere is increasing evidence for a latitudinal and altitudinal shift in the distribution range of Ixodes ricinus. The reported incidence of tick-borne disease in humans is on the rise in many European countries and has raised political concern and attracted media attention. It is disputed which factors are responsible for these trends, though many ascribe shifts in distribution range to climate changes. Any possible climate effect would be most easily noticeable close to the tick's geographical distribution limits. In Norway- being the northern limit of this species in Europe- no documentation of changes in range has been published. The objectives of this study were to describe the distribution of I. ricinus in Norway and to evaluate if any range shifts have occurred relative to historical descriptions.MethodsMultiple data sources - such as tick-sighting reports from veterinarians, hunters, and the general public - and surveillance of human and animal tick-borne diseases were compared to describe the present distribution of I. ricinus in Norway. Correlation between data sources and visual comparison of maps revealed spatial consistency. In order to identify the main spatial pattern of tick abundance, a principal component analysis (PCA) was used to obtain a weighted mean of four data sources. The weighted mean explained 67% of the variation of the data sources covering Norway's 430 municipalities and was used to depict the present distribution of I. ricinus. To evaluate if any geographical range shift has occurred in recent decades, the present distribution was compared to historical data from 1943 and 1983.ResultsTick-borne disease and/or observations of I. ricinus was reported in municipalities up to an altitude of 583 metres above sea level (MASL) and is now present in coastal municipalities north to approximately 69°N.ConclusionI. ricinus is currently found further north and at higher altitudes than described in historical records. The approach used in this study, a multi-source analysis, proved useful to assess alterations in tick distribution.

title: Changes in the geographical distribution and abundance of the tick Ixodes ricinus during the past 30 years in Sweden
abstract: BackgroundIxodes ricinus is the main vector in Europe of human-pathogenic Lyme borreliosis (LB) spirochaetes, the tick-borne encephalitis virus (TBEV) and other pathogens of humans and domesticated mammals. The results of a previous 1994 questionnaire, directed at people living in Central and North Sweden (Svealand and Norrland) and aiming to gather information about tick exposure for humans and domestic animals, suggested that Ixodes ricinus ticks had become more widespread in Central Sweden and the southern part of North Sweden from the early 1980s to the early 1990s. To investigate whether the expansion of the tick's northern geographical range and the increasing abundance of ticks in Sweden were still occurring, in 2009 we performed a follow-up survey 16 years after the initial study.MethodsA questionnaire similar to the one used in the 1994 study was published in Swedish magazines aimed at dog owners, home owners, and hunters. The questionnaire was published together with a popular science article about the tick's biology and role as a pathogen vector in Sweden. The magazines were selected to get information from people familiar with ticks and who spend time in areas where ticks might be present.ResultsAnalyses of data from both surveys revealed that during the near 30-year period from the early 1980s to 2008, I. ricinus has expanded its distribution range northwards. In the early 1990s ticks were found in new areas along the northern coastline of the Baltic Sea, while in the 2009 study, ticks were reported for the first time from many locations in North Sweden. This included locations as far north as 66°N and places in the interior part of North Sweden. During this 16-year period the tick's range in Sweden was estimated to have increased by 9.9%. Most of the range expansion occurred in North Sweden (north of 60°N) where the tick's coverage area doubled from 12.5% in the early 1990s to 26.8% in 2008. Moreover, according to the respondents, the abundance of ticks had increased markedly in LB- and TBE-endemic areas in South (Götaland) and Central Sweden.ConclusionsThe results suggest that I. ricinus has expanded its range in North Sweden and has become distinctly more abundant in Central and South Sweden during the last three decades. However, in the northern mountain region I. ricinus is still absent. The increased abundance of the tick can be explained by two main factors: First, the high availability of large numbers of important tick maintenance hosts, i.e., cervids, particularly roe deer (Capreolus capreolus) during the last three decades. Second, a warmer climate with milder winters and a prolonged growing season that permits greater survival and proliferation over a larger geographical area of both the tick itself and deer. High reproductive potential of roe deer, high tick infestation rate and the tendency of roe deer to disperse great distances may explain the range expansion of I. ricinus and particularly the appearance of new TBEV foci far away from old TBEV-endemic localities. The geographical presence of LB in Sweden corresponds to the distribution of I. ricinus. Thus, LB is now an emerging disease risk in many parts of North Sweden. Unless countermeasures are undertaken to keep the deer populations, particularly C. capreolus and Dama dama, at the relatively low levels that prevailed before the late 1970s - especially in and around urban areas where human population density is high - by e.g. reduced hunting of red fox (Vulpes vulpes) and lynx (Lynx lynx), the incidences of human LB and TBE are expected to continue to be high or even to increase in Sweden in coming decades.

title: Changing distributions of ticks: causes and consequences
abstract: Today, we are witnessing changes in the spatial distribution and abundance of many species, including ticks and their associated pathogens. Evidence that these changes are primarily due to climate change, habitat modifications, and the globalisation of human activities are accumulating. Changes in the distribution of ticks and their invasion into new regions can have numerous consequences including modifications in their ecological characteristics and those of endemic species, impacts on the dynamics of local host populations and the emergence of human and livestock disease. Here, we review the principal causes for distributional shifts in tick populations and their consequences in terms of the ecological attributes of the species in question (i.e. phenotypic and genetic responses), pathogen transmission and disease epidemiology. We also describe different methodological approaches currently used to assess and predict such changes and their consequences. We finish with a discussion of new research avenues to develop in order to improve our understanding of these host–vector–pathogen interactions in the context of a changing world.

title: Transmission cycles of Borrelia burgdorferi sensu lato involving Ixodes ricinus and/or I. hexagonus ticks and the European hedgehog, Erinaceus europaeus, in suburban and urban areas in Switzerland.
abstract: The European hedgehog, Erinaceus europaeus Linnaeus, 1758, is a common host of Ixodes ricinus L. and I. hexagonus Leach, vectors of the Lyme disease spirochaete, Borrelia burgdorferi sensu lato. To investigate whether hedgehogs are reservoirs for B. burgdorferi, hedgehogs were captured in a suburban area suitable for both tick species and in an urban area where I. ricinus is absent. The infection status of the hedgehogs was determined by xenodiagnosis using I. ricinus and I. hexagonus larvae. I. hexagonus and/or I. ricinus were found on all hedgehogs (n = 8) from the suburban area. In contrast, only I. hexagonus was infesting animals (n = 5) from the urban area. A total of 12/13 hedgehogs harboured B. burgdorferi infected ticks. Xenodiagnostic I. ricinus and I. hexagonus larvae that fed on hedgehogs became infected. The results clearly show that European hedgehogs are reservoir hosts of the Lyme disease spirochetes. DNA of B. burgdorferi sensu stricto, B. garinii and B. afzelii was detected in culture from ear biopsy and needle aspiration material and characterized by using a genospecies-specific PCR assay. One hedgehog presented a mixed infection of the skin with B. burgdorferi sensu stricto and B. garinii. This study also identifies an enzootic transmission cycle in an urban area involving E. europaeus and I. hexagonus. The close association of I. hexagonus with the burrows of its hosts mean that the risks of contact between I. hexagonus and humans may be low.

title: The ixodid tick species attaching to domestic dogs and cats in Great Britain and Ireland
abstract: The species of ixodid ticks, attached to dogs and cats presented to veterinary practices in Great Britain and Ireland were identified. Most host animals carried only one tick species with Ixodes ricinus Linné (Acari: Ixodidae) being the most common, identified on 52% of animals, Ixodes hexagonus Leach (Acari: Ixodidae) the second most common (on 39%) and Ixodes canisuga Johnston (Acari: Ixodidae) the third most common (on 11%). A significantly higher proportion of dogs than cats carried I. ricinus, while I. hexagonus was more frequently carried by cats. One animal carried a single specimen of Haemaphysalis punctata Canestrini & Fanzago (Acari: Ixodidae), one carried a Dermacentor reticulatus Fabricius (Acari: Ixodidae) but none carried Rhipicephalus sanguineus Latreille (Acari: Ixodidae). This indicates that the latter two species, vectors of ‘exotic’ tick‐borne diseases, remain at low densities in Great Britain and Ireland. Retrospective information on exposure of the animals to different habitats and geographic regions was collected by questionnaire and subject to contingency table and logistic regression analysis. Woodlands and moorlands were habitats significantly associated with I. ricinus attachment. Exposure to urban parks was significantly associated with I. hexagonus attachment and exposure to boarding kennels and catteries was significantly associated with I. canisuga attachment. Ixodes hexagonus, rather than I. ricinus, was the ixodid tick species most likely to be encountered by urban populations of dogs and cats and, by inference, possibly also humans. The implications of these findings, for the transmission of tick‐borne pathogens to dogs, cats and humans are discussed.

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

title: Borrelia burgdorferi infection prevalences in questing Ixodes ricinus ticks (Acari: Ixodidae) in urban and suburban Bonn, western Germany
abstract: From March to October 2003, a total of 2,518 host-seeking Ixodes ricinus ticks (1,944 nymphs, 264 females, 310 males) were collected by blanket dragging at 45 sites all over the city area of Bonn, western Germany, to be checked for Borrelia burgdorferi infection. The collection sites included 20 private gardens, nine public recreational parks, the boundaries of 14 sylvatic suburban areas and two footpaths between suburban farmed fields. Generally, numbers of specimens collected along sylvatic suburban areas and at urban sites with dense tree populations were significantly higher than at the other collection sites. Out of 1,394 specimens (865 nymphs, 241 females, 288 males) that were randomly chosen for Borrelia analysis by a simple PCR, 250 (17.9 %) were found to be infected with B. burgdorferi sensu lato. While the infection prevalences varied significantly between females (26.6%), males (12.5%) and nymphs (17.3%), there were no striking differences between sylvatic and unwooded sites. A total of 92.8% of the ticks Borrelia-positive by the simple PCR were also positive in a diagnostic nested PCR. Using genospecies-specific oligonucleotide probes, single Borrelia genospecies infections (91.4%) could be assigned to B. afzelii (39.5%), B. garinii (27.9%), B. burgdorferi sensu stricto (15.6%) and B. valaisiana (8.6%) by DNA hybridization. Various combinations of double infections were observed in 4.3% of the infected ticks. Another 4.3% of the Borrelia infections were untypeable. The B. burgdorferi genospecies distribution in the city area was shown to be variable from site to site and, even more, it was distinct from rural collection sites near Bonn. This is ascribed to a different spectrum of reservoir hosts. Taking into account the infection prevalences of host-seeking ticks in the forested surroundings of Bonn, our study demonstrates that the risk of acquiring Lyme disease after a tick bite in urban/suburban areas is comparably as high as in woodlands outside of the city.

title: Occurrence of Babesia spp., Rickettsia spp. and Bartonella spp. in Ixodes ricinus in Bavarian public parks, Germany
abstract: BackgroundOnly limited information is available about the occurrence of ticks and tick-borne pathogens in public parks, which are areas strongly influenced by human beings. For this reason, Ixodes ricinus were collected in public parks of different Bavarian cities in a 2-year survey (2009 and 2010) and screened for DNA of Babesia spp., Rickettsia spp. and Bartonella spp. by PCR. Species identification was performed by sequence analysis and alignment with existing sequences in GenBank. Additionally, coinfections with Anaplasma phagocytophilum were investigated.ResultsThe following prevalences were detected: Babesia spp.: 0.4% (n = 17, including one pool of two larvae) in 2009 and 0.5 to 0.7% (n = 11, including one pool of five larvae) in 2010; Rickettsia spp.: 6.4 to 7.7% (n = 285, including 16 pools of 76 larvae) in 2009. DNA of Bartonella spp. in I. ricinus in Bavarian public parks could not be identified. Sequence analysis revealed the following species: Babesia sp. EU1 (n = 25), B. divergens (n = 1), B. divergens/capreoli (n = 1), B. gibsoni-like (n = 1), R. helvetica (n = 272), R. monacensis IrR/Munich (n = 12) and unspecified R. monacensis (n = 1). The majority of coinfections were R. helvetica with A. phagocytophilum (n = 27), but coinfections between Babesia spp. and A. phagocytophilum, or Babesia spp. and R. helvetica were also detected.ConclusionsI. ricinus ticks in urban areas of Germany harbor several tick-borne pathogens and coinfections were also observed. Public parks are of particularly great interest regarding the epidemiology of tick-borne pathogens, because of differences in both the prevalence of pathogens in ticks as well as a varying species arrangement when compared to woodland areas. The record of DNA of a Babesia gibsoni-like pathogen detected in I. ricinus suggests that I. ricinus may harbor and transmit more Babesia spp. than previously known. Because of their high recreational value for human beings, urban green areas are likely to remain in the research focus on public health issues.

title: Longitudinal Analysis of Tick Densities and Borrelia, Anaplasma, and Ehrlichia Infections of Ixodes ricinus Ticks in Different Habitat Areas in The Netherlands
abstract: From 2000 to 2004, ticks were collected by dragging a blanket in four habitat areas in The Netherlands: dunes, heather, forest, and a city park. Tick densities were calculated, and infection with Borrelia burgdorferi and Anaplasma and Ehrlichia species was investigated by reverse line blot analysis. The lowest tick density was observed in the heather area (1 to 8/100 m2). In the oak forest and city park, the tick densities ranged from 26 to 45/100 m2. The highest tick density was found in the dune area (139 to 551/100 m2). The infection rates varied significantly for the four study areas and years, ranging from 0.8 to 11. 5% for Borrelia spp. and 1 to 16% for Ehrlichia or Anaplasma (Ehrlichia/Anaplasma) spp. Borrelia infection rates were highest in the dunes, followed by the forest, the city park, and heather area. In contrast, Ehrlichia/Anaplasma was found most often in the forest and less often in the city park. The following Borrelia species were found: Borrelia sensu lato strains not identified to the species level (2.5%), B. afzelii (2.5%), B. valaisiana (0.9%), B. burgdorferi sensu stricto (0.13%), and B. garinii (0.13%). For Ehrlichia/Anaplasma species, Ehrlichia and Anaplasma spp. not identified to the species level (2.5%), Anaplasma schotti variant (3.5%), Anaplasma phagocytophilum variant (0.3%), and Ehrlichia canis (0.19%) were found. E. canis is reported for the first time in ticks in The Netherlands in this study. Borrelia lusitaniae, Ehrlichia chaffeensis, and the human granylocytic anaplasmosis agent were not detected. About 1.6% of the ticks were infected with both Borrelia and Ehrlichia/Anaplasma, which was higher than the frequency predicted from the individual infection rates, suggesting hosts with multiple infections or a possible selective advantage of coinfection.

title: Activity of the Tick Ixodes ricinus Monitored in a Suburban Park in Brno, Czech Republic, in Association with the Evaluation of Selected Repellents
abstract: The ever-increasing number of Lyme borreliosis patients led us to consider more effective procedures for disease prevention. The aim of our study was to monitor the annual activity and infectivity of Ixodes ricinus ticks in the Pisárky region, City of Brno, CR, and to test the responses of the locally-captured ticks to selected repellents. The result of regular one-hourper-week monitoring in 2011 was the collection of ticks that directly reflected the highest number of Lyme disease patients (4,835) detected throughout the period of recording in the Czech Republic. The ticks were examined for spirochaetes by dark field microscopy. The positive samples were identified by PCR analysis, confirming that 76% of these were infected with Borrelia burgdorferi sensu lato. Ticks were most abundant in May and June, with August having the highest risk for spirochaetal infection. Tick activity was statistically correlated with temperature. The moving-object-bioassay was used to study repellent efficiency on the Ixodes ricinus nymphs captured in the above-mentioned suburban park. Five selected commercial repellents based on DEET (N, N-diethyl-3-methylbenzamide) showed statistically different effects on the non-repellent control group.

title: Ixodes ricinus abundance and its infection with the tick-borne pathogens in urban and suburban areas of Eastern Slovakia
abstract: BackgroundRaising 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.MethodsQuesting 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.ResultsIn 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.ConclusionsOur 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.

title: Rickettsiaceae and Anaplasmataceae infections in Ixodes ricinus ticks from urban and natural forested areas of Poland
abstract: BackgroundIxodes ricinus is a major vector for a range of microbial pathogens and the most prevalent and widely distributed tick species on the European continent, occurring in both natural and urban habitats. Nevertheless, little is known about the relative density of ticks in these two ecologically distinct habitats and the diversity of tick-borne pathogens that they carry.MethodsWe compared densities of questing I. ricinus nymphs and adults in urban and natural habitats in Central and Northeastern Poland, assessed the prevalence and rate of co-infection with A. phagocytophilum, Rickettsia, Ehrlichia and ‘Ca. Neoehrlichia spp.’ in ticks, and compared the diversity of tick-borne pathogens using molecular assays (PCR).ResultsOf the 1325 adults and nymphs, 6.2% were infected with at least one pathogen, with 4.4%, 1.7% and less than 0.5% being positive for the DNA of Rickettsia spp., A. phagocytophilum, Ehrlichia spp. and Ca. N. mikurensis, respectively. Although tick abundance was higher in natural habitats, the prevalence of the majority of pathogens was higher in urban forested areas.ConclusionWe conclude that: (i) zoonotic genetic variants of A. phagocytophilum are widely distributed in the Polish tick population, (ii) although the diversity of tick borne pathogens was higher in natural habitats, zoonotic species/strains were detected only in urban forests, (iii) and we provide the first description of Ca. N. mikurensis infections in ticks in Poland.

title: Big city life: carnivores in urban environments
abstract: Cities may represent one of the most challenging environments for carnivorous mammals. For example, cities have a dearth of vegetation and other natural resources, coupled with increased habitat fragmentation and an abundance of roads as well as altered climate (e.g. temperature, light, rainfall and water runoff). It is therefore intriguing that several carnivore species have become established in cities across the globe. Medium-sized carnivores such as the red fox, coyote, Eurasian badger and raccoon not only survive in cities but also have managed to exploit anthropogenic food sources and shelter to their significant advantage, achieving higher population densities than are found under natural conditions. In addition, although they may not live permanently within cities, even large carnivores such as bears, wolves and hyaenas derive significant benefit from living adjacent to urbanized areas. In this review, we examine the history of urban adaptation by mammalian carnivores, explore where they are living, what they eat, what kills them and the behavioural consequences of living in urban areas. We review the biology of urban carnivores, exploring traits such as body size and dietary flexibility. Finally, we consider the consequences of having populations of carnivores in urbanized areas, both for humans and for these charismatic mammals. In conclusion, in a time of massive environmental change across the globe, the continuing encroachment of urbanization upon wilderness areas is substantially reducing the availability of natural habitats for many species; therefore, understanding the biology of any taxon that is able to adapt to and exploit anthropogenically disturbed systems must aid us in both controlling and developing suitable conservation measures for the future of such species.

title: The role of rodents in the ecology of Ixodes ricinus and associated pathogens in Central and Eastern Europe
abstract: Rodents comprise more species than any other mammal order. Most rodents are considered keystone species in their ecological communities, hence the survival of many other species in the ecosystem depend on them. From medical point of view, this is particularly important for rodent-dependent pathogens. In the particular case of tick-borne diseases, rodents are important as hosts for vector ticks and as reservoir hosts (Lyme borreliosis, human granulocytic anaplasmosis, Crimean-Congo hemorrhagic fever, Tick-borne relapsing fevers, tick-borne rickettsioses, babesiosis). Community and population ecology of rodents was shown to be correlated with disease ecology in the case of many tick-borne diseases. In Eastern Europe, several adult hard-tick species use rodents as their principal hosts: Ixodes apronophorus, I. crenulatus, I. laguri, I. redikorzevi, I. trianguliceps. However, the majority of ticks feeding on rodents are immature stages of ticks which as adults are parasitic on larger mammals. Larvae and nymphs of Ixodes ricinus, the most abundant and medically important tick from Europe, are commonly found on rodents. This is particularly important, as many rodents are synanthropic and, together with other micromammals and birds are often the only available natural hosts for ticks in urban environments. This work reviews the correlated ecology of rodents and I. ricinus.

title: Life History and Demographic Drivers of Reservoir Competence for Three Tick-Borne Zoonotic Pathogens
abstract: Animal and plant species differ dramatically in their quality as hosts for multi-host pathogens, but the causes of this variation are poorly understood. A group of small mammals, including small rodents and shrews, are among the most competent natural reservoirs for three tick-borne zoonotic pathogens, Borrelia burgdorferi, Babesia microti, and Anaplasma phagocytophilum, in eastern North America. For a group of nine commonly-infected mammals spanning >2 orders of magnitude in body mass, we asked whether life history features or surrogates for (unknown) encounter rates with ticks, predicted reservoir competence for each pathogen. Life history features associated with a fast pace of life generally were positively correlated with reservoir competence. However, a model comparison approach revealed that host population density, as a proxy for encounter rates between hosts and pathogens, generally received more support than did life history features. The specific life history features and the importance of host population density differed somewhat between the different pathogens. We interpret these results as supporting two alternative but non-exclusive hypotheses for why ecologically widespread, synanthropic species are often the most competent reservoirs for multi-host pathogens. First, multi-host pathogens might adapt to those hosts they are most likely to experience, which are likely to be the most abundant and/or frequently bitten by tick vectors. Second, species with fast life histories might allocate less to certain immune defenses, which could increase their reservoir competence. Results suggest that of the host species that might potentially be exposed, those with comparatively high population densities, small bodies, and fast pace of life will often be keystone reservoirs that should be targeted for surveillance or management.

title: Public health significance of urban pests
abstract: of Urban Pests P ublic H ealth S ignicance of U ran P ests Xavier Bonnefoy Helge Kampen Kevin Sweeney The second half of the 20th century and the beginning of the 21st century have witnessed important changes in ecology, climate and human behaviour that favour the development of urban pests. Most alarmingly, urban planners are faced now with the dramatic expansion of urban sprawl, where the suburbs of our cities are growing into the natural habitats of ticks, rodents and other pests. Also, many city managers now erroneously assume that pest-borne diseases are relics that belong to the past.

title: Hantaviruses and their hosts in Europe: reservoirs here and there, but not everywhere?
abstract: Five hantaviruses are known to circulate among rodents in Europe, and at least two among insectivores. Four (Dobrava, Saaremaa, Seoul, and Puumala [PUUV] viruses) are clearly associated with hemorrhagic fever with renal syndrome (HFRS). PUUV, the most common etiological agent of HFRS in Europe, is carried by the bank vole (Myodes glareolus), one of the most widespread and abundant mammal species in Europe. This host-virus system is among hantaviruses also the most studied one in Europe. However, HFRS incidence varies throughout the continent. The spatial as well as temporal variation in the occurrence of HFRS is linked to geographic differences in the population dynamics of the reservoir rodents in different biomes of Europe. While rodent abundance may follow mast seeding events in many parts of temperate Europe, in northern (N) Europe multiannual cycles in population density exist as the result of the interaction between rodent populations and specialist predator populations in a delayed density-dependent manner. The spatial distribution of hantaviruses further depends on parameters such as forest patch size and connectivity of the most suitable rodent habitats, and the conditions for the survival of the virus outside the host, as well as historical distribution patterns (phylogeographies) of hosts and viruses. In multiannually fluctuating populations of rodents, with population increases of great amplitude, one should expect a simultaneous build-up of recently hantavirus-infected (shedding) rodents. The increasing number of infectious, virus-shedding rodents leads to a rapid transmission of hantavirus across the rodent population, and to humans. Our review discusses these aspects for PUUV, the only European hantavirus for which there is a reasonable, yet still far from complete, ecological continental-wide understanding. We discuss how this information could translate to other European hantavirus-host systems, and where the most important questions lie for further research.

title: Reservoir Competence of Various Rodents for the Lyme Disease Spirochete Borrelia spielmanii
abstract: To determine whether the pathogenic Lyme disease spirochete Borrelia spielmanii is adapted exclusively to garden dormice, we compared the reservoir competence of various rodent species for this spirochete, including sympatric and peridomestic rodents. The different kinds of rodents varied in their attractiveness to nymphal ticks and their level of susceptibility to tick-borne B. spielmanii infection, but only the edible dormouse appeared to be refractory. Although hazel dormice and Norway rats became infectious to ticks somewhat later than did garden dormice, they remained infectious for a longer period of time. During the course of a tick season, garden and hazel dormice contributed theoretically more than twice as many B. spielmanii-infected ticks than the somewhat less susceptible Norway rats and wood or yellow-necked mice. Hazel dormice appeared to be extraordinarily competent as reservoir hosts for B. spielmanii. Because peridomestic rodents proved to be reservoir competent, urban foci of transmission of B. spielmanii may affect the health of townspeople.

title: Competence of urban rats as reservoir hosts for Lyme disease spirochetes.
abstract: To determine whether urban rats serve efficiently as reservoir hosts for the agent of Lyme disease, we recorded the frequency of infection in nymphal Ixodes ricinus (L.) ticks that had fed as larvae on experimentally infected Norway rats, Rattus norvegicus (Berkenhout), or on black rats, R. rattus (L.), and evaluated the nidicolous venue of transmission. Subadult vector ticks attached readily to Norway rats as well as black rats and virtually all became infected in the course of feeding. Larval ticks detached when these nocturnally active hosts were at rest. Rats appeared to be competent reservoir hosts of Lyme disease spirochetes in a transmission cycle in urban sites.

title: Occurrence of different Borrelia burgdorferi sensu lato genospecies including B. afzelii, B. bavariensis, and B. spielmanii in hedgehogs (Erinaceus spp.) in Europe.
abstract: In order to determine whether European hedgehogs (Erinaceus europaeus and E. roumanicus) play a role in the epidemiological cycle of Borrelia burgdorferi sensu lato in Central Europe and Great Britain, tissue samples of hedgehogs from Germany (n=211), Austria (n=4), the Czech Republic (n=22), and the U.K. (n=32) were tested for the presence of these tick-borne pathogens. PCR for amplification of the B. burgdorferi s.l.-specific 5S-23S intergenic spacer region as well as the outer surface protein A (ospA) gene were used. B. burgdorferi s.l. DNA was detected in 35 of the 259 E. europaeus and in 2 of 10 E. roumanicus. B. burgdorferi prevalences in E. europaeus ranged from 0% (U.K.) to 37.5% (Czech Republic), for E. roumanicus from 0% (Czech Republic) to 50.0% (Austria). Sequencing revealed the occurrence of 3 different B. burgdorferi genospecies in E. europaeus: B. afzelii was the dominant genospecies, followed by B. bavariensis (previously B. garinii OspA serotype 4) and B. spielmanii, the latter was detected for the first time in Hamburg (Germany). B. afzelii and B. bavariensis were also found in E. roumanicus. Our results suggest that hedgehogs modulate the epidemiology of certain species of the B. burgdorferi s.l. complex, potentially affecting the distribution and abundance of individual B. burgdorferi s.l. genospecies in various habitats. We hypothesise that juvenile or individuals with low immune competence in particular, have a high reservoir potential for the 3 genospecies identified here.

title: Role of grey squirrels and pheasants in the transmission of Borrelia burgdorferi sensu lato, the Lyme disease spirochaete, in the U.K.
abstract: In Britain, grey squirrels (Sciurus carolinensis Gmelin) and pheasants (Phasianus colchicus Linnaeus) are important hosts of larvae and nymphs of Ixodes ricinus L., the principal European vector of the Lyme disease spirochaete, Borrelia burgdorferi sensu lato. To test whether squirrels are competent hosts of B. burgdorferi s.l., three females were trapped in the wild and then held in captivity. Following treatment, each animal was exposed to uninfected xenodiagnostic I. ricinus ticks. Squirrel A (an adult) which was inoculated experimentally with B. burgdorferi s.l., transmitted the infection to xenodiagnostic ticks. In contrast, squirrel B (a juvenile that was not inoculated)-showed no evidence of infection. Xenodiagnostic ticks that fed on control squirrel C (an adult) became infected and subsequently transmitted the infection experimentally to an uninfected hamster. The results indicated that squirrel C had a disseminated infection acquired in the wild and which persisted for at least 11 weeks. These data clearly demonstrate that grey squirrels are amplifying and reservoir hosts of B. burgdorferi s.l. The strain associated with squirrels was related to the B. afzelii genotype. Two observations implicated pheasants in a similar role: (i) a high prevalence of infection in engorged larvae collected from trapped pheasants, and (ii) the detection of B. burgdorferi s.l. (B. garinii genotype) in the wattle of 1/10 pheasants using PCR. Xenodiagnostic experiments similar to those undertaken with the squirrels are needed to confirm the role of pheasants in the transmission cycle of Lyme disease spirochaetes.

title: Distribution and activity of the introduced Tamias sibiricus (Laxmann 1769) in an urban park in Rome, Italy

title: Native and alien squirrels in Italy
abstract: In Italy there are four species of squirrels: the native red squirrel and other three species recently introduced. The red squirrel is present in the Italian peninsula with three subspecies, and is missing only in Salento, and Italian islands. This species is common on Alps and Apennines, while in the plains it is declining because of the habitat loss. Competition with the grey squirrel and habitat fragmentation are considered the major threats to the survival of the red squirrel. The grey squirrel is present in Piedmont and Liguria. A study on the Piedmontese colony showed that the red squirrel is disappearing from the area colonised by the grey squirrel and the damage due to bark-stripping and feeding is considerable. Free-ranging populations of the Siberian chipmunk live in Belluno, Verona, and Rome, but records of single animals were reported for other areas. The Finlayson's squirrel is pre- sent with a small nucleus in an urban area of Piedmont. Here, the impact of this species on the veg- etation appears dramatic. The eradication of the grey squirrel is a priority for the conservation of the red squirrel, but control plans for the other introduced species are also needed.

title: Introduced Siberian chipmunks are more heavily infested by ixodid ticks than are native bank voles in a suburban forest in France.
abstract: By serving as hosts for native vectors, introduced species can surpass native hosts in their role as major reservoirs of local pathogens. During a 4-year longitudinal study, we investigated factors that affected infestation by ixodid ticks on both introduced Siberian chipmunks Tamias sibiricus barberi and native bank voles Myodes glareolus in a suburban forest (Forêt de Sénart, Ile-de-France). Ticks were counted on adult bank voles and on adult and young chipmunks using regular monthly trapping sessions, and questing ticks were quantified by dragging. At the summer peak of questing Ixodes ricinus availability, the average tick load was 27-69 times greater on adult chipmunks than on adult voles, while average biomass per hectare of chipmunks and voles were similar. In adult chipmunks, individual effects significantly explained 31% and 24% of the total variance of tick larvae and nymph burdens, respectively. Male adult chipmunks harboured significantly more larvae and nymphs than adult females, and than juveniles born in spring and in summer. The higher tick loads, and more specifically the ratio of nymphs over larvae, observed in chipmunks may be caused by a higher predisposition--both in terms of susceptibility and exposure--to questing ticks. Tick burdens were also related to habitat and seasonal variation in age- and sex-related space use by both rodents. Introduced chipmunks may thus have an important role in the dynamics of local vector-borne pathogens compared with native reservoir hosts such as bank voles.

