The role of autophagy in tick-endosymbiont interactions: insights from Ixodes scapularis and Rickettsia buchneri.

IMPORTANCE: Ticks are second only to mosquitoes in their importance as vectors of disease agents; however, tick-borne diseases (TBDs) account for the majority of all vector-borne disease cases in the United States (approximately 76.5%), according to Centers for Disease Control and Prevention reports. Newly discovered tick species and their associated disease-causing pathogens, and anthropogenic and demographic factors also contribute to the emergence and re-emergence of TBDs. Thus, incorporating different tick control approaches based on a thorough knowledge of tick biology has great potential to prevent and eliminate TBDs in the future. Here we demonstrate that replication of a transovarially transmitted rickettsial endosymbiont depends on the tick's autophagy machinery but not on apoptosis. Our findings improve our understanding of the role of symbionts in tick biology and the potential to discover tick control approaches to prevent or manage TBDs.

Detection of Babesia species in questing Ixodes ricinus ticks in England and Wales.

Babesiosis, a disease in humans and animals is caused by piroplasms from the genus Babesia and is transmitted by ixodid ticks. Bovine babesiosis, commonly called redwater fever, is reported in cattle from many regions of the British Isles. The presence of Babesia in questing ticks in the United Kingdom (UK) and its potential impact on public and animal health has not been widely studied. Therefore, this study aimed to assess the presence of Babesia spp. in England and Wales using ticks collected over a six-year period. Questing Ixodes ricinus nymphs were collected at 20 recreational areas between 2014 and 2019 and screened for Babesia. Of 3912 nymphs tested, Babesia spp. were detected in 15, giving an overall prevalence of 0.38% [95%CI: 0.21-0.63%]. A number of Babesia species were identified including B. venatorum (n = 9), B. divergens/capreoli (n = 5) and B. odocoilei-like species (n = 1). Based on the low prevalence of Babesia detected in questing I. ricinus nymphs in the recreational areas studied, the likelihood of exposure to Babesia-infected ticks is lower compared to other pathogens more widely studied in the UK (e.g. Borrelia burgdorferi s.l.). However, localized areas of elevated risk may occur in pockets in England and Wales.

Methods for the Study of Ticks, Mosquitoes, and their Transmitted Pathogens: Toward a Greater Understanding of Vector Biology and Arthropod-Microbe Interactions.

ARTICLES DISCUSSED: Chen, L., Xiao, Q., Shi, M., Cheng, J., Wu, J. Detecting Wolbachia strain wAlbB in Aedes albopictus cell lines. Journal of Visualized Experiments. (184), e63662 (2022). Haziqah-Rashid, A. et al. Determining temperature preference of mosquitoes and other ectotherms. Journal of Visualized Experiments. (187), e64356 (2022). Huang, D. et al. Mosquito-associated virus isolation from field-collected mosquitoes. Journal of Visualized Experiments. (186), e63852 (2022). Khoo, B., Cull, B., Oliver, J. D. Tick artificial membrane feeding for Ixodes scapularis. Journal of Visualized Experiments. 64553 (2022). Leal-Galvan, B., Harvey, C., Thomas, D., Saelao, P., Oliva Chavez, A. S. Isolation of microRNAs from tick ex vivo salivary gland cultures and extracellular vesicles. Journal of Visualized Experiments. (182), e63618 (2022). Liang, Q. et al. Control of Aedes albopictus mosquito larvae with Carpesium abrotanoides L. Journal of Visualized Experiments. (186), e63976 (2022). Wang, F. et al. Experimental viral infection in adult mosquitoes by oral feeding and microinjection. Journal of Visualized Experiments. (185), e63830 (2022). Wang, X. R., Burkhardt, N. Y., Price, L. D., Munderloh, U. G. An electroporation method to transform Rickettsia spp. with a fluorescent protein-expressing shuttle vector in tick cell lines. Journal of Visualized Experiments. (188), e64562 (2022).

Impact of green space connectivity on urban tick presence, density and Borrelia infected ticks in different habitats and seasons in three cities in southern England.

