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PubMed1999

Dermacentor hunteri (Acari: Ixodidae): an experimental vector of Anaplasma marginale and A. ovis (Rickettsiales: Anaplasmataceae) to calves and sheep.

The experimental vector competence of laboratory-reared Dermacentor hunteri Bishopp for Anaplasma marginale Theiler and Anaplasma ovis Lestoquard was evaluated by delayed transfer of male ticks from infected to susceptible Holstein calves and from infected to susceptible domestic sheep, respectively. After feeding for 4 or 5 d on rickettsemic acquisition hosts, the ticks were held off the host at 26 degrees C, approximately 93% RH, and a photoperiod of 14:10 (L:D) h for 7 or 8 d, then test fed for 5 or 7 d. Additionally, ticks test-fed for 5 d on 2 susceptible calves were removed, held off the host for 7 d, and test-fed for 5 d on a 3rd susceptible calf to test the tick's ability to transmit A. marginale by delayed serial transfer. Tick transmission of A. marginale to 3 test calves and A. ovis to 3 test sheep was demonstrated by blood smear and indirect immunofluorescence serology. These data indicate that males of D. hunteri, a tick commonly found on desert bighorn, Ovis canadensis Shaw, in the southwestern United States and northern Mexico, may be competent natural vectors of these organisms present in desert bighorn populations.

Anaplasma↗
PubMed2002

Conservation of the unique rickettsial rRNA gene arrangement in Anaplasma.

The organization of the rRNA genes of Anaplasma marginale, the type species of the genus Anaplasma, was identified to determine if the atypical rRNA gene arrangement identified in rickettsiae preceded divergence of the order Rickettsiales into the families Anaplasmataceae and Rickettsiaceae. The rRNA genes are encoded by two unlinked units, each present in a single copy per A. marginale genome. The 16S rRNA gene is separated from the linked 23S and 5S rRNA genes by a minimum of 100 kb. Similar to species belonging to the genus Rickettsia, the typical bacterial 16S-23S spacer region containing tRNA genes has been lost in A. marginale. In contrast, the fmt gene located upstream of the 23S rRNA gene in most Rickettsia spp. is not maintained in A. marginale, consistent with the fmt arrangement being a relatively late event in the evolution of rickettsial species.

Alphaproteobacteria↗
PubMed2005

Structural basis for segmental gene conversion in generation of Anaplasma marginale outer membrane protein variants.

Bacterial pathogens in the genus Anaplasma generate surface coat variants by gene conversion of chromosomal pseudogenes into single-expression sites. These pseudogenes encode unique surface-exposed hypervariable regions flanked by conserved domains, which are identical to the expression site flanking domains. In addition, Anaplasma marginale generates variants by recombination of oligonucleotide segments derived from the pseudogenes into the existing expression site copy, resulting in a combinatorial increase in variant diversity. Using the A. marginale genome sequence to track the origin of sequences recombined into the msp2 expression site, we demonstrated that the complexity of the expressed msp2 increases during infection, reflecting a shift from recombination of the complete hypervariable region of a given pseudogene to complex mosaics with segments derived from hypervariable regions of different pseudogenes. Examination of the complete set of 1183 variants with segmental changes revealed that 99% could be explained by one of the recombination sites occurring in the conserved flanking domains and the other within the hypervariable region. Consequently, we propose an 'anchoring' model for segmental gene conversion whereby the conserved flanking sequences tightly align and anchor the expression site sequence to the pseudogene. Associated with the recombination sites were deletions, insertions and substitutions; however, these are a relatively minor contribution to variant generation as these occurred in less than 2% of the variants. Importantly, the anchoring model, which can account for more variants than a strict segmental sequence identity mechanism, is consistent with the number of msp2 variants predicted and empirically identified during persistent infection.

Amino Acid Sequence↗
PubMed2001

Use of repetitive DNA elements to define genetic relationships among Anaplasma marginale isolates.