title: Introduced Siberian Chipmunks (Tamias sibiricus barberi) Harbor More-Diverse Borrelia burgdorferi Sensu Lato Genospecies than Native Bank Voles (Myodes glareolus)
abstract: Little attention has been given in scientific literature to how introduced species may act as a new host for native infectious agents and modify the epidemiology of a disease. In this study, we investigated whether an introduced species, the Siberian chipmunk (Tamias sibiricus barberi), was a potentially new reservoir host for Borrelia burgdorferi sensu lato, the causative agent of Lyme disease. First, we ascertained whether chipmunks were infected by all of the B. burgdorferi sensu lato genospecies associated with rodents and available in their source of infection, questing nymphs. Second, we determined whether the prevalence and diversity of B. burgdorferi sensu lato in chipmunks were similar to those of a native reservoir rodent, the bank vole (Myodes glareolus). Our research took place between 2006 and 2008 in a suburban French forest, where we trapped 335 chipmunks and 671 voles and collected 743 nymphs of ticks that were questing for hosts by dragging on the vegetation. We assayed for B. burgdorferi sensu lato with ear biopsy specimens taken from the rodents and in nymphs using PCR and restriction fragment length polymorphism (RFLP). Chipmunks were infected by the three Borrelia genospecies that were present in questing nymphs and that infect rodents (B. burgdorferi sensu stricto, B. afzelii, and B. garinii). In contrast, voles hosted only B. afzelii. Furthermore, chipmunks were more infected (35%) than voles (16%). These results may be explained by the higher exposure of chipmunks, because they harbor more ticks, or by their higher tolerance of other B. burgdorferi sensu lato genospecies than of B. afzelii. If chipmunks are competent reservoir hosts for B. burgdorferi sensu lato, they may spill back B. burgdorferi sensu lato to native communities and eventually may increase the risk of Lyme disease transmission to humans.

title: Maintenance by hares of European Borrelia burgdorferi in ecosystems without rodents.
abstract: During May-October 1991 shrews (Sorex spp.), small rodents (Apodemus spp., Clethrionomys glareolus [Schreber]), and hares (Lepus spp.) were sampled near Stockholm, Sweden. Nymphal Ixodes ricinus (L.) derived from blood-engorged larvae collected from these mammals were investigated by phase-contrast microscopy and immunofluorescence assay (IFA) using the monoclonal antibody H6831 directed against the OspB protein of Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner. Three rodent species, previously known as vertebrate reservoirs for B. burgdorferi in Switzerland, are competent reservoirs of Lyme borreliosis also in Sweden. The first data are presented showing Lyme disease reservoir competency of European insectivores (Sorex araneus L., S. minutus L.) and lagomorphs (Lepus europaeus Pallas, L. timidus L.). Lagomorphs are the only European vertebrates known to be both competent reservoirs for B. burgdorferi and a source of blood for all stages of I. ricinus. We propose that Lyme borreliosis can be maintained for prolonged periods on islands, without rodents and insectivores, if lagomorphs are present; and, in such ecosystems, fluctuations of lagomorph population levels may influence the numbers of B. burgdorferi-infected ticks and hence the risk of human Lyme disease infection.

title: Parasites of wild rabbits (Oryctolagus cuniculus) from an urban area in Germany, in relation to worldwide results
abstract: The European rabbit (Oryctolagus cuniculus) belongs to the most invasive and successful mammalian species, which is distributed nearly worldwide. In Europe, they inhabit broad parts of the mainland and subsequently reached several European islands via anthropogenic diversion. Rabbits can also serve as hosts for numerous parasite species. The parasite and pathogen fauna of O. cuniculus have been well documented in various European countries, although studies in Germany are scarce. Until now, a comprehensive survey combining recent international studies over parasite fauna of wild rabbits had not been conducted. We examined 50 wild rabbits from an urban area near Aachen (Germany) to identify their metazoan parasite fauna, and then compared our findings to previous international investigations. A total of nine parasite species were isolated consisting of four endoparasite species (Cittotaenia denticulata, Graphidium strigosum, Passalurus ambiguus, and Trichostrongylus retortaeformis) and five ectoparasite species (Cheyletiella parasitivorax, Ixodes ricinus, Leporacarus gibbus, Haemodipsus ventricosus, and Spilopsyllus cuniculi). Among the ectoparasites were two verifiable human pathogenic species and two potentially pathogenic species. In comparison to previous studies, a high number of similarities in composition of helminth species fauna were revealed. Furthermore, our results showed partial agreement with international surveys in prevalence and mean intensity of the parasites C. denticulata, G. strigosum, P. ambiguus, and T. retortaeformis.

title: The patterns of seasonal activity of Ixodes vespertilionis (Acari: Ixodidae) on Rhinolophus hipposideros in nursery colonies.
abstract: The aim of this study was to describe the dynamics of the long-legged bat tick Ixodes vespertilionis infestation on the lesser horseshoe bat Rhinolophus hipposideros in 2 nursery colonies roosting in attics. Out of a total of 810 lesser horseshoe bats examined, 217 (26.8%) were found to be infested with a total of 464 I. vespertilionis individuals. The developmental stage most frequently found was the larva, followed by the nymph, and the adult female. Bats were significantly more frequently infested with I. vespertilionis ticks in the period April to May than in other months. In these months, all tick developmental stages were observed. During summer and autumn, only immature developmental stages were recorded, whilst in September and October larvae predominated. Considerable differences in tick load between nursery colonies were observed. The length of seasonal presence on bats, prevalence, and infestation intensity of I. vespertilionis on lesser horseshoe bats were higher in the nursery colony situated in close vicinity of a cave than in the colony situated far from the caves. The results suggest that the pattern of seasonal infestation of ticks on bats roosting in nursery colonies coincides with the seasonal activity of Rh. hipposideros in the caves. The first case of mixed infestation of the lesser horseshoe bat with I. vespertilionis and I. ricinus were also recorded.

title: Tick-borne viruses in Europe
abstract: The aim of this review is to present briefly background information on 27 tick-borne viruses (“tiboviruses”) that have been detected in Europe, viz flaviviruses tick-borne encephalitis (TBEV), louping-ill (LIV), Tyuleniy (TYUV), and Meaban (MEAV); orthobunyaviruses Bahig (BAHV) and Matruh (MTRV); phleboviruses Grand Arbaud (GAV), Ponteves (PTVV), Uukuniemi (UUKV), Zaliv Terpeniya (ZTV), and St. Abb's Head (SAHV); nairoviruses Soldado (SOLV), Puffin Island (PIV), Avalon (AVAV), Clo Mor (CMV), Crimean-Congo hemorrhagic fever (CCHFV); bunyavirus Bhanja (BHAV); coltivirus Eyach (EYAV); orbiviruses Tribec (TRBV), Okhotskiy (OKHV), Cape Wrath (CWV), Mykines (MYKV), Tindholmur (TDMV), and Bauline (BAUV); two thogotoviruses (Thogoto THOV, Dhori DHOV); and one asfivirus (African swine fever virus ASFV). Emphasis is laid on the taxonomic status of these viruses, range of their ixodid or argasid vectors and vertebrate hosts, pathogenicity for vertebrates including humans, and relevance to public health. In general, three groups of tibovirus diseases can be recognized according to main clinical symptoms produced: (i) febrile illness—usually with a rapid onset, fever, sweating, headache, nausea, weakness, myalgia, arthralgia, sometimes polyarthritis and rash; (ii) the CNS affection—meningitis, meningoencephalitis or encephalomyelitis with pareses, paralysis and other sequelae; (iii) hemorrhagic disease. Several “European” tiboviruses cause very serious human (TBEV, CCHFV) or animal (LIV, ASFV) diseases. Other arboviruses play definite role in human or animal pathology though the disease is usually either less serious or infrequently reported (TYUV, BHAV, AVAV, EYAV, TRBV, DHOV, THOV). The other European arboviruses are “orphans” without a proven medical or veterinary significance (BAHV, MTRV, MEAV, GAV, PTVV, ZTV, SAHV, UUKV, SOLV, PIV, AVAV, CMV, OKHV, CWV, MYKV, TDMV, BAUV). However, certain arbovirus diseases of free-living vertebrates (but also those of domestic animals and even man) may often pass unnoticed or misdiagnosed and eventually, they might potentially appear as emerging diseases. Active search for new tiboviruses or for new, pathogenic variants of the known tiboviruses in Europe should therefore continue.

title: Tick infestation on roe deer in relation to geographic and remotely sensed climatic variables in a tick-borne encephalitis endemic area
abstract: Roe deer Capreolus capreolus are among the most important feeding hosts for the sheep tick Ixodes ricinus, thus contributing to the occurrence of tick-borne diseases in Europe. Tick-borne encephalitis (TBE), which is transmitted by co-feeding of larvae and nymphs on rodents, requires precise climatic conditions to occur. We used roe deer as sentinels for potential circulation of TBE virus in Northern Italy, by examining the association between tick infestation, occurrence of TBE human cases, geographical and climatic parameters. Tick infestation on roe deer, and particularly frequency of co-feeding, was clearly associated with the geographic location and the autumnal cooling rate. Consistently, TBE occurrence in humans was geographically related to co-feeding tick abundance. The surveillance of tick infestation on roe deer, combined with remotely sensed climatic data, could therefore be used as an inexpensive early risk assessment tool of favourable conditions for TBE emergence and persistence in humans.

title: How does climate change influence demographic processes of widespread species? Lessons from the comparative analysis of contrasted populations of roe deer.
abstract: How populations respond to climate change depends on the interplay between life history, resource availability, and the intensity of the change. Roe deer are income breeders, with high levels of allocation to reproduction, and are hence strongly constrained by the availability of high quality resources during spring. We investigated how recent climate change has influenced demographic processes in two populations of this widespread species. Spring began increasingly earlier over the study, allowing us to identify 2 periods with contrasting onset of spring. Both populations grew more slowly when spring was early. As expected for a long-lived and iteroparous species, adult survival had the greatest potential impact on population growth. Using perturbation analyses, we measured the relative contribution of the demographic parameters to observed variation in population growth, both within and between periods and populations. Within periods, the identity of the critical parameter depended on the variance in growth rate, but variation in recruitment was the main driver of observed demographic change between periods of contrasting spring earliness. Our results indicate that roe deer in forest habitats cannot currently cope with increasingly early springs. We hypothesise that they should shift their distribution to richer, more heterogeneous landscapes to offset energetic requirements during the critical rearing stage.

title: Spatial risk assessment for Lyme borreliosis in Denmark.
abstract: A study of nymphal and adult Ixodes ricinus density was performed in well-defined spruce and beech forest habitats with different levels of roe deer abundance and soil water capacity. In 35 habitats, a total of 489 larvae, 1,611 nymphs and 193 adult I. ricinus ticks were collected. Tick density was found to be influenced by roe deer abundance and soil water capacity. Based on this evaluation, a model predicting increasing number of ticks with increasing roe deer abundance and soil water capacity was suggested. A total of 1,045 nymphs and 106 adult ticks were tested for infection with Borrelia burgdorferi sensu lato Of these, 53 nymphs and 6 adults were found to be infected, leading to an general infection rate of 5% and 6% for nymphs and adults, respectively. Prevalences of Borrelia burgdorferi sensu lato in nymphal I. ricinus were found to be independent of roe deer abundance and soil water capacity. The correlation between human neuroborreliosis incidence and the estimated number of I. ricinus based on roe deer abundance and soil water capacity was examined. Differences in human neuroborreliosis incidence were found to correspond with the expected spatial differences in tick density in 12 counties in Denmark.

title: Forest Structure and Roe Deer Abundance Predict Tick-Borne Encephalitis Risk in Italy
abstract: The Western Tick-borne encephalitis (TBE) virus often causes devastating or lethal disease. In Europe, the number of human TBE cases has increased dramatically over the last decade, risk areas are expanding and new foci are being discovered every year. The early localisation of new TBE foci and the identification of the main risk factors associated with disease emergence represent a priority for the public health community. Although a number of socio-economic parameters have been suggested to explain TBE upsurges in eastern Europe, the principal driving factors in relatively stable western European countries have not been identified.  In this paper, we analyse the correlation between the upsurge of TBE in 17 alpine provinces in northern Italy from 1992 to 2006 with climatic variables, forest structure (as a proxy for small mammal reservoir host abundance), and abundance of the principal large vertebrate tick host (roe deer), using datasets available for the last 40 years. No significant differences between the pattern of changes in climatic variables in provinces where TBE has emerged compared to provinces were no clinical TBE cases have been observed to date. Instead, the best model for explaining the increase in TBE incidence in humans in this area include changes in forest structure, in particular the ratio of coppice to high stand forest, and the density of roe deer.  Substantial changes in vegetation structure that improve habitat suitability for the main TBE reservoir hosts (small mammals), as well as an increase in roe deer abundance due to changes in land and wildlife management practices, are likely to be among the most crucial factors affecting the circulation potential of Western TBE virus and, consequently, the risk of TBE emergence in humans in western Europe. We believe our approach will be useful in predicting TBE risk on a wider scale.

title: Landscape Level Variation in Tick Abundance Relative to Seasonal Migration in Red Deer
abstract: Partial migration is common among northern ungulates, typically involving an altitudinal movement for seasonally migratory individuals. The main driving force behind migration is the benefit of an extended period of access to newly emerged, high quality forage along the green up gradient with increasing altitude; termed the forage maturation hypothesis. Any other limiting factor spatially correlated with this gradient may provide extra benefits or costs to migration, without necessarily being the cause of it. A common ectoparasite on cervids in Europe is the sheep tick (Ixodes ricinus), but it has not been tested whether migration may lead to the spatial separation from these parasites and thus potentially provide an additional benefit to migration. Further, if there is questing of ticks in winter ranges in May before spring migration, deer migration may also play a role for the distribution of ticks. We quantified the abundance of questing sheep tick within winter and summer home ranges of migratory (n = 42) and resident red deer (Cervus elaphus) individuals (n = 32) in two populations in May and August 2009–2012. Consistent with predictions, there was markedly lower abundance of questing ticks in the summer areas of migrating red deer (0.6/20 m2), both when compared to the annual home range of resident deer (4.9/20 m2) and the winter home ranges of migrants (5.8/20 m2). The reduced abundances within summer home ranges of migrants were explained by lower abundance of ticks with increasing altitude and distance from the coast. The lower abundance of ticks in summer home ranges of migratory deer does not imply that ticks are the main driver of migration (being most likely the benefits expected from forage maturation), but it suggests that ticks may add to the value of migration in some ecosystems and that it may act to spread ticks long distances in the landscape.

title: Detection of tick-borne pathogens in roe deer (Capreolus capreolus), in questing ticks (Ixodes ricinus), and in ticks infesting roe deer in southern Germany.
abstract: The hard tick Ixodes ricinus is the most common tick in Central Europe and plays an important role as a vector of several pathogens. In the complex life cycles of these pathogens, the role of wild animals as natural reservoirs has been discussed. The aims of this study were to investigate the role of roe deer (Capreolus capreolus) as a potential reservoir host for Babesia spp., Anaplasma phagocytophilum, and Rickettsia spp. Therefore, we explored the differences in the infection rates of roe deer and engorged and questing ticks with these pathogens from a single forest site with special attention to coinfection. Blood, spleen, and skin samples of a total of 95 roe deer individuals were screened by molecular methods for these pathogens from September 2010 to January 2012 in the 'Angelberger Forst' (Bavaria, Germany). Moreover, 331 engorged ticks from 44 roe deer individuals and 199 host-seeking ticks from the same area were screened. Altogether, the following prevalence rates and a high diversity of species were detected for the respective pathogens in individual animals and ticks: (i) Babesia spp.: roe deer, 89.5%; engorged ticks, 7.3%; questing ticks: adults, 2.5%, nymphs, 3.3%. Sequencing revealed B. venatorum, B. capreoli, and B. microti. (ii) A. phagocytophilum: roe deer 98.9%; engorged ticks, 86.1%; questing ticks: adults, 8.9%, nymphs, 0.8%. (iii) Rickettsia spp.: roe deer, 0%; engorged ticks, 16.6%; questing ticks: adults, 13.9%, nymphs, 17.5%. Sequencing revealed R. helvetica. Furthermore, several coinfections were detected in both roe deer and ticks. The high prevalence rates of B. capreoli and A. phagocytophilum in roe deer support their role as reservoir hosts for these pathogens, but no evidence for a role of roe deer in the life cycle of R. helvetica could be provided.

title: Tick burden on European roe deer (Capreolus capreolus)
abstract: In our study we assessed the tick burden on roe deer (Capreolus capreolus L.) in relation to age, physical condition, sex, deer density and season. The main objective was to find predictive parameters for tick burden. In September 2007, May, July, and September 2008, and in May and July 2009 we collected ticks on 142 culled roe deer from nine forest departments in Southern Hesse, Germany. To correlate tick burden and deer density we estimated deer density using line transect sampling that accounts for different detectability in March 2008 and 2009, respectively. We collected more than 8,600 ticks from roe deer heads and necks, 92.6% of which were Ixodes spp., 7.4% Dermacentor spp. Among Ixodes, 3.3% were larvae, 50.5% nymphs, 34.8% females and 11.4% males, with significant seasonal deviation. Total tick infestation was high, with considerable individual variation (from 0 to 270 ticks/deer). Adult tick burden was positively correlated with roe deer body indices (body mass, age, hind foot length). Significantly more nymphs were found on deer from forest departments with high roe deer density indices, indicating a positive correlation with deer abundance. Overall, tick burden was highly variable. Seasonality and large scale spatial characteristics appeared to be the most important factors affecting tick burden on roe deer.

title: Blood feeding on large grazers affects the transmission of Borrelia burgdorferi sensu lato by Ixodes ricinus.
abstract: The presence of Ixodes ricinus and their associated Borrelia infections on large grazers was investigated. Carcases of freshly shot red deer, mouflon and wild boar were examined for the presence of any stage of I. ricinus. Questing ticks were collected from locations where red deer and wild boar are known to occur. Presence of Borrelia burgdorferi s.l. DNA was examined in a fraction of the collected ticks. Larvae, nymphs and adult ticks were found on the three large grazers. Red deer had the highest tick burden, with many of the nymphs and adult females attached for engorgement. Most larvae had not attached. The mean number of ticks on the animals varied from 13 to 67. Ticks were highly aggregated amongst the animals: some animals had no ticks, while others had high numbers. Larvae and nymphs were mostly found on the ears, while adult ticks were attached to the axillae. The Borrelia infection rate of questing nymphs was 8.5%. Unengorged wandering nymphs on deer had a Borrelia infection rate of 12.5%, while only 0.9% of feeding nymphs carried a Borrelia infection. The infection rate of unengorged adult male ticks was 4.5%, and that of feeding female ticks was 0.7%. The data suggest that ticks feeding on red deer and wild boar lose their Borrelia infections. The implications of the results are discussed with respect to Borrelia epidemiology and maintenance of a Borrelia reservoir as well as the role of reproductive hosts for Ixodes ricinus.

title: Diet of wild boar Sus scrofa in Western Europe, with particular reference to consumption of agricultural crops
abstract: The diet of wild boar Sus scrofa in Western Europe is reviewed, paying particular attention to the consumption of agricultural crops and the implications of this from the point of view of crop damage. Data were taken mainly from 11 studies that provide quantitative informa- tion about the consumption of different food types, but we also list all the foods reported as being eaten by wild boar in a total of 21 studies. 2. Vegetable foods occurred more frequently in the diet than animal foods, and also consti- tuted the bulk of the food ingested. Overall, there were four major vegetable food categories: mast, roots, green plant matter and agricultural crops. Depending on the study area, wild boar always consumed at least one energy-rich plant food such as acorns, beechnuts, chest- nuts, pine seeds, olives, cereal grains or other crops. The number and types of agricultural crops consumed varied between study areas but crops represent an important component of wild boar diet throughout its Western European range. Among animal foods, insects, earthworms, birds and mammals were eaten most consistently but the diet also included amphibians, reptiles, gastropods and myriapods. 3. Seasonal, interannual and regional differences in the diet, together with its striking overall breadth, indicate that wild boar are opportunistic omnivores whose diet, in any particular instance, is largely determined by the relative availability of different food types. Dependence on energy-rich plant material as a major component of the diet, coupled with large body size and a propensity to trample crops as well as consume them, means that wild boar cause significant agricultural damage.

title: Ecology and epidemiology of Echinococcus multilocularis in Europe.
abstract: Human alveolar echinococcosis (AE), caused by the larval stage of Echinococcus multilocularis has a high mortality rate in untreated patients. The life-cycle of E. multilocularis in Europe predominantly involves foxes as definitive hosts. However, experimental studies demonstrated a comparable biotic potential of E. multilocularis in dogs and raccoon dogs but an insignificant potential in cats. AE occurs in central and eastern Europe at low incidence rates. Recent studies in foxes have shown that E. multilocularis has a wider geographic range (including Italy) than previously thought. In recent years, increases in fox populations have been observed in many European countries, especially in urban areas. As a result, the E. multilocularis cycle is now established in the urban environment. This presents an increased risk of infection for a large human population. Based on these facts and new epidemiological data, possible intervention strategies are presented.

title: Changes in the Distribution of Red Foxes (Vulpes vulpes) in Urban Areas in Great Britain: Findings and Limitations of a Media-Driven Nationwide Survey
abstract: Urbanization is one of the major forms of habitat alteration occurring at the present time. Although this is typically deleterious to biodiversity, some species flourish within these human-modified landscapes, potentially leading to negative and/or positive interactions between people and wildlife. Hence, up-to-date assessment of urban wildlife populations is important for developing appropriate management strategies. Surveying urban wildlife is limited by land partition and private ownership, rendering many common survey techniques difficult. Garnering public involvement is one solution, but this method is constrained by the inherent biases of non-standardised survey effort associated with voluntary participation. We used a television-led media approach to solicit national participation in an online sightings survey to investigate changes in the distribution of urban foxes in Great Britain and to explore relationships between urban features and fox occurrence and sightings density. Our results show that media-based approaches can generate a large national database on the current distribution of a recognisable species. Fox distribution in England and Wales has changed markedly within the last 25 years, with sightings submitted from 91% of urban areas previously predicted to support few or no foxes. Data were highly skewed with 90% of urban areas having <30 fox sightings per 1000 people km−2. The extent of total urban area was the only variable with a significant impact on both fox occurrence and sightings density in urban areas; longitude and percentage of public green urban space were respectively, significantly positively and negatively associated with sightings density only. Latitude, and distance to nearest neighbouring conurbation had no impact on either occurrence or sightings density. Given the limitations associated with this method, further investigations are needed to determine the association between sightings density and actual fox density, and variability of fox density within and between urban areas in Britain.

title: Ixodid ticks in red foxes (Vulpes vulpes) from Romania
abstract: Among wildlife, the red foxes (Vulpes vulpes) are one of the most adapted wild species to anthropic ecosystems. They are well recognized as important reservoirs for a large number of zoonotic agents in Europe, including ticks and tick-borne pathogens. Currently, there are few available data on the importance of red foxes in the ecoepidemiology of vector-borne diseases. Therefore, the aim of this study was to screen the dynamics of tick infestation in 357 red foxes from 12 Romanian counties. Tick identification was performed using the morphological keys. The overall prevalence of tick infestation was 43.7%. The 5753 collected ticks belonged to five species: Ixodes hexagonus (on 113 out of 156 foxes; prevalence 72.44%), I. ricinus (28.84%), I. crenulatus (7.7%), Dermacentor marginatus (7.05%) and Haemaphysalis punctata (0.64%). Coinfestation occurred in 24 foxes (22 with 2 tick species; 2 with 3 tick species) with the following associations: I. ricinus + I. hexagonus (n = 10), I. hexagonus + D. marginatus (n = 5), I. ricinus + I. crenulatus (n = 4), I. ricinus + D. marginatus (n = 2), I. hexagonus + I. crenulatus (n = 1), D. marginatus + I. hexagonus + I. ricinus (n = 1), and H. punctata + I. hexagonus + I. ricinus (n = 1). This study indicates that foxes are hosts to a relevant number of tick species with recognized vectorial role. The provided information can facilitate the understanding of the ecology of ticks and can be the basis for studies on the epidemiology of tick-borne diseases. The high prevalence of tick infestation in red foxes, coupled with the increasing presence of this species in synanthropic environments and a more nature-oriented lifestyle of people, can pose a significant threat to human health, through the emergence of tick-borne diseases. Moreover, foxes represent a good model of sentinel species. 
 
This research was performed as part of project IDEI PCE 236/2011.

title: Diversity and seasonal patterns of ticks parasitizing wild birds in western Portugal
abstract: The diversity and abundance of questing ticks and ticks parasitizing birds was assessed during 1 year in two recreational forests in western Portugal, a suburban forest and an enclosed game area. The aim of this study was to assess the distribution and seasonality of tick species and to understand the role of bird species as hosts for ticks. Ixodes ricinus was the most abundant questing tick collected in the enclosed game area, whereas in the suburban forest, only three ticks were collected by blanket dragging. Tick species parasitizing birds included I. ricinus, I. frontalis, I. arboricola, I. acuminatus, Haemaphysalis punctata, Hyalomma marginatum and H. lusitanicum. This is the first record of I. arboricola in Portugal. Tick prevalence and intensity of infestation differed between study areas and was higher in birds from the game area where a large population of deer and wild boar may support tick populations. Ground and shrub dwelling bird species such as Turdus merula, Erithacus rubecula and Sylvia melanocephala were the most heavily parasitized by ticks, but the importance of different bird species as hosts of larvae and nymphs of I. ricinus and I. frontalis differed. Therefore, different bird species may contribute differently for tick population maintenance.

title: Blackbirds and Song Thrushes Constitute a Key Reservoir of Borrelia garinii, the Causative Agent of Borreliosis in Central Europe
abstract: Turdus merula) and song thrushes (Turdus philomelos) were found to carry 95% of all spirochete-infected tick larvae among 40 bird species captured in Central Europe. More than 90% of the infections were typed as Borrelia garinii and Borrelia valaisiana. We conclude that thrushes are key players in the maintenance of these spirochete species in this region of Central Europe.

title: The potential role of migratory birds in transmission cycles of Babesia spp., Anaplasma phagocytophilum, and Rickettsia spp.
abstract: Babesia spp., Anaplasma phagocytophilum, and Rickettsia spp. are potentially emerging tick-borne pathogens, whereas many issues about their ecology, e.g. reservoir host specificity, are still unclear. In spring 2007, we collected 191 feeding Ixodes ricinus ticks from 99 birds of 11 different species on a German bird conservation island in the Baltic Sea. Babesia spp. were detected in 4.7% (9/191), A. phagocytophilum was present in 2.6% (5/191), and Rickettsia spp. were identified in 7.3% (14/191) of the investigated ticks. Further characterization of Babesia spp. infections resulted in B. divergens and B. microti. Among the Rickettsia spp. infections, we identified at least 2 different species: R. monacensis and R. helvetica. Furthermore, 2 ticks harboured mixed infections. Our study provides first interesting insights into the role of migratory birds in the distribution of several emerging tick-borne pathogens.

title: Transport of ixodid ticks and tick-borne pathogens by migratory birds
abstract: Birds, particularly passerines, can be parasitized by Ixodid ticks, which may be infected with tick-borne pathogens, like Borrelia spp., Babesia spp., Anaplasma, Rickettsia/Coxiella, and tick-borne encephalitis virus. The prevalence of ticks on birds varies over years, season, locality and different bird species. The prevalence of ticks on different species depends mainly on the degree of feeding on the ground. In Europe, the Turdus spp., especially the blackbird, Turdus merula, appears to be most important for harboring ticks. Birds can easily cross barriers, like fences, mountains, glaciers, desserts and oceans, which would stop mammals, and they can move much faster than the wingless hosts. Birds can potentially transport tick-borne pathogens by transporting infected ticks, by being infected with tick-borne pathogens and transmit the pathogens to the ticks, and possibly act as hosts for transfer of pathogens between ticks through co-feeding. Knowledge of the bird migration routes and of the spatial distribution of tick species and tick-borne pathogens is crucial for understanding the possible impact of birds as spreaders of ticks and tick-borne pathogens. Successful colonization of new tick species or introduction of new tick-borne pathogens will depend on suitable climate, vegetation and hosts. Although it has never been demonstrated that a new tick species, or a new tick pathogen, actually has been established in a new locality after being seeded there by birds, evidence strongly suggests that this could occur.

title: Spatiotemporal Variation in Avian Migration Phenology: Citizen Science Reveals Effects of Climate Change
abstract: A growing number of studies have documented shifts in avian migratory phenology in response to climate change, and yet there is a large amount of unexplained variation in the magnitude of those responses across species and geographic regions. We use a database of citizen science bird observations to explore spatiotemporal variation in mean arrival dates across an unprecedented geographic extent for 18 common species in North America over the past decade, relating arrival dates to mean minimum spring temperature. Across all species and geographic locations, species shifted arrival dates 0.8 days earlier for every °C of warming of spring temperature, but it was common for some species in some locations to shift as much as 3-6 days earlier per °C. Species that advanced arrival dates the earliest in response to warming were those that migrate more slowly, short distance migrants, and species with broader climatic niches. These three variables explained 63% of the interspecific variation in phenological response. We also identify a latitudinal gradient in the average strength of phenological response, with species shifting arrival earlier at southern latitudes than northern latitudes for the same degree of warming. This observation is consistent with the idea that species must be more phenologically sensitive in less seasonal environments to maintain the same degree of precision in phenological timing.

title: Changes in migration behaviour of Blackbirds Turdus merula from the Netherlands
abstract: Capsule Dutch Blackbirds from all regions had almost ceased migrating in the period between 1955 and 1990, probably as a result of climate change.

title: Do the Ticks of Birds at an Important Migratory Hotspot Reflect the Seasonal Dynamics of Ixodes ricinus at the Migration Initiation Site? A Case Study in the Danube Delta
abstract: Migratory birds play important roles as distributors of ticks within and between continents. In the Old World, the most important migratory route of birds links Asia, Europe and Africa. During their migration, birds use various stopover sites, where they feed and rest and where ticks may attach or detach, creating new natural foci for vector-borne diseases. Danube Delta is one of the most important migration hotspots and so far no studies were focused on ticks of migratory birds herein. The aim of the present study was to assess the species diversity and seasonal dynamics of ticks parasitizing migratory birds in Danube Delta Biosphere Reserve. Migratory birds were trapped on Grindul Lupilor (44°41′N; 28°56′E) using mist nets during 4 migratory seasons (2 spring and 2 autumn) in 2011 and 2012. From each bird, all the ticks were collected and identified based on morphological features. Epidemiological parameters (prevalence, mean abundance, mean intensity) were calculated and all data were analysed statistically based on the season (spring and autumn), regional status of birds (migrants and breeding) and foraging behaviour (ground feeders, reed-bed feeders, foliage feeders). A total of 1434 birds (46 species) were captured. Ticks were found on 94 birds (10 species). Significantly more migratory birds hosted ticks, compared to resident birds. The 400 collected ticks belonged to four species: Ixodes ricinus (92.25%), I. arboricola (6.25%), I. redikorzevi (1.00%) and Haemaphysalis punctata (0.50%). A higher prevalence was found for I. ricinus in spring, with higher prevalence of nymphs in this season, while larvae occurred with the same prevalence in both seasons. Larval intensity was higher during spring and nymphs were more abundant during autumn. The seasonal differences in our study may be related not to the local seasonal dynamics of ticks, but on the seasonal dynamics at the site of migration initiation.

