Microbiota in disease-transmitting vectors.

Haematophagous arthropods, including mosquitoes, ticks, flies, triatomine bugs and lice (here referred to as vectors), are involved in the transmission of various pathogens to mammals on whom they blood feed. The diseases caused by these pathogens, collectively known as vector-borne diseases (VBDs), threaten the health of humans and animals. Although the vector arthropods differ in life histories, feeding behaviour as well as reproductive strategies, they all harbour symbiotic microorganisms, known as microbiota, on which they depend for completing essential aspects of their biology, such as development and reproduction. In this Review, we summarize the shared and unique key features of the symbiotic associations that have been characterized in the major vector taxa. We discuss the crosstalks between microbiota and their arthropod hosts that influence vector metabolism and immune responses relevant for pathogen transmission success, known as vector competence. Finally, we highlight how current knowledge on symbiotic associations is being explored to develop non-chemical-based alternative control methods that aim to reduce vector populations, or reduce vector competence. We conclude by highlighting the remaining knowledge gaps that stand to advance basic and translational aspects of vector-microbiota interactions.

A systematic review and meta-analysis of the potential non-human animal reservoirs and arthropod vectors of the Mayaro virus.

Improving our understanding of Mayaro virus (MAYV) ecology is critical to guide surveillance and risk assessment. We conducted a PRISMA-adherent systematic review of the published and grey literature to identify potential arthropod vectors and non-human animal reservoirs of MAYV. We searched PubMed/MEDLINE, Embase, Web of Science, SciELO and grey-literature sources including PAHO databases and dissertation repositories. Studies were included if they assessed MAYV virological/immunological measured occurrence in field-caught, domestic, or sentinel animals or in field-caught arthropods. We conducted an animal seroprevalence meta-analysis using a random effects model. We compiled granular georeferenced maps of non-human MAYV occurrence and graded the quality of the studies using a customized framework. Overall, 57 studies were eligible out of 1523 screened, published between the years 1961 and 2020. Seventeen studies reported MAYV positivity in wild mammals, birds, or reptiles and five studies reported MAYV positivity in domestic animals. MAYV positivity was reported in 12 orders of wild-caught vertebrates, most frequently in the orders Charadriiformes and Primate. Sixteen studies detected MAYV in wild-caught mosquito genera including Haemagogus, Aedes, Culex, Psorophora, Coquillettidia, and Sabethes. Vertebrate animals or arthropods with MAYV were detected in Brazil, Panama, Peru, French Guiana, Colombia, Trinidad, Venezuela, Argentina, and Paraguay. Among non-human vertebrates, the Primate order had the highest pooled seroprevalence at 13.1% (95% CI: 4.3-25.1%). From the three most studied primate genera we found the highest seroprevalence was in Alouatta (32.2%, 95% CI: 0.0-79.2%), followed by Callithrix (17.8%, 95% CI: 8.6-28.5%), and Cebus/Sapajus (3.7%, 95% CI: 0.0-11.1%). We further found that MAYV occurs in a wide range of vectors beyond Haemagogus spp. The quality of evidence behind these findings was variable and prompts calls for standardization of reporting of arbovirus occurrence. These findings support further risk emergence prediction, guide field surveillance efforts, and prompt further in-vivo studies to better define the ecological drivers of MAYV maintenance and potential for emergence.

Arboviruses: How Saliva Impacts the Journey from Vector to Host.

Arthropod-borne viruses, referred to collectively as arboviruses, infect millions of people worldwide each year and have the potential to cause severe disease. They are predominately transmitted to humans through blood-feeding behavior of three main groups of biting arthropods: ticks, mosquitoes, and sandflies. The pathogens harbored by these blood-feeding arthropods (BFA) are transferred to animal hosts through deposition of virus-rich saliva into the skin. Sometimes these infections become systemic and can lead to neuro-invasion and life-threatening viral encephalitis. Factors intrinsic to the arboviral vectors can greatly influence the pathogenicity and virulence of infections, with mounting evidence that BFA saliva and salivary proteins can shift the trajectory of viral infection in the host. This review provides an overview of arbovirus infection and ways in which vectors influence viral pathogenesis. In particular, we focus on how saliva and salivary gland extracts from the three dominant arbovirus vectors impact the trajectory of the cellular immune response to arbovirus infection in the skin.

