Questing Ixodes Ricinus Ticks Research Articles

Tick-Borne pathogens and defensin genes expression: A closer look at Ixodes ricinus and Dermacentor reticulatus

The immune system of ticks, along with that of other invertebrates, is comparatively simpler than that of vertebrates, relying solely on innate immune responses. Direct antimicrobial defence is provided by the synthesis of antimicrobial peptides (AMPs), including defensins. The aim of this study was to investigate the differences in defensin genes expression between questing and engorged Ixodes ricinus (def1 and def2) and Dermacentor reticulatus (defDr) ticks, in the presence of selected pathogens: Borrelia spp., Rickettsia spp., Babesia spp., Anaplasma phagocytophilum, and Neoehrlichia mikurensis in the natural environment. After pathogen screening by PCR/qPCR, the expression of defensin genes in pathogen positive ticks and ticks without any of the tested pathogens, was analysed by reverse transcription qPCR. The results showed an increased expression of defensin genes in I. ricinus ticks after blood feeding and I. ricinus and D. reticulatus ticks during in cases of co-infection. In particular, the expression of defensins genes was higher in questing D. reticulatus than in questing and engorged I. ricinus ticks, when borreliae were detected. This study contributes to uncovering the expression patterns of defensin genes in the presence of several known tick pathogens, the occurrence of these pathogens and possible regulatory mechanisms of defensins in tick vector competence.

Pathogens Detected in Questing Ixodes ricinus Ticks in a Mountainous Area in Greece.

Ixodes ricinus ticks are vectors of a plethora of pathogens. The purpose of this study was to screen 398 I. ricinus ticks for a variety of pathogens. Following the pooling, homogenization, and extraction of total nucleic acids, a real-time PCR was applied for the detection of a panel of tick-borne pathogens, while additional conventional PCRs combined with Sanger sequencing were applied for the detection of viruses and typing of Rickettsia and Borrelia species. At least one pathogen was detected in 60 of the 80 (75%) tick pools. Rickettsia spp. predominated, as it was detected in 63.75% of the pools (51/80; MIR 12.81%), followed by Borrelia spp. (35 pools (45%); MIR 8.79%), while Anaplasma phagocytophilum was detected in 2 pools (2.5%, MIR 0.5%). The ticks of six Rickettsia-positive pools were tested individually (from stored half-ticks); all sequences were identical to those of R. monacensis. Similarly, the ticks of six Borrelia-positive pools were tested individually, and it was shown that four belonged to the genospecies Borrelia garinii and two to Borrelia valaisiana. Phleboviruses were detected in 3 pools (3.75%; MIR 0.75%), with sequences clustering in the Ixovirus genus, while nairoviruses were detected in 7 pools (8.75%; MIR 1.76%), with one sequence clustering in the Orthonairovirus genus, and six clustering in the Norwavirus genus. Although a small number of ticks from only one area in Greece were tested, a variety of pathogens together with recently identified viruses were detected, prompting further studies in ticks and surveillance studies in humans.

Features in and around residential gardens affecting the presence and abundance of questing Ixodes ricinus ticks

ABSTRACT People may be exposed to questing Ixodes ricinus ticks in urban settings, e.g. residential gardens. Little is known about the garden characteristics that support a tick population. To determine which features in and around residential gardens support or limit the occurrence and abundance of questing I. ricinus ticks, we sampled them in residential gardens in the Braunschweig region that differed in various intrinsic and extrinsic parameters. We recorded the number of questing nymphal and adult ticks on transects, and by using mixed-effects generalized linear regression models, we related their occurrence and abundance to garden characteristics, meteorological covariates, and landscape features in the vicinity. We detected questing I. ricinus ticks in about 90% of the 103 surveyed gardens. Our occurrence model (marginal R2 = 0.31) predicted the highest probability of questing ticks on transects with hedges or groundcover in gardens, which are located in neighborhoods with large proportions of forest. The abundance of questing ticks was similarly influenced. We conclude that I. ricinus ticks are frequent in residential gardens in Northern Germany and likely associated with intrinsic garden characteristics on a small scale, such as hedges, as well as extrinsic factors on a local scale, such as the proportion of nearby woodland.