title: Genetic and Ecologic Variability among Anaplasma phagocytophilum Strains, Northern Italy
abstract: The tick-borne pathogen Anaplasma phagocytophilum is an increasing potential public health threat across Europe. Its intraspecific genetic variability is associated with different reservoir host and vector tick species (1–4); however, the roles of various vertebrates as competent reservoirs of A. phagocytophilum in Europe need clarification (1). During March 2011–June 2013, we studied the prevalence and genetic variability of A. phagocytophilum in 821 questing Ixodes ricinus ticks (155 adults [A], 666 nymphs [N] collected by standard blanket dragging) and 284 engorged ixodid ticks (61A, 191N, 21 larvae [L]) collected from humans, dogs, sheep, hunted wild ungulates, live-trapped birds, and rodents. Blood samples from 1,295 rodents (yellow-necked mice [Apodemus flavicollis]), bank voles [Myodes glareolus], and harvest mice [Moscardinus avellanarius]) were also analyzed. All animal-handling procedures and ethical issues were approved by the Provincial Wildlife Management Committee (authorization n. 595 issued on 04.05.2011). The study site, Valle dei Laghi (northeastern Italian Alps), is a well-studied focus of emerging tick-borne pathogens in northern Italy (4).

title: Prevalence of tick-borne pathogens in ticks collected from migratory birds in Latvia.
abstract: Migratory birds act as hosts and long-distance vectors for several tick-borne infectious agents. Here, feeding Ixodes ticks were collected from migratory birds during the autumn migration period in Latvia and screened for the presence of epidemiologically important non-viral pathogens. A total of 93 DNA samples of ticks (37 larvae and 56 nymphs) removed from 41 birds (order Passeriformes, 9 species) was tested for Lyme borreliosis spirochaetes, Anaplasma phagocytophilum, Rickettsia spp., and Babesia spp. Borrelia burgdorferi DNA was detected in 18% of the tick samples, and a majority of infected ticks were from thrush (Turdus spp.) birds. Among the infected ticks, Borrelia valaisiana was detected in 41% of cases, Borrelia garinii in 35%, and mixed Bo. valaisiana and Bo. garinii infection in 24%. Anaplasma phagocytophilum DNA was detected in 2% of ticks, R. helvetica in 12%, and Babesia spp. pathogens in 4% of ticks. Among these samples, 3 Babesia species were identified: Ba. divergens, Ba. microti, and Ba. venatorum. Coinfection with different pathogens that included mixed infections with different Borrelia genospecies was found in 20% of nymphal and 3% of larval Ixodes ticks. These results suggest that migratory birds may support the circulation and spread of medically significant zoonoses in Europe.

title: Birds as potential reservoirs of tick-borne pathogens: first evidence of bacteraemia with Rickettsia helvetica
abstract: BackgroundBirds 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.MethodsBlood samples and ticks of birds were analysed with TaqMan real-time PCRs and conventional PCR followed by sequencing.ResultsDuring 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.ConclusionBased 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.

title: The lizards Lacerta agilis and L. vivipara as hosts to larvae and nymphs of the tick Ixodes ricinus
abstract: In this paper we provide quantitative information on the occurrence of larvae and nymphs of the tick Ixodes ricinus in populations of the lizards Lacerta agilis and L. vivipara. Levels of infestation were rather low, at least when compared with those of small mammals and sheep. Hence we suppose that lizards feed only a minor fraction of the total tick population.

title: Perpetuation of the Lyme Disease Spirochete Borrelia lusitaniae by Lizards
abstract: To determine whether the Lyme disease spirochete Borrelia lusitaniae is associated with lizards, we compared the prevalence and genospecies of spirochetes present in rodent- and lizard-associated ticks at a site where this spirochete frequently infects questing ticks. Whereas questing nymphal Ixodes ricinus ticks were infected mainly by Borrelia afzelii, one-half of the infected adult ticks harbored B. lusitaniae at our study site. Lyme disease spirochetes were more prevalent in sand lizards (Lacerta agilis) and common wall lizards (Podarcis muralis) than in small rodents. Although subadult ticks feeding on rodents acquired mainly B. afzelii, subadult ticks feeding on lizards became infected by B. lusitaniae. Genetic analysis confirmed that the spirochetes isolated from ticks feeding on lizards are members of the B. lusitaniae genospecies and resemble type strain PotiB2. At our central European study site, lizards, which were previously considered zooprophylactic for the agent of Lyme disease, appear to perpetuate B. lusitaniae.

title: Role of sand lizards in the ecology of Lyme and other tick-borne diseases in the Netherlands
abstract: BackgroundLizards are considered zooprophylactic for almost all Borrelia burgdorferi species, and act as dilution hosts in parts of North America. Whether European lizards significantly reduce the ability of B. burgdorferi to maintain itself in enzootic cycles, and consequently decrease the infection rate of Ixodes ricinus ticks for B. burgdorferi and other tick-borne pathogens in Western Europe is not clear.ResultsTicks were collected from sand lizards, their habitat (heath) and from the adjacent forest. DNA of tick-borne pathogens was detected by PCR followed by reverse line blotting. Tick densities were measured at all four locations by blanket dragging. Nymphs and adult ticks collected from lizards had a significantly lower (1.4%) prevalence of B. burgdorferi sensu lato, compared to questing ticks in heath (24%) or forest (19%). The prevalence of Rickettsia helvetica was significantly higher in ticks from lizards (19%) than those from woodland (10%) whereas neither was significantly different from the prevalence in ticks from heather (15%). The prevalence of Anaplasma and Ehrlichia spp in heather (12%) and forest (14%) were comparable, but significantly lower in ticks from sand lizards (5.4%). The prevalence of Babesia spp in ticks varied between 0 and 5.3%. Tick load of lizards ranged from 1 - 16. Tick densities were ~ 5-fold lower in the heather areas than in woodlands at all four sites.ConclusionsDespite their apparent low reservoir competence, the presence of sand lizards had insignificant impact on the B. burgdorferi s.l. infection rate of questing ticks. In contrast, sand lizards might act as reservoir hosts for R. helvetica. Remarkably, the public health risk from tick-borne diseases is approximately five times lower in heather than in woodland, due to the low tick densities in heather.

title: The role of the sand lizard (Lacerta agilis) in the transmission cycle of Borrelia burgdorferi sensu lato
abstract: In order to examine the role of Lacerta agilis in the transmission cycle of Borrelia burgdorferi sensu lato, lizards were captured in three different localities in Slovakia, Poland, and Romania. Skin biopsy specimens from collar scales and ticks feeding on the lizards at the time of capture were collected. In total, 87 individuals (11 in Slovakia, 48 in Poland, 28 in Romania) of L. agilis were captured. Altogether, 245 (74, 74, 97) larvae and 191 (78, 113, 0) nymphs were removed from captured lizards. Borrelial infection was detected by PCR amplifying a fragment of the 5S–23S rDNA intergenic spacer and genotyping by restriction fragment length polymorphism (RFLP). When examining the presence of borreliae in biopsy specimens, striking differences between separate populations were observed. Whilst none of the biopsy specimens from L. agilis from Poland were positive for B. burgdorferi s.l., 45% of the sand lizards from Slovakia and 57% from Romania were positive. B. lusitaniae was confirmed in all positive biopsy specimens. The prevalence of borreliae in ticks that had fed on lizards was 6% in Poland, 21% in Slovakia, and 13% in Romania. While B. lusitaniae was the only genospecies in ticks from Slovakia (except for 2 larvae infected with B. afzelii) and Romania, it represented 64% and B. valaisiana 27% of the borrelia infections in ticks from lizards captured in Poland. The highest probability of ticks to get infected expressed as specific infectivity of lizards was recorded in Slovakia (0.102) and the lowest in Poland (0.003). Our findings underline the importance of the sand lizard in the transmission cycle of B. lusitaniae.

title: Tick-borne encephalitis.
abstract: Tick-borne encephalitis (TBE) is one of the most dangerous human infections occurring in Europe and many parts of Asia. The etiological agent Tick-borne encephalitis virus (TBEV), is a member of the virus genus Flavivirus, of the family Flaviviridae. TBEV is believed to cause at least 11,000 human cases of encephalitis in Russia and about 3000 cases in the rest of Europe annually. Related viruses within the same group, Louping ill virus (LIV), Langat virus (LGTV) and Powassan virus (POWV), also cause human encephalitis but rarely on an epidemic scale. Three other viruses within the same group, Omsk hemorrhagic fever virus (OHFV), Kyasanur Forest disease virus (KFDV) and Alkhurma virus (ALKV), are closely related to the TBEV complex viruses and tend to cause fatal hemorrhagic fevers rather than encephalitis. This review describes the clinical manifestations associated with TBEV infections, the main molecular-biological properties of these viruses, and the different factors that define the incidence and severity of disease. The role of ticks and their local hosts in the emergence of new virus variants with different pathogenic characteristics is also discussed. This review also contains a brief history of vaccination against TBE including trials with live attenuated vaccine and modern tendencies in developing of vaccine virus strains.

title: Infections with the tick-borne bacterium "Candidatus Neoehrlichia mikurensis" mimic noninfectious conditions in patients with B cell malignancies or autoimmune diseases.
abstract: Candidatus Neoehrlichia mikurensis is a newly discovered noncultivatable bacterium spread among ticks and rodents in Europe and Asia that can infect humans, particularly immunocompromised patients.



We compiled clinical and laboratory data from 11 patients with hematological malignances or autoimmune diseases who were diagnosed with Candidatus N. mikurensis infection in Europe 2010-2013. Both published (6) and unpublished cases (5) were included.



The patients had a median age of 67, were mostly male (8/11), and resided in Sweden, Switzerland, Germany, and the Czech Republic. All but one had ongoing or recent immune suppressive treatment and a majority were splenectomized (8/11). Less than half of them recalled tick exposure. The most frequent symptoms were fever (11/11), localized pain afflicting muscles and/or joints (8/11), vascular and thromboembolic events (6/11), that is, deep vein thrombosis (4), transitory ischemic attacks (2), pulmonary embolism (1), and arterial aneurysm (1). Typical laboratory findings were elevated C-reactive protein, leukocytosis with neutrophilia, and anemia. Median time from onset of symptoms to correct diagnosis was 2 months. In at least 4 cases, the condition was interpreted to be due to the underlying disease, and immunosuppressive therapy was scheduled. All patients recovered completely when doxycycline was administered.



Candidatus N. mikurensis is an emerging tick-borne pathogen that may give rise to a systemic inflammatory syndrome in persons with hematologic or autoimmune diseases that could be mistaken for recurrence of the underlying disease and/or unrelated arteriosclerotic vascular events. Awareness of this new pathogen is warranted among rheumatologists, hematologists, oncologists, and infectious disease specialists.

title: Human Infection with Candidatus Neoehrlichia mikurensis, China
abstract: To identify Candidatus Neoehrlichia mikurensis infection in northeastern China, we tested blood samples from 622 febrile patients. We identified in 7 infected patients and natural foci for this bacterium. Field surveys showed that 1.6% of ticks and 3.8% of rodents collected from residences of patients were also infected.

title: Update on Tick-Borne Rickettsioses around the World: a Geographic Approach
abstract: Tick-borne rickettsioses are caused by obligate intracellular bacteria belonging to the spotted fever group of the genus Rickettsia. These zoonoses are among the oldest known vector-borne diseases. However, in the past 25 years, the scope and importance of the recognized tick-associated rickettsial pathogens have increased dramatically, making this complex of diseases an ideal paradigm for the understanding of emerging and reemerging infections. Several species of tick-borne rickettsiae that were considered nonpathogenic for decades are now associated with human infections, and novel Rickettsia species of undetermined pathogenicity continue to be detected in or isolated from ticks around the world. This remarkable expansion of information has been driven largely by the use of molecular techniques that have facilitated the identification of novel and previously recognized rickettsiae in ticks. New approaches, such as swabbing of eschars to obtain material to be tested by PCR, have emerged in recent years and have played a role in describing emerging tick-borne rickettsioses. Here, we present the current knowledge on tick-borne rickettsiae and rickettsioses using a geographic approach toward the epidemiology of these diseases.

title: Zoonotic babesiosis: overview of the disease and novel aspects of pathogen identity.
abstract: Babesiosis is a zoonosis caused by tick-transmitted intraerythrocytic protozoa of the Phylum Apicomplexa. The disease mostly occurs in the USA, but cases have also been reported in several European countries, in Egypt, India, Japan, Korea, Taiwan, and South Africa. The main pathological event is lysis of erythrocytes resulting in haemolytic anaemia, which in severe cases may lead to organ failure and death, particularly in immunocompromised patients. The 2 groups of parasites involved, Babesia microti-like and Babesia sensu stricto (s.s.) species, differ in their life cycle characteristics and susceptibility to antibabesial drugs. Molecular taxonomy is now making a major contribution to the identification of novel pathogens within both groups. Effective treatment of severe cases was initially hampered by the lack of specific antibabesial drugs for human use, but increased use of supportive measures and of the recently developed antimalarial, atovaquone, particularly in combination with azithromycin, has improved the prospects for management of acute disease especially when caused by Babesia s.s. species. Prevention should be based primarily on increasing the awareness of physicians and the public to the risks, but infection from blood transfusions is particularly difficult to prevent. Expanding deer populations, resulting in wider distribution and greater abundance of ticks, heightened medical awareness, and growing numbers of immunocompromised patients are likely to result in a continuing rise of reported cases.

title: Update on tick-borne bacterial diseases in Europe.
abstract: In recent years, the prevalence of tick-borne bacterial diseases has significantly increased in European countries. The emergence and reemergence of these illnesses are attributed to changes in the environment and human behavior. Several diseases are caused by bacteria initially isolated from ticks and subsequently considered pathogenic. It is necessary to consider the bacteria found in arthropods capable of biting humans as potential human pathogens. Here we review the clinical and epidemiological data on bacterial tick-borne diseases in European countries. We focus on the epidemiological and clinical aspects of tick-borne rickettsioses and give an overview of other tick-borne illnesses as well as the emergence and re-emergence of these diseases.

title: Tick-borne encephalitis--a notifiable disease: report of the 15th Annual Meeting of the International Scientific Working Group on Tick-Borne Encephalitis (ISW-TBE).
abstract: The 15th Meeting of the International Scientific Working Group on Tick-Borne Encephalitis (ISW-TBE)--a group of neurologists, general practicioners, clinicians, travel physicans, virologists, pediatricians, and epidemiologists--was held under the title "Tick-Borne Encephalitis--a notifiable disease". With the inclusion of TBE in the list of notifiable diseases, an important measure was established to continue improving the level of evidence on TBE in Europe to better help guide policies and methods to lower the burden of this disease. Due to differences in diagnosis, case definition, and reporting in European countries, the overall epidemiology and burden of TBE remains unclear. During the meeting, important issues regarding epidemiology, risk areas, vaccination rates, and latest news on vaccination were presented and extensively discussed. A poster session provided an overview of the epidemiological situation 2012 in 13 European countries.

title: Epidemiology and distribution of tick-borne encephalitis
abstract: SummaryTick-borne encephalitis (TBE) is the main tick-borne virus infection in Eurasia. It is prevalent across the entire continent from Japan to France and occurs in endemic foci. Expansion of prevalence in areas including northern Russia, Sweden, and Finland has been observed in recent years. Ticks are the most important vectors and may transmit the TBE virus to animals and humans. TBE can also be transmitted to humans in milk containing the virus. TBE has been implicated as a travel-acquired illness and there are isolated reports of its occurrence in countries outside the known areas of prevalence. Therefore, TBE should be included in the differential diagnosis for all central nervous system diseases inside or outside endemic areas.ZusammenfassungDie Frühsommer-Meningoenzephalitis (FSME) ist die medizinisch wichtigste durch Zecken übertragene Virusinfektion in Eurasien. Ihr Verbreitungsgebiet reicht von Japan im Osten Asiens bis nach Frankreich im Westen Europas. In den zurückliegenden Jahren wurde ein Ausweitung der Verbreitungsgebiete in nördlicher Richtung in Nord-Russland, Schweden und Finnland beobachtet. Zecken spielen die wichtigste Rolle in der Übertragung des FSME-Virus auf Mensch und Tier. Allerdings kann die Infektion auch durch Virus-haltige Milch erfolgen. Die FSME spielt eine zunehmende Rolle in der Reisemedizin. Erkrankungsfälle treten in Regionen außerhalb der bekannten FSME-Verbreitungsgebiete auf. Daher sollte die FSME grundsätzlich bei allen entzündlichen Erkrankungen des Zentralen Nervensystems differenzialdiagnostisch ausgeschlossen werden.

title: Epidemiology of tick-borne encephalitis (TBE) in Europe and its prevention by available vaccines
abstract: Tick-borne Encephalitis (TBE), which is caused by a Flavivirus, is the most common tick-transmitted disease in Central and Eastern Europe and Russia. Today, TBE is endemic in 27 European countries, and has become an international public health problem. The epidemiology of TBE is changing owing to various factors, such as improvements in diagnosis and case reporting, increased recreational activities in areas populated by ticks, and changes in climatic conditions affecting tick habitats. Vaccination remains the most effective protective measure against TBE for people living in risk zones, occupationally exposed subjects and travelers to endemic areas. The vaccines currently in use are FSME-Immun®, Encepur®, EnceVir® and TBE vaccine Moscow®. The numerous studies performed on the efficacy and safety of these vaccines have shown a high level of immunogenicity and an excellent safety profile. Several studies have also shown a high level of cross-protection among strains belonging to different subtypes. In the present paper we attempted to describe the continuously changing epidemiology of TBE in European States and to overview clinical development of available vaccines paying particular attention on cross-protection elicited by the vaccines.

title: Integration of a tick-borne encephalitis virus and Borrelia burgdorferi sensu lato into mountain ecosystems, following a shift in the altitudinal limit of distribution of their vector, Ixodes ricinus (Krkonose mountains, Czech Republic).
abstract: The altitudinal shift in the limit of Ixodes ricinus occurrence above the previously established altitude of 750 m above sea level has been monitored over the long-term (2002-2008) in the Krkonose Mts. (Giant Mts.), the highest in the Czech Republic, along two vertical transects in their eastern and central parts (600-1020 and 600-1270 m). Ticks were collected by flagging three times annually, and examined individually by PCR or RT-PCR for the presence of Borrelia burgdorferi sensu lato or tick-borne encephalitis virus (TBEV). A total of 5999 I. ricinus ticks were tested. TBEV RNA was detected in 26 ticks at up to 1140 m. Demonstration of TBEV in two larvae of I. ricinus indicates transovarial transmission. Similar infection rates in larvae and nymphs show vertical transmission in TBEV circulation to be very important under these mountain conditions. B. burgdorferi sensu stricto was found at up to 1040-1065 m, Borrelia garinii and Borrelia afzelii up to 1080-1140 m, and Borrelia valaisiana up to 1270 m. The total infection rates of nymphs and larvae were 7.3% and 2%, respectively. B. garinii was the most prevalent (37%), followed by B. afzelii (29%), B. burgdorferi s.s. (11%), and B. valaisiana (9%). Double to quadruple coinfections were detected in 32% of the infected ticks, most frequently B. garinii/B. afzelii. Predominance of B. garinii and B. valaisiana over B. afzelii suggests that small passerine birds moving on the ground are responsible for permanent local populations of I. ricinus in mountain localities with low numbers of small terrestrial mammals. The detection of B. burgdorferi sensu lato and TBEV in host-seeking larvae indicates an autochthonic infection. Upon analysis of the local climate we consider climate warming to be responsible for the spreading of ticks and tick-transmitted pathogens to higher altitudes.

title: The shifting landscape of tick-borne zoonoses: tick-borne encephalitis and Lyme borreliosis in Europe.
abstract: The two major vector-borne diseases of northern temperate regions, tick-borne encephalitis (TBE) and Lyme borreliosis (LB), show very different epidemiological patterns, but both have increased significantly in incidence since the 1980s. Insight into the temporal dynamics of TBE, gained from statistical analysis of spatial patterns integrated with biological explanation, suggests that the recent increases in TBE cases in Central Europe and the Baltic States may have arisen largely from changes in human behaviour that have brought more people into contact with infected ticks. Under forecast climate change scenarios, it is predicted that enzootic cycles of TBE virus may not survive along the southern edge of their present range, e.g. in Slovenia, Croatia and Hungary, where case numbers are indeed decreasing. New foci, however, are predicted and have been observed in Scandinavia. At the same time, human impact on the landscape, increasing both the habitat and wildlife hosts of ticks, has allowed tick populations to multiply significantly. This probably accounts for a genuine emergence of LB, with its high potential transmission rate, in both the USA and Europe, although the rate of emergence has been exaggerated by improved surveillance and diagnosis.

title: Evidence that climate change has caused 'emergence' of tick-borne diseases in Europe?
abstract: Even though tick-borne disease systems are highly susceptible to climatic influences, climate change to date is not necessarily the cause of the marked increased incidence of a variety of tick-borne diseases in many parts of Europe over the past two decades. To test for causality, rather than coincidence, we need to examine whether the right sorts of climate change have occurred at the right time and in the right places to account for the observed heterogeneous temporal and spatial patterns of tick-borne disease 'emergence'. Tick-borne encephalitis (TBE) incidence, for example, showed a 3-fold step increase from 1983 to 1986 in Sweden, doubled in 1993 in the Czech Republic, increased even more dramatically in the same year in Lithuania and Poland, but declined markedly in 1997 in Hungary, Croatia and Slovenia. Within each country, TBE incidence has changed to different degrees in different regions. Because other tick-borne diseases, notably Lyme borreliosis, has commonly 'emerged' in parallel with TBE, we should first examine climate variables predicted to have a general effect on tick abundance, which has indeed increased in the past decade. These include temperature and moisture stress, which have seasonally differential impacts. Monthly mean records for 1960-2000 from the UK Climate Research Unit's interpolated global climate surface reveal that mean spring, spring-autumn and winter temperatures have all increased gradually over the past 40 years, but apparently most sharply in the late 1980s, when moisture stress also increased. These climate data do not reveal any obvious differences between sites where TBE did or did not 'emerge', and in Sweden increases in TBE pre-dated the onset of warmer springs and winters. If recorded climate changes cannot yet satisfactorily explain the temporal and spatial patterns of tick-borne disease change in Europe, the impact of biotic factors, such as increases in deer abundance and changing habitat structure, and of socio-political changes following the end of communist rule, demand more detailed quantitative analyses.

title: Tick-borne encephalitis incidence in Central and Eastern Europe: consequences of political transition.
abstract: The variable, often dramatic, upsurge in tick-borne encephalitis in Central and Eastern Europe can best be understood as the result of a multi-factorial system of causes, including abiotic and biotic environmental changes, and human behaviour determined by socio-economic conditions. Many of these stem from the political transition with the end of Soviet rule.

title: Mapping Climate Change Vulnerabilities to Infectious Diseases in Europe
abstract: The incidence, outbreak frequency, and distribution of many infectious diseases are generally expected to change as a consequence of climate change, yet there is limited regional information available to guide decision making.  We surveyed government officials designated as Competent Bodies for Scientific Advice concerning infectious diseases to examine the degree to which they are concerned about potential effects of climate change on infectious diseases, as well as their perceptions of institutional capacities in their respective countries.  In 2007 and 2009/2010, national infectious disease experts from 30 European Economic Area countries were surveyed about recent and projected infectious disease patterns in relation to climate change in their countries and the national capacity to cope with them.  A large majority of respondents agreed that climate change would affect vector-borne (86% of country representatives), food-borne (70%), water-borne (68%), and rodent-borne (68%) diseases in their countries. In addition, most indicated that institutional improvements are needed for ongoing surveillance programs (83%), collaboration with the veterinary sector (69%), management of animal disease outbreaks (66%), national monitoring and control of climate-sensitive infectious diseases (64%), health services during an infectious disease outbreak (61%), and diagnostic support during an epidemic (54%).  Expert responses were generally consistent with the peer-reviewed literature regarding the relationship between climate change and vector- and water-borne diseases, but were less so for food-borne diseases. Shortcomings in institutional capacity to manage climate change vulnerability, identified in this assessment, should be addressed in impact, vulnerability, and adaptation assessments.

title: Socio‐economic factors in the differential upsurge of tick‐borne encephalitis in central and Eastern Europe
abstract: Tick‐borne encephalitis (TBE), the most serious widespread vector‐borne disease of humans in Europe, increased from 2‐ to 30‐fold in many Central and Eastern European countries from 1992 to 1993, coinciding with independence from Soviet rule. Unemployment and low income have been shown in Latvia to be statistically associated with high‐risk behaviour involving harvest of wild foods from tick‐infested forests, and also with not being vaccinated against TBE. Archival data for 1970‐‐2005 record major changes in the agricultural and industrial sectors, and consequent changes in the abiotic and biotic environment and socio‐economic conditions, which could have increased the abundance of infected ticks and the contact of humans with those ticks. For example, abandoned agricultural fields became suitable for rodent transmission hosts; use of pesticides and emissions of atmospheric industrial pollutants plummeted; wildlife hosts for ticks increased; tick populations appear to have responded; unemployment and inequality increased in all countries. These factors, by acting synergistically but differentially between and within each country, can explain the marked spatio‐temporal heterogeneities in TBE epidemiology better than can climate change alone, which is too uniform across wide areas. Different degrees of socio‐economic upheaval caused by political transition in Estonia, Latvia, Lithuania, Slovenia and the Czech Republic can apparently explain the marked variation in TBE upsurge. Causal linkage between national socio‐economic conditions and epidemiology is strongly indicated by striking correlations across eight countries between the degree of upsurge of TBE and both poverty and household expenditure on food (R2 = 0.533 and 0.716, respectively).

title: Tick-borne Encephalitis from Eating Goat Cheese in a Mountain Region of Austria
abstract: We report transmission of tick-borne encephalitis virus (TBEV) in July 2008 through nonpasteurized goat milk to 6 humans and 4 domestic pigs in an alpine pasture 1,500 m above sea level. This outbreak indicates the emergence of ticks and TBEV at increasing altitudes in central Europe and the efficiency of oral transmission of TBEV.

title: Tick-borne encephalitis outbreak in Hungary due to consumption of raw goat milk.
abstract: A tick-borne encephalitis outbreak involving 25 patients of 154 exposed persons occurred in Hungary in August 2007. None of the patients had a history of tick-bite, however all of them drank unpasteurized raw goat milk from the same farm. The aim of this study was to identify the goats on the farm which could have spread the infection through their milk. Blood samples were taken from 75 goats on the farm and were examined by various serological methods, namely indirect immunofluorescent assay, hemagglutination inhibition, microneutralization and an ELISA adapted to testing material from goats, to determine antibody levels in the serum. The four methods have proved different levels of specificity. The least specific was the indirect immunofluorescent assay, which showed a low titre in all sera. Comparison of the results of the other three methods indicates that two sera were positive for anti-TBEV IgG and one for anti-TBEV IgM. The goat with the IgM positive serum sample could have been a source of the infected milk. It has been concluded that serological results for goats by the different methods should be compared before final diagnosis because the specificity of methods in use can differ significantly.

title: Spatial distribution of a population at risk: an important factor for understanding the recent rise in tick-borne diseases (Lyme borreliosis and tick-borne encephalitis in the Czech Republic).
abstract: Recent rise in tick-borne diseases in many parts of Europe is a phenomenon in need of an explanation. We analyzed temporal trends in spatial distribution of a population at risk of Lyme borreliosis, tick-borne encephalitis, and as a control, also of a 'non-tick-borne disease' in the Czech Republic in 1997-2010. Analysis revealed that the population's exposure had been increasingly confined to the nearest surroundings of residences or in totally residential locations and that the incidence of the diseases depended in some causal way on how close to residences people exposed themselves to the risk. The rise in Lyme borreliosis and tick-borne encephalitis was solely due to infections acquired at or near patients' homes (<5 km), while the number of cases acquired further away was decreasing. The detected patterns in the data question some of the hypotheses which may be applicable in explaining the rise in disease incidences in the Czech Republic including the effect of climate change. Potentially causal factors are discussed.

title: New endemic foci of tick-borne encephalitis (TBE) identified in districts where testing for TBE was not available before 2009 in Poland
abstract: BackgroundTick-borne encephalitis (TBE) is found in limited endemic foci in Poland. Lack of diagnosis limits disease detection in non-endemic provinces.MethodsIn 2009, we enhanced TBE surveillance to confirm the location of endemic foci and inform vaccination policy. In 105 hospitals located in 11/16 provinces, we identified suspected TBE cases through admission ICD-10 codes indicating aseptic meningo-encephalitis or from specimens tested for TBE. The National Reference Laboratory confirmed cases at no cost, by testing serum and/or cerebrospinal fluid using ELISA method. We calculated TBE reported rates as the number of confirmed TBE cases per 100,000 inhabitants. Adjusting to neighbouring districts, we classified districts as non-endemic (<0.1 cases per 100,000 inhabitants), low endemic (> = 0.1 to <1), moderately endemic (> = 1 to <5) and highly endemic (> = 5). We compared surveillance data obtained in 2009 with 2004–2008 baseline data.ResultsAmong 166,099 admissions, we identified 1,585 suspected TBE cases of which 256 were confirmed. Physicians reported more suspected cases among patients <40 years old (12 cases per 1,000 admissions) than among older patients (8 cases per 1,000 admissions). However, patients <40 years of age were confirmed less frequently (16%), than older patients (35%). Physicians reported more suspected cases in districts classed as endemic during 2004–2008 (12 cases per 1,000 admissions, 77% tested for TBE) than in districts classed as non-endemic (7 cases per 1,000 admissions, 59% tested). Of the 38 newly identified endemic districts, 31 were adjacent to 2004–2008 endemic districts and 7 were isolated.ConclusionsEnhanced surveillance detected 38 new endemic districts to be considered for TBE vaccination. However, lack of consistent testing in districts believed to be TBE-free remained an obstacle for mapping TBE risk. Although the disease affects mostly older adults and the elderly, more attention is given to the diagnosis of TBE in young patients. Solutions need to be identified to sustain sensitive, acceptable and affordable TBE surveillance in all districts of Poland. Also, higher attention should be given to the diagnosis of TBE in the elderly.

title: Occurrence of ixodid ticks--the main vectors of tick-borne encephalitis virus in urbanized territory.
abstract: The influence of urbanization on ticks I. ricinus and I. persulcatus--the main vectors of tick-borne encephalitis virus is discussed on the basis of concrete examples. It has been revealed that under favourable conditions the populations of these tick species can exist for a long time not only in towns and new housing estates, but in the old residential districts as well. The necessity of studying the ecology of urban populations of ixodid ticks is pointed out.

title: Dynamics of infection in tick vectors and at the tick-host interface.
abstract: Tick-borne flaviviruses are common, widespread, and successfully adapted to their mode of transmission. Most tick vectors of flaviviruses are ixodid species. These ticks are characterized by a comparatively long life cycle, lasting several years, during which the infecting virus may be maintained from one developmental stage of the tick to the next. Hence ticks act as highly efficient reservoirs of flaviviruses. Many tick-borne flaviviruses are transmitted vertically, from adult to offspring, although the frequency is too low to maintain the viruses solely in the tick population. Instead, the survival of tick-borne flaviviruses is dependent on horizontal transmission, both from an infected tick to a susceptible vertebrate host and from an infected vertebrate to uninfected ticks feeding on the animal. The dynamics of transmission and infection have traditionally been considered in isolation: in the tick, following virus uptake in the infected blood meal, infection of the midgut, passage through the hemocoel to the salivary glands, and transmission via the saliva; and in the vertebrate host, virus delivery into the skin at the site of tick feeding, infection of the draining lymph nodes, and dissemination to target organs. However, there is now compelling evidence of a complex interaction between the tick vector and its vertebrate host that affects virus transmission profoundly. The feeding site in the skin is a battleground in which the hemostatic, inflammatory, and immune responses of the host are countered by antihemostatic, anti-inflammatory, and immunomodulatory molecules (mostly proteins and peptides) secreted in tick saliva. Here we speculate that exploitation of the tick pharmacopeia, rather than development of viremia, is the key step in successful tick-borne flavivirus transmission.

title: Tick-borne encephalitis virus transmission between ticks cofeeding on specific immune natural rodent hosts.
abstract: To determine whether the portion of a vertebrate host population having specific immunity to tick-borne encephalitis (TBE) virus can participate in the TBE virus transmission cycle, natural hosts immunized against TBE virus were challenged with infected and uninfected ticks. Yellow-necked field mice (Apodemus flavicollis) and bank voles (Clethrionomys glareolus) were either immunized with TBE virus by subcutaneous inoculation of the virus, or they were exposed to virus-infected Ixodes ricinus ticks. One month later, when serum neutralizing antibody was detectable, the animals were infested with infected (donor) adult female ticks and uninfected (recipient) nymphal ticks; recipients were allowed to feed either in close contact (chamber 1) or physically separated (chamber 2) from the infected donor ticks. Following challenge with infected (and uninfected) ticks, viremia developed in all the control, nonimmune animals, whereas viremia was undetectable in all those animals naturally immunized by previous exposure to infected ticks. Despite the presence of neutralizing antibodies in all the immunized animals, 89% (24/ 27) immune animals supported virus transmission between infected and uninfected cofeeding ticks. Most transmission was localized, occurring within chamber 1; disseminated transmission from chamber 1 to chamber 2 was reduced. Immunization by tick bite was more effective than immunization by syringe inoculation in blocking cofeeding virus transmission. Nevertheless 76% (9/12) animals with "natural" immunity still supported transmission. The results demonstrate that natural hosts having neutralizing antibodies to TBE virus (and no detectable viremia) can still support virus transmission between infected and uninfected ticks feeding closely together on the same animal. These observations have important epidemiological implications relating to the survival of TBE virus in

title: Tick-borne encephalitis 2010: epidemiology, risk areas, and virus strains in Europe and Asia-an overview.
abstract: Over the past decades, tick-borne encephalitis (TBE) has become a growing public health concern in Europe and Asia and is the most important viral tick-borne disease in Europe. It is also important in the Far East and in other parts of Asia. Vaccination can effectively prevent TBE. It is indicated for persons inhabiting or visiting a TBE endemic area who are likely to be exposed to tick bites. For this purpose, it is necessary to know where TBE virus (TBEV) occurs, where vectors are a potential hazard, and where as a consequence autochthonous TBE cases have been registered. Natural changes in the known TBE foci and the detection of new foci necessitate a summarizing data update to optimize the protection of inhabitants and visitors to TBE endemic areas. The present review includes an overview of the epidemiological data on TBE in Europe and Asia gathered between 1990 and 2009 and describes old and especially new TBE risk areas in Europe and Asia.

title: Rodents as sentinels for the prevalence of tick-borne encephalitis virus.
abstract: Tick-borne encephalitis virus (TBEV) causes one of the most important flavivirus infections of the central nervous system, affecting humans in Europe and Asia. It is mainly transmitted by the bite of an infected tick and circulates among them and their vertebrate hosts. Until now, TBE risk analysis in Germany has been based on the incidence of human cases. Because of an increasing vaccination rate, this approach might be misleading, especially in regions of low virus circulation.



