Analysis of Amblyomma americanum microRNAs in response to Ehrlichia chaffeensis infection and their potential role in vectorial capacity.

Background: MicroRNAs (miRNAs) represent a subset of small noncoding RNAs and carry tremendous potential for regulating gene expression at the post-transcriptional level. They play pivotal roles in distinct cellular mechanisms including inhibition of bacterial, parasitic, and viral infections via immune response pathways. Intriguingly, pathogens have developed strategies to manipulate the host's miRNA profile, fostering environments conducive to successful infection. Therefore, changes in an arthropod host's miRNA profile in response to pathogen invasion could be critical in understanding host-pathogen dynamics. Additionally, this area of study could provide insights into discovering new targets for disease control and prevention. The main objective of the present study is to investigate the functional role of differentially expressed miRNAs upon Ehrlichia chaffeensis, a tick-borne pathogen, infection in tick vector, Amblyomma americanum. Methods: Small RNA libraries from uninfected and E. chaffeensis-infected Am. americanum midgut and salivary gland tissues were prepared using the Illumina Truseq kit. Small RNA sequencing data was analyzed using miRDeep2 and sRNAtoolbox to identify novel and known miRNAs. The differentially expressed miRNAs were validated using a quantitative PCR assay. Furthermore, a miRNA inhibitor approach was used to determine the functional role of selected miRNA candidates. Results: The sequencing of small RNA libraries generated >147 million raw reads in all four libraries and identified a total of >250 miRNAs across the four libraries. We identified 23 and 14 differentially expressed miRNAs in salivary glands, and midgut tissues infected with E. chaffeensis, respectively. Three differentially expressed miRNAs (miR-87, miR-750, and miR-275) were further characterized to determine their roles in pathogen infection. Inhibition of target miRNAs significantly decreased the E. chaffeensis load in tick tissues, which warrants more in-depth mechanistic studies. Conclusions: The current study identified known and novel miRNAs and suggests that interfering with these miRNAs may impact the vectorial capacity of ticks to harbor Ehrlichia. This study identified several new miRNAs for future analysis of their functions in tick biology and tick-pathogen interaction studies.

Unraveling the Complexity: Ehrlichiosis-Induced Septic Shock and Acute Respiratory Distress Syndrome.

Human monocytic ehrlichiosis typically presents with nonspecific cold-like symptoms and a history of recent tick exposure, often responding well to early treatment. Here, we present the case of a 67-year-old immunocompetent male who initially presented with fevers, chills, dysuria, and hematuria, leading to admission to the intensive care unit with septic shock and acute respiratory distress syndrome (ARDS), which was later attributed to Ehrlichia chaffeensis infection. Prompt treatment with doxycycline resulted in a full clinical recovery. This case highlights the rare occurrence of severe ehrlichiosis and provides insights into its effective management based on updated literature.

Ehrlichiosis: A Unique Presentation of Fatal Sepsis in an Immunocompetent Adult.

Ehrlichiosis is a vector-borne illness transmitted by the lone star tick (Amblyomma americanum). Most patients have risk factors for tick exposure, such as hobbies or careers involving hunting, camping, and hiking. This case exposes a rare case of severe ehrlichiosis, ultimately resulting in fatal sepsis, in an elderly patient without any reported tick bites or exposures. This patient had a history of cognitive impairment, which was confounded by acute encephalopathy at presentation. Unfortunately, this hindered his ability to report any known tick exposures, which posed a challenge in the diagnosis and ultimately delayed treatment as there were no clear findings of a tick bite or known exposures.

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.

Alpha-Gal IgE Prevalence Patterns in the United States: An Investigation of 3,000 Military Recruits.

BACKGROUND: IgE to the oligosaccharide galactose-alpha-1,3-galactose (alpha-gal) is an important cause of allergic reactions to mammalian meat. The "alpha-gal syndrome" is strongly associated with a preceding history of tick bites and in the United States is most commonly reported in parts of the southeast, but there has been limited investigation into national alpha-gal sensitization patterns and the relevance of other risk factors. OBJECTIVE: To systematically investigate alpha-gal IgE prevalence, regional patterns, and risk factors. METHODS: Alpha-gal IgE was measured by ImmunoCAP in biobanked serum samples collected from 3000 service members who presented for intake to 1 of 10 military bases in the central/eastern United States. Alpha-gal IgE sensitization (cutoff 0.1 international units/mL) was related to home of record at enlistment. RESULTS: Of the cohort, 2456 (81.9%) subjects were male, median age was 19 years (interquartile range: 18-22 years), and alpha-gal IgE was detected in 179 (6.0%). Home of record spanned all 50 states, with a median of 36 recruits per state (range: 3-261). The highest prevalence rates were in Arkansas (39%), Oklahoma (35%), and Missouri (29%), with several other southeastern states >10%. Granular mapping revealed sensitization patterns that closely mimicked county-level Amblyomma americanum reports and Ehrlichia chaffeensis infections. Sensitization was associated with male sex, rural residence, and White race in univariate and multivariable models. CONCLUSIONS: In this systematic survey, the prevalence of alpha-gal IgE among incoming military personnel was 6.0%. There were significant regional differences, with an overall pattern consistent with the known range of the lone star tick (A. americanum) and highest frequency in an area including Arkansas, Oklahoma, and Missouri.