Anaplasma marginale genomic DNA was tested for the presence of repetitive extragenic palindromic (REP) and enterobacterial repetitive intergenic consensus (ERIC)-like sequences in order to evaluate the genetic diversity of multiple A. marginale isolates. A. marginale isolates were obtained from cattle of six different states of Brazil, from the US and an Anaplasma centrale strain was obtained from Uruguay. Patterns obtained from A. marginale isolates varied from 14 to 17 fragments by REP-polymerase chain reaction (PCR) and 6 to 14 fragments by ERIC-PCR. All A. marginale isolates presented a 0.75-kb fragment by REP and two common fragments (0.38 and 1.0 kb) by ERIC-PCR. These two fragments were not detectable in A. centrale. Both methods produced similar patterns (80%) among A. marginale isolates obtained from the same region, although some isolates within regions shared less similarity. Isolates from Parana and Pernambuco, were differentiated by these methods. The study demonstrates the presence of ERIC and REP-like elements in A. marginale isolates and shows that A. marginale isolates and strains can be differentiated by these methods.

Anaplasma↗
PubMed2005

Anaplasma phagocytophilum infection in a dog: identifying the causative agent using PCR.

A diagnosis of Anaplasma phagocytophilum infection was confirmed in a two-year-old male golden retriever displaying few clinical and haematological abnormalities. This was achieved by demonstrating ehrlichial organisms in circulating neutrophils, by indirect immunofluorescence assay using A phagocytophilum as an antigen, and by detecting DNA specific for the 16S rRNA gene of granulocytic Anaplasma by PCR. After treatment with doxycycline for 10 days the dog showed improvement and the laboratory values returned to normal.

Anaplasma phagocytophilum↗
PubMed1996

Strategies to interrupt the development of Anaplasma marginale in its tick vector. The effect of bovine-derived antibodies.

Anaplasma marginale is a rickettsia transmitted by ticks that invades and multiplies in bovine erythrocytes causing the disease anaplasmosis. A complex developmental cycle occurs within ticks that begins in midgut cells, with subsequent infection in gut muscle cells. Final development occurs in salivary glands from where the rickettsia is transmitted to the vertebrate host. At each site of development, A. marginale multiplies within membrane-bound inclusions. Attempts to control anaplasmosis have focused on cattle and have included immunization and prophylactic treatment with tetracyclines. New strategies for control of anaplasmosis are being focused on the tick vector. Development of vaccines against hemoparasites in ticks may be feasible because vertebrate host immunoglobulins appear to cross the midgut epithelium of invertebrates and enter the hemolymph without breakdown. We tested the effect of A. marginale antibodies ingested by ticks with the bloodmeal on infections in ticks. Cattle were immunized with purified outer membrane proteins of erythrocytic-derived parasites. Infections in ticks exposed to the immunized cattle were determined using an Anaplasma-specific DNA probe, light and electron microscopy, and tick transmission studies. Vaccine-derived antibodies did not appear to affect the development and transmission of A. marginale in ticks. Further studies are needed to determine if bovine antibodies remain intact within ticks and whether the tick stage of A. marginale has unique surface antigens from the erythrocytic stage.

Anaplasma↗
PubMed1998

Prospective study for the detection of Anaplasma marginale Theiler, 1911 (Rickettsiales: Anaplasmataceae) in Costa Rica.

A prospective study was conducted to assess the dynamics of the infection and host response to Anaplasma marginale in one closed herd in the dry tropical forest of Costa Rica. The study subjects were the dams and their calves born during 1 breeding season (1995-1996). All cows were sampled at 3 month intervals for antibody detection using a competitive ELISA (cELISA) and for antigen detection using PCR/nonradioactive probe assay. All 24 calves born during the study were individually identified at birth and subsequently sampled each month for PCR and cELISA. Ticks were identified from all animals throughout the entire study period. The results from this study confirmed that the cELISA is a reliable assay for identifying new and carrier infections and that carrier infections can exist at levels below that detectable by PCR. In addition, it was demonstrated that calves born in this region will most likely be exposed to Anaplasma within the first 6 months of age.

Anaplasma↗
PubMed1998

Evaluation of Anaplasma marginale from tick cell culture as an immunogen for cattle.