To test the suitability of rodents as a surrogate marker for virus spread, laboratory-bred Microtus arvalis voles were experimentally infected with TBEV and analyzed over a period of 100 days by real-time (RT)-quantitative polymerase chain reaction. Further, the prevalence of TBEV in rodents trapped in Brandenburg, a rural federal state in northeastern Germany with autochthonous TBE cases, was determined and compared with that in rodents from German TBE risk areas as well as TBE nonrisk areas.



In experimentally infected M. arvalis voles, TBEV was detectable in different organs for at least 3 months and in blood for 1 month. Ten percent of all rodents investigated were positive for TBEV. However, in TBE risk areas, the infection rate was higher compared with that of areas with only single human cases or of nonrisk areas. TBEV was detected in six rodent species: Apodemus agrarius, Apodemus flavicollis, Apodemus sylvaticus, Microtus agrestis, Microtus arvalis, and Myodes glareolus. M. glareolus showed a high infection rate in all areas investigated.



The infection experiments proved that TBEV can be reliably detected in infected M. arvalis voles. These voles developed a persistent TBE infection without clinical symptoms. Further, the study showed that rodents, especially M. glareolus, are promising sentinels particularly in areas of low TBEV circulation.

title: Non-viraemic transmission of tick-borne encephalitis virus: a mechanism for arbovirus survival in nature
abstract: The vectors of arthropod-borne viruses (arboviruses) become infected by feeding on the viraemic blood of an infected animal. This theory is based on transmission studies involving artificial infection of vertebrate hosts by syringe inoculation. To reproduce natural conditions of virus transmission, infected and uninfected vectors (ticks) of tick-borne encephalitis virus, the most important arbovirus in Europe, were allowed to feed together on uninfected wild vertebrate hosts. The greatest numbers of infected ticks were obtained from susceptible host species that had undetectable or very low levels of viraemia. The results suggest that ‘nonviremic transmission’ is an important mechanism for the survival of certain arboviruses in nature.

title: Incidence from coincidence: patterns of tick infestations on rodents facilitate transmission of tick-borne encephalitis virus.
abstract: Tick-borne encephalitis (TBE) virus has a highly focal distribution through Eurasia. Endemic cycles appear to depend on the transmission of non-systemic infections between ticks co-feeding on the same rodent hosts. The particular features of seasonal dynamics and infestation patterns of larval and nymphal Ixodes ricinus, but not Dermacentor reticulatus, from 4 regions within TBE foci in Slovakia, are such as to promote TBE virus transmission. The distributions of larvae and nymphs on their principal rodent hosts are highly aggregated and, rather than being independent, the distributions of each stage are coincident so that the same ca. 20% of hosts feed about three-quarters of both larvae and nymphs. This results in twice the number of infectible larvae feeding alongside potentially infected nymphs compared with the null hypothesis of independent distributions. Overall, co-feeding transmission under these circumstances brings the reproductive number (R0) for TBE virus to a level that accounts quantitatively for maintained endemic cycles. Essential for coincident aggregated distributions of larvae and nymphs is their synchronous seasonal activity. Preliminary comparisons support the prediction of a greater degree of coincident seasonality within recorded TBE foci than outside. This identifies the particular climatic factors that permit such patterns of tick seasonal dynamics as the primary predictors for the focal distribution of TBE.

title: Migrating Birds and Tickborne Encephalitis Virus
abstract: During spring and autumn 2001, we screened 13,260 migrating birds at Ottenby Bird Observatory, Sweden, and found 3.4% were infested with ticks. Four birds, each a different passerine species, carried tickborne encephalitis virus (TBEV)–infected ticks (Ixodes ricinus). Migrating birds may play a role in the geographic dispersal of TBEV-infected ticks.

title: Tick-borne pathogens in ticks collected from breeding and migratory birds in Switzerland.
abstract: From 2007 to 2010, 4558 migrating and breeding birds of 71 species were caught and examined for ticks in Switzerland. A total of 1205 specimens were collected; all were Ixodes ricinus ticks except one Ixodes frontalis female, which was found on a common chaffinch (Fringilla coelebs) for the first time in Switzerland. Each tick was analysed individually for the presence of Borrelia spp., Rickettsia spp., Anaplasma phagocytophilum and tick-borne encephalitis virus (TBEV). Altogether, 11.4% of birds (22 species) were infested by ticks and 39.8% of them (15 species) were carrying infected ticks. Bird species belonging to the genus Turdus were the most frequently infested with ticks and they were also carrying the most frequently infected ticks. Each tick-borne pathogen for which we tested was identified within the sample of bird-feeding ticks: Borrelia spp. (19.5%) and Rickettsia helvetica (10.5%) were predominantly detected whereas A. phagocytophilum (2%), Rickettsia monacensis (0.4%) and TBEV (0.2%) were only sporadically detected. Among Borrelia infections, B. garinii and B. valaisiana were largely predominant followed by B. afzelii, B. bavariensis, B. miyamotoi and B. burgdorferi ss. Interestingly, Candidatus Neoehrlichia mikurensis was identified in a few ticks (3.3%), mainly from chaffinches. Our study emphasizes the role of birds in the natural cycle of tick-borne pathogens that are of human medical and veterinary relevance in Europe. According to infection detected in larvae feeding on birds we implicate the common blackbird (Turdus merula) and the tree pipit (Anthus trivialis) as reservoir hosts for Borrelia spp., Rickettsia spp. and A. phagocytophilum.

title: Determinants of tick-borne encephalitis in counties of southern Germany, 2001-2008
abstract: BackgroundTick-borne encephalitis (TBE) virus can cause severe symptoms in humans. The incidence of this vector-borne pathogen in humans is characterised by spatial and temporal heterogeneity. To explain the variation in reported human TBE cases per county in southern Germany, we designed a time-lagged, spatially-explicit model that incorporates ecological, environmental, and climatic factors.ResultsWe fitted a logistic regression model to the annual counts of reported human TBE cases in each of 140 counties over an eight year period. The model controlled for spatial autocorrelation and unexplained temporal variation. The occurrence of human TBE was found to be positively correlated with the proportions of broad-leafed, mixed and coniferous forest cover. An index of forest fragmentation was negatively correlated with TBE incidence, suggesting that infection risk is higher in fragmented landscapes. The results contradict previous evidence regarding the relevance of a specific spring-time temperature regime for TBE epidemiology. Hunting bag data of roe deer (Capreolus capreolus) in the previous year was positively correlated with human TBE incidence, and hunting bag density of red fox (Vulpes vulpes) and red deer (Cervus elaphus) in the previous year were negatively correlated with human TBE incidence.ConclusionsOur approach suggests that a combination of landscape and climatic variables as well as host-species dynamics influence TBE infection risk in humans. The model was unable to explain some of the temporal variation, specifically the high counts in 2005 and 2006. Factors such as the exposure of humans to infected ticks and forest rodent population dynamics, for which we have no data, are likely to be explanatory factors. Such information is required to identify the determinants of TBE more reliably. Having records of TBE infection sites at a finer scale would also be necessary.

title: Tick-borne encephalitis transmission risk: its dependence on host population dynamics and climate effects.
abstract: Tick-borne encephalitis (TBE) is a human disease caused by a flavivirus that is spread by ticks (Ixodes ricinus). In 2011 and 2012, the highest TBE incidence ever was recorded in Sweden. It has been proposed that warmer spring temperatures result in higher survival of ticks and thus high incidence of TBE. Here, analyses were done of time series of TBE for 1976-2011 in relation to the North Atlantic Oscillation (NAO), mean summer temperatures, and yearly number of harvested European hare (Lepus europeaus), roe deer (Capreolus capreolus), and red fox (Vulpes vulpes) in the County of Stockholm, the area with most TBE cases in recent years in Sweden. The results show that the winter NAO index or winter temperature has no significant effect on the variation in wildlife numbers harvested or TBE cases over time. Mean summer temperature above 12°C had a slight effect, but a multivariate model revealed that only the numbers of European hare and red fox remained in the model and explained 64.4% of the variation in TBE cases. Ticks do not seem to be as sensitive to climate variations as anticipated, even though that summer temperature has increased by 2°C during the time period studied here. Instead, TBE cases seem to be more dependent on host population dynamics than on climate factors.

title: Detection and genetic characterization of tick-borne encephalitis virus (TBEV) derived from ticks removed from red foxes (Vulpes vulpes) and isolated from spleen samples of red deer (Cervus elaphus) in Croatia.
abstract: Tick-borne encephalitis (TBE) is a growing public health concern in central and northern European countries. Even though TBE is a notifiable disease in Croatia, there is a significant lack of information in regard to vector tick identification, distribution as well as TBE virus prevalence in ticks or animals. The aim of our study was to identify and to investigate the viral prevalence of TBE virus in ticks removed from red fox (Vulpes vulpes) carcasses hunted in endemic areas in northern Croatia and to gain a better insight in the role of wild ungulates, especially red deer (Cervus elaphus) in the maintenance of the TBE virus in the natural cycle. We identified 5 tick species (Ixodes ricinus, Ixodes hexagonus, Haemaphysalis punctata, Dermacentor reticulatus, Rhipicephalus sanguineus) removed from 40 red foxes. However, TBE virus was isolated only from adult I. ricinus and I. hexagonus ticks showing a viral prevalence (1.6%) similar to or higher than reported in endemic areas of other European countries. Furthermore, 2 positive spleen samples from 182 red deer (1.1%) were found. Croatian TBE virus isolates were genetically analyzed, and they were shown to be closely related, all belonging to the European TBE virus subgroup. However, on the basis of nucleotide and amino acid sequence analysis, 2 clusters were identified. Our results show that further investigation is needed to understand the clustering of isolates and to identify the most common TBE virus reservoir hosts in Croatia. Sentinel surveys based on wild animal species would give a better insight in defining TBE virus-endemic and possible risk areas in Croatia.

title: Goats and sheep as sentinels for tick-borne encephalitis (TBE) virus--epidemiological studies in areas endemic and non-endemic for TBE virus in Germany.
abstract: The aim of the study was to examine grazing goats and sheep as specific sentinels for characterization of the tick-borne encephalitis virus (TBEV)-related risk in an area by means of serosurveillance tests in the German federal states Baden-Wuerttemberg, Bavaria, Thuringia, North Rhine-Westphalia, Lower Saxony, Schleswig-Holstein, and Mecklenburg-West Pomerania. A total of 3590 sheep sera and 3793 goat sera was collected in 2003 and 2006-2009 and were examined by ELISA screening and confirmed by serum neutralization test. Considerable differences in seroprevalence were seen between single flocks in districts in Baden-Wuerttemberg, Bavaria, and Thuringia with values between 0 and 43% which confirmed the patchy pattern of TBEV foci that can range in size from very small to large. The here described serological screening may be a helpful tool for an early warning system of a potential TBEV risk. Testing of 1700 ticks by real-time RT-PCR in two districts in Baden-Wuerttemberg revealed only one positive tick, thus illustrating the problems of expensive and time-consuming tick collection.

title: Tick-borne encephalitis virus in dogs - is this an issue?
abstract: The last review on Tick-borne encephalitis (TBE) in dogs was published almost ten years ago. Since then, this zoonotic tick-borne arbovirus has been geographically spreading and emerging in many regions in Eurasia and continues to do so. Dogs become readily infected with TBE virus but they are accidental hosts not capable to further spread the virus. They seroconvert upon infection but they seem to be much more resistant to the clinical disease than humans. Apart from their use as sentinels in endemic areas, however, an increasing number of case reports appeared during the last decade thus mirroring the rising public health concerns. Owing to the increased mobility of people travelling to endemic areas with their companion dogs, this consequently leads to problems in recognizing and diagnosing this severe infection in a yet non-endemic area, simply because the veterinarians are not considering TBE. This situation warrants an update on the epidemiology, clinical presentation and possible preventions of TBE in the dog.

title: Early detection of tick-borne encephalitis virus spatial distribution and activity in the province of Trento, northern Italy.
abstract: New human cases of tick-borne encephalitis (TBE) have recently been recorded outside the recognised foci of this disease, i.e. in the province of Trento in northern Italy. In order to predict the highest risk areas for increased TBE virus activity, we have combined cross-sectional serological data, obtained from 459 domestic goats, with analysis of the autumnal cooling rate based on Moderate Resolution Imaging Spectroradiometer (MODIS) land surface temperature (LST) data. A significant relationship between finding antibodies against the virus in serum (seroprevalence) in goats and the autumnal cooling rate was detected, indicating that the transmission intensity of the virus does not only vary spatially, but also in relation to climatic factors. Virus seroprevalence in goats was correlated with the occurrence of TBE in humans and also with the average number of forestry workers' tick bites, demonstrating that serological screening of domestic animals, combined with an analysis of the autumnal cooling rate, can be used as early-warning predictors of TBE risk in humans.

title: Tick-borne encephalitis.
abstract: Tick-borne encephalitis (TBE), a zoonotic arbovirosis caused by tick-borne encephalitis virus (TBEV), is an increasing public health concern. Infections result in neurological symptoms in humans and the virus has rapidly expanded to new geographical areas. Three subtypes are currently present in different parts of Europe and Asia. The virus is transmitted by ticks, mainly Ixodes spp., between small mammals such as rodents, which serve as virus amplifying hosts. Humans are infected sporadically, either by a tick bite or by ingestion of infected milk or milk products. Other mammals (e.g. ruminants) can also be infected, but most of the time do not show clinical signs. In contrast to rodents, other wild and domestic mammals probably play only a very small direct role in maintaining TBEV in an area, but they might play an important role as hosts in sustaining a large tick population. Therefore, the virus prevalence and the occurrence of TBE can be influenced by several environmental, genetic and behavioural factors associated with the virus, the vectors or the hosts, and understanding these factors is essential for implementation of effective control measures. This article reviews virus characteristics and the epidemiological and clinical aspects of TBEV infections and examines pathogenesis, diagnostic approaches and control measures.

title: Virus Detection in Questing Ticks is not a Sensitive Indicator for Risk Assessment of Tick‐Borne Encephalitis in Humans
abstract: Tick‐borne encephalitis virus (TBEV) is the most important tick‐transmitted arbovirus causing human disease in Europe, but information on its endemic occurrence varies between countries because of differences in surveillance systems. Objective data are necessary to ascertain the disease risk for vaccination recommendations and other public health interventions. In two independent, separately planned projects, we used real‐time RT‐PCR to detect TBE virus in questing ticks. In Poland, 32 sampling sites were selected in 10 administrative districts located in regions where sporadic TBE cases were reported. In Germany, 18 sampling sites were selected in two districts located in a region with high TBE incidence. Altogether, >16 000 ticks were tested by real‐time RT‐PCR, with no sample testing positive for TBEV. A systematic search for published studies on TBEV prevalence in ticks in Poland and Germany also suggested that testing large numbers of collected ticks could not consistently assure virus detection in known endemic foci. Although assignment of results to administrative regions is essential for TBE risk mapping, this was possible in only 10 (investigating 22 417 ticks) of 15 published studies (>50 000 ticks) identified. We conclude that the collection and screening of ticks by real‐time RT‐PCR cannot be recommended for assessment of human TBE risk. Alternative methods of environmental TBEV monitoring should be considered, such as serological monitoring of rodents or other wildlife.

title: Tick-borne encephalitis virus prevalence in Ixodes ricinus ticks collected in high risk habitats of the South-Bohemian region of the Czech Republic
abstract: A survey for tick-borne encephalitis virus in Ixodes ricinus ticks was conducted in May 2000 in two districts of the South-Bohemian region of the Czech Republic with a high occurrence of tick-borne encephalitis (TBE) in humans. Homogenized ticks were tested on PS cells, which were examined for any cytopathic effect, plaque assay and indirect fluorescent antibody test (IFT). The IFT has proved to be the most sensitive and most rapid test to demonstrate the virus in ticks. TBE virus was found in 17 of 187 pooled samples, consisting of a total of 2,968 ticks. The mean minimum infection rate was 0.6% for all tick stages combined. Infection rates in nymphs collected indifferent locations varied between 0.2 and 1.3% and between 5.9 and 11.1% in adult ticks. The observed TBE prevalence in ticks was compared with data obtained elsewhere in the Czech Republic. It is concluded that screening of ticks for TBE virus prevalence using IFT is a valuable indicator for the degree of risk to contract TBE in as particular habitat.

title: Prevalence and genetic variability of tick-borne encephalitis virus in host-seeking Ixodes ricinus in northern Italy.
abstract: Tick-borne encephalitis (TBE) is a severe disease that has been endemic in north-east Italy since 1992. Over the past two decades, there has been an increase in the number of human cases reported in many European countries, including Italy. To assess the current TBE infection risk, questing ticks were collected from known TBE foci, as well as from a site in northern Italy where no human infections have been reported previously. A total of 1739 Ixodes ricinus (1485 nymphs and 254 adults) was collected and analysed for TBEV prevalence by a real-time RT-PCR targeting the 3' untranslated region. Phylogenetic analyses of the partial envelope gene were conducted on two newly sequenced TBE virus (TBEV) strains and 28 previously published sequences to investigate the genealogical relationships of the circulating TBEV strains. These phylogenetic analyses confirmed a previous report that the European TBEV subtype is the only subtype circulating within the TBE foci in north-east Italy. Interestingly, nucleotide sequence analysis revealed a high degree of divergence (mean 2.54 %) between the TBEV strains recovered in the Italian province of Trento, despite the circulation of a single TBEV subtype. This elevated genetic variability within a single TBE focus may reflect local differences in the long-standing evolutionary dynamics of TBEV at this site relative to previously characterized sites, or more recent and continuous reintroduction of various TBEV strains.

title: Prevalence and Genotyping of Tick-Borne Encephalitis Virus in Questing Ixodes ricinus Ticks in a New Endemic Area in Western Switzerland
abstract: Tick-borne encephalitis virus (TBEV) is the causative agent of tick-borne encephalitis (TBE) and causes neurological disease in humans in Eurasia. TBEV is transmitted by ticks of the genus Ixodes. Currently 10,000–12,000 clinical cases are reported annually in ≈30 TBE endemic countries. Since 1990 the epidemiology of TBE is characterized by a global increase of clinical cases and an expansion of risk areas. Similar trends are also observed in Switzerland but few studies confirmed the emergence of new TBE foci by detecting viral RNA in field-collected ticks. In this study, free-living Ixodes ricinus (L.) ticks from one nonendemic and three new TBE endemic regions located in the Western part of Switzerland were screened during four consecutive years (2007–2010) for the presence of TBEV. A total of 9,868 I. ricinus ticks (6,665 nymphs and 3,203 adults) were examined in pools for TBEV by real-time reverse transcription polymerase chain reaction. Our results confirmed the presence of viral RNA in 0.1% (6/6120) of questing ticks collected in one new endemic region. Among TBE endemic sites, the minimal infection rate per 100 ticks tested ranged from 0.21 (1/477) to 0.95 (1/105). Four positive samples were sequenced and phylogenetic analysis of the NS5 gene showed that all TBEV nucleotide sequences belonged to the European subtype and were split into two distinct lineages originating probably independently from two distinct foci located North-East and East of the study region.

title: Prevalence of tick-borne encephalitis virus (TBEV) RNA in Dermacentor reticulatus ticks from natural and urban environment, Poland
abstract: Tick-borne encephalitis virus (TBEV) (Flaviviridae, Flavivirus) is an arthropod-borne virus, an etiologic agent of tick-borne encephalitis (TBE), a human infection involving the central nervous system. The disease is endemic in a large region in Eurasia, where it is transmitted mainly by Ixodes ricinus and Ixodes persulcatus ticks. It is known that also Dermacentorreticulatus is involved in circulation of TBEV. However, the current knowledge of D. reticulatus importance in TBE epidemiology is still insufficient. A total of 471 adult D.reticulatus ticks were collected by flagging vegetation in the Białowieża Primeval Forest, Biebrza National Park, Masurian Landscape Park (North-Eastern Poland) and in the city of Warsaw in the years 2007–2010. All collected ticks were examined individually for the presence of RNA of TBEV using nested RT-PCR assay. Positive results were noted in all investigated localities with the infection rate ranging from 0.99 to 12.5 % with a total mean of 2.12 %. The difference in the percentage of infective males and females was not statistically significant.

title: Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes
abstract: In little more than 30 years, Lyme disease, which is caused by the spirochaete Borrelia burgdorferi, has risen from relative obscurity to become a global public health problem and a prototype of an emerging infection. During this period, there has been an extraordinary accumulation of knowledge on the phylogenetic diversity, molecular biology, genetics and host interactions of B. burgdorferi. In this Review, we integrate this large body of information into a cohesive picture of the molecular and cellular events that transpire as Lyme disease spirochaetes transit between their arthropod and vertebrate hosts during the enzootic cycle.

title: Epidemiology of lyme borreliosis.
abstract: Lyme borreliosis (LB) is the most frequent ixodid tick-borne human disease in the world, with an estimated 85,500 patients annually (underlying data presented in this review: Europe 65,500, North America 16,500, Asia 3,500, North Africa 10; approximate figures). This chapter summarizes the up-to-date knowledge about facts and factors important in the epidemiology of LB all over the world. Individual sections briefly describe geographic (latitudinal and altitudinal) distribution and incidence rates of LB in individual countries; seasonal distribution of the disease; effects of patients' age, sex, and profession; comparison of urban versus rural settings; weather-related effects on LB incidence; risk factors for LB acquisition by humans; and risk assessment. This chapter finishes by recommending a more thorough epidemiological surveillance for LB, including morbidity notification in some additional countries where it has not yet been fully implemented.

title: The expanding Lyme Borrelia complex--clinical significance of genomic species?
abstract: Ten years after the discovery of spirochaetes as agents of Lyme disease in 1982 in the USA, three genomic species had diverged from the phenotypically heterogeneous strains of Borrelia burgdorferi isolated in North America and Europe: Borrelia afzelii, B. burgdorferi sensu stricto (further B. burgdorferi), and Borrelia garinii. Whereas B. burgdorferi remained the only human pathogen in North America, all three species are aetiological agents of Lyme borreliosis in Europe. Another seven genospecies were described in the 1990s, including species from Asia (Borrelia japonica, Borrelia turdi, and B. tanukii), North America (Borrelia andersonii), Europe (Borrelia lusitaniae and Borrelia valaisiana), and from Europe and Asia (Borrelia bissettii). Another eight species were delineated in the years up to 2010: Borrelia sinica (Asia), Borrelia spielmanii (Europe), Borrelia yangtze (Asia), Borrelia californiensis, Borrelia americana, Borrelia carolinensis (North America), Borrelia bavariensis (Europe), and Borrelia kurtenbachii (North America). Of these 18 genomic species B. afzelii, B. burgdorferi and B. garinii are the confirmed agents of localized, disseminated and chronic manifestations of Lyme borreliosis, whereas B. spielmanii has been detected in early skin disease, and B. bissettii and B. valaisiana have been detected in specimens from single cases of Lyme borreliosis. The clinical role of B. lusitaniae remains to be substantiated.

title: Exploring gaps in our knowledge on Lyme borreliosis spirochaetes--updates on complex heterogeneity, ecology, and pathogenicity.
abstract: The Lyme borreliosis complex is a heterogeneous group of tick-borne spirochaetes of the genus Borrelia (Spirochaetales: Spirochaetaceae) that are distributed all over the temperate zone of the northern hemisphere. Due to the usage of new methods for phylogenetic analysis, this group has expanded rapidly during the past 5 years. Along with this development, the number of Borrelia spp. regarded as pathogenic to humans also increased. Distribution areas as well as host and vector ranges of Lyme borreliosis agents turned out to be much wider than previously thought. Furthermore, there is evidence that ticks, reservoir hosts, and patients can be coinfected with multiple Borrelia spp. or other tick-borne pathogens, which indicates a need to establish new and well-defined diagnostic and therapeutic standards for Lyme borreliosis. This review gives a broad overview on the occurrence of Lyme borreliosis spirochaetes worldwide with particular emphasis on their vectors and vertebrate hosts as well as their pathogenic potential and resultant problems in diagnosis and treatment. Against the background that many issues regarding distribution, species identity, ecology, pathogenicity, and coinfections are still unsolved, the purpose of this article is to reveal directions for future research on the Lyme borreliosis complex.

title: Transmission cycles of Borrelia burgdorferi sensu lato involving Ixodes ricinus and/or I. hexagonus ticks and the European hedgehog, Erinaceus europaeus, in suburban and urban areas in Switzerland.
abstract: The European hedgehog, Erinaceus europaeus Linnaeus, 1758, is a common host of Ixodes ricinus L. and I. hexagonus Leach, vectors of the Lyme disease spirochaete, Borrelia burgdorferi sensu lato. To investigate whether hedgehogs are reservoirs for B. burgdorferi, hedgehogs were captured in a suburban area suitable for both tick species and in an urban area where I. ricinus is absent. The infection status of the hedgehogs was determined by xenodiagnosis using I. ricinus and I. hexagonus larvae. I. hexagonus and/or I. ricinus were found on all hedgehogs (n = 8) from the suburban area. In contrast, only I. hexagonus was infesting animals (n = 5) from the urban area. A total of 12/13 hedgehogs harboured B. burgdorferi infected ticks. Xenodiagnostic I. ricinus and I. hexagonus larvae that fed on hedgehogs became infected. The results clearly show that European hedgehogs are reservoir hosts of the Lyme disease spirochetes. DNA of B. burgdorferi sensu stricto, B. garinii and B. afzelii was detected in culture from ear biopsy and needle aspiration material and characterized by using a genospecies-specific PCR assay. One hedgehog presented a mixed infection of the skin with B. burgdorferi sensu stricto and B. garinii. This study also identifies an enzootic transmission cycle in an urban area involving E. europaeus and I. hexagonus. The close association of I. hexagonus with the burrows of its hosts mean that the risks of contact between I. hexagonus and humans may be low.

title: Abundance and Borrelia burgdorferi-infection Prevalence of Nymphal Ixodes scapularis Ticks along Forest–Field Edges
abstract: More than 19,000 human cases of Lyme disease (LD) are reported each year in the United States. Lyme disease cases occur when humans are exposed to the bacterium Borrelia burgdorferi through the bite of an infected ixodid tick. The probability of human exposure to infected ticks results from a combination of human behaviors and entomological risk. Human behaviors include use of tick habitats, use of protective clothing, and grooming for tick removal. Entomological risks include the density of ticks in a habitat and the proportion of these that are infected with B. burgdorferi. Recent studies have suggested that humans are at higher risk of exposure to B. burgdorferi near edges between forests and herbaceous communities, including lawns and old fields, but whether this increased risk is a function of human behaviors, entomological risk, or both, is unknown. We assessed entomological risk across forest–old field edges in Dutchess County, NY. Densities of ticks and of infected ticks were considerably higher within forests than at forest–field edges, and were lowest within fields. Thus, edges between forests and fields do not pose a higher entomological risk than do the forests themselves, although risk at the edge is higher than in herbaceous habitat. Landscapes with abundant edges between forested and herbaceous habitat, and roughly even proportions of both, might attract both heavy human use and pose moderately high entomological risk, and thus could be targeted for mitigation. We suggest that determining appropriate methods for reducing human exposure to LD requires differentiating entomological risk from human behaviors.

title: Environmental risk and prevention of Lyme disease.
abstract: An understanding of the environmental determinants of Lyme disease risk is essential to evaluate human disease potential and to recommend strategies for disease prevention. Tick vectors of Lyme disease require a vertebrate blood meal during each of three motile developmental stages (larva, nymph, and adult). Although the immature stages (larvae and nymphs) exhibit broad and overlapping host ranges, adult ticks are primarily dependent on deer for feeding and reproduction. Consequently, the distribution and abundance of these ticks often reflect those of deer. Several species of smaller mammals and some birds that host immature ticks are also competent reservoirs for Borrelia burgdorferi, which becomes established within the tick population during feeding. The relative abundance of these host species and their ability to infect ticks are key factors in determining the distribution of Lyme disease risk in the local environment. Available methods for reducing the risk of Lyme disease in the environment include the application of insecticides and use of deer fencing, which have been shown to be 83-97% effective in reducing risk. However, the adverse environmental impact of insecticides and high cost of deer fencing limit these methods to high-risk areas where human exposure is constant and unavoidable (i.e., residential or occupational). Personal protection in high-risk areas can lessen the likelihood of contact with ticks. Future prevention methods may include host-targeted insecticides, environmental alteration, and biologic control. Moreover, research is currently directed to identifying antigens for use in recombinant human vaccines.

