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Mammalian Host Cell Reservoirs during Anaplasma Infection

Permanent Link: http://ufdc.ufl.edu/UFE0024351/00001

Material Information

Title: Mammalian Host Cell Reservoirs during Anaplasma Infection
Physical Description: 1 online resource (92 p.)
Language: english
Creator: Wamsley, Heather
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: amplification, anaplasma, bovine, canine, cattle, cell, chronic, circle, cow, dna, dogs, doxycycline, electron, endothelial, fluorescent, host, immunofluorescence, immunofluorescent, infection, marginale, microscopy, padlock, phagocytophilum, rolling, situ
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Endothelial cell culture and preliminary immunofluorescent staining of Anaplasma-infected tissues suggest that endothelial cells may be an in vivo nidus of mammalian infection. To investigate endothelial cells and other potential sites of Anaplasma infection in mammalian tissues, a sensitive and specific, in situ rolling-circle amplification technique to detect localized Anaplasma gene sequences was developed. Via the technique described here and von Willebrand factor immunofluorescence, A. phagocytophilum and A. marginale were successfully localized in situ within cultured mammalian cells. This is the first application of this in situ method for detection of a microorganism and forms the foundation for applications of this technique to detect, localize, and analyze Anaplasma nucleotide sequences in the tissues of infected hosts and in cell cultures. Three Anaplasma-infection trials using immunocompetent dogs and cattle were performed to investigate different aspects of endothelial cells as they relate to Anaplasma life cycles and to further describe clinical aspects of Anaplasma pathogenesis. Four Beagles were inoculated with A. phagocytophilum from different sources, allowed to develop chronic infection, and treated with doxycycline. Regardless of isolate or duration of doxycycline treatment, A. phagocytophilum DNA remained detectable for several months in blood and tissues, though organisms were not identified microscopically. This is the first infection of dogs using cultured endothelial cells as the source of inoculum, the first demonstration of molecular evidence of chronic, persistent infection in blood and tissues of subclinical dogs despite doxycycline treatment, and the first investigation of endothelial cells as a potential in vivo source of A. phagocytophilum during chronic canine infection using in situ rolling-circle amplification. Two steers were inoculated with A. marginale by tick-feeding transmission and were euthanized at different points within the parasitemic cycle. The tissue distribution of A. marginale during peak and trough parasitemia was described using real-time PCR, though organisms were not identified in tissues microscopically. This is the first survey of A. marginale tissue distribution after tick-transmission and the first investigation in immunocompetent cattle of endothelial cells as a potential in vivo source of A. marginale in tick-bite sites and distant tissues using three techniques (immunofluorescence, electron microscopy, in situ rolling-circle amplification).
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Heather Wamsley.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Barbet, Anthony F.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024351:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024351/00001

Material Information

Title: Mammalian Host Cell Reservoirs during Anaplasma Infection
Physical Description: 1 online resource (92 p.)
Language: english
Creator: Wamsley, Heather
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: amplification, anaplasma, bovine, canine, cattle, cell, chronic, circle, cow, dna, dogs, doxycycline, electron, endothelial, fluorescent, host, immunofluorescence, immunofluorescent, infection, marginale, microscopy, padlock, phagocytophilum, rolling, situ
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Endothelial cell culture and preliminary immunofluorescent staining of Anaplasma-infected tissues suggest that endothelial cells may be an in vivo nidus of mammalian infection. To investigate endothelial cells and other potential sites of Anaplasma infection in mammalian tissues, a sensitive and specific, in situ rolling-circle amplification technique to detect localized Anaplasma gene sequences was developed. Via the technique described here and von Willebrand factor immunofluorescence, A. phagocytophilum and A. marginale were successfully localized in situ within cultured mammalian cells. This is the first application of this in situ method for detection of a microorganism and forms the foundation for applications of this technique to detect, localize, and analyze Anaplasma nucleotide sequences in the tissues of infected hosts and in cell cultures. Three Anaplasma-infection trials using immunocompetent dogs and cattle were performed to investigate different aspects of endothelial cells as they relate to Anaplasma life cycles and to further describe clinical aspects of Anaplasma pathogenesis. Four Beagles were inoculated with A. phagocytophilum from different sources, allowed to develop chronic infection, and treated with doxycycline. Regardless of isolate or duration of doxycycline treatment, A. phagocytophilum DNA remained detectable for several months in blood and tissues, though organisms were not identified microscopically. This is the first infection of dogs using cultured endothelial cells as the source of inoculum, the first demonstration of molecular evidence of chronic, persistent infection in blood and tissues of subclinical dogs despite doxycycline treatment, and the first investigation of endothelial cells as a potential in vivo source of A. phagocytophilum during chronic canine infection using in situ rolling-circle amplification. Two steers were inoculated with A. marginale by tick-feeding transmission and were euthanized at different points within the parasitemic cycle. The tissue distribution of A. marginale during peak and trough parasitemia was described using real-time PCR, though organisms were not identified in tissues microscopically. This is the first survey of A. marginale tissue distribution after tick-transmission and the first investigation in immunocompetent cattle of endothelial cells as a potential in vivo source of A. marginale in tick-bite sites and distant tissues using three techniques (immunofluorescence, electron microscopy, in situ rolling-circle amplification).
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Heather Wamsley.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Barbet, Anthony F.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024351:00001


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MAMMALIAN HOST CELL RESERVOIRS DURING ANAPLASMA INFECTION By HEATHER L. WAMSLEY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009 1

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2009 Heather L. Wamsley 2

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TABLE OF CONTENTS page LIST OF TABLES................................................................................................................. ..........5LIST OF FIGURES.........................................................................................................................6ABSTRACT.....................................................................................................................................8CHAPTER 1 BACKGROUND AND SIGNIFICANCE..............................................................................10Overview and Long-Term Objectives....................................................................................10Anaplasma Background..........................................................................................................10Anaplasma phagocytophilum and Anaplasma marginale as Significant Pathogens within Anaplasmataceae ..............................................................................................10Anaplasma Life Cycle versus the Natural Disease Course of Anaplasmosis.................12Anaplasma phagocytophilum ...................................................................................12Anaplasma marginale ..............................................................................................14Central Hypothesis............................................................................................................. .....152 IN SITU DETECTION OF ANAPLASMA BY DNA TARGET-PRIMED ROLLINGCIRCLE AMPLIFICATION OF A PADLOCK PROBE AND INTRACELLULAR COLOCALIZATION WITH IMMUNOFLUOR ESCENTLY LABELED HOST CELL VON WILLEBRAND FACTOR...........................................................................................17Introduction................................................................................................................... ..........17Materials and Methods...........................................................................................................17Cultivation of Anaplasma spp.........................................................................................17In Situ Rolling-Circle Amplification of Padlock Probes.................................................18Padlock Probe Design......................................................................................................21Indirect Immunofluorescent Staining of von Willebrand Factor in Cultured Endothelial cells...........................................................................................................22Results.....................................................................................................................................23In Situ Detection of Anaplasma spp. by DNA Target-Primed Rolling-Circle Amplification of Padlock Probes.................................................................................23In situ Intracellular Colo calization of the A. phagocytophilum Rolling-Circle Amplification Product and von Willebr and Factor Immunofluorescence...................24Discussion...............................................................................................................................253 EXPERIMENTAL INOCULATION OF DOGS WITH ANAPLASMA PHAGOCYTOPHILUM MOLECULAR EVIDENCE OF PERSISTENT INFECTION FOLLOWING DOXYCYCLINE THERAP Y, AND INVESTIGATION OF ENDOTHELIAL CELLS AS SOURCE OF INFECTIOUS INOCULUM AND A REPOSITORY OF CHRONI C INFECTION IN DOGS.......................................................33 3

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Introduction................................................................................................................... ..........33Materials and Methods...........................................................................................................34Animals, Inocula, and Monitoring for Development of Infection...................................34Serologic Assays for A. phagocytophilum Infection.......................................................36PCR Detection of A. phagocytophilum DNA in Blood and Tissues...............................37Immunosuppression, Antibiotic Tr eatment, and Euthanasia...........................................38In Situ DNA Target-Primed Rolling-Circle Amp lification of a Padlock Probe for Detection of Anaplasma phagocytophilum ..................................................................39Results.....................................................................................................................................39Physical Exam, Hematopathol ogy, and Clinical Chemistry...........................................39Serologic Evidence of Infection......................................................................................40Molecular Evidence of Infection.....................................................................................40NY18-infected dogs.................................................................................................40Canine isolate-infected dogs....................................................................................41In Situ Detection of A. phagocytophilum ........................................................................42Discussion...............................................................................................................................424 EXPERIMENTAL INFECTION OF CATTLE WITH ANAPLASMA MARGINALE DISTRIBUTION OF ORGANI SMS IN BOVINE TISSUES, AND INVESTIGATION OF ENDOTHELIAL CELLS AS A NIDUS OF ACUT E AND CHRONIC INFECTION...........................................................................................................................53Introduction................................................................................................................... ..........53Materials and Methods...........................................................................................................54Animals, Inoculum, Monitoring Infection and Parasitemia, and Euthanasia..................54Tissue Sample Collection and Processing.......................................................................55PCR Detection of A. marginale DNA in Blood and Tissues..........................................56Microscopic Detection of A. marginale Organisms in Tissues.......................................57Dual indirect immuno fluorescent staining of A. marginale and endothelial cell antigens.................................................................................................................57Electron microscopy.................................................................................................58In Situ DNA target-primed rolling-circle am plification of a padlock probe for detection of Anaplasma marginale .......................................................................59Statistical Analysis..........................................................................................................6 0Results.....................................................................................................................................60Seroconversion, Parasitemia, and Anemia......................................................................60Tissue Distribution of A. marginale ................................................................................60Microscopic Examination for A. marginale in Tissues...................................................61Discussion...............................................................................................................................635 CONCLUSIONS AND FUTURE RESEARCH....................................................................78LIST OF REFERENCES...............................................................................................................81BIOGRAPHICAL SKETCH.........................................................................................................92 4

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LIST OF TABLES Table page 3-1 Treatments administered during two infection trials using different A. phagocytophilum isolates...................................................................................................453-2 Comparison of A. phagocytophilum SNAP4Dx seroconversion between the two canine experimental inoculation studies (NY18 isolate vs. canine isolate).......................453-3 Detection of A. phagocytophilum DNA by nestedand real-time PCR in the blood of the NY18 isolate-infected dogs pre-infection through day 340 post-infection..................46 5

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LIST OF FIGURES Figure page 2-1 Padlock probes and fluorescent oligonucleotides..............................................................292-2 Detection of A. phagocytophilum using in situ DNA target-primed rolling-circle amplification of a padlock probe.......................................................................................302-3 Detection of A. marginale using in situ DNA target-primed rolling-circle amplification of a padlock probe.......................................................................................312-4 Detection of A. phagocytophilum using in situ DNA target-primed rolling-circle amplification of a padlock probe and concur rent indirect immunof luorescent staining of von Willebrand Factor...................................................................................................323-1 PCR primers and fluores ceinated oligonucleotide probe...................................................473-2 Body temperature of one of the A. phagocytophilum canine isolate-infected dogs during the infection trial.....................................................................................................483-3 Immunoblot of polyclonal sera against A. phagocytophilum NY18 isolate demonstrates immuonoreactivity (seropositivity) in the canine isolate-infected dogs......493-4 Southern blot of the A. phagocytophilum msp2 -based nested-PCR products from the NY18 isolate-infected dogs................................................................................................503-5 Southern blots of the A. phagocytophilum msp2 -based nested-PCR products from the canine isolate-infected d ogs that received doxycycline from day 155 to 183 postinoculation..........................................................................................................................513-6 In situ DNA target-primed rolling-circle amplif ication of a padlock probe in canine isolate-infected Dog 1 kidney day 233 post-infection.......................................................524-1 Steer from A. marginale tick-feeding transmission trial....................................................664-2 Competitive ELISA results for steers during the A. marginale tick-feeding transmission trial............................................................................................................. ...674-3 Packed cell volume and microscopi c parasitemia for steers during the A. marginale tick-feeding transmission trial............................................................................................684-4 Real-time quantitative PCR parasitemia detected as opag2 copies per microliter of peripheral blood of steers during the A. marginale tick-feeding transmission trial...........694-5 Real-time quantitative PCR opag2 copies per milligram of tissue in the infected steer (5237) that was euthanized during the first peak in parasitemia day 41 post-infection....70 6

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4-6 Real-time quantitative PCR opag2 copies per milligram of tissue in the infected steer (3102) that was euthanized during the first trough in parasitemia day 64 postinfection.............................................................................................................................714-7 Dual indirect im munofluorescent staining of A. marginale MSP5 (green) and von Willebrand factor (red) in uninfected (steer 5180) and infected (steer 5237) tick-bite site dermal punch biopsies day 8 post-infection................................................................724-8 Toluidine blue-stained and transmissi on electron microscopy tick-bite site dermal punch biopsies day 6 post-infection...................................................................................734-9 Toluidine blue-stained and transmi ssion electron microscopy lung from the steer (3102) that was euthanized during the first trough in parasitemia day 64 postinfection.............................................................................................................................744-10 In situ DNA target-primed rolling-circle amp lification of a padlock probe using sonicated fish sperm in the oligonucleotide hybridization solutions in A. marginale infected (steer 3102) tick-bite si te dermis day 6 post-infection.........................................754-11 In situ DNA target-primed rolling-circle amp lification of a padlock probe using sonicated fish sperm in the oligonucleotide hybridization solutions in lung from the A. marginale -infected (steer 5237) th at was euthanized during the first peak in parasitemia day 41 post-infection......................................................................................764-12 In situ DNA target-primed rolling-circle am plification of a nonspecific padlock probe using sheared calf thymus DNA in the oligonucleotide hybr idization solutions in A. marginale -infected tick-bite site dermis....................................................................77 7

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ABSTRACT OF DISSERTATION PRES ENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY MAMMALIAN HOST CELL RESERVOIRS DURING ANAPLASMA INFECTION By Heather L. Wamsley May 2009 Chair: Anthony F. Barbet Major: Veterinary Medical Sciences Endothelial cell culture and prelim inary immunofluorescent staining of Anaplasma infected tissues suggest that endothelial cells may be an in vivo nidus of mammalian infection. To investigate endothelial cells and other potential sites of Anaplasma infection in mammalian tissues, a sensitive and specific, in situ rolling-circle amplification technique to detect localized Anaplasma gene sequences was developed. Via th e technique described here and von Willebrand factor immunofluorescence, A. phagocytophilum and A. marginale were successfully localized in situ within cultured mammalian cells. This is the first application of this in situ method for detection of a microorganism and fo rms the foundation for applications of this technique to detect, localize, and analyze Anaplasma nucleotide sequences in the tissues of infected hosts and in cell cultures. Three Anaplasma -infection trials using immunocompetent dogs and cattle were performed to investigate different aspects of endothelial cells as they relate to Anaplasma life cycles and to further describe clinical aspects of Anaplasma pathogenesis. Four Beagle s were inoculated with A. phagocytophilum from different sources, allowed to develop chronic infection, and treated with doxycycline. Regardless of isolat e or duration of doxycycline treatment, A. phagocytophilum DNA remained detectable for severa l months in blood and tissues, though 8