Interactions between sympatric invasive European fire ants (Myrmica rubra) and blacklegged ticks (Ixodes scapularis).

The blacklegged tick (Ixodes scapularis) and the invasive European fire ant (Myrmica rubra) are both expanding throughout their sympatric range in coastal New England. Ixodes scapularis is the primary vector of the bacterium Borrelia burgdorferi, which is the causative agent of Lyme disease, and Mount Desert Island, Maine, home to Acadia National Park, currently is affected by a high Lyme disease burden. Ticks have many natural predators, including ants, although no previous studies have investigated interactions between these two species. To test the hypothesis that the presence of M. rubra alters I. scapularis abundance, we collected ticks by drag-sampling at eight ant-infested sites and eight uninfested control sites in Acadia National Park. We found that nymph density was significantly higher at ant-infested sites, while larval density was significantly higher at control sites. In addition, we conducted a laboratory bioassay to measure M. rubra aggression against I. scapularis larvae, nymphs, and adults and Dermacentor variabilis adults, and found that ant aggression was significantly higher against D. variabilis adults than I. scapularis adults. Our findings support the hypothesis that M. rubra has divergent effects across I. scapularis life stages, and we discuss possible ecological mechanisms, including optimal microclimate and predation, that could promote density of nymphs while inhibiting density of larvae.

Phytoene desaturase-silenced citrus as a trap crop with multiple cues to attract Diaphorina citri, the vector of Huanglongbing.

Huanglongbing (HLB) is one of the most destructive diseases in citrus worldwide. Unfortunately, HLB has no cure and management relies on insecticides to reduce populations of the vector, Diaphorina citri Kuwayama (Hemiptera: Liviidae). We propose an attract-and-kill strategy using a trap crop as an alternative to vector control to reduce transmission of the pathogen, 'Candidatus Liberibacter asiaticus'. We evaluated vector response to phytoene desaturase-silenced citrus trees using virus-induced gene silencing technology. Citrus tristeza virus (CTV) was used to produce a phytoene desaturase-silenced citrus (CTV-tPDS) that expresses visual, olfactory, and gustatory cues to attract D. citri. We found that D. citri were more attracted to CTV-tPDS plants with noticeably better fecundity and overall population fitness than on control plants. Moreover, rearing D. citri on CTV-tPDS plants significantly increased their survival probability compared with those reared on control plants. CTV-tPDS plants possessed reduced content of both carotenoid and chlorophyll pigments resulting in a consistent photobleached phenotype on citrus leaves which provided a sufficient close-range visual attractant to stimulate D. citri landing. Additionally, CTV-tPDS plants exhibited an enriched profile of volatile organic compounds (VOCs), which offered adequate olfactory cues to attract psyllid from long-range. Finally, CTV-tPDS plants exhibited an enriched metabolite content of phloem sap and leaves which offered appropriate gustatory cues that influenced probing/feeding behavior. We believe that introducing CTV-tPDS plants (as a trap crop) to D. citri-infested orchards will attract and congregate psyllids to facilitate their removal from the target crop with insecticides or by other means. This new strategy could be deployed relatively quickly and economically to HLB-impacted citrus industries. Moreover, it is an eco-friendly strategy because it should partially reduce the input of chemical insecticides ameliorating the indirect cost of HLB infection.

Potential for online crowdsourced biological recording data to complement surveillance for arthropod vectors.

