No molecular detection of tick-borne pathogens in the blood of patients with erythema migrans in Belgium.

BACKGROUND: A number of tick-borne pathogens circulate in the Belgian tick population in addition to the causative agent of Lyme borreliosis. However, so far, only a few patients with tick-borne diseases other than Lyme borreliosis have been reported in Belgium. The aim of this study was to investigate the occurrence of other human tick-borne infections in Belgium and their possible clinical manifestation. METHODS: Patients with fever (> 37.5 °C) after a tick bite or those with erythema migrans (EM) were included in the study. EDTA-blood samples were screened for the presence of DNA from Borrelia burgdorferi sensu lato, Borrelia miyamotoi, Anaplasma phagocytophilum, Neoehrlichia mikurensis, spotted fever group rickettsiae (genus Rickettsia), Babesia spp., Bartonella spp., Spiroplasma ixodetis and tick-borne encephalitis virus, using multiplex PCR methods. A questionnaire on, among others, demographics and clinical symptoms, was also filled in. RESULTS: Over a period of 3 years, 119 patients with EM and 14 patients with fever after a recent tick bite were enrolled in the study. Three samples initially tested positive for N. mikurensis by quantitative PCR (qPCR), but the results could not be confirmed by other PCR methods, and repetition of the DNA extraction procedure and qPCR test was not successful. The qPCR test results for the other tick-borne pathogens were negative. CONCLUSIONS: In general, only a few patients with fever after a tick bite could be identified. Although no tick-borne pathogens were detected, their occurrence cannot be excluded based on the limited number of patients and the limitations inherent to current methodologies. This study underscores the possibility of false-positive PCR results and the necessity for the development of multiple independent tools for the sensitive and specific detection of emerging tick-borne pathogens.

Circulation of Babesia Species and Their Exposure to Humans through Ixodes Ricinus.

Human babesiosis in Europe has been attributed to infection with Babesia divergens and, to a lesser extent, with Babesia venatorum and Babesia microti, which are all transmitted to humans through a bite of Ixodes ricinus. These Babesia species circulate in the Netherlands, but autochthonous human babesiosis cases have not been reported so far. To gain more insight into the natural sources of these Babesia species, their presence in reservoir hosts and in I. ricinus was examined. Moreover, part of the ticks were tested for co-infections with other tick borne pathogens. In a cross-sectional study, qPCR-detection was used to determine the presence of Babesia species in 4611 tissue samples from 27 mammalian species and 13 bird species. Reverse line blotting (RLB) and qPCR detection of Babesia species were used to test 25,849 questing I. ricinus. Fragments of the 18S rDNA and cytochrome c oxidase subunit I (COI) gene from PCR-positive isolates were sequenced for confirmation and species identification and species-specific PCR reactions were performed on samples with suspected mixed infections. Babesia microti was found in two widespread rodent species: Myodes glareolus and Apodemus sylvaticus, whereas B. divergens was detected in the geographically restricted Cervus elaphus and Bison bonasus, and occasionally in free-ranging Ovis aries. B. venatorum was detected in the ubiquitous Capreolus capreolus, and occasionally in free-ranging O. aries. Species-specific PCR revealed co-infections in C. capreolus and C. elaphus, resulting in higher prevalence of B. venatorum and B. divergens than disclosed by qPCR detection, followed by 18S rDNA and COI sequencing. The non-zoonotic Babesia species found were Babesia capreoli, Babesia vulpes, Babesia sp. deer clade, and badger-associated Babesia species. The infection rate of zoonotic Babesia species in questing I. ricinus ticks was higher for Babesia clade I (2.6%) than Babesia clade X (1.9%). Co-infection of B. microti with Borrelia burgdorferi sensu lato and Neoehrlichia mikurensis in questing nymphs occurred more than expected, which reflects their mutual reservoir hosts, and suggests the possibility of co-transmission of these three pathogens to humans during a tick bite. The ubiquitous spread and abundance of B. microti and B. venatorum in their reservoir hosts and questing ticks imply some level of human exposure through tick bites. The restricted distribution of the wild reservoir hosts for B. divergens and its low infection rate in ticks might contribute to the absence of reported autochthonous cases of human babesiosis in the Netherlands.

