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Taxonomy of Rickettsiae

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Taxonomy of Rickettsiae Library Rickettsiae: Lecture Bacteriology and Taxonomy author Onlineby Marina© E. Eremeeva Centers for Disease Control and Prevention, Atlanta, USA Post Graduate Educational Course: Intracellular bacteria – from biology to clinic ESCMID2-5 November 2010, Sousse-Tunisia Definition • Taxonomy is the practice and scienceLibrary of classification • The word finds its roots in the Greek τάξις, taxis (meaning 'order' or 'arrangement') and νόμος, nomos (meaning 'law' or 'science')Lecture • Taxonomy uses taxonomic units, known as taxa author (singular taxon) • Taxonomy, or taxonomicOnlineby scheme, is also used as a © particular classification ("the taxonomy of ..."), arranged in a hierarchical structure ESCMID Different Forms of Taxonomy • Biological classification (Linnaean taxonomy)Library a. The best known form of taxonomy b. Applies empirical science to classifying - only the final step of a process Lecture c. Includes the prediction, discovery, description and (re)defining of taxa author d. Uses taxonomic ranks (Kingdom, Phylum, Class, Order, Family, Genus, Species)Onlineby © • Phylogenetic systematics (Phylogenetics) a. Predicts trees of descent b. AnalyzesESCMID relationships among groups Outline Library • Bacteriology Nature and properties of rickettsiae • Phylogeny Lecture History of the evolution of a species or group author • Taxonomy Onlineby Classification of© rickettsiae ESCMID It sounds very similar… Library Rickettsiae = rickets Lecture Obligate authorBone softening due to vitamin intracellularOnline by bacteria © D deficiency ESCMID Howard Taylor Ricketts, M.D. (1871-1910) Library • Described etiological agent of RMSF • Recognized tick as a vector • DiscoveredLecture low prevalence of infected ticks in nature • Suggestedauthor tick control program • Proposed serum therapy or Onlinevaccinationby © ESCMID The term “rickettsia” has forLibrary many years been loosely applied to a very wide range of Gram-negative bacteria simply because of their obligateLecture association with arthropods and their hosts,author their size and their intracellular habitat… Onlineby © ESCMID However, The rickettsiae are Library a highly heterogeneous group that historically included: Genera Rickettsia & Orientia Genera Anaplasma, EhrlichiaLecture, Neorickettsia , Wolbachia author Genera BartonellaOnline (Rochalimaea)by & Grahamella Genera Haemobartonella© & Eperythrozoon Genera Coxiella, Rickettsiella & Piscirickettsia ESCMID 16S rRNA gene Dr. Brouqui Library Lecture Dr. Birtles author Dr. Greub Onlineby © ESCMID Formal Taxonomy of Rickettsia Library Order: Rickettsiales Family: Rickettsiaceae Lecture Genus: Rickettsia author Onlineby Species: > 22 formally© described species ESCMID Further Division Library Genus Rickettsia In silico genome data typhus group typhus group Lecturetransition group spotted fever group spotted fever group author ancestral group Onlineby •Antigene structure © •Temperature requirements •Growth characteristics •DNA ESCMIDproperties Rickettsia: Formal Description Library • Obligate intracellular rod-shaped bacteria • 0.3-0.5 x 0.8-2.0 m • Cell culture or chicken embryos for growth • Slow growing Lecture • Surrounded by typical Gramauthor –negative membrane a. Not stainedOnline well byby the Gram method b. Retain basic fuschin© when stained by Gimenez ESCMID Gimienez Staining of Rickettsia Library Lecture author Onlineby © •Rickettsia primarily infect endothelial cells in vivo •Infect arthropod cells •Very promiscuous in cell culture •TemperatureESCMID range : 28-37oC Electron Microscopic Structure of Rickettsia Library Lecture author Onlineby © •Grow free in the cytoplasm but some can enter nucleus •Multiply by binary fission but have a slow generation time •SurroundedESCMID by protein S-layer Library Lecture author Intracellular Cycle of Onlineby Rickettsia and Orientia © ESCMID Spotted fever group rickettsiae Library Lecture SpottedAssociation fever with Cellular Cytoskeleton group author Typhus group: OnlineR. prowazekiiby © ESCMID Orientia Antigens and Proteins Library Lecture author Onlineby Serotyping antigens: © •OmpA & OmpB, Sca •17 kDa lipoprotein •Heat shock proteins ESCMID OmpA and OmpB western blotting • Smooth type LPS Genetic Characteristics Library • 32-33% G+C, spotted fever group • 29% G+C, typhus group • Genome 1.1-1.6 Mb Lecture • One circular chromosome author • 0-3 plasmids: 12,377-67,829 bp Onlineby © ESCMID Different Systems for Classification • Classic system Library a.Type of disease b.Vector association c.Geographic distributionLecture d.Cell association characteristics author • Serotyping with mouse hyperimmune serum Onlineby • SDS-PAGE and PFGE© • Comparison of nucleotide sequences a.16S rRNA gene vs protein genes ESCMID Generic