Anaplasma marginale has been propagated and continuously passaged in an Ixodes scapularis cell line. Anaplasma development was characterized and cultures with a high density of rickettsiae were harvested at a predictable rate. Culture-derived A. marginale (CAM) remained infective for cattle and was used effectively as antigen in diagnostic tests with the sensitivity to identify bovine carriers of A. marginale. This study presents results of an initial trial using the CAM as an immunogen for cattle. CAM was mechanically disrupted, frozen at -70 degrees C, and inactivated with beta-propiolactone. Two intact yearling cattle were immunized with CAM and Freund's adjuvant, receiving 4 subcutaneous injections at 3-4 week intervals. Two control yearling cattle received adjuvant and PBS. Serum samples were evaluated by competitive ELISA (C-ELISA) using CAM as antigen and the standard complement fixation test (CFT). All cattle were subsequently challenged with A. marginale-infected blood from a carrier cow. An additional intact calf was inoculated with live CAM from the same passage and screened by C-ELISA and CFT. Sera collected from immunized cattle were negative or suspicious by CFT throughout the immunization study. The same sera were strongly positive by C-ELISA two weeks after the first injection and throughout the study. All cattle became infected following challenge-exposure with blood, but immunized cattle exhibited longer prepatent periods as well as lower parasitemias and percent reduction of packed cell volumes as compared with the controls. The calf receiving live CAM became infected and underwent a mild clinical reaction with positive C-ELISA and CFT results and did not become clinically ill following blood challenge. This preliminary study suggests that the CAM antigen is highly immunogenic in cattle. Furthermore, the CFT did not identify immunized animals whereas the C-ELISA (using CAM) was highly sensitive for detection of both immunized and infected animals.

Anaplasma↗
PubMed1998

Use of tick cell culture-derived Anaplasma marginale antigen in a competitive ELISA for serodiagnosis of anaplasmosis.

Anaplasma marginale was propagated in a continuous tick cell line and detergent-solubilized infected cells were used as antigen in a competitive ELISA (C-ELISA) for detection of Anaplasma-specific antibody in bovine sera. Positive control sera competed well (> or = 35% inhibition) with an A. marginale-specific monoclonal antibody for binding to this antigen, while negative sera failed to compete (< 35% inhibition). The C-ELISA was compared to the standard complement-fixation test (CFT) using 2,208 bovine sera. Overall, C-ELISA was more sensitive than CFT (24.9% versus 9.4%), mainly because CFT yielded "suspicious" or "anti-complementary" results in 10.5% of the sera and also failed to identify several vaccinated and carrier cattle that were C-ELISA-positive. The apparent agreement between CFT and C-ELISA was 89.6% and the kappa value was 0.6. These results show that this C-ELISA would be a suitable replacement of the CFT as the standard test for detection of A. marginale antibody.

Anaplasma↗
PubMed1999

Effect of breed of cattle on innate resistance to infection with Anaplasma marginale transmitted by Boophilus microplus.

OBJECTIVE: To assess the innate resistance of and transmission in naive Bos taurus cross Bos indicus and purebred Bos indicus cattle when placed in a paddock with cattle infected with Anaplasma marginale and carrying Boophilus microplus ticks. DESIGN: A group of 49 purebred B indicus, and 48 B indicus cross B taurus (50%, F1 generation) 24-month-old steers were kept in the same paddock with cattle artificially infected with a virulent isolate of A marginale and Boophilus microplus. The cattle were seronegative for A marginale at the start of the trial but had previously been exposed to Babesia bovis and B bigemina. PROCEDURE: Cattle were inspected twice weekly for 118 days. Whole blood, blood smears and serum samples were collected from the cattle on day 37 after exposure and then at regular intervals to day 83 after exposure to measure packed-cell volumes, parasitaemias and antibody titres to A marginale. Any animals that met preset criteria were treated for anaplasmosis. On day 83 all cattle were treated with an acaricide and cattle infected with A marginale were removed from the rest of the group. RESULTS: A marginale was detected in blood smears from 14 crossbred and 9 B indicus steers between days 56 and 72 after exposure. Five and two of the infected crossbred and B indicus steers required treatment, respectively. One of the Bos indicus cattle died as a result of the A marginale infection despite treatment. Antibodies to A marginale were detected in the 23 infected cattle. The mean packed-cell volume depression was 40 and 37% in the affected crossbred and Bos indicus groups, respectively. There was no significant difference detected in susceptibility between these two groups. CONCLUSIONS: Innate resistance of purebred B indicus and crossbred cattle was not significantly different. The results confirm that purebred B indicus and crossbred cattle are sufficiently susceptible to warrant the use of vaccination against Anaplasma infections.

