Emergence of Anaplasma marginale antigenic variants during persistent rickettsemia.
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Anaplasma marginale is an ehrlichial pathogen of cattle, in the order Rickettsiales, that establishes persistent cyclic rickettsemia in the infected host. Within each rickettsemic cycle, A. marginale expressing antigenically variant major surface protein 2 (MSP2) emerge. By cloning 17 full-length msp2 transcripts expressed during cyclic rickettsemia, we determined that emergent variants have a single, central hypervariable region encoding variant B-cell epitopes. The N- and C-terminal regions are highly conserved among the expressed A. marginale variants, and similar sequences define the MSP2 homologues in the agent of human granulocytic ehrlichiosis (HGE). This is in contrast to the MSP2 homologues in ehrlichial genogroup I pathogens, Ehrlichia chaffeensis, Ehrlichia canis, and Cowdria ruminantium, that have multiple hypervariable regions. By defining the variable and conserved regions, we were able to show that the single hypervariable region of A. marginale MSP2 encodes epitopes that are immunogenic and induce variant-specific antibody responses during persistent infection. These findings demonstrate that the MSP2 structural variants that emerge during each cycle of persistent rickettsemia are true antigenic variants, consistent with MSP2 antigenic variation as a mechanism of A. marginale persistence. (+info)
Strain composition of the ehrlichia Anaplasma marginale within persistently infected cattle, a mammalian reservoir for tick transmission.
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Tick-borne ehrlichial pathogens of animals and humans require a mammalian reservoir of infection from which ticks acquire the organism for subsequent transmission. In the present study, we examined the strain structure of Anaplasma marginale, a genogroup II ehrlichial pathogen, in both an acute outbreak and in persistently infected cattle that serve as a reservoir for tick transmission. Using the msp1alpha genotype as a stable strain marker, only a single genotype was detected in a disease outbreak in a previously uninfected herd. In contrast, a diverse set of genotypes was detected in a persistently infected reservoir herd within a region where A. marginale is endemic. Genotypic diversity did not appear to be rapidly generated within an individual animal, because only a single genotype, identical to that of the inoculating strain, was detected at time points up to 2 years after experimental infection, and only a single identical genotype was found in repeat sampling of individual naturally infected cattle. Similarly, only a single genotype, identical to that of the experimentally inoculated St. Maries or South Idaho strain, was identified in the bloodmeal taken by Dermacentor andersoni ticks, in the midgut and salivary glands of the infected ticks, and in the blood of acutely infected cattle following tick transmission. The results show that mammalian reservoirs harbor genetically heterogeneous A. marginale and suggest that different genotypes are maintained by transmission within the reservoir population. (+info)
Sensitivity and specificity of the complement fixation test for detection of cattle persistently infected with Anaplasma marginale.
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The complement fixation (CF) test commonly is used to identify cattle infected with Anaplasma marginale prior to interstate or international movement. Estimates of the accuracy of the CF test in detecting animals persistently infected with A. marginale vary widely. In this study, the sensitivity and specificity of the CF test for detection of carrier animals was determined using serum from 232 cattle previously defined as A. marginale positive or negative by nested polymerase chain reaction methods and hybridization. Considering results from 2 independent laboratories and interpreting a 1:5 suspect reaction as positive, the best estimate of CF test sensitivity was 20%, with a specificity of 98%. Using a 1:10 cutoff, sensitivity decreased to 14% and specificity increased to 99%. Results of this study indicate that the CF test is ineffective for identifying cattle persistently infected with A. marginale and thus is inadequate for anaplasmosis regulatory and surveillance programs. (+info)
Selective in vivo depletion of CD4(+) T lymphocytes with anti-CD4 monoclonal antibody during acute infection of calves with Anaplasma marginale.
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To investigate the in vivo role of CD4(+) T lymphocytes during acute anaplasmosis, thymectomized calves were selectively depleted of CD4(+) T lymphocytes by treatment with anti-CD4 monoclonal antibody (MAb) and were then infected with the Florida strain of Anaplasma marginale in two sequential experiments (experiments 1 and 2). Treatment of thymectomized calves with a total of 5.0 mg of anti-CD4 MAb/kg of body weight during the 1st week followed by 0.3-mg/kg doses administered twice weekly for 7 weeks resulted in significant depletion of CD3(+) CD4(+) and CD4(+) CD45R(+) (naive) T lymphocytes from blood, spleen, and peripheral lymph nodes for the duration of the 8-week study, compared to the results for thymectomized control calves treated with a subclass-matched MAb. All calves became parasitemic and pyretic following experimental infection with A. marginale, and decreases in packed cell volume (PCV) coincided with peak parasitemia. No significant differences in PCV or parasitemia were observed between treatment groups. Thymectomized calves treated with anti-CD4 MAb were able to mount an anti-A. marginale antibody response, although in experiment 2, anti-CD4 MAb-treated calves had four- to sixfold lower immunoglobulin G1 (IgG1) and no detectable IgG2 anti-A. marginale major surface protein 2-specific antibody titers compared to thymectomized control calves treated with a subclass-matched MAb. At the level of CD4(+)-T-lymphocyte depletion achieved and experimental anaplasmosis induced, thymectomized anti-CD4 MAb-treated calves were able to control acute anaplasmosis. This was in contrast to the prediction that significant depletion of CD4(+) T lymphocytes would abrogate resistance to acute infection. (+info)
Seroprevalence of antibodies that react with Anaplasma phagocytophila, the agent of human granulocytic ehrlichiosis, in different populations in Westchester County, New York.
