First detection of Ehrlichia chaffeensis, Ehrlichia canis, and Anaplasma ovis in Rhipicephalus bursa ticks collected from sheep, Turkey.

Anaplasmosis and ehrlichiosis are important tick-borne rickettsial diseases of medical and veterinary importance that cause economic losses in livestock. In this study, the prevalence of Anaplasma ovis, Ehrlichia canis and Ehrlichia chaffeensis was investigated in ticks collected from sheep in various farms in Van province, which is located in the Eastern Anatolian Region of Turkey. The ticks used in this study were collected by random sampling in 26 family farm business in 13 districts of Van province. A total of 688 ticks were collected from 88 sheep and 88 tick pools were created. All ticks identified morphologically as Rhipicephalus bursa. Phylogenetic analysis of Chaperonin and 16S rRNA gene sequences confirmed A. ovis, E. canis and E. chaffeensis in this study. Of the 88 tick pools tested, 28.41% (25/88) were positive for at least one pathogen. Anaplasma DNA was detected in five of the 88 pools (5.68%), E. canis DNA was detected in 19 of the 88 pools (21.59%), and E. chaffeensis DNA was detected in one of the 88 pools (1.14%) of R. bursa ticks. To our knowledge, this is the first report describing the presence of A. ovis, E. canis, and E. chaffeensis in R. bursa ticks collected from sheep in Turkey. Further studies are needed to investigate other co-infections in sheep in Turkey.

Ehrlichia chaffeensis Etf-3 Induces Host RAB15 Upregulation for Bacterial Intracellular Growth.

Ehrlichia chaffeensis infects human monocytes or macrophages and causes human monocytic ehrlichiosis (HME), an emerging life-threatening zoonosis. After internalization, E. chaffeensis resides in membrane-bound inclusions, E. chaffeensis-containing vesicles (ECVs), which have early endosome-like characteristics and fuse with early autophagosomes but not lysosomes, to evade host innate immune microbicidal mechanisms and obtain nutrients for bacterial intracellular growth. The mechanisms exploited by E. chaffeensis to modulate intracellular vesicle trafficking in host cells have not been comprehensively studied. Here, we demonstrate that E. chaffeensis type IV secretion system (T4SS) effector Etf-3 induces RAB15 upregulation in host cells and that RAB15, which is localized on ECVs, inhibits ECV fusion with lysosomes and induces autophagy. We found that E. chaffeensis infection upregulated RAB15 expression using qRT-PCR, and RAB15 was colocalized with E. chaffeensis using confocal microscopy. Silence of RAB15 using siRNA enhanced ECV maturation to late endosomes and fusion with lysosomes, as well as inhibited host cell autophagy. Overexpression of Etf-3 in host cells specifically induced RAB15 upregulation and autophagy. Our findings deepen the understanding of E. chaffeensis pathogenesis and adaptation in hosts as well as the function of RAB15 and facilitate the development of new therapeutics for HME.

Science abhors a surveillance vacuum: Detection of ticks and tick-borne pathogens in southern New Mexico through passive surveillance.

Robust tick surveillance enhances diagnosis and prevention of tick-borne pathogens, yet surveillance efforts in the United States are highly uneven, resulting in large surveillance vacuums, one of which spans the state of New Mexico. As part of a larger effort to fill this vacuum, we conducted both active and passive tick sampling in New Mexico, focusing on the southern portion of the state. We conducted active tick sampling using dragging and CO2 trapping at 45 sites across Hidalgo, Doña Ana, Otero, and Eddy counties between June 2021 to May 2022. Sampling occurred intermittently, with at least one sampling event each month from June to October 2021, pausing in winter and resuming in March through May 2022. We also conducted opportunistic, passive tick sampling in 2021 and 2022 from animals harvested by hunters or captured or collected by researchers and animals housed in animal hospitals, shelters, and farms. All pools of ticks were screened for Rickettsia rickettsii, Rickettsia parkeri, Rickettsia amblyommatis, Ehrlichia ewingii, and Ehrlichia chaffeensis. Active sampling yielded no ticks. Passive sampling yielded 497 ticks comprising Carios kelleyi from pallid bats, Rhipicephalus sanguineus from dogs, mule deer, and Rocky Mountain elk, Otobius megnini from dogs, cats, horses, and Coues deer, Dermacentor parumapertus from dogs and black-tailed jackrabbits, Dermacentor albipictus from domesticated cats, mule deer and Rocky Mountain elk, and Dermacentor spp. from American black bear, Rocky Mountain elk, and mule deer. One pool of D. parumapterus from a black-tailed jackrabbit in Luna County tested positive for R. parkeri, an agent of spotted fever rickettsiosis. Additionally, a spotted fever group Rickettsia was detected in 6 of 7 C. kelleyi pools. Two ticks showed morphological abnormalities; however, these samples did not test positive for any of the target pathogens, and the cause of the abnormalities is unknown. Passive surveillance yielded five identified species of ticks from three domestic and six wild mammal species. Our findings update tick distributions and inform the public, medical, and veterinary communities of the potential tick-borne pathogens present in southern New Mexico.

