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Stephen J Dumler - One of the best experts on this subject based on the ideXlab platform.
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use of peptide based enzyme linked immunosorbent assay followed by immunofluorescence assay to document ehrlichia chaffeensis as a cause of febrile illness in nicaragua
Journal of Clinical Microbiology, 2016Co-Authors: Ijeuru Chikeka, Stephen J Dumler, Armando Matute, Christopher W Woods, Orlando Mayorga, Megan E. RellerAbstract:Ehrlichia chaffeensis, the etiologic agent of Human Monocytic Ehrlichiosis (HME), has been extensively studied as a cause of acute febrile illness and an emerging tick-borne zoonosis in the United States. Limited data suggest its presence in other regions, including Central and South America but not Nicaragua to date. Diagnosis of E. chaffeensis infection by indirect immunofluorescence assay (IFA) is the reference standard due to its presumed high sensitivity and specificity, but IFA is impractical, variably reproducible, and cumbersome for large epidemiologic studies and for clinical diagnosis in resource-poor regions. We evaluated a high-throughput, objective peptide-based enzyme-linked immunosorbent assay (ELISA) for use alone or in combination with IFA. We found that it performed best as a screening test (sensitivity, 100%; specificity, 84%) to reduce the proportion of serum samples that were required by the more cumbersome and subjective IFA testing to <20%. Using a two-step diagnostic approach (IFA is performed if the ELISA is positive), we identified E. chaffeensis or a serologically and antigenically similar organism as a heretofore unrecognized cause of acute febrile illness in Humans in Nicaragua and demonstrated the utility of the peptide ELISA as a screening tool for large-scale clinical studies.
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hepatic pathology in Human Monocytic Ehrlichiosis
American Journal of Clinical Pathology, 2016Co-Authors: Ann Smith E Sehdev, Stephen J DumlerAbstract:Ehrlichia chaffeensis causes Human Monocytic Ehrlichiosis (HME) that usually includes fever, myalgias, and pancytopenia and, in 80% to 90% of patients, elevations in serum transaminase levels. Thus, the pathology of liver injury was studied in liver tissues from 7 patients with laboratory-confirmed HME. H&E and immunohistochemical stains for E chaffeensis and leukocyte markers were examined. Scattered lobular lymphohistiocytic foci and diffuse lymphohistiocytic infiltration and Kupffer cell hyperplasia with increased phagocytosis frequently were present. Various degrees of liver cell injury and death were observed. Cholestasis was evident in 6 cases, sometimes with bile duct epithelial injury. Rare to abundant E chaffeensis –infected mononuclear cells infiltrating lobules or portal regions or in Kupffer cells were observed in 5 patients. The inflammation was out of proportion to the infection in 6 cases. In the absence of infected hepatocytes or biliary epithelial cells, these findings suggest that host inflammatory or immune responses contribute to the liver injury seen in HME.
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current management of Human granulocytic anaplasmosis Human Monocytic Ehrlichiosis and ehrlichia ewingii Ehrlichiosis
Expert Review of Anti-infective Therapy, 2009Co-Authors: Rachael J Thomas, Stephen J Dumler, Jason A CarlyonAbstract:Anaplasma phagocytophilum, Ehrlichia chaffeensis and Ehrlichia ewingii are emerging tick-borne pathogens and are the causative agents of Human granulocytic anaplasmosis, Human Monocytic Ehrlichiosis and E. ewingii Ehrlichiosis, respectively. Collectively, these are referred to as Human ehrlichioses. These obligate intracellular bacterial pathogens of the family Anaplasmataceae are transmitted by Ixodes spp. or Amblyomma americanum ticks and infect peripherally circulating leukocytes to cause infections that range in clinical spectra from asymptomatic seroconversion to mild, severe or, in rare instances, fatal disease. This review describes: the ecology of each pathogen; the epidemiology, clinical signs and symptoms of the Human diseases that each causes; the choice methods for diagnosing and treating Human ehrlichioses; recommendations for patient management; and is concluded with suggestions for potential future research.