Understanding the effects of local habitat and wider landscape connectivity factors on tick presence, nymph density and Borrelia species (spp.) prevalence in the tick population is important for identifying the public health risk from Lyme borreliosis. This multi-city study collected data in three southern England cities (Bath, Bristol, and Southampton) during spring, summer, and autumn in 2017. Focusing specifically on urban green space used for recreation which were clearly in urbanised areas, 72 locations were sampled. Additionally, geospatial datasets on urban green space coverage within 250 m and 1 km of sampling points, as well as distance to woodland were incorporated into statistical models. Distance to woodland was negatively associated with tick presence and nymph density, particularly during spring and summer. Furthermore, we observed an interaction effect between habitat and season for tick presence and nymph density, with woodland habitat having greater tick presence and nymph density during spring. Borrelia spp. infected Ixodes ricinus were found in woodland, woodland edge and under canopy habitats in Bath and Southampton. Overall Borrelia spp. prevalence in nymphs was 2.8%, similar to wider UK studies assessing prevalence in Ixodes ricinus in rural areas. Bird-related Borrelia genospecies dominated across sites, suggesting bird reservoir hosts may be important in urban green space settings for feeding and infecting ticks. Whilst overall density of infected nymphs across the three cities was low (0.03 per 100 m2), risk should be further investigated by incorporating data on tick bites acquired in urban settings, and subsequent Lyme borreliosis transmission.

Mapping and monitoring tick (Acari, Ixodida) distribution, seasonality, and host associations in the United Kingdom between 2017 and 2020.

Tick-borne disease risk is intrinsically linked to the distribution of tick vector species. To assess risk and anticipate disease emergence, an understanding of tick distribution, host associations, and seasonality is needed. This can be achieved, to some extent, using passive surveillance supported by engagement with the public, animal health, and public health experts. The Tick Surveillance Scheme (TSS) collects data and maps tick distribution across the United Kingdom (UK). Between 2017 and 2020, 3720 tick records were received and 39 tick species were detected. Most records were acquired in the UK, with a subset associated with recent overseas travel. The dominant UK acquired species was Ixodes ricinus (Ixodida: Ixodidae, Linnaeus), the main vector of Lyme borreliosis. Records peaked during May and June, highlighting a key risk period for tick bites. Other key UK species were detected, including Dermacentor reticulatus (Ixodida: Ixodidae, Fabricius) and Haemaphysalis punctata (Ixodida: Ixodidae, Canestrini & Fanzago) as well as several rarer species that may present novel tick-borne disease risk to humans and other animals. Updated tick distribution maps highlight areas in the UK where tick exposure has occurred. There is evidence of increasing human tick exposure over time, including during the COVID-19 pandemic, but seasonal patterns remain unchanged.

Tick Artificial Membrane Feeding for Ixodes scapularis.

Ticks and their associated diseases are an important topic of study due to their public health and veterinary burden. However, the feeding requirements of ticks during both study and rearing can limit experimental questions or the ability of labs to research ticks and their associated pathogens. An artificial membrane feeding system can reduce these problems and open up new avenues of research that may not have been possible with traditional animal feeding systems. This study describes an artificial membrane feeding system that has been refined for feeding and engorgement success for all Ixodes scapularis life stages. Moreover, the artificial membrane feeding system described in this study can be modified for use with other tick species through simple refinement of the desired membrane thickness. The benefits of an artificial membrane feeding system are counterbalanced by the labor intensiveness of the system, the additional environmental factors that may impact feeding success, and the need to refine the technique for each new species and life stage of ticks.

Monitoring Trends in Distribution and Seasonality of Medically Important Ticks in North America Using Online Crowdsourced Records from iNaturalist.

Recent increases in the incidence and geographic range of tick-borne diseases in North America are linked to the range expansion of medically important tick species, including Ixodes scapularis, Amblyomma americanum, and Amblyomma maculatum. Passive tick surveillance programs have been highly successful in collecting information on tick distribution, seasonality, host-biting activity, and pathogen infection prevalence. These have demonstrated the power of citizen or community science participation to collect country-wide, epidemiologically relevant data in a resource-efficient manner. This study examined tick observations from the online image-based biological recording platform iNaturalist to evaluate its use as an effective tool for monitoring the distributions of A. americanum, A. maculatum, I. scapularis, and Dermacentor in the United States and Canada. The distribution and seasonality of iNaturalist tick observations were found to accurately represent those of the studied species. County-level iNaturalist tick occurrence data showed good agreement with other data sources in documented areas of I. scapularis and A. americanum establishment, and highlighted numerous previously unreported counties with iNaturalist observations of these species. This study supports the use of iNaturalist data as a highly cost-effective passive tick surveillance method that can complement existing surveillance strategies to update tick distributions and identify new areas of tick establishment.