Ehrlichia chaffeensis Co-Opts Phagocytic Hemocytes for Systemic Dissemination in the Lone Star Tick, Amblyomma americanum.

INTRODUCTION: Hematophagous arthropods can acquire and transmit several pathogens of medical importance. In ticks, the innate immune system is crucial in the outcome between vector-pathogen interaction and overall vector competence. However, the specific immune response(s) elicited by the immune cells known as hemocytes remains largely undefined in Ehrlichia chaffeensis and its competent tick vector, Amblyomma americanum. METHODS: We utilized injection of clodronate liposome to deplete tick granulocytes combined with infection with E. chaffeensis to demonstrate their essential role in microbial infection. RESULTS: Here, we show that granulocytes, professional phagocytic cells, are integral in eliciting immune responses against commensal and pathogen infection. The chemical depletion of granulocytes led to decreased phagocytic efficiency of tissue-associated hemocytes. We demonstrate that E. chaffeensis can infect circulating hemocytes, and both cell-free plasma and hemocytes from E. chaffeensis-infected ticks can establish Ehrlichia infection in recipient ticks. Lastly, we provide evidence to show that granulocytes play a dual role in E. chaffeensis infection. Depleting granulocytic hemocytes increased Ehrlichia load in the salivary gland and midgut tissues. In contrast, granulocyte depletion led to a reduced systemic load of Ehrlichia. CONCLUSION: This study has identified multiple roles for granulocytic hemocytes in the control and systemic dissemination of E. chaffeensis infection.

Mathematical model of Ehrlichia chaffeensis transmission dynamics in dogs.

Ehrlichia chaffeensis is a tick-borne disease transmitted by ticks to dogs. Few studies have mathematical modelled such tick-borne disease in dogs, and none have developed models that incorporate different ticks' developmental stages (discrete variable) as well as the duration of infection (continuous variable). In this study, we develop and analyze a model that considers these two structural variables using integrated semigroups theory. We address the well-posedness of the model and investigate the existence of steady states. The model exhibits a disease-free equilibrium and an endemic equilibrium. We calculate the reproduction number (T0). We establish a necessary and sufficient condition for the bifurcation of an endemic equilibrium. Specifically, we demonstrate that a bifurcation, either backward or forward, can occur at T0=1, leading to the existence, or not, of an endemic equilibrium even when T0<1. Finally, numerical simulations are employed to illustrate these theoretical findings.

Seasonality of Amblyomma americanum (Acari: Ixodidae) Activity and Prevalence of Infection with Tick-Borne Disease Agents in North Central Oklahoma.