title: Epizootiologic background of dissimilar distribution of human cases of Lyme borreliosis and tick-borne encephalitis in a joint endemic area.
abstract: Lyme borreliosis (LB) and tick-borne encephalitis (TBE) are co-endemic in some parts of Europe, however, their distribution differs despite a common tick vector and comparable animal hosts. A serosurvey of game and small mammals was made in a highly endemic area and compared with historical data in human cases; the epidemiologic risk and the population density of game were modelled using a geographic information system. While LB-risk corresponded with an overall population density of game (red deer, roe deer, mouflon, wild boar) regardless of mouse abundance, TBE-risk suggested a dependence on the abundance of mice on the one hand, and game, particularly roe deer, on the other. While the prevalence of TBE-antibodies generally grew with the game's age, it was virtually constant at about 65% in LB irrespective of species. It implies a cumulation of scarce TBE-infection histories during the game's lifetime, and thus a limited size of TBE-foci relative to the living space of these animals, as well as omnipresent LB-foci, in which the animals became continuously re-infected. More ecological prerequisites seem necessary to keep TBE circulating in nature which may be responsible for its confined distribution.

title: Risk assessment in Lyme borreliosis.
abstract: A 3-year EU-funded project (EUCALB), initially involving 14 countries and more than 30 scientists and physicians, was undertaken with the main objective of identifying practical risk assessment criteria for Lyme borreliosis. A major part of the project was dedicated to the improvement of data quality. European case definitions were formulated and quality assurance schemes were developed for serological diagnosis and the detection of infection in ticks. Studies on the standardisation of immunoblot interpretation criteria are still in progress. Data on the clinical risk from tick bites were obtained and considerable progress was made in elucidating the complex ecology of the disease. A study on habitat assessment throughout Europe concluded that high risk was associated with highly heterogeneous recreational woodland and case data from both high and low incidence countries suggested that most infections were acquired in recreational areas. Considerable work is still required to relate clinical data to the epidemiology and ecology of the disease in order to assess risk in Lyme borreliosis.

title: Characteristics of Garden Dormice That Contribute to Their Capacity as Reservoirs for Lyme Disease Spirochetes
abstract: To describe the contribution of garden dormice to the epizootiology of Lyme disease, we compared their reservoir capacity for these pathogens to that of other sympatric hosts. Garden dormice are trapped most abundantly during early spring and again during midsummer, when their offspring forage. They are closely associated with moist forests. Garden dormice serve as hosts to nymphal ticks far more frequently than do other small mammals. Spirochetal infection is most prevalent in dormice, and many more larval ticks acquire infection in the course of feeding on these than on other rodents in the study site. Mature dormice appear to contribute more infections to the vector population than juveniles do. Replete larval ticks generally detach while their dormouse hosts remain within their nests. The population of garden dormice contributes five- to sevenfold more infections to the vector population than the mouse population does. Their competence, nymphal feeding density, and preference for a tick-permissive habitat combine to favor garden dormice over other putative reservoir hosts of Lyme disease spirochetes.

title: Risk of urban Lyme disease enhanced by the presence of rats.
abstract: To determine whether Norway rats contribute to the risk of human Lyme disease in a central European city park, densities of endemic rodents were compared as were feeding densities of vector ticks and prevalence of infection by the Lyme disease spirochete. Only Norway rats and yellow-necked mice were abundant, and three times as many mice as rats were present. More larval ticks fed on rats than on mice, and far more nymphs engorged on the rats. All rats but only about half of the mice infected ticks. Each rat was more infectious than each infectious mouse. Infected rats were distributed throughout the city. Spirochetes infected about a quarter of the questing nymphal ticks. The capacity of rats to serve as reservoir hosts for the Lyme disease spirochete, therefore, increases risk of infection among visitors to this and other urban parks.

title: Ixodes ricinus immatures on birds in a focus of Lyme borreliosis.
abstract: The infestation of birds by immature Ixodes ricinus was studied during 6 months in a Swiss woodland, where Lyme borreliosis is endemic. Thirteen passerine species were found to be parasitized by I. ricinus subadults and specially Turdus merula, T. philomelos and Erithacus rubecula. Overall, 300 larvae and 162 nymphs were collected on 95 avian hosts. Prevalence of infestation of nymphs on birds was higher in spring; larvae peaked in summer. The infection of birds by Borrelia burgdorferi was also studied using blood cultivation and examination of ticks. Motionless spirochetes were isolated from two E. rubecula. Infected ticks were removed from five species of passerines, and mainly three species of Turdidae (T. merula, T. philomelos and E. rubecula). Infection rate of larvae and nymphs by spirochetes averaged 16.3% and 21.7%, respectively. These percentages, compared to the infection rate of questing ticks collected through dragging, suggest that some Turdidae may play a role as amplifying hosts for spirochetes in the focus.

title: An avian reservoir (Turdus merula) of the Lyme borreliosis spirochetes.
abstract: The reservoir competence of passerine birds for the Lyme borreliosis spirochetes was studied in an enzootic focus in Switzerland. Skin aspirates and skin biopsies were used to isolate Borrelia spirochetes from Turdus species. B. burgdorferi sensu lato was isolated and/or PCR-detected in BSK medium containing skin biopsy or skin aspirate from 5 blackbirds (T. merula) and one song thrush (T. philomelos). Seven isolates were obtained from 3 different blackbirds. Either B. garinii or Borrelia from the genomic group VS116 was found in bird skin samples. Mixed infection occurred in 2 cases. Tick xenodiagnosis was used to determine whether blackbirds transmitted Borrelia to ticks. Five xenodiagnoses were performed on 3 different blackbirds. Borrelia DNA was detected in BSK medium inoculated with xenodiagnostic ticks from all the passerines tested. Isolates cultured from xenodiagnostic ticks were obtained from 2 blackbirds. Isolates belonged to group VS116 (n = 10) and to B. garinii (n = 1). Our study has shown that Turdus sp. are infected by B. garinii and by Borrelia from group VS116 and that blackbirds are implicated as reservoirs for these 2 genomic groups of Borrelia, as they transmit living borreliae to ticks. An association seems to exist between birds and Borrelia VS116, and to a lesser extent, B. garinii, similar to the association existing between small rodents and B. afzelii. Our observations emphasize the fact that different enzootic cycles maintain Lyme borreliosis spirochetes in nature.

title: Differential Role of Passerine Birds in Distribution of Borrelia Spirochetes, Based on Data from Ticks Collected from Birds during the Postbreeding Migration Period in Central Europe
abstract: Borrelia spirochetes in bird-feeding ticks were studied in the Czech Republic. During the postbreeding period (July to September 2005), 1,080 passerine birds infested by 2,240 Ixodes ricinus subadult ticks were examined. Borrelia garinii was detected in 22.2% of the ticks, Borrelia valaisiana was detected in 12.8% of the ticks, Borrelia afzelii was detected in 1.6% of the ticks, and Borrelia burgdorferi sensu stricto was detected in 0.3% of the ticks. After analysis of infections in which the blood meal volume and the stage of the ticks were considered, we concluded that Eurasian blackbirds (Turdus merula), song thrushes (Turdus philomelos), and great tits (Parus major) are capable of transmitting B. garinii; that juvenile blackbirds and song thrushes are prominent reservoirs for B. garinii spirochetes; that some other passerine birds investigated play minor roles in transmitting B. garinii; and that the presence B. afzelii in ticks results from infection in a former stage. Thus, while B. garinii transmission is associated with only a few passerine bird species, these birds have the potential to distribute millions of Lyme disease spirochetes between urban areas.

title: Synanthropic Birds Influence the Distribution of Borrelia Species: Analysis of Ixodes ricinus Ticks Feeding on Passerine Birds
abstract: Ixodes ricinus ticks collected from 835 birds and from vegetation in the Czech Republic were analyzed. Host-seeking ticks (n = 427) were infected predominantly by Borrelia afzelii (25%). Ticks (n = 1,012) from songbirds (Passeriformes) were infected commonly by Borrelia garinii (12.1%) and Borrelia valaisiana (13.4%). Juveniles of synanthropic birds, Eurasian blackbirds (Turdus merula) and song thrushes (Turdus philomelos), were major reservoir hosts of B. garinii.

title: Host association of Borrelia burgdorferi sensu lato--the key role of host complement.
abstract: Borrelia burgdorferi sensu lato (s.l.), the tick-borne agent of Lyme borreliosis, is a bacterial species complex comprising 11 genospecies. Here, we discuss whether the delineation of genospecies is ecologically relevant. We provide evidence that B. burgdorferi s.l. is structured ecologically into distinct clusters that are host specific. An immunological model for niche adaptation is proposed that suggests the operation of complement-mediated selection in the midgut of the feeding tick. We conclude that vertebrate hosts rather than tick species are the key to Lyme borreliosis spirochaete diversity.

title: Competence of pheasants as reservoirs for Lyme disease spirochetes.
abstract: Pheasants, Phasianus colchicus L., constitute a major part of the ground-feeding avifauna of England and Wales and are important hosts to immature stages of Ixodes ricinus L., the principal tick vector of Lyme borreliosis spirochetes in Europe. Therefore, their competence as hosts for Borrelia burgdorferi Johnson, Schmid, Steigerwalt & Brenner sensu lato was investigated. One group of pheasants was inoculated by needle with 1 x 10(6) cultured B. burgdorferi s.s. organisms, and a 2nd group of birds was infested with I. ricinus nymphs collected from a focus of Lyme borreliosis in southern England. Both bird groups were subjected to xenodiagnoses using uninfected I. ricinus nymphs. All recovered engorged ticks, as well as pheasant skin biopsies, were analyzed by a nested polymerase chain reaction targeting the 5S-23S rRNA genes of B. burgdorferi s.l. Both groups proved to be infective for ticks. The birds that were infected by tick bites proved to be significantly more infective for ticks (23% of the xenodiagnostic ticks positive) than those infected by needle (5%). The results show that pheasants can be infected experimentally with B. burgdorferi s.l., that they can pass the spirochetes to ticks and that their infectivity for ticks may persist as long as 3 mo. We conclude that pheasants are reservoir competent for Lyme borreliosis spirochetes and potentially play an important role in the maintenance of B. burgdorferi s.l. in England and Wales.

title: Transmission dynamics of Borrelia burgdorferi s.l. in a bird tick community.
abstract: We examined the Borrelia burgdorferi sensu lato circulation in a tick community consisting of three species (Ixodes ricinus, I. frontalis, I. arboricola) with contrasting ecologies, but sharing two European songbird hosts (Parus major and Cyanistes caeruleus). Parus major had the highest infestation rates, primarily due to larger numbers of I. ricinus, and probably because of their greater low-level foraging. The prevalence of Borrelia in feeding ticks did not significantly differ between the two bird species; however, P. major in particular hosted large numbers of Borrelia-infected I. frontalis and I. ricinus larvae, suggesting that the species facilitates Borrelia transmission. The low but significant numbers of Borrelia in questing I. arboricola ticks also provides the first field data to suggest that it is competent in maintaining Borrelia. Aside from Borrelia garinii, a high number of less dominant genospecies was observed, including several mammalian genospecies and the first record of Borrelia turdi for North-Western Europe. Borrelia burgdorferi sensu lato IGS genotypes were shared between I. arboricola and I. ricinus and between I. frontalis and I. ricinus, but not between I. arboricola and I. frontalis. This suggests that the Borrelia spp. transmission cycles can be maintained by bird-specific ticks, and bridged by I. ricinus to other hosts outside bird-tick cycles.

title: Lyme disease in California: interrelationship of ixodid ticks (Acari), rodents, and Borrelia burgdorferi.
abstract: The association of immature ixodid ticks, several species of rodents, and the Lyme disease spirochete, Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner, was studied in two habitats in northern California in spring and summer 1985 and year-round in 1986. A total of 428 rodents were collected from ecotonal chaparral and a woodland-grass-rock outcrop; the former habitat yielded six species, the latter three species. The deer mouse, Peromyscus maniculatus (Wagner), and the piñon mouse, P. truei (Shufeldt), were the dominant species year-round and collectively comprised 78% of rodents captured within chaparral and 87% from the rock outcrop in 1986. In both habitats, rodents were trapped most frequently in winter and spring, and least often in summer and fall. A total of 306 rodent blood films from all six species were assayed for spirochetes by direct immunofluorescence; of these, only one film prepared from P. truei (n = 123 films from 53 individual mice) was found to contain spirochetes. Immature western black-legged ticks, Ixodes pacificus Cooley & Kohls, and Pacific Coast ticks, Dermacentor occidentalis Marx, were collected from each species of rodent. Larvae of I. pacificus infested P. maniculatus and P. truei in low numbers year-round, but nymphs of this tick rarely parasitized these rodents. D. occidentalis larvae infested P. maniculatus and P. truei in spring and particularly in summer; nymphal ticks infested these mice primarily in summer. The efficiency of visual inspection for collecting immatures of these ticks from P. maniculatus ranged from 45 to 69% in spring and summer, whereas the efficiency of a drop-off technique appeared to be 100%. Spirochetes were detected in <1% of D. occidentalis larvae (n = 310) and nymphs (n = 120), and in approximately 4% of I. pacificus larvae (n = 75) derived from these hosts. The potential significance od these findings in the enzootiology of B. burgdorferi is discussed.

title: Ixodes ricinus as vector and reservoir of Borrelia burgdorferi in an urban environment
abstract: As specific arthropods, ticks in urban environments transmit the agents of dangerous contagious diseases endangering human and animal health. They are vectors of numerous infective agents that cause serious diseases of animals such as babesiosis and ehrlichiosis. Tick populations maintain and transmit an increas­ing number of different infective agents. In regard to the human population, they transmit the agents of tick encephalitis, Omsk hemorrhagic disease, Crim-Congo hemorrhagic disease, Kyasa-nur forest fever, ixodid fever, Q fever, Rocky Mountain spotted fever, tularemia, Siberian tick typhus and Lyme disease the last of which according to acarological references has become the dominant zoonosis in over 140 of the world's countries. In urban environments ticks represent problem both for their epidemiological significance and as organisms whose presence disturbs people during their work and rest. In Europe, the two most fre­quent species and Ixodes ricinus and Ixodes persulcatus. Both species have a wide spectrum of hosts.

title: [Borrelia burgdorferi sensu lato in ixodid ticks from Ostrava slag heaps].
abstract: In 2005 and 2006, Ixodes ricinus ticks were collected on two slag (waste rock) heaps from coal mines in the Ostrava area (North Moravia/Silesia, Czech Republic), Oskar (site A) and Emma (site B), partially covered by vegetation including trees, and at a control forest site near Hlucín (site C). The mean numbers of L. ricinus nymphs and imagoes flagged per person-hour were high: 35.3 nymphs and 12.7 imagoes, at site A, 23.3 and 26.0, respectively, at site B, and 25.4 and 16.8, respectively, at control site C. Using dark-field microscopy, 100 nymphs and 100 imagoes (50 females and 50 males) from each site were examined for borreliae. The mean prevalence rates of Borrelia burgdorferi sensu lato in nymphs and imagoes were 10.0% and 12.0%, respectively, at site A, 10.0% and 24.0%, respectively, at site B, and 13.0% and 17.0%, respectively, at site C. Differences in the prevalence of borreliae in nymphal and adult ticks from the slag heaps and control site were insignificant, but adult ticks from site B compared to site A contained borreliae significantly more frequently. The mean numbers of nymphs and imagoes infected with borreliae flagged per person-hour were 3.3 and 1.2, respectively at site A, 1.5 and 2.9, respectively, at site B, and 3.1 and 2.6, respectively, at site C. Isolation experiments for borreliae were carried out only in 16 ticks containing higher numbers of borreliae, with eight of these being culture-positive. The cultured borreliae were identified by PCR-RFLP as B. garinii (3 isolates: two from site B, one from site C), B. afzelii (4 isolates: one from site A, three from site B) and B. burgdorferi s.s. (one isolate from site A). Surprisingly, the results suggest that slag heaps, when covered by woody vegetation and frequented by humans, could theoretically pose roughly the same LB transmission risk to humans as common forest biotopes.

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

title: Urban and Rural Risks of Lyme Disease in the Scottish Highlands
abstract: This paper investigates the pattern of Lyme disease testing and infection within the Highland region of Scotland.  Data from all Highland samples tested during 2004-2006 were analysed according to result and patient's residence in relation to the eight fold Scottish Executive's urban/rural classification, and distance from woodland.  In total, 1602 patients were tested for Lyme disease, 0.71% of the Highland population. From these, 104 (6.5%) were seropositive. There were more patients tested, and seropositive patients from rural than urban locations, 1113 vs 489, and 79 vs 25 respectively. There were also significantly more seropositive patients per patients tested from rural locations (χ2, p < 0.0001). The number of patients tested and seropositive patients increased as the rural areas become more remote. The likelihood of being tested for Lyme disease also increased as the distance between a patient's residence and woodland decreased. The relative risk of being tested elevated by 74% for those patients living within 200 metres of woodland.  Those living in the most rural areas of Highland and those living closest to woodland have an increased risk of being tested and having Lyme disease.

title: Peri-urbanisation, counter-urbanisation, and an extension of residential exposure to ticks: a clue to the trends in Lyme borreliosis incidence in the Czech Republic?
abstract: The incidence of tick-borne human diseases (TBD) in the Czech Republic (CZ) is on the increase, driven by infections increasingly acquired in residential locations, earlier in spring and later in autumn, and among children and the elderly. To interpret these trends, data on Lyme borreliosis (LB) incidence between 1997 and 2010 were analysed in the context of population migration flows registered in the CZ during the same period. Analysis showed that a migration stream of families with children, and of the elderly, flowed from more urbanized and densely populated localities to those more rural and less populated, where the chance of acquiring LB in the home vicinity was greater than in the urban settings. By contrast, a stream of people in the life phase between early adulthood and family formation flowed reversely, corresponding to a prominent absence of this age category from the patient spectrum. The data further showed that the more the residential exposure became prevalent, the more people were in year-round (rather than in summertime only) contact with ticks, which accounts for an extension of the cases' seasonal distribution as well as for an overall increase in case numbers. Finally, the fact that majority of the urban-to-rural migrants could be categorised as wealthier people could explain the previously noticed lack of low-status people among TBD patients in the CZ.

title: Anaplasma phagocytophilum—a widespread multi-host pathogen with highly adaptive strategies
abstract: The bacterium Anaplasma phagocytophilum has for decades been known to cause the disease tick-borne fever (TBF) in domestic ruminants in Ixodes ricinus-infested areas in northern Europe. In recent years, the bacterium has been found associated with Ixodes-tick species more or less worldwide on the northern hemisphere. A. phagocytophilum has a broad host range and may cause severe disease in several mammalian species, including humans. However, the clinical symptoms vary from subclinical to fatal conditions, and considerable underreporting of clinical incidents is suspected in both human and veterinary medicine. Several variants of A. phagocytophilum have been genetically characterized. Identification and stratification into phylogenetic subfamilies has been based on cell culturing, experimental infections, PCR, and sequencing techniques. However, few genome sequences have been completed so far, thus observations on biological, ecological, and pathological differences between genotypes of the bacterium, have yet to be elucidated by molecular and experimental infection studies. The natural transmission cycles of various A. phagocytophilum variants, the involvement of their respective hosts and vectors involved, in particular the zoonotic potential, have to be unraveled. A. phagocytophilum is able to persist between seasons of tick activity in several mammalian species and movement of hosts and infected ticks on migrating animals or birds may spread the bacterium. In the present review, we focus on the ecology and epidemiology of A. phagocytophilum, especially the role of wildlife in contribution to the spread and sustainability of the infection in domestic livestock and humans.

title: Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease
abstract: Six patients from northern Minnesota and Wisconsin with a febrile illness accompanied by granulocytic cytoplasmic morulae suggestive of ehrlichial infection were identified. Two patients died, and splenic granulocytes of one patient contained cytoplasmic vacuoles with organisms ultrastructurally characteristic of ehrlichiae. From one patient, a 1.5-kb DNA product was amplified by PCR with universal eubacterial primers of 16S rDNA. Analysis of the nucleotide sequence of the amplified product revealed 99.9 and 99.8% similarities with E. phagocytophila and E. equi, respectively, neither of which has previously been known to infect humans. From the variable regions of the determined sequence, a forward primer specific for three organisms (human granulocytic ehrlichia, E. phagocytophila, and E. equi) and a reverse primer for these ehrlichiae and E. platys were designed. By nested PCR with amplification by the universal primers and then reamplification with the specific primers described above, the expected 919-bp product was generated from the blood of the index patient and three additional patients. Blood from these four patients and two more patients with granulocytic morulae contained DNA which was amplified by nested PCR involving a combination of a universal primer and the human granulocytic ehrlichia-E. phagocytophila-E. equi-E. platys group-specific primer. This apparently vector-borne human granulocytic ehrlichia has only 92.5% 16S rDNA homology with E. chaffeensis. Nested PCR with group-specific primers did not amplify E. chaffeensis DNA, and E. chaffeensis-specific primers did not amplify DNAs of the human granulocytic ehrlichia. Thus, six patients were shown to be infected by an Ehrlichia species never previously reported to infect humans.

title: Anaplasma phagocytophilum infection in a domestic cat in Finland: Case report
abstract: BackgroundAnaplasmosis is a vectorborne disease caused by the gram-negative bacterium Anaplasma phagocytophilum. This species displays positive tropism to granulocytes and can cause illness in several mammalian species, including cats, dogs, and humans. It is considered as an emerging disease in Europe. The clinical signs are nonspecific and include fever, lethargy, and inappetence. The most typical hematologic abnormality is thrombocytopenia. A tentative diagnosis can be made by detecting intracytoplasmic morulae inside neutrophils. The diagnosis is confirmed by PCR and serology in paired serum samples. A sample for PCR analysis should be taken before treatment. Anaplasmosis is treated with doxycycline.Case presentationA feline case of anaplasmosis is presented. The history, clinical presentation, diagnostics, treatment, and follow-up are discussed.ConclusionsThis case indicates that Anaplasma phagocytophilum infects cats in Finland. To provide accurate treatment, anaplasmosis should be listed as a differential diagnosis in cats suffering from acute febrile illness with previous tick exposure.

title: Clinical features of canine granulocytic anaplasmosis in 18 naturally infected dogs.
abstract: Anaplasma phagocytophilum, the causative agent of canine granulocytic anaplasmosis (CGA), is a Gram-negative intracellular organism transmitted by ixodid ticks. Thus far, only a few clinical studies evaluating dogs with CGA have been published.



Evaluation of dogs naturally infected with A. phagocytophilum in which known co-infections were excluded.



Eighteen dogs with CGA.



Prospective study. The diagnosis of CGA was based on a positive PCR test result; dogs with co-infections were excluded. History, clinical findings, CBC, clinical biochemistry, infectious disease screening, diagnostic imaging, and the course of disease were evaluated.



CGA was diagnosed based on a positive PCR test for A. phagocytophilum; 10 dogs also had morulae in neutrophils. Six of 18 dogs were seronegative to A. phagocytophilum, the others were seropositive. All dogs were acutely ill. The most common clinical findings were lethargy, inappetence, fever, and splenomegaly. Abnormal laboratory results included thrombocytopenia, anemia, lymphopenia, hypoalbuminemia, and abnormally high plasma alkaline phosphatase activity. In 6 of 10 dogs tested, the platelet-bound antibody test was positive; Coombs' test was negative in 9 dogs. All dogs were treated with doxycycline and recovered. PCR testing as well as blood smear analysis for morulae were negative in 14 tested dogs 2-8 weeks after beginning treatment.



Clinical findings in dogs with CGA were nonspecific. Positive platelet-bound antibody test results suggest immune-mediated platelet destruction as an important pathogenic mechanism. With correct diagnosis and treatment, prognosis is good.

title: Emergence of human granulocytic anaplasmosis in France.
abstract: In France, only one case of tick-borne human granulocytic anaplasmosis has been described in the literature (in 2003). Here, we report 5 new human cases of Anaplasma phagocytophilum infection from north-eastern France diagnosed in our laboratory in south-eastern France by serology and molecular biology. This increase is directly related to more physician interest in this disease and the implementation of a new PCR tool.

title: Human granulocytic ehrlichiosis in Europe: clinical and laboratory findings for four patients from Slovenia.
abstract: Febrile illnesses following a tick bite in patients from Slovenia were evaluated for an ehrlichial etiology. A case of acute human granulocytic ehrlichiosis (HGE) was confirmed by seroconversion to the HGE agent or molecular identification of ehrlichial organisms. Acute infection with the HGE agent was confirmed in four patients. None of the patients had detectable antibodies to the HGE agent at their first visit, but polymerase chain reaction analysis was positive for three patients. All four patients subsequently seroconverted to the HGE agent as shown by high titers of antibody. Clinical features and laboratory findings were similar to those in reports from the United States, although the disease course was relatively mild in the Slovenian cases. All patients recovered rapidly and without sequelae, although only two received antibiotic therapy (of whom only one was treated with doxycycline). HGE is an emerging tick-borne disease in the United States and should now be included in the differential diagnosis of febrile illnesses occurring after a tick bite in Europe.

title: Human granulocytic ehrlichiosis in western Europe.
abstract: Human granulocytic ehrlichiosis was first reported in the United States in 1994.1 Over the past five years at least several hundred cases have been reported, mostly in the upper Midwest and Northeast, regions where other diseases transmitted by Ixodes scapularis ticks, such as Lyme disease and babesiosis, are common. Four cases of human granulocytic ehrlichiosis have also recently been documented in Slovenia.2 We describe a case of human granulocytic ehrlichiosis in western Europe. In September 1998, a 58-year-old Dutch man presented with a seven-day history of fever, chills, and diarrhea. He often camped in Gelderland, a region . . .

title: First European Pediatric Case of Human Granulocytic Ehrlichiosis
abstract: Herein we report on the first confirmed pediatric case of acute human granulocytic ehrlichiosis in Europe. Presentation in this 11-year-old girl was comparable to clinical findings seen in adult European patients with human granulocytic ehrlichiosis; i.e., she had self-limited febrile illness with leukopenia, thrombocytopenia, and elevated serum C-reactive protein concentration. It is of interest that the patient not only had a fourfold change in antibody titer toEhrlichia phagocytophila but also developed antibodies to Ehrlichia chaffeensis and that her PCR test result was positive on the third as well as on the 22nd day after the onset of illness, that is, 16 days after spontaneous defervescence.

title: Human granulocytic ehrlichiosis--a clinical case in Scandinavia.
abstract: A clinical case of human granulocytic ehrlichiosis in Scandinavia is presented. The patient developed high fever, myalgia, headache and dyspnoea. Doxycycline treatment resulted in a dramatic improvement. Laboratory confirmation included a fourfold change in anti-Ehrlichia equi IFA titre and a positive PCR confirmed by gene sequence analysis.

title: Clinical Diagnosis and Treatment of Human Granulocytotropic Anaplasmosis
abstract: Tick‐borne rickettsiae in the genera Ehrlichia and Anaplasma are intracellular bacteria that infect wild and domestic mammals and, more recently, man. The increased desire of humans for recreational activities outdoors has increased the exposure to potential human pathogens that previously cycled almost exclusively within natural, nonhuman enzootic hosts. Anaplasma phagocytophilum causes an acute, nonspecific febrile illness of humans previously known as human granulocytotropic ehrlichiosis (HGE) and now called human granulocytotropic anaplasmosis (HGA). The first patient to have recognized HGA was hospitalized at St Mary's Hospital in Duluth, Minnesota, USA in 1990. However, the clinical and laboratory presentation of this infection remained undefined until 1994, when Bakken and collaborators published their experience with 12 patients who had HGA. By the end of December 2004, at least 2,871 cases of HGA had been reported from 13 U.S. states to the Centers for Disease Control and Prevention (CDC). A limited number of laboratory‐confirmed cases have been reported from countries in Europe, including Austria, Italy, Latvia, the Netherlands, Norway, Poland, Slovenia, Spain, and Sweden. Ixodes persulcatus‐complex ticks are the arthropod hosts for Borrelia burgdorferi, the agent of Lyme borreliosis, and are also the arthropod hosts for A. phagocytophilum. Most cases of HGA have been contracted in geographic regions that are endemic for Lyme borreliosis. Male patients outnumber female patients by a factor of 3 to 1 and as many as 75% of patients with HGA have had a tick bite prior to their illness. Seroepidemiologic studies have demonstrated that HGA for the most part is a mild or even asymptomatic illness. However, older individuals and patients who are immunocompromised by natural disease processes or medications may develop an acute, influenza‐like illness characterized by high fever, rigors, generalized myalgias, and severe headache. Local skin reactions at the site of the tick bite have not been described, and nonspecific skin rashes have been reported only occasionally. Anaplasmosis is associated with variable but suggestive changes in routine laboratory test parameters. Most patients develop transient reductions in total leukocyte and platelet concentrations. Relative granulocytosis accompanied by a left shift and lymphopenia during the first week of illness has been reported frequently. Serum hepatic transaminase concentrations usually increase two‐ to fourfold, and inflammatory markers, such as C‐reactive protein and the erythrocyte sedimentation rate, rise during the acute phase. Abnormal laboratory findings may return toward normal range for patients who have been ill for more than 7 days, which may obfuscate the clinical decision making. Characteristic clusters of bacteria (morulae) are observed in the cytoplasm of peripheral blood granulocytes in 20% to 80% of infected patients during the acute phase of illness. The clinical diagnosis may be confirmed retrospectively by specific laboratory tests, which include positive polymerase chain reaction (PCR), identification of A. phagocytophilum in culture of acute‐phase blood, or the detection of specific antibodies to A. phagocytophilum in convalescent serum. Virtually all patients have developed serum antibodies to A. phagocytophilum after completion of antibiotic therapy, and demonstration of seroconversion by indirect immunofluorescent antibody testing of acute‐phase and convalescent‐phase serum samples is currently the most sensitive and specific tool for laboratory confirmation of HGA. Treatment with doxycycline usually results in rapid improvement and cure. Most patients with HGA have made an uneventful recovery even without specific antibiotic therapy. However, delayed diagnosis in older and immunocompromised patients may place those individuals at risk for an adverse outcome, including death. Thus, prompt institution of antibiotic therapy is advocated for any patient who is suspected to have HGA and for all patients who have confirmed HGA.

title: Human granulocytic ehrlichiosis in Europe.
abstract: Ehrlichiosis comprises a group of emerging tick-borne infectious diseases caused by obligate intracellular Gram-negative bacteria that infect leukocytes. Infections caused by members of the genus Ehrlichia have been described in animals and humans, but to date there are no convincing reports of the presence of other types of human ehrlichiosis different from human granulocytic ehrlichiosis (HGE) in Europe. The European vector is the same as that of Lyme borreliosis, the hard tick Ixodes ricinus, and HGE has a similar epidemiology to that of Borrelia burgdorferi infection. Across Europe, I. ricinus is infected to a variable extent (0.4-66.7%) with the causative agent Ehrlichia (Anaplasma) phagocytophila genogroup, and since its first description in Slovenia in 1997, details of 15 patients have been published. Diagnosis requires careful consideration of all circumstances and symptoms (history of tick bite and the presence of a flu-like syndrome with variable degrees of anemia, thrombocytopenia, and leukopenia, and elevated liver enzymes). Some differences can be seen between US and European HGE patients. European HGE cases have a less severe course, and the presence of morulae is uncommon. In Europe, verification of HGE has been based on PCR and immunofluorescence antibody tests, because no isolation from humans has been reported.

title: First Cases of Acute Human Granulocytic Ehrlichiosis in Poland
abstract: The first three cases of acute human granulocytic ehrlichiosis in Poland are described. Blood samples were tested by an indirect immunofluorescence method to detect specific serum antibodies, and the polymerase chain reaction was used to detect ehrlichial DNA. Additionally, peripheral blood smears were examined for the presence of morulae. According to criteria of the Centers for Disease Control and Prevention, all three cases can be classified as confirmed granulocytic ehrlichiosis. Using the criteria recommended by a consensus group, however, two cases can be classified as confirmed granulocytic ehrlichiosis and one case as probable granulocytic ehrlichiosis.

title: First documented case of human granulocytic ehrlichiosis in Austria
abstract: ZusammenfassungWir beschreiben den ersten Fall von Humaner Granulozytärer Ehrlichiose, in Österreich, wobei die Infektion wahrscheinlich durch einen Zeckenstich in der Nähe von Arzl im Bezirk Innsbruck Land (Nordtirol) erfolgte. Die Krankheit verlief biphasisch: 8 Tage nach dem Zeckenstich begann die Patientin an fronto-occipitalen Kopfschmerzen, diffusen Arthralgien. Myalgien, allgemeinem Unwohlsein und subfebrilen Temperaturen zu leiden, nach 3 beschwerdefreien Tagen setzte am 20. Tag akut Fieber bis 39,5°C ein, das vier Tage später eine stationäre Aufnahme notwendig machte. Die klinischen und labormedizinischen Befunde entsprachen den Beobachtungen bei anderen erwachsenen europäischen Patienten, allerdings wurden auch transiente Antikörper gegen Thrombozyten nachgewiesen.Bei der stationären Aufnahme wies die Patientin hohe Antikörpertiter gegenanaplasma phagocytophilum auf (IgG 1∶1024, IgM 1∶640), 6 Wochen später war der IgG-Titer auf 1∶2048 gestiegen, die IgM-Konzentration war auf <1∶40 abgesunken.Auch in Österreich sollte bei febrilen Thrombozytopenien die Humane Granulozytäre Ehrlichiose bedacht werden, besonders, wenn ein Zeckenstich in der Anamnese eruierbar ist.SummaryWe report the first documented case of human granulocytic ehrlichiosis (HGE) in Austria. The infection was acquired near Arzl in the surroundings of Innsbruck in northern Tyrol. Except for a biphasic course of illness, presentation in this 33-year-old female patient was comparable to clinical findings observed in other European adults with high fever, transient thrombocytopenia, elevated levels of CRP and LDH, arthralgias, myalgias, fatigue and subfebrile temperature. Flu-like symptoms started about seven days after a tick bite and lasted for ten days, followed by an asymptomatic interval of three days and an acute onseet of fever up to 39.5°C on day 20. On admission, the patient showed high antibody titres againstAnaplasma phagocytophilum (IgG 1∶1024, IgM 1∶640); six weeks later the IgG-titre had risen to 1∶2048, and IgM-levels had fallen below 1∶40. The demonstration of anti-platelet antibodies in acute-phase serum was noteworthy.We conclude that also in Austria HGE should be considered in patients with febrile thrombocytopenia, especially when the medical history reveals recent tick exposure.

title: Factors contributing to emergence of Ehrlichia and Anaplasma spp. as human pathogens.
abstract: Ehrlichioses and anaplasmoses are caused by alpha-proteobacteria within the family of Anaplasmataceae. These diseases have been known for a long time in veterinary medicine and recently in human medicine. These tick-borne zoonoses are considered as emerging diseases. The first case of human monocytotropic ehrlichiosis occurred in 1986. Human granulocytic anaplasmosis was described as a separate entity in 1994 and ehrlichiosis caused by Ehrlichia ewingii was reported in humans in 1999. The number of cases has been rising steadily due to better diagnostic techniques and better surveillance worldwide. In this review, we will present human and animal ehrlichioses and anaplasmoses as emerging diseases and present candidate(s) for the future.

title: First detection of the Anaplasma phagocytophilum groEL-A genotype in man.
abstract: Human Granulocytic Anaplasmosis (HGA) is an emerging disease caused by the gram-negative bacterium Anaplasma phagocytophilum which is transmitted by ticks of the genus Ixodes ricinus. For molecular detection of the pathogen by PCR, a conserved portion of the groEL gene within the groESL operon is frequently used as a target. A single G/A polymorphism in this region allows to discriminate between two genotypes, groEL-G and groEL-A.



