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      Protein and DNA Biosynthesis Demonstrated in Host Cell-Free Phagosomes Containing Anaplasma phagocytophilum or Ehrlichia chaffeensis in Axenic Media

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          Abstract

          Rickettsiae belong to the Anaplasmataceae family, which includes mostly tick-transmitted pathogens causing human, canine, and ruminant diseases. Biochemical characterization of the pathogens remains a major challenge because of their obligate parasitism.

          ABSTRACT

          Rickettsiae belong to the Anaplasmataceae family, which includes mostly tick-transmitted pathogens causing human, canine, and ruminant diseases. Biochemical characterization of the pathogens remains a major challenge because of their obligate parasitism. We investigated the use of an axenic medium for growth of two important pathogens— Anaplasma phagocytophilum and Ehrlichia chaffeensis—in host cell-free phagosomes. We recently reported that the axenic medium promotes protein and DNA biosynthesis in host cell-free replicating form of E. chaffeensis, although the bacterial replication is limited. We now tested the hypothesis that growth on axenic medium can be improved if host cell-free rickettsia-containing phagosomes are used. Purification of phagosomes from A. phagocytophilum- and E. chaffeensis-infected host cells was accomplished by density gradient centrifugation combined with magnet-assisted cell sorting. Protein and DNA synthesis was observed for both organisms in cell-free phagosomes with glucose-6-phosphate and/or ATP. The levels of protein and DNA synthesis were the highest for a medium pH of 7. The data demonstrate bacterial DNA and protein synthesis for the first time in host cell-free phagosomes for two rickettsial pathogens. The host cell support-free axenic growth of obligate pathogenic rickettsiae will be critical in advancing research goals in many important tick-borne diseases impacting human and animal health.

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          Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila.

          The genera Anaplasma, Ehrlichia, Cowdria, Neorickettsia and Wolbachia encompass a group of obligate intracellular bacteria that reside in vacuoles of eukaryotic cells and were previously placed in taxa based upon morphological, ecological, epidemiological and clinical characteristics. Recent genetic analyses of 16S rRNA genes, groESL and surface protein genes have indicated that the existing taxa designations are flawed. All 16S rRNA gene and groESL sequences deposited in GenBank prior to 2000 and selected sequences deposited thereafter were aligned and phylogenetic trees and bootstrap values were calculated using the neighbour-joining method and compared with trees generated with maximum-probability, maximum-likelihood, majority-rule consensus and parsimony methods. Supported by bootstrap probabilities of at least 54%, 16S rRNA gene comparisons consistently clustered to yield four distinct clades characterized roughly as Anaplasma (including the Ehrlichia phagocytophila group, Ehrlichia platys and Ehrlichia bovis) with a minimum of 96.1% similarity, Ehrlichia (including Cowdria ruminantium) with a minimum of 97.7% similarity, Wolbachia with a minimum of 95.6% similarity and Neorickettsia (including Ehrlichia sennetsu and Ehrlichia risticii) with a minimum of 94.9% similarity. Maximum similarity between clades ranged from 87.1 to 94.9%. Insufficient differences existed among E. phagocytophila, Ehrlichia equi and the human granulocytic ehrlichiosis (HGE) agent to support separate species designations, and this group was at least 98.2% similar to any Anaplasma species. These 16S rRNA gene analyses are strongly supported by similar groESL clades, as well as biological and antigenic characteristics. It is proposed that all members of the tribes Ehrlichieae and Wolbachieae be transferred to the family Anaplasmataceae and that the tribe structure of the family Rickettsiaceae be eliminated. The genus Anaplasma should be emended to include Anaplasma (Ehrlichia) phagocytophila comb. nov. (which also encompasses the former E. equi and the HGE agent), Anaplasma (Ehrlichia) bovis comb. nov. and Anaplasma (Ehrlichia) platys comb. nov., the genus Ehrlichia should be emended to include Ehrlichia (Cowdria) ruminantium comb. nov. and the genus Neorickettsia should be emended to include Neorickettsia (Ehrlichia) risticii comb. nov. and Neorickettsia (Ehrlichia) sennetsu comb. nov.
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            Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease.

