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1.  Circulation of four Anaplasma phagocytophilum ecotypes in Europe 
Parasites & Vectors  2014;7(1):365.
Anaplasma phagocytophilum is the etiological agent of granulocytic anaplasmosis in humans and animals. Wild animals and ticks play key roles in the enzootic cycles of the pathogen. Potential ecotypes of A. phagocytophilum have been characterized genetically, but their host range, zoonotic potential and transmission dynamics has only incompletely been resolved.
The presence of A. phagocytophilum DNA was determined in more than 6000 ixodid ticks collected from the vegetation and wildlife, in 289 tissue samples from wild and domestic animals, and 69 keds collected from deer, originating from various geographic locations in The Netherlands and Belgium. From the qPCR-positive lysates, a fragment of the groEL-gene was amplified and sequenced. Additional groEL sequences from ticks and animals from Europe were obtained from GenBank, and sequences from human cases were obtained through literature searches. Statistical analyses were performed to identify A. phagocytophilum ecotypes, to assess their host range and their zoonotic potential. The population dynamics of A. phagocytophilum ecotypes was investigated using population genetic analyses.
DNA of A. phagocytophilum was present in all stages of questing and feeding Ixodes ricinus, feeding I. hexagonus, I. frontalis, I. trianguliceps, and deer keds, but was absent in questing I. arboricola and Dermacentor reticulatus. DNA of A. phagocytophilum was present in feeding ticks and tissues from many vertebrates, including roe deer, mouflon, red foxes, wild boar, sheep and hedgehogs but was rarely found in rodents and birds and was absent in badgers and lizards. Four geographically dispersed A. phagocytophilum ecotypes were identified, that had significantly different host ranges. All sequences from human cases belonged to only one of these ecotypes. Based on population genetic parameters, the potentially zoonotic ecotype showed significant expansion.
Four ecotypes of A. phagocytophilum with differential enzootic cycles were identified. So far, all human cases clustered in only one of these ecotypes. The zoonotic ecotype has the broadest range of wildlife hosts. The expansion of the zoonotic A. phagocytophilum ecotype indicates a recent increase of the acarological risk of exposure of humans and animals.
Electronic supplementary material
The online version of this article (doi:10.1186/1756-3305-7-365) contains supplementary material, which is available to authorized users.
PMCID: PMC4153903  PMID: 25127547
Anaplasma phagocytophilum; Zoonoses; Ixodes ricinus; Wildlife; Epidemiology
2.  Human Exposure to Tickborne Relapsing Fever Spirochete Borrelia miyamotoi, the Netherlands 
Emerging Infectious Diseases  2014;20(7):1244-1246.
PMCID: PMC4073841  PMID: 24963562
Tickborne diseases; zoonoses; relapsing fever; Lyme disease; Borrelia; borreliosis; miyamotoi; B. burgdorferi sensu lato; the Netherlands
3.  Sensitivity of a point of care tick-test for the development of Lyme borreliosis 
Parasites & Vectors  2013;6:338.
A commercially available self-test for the detection of Borrelia burgdorferi sensu lato in ticks was evaluated for its ability to predict erythema migrans formation.
The self-test was performed on 127 Ixodes ricinus from 122 humans that reported tick bites at enrolment and occurrence of symptoms during follow-up. The self-test gave negative results on all the 122 individuals, 14 of whom reported erythema migrans (EM) at follow-up of which 10 were confirmed by their GP. The estimated sensitivity of the self-test for prediction of EM formation is 0% (95% CI: 0%-28%).
This self-test is not suitable for reducing the number needed to treat in a post-exposure prophylaxis setting as it already missed all the obvious early Lyme borreliosis cases.
PMCID: PMC4235040  PMID: 24304944
Lyme borreliosis; Self-test; Ixodes ricinus; Validation; Post-exposure prophylaxis
4.  A Prospective Study among Patients Presenting at the General Practitioner with a Tick Bite or Erythema Migrans in the Netherlands 
PLoS ONE  2013;8(5):e64361.
We performed a nationwide prospective study on the transmission risk for Borrelia to humans, investigating symptoms and serology at enrolment and three months after tick bites, and after standard treatment for erythema migrans (EM). Aiming to quantify the infection risk at point of care by physicians, we explored risk factors such as tick testing for Borrelia and assessment of the duration of the tick's blood meal.