Voluntary contributions by citizen scientists can gather large datasets covering wide geographical areas, and are increasingly utilized by researchers for multiple applications, including arthropod vector surveillance. Online platforms such as iNaturalist accumulate crowdsourced biological observations from around the world and these data could also be useful for monitoring vectors. The aim of this study was to explore the availability of observations of important vector taxa on the iNaturalist platform and examine the utility of these data to complement existing vector surveillance activities. Of ten vector taxa investigated, records were most numerous for mosquitoes (Culicidae; 23,018 records, 222 species) and ticks (Ixodida; 16,214 records, 87 species), with most data from 2019-2020. Case studies were performed to assess whether images associated with records were of sufficient quality to identify species and compare iNaturalist observations of vector species to the known situation at the state, national and regional level based on existing published data. Firstly, tick data collected at the national (United Kingdom) or state (Minnesota, USA) level were sufficient to determine seasonal occurrence and distribution patterns of important tick species, and were able to corroborate and complement known trends in tick distribution. Importantly, tick species with expanding distributions (Haemaphysalis punctata in the UK, and Amblyomma americanum in Minnesota) were also detected. Secondly, using iNaturalist data to monitor expanding tick species in Europe (Hyalomma spp.) and the USA (Haemaphysalis longicornis), and invasive Aedes mosquitoes in Europe, showed potential for tracking these species within their known range as well as identifying possible areas of expansion. Despite known limitations associated with crowdsourced data, this study shows that iNaturalist can be a valuable source of information on vector distribution and seasonality that could be used to supplement existing vector surveillance data, especially at a time when many surveillance programs may have been interrupted by COVID-19 restrictions.

Comparison of the hemolysis machinery in two evolutionarily distant blood-feeding arthropod vectors of human diseases.

Host blood protein digestion plays a pivotal role in the ontogeny and reproduction of hematophagous vectors. The gut of hematophagous arthropods stores and slowly digests host blood and represents the primary gateway for transmitted pathogens. The initial step in blood degradation is induced lysis of host red blood cells (hemolysis), which releases hemoglobin for subsequent processing by digestive proteolytic enzymes. The activity cycles and characteristics of hemolysis in vectors are poorly understood. Hence, we investigated hemolysis in two evolutionarily distant blood-feeding arthropods: The mosquito Culex pipiens and the soft tick Argas persicus, both of which are important human and veterinary disease vectors. Hemolysis in both species was cyclical after blood meal ingestion. Maximum digestion occurs under slightly alkaline conditions in females. Hemolytic activity appears to be of lipoid origin in C. pipiens and enzymatic activity (proteolytic) in A. persicus. We have assessed the effect of pH, incubation time, and temperature on hemolytic activity and the hemolysin. The susceptibility of red blood cells from different hosts to the hemolysin and the effect of metabolic inhibition of hemolytic activity were assessed. We conclude that in C. pipiens and A. persicus midgut hemolysins control the amplitude of blood lysis step to guarantee an efficient blood digestion.

Ticks (Acari: Ixodoidea) associated with mammals in Colombia: a historical review, molecular species confirmation, and establishment of new relationships.

Ticks are considered the second most important vectors of pathogens worldwide, after mosquitoes. This study provides a systematic review of vector-host relationships between ticks and mammals (domestic and wild) and consolidates information from studies conducted in Colombia between 1911 and 2020. Using the PRISMA method, 71 scientific articles containing records for 51 tick species (Argasidae and Ixodidae) associated with mammals are reported. The existing information on tick-mammal associations in Colombia is scarce, fragmented, or very old. Moreover, 213 specimens were assessed based on morphological and molecular analyses, which allowed confirming eight tick species associated with mammals: Amblyomma calcaratum, Amblyomma dissimile, Amblyomma mixtum, Amblyomma nodosum, Amblyomma ovale, Amblyomma varium, Ixodes luciae, and Ixodes tropicalis. Several tick species are molecularly confirmed for Colombia and nine new relationships between ticks and mammals are reported. This research compiles and confirms important records of tick-mammal associations in Colombia.

Predicting the current and future distribution of the western black-legged tick, Ixodes pacificus, across the Western US using citizen science collections.

