A Streamlined Method for Transposon Mutagenesis of Rickettsia Parkeri

Total Page:16

File Type:pdf, Size:1020Kb

A Streamlined Method for Transposon Mutagenesis of Rickettsia Parkeri bioRxiv preprint doi: https://doi.org/10.1101/277160; this version posted March 8, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 A streamlined method for transposon mutagenesis of 2 Rickettsia parkeri yields numerous mutations that 3 impact infection 4 5 Rebecca L. Lamason1,#a,*, Natasha M. Kafai1,#b, and Matthew D. Welch1* 6 7 8 1 Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, 9 CA 10 #a Current address: Department of Biology, Massachusetts Institute of Technology, 11 Cambridge, MA 12 #b Current address: Medical Scientist Training Program, Washington University in St. 13 Louis School of Medicine, St. Louis, MO 14 15 16 17 18 * Co-corresponding authors 19 E-mail: [email protected], [email protected] 20 1 bioRxiv preprint doi: https://doi.org/10.1101/277160; this version posted March 8, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 21 Abstract 22 The rickettsiae are obligate intracellular alphaproteobacteria that exhibit a complex 23 infectious life cycle in both arthropod and mammalian hosts. As obligate intracellular 24 bacteria, Rickettsia are highly adapted to living inside a variety of host cells, including 25 vascular endothelial cells during mammalian infection. Although it is assumed that the 26 rickettsiae produce numerous virulence factors that usurp or disrupt various host cell 27 pathways, they have been challenging to genetically manipulate to identify the key 28 bacterial factors that contribute to infection. Motivated to overcome this challenge, we 29 sought to expand the repertoire of available rickettsial loss-of-function mutants, using an 30 improved mariner-based transposon mutagenesis scheme. Here, we present the 31 isolation of over 100 transposon mutants in the spotted fever group species Rickettsia 32 parkeri. These mutants targeted genes implicated in a variety of pathways, including 33 bacterial replication and metabolism, hypothetical proteins, the type IV secretion 34 system, as well as factors with previously established roles in host cell interactions and 35 pathogenesis. Given the need to identify critical virulence factors, forward genetic 36 screens such as this will provide an excellent platform to more directly investigate 37 rickettsial biology and pathogenesis. 2 bioRxiv preprint doi: https://doi.org/10.1101/277160; this version posted March 8, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 38 Introduction 39 The Rickettsia are a genus of obligate intracellular alphaproteobacteria that are 40 divided into four groups - the spotted fever group (SFG), typhus group (TG), ancestral 41 group (AG), and transitional group (TRG) [1]. They inhabit arthropods (ticks, fleas, and 42 mites), and many can be transmitted to humans and other mammals. Pathogenic 43 species primarily target endothelial cells in the vasculature, causing a variety of vascular 44 diseases such as typhus and Rocky Mountain spotted fever [2]. Despite the prevalence 45 of rickettsial diseases throughout the world, we know little about the bacterial factors 46 required for infection and pathogenesis. 47 The SFG species Rickettsia parkeri, a tick-borne pathogen that causes a mild 48 form of spotted fever in humans [3,4], is emerging as a model organism to study SFG 49 rickettsial pathogenesis. R. parkeri can be studied under BL2 conditions and has animal 50 models of pathogenesis that mimic aspects of human infection [5,6]. Furthermore, the 51 R. parkeri life cycle closely matches that of the more virulent SFG species R. rickettsii 52 [7,8], the causative agent of Rocky Mountain spotted fever. Like its more virulent 53 relative, R. parkeri invades non-phagocytic cells and is taken into a primary phagocytic 54 vacuole [9]. They then break out of this vacuole and enter the cytosol to replicate and 55 grow [10]. R. parkeri and many other Rickettsia species also hijack the host cell actin 56 cytoskeleton to polymerize actin tails and undergo actin-based motility [11-13]. During 57 spread, motile R. parkeri move to a host cell-cell junction but then lose their actin tails 58 before entering into a short (~1 µm) plasma membrane protrusion that is subsequently 59 engulfed into the neighboring cell. The bacterium then lyses the double-membrane 60 secondary vacuole to enter the neighboring cell cytosol and begin the process again 3 bioRxiv preprint doi: https://doi.org/10.1101/277160; this version posted March 8, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 61 [14]. Because of its experimental tractability and the fact that its lifecycle is 62 indistinguishable from more virulent species, R. parkeri provides an attractive system for 63 investigating rickettsial host-pathogen interactions. 