Human Monocytic Ehrlichiosis Research Articles

МОНИТОРИНГ ПРИРОДНО-ОЧАГОВЫХ ИНФЕКЦИЙ НА ЮГЕ ЕВРОПЕЙСКОЙ ЧАСТИ РОССИИ В 2016 ГОДУ

The results of epizootological monitoring of the Southern European part of Russia (Southern and North- Caucasian Federal Districts), which has been tested for 18 nosological forms of natural focal infections are presented. There have been tested the laboratory studies of 45 615 samples of field material, the markers of pathogens of 15 nosological forms are identified. The circulation of Crimean-Congo hemorrhagic fever virus is identified in 10 subjects, the pathogen of tularemia – in eight regions, Lyme borreliosis – in seven, Q fever – in six; pathogens of hemorrhagic fever with renal syndrome, West Nile fever, leptospirosis and human granulocytic anaplasmosis – in five subjects. The markers of pathogens of human monocytic ehrlichiosis and tick-borne viral encephalitis were detected in four subjects, pseudotuberculosis and intestinal yersiniosis – in three and Arboviruses (Sindbis, Inkoо, Tahyna) – in one region.

An unusual case of Human Monocytic Ehrlichiosis

An unusual case of Human Monocytic Ehrlichiosis

PCR Results of Vector and Carrier Investigations for the Presence of Tick-Borne Borreliosis, Human Monocytic Ehrlichiosis, and Human Granulocytic Anaplasmosis Agents in Natural Foci of the Rostov Region

Objective of the study is to detect the circulation of the agents of ixodic tick-borne borreliosis (ITB), human monocytic ehrlichiosis (HME), and human granulocytic anaplasmosis (HGA) in samples collected from carriers and vectors of transmissible tick-borne infections using PCR-assay. Materials and methods. Field material was studied in PCR for evaluating the rate of infection by causative agents of tick-borne infections (tick-borne borrelioses, ehrlichioses, anaplasmosis) in combined natural foci on the territory of the Rostov Region. Results and conclusions. The data obtained during epizootiological investigations carried out in 21 administrative districts and 10 cities of the Rostov Region between 2014 and 2016 were used in the study. The results were indicative of spontaneous infection in ixodic ticks by ITB causative agent on the territory of the Rostov Region, as well as of presence of corresponding natural focus. Since 2013 the activity of different components of ITB natural focus was confirmed by the detection and official registration of patients. Moreover, in the Rostov Region for the first time ever the circulation of HME and HGA causative agents was recorded and territorially combined foci of HME and HGA with ITB were identified. The results obtained allow assuming the genesis of a focus of ixodic tick-borne borreliosis and monocytic and granulocytic ehrlichioses owing to annual expansion of the areal of ticks, infected by these pathogens, and animals on which the ticks are feeding. PCR-analysis is of significant value for assessment of spread of causative agents of the “tick-borne” infections in the Rostov Region, forecasting of epidemiological situation, as well as implementation of preventive measures in the region.

A genetic system for targeted mutations to disrupt and restore genes in the obligate bacterium, Ehrlichia chaffeensis

Obligate intracellular bacteria (obligates) belonging to Rickettsiales and Chlamydiales cause diseases in hundreds of millions of people worldwide and in many animal species. Lack of an efficient system for targeted mutagenesis in obligates remains a major impediment in understanding microbial pathogenesis. Challenges in creating targeted mutations may be attributed to essential nature of majority of the genes and intracellular replication dependence. Despite success in generating random mutations, a method that works well in creating mutations in specific genes of interest followed by complementation remains problematic for obligates and is a highly sought-after goal. We describe protocols to generate stable targeted mutations by allelic exchange in Ehrlichia chaffeensis, an obligate intracellular tick-borne bacterium responsible for human monocytic ehrlichiosis. Targeted mutations in E. chaffeensis were created to disrupt two genes, and also to restore one gene by another allelic exchange mutation leading to the restoration of transcription and protein expression from the inactivated gene and the recovered organisms also express mCherry, which distinguishes from the wild type. We expect that the methods developed are broadly applicable to other obligates, particularly to rickettsial pathogens, to routinely perform targeted mutations to enable studies focused on protein structure-function analyses, host-pathogen interactions and in developing vaccines.