In the twenty-first century, ticks and tick-borne diseases have expanded their ranges and impact across the US. With this spread, it has become vital to monitor vector and disease distributions, as these shifts have public health implications. Typically, tick-borne disease surveillance (e.g., Lyme disease) is passive and relies on case reports, while disease risk is calculated using active surveillance, where researchers collect ticks from the environment. Case reports provide the basis for estimating the number of cases; however, they provide minimal information on vector population or pathogen dynamics. Active surveillance monitors ticks and sylvatic pathogens at local scales, but it is resource-intensive. As a result, data are often sparse and aggregated across time and space to increase statistical power to model or identify range changes. Engaging public participation in surveillance efforts allows spatially and temporally diverse samples to be collected with minimal effort. These citizen-driven tick collections have the potential to provide a powerful tool for tracking vector and pathogen changes. We used MaxEnt species distribution models to predict the current and future distribution of Ixodes pacificus across the Western US through the use of a nationwide citizen science tick collection program. Here, we present niche models produced through citizen science tick collections over two years. Despite obvious limitations with citizen science collections, the models are consistent with previously-predicted species ranges in California that utilized more than thirty years of traditional surveillance data. Additionally, citizen science allows for an expanded understanding of I. pacificus distribution in Oregon and Washington. With the potential for rapid environmental changes instigated by a burgeoning human population and rapid climate change, the development of tools, concepts, and methodologies that provide rapid, current, and accurate assessment of important ecological qualities will be invaluable for monitoring and predicting disease across time and space.

Canine leishmaniosis in the Italian northeastern Alps: A survey to assess serological prevalence in dogs and distribution of phlebotomine sand flies in the Autonomous Province of Bolzano - South Tyrol, Italy.

The Autonomous Province of Bolzano-South Tyrol (APB), located in the northernmost territory of the Italian eastern Alps, is still considered non-endemic for canine leishmaniosis (CanL) despite clinical cases being observed and a competent Leishmania infantum vector (Phlebotomus perniciosus) having been recorded since 2008. A serological survey of leishmaniosis among a randomly-selected subpopulation of registered owned dogs was carried in 2018, followed by entomological investigations performed in 2019 and driven by canine survey results. A total of 457 resident dogs from all over the APB territory were examined through IFAT for antibodies against L.infantum, of which 63 (13.8%) tested positive. Thirty-five seropositive cases (7.7%) were considered autochthonous to APB, i.e. dogs born and lived in the province, or imported dogs with no travel history in the past 5 years. Most of these animals showed an antibody titre at the threshold level of 1:40, suggesting a low degree of parasite transmission/contacts. In 2 autochthonous cases with moderately high IFAT titre, the infection was confirmed by nested-PCR in peripheral blood. Thirty-one georeferenced sites were monitored for sand flies by means of interception (sticky papers) and attraction (CDC miniature light traps) collection devices. Traps were set during summer approximately on monthly basis, and extended up to October for positive sites. Only 2 sites were found positive for a total of 317 phlebotomine specimens collected by sticky traps, which included a previously known P. perniciosus-endemic site near Bolzano town. Sergentomyia minuta was by far the most prevalent (98.1%) and the only recorded sand fly species in the most northerly Italian site ever investigated (Coldrano municipality in Venosta valley). For the first time, Leishmania serology and n-PCR positive dogs autochthonous to APB were identified, however the spread of sand flies competent for L. infantum transmission could not be demonstrated in several places where endemic seropositive cases were recorded. APB can be considered a territory of low CanL endemicity, however awareness and continuous monitoring are needed to detect changes in the epidemiological status of the zoonosis.

Climate change could shift disease burden from malaria to arboviruses in Africa.

Malaria is a long-standing public health problem in sub-Saharan Africa, whereas arthropod-borne viruses (arboviruses) such as dengue and chikungunya cause an under-recognised burden of disease. Many human and environmental drivers affect the dynamics of vector-borne diseases. In this Personal View, we argue that the direct effects of warming temperatures are likely to promote greater environmental suitability for dengue and other arbovirus transmission by Aedes aegypti and reduce suitability for malaria transmission by Anopheles gambiae. Environmentally driven changes in disease dynamics will be complex and multifaceted, but given that current public efforts are targeted to malaria control, we highlight Ae aegypti and dengue, chikungunya, and other arboviruses as potential emerging public health threats in sub-Saharan Africa.

Considerations for Human Blood-Feeding and Arthropod Exposure in Vector Biology Research: An Essential Tool for Investigations and Disease Control.