Background: Amblyomma americanum is the most common tick infesting both animals and humans in the southern United States and transmits a variety of zoonotic agents. The rise in tick-borne diseases (TBD) globally imparts a need for more active surveillance of tick populations to accurately quantify prevalence and risk of tick-borne infectious organisms. To better understand TBD risk in north central Oklahoma, this study aimed to describe the current seasonal activity of A. americanum in this region and investigate the seasonality of tick-borne infectious agents. Materials and Methods: Tick collections were performed twice a month for a duration of 2 years at a field site in Payne County, Oklahoma. Total nucleic acid was extracted from a subset of adult A. americanum and tested for Rickettsia spp., Ehrlichia spp., and Borrelia spp. using established PCR protocols. Results: Peak activity times for each life stage were observed, with adults primarily active 1 month earlier than historical seasonal trends describe, and male A. americanum active earlier in the year than female A. americanum. Rickettsia spp., Ehrlichia chaffeensis, Ehrlichia ewingii, and Borrelia lonestari were found in 26.4%, 6.1%, 2.5%, and 1.1% of adult A. americanum, respectively. No seasonal trend in spotted fever group Rickettsia spp. (SFGR) was observed in peak activity months. Conclusions: This study found an apparently shifting phenology for A. americanum adults in Oklahoma. While these results did not show a trend in SFGR, further investigation is needed to better understand the potential seasonality of infection prevalence within A. americanum across the expanding range of this vector, especially considering the extended activity of males in winter months.

Establishing the intracellular niche of obligate intracellular vacuolar pathogens.

Obligate intracellular pathogens occupy one of two niches - free in the host cell cytoplasm or confined in a membrane-bound vacuole. Pathogens occupying membrane-bound vacuoles are sequestered from the innate immune system and have an extra layer of protection from antimicrobial drugs. However, this lifestyle presents several challenges. First, the bacteria must obtain membrane or membrane components to support vacuole expansion and provide space for the increasing bacteria numbers during the log phase of replication. Second, the vacuole microenvironment must be suitable for the unique metabolic needs of the pathogen. Third, as most obligate intracellular bacterial pathogens have undergone genomic reduction and are not capable of full metabolic independence, the bacteria must have mechanisms to obtain essential nutrients and resources from the host cell. Finally, because they are separated from the host cell by the vacuole membrane, the bacteria must possess mechanisms to manipulate the host cell, typically through a specialized secretion system which crosses the vacuole membrane. While there are common themes, each bacterial pathogen utilizes unique approach to establishing and maintaining their intracellular niches. In this review, we focus on the vacuole-bound intracellular niches of Anaplasma phagocytophilum, Ehrlichia chaffeensis, Chlamydia trachomatis, and Coxiella burnetii.

Ehrlichia Notch signaling induction promotes XIAP stability and inhibits apoptosis.

Ehrlichia chaffeensis has evolved multiple strategies to evade innate defenses of the mononuclear phagocyte. Recently, we reported the E. chaffeensis tandem repeat protein (TRP)120 effector functions as a Notch ligand mimetic and a ubiquitin ligase that degrades the nuclear tumor suppressor, F-box and WD repeat domain-containing 7, a negative regulator of Notch. The Notch intracellular domain (NICD) is known to inhibit apoptosis primarily by interacting with X-linked inhibitor of apoptosis protein (XIAP) to prevent degradation. In this study, we determined that E. chaffeensis activation of Notch signaling increases XIAP levels, thereby inhibiting apoptosis through both the intrinsic and executioner pathways. Increased NICD and XIAP levels were detected during E. chaffeensis infection and after TRP120 Notch ligand mimetic peptide treatment. Conversely, XIAP levels were reduced in the presence of Notch inhibitor DAPT. Cytoplasmic and nuclear colocalization of NICD and XIAP was observed during infection and a direct interaction was confirmed by co-immunoprecipitation. Procaspase levels increased temporally during infection, consistent with increased XIAP levels; however, knockdown (KD) of XIAP during infection significantly increased apoptosis and Caspase-3, -7, and -9 levels. Furthermore, treatment with SM-164, a second mitochondrial activator of caspases (Smac/DIABLO) antagonist, resulted in decreased procaspase levels and increased caspase activation, induced apoptosis, and significantly decreased infection. In addition, RNAi KD of XIAP also decreased infection and significantly increased apoptosis. Moreover, ectopic expression of TRP120 HECT Ub ligase catalytically defective mutant in HeLa cells decreased NICD and XIAP levels and increased caspase activation compared to HeLa cells with functional HECT Ub ligase catalytic activity (TRP120-WT). This investigation reveals a mechanism whereby E. chaffeensis modulates Notch signaling to stabilize XIAP and inhibit apoptosis.