Sudden Sensorineural Hearing Loss in Human Monocytic Ehrlichiosis

The hallmarks of symptomatic human monocytic ehrlichiosis (HME) include fever, headache, myalgia, nausea, malaise, transaminitis, and blood cell abnormalities. Previous case reports have described isolated cranial nerve palsies in infected patients but not hearing loss. We describe the onset of sudden sensorineural hearing loss in 2 patients with HME-a 31-year-old woman and an 82-year-old woman. The older patient experienced objective and subjective improvement in her hearing after treatment with an antibiotic and steroid taper; the younger patient was lost to audiologic follow-up. Additionally, we discuss the possible mechanisms of the hearing loss in these patients.

Human Monocytic Ehrlichiosis presenting as Guillain–Barré syndrome (P3.315)

Human Monocytic Ehrlichiosis presenting as Guillain–Barré syndrome (P3.315)

Detection of Borrelia miyamotoi and other tick-borne pathogens in human clinical specimens and Ixodes scapularis ticks in New York State, 2012–2015

Borrelia miyamotoi (Bm) is a recently emerging bacterial agent transmitted by several species of ixodid ticks. Diagnosis of Bm infection can be challenging, as the organism is not easily cultivable. We have developed and validated a multiplex real-time PCR to simultaneously identify Bm infection and the agents causing human granulocytic anaplasmosis and human monocytic ehrlichiosis, Anaplasma phagocytophilum and Ehrlichia chaffeensis, respectively. The assay is 100% specific; highly sensitive, detecting 11 gene copies of Bm DNA in both whole blood and cerebral spinal fluid; and provides rapid results in less than two hours. A retrospective study of 796 clinical specimens collected between the years 2012 and 2014 and a prospective study of 366 clinical specimens were performed utilizing this novel assay to evaluate the frequency of Bm infection in New York State (NYS). Eight clinical specimens (1%) were found to be positive for Bm, 216 were positive for A. phagocytophilum, and 10 were positive for E. chaffeensis. Additionally, we tested 411 I. scapularis ticks collected in NYS during 2013 and 2014 in a separate multiplex real-time PCR to determine the prevalence of Bm, A. phagocytophilum, Borrelia burgdorferi s.s., and Borrelia species. Our results indicated rates of 1.5%, 27%, 19.7%, and 8.8% respectively. The ability to monitor both the frequency and geographic distribution of Bm cases and the prevalence and geographic distribution of Bm in ticks will help create a better understanding of this emerging tick-borne pathogen.

Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing.

Ehrlichioses: An Important One Health Opportunity.

Ehrlichioses are caused by obligately intracellular bacteria that are maintained subclinically in a persistently infected vertebrate host and a tick vector. The most severe life-threatening illnesses, such as human monocytotropic ehrlichiosis and heartwater, occur in incidental hosts. Ehrlichia have a developmental cycle involving an infectious, nonreplicating, dense core cell and a noninfectious, replicating reticulate cell. Ehrlichiae secrete proteins that bind to host cytoplasmic proteins and nuclear chromatin, manipulating the host cell environment to their advantage. Severe disease in immunocompetent hosts is mediated in large part by immunologic and inflammatory mechanisms, including overproduction of tumor necrosis factor α (TNF-α), which is produced by CD8 T lymphocytes, and interleukin-10 (IL-10). Immune components that contribute to control of ehrlichial infection include CD4 and CD8 T cells, natural killer (NK) cells, interferon-γ (IFN-γ), IL-12, and antibodies. Some immune components, such as TNF-α, perforin, and CD8 T cells, play both pathogenic and protective roles. In contrast with the immunocompetent host, which may die with few detectable organisms owing to the overly strong immune response, immunodeficient hosts die with overwhelming infection and large quantities of organisms in the tissues. Vaccine development is challenging because of antigenic diversity of E. ruminantium, the necessity of avoiding an immunopathologic response, and incomplete knowledge of the protective antigens.

Granulomatosis with Polyangiitis Occurring Concurrently With Human Monocytic Ehrlichiosis, a Causative Relationship or Coincidence

The relationship between some infections and vasculitides especially antineutrophil cytoplasmic antibodies (ANCA) mediated disease is well established. However, the mechanisms by which infection triggers ANCA formation are not fully understood. We report a case of granulomatosis with polyangiitis (GPA) occurring concurrently with human monocytic ehrlichiosis (HME) and discuss the possible relation between the two disease processes. A 49 year old Caucasian female presented with septic shock with multiple organ dysfunction syndrome that was attributed to HME. The patient was noted to have a small crusted lesion on the left nasolabial fold. Over the course of her hospitalization, it gradually expanded and eroded into the nasal cavity. After appropriate workup, she was diagnosed with GPA. HME hasn’t been clearly linked to any type of vasculitis. Review of the literature revealed scarce evidence limited to case reports and retrospective studies describing the association between GPA and HME. We highlight in this case a possible link between HME and GPA, however, the exact relationship between HME and GPA remains unclear and warrants further study.

