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PubMed2004

Anaplasma marginale (Rickettsiales: Anaplasmataceae): recent advances in defining host-pathogen adaptations of a tick-borne rickettsia.

The tick-borne intracellular pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) develops persistent infections in cattle and tick hosts. While erythrocytes appear to be the only site of infection in cattle, A. marginale undergoes a complex developmental cycle in ticks and transmission occurs via salivary glands during feeding. Many geographic isolates occur that vary in genotype, antigenic composition, morphology and infectivity for ticks. In this chapter we review recent research on the host-vector-pathogen interactions of A. marginale. Major surface proteins (MSPs) play a crucial role in the interaction of A. marginale with host cells. The MSP1a protein, which is an adhesin for bovine erythrocytes and tick cells, is differentially regulated and affects infection and transmission of A. marginale by Dermacentor spp. ticks. MSP2 undergoes antigenic variation and selection in cattle and ticks, and contributes to the maintenance of persistent infections. Phylogenetic studies of A. marginale geographic isolates using msp4 and msp1alpha provide information about the biogeography and evolution of A. marginale: msp1alpha genotypes evolve under positive selection pressure. Isolates of A. marginale are maintained by independent transmission events and a mechanism of infection exclusion in cattle and ticks allows for only the infection of one isolate per animal. Prospects for development of control strategies by use of pathogen and tick-derived antigens are discussed. The A. marginale/vector/host studies described herein could serve as a model for research on other tick-borne rickettsiae.

Anaplasma marginale↗
PubMed2002

Adaptations of the tick-borne pathogen, Anaplasma marginale, for survival in cattle and ticks.

The tick-borne cattle pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) multiplies within membrane-bound inclusions in host cell cytoplasm. Many geographic isolates of A. marginale occur that vary in genotype, antigenic composition, morphology and infectivity for ticks. A tick cell culture system for propagation of A. marginale proved to be a good model for study of tick-pathogen interactions. Six major surface proteins (MSPs) identified on A. marginale from bovine erythrocytes were conserved on A. marginale derived from tick cells. MSP1a and MSP1b were adhesins for bovine erythrocytes, while only MSP1a was bound to be an adhesin for tick cells. The tandemly repeated portion of MSP1a was found to be necessary and sufficient for adhesion to both tick cells and bovine erythrocytes. Infectivity of A. marginale isolates for ticks was dependent on the adhesive capacity of the isolate MSP1a, which was found to involve both the adhesive properties and sequence of the repeated peptides. Cattle immunized with A. marginale derived from bovine erythrocytes or tick cells demonstrated a differential antibody response to MSP1a and MSP1b that resulted from the differential expression of these proteins in cattle and ticks cells. MSP2, derived from a multigene family, was found to undergo antigenic variation in cattle and ticks and may contribute to establishment of persistent A. marginale infections. MSP1a has been used as a stable genetic marker for geographic isolates because the molecular weight varies due to differing numbers of the tandem repeats. However, phylogenetic studies of A. marginale isolates from North America using MSP1a and MSP4 demonstrated that MSP4 was a good biogeographic marker, while MSP1a varied greatly among and within geographic areas. Infection and development of A. marginale in cattle and tick cells appears to differ and to be mediated by several surface proteins encoded from the small genome.

Anaplasma marginale↗
PubMed2002

Reduced weight gain due to subclinical Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila) infection.

Tick-borne fever (TBF) is caused by the rickettsia Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila) and is a common disease in sheep in areas of Norway infested by Ixodes ricinus ticks. TBF can cause both direct and indirect losses to sheep kept on tick-infested pastures. In the present work we studied a sheep flock of 26 ewes and 50 lambs on pasture from May until September. No cases of TBF had earlier been observed on this pasture. Blood samples from lambs with a reduced weekly weight gain were collected and analysed for A. phagocytophilum infection by blood smear examination. In addition, at the end of the study, sera from all lambs were analysed by an indirect fluorescent antibody assay (IFA) to determine the antibody titre to E. equi. No clinical signs of tick-borne infections were observed, except in one lamb. However, 30 (60%) of the lambs grazing on this pasture became infected with A. phagocytophilum, and the infected lambs had a reduced weight gain (mean) of 3.8 kg compared with the uninfected lambs. The present study indicates that A. phagocytophilum infection may be widespread and contribute to considerable productivity losses even on pastures with no apparent tick infestation.

Anaplasma phagocytophilum↗
PubMed2002

A review of studies on the transmission of Anaplasma phagocytophilum from sheep: implications for the force of infection in endemic cycles.