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We determined the frequencies of antibodies to Anaplasma phagocytophila, the agent of human granulocytic ehrlichiosis (HGE), in different groups of adults and children from Westchester County, New York. The groups included 159 adult blood donors and 215 children who were seronegative for Borrelia burgdorferi antibodies, 118 adult patients and 57 children who were seropositive for B. burgdorferi antibodies, and 42 adult patients with culture-confirmed erythema migrans. Eighteen (11.3%) of the blood donors and 11 (5.1%) of the B. burgdorferi-seronegative children were found to have A. phagocytophila antibodies by indirect immunofluorescent-antibody assay (IFA). Nine of 42 (21.4%) patients with culture-confirmed erythema migrans tested at the baseline visit, 42 of 118 (35.6%) adults, and 3 of 57 (5.3%) children whose sera were reactive for B. burgdorferi antibodies also tested positive for A. phagocytophila antibodies. The geometric mean titer ranged from 219 to 315 for all groups, and the differences in titers among the groups were not statistically significant. Only one-third of the healthy blood donors reactive by IFA were confirmed to be positive by immunoblotting. We conclude that a significant proportion of adults and children without clinical evidence of HGE will test positive for A. phagocytophila antibodies when the conventional cutoff titer of 80 is used in the IFA. This information must be considered in interpretation of test results. (+info)
Superoxide anion production during Anaplasma phagocytophila infection.
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Anaplasma phagocytophila persists within neutrophils and prevents the respiratory burst by inhibiting gp91(phox). Mutations in gp91(phox) result in chronic granulomatous disease (CGD), which is diagnosed by use of the nitroblue tetrazolium (NBT) and Fc-Oxyburst assays that examine whether cells produce O2-. This study assessed whether the NBT and Fc-Oxyburst assays could detect a respiratory burst during A. phagocytophila infection. O2- production was inhibited in HL-60 cells and neutrophils infected with A. phagocytophila. In a mouse model of A. phagocytophila infection, 15%+/-4% (mean+/-SD) of polymorphonuclear leukocytes from infected mice had an ineffective respiratory burst compared with 1%+/-1% (mean+/-SD) of the neutrophils from uninfected animals. A population of neutrophils that did not produce O2- was also detected in 2 patients with A. phagocytophila infection. These data demonstrate respiratory burst inhibition by A. phagocytophila in vivo and on an individual cell basis by use of assays designed to evaluate CGD. (+info)
Analysis of sequences and loci of p44 homologs expressed by Anaplasma phagocytophila in acutely infected patients.
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Anaplasma phagocytophila is an obligatory intragranulocytic bacterium that causes human granulocytic ehrlichiosis. Immunodominant 44-kDa outer membrane proteins of A. phagocytophila are encoded by a p44 multigene family. In the present study, expression profiles of p44 genes in the blood of acutely infected patients in the year 2000 were characterized. A single p44 gene was predominantly expressed in peripheral blood leukocytes from one patient, while up to 17 different p44 genes were transcribed without a single majority in the other two patients. The cDNA sequences of the central hypervariable region of several p44 genes were identical among the isolates from the three patients and a 1995 A. phagocytophila isolate. A. phagocytophila was isolated by cell culture from all of the three 2000 patients. Genomic Southern blot analysis of the three 2000 and two 1995 A. phagocytophila isolates with probes specific to the most dominant p44 transcript in each patient showed that the p44 loci in the A. phagocytophila genome were conserved. Analysis of the predicted amino acid sequences of 43 different p44 genes including 19 new sequences found in the present study, revealed that five amino acids were absolutely conserved. The hypervariable region was subdivided into five domains, including three extremely hypervariable central domains. These results suggest that variations in the sequences of p44 are not random but are restricted. Furthermore, several p44 genes are not hypermutatable in nature, based on the conservation of gene sequences and loci among isolates obtained 5 years apart. (+info)
Serologic and molecular detection of Ehrlichia chaffeensis and Anaplasma phagocytophila (human granulocytic ehrlichiosis agent) in Korean patients.
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Sera from 491 Korean patients with acute febrile diseases were tested for Ehrlichia chaffeensis and Anaplasma phagocytophila antibodies by indirect immunofluorescence assay (IFA), Western blotting, and TaqMan real-time PCR. Overall, 0.4% of sera reacted with E. chaffeensis, and 1.8% reacted with A. phagocytophila in IFAs. This is the first report of detection of antibodies to A. phagocytophila and E. chaffeensis in Korea and suggests the presence of A. phagocytophila and E. chaffeensis or antigenically similar species. (+info)