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.

Tick extracellular vesicles impair epidermal homeostasis through immune-epithelial networks during hematophagy.

Hematophagous ectoparasites, such as ticks, rely on impaired wound healing for skin attachment and blood feeding. Wound healing has been extensively studied through the lens of inflammatory disorders and cancer, but limited attention has been given to arthropod-borne diseases. Here, we used orthogonal approaches combining single-cell RNA sequencing (scRNAseq), flow cytometry, murine genetics, and intravital microscopy to demonstrate how tick extracellular vesicles (EVs) disrupt networks involved in tissue repair. Impairment of EVs through silencing of the SNARE protein vamp33 negatively impacted ectoparasite feeding and survival in three medically relevant tick species, including Ixodes scapularis. Furthermore, I. scapularis EVs affected epidermal γδ T cell frequencies and co-receptor expression, which are essential for keratinocyte function. ScRNAseq analysis of the skin epidermis in wildtype animals exposed to vamp33-deficient ticks revealed a unique cluster of keratinocytes with an overrepresentation of pathways connected to wound healing. This biological circuit was further implicated in arthropod fitness when tick EVs inhibited epithelial proliferation through the disruption of phosphoinositide 3-kinase activity and keratinocyte growth factor levels. Collectively, we uncovered a tick-targeted impairment of tissue repair via the resident γδ T cell-keratinocyte axis, which contributes to ectoparasite feeding.

Environmental Drivers of Immature Ixodes scapularis in Minnesota's Metro Area.

Research on the public health significance of Ixodes scapularis ticks in the Midwest seldom focuses on extreme weather conditions that can modulate their population dynamics and ability to transmit pathogenic organisms. In this study, we assessed whether the distributional abundance of I. scapularis immatures is associated with current and time-lagged climatic determinants either directly or indirectly. We analyzed a 20-year longitudinal small mammal live-trapping dataset within a seven-county metropolitan area in Minnesota (1998-2016) using yearly tick counts at each site to assess whether inter- and intra-annual variation in immature I. scapularis counts is associated with climate and land-use conditions. We found that (1) immature I. scapularis ticks infesting mammals expanded southwesterly over the study period, (2) eastern chipmunks, Tamias striatus, supplied a substantial proportion of nymphal blood meals, (3) a suite of climatological variables are demonstrably associated with I. scapularis presence, and abundance across sites, most notably summer vapor pressure deficit, and (4) immature I. scapularis display an affinity for deciduous forests in metro areas. Our results suggest that climatic and land-type conditions may impact host-seeking I. scapularis ticks through numerous mechanistic avenues. These findings extend our understanding of the abiotic factors supporting I. scapularis populations in metro areas of the upper Midwest with strong implications for discerning future tick-borne pathogen risk.

An Anaplasma phagocytophilum T4SS effector, AteA, is essential for tick infection.

IMPORTANCE: Ticks are the number one vector of pathogens for livestock worldwide and for humans in the United States. The biology of tick transmission is an understudied area. Understanding this critical interaction could provide opportunities to affect the course of disease spread. In this study, we examined the zoonotic tick-borne agent Anaplasma phagocytophilum and identified a secreted protein, AteA, which is expressed in a tick-specific manner. These secreted proteins, termed effectors, are the first proteins to interact with the host environment. AteA is essential for survival in ticks and appears to interact with cortical actin. Most effector proteins are studied in the context of the mammalian host; however, understanding how this unique set of proteins affects tick transmission is critical to developing interventions.

Metagenomic surveillance for bacterial tick-borne pathogens using nanopore adaptive sampling.