We review the findings of a longitudinal study of transmission of the intracellular tick-borne bacterium Anaplasma phagocytophilum from sheep to Ixodes ricinus ticks under natural conditions of tick attachment in the UK. In this study, sheep-to-tick transmission efficiency varied in a quadratic relationship with the number of adult ticks that were feeding on the sheep. We raise the hypothesis that this relationship may be due to conflicting effects of the density of ticks on bacterial survival and target cell (neutrophil) fluxes at the tick-host interface: in the same sheep at the same time, resistance to ticks was progressively inhibited with increasing number of feeding adult ticks, and investigation of serological responses to tick antigens suggesting loss of resistance may be associated with polarisation of host Th1 to Th2 type responses to ticks. We also raise the hypothesis that these properties, with superimposed effects on tick survival, may mean that variation in tick density is an important causal factor of observed variations in the force of A. phagocytophilum infection amongst different geographic foci.

Anaplasma phagocytophilum↗
PubMed2004

The effect of management factors on the seroprevalence of Anaplasma marginale in Bos indicus cattle in the Mexican tropics.

A cross-sectional study with a two-stage design and proportional distribution was carried out to determine the effect of management factors on the seroprevalence of Anaplasma marginale in Bos indicus cattle in the Mexican tropics. Serum was obtained from 384 cattle aged 1-2 years on 92 farms. The number of samples was proportionally distributed according to the number of farms in eastern Yucatan. Antibody activity against A. marginale was assessed using the card agglutination test. A primary screening using a 2 x K contingency table of the exposed variables with the outcomes was performed. All variables for which p < 0.20 were included in a fixed-effects log regression. The seroprevalence in the cattle was 69.75% (SE +/- 0.02). Sixty-four per cent of the farms had a seroprevalence > or = 75%. The risks related to managemental factors were stocking density ( > or = 1 animal/ha, OR = 10.94), type of acaricide (pyrethroids, OR = 3.8), dipping interval ( > 60 days, OR = 0.13) and type of veterinary instruments used (needles, scalpels, ear tattoos, and dehorners, OR = 0.17).

Agglutination Tests↗
PubMed2005

Serological survey of Babesia bovis and Anaplasma marginale in cattle in Tete Province, Mozambique.

A serological survey of bovine babesiosis and anaplasmosis in communal cattle was conducted in the northwestern province of Tete, Mozambique. Blood was collected from cattle ranging from 4 to 15 months old from randomly selected farms from six districts. Thirty-nine per cent of all 478 calves tested in Tete Province were seropositive to the ELISA for Babesia bovis antibodies and 63% of all calves were seropositive in the card agglutination test for Anaplasma marginale. Seroprevalence of B. bovis ranged from 22.8% in Tete City District to 48.1% in Angonia District. For A. marginale, it ranged from 34.4% in Angonia District to 87.3% in Moatize District. The dominant factor affecting seroprevalence for both haemoparasites was district and there was a trend for higher intensity of tick control to be associated with a higher seroprevalence of B. bovis and a lower seroprevalence of A. marginale. The obvious differences were the low prevalence of B. bovis in Tete City Council District and the low prevalence of A. marginale in Angonia District. The levels of exposure to B. bovis seen in our study are well below any that could be considered to be consistent with endemic stability, yet they are sufficiently high to ensure that clinical disease would be a risk. The seroprevalence of A. marginale, however, suggests that endemic stability with respect to this disease could exist in districts other than Angonia. There was no strong and consistent relationship between the intensity of control and the likelihood of seropositivity to either of the diseases.

Agglutination Tests↗
PubMed2002

Antigenic variation of Anaplasma marginale msp2 occurs by combinatorial gene conversion.

The rickettsial pathogen Anaplasma marginale establishes lifelong persistent infection in the mammalian reservoir host, during which time immune escape variants continually arise in part because of variation in the expressed copy of the immunodominant outer membrane protein MSP2. A key question is how the small 1.2 Mb A. marginale genome generates sufficient variants to allow long-term persistence in an immunocompetent reservoir host. The recombination of whole pseudogenes into the single msp2 expression site has been previously identified as one method of generating variants, but is inadequate to generate the number of variants required for persistent infection. In the present study, we demonstrate that recombination of a whole pseudogene is followed by a second level of variation in which small segments of pseudogenes recombine into the expression site by gene conversion. Evidence for four short sequential changes in the hypervariable region of msp2 coupled with the identification of nine pseudogenes from a single strain of A. marginale provides for a combinatorial number of possible expressed MSP2 variants sufficient for lifelong persistence.