Characteristics Used for Classification of Rickettsiae Library Geography & Vector Disease Impact • Louse-borne • TyphusLecture • Epidemic • Flea-borne • Spotted fever • Endemic author • Mite-borne • Scrub typhus • Sporadic • Tick-borne Onlineby © ESCMID Serotyping of Rickettsia • LPS cross-reactive epitopes Library a.Proteus vulgaris OX19, OX2 b.Proteus mirabilis OXKLecture • Group-reactive LPS epitopes • Group and species specificauthor protein epitopes a. 17 kDa proteinOnline antigenby (lipoprotein) © b. S-layer proteins and SCA proteins c. Heat shock proteins ESCMID Indirect Immunofluorescence Assay: Mouse Serum Serotyping, Philip et al. 1978 Library SPecificity Difference= (Aa + Bb) – (Ab + Ba) SPD >Lecture3 : different serotypes SPD < 3 : the same serotypes Aa & Bb, titerauthor of serum with homologous antigen Ab & Ba, titer of serum with heterologous antigen Onlineby © ESCMID Antigenic Diversity of Rickettsia Library Lecture author Onlineby © A: Antigenic diversity estimated based on ESCMID monoclonal antibody panel reactivity Xu and Raoult, 1998 B. ompA sequence diversity Limitations • Hard to standardize Library • Subjective • Need multiple controlsLecture author Onlineby © ESCMID Library Chromosome Typing: RFLP and PFGE Lecture • Infrequent cutter restriction endonuclease analysis • Pulsed authorfield gel electrophoresis Onlineby © ESCMID Roux and Raoult 1995; Eremeeva et al. 1994 Library Lecture Not applicableauthor to uncultivated Onlineby © Limits ESCMID Rickettsia Taxonomy: Molecular Criteria Criteria Free livingLibrary bacteria DNA-DNA hybridization Cut off 70% Average nucleotide identity LectureCut off 94% 16S rRNA gene sequence author Cut off 97% similarity Onlineby © Common standards of species definition are not useful for classification of rickettsiae ESCMID Comparison of Nucleotide Sequences Genus level •16S rRNA gene (rrs) Library •Citrate synthase gene (gltA) •17 kDa protein gene Species level Lecture •Citrate synthase gene (gltA) •OmpA proteinauthor gene (ompA) Online•OmpBby protein gene (ompB) •Sca4© protein gene (sca4) Isolate level Intergenic regions ESCMID Multiple Gene Sequence Analysis gltA ompA ompB scaLibrary4 Group R. rickettsii, R. conorii Strain S, R. africae R. parkeri, R. sibirica R. mongolotimonae RrickG RrickG RrickG RrickG R. slovaca, R. honei Rjapn R. japonica Rjapn Rjapn Rjapn Rmont R. montanenesis Rmont Rmont LectureRmont RmasG RmasG R. massiliae, Bar 29 RmasG RmasG R. rhipicephali Rhelv R. aeschlimannii Rhelv Rhelv author R. felis Rfel Rfel Rfel Rfel RakrG RakrG RakrG RakrG R. akari Onlineby R. australis RproG RproG RproG R. prowazekii R. typhi AB © AB AB bacterium Rcan Rcan R. canadensis Rbel Rbel R. bellii ESCMID Adapted from Z. Sekeyova, V. Roux & D. Raoult, 2001 Library Phylogeny of Individual Coding Sequences Match Lecture author Phylogeny of Whole Genome Onlineby © ESCMID Concatenated Genes for Type Four Secretion System (left) and Whole Genome (right) Library Lecture author Onlineby © ESCMID Multiple Locus Sequence Analysis Library Gene target Cut off Classification 16S rRNA gene 98.1% Rickettsia sp. gltA 86.5% LectureRickettsia sp. 99.9% New or validated Rickettsia sp. author ompA 98.8% New or validated Rickettsia sp. Onlineby ompB 99.2%© New or validated Rickettsia sp. sca4 99.3% New or validated Rickettsia sp. ESCMID Fournier et al., 2003 Defining a Status of New Rickettsia • New sequences, but it does not growLibrary – Candidatus Rickettsia sp. • New sequences, sustained isolate, unique phenotypic features – LectureNEW Rickettsia sp. • Sequences similar to a validated species, but the isolate is antigenically and epidemiologicallyauthor different from type strain Online– by New subspecies© of the known Rickettsia sp. • Sequences and antigenic characteristics are similar to a known Rickettsia sp. – NEW isolate (strain) ESCMID Characterizing New Rickettsia • Sequence 16S rDNA and gltA Library • Sequence ompA, ompB, sca4 • Determine genetic similarity to the nearest neighbors • Obtain sustained cell cultureLecture isolate • Describe phenotypic characteristicsauthor • Deposit type strain into two culture collections Onlineby • Publish in International© Journal of Systematic and Evolutionary Microbiology New species name should appear on the ESCMIDApproved List of Bacterial Species Library Lecture author Onlineby © ESCMID New Rickettsial Disease Library • New disease can be named with a description of single patient case • No requirements thatLecture agent is characterized or cultivatedauthor • Specific nameOnline shouldby be given to each disease caused by© each rickettsial species • Each disease should have a single name ESCMID R.sibirica R. parkeri R. africae Genus Rickettsia: R. conorii R. marmionii R. japonica R. heilongjiangii 16S rRNA gene R. peacockii R. rickettsii R. honei Library R. slovaca Endosymbiont
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