Detection of pathogens in Dermacentor reticulatus in northwestern Europe: evaluation of a high-throughput array.

BACKGROUND: The geographic distribution of Dermacentor reticulatus is expanding in Europe. Surveillance of this tick species and its pathogens is desirable, as it transmits pathogens of public and veterinary importance. A high-throughput real-time PCR-based array was used to screen 1.741 D. reticulatus ticks from Belgium, Germany, The Netherlands, and Great Britain for the presence of 28 tick-borne bacteria and twelve protozoan parasites. The presence of pathogen DNA was confirmed by conventional PCR followed by sequencing. RESULTS: The array detected the presence of DNA from Borrelia spp. (7%), B. afzelii (0.1%), B. garinii (0.1%), B. spielmanii (0.1%), B. miyamotoi (0.2%), Anaplasma marginale (0.1%), A. phagocytophilum (0.1%), Ehrlichia canis (2%), Rickettsia helvetica (0.2%), spotted fever group Rickettsia (9.6%), Francisella tularensis or Francisella-like endosymbionts (95%), Coxiella burnettii (0.1%), Babesia divergens (0.2%), B. canis (0.9%) B. vogeli (5.6%), and Theileria equi (0.1%). Only the presence of B. canis and spotted fever group Rickettsia could be confirmed by conventional PCR and sequencing. The spotted fever Rickettsia-positive samples were all identified as R. raoultii. CONCLUSIONS: We successfully detected and determined the prevalence of B. canis and R. raoultii in D. reticulatus. An high-throughput array that allows fast and comprehensive testing of tick-borne pathogens is advantageous for surveillance and future epidemiological studies. The importance of thorough validation of real-time PCR-based assays and careful interpretation is evident.

Road-killed mammals provide insight into tick-borne bacterial pathogen communities within urban habitats.

Small- and medium-sized mammals play an important role in the life cycle of tick-borne pathogens in urban habitats. Our aim was to apply the general protocol, DAMA (documentation-assessment-monitoring-action), which is an integrated proposal to build a proactive capacity to understand, anticipate, and respond to the outcomes of accelerating environmental change. Here we tested whether road-killed carcasses in urban areas are useful sources of tissue and parasite samples to investigate these species' contribution to the epidemiology of vector-borne diseases. We collected 29 road-killed and 6 carcasses with different causes of mortality (23 northern white-breasted hedgehogs and 12 from seven other mammal species) mainly from Budapest, Hungary. We used quantitative and conventional PCRs to determine pathogens in 90 collected tissues (52 from hedgehogs; 38 from other species) and 417 ticks that were only found on hedgehogs. Tissue samples revealed a wide range of bacteria including human zoonotic pathogens identified as Anaplasma phagocytophilum ecotype I, Borrelia afzelii, B. spielmanii, Borrelia miyamotoi, Rickettsia helvetica, and Bartonella species. Among the 23 collected hedgehog carcasses, 17 (74%) were infected with A. phagocytophilum, 6 (26%) with Borrelia burgdorferi s.l., 12 (52%) with R. helvetica, and 15 (65%) with Rickettsia sp. Furthermore, we report the first detection of Rickettsia sp. infection in European moles and lesser weasel and R. helvetica in stone marten. Through sequencing B. afzelii, R. helvetica, R. monacensis and A. phagocytophilum ecotype I were identified in the ticks removed from the carcasses. We showed that road-killed urban mammal species are exposed to multiple tick-borne pathogens but further studies have to clarify whether they, in fact, also have a role in their maintenance and spread. Our study also demonstrates that roadkill can be used in the risk assessment of potential human infection and in the implementation of the DAMA protocol.

Pathogen communities of songbird-derived ticks in Europe's low countries.

