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F S M T ESCMID Postgraduate Education Course P S Intra cellular Bacteria : From Biology to Clinic I 2 - 5 November 2010, Sousse - TunisiaLibrary

Ecology and Lectureepidemiology of Anaplasmaauthor Onlineby ©

DR. Ali BOUATTOUR, PhD ESCMID Medical entomology laboratory IPT GENUS ANAPLASMA

¾Kingdom : BACTERIA Library ¾ Phylum: PROTEOBACTERIA

¾ Class: ALPHAPROTEOBACTERIALecture ¾ Order: RICKETTSIALESauthor Onlineby © ¾ Family : ANAPLASMATACEAE ESCMID

ESCMID Postgraduate Education Course Intra cellular Bacteria : From Biology to Clinic SOUSSE- 2-5 /11/2010 Library

Lecture author Onlineby ©

Using ESCMIDthe 16S rRNA gene and the groESL operon sequence analysis, the genus Anaplasma forms a clade distinct from Ehrlichia,Wolbachia, Neorickettsia, Rickettsia and Orientia in the Alphaproteobacteria, Order Rickettsiales.Organisms possess not less than 96% 16S rRNA gene similarity. Rikihisa, 2010, Microbiology List of species of the genus ANAPLASMA (1)

Species Hosts/ Host cell Vectors Distribution reservoir A. marginale Cattle, wild Erythrocytes Boophilus microplus,Library Worldwide ruminants and Dermacentor cervids (deer, elk, andersoni & water buffalo , mecanical vectors sheep) A. bovis Cattle MonocytesLectureRh. appendiculatus, Africa, (E. bovis) (Benign) A. variegatum Asia, A. cajennense America ? authorI. scapularus Onlineby Hae. liltileporispalustris © Hy. excavatum A. centrale Cattle Erythrocytes Rh. appendiculatus, Africa A. variegatum ESCMID

ESCMID Postgraduate Education Course Intra cellular Bacteria : From Biology to Clinic SOUSSE- 2-5 /11/2010 List of species of the genus ANAPLASMA (2)

Species Hosts/ Host cell Vectors Distribution reservoir A. ovis Sheep and goats Erythrocytes Rh. bursaLibrary Worldwide ? Dermacentor sp A. platys Dogs Platelets Rh. sanguineus Europe (E. platys) Infectious cyclic North America thrombocytopenia Lecture Tunisia Taiwan A. mesaeterum sheep Erythrocytes Ixodes ricinus Europe authorHae punctata Onlineby A. caudatum Cattle Often found in © mixed infection with A. ESCMID marginale

ESCMID Postgraduate Education Course Intra cellular Bacteria : From Biology to Clinic SOUSSE- 2-5 /11/2010 List of species of the genus ANAPLASMA (3) Species Hosts/ Host cell Geographical disribution reservoir A. phagocytophilum Humans GranulocytesLibrary Europe (agent of HGE) Dogs North America (Human granulocytic ehrlichiois)Lecture

A. phagocytophilum Horses Granulocytes Worldwide (Ehrlichia equi) (Equine author Onlinegranulocytic ehrlichiosis)by A. phagocytophilum ©Ruminants Granulocytes Europe ( Ehrlichia phagocytophila) (tick-borne fever) ESCMID

ESCMID Postgraduate Education Course Intra cellular Bacteria : From Biology to Clinic SOUSSE- 2-5 /11/2010 List of species of the genus ANAPLASMA (4)

¾ In 2001, taxonomic reclassification Species grouped all as a single species : A. phhthilagocytopLibraryhilum A. phagocytophilum (agent of HGE) ¾ Variants of A phagocytophilum. phagocytophilum present A. phagocytophilum minor differences in the sequences of the (Ehrlichia equi) 16S rRNA orLecture the msp4 (major surface proteins) genes. A. phagocytophilum ¾ Some variants are antigenically similar, ( Eh. phagocytophila) author Onlineif not byidentical, ¾ but there© are a very little information about the cross -protective effect of the different variants ESCMIDThis bacteria is the agent of Human Granulocytic Anaplasmosis (HGA) Considered as an emerging tick-borne disease HUMAN GRANULOCYTIC ANAPLASMOSIS (HGA)

-The first case of clinically recognized HGA was described in the USA (Ch en et al ., 1994) . Library

-The life cycle of A phagocytophilum. phagocytophilum is not fully understood but has been adequately investigated.Lecture

- A. phagocytophilum is naturally maintainedauthor in a zoonotic cycle between : Onlineby ) mammalian© reservoir hosts and ESCMID) tick vectors

ESCMID Postgraduate Education Course Intra cellular Bacteria : From Biology to Clinic SOUSSE- 2-5 /11/2010 What is the reservoir of A. phagocytophilum IN EUROPE ?