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organisms were not identified microscopically. Th is is the first infecti on of dogs using cultured endothelial cells as the source of inoculum, the first demonstration of molecular evidence of chronic, persistent infecti on in blood and tissues of subc linical dogs despite doxycycline treatment, and the first investigation of endothelial cells as a potential in vivo source of A. phagocytophilum during chronic canine infection using in situ rolling-circle amplification. Two steers were inoculated with A. marginale by tick-feeding transmission and were euthanized at different points within th e parasitemic cycle. The tissue distribution of A. marginale during peak and trough parasitemia was descri bed using real-time PCR, though organisms were not identified in tissues microscopically. Th is is the first survey of A. marginale tissue distribution after ticktransmission and the first invest igation in immunocompetent catt le of endothelial cells as a potential in vivo source of A. marginale in tick-bite sites and di stant tissues using three techniques (immunofluores cence, electron microscopy, in situ rolling-circle amplification). 9

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CHAPTER 1 BACKGROUND AND SIGNIFICANCE Overview and Long-Term Objectives The order Rickettsiales is comprised of many important zoonotic pathogens, including the agent of human granulocytotropic anaplasmosis, Anaplasma phagocytophilum the agent of Rocky Mountain spotted fever, Rickettsia rickettsii and the agent of human monocytotropic ehrlichiosis, Ehrlichia chaffeensis (33). In vivo, endothelial cell infection in mammals has been suggested or demonstrated for many pat hogens within this order, such as, A. phagocytophilum A. marginale R. rickettsii R. typhi R. conori R. prowazekii E. ruminantium (24, 35, 54, 94, 100, 112). The importance of endothelial cells duri ng the pathogenesis of these rickettsial and ehrlichial diseases and their inte ractions with common co-infecting tick-borne organisms, such as the agent of Lyme disease, Borrelia burgdorferi is beginning to be recognized (24, 54, 81, 88, 100, 112). The long-term objectives of this work are to develop a more complete understanding of the contribution of mammalian endothelial cells to the mechanisms leading to the establishment and persistence of Anaplasma infection in mammals underlying host cell-specific adaptations by Anaplasma during different life cycle stages. This knowledge will guide future investigations aimed at development of effective treatment and control strategies for these ehrlichial pathogens of humans and animals. Anaplasma Background Anaplasma phagocytophilum and Anaplasma marginale as Significant Pathogens within Anaplasmataceae Since initially reported in 1994 (25), the number of human A. phagocytophilum infections reported to the CDC has substantially increase d, rendering it the third most common tick-borne infection of humans in th e United States (8, 34). A. phagocytophilum infections are likely under10

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reported since subclinical infections or mild, co ld-like disease are probable given the relatively high seroprevalence among hu mans residing in endemic regions (7, 8, 34). When A. phagocytophilum infections are diagnosed, over half of the individua ls with this diagnosis require potentially costly hospitalization, and 7% require admission to the intensive care unit (34). When diagnosed early, most patients exhi bit rapid clinical impr ovement after doxycycline treatment; however, protracted disease course or death is possible among those who are heavily infected, elderly, or otherwise immunocompromised (8, 25, 34). Globally, A. marginale is the most prevalent tick-transmitted pathogen of cattle and other ruminants. It is the etiologic agent of bovi ne anaplasmosis, an economically significant, arthropod-borne hemolytic disease of cattle that engenders substantial morbidity and mortality in United States bovine livestock, with estimated annual losses exceeding 300 million dollars (1986 U.S. dollars) (63). In addition to being an important pathogen of ruminant livestock, A. marginale is the type species of the family Anaplasmataceae, which encompasses several human and veterinary pathogens with zoonotic potential (33). Previous studies of A. marginale infections of cattle have revealed important features of the biology of these organisms including the nature and length of persistent infections (39, 91), the im munologic and molecular basis of persistence (10,18, 44, 45, 80), and transmission dynamics in the mammalian host and tick vectors (62, 92, 93, 103, 104). Anaplasmataceae are obligate intracellular parasites of eukaryotic hosts. Anaplasma are all maintained in a cycle between mammalian cel ls of myeloid lineage and tick epithelial cells (33). Beyond similar morphologic and life cycl e features, there is molecular biological homology between A. marginale and A. phagocytophilum Based upon housekeeping gene 11

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sequence, there is 96.1% or greater similarity between all of the members of Anaplasmataceae ; and Anaplasma share several common surface protein antige ns (33). Phylogeographic clades of A. marginale and A. phagocytophilum can be established based upon comparison of the genetic sequence for one of the surface proteins, major surface protein 4 (MSP4), which is common to both organisms (28, 29). These two organi sms and other members of the family Anaplasmataceae employ host cell-specific differential expression of surface antigens (11, 15, 16, 46, 56, 58, 74, 75, 83, 87, 106, 109, 110, 119), likely repr esenting an adaptation to improve cell-specific infectivity or mammalian host immune evasion. A. marginale and A. phagocytophilum exhibit similar operon structure and promoter regulation for the immunodominant antigen common to both organism s (MSP2 and MSP2 (p44), respectively) (9, 11, 12). Both organisms display a similar, plasmid-independent adaptation to evade the mammalian host immune system via sequential ge neration of antigenic variants by segmental gene conversion using a single ch romosomal MSP2 expression site and a system of functional chromosomal pseudogenes (9, 11, 18, 45, 71, 72, 73, 114, 120); this facilitates persistent mammalian infection, an esse ntial component of the Anaplasma life cycle. Anaplasma Life Cycle versus the Natural Disease Course of Anaplasmosis Anaplasma phagocytophilum High seroprevalence in the canine (78, 98), equine (22, 77), and human (7,8) populations suggests that subclinical A. phagocytophilum infection or mild disease due to infection is common in endemic regions where ixodi d tick density is high. Chronic A. phagocytophilum infection has been reported in dogs (36, 37) cats (67), rodents ( 55, 108), horses (42, 89, 97), lambs (107), and sheep (41) and is suspected in some human cases (30, 31, 95, 96). Persistent infection by Ehrlichia and other Anaplasma species is commonly observed in animals and humans (5, 26, 27, 32, 39, 52, 59, 82, 91, 97, 99, 118). 12

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Doxycycline is considered the optimal treatment choice for most cases of A. phagocytophilum infection (1, 6, 34, 50, 79), and rapid clin ical improvement within one to two days of treatment is typically observed in most uncomplicated cases (1, 6, 21, 34, 36, 50, 67). However, doxycycline is bacteriostatic against A. phagocytophilum (79). Controlled studies investigating the optimal duration of treatment or the dosage regime required to completely clear viable A. phagocytophilum organisms from infected individuals, rather than to ameliorate clinical signs and potentially induce a carrier state of infection, have not been reported. This represents a gap in the current understanding of this zoonotic pathogen. Further, there is evidence that A. phagocytophilum deoxyribonucleic acid (DNA) in domestic cats (67), E. chaffeensis DNA in dogs (21), and E. canis DNA and viable organisms in dogs (51, 57, 102, 116) persist in blood or organs despite doxycycline administration at a do se and duration generally considered effective against Ehrlichiae. It is unknown whether or not A. phagocytophilum DNA can be detected in tissues of infected humans after treatment with doxycycline since tissues have not been specifically examined for this purpose in cases of human granulocytotropic anaplasmosis (34). A. phagocytophilum -infected neutrophils have delayed apoptosis and prolonged half-life compared to uninfected neutrophils, which norma lly have a half-life of 10 to 12 hours (23, 47, 69, 101, 117). The observation that A. phagocytophilum establishes chronic infections in animals on the order of several months (37, 41, 89) is incongruous with the th eory that terminally differentiated circulating gr anulocytes, which have a relatively br ief, finite half-life, are the only site of infection in mammals. Als o, though it is theoretically possible that A. phagocytophilum may transfer between terminally differentiated granulocytes in the peri pheral blood (34), it is questionable whether these peripheral blood cells are the sole source of microorganisms required for sustained infection. 13

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Based upon initial in vitro cultivation of A. phagocytophilum in undifferentiated bone marrow hematopoietic precursors (CD34+, HL A-DR+), it was suggested that bone marrow progenitors may serve as a continuous source of microorganisms during ch ronic infection (60). However, that study and subse quent studies suggest that A. phagocytophilum preferentially infects cells with mature myeloid or neutrophil-lik e differentiation rather than less differentiated precursor cells (14, 60, 61). It is currently unknown whether A. phagocytophilum -infected granulocytes that are identified in peripheral blood are infected as immature precursors within the bone marrow or if they are infected while in pe ripheral circulation (14). More recent reports indicate that endothelial cells may serve as a nidus of infection in mammals and as a source of organisms to infect granulocytes circulat ing in peripheral blood. A. phagocytophilum can be continuously cultivated in feta l primate endothelial cells (81). And, immunofluorescence has been used to coloca lize an endothelial cell antigen and an A. phagocytophilum surface protein within the cardiac and hepatic microvasculature of mice with severe combined immunodeficiency (SCID) after 7 weeks of infection. However, photomicrographs were not published (54). Anaplasma marginale The only recognized mammalian life cycle stage of A. marginale is within terminally differentiated, circulating mature erythrocytes (33), which have a half-life of approximately 130 days (64). However, bovine anaplasmosis is ch aracterized by obligatory, pe rsistent infection of ruminant hosts associated with chronic, sub-micr oscopic, cyclic parasite mias due to antigenic variation of the surface proteins, MSP2 and MSP3 (3, 9, 10, 17, 18, 19, 38, 39, 43, 44, 59, 80, 90, 91). There are disparities between this mamma lian life cycle model and the natural disease course of anaplasmosis. While, A. marginale organisms may transfer between terminally differentiated erythrocytes (48, 111), it is equivocal whether thes e peripheral blood cells are the 14

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sole source of microorganisms required for sustaine d and proliferative infec tion. The finite halflife of mature erythrocytes sugge sts that other, secondarily in fected mammalian cells may be required to produce the chronic disease course and cyclic paras itemias of bovine anaplasmosis. Electron microscopy has revealed seve ral intact initial bodies within A. marginale -infected erythrocytes co-cultured with endothelial cells, whereas, only de generating initial bodies were observed in erythrocytes cultured in the absen ce of endothelial cells (111). Recent experiments have documented that A. marginale can be successfully propagate d within primate endothelial cells in long term cell culture (81). Prelim inary observation of imm unofluorescently stained tissue has suggested the presence of A. marginale within renal endothelial cells of a splenectomized steer, though negative control tissues were not examined (24). Central Hypothesis The life cycle of A. phagocytophilum and related ehrlichial organisms is complex, involving peripheral blood cells and epithelia l cells of mammalian and arthropod hosts, respectively (33). However, steps in the establishment and persistence of A. phagocytophilum and A. marginale infection within mammalian hosts are incompletely characterized. The current paradigm indicates that within mammalian hosts, Anaplasma infection only occurs within mature, terminally-differentiated blood cells (33) These well-differentiated blood cells that are infected by Anaplasma have a finite half-life (53); this creates an incongruity between the current life cycle paradigm and the natural disease cour se of anaplasmosis, which is characterized by persistent infection of humans and animals (30, 36, 37, 41, 42, 55, 59, 67, 89, 91, 95, 96, 97, 107, 108). It is questionable whether or not termin ally differentiated peripheral blood cells are the sole source of microorganisms required for sustained, if not proliferativ e, infection. This disparity between the present understanding of Anaplasma life cycles and the observed natural course of anaplasmosis represen ts a gap in current knowledge. 15

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The central hypothesis of this work is th at endothelial cells serve as a nidus of A. phagocytophilum and A. marginale infection in dogs and cattle, respectively. The central hypothesis is founded on the following four observations. First, A. phagocytophilum and A. marginale establish chronic infection in ma mmalian hosts (19, 36, 37, 39, 41, 42, 55, 59, 89, 91, 97, 107, 108). Second, A. phagocytophilum and A. marginale can attach in vitro to bovine and primate endothelial cells, invade, and replicate (81). Third, preliminary observations of immunofluorescently stained postmortem tissues suggest that A. phagocytophilum and A. marginale may invade endothelial cells in SCID mice (54) and a splenectomized steer (24). Fourth, an endothelial cell life cy cle stage has been confirmed for another pathogen in the family Anaplasmataceae E. ruminantium which exhibits a vegetative gr owth stage within endothelial cells (94). The work described here s eeks to further define the life cycle of Anaplasma through investigations designed to complete the following: discover whether or not A. phagocytophilum persists within canine endothelial cells during chronic infection determine whether or not A. marginale invades bovine endothelial cells at tick attachment sites during initial infect ion and whether or not A. marginale persists within bovine endothelial cells during cycl ic parasitemia (peak parasitemia and during a trough). By developing a more thorough understanding of Anaplasma life cycles, this work will lay the foundation for future comparative studies of host cell-specific adaptations in Anaplasmataceae and guide the development of effective treatment and control strategies for both the zoonotic pathogen, A. phagocytophilum and the significant ruminant pathogen, A. marginale. 16

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CHAPTER 2 IN SITU DETECTION OF ANAPLASMA BY DNA TARGET-PRIMED ROLLING-CIRCLE AMPLIFICATION OF A PADLOCK PROBE AND INTRACELLULAR COLOCALIZATION WITH IMMUNOFLUORESCENTLY LAB ELED HOST CELL VON WILLEBRAND FACTOR Introduction The life cycle of Anaplasma spp. involves mammalian periph eral blood cells and arthropod epithelial cells (33). Steps in the tick feeding-associated establishment and persistence of Anaplasma infection within mammalian hosts are incompletely characterized. Recent in vitro and preliminary in vivo immunofluorescence studies suggest th at endothelial cells may be a nidus of Anaplasma infection in mammals and a source of organisms to infect circulating blood cells (24, 54, 81). The purpose of this investigation was to develop a specific and sensitive technique for in situ detection of Anaplasma within tissues of infected hosts with special attention to mammalian endothelial cells. In the future, this met hod could be used to determine the in vivo cellular localization of potentially cryptic infection nidi and to provide nucleotide sequence information in situ Materials and Methods Cultivation of Anaplasma spp. The NY18 isolate of A. phagocytophilum or the Virginia isolate of A. marginale was cultivated in fetal rhesus monkey ( Macaca mulatta ) RF/6A endothelial cells (American Type Culture Collection, ATCC CRL-1780, Manassas, VA), and the HZ isolate of A. phagocytophilum was cultivated in human ( Homo sapiens ) HL-60 myeloblastic leukemia cells (American Type Culture Collection, ATCC CCL-240, Manassas, VA) as described (49, 81) (DMEM medium for the RF/6A endothelial cell s (HyClone, Logan, UT) or RPMI-1640 medium for the HL-60 myeloblastic leukemia cells (HyC lone, Logan, UT) supplemented with 10% heatinactivated fetal bovine serum (HyClone, L ogan, UT), 2 mM Gibco L-Glutamine-200 mM 17

