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Leishmania spp. are sand fly-borne protozoan parasites causing leishmaniasis in humans and animals. The aim of the study was to analyse the epidemiology of leishmaniasis in Turkey, Azerbaijan and Georgia from 2005 to 2020 and evaluate the associated risk for disease emergence in European countries. It is based on an analysis of WHO and OIE reported cases between 2005 and 2020, a review of scientific articles published in SCOPUS between 2009 and 2020 and a questionnaire survey to public health and veterinary authorities in these countries. Endemic Leishmania spp. include L. infantum in the three countries, L. major in Azerbaijan and Turkey and L. tropica and L. donovani in Turkey. Leishmaniasis is reported in humans, animals and sand flies and incidence is spatially and temporarily variable. In the southern Caucasus and particularly in Georgia, reported incidence of human visceral leishmaniasis by L. infantum remains high. However, whilst Georgia experienced a gradual decrease from >4.0 cases per 100,000 population in 2005-09 to 1.13 cases per 100,000 population in 2020, the period with highest incidence in Azerbaijan, which ranged between 0.40 and 0.61 cases per 100,000 population, was 2016-2019, and no cases have so far been reported for 2020. Visceral leishmaniasis in the Southern Caucasus affects mostly young children from deprived urban areas and its closely associated to canine leishmaniasis. Turkey reported cases of visceral leishmaniasis between 2005 and 2012 and in 2016 only, and incidence ranged between 0.02 and 0.05 per 100,000 population. In contrast, the reported annual incidence of cutaneous leishmaniasis in Turkey was much greater and peaked at 7.02 cases per 100,000 population in 2013, associated to imported cases from cutaneous leishmaniasis endemic Syria. Leishmaniasis by L. infantum in Azerbaijan and Georgia represents a regional public and animal health challenge that requires support to improve diagnosis, treatment and control. The unprecedented rise of cutaneous leishmaniasis and the spread of L. tropica and L. donovani in Turkey is an important risk factor for their emergence in Europe, especially in Mediterranean countries where competent vectors are widespread., European Centre for Disease Prevention and Control (ECDC) [NP/2020/DPR/11745], This manuscript was commissioned by the European Centre for Disease Prevention and Control (ECDC) under the negotiated procedure reference NP/2020/DPR/11745.

The aim of this study was to determine the prevalence of hemoplasma species in cats by real-time PCR and to determine the distribution of the species. Furthermore, it was aimed to evaluate factors that are thought to be important in the epidemiology of the disease in cats statistically. For this purpose, blood samples from 246 cats were examined for Candidatus Mycoplasma haemominitum (CMhm), Mycoplasma haemofelis (Mhf) and Candidatus Mycoplasma turicensis (CMt) by real-time PCR. CMhm was detected in 20 (8.13%), CMt was detected in 3 (1.22%), and Mhf was found in 2 (0.81%) of 246 cats. At least one of the hemoplasma species was detected in 21 (8.54%) of 246 cats, and two different hemoplasma species were detected in 4 (19.05%) of 21 cats diagnosed with hemoplasma. The relationship between PCR positivity, the contact of cats with other cats (p = 0.02) and the detection of the intraoral wound (p = 0.001) was found to be statistically significant. The statistical significance of contact with other cats in the formation of the disease was revealed. The strong relationship between the presence of intraoral lesions and hemoplasma infection was revealed for the first time. Studies involving epidemiological data and their relationship with clinical findings should be continued. Scientific Research Project Coordination Unit of Istanbul University-Cerrahpasa [TSA-2017-25305] This study was supported by the Scientific Research Project Coordination Unit of Istanbul University-Cerrahpasa (Project code: TSA-2017-25305). The authors would like to thank Dr. Severine Tasker from Bristol Veterinary School, University of Bristol, for the positive controls.