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current management of Human granulocytic anaplasmosis Human Monocytic Ehrlichiosis and ehrlichia ewingii Ehrlichiosis
Expert Review of Anti-infective Therapy, 2009Co-Authors: Rachael J Thomas, Stephen J Dumler, Jason A CarlyonAbstract:Anaplasma phagocytophilum, Ehrlichia chaffeensis and Ehrlichia ewingii are emerging tick-borne pathogens and are the causative agents of Human granulocytic anaplasmosis, Human Monocytic Ehrlichiosis and E. ewingii Ehrlichiosis, respectively. Collectively, these are referred to as Human ehrlichioses. These obligate intracellular bacterial pathogens of the family Anaplasmataceae are transmitted by Ixodes spp. or Amblyomma americanum ticks and infect peripherally circulating leukocytes to cause infections that range in clinical spectra from asymptomatic seroconversion to mild, severe or, in rare instances, fatal disease. This review describes: the ecology of each pathogen; the epidemiology, clinical signs and symptoms of the Human diseases that each causes; the choice methods for diagnosing and treating Human ehrlichioses; recommendations for patient management; and is concluded with suggestions for potential future research.
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lymph node hemophagocytosis in rickettsial diseases a pathogenetic role for cd8 t lymphocytes in Human Monocytic Ehrlichiosis hme
BMC Infectious Diseases, 2006Co-Authors: Kerry L. Dierberg, Stephen J DumlerAbstract:Human Monocytic Ehrlichiosis (HME) and Rocky Mountain spotted fever (RMSF) are caused by Ehrlichia chaffeensis and Rickettsia rickettsii, respectively. The pathogenesis of RMSF relates to rickettsia-mediated vascular injury, but it is unclear in HME. To study histopathologic responses in the lymphatic system for correlates of immune injury, lymph nodes from patients with HME (n = 6) and RMSF (n = 5) were examined. H&E-stained lymph node tissues were examined for five histopathologic features, including hemophagocytosis, cellularity, necrosis, and vascular congestion and edema. The relative proportions of CD68 macrophages, CD8 and CD4 T lymphocytes, and CD20 B lymphocytes were evaluated by immunohistochemical staining. Hemophagocytosis was similar in HME and RMSF, and was greater than in control cases (p = .015). Cellularity in HME was not different from controls, whereas RMSF lymph nodes were markedly less cellular (p < 0.002). E. chaffeensis-infected mononuclear phagocytes were infrequent compared to R. rickettsii-infected endothelial cells. More CD8 cells in lymph nodes were observed with HME (p < .001), but no quantitative differences in CD4 lymphocytes, macrophages, or B lymphocytes were identified. Hemophagocytosis, CD8 T cell expansion, and the paucity of infected cells in HME, suggest that E. chaffeensis infection leads to macrophage activation and immune-mediated injury.
Roman R Ganta - One of the best experts on this subject based on the ideXlab platform.
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a genetic system for targeted mutations to disrupt and restore genes in the obligate bacterium ehrlichia chaffeensis
Scientific Reports, 2017Co-Authors: Ying Wang, Chuanmin Cheng, Huitao Liu, Lanjing Wei, Roman R GantaAbstract: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.
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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
DNA Research, 2016Co-Authors: Huitao Liu, Tonia Von Ohlen, Laxmi U M R Jakkula, Roman R GantaAbstract: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.
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Transmission electron microscopy reveals distinct macrophage- and tick cell-specific morphological stages of Ehrlichia chaffeensis. PLoS One 2012
2016Co-Authors: Sarah E. Dedonder, Chuanmin Cheng, Lloyd H. Willard, Daniel L. Boyle, Roman R GantaAbstract:Background: Ehrlichia chaffeensis is an emerging tick-borne rickettsial pathogen responsible for Human Monocytic Ehrlichiosis. Despite the induction of an active host immune response, the pathogen has evolved to persist in its vertebrate and tick hosts. Understanding how the organism progresses in tick and vertebrate host cells is critical in identifying effective strategies to block the pathogen transmission. Our recent molecular and proteomic studies revealed differences in numerous expressed proteins of the organism during its growth in different host environments. Methodology/Principal Findings: Transmission electron microscopy analysis was performed to assess morphological changes in the bacterium within macrophages and tick cells. The stages of pathogen progression observed included the attachment of the organism to the host cells, its engulfment and replication within a morulae by binary fission and release of the organisms from infected host cells by complete host cell lysis or by exocytosis. E. chaffeensis grown in tick cells was highly pleomorphic and appears to replicate by both binary fission and filamentous type cell divisions. The presence of Ehrlichia-like inclusions was also observed within the nucleus of both macrophages and tick cells. This observation was confirmed by confocal microscopy and immunoblot analysis. Conclusions/Significance: Morphological differences in the pathogen’s progression, replication, and processing within macrophages and tick cells provide further evidence that E. chaffeensis employs unique host-cell specific strategies i
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attenuated mutants of ehrlichia chaffeensis induce protection against wild type infection challenge in the reservoir host and in an incidental host
Infection and Immunity, 2015Co-Authors: Arathy D S Nair, Chuanmin Cheng, Deborah C Jaworski, Suhasini Ganta, Michael W Sanderson, Roman R GantaAbstract:Ehrlichia chaffeensis, a tick-borne rickettsial organism, causes the disease Human Monocytic Ehrlichiosis. The pathogen also causes disease in several other vertebrates, including dogs and deer. In this study, we assessed two clonally purified E. chaffeensis mutants with insertions within the genes Ech_0379 and Ech_0660 as vaccine candidates in deer and dogs. Infection with the Ech_0379 mutant and challenge with wild-type E. chaffeensis 1 month following inoculation with the mutant resulted in the reduced presence of the organism in blood compared to the presence of wild-type infection in both deer and dogs. The Ech_0660 mutant infection resulted in its rapid clearance from the bloodstream. The wild-type infection challenge following Ech_0660 mutant inoculation also caused the pathogen's clearance from blood and tissue samples as assessed at the end of the study. The Ech_0379 mutant-infected and -challenged animals also remained positive for the organism in tissue samples in deer but not in dogs. This is the first study that documents that insertion mutations in E. chaffeensis that cause attenuated growth confer protection against wild-type infection challenge. This study is important in developing vaccines to protect animals and people against Ehrlichia species infections.
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bayesian spatio temporal analysis and geospatial risk factors of Human Monocytic Ehrlichiosis
PLOS ONE, 2014Co-Authors: Ram Raghavan, Daniel Neises, Douglas G Goodin, Daniel Andresen, Roman R GantaAbstract:Variations in spatio-temporal patterns of Human Monocytic Ehrlichiosis (HME) infection in the state of Kansas, USA were examined and the relationship between HME relative risk and various environmental, climatic and socio-economic variables were evaluated. HME data used in the study was reported to the Kansas Department of Health and Environment between years 2005–2012, and geospatial variables representing the physical environment [National Land cover/Land use, NASA Moderate Resolution Imaging Spectroradiometer (MODIS)], climate [NASA MODIS, Prediction of Worldwide Renewable Energy (POWER)], and socio-economic conditions (US Census Bureau) were derived from publicly available sources. Following univariate screening of candidate variables using logistic regressions, two Bayesian hierarchical models were fit; a partial spatio-temporal model with random effects and a spatio-temporal interaction term, and a second model that included additional covariate terms. The best fitting model revealed that spatio-temporal autocorrelation in Kansas increased steadily from 2005–2012, and identified poverty status, relative humidity, and an interactive factor, ‘diurnal temperature range x mixed forest area’ as significant county-level risk factors for HME. The identification of significant spatio-temporal pattern and new risk factors are important in the context of HME prevention, for future research in the areas of ecology and evolution of HME, and as well as climate change impacts on tick-borne diseases.
Yasuko Rikihisa - One of the best experts on this subject based on the ideXlab platform.