Eventually there may be a broadly acceptable, even perfected, substitute for the human host requirement for direct feeding experiments by arthropods, most notably mosquitoes. However, for now, direct and indirect feeding on human volunteers is an important, if not essential, tool in vector biology research (VBR). This article builds on the foundational publication by Achee et al. (2015) covering considerations for the use of human participants in VBR pursuits. The authors introduced methods involving human participation in VBR, while detailing human-landing collections (catches) as a prime example. Benedict et al. (2018) continued this theme with an overview of human participation and considerations for research that involves release of mosquito vectors into the environment. In this study, we discuss another important aspect of human use in VBR activities: considerations addressing studies that require an arthropod to feed on a live human host. Using mosquito studies as our principal example, in this study, we discuss the tremendous importance and value of this approach to support and allow study of a wide variety of factors and interactions related to our understanding of vector-borne diseases and their control. This includes establishment of laboratory colonies for test populations, characterization of essential nutrients that contribute to mosquito fitness, characterization of blood-feeding (biting) behavior and pathogen transmission, parameterization for modeling transmission dynamics, evaluation of human host attraction and/or agents that repel, and the effectiveness of antivector or parasite therapeutic drug studies.

Putative Role of Arthropod Vectors in African Swine Fever Virus Transmission in Relation to Their Bio-Ecological Properties.

African swine fever (ASF) is one of the most important diseases in Suidae due to its significant health and socioeconomic consequences and represents a major threat to the European pig industry, especially in the absence of any available treatment or vaccine. In fact, with its high mortality rate and the subsequent trade restrictions imposed on affected countries, ASF can dramatically disrupt the pig industry in afflicted countries. In September 2018, ASF was unexpectedly identified in wild boars from southern Belgium in the province of Luxembourg, not far from the Franco-Belgian border. The French authorities rapidly commissioned an expert opinion on the risk of ASF introduction and dissemination into metropolitan France. In Europe, the main transmission routes of the virus comprise direct contact between infected and susceptible animals and indirect transmission through contaminated material or feed. However, the seasonality of the disease in some pig farms in Baltic countries, including outbreaks in farms with high biosecurity levels, have led to questions on the possible involvement of arthropods in the transmission of the virus. This review explores the current body of knowledge on the most common arthropod families present in metropolitan France. We examine their potential role in spreading ASF-by active biological or mechanical transmission or by passive transport or ingestion-in relation to their bio-ecological properties. It also highlights the existence of significant gaps in our knowledge on vector ecology in domestic and wild boar environments and in vector competence for ASFV transmission. Filling these gaps is essential to further understanding ASF transmission in order to thus implement appropriate management measures.

Humoral immune response of pigs, Sus scrofa domesticus, upon repeated exposure to blood-feeding by Ornithodoros turicata Duges (Ixodida: Argasidae).

BACKGROUND: Ornithodoros turicata is an important vector of both human and veterinary pathogens. One primary concern is the global spread of African swine fever virus and the risk of its re-emergence in the Americas through potential transmission by O. turicata to domestic pigs and feral swine. Moreover, in Texas, African warthogs were introduced into the state for hunting purposes and evidence exists that they are reproducing and have spread to three counties in the state. Consequently, it is imperative to develop strategies to evaluate exposure of feral pigs and African warthogs to O. turicata. RESULTS: We report the development of an animal model to evaluate serological responses of pigs to O. turicata salivary proteins after three exposures to tick feeding. Serological responses were assessed for ~ 120 days by enzyme-linked immunosorbent assay and immunoblotting using salivary gland extracts from O. turicata. CONCLUSIONS: Our findings indicate that domestic pigs seroconverted to O. turicata salivary antigens that is foundational toward the development of a diagnostic assay to improve soft tick surveillance efforts.

Impact of Unexplored Data Sources on the Historical Distribution of Three Vector Tick Species in Illinois.