BACKGROUND: Birds play a major role in the maintenance of enzootic cycles of pathogens transmitted by ticks. Due to their mobility, they affect the spatial distribution and abundance of both ticks and pathogens. In the present study, we aim to identify members of a pathogen community [Borrelia burgdorferi (s.l.), B. miyamotoi, 'Ca. Neoehrlichia mikurensis', Anaplasma phagocytophilum and Rickettsia helvetica] in songbird-derived ticks from 11 locations in the Netherlands and Belgium (2012-2014). RESULTS: Overall, 375 infested songbird individuals were captured, belonging to 35 species. Thrushes (Turdus iliacus, T. merula and T. philomelos) were trapped most often and had the highest mean infestation intensity for both Ixodes ricinus and I. frontalis. Of the 671 bird-derived ticks, 51% contained DNA of at least one pathogenic agent and 13% showed co-infections with two or more pathogens. Borrelia burgdorferi (s.l.) DNA was found in 34% of the ticks of which majority belong to so-called avian Borrelia species (distribution in Borrelia-infected ticks: 47% B. garinii, 34% B. valaisiana, 3% B. turdi), but also the mammal-associated B. afzelii (16%) was detected. The occurrence of B. miyamotoi was low (1%). Prevalence of R. helvetica in ticks was high (22%), while A. phagocytophilum and 'Ca. N. mikurensis' prevalences were 5% and 4%, respectively. The occurrence of B. burgdorferi (s.l.) was positively correlated with the occurrence of 'Ca. N. mikurensis', reflecting variation in susceptibility among birds and/or suggesting transmission facilitation due to interactions between pathogens. CONCLUSIONS: Our findings highlight the contribution of European songbirds to co-infections in tick individuals and consequently to the exposure of humans to multiple pathogens during a tick bite. Although poorly studied, exposure to and possibly also infection with multiple tick-borne pathogens in humans seems to be the rule rather than the exception.

Predicting the risk of Lyme borreliosis after a tick bite, using a structural equation model.

BACKGROUND: Understanding and quantification of the risk of Lyme borreliosis after a tick bite can aid development of prevention strategies against Lyme borreliosis. METHODS: We used 3,525 single tick bite reports from three large prospective studies on the transmission risk of tick-borne pathogens to humans, with 50 reports of Lyme borreliosis during the follow-up period, among 1,973 reports with known outcome. A structural equation model was applied to estimate the risk of Lyme borreliosis after a tick bite, and quantify the influence of: developmental stage of the tick, detection of Borrelia burgdorferi s.l. DNA in the tick by PCR, tick engorgement, patient-estimated duration of tick attachment, and patient age. RESULTS: The overall risk of developing Lyme borreliosis after a tick bite was 2.6% (95%CI 1.4-5.1). The risk increased with: - Tick engorgement: 1.4% (95%CI 0.7%-2.3%) for low engorgement to 5.5% (95%CI 2.8%-9.2%) for substantially engorged ticks;- Rising patient-estimated tick attachment duration: 2.0% (95%CI 1.3%-2.8%) after <12 hours, to 5.2% (95%CI 3.0%-8.9%) after ≥4 days;- Detection of Borrelia burgdorferi s.l. DNA in ticks: 6.7% (95%CI 3.6%-13.5%), versus 1.4% (95%CI 0.7%-2.9%) when ticks tested negative.The highest observed risk of Lyme borreliosis was 14.4% (95%CI 6.8%-24.6%) after one tick bite of a substantially engorged tick that tested positive for Borrelia burgdorferi s.l. DNA, which corresponds to one new case of Lyme borreliosis per 7 (95%CI 4-15) of such tick bites. CONCLUSIONS: An individual's risk of Lyme borreliosis after a tick bite can be predicted with tick engorgement, patient-estimated duration of tick attachment, and detection of Borrelia burgdorferi s.l. DNA in the tick.

New foci of Haemaphysalis punctata and Dermacentor reticulatus in the Netherlands.