Spatial and temporal heterogeneity of the density of Borrelia burgdorferi-infected Ixodes ricinus ticks across a landscape: A 5-year study in southern England.

The density of Borrelia burgdorferi-infected Ixodes ricinus nymphs (DIN) was investigated during 2013-2017 across a Lyme disease-endemic landscape in southern England. The density of nymphs (DON), nymph infection prevalence (NIP), and DIN varied across five different natural habitats, with the highest DIN in woodland edge and high biodiversity woodlands. DIN was significantly lower in scrub grassland compared to the woodland edge, with low DON and no evidence of infection in ticks in non-scrub grassland. Over the 5 years, DON, NIP and DIN were comparable within habitats, except in 2014, with NIP varying three-fold and DIN significantly lower compared to 2015-2017. Borrelia garinii was most common, with bird-associated Borrelia (B. garinii/valaisiana) accounting for ~70% of all typed sequences. Borrelia burgdorferi sensu stricto was more common than B. afzelii. Borrelia afzelii was more common in scrub grassland than woodland and absent in some years. The possible impact of scrub on grazed grassland, management of ecotonal woodland margins with public access, and the possible role of birds/gamebirds impacting NIP are discussed. Mean NIP was 7.6%, highlighting the potential risk posed by B. burgdorferi in this endemic area. There is a need for continued research to understand its complex ecology and identify strategies for minimizing risk to public health, through habitat/game management and public awareness.

Prevalence of Anaplasma phagocytophilum in questing Ixodes ricinus nymphs across twenty recreational areas in England and Wales.

Human granulocytic anaplasmosis and tick-borne fever, affecting livestock, are diseases caused by an infection with the bacterium Anaplasma phagocytophilum. Its transmission dynamics between vertebrate hosts and ticks remain largely unknown and the potential impact on public health in the United Kingdom is unclear. This study aimed to assess the distribution and estimate the prevalence of A. phagocytophilum in questing Ixodes ricinus at recreational locations across England and Wales over six years. An additional objective was to investigate possible associations between prevalence, habitat and presence of ruminant hosts. Ixodes ricinus ticks were collected each spring at 20 recreational locations across England and Wales between 2014 and 2019. Nymphs were tested for infection with A. phagocytophilum by detection of bacterial genome in DNA extracts, targeting the msp2 gene locus. Positive samples were further investigated for the presence of different ecotypes based on the GroEL region. Of 3,919 nymphs tested, the mean infection prevalence was 3.6% [95%CI: 3.1-4.3] and ranged from 0 to 20.4%. Northern England had a higher overall prevalence (4.7% [95%CI: 3.4-6.4]) compared to Southern England (1.8% [95%CI: 1.3-2.5]) and the presence of sheep was associated with higher A. phagocytophilum prevalence (8.4% [95%CI: 6.9-10.1] vs 1.2% [95%CI: 0.8-1.7] when absent). There was also a negative correlation with the prevalence of Borrelia burgdorferi s.l. (causing Lyme borreliosis). When investigating the diversity of A. phagocytophilum, ecotype I accounted for 86.8% of samples and ecotype II for 13.2%. Our study presents an overview of A. phagocytophilum prevalence in questing I. ricinus in recreational areas across England and Wales and discusses the potential public and veterinary health relevance.

Apoptosis and Autophagy: Current Understanding in Tick-Pathogen Interactions.