We updated the Illinois historical (1905-December 2017) distribution and status (not reported, reported or established) maps for Amblyomma americanum (L.) (Acari: Ixodidae), Dermacentor variabilis (Say) (Acari: Ixodidae), and Ixodes scapularis (Say) (Acari: Ixodidae) by compiling publicly available, previously unexplored or newly identified published and unpublished data (untapped data). Primary data sources offered specific tick-level information, followed by secondary and tertiary data sources. For A. americanum, D. variabilis, and I. scapularis, primary data contributed to 90% (4,045/4,482), 80% (2,124/2,640), and 32% (3,490/10,898) tick records vs 10%, 20%, and 68%, respectively from secondary data; primary data updated status in 95% (62/65), 94% (51/54) and in 90% (9/10) of the updated counties for each of these tick species; by 1985 there were tick records in 6%, 68%, and 0% of the counties, compared to 20%, 72%, and 58% by 2004, and 77%, 96%, and 75% of the counties by 2017, respectively for A. americanum, D. variabilis, and I. scapularis. We document the loss of tick records due to unidentified, not cataloged tick collections, unidentified ticks in tick collections, unpublished data or manuscripts without specific county location, and tick-level information, to determine distribution and status. In light of the increase in tick-borne illnesses, updates in historical distributions and status maps help researchers and health officials to identify risk areas for a tick encounter and suggest targeted areas for public outreach and surveillance efforts for ticks and tick-borne diseases. There is a need for a systematic, national vector surveillance program to support research and public health responses to tick expansions and tick-borne diseases.

Minimal Duration of Tick Attachment Sufficient for Transmission of Infectious Rickettsia rickettsii (Rickettsiales: Rickettsiaceae) by Its Primary Vector Dermacentor variabilis (Acari: Ixodidae): Duration of Rickettsial Reactivation in the Vector Revisited.

It has been reported that starving ticks do not transmit spotted fever group Rickettsia immediately upon attachment because pathogenic bacteria exist in a dormant, uninfectious state and require time for 'reactivation' before transmission to a susceptible host. To clarify the length of reactivation period, we exposed guinea pigs to bites of Rickettsia rickettsii-infected Dermacentor variabilis (Say) and allowed ticks to remain attached for predetermined time periods from 0 to 48 h. Following removal of attached ticks, salivary glands were immediately tested by PCR, while guinea pigs were observed for 10-12 d post-exposure. Guinea pigs in a control group were subcutaneously inoculated with salivary glands from unfed D. variabilis from the same cohort. In a parallel experiment, skin at the location of tick bite was also excised at the time of tick removal to ascertain dissemination of pathogen from the inoculation site. Animals in every exposure group developed clinical and pathological signs of infection. The severity of rickettsial infection in animals increased with the length of tick attachment, but even attachments for less than 8 h resulted in clinically identifiable infection in some guinea pigs. Guinea pigs inoculated with salivary glands from unfed ticks also became severely ill. Results of our study indicate that R. rickettsii residing in salivary glands of unfed questing ticks does not necessarily require a period of reactivation to precede the salivary transmission and ticks can transmit infectious Rickettsia virtually as soon as they attach to the host.

A new locality of the Haemaphysalis concinna tick (Koch, 1844) in Poland and its role as a potential vector of infectious diseases

The Haemaphysalis concinna tick is a rare species in Poland. To date, it was found only once a few decades ago. During tick collection for epidemiological studies, a stable population of this arachnid was found in a military training area near Nowa Deba. This report is particularly important, given the role of Haemaphysalis concinna in the spread of dangerous vector-borne diseases.

Evidence for Host Epigenetic Signatures Arising From Arbovirus Infections: A Systematic Review.

Background: Arbovirus infections have steadily become a major pandemic threat. This study aimed at investigating the existence of host epigenetic markers arising from the principal arboviruses infections impacting on human health. We set to systematically review all published evidence describing any epigenetic modifications associated with infections from arboviruses, including, but not limited to, microRNAs, DNA methylation, and histone modifications. Methods: A comprehensive search was conducted using the electronic databases PubMed, Science Direct and Cochrane Library from inception to January 4th, 2018. We included reports describing original in vivo or in vitro studies investigating epigenetic changes related to arbovirus infections in either clinical subjects or human cell lines. Studies investigating epigenetic modifications related to the virus or the arthropod vector were excluded. A narrative synthesis of the findings was conducted, contextualizing comparative evidence from in vitro and in vivo studies. Results: A total of 853 unique references were identified and screened by two independent researchers. Thirty-two studies met the inclusion criteria and were reviewed. The evidence was centered mainly on microRNA and DNA methylation signatures implicated with secondary Dengue fever. Evidence for recent epidemic threats, such as the infections by Zika or Chikungunya viruses is still scant. Conclusions: Major epigenetic alterations found on arboviruses infections were miR-146, miR-30e and the Dicer complex. However, existing studies frequently tested distinct hypotheses resulting in a heterogeneity of methodological approaches. Whilst epigenetic signatures associated with arbovirus infections have been reported, existing studies have largely focused on a small number of diseases, particularly dengue. Validation of epigenetic signatures have an untapped potential, but concerted investigations are certainly required to deliver robust candidates of clinical utility for diagnosis, staging and prognosis of specific arboviral diseases.