The first detection of relapsing fever spirochete Borrelia miyamotoi in Ixodes ricinus ticks from the northeast Czech Republic

Borrelia miyamotoi, a relapsing fever spirochete, is considered a human pathogen. Knowledge of this borrelia is currently limited. Data about its potential impact on public health, circulation in nature, or its occurrence in natural environments are insufficient. For our study, a total of 505 questing Ixodes ricinus ticks (337 nymphs, 85 females and 83 males) from Hradec Králové Region in the Czech Republic were collected. Additionally, 160 winged Lipoptena deer keds from Hradec Králové Region, from Pardubice Region, Czech Republic, and from one location in western Slovakia were collected. The presence of B. miyamotoi in ticks and deer keds was determined using polymerase chain reaction (PCR) targeting a gene encoding glycerophosphodiester phosphodiesterase (glpQ), antigenic protein specific to the relapsing fever spirochetes. Borrelia miyamotoi was identified in six nymphs and four females of I. ricinus ticks. The overall prevalence was 2%. None of the examined Lipoptena specimens were found to be infected. Although no human case of infection with B. miyamotoi has been reported in the Czech Republic yet, this spirochete is widespread in ticks, and therefore the risk of human infection exists.

Questing Ixodes ricinus ticks (Acari, Ixodidae) as a vector of Borrelia burgdorferi sensu lato and Borrelia miyamotoi in an urban area of north-eastern Poland

Green areas located within large cities, as natural ecotypes, are a convenient habitat for ticks and their use as recreational areas is associated with the potential risk of acquiring tick-borne diseases. This study estimated the I. ricinus tick density, prevalence of infection with Borrelia species and the diversity of these bacteria in a green urban area (Olsztyn) of north-eastern Poland, an endemic region of tick-borne diseases. The ticks were collected during spring and autumn of 2015, at sites differing in the degree of human pressure and habitat. Borrelia species detection, typing and a molecular phylogenetic analysis were carried out based on the sequenced flaB gene. The overall mean abundance of I. ricinus was 2.0 ± 1.55 ticks per 100 m2. The density of I. ricinus did not vary significantly between sites. According to semi-qualitative tick abundance categories, the collection sites were classified as ‘very low’ and ‘low’ tick abundance category. The overall infection rate of I. ricinus with Borrelia spirochaetes was 27.4%. The infection rate of adult ticks (42.0%) was three times higher than with nymphs (14.3%). Based on the restriction patterns and sequencing, B. afzelii (93.1%; 27/29), B. valaisiana 3.5% (1/29) and B. miyamotoi (3.5%; 1/29), related to the relapsing fever (RF) spirochaetes, were detected. No co-infections were found. Borrelia miyamotoi, detected for the first time in ticks in the north-eastern urban areas of Poland, was identical to isolates described as European-type. The Borrelia spirochaete infection rate of I. ricinus ticks in an urban area indicated a high risk of LB. Physicians should also be aware of B. miyamotoi infections among patients with a history of tick-bites in north-eastern Poland.

Simultaneous Occurrence of Borrelia miyamotoi, Borrelia burgdorferi Sensu Lato, Anaplasma phagocytophilum and Rickettsia helvetica in Ixodes ricinus Ticks in Urban Foci in Bratislava, Slovakia.

Questing Ixodes ricinus ticks were collected in two urban parks (Železná studienka and Horský park) of the capital city of Slovakia, Bratislava, during two consecutive years in 2011 and 2012. Atotal of 932 ticks were analyzed for the presence of tick-borne agents: B. miyamotoi, B. burgdorferi s.l., A. phagocytophilum and R. helvetica. PCR analysis confirmed the presence of all pathogens at both localities. The overall infection prevalence of B. miyamotoi, B. burgdorferi s.l., A. phagocytophilum and R. helvetica was 0.75, 13.2, 5.6 and 8.9%, respectively. B. burgdorferi s.l. positive samples were represented by six genospecies. The most frequent one was B. afzelii followed by B. garinii and B. valaisiana. Our study confirms the presence of I. ricinus ticks and at least nine tick-borne bacterial agents in city forest parks, which are used for recreational purposes. Ordination analysis revealed significant differences in the composition of pathogens with respect to study site location, time of season and ambient temperature, despite the fact that both sites are located relatively close to one another within the city.