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an entry triggering protein of ehrlichia is a new vaccine candidate against tick borne Human Monocytic Ehrlichiosis
Mbio, 2020Co-Authors: Khemraj Budachetri, Omid Teymournejad, Mingqun Lin, Qi Yan, Mariella Mestresvillanueva, Guy Brock, Yasuko RikihisaAbstract:ABSTRACT Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes Human Monocytic Ehrlichiosis, an emerging disease transmitted by the Lone Star tick, Amblyomma americanum. E. chaffeensis outer membrane protein entry triggering protein of Ehrlichia (EtpE) is necessary for bacterial entry into Human cells. We investigated the role of EtpE in transmission of the bacteria from tick to Human cells and whether or not vaccination with EtpE can prevent transmission of ehrlichiae from ticks to mammals. An antiserum against the recombinant C terminus of EtpE (rEtpE-C), which binds a mammalian cell-surface receptor and triggers bacterial entry, significantly inhibited E. chaffeensis transmission from infected tick cells to Human monocytes in culture. Each of five specific-pathogen-free dogs were vaccinated with rEtpE-C along with an immunostimulating complex or were sham vaccinated with the complex alone. Dogs vaccinated with rEtpE-C developed high antibody titers against rEtpE-C and produced interferon-γ-secreting cells, as assessed with the ELISpot assay. All 10 dogs were challenged with A. americanum adult ticks infected as nymphs by syringe inoculation with E. chaffeensis. Upon challenge, both the vaccinated and control dogs became infected by day 1 post-tick attachment, but the majority of rEtpE-C-vaccinated dogs rapidly cleared the infection from the bloodstream as soon as day 7, whereas most of sham-vaccinated dogs remained infected at day 35. Peripheral blood leukocytes from vaccinated dogs had significantly elevated interferon-γ mRNA levels and secreted significantly elevated interferon-γ soon after tick attachment. Thus, the EtpE-C vaccine represents the first ehrlichial protein vaccine demonstrated to reduce bacterial infection in mammals upon challenge with infected ticks. IMPORTANCE The incidence of tick-borne diseases has risen dramatically in the past two decades and continues to rise. Discovered in 1986 and designated a nationally notifiable disease in 1998 by the Centers for Disease Control and Prevention, Human Monocytic Ehrlichiosis, which is caused by the bacterium Ehrlichia chaffeensis, is one of the most prevalent, life-threatening, emerging tick-borne zoonoses in the United States. We investigated the role of the E. chaffeensis protein EtpE in transmission of the bacterium from tick to Human cells and in vaccinated dogs with EtpE to assess the efficacy of vaccination against E. chaffeensis-infected tick challenge. Our results help fill gaps in our understanding of E. chaffeensis-derived protective antigens that could be used in a candidate vaccine for immunization of Humans to counter tick-transmitted Ehrlichiosis.
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An Entry-Triggering Protein of Ehrlichia Is a New Vaccine Candidate against Tick-Borne Human Monocytic Ehrlichiosis
'American Society for Microbiology', 2020Co-Authors: Khemraj Budachetri, Omid Teymournejad, Mingqun Lin, Qi Yan, Mariella Mestres-villanueva, Guy Nathaniel Brock, Yasuko RikihisaAbstract:The incidence of tick-borne diseases has risen dramatically in the past two decades and continues to rise. Discovered in 1986 and designated a nationally notifiable disease in 1998 by the Centers for Disease Control and Prevention, Human Monocytic Ehrlichiosis, which is caused by the bacterium Ehrlichia chaffeensis, is one of the most prevalent, life-threatening, emerging tick-borne zoonoses in the United States. We investigated the role of the E. chaffeensis protein EtpE in transmission of the bacterium from tick to Human cells and in vaccinated dogs with EtpE to assess the efficacy of vaccination against E. chaffeensis-infected tick challenge. Our results help fill gaps in our understanding of E. chaffeensis-derived protective antigens that could be used in a candidate vaccine for immunization of Humans to counter tick-transmitted Ehrlichiosis.Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes Human Monocytic Ehrlichiosis, an emerging disease transmitted by the Lone Star tick, Amblyomma americanum. E. chaffeensis outer membrane protein entry triggering protein of Ehrlichia (EtpE) is necessary for bacterial entry into Human cells. We investigated the role of EtpE in transmission of the bacteria from tick to Human cells and whether or not vaccination with EtpE can prevent transmission of ehrlichiae from ticks to mammals. An antiserum against the recombinant C terminus of EtpE (rEtpE-C), which binds a mammalian cell-surface receptor and triggers bacterial entry, significantly inhibited E. chaffeensis transmission from infected tick cells to Human monocytes in culture. Each of five specific-pathogen-free dogs were vaccinated with rEtpE-C along with an immunostimulating complex or were sham vaccinated with the complex alone. Dogs vaccinated with rEtpE-C developed high antibody titers against rEtpE-C and produced interferon-γ-secreting cells, as assessed with the ELISpot assay. All 10 dogs were challenged with A. americanum adult ticks infected as nymphs by syringe inoculation with E. chaffeensis. Upon challenge, both the vaccinated and control dogs became infected by day 1 post-tick attachment, but the majority of rEtpE-C-vaccinated dogs rapidly cleared the infection from the bloodstream as soon as day 7, whereas most of sham-vaccinated dogs remained infected at day 35. Peripheral blood leukocytes from vaccinated dogs had significantly elevated interferon-γ mRNA levels and secreted significantly elevated interferon-γ soon after tick attachment. Thus, the EtpE-C vaccine represents the first ehrlichial protein vaccine demonstrated to reduce bacterial infection in mammals upon challenge with infected ticks