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(100X) liquid (final concen tration 4 mM) (Invitrogen, Ca rlsbad, CA), 0.25% NaHCO3 (Sigma, St. Louis, MO), 25 mM HEPES (Sigma, St. Louis, MO), [pH 7.5], 37C, 5% CO2). Uninfected RF/6A endothelial cells and uninfected HL-60 myeloblastic le ukemia cells were similarly maintained. When at least 80% of the cells were A. phagocytophilum -infected or at least 20% of the cells were A. marginale -infected as determined by light microscopy, cell suspensions were diluted and cytocentrifuged on to Bond-Rite glass microscope slid es (Richard-Allan Scientific, Kalamazoo, MI). To form a cell suspension, the RF /6A endothelial cell cu lture monolayers were detached from the culture flask using 0.25% trypsin (HyClone, Logan, UT). The HL-60 myeloblastic leukemia cell line is a nonadherent cell line, which grows as a cell suspension; therefore, treatment with trypsin was not necessary. In Situ Rolling-Circle Amplific ation of Padlock Probes In situ DNA target-primed rolling-circle amplif ication of padlock probes was performed with modifications of a previously described technique (68). All reactions were performed on microscope slides without coverslips. The fina l volume of all reactions was 40 L. All heated reactions were performed in a 16/16 dual bloc k slide chamber mounted on a DNA Engine (PTC200) Peltier Thermal Cycler (Bio-R ad Laboratories, Hercules, CA). Cytospin culture material on the microscope slides was uniformly treated as follows. The slides were washed twice in 1X phospha te-buffered saline (PBS ) ([pH 7.4], 2 min), fixed in 70% denatured ethanol ( 20 min), and subsequently washed twice in 1X PBS (2 min). The cells were permeabilized in HCl ([pH 3.6], 37C, RF/6A endothelial cells: 3 min; HL-60 myeloblastic leukemia cells: 1.5 min). Afterward, the slides were washed three times in 1X PBS (2 min). The bacterial genome was made irreversib ly linear by endonuclease digestion ( A. phagocytophilum : Afe I (New England BioLabs, Ipswich, MA); A. marginale : Zra I (New 18

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England BioLabs, Ipswich, MA), 0.5 U/L in 1X supplied enzyme buffer plus 0.2 g/L bovine serum albumin (New England BioLabs, Ipswich, MA), 37C, 30 min) followed by a brief rinse in buffer A (0.1 M Tris-HCl [pH 7.5], 0.15 M NaCl, and 0.05% Tween-20). The genomic DNA target was made single-stranded by 5 to 3 exonucleolysis (Lambda E xonuclease (New England BioLabs, Ipswich, MA), 0.2 U/L in 1X supplied enzyme buffer plus 0.2 g/L bovine serum albumin and 10% glycerol (Sigma, St. Louis, MO ), 37C, 15 min) followed by a brief rinse in buffer A. The genomic DNA target was detected by hybr idization to a circ ularizable, linear, oligonucleotide padlock probe (Figure 2-1) (either an A. phagocytophilum -specific probe, an A. marginale-specific probe, or a nonsp ecific probe, 0.10 M in 2X SSC [pH 7.0], 20% formamide (Fisher Scientific, Pittsburg, PA), and 0.5 g/ L sonicated fish sperm (DNA MB-grade, Roche Applied Science, Indianapolis, IN), 37C, 15 min) Subsequently, the slides were washed in prewarmed buffer B (2X SSC [pH 7.0] and 0.05% Tween-20, 37C, 5 min) and rinsed briefly in buffer A. The A. phagocytophilum -specific probe or the A. marginale -specific padlock probe, which should hybridize as a nicked ci rcle to its complementary genomic DNA target, was then irreversibly locked into place by enzymatic formation of a phosphodiester bond between the juxtaposed 5' phosphate and 3' hydroxyl termini of the padlock probe (T4 ligase (New England BioLabs, Ipswich, MA), 100 U/L in 1X supplied enzyme buffer plus 0.2 g/L bovine serum albumin, 16C, 15 min). The nonspecific padlock probe should remain linear and be washed away during this and subsequent steps since this probe should fail to form the appropriate conformation required for ligase re cognition and activity. Afterward, the slides were washed in 19

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prewarmed buffer B (37C, 5 min), rinsed once in buffer A, and dehydrated in graded denatured ethanols (75%, 85%, 100%; 3 min each). After exonucleolysis of any remaining 3 single-stranded genomic DNA, the genomic DNA target was used to prime isothermal, in situ rolling-circle amplification of the specifically bound padlock probe (phi29 DNA Polymerase (New England BioLabs, Ipswich, MA), 1.0 U/L in 1X supplied enzyme buffer plus 0.25 mM of each dNTP (Deoxynucleotide Solution Mix, New England BioLabs, Ipswich, MA), 0.2 g/L bovine serum albumin, and 10% glycerol, 37C, 30 min). During the 30 min DNA polymerization, the slides were removed from the slide chamber every 10 min to gently agitate the reaction mixture. The slides were then rinsed briefly in buffer A before the single-stranded, l oosely coiled, concatameric amplif ication product was hybridized to a fluorescently labeled, linear oligonucleotide tag (oligonucleotide AB1252 (green) or oligonucleotide AB1279 (red), 0.25 M in 2X SSC 20% formamide, and 0.5 g/L sonicated fish sperm, 37C, 15 min). Afterward, the slides were rinsed briefly in buffer A, dehydrated in graded denatured ethanols (75%, 85%, 100%; 3 min each), and either immunofluorescently stained for von Willebrand factor or immediately air-dried and coverslip-mounted using VECTASHIELD HardSet Mounting Medium with 1.5 g/mL DAPI (4',6-diamidino-2-phenylindole) (Vector Laboratories, Burlingame, CA). The slides we re examined using a Leica DMI 3000B inverted microscope fitted for epifluorescence and equipped with a digital camera (Micropublisher 3.3 RTV, QImaging Corporation, Surrey, BC Canada) and then stored at 4C. Digital images were collected using QCapture Pro 5.1.1.14 (QImag ing Corporation, Surrey, BC Canada) and uniformly processed using SPOT Advanced Wi ndows Version 4.0.9 (Dia gnostic Instruments, 20

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Sterling Heights, MI) and Adobe Photoshop Elements 2.0 (Adobe Systems Incorporated, San Jose, CA). Padlock Probe Design Three circularizable, lin ear, oligonucleotide padlock probes were used (Figure 2-1) (MWG Biotech, High Point, NC). The A. phagocytophilum -specific probe (AB1251) was designed to include an A. phagocytophilum genomic DNA target-specificsequence whose cognate is a conserved 5 region of msp2 ( p44) that is present in the expr ession site of three geographic isolates (HZ, United States (35) Norway (13), and Sweden (13) and 81 different pseudogenes of the HZ isolate (35). Of thes e target sequences within the A. phagocytophilum genome (HZ isolate), there are 29 potentia l targets (the expression site and 28 pseudogenes) that are associated with a 3 AfeI endonuclease recognition site within 100 base pairs (bp) of the genomic target and could, therefore, be detect ed by the technique described here. The A. marginale -specific probe (AB1270) was designed to include an A. marginale genomic DNA target-specificsequence whose cognate is orfY that is repeated 8 times in the genome (20). Of these target sequences within the A. marginale genome (St. Maries isolate), there are 7 potential targets th at are associated with a 3 Zra I endonuclease recognition site within 1 bp of the genomic target and could, therefore, be dete cted by the technique described here. The use of repetitive sequences ( A. phagocytophilum : msp2 ( p44); A. marginale : orfY ) as genomic DNA targets for padlock probe hybridi zation was expected to increase sensitivity beyond that provided by rolling-ci rcle amplification alone. The target-specific-sequence of th e padlock probe is within the 5 and 3 arms of the probe, which are joined by an intervening linker region. The rolling-circle am plification product is detected by a fluorescently labeled oligonucle otide tag (AB1252 or AB1279) that is the complement of the padlock probe linker region amplification product. The linker region of the 21

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nonspecific padlock probe (AB 1253) was identical to the A. phagocytophilum -specific and A. marginale-specific padlock probes. The 5 and 3 arms of the nonspecific probe contained the same nucleotide composition as the A. phagocytophilum -specific probe; how ever, the sequence of the nucleotides was randomized. Indirect Immunofluorescent Staining of von Willebrand Factor in Cultured Endothelial cells After the final graded al cohol dehydration of the in situ rolling-circle amplification procedure, slides that had been reacted with the A. phagocytophilum -specific padlock probe or the nonspecific padlock probe we re immunofluorescently stained fo r von Willebrand factor. All reactions were performed on micr oscope slides without coversli ps; the final volume of all reactions was 150 L. The cyto spin culture material was bl ocked with normal rabbit serum (X0902 (Dako, Carpinteria, CA) 5% in 1X PBS [p H 7.4], 30 min) and subsequently washed once in 1X PBS (5 min). The cytospin culture mate rial was then incubated with either antibody-free diluent or rabbit polyclonal anti-human von Willebrand factor antibody (N1505 (Dako, Carpinteria, CA), 1 to 2 dilution of the proprie tary antibody solution in 0.05 M Tris-HCl [pH 7.5] and 1% bovine serum albumin fraction V ((Fisher Scientific, Pittsburg, PA), 30 min). The slides were washed twice in 1X PBS (5 min) prio r to secondary antibody labeling. The cytospin culture material was subsequently incubated w ith a highly cross-adsorbed goat polyclonal antirabbit-IgG antibody-Alexa Fluor 568 conjugate (A11036 (Invitrogen Molecular Probes, Carlsbad, CA), 2 g/mL in 0.05 M Tris-HCl [pH 7.5] and 1% bovine serum albumin fraction V, 30 min). Afterward, the slides were washed tw ice in 1X PBS (5 min), air-dried, coverslipmounted using VECTASHIELD HardSet Mounting Medium with DAPI, and examined as above described. 22

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Results In Situ Detection of Anaplasma spp. by DNA Target-Primed Rolling-Circle Amplification of Padlock Probes During persistent, latent infec tions that can be caused by Anaplasma spp., it would be valuable to sensitively and specifi cally detect organisms in infected tissues using an isothermal DNA amplification technique that has the potent ial to provide information about organism genotype and host-cellular localizati on. To determine whether such a technique could be used to detect A. phagocytophilum and A. marginale, in situ DNA target-primed rolling-circle amplification of padlock probes was developed based upon previous in situ genotyping of human mitochondrial DNA using rolling-circle amp lification of padlock probes (68). Cytospin preparations of uninfected or A. phagocytophilum HZ-infected human myeloblastic leukemia cell cultures and a padloc k probe, either a nonspe cific probe or an A. phagocytophilum -specific probe, were used for in situ DNA target-primed rolling-circle amplification. When A. phagocytophilum HZ-infected culture cytospins were microscopically examined after in situ DNA target-primed rolling-circ le amplification of an A. phagocytophilum specific padlock probe, numerous aggregates of stippled, green or red fluorescence, which represented the fluorescently labe led, localized amplifi cation product, were frequently identified within intact cultured myeloblastic leukemia cells The intracellular lo cation of fluorescence correlated well with microscopic observations of A. phagocytophilum morulae (mulberry-like aggregates of bacteria) within intact myeloblas tic leukemia cells in a Wrights-Giemsa-stained infected culture cytospin (Figure 2-2 A, B, a nd D). Similar fluorescence was not observed when either an uninfected culture cytospin (not s hown) or a nonspecific padlock probe was used (Figure 2-2 C and E). The results were c onfirmed in five independent experiments. 23

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Cytospin preparations of uninfected or A. marginale Virginia-infected fetal rhesus monkey endothelial cell cultures and a padlock pr obe, either a nonspecific probe or an A. marginale specific probe, were used for in situ DNA target-primed rolling-circ le amplification. When A. marginale Virginia-infected culture cytospins were microscopically examined after in situ DNA target-primed rolling-circle amplification of an A. marginale -specific padlock probe, round aggregates of stippled, green or red fluorescen ce, which represented the fluorescently labeled, localized amplification product, were frequently identified within int act cultured endothelial cells. The intracellular location of fluorescence correlated well with microscopic observations of A. marginale morulae within intact endothelial cells in a Wrights-Giemsa-stained infected culture cytospin (Figure 2-3 A, B, and D). Sim ilar fluorescence was not obs erved when either an uninfected culture cytospin (not shown) or a nonspecific padlock probe was used (Figure 2-3 C and E). The results were confirmed in three independent experiments. In situ Intracellular Colocalization of the A. phagocytophilum Rolling-Circle Amplification Product and von Willebrand Factor Immunofluorescence Since A. phagocytophilum can be continuously cultivated in endothelial cells (81) and preliminary immunofluorescent st aining of SCID mouse tissues suggests endothelial cells may also be infected in vivo (54), there has been heightened interested in more conclusively determining whether endothelial cells are an in vivo nidus of A. phagocytophilum in naturally or experimentally infected, immunocom petent mammals. To that end, in situ A. phagocytophilum DNA target-primed rolling-circle amplification of a padlock probe was combined with indirect immunofluorescent staining of von Willebrand fact or, which is present within Weibel-Palade bodies of endothelial cells (115). Cyto spin preparations of uninfected or A. phagocytophilum NY18-infected fetal rhesus monkey endothelial ce ll cultures, two padlock probes (a nonspecific probe or an A. phagocytophilum -specific probe), and two antibody staining variations (secondary 24

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antibody only or primary and secondary anti body) were examined using these combined techniques. When A. phagocytophilum NY18-infected culture cyto spins were microscopically examined after in situ DNA target-primed rolling-circ le amplification of an A. phagocytophilum specific padlock probe, focal aggregates of stip pled, green fluorescence, which represented the fluorescently labeled, localized amp lification product, were often identified perinuclearly within intact cultured endothelial cells The intracellular location of green fluorescence correlated well with microscopic observations of A. phagocytophilum morulae within intact endothelial cells in a Wrights-Giemsa-stained infected culture cyto spin (Figure 2-4 A and B). Similar green fluorescence was not observed when either an uninfected culture cytospin (not shown) or a nonspecific padlock probe was us ed (Figure 2-4 C). The resu lts were confirmed in five independent experiments. When A. phagocytophilum -infected culture cytospins were examined microscopically after combined in situ DNA target-primed rolling-ci rcle amplification of an A. phagocytophilum specific padlock probe and indi rect immunofluorescent staining of von Willebrand factor, focal aggregates of stippled, green fl uorescence were identified perinuc learly juxtaposed with focal areas of red fluorescence, which represented th e fluorescently labeled von Willebrand factor within Weibel-Palade bodies (Figure 2-4 D). Similar green and red fluorescence were not observed when the combined procedures were performed using a nonspecific padlock probe and only the fluorescently labeled se condary antibody (Figure 2-4 E). Discussion The in situ DNA target-primed rolling-circle amplif ication of a padlock probe technique adapted here for Anaplasma detection was first described as a method to distinguish single nucleotide polymorphisms in human mitochondrial DNA (68). We considered it likely that this 25

