Cytokine gene expression by peripheral blood leukocytes in horses experimentally infected with Anaplasma phagocytophila.
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Human granulocytic ehrlichiosis (HGE), a tick-borne zoonosis, is caused by an obligatory intragranulocytic bacterium, the HGE agent, a strain of Anaplasma phagocytophila. The equine model of HGE is considered valuable in understanding pathogenic and immune mechanisms of HGE. In the present study, cytokine mRNA expression by peripheral blood leukocytes (PBLs) in horses was examined during the course of infection by intravenous inoculation of A. phagocytophila or by allowing feeding by infected ticks. The p44 genes encoding the major outer membrane protein P44s of A. phagocytophila were detected by PCR in PBLs of all four horses from 4 to 20 days postexposure. During the 20-day infection period, interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) mRNA expression was upregulated in PBLs of all four horses, and IL-8 mRNA expression was upregulated in three horses. Gamma interferon, IL-10, and IL-12 p35 mRNAs were weakly expressed in only one horse each. IL-2, IL-4, IL-6, and IL-12 p40 mRNA expression, however, could not be detected in the PBLs of any of the four horses. These results suggest that IL-1beta, TNF-alpha, and IL-8 generation during A. phagocytophila infection has a primary role in HGE pathogenesis and immunomodulation. (+info)
Repression of rac2 mRNA expression by Anaplasma phagocytophila is essential to the inhibition of superoxide production and bacterial proliferation.
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Anaplasma phagocytophila, the etiologic agent of human granulocytic ehrlichiosis, is an emerging bacterial pathogen that invades neutrophils and can be cultivated in HL-60 cells. Infected neutrophils and HL-60 cells fail to produce superoxide anion (O(2)(-)), which is partially attributable to the fact that A. phagocytophila inhibits transcription of gp91(phox), an integral component of NADPH oxidase. cDNA microarray and RT-PCR analyses demonstrated that transcription of the gene encoding Rac2, a key component in NADPH oxidase activation, was down-regulated in infected HL-60 cells. Quantitative RT-PCR demonstrated that rac2 mRNA expression was reduced 7-fold in retinoic acid-differentiated HL-60 cells and 50-fold in neutrophils following A. phagocytophila infection. Rac2 protein expression was absent in infected HL-60 cells. Rac1 and Rac2 are interchangeable in their abilities to activate NADPH oxidase. HL-60 cells transfected to express myc-tagged rac1 and gp91(phox) from the CMV immediate early promoter maintained the ability to generate O(2)(-) 120 h postinfection. A. phagocytophila proliferation was severely inhibited in these cells. These results directly attribute the inhibition of rac2 and gp91(phox) transcription to the loss of NADPH oxidase activity in A. phagocytophila-infected cells and demonstrate its importance to bacterial intracellular survival. (+info)
Roles of neutrophil beta 2 integrins in kinetics of bacteremia, extravasation, and tick acquisition of Anaplasma phagocytophila in mice.
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Tick saliva contains anti-inflammatory and immunosuppressive substances that facilitate blood feeding and enhance tick-vectored pathogen transmission, including Anaplasma phagocytophila an etiologic agent of granulocytic ehrlichiosis. As such, inflammation at a tick-feeding site is strikingly different than that typically observed at other sites of inflammation. Up-regulation of CD11b/CD18 occurs in host granulocytes following interaction or infection with A phagocytophila, and the absence of CD11b/CD18 results in early increases in bacteremia. We hypothesized that beta 2 integrin-dependent infection kinetics and leukocyte extravasation are important determinants of neutrophil trafficking to, and pathogen acquisition at, tick-feeding sites. A phagocytophila infection kinetics were evaluated in CD11a/CD18, CD11b/CD18, and CD18 knock-out mice using quantitative polymerase chain reaction (PCR) of blood, ticks, and skin biopsies in conjunction with histopathology. A marked increase in the rate of A phagocytophila infection of neutrophils and pathogen burden in blood followed tick feeding. Infection kinetics were modified by beta 2 integrin expression and systemic neutrophil counts. Significant neutrophil-pathogen trafficking was observed to both suture and tick sites. Despite the prominent role for beta 2 integrins in neutrophil arrest in flowing blood, successful pathogen acquisition by ticks occurred in the absence of beta 2 integrins. Establishment of feeding pools that rely less on leukocyte trafficking and more on small hemorrhages may explain the ready amplification of A phagocytophila DNA from ticks infested on CD11/CD18-deficient mouse strains. (+info)
Antibiotic susceptibilities of Anaplasma (Ehrlichia) phagocytophilum strains from various geographic areas in the United States.