64 As an obligate intracellular pathogen, R. parkeri must produce virulence factors 65 that usurp or disrupt various host cell pathways. However, due to their obligate growth 66 requirement, it has been challenging to genetically manipulate the rickettsiae to identify 67 the key bacterial factors that contribute to infection [15]. Fortunately, recent advances 68 have expanded the genetic toolkit that can be used in the rickettsiae, allowing us to 69 more directly peer into the molecular mechanisms that drive rickettsial biology. Chief 70 among these advances was the development of a Himar1 mariner-based transposon 71 system for random mutagenesis of rickettsial genomes [16]. To date, smaller-scale 72 mutagenesis studies have been completed in the TG species R. prowazekii [16-18] and 73 the SFG species R. rickettsii [18,19]. 74 Despite these advances, we still do not know all of the critical bacterial factors 75 that mediate interactions with the host. Moreover, many of the genes in R. parkeri are 76 annotated to encode hypothetical proteins, which limits our ability to rationally explore 77 their functions. Therefore, we set out to expand the repertoire of available R. parkeri 78 mutants using a forward genetic screen. We used the mariner-based transposon 79 system [16] and developed a more streamlined protocol to rapidly isolate R. parkeri 80 mutants that alter plaque size. To date, we have isolated over 100 mutants that disrupt 81 genes predicted to function in a variety of pathways. We have previously published our 82 detailed analysis of three mutants – in sca2, rickA, and sca4 [14,20]. Here, we present 4 bioRxiv preprint doi: https://doi.org/10.1101/277160; this version posted March 8, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 83 the full panel of mutants to demonstrate the potential and ease of developing rickettsial 84 transposon libraries. 85 86 Materials and methods 87 Cell lines 88 Vero cells (monkey, kidney epithelial) were obtained from the University of 89 California, Berkeley tissue culture facility and grown in Dulbecco’s modified Eagle’s 90 medium (DMEM) (Invitrogen) containing 5% fetal bovine serum (FBS) at 37˚C in 5% 91 CO2. 92 93 Transposon mutagenesis in R. parkeri 94 R. parkeri Portsmouth strain was a gift from Dr. Chris Paddock (Centers for 95 Disease Control and Prevention). Wild-type R. parkeri were expanded and purified by 96 centrifugation through a 30% MD-76R solution, as previously described [14]. The 97 pMW1650 plasmid carrying the Himar1 mariner-based transposon [16] (a gift from Dr. 98 David Wood, University of South Alabama) was used to generate R. parkeri strains 99 carrying transposon insertions. To isolate small plaque mutants, a small-scale 100 electroporation protocol was designed. First, a T75 cm2 flask of confluent Vero cells was 101 infected with approximately 107 WT R. parkeri. Three days post-infection, when Vero 102 cells were at least 90% rounded, they were scraped from the flask. Infected cells were 103 spun down for 5 min at 1800 x g at 4˚C and resuspended in 3-6 ml K-36 buffer. To 104 mechanically disrupt infected cells and release bacteria, cells were either passed 5 bioRxiv preprint doi: https://doi.org/10.1101/277160; this version posted March 8, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 105 through a 27.5 gauge syringe needle 10 times, or vortexed at ~2900 rpm using a Vortex 106 Genie 2 (Scientific Industries Inc.) in a 15 ml conical tube containing 2 g of 1 mm glass 107 beads, with two 30 s pulses and 30 s incubations in ice after each pulse. This bead 108 disruption procedure was adopted for a majority of the screen because it was faster and 109 reduced the possibility of a needle stick. Host cell debris was pelleted for 5 min at 200 x 110 g at 4˚C. The supernatant containing R. parkeri was transferred to 1.5 ml 111 microcentrifuge tubes and spun for 2 min at 9000 x g at 4˚C. The bacterial pellets were 112 then washed three times in cold 250 mM sucrose, resuspended in 50 µl cold 250 mM 113 sucrose, mixed with 1 µg of pMW1650 plasmid, placed in a 0.1 cm cuvette, and 114 electroporated at 1.8 kV, 200 ohms, 25 µF, 5 ms using a Gene Pulser Xcell (Bio-Rad). 115 Bacteria were immediately recovered in 1.2 ml brain heart infusion (BHI) media. To 116 infect confluent Vero cells in 6-well plates, media was removed from each well, and 117 cells were washed with phosphate-buffered saline (PBS).