Total DNA from peripheral blood samples of two HGA patients was analysed by RealTime PCR, employing a protocol designed for genotyping groEL-G- and groEL-A variants of A. phagocytophilum.



We confirmed two clinical cases of HGA by PCR; in one patient, and for the first time in a human host, the groEL-A variant was detected, in the other case the pathogen was recognised as the groEL-G variant, up to now representing the only genotype reported in man.



It is documented that HGA infections can be caused by two A. phagocytophilum groEL genotypes. At present, the preference of the A. phagocytophilum groEL-G genotype in humans remains unclear, as we describe the first patient with HGA caused by the groEL-A variant. For a conclusive interpretation, more data from HGA patients will be required.

title: Human granulocytic anaplasmosis in eastern France: clinical presentation and laboratory diagnosis.
abstract: Human granulocytic anaplasmosis (HGA) is a tick-borne infection characterised by an acute, nonspecific febrile illness. To date, few clinical cases have been supported by both a positive polymerase chain reaction (PCR) assay and subsequent seroconversion against Anaplasma phagocytophilum antigen all over Europe. We report here 3 consecutive cases of HGA that occurred during the summer of 2009 which fulfilled the epidemiologic, clinical, and biological criteria for HGA. These data highlight PCR assay on ethylenediaminetetraacetic acid blood rather than serology as the diagnostic test of choice during the acute phase of the disease. In endemic areas, HGA should be investigated in patients presenting an undifferentiated febrile illness with cytopenia, elevated rates of liver enzymes, and increased C-reactive protein values.

title: First case of human granulocytic anaplasmosis from Slovakia.
abstract: In order to evaluate the potential risks of human granulocytic anaplasmosis in Slovakia, blood and serum samples of hunters and foresters from the northern parth of Slovakia were tested. We present the first case of HGA from Slovakia confirmed by nested PCR amplification of the 16S rRNA gene fragment of A. phagocytophilum.

title: First described case of human granulocytic anaplasmosis in a patient in Eastern Austria
abstract: ZusammenfassungEin 64-jähriger Patient gelangt zur Aufnahme mit Fieber, Thrombozytopenie, erhöhten Leberenzymen und einem Exanthem am Stamm. Anamnestisch berichtet der Patient über einen Zeckenbiss vier Wochen vor Beginn der Symptome. Eine Erkrankung mit Meningokokken oder eine hämatologische Grunderkrankung wurden ausgeschlossen. Im Anschluss wurden serologische Untersuchungen hinsichtlich des Zeckenbisses durchgeführt. Die Ergebnisse zeigten einen hohen IgG-Antikörper-Titer für Anaplasma phagozytophilia. Nach einer Woche normalisierten sich alle Laborparameter und Symptome bildeten sich zurück. Der Patient erhielt keine Therapie und erholte sich komplett. Wir berichten über den ersten Fall einer Anaplasmose in Ost-ÖsterreichSummaryA 64-year-old otherwise healthy patient presented with high fever, thrombocytopenia, elevated liver enzymes and an erythema on the belly. The patient remembered a tick bite four weeks ago when walking with his dog before the specific symptoms started. A meningococcal disease or hematological illness was excluded. The serological results for tick-borne diseases showed a high IgG antibody titer for Anaplasma phagocytophila. All symptoms and laboratory parameters normalized after one week of hospitalization. The patient received no treatment and recovered completely. This is the first confirmed case of human granulocytic anaplasmosis (HGA) in Eastern Austria.

title: Rare neurological presentation of human granulocytic anaplasmosis
abstract: Human granulocytic anaplasmosis (HGA) is a tick-borne, 
illness with increasing incidence . It is caused by
Anaplasma phagocytophilum proved to have a tick vector Ixodes
ricinus in Europe. HGA is usually a mild or even asymptomatic
illness with non-specific influenza-like symptoms, and is
associated with variable but suggestive laboratory alterations.
When comparing HGA with human ehrlichiosis caused by Ehrlichia
chaffeensis, beside differences in geographical distribution,
there is a close association with higher hospitalization rate
(36% vs. 48.6%), morbidity and life-threatening complications
(e.g. adult respiratory distress syndrome, disseminated
intravascular coagulopathy, meningoencephalitis, renal failure)
and mortality (0.6% vs. 1.9%) in E. chaffeensis cases . A
rush, as a clinical presentation, is also more frequently
reported in patients with E. chaffeensis. HGA occurs in
previously healthy individuals, and the disease severity is
associated with the degree of compromised immunity of infected
patients. The diagnosis is confirmed retrospectively using
specific laboratory tests, which include positive polymerase
chain reaction (PCR), identification of A. phagocytophilum in a
culture of acute-phase blood, or the detection of specific
antibodies to A. phagocytophilum in convalescent-phase serum
samples. Doxycycline provides rapid and effective treatment.

title: Epidemiology and control of human granulocytic anaplasmosis: a systematic review.
abstract: Granulocytic anaplasmosis caused by Anaplasma phagocytophilum is an emerging tick-borne zoonosis worldwide. The obligate intracellular pathogen is transmitted by Ixodes ticks and infects neutrophils in humans and animals, resulting in clinical symptoms ranging from asymptomatic seroconversion to mild, severe, or fatal disease. Since the initial description of human granulocytic anaplasmosis (HGA) in the United States in 1990, HGA has been increasingly recognized in America, Europe, and Asia. This review describes the epidemiology, diagnosis, and treatment of HGA and provides background information on the potential vectors and reservoirs of A. phagocytophilum.

title: Detection of Anaplasma phagocytophilum in animals by real‐time polymerase chain reaction
abstract: The aim of this study was to detect Anaplasma phagocytophilum in wild and domesticated animals and to identify the phylogenetic relationships of different strains of this bacterium. We adapted six published conventional methods targeting 16S fragments for real‐time polymerase chain reaction. Initial screening of samples from 419 animals found 37 Anaplasma positives, later confirmed with several different primers and a TaqMan probe. We also performed DNA quantification and melting curve analysis. The nucleic acid of Anaplasma sp. was detected in a higher percentage of cases in members of the deer family, hares, bank voles and mice (12.5∼15%) than in foxes, boars, cows, and horses (around 4∼6%). We also performed blood analysis of cows, horses, mice, and ticks removed from animals, evaluating the presence of antibodies against granulocytic Anaplasma sp. Finally, we subjected 11 randomly selected PCR amplified products to direct sequencing and we constructed the corresponding phylogenetic tree with respect to the Ehrlichia equi sequence, homologous to the human granulocytic ehrlichiosis agent. Mutual identity of the sequencing ranged from 99% to 100%.

title: Distinct Host Species Correlate with Anaplasma phagocytophilum ankA Gene Clusters
abstract: Anaplasma phagocytophilum is a Gram-negative, tick-transmitted, obligate intracellular bacterium that elicits acute febrile diseases in humans and domestic animals. In contrast to the United States, human granulocytic anaplasmosis seems to be a rare disease in Europe despite the initial recognition of A. phagocytophilum as the causative agent of tick-borne fever in European sheep and cattle. Considerable strain variation has been suggested to occur within this species, because isolates from humans and animals differed in their pathogenicity for heterologous hosts. In order to explain host preference and epidemiological diversity, molecular characterization of A. phagocytophilum strains has been undertaken. Most often the 16S rRNA gene was used, but it might be not informative enough to delineate distinct genotypes of A. phagocytophilum. Previously, we have shown that A. phagocytophilum strains infecting Ixodes ricinus ticks are highly diverse in their ankA genes. Therefore, we sequenced the 16S rRNA and ankA genes of 194 A. phagocytophilum strains from humans and several animal species. Whereas the phylogenetic analysis using 16S rRNA gene sequences was not meaningful, we showed that distinct host species correlate with A. phagocytophilum ankA gene clusters.

title: Segregation of genetic variants of Anaplasma phagocytophilum circulating among wild ruminants within a Bohemian forest (Czech Republic)
abstract: Seroprevalence, infection rate, and genetic variants of Anaplasma phagocytophilum were studied in a synusia of large mammals (deer, moufflon, and wild boar) which occupied a natural, largely broad-leaved forest in Central Bohemia. Almost 250 animals were harvested during 1999–2002. Their serum and/or full blood as well as spleen tissues were sampled. The sera were tested for the presence of anti- A. phagocytophilum antibodies with IFA, and the blood and spleen DNA extracts were examined with nested PCR. Selected positive samples were sequenced in both directions using BigDye chemistry. Whenever a mixture was encountered, the PCR product was cloned, and several randomly selected clones were individually sequenced, to make the mixture's constituents clear. The established sequences were compared with each other, among host species, and also with the GenBank database. All 154 ruminant sera (moufflon, red deer, roe deer, fallow deer, and sika deer) showed reactive antibodies to A. phagocytophilum , with titres ranging from 1:320 to 1:10,240. By contrast, only 8 out of 34 (24%) wild boar sera showed elevated titres, from 1:80 to 1:1280. A. phagocytophilum DNA was detected in the blood samples ( n =66) of all ruminant species. The highest prevalence was shown in red deer (18/21) and the lowest in moufflon (1/28). The difference is statistically significant ( p =0.01). Two samples of wild boar blood tested negative, but one spleen sample was positive. The paired blood/spleen samples ( n =17) showed 71% correspondence in the PCR tests. Eleven extracts of DNA of A. phagocytophilum (6 red deer, 4 roe deer, 1 moufflon) were analysed in detail, and 4 genetic variants were distinguished based on partial 16S rRNA sequence. All red deer showed dual infections with a variant already reported from this host (DV), accompanied by a variant which was undistinguishable from that associated with horse, dog, and human infections in Europe (HV). All the roe deer hosted a variant peculiar to that species (RV); this variant was either found singly or mixed with HV. The unique moufflon isolate corresponded to a variant reported from sheep in Europe (SV). Any genetic variant, with the exception of HV, was only detected within a single ruminant species. Fisher's exact test proved that the association of RV and DV with the respective roe and red deer is statistically significant ( p =0.005). This study indicates that while circulating in nature, the variants of A. phagocytophilum in wild ruminants tend to be segregated from one another by species barriers despite the close relationships of their hosts, their sympatric ranges as well as a shared population of vector ticks.

title: Wild Boars as Hosts of Human-Pathogenic Anaplasma phagocytophilum Variants
abstract: To investigate the potential of wild boars to host Anaplasma phagocytophilum, we analyzed bacterial 16S rRNA and ank genes. DNA sequencing identified several A. phagocytophilum variants, including a predominance of strains known to cause human disease. Boars are thus hosts for A. phagocytophilum, notably, strains associated with human granulocytic anaplasmosis.

title: Equine and Canine Anaplasma phagocytophilum Strains Isolated on the Island of Sardinia (Italy) Are Phylogenetically Related to Pathogenic Strains from the United States
abstract: The presence of Anaplasma phagocytophilum, a tick-transmitted zoonotic pathogen, was investigated in Sardinia using a molecular approach. Phylogenetic analysis revealed that Sardinian strains are genetically distinct from the two lineages previously described in Europe and are closely related to strains isolated in different areas of the United States.

title: Genetic variants of Anaplasma phagocytophilum from 14 equine granulocytic anaplasmosis cases
abstract: BackgroundEquine 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).ResultsAll 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.ConclusionsThe 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.

title: Genetic variants of Anaplasma phagocytophilum in wild caprine and cervid ungulates from the Alps in Tyrol, Austria.
abstract: The occurrence of genetic variants of Anaplasma phagocytophilum was studied in wild ungulates from the northern and central eastern Alps in Tyrol, Austria. For this purpose, spleen samples collected from 53 game animals during the hunting season 2008/2009 (16 roe deer [Capreolus capreolus], 10 red deer [Cervus elaphus], 16 Alpine chamois [Rupicapra r. rupicapra], 7 Alpine ibex [Capra i. ibex], and 4 European mouflons [Ovis orientalis musimon]) were analyzed. Thirty-five animals originated from the Karwendel mountains, 12 from the Kaunertal area (Ötztal Alps), and the remaining from other mountainous areas in Tyrol. DNA extracts were screened with a real-time polymerase chain reaction targeting the msp2 gene of A. phagocytophilum. A total of 23 (43.4%) samples, from all ungulate species studied, were A. phagocytophilum positive. As of the date of this article, A. phagocytophilum has not been reported in the Alpine ibex. The positive samples were investigated further with polymerase chain reactions for amplification of the partial 16S rRNA, groEL, and msp4 genes. Sequence analysis using forward and reverse primers revealed seven different 16S rRNA gene variants. No variant could be attributed to any particular ungulate species. The groEL gene revealed 11 different variants, which grouped in the phylogenetic analysis into two distinct clusters: one cluster contained the sequences from roe deer, whereas the sequences of the other species formed the second cluster. The msp4 gene showed a high degree of variability in the amplified part with a total of 10 different sequence types. The results show that the wild mountain ungulates were infected to a considerable extent with various variants of A. phagocytophilum. The pathogenicity of the variants and the reservoir competence of the species investigated in this study deserve further attention in future studies.

title: Analysis of the Population Structure of Anaplasma phagocytophilum Using Multilocus Sequence Typing
abstract: Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophils. It is transmitted via tick-bite and causes febrile disease in humans and animals. Human granulocytic anaplasmosis is regarded as an emerging infectious disease in North America, Europe and Asia. However, although increasingly detected, it is still rare in Europe. Clinically apparent A. phagocytophilum infections in animals are mainly found in horses, dogs, cats, sheep and cattle. Evidence from cross-infection experiments that A. phagocytophilum isolates of distinct host origin are not uniformly infectious for heterologous hosts has led to several approaches of molecular strain characterization. Unfortunately, the results of these studies are not always easily comparable, because different gene regions and fragment lengths were investigated. Multilocus sequence typing is a widely accepted method for molecular characterization of bacteria. We here provide for the first time a universal typing method that is easily transferable between different laboratories. We validated our approach on an unprecedented large data set of almost 400 A. phagocytophilum strains from humans and animals mostly from Europe. The typability was 74% (284/383). One major clonal complex containing 177 strains was detected. However, 54% (49/90) of the sequence types were not part of a clonal complex indicating that the population structure of A. phagocytophilum is probably semiclonal. All strains from humans, dogs and horses from Europe belonged to the same clonal complex. As canine and equine granulocytic anaplasmosis occurs frequently in Europe, human granulocytic anaplasmosis is likely to be underdiagnosed in Europe. Further, wild boars and hedgehogs may serve as reservoir hosts of the disease in humans and domestic animals in Europe, because their strains belonged to the same clonal complex. In contrast, as they were only distantly related, roe deer, voles and shrews are unlikely to harbor A. phagocytophilum strains infectious for humans, domestic or farm animals.

title: Human Granulocytic Anaplasmosis Acquired in Scotland, 2013
abstract: Human granulocytic anaplasmosis is a tick-borne disease caused by Anaplasma phagocytophilum, an obligate intracellular gram-negative bacterium that infects granulocytes. The usual clinical signs and symptoms include nonspecific fever, chills, headache, and myalgia. Infection is usually mild or asymptomatic, but severe systemic complications can occur, leading to a need for intensive care and estimated fatality rates of 0.5%–1.0% (1,2). 
 
A. phagocytophilum was first described in 1932 in Scotland as the causative agent of tick-borne fever in sheep (3). Although some clinical cases of human granulocytic anaplasmosis have been reported in Europe, mostly from Slovenia, Sweden, and Poland (4), most cases have occurred in the United States. This difference cannot be explained by the prevalence of the pathogen in ticks or human exposure to the pathogen because the 3% prevalence of A. phagocytophilum among Ixodes ricinus ticks in Europe seems to be nearly as high as that among ticks in the United States (2). The median seroprevalence rate for A. phagocytophilum infection among humans in Europe is 6.2%, reaching up to 21% (2). This incongruence between seroprevalence rate and number of human cases might be associated with underdiagnosis of cases (2), a high rate of asymptomatic disease (5), or cross-reactivities in serologic tests that might lead to overestimation of seroprevalence rate (5). 
 
In August 2013, an immunocompetent 40-year-old man sought treatment for fever (≈39°C) and other nonspecific symptoms such as malaise, myalgia, and severe headache 3 days after becoming aware of several tick bites received while on a hiking vacation in Scotland. The man had removed the 3 tick nymphs from his legs immediately after their discovery and stored them in a plastic container; they were later sent to the Consultant Laboratory for Tick-borne Encephalitis in Berlin, Germany, for analysis. When the patient returned to Germany, 5 days after the onset of symptoms and 8 days after tick removal, a blood sample was collected (sample 1) and the bite sites were swabbed with a sterile cotton bud. By that time, the fever was gone, but malaise and other symptoms persisted. The patient began taking doxycycline, and within 2 days all symptoms subsided and the patient recovered completely. 
 
A second blood sample was collected 28 days after tick removal (sample 2). Complete blood counts and chemistry panels were performed for both samples. All values were within the reference range except that for lactate dehydrogenase (248 U/L), which was moderately increased over the baseline value of <245 U/L in sample 1. Values did not differ substantially between the 2 samples. 
 
DNA from whole-blood samples and swabs was extracted (QIAamp DNA Blood Mini Kit; QIAGEN, Hilden, Germany) and tested for A. phagocytophilum, Babesia spp., Borrelia spp., and Rickettsia spp. by using commercially available rapidSTRIPE assays for Anaplasma, Babesia, Borrelia, Rickettsia (all Analytik Jena AG; Jena, Germany). DNA extracted from blood and swab samples was negative for all tested pathogens. 
 
After the tick specimens were taxonomically identified as I. ricinus, DNA/RNA was extracted (blackPREP Tick DNA/RNA Kit;  and tested for the same pathogens. All 3 ticks were negative for Babesia spp., Borrelia spp., and Rickettsia spp., but 2 were positive for A. phagocytophilum. 
 
Indirect immunofluorescence assays (Focus Diagnostics, Cypress, CA, USA) performed on the paired serum samples revealed an increased A. phagocytophilum–specific IgM titer, from 20 at 5 days after symptom onset to 80 at 20 days later; the A. phagocytophilum–specific IgG titer rose from a high titer of 800 to >3,200 over this period. 
 
The presence and 4-fold increase of A. phagocytophilum–specific IgM and IgG in paired serum samples confirmed the diagnosis of human granulocytic anaplasmosis in accordance with Centers for Disease Control and Prevention criteria (6). As described previously for several cases of human granulocytic anaplasmosis, patient blood counts were within reference limits but serum lactate dehydrogenase level was elevated (7). The diagnosis was further corroborated by detection of A. phagocytophilum DNA in 2 of the 3 ticks removed from the patient’s skin. PCR amplification failed to detect A. phagocytophilum DNA in the patient’s blood, consistent with previous studies documenting frequent lack of A. phagocytophilum DNA detection in whole blood and a substantial drop in PCR positivity after the acute phase of illness (8). 
 
Human granulocytic anaplasmosis is not usually reported in Scotland like it is in the rest of Europe. The case originated from an area with long-established disease occurrence in ruminants, but the literature reports only 1 case of human infection in southwestern Scotland (9), ≈500 km from where this infection was probably acquired. Correct diagnosis would have been difficult had the patient not conserved the ticks and contacted the Consultant Laboratory for Tick-borne Encephalitis immediately after returning to Germany. A large number of human granulocytic anaplasmosis cases might be missed because general practitioners may not be aware of the pathogen’s existence or its distribution.

title: Ultrastructure and phylogenetic analysis of 'Candidatus Neoehrlichia mikurensis' in the family Anaplasmataceae, isolated from wild rats and found in Ixodes ovatus ticks.
abstract: A novel bacterium that infects laboratory rats was isolated from wild Rattus norvegicus rats in Japan. Transmission electron microscopy of the spleen tissue revealed small cocci surrounded by an inner membrane and a thin, rippled outer membrane in a membrane-bound inclusion within the cytoplasm of endothelial cells. Phylogenetic analysis of the 16S rRNA gene sequence of the bacterium found in R. norvegicus rats and Ixodes ovatus ticks in Japan revealed that the organism represents a novel clade in the family Anaplasmataceae, which includes the Schotti variant found in Ixodes ricinus ticks in the Netherlands and the Ehrlichia-like Rattus strain found in R. norvegicus rats from China. The novel clade was confirmed by phylogenetic analysis of groESL sequences found in R. norvegicus rats and Ixodes ovatus ticks in Japan. No serological cross-reactivity was detected between this bacterium and members of the genera Anaplasma, Ehrlichia or Neorickettsia in the family Anaplasmataceae. It is proposed that this new cluster of bacteria should be designated 'Candidatus Neoehrlichia mikurensis'.

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

title: Identification of Ehrlichia spp. andBorrelia burgdorferi in Ixodes Ticks in the Baltic Regions of Russia
abstract: The presence and distribution of Ehrlichia spp. andBorrelia burgdorferi sensu lato was demonstrated among ixodid ticks collected in the Baltic regions of Russia, where Lyme borreliosis is endemic. A total of 3,426 Ixodes ricinusand 1,267 Ixodes persulcatus specimens were collected, and dark-field microscopy showed that 265 (11.5%) I. ricinus and 333 (26.3%) I. persulcatus ticks were positive. From these samples, 472 dark-field-positive and 159 dark-field-negative ticks were subjected to PCR and subsequent reverse line blot hybridization. Fifty-four ticks (8.6%) carriedEhrlichia species, and 4 (0.6%) carried ehrlichiae belonging to the Ehrlichia phagocytophila complex, which includes the human granulocytic ehrlichiosis agent. The E. phagocytophila complex and anEhrlichia-like species were detected only in I. ricinus whereas Ehrlichia muris was found exclusively in I. persulcatus, indicating a possible vector-specific infection. Borrelia garinii was found predominantly in I. persulcatus, but Borrelia afzelii was evenly distributed among the two tick species. Only two I. ricinus ticks carried B. burgdorferi sensu stricto, while Borrelia valaisiana and a newly identified B. afzelii-like species were found in 1.7 and 2.5% of all ticks, respectively. Of the dark-field-positive ticks, only 64.8% yielded aBorrelia PCR product, indicating that dark-field microscopy may detect organisms other than B. burgdorferi sensu lato. These observations show that the agent of human granulocytic ehrlichiosis may be present in ticks in the Baltic regions of Russia and that clinicians should be aware of this agent as a cause of febrile disease.

title: Genetic Diversity of Bacterial Agents Detected in Ticks Removed from Asymptomatic Patients in Northeastern Italy
abstract: A total of 360 ticks were removed from 353 asymptomatic subjects in Belluno Province, Italy and surrounding areas, from 1998 to 2001. Ticks were identified as Ixodes ricinus (357), Ixodes hexagonus (1), Rhipicephalus sanguineus (1), and Ixodes ventalloi (1). Tick DNA was investigated by PCR and subsequent sequencing of amplified products to identity associated bacterial agents. Primers targeting different genes of Rickettsia (gltA and OmpA), Borrelia (16S rDNA, rpoB), Francisella (16S rDNA), and all genera members of the Anaplasmataceae (16S rDNA), were used. DNA of bacterial agents was identified in 28 Ixodes ricinus specimens (7.8%). Rickettsia helvetica was detected in 7 ticks. Rickettsia sp. IRS4 and Borrelia afzelii was detected in 4 ticks each. B. garinii and B. valaisiana were identified in one tick each. Anaplasma phagocytophilum, the agent of human granulocytic ehrlichiosis, was identified in 1 specimen of I. ricinus. A new Ehrlichia sp. (‘Candidatus Ehrlichia walkerii’, sp. nov.) was identified in 10 I. ricinus specimens.

title: High Diversity of ankA Sequences of Anaplasma phagocytophilum among Ixodes ricinus Ticks in Germany
abstract: In Germany humans with acute granulocytic ehrlichiosis have not yet been described. Here, we characterized three different genes of Anaplasma phagocytophilum strains infecting German Ixodes ricinus ticks in order to test whether they differ from strains in other European countries and the United States. A total of 1,022 I. ricinus ticks were investigated for infection with A. phagocytophilum by nested PCR and sequence analysis. Forty-two (4.1%) ticks were infected. For all positive ticks, parts of the 16S rRNA and groESL genes were sequenced. The complete coding sequence of the ankA gene could be determined in 24 samples. The 16S rRNA and groESL gene sequences were as much as 100% identical to known sequences. Fifteen ankA sequences were ≥99.37% identical to sequences derived from humans with granulocytic ehrlichiosis in Europe and from a horse with granulocytic ehrlichiosis in Germany. Thus, German I. ricinus ticks most likely harbor A. phagocytophilum strains that can cause disease in humans. Nine additional sequences were clearly different from known ankA sequences. Because these newly described sequences have never been obtained from diseased humans or animals, their biological significance is currently unknown. Based on this unexpected sequence heterogeneity, we propose to use the ankA gene for further phylogenetic analyses of A. phagocytophilum and to investigate the biology and pathogenicity of strains that differ in the ankA gene.

title: Ehrlichia‐like Organism Gene Found in Small Mammals in the Suburban District of Guangzhou of China
abstract: The 16S rRNA gene of Ehrlichia genus was examined from the blood and spleen of 26 wild small mammals that were captured at a canine feed lot where the first case of canine ehrlichiosis was found in south of China in 1998. Samples (3 blood and 1 spleen) from Rattus norvegicus were positive by polymerase chain reaction. The amplimers were then cloned and sequenced. All of the 16S rRNA gene sequence of these samples were found to be same and unique when compared with the other members of genus Ehrlichia. The most closely related species was Ehrlichia‐like sp. Schotti variant (99.03% related), which was detected and identified in Dutch Ixodes ricinus ticks by Schouls in 1999. The organism found on these animals was obviously not the species of E. muris, which was isolated and identified from a wild mouse in Japan in 1995, since the relatedness of this gene between the former and the latter was 93.28%. It is unclear whether this organism can cause disease in other mammals, including human beings.

title: Prevalence of Neoehrlichia mikurensis in ticks and rodents from North-west Europe
abstract: BackgroundNeoehrlichia 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.MethodsThree 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.ResultsThe 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.ConclusionsEhrlichia 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.

title: Molecular detection of tick-borne pathogens in Ixodes ricinus from Moldova collected in 1960.
abstract: This study is the first report about the prevalence of tick-borne pathogens, as well as their (co-)infection rates, in the museum-archived I. ricinus female ticks collected in Moldova in 1960. A total of 16.7% (21/126) ticks was mono-infected. Borrelia burgdorferi sensu stricto was revealed as the most abundant species (4.8%) followed by B. garinii (1.6%), B. afzelii (0.8%), B. valaisiana (0.8%), and B. lusitaniae (0.8%). DNA of Rickettsia helvetica (2.4%), R. monacensis (2.4%), Anaplasma phagocytophilum (2.4%), 'Candidatus Neoehrlichia mikurensis' (0.8%), and Babesia microti (0.8%) were also detected, indicating the occurrence of these emerging tick-borne microorganisms in Moldova since 1960 at least. In this study, we detected a co-infection (0.8%; 1/126 tested ticks) between B. microti and R. helvetica. Additional investigations are warranted to further characterize a historical snapshot of the distribution of tick-borne pathogens in Europe.

title: Absence of Borrelia spp., Candidatus Neoehrlichia mikurensis, and Anaplasma phagocytophilum in questing adult Dermacentor reticulatus ticks
abstract: To determine whether Dermacentor reticulatus ticks are infected by Borrelia spp., Candidatus Neoehrlichia mikurensis, or Anaplasma phagocytophilum, we collected questing adults in the outskirts of Berlin, Germany, examined them for the presence of DNA of these pathogens, and compared the infection rates to those of sympatric Ixodes ricinus ticks. Questing D. reticulatus adults appeared not to harbor the bacterial pathogens that are prevalent in I. ricinus ticks. Based on our sample size, the estimated prevalence of each of these pathogens in D. reticulatus ticks would be well below three tenth of a percent (<0.3 %). For pathogens which so rarely infect D. reticulatus ticks, this tick likely plays no epidemiologic vector role for either their enzootic transmission cycle in nature or their transmission to people.

title: Candidatus Neoehrlichia mikurensis in rodents in an area with sympatric existence of the hard ticks Ixodes ricinus and Dermacentor reticulatus, Germany
abstract: BackgroundCandidatus 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.MethodsBlood, 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.Results52.7% 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.ConclusionThe 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.

title: Candidatus Neoehrlichia mikurensis and Anaplasma phagocytophilum in Urban Hedgehogs
abstract: Candidatus Neoehrlichia mikurensis is a member of the order Rickettsiales, family Anaplasmataceae (1). Manifestations of infection with these bacteria are atypical and severe and include cough, nausea, vomiting, anemia, headache, pulmonary infiltration, malaise, myalgia, arthralgia, fatigue, recurrent fever for ≤8 months, and/or death (2–5). Candidatus N. mikurensis has been detected in Ixodes ovatus, I. persulcatus, and Haemaphysalis concinna ticks in Asia (1,5). 
 