Domestic felines' re-emerging infectious and neglected zoonotic diseases are a significant focus of global "One Health" efforts. This study aimed to rapidly diagnose 14 pathogens, including zoonoses by using PCR primers in 167 client-owned symptomatic cats, routinely accepted to the Veterinary Clinics of Tekirdag. The prevalence of pathogens investigated were as follows: Babesia canis canis (24%), Babesia microti (2.4%), Hepatozoon felis (10.8%), Cytauxzoon felis (6.6%), Bartonella henselae (40.1%), Anaplasma platys (30.5%), Anaplasma phagocytophilum (7.2%), Rickettsia felis (26.3%), Borrelia burgdorferi (21%), and hemotropic Mycoplasma sp. (11.4%). There was a significant difference between the prevalence of the pathogens (?2 = 152.26, df = 9, p &lt, 0.001). There was also a statistical difference between the gender of the cats in terms of the prevalence of all pathogens considered together (?2 = 4.80, df = 1, p = 0.028), where the female cats showed a higher prevalence. This was not the case for the different age groups (?2 = 2.92, df = 1, p=0.088). The lowest infection was observed for B. microti (p &lt, 0.001), while the highest infection was observed for B. henselae (p &lt, 0.01). Leishmania donovani, Plasmodium spp., Ehrlichia chaffeensis, and Neoehrlichia mikurensis PCR test results were negative in all samples. In conclusion, house cats of Tekirdag are apparently highly susceptible to some neglected zoonoses important for "One Health," and their prevalence in the region is most probably underestimated. Hence, applying PCR tests to assist fast clinic diagnosis in routine, may be an efficient option to protect the public as well as the cats from severe diseases.

How multitrophic relationships between wildlife communities and their ectoparasitic vectors interact to shape the diversity of vector-borne microorganisms is poorly understood. Nested levels of dependence among microbes, vectors, and vertebrate hosts may have complicated effects on both microbial community assembly and evolution. We examined Bartonella sequences from European bats and their ectoparasites with a combination of network analysis, Bayesian phylogenetics, tip-association and cophylogeny tests, and linear regression to understand the ecological and evolutionary processes that shape parasite communities. We detected seven bat–ectoparasite–Bartonella communities that can be differentiated based on bat families and roosting patterns. Tips of the Bartonella tree were significantly clustered by host taxonomy and geography. We also found significant evidence of evolutionary congruence between bat host and Bartonella phylogenies, indicating that bacterial species have evolved to infect related bat species. Exploring these ecological and evolutionary associations further, we found that sharing of Bartonella species among bat hosts was strongly associated with host phylogenetic distance and roost sharing and less strongly with geographic range overlap. Ectoparasite sharing between hosts was strongly predicted by host phylogenetic distance, roost sharing, and geographic overlap but had no additive effect on Bartonella sharing. Finally, historical Bartonella host-switching was more frequent for closely related bats after accounting for sampling bias among bat species. This study helps to disentangle the complex ecology and evolution of Bartonella bacteria in bat species and their arthropod vectors. Our work provides insight into the important mechanisms that partition parasite communities among hosts, particularly the effect of host phylogeny and roost sharing, and could help to elucidate the evolutionary patterns of other diverse vector-borne microorganisms.

15 pages; International audience; How food webs are structured has major implications for their stability and dynamics. While poorly studied to date, arctic food webs are commonly assumed to be simple in structure, with few links per species. If this is the case, then different parts of the web may be weakly connected to each other, with populations and species united by only a low number of links. We provide the first highly resolved description of trophic link structure for a large part of a high-arctic food web. For this purpose, we apply a combination of recent techniques to describing the links between three predator guilds (insectivorous birds, spiders, and lepidopteran parasitoids) and their two dominant prey orders (Diptera and Lepidoptera). The resultant web shows a dense link structure and no compartmentalization or modularity across the three predator guilds. Thus, both individual predators and predator guilds tap heavily into the prey community of each other, offering versatile scope for indirect interactions across different parts of the web. The current description of a first but single arctic web may serve as a benchmark toward which to gauge future webs resolved by similar techniques. Targeting an unusual breadth of predator guilds, and relying on techniques with a high resolution, it suggests that species in this web are closely connected. Thus, our findings call for similar explorations of link structure across multiple guilds in both arctic and other webs. From an applied perspective, our description of an arctic web suggests new avenues for understanding how arctic food webs are built and function and of how they respond to current climate change. It suggests that to comprehend the community-level consequences of rapid arctic warming, we should turn from analyses of populations, population pairs, and isolated predator-prey interactions to considering the full set of interacting species.

