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PubMed2004

Anaplasma phagocytophilum utilizes multiple host evasion mechanisms to thwart NADPH oxidase-mediated killing during neutrophil infection.

Anaplasma phagocytophilum, the etiologic agent of human anaplasmosis, is a bacterial pathogen that specifically colonizes neutrophils. Neutrophils utilize the NADPH oxidase complex to generate superoxide (O(2)(-)) and initiate oxidative killing of microorganisms. A. phagocytophilum's unique tropism for neutrophils, however, indicates that it subverts and/or avoids oxidative killing. We therefore examined the effects of A. phagocytophilum infection on neutrophil NADPH oxidase assembly and reactive oxygen species (ROS) production. Following neutrophil binding, Anaplasma invasion requires at least 240 min. During its prolonged association with the neutrophil plasma membrane, A. phagocytophilum stimulates NADPH oxidase assembly, as indicated by increased cytochrome b(558) mobilization to the membrane, as well as colocalization of Rac and p22(phox). This initial stimulation taxes the host neutrophil's finite oxidase reserves, as demonstrated by time- and bacterial-dose-dependent decreases in secondary activation by N-formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol myristate acetate (PMA). This stimulation is modest, however, and does not diminish oxidase stores to nearly the extent that Escherichia coli, serum-opsonized zymosan, FMLP, or PMA do. Despite the apparent activation of NADPH oxidase, no change in ROS-dependent chemiluminescence is observed upon the addition of A. phagocytophilum to neutrophils, indicating that the bacterium may scavenge exogenous O(2)(-). Indeed, A. phagocytophilum rapidly detoxifies O(2)(-) in a cell-free system. Once internalized, the bacterium resides within a protective vacuole that excludes p22(phox) and gp91(phox). Thus, A. phagocytophilum employs at least two strategies to protect itself from neutrophil NADPH oxidase-mediated killing.

Anaplasma phagocytophilum↗
PubMed1974

In vitro uptake of 14C-labeled amino acids by preparations of partially purified Anaplasma marginale bodies.

Studies on the incorporation of three (14)C-labeled amino acids into preparations of partially purified Anaplasma marginale were conducted. Relatively high levels of uptake of radioactive isoleucine, glycine, and methionine were detected in both trichloroacetic acid-soluble and -insoluble fractions of these preparations, indicating that the parasite is capable of protein synthesis outside its erythrocytic environment. When erythrocytes (red blood cells) from infected calves were incubated with [(14)C]methionine and Anaplasma bodies were subsequently purified, it was found that some of the label detected was associated with the marginal bodies.

Amino Acids↗
PubMed1987

Detection of antibodies to Anaplasma marginale by an improved enzyme-linked immunosorbent assay with sodium dodecyl sulfate-disrupted antigen.

Sensitivities of two enzyme-linked immunosorbent assays (ELISAs) with particulate and sodium dodecyl sulfate (SDS)-disrupted Anaplasma marginale antigen were compared. The quotient of positive reference sera divided by the absorbance quotient of a negative reference serum at identical dilution was termed the signal-to-noise ratio. Optimal signal-to-noise ratios were dependent on both pretreatment of antigen and antigen concentration. SDS disruption of anaplasmal antigen resulted in a markedly improved signal-to-noise ratio of ELISA compared with ELISA with untreated antigen at identical antigen and serum dilutions. This represented higher sensitivity and lower background absorbance of the ELISA with disrupted antigen. SDS-disrupted A. marginale antigen was standardized by protein determination, and antigen, as well as precoated microtiter wells, was stored frozen without apparent loss of antigenic properties. ELISA results were in agreement with results of positive and negative control sera tested by the complement fixation test or by light microscopy Anaplasma diagnosis in Giemsa-stained blood films.

Anaplasma↗
PubMed1996

Evaluation of Anaplasma marginale major surface protein 3 (MSP3) as a diagnostic test antigen.

