Specific expression of Anaplasma marginale major surface protein 2 salivary gland variants occurs in the midgut and is an early event during tick transmission.
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Infectivity of Anaplasma spp. develops when infected ticks feed on a mammalian host (transmission feed). Specific Anaplasma marginale major surface protein 2 (MSP2) variants are selected for within the tick and are expressed within the salivary glands. The aims of this study were to determine when and where MSP2 variant selection occurs in the tick, how MSP2 expression is regulated in salivary glands of transmission-feeding ticks, and whether the number of A. marginale organisms per salivary gland is significantly increased during transmission feeding. The South Idaho strain of A. marginale was used, as MSP2 expression is restricted to two variants, SGV1 and SGV2, in Dermacentor andersoni. Using Western blot, real-time PCR, and DNA sequencing analyses it was shown that restriction and expression of MSP2 occurs early in the midgut within the first 48 h of the blood meal, when ticks acquire infection. A. marginale is present in the tick salivary glands before transmission feeding is initiated, but the msp2 mRNA and MSP2 protein levels per A. marginale organism increase only minimally and transiently in salivary glands of transmission-feeding ticks compared to that of unfed ticks. A. marginale numbers per tick increase gradually in salivary glands of both transmission-fed and unfed ticks. It is concluded that MSP2 variant selection is an early event in the tick and that MSP2 variants SGV1 and SGV2 are expressed both in the midgut and salivary glands. While MSP2 may be required for infectivity, there is no strict temporal correlation between MSP2 expression and the development of infectivity. (+info)
Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila.
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The genera Anaplasma, Ehrlichia, Cowdria, Neorickettsia and Wolbachia encompass a group of obligate intracellular bacteria that reside in vacuoles of eukaryotic cells and were previously placed in taxa based upon morphological, ecological, epidemiological and clinical characteristics. Recent genetic analyses of 16S rRNA genes, groESL and surface protein genes have indicated that the existing taxa designations are flawed. All 16S rRNA gene and groESL sequences deposited in GenBank prior to 2000 and selected sequences deposited thereafter were aligned and phylogenetic trees and bootstrap values were calculated using the neighbour-joining method and compared with trees generated with maximum-probability, maximum-likelihood, majority-rule consensus and parsimony methods. Supported by bootstrap probabilities of at least 54%, 16S rRNA gene comparisons consistently clustered to yield four distinct clades characterized roughly as Anaplasma (including the Ehrlichia phagocytophila group, Ehrlichia platys and Ehrlichia bovis) with a minimum of 96.1% similarity, Ehrlichia (including Cowdria ruminantium) with a minimum of 97.7% similarity, Wolbachia with a minimum of 95.6% similarity and Neorickettsia (including Ehrlichia sennetsu and Ehrlichia risticii) with a minimum of 94.9% similarity. Maximum similarity between clades ranged from 87.1 to 94.9%. Insufficient differences existed among E. phagocytophila, Ehrlichia equi and the human granulocytic ehrlichiosis (HGE) agent to support separate species designations, and this group was at least 98.2% similar to any Anaplasma species. These 16S rRNA gene analyses are strongly supported by similar groESL clades, as well as biological and antigenic characteristics. It is proposed that all members of the tribes Ehrlichieae and Wolbachieae be transferred to the family Anaplasmataceae and that the tribe structure of the family Rickettsiaceae be eliminated. The genus Anaplasma should be emended to include Anaplasma (Ehrlichia) phagocytophila comb. nov. (which also encompasses the former E. equi and the HGE agent), Anaplasma (Ehrlichia) bovis comb. nov. and Anaplasma (Ehrlichia) platys comb. nov., the genus Ehrlichia should be emended to include Ehrlichia (Cowdria) ruminantium comb. nov. and the genus Neorickettsia should be emended to include Neorickettsia (Ehrlichia) risticii comb. nov. and Neorickettsia (Ehrlichia) sennetsu comb. nov. (+info)
Expression of major surface protein 2 variants with conserved T-cell epitopes in Anaplasma centrale vaccinates.