In 2014 Haemaphysalis punctata was found in several locations on the mainland of the Netherlands for the first time since 1897. In the same areas Dermacentor reticulatus and Ixodes ricinus were found. Haemaphysalis punctata and D. reticulatus were tested for presence of Babesia spp. and Rickettsia spp. by PCR. Babesia spp. and spotted fever Rickettsiae were not detected in any of the collected H. punctata, while several D. reticulatus (6%) collected from the same areas were found to be positive for R. raoultii, a causative agent of tick-borne lymphadenopathy. We discuss the role of free-ranging domestic animals in maintaining H. punctata and D. reticulatus populations in dune areas in the Netherlands.

Prevalence and diversity of human pathogenic rickettsiae in urban versus rural habitats, Hungary.

Tick-borne rickettsioses belong to the important emerging infectious diseases worldwide. We investigated the potential human exposure to rickettsiae by determining their presence in questing ticks collected in an urban park of Budapest and a popular hunting and recreational forest area in southern Hungary. Differences were found in the infectious risk between the two habitats. Rickettsia monacensis and Rickettsia helvetica were identified with sequencing in questing Ixodes ricinus, the only ticks species collected in the city park. Female I. ricinus had a particularly high prevalence of R. helvetica (45%). Tick community was more diverse in the rural habitat with Dermacentor reticulatus ticks having especially high percentage (58%) of Rickettsia raoultii infection. We conclude that despite the distinct eco-epidemiological traits, the risk (hazard and exposure) of acquiring human pathogenic rickettsial infections in both the urban and the rural study sites exists.

Extended spectrum ß-lactamase- and constitutively AmpC-producing Enterobacteriaceae on fresh produce and in the agricultural environment.

The attribution of fresh produce to the overall community-associated exposure of humans to ESBL- or AmpC-producing bacteria is currently unknown. To address this issue, the prevalence of ESBL- and AmpC-producing Enterobacteriaceae on fresh produce produced in the Netherlands was determined. Seven vegetable types that are consumed raw were selected: blanched celery, bunched carrots, chicory, endive, iceberg lettuce, mushrooms, and radish. The vegetables were mostly obtained from supermarkets. To determine whether the agricultural environment is the source of ESBL-producing Enterobacteriaceae on fresh produce, iceberg lettuce was also obtained directly from three farms, in conjunction with soil and irrigation water. ESBL-producing Enterobacteriaceae isolated from vegetables and environment were all environmental species: Rahnella aquatilis (n = 119), Serratia fonticola (n = 45) and Pantoea agglomerans (n = 1). ESBL genes of R. aquatilis and S. fonticola were identified as blaRAHN-1 and blaRAHN-2 and blaFONA-1, blaFONA-2, blaFONA-3/6 and blaFONA-5, respectively. For R. aquatilis and S. fonticola, different prevalence numbers were observed using different isolation methods, which could at least partially be explained by an inverse correlation between the level of cefotaxime resistance of these species and incubation temperature. R. aquatilis was isolated from 0 to 46% of soil samples and 11 to 83% of vegetable samples, and S. fonticola from 2 to 60% of soil samples and 0 to 1.3% of vegetable samples. Third generation cephalosporin-resistant faecal Enterobacteriaceae were isolated from 2.7%, 1.3% and 1.1% of supermarket vegetables, iceberg lettuce from farms, and agricultural soil respectively. Faecal Enterobacteriaceae were all identified as Citrobacter and Enterobacter species and, with the exception of one Citrobacter koseri strain, all had phenotypes indicative of constitutive AmpC production. Comparison of fresh produce and its agricultural environment indicates that the Enterobacteriaceae population on fresh produce reflects that of the soil it is grown in. Public health risks associated with exposure to ESBL- and AmpC-producing bacteria through consumption of uncooked fresh produce are diverse. They range from occasional ingestion of 3GC-resistant opportunistic pathogens which may result in difficult-to-treat infections, to frequent ingestion of relatively harmless ESBL-producing environmental bacteria that may therewith constitute a continuously replenished intestinal reservoir facilitating dissemination of ESBL genes to (opportunistic) pathogens.

Sensitivity of a point of care tick-test for the development of Lyme borreliosis.

BACKGROUND: 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. FINDINGS: 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%). CONCLUSIONS: 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.