Tick-borne encephalitis virus, Borrelia burgdorferi sensu lato, Borrelia miyamotoi, Anaplasma phagocytophilum and Candidatus Neoehrlichia mikurensis in Ixodes ricinus ticks collected from recreational islands in southern Norway

The aim of this study was to determine the occurrence of tick-borne pathogens of medical importance in questing ticks collected from five recreationally used islands along the Norwegian coastline. Furthermore, since coinfection may affect the disease severity, this study aimed to determine the extent of coinfection in individual ticks or co-localization of tick-borne pathogens. In all, 4158 questing Ixodes ricinus ticks were analyzed. For detection of tick-borne encephalitis virus (TBEV), nymphs (3690) were analyzed in pools of ten. To detect Borrelia burgdorferi sensu lato, B. miyamotoi, Anaplasma phagocytophilum and Candidatus Neoehrlichia mikurensis, 468 nymphs were analyzed individually. A total of five nymph pools was infected with TBEV, giving an overall prevalence of 0.14%. In the individually analyzed ticks, B. burgdorferi s. l. (15.6%), Candidatus N. mikurensis (11%), A. phagocytophilum (1.4%) and B. miyamotoi (0.9%) were detected. Coinfection was found in 3.3% of the ticks, and the only dual infection observed was with B. afzelii and Candidatus N. mikurensis. This association was significantly higher than what would occur by random chance.

Molecular evidence for Toxoplasma gondii in feeding and questing Ixodes ricinus ticks

The aim of the present study was to detect Toxoplasma gondii in ticks collected from ponies and field vegetation and to determine the role of Shetland ponies as a potential reservoir host for T. gondii. A total of 1737 feeding Ixodes ricinus collected from 49 horses and 371 questing ticks were tested by PCR and sequencing for the presence and genotyping of T. gondii. All ticks were examined in a previous study to detect and identify pathogenic bacterial species. The aim of this study was also to detect co-infection of ticks with these bacteria and T. gondii. Genotyping of the sequenced B1 gene revealed that detected T. gondii strains represented genotype I, which is pathogenic for humans. T. gondii genotype I was detected in 4.5% of all I. ricinus, including in 2.99% of feeding ticks and in 10.24% of questing ticks; this difference was statistically significant. Thus, the above results indicate that ponies probably are not an essential host for the detected sporozoan. Infections with more than one pathogenic species were rare and involved mostly T. gondii and Borrelia burgdorferi sensu lato. Our results confirmed the presence of T. gondii in I. ricinus and showed a new geographical habitat of T. gondii occurring in I. ricinus ticks in Poland.

Infections and mixed infections with the selected species of Borrelia burgdorferi sensu lato complex in Ixodes ricinus ticks collected in eastern Poland: a significant increase in the course of 5 years.

In the years 2008–2009 and 2013–2014, 1620 and 1500 questing Ixodes ricinus ticks, respectively, were examined on the territory of the Lublin province (eastern Poland). The presence of three pathogenic species causing Lyme disease was investigated: Borrelia burgdorferi sensu stricto, B. afzelii and B. garinii. The proportion of I. ricinus ticks infected with B. burgdorferi sensu lato showed a highly significant increase between 2008–2009 and 2013–2014, from 6.0 to 15.3 %. A significant increase was noted with regard to all types of infections with individual species: single (4.7–7.8 %), dual (1.2–6.6 %), and triple (0.1–0.9 %). When expressed as the percent of all infections, the frequency of mixed infections increased from 21.4 to 49.2 %. Statistical analysis performed with two methods (by calculating of odds ratios and by Fisher’s exact test) showed that the frequencies of mixed infections in most cases proved to be significantly greater than expected. The strongest associations were found between B. burgdorferi s. s. and B. afzelii, and between B. burgdorferi s. s. and B. garinii. They appeared to be highly significant (P < 0.0001) when assessed by two methods for 2013–2014, and for the sum of findings for both time periods. The proportions of the individual species detected in the mixed infections in 2008–2009 and 2013–2014 revealed highly significant increases for B. burgdorferi s. s. and B. garinii (from 33.9 to 71.1 % and from 18.2 to 82.9 %, respectively), and an insignificant decrease for B. afzelii (from 51.4 to 41.6 %). The proportions of the species B. burgdorferi s. s., B. afzelii and B. garinii (with combined single and mixed infections) for 2008–2009 and 2013–2014 were: 51.2/44.0 %, 30.6/24.9 % and 18.2/31.1 %, respectively. In conclusion, our results seem to indicate the detrimental trend of the increasing infection rate of I. ricinus ticks with B. burgdorferi s. l. in eastern Poland, and dramatic enhancement of mixed infections with individual species, which may result in mixed infections of humans and exacerbation of the clinical course of Lyme disease cases on the studied area.