Methods and Findings
Questionnaires, blood samples and ticks from patients who consulted one of 307 general practitioners for tick bites (n = 327) or EM (n = 283) in 2007 and 2008, were collected at enrolment and three months later at follow-up. Borrelia burgdorferi sensu lato DNA was detected in 29.3% of 314 ticks, using PCR/reverse line blot and real-time PCR on the OspA gene. Seroconversion in C6 ELISA, IgM or IgG immunoblots for Borrelia-specific antibodies was observed in 3.2% of tick bite cases. Fourteen tick bite cases had evidence of early Borrelia infection, of which EM developed among seven cases. The risk of developing EM after tick bites was 2.6% (95%CI: 1.1%–5.0%), and the risk of either EM or seroconversion was 5.1% (95%CI: 2.9%–8.2%). Participants with Borrelia-positive ticks had a significantly higher risk of either EM or seroconversion (odds ratio 4.8, 95%CI: 1.1–20.4), and of seroconversion alone (odds ratio 11.1, 95%CI: 1.1–108.9). A third (34%) of the cases enrolled with EM did not recall preceding tick bites. Three EM cases (1%) reported persisting symptoms, three months after standard antibiotic treatment for EM.
One out of forty participants developed EM within three months after tick bites. The infection risk can be assessed by tick testing for Borrelia at point of care by physicians. However, further refining is needed considering sensitivity and specificity of tick tests, accuracy of tick attachment time and engorgement.
PMCID: PMC3655959  PMID: 23696884
5.  Spatiotemporal dynamics of emerging pathogens in questing Ixodes ricinus 
Ixodes ricinus transmits Borrelia burgdorferi sensu lato, the etiological agent of Lyme disease. Previous studies have also detected Rickettsia helvetica, Anaplasma phagocytophilum, Neoehrlichia mikurensis, and several Babesia species in questing ticks in The Netherlands. In this study, we assessed the acarological risk of exposure to several tick-borne pathogens (TBPs), in The Netherlands. Questing ticks were collected monthly between 2006 and 2010 at 21 sites and between 2000 and 2009 at one other site. Nymphs and adults were analysed individually for the presence of TBPs using an array-approach. Collated data of this and previous studies were used to generate, for each pathogen, a presence/absence map and to further analyse their spatiotemporal variation. R. helvetica (31.1%) and B. burgdorferi sensu lato (11.8%) had the highest overall prevalence and were detected in all areas. N. mikurensis (5.6%), A. phagocytophilum (0.8%), and Babesia spp. (1.7%) were detected in most, but not all areas. The prevalences of pathogens varied among the study areas from 0 to 64%, while the density of questing ticks varied from 1 to 179/100 m2. Overall, 37% of the ticks were infected with at least one pathogen and 6.3% with more than one pathogen. One-third of the Borrelia-positive ticks were infected with at least one other pathogen. Coinfection of B. afzelii with N. mikurensis and with Babesia spp. occurred significantly more often than single infections, indicating the existence of mutual reservoir hosts. Alternatively, coinfection of R. helvetica with either B. afzelii or N. mikurensis occurred significantly less frequent. The diversity of TBPs detected in I. ricinus in this study and the frequency of their coinfections with B. burgdorferi s.l., underline the need to consider them when evaluating the risks of infection and subsequently the risk of disease following a tick bite.
PMCID: PMC3726834  PMID: 23908971
vector-borne disease; Borrelia burgdorferi; Candidatus Neoehrlichia mikurensis; Rickettsia helvetica; Rickettsia conorii; Anaplasma phagocytophilum; Babesia; Ixodes ricinus
6.  Prevalence of Neoehrlichia mikurensis in ticks and rodents from North-west Europe 
Parasites & Vectors  2012;5:74.
Neoehrlichia mikurensis s an emerging and vector-borne zoonosis: The first human disease cases were reported in 2010. Limited information is available about the prevalence and distribution of Neoehrlichia mikurensis in Europe, its natural life cycle and reservoir hosts. An Ehrlichia-like schotti variant has been described in questing Ixodes ricinus ticks, which could be identical to Neoehrlichia mikurensis.