Anaplasma↗
PubMed2001

Analysis of the 16S rRNA gene sequence of Anaplasma centrale and its phylogenetic relatedness to other ehrlichiae.

The nucleotide sequence of the Anaplasma centrale 16S rRNA gene was determined and compared with the sequences of ehrlichial bacteria. The sequence of A. centrale was closely related to Anaplasma marginale by both level-of-similarity (98.08% identical) and distance analysis. A species-specific PCR was developed based upon the alignment data. The PCR can detect A. centrale DNA extracted from 10 infected bovine red blood cells in a reaction mixture. A. centrale DNA was amplified in the reaction, but not other related ehrlichial species.

Anaplasma↗
PubMed2003

Antigens and alternatives for control of Anaplasma marginale infection in cattle.

Anaplasmosis, a tick-borne cattle disease caused by the rickettsia Anaplasma marginale, is endemic in tropical and subtropical areas of the world. The disease causes considerable economic loss to both the dairy and beef industries worldwide. Analyses of 16S rRNA, groESL, and surface proteins have resulted in the recent reclassification of the order Rickettsiales. The genus Anaplasma, of which A. marginale is the type species, now also includes A. bovis, A. platys, and A. phagocytophilum, which were previously known as Ehrlichia bovis, E. platys, and the E. phagocytophila group (which causes human granulocytic ehrlichiosis), respectively. Live and killed vaccines have been used for control of anaplasmosis, and both types of vaccines have advantages and disadvantages. These vaccines have been effective in preventing clinical anaplasmosis in cattle but have not blocked A. marginale infection. Thus, persistently infected cattle serve as a reservoir of infective blood for both mechanical transmission and infection of ticks. Advances in biochemical, immunologic, and molecular technologies during the last decade have been applied to research of A. marginale and related organisms. The recent development of a cell culture system for A. marginale provides a potential source of antigen for the development of improved killed and live vaccines, and the availability of cell culture-derived antigen would eliminate the use of cattle in vaccine production. Increased knowledge of A. marginale antigen repertoires and an improved understanding of bovine cellular and humoral immune responses to A. marginale, combined with the new technologies, should contribute to the development of more effective vaccines for control and prevention of anaplasmosis.

Anaplasma marginale↗
PubMed1978

Synthesis of DNA and protein by Anaplasma marginale in bovine erythrocytes during short-term culture.

Bovine erythrocytes infected with Anaplasma marginale were cultured for 1 to 5 days in a CO2 incubation chamber, pulse-labeled with [3H]thymidine and [14C]methionine, lysed, and fractionated by differential centrifugation and continuous density gradient centrifugation in Renografin. Anaplasma and associated fragments of stroma formed two distinct bands in the dense region of the gradient. Electron microscopic examination of pelleted material from the bands from cells cultured for 1 day revealed the presence of organisms that were morphologically intact or in various states of degeneration. Examination of fractions from the gradient for incorporation of label revealed that analplasma present in erythrocytes can incorporate both [3H]thymidine and [14C]methionine. Subsequent experiments demonstrated that organisms cultured for 3 and 5 days incorporated the radiolabeled compounds also, but to a lesser extent. The experiments demonstrate that it is possible to culture analplasma in vitro for short periods of time and monitor their growth characteristics.

Anaplasma↗
PubMed1984

Common and isolate-restricted antigens of Anaplasma marginale detected with monoclonal antibodies.