Tick-borne diseases are a significant threat to human and animal health throughout the world. How tick-borne pathogens successfully infect and disseminate in both their vertebrate and invertebrate hosts is only partially understood. Pathogens have evolved several mechanisms to combat host defense systems, and to avoid and modulate host immunity during infection, therefore benefitting their survival and replication. In the host, pathogens trigger responses from innate and adaptive immune systems that recognize and eliminate invaders. Two important innate defenses against pathogens are the programmed cell death pathways of apoptosis and autophagy. This Mini Review surveys the current knowledge of apoptosis and autophagy pathways in tick-pathogen interactions, as well as the strategies evolved by pathogens for their benefit. We then assess the limitations to studying both pathways and discuss their participation in the network of the tick immune system, before highlighting future perspectives in this field. The knowledge gained would significantly enhance our understanding of the defense responses in vector ticks that regulate pathogen infection and burden, and form the foundation for future research to identify novel approaches to the control of tick-borne diseases.

17-AAG-Induced Activation of the Autophagic Pathway in Leishmania Is Associated with Parasite Death.

The heat shock protein 90 (Hsp90) is thought to be an excellent drug target against parasitic diseases. The leishmanicidal effect of an Hsp90 inhibitor, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), was previously demonstrated in both in vitro and in vivo models of cutaneous leishmaniasis. Parasite death was shown to occur in association with severe ultrastructural alterations in Leishmania, suggestive of autophagic activation. We hypothesized that 17-AAG treatment results in the abnormal activation of the autophagic pathway, leading to parasite death. To elucidate this process, experiments were performed using transgenic parasites with GFP-ATG8-labelled autophagosomes. Mutant parasites treated with 17-AAG exhibited autophagosomes that did not entrap cargo, such as glycosomes, or fuse with lysosomes. ATG5-knockout (Δatg5) parasites, which are incapable of forming autophagosomes, demonstrated lower sensitivity to 17-AAG-induced cell death when compared to wild-type (WT) Leishmania, further supporting the role of autophagy in 17-AAG-induced cell death. In addition, Hsp90 inhibition resulted in greater accumulation of ubiquitylated proteins in both WT- and Δatg5-treated parasites compared to controls, in the absence of proteasome overload. In conjunction with previously described ultrastructural alterations, herein we present evidence that treatment with 17-AAG causes abnormal activation of the autophagic pathway, resulting in the formation of immature autophagosomes and, consequently, incidental parasite death.

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.

Mitochondrion-Dependent Apoptosis Is Essential for Rickettsia parkeri Infection and Replication in Vector Cells.

Apoptosis is an innate immune response induced by infection in eukaryotes that contributes significantly to protection from pathogens. However, little is known about the role of apoptosis in the interactions of arthropod vectors with the rickettsiae that they transmit. Rickettsia spp. are vector-borne obligately intracellular bacteria and display different degrees of virulence in their eukaryotic hosts. In this study, we found that infection with Rickettsia parkeri (Rp) activated the apoptosis pathway in an Amblyomma americanum tick cell line (AAE2), as evidenced by the loss of phospholipid membrane asymmetry and DNA fragmentations. Additionally, infection with Rp also led to apoptosis activation in cell lines of different tick species. Interestingly, suppressing apoptosis decreased Rp infection and replication, while the activation of apoptosis increased Rp accumulation at the early stage of infection. Moreover, mitochondrion-dependent apoptosis was essential for Rp infection and replication in vector cells, and apoptosis induction required intracellular rickettsia replication. We further showed that Rp utilizes two different survival strategies to modulate apoptosis in the arthropod vectors and mammalian host cells. There was no direct correlation between apoptosis activation in vector cells and rickettsial pathogenicity. These novel findings indicate a possible mechanism whereby apoptosis facilitates infection and replication of a Rickettsia sp. in an arthropod vector. These results contribute to our understanding of how the vector's responses to pathogen infection affect pathogen replication and therefore transmission.IMPORTANCE Rickettsioses, infections caused by the genus Rickettsia, are among the oldest known infectious diseases. Ticks are essential arthropod vectors for rickettsiae, and knowledge about the interactions between ticks, their hosts, and pathogens is fundamental for identifying drivers of tick-borne rickettsioses. Despite the rapid development in apoptosis research with rickettsiae, little is known regarding the role of apoptosis in the interactions between Rickettsia spp., vertebrate hosts, and arthropod vectors. Here, we demonstrated that mitochondrion-dependent apoptosis is essential for rickettsial infection and replication in vector cells and that apoptosis induction requires intracellular rickettsial replication. However, rickettsial pathogenicity is not linked with apoptosis activation in tick cells. Our findings improve understanding of the apoptosis mechanism in arthropods exploited by rickettsiae and also the potential to discover specific targets for new vaccines and drugs to prevent or treat rickettsial infections.