Background Rapid and accurate diagnosis of neonatal sepsis is highly warranted because of high associated morbidity and mortality. The aim of this study was to evaluate the performance of a novel multiplex PCR assay for diagnosis of late-onset sepsis and to investigate the value of bacterial DNA load (BDL) determination as a measure of infection severity. Methods This cross-sectional study was conducted in a neonatal intensive care unit. Preterm and/or very low birth weight infants suspected for late-onset sepsis were included. Upon suspicion of sepsis, a whole blood sample was drawn for multiplex PCR to detect the eight most common bacteria causing neonatal sepsis, as well as for blood culture. BDL was determined in episodes with a positive multiplex PCR. Results In total, 91 episodes of suspected sepsis were investigated, and PCR was positive in 53 (58%) and blood culture in 60 (66%) episodes, yielding no significant difference in detection rate (p = 0.17). Multiplex PCR showed a sensitivity of 77%, specificity of 81%, positive predictive value of 87%, and negative predictive value of 68% compared with blood culture. Episodes with discordant results of PCR and blood culture included mainly detection of coagulase-negative staphylococci (CoNS). C-reactive protein (CRP) level and immature to total neutrophil (I/T) ratio were lower in these episodes, indicating less severe disease or even contamination. Median BDL was high (4.1 log10 cfu Eq/ml) with a wide range, and was it higher in episodes with a positive blood culture than in those with a negative blood culture (4.5 versus 2.5 log10 cfu Eq/ml; p < 0.0001). For CoNS infection episodes BDL and CRP were positively associated (p = 0.004), and for Staphylococcus aureus infection episodes there was a positive association between BDL and I/T ratio (p = 0.049). Conclusions Multiplex PCR provides a powerful assay to enhance rapid identification of the causative pathogen in late-onset sepsis. BDL measurement may be a useful indicator of severity of infection. Electronic supplementary material The online version of this article (10.1186/s13054-018-2010-4) contains supplementary material, which is available to authorized users.

This manuscript is part of a series of reviews that aim to cover published research on Crimean-Congo hemorrhagic fever (CCHF) and its etiological agent, CCHF virus (CCHFV). The virus is maintained and transmitted in a vertical and horizontal transmission cycle involving a variety of wild and domestic vertebrate species that act as amplification hosts, without showing signs of illness. These vertebrates have traditionally been considered reservoirs of CCHFV, but in fact they develop only a transient viremia, while the virus can persist in ticks for their entire lifespan, and can also be transmitted vertically to the next generation. As a result, ticks are now considered to be both the vector and the reservoir for the virus. CCHFV has been detected in a wide range of tick species, but only a few have been proven to be vectors and reservoirs, mainly because most published studies have been performed under a broad variety of conditions, precluding definitive characterization. This article reviews the published literature, summarizes current knowledge of the role of ticks in CCHFV maintenance and transmission and provides guidance for how to fill the knowledge gaps. Special focus is given to existing data on tick species in which vertical passage has been demonstrated under natural or experimental conditions. At the same time, we identify earlier reports that used unreliable methods and perceptions to ascribe a vector role to some species of ticks, and have contributed to confusion regarding viral transmission. We also examine epidemiological pathways of CCHFV circulation and discuss priority areas for future research. (C) 2017 Elsevier B.V. All rights reserved.

The objective of this study was to follow Nosema infection levels and species under hygienic bee breeding program for resistance to American foulbrood (Paenibacillus larvae). The incidence of Nosema parasite infection levels and detection of the species of Nosema were evaluated in 5 periods during 2012-2014 for Mugla honey bees known as an ecotype of Apis mellifera anatoliaca in the hygienic bee breeding program. During the hygienic breeding program, no organic or synthetic chemical treatments were applied against nosemosis in the colonies. The incidences of Nosema spores were followed in 123 colonies at five time periods. Although the correlations were negative for between spores-temperature ( r = - 0.115; P> 0.01) and positive for spores-humidity ( r = 0.013; P> 0.01) but not significant statistically. Molecular diagnosis showed that only N. ceranae spores were detected from samples during 5 seasons. In conclusion, nosema infection levels decreased under hygienic bee breeding programme but further monitoring studies should be performed in order to decide whether the nosema spores decrease due to hygienic behavior. To our knowledge, this is the first long-term and unique study for observation of Nosema during breeding program in Turkey so far.