Amino Acid Sequence↗
PubMed2002

Activation of a p44 pseudogene in Anaplasma phagocytophila by bacterial RNA splicing: a novel mechanism for post-transcriptional regulation of a multigene family encoding immunodominant major outer membrane proteins.

Immunodominant 44 kDa major outer membrane proteins of Anaplasma phagocytophila (human granulocytic ehrlichiosis agent) are encoded by the p44 multigene family. One of the paralogues, p44-18 is predominantly expressed by A. phagocytophila in mammalian hosts, but is downregulated in the arthropod vector. The expression of p44-18 was upregulated in A. phagocytophila cultivated in HL-60 cells at 37 degrees C compared with 24 degrees C. However, the molecular mechanism of such gene expression was unclear, as p44-18 has a pseudogene-like structure, i.e. it lacks an AUG start codon and is out of frame with an upstream overlapping paralogue, p44-1. In the present study, we found that an amplicon detected by reverse transciption-polymerase chain reaction (RT-PCR) [808 basepair (bp)] for the p44-1/p44-18 gene locus was smaller than that detected by PCR with the genomic DNA (1652 bp) in the A. phagocytophila-infected HL-60 cells cultured at 37 degrees C. A circularized RNA molecule corresponding to the 844 bp region missing from the locus in the RT-PCR product was detected by inverse RT-PCR, indicating that this is an intron (designated p44-1 intervening sequence, p44-1 IVS). The splicing event of p44-1 IVS was also observed when the p44-1 IVS-carrying plasmid was introduced into Escherichia coli, suggesting that the splicing is sequence-dependent. Structural analysis and in vitro splicing experiments of p44-1 IVS suggested that this is likely to represent a new class of introns in eubacteria. The primer extension analysis showed the presence of a putative sigma(32)-type promoter in region upstream from p44-1. Collectively, the novel RNA splicing and the temperature-dependent transcription may account for the dominant p44-18 expression in mammals.

Amino Acid Sequence↗
PubMed2003

Conservation of a gene conversion mechanism in two distantly related paralogues of Anaplasma marginale.

Anaplasmataceae, the causative agents of anaplasmosis and ehrlichiosis, persist in the bloodstream of their mammalian hosts, allowing acquisition and transmission by tick vectors. Anaplasma marginale establishes persistent infection characterized by sequential cycles of rickettsaemia in which new antigenic variants emerge. The two most immunodominant outer membrane proteins, MSP2 and MSP3, are paralogues, each encoded by a distinct family of related genes. This study demonstrates that, although the two gene families have diverged substantially, each has maintained a similar mechanism to generate structurally and antigenically polymorphic surface antigens. Like MSP2, MSP3 is expressed from a single locus in which variation of the expressed msp3 gene is generated by recombination using msp3 pseudogenes. Each of the msp3 pseudogenes encodes a unique central variable region (CVR) flanked by conserved 5' and 3' regions. Changes in the CVR of the expressed msp3, concomitant with invariance of the pseudogenes, indicate that expression site variation is generated using gene conversion. A. marginale thus maintains two large, separate systems within its small genome to generate antigenic variation of its surface proteins, while analogous structural elements indicate a common mechanism.

Acute Disease↗
PubMed2000

Development of an ELISA system for detection of anti-Anaplasma marginale antibodies in cattle in Brazil.

An ELISA test was developed for detecting antibodies against Anaplasma marginale in bovine sera. Four antigenic preparations were produced from infected red blood cells. Some aliquots of this preparation were stored at -70 degrees C with 30% DMSO in phosphate-buffered saline (PBS) and others were lysed with 0.9% NH4Cl and stored at -20 degrees C. Typical anaplasmal structures were seen by electron microscopy in the antigenic preparations containing the erythrocytes that had been stored with DMSO. The performance of the ELISA test was evaluated by testing 298 positive serum samples collected from immunized cattle, 39 negative serum samples collected from cattle imported from areas free of A. marginale and 50 samples collected from cattle naturally infected in the field. The test gave a specificity of 94.87% and a sensitivity of 100%.

Anaplasma↗
PubMed2002

Transmission of Anaplasma phagocytophila (human granulocytic ehrlichiosis agent) in horses using experimentally infected ticks (Ixodes scapularis).