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ehrlichia type iv secretion effector ech0825 is translocated to mitochondria and curbs ros and apoptosis by upregulating host mnsod
Cellular Microbiology, 2012Co-Authors: Hongyan Liu, Mingqun Lin, Weichao Bao, Hua Niu, Yasuko RikihisaAbstract:Summary Ehrlichia chaffeensis infects monocytes/macrophages and causes Human Monocytic Ehrlichiosis. To determine the role of type IV secretion (T4S) system in infection, candidates for T4S effectors were identified by bacterial two-hybrid screening of E. chaffeensis hypothetical proteins with positively charged C-terminus using E. chaffeensis VirD4 as bait. Of three potential T4S effectors, ECH0825 was highly upregulated early during exponential growth in a Human Monocytic cell line. ECH0825 was translocated from the bacterium into the host-cell cytoplasm and localized to mitochondria. Delivery of anti-ECH0825 into infected host cells significantly reduced bacterial infection. Ectopically expressed ECH0825 also localized to mitochondria and inhibited apoptosis of transfected cells in response to etoposide treatment. In double transformed yeast, ECH0825 localized to mitochondria and inhibited Human Bax-induced apoptosis. Mitochondrial manganese superoxide dismutase (MnSOD) was increased over ninefold in E. chaffeensis-infected cells, and the amount of reactive oxygen species (ROS) in infected cells was significantly lower than that in uninfected cells. Similarly, MnSOD was upregulated and the ROS level was reduced in ECH0825-transfected cells. These data suggest that, by upregulating MnSOD, ECH0825 prevents ROS-induced cellular damage and apoptosis to allow intracellular infection. This is the first example of host ROS levels linked to a bacterial T4S effector.
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insights into the ctra regulon in development of stress resistance in obligatory intracellular pathogen ehrlichia chaffeensis
Molecular Microbiology, 2011Co-Authors: Zhihui Cheng, Vsevolod L Popov, Koshiro Miura, Yumi Kumagai, Yasuko RikihisaAbstract:Ehrlichia chaffeensis is an obligate intracellular bacterium that causes Human Monocytic Ehrlichiosis. Ehrlichiae have a biphasic developmental cycle consisting of dense-cored cells (DCs) and reticulate cells (RCs). Isolated DCs are more stress resistant and infectious than RCs. Here, we report that a response regulator, CtrA was upregulated in Human monocytes at the late growth stage when DCs develop. E. chaffeensis CtrA bound to the promoters of late-stage transcribed genes: ctrA, ompA (peptidoglycan-associated lipoprotein), bolA (stress-induced morphogen), and surE (stationary phase survival protein), which contain CtrA-binding motifs, and transactivated ompA, surE, and bolA promoter-lacZ fusions in Escherichia coli. OmpA was predominantly expressed in DCs. E. chaffeensis binding to and subsequent infection of monocytes were inhibited by anti-OmpA IgG. E. chaffeensis BolA bound to the promoters of genes encoding outer surface proteins TRP120 and ECH_1038, which were expressed in DCs, and transactivated trp120 and ECH_1038 promoter-lacZ fusions. E. chaffeensis bolA complemented a stress-sensitive E. coli bolA mutant. E. coli expressing E. chaffeensis surE exhibited increased resistance to osmotic stress. Our results suggest that E. chaffeensis CtrA plays a role in coordinating development of the stress resistance for passage from the present to the next host cells through its regulon.
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Human infection with ehrlichia canis accompanied by clinical signs in venezuela
Annals of the New York Academy of Sciences, 2006Co-Authors: Miriam Perez, Mairim Bodor, Chunbin Zhang, Qingmin Xiong, Yasuko RikihisaAbstract:A total of 20 Human patients with clinical signs compatible with Human Monocytic Ehrlichiosis (HME), who were admitted to the emergency clinic in Lara State, Venezuela, were studied. Thirty percent (6/20) patients were positive for Ehrlichia canis 16S rRNA on gene-specific polymerase chain reaction (PCR). Compared with the U.S. strains, 16S rRNA gene sequences from all six patients had the same base mutation as the sequence of the E. canis Venezuelan Human Ehrlichia (VHE) strain previously isolated from an asymptomatic Human. This study is the first report of E. canis infection of Human patients with clinical signs of HME.
Didier Raoult - One of the best experts on this subject based on the ideXlab platform.
