Library Lecture Anaplasmataceae author Onlineby Marina E. Eremeeva,© MD, PhD Centers for Disease Control and Prevention, Atlanta, USA Post Graduate Educational Course: Intracellular bacteria – from biology to clinic ESCMID2-5 November 2010, Sousse-Tunisia 16S rDNA Taxonomy and Biology Library Erythrocytes Platelets Neutrophiles Lecture and granulocytes Monocytes and author macrophages Onlineby Endothelial © cells Insect and helminth cells ESCMID http://www.udel.edu/biology; courtesy of J.S. Dumler Staining of Ehrlichia and Anaplasma Library Lecture author Onlineby © •Ehrlichia and Anaplasma infect blood cells •Grow in membrane-bound inclusions, morulae •RomanowskyESCMID stains (Giemsa, Wright, Dif-Quik, LeukoStat) Goodman et al., 1996; Pritt et al., 2011 Electron Microscopic Structure of Ehrlichia and Anaplasma Library Lecture author Onlineby © A. Ehrlichia canis B. Ehrlichia chaffeensis ESCMIDC. Anaplasma phagocytophilum D. Ehrlichia muris Popov et al. 1998, Karpathy et al., 2010 Library Lecture authorLife Cycle and Pathogenesis Onlineby © ESCMID Adapted from Ristic, 1990 Animal Diseases • E. canis (1935) Canine ehrlichiosisLibraryEhrlichia • E. equi (1988) Equine ehrlichiosis • E. ewingii (1992) Canine granulocytic ehrlichiosis • E. muris (1995) Persisting ehrlichiosis • E. ruminantium (1925) HeartLecture water • A. phagocytophilum (1949) Tick-borne fever Anaplasma • A. bovis (1936) Bovineauthor ehrlichiosis • A. ondiri Bovine petechial fever • A. ovis (1924) OnlinebyOvine ehrlichiosis • A. platys (1983) © Canine cyclic thrombocytopenia • N. risticii (1985) Potomac horse fever • N. helminthoeca (1953) Salmon poisoning disease ESCMID Neorickettsia Recognition of Human Pathogens Library •1954: Neorickettsia sennetsu •1987: Ehrlichia chaffeensisLecture (human monocytic ehrlichiosis) •1994: Anaplasma phagocytophilumauthor (human anaplasmosis)Onlineby •1999: Ehrlichia© ewingii (E. ewingii ehrlichiosis) ESCMID Vectors and Distribution in the USA Library Amblyomma Ixodes scapularisLectureIxodes pacificus americanum Black-legged tick Western black-legged tick Lone star tick author Onlineby © E. chaffeensis & E. ewingii A. phagocytophilum ESCMID Life Cycle of Ticks and Transmission of Infected or Uninfected AdultAnaplasmataceaeUninfected Uninfected 1st Blood Meal Female Ixodes scapularis Eggs Larvae Library Lecture 3rd Blood Meal 2nd Blood Meal Adult Mating author Infected Onlineby Nymph © Infected Infected InfectionESCMID route Adult Human Clinical Presentations Library Symptoms %HME %HGA Fever 97 98 Malaise 84 95 Myalgia/Arthralgia 68Lecture 93 Chills, Rigor 61 80 Headache 81author 78 Nausea Onlineby48 29 Cough © 26 26 Confusion 20 13 Bakken et al, 1996;ESCMID Aguero-Rosenfield et al. 1996; HME-human monocytic ehrlichiosis; Fishbein et al., 1994; Steere et al., 1983 HGA- human granulocytic anaplasmosis Clinical Features HME LibraryHGA Interval to medical 4 d 4 - 8 d attention Incubation period 0-34 d (9 d median) 1-60 d (6-10 d median) Tick exposure 80%Lecture 75-85% Severity author - hospitalization 40-62% 56% (7% ICU) - length of illness Online23 d (median)by 6 d (hospital days) - case fatality rate ©3.0% 0.5-1.7% ESCMID Laboratory Diagnosis Library Routine laboratory blood test leucopenia, lymphocytopenia, low platelets and elevated liver enzymes Lecture •Detection of morulae authorin blood smear •Culture isolationOnlineby •Immunohistochemistry© •PCR detection of bacterial DNA •Serology ESCMID Peripheral Blood Findings in Patients with Ehrlichioses and Anaplasmosis LibraryHME • Morulae: intraleukocytic bacterial clusters (2-5 microns) peripheral • bloodPercentage of infected cells generally low (<1%) Lecture • E. chaffeensis: monocytes, macrophages, lymphocytes author • A. phagocytophilum: neutrophils, bands Onlineby • E. ewingii: neutrophils,© bands, eosinophils HGA HME – 2 to 38% sensitive ESCMID HGA – 25 to 75% sensitive Diagnosis of E. chaffeensis and A. phagocytophilum Infection by Culture Isolation • Generally reserved for specialized public health reference or research laboratories Library • Biosafety concerns much lower than for Rickettsia • Samples – Heparin-, citrate-, or EDTA-anticoagulated blood, buffy coat, clotted blood, leukocyte-rich plasma, or purified leukocytes Lecture – CSF • Mammalian cells author – E. chaffeensis: DH-82, THP-1, U937, HMEC-1 cells – A. phagocytophilumOnline: HL-60,by THP-1, KG-1, HMEC-1… • Invertebrate cells © – IDE8, ISE6, or AAE2 tick cells • Culture methods – Standard flask-based cell culture • TimeESCMID to detection: 3 to 90 days Courtesy of J.S. Dumler Library Lecture author Onlineby Pathology Findings © ESCMID Immunohistochemical detection of HME in lung tissue Courtesy of C. Paddock IFA Testing: Guidelines for InterpretationLibrary • Confirmation of disease (e.g. ehrlichiosis or anaplasmosis) requires a four-fold rise in titer AND a clinically compatible illnessLecture • Four-fold rise in titer : “recent” exposure to Ehrlichia or Anaplasma author • Single titer > 64: exposureOnlineby to Ehrlichia or Anaplasma at some undetermined© time • Single titer < 64: no significant antibody detected ESCMID IFA Testing : Limitations Library • Diagnostic titer may not appear for 7-14 days; blunted or delayed response in settings of early testing • Persistence of antibody: elevated IgG may last > 2.5 years; IgM > 1.5 years after Lectureacute illness • Serologic cross-reactivity (especially IgG antibodies) among species of Anaplasmataceae:author - E. chaffeensisOnlineand E.by ewingii - E. chaffeensis and© A. phagocytophilum - other microorganisms • IgM antibody may help discriminate ESCMID Laboratory Diagnosis: test comparison Library Sensitivity Test HME HGA Blood smear (acute phase only) Lecture2-38% 25-75% Whole blood PCR (acute only) 50-60% 50-60% Culture (acute only) author50-95% 55-95% IFA Serology (paired sera)Online IgMby 86% 27-37% ©IgG 88% 82-100% Ehrlichia ewingii infection: - neutrophil inclusions (HGA-like) - serologically cross-reacts with HME ESCMID - genetically distinct (PCR) - not cultivated Example 1: Febrile Illness in Missouri Library • Cluster of 28 patients from different parts of the same county visiting the same hospital (15 , 13 ) 29-83 yr old (median 60 yr old) Lecture • Fever 101-102oF, myalgia, malaise, fatigue, headache, vomiting, thrombocytopeniaauthor and leukopenia Onlineby • Most patients reported© or suspected a tick bite prior to disease onset (June-September) ESCMID PCR Results Library Lecture author Onlineby © Nested PCR: 8/28 positive for Ehrlichia chaffeensis VLPT EDTA 2/28 positive for Ehrlichia ewingii 16S rDNA whole bloodESCMID 28/28 negative for SFG rickettsia 17 kDa antigen gene PCR vs IFA for Laboratory Confirmed Cases from Missouri Library Test N Performance Results HME (8) PCR 10 100% LectureE . ewingii (2) Acute serum [8/10] <32 10 20%author submitted [2/10] >1024 HME Onlineby Convalescent >1024 HME serum 1 © 10% submitted ESCMID Example 2 • 51-yr male from Eau Claire Co., WI became ill in June, 2009 • Fever, fatique, headache, leucopenia, lymphocytopenia,Library low platelets and elevated liver enzymes • Reported exposure to ticks • Acute blood sample was PCR positive for Ehrlichia sp. at Mayo Clinic Laboratory • Treated with doxycycline and recoveredLecture author Patient sample Onlineby © Detection by FRET PCR groEL DNA detected in clinical sample belongs ESCMID to an Ehrlichia agent very similar to Ehrlichia L. Sloan and B. Pritt, MCL muris from Asia Vector, Reservoir and Prevalence of E. muris and E. muris-like agent Library Characteristics Old World USA Geographic range Western Europe MN, WI to JapanLecture Ticks I. persulcatus, I. scapularis I. ovatus, I. authorricinus Reservoir Small rodents, Peromyscus Onlinebydeer leucopus Prevalence (rodent) ©5.5 -63% ND Prevalence (human) 1.1-4.5% ND ESCMID Vorobyeva et al 2002, Kawahara et al 1999 Ehrlichia canis Library • Affects dogs, wolves and other canids • Tracker dog disease, tropical canine pancytopenia, canine hemorrhagic fever and canine typhus • Transmitted by Rh. sanguineusLecture, the brown dog tick • Acute stage (fever, malaise, lymphadenomegaly, splenomegaly, thrombocytopenia,author leukopenia, and nonregenerativeOnline anemia)by • Asymptomatic persistent© and chronic infection (ocular disease and meningitis) ESCMID E. canis as a Cause of Human Infection Library Lecture author Onlineby © • 20 febrile patients with symptoms resembling HME • ESCMID6/20 PCR positive for E. canis Perez et al, 1996 Tick-transmitted Ehrlichia sp. P-Mt from Georgia, USA, that is closely related to E. ruminantium • First detected in 1.6% A. Library americanum ticks from Panola Mountain State Park, GA • Maintained transstadially in Lecture ticks • Causes febrile illness in experimentally infected goats author • Causes infection in deerOnlineby • Genetic variations detected© in map1 16S rRNA gene phylogenetic tree ESCMID Loftis et al, 2006; Loftis et al, 2010 ASR, Yabsley et al 2008 Ehrlichia ruminantium • Endemic to Africa Library • Transmitted by Amblyomma ticks • Infects cattle, sheep, goats, antelope and water buffalo Lecture • Non-ruminant carriers: guinea-fowl, leopard tortoises, scrub hareauthor • Symptoms: fever, nervousby signs, hydropericardium,Online hydrothorax, ascites, © Hydropericardium edema of lungs, and high mortality from www.vet.uga.edu • Several reports describe human cases caused by E. ruminantium ESCMID Allsopp et al. 2005; Louw et al. 2005 Panola Mountain Ehrlichia • Wide distribution range Library Collection State Questing ticks Human-biting ticks Florida 0.66% (151) 0%(7) Georgia