Most human granulocytic ehrlichiosis (HGE) studies carried out in horses use needle inoculation of infected leucocytes or cell cultures. This route of inoculation does not accurately reflect natural infection of the tick-borne agent. To investigate whether tick transmission influences the course of granulocytic ehrlichiosis in the horse model, experimental transmission through infected laboratory-reared Ixodes scapularis ticks was attempted into two healthy horses. One additional horse served as negative control and was exposed to uninfected ticks. Eleven days after exposure to nymphal or adult ticks infected with Anaplasma phagocytophila (HGE agent) the two horses developed severe clinical and laboratory signs consistent with granulocytic ehrlichiosis. Bacteraemia was determined at various time points in the two horses by observation of morulae within neutrophils and by detection of A. phagocytophila genomic DNA by PCR of peripheral blood leucocytes. Further, both horses seroconverted. In contrast the control horse stayed uninfected. The results demonstrate that A. phagocytophila can be experimentally transmitted by infected nymphal and adult ticks and that the agent is able to produce a severe disease, similar to naturally occurring cases. Therefore, tick transmission is highly reproducible and can be successfully used in the equine animal model in order to study HGE.

Anaplasma phagocytophilum↗
PubMed2003

Obligatory intracellular parasitism by Ehrlichia chaffeensis and Anaplasma phagocytophilum involves caveolae and glycosylphosphatidylinositol-anchored proteins.

Obligatory intracellular, human ehrlichiosis agents Ehrlichia chaffeensis and Anaplasma phagocytophilum create unique replicative compartments devoid of lysosomal markers in monocytes/macrophages and granulocytes respectively. The entry of these bacteria requires host phospholipase C (PLC)-gamma2 and protein tyrosine kinases, but their entry route is still unclear. Here, using specific inhibitors, double immunofluorescence labelling and the fractionation of lipid rafts, we demonstrate that bacterial entry and intracellular infection involve cholesterol-rich lipid rafts or caveolae and glycosylphosphatidylinositol (GPI)-anchored proteins. By fluorescence microscopy, caveolar marker protein caveolin-1 was co-localized with both early and replicative bacterial inclusions. Additionally, tyrosine-phosphorylated proteins and PLC-gamma2 were found in bacterial early inclusions. In contrast, clathrin was not found in any inclusions from either bacterium. An early endosomal marker, transferrin receptor, was not present in the early inclusions of E. chaffeensis, but was found in replicative inclusions of E. chaffeensis. Furthermore, several bacterial proteins from E. chaffeensis and A. phagocytophilum were co-fractionated with Triton X-100-insoluble raft fractions. The formation of bacteria-encapsulating caveolae, which assemble and retain signalling molecules essential for bacterial entry and interact with the recycling endosome pathway, may ensure the survival of these obligatory intracellular bacteria in primary host defensive cells.

Anaplasma phagocytophilum↗
PubMed2003

Invasion and survival strategies of Anaplasma phagocytophilum.

Anaplasma phagocytophilum is an aetiological agent of human granulocytic ehrlichiosis, an emerging tick-borne zoonosis in the United States and Europe. This obligate intracellular bacterium is unique in that it colonizes polymorphonuclear leucocytes (neutrophils). Neutrophils are key players in innate immunity. These short-lived phagocytes ingest invading microorganisms and destroy them by various means, which include fusing the bacteria-containing phagosome with acidic lysosomes as well as directing toxic oxidative and proteolytic compounds into the phagosomal lumen. Its tropism for neutrophils indicates that A. phagocytophilum uses strategies for evading and/or neutralizing these microbicidal activities. This review focuses on some of the mechanisms that A. phagocytophilum uses for neutrophil adhesion, surviving within the hostile intracellular environment of its host neutrophil and for effectively disseminating to naïve host cells.

Anaplasma phagocytophilum↗
PubMed2002

Relationship between tick bites and the seroprevalence of Babesia microti and Anaplasma phagocytophila (previously Ehrlichia sp.) in blood donors.

BACKGROUND: Tick-borne diseases, particularly babesiosis and ehrlichiosis, represent recently emerging infections. Despite an increased recognition of the threat tick-borne agents pose to blood safety, our understanding of the prevalence and transmissibility of these agents in blood donors is limited. STUDY DESIGN AND METHODS: Babesia microti and Anaplasma phagocytophila (previously Ehrlichia sp.) seroprevalence was determined in random Connecticut and Wisconsin donors, and subsequently in Connecticut donors reporting tick bites. In the interim, a postcard survey regarding tick bites during the previous 6 months was sent to 6,000 random donors in six geographically distinct collection regions. RESULTS: In total, 3 of 999 Wisconsin donors (0.3%) and 6 of 1,007 Connecticut donors (0.6%) had antibodies to B. microti. Of 992 donors tested for A. phagocytophila, 5 Wisconsin donors (0.5%) and 35 Connecticut donors (3.5%) were seropositive. A total of 2,482 donors (41.4%) completed the survey; 103 (4.1%) reported a tick bite. Of 848 Connecticut donors (0.4%) reporting tick bites, 3 had B. microti antibodies, while 8 (0.9%) had A. phagocytophila antibodies. These rates were not significantly different from control donors. CONCLUSION: Blood donors seropositive for B. microti and A. phagocytophila are present in Connecticut and Wisconsin. Donors readily recall previous tick bites, but self-reported bites are not reliable indicators of serologic status. The exposure of blood donors to tick-borne pathogens does suggest a need to better understand the transfusion transmission potential of these agents.