Ehrlichia Wnt SLiM ligand mimic deactivates the Hippo pathway to engage the anti-apoptotic Yap-GLUT1-BCL-xL axis.

Ehrlichia chaffeensis TRP120 effector has evolved short linear motif (SLiM) ligand mimicry to repurpose multiple evolutionarily conserved cellular signaling pathways, including Wnt, Notch, and Hedgehog. In this investigation, we demonstrate that E. chaffeensis and recombinant TRP120 deactivate Hippo signaling, resulting in the activation of Hippo transcription coactivator Yes-associated protein (Yap). Moreover, a homologous 6 amino acid (QDVASH) SLiM shared by TRP120 and Wnt3a/5a ligands phenocopied Yap and ß-catenin activation induced by E. chaffeensis, rTRP120, and Wnt5a. Similar Hippo gene expression profiles were also stimulated by E. chaffeensis, rTRP120, SLiM, and Wnt5a. Single siRNA knockdown of Hippo transcription co-activator/factors, Yap, and transcriptional enhanced associate domain (TEAD) significantly decreased E. chaffeensis infection. Yap activation was abolished in THP-1 Wnt Frizzled-5 (Fzd5) receptor knockout cells (KO), demonstrating Fzd5 receptor dependence. In addition, the TRP120-Wnt-SLiM antibody blocked Hippo deactivation (Yap activation). Expression of anti-apoptotic Hippo target gene SLC2A1 (encodes glucose transporter 1; GLUT1) was upregulated by E. chaffeensis and corresponded to increased levels of GLUT1. Conversely, siRNA knockdown of SLC2A1 significantly inhibited infection. Higher GLUT1 levels correlated with increased B cell lymphoma-extra large (BCL-xL) and decreased BCL2-associated X, apoptosis regulator (Bax) levels. Moreover, blocking Yap activation with the inhibitor Verteporfin induced apoptosis that corresponded to significant reductions in GLUT1 and BCL-xL levels and activation of Bax and Caspase-3 and -9. This study identifies a novel shared Wnt/Hippo SLiM ligand mimic and demonstrates that E. chaffeensis deactivates the Hippo pathway to engage the anti-apoptotic Yap-GLUT1-BCL-xL axis.

Multiple Ehrlichia chaffeensis genes critical for persistent infection in a vertebrate host are identified as nonessential for its growth in the tick vector; Amblyomma americanum.

Ehrlichia chaffeensis is a tick-transmitted monocytic ehrlichiosis agent primarily causing the disease in people and dogs. We recently described the development and characterization of 55 random mutations in E. chaffeensis, which aided in defining the critical nature of many bacterial genes for its growth in a physiologically relevant canine infection model. In the current study, we tested 45 of the mutants for their infectivity ability to the pathogen's tick vector; Amblyomma americanum. Four mutations resulted in the pathogen's replication deficiency in the tick, similar to the vertebrate host. Mutations causing growth defects in both vertebrate and tick hosts included in genes coding for a predicted alpha/beta hydrolase, a putative dicarboxylate amino acid:cation symporter, a T4SS protein, and predicted membrane-bound proteins. Three mutations caused the bacterial defective growth only in the tick vector, which represented putative membrane proteins. Ten mutations causing no growth defect in the canine host similarly grew well in the tick vector. Mutations in 28 genes/genomic locations causing E. chaffeensis growth attenuation in the canine host were recognized as non-essential for its growth in the tick vector. The tick non-essential genes included genes coding for many metabolic pathway- and outer membrane-associated proteins. This study documents novel vector- and host-specific differences in E. chaffeensis for its functional gene requirements.

Capybara density and climatic factors as modulators of Ehrlichia prevalence in questing ticks in the Iberá wetlands, Argentina.