An immunodominant surface protein, major surface protein 3 (MSP3), has been proposed as an antigen suitable for use in the diagnosis of bovine anaplasmosis. We further characterized MSP3 to examine its potential as a test antigen for the serological diagnosis of carrier cattle. The specificity of this antigen in detecting infected cattle as well as the conservation of MSP3 between strains of Anaplasma marginale was evaluated by using immunoblots of A. marginale proteins separated by one- and two-dimensional polyacrylamide gel electrophoreses. Immune sera from animals infected with Anaplasma ovis, Ehrlichia risticii, and Ehrlichia ewingii reacted with the MSP3 antigen of A. marginale. One-dimensional gel electrophoresis of A. marginale proteins demonstrated size polymorphism of MSP3 between different geographic isolates. Two-dimensional gel electrophoresis revealed at least three different antigens migrating at the 86-kDa molecular size, and sera from animals infected with different strains of A. marginale reacted with different 86-kDa antigens. These results indicate that MSP3 may share cross-reactive epitopes with antigens found in A. ovis and some Ehrlichia spp. In addition, MSP3 is not conserved between different isolates of A. marginale, and at least in the isolate from Florida, what was previously identified as MSP3 is actually a group of three or more 86-kDa antigens with different isoelectric points. The cross-reactivity of MSP3 with some Ehrlichia spp., the variability of MSP3 between isolates, and the multiple 86-kDa antigens recognized by various sera suggest that MSP3 is not a suitable candidate for use as a recombinant test antigen.

Anaplasma↗
PubMed2003

Comparison of PCR assays for detection of the agent of human granulocytic ehrlichiosis, Anaplasma phagocytophilum.

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 <or=2.5 infected cells was tested by the use of template DNA extracted from Ehrlichia chaffeensis, Rickettsia rickettsii, and Bartonella henselae. The assays with the lowest limits of detection were shown to be a nested assay that amplifies the 16S rRNA gene (primer pairs ge3a-ge10 [primary] and ge9-ge3 [nested]; detects 0.25 infected cell), a direct assay that amplifies the major surface protein gene msp2 (primer pair msp2-3f-msp2-3r; detects 0.25 infected cell), and a direct assay that amplifies the 16S rRNA gene (primer pair ehr521-ehr790; detects 0.25 infected cell). The specificity and limit of detection of the MSP2 and 16S rRNA direct assays were further tested by use of A. phagocytophilum template DNA from both North America and Europe and from human, tick, white-footed mouse, equine, deer, bovine, and wood rat samples and of template DNA from closely related species (Anaplasma marginale, the white-tailed deer agent, and additional E. chaffeensis-positive samples). Three manufacturers' PCR kits were tested and showed distinct variations in the limit of detection, specificity, and nonspecific background amplification. The importance of these results for the molecular diagnosis of human granulocytic ehrlichiosis is discussed.

Anaplasma phagocytophilum↗
PubMed2003

Enzootic transmission of Anaplasma bovis in Nantucket cottontail rabbits.

Serological studies of cottontail rabbits sampled from Nantucket Island, Mass., have suggested exposure to at least two ehrlichiae. The agent of human granulocytic ehrlichiosis (Anaplasma phagocytophilum) is intensely enzootic in rabbits there, but the identity of the other ehrlichial infection remains undescribed. We sampled rabbits over five transmission seasons and tested their blood and tissues for evidence of infection using PCR targeting an Ehrlichia genus-wide 16S rDNA target. Sequence analysis of positive amplicons revealed the presence of Anaplasma bovis, an agent not known to be present in North America. The average annual prevalence of A. bovis within rabbits, as determined by PCR of blood samples, was 18%. Haemaphysalis leporispalustris appears to serve as vector. The public health (human or veterinary) significance of this finding remains speculative.

Anaplasma↗
PubMed2004

Multiplex real-time PCR for detection of anaplasma phagocytophilum and Borrelia burgdorferi.

A multiplex real-time PCR assay was developed for the simultaneous detection of Anaplasma phagocytophilum and Borrelia burgdorferi. The assay was tested on various Anaplasma, Borrelia, Erhlichia, and Rickettsia species, as well as on Bartonella henselae and Escherichia coli, and the assay was found to be highly specific for A. phagocytophilum and the Borrelia species tested (B. burgdorferi, B. parkeri, B. andersonii, and B. bissettii). The analytical sensitivity of the assay is comparable to that of previously described nested PCR assays (A. phagocytophilum, 16S rRNA; B. burgdorferi, fla gene), amplifying the equivalent of one-eighth of an A. phagocytophilum-infected cell and 50 borrelia spirochetes. The dynamic range of the assay for both A. phagocytophilum and B. burgdorferi was >/=4 logs of magnitude. Purified DNA from A. phagocytophilum and B. burgdorferi was spiked into DNA extracted from uninfected ticks and from negative control mouse and human bloods, and these background DNAs were shown to have no significant effect on sensitivity or specificity of the assay. The assay was tested on field-collected Ixodes scapularis ticks and shown to have 100% concordance compared to previously described non-probe-based PCR assays. To our knowledge, this is the first report of a real-time multiplex PCR assay that can be used for the simultaneous and rapid screening of samples for A. phagocytophilum and Borrelia species, two of the most common tick-borne infectious agents in the United States.