Human monocytic ehrlichiosis complicated by hemophagocytic lymphohistiocytosis and multi-organ dysfunction syndrome

Human monocytic ehrlichiosis can manifest as a disease causing multi-organ failure. Rarely, it can cause secondary hemophagocytic lymphohistiocytosis (HLH). Early diagnosis and initiation of treatment for both ehrlichiosis and HLH is lifesaving. Therefore, clinical suspicion of HLH must remain high in the setting of an ehrlichiosis infection.

Sequence determinants spanning -35 motif and AT-rich spacer region impacting Ehrlichia chaffeensis Sigma 70-dependent promoter activity of two differentially expressed p28 outer membrane protein genes.

Ehrlichia chaffeensis is an obligate intracellular tick-borne bacterium which causes the disease, human monocytic ehrlichiosis. Ehrlichia chaffeensis contains only two sigma factors, σ32 and σ70. It is difficult to study E. chaffeensis gene regulation due to lack of a transformation system. We developed an Escherichia coli-based transcription system to study E. chaffeensis transcriptional regulation. An E. coli strain with its σ70 repressed with trp promoter is used to express E. chaffeensis σ70. The E. coli system and our previously established in vitro transcription system were used to map transcriptional differences of two Ehrlichia genes encoding p28-outer membrane proteins 14 and 19. We mapped the -10 and -35 motifs and the AT rich spacers located between the two motifs by performing detailed mutational analysis. Mutations within the -35 motif of the genes impacted transcription differently, while -10 motif deletions had no impact. The AT-rich spacers also contributed to transcriptional differences. We further demonstrated that the domain 4.2 of E. chaffeensis σ70 is important for regulating promoter activity and the deletion of region 1.1 of E. chaffeensis σ70 causes enhancement of the promoter activity. This is the first study defining the promoters of two closely related E. chaffeensis genes.

Ehrlichia chaffeensis TRP120 Activates Canonical Notch Signaling To Downregulate TLR2/4 Expression and Promote Intracellular Survival.

ABSTRACTEhrlichia chaffeensis preferentially targets mononuclear phagocytes and survives through a strategy of subverting innate immune defenses, but the mechanisms are unknown. We have shown E. chaffeensis type 1 secreted tandem repeat protein (TRP) effectors are involved in diverse molecular pathogen-host interactions, such as the TRP120 interaction with the Notch receptor-cleaving metalloprotease ADAM17. In the present study, we demonstrate E. chaffeensis, via the TRP120 effector, activates the canonical Notch signaling pathway to promote intracellular survival. We found that nuclear translocation of the transcriptionally active Notch intracellular domain (NICD) occurs in response to E. chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes, and hey. Significant differences in canonical Notch signaling gene expression levels (>40%) were observed during early and late stages of infection, indicating activation of the Notch pathway. We linked Notch pathway activation specifically to the TRP120 effector, which directly interacts with the Notch metalloprotease ADAM17. Using pharmacological inhibitors and small interfering RNAs (siRNAs) against γ-secretase enzyme, Notch transcription factor complex, Notch1, and ADAM17, we demonstrated that Notch signaling is required for ehrlichial survival. We studied the downstream effects and found that E. chaffeensis TRP120-mediated activation of the Notch pathway causes inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways required for PU.1 and subsequent Toll-like receptor 2/4 (TLR2/4) expression. This investigation reveals a novel mechanism whereby E. chaffeensis exploits the Notch pathway to evade the host innate immune response for intracellular survival.

Hacker within! Ehrlichia chaffeensis Effector Driven Phagocyte Reprogramming Strategy.

Ehrlichia chaffeensis is a small, gram negative, obligately intracellular bacterium that preferentially infects mononuclear phagocytes. It is the etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. Mechanisms by which E. chaffeensis establishes intracellular infection, and avoids host defenses are not well understood, but involve functionally relevant host-pathogen interactions associated with tandem and ankyrin repeat effector proteins. In this review, we discuss the recent advances in our understanding of the molecular and cellular mechanisms that underlie Ehrlichia host cellular reprogramming strategies that enable intracellular survival.