Candidatus N. mikurensis has been identified as one of the most prevalent pathogenic agents in I. ricinus ticks throughout Europe (2,3,6). Rodents of diverse species and geographic origins have been shown to carry these bacteria, but transmission experiments have not been conducted to unambiguously identify natural vertebrate reservoirs (1–3,5–7). This emerging tickborne pathogen has been detected mainly in immunocompromised patients in Sweden (n = 1), Switzerland (n = 3), Germany (n = 2), and the Czech Republic (n = 2) and in immunocompetent patients in China (n = 7) (2–5). 
 
Anaplasma phagocytophilum is an obligate, intracellular, tickborne bacterium of the family Anaplasmataceae and causes granulocytic anaplasmosis in humans and domestic animals. In Europe, I. ricinus ticks are its major vector, and red deer, roe deer, rodents, and European hedgehogs (Erinaceus europaeus) are suspected reservoir hosts (8). 
 
Northern white-breasted hedgehogs (Erinaceus roumanicus) are urban-dwelling mammals (order Eulipotyphla, family Erinaceidae) that serve as major maintenance hosts for the 3 stages of I. ricinus ticks (9). However, E. roumanicus hedgehogs have not been studied for their ability to carry A. phagocytophilum. In addition, no suspected reservoirs other than rodents have been investigated for Candidatus N. mikurensis. The purpose of this study was to determine whether this hedgehog is a potential reservoir of these 2 bacteria. 
 
We conducted an ecoepidemiologic study during 2009–2011 to obtain information about ticks and tickborne pathogens of urban hedgehogs in a park on Margaret Island in central Budapest, Hungary (9). Ear tissue samples were obtained from hedgehogs anesthetized with intramuscular ketamine (5 mg/kg) and dexmedetomidine (50 µg/kg). 
 
DNA was extracted from samples by using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) or the Miniprep Express Matrix protocol (MP Biomedicals, Santa Ana, CA, USA). We used quantitative real-time PCRs that partially amplify the heat shock protein gene (groEL) of Candidatus N. mikurensis and the merozoite surface protein 2 gene (msp2) of A. phagocytophilum (3). PCR was performed in a 20-μL volume containing iQ Multiplex Powermix (Bio-Rad Laboratories, Hercules, CA, USA) in a LightCycler 480 Real-Time PCR System (F. Hoffmann-La Roche, Basel, Switzerland). Final PCR concentrations were 1× iQ Powermix, 250 nmol/L of primers ApMSP2F and ApMSP2R, 125 nmol/L of probe ApMSP2P-FAM, 250 nmol/L of primers NMikGroEL-F2a and NMikGroEL-R2b, 250 nmol/L of probe NMikGroEL-P2a-RED, and 3 μL of template DNA. 
 
To confirm quantitative PCR results, we performed conventional PCRs in a Px2 Thermal Cycler (Thermo Electron Corporation, Waltham, MA, USA) on selected PCR-positive samples for both pathogens (3). Sequences obtained were submitted to GenBank under accession nos. {"type":"entrez-nucleotide","attrs":{"text":"KF803997","term_id":"576637860","term_text":"KF803997"}}KF803997 (groEL gene of Candidatus N. mikurensis) and {"type":"entrez-nucleotide","attrs":{"text":"KF803998","term_id":"576637863","term_text":"KF803998"}}KF803998 (groEL gene of A. phagocytophilum). 
 
Candidatus N. mikurensis was detected in 2 (2.3%) of 88 hedgehog tissue samples. Formerly, rodents were the only wild mammals found to act as potential reservoirs for this pathogen. Results of studies that attempted to detect these bacteria in common shrews (Sorex araneus), greater white-toothed shrews (Crocidura russula) (2,3), or common moles (Talpa europaea) (2) were negative. However, our results indicate that northern white-breasted hedgehogs might be a non-rodent reservoir for Candidatus N. mikurensis. 
 
The low pathogen prevalence observed in this urban hedgehog population compared with that in rodents in other locations (2,3) might be caused by use of skin samples. Skin samples from rodents showed only 1.1% positivity in a study in Germany; however, average prevalence of Candidatus N. mikurensis in transudate, spleen, kidney, and liver samples from the same animals was 37.8%–51.1% (2). Although we did not test other organs, we hypothesize that prevalence of Candidatus N. mikurensis infection urban hedgehogs is probably >2.3%. 
 
We detected A. phagocytophilum in 67 (76.1%) of 88 urban hedgehogs. This prevalence was similar to that found among European hedgehogs in Germany (8). I. ricinus ticks are more common than I. hexagonus ticks in this urban hedgehog population (9). Thus, I. ricinus ticks can acquire these bacteria when feeding on hedgehogs and the risk for human infection with A. phagocytophilum in this park in Budapest is relatively high. 
 
Neoehrlichiosis and granulocytic anaplasmosis have not been diagnosed in humans in Hungary. This finding is probably caused by diagnostic difficulties rather than absence of these pathogens in the environment. Infection with Candidatus N. mikurensis and A. phagocytophilum cause predominantly noncharacteristic symptoms. Laboratory cultivation and serologic detection of Candidatus N. mikurensis has not been successful, and this pathogen has not been identified in blood smears. Thus, accurate diagnosis of suspected cases requires suitable molecular methods. 
 
Parks can be considered points of contact for reservoir animals, pathogens, ticks, and humans. Our results indicate that E. roumanicus hedgehogs play a role in urban ecoepidemiology of ≥2 emerging human pathogens. To better understand the urban cycle of these pathogens, potential reservoir hosts, ticks collected from these hosts, and vegetation in parks should be investigated.

title: “Candidatus Neoehrlichia mikurensis” Infection in a Dog from Germany
abstract: 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.

title: Molecular Detection and Characterization of Tick-borne Pathogens in Dogs and Ticks from Nigeria
abstract: 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.  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.  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.

title: Tick-Borne Pathogen – Reversed and Conventional Discovery of Disease
abstract: Molecular methods have increased the number of known microorganisms associated with ticks significantly. Some of these newly identified microorganisms are readily linked to human disease while others are yet unknown to cause human disease. The face of tick-borne disease discovery has changed with more diseases now being discovered in a “reversed way,” detecting disease cases only years after the tick-borne microorganism was first discovered. Compared to the conventional discovery of infectious diseases, reverse order discovery presents researchers with new challenges. Estimating public health risks of such agents is especially challenging, as case definitions and diagnostic procedures may initially be missing. We discuss the advantages and shortcomings of molecular methods, serology, and epidemiological studies that might be used to study some fundamental questions regarding newly identified tick-borne diseases. With increased tick-exposure and improved detection methods, more tick-borne microorganisms will be added to the list of pathogens causing disease in humans in the future.

title: Septicemia Caused by Tick-borne Bacterial Pathogen Candidatus Neoehrlichia mikurensis
abstract: 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.

title: Detection of “Candidatus Neoehrlichia mikurensis” in Two Patients with Severe Febrile Illnesses: Evidence for a European Sequence Variant
abstract: 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.

title: First Case of Human “Candidatus Neoehrlichia mikurensis” Infection in a Febrile Patient with Chronic Lymphocytic Leukemia
abstract: 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.”

title: Close Geographic Association of Human Neoehrlichiosis and Tick Populations Carrying “Candidatus Neoehrlichia mikurensis” in Eastern Switzerland
abstract: 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.

title: Rickettsioses as paradigms of new or emerging infectious diseases
abstract: Rickettsioses are caused by species of Rickettsia, a genus comprising organisms characterized by their strictly intracellular location and their association with arthropods. Rickettsia species are difficult to cultivate in vitro and exhibit strong serological cross-reactions with each other. These technical difficulties long prohibited a detailed study of the rickettsiae, and it is only following the recent introduction of novel laboratory methods that progress in this field has been possible. In this review, we discuss the impact that these practical innovations have had on the study of rickettsiae. Prior to 1986, only eight rickettsioses were clinically recognized; however, in the last 10 years, an additional six have been discovered. We describe the different steps that resulted in the description of each new rickettsiosis and discuss the influence of factors as diverse as physicians' curiosity and the adoption of molecular biology-based identification in helping to recognize these new infections. We also assess the pathogenic potential of rickettsial strains that to date have been associated only with arthropods, and we discuss diseases of unknown etiology that may be rickettsioses.

title: Are Apodemus spp. mice and Myodes glareolus reservoirs for Borrelia miyamotoi, Candidatus Neoehrlichia mikurensis, Rickettsia helvetica, R. monacensis and Anaplasma phagocytophilum?
abstract: In Europe, in addition to Borrelia burgdorferi sensu lato and tick-borne encephalitis (TBE) virus, other zoonotic pathogens, like B. miyamotoi, a species related to the relapsing fever spirochaetes, Candidatus Neoehrlichia mikurensis (N. mikurensis), Rickettsia helvetica, Rickettsia monacensis, and Anaplasma phagocytophilum have been reported in the ixodid tick Ixodes ricinus. No study was conducted to identify reservoir hosts for these pathogens. Here, we investigated the role played by wild rodents in the natural transmission cycle of B. miyamotoi, N. mikurensis, R. helvetica, R. monacensis, and A. phagocytophilum in Switzerland. In 2011 and 2012, small mammals were captured in an area where these pathogens occur in questing ticks. Ixodes ricinus ticks infesting captured small mammals were analysed after their moult by PCR followed by reverse line blot to detect the different pathogens. Xenodiagnostic larvae were used to evaluate the role of rodents as reservoirs and analysed after their moult. Most of the 108 captured rodents (95.4%) were infested by I. ricinus ticks; 4.9%, 3.9%, 24.0%, and 0% of the rodents were infested by Borrelia, N. mikurensis, Rickettsia spp., and A. phagocytophilum-infected larvae, respectively. Borrelia afzelii, B. miyamotoi, N. mikurensis, Rickettsia spp., and A. phagocytophilum were detected in 2.8%, 0.17%, 2.6%, 6.8%, and 0% of the ticks attached to rodents, respectively. Borrelia afzelii was transmitted by 4 rodents to 41.2% of the xenodiagnostic ticks, B. miyamotoi by 3 rodents to 23.8%, and N. mikurensis was transmitted by 6 rodents to 41.0% of the xenodiagnostic ticks. None of the tested rodent transmitted Rickettsia spp. or A. phagocytophilum to I. ricinus xenodiagnostic larvae. This study showed that rodents are reservoir hosts for B. miyamotoi and N. mikurensis in Europe.

title: Isolation of Rickettsia akari from a patient in a region where Mediterranean spotted fever is endemic.
abstract: Rickettsia akari was isolated from blood collected from a patient in Croatia in 1991. We believe this is the first human isolate of R. akari to be reported in more than 40 years and the first ever from southern Europe. The Croatian isolate was antigenically and genetically indistinguishable from the prototype American strain and a Ukrainian strain. In all probability, rickettsialpox would be diagnosed more frequently and over a wider geographic area if physicians gave greater consideration to the diagnosis and if laboratory diagnostic methods were better able to distinguish among spotted fever group rickettsioses.

title: Rickettsiosis in Europe
abstract: In Europe, rickettsioses are long‐known infectious diseases. Until recently, it was thought that Mediterranean spotted fever due to Rickettsia conorii was the only tick‐borne rickettsiosis in Europe. In the last decade new Rickettsia spp. have been implicated in human pathology (R. slovaca, R. sibirica mongolotimonae, R. helvetica). Furthermore, cases of infection due to flea‐borne rickettsioses (R. typhi, R. felis) have been described. Finally, although no outbreak of epidemic typhus has been reported yet in central and southern Europe, we should be aware of the possibility of reemergence of this disease in Europe. Other rickettsioses exist that have not yet been implicated in human pathology. We should consider that climate changes and other factors could contribute to the emergence and reemergence of other new diseases.

title: Tick-borne rickettsioses in Europe.
abstract: Rickettsioses are caused by obligate intracellular bacteria within the genus Rickettsia, mainly transmitted by arthropods. Until recently, Mediterranean spotted fever (MSF) caused by Rickettsia conorii was considered the only tick-borne rickettsiosis in Europe. However, 'new' TBR have been described in Europe during last years. For instance, other subspecies such as R. conorii caspia and R. conorii israelensis have been involved in MSF. Dermacentor-borne necrosis erythema and lymphadenopathy/tick-borne lymphadenopathy (DEBONEL/TIBOLA) cases caused by Rickettsia slovaca, Rickettsia raoultii, and Rickettsia rioja been described in several countries where Dermacentor marginatus ticks (the mainly implicated vector) are present. Rickettsia helvetica has also been involved as a human pathogen in cases of fever with and without rash and in patients with meningitis and carditis. Other TBR such as lymphangitis-associated rickettsioses (LAR), caused by Rickettsia sibirica mongolitimonae, have been diagnosed in different European countries (France, Spain, Portugal and Greece). Rickettsia massiliae is considered an etiological agent of MSF-like illness in the Mediterranean basin. Furthermore, Rickettsia monacensis that is distributed all along Europe has been isolated from patients with MSF-like illness in Spain. Although Rickettsia aeschlimannii has been associated with MSF-like in Africa and is distributed in the Mediterranean area, no autochthonous human cases have been reported for Europe. Other Rickettsia species detected in ticks and unrelated to human disease (Candidatus Rickettsia kotlanii, Candidatus Rickettsia barbariae, Candidatus Rickettsia vini) could be potentially involved in the next years. Climate changes, among other factors, may contribute to the emergence of other rickettsioses or change their distribution. Lastly, African tick-bite fever (ATBF), caused by Rickettsia africae, is frequently diagnosed in Europe in patients returning from endemic areas.

title: Rickettsiae of the spotted-fever group in ixodid ticks from Hungary: identification of a new genotype ('Candidatus Rickettsia kotlanii').
abstract: Three common European 'anthrophilic' ticks, Ixodes ricinus, Haemaphysalis concinna and Dermacentor reticulatus, were collected in Hungary and tested, in assays based on nested PCR, for rickettsiae of the spotted-fever group. Low percentages of I. ricinus (2.7%) and H. concinna (1.0%) and a high percentage of D. reticulatus (26.8%) were found to be infected. The rickettsiae in the ticks were then identified, by sequencing of the genes coding for 16S ribosomal RNA (16S rDNA), citrate synthase (gltA) and the rOmpA outer-membrane protein (ompA), as Rickettsia helvetica, Rickettsia monacensis, Rickettsia sp. RpA4, or what is probably a newly recognized Rickettsia species ('Candidatus Rickettsia kotlanii'). These results raise the possibility that rickettsiae other than Rickettsia slovaca are involved in human disease in Hungary. Current knowledge on the distributions of the rickettsiae of the spotted-fever group that are emerging in Europe is also summarized.

title: Ticks and tickborne bacterial diseases in humans: an emerging infectious threat.
abstract: Ticks are currently considered to be second only to mosquitoes as vectors of human infectious diseases in the world. Each tick species has preferred environmental conditions and biotopes that determine the geographic distribution of the ticks and, consequently, the risk areas for tickborne diseases. This is particularly the case when ticks are vectors and reservoirs of the pathogens. Since the identification of Borrelia burgdorferi as the agent of Lyme disease in 1982, 15 ixodid-borne bacterial pathogens have been described throughout the world, including 8 rickettsiae, 3 ehrlichiae, and 4 species of the Borrelia burgdorferi complex. This article reviews and illustrate various aspects of the biology of ticks and the tickborne bacterial diseases (rickettsioses, ehrlichioses, Lyme disease, relapsing fever borrelioses, tularemia, Q fever), particularly those regarded as emerging diseases. Methods are described for the detection and isolation of bacteria from ticks and advice is given on how tick bites may be prevented and how clinicians should deal with patients who have been bitten by ticks.

title: Rickettsia massiliae in ticks removed from humans in Castilla y León, Spain
abstract: Rickettsia massiliae, a spotted fever group (SFG) rickettsia, was first isolated from Rhipicephalus sanguineus collected in Marseille in 1992 . Since then, this rickettsia has been commonly detected by molecular tools in Rhipicephalus ticks from France, Greece, Portugal, Switzerland, Spain, Central Africa and Mali . Recently, R. massiliae has been reported as a human pathogen . This strain was first isolated in 1985 from a Sicilian patient who was hospitalized for fever and a rash. He presented with a necrotic eschar on his right ankle, a maculopapular rash on his palms and soles, and slight hepatomegaly. The strain was stored for 20 years and definitively identified as R. massiliae in 2005. From 1997 to 2003, as part of a currently ongoing study, we analyzed 4,049 ticks that had been removed from 3,685 asymptomatic patients seen for tick bites at hospitals in the region of Castilla y Leon (northwestern Spain). All of these ticks were sent to our laboratory for specific identification and for PCR analysis to look for DNA of Anaplasma phagocytophilum, Borrelia burgdorferi, Francisella tularensis and Rickettsia spp. In this way, we found R. massiliae not only in Rhipicephalus ticks but also in Ixodes ricinus ticks. The 4,059 ticks examined belonged to 14 ixodid and one argasid species. After tick identification, DNA from each individual tick was extracted and analysed by PCR. Rickettsial DNA was detected in these tick samples with primers specific for rickettsiae (i.e., RpCS.877-RpCS.1258, which amplify a 380–397 bp fragment of the gltA gene, and Rr190.70-Rr190.701, which amplify a 629–632 bp fragment of the ompA gene), as described elsewhere [4, 5]. The rickettsiae detected were then identified by sequencing the amplified fragments and comparing the sequences obtained with those contained in gene databases. DNA contamination and carry-over of amplified products were prevented by using sterile tools at all times and performing each step of the analysis in separate work areas. Two negative controls (ultrapure water and pathogen-free tick DNA) were included in each amplification trial. These controls never amplified. Among the 4,049 ticks analyzed, a total of 320 (7.9%) ticks belonging to ten species were PCR-positive for rickettsiae, as indicated by the successful amplification of the gltA, the ompA, or both fragments. In 48 of these 320 PCR-positive ticks, namely, 37 Rhipicephalus turanicus, six R. sanguineus, four I. ricinus and one Rhipicephalus pusillus, the sequences of the gltA amplicons obtained were 99.71–100% identical to the gltA of R. massiliae/Bar29 (GenBank, RMU59719/RSU59720). Additionally, in 40 of these 48 ticks that were gtlA-positive for R. massiliae/Bar 29 (30 R. turanicus, six R. sanguineus and four I. ricinus ticks), the ompA amplicon was also obtained. Due to reasons as yet unknown, the ompA amplification failed in  (2006) 25:811–813 DOI 10.1007/s10096-006-0217-9

title: Dissemination of Spotted Fever Rickettsia Agents in Europe by Migrating Birds
abstract: 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.

title: Ixodes ricinus: vector of a hitherto undescribed spotted fever group agent in Switzerland.
abstract: A tick/rickettsial survey in various parts of Switzerland revealed the presence of a new, hitherto undescribed spotted fever group rickettsia ("Swiss agent") in up to 11.7% of I. ricinus collected off vegetation. Infection in ticks was found to be generalized with rickettsiae developing intracellularly and occasionally also intranuclearly. As a result of massive growth in ovarial tissues, including the germinative cells, the rate of transovarial and filial infection was 100%. The "Swiss agent" appears to be nonpathogenic for guinea pigs, domestic rabbits, and Swiss mice, but in male meadow voles (Microtus pennsylvanicus) it produces a microscopically detectable infection in the tunica vaginalis. The rickettsia grows well in tissue culture systems including chick embryo fibroblast, Vero, and vole tissue cells, when inoculated via yolk sac into 5-day-old hens' eggs, it kills 100% of the embryos after 5 to 7 days. Antigenic relatedness of the "Swiss agent" to rickettsiae of the spotted fever group was indicated by indirect and direct fluorescent antibody staining. Preliminary serologic typing by microimmunofluorescence and by microagglutination indicated that the "Swiss agent" differs from all prototype strains of spotted fever group rickettsiae studied so far.

title: Rickettsia massiliae sp. nov., a new spotted fever group Rickettsia.
abstract: We propose the name Rickettsia massiliae sp. nov. (type strain, Mtu1 in the Collection of the World Health Organization Collaborative Center for Rickettsial Reference, Marseille, France) for a spotted fever group rickettsia determined to be distinct from previously recognized species by the serotyping method (L. Beati, J.-P. Finidori, B. Gilot, and D. Raoult, 1930, 1992). This rickettsia has biological characteristics similar to those of the other spotted fever group rickettsiae. In addition, a sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein analysis, a polymerase chain reaction followed by a restriction fragment length polymorphism analysis of DNA fragments, and pulsed-field electrophoresis of the genome of R. massiliae revealed unique migration patterns distinct from those of all previously recognized spotted fever group rickettsiae. These additional characteristics (Beati et al., 1930, 1992), together with the data usually considered sufficient for description of rickettsiae, are crucial to the proposal of this new species and should be helpful in species identification.

title: Rickettsia helvetica in Dermacentor reticulatus Ticks
abstract: We report on the molecular evidence that Dermacentor reticulatus ticks in Croatia are infected with Rickettsia helvetica (10%) or Rickettsia slovaca (2%) or co-infected with both species (1%). These findings expand the knowledge of the geographic distribution of R. helvetica and D. reticulatus ticks.

title: Ixodes ricinus ticks are reservoir hosts for Rickettsia helvetica and potentially carry flea-borne Rickettsia species
abstract: BackgroundHard 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.ResultsThe 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.ConclusionBesides 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.

title: Retracted: Established and emerging pathogens in Ixodes ricinus ticks collected from birds on a conservation island in the Baltic Sea
abstract: The following article from Medical and Veterinary Entomology, ‘Established and emerging pathogens in Ixodes ricinus ticks collected from birds on a conservation island in the Baltic Sea’ by J. Franke, F. Meier, A. Moledenhauer, E. Straube, W. Dorne and A. Hildebrandt, published online on 26 September 2010 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editors, Doug D. Colwell, Mary Cameron, Domenico Otranto and Hilary Ranson and Blackwell Publishing Ltd. and with the knowledge of the authors. The retraction has been agreed due to overlap between this article and the following articles published in the journal Ticks and Tick‐borne Diseases:

title: Seasonal and habitat variation in the prevalence of Rickettsia helvetica in Ixodes ricinus ticks from Denmark.
abstract: A total of 704 unfed ticks of the species Ixodes ricinus collected in Denmark were screened for Rickettsia DNA by a genus-specific real-time PCR. Of the nymphs, 4.7% (31/662) were positive for rickettsial DNA. Among the positive ticks, we observed a seasonal and habitat variation. The infection rate was highest in May as compared to July, August, and October. Ecotone (high tick density) showed an elevated prevalence as compared to spruce or beech forests. Sequencing revealed only DNA from R. helvetica.

title: Association of Rickettsia helvetica with chronic perimyocarditis in sudden cardiac death
abstract: Rickettsia helvetica is the only non-imported rickettsia found in Scandinavia. It was first detected in Ixodes ricinus ticks, but has never been linked to human disease. We studied two young Swedish men who died of sudden cardiac failure during exercise, and who showed signs of perimyocarditis similar to those described in rickettsial disease.



Samples from the heart and other organs were analysed by PCR and DNA sequencing. May-Grünwald-Giemsa, Grocott, and acridine-orange stains were used for histopathological examinations. Staining of R. helvetica grown on shell-vials in vero cells, and the early descriptions of R. rickettsii by H T Ricketts and S B Wohlbach served as controls. Immunohistochemistry was done with Proteus OX-19 rabbit antisera as the primary antibody. The structure of rickettsia-like organisms was investigated by transmission electron microscopy. Serological analyses were carried out by indirect immunofluorescence with R. helvetica as the antigen.



By use of a semi-nested PCR, with primers specific for the 16S rRNA and 17-kDa outer-membrane-protein genes, and sequence analysis of the amplified products, genetic material from R. helvetica was detected in the pericardium and in a lymph node from the pulmonary hilum in case 1, and in a coronary artery and the heart muscle in case 2. A serological response in case 1 revealed an endpoint titre for R. helvetica of 1/320 (1/256 with R. rickettsii as the antigen). Examination of PCR-positive tissue showed chronic interstitial inflammation and the presence of rickettsia-like organisms predominantly located in the endothelium. These organisms reacted with Proteus OX-19 antisera, and their size and form were consistent with rickettsia. Electron microscopy confirmed that the appearance of the organisms was similar to that described for spotted-fever rickettsia.



R. helvetica, transmitted by I. ricinus ticks, may be an important pathogen in the aetiology of perimyocarditis, which can result in sudden unexpected cardiac death in young people.

title: Is sarcoidosis a rickettsiosis? An archival study
abstract: Based on earlier research, Rickettsia helvetica could possibly be involved in the pathogenesis of sarcoidosis. Rickettsiae are transmitted to humans by a tick vector, Ixodes ricinus; this tick is highly prevalent in Northern Europe. We aimed to investigate the association between evidence of rickettsiae and sarcoidosis in histological samples.  We included formalin-fixed, paraffin-embedded mediastinal lymph node biopsies from 52 ethnic Danish patients with sarcoidosis and compared these with 50 biopsies from ethnic Danish patients with mediastinal lymphadenopathy of other causes. Samples were analysed for: (1) rickettsial DNA by real-time polymerase chain reaction (PCR) and (2) rickettsial rDNA (ribosomal DNA) by a specific fluorescence in situ hybridization technique (FISH).  Rickettsia was not detected in biopsies by real-time PCR and/or FISH analyses.  Our results do not support the hypothesis that Rickettsia is involved in the pathogenesis of sarcoidosis.

title: Aneruptive Fever Associated with Antibodies to Rickettsia helvetica in Europe and Thailand
abstract: We report that eight patients from France, Italy, and Thailand had serological evidence of Rickettsia helvetica infection. The infection presented as a mild disease in the warm season and was associated with fever, headache, and myalgia but not with a cutaneous rash. R. helvetica should be suspected in patients with unexplained fever, especially following a bite from an Ixodes sp. tick.

title: Tick-Borne Rickettsioses around the World: Emerging Diseases Challenging Old Concepts
abstract: During most of the 20th century, the epidemiology of tick-borne rickettsioses could be summarized as the occurrence of a single pathogenic rickettsia on each continent. An element of this paradigm suggested that the many other characterized and noncharacterized rickettsiae isolated from ticks were not pathogenic to humans. In this context, it was considered that relatively few tick-borne rickettsiae caused human disease. This concept was modified extensively from 1984 through 2005 by the identification of at least 11 additional rickettsial species or subspecies that cause tick-borne rickettsioses around the world. Of these agents, seven were initially isolated from ticks, often years or decades before a definitive association with human disease was established. We present here the tick-borne rickettsioses described through 2005 and focus on the epidemiological circumstances that have played a role in the emergence of the newly recognized diseases.

title: Rickettsia monacensis sp. nov., a Spotted Fever Group Rickettsia, from Ticks (Ixodes ricinus) Collected in a European City Park
abstract: We describe the isolation and characterization of Rickettsia monacensis sp. nov. (type strain, IrR/MunichT) from an Ixodes ricinus tick collected in a city park, the English Garden in Munich, Germany. Rickettsiae were propagated in vitro with Ixodes scapularis cell line ISE6. BLAST analysis of the 16S rRNA, the citrate synthase, and the partial 190-kDa rickettsial outer membrane protein A (rOmpA) gene sequences demonstrated that the isolate was a spotted fever group (SFG) rickettsia closely related to several yet-to-be-cultivated rickettsiae associated with I. ricinus. Phylogenetic analysis of partial rompA sequences demonstrated that the isolate was genotypically different from other validated species of SFG rickettsiae. R. monacensis also replicated in cell lines derived from the ticks I. ricinus (IRE11) and Dermacentor andersoni (DAE100) and in the mammalian cell lines L-929 and Vero, causing cell lysis. Transmission electron microscopy of infected ISE6 and Vero cells showed rickettsiae within the cytoplasm, pseudopodia, nuclei, and vacuoles. Hamsters inoculated with R. monacensis had immunoglobulin G antibody titers as high as 1:16,384, as determined by indirect immunofluorescence assay. Western blot analyses demonstrated that the hamster sera cross-reacted with peptides from other phylogenetically distinct rickettsiae, including rOmpA. R. monacensis induced actin tails in both tick and mammalian cells similar to those reported for R. rickettsii. R. monacensis joins a growing list of SFG rickettsiae that colonize ticks but whose infectivity and pathogenicity for vertebrates are unknown.

title: Characterization of a New Spotted Fever Group Rickettsia Detected in Ixodes ricinus (Acari: Ixodidae) Collected in Slovakia
abstract: Two previously undescribed rickettsiae were detected in Ixodes ricinus Ricketts by polymerase chain reaction. Ixodes ricinus Slovakia (IRS)3 and IRS4 were identified in ticks collected in northeastern and southwestern Slovakia, respectively. Sequences of the 16S rRNA citrate synthase (gltA) and outer membrane protein rOmpA (ompA) encoding genes of both strains were nearly identical but were distinct from those of all other known rickettsiae. Phylogenetic relationships inferred from the comparison of these sequences with those of other members of the genus Rickettsia indicate that IRS3 and IRS4 constitute a new rickettsial genotype and form a separate cluster among the spotted fever group rickettsiae.