Anaplasma phagocytophilum↗
PubMed2005

Sequence analysis of the msp4 gene of Anaplasma phagocytophilum strains.

The causative agent of human granulocytic ehrlichiosis was recently reclassified as Anaplasma phagocytophilum, unifying previously described bacteria that cause disease in humans, horses, dogs, and ruminants. For the characterization of genetic heterogeneity in this species, the homologue of Anaplasma marginale major surface protein 4 gene (msp4) was identified, and the coding region was PCR amplified and sequenced from a variety of sources, including 50 samples from the United States, Germany, Poland, Norway, Italy, and Switzerland and 4 samples of A. phagocytophilum-like organisms obtained from white-tailed deer in the United States. Sequence variation between strains of A. phagocytophilum (90 to 100% identity at the nucleotide level and 92 to 100% similarity at the protein level) was higher than in A. marginale. Phylogenetic analyses of msp4 sequences did not provide phylogeographic information but did differentiate strains of A. phagocytophilum obtained from ruminants from those obtained from humans, dogs, and horses. The sequence analysis of the recently discovered A. phagocytophilum msp2 gene corroborated these results. The results reported here suggest that although A. phagocytophilum-like organisms from white-tailed deer may be closely related to A. phagocytophilum, they could be more diverse. These results suggest that A. phagocytophilum strains from ruminants could share some common characteristics, including reservoirs and pathogenicity, which may be different from strains that infect humans.

Amino Acid Sequence↗
PubMed2004

Anaplasma phagocytophilum: an emerging tick-borne pathogen in Hungary and Central Eastern Europe.

Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila, Ehrlichia equi and Anaplasma phagocytophila) is the causative agent of granulocytic ehrlichiosis (anaplasmosis) in humans, horses, sheep, cattle, dogs and cats. In the present study, 452 European sheep ticks (Ixodes ricinus) collected from 100 red foxes (Vulpes vulpes) in Hungary were tested for the pathogen, as 112 pools each containing five or fewer ticks from one fox. Six of the pools, representing ticks from six different foxes, were found infected in the PCR-based test employed. This is the first time that A. phagocytophilum has been reported in Hungary. A summary of the information available from Central Eastern Europe on the prevalence of A. phagocytophilum in ticks, its seroprevalence in humans, and the number of human cases of granulocytic ehrlichiosis known in the region is presented.

Anaplasma phagocytophilum↗
PubMed2004

Immunization of bovines using a DNA vaccine (pcDNA3.1/MSP1b) prepared from the Jaboticabal strain of Anaplasma marginale.

Anaplasma is a tick-borne ehrlichial pathogen of cattle that causes the disease, anaplasmosis. In the present study, a total of 11 Anaplasma marginale seronegative calves were assigned into two groups: one immunized (G1, n = 6) and one nonimmunized-control (G2, n = 5). Six calves were immunized by using a DNA vaccine containing the gene of a major surface protein, MSP1b, encoded by the plasmid identified as pcDNA3.1/MSP1b. Calves received three intramuscular inoculations of 100 microg of pcDNA3.1/MSP1b at a 20-day interval. The control group received buffer phosphate at the same schedule as the experimental group. The immune response elicited by immunization with pcDNA3.1/MSP1b was evaluated in mice and calves. Twenty days following initial immunization, specific serum antibody from four BALB/c mice bound MSP1b in immunoblots. Sixty days after the last immunization, all calves were challenged with cryopreserved A. marginale at a dose of 10(4) parasites/mL/animal by intravenous injection. Results of packed cell volume (PCV) and detection of infected erythrocytes in all experimental groups revealed that the decrease of PCV and detection of infected erythrocytes occurred at 28 to 42 days after challenge. Mean temperature values did not increase over 39.85 degrees C. Antibodies developed by immunized bovines from G2 were detected 14 days after challenge. MSP1b was characterized during the immunization period and MSP2 was the most predominant polypeptide at the challenge period. DNA of A. marginale was detected in all groups just after challenge by nested PCR assay. It can be concluded that all immunized bovines were partially protected against homologous challenge.