MyD88 suppresses IL-10 and IL-17 production in response to obligate intracellular Ehrlichia infection

Abstract Ehrlichia is an obligate intracellular gram negative bacterium that target macrophages, and causes the potentially fatal human monocytic ehrlichiosis (HME). In murine models of fatal ehrlichiosis (FE), Ixodes ovatus Ehrlichia (IOE) inhibits autophagy, suppresses protective acquired immunity, and causes immunopathology and toxic shock syndrome. Innate Ehrlichia detection by TLR2 mediates host resistance, while NOD2 signaling confers host susceptibility to FE. Recently, we have shown that deficiency of type I interferon receptor (IFNaR) phenocopy NOD2 deficiency in IOE-infected mice, implying a contribution of both NOD2-IFNaR pathways to susceptibility to FE. However, how TLR2 mediates protective function against Ehrlichia is not clearly defined. Here, we investigated the role of MyD88, the main adaptor molecule for the TLRs in host response to virulent IOE using MyD88 knockout mice (MyD88−/−). Our data showed that, MyD88−/− mice have an impaired bacterial clearance, increased liver injury, and a heightened mortality when compared to infected wild type (WT) mice. Increased bacterial load in MYD88−/− mice correlates with decreased levels of TNF-α in both in vivo and in vitro {serum and IOE- infected bone marrow-derived macrophages (BMM)}, and increased frequency of splenic CD4+ T cells producing IL-10 and IL-17. Protective anti-Ehrlichia immunity and minimal pathology in WT mice infected with low virulent Ehrlichia species correlates with a high Th1 response, but a low frequency of IL-10 and IL-17 producing splenic T cells, suggesting that IL-10 and IL-17 may play pathogenic roles in host defense against Ehrlichia. Together these data suggest that MYD88 plays a protective role in ehrlichiosis via suppression of IL-10 and IL-17.

EPIDEMIOLOGIC SITUATION BY NATURAL-FOCI INFECTIONS IN THE CRIMEA FEDERAL DISTRICT IN 2014 - 2015

Analysis of epidemic manifestations of natural-foci infections (NFI), clarification of spectrum of their causative agents, determination of epizootic activity of natural foci in the Crimea Federal District (KFD). Epizootologic examination of 10 administrative districts of KDF was carried out. 291 pools (2705 specimens) of ixodes ticks and 283 samples of organs of small mammals were studied by PCRmethod for the presence of DNA/RNA of causative agents of a number of NFI. Morbidity by NFI in KFD was registered by 6 nosologies: Lyme borreliosis, Marseilles fever, leptospirosis; tularemia, intestine yersiniosis and tick-borne viral encephalitis, wherein, transmissive infections made up 91.6%. Circulation of causative agents of Crimea hemorrhagic fever, Q fever, group of tick-borne spotted fever, Lyme borreliosis, human granulocytic anaplasmosis, human monocytic ehrlichiosis, hemorrhagic fever with renal syndrome, West Nile fever, tularemia and leptospirosis was established. Due to activity of natural foci of NFI further monitoring of epidemiologic and epizootologic manifestations of these infections in the Crimea, including using genetic methods of analysis, is necessary for ensuring sanitary-epidemiologic welfare of KFD population.

NK Cell-Mediated Regulation of Protective Memory Responses against Intracellular Ehrlichial Pathogens.

Ehrlichiae are gram-negative obligate intracellular bacteria that cause potentially fatal human monocytic ehrlichiosis. We previously showed that natural killer (NK) cells play a critical role in host defense against Ehrlichia during primary infection. However, the contribution of NK cells to the memory response against Ehrlichia remains elusive. Primary infection of C57BL/6 mice with Ehrlichia muris provides long-term protection against a second challenge with the highly virulent Ixodes ovatus Ehrlichia (IOE), which ordinarily causes fatal disease in naïve mice. Here, we show that the depletion of NK cells in E. muris-primed mice abrogates the protective memory response against IOE. Approximately, 80% of NK cell-depleted E. muris-primed mice succumbed to lethal IOE infection on days 8–10 after IOE infection, similar to naïve mice infected with the same dose of IOE. The lack of a recall response in NK cell-depleted mice correlated with an increased bacterial burden, extensive liver injury, decreased frequency of Ehrlichia-specific IFN-γ-producing memory CD4+ and CD8+ T-cells, and a low titer of Ehrlichia-specific antibodies. Intraperitoneal infection of mice with E. muris resulted in the production of IL-15, IL-12, and IFN-γ as well as an expansion of activated NKG2D+ NK cells. The adoptive transfer of purified E. muris-primed hepatic and splenic NK cells into Rag2-/-Il2rg-/- recipient mice provided protective immunity against challenge with E. muris. Together, these data suggest that E. muris-induced memory-like NK cells, which contribute to the protective, recall response against Ehrlichia.