Recommended publications
  • (Batch Learning Self-Organizing Maps), to the Microbiome Analysis of Ticks
    Title A novel approach, based on BLSOMs (Batch Learning Self-Organizing Maps), to the microbiome analysis of ticks Nakao, Ryo; Abe, Takashi; Nijhof, Ard M; Yamamoto, Seigo; Jongejan, Frans; Ikemura, Toshimichi; Sugimoto, Author(s) Chihiro The ISME Journal, 7(5), 1003-1015 Citation https://doi.org/10.1038/ismej.2012.171 Issue Date 2013-03 Doc URL http://hdl.handle.net/2115/53167 Type article (author version) File Information ISME_Nakao.pdf Instructions for use Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP A novel approach, based on BLSOMs (Batch Learning Self-Organizing Maps), to the microbiome analysis of ticks Ryo Nakao1,a, Takashi Abe2,3,a, Ard M. Nijhof4, Seigo Yamamoto5, Frans Jongejan6,7, Toshimichi Ikemura2, Chihiro Sugimoto1 1Division of Collaboration and Education, Research Center for Zoonosis Control, Hokkaido University, Kita-20, Nishi-10, Kita-ku, Sapporo, Hokkaido 001-0020, Japan 2Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan 3Graduate School of Science & Technology, Niigata University, 8050, Igarashi 2-no-cho, Nishi- ku, Niigata 950-2181, Japan 4Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Königsweg 67, 14163 Berlin, Germany 5Miyazaki Prefectural Institute for Public Health and Environment, 2-3-2 Gakuen Kibanadai Nishi, Miyazaki 889-2155, Japan 6Utrecht Centre for Tick-borne Diseases (UCTD), Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands 7Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, 0110 Onderstepoort, South Africa aThese authors contributed equally to this work. Keywords: BLSOMs/emerging diseases/metagenomics/microbiomes/symbionts/ticks Running title: Tick microbiomes revealed by BLSOMs Subject category: Microbe-microbe and microbe-host interactions Abstract Ticks transmit a variety of viral, bacterial and protozoal pathogens, which are often zoonotic.
    [Show full text]
  • Characterization of the Interaction Between R. Conorii and Human
    Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 4-5-2018 Characterization of the Interaction Between R. Conorii and Human Host Vitronectin in Rickettsial Pathogenesis Abigail Inez Fish Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Bacteria Commons, Bacteriology Commons, Biology Commons, Immunology of Infectious Disease Commons, and the Pathogenic Microbiology Commons Recommended Citation Fish, Abigail Inez, "Characterization of the Interaction Between R. Conorii and Human Host Vitronectin in Rickettsial Pathogenesis" (2018). LSU Doctoral Dissertations. 4566. https://digitalcommons.lsu.edu/gradschool_dissertations/4566 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. CHARACTERIZATION OF THE INTERACTION BETWEEN R. CONORII AND HUMAN HOST VITRONECTIN IN RICKETTSIAL PATHOGENESIS A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Interdepartmental Program in Biomedical and Veterinary Medical Sciences Through the Department of Pathobiological Sciences by Abigail Inez
    [Show full text]
  • Transition of Serum Cytokine Concentration in Rickettsia Japonica Infection
    Article Transition of Serum Cytokine Concentration in Rickettsia japonica Infection Makoto Kondo, Yoshiaki Matsushima, Kento Mizutani, Shohei Iida, Koji Habe and Keiichi Yamanaka * Department of Dermatology, Mie University Graduate School of medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan; [email protected] (M.K.); [email protected] (Y.M.); [email protected] (K.M.); [email protected] (S.I.); [email protected] (K.H.) * Correspondence: [email protected]; Tel.: +81-59-231-5025; Fax: +81-59-231-5206 Received: 31 October 2020; Accepted: 8 December 2020; Published: 11 December 2020 Abstract: (1) Background. Rickettsia japonica (R. japonica) infection induces severe inflammation, and the disappearance of eosinophil in the acute stage is one of the phenomena. (2) Materials and Methods. In the current study, we measured the serum concentrations of Th1, Th2, and Th17 cytokines in the acute and recovery stages. (3) Results. In the acute phase, IL-6 and IFN-γ levels were elevated and we speculated that they played a role as a defense mechanism against R. japonica. The high concentration of IFN-γ suppressed the differentiation of eosinophil and induced apoptosis of eosinophil, leading to the disappearance of eosinophil. On day 7, IL-6 and IFN-γ concentrations were decreased, and Th2 cytokines such as IL-5 and IL-9 were slightly increased. On day 14, eosinophil count recovered to the normal level. The transition of serum cytokine concentration in R. japonica infection was presented. (4) Conclusions.