Technological and computational advancements in the fields of genomics and bioinformatics are providing exciting new opportunities for pathogen discovery and genomic surveillance. In particular, single-molecule nucleotide sequence data originating from Oxford Nanopore Technologies (ONT) sequencing platforms can be bioinformatically leveraged, in real-time, for enhanced biosurveillance of a vast array of zoonoses. The recently released nanopore adaptive sampling (NAS) strategy facilitates immediate mapping of individual nucleotide molecules to a given reference as each molecule is being sequenced. User-defined thresholds then allow for the retention or rejection of specific molecules, informed by the real-time reference mapping results, as they are physically passing through a given sequencing nanopore. Here, we show how NAS can be used to selectively sequence DNA of multiple bacterial tick-borne pathogens circulating in wild populations of the blacklegged tick vector, Ixodes scapularis.

Positive associations matter: Microbial relationships drive tick microbiome composition.

Untangling how factors such as environment, host, associations among bacterial species and dispersal predict microbial composition is a fundamental challenge. In this study, we use complementary machine-learning approaches to quantify the relative role of these factors in shaping microbiome variation of the blacklegged tick Ixodes scapularis. I. scapularis is the most important vector for Borrelia burgdorferi (the causative agent for Lyme disease) in the U.S. as well as a range of other important zoonotic pathogens. Yet the relative role of the interactions between pathogens and symbionts compared to other ecological forces is unknown. We found that positive associations between microbes where the occurrence of one microbe increases the probability of observing another, including between both pathogens and symbionts, was by far the most important factor shaping the tick microbiome. Microclimate and host factors played an important role for a subset of the tick microbiome including Borrelia (Borreliella) and Ralstonia, but for the majority of microbes, environmental and host variables were poor predictors at a regional scale. This study provides new hypotheses on how pathogens and symbionts might interact within tick species, as well as valuable predictions for how some taxa may respond to changing climate.

An Anaplasma phagocytophilum T4SS effector, AteA, is essential for tick infection.

Pathogens must adapt to disparate environments in permissive host species, a feat that is especially pronounced for vector-borne microbes, which transition between vertebrate hosts and arthropod vectors to complete their lifecycles. Most knowledge about arthropod-vectored bacterial pathogens centers on their life in the mammalian host, where disease occurs. However, disease outbreaks are driven by the arthropod vectors. Adapting to the arthropod is critical for obligate intracellular rickettsial pathogens, as they depend on eukaryotic cells for survival. To manipulate the intracellular environment, these bacteria use Type IV Secretion Systems (T4SS) to deliver effectors into the host cell. To date, few rickettsial T4SS translocated effectors have been identified and have only been examined in the context of mammalian infection. We identified an effector from the tick-borne rickettsial pathogen Anaplasma phagocytophilum , HGE1_02492, as critical for survival in tick cells and acquisition by ticks in vivo . Conversely, HGE1_02492 was dispensable during mammalian cell culture and murine infection. We show HGE1_02492 is translocatable in a T4SS-dependent manner to the host cell cytosol. In eukaryotic cells, the HGE1_02492 localized with cortical actin filaments, which is dependent on multiple sub-domains of the protein. HGE1_02492 is the first arthropod-vector specific T4SS translocated effector identified from a rickettsial pathogen. Moreover, the subcellular target of HGE1_02492 suggests that A. phagocytophilum is manipulating actin to enable arthropod colonization. Based on these findings, we propose the name AteA for Anaplasma ( phagocytophilum ) tick effector A. Altogether, we show that A. phagocytophilum uses distinct strategies to cycle between mammals and arthropods. Importance: Ticks are the number one vector of pathogens for livestock worldwide and for humans in the US. The biology of tick transmission is an understudied area. Understanding this critical interaction could provide opportunities to affect the course of disease spread. In this study we examined the zoonotic tick-borne agent Anaplasma phagocytophilum and identified a secreted protein, AteA, that is expressed in a tick-specific manner. These secreted proteins, termed effectors, are the first proteins to interact with the host environment. AteA is essential for survival in ticks and appears to interact with cortical actin. Most effector proteins are studied in the context of the mammalian host; however, understanding how this unique set of proteins affect tick transmission is critical to developing interventions.

Nanopore adaptive sampling for targeted mitochondrial genome sequencing and bloodmeal identification in hematophagous insects.