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technique could be used for in situ detection of intracellular mi croorganisms. Here it is demonstrated that it is possi ble to detect and localize Anaplasma spp. within intact cultured mammalian cells using in situ DNA target-primed rolling-circle amplification of padlock probes and that this technique can be combined with immunofluorescent staining to identify A. phagocytophilum and an endothelial cell antigen within a single cultured endothelial cell. These observations suggest that this tec hnique could be applied to natural Anaplasma spp. isolates in tissues obtained from naturally or experiment ally infected mammalian or arthropod hosts to determine the cellular localization of potentially cryptic infections in vivo and to provide nucleotide sequence information in situ The specificity of this technique arises fr om use of a padlock probe, which depends upon specific hybridization to the genomic DNA target sequence. The use of ligase allows interrogation of the hybrid ization quality between the target-specific-sequence of the padlock probe and the genomic target. If the quality of the hybr idization is not opti mal, ligation of the padlock probe to form a closed, partially double-str anded circle involving th e genomic target will not occur. The use of ligase in this procedur e is the basis for distin ction of genomic single nucleotide polymorphisms based on the quality of hybridization with the 3 terminus of the padlock probe (65, 66, 68, 84, 85, 86). The e xponential amplification of the bound padlock probe is one basis for the sensi tivity of this technique (68, 86 ). The amplification product remains bound to the target sequence and is subs equently detected by the addition of a second fluorescently labeled oligonucleotide tag that specifically binds repeated linker sequence within the concatameric amplification product. The f act that the amplification product remains tightly bound as a 3 extension of the genomic targ et limits background fluorescence, which also 26

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contributes to the sensitivity a nd specificity of this technique and is another improvement over other in situ nucleotide detection techniques (68, 85). An additional benefit of the in situ DNA amplification technique applied here over other methods of in situ DNA detection is that the reactions ar e isothermal, which preserves tissue architecture and allows subseque nt immunostaining of host cell antig ens, such as the endothelial cell antigen, von Willebrand factor. This is of particular import given the recently heightened interest in endothelial cells as a potential natural mammalia n infection nidus in light of the ability to continuously cultivate A. phagocytophilum and A. marginale in fetal primate endothelial cells (81) and preliminary immunofluorescent colocalization of A. phagocytophilum or A. marginale with endothelial cell antigens in vivo (24, 54). Transformation of A. phagocytophilum rendering it able to express green fluorescent protein and subsequent experime ntal infection of mice with A. phagocytophilum transformants have been recently described (40). Such transf ormants may facilitate temporal tracking of organism tissue distribution duri ng experimental infection, including host cellular binding, entry, and intracellular development; however, their us e is dependent upon an e xperimental model of mammalian infection and the ability to maintain stable transformants in long-term culture and during the full course of acute and chronic infection. The fluores cence microscopy-based Anaplasma spp. detection method described here doe s not depend upon a genetic transformant that may be unstable or attenuated and could be a pplied to naturally or ex perimentally infected mammalian or arthropod host tissues to detect poten tially cryptic sites of infection. Additionally, with knowledge of variable Anaplasma genomic DNA sequences that may potentially be present in host tissues during an infection, multiple padlock probes that bind to differentially labeled 27

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fluorescent oligonucleotide tags c ould be designed and used in a single isothermal reaction to provide information about Anaplasma nucleotide sequence in situ In summary, the observations presented here show that in situ DNA target-primed rollingcircle amplification of padlock probes can be used to detect Anaplasma spp. within intact cultured mammalian cells. This demonstrates that the amplification technique can be used for in situ detection of an intracellula r microorganism. Also, this technique can be combined with immunofluorescent staining in orde r to identify a host cell antigen and the fluorescently labeled rolling-circle amplification pr oduct within a single cell. This work forms the foundation for future applications of this techni que to detect, localize, and analyze Anaplasma nucleotide sequences in the tissues of infected hosts and in cell cultures. There is also the potential to apply the technique described here to investigate prospective cryptic host-cellular localization of other microorganisms and further elucidate the etiopat hogenesis of disease associated with infection by other obligate or facultativ e intracellular microorganisms. 28

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Figure 2-1. Padlock probes and fluorescent oligonucleotides. Th ree padlock probes were used, AB1251, A. phagocytophilum -specific, AB1270, A. marginale -specific, or AB1253, nonspecific. The genomic DNA target-s pecific-sequences of AB1251 and AB1270 are within the underlin ed 5 and 3 arms of the padlock probes. When the singlestranded genomic complement of the probes target-specific-sequence is detected, the probe hybridizes as a nicked circle, which is subsequently locked in place by ligase as a partially double helical, cl osed circle. The circular ized padlock probe is the template for in situ DNA target-primed rolling-circle amplification. The 5 and 3 arms of the nonspecific probe, AB1253, cont ained the same nucleotide composition as AB1251; however, the sequence of the nuc leotides was randomized within the underlined regions. The 5 and 3 arms of the padlock probes were joined by an identical intervening linker region. The amplif ication product of the italicized portion of the probe linker region is the complement of the fluorescently-labeled oligonucleotide tags, AB1252 or AB1279 (also known as Lin 33 [68]). All oligonucleotides contained a 5 modification designated as P which stands for phosphate, FAM which stands for phosphoramidite coupled fluorescein, or Alexa 555, which stands for Alexa Fluor 555 (Invitrogen, Carlsbad, CA). 29

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Figure 2-2. Detection of A. phagocytophilum using in situ DNA target-primed rolling-circle amplification of a padlock probe. A) Wr ight-Giemsa-stained cytospin preparation 63X objective, bright field, B) in situ rolling-circle amplification product within cytospin preparation 63X objective, differen tial interference contrast with blue and green emissions, C) negative co ntrol for reaction shown in B in situ rolling-circle amplification product within cytospin preparation 63X objective, differential interference contrast with bl ue and green emissions, D) in situ rolling-circle amplification product within cytospin preparation 63X objective, differential interference contrast with blue and red em issions, E) negative control for reaction shown in D in situ rolling-circle amplification product within cyto spin preparation 63X objective, differential interference c ontrast with blue and red emissions. Numerous intracellular morulae were observed within heavily A. phagocytophilum HZ-infected, intact human myeloblasts in cy tospin preparations (A). Morulae were also detected as the stippled, green fluor escent product or red fluorescent product of in situ DNA target-primed rolling-ci rcle amplification of an A. phagocytophilum specific padlock probe (B and D). Simila r fluorescence was not detected when a nonspecific padlock probe was used in the otherwise identical procedure (C and E). 30

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Figure 2-3. Detection of A. marginale using in situ DNA target-primed rolling-circle amplification of a padlock probe. A) Wr ight-Giemsa-stained cytospin preparation 63X objective, bright field, B) in situ rolling-circle amplification product within cytospin preparation 63X objective, differen tial interference contrast with blue and green emissions, C) negative co ntrol for reaction shown in B in situ rolling-circle amplification product within cytospin preparation 63X objective, differential interference contrast with bl ue and green emissions, D) in situ rolling-circle amplification product within cytospin preparation 63X objective, differential interference contrast with blue and red em issions, E) negative control for reaction shown in D in situ rolling-circle amplification product within cyto spin preparation 63X objective, differential interference c ontrast with blue and red emissions. Intracellular morulae were observed within A. marginale -infected, intact fetal rhesus monkey endothelial cells in cyto spin preparations (A). Moru lae were also detected as the stippled, green fluorescent product or red fluorescent product of in situ DNA target-primed rolling-circle amplification of an A. marginale -specific padlock probe (B and D). Similar fluorescence was not de tected when a nonspecific padlock probe was used in the otherwise identi cal procedure (C and E). 31

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Figure 2-4. Detection of A. phagocytophilum using in situ DNA target-primed rolling-circle amplification of a padlock probe and concur rent indirect immunof luorescent staining of von Willebrand Factor. A) Wright-G iemsa-stained cytospin preparation 100X objective, bright field, B) in situ rolling-circle amplific ation product within cytospin preparation 63X objective, differential inte rference contrast with blue and green emissions, C) negative control for reaction shown in B in situ rolling-circle amplification product within cy tospin preparation 40X objective, phase contrast with blue and green emissions, D) in situ rolling-circle amplif ication product and indirect immunofluorescent labeling of von Willebrand factor within cytospin preparation 40X objective, blue, red, and green emissi ons, E) negative control for reaction shown in D in situ rolling-circle amplification produc t and secondary antibody labeling only within cytospin preparation 40X objective, blue, red, and green emissions. A few perinuclear morulae were observed within intact A. phagocytophilum NY18-infected fetal rhesus monkey endothelial cells in cytospin prepara tions (A). Morulae were also detected as the stippled, green fluorescent product of in situ DNA target-primed rolling-circle amplification of an A. phagocytophilum -specific padlock probe (B). A similar green fluorescent product was not de tected when a nonspecific padlock probe was used in the otherwise identical procedure (C). A. phagocytophilum -infected fetal rhesus monkey endothelial cells in cytospin preparations were subjected to combined in situ DNA target-primed rolling-ci rcle amplification of an A. phagocytophilum specific padlock probe and indirect immunofluorescent staining of von Willebrand factor. Focal aggregates of stippled, green fluorescence we re identified perinuclearly and juxtaposed with focal areas of red fluorescence, which represented the fluorescently labeled von Willebrand factor (D). Similar green and red fluorescence were not observed when the combined pro cedure was performed using a nonspecific padlock probe and only the fluorescen tly labeled secondary antibody (E). 32

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CHAPTER 3 EXPERIMENTAL INOCULATION OF DOGS WITH ANAPLASMA PHAGOCYTOPHILUM MOLECULAR EVIDENCE OF PERSISTENT INFECTION FOLLOWING DOXYCYCLINE THERAPY, AND INVESTIGATION OF EN DOTHELIAL CELLS AS SOURCE OF INFECTIOUS INOCULUM AND A REPOSITO RY OF CHRONIC INFECTION IN DOGS Introduction A. phagocytophilum is the third most common tick-bor ne infection of humans in the United States (8, 34). When diagnosed early, most patients exhibit rapi d clinical improvement within one to two days of treatment; however, pr otracted disease course or death is possible among those who are heavily infected, elderly, or otherwise immunocompromised (8, 25, 34). Doxycycline, which is bacteriostatic against A. phagocytophilum is considered the optimal treatment choice for most cases of A. phagocytophilum infection (1, 6, 34, 50, 79). Though, it is uncertain whether currently recommended treatment regimes merely alleviate clinical signs and potentially induce a carrier state of infection since controlled st udies investigating the optimal duration of treatment or the dosage re quired to completely clear viable A. phagocytophilum organisms from infected individu als have not been reported. Chronic A. phagocytophilum infection has been described in several animals: dogs (36, 37), cats (67), rodents (55, 108), horses (42, 89, 97), lambs (107), and sheep (41). Chronic infection is suspected in some human cases (30, 31, 95, 96). Also, there is molecular evidence for persistent infection by A. phagocytophilum and related organisms after treatment with doxycycline. A. phagocytophilum DNA in domestic cats (67), E. chaffeensis DNA in dogs (21), and E. canis DNA and viable organisms in dogs (51, 57, 102, 116) have been detected in blood or organs despite doxycycline administration at a dose and dur ation generally considered effective against Ehrlichiae. Previously described experimental infection of dogs with A. phagocytophilum have depended upon use of infected blood from a naturally occurring clinical ca se or cultivation of 33

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organisms in human ( Homo sapiens ) HL-60 myeloblastic leukemia cells (American Type Culture Collection, ATCC CCL-240, Manassas, VA) or autologous neutrophils (70, 76, 105). These culture systems rely upon mammalian cells of myeloid origin which are known to harbor A. phagocytophilum infection in vivo Determining whether or not endothelial cells can serve as a source of viable inoculum capable of establishing infection in dogs represents an initial step in the investigation of whether or not endothelial cells may be a cell that is involved in the mammalian life cycle stages of this microorga nism. Also, should e ndothelial cell-derived inoculum prove capable of establishing mamma lian infection, this would have important implications upon future vaccine development. As opposed to previously used in vitro cell lines, which are loosely adherent or nonadherent, endothelial cells are a tightly adherent cell line. The tightly adherent nature of cultu red endothelial cells facilitates their use in genetic manipulation of Anaplasma and clonal selection by pl aque purification (81). The purposes of this investigation were to determine if experimental inoculation of different A. phagocytophilum isolates could establish chr onic canine infection, to observe whether or not infection persisted in spite of antimicrobial treatme nt, and to discover whether or not cultured endothelial cells could be a source of infecti ous inoculum in dogs and whether or not infection of endot helial cells occurred in vivo in chronically infected dogs. Materials and Methods Animals, Inocula, and Monitoring for Development of Infection Four adult, intact male, Sprague Dawley B eagles, confirmed by polymerase chain reaction (PCR) or serology (immunofluorescent antibody testing or SNAP4Dx, IDEXX, Westbrook, ME) to be negative for common arthropod-borne diseases ( A. phagocytophilum A. platys E. canis E. chaffeensis B. burgdorferi Babesia gibsoni Bartonella henselae Dirofilaria immitis ) were inoculated intravenously with A. phagocytophilum from one of two sources. Two dogs 34

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were inoculated with a huma n isolate, NY18 strain of A. phagocytophilum cultivated in fetal rhesus monkey endothelial cells ; the other two dogs were inocul ated with a canine isolate via injection of stabilate prepared from parasi temic blood of a naturally infected dog from Minnesota. The NY18 isolate of A. phagocytophilum was cultivated in fetal rhesus monkey ( Macaca mulatta ) RF/6A endothelial cells (American Type Culture Collection, ATCC CRL-1780, Manassas, VA) (81) (DMEM medium (HyClone Logan, UT) supplemented with 10% heatinactivated fetal bovine serum (HyClone, Logan, UT), 0.25% NaHCO3 (Sigma, St. Louis, MO), 25 mM HEPES (Sigma, St. Louis, MO), [pH 7.5], 37C, 5% CO2). Two 25 mL infected-tissue culture flasks were propagated (one for each dog). When at least 35% of the cells in each flask were A. phagocytophilum -infected as determined by light microscopic examination of cytospin culture material on microscope slides, the entir e cultures were harveste d by scraping the bottom of the flasks using a sterile cell scraper (Fisher Scientific, Pittsburg, PA) to form a cell suspension within the culture medium. The cell suspension from each flask was transferred into individual tubes and was centrif uged at 300 x g for 10 min at 25C. The supernatant medium was removed from each tube and used to inocul ate other ongoing cell cultures. Each of the resultant cellular pellets was individually washed in 5 mL of fresh, sterile 1X Hanks Balanced Salt Solution (HBSS) (20-021-CV, Mediatech, Inc., Manassas, VA) and centrifuged at 300 x g for 10 min at 25C twice. After the second was h, each cellular pellet was resuspended in 5 mL of fresh HBSS giving a fina l concentration of 2.5 x 105 cells/mL. Four milliliters of each infected cell suspension (containing both intr acellular and free e ndothelial cell-derived A. phagocytophilum ) was used to intravenously inoculate a dog (2 dogs total, 4 mL per dog); 1 mL of each infected cell suspension was stored at -8 0C. Four milliliters of parasitemic blood from a 35