    [Show full text]
  • Phenotypic and Genomic Analyses of Burkholderia Stabilis Clinical Contamination, Switzerland Helena M.B
    RESEARCH Phenotypic and Genomic Analyses of Burkholderia stabilis Clinical Contamination, Switzerland Helena M.B. Seth-Smith, Carlo Casanova, Rami Sommerstein, Dominik M. Meinel,1 Mohamed M.H. Abdelbary,2 Dominique S. Blanc, Sara Droz, Urs Führer, Reto Lienhard, Claudia Lang, Olivier Dubuis, Matthias Schlegel, Andreas Widmer, Peter M. Keller,3 Jonas Marschall, Adrian Egli A recent hospital outbreak related to premoistened gloves pathogens that generally fall within the B. cepacia com- used to wash patients exposed the difficulties of defining plex (Bcc) (1). Burkholderia bacteria have large, flexible, Burkholderia species in clinical settings. The outbreak strain multi-replicon genomes, a large metabolic repertoire, vari- displayed key B. stabilis phenotypes, including the inabil- ous virulence factors, and inherent resistance to many anti- ity to grow at 42°C; we used whole-genome sequencing to microbial drugs (2,3). confirm the pathogen was B. stabilis. The outbreak strain An outbreak of B. stabilis was identified among hos- genome comprises 3 chromosomes and a plasmid, shar- ing an average nucleotide identity of 98.4% with B. stabilis pitalized patients across several cantons in Switzerland ATCC27515 BAA-67, but with 13% novel coding sequenc- during 2015–2016 (4). The bacterium caused bloodstream es. The genome lacks identifiable virulence factors and has infections, noninvasive infections, and wound contamina- no apparent increase in encoded antimicrobial drug resis- tions. The source of the infection was traced to contaminat- tance, few insertion sequences, and few pseudogenes, ed commercially available, premoistened washing gloves suggesting this outbreak was an opportunistic infection by used for bedridden patients. After hospitals discontinued an environmental strain not adapted to human pathogenic- use of these gloves, the outbreak resolved.
    [Show full text]
  • Rickettsia Prowazekii Common Human Exposure Routes
    APPENDIX 2 Rickettsia prowazekii Common Human Exposure Routes: Disease Agent: • Exposure to the feces of infected body lice. The lice are infected by a human blood meal. The rickettsiae • Rickettsia prowazekii reproduce in the louse gut epithelium. Infection occurs when louse feces are scratched into the skin, Disease Agent Characteristics: inoculated onto mucous membrane or inhaled. • As a bioweapon, the agent can be aerosolized, with • Rickettsiae are obligate intracellular Gram-negative intent of infection through inhalation. bacteria. • Sporadic cases occur after exposure to flying squir- • Order: Rickettsiales; Family: Rickettsiaceae rels, most likely as a result of exposure to squirrel flea • Size: 0.3 ¥ 1.0 mm intracellular bacteria that take up feces. The organism has also been identified in ticks Gram stain poorly feeding on livestock in Africa. • Nucleic acid: Rickettsial genomes are among the Likelihood of Secondary Transmission: smallest of bacteria at 1000-1600 kb. The R. prowazekii genome is 1100 kb. • No evidence of direct person-to-person transmission • Physicochemical properties: Susceptible to 1% • Under crowded conditions, where bathing and sodium hypochlorite, 70% ethanol, glutaraldehyde, washing clothes are difficult, and where lice are formaldehyde and quaternary ammonium disinfec- present, typhus can spread explosively. tants. Sensitive to moist heat (121°C for at least • Recent outbreaks have occurred in a number of areas 15 min) and dry heat (160-170°C for at least 1 hour). in the world under conditions of war and population The organism is stable in tick tissues or blood under displacement. ambient environmental conditions, surviving up to 1 year; sensitive to drying (feces of infected ticks At-Risk Populations: quickly lose their infectivity on drying).