We evaluated the presence of Ehrlichia spp. in unfed capybara ticks, Amblyomma dubitatum, and explored its association with capybaras density, ticks density and environmental variables. We observed that in the Iberá wetlands ecoregion A. dubitatum is infected by "Candidatus Ehrlichia hydrochoerus" and in a lesser extent with an Ehrlichia species closely related to Ehrlichia chaffeensis. The frequency of "Ca. Ehrlichia hydrochoerus" presence in A. dubitatum was not associated with vector abundance, but the probability of finding "Ca. Ehrlichia hydrochoerus"-infected ticks increased when the density of capybaras was low two months before. We hypothesize that when the density of capybaras decreases, A. dubitatum immature stages may seek out alternative hosts one of which could exhibit high realized reservoir competence for "Ca. Ehrlichia hydrochoerus", leading to an increased prevalence of this ehrlichiae in questing A. dubitatum. High minimum temperatures and high cumulative rainfall in the time period previous to tick collection (15 to 60 days) were positively correlated with the prevalence of "Ca. Ehrlichia hydrochoerus" infection in A. dubitatum. Our results suggest that a combination of factors (both biological and abiotic) could raise the risk of human exposure to tick-borne Ehrlichia in the Iberá wetlands ecoregion.

First report of pediatric ehrlichiosis in Mexico.

BACKGROUND: Ehrlichia chaffeensis is responsible for most cases of human ehrlichiosis, an acute febrile tick-borne disease. This clinical entity is more commonly reported in adults from the United States. Therefore, it is of special interest to characterize this disease in children, given that very few cases in children have been reported outside of this country. CASE REPORT: We describe the case of a 15-year-old female from northeastern Mexico with a five-day history of myalgias, arthralgias, fever, abdominal pain, rash, and somnolence. The possibility of tick-borne disease was suspected considering that she lived with three tick-infested dogs that had recently died and a neighbor with similar symptoms who deteriorated rapidly and died a week earlier. Ehrlichia spp. was detected in blood samples by polymerase chain reaction. The patient completed a seven-day course of doxycycline and was discharged with complete resolution of symptoms. CONCLUSIONS: This case is the first report of ehrlichiosis in a pediatric patient in Mexico, illustrating the importance of considering tick-borne diseases as a differential diagnosis in patients with rash, fever, and altered level of consciousness. This initial clinical presentation may be indistinct from other conditions such as dengue, meningococcemia, and multisystem inflammatory syndrome in children (MIS-C), among others.

INTRODUCCIÓN: Ehrlichia chaffeensis es responsable de la mayoría de los casos de ehrlichiosis humana, una enfermedad febril aguda transmitida por garrapatas. Esta entidad clínica se reporta con mayor frecuencia en adultos de Estados Unidos. Por lo tanto, es de especial interés caracterizarla en niños, dado que se han reportado muy pocos casos en niños fuera de este país. CASO CLÍNICO: Se describe el caso de una paciente de sexo femenino de 15 años, originaria y residente del noreste de México con una historia de cinco días de mialgias, artralgias, fiebre, dolor abdominal, erupción cutánea y somnolencia. Se sospechó la posibilidad de una enfermedad transmitida por garrapatas considerando que convivió con tres perros infestados de garrapatas que habían muerto recientemente y una vecina con síntomas similares, quien se deterioró rápidamente y murió una semana antes. Ehrlichia spp. se detectó en una muestra sérica mediante reacción en cadena de la polimerasa. La paciente completó un curso de siete días de doxiciclina y fue dada de alta con resolución de los síntomas. CONCLUSIONES: Este caso es el primer reporte de ehrlichiosis en un paciente pediátrico en México que ilustra la importancia de considerar enfermedades transmitidas por garrapatas dentro del diagnóstico diferencial de pacientes con exantema, fiebre y alteración del estado de conciencia. Esta presentación clínica inicial puede ser indistinguible de otras entidades como dengue, meningococcemia y síndrome multisistémico inflamatorio, entre otras.

Molecular detection and genetic characterization of Ehrlichia canis and Ehrlichia sp. in neotropical primates from Brazil.