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tick and flea borne rickettsial emerging zoonoses
Veterinary Research, 2005Co-Authors: Philippe Parola, Bernard Davoust, Didier RaoultAbstract:Between 1984 and 2004, nine more species or subspecies of spotted fever rickettsiae were identified as emerging agents of tick-borne rickettsioses throughout the world. Six of these species had first been isolated from ticks and later found to be pathogenic to Humans. The most recent example is Rickettsia parkeri, recognized as a Human pathogen more than 60 years after its initial isolation from ticks. A new spotted fever rickettsia, R. felis was also found to be associated with fleas and to be a Human pathogen. Similarly, bacteria within the family Anaplasmataceae have been considered to be of veterinary importance only, yet three species have been implicated in Human diseases in recent years, including Ehrlichia chaffeensis, the agent of Human Monocytic Ehrlichiosis, Anaplasma phagocytophilum, the agent of Human anaplasmosis (formerly known as Human granulocytic Ehrlichiosis agent, E. equi and E. phagocytophila), and finally Ehrlichia ewingii, which causes granulocytic Ehrlichiosis in Humans. We present here an overview of the various tick- and flea-borne rickettsial zoonoses described in the last 20 years, focusing on the ecological, epidemiological and clinical aspects.
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serologic diagnosis of Human Monocytic Ehrlichiosis by immunoblot analysis
Clinical and Vaccine Immunology, 1994Co-Authors: P Brouqui, C Lecam, J Olson, Didier RaoultAbstract:Human Monocytic Ehrlichiosis is caused by Ehrlichia chaffeensis, an intracellular bacterium probably transmitted by the tick Amblyomma americanum in the United States. Despite its lack of specificity in discriminating among infections by closely related Ehrlichia spp., immunofluorescence assay (IFA) is the most frequently used serological diagnostic method. To improve the specificity of the serological diagnosis, we compared antigenic profile of E. canis and E. chaffeensis antigen with homologous and heterologous sera, searching for the specificity of the presence of low-molecular-weight proteins. Western immunoblot analysis of IFA-positive Human sera revealed 27- and 29-kDa proteins which are not found in E. canis IFA-positive sera from dogs. IFA-positive sera from dogs revealed a low-molecular-weight group of proteins (20 to 28 kDa) which were not found in Human E. chaffeensis-positive sera except for a weak band at 22 kDa. The presence o antibodies directed against the 27- and 29-kDa proteins on Western blots is specific for E. chaffeensis infection, and we suggest that the Western blot might complete IFA in cases with low positive predictive value.
Chuanmin Cheng - One of the best experts on this subject based on the ideXlab platform.
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a genetic system for targeted mutations to disrupt and restore genes in the obligate bacterium ehrlichia chaffeensis
Scientific Reports, 2017Co-Authors: Ying Wang, Chuanmin Cheng, Huitao Liu, Lanjing Wei, Roman R GantaAbstract: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.
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Transmission electron microscopy reveals distinct macrophage- and tick cell-specific morphological stages of Ehrlichia chaffeensis. PLoS One 2012
2016Co-Authors: Sarah E. Dedonder, Chuanmin Cheng, Lloyd H. Willard, Daniel L. Boyle, Roman R GantaAbstract:Background: Ehrlichia chaffeensis is an emerging tick-borne rickettsial pathogen responsible for Human Monocytic Ehrlichiosis. Despite the induction of an active host immune response, the pathogen has evolved to persist in its vertebrate and tick hosts. Understanding how the organism progresses in tick and vertebrate host cells is critical in identifying effective strategies to block the pathogen transmission. Our recent molecular and proteomic studies revealed differences in numerous expressed proteins of the organism during its growth in different host environments. Methodology/Principal Findings: Transmission electron microscopy analysis was performed to assess morphological changes in the bacterium within macrophages and tick cells. The stages of pathogen progression observed included the attachment of the organism to the host cells, its engulfment and replication within a morulae by binary fission and release of the organisms from infected host cells by complete host cell lysis or by exocytosis. E. chaffeensis grown in tick cells was highly pleomorphic and appears to replicate by both binary fission and filamentous type cell divisions. The presence of Ehrlichia-like inclusions was also observed within the nucleus of both macrophages and tick cells. This observation was confirmed by confocal microscopy and immunoblot analysis. Conclusions/Significance: Morphological differences in the pathogen’s progression, replication, and processing within macrophages and tick cells provide further evidence that E. chaffeensis employs unique host-cell specific strategies i