BACKGROUND: Blood-feeding insects are important vectors for an array of zoonotic pathogens. While previous efforts toward generating molecular resources have largely focused on major vectors of global medical and veterinary importance, molecular data across a large number of hematophagous insect taxa remain limited. Advancements in long-read sequencing technologies and associated bioinformatic pipelines provide new opportunities for targeted sequencing of insect mitochondrial (mt) genomes. For engorged hematophagous insects, such technologies can be leveraged for both insect mitogenome genome assembly and identification of vertebrate blood-meal sources. METHODS: We used nanopore adaptive sampling (NAS) to sequence genomic DNA from four species of field-collected, blood-engorged mosquitoes (Aedes and Culex spp.) and one deer fly (Chrysops sp.). NAS was used for bioinformatical enrichment of mtDNA reads of hematophagous insects and potential vertebrate blood-meal hosts using publically available mt genomes as references. We also performed an experimental control to compare results of traditional non-NAS nanopore sequencing to the mt genome enrichment by the NAS method. RESULTS: Complete mitogenomes were assembled and annotated for all five species sequenced with NAS: Aedes trivittatus, Aedes vexans, Culex restuans, Culex territans and the deer fly, Chrysops niger. In comparison to data generated during our non-NAS control experiment, NAS yielded a substantially higher proportion of reference-mapped mtDNA reads, greatly streamlining downstream mitogenome assembly and annotation. The NAS-assembled mitogenomes ranged in length from 15,582 to 16,045 bp, contained between 78.1% and 79.0% A + T content and shared the anticipated arrangement of 13 protein-coding genes, two ribosomal RNAs, and 22 transfer RNAs. Maximum likelihood phylogenies were generated to further characterize each insect species. Additionally, vertebrate blood-meal analysis was successful in three samples sequenced, with mtDNA-based phylogenetic analyses revealing that blood-meal sources for Chrysops niger, Culex restuans and Aedes trivittatus were human, house sparrow (Passer domesticus) and eastern cottontail rabbit (Sylvilagus floridanus), respectively. CONCLUSIONS: Our findings show that NAS has dual utility to simultaneously molecularly identify hematophagous insects and their blood-meal hosts. Moreover, our data indicate NAS can facilitate a wide array of mitogenomic systematic studies through novel 'phylogenetic capture' methods. We conclude that the NAS approach has great potential for broadly improving genomic resources used to identify blood-feeding insects, answer phylogenetic questions and elucidate complex pathways for the transmission of vector-borne pathogens.

Dome1-JAK-STAT signaling between parasite and host integrates vector immunity and development.

Ancestral signaling pathways serve critical roles in metazoan development, physiology, and immunity. We report an evolutionary interspecies communication pathway involving a central Ixodes scapularis tick receptor termed Dome1, which acquired a mammalian cytokine receptor motif exhibiting high affinity for interferon-gamma (IFN-γ). Host-derived IFN-γ facilitates Dome1-mediated activation of the Ixodes JAK-STAT pathway. This accelerates tick blood meal acquisition and development while upregulating antimicrobial components. The Dome1-JAK-STAT pathway, which exists in most Ixodid tick genomes, regulates the regeneration and proliferation of gut cells-including stem cells-and dictates metamorphosis through the Hedgehog and Notch-Delta networks, ultimately affecting Ixodes vectorial competence. We highlight the evolutionary dependence of I. scapularis on mammalian hosts through cross-species signaling mechanisms that dually influence arthropod immunity and development.

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.

Epidemiology, Distribution and Identification of Ticks on Livestock in Pakistan.