The Anaplasmataceae family includes obligate, arthropod-transmitted intracellular bacteria that can be zoonotic and potentially fatal. Studies focusing on the interaction between neotropical primates and the agents of this family are scarce. The present study aimed to identify agents of the Anaplasmataceae family in the whole blood of free-living and captive neotropical primates in the State of Mato Grosso, Central-West Brazil. Thirty-eight samples of six nonhuman primate (NHP) species were collected in seven municipalities and analysed through polymerase chain reaction (PCR), nucleotide sequencing, and phylogenetic analysis of the dsb, groEL, 16S rRNA, and gltA genes. DNA fragments similar to those of Ehrlichia canis were detected in Sapajus apella and Ehrlichia chaffeensis from Mico melanurus. The sequences generated in this study and homologous sequences retrieved from GenBank® were used for phylogenetic analyses to characterize the Ehrlichial agents detected in NHPs. The agents were then grouped into clades corresponding to different isolates from the NHP species. In addition, an Anaplasma sp. closely related to Anaplasma marginale was identified in two S. apella individuals. These findings shed light on the susceptibility of neotropical NHPs to Anaplasmataceae agents. These bacteria are known to be transmitted by ticks, which can also serve as possible sources of infection for other animals, including humans.

Ehrlichia effector SLiM-icry: Artifice of cellular subversion.

As an obligately intracellular bacterial pathogen that selectively infects the mononuclear phagocyte, Ehrlichia chaffeensis has evolved sophisticated mechanisms to subvert innate immune defenses. While the bacterium accomplishes this through a variety of mechanisms, a rapidly expanding body of evidence has revealed that E. chaffeensis has evolved survival strategies that are directed by the versatile, intrinsically disordered, 120 kDa tandem repeat protein (TRP120) effector. E. chaffeensis establishes infection by manipulating multiple evolutionarily conserved cellular signaling pathways through effector-host interactions to subvert innate immune defenses. TRP120 activates these pathways using multiple functionally distinct, repetitive, eukaryote-mimicking short linear motifs (SLiMs) located within the tandem repeat domain that have evolved in nihilo. Functionally, the best characterized TRP120 SLiMs mimic eukaryotic ligands (SLiM-icry) to engage pathway-specific host receptors and activate cellular signaling, thereby repurposing these pathways to promote infection. Moreover, E. chaffeensis TRP120 contains SLiMs that are targets of post-translational modifications such as SUMOylation in addition to many other validated SLiMs that are curated in the eukaryotic linear motif (ELM) database. This review will explore the extracellular and intracellular roles TRP120 SLiM-icry plays during infection - mediated through a variety of SLiMs - that enable E. chaffeensis to subvert mononuclear phagocyte innate defenses.

Genome-Wide Subtraction Analysis and Reverse Vaccinology to Detect Novel Drug Targets and Potential Vaccine Candidates Against Ehrlichia chaffeensis.

Human monocytotropic ehrlichiosis is an emerging tick-borne infection caused by the obligate intracellular pathogen, Ehrlichia chaffeensis. The non-specific symptoms can range from a self-limiting fever to a fatal septic-like syndrome and may be misdiagnosed. The limited treatment choices including doxycycline are effective only in the initiation phase of the infection. It seems that novel therapeutic targets and new vaccine strategies could be effective to control this pathogen. This study is comprised of two major phases. First, the common proteins retrieved through subtractive analysis and potential drug targets were evaluated by subcellular localization, homology prediction, metabolic pathways, druggability, essentiality, protein-protein interaction networks, and protein data bank availability. In the second phase, surface-exposed proteins were assessed based on antigenicity, allergenicity, physiochemical properties, B cell and T cell epitopes, conserved domains, and protein-protein interaction networks. A multi-epitope vaccine was designed and characterized using molecular dockings and immune simulation analysis. Six proteins including WP_011452818.1, WP_011452723.1, WP_006010413.1, WP_006010278.1, WP_011452938.1, and WP_006010644.1 were detected. They belong to unique metabolic pathways of E. chaffeensis that are considered as new essential drug targets. Based on the reverse vaccinology, WP_011452702.1, WP_044193405.1, WP_044170604.1, and WP_006010191.1 proteins were potential vaccine candidates. Finally, four B cell epitopes, including SINNQDRNC, FESVSSYNI, SGKKEISVQSN, and QSSAKRKST, were used to generate the multi-epitope vaccine based on LCL platform. The vaccine showed strong interactions with toll-like receptors and acceptable immune-reactivity by immune simulation analysis. The findings of this study may represent a turning point in developing an effective drug and vaccine against E. chaffeensis. However, further experimental analyses have remained.