The Ixodes scapularis Symbiont Rickettsia buchneri Inhibits Growth of Pathogenic Rickettsiaceae in Tick Cells: Implications for Vector Competence.

Ixodes scapularis is the primary vector of tick-borne pathogens in North America but notably does not transmit pathogenic Rickettsia species. This tick harbors the transovarially transmitted endosymbiont Rickettsia buchneri, which is widespread in I. scapularis populations, suggesting that it confers a selective advantage for tick survival such as providing essential nutrients. The R. buchneri genome includes genes with similarity to those involved in antibiotic synthesis. There are two gene clusters not found in other Rickettsiaceae, raising the possibility that these may be involved in excluding pathogenic bacteria from the tick. This study explored whether the R. buchneri antibiotic genes might exert antibiotic effects on pathogens associated with I. scapularis. Markedly reduced infectivity and replication of the tick-borne pathogens Anaplasma phagocytophilum, R. monacensis, and R. parkeri were observed in IRE11 tick cells hosting R. buchneri. Using a fluorescent plate reader assay to follow infection dynamics revealed that the presence of R. buchneri in tick cells, even at low infection rates, inhibited the growth of R. parkeri by 86-100% relative to R. buchneri-free cells. In contrast, presence of the low-pathogenic species R. amblyommatis or the endosymbiont R. peacockii only partially reduced the infection and replication of R. parkeri. Addition of host-cell free R. buchneri, cell lysate of R. buchneri-infected IRE11, or supernatant from R. buchneri-infected IRE11 cultures had no effect on R. parkeri infection and replication in IRE11, nor did these treatments show any antibiotic effect against non-obligate intracellular bacteria E. coli and S. aureus. However, lysate from R. buchneri-infected IRE11 challenged with R. parkeri showed some inhibitory effect on R. parkeri infection of treated IRE11, suggesting that challenge by pathogenic rickettsiae may induce the antibiotic effect of R. buchneri. This research suggests a potential role of the endosymbiont in preventing other rickettsiae from colonizing I. scapularis and/or being transmitted transovarially. The confirmation that the observed inhibition is linked to R. buchneri's antibiotic clusters requires further investigation but could have important implications for our understanding of rickettsial competition and vector competence of I. scapularis for rickettsiae.

The Unexpected Holiday Souvenir: The Public Health Risk to UK Travellers from Ticks Acquired Overseas.

Overseas travel to regions where ticks are found can increase travellers' exposure to ticks and pathogens that may be unfamiliar to medical professionals in their home countries. Previous studies have detailed non-native tick species removed from recently returned travellers, occasionally leading to travel-associated human cases of exotic tick-borne disease. There are 20 species of tick endemic to the UK, yet UK travellers can be exposed to many other non-native species whilst overseas. Here, we report ticks received by Public Health England's Tick Surveillance Scheme from humans with recent travel history between January 2006 and December 2018. Altogether, 16 tick species were received from people who had recently travelled overseas. Confirmed imports (acquired outside of the UK) were received from people who recently travelled to 22 countries. Possible imports (acquired abroad or within the UK) were received from people who had recently travelled to eight European countries. Species-specific literature reviews highlighted nine of the sixteen tick species are known to vector at least one tick-borne pathogen to humans in the country of acquisition, suggesting travellers exposed to ticks may be at risk of being bitten by a species that is a known vector, with implications for novel tick-borne disease transmission to travellers.

Identification and RNAi Profile of a Novel Iflavirus Infecting Senegalese Aedes vexans arabiensis Mosquitoes.