    [Show full text]
  • Article/25/5/18-0438-App1.Pdf)
    RESEARCH LETTERS Pathology. 2011;43:58–63. http://dx.doi.org/10.1097/ variabilis ticks can transmit the causative agent of Rocky PAT.0b013e328340e431 Mountain spotted fever, and Ixodes scapularis ticks can 8. Rodriguez-Lozano J, Pérez-Llantada E, Agüero J, Rodríguez-Fernández A, Ruiz de Alegria C, Martinez-Martinez L, transmit the causative agents of Lyme disease, babesiosis, et al. Sternal wound infection caused by Gordonia bronchialis: and human granulocytic anaplasmosis (1). Although less identification by MALDI-TOF MS. JMM Case Rep. 2016;3: common in the region, A. maculatum ticks are dominant e005067. in specific habitats and can transmit the causative agent of Rickettsia parkeri rickettsiosis (1). Address for correspondence: Rene Choi, Department of Ophthalmology, Persons who have occupations that require them to be Casey Eye Institute, Oregon Health and Science University, 3375 SW outside on a regular basis might have a greater risk for ac- Terwilliger Blvd, Portland, OR 97239, USA; email: [email protected] quiring a tickborne disease (2). Although numerous stud- ies have been conducted regarding risks for tickborne dis- eases among forestry workers in Europe, few studies have been performed in the United States (2,3). The studies that have been conducted in the United States have focused on forestry workers in the northeastern region (2). However, because of variable phenology and densities of ticks, it is useful to evaluate tick activity and pathogen prevalence in Rickettsiales in Ticks various regions and ecosystems. Burn-tolerant and burn-dependent ecosystems, such as Removed from Outdoor pine (Pinus spp.) and mixed pine forests commonly found Workers, Southwest Georgia in the southeastern United States, have unique tick dynam- and Northwest Florida, USA ics compared with those of other habitats (4).
    [Show full text]
  • Yersinia Enterocolitica
    Li et al. BMC Genomics 2014, 15:201 http://www.biomedcentral.com/1471-2164/15/201 RESEARCH ARTICLE Open Access Gene polymorphism analysis of Yersinia enterocolitica outer membrane protein A and putative outer membrane protein A family protein Kewei Li1†, Wenpeng Gu2†, Junrong Liang1†, Yuchun Xiao1, Haiyan Qiu1, Haoshu Yang1, Xin Wang1 and Huaiqi Jing1* Abstract Background: Yersinia enterocolitica outer membrane protein A (OmpA) is one of the major outer membrane proteins with high immunogenicity. We performed the polymorphism analysis for the outer membrane protein A and putative outer membrane protein A (p-ompA) family protein gene of 318 Y. enterocolitica strains. Results: The data showed all the pathogenic strains and biotype 1A strains harboring ystB gene carried both ompA and p-ompA genes; parts of the biotype 1A strains not harboring ystB gene carried either ompA or p-ompA gene. In non-pathogenic strains (biotype 1A), distribution of the two genes and ystB were highly correlated, showing genetic polymorphism. The pathogenic and non-pathogenic, highly and weakly pathogenic strains were divided into different groups based on sequence analysis of two genes. Although the variations of the sequences, the translated proteins and predicted secondary or tertiary structures of OmpA and P-OmpA were similar. Conclusions: OmpA and p-ompA gene were highly conserved for pathogenic Y. enterocolitica. The distributions of two genes were correlated with ystB for biotype 1A strains. The polymorphism analysis results of the two genes probably due to different bio-serotypes of the strains, and reflected the dissemination of different bio-serotype clones of Y. enterocolitica. Keywords: Yersinia enterocolitica, ompA, p-ompA, ystB Background are mainly referred to type III secretion system (TTSS) Y.