County-level surveillance for the lone star tick, Amblyomma americanum, and its associated pathogen, Ehrlichia chaffeensis, in Kentucky.

Kentucky experiences some of the highest incidence rates for ehrlichiosis nationwide. Ehrlichiosis is a bacterial infection caused primarily by the pathogen Ehrlichia chaffeensis and can be transmitted to humans through the bite of an infected tick, notably Amblyomma americanum. Amblyomma americanum, the lone star tick, is common to Kentucky and much of the southeast, but has expanded farther north in recent years. As an abundant and aggressive nondiscriminatory biter, this species is of major public health concern for transmission of pathogens to humans. As this vector's range expands, surveillance remains a necessary tool providing data that allows researchers to track this expansion over time. The historical information on tick distribution in Kentucky is variable with very little data on a statewide scale. From January 2019 to December 2020, we conducted surveillance for A. americanum in Kentucky through field collections and the establishment of a statewide tick submission program with the help of the Kentucky Department for Public Health and screened for E. chaffeensis on a county-level throughout the state. We collected 5,726 A. americanum ticks in 77 counties and detected E. chaffeensis in 32 counties. The minimum infection rate was 1.8%. With the expansion of A. americanum and increasing cases of tick-borne diseases, future surveillance is needed to monitor this important tick vector over time.

Tick innate immune responses to hematophagy and Ehrlichia infection at single-cell resolution.

Introduction: Ticks rely on robust cellular and humoral responses to control microbial infection. However, several aspects of the tick's innate immune system remain uncharacterized, most notably that of the immune cells (called hemocytes), which are known to play a significant role in cellular and humoral responses. Despite the importance of hemocytes in regulating microbial infection, our understanding of their basic biology and molecular mechanisms remains limited. Therefore, we believe that a more detailed understanding of the role of hemocytes in the interactions between ticks and tick-borne microbes is crucial to illuminating their function in vector competence and to help identify novel targets for developing new strategies to block tick-borne pathogen transmission. Methods: This study examined hemocytes from the lone star tick (Amblyomma americanum) at the transcriptomic level using the 10X genomics single-cell RNA sequencing platform to analyze hemocyte populations from unfed, partially blood-fed, and Ehrlichia chaffeensis-infected ticks. The functional role of differentially expressed hemocyte markers in hemocyte proliferation and Ehrlichia dissemination was determined using an RNA interference approach. Results and discussion: Our data exhibit the identification of fourteen distinct hemocyte populations. Our results uncover seven distinct lineages present in uninfected and Ehrlichia-infected hemocyte clusters. The functional characterization of hemocytin, cystatin, fibronectin, and lipocalin demonstrate their role in hemocyte population changes, proliferation, and Ehrlichia dissemination. Conclusion: Our results uncover the tick immune responses to Ehrlichia infection and hematophagy at a single-cell resolution. This work opens a new field of tick innate immunobiology to understand the role of hemocytes, particularly in response to prolonged blood-feeding (hematophagy), and tick-microbial interactions.

Antibody reactive immunomes of Ehrlichia chaffeensis and E. canis are diverse and defined by conformational antigenic determinants.