The role of juvenile Dermacentor reticulatus ticks as vectors of microorganisms and the problem of 'meal contamination'.

Juvenile Dermacentor reticulatus ticks inhabit nests and burrows of their rodent hosts and cannot be collected from vegetation. To detect vertical transmission of Babesia canis in D. reticulatus, we studied larvae and nymphs collected from rodents. However, the molecular techniques used for detection of pathogen DNA are sensitive enough to detect not only pathogens vectored by ticks but also those taken up with current or previous blood meals ('meal contamination') or just present in the environment and on the tick or host surface ('environmental contaminations'). Thus, an additional aim of our study was to evaluate the extent of such contamination while studying feeding ticks collected from rodents. Juvenile D. reticulatus were collected from 140 rodents: 91 bank voles trapped in two forest sites in the Mazury Lake District and 49 rodents (Apodemus and Microtus spp.) from an open habitat near the town of Bialobrzegi in Central Poland. Altogether 504 D. reticulatus ticks, comprising 266 individually evaluated nymphs and 238 larvae assigned to 50 larval pools, were studied for the presence of Babesia, Bartonella and Rickettsia spp. DNA. Statistical analyses were conducted to (1) evaluate the effect of rodent host factors (species, sex and age) on prevalence of infection in ticks, and (2) to compare the frequency of positive samples between groups of pathogen-positive and pathogen-negative rodent hosts. To complete the last aim, blood samples obtained from 49 rodents from Bialobrzegi were studied for the presence of Babesia and Bartonella DNA. Infestation of rodent hosts with juvenile ticks ranged between 46 and 78%, with a mean abundance of 3.6 ticks/rodent for D. reticulatus and 4.8 ticks/rodent for Ixodes ricinus. The highest prevalence of PCR-positive D. reticulatus samples was obtained for Rickettsia spp. (28%) and R. raoultii was identified in 22 sequenced PCR products. Babesia DNA was detected in 20 (7.5%), including B. microti in 18 (6.8%) and B. canis in two (0.8%) of 266 D. reticulatus nymphs that were analyzed. Babesia microti DNA was also detected in four pools of D. reticulatus larvae (4/50 pools = 8%). The detection success of B. microti in D. reticulatus was associated with the species of the rodent hosts of the ticks (much higher for typical B. microti-host-species such as Microtus spp. than for Apodemus spp.) and host age (3 × higher in ticks collected from adult hosts in comparison to juvenile ones). Moreover, the DNA of B. microti was detected in 68% of D. reticulatus nymphs collected from B. microti-positive rodents in comparison to only 1.6% of nymphs collected from B. microti-negative rodents. Bartonella DNA was detected in 18% of D. reticulatus tick samples (38% of larval pools, 14% of nymphs). Again, host factors played important roles for 'tick positivity'-the highest prevalence of positive ticks was on Apodemus spp., which are regarded as Bartonella reservoirs. Bartonella DNA was detected in 42% of nymphs and 57% of larval pools collected from Bartonella-positive rodents in comparison to 28% of nymphs and 11% of larvae collected from Bartonella-negative rodents. Vertical transmission of B. canis in D. reticulatus ticks was confirmed in the field. Additionally, we demonstrated that 'meal contamination' generates a confounding signal in molecular detection of pathogen DNA extracted from ticks collected from infected hosts and must be taken into account in evaluating the competence of tick species as vectors.