    [Show full text]
  • Health: Epidemiology Subchapter 41A
    CHAPTER 41 – HEALTH: EPIDEMIOLOGY SUBCHAPTER 41A – COMMUNICABLE DISEASE CONTROL SECTION .0100 – REPORTING OF COMMUNICABLE DISEASES 10A NCAC 41A .0101 REPORTABLE DISEASES AND CONDITIONS (a) The following named diseases and conditions are declared to be dangerous to the public health and are hereby made reportable within the time period specified after the disease or condition is reasonably suspected to exist: (1) acquired immune deficiency syndrome (AIDS) - 24 hours; (2) anthrax - immediately; (3) botulism - immediately; (4) brucellosis - 7 days; (5) campylobacter infection - 24 hours; (6) chancroid - 24 hours; (7) chikungunya virus infection - 24 hours; (8) chlamydial infection (laboratory confirmed) - 7 days; (9) cholera - 24 hours; (10) Creutzfeldt-Jakob disease - 7 days; (11) cryptosporidiosis - 24 hours; (12) cyclosporiasis - 24 hours; (13) dengue - 7 days; (14) diphtheria - 24 hours; (15) Escherichia coli, shiga toxin-producing - 24 hours; (16) ehrlichiosis - 7 days; (17) encephalitis, arboviral - 7 days; (18) foodborne disease, including Clostridium perfringens, staphylococcal, Bacillus cereus, and other and unknown causes - 24 hours; (19) gonorrhea - 24 hours; (20) granuloma inguinale - 24 hours; (21) Haemophilus influenzae, invasive disease - 24 hours; (22) Hantavirus infection - 7 days; (23) Hemolytic-uremic syndrome – 24 hours; (24) Hemorrhagic fever virus infection - immediately; (25) hepatitis A - 24 hours; (26) hepatitis B - 24 hours; (27) hepatitis B carriage - 7 days; (28) hepatitis C, acute - 7 days; (29) human immunodeficiency
    [Show full text]
  • Pdfs/ Ommended That Initial Cultures Focus on Common Pathogens, Pscmanual/9Pscssicurrent.Pdf)
    Clinical Infectious Diseases IDSA GUIDELINE A Guide to Utilization of the Microbiology Laboratory for Diagnosis of Infectious Diseases: 2018 Update by the Infectious Diseases Society of America and the American Society for Microbiologya J. Michael Miller,1 Matthew J. Binnicker,2 Sheldon Campbell,3 Karen C. Carroll,4 Kimberle C. Chapin,5 Peter H. Gilligan,6 Mark D. Gonzalez,7 Robert C. Jerris,7 Sue C. Kehl,8 Robin Patel,2 Bobbi S. Pritt,2 Sandra S. Richter,9 Barbara Robinson-Dunn,10 Joseph D. Schwartzman,11 James W. Snyder,12 Sam Telford III,13 Elitza S. Theel,2 Richard B. Thomson Jr,14 Melvin P. Weinstein,15 and Joseph D. Yao2 1Microbiology Technical Services, LLC, Dunwoody, Georgia; 2Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota; 3Yale University School of Medicine, New Haven, Connecticut; 4Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, Maryland; 5Department of Pathology, Rhode Island Hospital, Providence; 6Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill; 7Department of Pathology, Children’s Healthcare of Atlanta, Georgia; 8Medical College of Wisconsin, Milwaukee; 9Department of Laboratory Medicine, Cleveland Clinic, Ohio; 10Department of Pathology and Laboratory Medicine, Beaumont Health, Royal Oak, Michigan; 11Dartmouth- Hitchcock Medical Center, Lebanon, New Hampshire; 12Department of Pathology and Laboratory Medicine, University of Louisville, Kentucky; 13Department of Infectious Disease and Global Health, Tufts University, North Grafton, Massachusetts; 14Department of Pathology and Laboratory Medicine, NorthShore University HealthSystem, Evanston, Illinois; and 15Departments of Medicine and Pathology & Laboratory Medicine, Rutgers Robert Wood Johnson Medical School, New Brunswick, New Jersey Contents Introduction and Executive Summary I.