The inland floodwater mosquito Aedes vexans (Meigen, 1830) is a competent vector of numerous arthropod-borne viruses such as Rift Valley fever virus (Phenuiviridae) and Zika virus (Flaviviridae). Aedes vexans spp. have widespread Afrotropical distribution and are common European cosmopolitan mosquitoes. We examined the virome of Ae. vexans arabiensis samples from Barkédji village, Senegal, with small RNA sequencing, bioinformatic analysis, and RT-PCR screening. We identified a novel 9494 nt iflavirus (Picornaviridae) designated here as Aedes vexans iflavirus (AvIFV). Annotation of the AvIFV genome reveals a 2782 amino acid polyprotein with iflavirus protein domain architecture and typical iflavirus 5' internal ribosomal entry site and 3' poly-A tail. Aedes vexans iflavirus is most closely related to a partial virus sequence from Venturia canescens (a parasitoid wasp) with 56.77% pairwise amino acid identity. Analysis of AvIFV-derived small RNAs suggests that AvIFV is targeted by the exogenous RNA interference pathway but not the PIWI-interacting RNA response, as ~60% of AvIFV reads corresponded to 21 nt Dicer-2 virus-derived small RNAs and the 24-29 nt AvIFV read population did not exhibit a "ping-pong" signature. The RT-PCR screens of archival and current (circa 2011-2020) Ae. vexans arabiensis laboratory samples and wild-caught mosquitoes from Barkédji suggest that AvIFV is ubiquitous in these mosquitoes. Further, we screened wild-caught European Ae. vexans samples from Germany, the United Kingdom, Italy, and Sweden, all of which tested negative for AvIFV RNA. This report provides insight into the diversity of commensal Aedes viruses and the host RNAi response towards iflaviruses.

Seasonality and anatomical location of human tick bites in the United Kingdom.

Tick bites on humans can occur in a variety of habitats and may result in the transmission of tick-borne pathogens, such as the causative agent of Lyme borreliosis (LB), Borrelia burgdorferi sensu lato. As the risk of transmission of this pathogen to the host increases with the duration of tick feeding, the recognition and removal of ticks as soon as possible following attachment is important for reducing the risk of infection. Performing a thorough body examination for ticks following potential exposure is recommended by tick awareness campaigns. Knowledge of where on the body feeding ticks are frequently found, and at which times of year peak tick exposure occurs, provides important information for public health messaging and may aid those bitten by ticks to engage more effectively with tick-checking behaviour. This paper summarizes human tick bites in the United Kingdom (UK) during 2013-2018 reported to Public Health England's passive Tick Surveillance Scheme and further examines the anatomical location and seasonality of bites from the most commonly encountered tick and LB vector Ixodes ricinus. A total of 1,328 tick records from humans were received of which 93% were I. ricinus. Humans were most commonly bitten by I. ricinus nymphs (70% bites). Tick bites were recorded on all parts of the body, but there were significant differences in their anatomical location on adults and children. Most tick bites on adults occurred on the legs (50%), whereas on children tick bites were mostly on the head and neck (43%). Bites from I. ricinus were recorded throughout the year but were most numerous during May to August. This study adds to the body of research on the seasonality and anatomical location of human tick bites in temperate Europe and highlights the importance of data collected through passive surveillance in addition to research and epidemiological studies.

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

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

Hyalomma rufipes on an untraveled horse: Is this the first evidence of Hyalomma nymphs successfully moulting in the United Kingdom?

During September 2018, a tick was submitted to Public Health England's Tick Surveillance Scheme for identification. The tick was sent from a veterinarian who removed it from a horse in Dorset, England, with no history of overseas travel. The tick was identified as a male Hyalomma rufipes using morphological and molecular methods and then tested for a range of tick-borne pathogens including; Alkhurma virus, Anaplasma, Babesia, Bhanja virus, Crimean-Congo Haemorrhagic fever virus, Rickettsia and Theileria. The tick tested positive for Rickettsia aeschlimannii, a spotted fever group rickettsia linked to a number of human cases in Africa and Europe. This is the first time H. rufipes has been reported in the United Kingdom (UK), and the lack of travel by the horse (or any in-contact horses) suggests that this could also be the first evidence of successful moulting of a Hyalomma nymph in the UK. It is postulated that the tick was imported into the UK on a migratory bird as an engorged nymph which was able to complete its moult to the adult stage and find a host. This highlights that passive tick surveillance remains an important method for the detection of unusual species that may present a threat to public health in the UK. Horses are important hosts of Hyalomma sp. adults in their native range, therefore, further surveillance studies should be conducted to check horses for ticks in the months following spring bird migration; when imported nymphs may have had time to drop off their avian host and moult to adults. The potential human and animal health risks of such events occurring more regularly are discussed.