Three genetic markers, 16S rDNA, gltA and GroEL, of Ehrlichia schotti-positive tick lysates were amplified, sequenced and compared to sequences from Neoehrlichia mikurensis. Based on these DNA sequences, a multiplex real-time PCR was developed to specifically detect Neoehrlichia mikurensis in combination with Anaplasma phagocytophilum in tick lysates. Various tick species from different life-stages, particularly Ixodes ricinus nymphs, were collected from the vegetation or wildlife. Tick lysates and DNA derived from organs of wild rodents were tested by PCR-based methods for the presence of Neoehrlichia mikurensis. Prevalence of Neoehrlichia mikurensis was calculated together with confidence intervals using Fisher's exact test.
The three genetic markers of Ehrlichia schotti-positive field isolates were similar or identical to Neoehrlichia mikurensis. Neoehrlichia mikurensis was found to be ubiquitously spread in the Netherlands and Belgium, but was not detected in the 401 tick samples from the UK. Neoehrlichia mikurensis was found in nymphs and adult Ixodes ricinus ticks, but neither in their larvae, nor in any other tick species tested. Neoehrlichia mikurensis was detected in diverse organs of some rodent species. Engorging ticks from red deer, European mouflon, wild boar and sheep were found positive for Neoehrlichia mikurensis.
Ehrlichia schotti is similar, if not identical, to Neoehrlichia mikurensis. Neoehrlichia mikurensis is present in questing Ixodes ricinus ticks throughout the Netherlands and Belgium. We propose that Ixodes ricinus can transstadially, but not transovarially, transmit this microorganism, and that different rodent species may act as reservoir hosts. These data further imply that wildlife and humans are frequently exposed to Neoehrlichia mikurensis-infected ticks through tick bites. Future studies should aim to investigate to what extent Neoehrlichia mikurensis poses a risk to public health.
PMCID: PMC3395572  PMID: 22515314
Vector-borne disease; Emerging zoonoses; Candidatus N. mikurensis; I. ricinus; Anaplasma phagocytophylum
7.  Absence of zoonotic Bartonella species in questing ticks: First detection of Bartonella clarridgeiae and Rickettsia felis in cat fleas in the Netherlands 
Parasites & Vectors  2011;4:61.
Awareness for flea- and tick-borne infections has grown in recent years and the range of microorganisms associated with these ectoparasites is rising. Bartonella henselae, the causative agent of Cat Scratch Disease, and other Bartonella species have been reported in fleas and ticks. The role of Ixodes ricinus ticks in the natural cycle of Bartonella spp. and the transmission of these bacteria to humans is unclear. Rickettsia spp. have also been reported from as well ticks as also from fleas. However, to date no flea-borne Rickettsia spp. were reported from the Netherlands. Here, the presence of Bartonellaceae and Rickettsiae in ectoparasites was investigated using molecular detection and identification on part of the gltA- and 16S rRNA-genes.
The zoonotic Bartonella clarridgeiae and Rickettsia felis were detected for the first time in Dutch cat fleas. B. henselae was found in cat fleas and B. schoenbuchensis in ticks and keds feeding on deer. Two Bartonella species, previously identified in rodents, were found in wild mice and their fleas. However, none of these microorganisms were found in 1719 questing Ixodes ricinus ticks. Notably, the gltA gene amplified from DNA lysates of approximately 10% of the questing nymph and adult ticks was similar to that of an uncultured Bartonella-related species found in other hard tick species. The gltA gene of this Bartonella-related species was also detected in questing larvae for which a 16S rRNA gene PCR also tested positive for "Candidatus Midichloria mitochondrii". The gltA-gene of the Bartonella-related species found in I. ricinus may therefore be from this endosymbiont.
We conclude that the risk of acquiring Cat Scratch Disease or a related bartonellosis from questing ticks in the Netherlands is negligible. On the other hand fleas and deer keds are probable vectors for associated Bartonella species between animals and might also transmit Bartonella spp. to humans.
PMCID: PMC3087693  PMID: 21501464
8.  Small risk of developing symptomatic tick-borne diseases following a tick bite in the Netherlands 
Parasites & Vectors  2011;4:17.