- AdApodemus sy ltilvaticus (d(wood Librarymouse ) - Clethrionomys glareolus (bank vole) - Sorex aranusLecture(common shrew) - Capreolus capreolus (roe deer) - Rupicapra rupicapraauthor (Chamois) Onlineby - Vulpes© vulpes (foxes) - Sus scrofa (()wild boar)

ESCMIDDomestic animals : - horses, cattle, sheep and dogs What is the reservoir of A. phagocytophilum TUNISIA ?

Partial amplification 16S rRNA gene using nested PCR Primers used : First amplification ÎLibrary900 bp Ge 10r 5 ’’--TCCGTTAAGAAGGATCTAATCTCC--333’ Ge 3a 5’5’--CACATGCAAGTCGAACGGATTATTCCACATGCAAGTCGAACGGATTATTC-Lecture -3’3’ Secon d amp lificati on Î 546 bp author Ge 9f 5’-5’-AACGGATTATTCTTTATAGCTTGCTAACGGATTATTCTTTATAGCTTGCT-Online -3’3’ Ge2 5’-5’-GGCAGTATTAAAAGCAGCTCCAGGGGCAGTATTAAAAGCAGCTCCAGG-by -3’3’ © nPCRnPCR DNA 546 pb546 pb ESCMID AlAnaplasma ph agocytophil um MASSUNG et al. 1998 What is the reservoir of A. phagocytophilum TUNISIA ?

Presence of DNA of A. phagocytophilum in cattle Library

Lecture author Onlineby © In 3 farms in the semi-arid zone : 66 cows : 3 positives ESCMID What is the reservoir of A. phagocytophilum TUNISIA ?

Presence of DNA of A. phagocytophilum in dogs Library DNA detection : 228 dogs, only 2 positives SlSeroprevalence of A phthilhagocytophilum Lecture25.2 % (286 dogs) author Onlineby A. platys© (10/226) E. canis (11/228) ESCMID What is the reservoir of A. phagocytophilum TUNISIA ?

Presence of DNA of A. phagocytophilum in horses Library

Serolocggyical survey in collaboration with Pr. Lilia Messadi Lecture Seroprevalence of A phagocytophilum 16.3author % (343 pure breed horses) Onlineby 60% (115 cross bdbreed h)horse) © ESCMID What is the reservoir of A. phagocytophilum IN NORTH AMERICA

- Peromyypscus leucopus (the white-footed mouse) is the a prominent reservoirLibrary

- Odocoileus virginianusLecture(white-tailed deer) - Procyon lotor (raccoons ) - Didelphis virginianaauthor (Virginia opossums) - SciurusOnline spp.by (squirrels) - Mephitis© mephitis (striped skunks ) - Neotoma fuscipes (dusky-footed woodrats ) ESCMID-Tamias senex (chipmunks) squirrel What is the vectors of A. phagocytophilum ?

A. phagocytophilum is naturally maintained in a zoonotic cycle between mammalian reservoir hosts and tickLibrary vectors

Lecture author Onlineby © ESCMID What is the vectors of A.phagocytophilum NORTH AFRICA Presence of DNA of A. phagocytophilum in ticksLibrary

Lecture author Onlineby © Hy excavatum (n=90) Ixodes ricinus Hyalomma detritum Hy marginatum (n=45) Prevalence 40 % adults Prevalence 6.12 % ESCMID10% nymphs

ESCMID Postgraduate Education Course Intra cellular Bacteria : From Biology to Clinic SOUSSE- 2-5 /11/2010 What is the vectors of A.phagocytophilum IN EUROPE AND NORTH AFRICA

Tick species A. ppgyphagocytophilum Geoggpraphical Prevalence of Librarydistribution infection IdIxodes riiicinus 2 to 45% Europe LectureTunisia/ Morocco IdIxodes tr iangu liceps (lldt)(small rodents) UK author Ixodes ventalloi Online(smallby rodents) Portugal Dermacentor reticulatus © France, Austria Ixodes persulcatus Russia and Asia ESCMID Hyalomma detritum Tunisia

ESCMID Postgraduate Education Course Intra cellular Bacteria : From Biology to Clinic SOUSSE- 2-5 /11/2010 Tick vectors of A. phagocytophilum IN USA

Ixodes scapularis ((gg)blackLibrary legged tick )

Lecture author Onlineby © Eastern &mid Western States

ESCMID- Affinity for humans is very high - Prevalence of A. phagocytophilum is 10 to 50 % Tick vectors of A. phagocytophilum IN USA

Ixodes pacificus Library Western Coastal areas Lecture author mountain Onlineby areas © Other species : - Ixodes sppppinipalpis ESCMID -Dermacentor variabilis