Background: Ticks are ectoparasites that transmit a variety of pathogens that cause many diseases in livestock which can result in skin damage, weight loss, anemia, reduced production of meat and milk, and mortality. Aim: The aim of this study was to identify tick species and the distribution on livestock hosts (sheep, goat, dairy cattle, and buffalo) of Punjab, Khyber Pakhtunkhwa Province and Islamabad from October 2019 to November 2020. Materials and Methods: Surveillance was performed to calculate the prevalence of ticks on livestock. Tick prevalence data (area, host, breed, gender, age, and seasonal infestation rate) was recorded and analyzed. Results: A total of 2080 animals were examined from selected farms, and, of these, 1129 animals were tick-infested. A total of 1010 male tick samples were identified to species using published keys. Haemaphysalis punctata, Haemaphysalis sulcata, Hyalomma anatolicum, Hyalomma detritum, Hyalomma dromedarii, Hyalomma excavatum, Hyalomma marginatum, Hyalomma rufipes, Rhipicephalus decoloratus Rhipicephalus microplus, and Rhipicephalus sanguineus were collected from goats, sheep, buffalo, and cattle. The overall rates of tick infestation on livestock were 34.83% (buffalo), 57.11% (cattle), 51.97% (sheep) and 46.94% (goats). Within each species, different breeds demonstrated different proportions of infestation. For cattle breeds, infestation proportions were as follows: Dhanni (98.73%), Jersey (70.84%) and the Australian breed of cattle (81.81%). The Neeli Ravi breed (40%) of buffalo and the Beetal breed (57.35%) of goats were the most highly infested for these species. Seasonally, the highest prevalence of infestation (76.78%) was observed in summer followed by 70.76% in spring, 45.29% in autumn, and 20% in winter. The prevalence of tick infestation in animals also varied by animal age. In goats, animals aged 4-6 years showed the highest prevalence (90%), but in cattle, the prevalence of ticks was highest (68.75%) in 6 months-1-year-old animals. 1-3 years old buffalo (41.07%) and 6 months-1 year sheep (65.78%) had the highest prevalence rate. Females had significantly higher infestation rates (61.12%, 55.56% and 49.26%, respectively) in cattle, sheep, and goats. In buffalo, males showed a higher prevalence (38.46%) rate. Conclusions: This study showed tick diversity, infestation rate, and numerous factors (season, age, and gender of host) influencing tick infestation rate in different breeds of cattle, sheep, goats, and buffalo in Punjab Province, Khyber Pakhtunkhwa Province, and Islamabad, Pakistan. Higher tick burdens and rates of tick-borne disease reduce production and productivity in animals. Understanding tick species' prevalence and distribution will help to develop informed control measures.

Persistence of the invasive bird-parasitic fly Philornis downsi over the host interbreeding period in the Galapagos Islands.

Many parasites of seasonally available hosts must persist through times of the year when hosts are unavailable. In tropical environments, host availability is often linked to rainfall, and adaptations of parasites to dry periods remain understudied. The bird-parasitic fly Philornis downsi has invaded the Galapagos Islands and is causing high mortality of Darwin's finches and other bird species, and the mechanisms by which it was able to invade the islands are of great interest to conservationists. In the dry lowlands, this fly persists over a seven-month cool season when availability of hosts is very limited. We tested the hypothesis that adult flies could survive from one bird-breeding season until the next by using a pterin-based age-grading method to estimate the age of P. downsi captured during and between bird-breeding seasons. This study showed that significantly older flies were present towards the end of the cool season, with ~ 5% of captured females exhibiting estimated ages greater than seven months. However, younger flies also occurred during the cool season suggesting that some fly reproduction occurs when host availability is low. We discuss the possible ecological mechanisms that could allow for such a mixed strategy.

Mosquito Guttersnipe: A New Sampling Tool for Roof Gutters, Tree Holes, and Other Elevated Mosquito Habitats.

Mosquitoes pose health risks to human populations by serving as vectors of diseases. Mosquito control organizations are responsible for inspecting and controlling vector populations to reduce the risk of infection of these diseases. Current sampling methods are effective for numerous types of mosquito habitat, but not conducive for sampling small overhead habitat such as roof gutters or tree holes. We have developed and tested a tool called the Mosquito GutterSnipe to sample these overhead habitats. Volumetric and larval capacity testing of the tool prototype demonstrated comparable sampling integrity to standard mosquito dipping methods. The GutterSnipe can be employed as a reliable way to sample previously overlooked mosquito habitat. Its current model is cost effective and easy to produce for mosquito control organizations and easy to use for inspectors.