Vaccination with an Attenuated Mutant of Ehrlichia chaffeensis Induces Pathogen-Specific CD4+ T Cell Immunity and Protection from Tick-Transmitted Wild-Type Challenge in the Canine Host.

Ehrlichia chaffeensis is a tick-borne rickettsial pathogen and the causative agent of human monocytic ehrlichiosis. Transmitted by the Amblyomma americanum tick, E. chaffeensis also causes disease in several other vertebrate species including white-tailed deer and dogs. We have recently described the generation of an attenuated mutant strain of E. chaffeensis, with a mutation in the Ech_0660 gene, which is able to confer protection from secondary, intravenous-administered, wild-type E. chaffeensis infection in dogs. Here, we extend our previous results, demonstrating that vaccination with the Ech_0660 mutant protects dogs from physiologic, tick-transmitted, secondary challenge with wild-type E. chaffeensis; and describing, for the first time, the cellular and humoral immune responses induced by Ech_0660 mutant vaccination and wild-type E. chaffeensis infection in the canine host. Both vaccination and infection induced a rise in E. chaffeensis-specific antibody titers and a significant Th1 response in peripheral blood as measured by E. chaffeensis antigen-dependent CD4+ T cell proliferation and IFNγ production. Further, we describe for the first time significant IL-17 production by peripheral blood leukocytes from both Ech_0660 mutant vaccinated animals and control animals infected with wild-type E. chaffeensis, suggesting a previously unrecognized role for IL-17 and Th17 cells in the immune response to rickettsial pathogens. Our results are a critical first step towards defining the role of the immune system in vaccine-induced protection from E. chaffeensis infection in an incidental host; and confirm the potential of the attenuated mutant clone, Ech_0660, to be used as a vaccine candidate for protection against tick-transmitted E. chaffeensis infection.

Development of Structure-Based Vaccines for Ehrlichiosis.

The obligate intracellular bacterium Ehrlichia chaffeensis that resides in mononuclear phagocytes is the etiologic agent of human monocytotropic ehrlichiosis (HME). HME is an emerging and often life-threatening zoonotic, tick-transmitted infectious disease in the USA. Lack of early diagnosis and treatment of HME are the main factors that lead to severe and fatal disease. Ehrlichia also causes diseases in companion animals and domesticated ruminants. E. chaffeensis and E. canis cause canine ehrlichioses in dogs, whereas E. ruminantium causes heartwater in cattle, sheep, and goats. As yet there are no commercially available vaccines to protect against these pathogens. This chapter describes a protocol to develop a structure-based vaccine for Ehrlichia based on the antigenic proteins P28-19 and Ehrlichia Hsp60.

Epidemiological Situation on Natural Focal Infectious Diseases in the Territory of the North-Caucasian Federal District in 2015

Objective of the study is to analyze epidemiological and epizootiological manifestations of natural-focal infections in the territory of the North-Caucasian Federal District (NCFD) in 2015. Materials and methods. Utilized were the reports of the Rospotrebnadzor Administrations, “Centers of Hygiene and Epidemiology” in the NCFD entities, Stavropol Research Anti-Plague Institute, Dagestan and Kabardino-Balkaria Plague Control Stations. Data processing was conducted using Excel Microsoft ware. Results and conclusions. Identified have been 8 nosological forms of natural-focal infections, including infections of bacterial etiology – 6 (75 %), and viral etiology – 2 (25 %). The majority of patients (164) are identified in the Stavropol Region: 67 (intestinal yersiniosis), 43 (Crimean–Congo hemorrhagic fever), 40 (Lyme borreliosis), 9 (leptospirosis), 2 (pseudotuberculosis), 1 (rabies), 1 (human granulocytic anaplasmosis) and 1 (human monocytic ehrlichiosis). The patients with 2 nosological forms of natural focal infections are identified in the Republic of Dagestan: 2 – Crimean–Congo hemorrhagic fever and one patient – tularemia. Also 2 nosological forms of natural focal infections are revealed in the Republic of Karachay-Cherkessia: one case of leptospirosis and one case of Crimean–Congo hemorrhagic fever (imported from the Stavropol region). 8502 field samples have been tested for the presence of markers of 12 nosological forms of natural-focal infections. Positive results for 10 of them have been obtained. The most comprehensive epizootiological monitoring has been carried out in the Stavropol Territory (10 nosological forms), whereat circulation of 8 pathogenic agents is registered.