Anaplasma marginale-infected erythrocytes were examined for the presence of maturation, isolate-restricted, and isolate-common antigens by indirect immunofluorescence with monoclonal antibodies. A panel of 18 monoclonal antibodies was used; none of the antibodies reacted with Anaplasma ovis, Babesia bigemina, babesia bovis, Trypanosoma brucei, Trypanosoma congolense, or uninfected bovine erythrocytes. Antigens common to all six A. marginale isolates were detected by nine antibodies. Single isolates from Florida, Southern Idaho, Northern Texas, and Virginia and two isolates from Washington state had four patterns of reactivity with a second panel of nine antibodies. Antigenically distinct stages were not detected, as sequential smears taken daily during acute infection had the same pattern of reactivity. The results demonstrate antigenic heterogeneity among isolates of A. marginale and the presence of common antigens. This information allows grouping of different isolates and, more importantly, provides a method for the identification and isolation of common antigens for diagnostic tests.

Anaplasma↗
PubMed1998

CD4(+) T-lymphocyte and immunoglobulin G2 responses in calves immunized with Anaplasma marginale outer membranes and protected against homologous challenge.

Protective immunity against the ehrlichial pathogen Anaplasma marginale has been hypothesized to require induction of immunoglobulin G2 (IgG2) antibody against outer membrane protein epitopes and coordinated activation of macrophages for phagocytosis and killing. In the present study, cell-mediated immune responses, including induction of IgG isotype switching, were characterized in calves immunized with purified outer membranes of the Florida strain of A. marginale. Importantly, these calves were subsequently shown to be protected upon experimental challenge with the Florida strain, and calves which developed the highest IgG2 titers were completely protected against infection. Peripheral blood mononuclear cells (PBMC) obtained after immunization proliferated strongly in response to both whole A. marginale homogenates and purified outer membranes, and this responsiveness persisted until the time of challenge. Responding cells were shown to be CD4(+) T cells, and CD4(+) T-cell lines cultured for 2 to 4 weeks also proliferated specifically in response to A. marginale and produced high titers of gamma interferon. The helper T-cell response included recognition of conserved epitopes, as PBMC proliferation was stimulated by the homologous Florida strain, four genetically distinct A. marginale strains, and Anaplasma ovis. The outer membrane proteins stimulating the PBMC responses in protected calves included major surface proteins (MSPs) MSP-1, MSP-2, and MSP-3, which were previously shown to induce partial protection against infection. These studies demonstrate, for the first time, potent helper T-cell responses in cattle protectively immunized with outer membranes against A. marginale challenge and identify three MSPs that are recognized by immune T cells. These experiments provide the basis for subsequent identification of the helper T-cell epitopes on MSP-1, MSP-2, and MSP-3 that are needed to evoke anamnestic antibody and effector T-cell responses elicited by protein or nucleic acid immunization.

Anaplasma↗
PubMed2002

Specific expression of Anaplasma marginale major surface protein 2 salivary gland variants occurs in the midgut and is an early event during tick transmission.

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.

Amino Acid Sequence↗
PubMed2003

Expression of multiple outer membrane protein sequence variants from a single genomic locus of Anaplasma phagocytophilum.

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.

Amino Acid Sequence↗
PubMed2004

Anaplasma marginale major surface protein 2 CD4+-T-cell epitopes are evenly distributed in conserved and hypervariable regions (HVR), whereas linear B-cell epitopes are predominantly located in the HVR.

Organisms in the genus Anaplasma express an immunodominant major surface protein 2 (MSP2), composed of a central hypervariable region (HVR) flanked by highly conserved regions. Throughout Anaplasma marginale infection, recombination results in the sequential appearance of novel MSP2 variants and subsequent control of rickettsemia by the immune response, leading to persistent infection. To determine whether immune evasion and selection for variant organisms is associated with a predominant response against HVR epitopes, T-cell and linear B-cell epitopes were localized by measuring peripheral blood gamma interferon-secreting cells, proliferation, and antibody binding to 27 overlapping peptides spanning MSP2 in 16 cattle. Similar numbers of MSP2-specific CD4(+) T-cell epitopes eliciting responses of similar magnitude were found in conserved and hypervariable regions. T-cell epitope clusters recognized by the majority of animals were identified in the HVR (amino acids [aa] 171 to 229) and conserved regions (aa 101 to 170 and 272 to 361). In contrast, linear B-cell epitopes were concentrated in the HVR, residing within hydrophilic sequences. The pattern of recognition of epitope clusters by T cells and of HVR epitopes by B cells is consistent with the influence of protein structure on epitope recognition.

Anaplasma marginale↗