Risk of exposure to ticks (Ixodidae) and the prevalence of tick-borne encephalitis virus (TBEV) in ticks in Southern Poland

The article presents the results of the first study on seasonal activity of ticks and prevalence of tick-borne encephalitis virus (TBEV) in nymphs from the Silesian Province (Southern Poland). Previous studies on the prevalence of TBEV in ticks in Poland have been conducted mostly in northern and eastern regions, but none in the Silesian Province itself. The aims of this study were to analyse the seasonal variation in tick populations and compare TBEV prevalence in nymphs from different geographical locations in the Silesia. A total of 5160 questing Ixodes ricinus ticks were collected by the flagging method from 23 localities in southern Poland in 2010. Micro-climatic parameters (air temperature and humidity) were measured in order to estimate their influence on tick population. The highest tick activity was recorded in spring and was positively correlated with relative air humidity (RH). TBEV in the Silesian Province was analysed in 1750 nymphs and an overall prevalence was 0.11% (2 pools out of 175 analysed). The results of this study show that TBEV pool prevalence in nymphs is low in accordance with the low number of TBE cases reported within the region.

Occurrence of Francisella spp. in Dermacentor reticulatus and Ixodes ricinus ticks collected in eastern Poland

A total of 530 questing Dermacentor reticulatus ticks and 861 questing Ixodes ricinus ticks were collected from Lublin province (eastern Poland) and examined for the presence of Francisella by PCR for 16S rRNA (rrs) and tul4 genes. Only one female D. reticulatus tick out of 530 examined (0.2%) was infected with Francisella tularensis subspecies holarctica, as determined by PCR of the rrs gene. None of 861 I. ricinus ticks were infected with F. tularensis. In contrast, the presence of Francisella-like endosymbionts (FLEs) was detected in more than half of the D. reticulatus ticks (50.4%) and 0.8% of the I. ricinus ticks. The nucleotide sequences of the FLEs detected in D. reticulatus exhibited 100% homology with the nucleotide sequence of the FLE strain FDrH detected in Hungary in D. reticulatus. In conclusion, our results suggest a low contribution of D. reticulatus and I. ricinus ticks to the circulation of F. tularensis in eastern Poland. This finding, however, needs to be confirmed by further studies in other areas. Our study confirmed the common infection of D. reticulatus with Francisella-like endosymbionts (FLEs) of unknown pathogenic potential and revealed, for the first time, a low grade of infection of I. ricinus with FLEs.

Detection of TBEV RNA in Ixodes ricinus ticks in north-eastern Poland.

TBEV (Flaviviridae, Flavivirus) is an etiologic agent of tick-borne encephalitis (TBE), the most important arboviral human infection involving the central nervous system. The disease is endemic in a zone extending from central and eastern Europe to Siberia and Japan, and corresponds to the distribution of the ixodid ticks, which act both as the vectors and reservoir of TBEV. Humans acquire infection mainly by the bite of an infected tick. A continuous increase of TBE cases throughout Europe has been observed over a period of 30 years. The objective of this study was a preliminary determination of the infection level of ticks collected in North-Eastern Poland, the endemic area of TBE. Questing Ixodes ricinus ticks (adults, nymphs and larvae) were collected by flagging the lower vegetation in 55 locations in Poland in 2006-2009. A total of 2075 ticks (676 females, 555 males, 799 nymphs and 45 larvae) were collected and examined for the presence of RNA TBEV by nested RT-PCR. The average number of ticks in one pool was 6.98. The minimum infection rate of ticks with TBEV was estimated in total area as 0.96%. TBEV RNA was detected in all of the investigated developmental stages of ticks. The prevalence of viral infection in ticks is a useful indicator of TBE virus circulation and may be used for risk assessment of the degree of natural focus activity and of the risk to contact TBE in a particular natural habitat.