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naturally infected dog was collect ed in ethylenediaminetetraacet ic acid (EDTA) and a stabilate was prepared by adding dimethyl sulfoxide (DMS O) to a final concentration of 10%. Two milliliters of infected blood stabilate was used to intravenously inoculate 2 different dogs (1 mL per dog). After inoculation, the dogs were examined a nd phlebotomized at regular intervals for the duration of the trial. Blood was collected into both EDTA-containing and plain sterile tubes, from which serum was harvested. Rectal temper ature, gait, and diarth rodial joints were monitored for abnormalities consistent with anapla smosis, such as pyrexia and polyarthropathy. Complete blood cell counts were monitored fo r the development of thrombocytopenia or leukopenia, and serum biochemistry profiles were monitored for elevations in hepatocellular leakage enzyme activities. Wrights Giemsa-s tained (Harleco, EM Science, Gibbstown, NJ) peripheral blood films were examined by light mi croscopy for the occurrence of parasitemia as evidenced by the presence of basophilic, stippled circular granulocytic inclusions consistent with intragranulocytic A. phagocytophilum morulae. Serologic Assays for A. phagocytophilum Infection To document the occurrence of se rum antibodies directed against A. phagocytophilum polyclonal sera were tested using a commercially availabl e sandwich ELISA based upon the MSP2 (p44) protein (SNAP4Dx, IDEXX, Westbrook, ME) and were reacted with immunoblots of sodium dodecyl sulfate-polyac rylamide gel electrophoresis (SDS-PAGE) gradient gels containing separate d, denatured proteins of whole A. phagocytophilum derived from RF/6A endothelial cell culture (81), as desc ribed (2). Preand post-infection polyclonal sera from the four A. phagocytophilum -inoculated dogs and from one PCR-positive, naturally A. phagocytophilum -infected human were used. The canin e sera were diluted 1:2000; the human sera were diluted 1:5000. Horseradish per oxidase-conjugated, speci es-specific secondary 36

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antibodies directed against canine or human IgG were diluted 1:75,000 (Sigma, St. Louis, MO). Chemiluminescence was detected using SuperSig nal West Dura Extended Duration Substrate (Pierce, Rockford, IL). PCR Detection of A. phagocytophilum DNA in Blood and Tissues DNA was extracted from anticoagulated, ED TA-whole blood and plain-frozen postmortem tissue samples using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA) in accord with the manufacturer instructions, which indicated the following amounts of biologic material be used for DNA extraction: 200 L of anticoagul ated whole blood, 25 mg of tissues other than spleen, or 10 mg of spleen. Control DNA sa mples, including positive control DNA extracted from A. phagocytophilum grown in fetal rhesus ( Macaca mulatta ) RF/6A endothelial cells (81) and negative control DNA extracted from an uninfected dog were similarly extracted. Extracted DNA was used as the template fo r nested-PCR amplifica tion of a conserved region of msp2. Increased sensitivity for detection of A. phagocytophilum beyond that which is expected from nested-PCR alone was derived fro m use of synthetic oligonucleotide primers (Figure 3-1) (MWG Biotech, High Point, NC) that were designed to detect nearly all genomic copies of this conserved region within msp2 including copies that are present in the single expression site and in most of th e approximately 100 pseudogenes. Fifty microliter DNA amplification reactions were run in a GeneAmp PCR System 9600 (Perkin Elmer, Waltham, MA) using 0.05 U/L AmpliTaq DNA Polymerase (Applied Biosystems Inc, Foster City, CA) in 1X supplied enzyme buffer plus 0.2 mM of each dNTP (Applied Biosystems Inc, Foster City, CA) and 0.2 M of each primer (forward and reverse) for the reaction (primary or nested) (Figure 3-1). The following thermo cycler conditions were used: hot start; 3 cycles of 97C for 15 sec denatura tion, 50C for 30 sec annealing, 72C for 30 sec polymerization; 47 cycles of 94C for 15 sec de naturation, 50C for 30 sec annealing, 72C for 37

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30 sec polymerization; 72C for 3 min final extensi on; hold at 4C. Two and a half microliters of the primary PCR product was used as the DNA te mplate in the subsequent nested-PCR. Use of the nested primers was expect ed to yield a 464 bp final amplific ation product. The identity of the amplification products was confirmed by fl uoresceinated-DNA oligonuc leotide (Figure 3-1, AB1214) probing of southern blotted PCR products that were separa ted on a 2% agarose gel, as described (9). Real-time PCR detection of two A. phagocytophilum genes, msp2 and the heat shock protein, groEL in anticoagulated, EDTA-whole bl ood samples collected from the A. phagocytophilum (NY18)-infected dogs was performed using a test offered by a commercial laboratory (IDEXX, Westbrook, ME). Immunosuppression, Antibiotic Treatment, and Euthanasia In order to see if post-infec tion parasitemia or clinical signs could be enhanced, all four dogs were administered an immunos uppressive dose of prednisone for approximately two weeks. After the period of immunosuppressi on, all four dogs were treated with doxycycline to determine if parasitemia or clinical signs could be abrogated. After the an tibiotic treatment, all four dogs were again administered an immunosuppressive dose of pre dnisone for approximately two weeks. The NY18 isolate-infected dogs were eu thanized 11.5 months post-infection; the canine isolate-infected dogs were euthanized 7.8 months post-infection (Table 3-1). The efficacy of two dosage regimes was investigated. The NY18 isolate-infected dogs were treated with doxycycline 10 mg/kg orally once daily for tw o weeks (21); the two canine isolate-infected dogs were treated with doxycyc line at the currently recommended dose (E. B. Breitschwerdt, personal communication) of 10 mg/ kg orally twice daily for four weeks (21). 38

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In Situ DNA Target-Primed Rolling-Circle Amplification of a Padlock Probe for Detection of Anaplasma phagocytophilum Post-mortem tissues from the canine isolate-infected dogs were cryopreserved immediately after collection. Tissue samples were initially stored and transported in sterile tissue culture medium (HyClone, Logan, UT) on i ce. Subsequently, samples were trimmed and cryoembedded in Neg-50 medium (Thermo Scientific Richard-Allan, Pittsburg, PA) by freezing in 2-methylbutane on liquid nitrogen. Frozen tissue blocks were stored at -80C until cryosectioned using a Leica cryos tat (Leica Microsystems, Bannockbur n, IL). Four micrometer tissue sections were prepared at -20C and immediat ely fixed in acetone on ice for 5 min. Sections were stored at -80C until examined using in situ DNA target-primed rolling-circle amplification of padlock probes for detection of A. phagocytophilum modified from the described (113) to use sheared calf thymus DNA (R&D Systems, Minneapolis, MN) as the DNA carrier in the oligonucleotide hyb ridization reactions. Sheared calf thymus DNA was used at 0.5 g/L in the padlock probe hybridization solution and was used at 6.0 g/L in the fluorescently labeled oligonucleotide hybridizat ion solution. Cytospins of A. phagocytophilum HZ isolate cultivated in human ( Homo sapiens ) HL-60 myeloblastic leukemia cells (American Type Culture Collection, ATCC CCL-240, Manassas, VA), as described (49), were also run with an A. phagocytophilum -specific padlock probe as a re agent positive control. Results Physical Exam, Hematopathol ogy, and Clinical Chemistry Hematologic abnormalities, clinical ch emistry abnormalities, or circulating intragranulocytic morulae were not observed during the two canine infection trials. The physical exam findings were unremarkable, except that one of the canine isolate-infected dogs was intermittently pyrexic (body temperature >102.5F) (Figure 3-2). There was an average of 26.1 39

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days (median 26.0 days) between instances or episodes (more than one consecutive measurement) of body temperatures >102.5F. Th e minimum number of days between febrile episodes was 8, and the maximum was 49 days. Serologic Evidence of Infection In the NY18-infected do gs, seroconversion to A. phagocytophilum -positive status was first detected about one week after infection using the MSP2 (p44) antigen-based IDEXX SNAP4Dx (Table 3-2). Subsequent samples that were collected on day 11 through day 316 post-infection remained seropositiv e (25 samples per dog tested). In the canine isolate-infected dogs, seroconversion to A. phagocytophilum -positive status was not detected until day 51 (dog 1) or day 59 (dog 2) after infecti on. Subsequent samples that were collected on day 71 through day 210 post-infection were seronegative by th is assay (27 samples per dog tested). The seropositive status of the canine isol ate-infected dogs was confirmed based upon immuonoreactivity detected when 37 day post-infection polyclonal sera was reacted with an immunoblot containing separate d, denatured proteins of A. phagocytophilum (NY18) (Figure 33). Serum from this day was selected based on knowledge of the temporally associated changes in immuonoreactivity detected by competitive-en zyme-linked immunosorbent assay (cELISA) in the NY18-infected dogs (4). Molecular Evidence of Infection NY18-infected dogs In the NY18-infected dogs, A. phagocytophilum DNA was readily detected by at least one of three assays (nested-PCR amplification of msp2 or real-time PCR amplification of msp2 or groEL ) for about 1 month after initial inf ection (Table 3-3). Subsequently, A. phagocytophilum DNA was rarely detected between day 36 and day 230 post-infection (3 out of 42 total tested samples were positive); PCR detection impr oved after immunosuppression. By day 15 post40

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immunosuppression (day 245 post-infection), both dogs were PCR-positive using nestedor realtime, msp2 -based PCR. Doxycycline treatment (10 mg/kg by mouth on ce daily for 2 weeks) did not eliminate molecular evidence of A. phagocytophilum infection in the NY18-infected dogs. A. phagocytophilum DNA was still detectable in samples collected 5.4 w eeks after treatment with doxycycline had ended (day 299 post-infection) Eight weeks afte r doxycycline therapy concluded, immunosuppression was performed a second time (days 316 through 325 postinfection). Subsequently, samples from the re-immunosuppressed dogs, which were collected for 11 weeks after doxycycline therapy, remained PCR positive. After the predicted time for prednisone to be eliminated had passed, the NY18-infected dogs were euthanized on day 344 post-infection (11.9 weeks after the last doxycycline dose, 2.7 weeks after the last prednisone dose). Hear t, lung, liver, spleen, and bone marrow were subjected to nested-PCR; the heart and spleen of Dog 1 were nested-PCR-positive (Figure 3-4). Canine isolate-infected dogs In the canine isolate-infected dogs, A. phagocytophilum DNA was detected by nested-PCR amplification of msp2 intermittently during the initial period of infection, prior to immunosuppression (Dog 1: days 16, 20, 39, and 72 post-infection; Dog 2: days 2, 48, and 62 post-infection). During the first period of immunosuppression (day 143 through 156 postinfection) (Table 3-1), both canine isolate-infected dogs were nested-PCR-positive within 6 days of initiating prednisone administ ration (Dog 1: days 149 and 153 post-infection; Dog 2: days 149 and 151 post-infection). Doxycycline treatment (10 mg/kg by mouth twic e daily for 4 weeks) did not eliminate molecular evidence of A. phagocytophilum infection in the canine isol ate-infected dogs. Prior to the second period of immunosuppression, A. phagocytophilum DNA was detected by nested41

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PCR in one of the dogs 3 days after doxycyc line administration ended (Dog 1, day 187 postinfection). One week after concluding doxycycline th erapy, immunosuppression was performed a second time (day 191 through 204 post-infection). Subsequently, one blood sample (Dog 2, day 213 post-infection) and one post-mortem ti ssue sample (Dog 1 right kidney, day 233 postinfection) were nested-PCR-positive (Figure 3-5). In Situ Detection of A. phagocytophilum When tissues from the canine isolate-infected dogs (Dog 1: right ki dney (Figure 3-6) and liver; Dog 2: lung) were mi croscopically examined after in situ DNA target-primed rollingcircle amplification, multifocal areas of autofluorescence were id entified regardless of whether the A.phagocytophilum -specific padlock probe or the nons pecific padlock probe was used. Fluorescently labeled, in situ rolling-circle amplifi cation products were no t identified in any of the tissues examined. Discussion Regardless of the two isolates tested (hum an or canine) or the two doxycycline dosages tested, A. phagocytophilum DNA remained detectable for several months in the peripheral blood and some post-mortem tissues (heart, spleen, kidn ey) from these four dogs. This is the first molecular evidence of chronic, persistent A. phagocytophilum infection in blood and tissues of subclinical dogs despite doxycyclin e treatment using the currently recommended dosage. This has critical implications since treatm ent with doxycycline may not eliminate A. phagocytophilum human or canine isolate infection fr om dogs. Dogs may become life-long A. phagocytophilum carriers, at risk for recrudescence with conc urrent illness or immunosupp ression (30, 95). Also, chronic carriers may act as an environmental source of orga nisms to ixodid ticks during 42

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acquisition-feeding. Future trials investigating different doxycycline dosages with precise quantitation of plasma drug concen tration may be warranted. There were isolate-dependent differences between the two A. phagocytophilum -infection trials. The NY18-infected dogs exhibited greater immuonoreactivity in the IDEXX SNAP4Dx test and on immunoblots compared to the canine isolate-infected dogs. In the case of the immunoblots, this difference may have been due to the antigen used in the immunoblot, A. phagocytophilum NY18. It is expected that the NY18infected dogs would demonstrate a more robust immune response against this antigen than the canine isolateinfected dogs. However, this explanation cannot be used to e xplain the difference in IDEXX SNAP4Dx immuonoreactivity since the test uses a highly cons erved region of MSP2 (p44) as the antigen. During the course of the trials, DNA was also more frequently detected in the blood of the NY18-infected dogs than in the blood of the canine isolate-infected dogs. In addition to a potential inherent variation between the isolates (e.g., degree of host -adaptation), the diff erence in IDEXX SNAP4Dx immuonoreactivity and DNA detection between the two infection trials may reflect a difference in the dose of organisms in th e inoculum, 4 mL of tissue cult ure containing approximately 350,000 infected cells at high multip licity of infection vs. 1 mL of blood stabilate from a parasitemic dog. It is also of not e that one of the canine isolate-in fected dogs exhibited cyclical periodicity in pyrexia episodes. This may have correlated with the emergence of new MSP2 variants (11), but this possibility wa s not investigated in these dogs. The A. phagocytophilum NY18-infection trial reported here represents the first infection of dogs using cultured endothelial cells as the source of inoculum. This is an important proof of concept as progress is made toward potenti al vaccines based upon genetically modified A. 43

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phagocytophilum (40) cultured in endothelial cells, which are a more tractable source of organisms compared to other in vitro systems (81). The A. phagocytophilum canine isolate-infection trial reported here is the first investigation of endothelial cells as a potential in vivo source of A. phagocytophilum during chronic canine infection. In SCID mice, dual indirect immunofl uorescent colocalization of an endothelial cell antigen and an A. phagocytophilum surface protein within the cardiac and hepatic microvasculature after 7 weeks of infection has been described; however, photomicrographs and rigorous controls were not published in th e report (54). Uneq uivocal evidence of in vivo endothelial cell infection in exam ined tissues (kidney, liver, lung) from chronically infected dogs was not identified using the in situ rolling-circle amplification t echnique presented here. There are a few potential explanations for why this might be so. First, endothelial cells may, in fact, not be involved in the in vivo A. phagocytophilum life cycle. Second, endothelial cells may be involved in the in vivo A. phagocytophilum life cycle, but at a level that is below the detection limit of the in situ rolling-circle amplification technique described here. Third, since positive control tissue sections were not available (cytospins of culture material were used for this purpose), the in situ rolling-circle technique described here may not have been properly optimized for detection of prokaryotic cells in fr ozen canine tissue sections. Future examination of A. phagocytophilum infection of endothelial cells in vivo using other techniques (e.g., monoclonal-based immunofluorescence, fluorescence in situ hybridization (FISH), or in situ PCR) and techniques modified to dimini sh autofluorescence may be useful. 44