ESCMID Postgraduate Education Course Intra cellular Bacteria : From Biology to Clinic SOUSSE- 2-5 /11/2010 Library

Lecture author Ixodes spp play an Onlineimportant role in transmitting A. phago : ¾ Live in heavily-forested or denseby brushy areas, wood border and pasture, not open areas. © ¾ Preferred hosts for immature ticks are birds and small mammals, primarily rodents but humans and dogs serve as good substitutes. ¾ ForESCMID adults, common hosts include livestock, dogs, and humans. Library

Ixodes on vegetation “questing behavior” Lecture collected b y flaggi ng author In North Tunisia, in theOnlinebiotope ofbyI . ricinus, vegetation is dominated by Quercus suber and Q. faginea© with an under tree cover with cirsium (Petridium aquitinum). In this area, the fauna is composed of domestic animals (cattle, sheep and goats) and wild animals, mainly lizards and wild boards.ESCMID

ESCMID Postgraduate Education Course Intra cellular Bacteria : From Biology to Clinic SOUSSE- 2-5 /11/2010 Life cycle of Ixodes ricinus in North Africa

Eggs deposited

Library cattle sheep.

LectureHGA Larvae adults May-July author October-Mars, Onlineby pick en © April-June November ESCMID NhNymphs Psammodromus algirus Turdus merula. Life cycle of Ixodes ricinus in Europe

larvesLaarvae Library Larvae feeds on Engorged larvae small animals

Summer 1 AUTUMN-WINTER 1

SPRING 1 Lecture Moult of larvae --> nymphs Adults (F) feeds on AUTUMN-WINTER 2 authorSPRING 2 large animals Online bySummer 2

Moult of nymphs © --> adults ESCMID NhfdNyLesmp nymphes hs fee ds on a secon dhtd host (small animals are preferred) F. Jonjean Library

Lecture author Onlineby © ESCMID

ESCMID Postgraduate Education Course Intra cellular Bacteria : From Biology to Clinic SOUSSE- 2-5 /11/2010 A. phagocytophilum is not transmitted transovarially from an adult female tick to her progeny and ticks must therefore acquire the bacterium through a blood meal.. Library c Larvae Engorged larvae Infected with A. pha Attach feed Lecture on 1st host and may acquire A. phago authorInfected Onlineby nymphs No transmission© to eggs IfInfec tdted a dltdults ESCMID The infectious agent of HGA is transmitted trans-stadially by ticks Adult ticks Nymphs Library

Adult Ixodes spp. ticks are active from October through February in the Maghreb andLecture some European countries. Nymph activity isauthor higher from April to JuneOnline. by The southern USA sees all stages, active from© November through May

ThereESCMID are also evidence of blood transfusion, nosocomial and perinatal transmissions of A. phagocytophilum. infected ticks Onset of HGA occurs 5–21 days after receiving a bite from an infected tick HGA ranges in sever ity from asymp tomaLibrary tic seroconversion to a mild or severe febrile illness. Lecture author During the period Onlineof bacteraemia, the main targets of infection are the neutrophilsby, eosinophils and monocytes, with the latter being infected© at the end of the primary bacteraemia. ESCMID

ESCMID Postgraduate Education Course Intra cellular Bacteria : From Biology to Clinic SOUSSE- 2-5 /11/2010 Clinical manifestations in patients with HGA

Library

Lecture author Onlineby ©

ESCMID Dumler et al 2005

ESCMID Postgraduate Education Course Intra cellular Bacteria : From Biology to Clinic SOUSSE- 2-5 /11/2010 LABORATORY DIAGNOSIS OF HGA 1/ Examination of peripheral blood smears - Microscopic examination of Wright- or Giemsa-stained peripheral blood smears is a rapid test after onsetLibrary of disease and before any ATB.

morulae Lecture

A.phagocytophilum authormorulae Online HL-60 humanby leukocytes of infected Canine neutrophil human patients promyelocytic© cells - Blood smear evaluation is more useful for HGA diagnosis, because 25%–75%ESCMID of reported cases in the United States have morulae in peripheral blood examinations, and sensitivity is higher during the first week of infection. Shaep. (Dumler et al,2007) LABORATORY DIAGNOSIS OF HGA

2/ Amplification of A. phagocytophilum DNA ÖThe polymerase chain reaction (PCR) is the most sensitive tool for detecting A. phagocytophilum duringLibrary acute infection.. - Sensitivity is 67–90% for detecting A. phago. DNA. -Usingthe: ¾16S rRNA gene, ¾ Msp 1a & 4 gene,Lecture ¾ ankAkaA gene ¾ the groESL operon,author - Several primers were Online used by to detect A. phagocyto in samples from human, animal or© ticks ÖMultiplex, real -time PCR , RLB can reduce the time spent waiting for results by combining the detection of two or more ehrlichiosis andESCMID anappglasmosis a gents into a sin gppgle experiment sample.