Prevalence of Rickettsiales (Anaplasma phagocytophilum and Rickettsia spp.) in hard ticks (Ixodes ricinus) in the city of Hamburg, Germany.

To narrow the gap of missing knowledge on Rickettsia spp. and Anaplasma phagocytophilum infections in ticks in northwestern Germany and, at the same time, to provide first prevalence data on these pathogens in the city of Hamburg, a total of 1,400 questing Ixodes ricinus ticks were collected at ten different public green areas from April until October 2011. Ticks were examined using probe-based quantitative real-time PCR. A percentage of 3.6% (51/1,400) ticks were tested positive for A. phagocytophilum infections divided into 2.1% (3/141) adults [1.7% (1/60) females and 2.5% (2/81) males] and 3.8% (48/1,259) nymphs. The percentage of infected ticks per sampling site varied statistically significantly from 0.7% (1/140) to 12.1% (17/140), whereas between sampling months, no statistically significant differences were observed (2.0-6.5%, 4-13/140). The overall Rickettsia spp. infection rate was 52.5% (735/1,400). In adult ticks, Rickettsia spp. infection rate was 56% (79/141) divided into 61.7% (37/60) infected females and 51.9% (42/81) infected males. Nymphs showed an infection rate of 52.1% (656/1,259). In contrast to A. phagocytophilum infections, no statistically significant differences in Rickettsia spp. infection rates among sampling sites (44.3-63.6%, 62-89/140) were observed, whereas seasonal variations were obvious: the percentage of Rickettsia-positive ticks was significantly lower in April (36.5%, 73/200) and May (29.5%, 59/200) compared to the summer and fall months (55.0-64.5%, 110-129/200). Rickettsia species differentiation via real-time pyrosequencing revealed Rickettsia helvetica as the only occurring species. Co-infections with both Rickettsia spp. and A. phagocytophilum were detected in 2.0% (28/1,400) of the ticks. The present study revealed that in the city of Hamburg, the tick infection rate with A. phagocytophilum is comparable with other German data, whereas the Rickettsia spp. infection rate of 52.5% is by far the highest prevalence detected in Germany so far. As the city of Hamburg has 1.8 million inhabitants and attracts millions of tourists every year, the potential health risk should not be underestimated.

Cat Scratch Disease Caused by Bartonella grahamii in an Immunocompromised Patient

Bartonella grahamii colonizes rodents worldwide and has been detected in questing Ixodes ricinus ticks. Here, the first human B. grahamii infection confirmed by multilocus sequence typing is reported. The route of transmission and clinical picture of the patient are similar to those seen in patients with cat scratch disease, which is typically diagnosed as a Bartonella henselae infection.

Prevalence of Tick-Borne Pathogens in Ixodes ricinus and Dermacentor reticulatus Ticks from Different Geographical Locations in Belarus

Worldwide, ticks are important vectors of human and animal pathogens. Besides Lyme Borreliosis, a variety of other bacterial and protozoal tick-borne infections are of medical interest in Europe. In this study, 553 questing and feeding Ixodes ricinus (n = 327) and Dermacentor reticulatus ticks (n = 226) were analysed by PCR for Borrelia, Rickettsia, Anaplasma, Coxiella, Francisella and Babesia species. Overall, the pathogen prevalence in ticks was 30.6% for I. ricinus and 45.6% for D. reticulatus. The majority of infections were caused by members of the spotted-fever group rickettsiae (24.4%), 9.4% of ticks were positive for Borrelia burgdorferi sensu lato, with Borrelia afzelii being the most frequently detected species (40.4%). Pathogens with low prevalence rates in ticks were Anaplasma phagocytophilum (2.2%), Coxiella burnetii (0.9%), Francisella tularensis subspecies (0.7%), Bartonella henselae (0.7%), Babesia microti (0.5%) and Babesia venatorum (0.4%). On a regional level, hotspots of pathogens were identified for A. phagocytophilum (12.5–17.2%), F. tularensis ssp. (5.5%) and C. burnetii (9.1%), suggesting established zoonotic cycles of these pathogens at least at these sites. Our survey revealed a high burden of tick-borne pathogens in questing and feeding I. ricinus and D. reticulatus ticks collected in different regions in Belarus, indicating a potential risk for humans and animals. Identified hotspots of infected ticks should be included in future surveillance studies, especially when F. tularensis ssp. and C. burnetii are involved.