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Table 3-1. Treatments administered duri ng two infection tria ls using different A. phagocytophilum isolates A. phagocytophilum isolate Human NY18 Canine natural Animals 2 adult, male Beagles 2 adult, male Beagles First immunosuppression Pre dnisone 2 mg/kg by mouth daily, 12 days (230-242 PI) Prednisone 2 mg/kg by mouth daily, 14 days (143-156 PI) Antibiotic treatment Doxycycline 10 mg/kg by mouth once daily, 14 days (247-261 PI) Doxycycline 10 mg/kg by mouth twice daily, 28 days (157-184 PI) Second immunosuppression Predni sone 2 mg/kg by mouth daily, 10 days (316-325 PI) Prednisone 2 mg/kg by mouth daily, 14 days (191-204 PI) Euthanasia Day 344 (11.5 months ) PI Day 233 (7.8 months) PI PI indicates post-infection Table 3-2. Comparison of A. phagocytophilum SNAP4Dx seroconversion between the two canine experimental inoculation studies (NY18 isolate vs. canine isolate) Inoculum Day post-infection first positive Days positive after initial positive NY18 Isolate Dogs 1 and 2: day 8 Dogs 1 and 2: days 11-316 Canine Isolate Dog 1: day 51 Dog 1: day 59 only Canine Isolate Dog 2: day 59 Dog 2: none (negative) 45

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Table 3-3. Detection of A. phagocytophilum DNA by nestedand real-time PCR in the blood of the NY18 isolate-infected dogs pre-infection through day 340 post-infection Dog 1 Dog 2 Days postinfection Nested msp2 Real-time msp2 Real-time groEL Nested msp2 Real-time msp2 Real-time groEL Pre Negative Negative 1 Negative Positive 4 Positive Negative 6 Positive Negative 8 Positive Positive Positive Positive Negative 11 Positive Positive 13 Positive 15 Positive Positive 20 Positive Positive 22 Positive 25 Negative Negative 32 Positive Negative 35 Negative Positive 36 through 230 Negative Negative 1 positive 210 PI 1 positive 98 PI 1 positive 119 PI Negative Immunosuppression for 12 days (Prednisone 2 mg/kg by mouth once daily, days 230-242) 231 through 242 Negative Negative 243 Positive Positive Negative Negative Positive Negative 245 Negative Positive Negative Positive Positive Negative Antibiotic treatment for 14 days (Doxycycline 10 mg/kg by mouth once daily, days 247-261) 299 Negative Positive Negative Negative Negative Positive 316 Positive Negative Positive Negative Immunosuppression for 10 days (Prednisone 2 mg/kg by mouth once daily, days 316-325) 327 Positive Negative Positive Negative 329 Positive Negative Positive Negative 333 Positive Negative Positive Negative 340 Positive Negative Positive Negative Between days 36 and 230 post-infection, samples fr om 21 sampling days were tested by nestedPCR, and samples from 4 sampling days (day s 69, 119, 210, and 230) were tested by real-time PCR. Cells in the table that are empty indicate that the assay was not run on a particular sample. 46

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Figure 3-1. PCR primers and fluoresceinated oligonucleotide probe. AB1210 (forward) and AB1211 (reverse) were used in the prim ary PCR reaction. AB1212 (forward) and AB1213 (reverse) were used in the nest ed-PCR reaction. AB1214 contained a 5 modification designated as FLU which stands for fluorescein. AB1211, AB1213, and AB1214 contained degenerate bases, which are equimolar mixtures of two or more different bases at a given positi on within the sequence, designated as D =A/G/T, M =A/C, R =A/G, W =A/T, or Y =C/T. 47

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Figure 3-2. Body temperature of one of the A. phagocytophilum canine isolate-infected dogs during the infection tr ial. The black hor izontal line at 102.5 F indicates the upper limit of the reference interval for canine body temperature. There was an average of 26.1 days (median 26.0 days) between ep isodes of body temperatures >102.5F. Elevated body temperature was first dete cted on day 20 PI. There were 31 days between next elevation, which occurred on da y 51 PI. There were 8 days between the next at day 59 PI. There were 20 days betw een the next at day 79 PI. There were 49 days between the next at day 128 PI. There were 15 days between the next at day 143 PI. There were 34 days between next episode at day 177 PI, during which the body temperature remained elevated for four consecutive measurements. There were 26 days between next episode at day 21 3 PI, during which the body temperature remained elevated for three consecutive measurements. 48

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Figure 3-3. Immunoblot of polyclonal sera against A. phagocytophilum NY18 isolate demonstrates immuonoreactivity (seropositivity) in the canine isolate-infected dogs. An SDS-PAGE gel containing sepa rated, denatured proteins of A. phagocytophilum (NY18) was reacted on an immunoblot using preand post-infection polyclonal sera from an NY18-infected human, one of the NY18-infected dogs, and both canine isolate-infected dogs. The antigen in the blot, A. phagocytophilum (NY18), was obtained from RF/6A endot helial cell cultures. 49

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Figure 3-4. Southern blot of the A. phagocytophilum msp2 -based nested-PCR products from the NY18 isolate-infected dogs. Th e southern blot reveals the msp2 product amplified by nested-PCR from peripheral blood (Dog 1 preand post-inoculation) and selected organs (uninfected control dog a nd Dog 1, day 344 post-infection). 50

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Figure 3-5. Southern blots of the A. phagocytophilum msp2 -based nested-PCR products from the canine isolate-infected d ogs that received doxycycline from day 155 to 183 postinoculation. The southe rn blots reveal the msp2 products amplified by nested-PCR from A) peripheral blood (Dog 2 preand pos t-inoculation) and B) selected organs (Dog 1, day 233 post-infection). 51

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Figure 3-6. In situ DNA target-primed rolling-circle amplif ication of a padlock probe in canine isolate-infected Dog 1 kidney day 233 post-infection. A) A.phagocytophilum specific padlock probe with DAPI nuclear counterstain, 63X objective, differential interference contrast with blue emission, B) A. phagocytophilum -specific padlock probe, 63X objective, green emission, C) A. phagocytophilum -specific padlock probe, 63X objective, red emission, D) A. phagocytophilum -specific padlock probe, 63X objective, red and green emissions, E) A. phagocytophilum -specific padlock probe, 63X objective, blue, red, and green emi ssions, F) nonspecific padlock probe with DAPI nuclear counterstain, 63X objective, differe ntial interference co ntrast with blue emission, G) nonspecific padlock probe, 63X objective, green emission, H) nonspecific padlock probe, 63X objective, red emission, I) nonspecific padlock probe, 63X objective, red and green emissions, J) nonspecific padlock probe, 63X objective, blue, red, and green emissions. Multifocal areas of autofluorescence were identified in the tissues regard less of whether an A.phagocytophilum -specific padlock probe or a nonspecific padlock probe was used and rega rdless of the incide nt excitation light wavelength. 52

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CHAPTER 4 EXPERIMENTAL INFECTION OF CATTLE WITH ANAPLASMA MARGINALE DISTRIBUTION OF ORGANISM S IN BOVINE TISSUES, AND INVESTIGATION OF ENDOTHELIAL CELLS AS A NIDUS OF ACUTE AND CHRONIC INFECTION Introduction Globally, A. marginale is the most prevalent tick-transmitted pathogen of cattle and other ruminants. It is the etiologic agent of bovi ne anaplasmosis, an economically significant, arthropod-borne hemolytic disease of cattle that engenders substantial morbidity and mortality in United States bovine livestock, with estimated annual losses exceeding 300 million dollars (1986 U.S. dollars) (63). The only re cognized mammalian life cycle stage of A. marginale is within terminally differentiated, circulating mature erythrocytes (33), which have a half-life of approximately 130 days (64). However, bovine anaplasmosis is characterized by obligatory, persistent, often life-long, infecti on of ruminant hosts associated with chronic, sub-microscopic, cyclic parasitemias due to antigenic variation of the surface proteins, MSP2 and MSP3 (3, 9, 10, 17, 18, 19, 38, 39, 43, 44, 59, 80, 90, 91). While, A. marginale organisms may transfer between term inally differentiated erythrocytes (48, 111) or shuttle between erythrocytes and the mononuclear phagocyte system, it is equivocal whether this is sufficient for sustained and pr oliferative infection. Recent experiments have documented that A. marginale can be successfully propagated w ithin primate endothelial cells in long term cell culture (81). Preliminary observa tion of immunofluorescen tly stained tissue has suggested the presence of A. marginale within renal endothelial cells of a splenectomized steer (24). The purposes of this investigation were to describe the tissue distribution of A. marginale in bovine tissues after tic k-feeding transmission, to determine whether or not A. marginale 53

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invades bovine endothelial cells at tick attachme nt sites during initial in fection, and to discover whether or not A. marginale persists within bovine endothelial cells during cyclic parasitemia. Materials and Methods Animals, Inoculum, Monitoring Infection and Parasitemia, and Euthanasia Three adult, field-sourced Holstein steers, confirmed by a commercially available cELISA (VMRD, Inc., Pullman, WA) to be negative for A. marginale were inoculated by tick-feeding transmission. A. marginale St. Maries-infected, male Dermacentor andersoni adult ticks were placed on two of the steers, while uninfected male Dermacentor andersoni adult ticks were placed on the one remaining steer, which served as a negative control (ticks provided by Guy H. Palmer Laboratory, Washington State University, Pu llman, WA). Prior to tick application, the steers were bathed three times to remove residu e of acaricide that might have previously been applied to the steers coats. On each of the steers, the hair in three 15 cm2 areas near the withers was clipped as close to the skin as possible. In each of the three shaved areas, a sleeve-like cylinder of fabric (1560 cm3) that was open on both ends was affi xed to the skin using livestock identification tag cement (Ketchum Mfg. Co, Inc., Lake Luzerne, NY). The sleeves were affixed perpendicularly to the skin so that one of the ope n ends was adhered to the skin, thus sealing the bottom of the cylinder. Ticks were placed on th e shaved skin enclosed within the sleeve-like cylinders of fabric (15 ticks per cylinder). The remaining ope n, top end of the cylinder was twisted and held closed usi ng a rubber band (Figure 4-1). After inoculation, the steers were examined a nd phlebotomized at regular intervals for the duration of the trial. Blood was collected into both EDTA-containing and plain sterile tubes, from which serum was harvested. Rectal temper ature, mucus membrane color, and respiratory rate were monitored for abnormalities consistent w ith anaplasmosis, such as pyrexia and signs of anemia. To document the occurrence of serum antibodies directed against A. marginale sera 54

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from the steers were tested using a commercially available cELISA (VMRD, Inc., Pullman, WA). The packed cell volume (P CV) percentage was monitored for the development of anemia. Wrights Giemsa-stained (Harleco, EM Scien ce, Gibbstown, NJ) peripheral blood films were examined by light microscopy for the occurrence of parasitemia as evidenced by the presence of basophilic, circular, marginally locate d erythrocytic inclusions consistent A. marginale parasitophorous vacuoles. A Miller ocular disc was used to facilitate enumeration of A marginale-parasitized erythrocytes. One of the A. marginale -infected steers was euthanized during the first peak parasitemia. The other A. marginale -infected steer and the uninfected steer were euthanized at the time of th e first trough in parasitemia. Tissue Sample Collection and Processing Dermal punch biopsies were collected from tic k-bite sites every day between days 2 and 10 post-attachment; thereafter, they were collected every other day unt il no ticks remained attached to the steers skin. Once weekly, dermal punc h biopsies were collected from each steer at a distant, non-tick-bite site area of normal skin as a noninflamed tissue control. Tissue samples from all body systems were also collected postmortem. A portion of all tissue samples was processed three ways: 1) 10% buffered forma lin-fixed for histology or electron microscopy, 2) Trumps fixative-fixed for electron micr oscopy, 3) cryopreserved for indirect immunofluorescence and in situ rolling circle amplification. A portion of the tissues collected post-mortem was also frozen plain wit hout added preservative at -20C. Immediately after collection, whole dermal punch biopsy tissue samples were initially stored and transported back to the laboratory for further processing in sterile tissue culture medium (HyClone, Logan, UT) on ice. The cylin der-shaped biopsy tissue wa s later cut in half longitudinally. One half was further quartere d. One quarter was pl aced in 10% buffered formalin; the other quarter was placed in Trumps fixative. Both quarters were stored at 4C. 55

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The remaining half of the dermal punch biops y was cryoembedded in Neg-50 medium (Thermo Scientific Richard-Allan, Pittsburg, PA) by freezing in 2-methylbutane on liquid nitrogen. Frozen tissue blocks were stored at -80C until cryosectioned. Four micrometer sections were prepared from cryoembedded tissue blocks us ing a Leica cryostat (Leica Microsystems, Bannockburn, IL) at -20C and immediately fixed in acetone on ice for 5 min. Sections were stored at -80C until examined Immediately after collection, a portion of each post-mortem tissue sample was placed directly in either 10% buffered formalin, Trump s fixative, sterile tissue culture medium on ice, or a plain container. Sample portions that we re placed in sterile tissue culture medium on ice were transported back to the lab and cryoembedded as above described. PCR Detection of A. marginale DNA in Blood and Tissues DNA was extracted from anticoagulated, ED TA-whole blood and plain-frozen postmortem tissue samples using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA) in accord with the manufacturer instructions, which indicated the following amounts of biologic material be used for DNA extraction: 200 L of anticoagul ated whole blood, 25 mg of tissues other than spleen, or 10 mg of spleen. Extracted DNA was used as the template for real-time quantitative PCR amplification of the single copy operon-associated gene 2 ( opag2), which is present in the 3.5 kilobase (kb) msp2 expression site of A. marginale (74). Though potentially reduced in sensitivity compared to amplifi cation of a multicopy gene, such as msp2 or orfx orfy the single copy opag2 was chosen to allow direct re presentation of the number of A. marginale genome copies that were detected in each DNA extract sample. Twenty-five microliter DNA amplificati on reactions were run in a MicroAmp Fast Optical 96-Well Reaction Plate with Barcode covered with MicroAmp 96-Well Optical Adhesive Film on a 7500 Fast Real-Time PCR System using 12.5 L TaqMan Fast Universal 56