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We tested the antibiotic susceptibilities of eight strains of Anaplasma phagocytophilum (the agent of human granulocytic ehrlichiosis) collected in various geographic areas of the United States, including Minnesota, Wisconsin, California, and New York. The results are homogeneous and show that doxycycline, rifampin, and levofloxacin are the most active antibiotics against these strains in vitro. (+info)
Seasonal dynamics of Anaplasma phagocytophila in a rodent-tick (Ixodes trianguliceps) system, United Kingdom.
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We investigated the reservoir role of European wild rodents for Anaplasma phagocytophila using polymerase chain reaction (PCR) analysis of blood collected from individually tagged rodents captured monthly over 2 years. The only tick species observed in the woodland study site was Ixodes trianguliceps, and ruminant reservoir hosts were not known to occur. A. phagocytophila infections were detected in both bank voles and wood mice but were restricted to periods of peak nymphal and adult tick activity. Most PCR-positive rodents were positive only once, suggesting that rodent infections are generally short-lived and that ticks rather than rodents may maintain the infection over winter. Bank voles were more likely to be PCR positive than wood mice, possibly because detectable infections are longer lived in bank voles. This study confirms that woodland rodents can maintain A. phagocytophila in Great Britain in the absence of other reservoir hosts and suggests that I. trianguliceps is a competent vector. (+info)
Comparison of PCR assays for detection of the agent of human granulocytic ehrlichiosis, Anaplasma phagocytophilum.
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Human granulocytic ehrlichiosis is an emerging infectious disease in the United States and Europe, and PCR methods have been shown to be effective for the diagnosis of acute infections. Numerous PCR assays and primer sets have been reported in the literature. The analytical sensitivities (limits of detection) of 13 published PCR primer sets were compared using DNA extracted from serial dilutions of Anaplasma phagocytophilum-infected HL-60 cells. The specificity of the assays that were able to detect +info)
Expression of multiple outer membrane protein sequence variants from a single genomic locus of Anaplasma phagocytophilum.
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Anaplasma phagocytophilum is the causative agent of an emerging tick-borne zoonosis in the United States and Europe. The organism causes a febrile illness accompanied by other nonspecific symptoms and can be fatal, especially if treatment is delayed. Persistence of A. phagocytophilum within mammalian reservoir hosts is important for ensuring continued disease transmission. In the related organism Anaplasma marginale, persistence is associated with antigenic variation of the immunoprotective outer membrane protein MSP2. Extensive diversity of MSP2 is achieved by combinatorial gene conversion of a genomic expression site by truncated pseudogenes. The major outer membrane protein of A. phagocytophilum, MSP2(P44), is homologous to MSP2 of A. marginale, has a similar organization of conserved and variable regions, and is also encoded by a multigene family containing some truncated gene copies. This suggests that the two organisms could use similar mechanisms to generate diversity in outer membrane proteins from their small genomes. We define here a genomic expression site for MSP2(P44) in A. phagocytophilum. As in A. marginale, the msp2(p44) gene in this expression site is polymorphic in all populations of organisms we have examined, whether organisms are obtained from in vitro culture in human HL-60 cells, from culture in the tick cell line ISE6, or from infected human blood. Changes in culture conditions were found to favor the growth and predominance of certain msp2(p44) variants. Insertions, deletions, and substitutions in the region of the genomic expression site encoding the central hypervariable region matched sequence polymorphisms in msp2(p44) mRNA. These data suggest that, similarly to A. marginale, A. phagocytophilum uses combinatorial mechanisms to generate a large array of outer membrane protein variants. Such gene polymorphism has profound implications for the design of vaccines, diagnostic tests, and therapy. (+info)
Anaplasma phagocytophilum reduces neutrophil apoptosis in vivo.
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Ovine neutrophils spontaneously underwent apoptosis during culture in vitro, as assessed by morphological changes and exposure of annexin V binding sites on their cell surfaces. The addition of conditioned medium from concanavalin A-treated ovine peripheral blood mononuclear cells (PBMC) could partially protect against this progression into apoptosis, but dexamethasone and sodium butyrate could not. Actinomycin D accelerated the rate at which ovine neutrophils underwent apoptosis. Neutrophils isolated from sheep experimentally infected with Anaplasma phagocytophilum showed significantly delayed apoptosis during culture ex vivo, and the addition of conditioned medium from PBMC to these cells could not delay apoptosis above the protective effects observed after in vivo infection. The ability of neutrophils from A. phagocytophilum-infected sheep to activate a respiratory burst was increased compared to the activity measured in neutrophils from uninfected sheep, but chemotaxis was decreased in neutrophils from infected sheep. These data are the first demonstration that in vivo infection with A. phagocytophilum results in changes in rates of apoptosis of infected immune cells. This may help explain how these bacteria replicate in these normally short-lived cells. (+info)