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.

Interspecies bacterial communication produces a delicate balance between Vibrio cholerae and the chironomid egg mass microbiome.

The evolution of antimicrobial resistance in bacterial pathogens is considered by the World Health Organization to be one of the ten most concerning public health threats facing humanity (World Health Organization, 2020). Bacterial diseases previously controllable by antibiotics are resurging and treatment options are dwindling. Cholera is one such disease. Human pathogenic strains of Vibrio cholerae cause as many as 4 million cases of disease resulting in over 100,000 deaths each year (Ali et al. 2015) and multidrug-resistant V. cholerae is now established where pandemic cholera persists. Vibrio cholerae is fundamentally an aquatic species thriving in brackish and estuarial waters. Its environmental prevalence, together with both extracellular and intracellular infection of alternative arthropod and mollusc hosts, produces a highly complex ecological milieu that is not well understood. With the absence of reliable antibiotic-based treatment options, it is necessary to build a better understanding of V. cholerae biology and ecology in order to develop alternative methods for risk modelling and disease control. In this issue of Molecular Ecology, authors Sela, Hammer, and Halpern experimentally investigated a mechanism by which V. cholerae pathogenicity is affected by interspecies quorum sensing involving an array of bacterial species from the microbiome of an alternative arthropod host, the egg mass of a chironomid midge (Diptera:Chironomidae) (Sela et al. 2020). Quorum sensing is a mechanism whereby bacteria communicate with each other using autoinducers and is known to be important, for example, in shaping virulence in a variety of pathogenic bacteria. The innovative methodologies they used, both in molecular and protein biology and reductive investigative microbiomics, are helping to develop the tools needed for understanding this understudied ecological system and fighting cholera in a post-antibiotic world.

Growth Dynamics and Antibiotic Elimination of Symbiotic Rickettsia buchneri in the Tick Ixodes scapularis (Acari: Ixodidae).

Rickettsia buchneri is the principal symbiotic bacterium of the medically significant tick Ixodes scapularis This species has been detected primarily in the ovaries of adult female ticks and is vertically transmitted, but its tissue tropism in other life stages and function with regard to tick physiology is unknown. In order to determine the function of R. buchneri, it may be necessary to produce ticks free from this symbiont. We quantified the growth dynamics of R. buchneri naturally occurring in I. scapularis ticks throughout their life cycle and compared it with bacterial growth in ticks in which symbiont numbers were experimentally reduced or eliminated. To eliminate the bacteria, we exposed ticks to antibiotics through injection and artificial membrane feeding. Both injection and membrane feeding of the antibiotic ciprofloxacin were effective at eliminating R. buchneri from most offspring of exposed females. Because of its effectiveness and ease of use, we have determined that injection of ciprofloxacin into engorged female ticks is an efficient means of clearing R. buchneri from the majority of progeny.IMPORTANCE This paper describes the growth of symbiotic Rickettsia buchneri within Ixodes scapularis through the life cycle of the tick and provides methods to eliminate R. buchneri from I. scapularis ticks.

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.

Plant-Derived Natural Compounds for Tick Pest Control in Livestock and Wildlife: Pragmatism or Utopia?

Ticks and tick-borne diseases are a significant economic hindrance for livestock production and a menace to public health. The expansion of tick populations into new areas, the occurrence of acaricide resistance to synthetic chemical treatments, the potentially toxic contamination of food supplies, and the difficulty of applying chemical control in wild-animal populations have created greater interest in developing new tick control alternatives. Plant compounds represent a promising avenue for the discovery of such alternatives. Several plant extracts and secondary metabolites have repellent and acaricidal effects. However, very little is known about their mode of action, and their commercialization is faced with multiple hurdles, from the determination of an adequate formulation to field validation and public availability. Further, the applicability of these compounds to control ticks in wild-animal populations is restrained by inadequate delivery systems that cannot guarantee accurate dosage delivery at the right time to the target animal populations. More work, financial support, and collaboration with regulatory authorities, research groups, and private companies are needed to overcome these obstacles. Here, we review the advancements on known plant-derived natural compounds with acaricidal potential and discuss the road ahead toward the implementation of organic control in managing ticks and tick-borne diseases.