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PCR Master Mix (Applied Biosystems Inc, Fo ster City, CA) plus 5 L of extracted DNA template, 0.8 M of each primer (forward and reverse), and 0.2 M of fluorescently labeled oligonucleotide probe (MWG Biotech, High Point, NC). The following primer set, which amplifies a 151 bp region of opag2, was used: AB1242 5AAA ACA GGC TTA CCG CTC CAA-3 (forward) and AB1243 5-GGC GTG T AG CTA GGC TCA AAG T3 (reverse). The oligonucleotide probe was labeled with 5 6-carb oxyfluorescein (FAM) as a reporter dye and 3 6-carboxy-N, N, N, N-tetramethylrhodamine (T AMRA) as a quencher. The sequence of the probe was AB1250 5-FAM-CTC TCC TCT GC T CAG GGC TCT GCGTAMRA-3. The thermocycler conditions used for quantifying each single copy gene were 95C for 20 sec activation followed by 40 cycles of 95C for 30 sec denaturation and 60C for 30 sec annealing, extension, and data collecti on. A serial dilution (109, 108, 107, 106, 105, 104, 103, 102, and 101 copies) of the plasmid, opag2/pCR4-TOPO which contains the full length opag2, were prepared for standard curve generation. Copy numbers for samples were calculated based on the standard curve by using the 7500 Fast Real-Time PCR System software (Applied Biosystems Inc, Foster City, CA). Microscopic Detection of A. marginale Organisms in Tissues Dual indirect immunofl uorescent staining of A. marginale and endothelial cell antigens Dual, indirect immuno fluorescent staining of A. marginale MSP5 and mammalian von Willebrand factor was performed on cryosectioned tissues based on the published procedure (24). The cryosectioned tissues were bl ocked with normal rabbit serum (X0902 (Dako, Carpinteria, CA) 5% in 1X PBS [pH 7.4], 30 min) and subsequen tly washed once in 1X PBS (5 min) and then blocked a second time with 5% normal goat serum and rinsed in 1X PBS, as before. The tissue sections were then incubated for 30 min with either antibody-free diluent or an equal part mixture of diluted mouse monoclonal antiA. marginale MSP5 antibody and diluted 57

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rabbit polyclonal anti-human von Willebrand factor antibody (antiA. marginale MSP5 antibody: ANAF16C1 (Guy H. Palmer Laboratory, Washin gton State University, Pullman, WA), 100 g/mL in 0.05 M Tris-HCl [pH 7.5] and 1% bovine serum albumin fraction V (Fisher Scientific, Pittsburg, PA); anti-human von Willebrand factor antibody: N1505 (Dako, Carpinteria, CA), 1 to 2 dilution of the proprietary antibody solution in 0.05 M Tris-HCl [pH 7.5] and 1% bovine serum albumin fraction V (Fisher Scientific, Pitts burg, PA)). The slides were washed twice in 1X PBS (5 min) prior to secondary antibody labe ling. The tissue sections were subsequently incubated for 30 min with either antibody-free diluent or an equal part mixture of diluted, highly cross-adsorbed, goat polyclona l anti-mouse-IgG antibody-Alexa Fluor 488 conjugate and diluted, highly cross-adsorbed, goat polyc lonal anti-rabbit-IgG antibody-Alexa Fluor 568 conjugate (anti-mouse-IgG antibody conjugate : A11029, anti-rabbit-IgG antibody conjugate: A11036 (Invitrogen Molecular Prob es, Carlsbad, CA), both antibod ies were diluted to 2 g/mL in 0.05 M Tris-HCl [pH 7.5] and 1% bovine se rum albumin fraction V (Fisher Scientific, Pittsburg, PA)). Afterward, the slides were washed twice in 1X PBS (5 min), air-dried, coverslip-mounted using VE CTASHIELD HardSet Mounting Medium with DAPI, and examined immediately using a Nikon Eclipse E800 microscope (N ikon Instruments, Inc., Kanagawa, Japan) fitted for epifluorescence and e quipped with a digital camera (Diagnostic Instruments, Sterling Heights, MI) and then stored at 4C. Digital images were collected using Spot Advanced software (Diagnostic Instruments, Sterli ng Heights, MI) and uniformly processed using SPOT Advanced Windows Version 4.0.9 (Dia gnostic Instruments, Sterling Heights, MI) and Adobe Photoshop Elements 2.0 (Adobe Systems Incorporated, San Jose, CA). Electron microscopy One square-millimeter subsamples of Trumps fixative-preserved tissues were trimmed-in and embedded in glycol methacrylate (Universit y of Florida, College of Medicine, Electron 58

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Microscopy Core). Prior to transmission elec tron microscopy, 0.5 m sections were prepared from the methacrylate-embedded tis sue blocks and stained with to luidine blue. Using routine light microscopy, the toluidine blue-stained secti ons were pre-screened to identify an area of interest within the tissue block faces. The observer performing the pre-screening of the toluidine blue-stained tissues was blind to the identity of the animal from which the tissue samples had been collected. The methacrylate tissue block fa ces were then further trimmed to focus upon the area of interest within the block face and, ther eby, reduce the surface ar ea of the block face to improve the quality of the ultrathin sections. Ultr athin sections were then cut, mounted on nickel grids, and stained with uranyl acetate and lead citrate. Sections were evaluated using a transmission electron microscope (Zeiss EM 10A, Carl Zeiss MicroImaging, Inc., Thornwood, NY) fitted with a digital camera (Finger Lakes Instrumentation, Lima, NY). In Situ DNA target-primed rolling-circle amplificat ion of a padlock probe for detection of Anaplasma marginale In situ DNA target-primed rolling-ci rcle amplification of padl ock probes for detection of A. marginale was performed on cryosectioned tissues by the published procedure (113) and by a modified version of the described procedure that used sheared calf thymus DNA (R&D Systems, Minneapolis, MN) as the DNA carrier in the o ligonucleotide hybridizatio n reactions. Sheared calf thymus DNA was used at 0.5 g/L in the padlock probe hybridization solution and was used at 6.0 g/L in the fluorescently labeled oligonucleotide hybridizati on solution. Cytospins of A. phagocytophilum HZ isolate cultivated in human (Homo sapiens ) HL-60 myeloblastic leukemia cells (American Type Culture Collection, ATCC CCL-240, Manassas, VA), as described (49), were also run with an A. phagocytophilum -specific padlock probe as a reagent positive control. 59

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Statistical Analysis Data analysis was performed and graphs we re created using Microsoft Office Excel 2007 (Microsoft Corporation, Re dmond, WA), SigmaStat for Windows Version 3.11, and SigmaPlot 2004 for Windows Version 9.01 (Systat Software, Inc., Chicago, IL). Results Seroconversion, Parasitemia, and Anemia Competitive ELISA revealed that the two A. marginale -infected steers became seropositive between two and three weeks post-infection, while the negative cont rol steer remained seronegative (Figure 4-2). A modest parasitemia peak occurred in the two A. marginale -infected steers about 7 weeks post-infection (steer 5237 day 33 post-infection 4.7%; steer 3102 day 36 post-infection 3.7%) (Figure 4-3). Microscopic observation of parasitemia corresponded well with the onset, peak, and resolution of parasite mia detected by real-time quantitative PCR (Figure 4-4). The maximum opag2 copy number detected occurred on the same day as peak microscopic parasitemia in both steers (steer 5237 day 33 post-infection 6.4 x 105 copies per L of blood; steer 3102 day 36 post-infection 3.2 x 105 copies per L blood). At the time of peak parasitemia, one of the steer s (5237) was pyrexic with body te mperature elevated above the upper limit of the reference interval for adu lt bovine body temperature (102.5F): day 33 postinfection (103.5F) and day 34 post-infection (1 02.8F). Both steers PCV became subnormal, dropping below 24%, the lower limit of the adult bovine PCV reference interval. The nadir PCV occurred on day 38 post-infection in both steers and was 21% in both steers. The steers did not manifest clinical signs of an emia during the trial. Tissue Distribution of A. marginale Real-time quantitative PCR revealed a variable number of opag2 copies distributed throughout the post-mortem tissues of the two A. marginale -infected steers (Figures 4-5 and 460

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6). The difference in the number of opag2 copies in each of the tissues was statistically significant, being greater than that expected simply due to chance (Kruskal-Wallis one way analysis of variance on ranks: steer 5237 P = 0.010; steer 3102 P = 0.003). Opag2 copies were not detected in the uninfected st eer. In the steer (5237) that was euthanized during the first peak in parasitemia, the greatest number of opag2 copies was found in one of the hemi-lungs and a perihilar hemal node, 1.32 x 104 and 1.31 x 104 opag2 copies per mg tissue, respectively. In the steer (3102) that was euthanized during the first trough in parasi temia, the greatest number of opag2 copies was found in the spleen, 0.51 x 104 opag2 copies per mg tissue. The left and right lung contained the ne xt most numerous opag2 copies per mg tissue in this steer, 0.09 x 104 and 0.08 x 104 opag2 copies per mg tissue, respectively. Microscopic Examination for A. marginale in Tissues In the A. marginale -infected tick-bite sites, dual indi rect immunofluores cent staining of tick-bite sites collected between days 6 and 8 pos t-infection revealed j uxtaposition of intensely green-fluorescent amorphous material ( A. marginale intended to be labeled green) and redfluorescent stippled structures in capillaries corresponding to Weibel-Palade bodies (von Willebrand factor intended to be labeled red). Similar intensity green fluorescence was absent from the uninfected tick-bite s ites during the corresponding time pe riod (Figure 4-7). However, when tick bite sites from later time points, day 10 and 22 post-infection, were examined, it became increasingly difficult or impossible to distinguish A. marginale -infected tick-bite sites from uninfected tick-bite sites using this method. It was only possible to distinguish normal, noninflamed skin to which ticks were not attached from tick-bite sites, re gardless of whether or not the ticks were A. marginale -infected. Day 6 post-infection tick b ite sites and post-mortem sp leen and lung samples were examined using transmission electron microsco py and light microsc opic pre-screening of 61

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toluidine blue-stained tissues (Figures 4.8 and 4.9). Using these techniques, it was not possible to distinguish A. marginale -infected tissues from uninfected tissues. The observer (HLW) performing the pre-screening of the toluidine blue-s tained tissues was blind to the identity of the animal from which the tissue samples had been co llected. Once the identity of the tissues was disclosed, it was apparent that da rk blue-staining structures of in terest had been identified in both A. marginale -infected and uninfected tissues during blin ded observation. These dark structures corresponded to intracytoplasmic granules that ar e normally present in mast cells (Figures 4-8 A and B and 4-9). A. marginale was not observed during close examination of capillaries in the tissue samples; though, mitochondria were frequently id entified (Figure 4-8 C). Dermal punch biopsies of tick-bite site s collected from the three steers (days 2, 5, 6, 7, 8, 14, and 22 post-infection) and post-mortem tissues collected from the steer that was euthanized during the first pa rasitemia peak with highest opag2 copy number per milligram of tissue (steer 5237; day 41 post-in fection; hemi-lung site 1, perihilar hemal node, abomasum, and spleen) were microscopi cally examined after in situ DNA target-primed rolling-circle amplification of a padlock probe for detection of A. marginale (Figures 4-10 and 4-11). Fluorescently labeled in situ rolling circle amplifi cation reaction product was not detected within capillaries or the rest of any of the tissue sections examined. It was not possible to distinguish A. marginale-infected tick-bite sites from uninfected tick-bite sites us ing this technique, nor was it possible to distinguish tissues th at had been hybridized with the A. marginale -specific padlock probe from tissues that had been hybridized with the nonspecific padlock probe. When sonicated fish sperm was used in the oligonucleotide hybr idization solutions, leukocyte granules were nonspecifically fluorescently labeled regardless of whether an A. marginale -specific padlock probe or a nonspecific padlock probe was use d. The severity of the nonspecific staining 62

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increased as the tick-bite sites became more infl amed and leukocyte-infiltrated with the passage of time. It was empirically determined that omission of the fluorescentl y labeled oligonucleotide probe from the in situ rolling-circle amplification protocol abrogated this nonspecific staining of leukocyte granules, and use of an alternative ca rrier-DNA (sheared calf thymus DNA) mitigated this nonspecific staining of leukoc ytes (Figure 4-12). The mitigation afforded by use of sheared calf thymus DNA was concentration dependent. The blocking was most effective when sheared calf thymus DNA was used in the fluorescent o ligonucleotide hybridizati on solution at 12X the original sonicated fish sperm concentration, which was the maximum increase in carrier-DNA possible given the concentration of the st ock solutions and final reaction volume. Discussion This study represents the first description of A. marginale tissue distributi on in cattle after tick-feeding transmission. The distribution within tissues was described during a peak and a trough in parasitemia. Lung and spleen contained a relatively high number of A. marginale gene copies regardless of the parasitemia phase. This finding may reflect the anatomic nature of these organs, which contain copious microva sculature and numerous macrophages. A. marginale may be residing in endothelial cells within microvasculature, may be shuttling between erythrocytes and macrophages, or simply, may be in greater number within these organs, which are normally heavily perfused, due to contamination by parasitized erythrocytes. This study represents the first i nvestigation of endothelial cell s as a potentia l repository of A. marginale in adult, immunocompetent cattle. The si ngle previously described investigation (24) used a highly parasitemic, splenectomized (therefore im munocompromised) calf. Using, simple, non-confocal epifluorescence microscopy, the authors suggested that juxtaposition of green epifluorescence and red epiflu orescence in renal tissue indicated in vivo colocalization of an A. marginale surface protein and von Willebrand factor within endothelial cells. This study 63

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had been performed as a proof of concept for the dual indirect immunof luorescence technique for A. marginale detection and was somewhat flawed. Th e authors did not examine tissues from a non-parasitemic, uninfected negative control animal in order to conf irm that tissue that should be negatively staining using this technique was in fact negatively staining. When this technique for in situ detection of A. marginale was applied to the tissues collected during the investigation reported here, inflamed A. marginale -infected tick bite sites coul d not be distinguished from inflamed uninfected tick bite sites. Ther e was suggestion of possi ble colocalization of A. marginale surface protein and von Willebrand factor wh en tick-bite sites from early collection days were examined. However, these observatio ns came to be viewed with skepticism as negative control tissues from later time points were processed. Endothelial cells were investigated using three techniques to detect A. marginale dual indirect immunofluorescence, tran smission electron microscopy, and in situ DNA target-primed rolling-circle amplification of a padlock probe. None of these techni ques yielded unequivocal evidence of A. marginale infection of endothelial cells in vivo In the future, tissues may be examined using other in situ techniques (e.g., FISH or in situ PCR), and another infection trial using live animals and endothelial cell-derived inoculum will be performed. Some future strategies for A. marginale vaccine development may use genetically modified A. marginale that has been cultivated in endothelia l cells since these cells are easier to manipulate and perform plaque isolation of mutants compared to other in vitro host cell types (81). The observations reported here, suggest that endothelial cells may not be a nidus of A. marginale infection in vivo Therefore, it becomes doubly importa nt to determine if it is possible to establish a viable, prolifera tive infection in ruminants using A. marginale cultivated long-term in endothelial cells. One of the A. phagocytophilum -infection trials desc ribed in Chapter 3 64

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demonstrated that this was possible with the NY18 isolate in dogs. However, at this point, it is uncertain whether or not the same thing is possible for A. marginale in cattle. To enhance the likelihood of successfully establishing an infection, splenectomized calves, which are known to be less resistant to A. marginale infection than spleen-intact calv es and adults (63, 91), will be subjected to a future infection trial using A. marginale cultivated in endot helial cells as the inoculum. The calves will be monitored for the development of infection and parasitemia using serology, light microscopy, and real-time quantita tive PCR. Blood samples collected during this trial will also form the basis for additional investigations aimed at developing a better understanding of host cell-specific adaptations of the proteome us ing isobaric tag for relative and absolute quantitation (iTRAQ) of protein a nd two-dimensional fluorescence difference gel electrophoresis (2-D DIGE). 65