Anaplasma marginale↗
PubMed2005

Platelet dysfunction after association with Anaplasma phagocytophilum in vitro.

Granulocytic anaplasmosis is a tick-borne, zoonotic disease caused by an obligate intracellular granulocytotropic bacterium, Anaplasma phagocytophilum. A. phagocytophilum and other Anaplasma and Ehrlichia spp. have been variably implicated with an associated hematologic alteration, platelet dysfunction, that can result in potentially serious clinical sequelae including hemorrhage. The objective of this work was to define an in vitro model to assess alterations in platelet function associated with A. phagocytophilum.

Anaplasma phagocytophilum↗
PubMed2006

Detection of members of the genera Rickettsia, Anaplasma, and Ehrlichia in ticks collected in the Asiatic part of Russia.

A total of 395 adult ixodid ticks from three genera (Dermacentor, Haemaphysalis, and Ixodes) collected from the Urals to the Far East of Russia were tested by PCR and sequencing for the presence of spotted fever rickettsiae, anaplasmae, and ehrlichiae. Four, pathogens recognized in humans were detected in ticks: Rickettsia sibirica, R. heilongjiangensis, R. helvetica, and Anaplasma phagocytophilum. In addition, rickettsiae and ehrlichiae of unknown pathogenicity were detected, including Rickettsia sp. RpA4, Rickettsia sp. DnS14, Rickettsia sp. DnS28, "Candidatus R. tarasevichiae," a rickettsia closely related to R. helvetica, A. bovis, Ehrlichia muris, "Ehrlichia-like" "Schotti variant," and bacterium "Montezuma." Our findings indicated the distribution of rickettsiae and ehrlichiae in hard ticks in Russia.

Anaplasma↗
PubMed2006

Multiplex detection of Ehrlichia and Anaplasma pathogens in vertebrate and tick hosts by real-time RT-PCR.

Tick-borne rickettsial infections are responsible for many emerging diseases in humans and several vertebrates. These include human infections with Ehrlichia chaffeensis, Ehrlichia ewingii and Anaplasma phagocytophilum. As single or co-infections can result from a tick bite, the availability of a rapid, multiplex molecular test will be valuable for timely diagnosis and treatment. We recently described a muliplex-molecular test that can detect single or co-infections with up to five Ehrlichia and Anaplasma species. We reported that the test has the sensitivity to identify single infections in the canine host with E. chaffeensis, E. canis, E. ewingii, A. phagocytophilum, and A. platys and co-infection with E. canis and A. platys. In this study, ticks were collected from different parts of the state of Kansas during summer months of the year 2003 and tested for the presence of infection using the molecular test. The analysis revealed a minimum of 3.66% of the ticks to be positive for either E. chaffeensis or E. ewingii in A. americanum and Dermacenter species. This assay will be valuable in monitoring infections in dogs and ticks, and with minor modifications it can be used for diagnosing infections in people and other vertebrates.

Anaplasma↗
PubMed2004

Detection of Anaplasma phagocytophilum DNA in Ixodes ticks (Acari: Ixodidae) from Madeira Island and Setubal District, mainland Portugal.

A total of 278 Ixodes ticks, collected from Madeira Island and Setubal District, mainland Portugal, were examined by polymerase chain reaction (PCR) for the presence of Anaplasma phagocytophilum. Six (4%) of 142 Ixodes ricinus nymphs collected in Madeira Island and 1 nymph and 1 male (2%) of 93 I. ventalloi collected in Setubal District tested positive for A. phagocytophilum msp2 genes or rrs. Infection was not detected among 43 I. ricinus on mainland Portugal. All PCR products were confirmed by nucleotide sequencing to be identical or to be most closely related to A. phagocytophilum. To our knowledge, this is the first evidence of A. phagocytophilum in ticks from Setubal District, mainland Portugal, and the first documentation of Anaplasma infection in I. ventalloi. Moreover, these findings confirm the persistence of A. phagocytophilum in Madeira Island's I. ricinus.

Anaplasma phagocytophilum↗
PubMed2003

The hypervariable region of Anaplasma marginale major surface protein 2 (MSP2) contains multiple immunodominant CD4+ T lymphocyte epitopes that elicit variant-specific proliferative and IFN-gamma responses in MSP2 vaccinates.