            Six patients from northern Minnesota and Wisconsin with a febrile illness accompanied by granulocytic cytoplasmic morulae suggestive of ehrlichial infection were identified. Two patients died, and splenic granulocytes of one patient contained cytoplasmic vacuoles with organisms ultrastructurally characteristic of ehrlichiae. From one patient, a 1.5-kb DNA product was amplified by PCR with universal eubacterial primers of 16S rDNA. Analysis of the nucleotide sequence of the amplified product revealed 99.9 and 99.8% similarities with E. phagocytophila and E. equi, respectively, neither of which has previously been known to infect humans. From the variable regions of the determined sequence, a forward primer specific for three organisms (human granulocytic ehrlichia, E. phagocytophila, and E. equi) and a reverse primer for these ehrlichiae and E. platys were designed. By nested PCR with amplification by the universal primers and then reamplification with the specific primers described above, the expected 919-bp product was generated from the blood of the index patient and three additional patients. Blood from these four patients and two more patients with granulocytic morulae contained DNA which was amplified by nested PCR involving a combination of a universal primer and the human granulocytic ehrlichia-E. phagocytophila-E. equi-E. platys group-specific primer. This apparently vector-borne human granulocytic ehrlichia has only 92.5% 16S rDNA homology with E. chaffeensis. Nested PCR with group-specific primers did not amplify E. chaffeensis DNA, and E. chaffeensis-specific primers did not amplify DNAs of the human granulocytic ehrlichia. Thus, six patients were shown to be infected by an Ehrlichia species never previously reported to infect humans.
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              Human ehrlichiosis and anaplasmosis.

              Human ehrlichiosis and anaplasmosis are acute febrile tick-borne diseases caused by various members of the genera Ehrlichia and Anaplasma (Anaplasmataceae). Human monocytotropic ehrlichiosis has become one of the most prevalent life-threatening tick-borne disease in the United States. Ehrlichiosis and anaplasmosis are becoming more frequently diagnosed as the cause of human infections, as animal reservoirs and tick vectors have increased in number and humans have inhabited areas where reservoir and tick populations are high. Ehrlichia chaffeensis, the etiologic agent of human monocytotropic ehrlichiosis (HME), is an emerging zoonosis that causes clinical manifestations ranging from a mild febrile illness to a fulminant disease characterized by multiorgan system failure. Anaplasma phagocytophilum causes human granulocytotropic anaplasmosis (HGA), previously known as human granulocytotropic ehrlichiosis. This article reviews recent advances in the understanding of ehrlichial diseases related to microbiology, epidemiology, diagnosis, pathogenesis, immunity, and treatment of the 2 prevalent tick-borne diseases found in the United States, HME and HGA. 2010 Elsevier Inc. All rights reserved.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                Infect Immun
                Infect Immun
                iai
                iai
                IAI
                Infection and Immunity
                American Society for Microbiology (1752 N St., N.W., Washington, DC )
                0019-9567
                1098-5522
                11 January 2021
                17 March 2021
                April 2021
                17 March 2021
                : 89
                : 4
                : e00638-20
                Affiliations
                [a ]Center of Excellence for Vector-Borne Diseases, Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA
                Yale University School of Medicine
                Author notes
                Address correspondence to Roman R. Ganta, rganta@ 123456vet.k-state.edu .
                [*]

                Present address: Li Chen, Yangzhou University, College of Veterinary Medicine, Yangzhou, China.

                Citation Zhang Y, Chen L, Kondethimmanahalli C, Liu H, Ganta RR. 2021. Protein and DNA biosynthesis demonstrated in host cell-free phagosomes containing Anaplasma phagocytophilum or Ehrlichia chaffeensis in axenic media. Infect Immun 89:e00638-20. https://doi.org/10.1128/IAI.00638-20.

                Author information
                https://orcid.org/0000-0001-9794-6158
                Article
                00638-20
                10.1128/IAI.00638-20
                8090944
                33431703
                b738c9f6-efe9-473d-92dc-2d779bd45c73
                Copyright © 2021 Zhang et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

                History
                : 10 October 2020
                : 17 November 2020
                : 23 December 2020
                Page count
                Figures: 5, Tables: 1, Equations: 0, References: 32, Pages: 11, Words: 6236
                Funding
                Funded by: HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID), https://doi.org/10.13039/100000060;
                Award ID: AI070908
                Award Recipient :
                Funded by: HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID), https://doi.org/10.13039/100000060;
                Award ID: AI152418
                Award Recipient :
                Categories
                Cellular Microbiology: Pathogen-Host Cell Molecular Interactions
                Custom metadata
                April 2021

                Infectious disease & Microbiology
                axenic medium,cell-free culture,phagosomes,a. phagocytophilum,e. chaffeensis,anaplasma,ehrlichia,host cell-free culture,obligate,rickettsiae,tick-borne pathogens

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