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Major surface protein 2 (MSP-2), identified as a protection-inducing immunogen against Anaplasma marginale challenge, is an immunodominant outer membrane protein with orthologues in all examined Anaplasma species. Although immunization with live Anaplasma centrale has long been used to induce protection against acute disease upon challenge with virulent A. marginale, its MSP-2 structure and whether MSP-2 variants are generated during persistence of the vaccine strain was unknown. In this study, we showed that the A. centrale vaccine strain persisted for a minimum of 4 years postvaccination and generated sequential MSP-2 variants. Comparison of amino acid sequences encoded by A. centrale msp-2 transcripts from the initial postimmunization period and from sequential time points during persistence of the vaccine strain revealed a central hypervariable domain flanked by conserved amino and carboxy-terminal regions. This structure corresponded to that shown in A. marginale MSP-2, where the central hypervariable region encodes variant B-cell epitopes in the extracellular domain and the flanking transmembrane domains are rich in CD4(+)-T-cell epitopes. Importantly, at least four CD4(+)-T-cell epitopes are conserved between the two species, a finding consistent with A. marginale challenge triggering a recall response of CD4(+) T cells induced by A. centrale vaccination. The genomic arrangement is conserved between A. centrale and A. marginale with multiple msp-2 pseudogenes and a single operon-linked expression site for the full-length msp-2. This conservation of both genomic structure for generating MSP-2 variants and the CD4(+)-T-cell epitopes between these two genetically distinct Anaplasma species indicates that they present a similar repertoire of MSP-2 epitopes to the immune system and that this similarity may be responsible for all or part of the A. centrale vaccine efficacy. (+info)
Transcript heterogeneity of the p44 multigene family in a human granulocytic ehrlichiosis agent transmitted by ticks.
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Human granulocytic ehrlichiosis (HGE) is an emerging tick-borne zoonosis caused by a strain of Anaplasma phagocytophila called the HGE agent, an obligatory intracellular bacterium. The agent expresses immunodominant 44-kDa outer membrane proteins (P44s) encoded by a multigene family. The present study established an experimental process for transmission of the HGE agent from infected mice (a reservoir model) to nymphal Ixodes scapularis ticks (a biological vector) and subsequently to horses (a patient model) by the adult infected ticks. Overall, a total of 20 different p44 transcripts were detected in the mammals, ticks, and cell cultures. Among them, a transcript from a p44-18 gene was major at acute stage in mice and horses but minor in ticks. Both mRNA and protein produced from the p44-18 gene were detected in the HGE agent cultivated in HL-60 cells at 37 degrees C, but their expression levels decreased in the organisms cultivated at 24 degrees C, suggesting that temperature is one of the factors that influence the expression of members of the p44 multigene family. Several additional p44 transcripts that were not detected in the mammals at the acute stage of infection were detected in ticks. Phylogenetic analysis of the 20 different p44 transcripts revealed that the major transcripts found in mammals and ticks were distinct, suggesting a difference in surface properties between populations of the HGE agent in different host environments. The present study provides new information for understanding the role of the p44 multigene family in transmission of the HGE agent between mammals and ticks. (+info)
Genetic variability and stability of Anaplasma phagocytophila msp2 (p44).
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Anaplasma (Ehrlichia) phagocytophila's major immunodominant surface protein antigen, Msp2 (P44, 44-kDa antigen), is encoded by a family of paralogous genes characterized by conserved sequences flanking a hypervariable region. The antigenic profiles of most strains of A. phagocytophila are different, and the differences are principally related to Msp2 expression. To date, multiple unique msp2 gene paralogs have been found in A. phagocytophila isolates, but the overall number in the genome of a single strain is not yet known. Changes in msp2 expression may be related to antigenic variability; thus, we examined the minimal complement of msp2 genes or pseudogenes in two strains of A. phagocytophila and the number of transcriptionally active msp2 gene paralogs during low-passage, steady-state, in vitro propagation. Of 15 BDS strain clones, 1 had a hypervariable region identical to the region in a clone obtained from a BDS strain genomic library previously prepared from organisms after only two horse passages. When 124 Webster strain clones were examined, 18 unique hypervariable regions were identified. Of 64 Webster strain cDNA clones, 56 (87.5%) were derived from a single gene, and transcripts from six additional msp2 genes were also identified. The sequences of several hypervariable regions that were > or = 97% similar to regions present in other strains were identified by performing a BLAST analysis of sequences deposited in the GenBank database. These findings suggest that antigenic variability results from transcription of one or a few of the multiple paralogs and not from genetic instability that results in random accumulated mutations, although the possibility that gene recombination plays a role cannot be eliminated. The predominant Msp2 pattern in vitro is determined by transcription from a single gene. (+info)
Effects of Anaplasma phagocytophila on NADPH oxidase components in human neutrophils and HL-60 cells.