In The Netherlands, the incidence of Lyme borreliosis is on the rise. Besides its causative agent, Borrelia burgdorferi s.l., other potential pathogens like Rickettsia, Babesia and Ehrlichia species are present in Ixodes ricinus ticks. The risk of disease associated with these microorganisms after tick-bites remains, however, largely unclear. A prospective study was performed to investigate how many persons with tick-bites develop localized or systemic symptoms and whether these are associated with tick-borne microorganisms.
In total, 297 Ixodes ricinus ticks were collected from 246 study participants who consulted a general practitioner on the island of Ameland for tick bites. Ticks were subjected to PCR to detect DNA of Borrelia burgdorferi s.l., Rickettsia spp., Babesia spp. or Ehrlichia/Anaplasma spp.. Sixteen percent of the collected ticks were positive for Borrelia burgdorferi s.l., 19% for Rickettsia spp., 12% for Ehrlichia/Anaplasma spp. and 10% for Babesia spp.. At least six months after the tick bite, study participants were interviewed on symptoms by means of a standard questionnaire. 14 out of 193 participants (8.3%) reported reddening at the bite site and 6 participants (4.1%) reported systemic symptoms. No association between symptoms and tick-borne microorganisms was found. Attachment duration ≥24 h was positively associated with reddening at the bite site and systemic symptoms. Using logistic regression techniques, reddening was positively correlated with presence of Borrelia afzelii, and having 'any symptoms' was positively associated with attachment duration.
The risk of contracting acute Lyme borreliosis, rickettsiosis, babesiosis or ehrlichiosis from a single tick bite was <1% in this study population.
PMCID: PMC3050846  PMID: 21310036
9.  Age-Related Toxoplasma gondii Seroprevalence in Dutch Wild Boar Inconsistent with Lifelong Persistence of Antibodies 
PLoS ONE  2011;6(1):e16240.
Toxoplasma gondii is an important zoonotic pathogen that is best known as a cause of abortion or abnormalities in the newborn after primary infection during pregnancy. Our aim was to determine the prevalence of T. gondii in wild boar to investigate the possible role of their meat in human infection and to get an indication of the environmental contamination with T. gondii. The presence of anti-T. gondii antibodies was determined by in-house ELISA in 509 wild boar shot in 2002/2003 and 464 wild boar shot in 2007. Most of the boar originated from the “Roerstreek” (n = 673) or the “Veluwe” (n = 241). A binormal mixture model was fitted to the log-transformed optical density values for wild boar up to 20 months old to estimate the optimal cut-off value (−0.685) and accompanying sensitivity (90.6%) and specificity (93.6%). The overall seroprevalence was estimated at 24.4% (95% CI: 21.1–27.7%). The prevalence did not show variation between sampling years or regions, indicating a stable and homogeneous infection pressure from the environment. The relation between age and seroprevalence was studied in two stages. Firstly, seroprevalence by age group was determined by fitting the binary mixture model to 200 animals per age category. The prevalence showed a steep increase until approximately 10 months of age but stabilized at approximately 35% thereafter. Secondly, we fitted the age-dependent seroprevalence data to several SIR-type models, with seropositives as infected (I) and seronegatives as either susceptible (S) or resistant (R). A model with a recovery rate (SIS) was superior to a model without a recovery rate (SI). This finding is not consistent with the traditional view of lifelong persistence of T. gondii infections. The high seroprevalence suggests that eating undercooked wild boar meat may pose a risk of infection with T. gondii.
PMCID: PMC3024411  PMID: 21283764
10.  Antibody response against Trichinella spiralis in experimentally infected rats is dose dependent 
Veterinary Research  2011;42(1):113.