For decades, the defined antibody reactive proteins of Ehrlichia chaffeensis and E. canis were limited to a small group with linear antibody epitopes. Recently, our laboratory has utilized an immunomics-based approach to rapidly screen and identify undefined Ehrlichia chaffeensis and E. canis antigenic proteins and antibody epitopes. In this study, we analyzed the remaining portion (~50%) of the E. chaffeensis and E. canis proteomes (n = 444 and n = 405 proteins, respectively), that were not examined in previous studies, to define the complete immunomes of these important pathogens. Almost half of the E. chaffeensis proteins screened (196/444) reacted with antibodies in convalescent HME patient sera, while only 43 E. canis proteins reacted with CME dog sera. New major immunoreactive proteins were identified in E. chaffeensis (n = 7) and E. canis (n = 1), increasing the total number of E. chaffeensis (n = 14) and E. canis proteins (n = 18) that exhibited antibody reactivity comparable to well-defined major antigenic proteins (TRP120 and TRP19). All of the E. chaffeensis but only some E. canis major immunoreactive proteins contained major conformation-dependent antibody epitopes. The E. chaffeensis immunoreactive proteins were generally small (< 250 amino acids; ~27kDa) and the E. canis proteins were slightly larger (> 320 amino acids; ~35 kDa). The majority of these new Ehrlichia major immunoreactive proteins were predicted to be type I secreted effectors, some of which contained transmembrane domains. Characterization of the immunomes of E. chaffeensis and E. canis and understanding the host specific Ehrlichia immune responses will facilitate identification of protective antigens and define the biophysical epitope characteristics vital to effective vaccine development for the ehrlichioses.

CtrA activates the expression of glutathione S-transferase conferring oxidative stress resistance to Ehrlichia chaffeensis.

Ehrlichia chaffeensis, the causative agent of human monocytic ehrlichiosis (HME), is a Gram-negative obligatory intracellular bacterium, which infects and multiplies in human monocytes and macrophages. Host immune cells produce reactive oxygen species (ROS) to eliminate E. chaffeensis upon infection. E. chaffeensis global transcriptional regulator CtrA activates the expression of GshA and GshB to synthesize glutathione (GSH), the most potent natural antioxidant, upon oxidative stress to combat ROS damage. However, the mechanisms exploited by E. chaffeensis to utilize GSH are still unknown. Here, we found that in E. chaffeensis CtrA activated the expression of glutathione S-transferase (GST) upon oxidative stress, and E. chaffeensis GST utilizes GSH to eliminate ROS and confers the oxidative stress resistance to E. chaffeensis. We found that CtrA bound to the promoter regions of 211 genes, including gst, in E. chaffeensis using chromatin immunoprecipitation coupled to deep sequencing (ChIP-seq). Recombinant E. chaffeensis CtrA directly bound to the gst promoter region determined with electrophoretic mobility shift assay (EMSA), and activated the gst expression determined with reporter assay. Recombinant GST showed GSH conjugation activity towards its typical substrate 2,4-dinitrochlorobenzene (CDNB) in vitro and peptide nucleic acid (PNA) transfection of E. chaffeensis, which can knock down the gst transcription level, reduced bacterial survival upon oxidative stress. Our results demonstrate that E. chaffeensis CtrA regulates GSH utilization, which plays a critical role in resistance to oxidative stress, and aid in the development of new therapeutics for HME.

Evaluating EcxR for Its Possible Role in Ehrlichia chaffeensis Gene Regulation.

Ehrlichia chaffeensis, a tick-transmitted intraphagosomal bacterium, is the causative agent of human monocytic ehrlichiosis. The pathogen also infects several other vertebrate hosts. E. chaffeensis has a biphasic developmental cycle during its growth in vertebrate monocytes/macrophages and invertebrate tick cells. Host- and vector-specific differences in the gene expression from many genes of E. chaffeensis are well documented. It is unclear how the organism regulates gene expression during its developmental cycle and for its adaptation to vertebrate and tick host cell environments. We previously mapped promoters of several E. chaffeensis genes which are recognized by its only two sigma factors: σ32 and σ70. In the current study, we investigated in assessing five predicted E. chaffeensis transcription regulators; EcxR, CtrA, MerR, HU and Tr1 for their possible roles in regulating the pathogen gene expression. Promoter segments of three genes each transcribed with the RNA polymerase containing σ70 (HU, P28-Omp14 and P28-Omp19) and σ32 (ClpB, DnaK and GroES/L) were evaluated by employing multiple independent molecular methods. We report that EcxR binds to all six promoters tested. Promoter-specific binding of EcxR to several gene promoters results in varying levels of gene expression enhancement. This is the first detailed molecular characterization of transcription regulators where we identified EcxR as a gene regulator having multiple promoter-specific interactions.