title: Isolation and Characterization of a New Rickettsia from Ixodes ricinus Ticks Collected in Slovakia
abstract: Ixodes ricinus is the most prevalent and widely distributed tick species in Central Europe, especially in Alpine regions. 1 Also known as the European sheep tick, I. ricinus is known to transmit various species of Borrelia, 2 Ehrlichia, 3,4 and Francisella, 5 agents of Lyme disease, human granulocytic ehrlichiosis, and tularemia, as well as a variety of viruses. Rickettsia helvetica , the Swiss rickettsia, was the first spotted fever group (SFG) rickettsia to be isolated from this tick. 6 We detected a newly recognized rickettsia in 2/152 I. ricinus ticks, using specific primers for 16S rDNA. IRS 3 ( Ixodes ricinus Slovakia) was detected in DNA from a tick collected in Northeastern Slovakia and IRS 4 was from a tick collected in Southwestern Slovakia. Cultivation yielded one isolate. Strain IRS 4 was isolated from internal organs of an infected tick inoculated onto L929 cells. No antibiotics were used after the inoculation of strain IRS 4 and the best growth temperature was 34 ° C. Light microscopy showed that maximum accumulation of rickettsiae was at days 5 − 7 and cell death was visible 7 − 10 days post infection. Giménez staining revealed intracellular multiplication of isolate IRS 4, which is common to SFG rickettsiae. Numerous bacteria were localized in the cytoplasm of L929 cells. Later stages of infection were characterized by necrotic effect of the infected cultures. Positive cultures exhibited a cytopathic effect, and immunofluorescent assay showed the presence of rickettsial antigens. Reactivity with sera against SFG and typhus group (TG) rickettsiae was noted. Sequence determination of 16S ribosomal DNA (16S rDNA), citrate synthase gene ( gltA ), and rOmpA outer membrane protein encoding gene ( ompA ), showed genetic similarity of DNA extracts of IRS 3 and IRS 4, although we detected several mutations within these genes, mutations in the three conserved genes being highly divergent from those of recognized rickettsial species. Phylogenetic analysis inferred from gltA and 16S rDNA sequence comparisons showed that this newly recognized rickettsial genotype is most closely related to R. helvetica , whereas the one inferred from ompA sequence clustered IRS 3 and IRS 4

title: Ticks and tick-borne diseases: a One Health perspective.
abstract: Tick-borne diseases are common occurrences in both the medical and veterinary clinical settings. In addition to the constraints related to their diagnosis and clinical management, the control and prevention of these diseases is often difficult, because it requires the disruption of a complex transmission chain, involving vertebrate hosts and ticks, which interact in a constantly changing environment. We provide a contemporary review of representative tick-borne diseases of humans and discuss aspects linked to their medical relevance worldwide. Finally, we emphasize the importance of a One Health approach to tick-borne diseases, calling physicians and veterinarians to unify their efforts in the management of these diseases, several of which are zoonoses.

title: Identification of Parasitic Communities within European Ticks Using Next-Generation Sequencing
abstract: Risk assessment of tick-borne and zoonotic disease emergence necessitates sound knowledge of the particular microorganisms circulating within the communities of these major vectors. Assessment of pathogens carried by wild ticks must be performed without a priori, to allow for the detection of new or unexpected agents.  We evaluated the potential of Next-Generation Sequencing techniques (NGS) to produce an inventory of parasites carried by questing ticks. Sequences corresponding to parasites from two distinct genera were recovered in Ixodes ricinus ticks collected in Eastern France: Babesia spp. and Theileria spp. Four Babesia species were identified, three of which were zoonotic: B. divergens, Babesia sp. EU1 and B. microti; and one which infects cattle, B. major. This is the first time that these last two species have been identified in France. This approach also identified new sequences corresponding to as-yet unknown organisms similar to tropical Theileria species.  Our findings demonstrate the capability of NGS to produce an inventory of live tick-borne parasites, which could potentially be transmitted by the ticks, and uncovers unexpected parasites in Western Europe.

title: Human Babesiosis in Europe: what clinicians need to know
abstract: Although best known as an animal disease, human babesiosis is attracting increasing attention as a worldwide emerging zoonosis. Humans are commonly infected by the bite of ixodid ticks. Rare ways of transmission are transplacental, perinatal and transfusion-associated. Infection of the human host can cause a very severe host-mediated pathology including fever, and hemolysis leading to anemia, hyperbilirubinuria, hemoglobinuria and possible organ failure. In recent years, apparently owing to increased medical awareness and better diagnostic methods, the number of reported cases in humans is rising steadily worldwide. Hitherto unknown zoonotic Babesia spp. are now being reported from geographic areas where babesiosis was not previously known to occur and the growing numbers of travelers and immunocompromised individuals suggest that the frequency of cases in Europe will also continue to rise. Our review is intended to provide clinicians with practical information on the clinical management of this rare, but potentially life-threatening zoonotic disease. It covers epidemiology, phylogeny, diagnostics and treatment of human babesiosis and the potential risk of transfusion-transmitted disease with a special focus on the European situation.

title: Babesiosis: recent insights into an ancient disease.
abstract: Ever since the discovery of parasitic inclusions in erythrocytes of cattle in Romania by Victor Babes at the end of the 19th century, newly recognised babesial pathogens continue to emerge around the world and the substantial public health impact of babesiosis on livestock and man is ongoing. Babesia are transmitted by ixodid ticks and infection of the host causes a host-mediated pathology and erythrocyte lysis, resulting in anemia, hyperbilirubinuria, hemoglobinuria, and possibly organ failure. Recently obtained molecular data, particularly for the 18S rRNA gene, has contributed significantly to a better understanding of the sometimes puzzling phylogenetic situation of the genus Babesia and new information has been added to help determine the taxonomic position of many species. Moreover, it seems that owing to higher medical awareness the number of reported cases in humans is rising steadily. Hitherto unknown zoonotic babesias are now being reported from geographical areas where babesiosis was not known to occur and the growing numbers of immunocompromised individuals suggest that the frequency of cases will continue to rise. This review covers recent insights into human babesiosis with regard to phylogeny, diagnostics and treatment in order to provide new information on well known as well as recently discovered parasites with zoonotic potential.

title: Molecular Characterization of a Non–Babesia divergens Organism Causing Zoonotic Babesiosis in Europe
abstract: In Europe, most reported human cases of babesiosis have been attributed, without strong molecular evidence, to infection with the bovine parasite Babesia divergens. We investigated the first known human cases of babesiosis in Italy and Austria, which occurred in two asplenic men. The complete 18S ribosomal RNA (18S rRNA) gene was amplified from specimens of their whole blood by polymerase chain reaction (PCR). With phylogenetic analysis, we compared the DNA sequences of the PCR products with those for other Babesia spp. The DNA sequences were identical for the organism from the two patients. In phylogenetic analysis, the organism clusters with B. odocoilei, a parasite of white-tailed deer; these two organisms form a sister group with B. divergens. This evidence indicates the patients were not infected with B. divergens but with an organism with previously unreported molecular characteristics for the 18S rRNA gene.

title: First case of human babesiosis in Germany - Clinical presentation and molecular characterisation of the pathogen.
abstract: Babesiosis is a common infection of animals and is gaining increasing attention as an emerging tick-borne zoonosis of humans in Europe. Here we report on the first case of human babesiosis in Germany in a 63-year-old splenectomised German patient with a relapse of nodular lymphocyte-predominant Hodgkin's lymphoma. After treatment with a chimeric anti-CD20 antibody preparation (Rituximab), the patient was hospitalised because of anaemia and dark urine from haemoglobinuria. Presumptive diagnosis of babesiosis was made based on piriform parasitic erythrocytic inclusions in peripheral blood smears and confirmed by Babesia-specific 18S rDNA PCR. Sequence analysis revealed a >99% homology of the amplicon with the recently described EU1 organism clustering within the Babesia divergens/Babesia odocoilei complex. Despite treatment with quinine and clindamycin the patient relapsed and developed chronic parasitaemia requiring re-treatment and long-term maintenance therapy with atovaquone before he eventually seroconverted and the parasite was cleared. Our findings suggest that human babesiosis occurs in Germany and can take a chronic course in immunocompromised individuals.

title: Babesia and its hosts: adaptation to long-lasting interactions as a way to achieve efficient transmission
abstract: Babesia, the causal agent of babesiosis, are tick-borne apicomplexan protozoa. True babesiae (Babesia genus sensu stricto) are biologically characterized by direct development in erythrocytes and by transovarial transmission in the tick. A large number of true Babesia species have been described in various vertebrate and tick hosts. This review presents the genus then discusses specific adaptations of Babesia spp. to their hosts to achieve efficient transmission. The main adaptations lead to long-lasting interactions which result in the induction of two reservoirs: in the vertebrate host during low long-term parasitemia and throughout the life cycle of the tick host as a result of transovarial and transstadial transmission. The molecular bases of these adaptations in vertebrate hosts are partially known but few of the tick-host interaction mechanisms have been elucidated.

title: Evaluation of an indirect fluorescence immunoassay for the detection of serum antibodies against Babesia divergens in humans
abstract: Since an indirect fluorescence immunoassay (IFA) for the detection of specific antibodies against Babesia divergens in human sera is not commercially available, an in-house prepared B. divergens IFA for the examination of bovine sera was established for serological studies in humans. To determine whether the described IFA is appropriate for such studies, 2 B. divergens antigens (of human or bovine origin) were tested using serum samples obtained from febrile human patients with a history of ‘tick bite’. Sera from other species of animals infected with B. divergens, Babesia EU1, B. microti or B. canis were also included for comparative purposes. All serum samples were also tested using a commercially available IFA for the detection of antibodies against B. microti, and the results compared with those obtained using blood smear and molecular techniques. This study showed that the evaluation and standardization of a B. divergens IFA for testing human sera is critical and that different B. divergens antigens provide different end-point titres of antibodies, leading to false negative or positive results. Serological cross-reactivity between B. divergens and Babesia EU1 needs to be taken into account when interpreting IFA results.

title: Babesia sp. EU1 from Roe Deer and Transmission within Ixodes ricinus
abstract: We report in vitro culture of zoonotic Babesia sp. EU1 from blood samples of roe deer in France. This study provides evidence of transovarial and transstadial transmission of the parasite within Ixodes ricinus, which suggests that this tick could be a vector and reservoir of EU1.

title: Natural Transmission of Zoonotic Babesia spp. by Ixodes ricinus Ticks
abstract: To determine characteristics of natural transmission of Babesia sp. EU1 and B. divergens by adult Ixodes ricinus ticks, we examined tick salivary gland contents. We found that I. ricinus is a competent vector for EU1 and that their sporozoites directly invade erythrocytes. We conclude that EU1 is naturally transmitted by I. ricinus.

title: Experimental in vitro transmission of Babesia sp. (EU1) by Ixodes ricinus
abstract: Babesia sp. (EU1), first characterized in 2003, has been implicated in human cases of babesiosis in Italy, Austria and Germany. It has been identified in roe deer and in its suspected tick vector, Ixodes ricinus, in several European countries. The aim of the present study was to validate the competence of I. ricinus as a vector of Babesia sp. (EU1) via experimental infections. For this purpose, a parasite strain isolated from roe deer was cloned in sheep erythrocytes. After experimental infections, parasite DNA was successfully amplified by PCR in both eggs and larvae originating from infected I. ricinus females and in the salivary glands of females exposed to Babesia sp. (EU1) as nymphs. We also demonstrate that infected females were able to transmit parasite DNA during a new blood meal. Together with previous epidemiological studies, these results validate I. ricinus as a competent vector for Babesia sp. (EU1).

title: Questing ticks in suburban forest are infected by at least six tick-borne pathogens.
abstract: The role of Ixodes ricinus ticks in the transmission of pathogens of public health importance such as Borrelia burgdorferi s.l. is widely recognized and is suspected in several emerging vector-borne pathogens in Europe. Here, we assess prevalence rates of several endemic and emerging zoonotic pathogens in tick populations in an area of high human population density in France, to contribute to a risk assessment for potential transmission to humans. Pathogen prevalence rates were evaluated by polymerase chain reaction detection and sequencing in questing ticks, individually for adults and in pools of 10 for nymphs. In addition to finding micro-organisms corresponding to symbionts, we found high prevalence rates of B. burgdorferi s.l. (32% of adult females and 10% of nymphs) and low to moderate ones of Anaplasma phagocytophilum (~1%), spotted fever group Rickettsia spp. (~6%), Babesia sp. EU1 (~1%), Bartonella birtlesii (0.1%), and Francisella tularensis (!1%). Our findings extend the knowledge of the geographical distribution of these endemic and emergent pathogens and support the conclusion that ticks are important vectors of pathogenic micro-organisms in suburban forests. Moreover, tick coinfection with multiple pathogens was found to occur frequently, which poses a serious challenge for diagnosis and appropriate treatment. The incrimination of these pathogens in potentially severe pathologies requires widespread surveillance to assess the risk of infection, thereby facilitating diagnosis and treatment, as well as raising local awareness of tick-borne diseases.

title: Babesia spp. and Anaplasma phagocytophilum in questing ticks, ticks parasitizing rodents and the parasitized rodents – Analyzing the host-pathogen-vector interface in a metropolitan area
abstract: BackgroundThe aims of this study were to evaluate the host-tick-pathogen interface of Babesia spp. and Anaplasma phagocytophilum in restored areas in both questing and host-attached Ixodes ricinus and Dermacentor reticulatus and their small mammalian hosts.MethodsQuesting ticks were collected from 5 sites within the city of Leipzig, Germany, in 2009. Small mammals were trapped at 3 of the 5 sites during 2010 and 2011. DNA extracts of questing and host-attached I. ricinus and D. reticulatus and of several tissue types of small mammals (the majority bank voles and yellow-necked mice), were investigated by PCR followed by sequencing for the occurrence of DNA of Babesia spp. and by real-time PCR for A. phagocytophilum. A selected number of samples positive for A. phagocytophilum were further investigated for variants of the partial 16S rRNA gene. Co-infection with Rickettsia spp. in the questing ticks was additionally investigated.Results4.1% of questing I. ricinus ticks, but no D. reticulatus, were positive for Babesia sp. and 8.7% of I. ricinus for A. phagocytophilum. Sequencing revealed B. microti, B. capreoli and Babesia spp. EU1 in Leipzig and sequence analysis of the partial 16S RNA gene of A. phagocytophilum revealed variants either rarely reported in human cases or associated with cervid hosts. The statistical analysis revealed significantly less ticks infected with A. phagocytophilum in a city park in Leipzig as compared to the other sampling sites. A. phagocytophilum-DNA was detected in 2 bank voles, DNA of B. microti in 1 striped field-mouse and of Babesia sp. EU1 in the skin tissue of a mole. Co-infections were detected.ConclusionOur results show the involvement of small mammals in the natural endemic cycles of tick-borne pathogens. A more thorough understanding of the interactions of ticks, pathogens and hosts is the essential basis for effective preventive control measures.

title: Diversity of Babesia and Rickettsia species in questing Ixodes ricinus: a longitudinal study in urban, pasture, and natural habitats.
abstract: In a previous study, our group investigated the Babesia spp. prevalence in questing Ixodes ricinus ticks from nine city parks in South Germany in the years 2009 and 2010. We showed predominant prevalence of B. venatorum (in previous literature also known as Babesia sp. EU1), especially in those parks in a more natural condition and with occurrence of large wild animals, such as roe deer. To obtain longitudinal data and to broaden the knowledge about this pathogen, further investigations were carried out in 2011 and 2012 in four of those city parks. Two additional habitat types were chosen for comparison of prevalence data and species analysis focusing on occurrence of potential reservoir hosts. A total of 10,303 questing I. ricinus were collected in four city parks, a pasture, and a natural area in Bavaria, and a representative number of samples were investigated for prevalence of DNA of Babesia spp. (n=4381) and Rickettsia spp. (n=2186) by PCR. In the natural and pasture area, a significantly higher Babesia spp. prevalence compared to the urban area was detected. The natural area revealed sequences of B. microti, B. venatorum, and B. capreoli. In the pasture and urban habitat, predominantly B. venatorum was found, whereas B. capreoli was less frequent and only one B. microti-infected tick was found. All B. microti sequences were 100% identical to the zoonotic Jena/Germany strain. For Rickettsia spp., the significantly highest prevalence was also detected in the natural and pasture areas, whereas lower prevalence was found in the urban area. Sequence analysis revealed R. helvetica (98%) and R. monacensis (2%). Prevalence rates and occurrence of Babesia spp. and Rickettsia spp. differed in urban, pasture and natural sites, most likely depending on the habitat structure (natural or cultivated) and therefore on the appearance and availability of reservoir hosts like roe deer or small mammals.

title: The first detection of Babesia EU1 and Babesia canis canis in Ixodes ricinus ticks (Acari, Ixodidae) collected in urban and rural areas in northern Poland.
abstract: Ixodes ricinus, the most commonly observed tick species in Poland, is a known vector of such pathogenic microorganisms as TBE viruses, Borrelia burgdoferi sensu lato, Anaplasma phagocytophilum, Rickettsia helvetica, Babesia divergens and B. microti in our country. Our study aimed to find out whether this tick can also transmit other babesiae of medical and veterinary importance. DNA extracts of 1392 ticks (314 nymphs, 552 male and 526 female ticks) collected in urban and rural areas in the Pomerania province (northern Poland), were examined by nested PCR for the detection of Babesia spp., using outer primers: 5-22F and 1661R, and inner primers: 455-479F and 793-772R, targeting specific fragment of 18S rRNA gene. Overall, at least 1.6% ticks were found to be infected with babesial parasites. In the case of nymphs, the minimal prevalence was 0.6%, and it was approx. 3-times lower than in adults (1.9%). Percentages of infected males and females were comparable (2.0% vs. 1.7%). Sequences of 15/22 PCR-derived fragments of 18S rRNA gene demonstrated 100% similarities with the sequence of Babesia EUI (proposed name B. venatorum) (acc. no. AY046575) (n = 13) and with B. canis canis (acc. no. AY321119) (n = 2), deposited in the GenBank database. The partial 18S rDNA sequences of Babesia EUI and B. c. canis obtained by us from I. ricinus have been deposited in GenBank, accession nos. GQ325619 and GQ325620, respectively. The results obtained suggest the possible role of I. ricinus as a source of microorganisms, which have been identified as agents of human and canine babesiosis, respectively, in Europe. To our knowledge this is the first report on the occurrence of Babesia EUI and B. c. canis in I. ricinus in Poland.

title: Diversity of Babesia in Ixodes ricinus ticks in Poland.
abstract: The aims of this study were: (1) to estimate Babesia prevalence in the most common species of tick in Poland, Ixodes ricinus, in two recreational areas (Urwitałt in the Mazury Lake District and Bielański Forest in Warsaw), and (2) to evaluate the molecular diversity of Babesia isolates in questing I. ricinus in Poland.



Questing ticks were collected from vegetation in forest areas in Urwitałt near Mikołajki and in Bielański Forest (Warsaw). Purified genomic DNA was used with specific primers to amplify a fragment of the Babesia spp. 18S rRNA gene.



Tick-drag indices for I. ricinus were high in both study areas, reaching somewhat higher values in Urwitałt than in Bielański Forest. The overall prevalence of Babesia spp. in examined ticks was 1.6%. In Urwitałt, two strains of B. microti were identified using rRNA sequences: the enzootic Munich strain and an isolate close to the zoonotic Jena strain. The proportion of infections due to these two strains in questing ticks reversed over a six-year period. During 3 years of study in Bielański Forest, all Babesia isolates obtained from I. ricinus were identical to Babesia sp. EU1 (B. venatorum), previously recognized as an agent of human babesiosis.



This study has confirmed the presence of enzoonotic and zoonotic Babesia species/strains in the abundant human-biting tick I. ricinus in recreational areas in Poland. It has also shown that the distribution of different genotypes has changed over time, however the reasons for these fluctuations still remain to be investigated.

title: Phylogeny and evolution of the Piroplasmida as inferred from 18S rRNA sequences.
abstract: The order Piroplasmida consists of several genera of tick-borne parasites that infect mammals, and to a lesser extent birds, and are therefore of medical and economic importance. Despite their importance, considerable confusion exists concerning the relationship among piroplasmid species, specifically concerning the number of genera and the intergeneric relationships. To examine evolutionary relationships among piroplasmids, we conducted phylogenetic analyses of 192 18S rDNA sequences from the genera Theileria, Babesia and Cytauxzoon. Our analyses revealed eight clades potentially representing distinct genera, and we distinguish the Duncani Group and Microti Group as genetically distinct groups of species requiring detailed analysis of morphology and life-history to allow formal generic description. The piroplasmid phylogeny revealed considerable host diversity and limited host specificity, suggesting piroplasmids have undergone frequent host switches during their evolution. Our analyses provide the first reported evolutionary timescale for piroplasmids independent of the assumption of parasite-host cospeciation, which is invalid for piroplasmids. Evolutionary rate analyses revealed considerable substitution rate heterogeneity, which we attribute to host switching and diversification. Finally, we call for a comprehensive phylogenetic, morphological and life-history analysis for these medically relevant taxa to resolve relationships and understand host specificity.

title: Babesia: a world emerging.
abstract: Babesia are tick-transmitted hemoprotozooans that infect mammals and birds, and which are acknowledged for their major impact on farm and pet animal health and associated economic costs worldwide. Additionally, Babesia infections of wildlife can be fatal if associated with stressful management practices; and human babesiosis, also transmitted by blood transfusion, is an increasing public-health concern. Due to the huge diversity of species reported to serve as Babesia hosts, all vertebrates might be potential carriers, as long as they are adequate hosts for Babesia-vector ticks. We here provide a comprehensive overview of the most relevant Babesia species, and a discussion of the classical taxonomic criteria. Babesia, Cytauxzoon and Theileria parasites are closely related and collectively referred to as piroplasmids. A possible scenario for the history of piroplasmids is presented in the context of recent findings, and its implications for future research avenues are outlined. Phylogenetic trees of all available 18S rRNA and hsp70 genes were generated, based on which we present a thoroughly revised molecular classification, comprising five monophyletic Babesia lineages, one Cytauxzoon clade, and one Theileria clade. Updated 18S rRNA and beta-tubulin gene trees of the B. microti isolates agree with those previously reported. To reconcile estimates of the origin of piroplasmids and ticks (~300 Ma, respectively), and mammalian radiation (60 Ma), we hypothesize that the dixenous piroplasmid life cycle evolved with the origin of ticks. Thus, the observed time gap between tick origin and mammalian radiation indicates the existence of hitherto unknown piroplasmid lineages and/or species in extant vertebrate taxa, including reptiles and possibly amphibians. The development and current status of the molecular taxonomy of Babesia, with emphasis on human-infecting species, is discussed. Finally, recent results from population genetic studies of Babesia parasites, and their implications for the development of pathogenicity, drug resistance and vaccines, are summarized.

title: Whole Genome Mapping and Re-Organization of the Nuclear and Mitochondrial Genomes of Babesia microti Isolates
abstract: Babesia microti is the primary causative agent of human babesiosis, an emerging pathogen that causes a malaria-like illness with possible fatal outcome in immunocompromised patients. The genome sequence of the B. microti R1 strain was reported in 2012 and revealed a distinct evolutionary path for this pathogen relative to that of other apicomplexa. Lacking from the first genome assembly and initial molecular analyses was information about the terminal ends of each chromosome, and both the exact number of chromosomes in the nuclear genome and the organization of the mitochondrial genome remained ambiguous. We have now performed various molecular analyses to characterize the nuclear and mitochondrial genomes of the B. microti R1 and Gray strains and generated high-resolution Whole Genome maps. These analyses show that the genome of B. microti consists of four nuclear chromosomes and a linear mitochondrial genome present in four different structural types. Furthermore, Whole Genome mapping allowed resolution of the chromosomal ends, identification of areas of misassembly in the R1 genome, and genomic differences between the R1 and Gray strains, which occur primarily in the telomeric regions. These studies set the stage for a better understanding of the evolution and diversity of this important human pathogen.

title: Transmission of Bartonella henselae by Ixodes ricinus
abstract: The causative agent of cat-scratch disease in humans can be transmitted by this tick through saliva.

title: Evidence of Bartonella sp. in questing adult and nymphal Ixodes ricinus ticks from France and co-infection with Borrelia burgdorferi sensu lato and Babesia sp.
abstract: Ticks are known vectors for a wide range of pathogenic microorganisms. Their role in the transmission of some others is so far only suspected. Ticks can transmit multiple pathogens, however, little is known about the co-existence of these pathogens within questing ticks. We looked for the presence of DNA from three micro-organisms, Bartonella sp., Borrelia burgdorferi sensu lato and Babesia sp. which are known or suspected tick-borne pathogens, using a cohort of 92 questing Ixodes ricinus ticks collected from pastures in northern France. DNA was extracted from each individual tick and the presence of the three pathogens was investigated using Polymerase Chain Reaction (PCR) amplification. Nine among 92 samples (9.8%) demonstrated PCR products using Bartonella specific primers, 3 among 92 (3.3%) using Borrelia burgdorferi sensu lato specific primers and 19 among 92 (20.6%) using Babesia specific primers. Seven among 92 samples (7.6%) were PCR positive for at least two of the pathogens and one sample was positive for all three. Adult ticks (12/18; 67%) showed significantly higher infection rates compared to nymphs (11/74; 15%) for all three pathogens (P < 0.001). This study is the demonstration of the simultaneous presence of Bartonella sp., Borrelia burgdorferi sensu lato and Babesia sp. in questing Ixodes ricinus ticks.

title: Vector Competence of the Tick Ixodes ricinus for Transmission of Bartonella birtlesii
abstract: Bartonella spp. are facultative intracellular vector-borne bacteria associated with several emerging diseases in humans and animals all over the world. The potential for involvement of ticks in transmission of Bartonella spp. has been heartily debated for many years. However, most of the data supporting bartonellae transmission by ticks come from molecular and serological epidemiological surveys in humans and animals providing only indirect evidences without a direct proof of tick vector competence for transmission of bartonellae. We used a murine model to assess the vector competence of Ixodes ricinus for Bartonella birtlesii. Larval and nymphal I. ricinus were fed on a B. birtlesii-infected mouse. The nymphs successfully transmitted B. birtlesii to naïve mice as bacteria were recovered from both the mouse blood and liver at seven and 16 days after tick bites. The female adults successfully emitted the bacteria into uninfected blood after three or more days of tick attachment, when fed via membrane feeding system. Histochemical staining showed the presence of bacteria in salivary glands and muscle tissues of partially engorged adult ticks, which had molted from the infected nymphs. These results confirm the vector competence of I. ricinus for B. birtlesii and represent the first in vivo demonstration of a Bartonella sp. transmission by ticks. Consequently, bartonelloses should be now included in the differential diagnosis for patients exposed to tick bites.

title: Next Generation Sequencing Uncovers Unexpected Bacterial Pathogens in Ticks in Western Europe
abstract: Ticks are highly susceptible to global environmental and socio-economical changes. Several tick-borne pathogens have been reported in new geographical regions while new species, strains or genetic variants of tick-borne microorganisms are continually being detected. However, tick-borne pathogens are still poorly understood, and it is estimated that half of all human tick-borne disease has an unknown origin. Therefore in order to prevent these diseases, more effort is required to identify unknown or unexpected tick-borne pathogens. Ixodes ricinus is the vector for a broad range of bacterial pathogens and the most prevalent tick in Europe. The aim of the present study was to evaluate the capability of Next Generation Sequencing (NGS) to extend the inventory of pathogenic bacteria carried by this species of tick in France.  RNA and DNA were extracted from 1450 I. ricinus questing nymphs collected by flagging in Alsace, France. RNA was pooled and used for NGS. Following de novo assembly, bacterial contigs were assigned to the closest known taxonomy. DNA was used for real time PCR to confirm taxonomic species assignment of NGS-derived contigs for the doubtful cases, and for determination of prevalence.  We have generated a global in-depth picture of tick-borne bacteria. We identified RNA from the main pathogenic bacterial species known to be transmitted by I. ricinus. In addition we also identified unanticipated bacterial species for which we have estimated the prevalence within those ticks inhabiting the studied areas.  The data obtained from this study has proven that NGS has an enormous potential to detect the unexpected and provides the means to monitor pathogen occurrence.

title: Scalp eschar and neck lymphadenopathy caused by Bartonella henselae after Tick Bite.
abstract: Rickettsia slovaca and Rickettsia raoultii have been associated with a syndrome characterized by scalp eschar and neck lymphadenopathy following tick bites. However, in many cases, the causative agent remains undetermined. We report 3 cases of this syndrome caused by Bartonella henselae, and we propose the term "SENLAT" to collectively describe this clinical entity.

title: Humans Infected with Relapsing Fever Spirochete Borrelia miyamotoi, Russia
abstract: Disease may occur throughout the world because of the widespread prevalence of this pathogen in ixodid ticks.

title: Human Borrelia miyamotoi infection in the United States.
abstract: A tickborne pathogen, Borrelia miyamotoi, has recently been shown to cause disease in humans. A serologic survey showed that infection with this organism is not uncommon in the Northeast.

title: Meningoencephalitis from Borrelia miyamotoi in an immunocompromised patient.
abstract: Ixodes ticks serve as vectors for Borrelia burgdorferi, the agent of Lyme disease. Globally, these ticks often concurrently harbor B. miyamotoi, a spirochete that is classified within the relapsing-fever group of spirochetes. Although humans presumably are exposed to B. miyamotoi, there are limited data suggesting disease attributable to it. We report a case of progressive mental deterioration in an older, immunocompromised patient, and even though Koch's postulates were not met, we posit B. miyamotoi as the cause, owing to its direct detection in cerebrospinal fluid (CSF) with the use of microscopy and a polymerase-chain-reaction (PCR) assay. It is likely that B. miyamotoi is an underrecognized cause of disease, especially in sites where Lyme disease is endemic.

title: Genetic characterization of the human relapsing fever spirochete Borrelia miyamotoi in vectors and animal reservoirs of Lyme disease spirochetes in France
abstract: BackgroundIn France as elsewhere in Europe the most prevalent TBD in humans is Lyme borreliosis, caused by different bacterial species belonging to Borrelia burgdorferi sensu lato complex and transmitted by the most important tick species in France, Ixodes ricinus. However, the diagnosis of Lyme disease is not always confirmed and unexplained syndromes occurring after tick bites have become an important issue. Recently, B. miyamotoi belonging to the relapsing fever group and transmitted by the same Ixodes species has been involved in human disease in Russia, the USA and the Netherlands. In the present study, we investigate the presence of B. miyamotoi along with other Lyme Borreliosis spirochetes, in ticks and possible animal reservoirs collected in France.MethodsWe analyzed 268 ticks (Ixodes ricinus) and 72 bank voles (Myodes glareolus) collected and trapped in France for the presence of DNA from B. miyamotoi as well as from Lyme spirochetes using q-PCR and specific primers and probes. We then compared the French genotypes with those found in other European countries.ResultsWe found that 3% of ticks and 5.55% of bank voles were found infected by the same B. miyamotoi genotype, while co-infection with other Lyme spirochetes (B. garinii) was identified in 12% of B. miyamotoi infected ticks. Sequencing showed that ticks and rodents carried the same genotype as those recently characterized in a sick person in the Netherlands.ConclusionsThe genotype of B. miyamotoi circulating in ticks and bank voles in France is identical to those already described in ticks from Western Europe and to the genotype isolated from a sick person in The Netherlands. This results suggests that even though no human cases have been reported in France, surveillance has to be improved. Moreover, we showed that ticks could simultaneously carry B. miyamotoi and Lyme disease spirochetes, increasing the problem of co-infection in humans.

title: A new phlebovirus associated with severe febrile illness in Missouri.
abstract: Two men from northwestern Missouri independently presented to a medical facility with fever, fatigue, diarrhea, thrombocytopenia, and leukopenia, and both had been bitten by ticks 5 to 7 days before the onset of illness. Ehrlichia chaffeensis was suspected as the causal agent but was not found on serologic analysis, polymerase-chain-reaction (PCR) assay, or cell culture. Electron microscopy revealed viruses consistent with members of the Bunyaviridae family. Next-generation sequencing and phylogenetic analysis identified the viruses as novel members of the phlebovirus genus. Although Koch's postulates have not been completely fulfilled, we believe that this phlebovirus, which is novel in the Americas, is the cause of this clinical syndrome.