Domestic pigs are the main representatives of the domestic cycle of Trichinella spiralis that play a role in transmission to humans. In Europe, backyard pigs of small household farms are the most important risks for humans to obtain trichinellosis. Rats might play a role in the transmission of Trichinella spiralis from domestic to sylvatic animals and vice versa. In order to be able to investigate the role of wild rats in the epidemiology of T. spiralis in The Netherlands, we studied the dynamics of antibody response after T. spiralis infections in experimental rats, using infection doses ranging from very low (10 muscle larvae, ML, per rat) to very high (16 000 ML per rat). To evaluate the feasibility of rats surviving high infection doses with T. spiralis, clinical and pathological parameters were quantified. Serological tools for detecting T. spiralis in rats were developed to quantitatively study the correlation between parasite load and immunological response. The results show that an infection dose-dependent antibody response was developed in rats after infection with as low as 10 ML up to a level of 10 000 ML. A positive correlation was found between the number of recovered ML and serum antibody levels, although specific measured antibody levels correspond to a wide range of LPG values. Serum antibodies of rats that were infected even with 10 or 25 ML could readily be detected by use of the T. spiralis western blot 2 weeks post infection. We conclude that based on these low infection doses, serologic tests are a useful tool to survey T. spiralis in wild rats.
PMCID: PMC3247182  PMID: 22129040
11.  Exotic Rickettsiae in Ixodes ricinus: fact or artifact? 
Parasites & Vectors  2010;3:54.
Several pathogenic Rickettsia species can be transmitted via Ixodes ricinus ticks to humans and animals. Surveys of I. ricinus for the presence of Rickettsiae using part of its 16S rRNA gene yield a plethora of new and different Rickettsia sequences. Interpreting these data is sometimes difficult and presenting these findings as new or potentially pathogenic Rickettsiae should be done with caution: a recent report suggested presence of a known human pathogen, R. australis, in questing I. ricinus ticks in Europe. A refined analysis of these results revealed that R. helvetica was most likely to be misinterpreted as R. australis. Evidence in the literature is accumulating that rickettsial DNA sequences found in tick lysates can also be derived from other sources than viable, pathogenic Rickettsiae. For example, from endosymbionts, environmental contamination or even horizontal gene transfer.
PMCID: PMC2904298  PMID: 20569494
12.  Role of sand lizards in the ecology of Lyme and other tick-borne diseases in the Netherlands 
Parasites & Vectors  2010;3:42.
Lizards 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.
Ticks 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.
Despite 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.
PMCID: PMC2890652  PMID: 20470386
13.  Ixodes ricinus ticks are reservoir hosts for Rickettsia helvetica and potentially carry flea-borne Rickettsia species 
Parasites & Vectors  2009;2:41.
Hard ticks have been identified as important vectors of rickettsiae causing the spotted fever syndrome. Tick-borne rickettsiae are considered to be emerging, but only limited data are available about their presence in Western Europe, their natural life cycle and their reservoir hosts. Ixodes ricinus, the most prevalent tick species, were collected and tested from different vegetation types and from potential reservoir hosts. In one biotope area, the annual and seasonal variability of rickettsiae infections of the different tick stages were determined for 9 years.
The DNA of the human pathogen R. conorii as well as R. helvetica, R. sp. IRS and R. bellii-like were found. Unexpectedly, the DNA of the highly pathogenic R. typhi and R. prowazekii and 4 other uncharacterized Rickettsia spp. related to the typhus group were also detected in I. ricinus. The presence of R. helvetica in fleas isolated from small rodents supported our hypothesis that cross-infection can occur under natural conditions, since R. typhi/prowazekii and R. helvetica as well as their vectors share rodents as reservoir hosts. In one biotope, the infection rate with R. helvetica was ~66% for 9 years, and was comparable between larvae, nymphs, and adults. Larvae caught by flagging generally have not yet taken a blood meal from a vertebrate host. The simplest explanation for the comparable prevalence of R. helvetica between the defined tick stages is, that R. helvetica is vertically transmitted through the next generation with high efficiency. The DNA of R. helvetica was also present in whole blood from mice, deer and wild boar.
Besides R. helvetica, unexpected rickettsiae are found in I. ricinus ticks. We propose that I. ricinus is a major reservoir host for R. helvetica, and that vertebrate hosts play important roles in the further geographical dispersion of rickettsiae.
PMCID: PMC2743653  PMID: 19732416
14.  Longitudinal Analysis of Tick Densities and Borrelia, Anaplasma, and Ehrlichia Infections of Ixodes ricinus Ticks in Different Habitat Areas in The Netherlands▿  
Applied and Environmental Microbiology  2006;72(12):7594-7601.
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.
PMCID: PMC1694262  PMID: 17028227

Results 1-14 (14)