These tests need : materiel, technician and expenses LABORATORY DIAGNOSIS OF HGA 3/ I n vi tro cultivati on of b act eri a i n cell cult ure

A. phagocytophilum can be cultivated in : Library

¾ human promyelocytic leukemia cell line HL -60 by direct inoculation of cell cultures (RPMI)Lecture with peripheral blood from a potentially infected patient. After 5 days, morulae can be detected, using Wright or Giemsa staining, in the cytoplasm of infected cells. author Onlineby ¾ Tick cell lines (IDE8 and© ISE6) derived from embryos of the Ixodes scapularis; (BME6) from Rh. microplus . ESCMID ¾ Human,,ypy monkey and capillary endothelial cells. LABORATORY DIAGNOSIS OF HGA

3/ In vitro cultivation of bacteria in cell culture Library ) Need up to 2 weeks or longer for a positive result. ) SliiiSo, cultivation is not generall llilblfly available or useful for clinical diagnosis at the timeLecture of active disease. ) Necessary training in cell cultureauthor techniques and biosafety L3 unit. Onlineby )Facilitates© studies on the mechanism. )Allows genera tion o f an tigens for immuno log ica l s tu dies. ESCMID

serodiagosis ESCMID Postgraduate Education Course Intra cellular Bacteria : From Biology to Clinic SOUSSE- 2-5 /11/2010 LABORATORY DIAGNOSIS OF HGA

4/ Serodiagnosis Serological detection of antibodies in patient serum Library or plasma by : ªIFA the most sensitive and widely used diagnostic method.

) It employs the detection of IgM andLecture IgG antibodies, together or separatltely, th thtat are reacti ve aga inst AhA. phagocy thiltophilum. author ) Infected tissue cultureOnline cells or by purified bacteria can be fixed to glass slides. © ) Sensitivity is high [ranges from 82-100% IgG]. ) But 2–4 weeks following disease onset. ESCMID ) Seropositi vit y agai nst AhA. phagocy thiltophilum, while d ec lini ng in titer, often lasts for months and sometimes years after initial exposure. Diagnosis of A. phago infection is confirmed by a ffldfourfold increase inantibo dy Library titers between acute and convalescent sera or a seroconversion to a titer of 128 or higher Lecture author Onlineby Positive sera by IFA False-positive can occur mainly© with Rocky Mountain spotted fever, typhus, Q fever, brucellosis, Lyme disease, Epstein–Barr virus and autoimmuneESCMID disorders that produce autoantibodies (ei. rheumatoid factor, antinuclear antibodies , …….) . 4/ Serodiagnosis Serological detection of antibodies in patient serum or plasma by : ª IFA ª ELISA Library Available ELISA kits for the detection of antibodies against A. marg A. centrale and AovisA. ovis are available . For A. phagocytophilum, no commercialLecture kit, but some laboratories use a h ouse ma de ELIS Atest - ELISA is sensitive, specific and rapid. - Easy to use. author Onlineby © ESCMID

ESCMID Postgraduate Education Course Intra cellular Bacteria : From Biology to Clinic SOUSSE- 2-5 /11/2010 CONCLUSION After a tick bit, there is a ‘grace period’ before which the pathogen is transferred to the host. After infection, there is a varyyging period of time before clinical syypmptoms or signs appear. Detection of the pathogen by direct visualization, molecular analysis,orcultivation is possible in the earlier phase of illness. Later, serology is most useful, and can be used for both clinical and seroepidemiological purposes. 5ClltiCollection of specimens shldhould be ttdtargeted to the appropritiatephase of the disease dldevelopmen t to ensure optimal results. Library

Lecture author Onlineby © ESCMID Case definitions were adopted in Europe by the ESCAR- ESCMID : Library Confirmed HGA definition requires :

5 febrile illness with a history ofLecture a tick bite or a tick exposure, 5 demonstration of A. phagocytophilum infection by : - seroconversion or > 4-foldauthor change in serum antibody titers, Onlineby - or PCR positive© plus sequencing Anaplasma- spp,ecific DNA in the blood, ESCMID- or isolation of A. phagocytophilum in blood culture.

Brouqui et al. CMI, 2005

ESCMID Postgraduate Education Course Intra cellular Bacteria : From Biology to Clinic SOUSSE- 2-5 /11/2010 Library

Lecture author Onlineby © ESCMID WE WISHE YOU A NICE STAY IN TUNSIA Library Detection of Anaplasma phagocytophilum phagocytophilum in ticks and cattleLecture in Tunisia author Onlineby © ESCMID