Anaplasma phagocytophilum↗
PubMed1994

Transmission of Anaplasma marginale with adult Boophilus microplus ticks fed as nymphs on calves with different levels of rickettsaemia.

Nine splenectomised calves were infested with dissimilar numbers of adult Boophilus microplus ticks 72 h after collection as engorged nymphs from three non splenectomised calves with different levels of Anaplasma marginale rickettsaemia. Successful transmission of A. marginale appeared to be more dependent on the level of rickettsaemia of the donor calves than on the number of ticks attaching to the splenectomised calves, since infection was transmitted only when the rickettsaemia was 0.3% or greater. Field transmission would thus depend on the rate of tick migration amongst susceptible hosts and the rickettsaemia level of cattle on which the ticks fed previously.

Anaplasma↗
PubMed2003

Purification of ovine neutrophils and eosinophils: Anaplasma phagocytophilum affects neutrophil density.

Studies on the functions of ovine granulocytes require pure and functionally active populations of neutrophils and eosinophils. This report describes an improved technique for the separation of neutrophils and eosinophils from the peripheral blood of sheep infected with Anaplasma phagocytophilum and from normal sheep. After centrifugation and discarding the buffy coat layer, which contains the bulk of mononuclear cells, neutrophils with a high degree of purity (94.87 [+/-1.7]%, n=9) and good yield (69 [+/-9]%, n=9) were obtained by density gradient centrifugation on Percoll with a density of 1.09 g/ml (65%). However, this density was not suitable for neutrophils obtained from sheep during the peak period of A. phagocytophilum bacteraemia. Improved purity of infected neutrophils was obtained when the leucocytes were separated on Percoll with a density of 1.08 g/ml (55%). Relatively good purity of eosinophils was obtained when leucocytes from normal sheep were separated on Percoll with a density of 1.10 g/ml (70%). Ovine eosinophils formed a distinct band just below the band of mononuclear cells when a continuous Percoll gradient with a density of 1.10 g/ml was used. The purity of the eosinophils obtained was 87.7 (+/-12.5)% (n=6; range 64.1-97.6%), with a mean recovery rate of 61.9 (+/-20.3)%.

Anaplasma phagocytophilum↗
PubMed2004

Complete genome sequencing of Anaplasma marginale reveals that the surface is skewed to two superfamilies of outer membrane proteins.

The rickettsia Anaplasma marginale is the most prevalent tick-borne livestock pathogen worldwide and is a severe constraint to animal health. A. marginale establishes lifelong persistence in infected ruminants and these animals serve as a reservoir for ticks to acquire and transmit the pathogen. Within the mammalian host, A. marginale generates antigenic variants by changing a surface coat composed of numerous proteins. By sequencing and annotating the complete 1,197,687-bp genome of the St. Maries strain of A. marginale, we show that this surface coat is dominated by two families containing immunodominant proteins: the msp2 superfamily and the msp1 superfamily. Of the 949 annotated coding sequences, just 62 are predicted to be outer membrane proteins, and of these, 49 belong to one of these two superfamilies. The genome contains unusual functional pseudogenes that belong to the msp2 superfamily and play an integral role in surface coat antigenic variation, and are thus distinctly different from pseudogenes described as byproducts of reductive evolution in other Rickettsiales.

Anaplasma marginale↗
PubMed1988

Detection of Anaplasma-marginale-infected tick vectors by using a cloned DNA probe.

Anaplasmosis is the most widely distributed of several important tick-borne diseases that constrain cattle production throughout much of the world. Evaluation of the effectiveness of disease control strategies that integrate vaccination with tick control requires the ability to monitor tick and cattle infection rates. To detect Anaplasma marginale in ticks and bovine erythrocytes, a 2-kilobase DNA fragment from a cloned A. marginale gene coding for a surface protein having a Mr of 105,000 was prepared and evaluated as a probe. The probe was species specific and detected A. marginale DNA derived from infected bovine erythrocytes and adult Dermacentor ticks infected either as nymphs or adults. Tick infection was confirmed by microscopy and test feeding on a susceptible calf. The sensitivity of the probe is suitable for detecting infected ticks in experimental and field epizootiology studies.

Anaplasma↗