Anaplasma phagocytophilum in Questing Ixodes ricinus Ticks: Comparison of Prevalences and Partial 16S rRNA Gene Variants in Urban, Pasture, and Natural Habitats

Urban, natural, and pasture areas were investigated for prevalences and 16S rRNA gene variants of Anaplasma phagocytophilum in questing Ixodes ricinus ticks. The prevalences differed significantly between habitat types, and year-to-year variations in prevalence and habitat-dependent occurrence of 16S rRNA gene variants were detected.

Infections and Coinfections of Questing Ixodes ricinus Ticks by Emerging Zoonotic Pathogens in Western Switzerland

In Europe, Ixodes ricinus is the vector of many pathogens of medical and veterinary relevance, among them Borrelia burgdorferi sensu lato and tick-borne encephalitis virus, which have been the subject of numerous investigations. Less is known about the occurrence of emerging tick-borne pathogens like Rickettsia spp., Babesia spp., "Candidatus Neoehrlichia mikurensis," and Anaplasma phagocytophilum in questing ticks. In this study, questing nymph and adult I. ricinus ticks were collected at 11 sites located in Western Switzerland. A total of 1,476 ticks were analyzed individually for the simultaneous presence of B. burgdorferi sensu lato, Rickettsia spp., Babesia spp., "Candidatus Neoehrlichia mikurensis," and A. phagocytophilum. B. burgdorferi sensu lato, Rickettsia spp., and "Candidatus Neoehrlichia mikurensis" were detected in ticks at all sites with global prevalences of 22.5%, 10.2%, and 6.4%, respectively. Babesia- and A. phagocytophilum-infected ticks showed a more restricted geographic distribution, and their prevalences were lower (1.9% and 1.5%, respectively). Species rarely reported in Switzerland, like Borrelia spielmanii, Borrelia lusitaniae, and Rickettsia monacensis, were identified. Infections with more than one pathogenic species, involving mostly Borrelia spp. and Rickettsia helvetica, were detected in 19.6% of infected ticks. Globally, 34.2% of ticks were infected with at least one pathogen. The diversity of tick-borne pathogens detected in I. ricinus in this study and the frequency of coinfections underline the need to take them seriously into consideration when evaluating the risks of infection following a tick bite.

Prevalence of Neoehrlichia mikurensis in ticks and rodents from North-west Europe

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.

Absence of zoonotic Bartonella species in questing ticks: First detection of Bartonella clarridgeiae and Rickettsia felis in cat fleas in the Netherlands

BackgroundAwareness 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.ResultsThe 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.ConclusionsWe 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.

Ixodes ricinus ticks (Acari: Ixodidae): vectors for Lyme disease spirochetes in Romania

In this study 1,868 questing Ixodes ricinus ticks (nymphs and adults), collected in six sites from three counties--Giurgiu, Sibiu, and Tulcea--in Romania, were examined by polymerase chain reaction (PCR) followed by reverse line blot (RLB) for detection of Borrelia burgdorferi sensu lato presence. The bacteria were found in 18% of the investigated ticks. The prevalence of infection did not differ significantly between nymphs (19.1%) and adults (15.4%). Three B. burgdorferi s.l. genospecies were detected: B. afzelii (61.1%), B. garinii (31.2%), and B. valaisiana (7.7%). No mixed infections were detected. The highest infection prevalence in nymphs was detected at Cristian (Sibiu County)--22.0%, whereas in adults it was at Comana (Giurgiu County)--19.8%. This preliminary study provides evidence that Lyme disease spirochetes are present in various regions of Romania, and at a relatively high prevalence in their vectors, thus posing a risk of infection to human subjects in the areas infested by ticks.