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The human granulocytic ehrlichiosis agent, Anaplasma phagocytophila, resides and multiplies exclusively in cytoplasmic vacuoles of granulocytes. A. phagocytophila rapidly inhibits the superoxide anion (O(2)(-)) generation by human neutrophils in response to various stimuli. To determine the inhibitory mechanism, the influence of A. phagocytophila on protein levels and localization of components of the NADPH oxidase were examined. A. phagocytophila decreased levels of p22(phox), but not gp91(phox), p47(phox), p67(phox), or P40(phox) reactive with each component-specific antibody in human peripheral blood neutrophils and HL-60 cells. Double immunofluorescence labeling revealed that p47(phox), p67(phox), Rac2, and p22(phox) did not colocalize with A. phagocytophila inclusions in neutrophils or HL-60 cells, and p22(phox) levels were also reduced. A. phagocytophila did not prevent either membrane translocation of cytoplasmic p47(phox) and p67(phox) or phosphorylation of p47(phox) upon stimulation by phorbol myristate acetate. The inhibitory signals for O(2)(-) generation was independent of several signals required for A. phagocytophila internalization. These results suggest that rapid alteration in p22(phox) induced by binding of A. phagocytophila to neutrophils is involved in the inhibition of O(2)(-) generation. Absence of colocalization of NADPH oxidase components with the inclusion further protects A. phagocytophila from oxidative damage. (+info)
Characterization and transcriptional analysis of gene clusters for a type IV secretion machinery in human granulocytic and monocytic ehrlichiosis agents.
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Anaplasma (Ehrlichia) phagocytophila and Ehrlichia chaffeensis, the etiologic agents of granulocytic and monocytic ehrlichioses, respectively, are obligatory intracellular bacteria that cause febrile systemic illness in humans. We identified and characterized clusters of genes for a type IV secretion machinery in these two bacteria, and analyzed their gene expression in cell culture and mammalian hosts. Eight virB and virD genes were found in each bacterial genome, and all of the genes were transcribed in cell culture. Although the gene order and orientation were similar to those found in other bacteria, the eight virB and virD genes were clustered at two separate loci in each genome. Five of the genes (virB8, virB9, virB10, virB11, and virD4) were located downstream from a ribA gene. These five genes in both A. phagocytophila and E. chaffeensis were polycistronically transcribed and controlled through at least two tandem promoters located upstream of the virB8 gene in human leukemia cell lines. The virB9 gene of A. phagocytophila was transcriptionally active in peripheral blood leukocytes from human ehrlichiosis patients and experimentally infected animals. Three of the remaining genes (virB3, virB4, and virB6) of both A. phagocytophila and E. chaffeensis were arranged downstream from a sodB gene and cotranscribed with the sodB gene through one or more sodB promoters in human leukocytes. This suggests that transcription of the three virB genes in these two Anaplasma and Ehrlichia spp. is regulated by factors that influence the sodB gene expression. This unique regulation of gene expression for the type IV secretion system may be associated with intracellular survival and replication of Anaplasma and Ehrlichia spp. in granulocytes or monocytes. (+info)
Infection of tick cells and bovine erythrocytes with one genotype of the intracellular ehrlichia Anaplasma marginale excludes infection with other genotypes.
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Anaplasma marginale, a tick-borne rickettsial pathogen of cattle, is endemic in several areas of the United States. Many geographic isolates of A. marginale that occur in the United States are characterized by the major surface protein 1a, which varies in sequence and molecular weight due to different numbers of tandem repeats of 28 or 29 amino acids. Recent studies (G. H. Palmer, F. R. Rurangirwa, and T. F. McElwain, J. Clin. Microbiol. 39:631-635, 2001) of an A. marginale-infected herd of cattle in an area of endemicity demonstrated that multiple msp1alpha genotypes were present but that only one genotype was found per individual bovine. These findings suggested that infection of cattle with other genotypes was excluded. The present study was undertaken to confirm the phenomenon of infection exclusion of A. marginale genotypes in infected bovine erythrocytes and cultured tick cells. Two tick-transmissible isolates of A. marginale, one from Virginia and one from Oklahoma, were used for these studies. In two separate trials, cattle inoculated with equal doses of the two isolates developed infection with only one genotype. Tick cell cultures inoculated with equal doses of the two isolates became infected with only the Virginia isolate of A. marginale. When cultures were inoculated with different ratios of the Oklahoma and Virginia isolates of A. marginale, the isolate inoculated in the higher ratio became established and excluded infection with the other. When cultures with established infections of one isolate were subsequently infected with the other, only the established isolate was detected. We documented infection exclusion during initial infection in cell culture by labeling each isolate with a different fluorescent dye. After 2 days in culture, only a single isolate was detected per cell by fluorescence microscopy. Finally, when Anaplasma ovis infections were established in cultures that were subsequently inoculated with the Virginia or Oklahoma isolate of A. marginale, A. marginale infection was excluded. These studies confirm that infection exclusion occurs with A. marginale in bovine erythrocytes and tick cells, resulting in the establishment of only one genotype, and appears to be the first report of infection exclusion for Anaplasma and Ehrlichia species. (+info)