Major surface protein 2 (MSP2) is an immunodominant outer membrane protein of Anaplasma marginale and Anaplasma phagocytophilum pathogens that cause bovine anaplasmosis and human granulocytic ehrlichiosis, respectively. MSP2 has a central hypervariable region (HVR) flanked by highly conserved amino and carboxyl termini. During A. marginale infection, dynamic and extensive amino acid sequence variation in MSP2 occurs through recombination of msp2 pseudogenes into the msp2 expression site, followed by sequential segmental gene conversions to generate additional variants. We hypothesized that MSP2 variation leads to significant changes in Th cell recognition of epitopes in the HVR. T cell epitopes were mapped using T cells from native MSP2-immunized cattle and overlapping peptides spanning the most abundant of five different MSP2 HVRs in the immunogen. Several epitopes elicited potent effector/memory Th cell proliferative and IFN-gamma responses, including those in three discreet blocks of sequence that undergo segmental gene conversion. Th cell clones specific for an epitope in the block 1 region of the predominant MSP2 variant type failed to respond to naturally occurring variants. However, some of these variants were recognized by oligoclonal T cell lines from MSP2 vaccinates, indicating that the variant sequences contain immunogenic CD4(+) T cell epitopes. In competition/antagonism assays, the nonstimulatory variants were not inhibitory for CD4(+) T cells specific for the agonist peptide. Dynamic amino acid sequence variation in MSP2 results in escape from recognition by some effector/memory MSP2-specific Th cells. Antigenic variation in MSP2 Th cell and B cell epitopes may contribute to immune evasion that allows long-term persistence of A. marginale in the mammalian reservoir.

Adjuvants, Immunologic↗
PubMed2005

Rapid and long-term disappearance of CD4+ T lymphocyte responses specific for Anaplasma marginale major surface protein-2 (MSP2) in MSP2 vaccinates following challenge with live A. marginale.

In humans and ruminants infected with Anaplasma, the major surface protein 2 (MSP2) is immunodominant. Numerous CD4(+) T cell epitopes in the hypervariable and conserved regions of MSP2 contribute to this immunodominance. Antigenic variation in MSP2 occurs throughout acute and persistent infection, and sequentially emerging variants are thought to be controlled by variant-specific Ab. This study tested the hypothesis that challenge of cattle with Anaplasma marginale expressing MSP2 variants to which the animals had been immunized, would stimulate variant epitope-specific recall CD4(+) T cell and IgG responses and organism clearance. MSP2-specific T lymphocyte responses, determined by IFN-gamma ELISPOT and proliferation assays, were strong before and for 3 wk postchallenge. Surprisingly, these responses became undetectable by the peak of rickettsemia, composed predominantly of organisms expressing the same MSP2 variants used for immunization. Immune responsiveness remained insignificant during subsequent persistent A. marginale infection up to 1 year. The suppressed response was specific for A. marginale, as responses to Clostridium vaccine Ag were consistently observed. CD4(+)CD25(+) T cells and cytokines IL-10 and TGF-beta1 did not increase after challenge. Furthermore, a suppressive effect of nonresponding cells was not observed. Lymphocyte proliferation and viability were lost in vitro in the presence of physiologically relevant numbers of A. marginale organisms. These results suggest that loss of memory T cell responses following A. marginale infection is due to a mechanism other than induction of T regulatory cells, such as peripheral deletion of MSP2-specific T cells.

Amino Acid Sequence↗
PubMed1987

Experimental infections of Anaplasma ovis in pronghorn antelope.

Anaplasma ovis was experimentally transmitted from sheep to pronghorn antelope (Antilocapra americana) and back to sheep. Anaplasma ovis was recovered in splenectomized sheep, from two of three spleen-intact pronghorns following their inoculation with blood from known A. ovis carrier sheep. These two pronghorns exhibited a 0.5% or higher A. ovis parasitemia within 48 days after exposure, and an anaplasmosis-positive serological response 91 days after exposure. Clinical signs of illness were not observed. Blood from the infected pronghorns produced disease in four splenectomized sheep.

Agglutination Tests↗
PubMed1988

Experimental anaplasmosis in mule deer: persistence of infection of Anaplasma marginale and susceptibility to A. ovis.

An experimental Anaplasma marginale infection was induced in a splenectomized mule deer (Odocoileus hemionus hemionus) which persisted subclinically at least 376 days as detected by subinoculation into susceptible cattle. Anaplasma ovis was experimentally transmitted from sheep to a splenectomized and a spleen-intact mule deer, and back to sheep. The pathogenesis in deer was very similar to that seen in sheep using ovine blood inoculations.

Anaplasma↗