Control of pathogens arising from humans, livestock and wild animals can be enhanced by genome-based investigation. Phylogenetically classifying and optimal construction of these genomes using short sequence reads are key to this process. We examined the mammal-infecting unicellular parasite Leishmania adleri belonging to the lizard-infecting Sauroleishmania subgenus. L. adleri has been associated with cutaneous disease in humans, but can be asymptomatic in wild animals. We sequenced, assembled and investigated the L. adleri genome isolated from an asymptomatic Ethiopian rodent (MARV/ET/75/HO174) and verified it as L. adleri by comparison with other Sauroleishmania species. Chromosome-level scaffolding was achieved by combining reference-guided with de novo assembly followed by extensive improvement steps to produce a final draft genome with contiguity comparable with other references. L. tarentolae and L. major genome annotation was transferred and these gene models were manually verified and improved. This first high-quality draft Leishmania adleri reference genome is also the first Sauroleishmania genome from a non-reptilian host. Comparison of the L. adleri HO174 genome with those of L. tarentolae Parrot-TarII and lizard-infecting L. adleri RLAT/KE/1957/SKINK-7 showed extensive gene amplifications, pervasive aneuploidy, and fission of chromosomes 30 and 36. There was little genetic differentiation between L. adleri extracted from mammals and reptiles, highlighting challenges for leishmaniasis surveillance.

Aims and background The major cause of death in breast cancer patients is metastasis. Various biomarkers have been used for the early detection of circulating tumor cells in the peripheral blood of breast cancer patients. The aims of the current study were to analyze circulating tumor cells in the blood of breast cancer patients by investigating EGFR, CK19, CK20 and HER2 expression profiles and to evaluate their prognostic importance. Methods CK19, CK20 and EGFR gene expression profiles were evaluated in the blood samples of 84 female patients with primary invasive ductal breast cancer and 20 healthy female volunteers using SYBR green-based real-time qPCR assays. HER2 expression analyses were conducted in 46 patients who had an HER2-positive primary tumor and in 30 healthy women to determine the cutoff level of positivity. Results The positive rates of CK20, EGFR, CK19 and HER2 mRNA expression in the peripheral blood were 28.57% (24/84), 20.23% (17/84), 5.95% (5/84) and 2.17% (1/46), respectively. The high positive ratio of CK20 mRNA expression in the peripheral blood of breast cancer was identified for the first time in the current study. Significant differences were identified in CK20 expression status and several clinical parameters related with aggressiveness of tumors using a binary logistic regression analysis. Higher CK20-positive levels were observed in patients who had lymph node metastasis and advanced-grade primary tumors, which were estrogen receptor-negative. We have demonstrated that CK20 may be a novel biomarker that is useful to identify circulating tumor cells and predict breast cancer progression. Conclusions The results suggest that the investigation of CK20 mRNA with other biomarkers in the peripheral blood of breast cancer patients may be useful to monitor the presence of disseminated tumor cells in the blood circulation and to predict the prognosis of breast cancer.

The aim of our study was to detect micrometastatic breast cancer by epithelial glycoprotein-2 (EGP-2) and cytokeratin 19 (CK19), using immunostaining and real time quantitative reverse transcriptase-polymerise chain reaction (qRT-PCR). Fifty-eight breast cancer patients, 52 primary tumors, 75 sentinel nodes (SN) and 149 peripheral blood (PB) samples (from before, during and 4 days after operation) were examined. Immunostaining was performed with antibodies directed against EGP-2 and CK19. Detection limits were one Michigan Cancer Foundation-7 (MCF-7) breast cancer cell line cell/2.10(6) leukocytes (immunostaining) and one MCF-7 cell/10(6) leukocytes qRT-PCR. Control noncancer lymph nodes (n = 10) showed nonspecific CK19 staining, but were qRT-PCR negative; control healthy volunteer PB (n = 11) was always negative. Primary tumor samples, all positive with immunostaining, showed a wide variation of EGP-2 (> 104 fold) and CK19 mRNA expression (> 10(3) fold). SN (n = 19) from 16 patients were tumor-positive with routine haematoxylin-eosin (H&E) and/or immunostaining. SN tumor presence was positively correlated to qRT-PCR expression, but 3 tumor-positive SN were false negative with qRTPCR. Three SN were qRT-PCR positive, while tumor negative with H&E and/or immunostaining. No immunostaining positive PB was observed, but 19 patients (33%) had one or more qRT-PCR positive PB samples. We concluded that primary tumors have varying expressions of EGP-2 and CK19 mRNA. Both markers can be used in qRT-PCR to obtain adequate sensitivity for single tumor cell detection. In SN, immunostaining appears more sensitive/specific than H&E or qRT-PCR for tumor detection. No immunostaining positivity was found in PB, while 33% of patients had qRTPCR positive PB. The clinical value of these findings will have to be clarified. (C) 2003 Wiley-Liss, Inc.