    [Show full text]
  • Ohio Department of Health, Bureau of Infectious Diseases Disease Name Class A, Requires Immediate Phone Call to Local Health
    Ohio Department of Health, Bureau of Infectious Diseases Reporting specifics for select diseases reportable by ELR Class A, requires immediate phone Susceptibilities specimen type Reportable test name (can change if Disease Name other specifics+ call to local health required* specifics~ state/federal case definition or department reporting requirements change) Culture independent diagnostic tests' (CIDT), like BioFire panel or BD MAX, E. histolytica Stain specimen = stool, bile results should be sent as E. histolytica DNA fluid, duodenal fluid, 260373001^DETECTED^SCT with E. histolytica Antigen Amebiasis (Entamoeba histolytica) No No tissue large intestine, disease/organism-specific DNA LOINC E. histolytica Antibody tissue small intestine codes OR a generic CIDT-LOINC code E. histolytica IgM with organism-specific DNA SNOMED E. histolytica IgG codes E. histolytica Total Antibody Ova and Parasite Anthrax Antibody Anthrax Antigen Anthrax EITB Acute Anthrax EITB Convalescent Anthrax Yes No Culture ELISA PCR Stain/microscopy Stain/spore ID Eastern Equine Encephalitis virus Antibody Eastern Equine Encephalitis virus IgG Antibody Eastern Equine Encephalitis virus IgM Arboviral neuroinvasive and non- Eastern Equine Encephalitis virus RNA neuroinvasive disease: Eastern equine California serogroup virus Antibody encephalitis virus disease; LaCrosse Equivocal results are accepted for all California serogroup virus IgG Antibody virus disease (other California arborviral diseases; California serogroup virus IgM Antibody specimen = blood, serum, serogroup
    [Show full text]
  • Mechanisms of Rickettsia Parkeri Invasion of Host Cells and Early Actin-Based Motility
    Mechanisms of Rickettsia parkeri invasion of host cells and early actin-based motility By Shawna Reed A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Microbiology in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Matthew D. Welch, Chair Professor David G. Drubin Professor Daniel Portnoy Professor Kathleen R. Ryan Fall 2012 Mechanisms of Rickettsia parkeri invasion of host cells and early actin-based motility © 2012 By Shawna Reed ABSTRACT Mechanisms of Rickettsia parkeri invasion of host cells and early actin-based motility by Shawna Reed Doctor of Philosophy in Microbiology University of California, Berkeley Professor Matthew D. Welch, Chair Rickettsiae are obligate intracellular pathogens that are transmitted to humans by arthropod vectors and cause diseases such as spotted fever and typhus. Spotted fever group (SFG) Rickettsia hijack the host actin cytoskeleton to invade, move within, and spread between eukaryotic host cells during their obligate intracellular life cycle. Rickettsia express two bacterial proteins that can activate actin polymerization: RickA activates the host actin-nucleating Arp2/3 complex while Sca2 directly nucleates actin filaments. In this thesis, I aimed to resolve which host proteins were required for invasion and intracellular motility, and to determine how the bacterial proteins RickA and Sca2 contribute to these processes. Although rickettsiae require the host cell actin cytoskeleton for invasion, the cytoskeletal proteins that mediate this process have not been completely described. To identify the host factors important during cell invasion by Rickettsia parkeri, a member of the SFG, I performed an RNAi screen targeting 105 proteins in Drosophila melanogaster S2R+ cells.
    [Show full text]
  • Scrub Typhus and Molecular Characterization of Orientia Tsutsugamushi from Central Nepal
    pathogens Article Scrub Typhus and Molecular Characterization of Orientia tsutsugamushi from Central Nepal Rajendra Gautam 1, Keshab Parajuli 1, Mythili Tadepalli 2, Stephen Graves 2, John Stenos 2,* and Jeevan Bahadur Sherchand 1 1 Department of Microbiology, Maharajgunj Medical Campus, Institute of Medicine, Kathmandu 44600, Nepal; [email protected] (R.G.); [email protected] (K.P.); [email protected] (J.B.S.) 2 Australian Rickettsial Reference Laboratory, Geelong, VIC 3220, Australia; [email protected] (M.T.); [email protected] (S.G.) * Correspondence: [email protected]; Tel.: +61-342151357 Abstract: Scrub typhus is a vector-borne, acute febrile illness caused by Orientia tsutsugamushi. Scrub typhus continues to be an important but neglected tropical disease in Nepal. Information on this pathogen in Nepal is limited to serological surveys with little information available on molecular methods to detect O. tsutsugamushi. Limited information exists on the genetic diversity of this pathogen. A total of 282 blood samples were obtained from patients with suspected scrub typhus from central Nepal and 84 (30%) were positive for O. tsutsugamushi by 16S rRNA qPCR. Positive samples were further subjected to 56 kDa and 47 kDa molecular typing and molecularly compared to other O. tsutsugamushi strains. Phylogenetic analysis revealed that Nepalese O. tsutsugamushi strains largely cluster together and cluster away from other O. tsutsugamushi strains from Asia and elsewhere. One exception was the sample of Nepal_1, with its partial 56 kDa sequence clustering Citation: Gautam, R.; Parajuli, K.; more closely with non-Nepalese O. tsutsugamushi 56 kDa sequences, potentially indicating that Tadepalli, M.; Graves, S.; Stenos, J.; homologous recombination may influence the genetic diversity of strains in this region.
    [Show full text]