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.

Ehrlichia-Induced Hemophagocytic Lymphohistiocytosis (HLH) With No Response to Doxycycline Treatment.

Hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening condition characterized by the persistent activation of antigen-presenting cells and multisystemic inflammation. Ehrlichiosis is a tick-born infection that primarily infects the white blood cells and can present with a variety of symptoms, including fever, fatigue, and multisystemic complications. Here, we present a 58-year-old female hospitalized for a urinary tract infection. Her hospital course was complicated by HLH, which was later discovered to be precipitated by an Ehrlichia chaffeensis infection. The patient did not respond to the doxycycline treatment, developed multiorgan failure, and passed away after a few weeks of treatment trials.

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.

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.

Inhibition of Ehrlichia chaffeensis infection by cell-permeable macrocyclic peptides that bind type IV secretion effector Etf-1.

Ehrlichia chaffeensis is an obligatory intracellular bacterium that infects monocytes and macrophages, and causes human monocytic ehrlichiosis, an emerging life-threatening infectious disease. Ehrlichia translocated factor-1 (Etf-1), a type IV secretion system effector, is essential for Ehrlichia infection of host cells. Etf-1 translocates to mitochondria to block host apoptosis; furthermore, it can bind Beclin 1 (ATG6) to induce cellular autophagy and localize to E. chaffeensis-inclusion membrane to obtain host-cell cytoplasmic nutrients. In this study, we screened a synthetic library of over 320,000 cell-permeable macrocyclic peptides, which consist of an ensemble of random peptide sequences in the first ring and a small family of cell-penetrating peptides in the second ring, for Etf-1 binding. Library screening followed by hit optimization identified multiple Etf-1-binding peptides (with K D values of 1-10 µM) that efficiently enter the cytosol of mammalian cells. Peptides B7, C8, B7-131-5, B7-133-3, and B7-133-8 significantly inhibited Ehrlichia infection of THP-1 cells. Mechanistic studies revealed that peptide B7 and its derivatives inhibited the binding of Etf-1 to Beclin 1, and Etf-1 localization to E. chaffeensis-inclusion membranes, but not Etf-1 localization to the mitochondria. Our results not only affirm the critical role of Etf-1 functions in E. chaffeensis infection, but also demonstrate the feasibility of developing macrocyclic peptides as powerful chemical probes and potential treatment of diseases caused by Ehrlichia and other intracellular pathogens.

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.

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.

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.

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.

Automated Real-Time PCR Detection of Tickborne Diseases Using the Panther Fusion Open Access System.

The incidence of tickborne infections in the United States has risen significantly. Automation is needed for the increasing demand for testing. The Panther Fusion (Fusion) has an Open Access functionality to perform lab developed tests (LDTs) on a fully automated system. Our laboratory adapted two LDTs on Fusion; a multiplex real-time PCR for Anaplasma phagocytophilum and Ehrlichia chaffeensis (AP/EC) and a Babesia microti (BM) PCR. Limits of detection (LODs) were performed with target region plasmid panels spiked into whole blood. The LODs for AP, BM, and EC on the Fusion were 11, 17, and 10 copies/reaction, respectively. The performance of AP/EC was evaluated with 80 whole blood specimens, including 50 specimens previously positive for AP by our test of record (TOR) and 30 specimens (including 20 AP positive) spiked with EC plasmid. AP was detected in 49 out of 50 positive specimens and EC was detected in all 30 spiked specimens. BM PCR on Fusion was evaluated with 75 whole blood samples, including 16 specimens previously shown to be positive for BM and 59 negative specimens, of which 29 were spiked with BM plasmid DNA. BM was detected in 45 samples as expected. AP/EC and BM PCRs were successfully developed and optimized on the Panther Fusion with performance characteristics comparable to our TOR. These assays complement each other and allow for a modular testing approach for tickborne diseases which have differing clinical presentation. Furthermore, automation of these assays will help the lab meet the increasing demand for testing. IMPORTANCE Since the incidence of tickborne diseases has been accelerating in the United States, automation for testing has become essential in affected regions. Unfortunately, because the need is regional, commercial test manufacturers have not yet provided answers for clinical laboratories. Here, we describe the development of PCR tests on the highly automated Panther Fusion for three tickborne diseases. The Panther Fusion assays were evaluated using 155 archived whole blood (WB) specimens previously tested for Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Babesia microti, while WB spiked with DNA from plasmid clones of the target regions were used for analytical sensitivity. We demonstrated that the Panther Fusion assays performed similar to the manual PCR tests used clinically in our laboratory and that automation of these tests had no adverse effect on the performance.

An Unusual Case of Ehrlichiosis Manifesting With Hyponatremia, Acute Encephalopathy, and Hemophagocytic Lymphohistiocytosis.