The worldwide spread of diseases is considered a major threat to biodiversity and a possible driver of the decline of pollinator populations, particularly when novel species or strains of parasites emerge. Previous studies have suggested that populations of introduced European honeybee (Apis mellifera) and bumblebee species (Bombus terrestris and Bombus ruderatus) in Argentina share the neogregarine parasite Apicystis bombi with the native bumblebee (Bombus dahlbomii). In this study we investigated whether A. bombi is acting as an emergent parasite in the non-native populations. Specifically, we asked whether A. bombi, recently identified in Argentina, was introduced by European, non-native bees. Using ITS1 and ITS2 to assess the parasite's intraspecific genetic variation in bees from Argentina and Europe, we found a largely unstructured parasite population, with only 15% of the genetic variation being explained by geographic location. The most abundant haplotype in Argentina (found in all 9 specimens of non-native species) was identical to the most abundant haplotype in Europe (found in 6 out of 8 specimens). Similarly, there was no evidence of structuring by host species, with this factor explaining only 17% of the genetic variation. Interestingly, parasites in native Bombus ephippiatus from Mexico were genetically distant from the Argentine and European samples, suggesting that sufficient variability does exist in the ITS region to identify continent-level genetic structure in the parasite. Thus, the data suggest that A. bombi from Argentina and Europe share a common, relatively recent origin. Although our data did not provide information on the direction of transfer, the absence of genetic structure across space and host species suggests that A. bombi may be acting as an emergent infectious disease across bee taxa and continents., Centro de Estudios Parasitológicos y de Vectores

International audience; Among the 33 confirmed Bartonella species to date, more than half are hosted by rodent species, and at least five of them have been involved in human illness causing diverse symptoms including fever, myocarditis, endocarditis, lymphadenitis and hepatitis. In almost all countries, wild rodents are infected by extremely diverse Bartonella strains with a high prevalence. In the present paper, in light of new knowledge on rodent-adapted Bartonella species genomics, we bring together knowledge gained in recent years to have an overview of the impact of rodent-adapted Bartonella infection on humans and to determine how diversity of Bartonella helps to understand their mechanisms of adaptation to rodents and the consequences on human health.

Since the last decade, unusually high honey bee colony losses have been reported mainly in North-America and Europe. Here, we report on a comprehensive bee pathogen screening in Belgium covering 363 bee colonies that were screened for 18 known disease-causing pathogens and correlate their incidence in summer with subsequent winter mortality. Our analyses demonstrate that, in addition to Varroa destructor, the presence of the trypanosomatid parasite Crithidia mellificae and the microsporidian parasite Nosema ceranae in summer are also predictive markers of winter mortality, with a negative synergy being observed between the two in terms of their effects on colony mortality. Furthermore, we document the first occurrence of a parasitizing phorid fly in Europe, identify a new fourth strain of Lake Sinai Virus (LSV), and confirm the presence of other little reported pathogens such as Apicystis bombi, Aphid Lethal Paralysis Virus (ALPV), Spiroplasma apis, Spiroplasma melliferum and Varroa destructor Macula-like Virus (VdMLV). Finally, we provide evidence that ALPV and VdMLV replicate in honey bees and show that viruses of the LSV complex and Black Queen Cell Virus tend to non-randomly co-occur together. We also noticed a significant correlation between the number of pathogen species and colony losses. Overall, our results contribute significantly to our understanding of honey bee diseases and the likely causes of their current decline in Europe. ispartof: PLoS One vol:8 issue:8 ispartof: location:United States status: published