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Figure 4-1. Steer from A. marginale tick-feeding transmission trial. Sleeve-like cylinders of fabric adhered to shaven areas about the w ithers contained the ticks. The outer end of the sleeve was twisted and held closed with a rubber band that could be removed and replaced repeatedly to allow access for tick-b ite site dermal punch bi opsy collection. 66

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Figure 4-2. Competitive ELISA results for steers during the A. marginale tick-feeding transmission trial. cELISA percent inhibition greater than 30% indicates a positive result. The black horizontal line de notes the 30% cut-off. The two A. marginale infected steers (5237, 3102) became seropositive between two and three weeks postinfection. 67

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Figure 4-3. Packed cell volume and micros copic parasitemia for steers during the A. marginale tick-feeding transmission trial. PCV percent below 24% is subnormal in adult bovines. The black horizontal line denotes the 24% lower limit of the PCV reference interval. The two A. marginale -infected steers (5237, 3102) became developed a modest microscopic parasitemia and subseque nt mild anemia at approximately seven weeks post-infection. 68

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Figure 4-4. Real-time quantitative PCR parasitemia detected as opag2 copies per microliter of peripheral blood of steers during the A. marginale tick-feeding transmission trial. The onset, peak, and resolution of parasitemia detected by real-time quantitative PCR corresponded well with observation of mi croscopic parasitemia (Figure 4-3). 69

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Figure 4-5. Real-time quantitative PCR opag2 copies per milligram of tissue in the infected steer (5237) that was euthanized during the first peak in parasitemia day 41 post-infection. During this parasitemia p eak, the greatest number of opag2 copies was found in one of the hemi-lungs and a perihilar hemal node. 70

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Figure 4-6. Real-time quantitative PCR opag2 copies per milligram of tissue in the infected steer (3102) that was euthanized during the first trough in parasitemia day 64 postinfection. Note the upper limit of the x-ax is is 6000 copies per milligram of tissue, whereas the upper limit of the x-axis in Figure 4-5 is 14,000 copies per milligram of tissue. During this parasitemi a trough, the greatest number of opag2 copies was found in the spleen. 71

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Figure 4-7. Dual indirect immunofluorescent staining of A. marginale MSP5 (green) and von Willebrand factor (red) in uninfected (steer 5180) and infected (steer 5237) tick-bite site dermal punch biopsies day 8 post-infecti on. A) Uninfected tick-bite site, 40X objective, red and green emissions, B) uninf ected tick-bite site with DAPI nuclear counterstain, 40X objective, differential in terference contrast with blue, red, and green emissions, C) A. marginale -infected tick-bite site, 40X objective, red and green emissions, D) A. marginale -infected tick-bite site w ith DAPI nuclear counterstain, 40X objective, differential inte rference contrast with blue red, and green emissions. Dermis from an uninfected tick-bite site and an A. marginale -infected tick-bite site punch biopsy exhibits stippled, intensely re d fluorescence along the lengths of the capillary lumina, which corresponds to labe led von Willebrand factor within WeibelPalade bodies. In panels B and D, the grey linear structures are bundles of dermal collagen, and the round, blue fluorescent stru ctures are DAPI-stained nuclei. Intense green fluorescence is absent from panels A and B; only, fine linear, pale green autofluorescence corresponding to subendothelial collagen is observed. In panels C and D, green intensely fluorescent amor phous material is found juxtaposed with fluorescently red-labeled von Willebrand factor. 72

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Figure 4-8. Toluidine blue-stained and transmission electron microscopy tick-bite site dermal punch biopsies day 6 post-infec tion. A) Toluidine blue-s tained uninfected (steer 5180) tick-bite site 40X obj ective, bright field, B) toluidine blue-stained A. marginale-infected (steer 3102) tick-bite site 40X objective, bright field, C) transmission electron photomicrogr aph of a capillary within an A. marginale -infected (steer 3102) tick-bite site, the bar in the in set image denotes 1 micron. Toluidine-blue pre-screening of the uninfected tick-bite site and A. marginale -infected tick-bite site revealed similar dark-blue, circular stru ctures near capillaries (arrows). These structures corresponded to mast cell granules that were identical to those shown in Figure 4-9 B. The circular structures observe d in association with the capillaries were mitochondria. 73

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Figure 4-9. Toluidine blue-stained and transm ission electron microsc opy lung from the steer (3102) that was euthanized during the first trough in parasitemia day 64 postinfection. A) Toluidin e blue-stained l ung 40X objective, bright field, B) transmission electron photom icrograph of lung, the bar in the inset image denotes 1 micron. The dark blue circul ar structures observed in the lung and in the dermis (Figure 4-8 A and B) were intracytoplasmic mast cell granules. 74

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Figure 4-10. In situ DNA target-primed rolling-circle amplification of a padlock probe using sonicated fish sperm in the oligonucleotide hybridization solutions in A. marginale infected (steer 3102) tick-bite site dermis day 6 post-infection. A) A. marginale specific padlock probe, 40X objective, di fferential interference contrast, B) A. marginale-specific padlock probe with DAPI nuclear counterstain, 40X objective, differential interference contra st with blue emission, C) A. marginale -specific padlock probe with DAPI nuclear count erstain, 40X objective, blue and red emissions, D) A. marginale -specific padlock probe, 40X objective, red emission, E) A. marginale -specific padlock probe, 40X objectiv e, green emission, F) nonspecific padlock probe, 40X objective, differentia l interference contrast, G) nonspecific padlock probe with DAPI nuclear count erstain, 40X objective, differential interference contrast with blue emissi on, H) nonspecific padlock probe with DAPI nuclear counterstain, 40X objective, blue and red emissions, I) nonspecific padlock probe, 40X objective, red emission, J) nons pecific padlock probe, 40X objective, green emission. Red fluorescent-labeled in situ rolling circle amplification reaction product was not detected within capillaries or the rest of the tissue section. Regardless of whether an A. marginale -specific padlock probe or a nonspecific padlock probe was used, leukocyte granul es were non-specifically labeled red. 75

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Figure 4-11. In situ DNA target-primed rolling-circle amplification of a padlock probe using sonicated fish sperm in the oligonucleotide hybridization solutions in lung from the A. marginale-infected (steer 5237) that was euthanized during the first peak in parasitemia day 41 post-infection. A) A. marginale -specific padlock probe, 63X objective, differential interference contrast, B) A. marginale -specific padlock probe with DAPI nuclear counterstain, 63X objective, differential interference contrast with blue emission, C) A. marginale -specific padlock probe with DAPI nuclear counterstain, 63X objective, bl ue and green emissions, D) A. marginale -specific padlock probe, 63X objective, green emission, E) A. marginale -specific padlock probe, 63X objective, red emission, F) nonspecific padlock probe, 63X objective, differential interference contrast, G) nons pecific padlock probe with DAPI nuclear counterstain, 63X objective, differential inte rference contrast with blue emission, H) nonspecific padlock probe with DAPI nuclear counterstain, 63X objective, blue and green emissions, I) nonspecific padlock probe, 63X objective, green emission, J) nonspecific padlock probe, 63X objective, red emission. Green fluorescent-labeled in situ rolling circle amplification reaction product was not detect ed within capillaries or the rest of the tissue secti on. Regardless of whether an A. marginale -specific padlock probe or a nonspecific padlock probe was used, leukocyte granules were nonspecifically labeled green. 76

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Figure 4-12. In situ DNA target-primed rolling-circle am plification of a nonspecific padlock probe using sheared calf thymus DNA in the oligonucleotide hybr idization solutions in A. marginale -infected tick-bite site dermis. A) 7 days post-infection, steer 3102, DAPI nuclear counterstain day, 40X objective, differential interference contrast and blue emission, B) 7 days post-infection, steer 3102, 40X objective, green emission, C) 7 days post-infection, steer 3102, 40X objective, red emission, D) 22 days postinfection, steer 5237, DAPI nuclear counter stain day, 40X objective, differential interference contrast and blue emission, E) 22 days post-infection, steer 5237, 40X objective, green emission, F) 22 days postinfection, steer 5237, 40X objective, red emission. Green fluorescent-labeled in situ rolling circle amplification reaction product was not detected. Use of the alte rnative DNA-carrier at a 12X concentration in the fluorescent oligonucleotide probe hybridization solution ameliorated nonspecific fluorescence of leukocyte granules. 77

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CHAPTER 5 CONCLUSIONS AND FUTURE RESEARCH The studies reported here de scribe a new technique for in situ detection of Anaplasma They also further elucidate the Anaplasma life cycle by exploring the pos sibility that endothelial cells are an in vivo repository of organisms in mammals a nd by describing a carrier-state of infection after antibiotic treatm ent. This lays the groundwork for future investigations of how endothelial cells fu nction in the natural and experimental Anaplasma life cycle, genetic modification of Anaplasma cultivated in endothelial ce lls for vaccine development, mammalian and arthropod host ce ll-specific adaptations. Endothelial cell culture and prelim inary immunofluorescent staining of Anaplasma infected tissues suggest that endothelial cells may be an in vivo nidus of mammalian infection. To investigate endothelial cells a nd other potential cryptic sites of Anaplasma spp. infection in mammalian tissues, a sensitive and specific, isothermal, in situ technique to detect localized Anaplasma gene sequences using rolling circle am plification of circularizable, linear, oligonucleotide probes (padlock probes) was devel oped. Cytospin preparations of uninfected or Anaplasma -infected cell cultures were examined us ing this technique. Via fluorescence microscopy, the technique described here, and a comb ination of differential interference contrast microscopy or von Willebrand factor immunofluorescence, A. phagocytophilum and A. marginale were successfully localized in situ within intact cultured mammalian cells. This work represents the first application of this in situ method for detection of a microorganism and forms the foundation for future applications of this technique to detect, localize, and analyze Anaplasma nucleotide sequences in the tissues of infected ma mmalian and arthropod hosts and in cell cultures. Three Anaplasma -infection trials using immunocompete nt dogs and cattle were performed to investigate different aspects of endothelial cells as they relate to Anaplasma life cycles and to 78

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further describe clinical aspects of Anaplasma pathogenesis. Four Beagle s were inoculated with A. phagocytophilum from two different sources, allowe d to develop chronic infection, and treated with doxycycline. Two dogs were inocul ated with a human isol ate, NY18 strain of A. phagocytophilum cultivated in fetal rhesus monkey e ndothelial cells; the other two dogs were inoculated with a canine isolate via injection of stabilate prepared from parasitemic blood of a naturally infected dog. Regardle ss of the two isolates tested (human or canine) or the two doxycycline dosages tested, A. phagocytophilum DNA remained detectable for several months in the peripheral blood and some post-mortem tissues (heart, spleen, kidney) from these four dogs. However, microorganisms were not identified in tissues (kidney, liver, lung) from the canine isolate-infected dogs using in situ rolling-circle amplification of padlock probes for detection of Anaplasma Two steers were inoculated with A. marginale by tick-feeding transmission and were euthanized at different points within the pa rasitemic cycle. The tissue distribution of A. marginale during peak and trough parasitemia was desc ribed using real-time quantitative PCR. Lung and spleen tissue samples contained the highest A. marginale gene copy number. However, organisms were not unequivocally identif ied in tissues (tick-bite sites during initial infection and distant tissues post-mortem) using three techniques (indir ect immunofluorescence, electron microscopy, in situ rolling-circle amplification of padlock probes for detection of Anaplasma ). Together these findings represent the following: the first infection of dogs using cultured e ndothelial cells as the source of inoculum, the first molecular evidence of chronic, persistent A. phagocytophilum infection in blood and tissues of subclinical dogs despite doxycycline treatment using the currently recommended dosage, 79

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the first description of A. marginale tissue distribution in cattle after tick-feeding transmission, the first investigation of endot helial cells as a potential in vivo source of Anaplasma in immunocompetent animals using dual indire ct immunofluorescence (cattle), transmission electron microscopy (cattle), and in situ DNA target-primed rolling-circle amplification of a padlock probe for detection of Anaplasma (dogs and cattle). Anaplasma were not unequivocally identified in ti ssues using the three listed microscopic techniques. Future examination of Anaplasma infection of endothelial cells in vivo using other techniques (e.g., monoclonal-base d immunofluorescence, FISH, or in situ PCR) and techniques modified to ameliorate autofluorescence may be useful. Some future strategies for Anaplasma vaccine development may use genetically modified bacteria that have been cultivated in endothelial cells since these cells are easier to manipulate and perform plaque isolation of mutants compared to other in vitro host cell types (81). One of the A. phagocytophilum -infection trials described in Chapte r 3 demonstrated that it was possible to infect dogs with A. phagocytophilum cultivated in endothelial cells. To investigate whether the same is possible for A. marginale in cattle, splenectomized calves, which are known to be less resistant to A. marginale infection than spleen-intact calves and adults (63, 91), will be used in a future infection trial using A. marginale cultivated in endothelial cells as the inoculum. Blood samples collected during this trial will also form the basis for additional investigations aimed at developing a deeper unde rstanding host cell-specific adapta tions of the proteome since it is known that these two organisms and other members of the family Anaplasmataceae employ host cell-specific differential expression of surface antigens (11, 15, 16, 46, 56, 58, 74, 75, 83, 87, 106, 109, 110, 119), which may be an adaptation to improve cell-specif ic infectivity or mammalian host immune evasion. A deeper unde rstanding of the mechanisms of disease pathogenesis will foster development of eff ective treatment and control strategies. 80

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BIOGRAPHICAL SKETCH Heather L. Wamsley graduated from Manatee High School in Bradenton, Florida, and then began undergraduate study as a chemistry major at New College of Florida in Sarasota, Florida. In her sophomore year, Heather transferred to University of WisconsinMadison in Madison, Wisconsin, where she completed a Bachelor of Science in natural sciences with honors, bacteriology major in May 1995. Heather con tinued studies at Univ ersity of Wisconsin Madison, earning a Doctorate of Veterinary Me dicine with honors in May 2000. She obtained state-licensure to practice as a veterinarian in Wisconsin and in New York. After veterinary school, Heather completed a total of four-years of specialty clinical training: one year as a Small Animal Medica l and Surgical Rotating Intern at The Animal Medical Center in New York, New York and th ree years as a Veterinary Clinical Pathology Resident at University of Flor ida in Gainesville, Florida. She was certified by the examination board of the American College of Veterinary Pathologists as a Diplomate of the American College of Veterinary Pathologists, Clin ical Pathology Specialty in September 2004. Heather began graduate studies in 2002 concurrent with he r Residency in Veterinary Clinical Pathology at University of Florida. After completing pathology boards in 2004, she continued her graduate research while working as a Clinical Instructor at the University of Florida, College of Veterinary Medicine, Department of Physiological Sciences. In the winter of 2008, she became an Assistant Professor in the Department of P hysiological Sciences as she finalized her graduate research training. Heather received he r Doctor of Philosophy from the University of Florida, College of Veterinary Me dicine, Department of Infectious Diseases and Pathology in the spring of 2009. 92