Ehrlichiosis is a tick-borne infection that has become increasingly more common in the United States in recent years. We present a case of a patient who was found to have confusion, hyponatremia, and hemophagocytic lymphohistiocytosis after contracting Ehrlichia chaffeensis following a tick exposure. This unusual presentation emphasizes the need for increased awareness of the varied symptoms of this infection and the importance of obtaining a complete history from patients at risk of vector-borne diseases.

Genetic characterization of a novel Ehrlichia chaffeensis genotype from an Amblyomma tenellum tick from South Texas, USA.

Ehrlichia chaffeensis is the causative agent of human monocytotropic ehrlichiosis (HME), a disease that ranges in severity from mild to fatal infection. Ehrlichia chaffeensis is maintained in a zoonotic cycle involving white-tailed deer (Odocoileus virginianus) as the main vertebrate reservoir and lone star ticks (Amblyomma americanum) as its principal vector. Through complete genomic analysis from human ehrlichial isolates and DNA sequences obtained from deer and tick specimens, nine strains of E. chaffeensis have been characterized. Few studies have examined the genetic diversity of E. chaffeensis in ticks, and some of these investigations have identified that the genetic sequences coincide with the circulating strains reported so far. Here, we report the first evidence of E. chaffeensis DNA from an unfed Amblyomma tenellum (formerly Amblyomma imitator) collected in South Texas. We characterized the genetic variation of this E. chaffeensis genotype using conserved gene markers such as rRNA, dsb, and groEL. We also used gene targets useful to distinguish genotypes, such as the variable length PCR target gene (VLPT) and 120-kDa gene, encoding the tandem-repeat proteins TRP32 and TRP120, respectively. Our results suggest a novel E. chaffeensis genotype that exhibited greater variability than other genotypes of E. chaffeensis and highlights the role for A. tenellum as a potential vector of E. chaffeensis.

Glutathione Synthesis Regulated by CtrA Protects Ehrlichia chaffeensis From Host Cell Oxidative Stress.

Ehrlichia chaffeensis, a small Gram-negative obligatory intracellular bacterium, infects human monocytes or macrophages, and causes human monocytic ehrlichiosis, one of the most prevalent, life-threatening emerging zoonoses. Reactive oxygen species are produced by the host immune cells in response to bacterial infections. The mechanisms exploited by E. chaffeensis to resist oxidative stress have not been comprehensively demonstrated. Here, we found that E. chaffeensis encodes two functional enzymes, GshA and GshB, to synthesize glutathione that confers E. chaffeensis the oxidative stress resistance, and that the expression of gshA and gshB is upregulated by CtrA, a global transcriptional regulator, upon oxidative stress. We found that in E. chaffeensis, the expression of gshA and gshB was upregulated upon oxidative stress using quantitative RT-PCR. Ehrlichia chaffeensis GshA or GshB restored the ability of Pseudomonas aeruginosa GshA or GshB mutant to cope with oxidative stress, respectively. Recombinant E. chaffeensis CtrA directly bound to the promoters of gshA and gshB, determined with electrophoretic mobility shift assay, and activated the expression of gshA and gshB determined with reporter assay. Peptide nucleic acid transfection of E. chaffeensis, which reduced the CtrA protein level, inhibited the oxidative stress-induced upregulation of gshA and gshB. Our findings provide insights into the function and regulation of the two enzymes critical for E. chaffeensis resistance to oxidative stress and may deepen our understanding of E. chaffeensis pathogenesis and adaptation in hosts.

Ehrlichiosis in Dogs: A Comprehensive Review about the Pathogen and Its Vectors with Emphasis on South and East Asian Countries.

Ehrlichiosis in dogs is an emerging vector borne rickettsial zoonotic disease of worldwide distribution. In general, three Ehrlichial species (Ehrlichia canis, E. ewingii, and E. chaffeensis) are involved in infecting dogs. Among them, E. canis is the well-known etiological pathogen affecting platelets, monocytes, and granulocytes. Dogs act as a reservoir, while the main vector responsible for disease transmission is Rhipicephalus sanguineus. However, in east Asian countries, Haemaphysalis longicornis is considered the principal vector for disease transmission. This disease affects multiple organs and systems and has three clinical manifestations, including acute, subclinical, and chronic. Definitive diagnosis involves visualization of morulae on cytology, detection of antibodies through an indirect immunofluorescence test (IFAT), and DNA amplification by polymerase chain reaction (PCR). In canine ehrlichiosis, no predilection of age or sex is observed; however, Siberian Huskies and German Shepherds are more likely to develop severe clinical manifestations. Doxycycline, rifampicin, and minocycline are proven to be effective drugs against canine ehrlichiosis. This review is intended to describe a brief overview of Ehrlichia infection in dogs, its reported prevalence in east and south Asian countries, and the latest knowledge regarding chemotherapy and associated vectors responsible for the disease transmission. This manuscript also identifies the prevailing knowledge gaps which merit further attention by the scientific community.