Ní Chualáin, C., and Robinson, M. 2011. Comparison of assessment methods used to diagnose Hematodinium sp. infections in Cancer pagurus. – ICES Journal of Marine Science, 68: . Endoparasitic dinoflagellates of the genus Hematodinium have recently gained attention as significant pathogens of the brown crab Cancer pagurus in Ireland. Patent infections, which are characterized by a hyperpigmented carapace and moribund condition, are limited to the discrete periods when macroscopic identification is possible. Three methods are assessed for diagnosing Hematodinium sp. infections in brown crab at the times when macroscopic identification is not always possible. Haemolymph smears, histological sections of gill, heart, midgut, hepatopancreas, muscle, and gonad, and a polymerase chain reaction (PCR) assay provided virtually equivalent accuracy in gauging infection prevalence, regardless of season. Sequences of PCR amplicons from the 18S ribosomal RNA gene confirmed the identity of the parasite as belonging to the genus Hematodinium. Infection intensity values (

Animal kobuviruses have been described in pigs, cattle, sheep and bats in countries in Asia and Europe. The virus can be detected in fecal and serum samples of infected animals with or without diarrhea, but most of the clinical as well as epidemiological features of kobuvirus infection are still unknown. This study reports the first detection of kobuvirus in farm animals from Brazil and the Netherlands and the molecular analysis of the detected strains. In Brazil, 53% (61/115) of the pigs (suckling, weaned and sows) were shedding porcine kobuvirus in feces, while in the Netherlands 16.7% (3/18) of the tested weaned pigs were infected. Kobuviruses detected in fecal samples of pigs in Brazil showed association (p=0.0002) with diarrhea. In pig serum, kobuvirus was detected at different ages (3, 21, 36, 60, 75, and 180days), with an overall rate of 76.7% (23/30). The sequencing of amplicons detected in serum of pigs of different ages suggested reinfection and no persistent infection. Kobuvirus was also detected in sheep and cattle feces from Brazil and the Netherlands, respectively. Phylogenetic analyses of Brazilian and Dutch kobuviruses from pig, cattle and sheep revealed genetic variability, particularly in one strain detected in sheep feces, which was more closely related to human Aichi virus. The molecular and phylogenetic analyses performed with other published kobuvirus strains and the strains presented in this study, showed that, in most of the cases, kobuvirus seems to group according to host species, but not to geographical region of origin. The data presented in this study contribute to the comprehension of kobuvirus epidemiology and also to the molecular identification of kobuvirus strains circulating worldwide.

Ioos, R., Fabre, B., Saurat, C., Fourrier, C., Frey, P., and Marcais, B. 2010. Development, comparison, and validation of real-time and conventional PCR tools for the detection of the fungal pathogens causing brown spot and red band needle blights of pine. Phytopathology 100:105-114. Dothistroma pini, D. septosporum, and Lecanosticta acicola are fungal pathogens that cause severe foliage diseases in conifers. All three pathogens are listed as quarantine organisms in numerous countries throughout the world and, thus, are subject to specific monitoring. Detection and identification of these pathogens still often relies on cumbersome and unsatisfactory methods that are based upon the morphological characterization of fungal fruiting bodies and conidia. In this study, we present the development of several new molecular tools that enable a rapid and specific in planta detection of each of these pathogens. Several DNA extraction procedures starting from infected needles were compared and four commercial DNA extraction kits provided DNA of satisfactory quality for amplification by polymerase chain reaction (PCR). In addition, we developed several sets of conventional PCR primers, dual-labeled probes (DLPs), and duplex-scorpion probes (DSPs), all of which targeted each pathogen. Their ability to detect the pathogens in a series of naturally infected needle samples was compared. The quadruplex DLP real-time assay proved to be more sensitive than the DSP assay and conventional PCR but the two real-time probe formats yielded identical results in the naturally infected samples. Both real-time assays proved to be significantly superior to the technique of humid chamber incubation, which often failed to produce spores for the accurate identification of the pathogens.

Background 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. Results 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. Conclusions 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.