Spatio-Temporal Dynamics of Tick-Borne Diseases in North-Central Wisconsin from 2000-2016.

Lyme disease is a well-recognized public health problem in the USA, however, other tick-borne diseases also have major public health impacts. Yet, limited research has evaluated changes in the spatial and temporal patterns of non-Lyme tick-borne diseases within endemic regions. Using laboratory data from a large healthcare system in north-central Wisconsin from 2000-2016, we applied a Kulldorf's scan statistic to analyze spatial, temporal and seasonal clusters of laboratory-positive cases of human granulocytic anaplasmosis (HGA), babesiosis, and ehrlichiosis at the county level. Older males were identified as the subpopulation at greatest risk for non-Lyme tick-borne diseases and we observed a statistically significant spatial and temporal clustering of cases (p < 0.05). HGA risk shifted from west to east over time (2000-2016) with a relative risk (RR) ranging from 3.30 to 11.85, whereas babesiosis risk shifted from south to north and west over time (2004-2016) with an RR ranging from 4.33 to 4.81. Our study highlights the occurrence of non-Lyme tick-borne diseases, and identifies at-risk subpopulations and shifting spatial and temporal heterogeneities in disease risk. Our findings can be used by healthcare providers and public health practitioners to increase public awareness and improve case detection.

Global Transcription Profiles of Anaplasma phagocytophilum at Key Stages of Infection in Tick and Human Cell Lines and Granulocytes.

The incidence of human diseases caused by tick-borne pathogens is increasing but little is known about the molecular interactions between the agents and their vectors and hosts. Anaplasma phagocytophilum (Ap) is an obligate intracellular, tick-borne bacterium that causes granulocytic anaplasmosis in humans, dogs, sheep, and horses. In mammals, neutrophil granulocytes are a primary target of infection, and in ticks, Ap has been found in gut and salivary gland cells. To identify bacterial genes that enable Ap to invade and proliferate in human and tick cells, labeled mRNA from Ap bound to or replicating within human and tick cells (lines HL-60 and ISE6), and replicating in primary human granulocytes ex vivo, was hybridized to a custom tiling microarray containing probes representing the entire Ap genome. Probe signal values plotted over a map of the Ap genome revealed antisense transcripts and unannotated genes. Comparisons of transcript levels from each annotated gene between test conditions (e.g., Ap replicating in HL-60 vs. ISE6) identified those that were differentially transcribed, thereby highlighting genes associated with each condition. Bacteria replicating in HL-60 cells upregulated 122 genes compared to those in ISE6, including numerous p44 paralogs, five HGE-14 paralogs, and 32 hypothetical protein genes, of which 47% were predicted to be secreted or localized to the membrane. By comparison, 60% of genes upregulated in ISE6 encoded hypothetical proteins, 60% of which were predicted to be secreted or membrane associated. In granulocytes, Ap upregulated 120 genes compared to HL-60, 33% of them hypothetical and 43% of those predicted to encode secreted or membrane associated proteins. HL-60-grown bacteria binding to HL-60 cells barely responded transcriptionally, while ISE6-grown bacteria binding to ISE6 cells upregulated 48 genes. HL-60-grown bacteria, when incubated with ISE6 cells, upregulated the same genes that were upregulated by ISE6-grown bacteria exposed to uninfected ISE6. Hypothetical genes (constituting about 29% of Ap genes) played a disproportionate role in most infection scenarios, and particular sets of them were consistently upregulated in bacteria binding/entering both ISE6 and HL-60 cells. This suggested that the encoded proteins played central roles in establishing infection in ticks and humans.