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attenuated mutants of ehrlichia chaffeensis induce protection against wild type infection challenge in the reservoir host and in an incidental host
Infection and Immunity, 2015Co-Authors: Arathy D S Nair, Chuanmin Cheng, Deborah C Jaworski, Suhasini Ganta, Michael W Sanderson, Roman R GantaAbstract:Ehrlichia chaffeensis, a tick-borne rickettsial organism, causes the disease Human Monocytic Ehrlichiosis. The pathogen also causes disease in several other vertebrates, including dogs and deer. In this study, we assessed two clonally purified E. chaffeensis mutants with insertions within the genes Ech_0379 and Ech_0660 as vaccine candidates in deer and dogs. Infection with the Ech_0379 mutant and challenge with wild-type E. chaffeensis 1 month following inoculation with the mutant resulted in the reduced presence of the organism in blood compared to the presence of wild-type infection in both deer and dogs. The Ech_0660 mutant infection resulted in its rapid clearance from the bloodstream. The wild-type infection challenge following Ech_0660 mutant inoculation also caused the pathogen's clearance from blood and tissue samples as assessed at the end of the study. The Ech_0379 mutant-infected and -challenged animals also remained positive for the organism in tissue samples in deer but not in dogs. This is the first study that documents that insertion mutations in E. chaffeensis that cause attenuated growth confer protection against wild-type infection challenge. This study is important in developing vaccines to protect animals and people against Ehrlichia species infections.
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Transcription of Ehrlichia chaffeensis Genes Is Accomplished by RNA Polymerase Holoenzyme
2013Co-Authors: Huitao Liu, Chuanmin Cheng, Tonia Von Ohlen, Bonto Faburay, Roman R GantaAbstract:Bacterial gene transcription is initiated by RNA polymerase containing a sigma factor. To understand gene regulation in Ehrlichia chaffeensis, an important tick-transmitted rickettsiae responsible for Human Monocytic Ehrlichiosis, we initiated studies evaluating the transcriptional machinery of several genes of this organism. We mapped the transcription start sites of 10 genes and evaluated promoters of five genes (groE, dnaK, hup, p28-Omp14 and p28-Omp19 genes). We report here that the RNA polymerase binding elements of E. chaffeensis gene promoters are highly homologous for its only two transcription regulators, sigma 32 and sigma 70, and that gene expression is accomplished by either of the transcription regulators. RNA analysis revealed that although transcripts for both sigma 32 and sigma 70 are upregulated during the early replicative stage, their expression patterns remained similar for the entire replication cycle. We further present evidence demonstrating that the organism’s-35 motifs are essential to transcription initiations. The data suggest that E. chaffeensis gene regulation has evolved to support the organism’s growth, possibly to facilitate its intraphagosomal growth. Considering the limited availability of genetic tools, this stud
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Transcription of Ehrlichia chaffeensis Genes Is Accomplished by RNA Polymerase Holoenzyme Containing either Sigma 32 or Sigma 70
2013Co-Authors: Huitao Liu, Chuanmin Cheng, Tonia Von Ohlen, Bonto Faburay, Roman R GantaAbstract:Bacterial gene transcription is initiated by RNA polymerase containing a sigma factor. To understand gene regulation in Ehrlichia chaffeensis, an important tick-transmitted rickettsiae responsible for Human Monocytic Ehrlichiosis, we initiated studies evaluating the transcriptional machinery of several genes of this organism. We mapped the transcription start sites of 10 genes and evaluated promoters of five genes (groE, dnaK, hup, p28-Omp14 and p28-Omp19 genes). We report here that the RNA polymerase binding elements of E. chaffeensis gene promoters are highly homologous for its only two transcription regulators, sigma 32 and sigma 70, and that gene expression is accomplished by either of the transcription regulators. RNA analysis revealed that although transcripts for both sigma 32 and sigma 70 are upregulated during the early replicative stage, their expression patterns remained similar for the entire replication cycle. We further present evidence demonstrating that the organism’s -35 motifs are essential to transcription initiations. The data suggest that E. chaffeensis gene regulation has evolved to support the organism’s growth, possibly to facilitate its intraphagosomal growth. Considering the limited availability of genetic tools, this study offers a novel alternative in defining gene regulation in E. chaffeensis and other related intracellular pathogens.