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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↗

PubMed1997

Anaplasma marginale major surface protein 3 is encoded by a polymorphic, multigene family.

The immunodominant surface protein, MSP3, is structurally and antigenically polymorphic among strains of Anaplasma marginale. In this study we show that a polymorphic multigene family is at least partially responsible for the variation seen in MSP3. The A. marginale msp3 gene msp3-12 was cloned and expressed in Escherichia coli. With msp3-12 as a probe, multiple, partially homologous gene copies were identified in the genomes of three A. marginale strains. These copies were widely distributed throughout the chromosome. Sequence analysis of three unique msp3 genes, msp3-12, msp3-11, and msp3-19, revealed both conserved and variant regions within the open reading frames. Importantly, msp3 contains amino acid blocks related to another polymorphic multigene family product, MSP2. These data, in conjunction with data presented in previous studies, suggest that multigene families are used to vary important antigenic surface proteins of A. marginale. These findings may provide a basis for studying antigenic variation of the organism in persistently infected carrier cattle.

Amino Acid Sequence↗

PubMed1985

Evaluation of jackrabbits as nonruminant hosts for Anaplasma marginale.

Two black-tailed jackrabbits (Lepus californicus), 1 splenectomized and 1 intact, were inoculated with 0.2 ml of a 1:5 dilution of a Florida Anaplasma marginale stabilate. Five months later, both hares were inoculated with 1 ml of whole blood from a calf with acute anaplasmosis. Neither hare developed any signs of clinical anaplasmosis. Pooled blood (7 ml) from these jackrabbits which was inoculated into 2 Anaplasma-susceptible, splenectomized calves failed to induce hematologic or serologic signs of anaplasmosis for at least 90 days. Two susceptible, splenectomized calves were inoculated with 35 ml of pooled whole blood from 9 wild-collected black-tailed jackrabbits from a known anaplasmosis enzootic area. Both steers remained free of anaplasmosis signs for 90 days.

Anaplasmosis↗

PubMed1992

Single-step technique for staining Anaplasma marginale in bovine blood smears.

Three available differential stains, Camco-Quik, Diff-Quik, and Wright-Giesma were compared for detection of intraerythrocytic Anaplasma marginale in bovine blood smears. In samples where < 1% to more than 51% of the RBC were infected, statistical analysis of the data indicated no significant difference in the detection of A marginale with Camco-Quik or Diff-Quik stains. However, a significantly lower percentage of infected RBC were detected when blood smears were stained with the Wright-Giemsa stain, compared with the other 2 methods.

Anaplasma↗

PubMed2004

A novel antigen from Anaplasma marginale: characterization, expression and preliminary evaluation of the recombinant protein.

Through a process of protein fractionation and vaccination we previously identified four native antigens that confer a degree of protection against challenge with Anaplasma marginale. One of these, Ana 29 has been successfully cloned and sequenced using degenerate primers designed to N-terminal and internal peptide sequences. The full-length gene codes for a protein with a theoretical molecular weight of 27 kDa and pI 8.6. The sequence is highly conserved, showing 99% identity between two Australian and an American isolate of A. marginale. The gene sequences from these isolates also share 99% identity with the strain of Anaplasma centrale used in the commercial Australian vaccine. Protein prediction algorithms suggest the native protein is an integral membrane protein. This protein has been over-expressed and purified from Escherichia coli and used in vaccination trials in cattle using two adjuvants. The initial results from the trial show a significant level of protection was obtained with one adjuvant; in comparison, the second adjuvant slightly aggravated the disease. Preliminary data suggests a good correlation between the induction of an IgG2 response and protection.

Amino Acid Sequence↗

PubMed1983

Demonstration of colonies of Anaplasma marginale in the midgut of Rhipicephalus simus.

Rhipicephalus simus nymphs were allowed to feed on a cow experimentally infected with the BW-strain of Anaplasma marginale from Republic of South Africa, and they were studied as adults. Colonies were demonstrated by light microscopy in midgut epithelial cells of adult ticks that were unfed (as adults), incubated, or prefed for 72 hours on a cow. The colonies occurred in 5 different morphologic types (1 to 5) that were similar to those described previously for a Virginia isolate of A marginale in Dermacentor andersoni. The colony density (number of colonies/0.001 mm2 midgut tissue examined) ranged from 0 to 2.0 and was highest in unfed ticks that were not incubated (mean 0.566). Colonies observed by light microscopy were sectioned for study with the electron microscope. The colonies contained both electron-dense forms and reticulated forms. The organisms in type 2 and 3 colonies appeared to be attached to one another, and those in type 4 and 5 colonies occurred separately. Small particles were seen within the limiting membrane of some organisms. A few colonies contained a dense matrix and were surrounded by many small electron-dense particles.

Anaplasma↗

PubMed1993

Detection of Anaplasma marginale (Rickettsiales: Anaplasmataceae) in secretagogue-induced oral secretions of Dermacentor andersoni (Acari: Ixodidae) with the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to detect Anaplasma marginale in secretagogue-induced oral secretions of male and female Dermacentor andersoni Stiles exposed as nymphs or adults by feeding on infected calves. A 409-bp DNA fragment derived from the A. marginale (Florida isolate) msp1 beta gene was amplified with oligonucleotide primers BAP-2 (5'-GTATGGCACGTAGTCTTGGGATCA-3') and AL34S (5'-CAGCAGCAGCAAGACCTTCA-3'). The target DNA was amplified in oral secretions of female ticks exposed to A. marginale as adults and stimulated to secrete by injection of dopamine. Conversely, A. marginale was detected in saliva from prefed female ticks exposed as nymphs only after stimulation with a combination of dopamine, gamma-aminobutyric acid, pilocarpine, and theophylline. Saliva from ticks exposed as nymphs and stimulated with ergot alkaloids did not contain the A. marginale target DNA. Saliva collected after 11 d of feeding from dopamine-stimulated male ticks contained A. marginale DNA. The results indicate that A. marginale is present in tick saliva and suggest that the parasite can be transmitted to cattle via saliva of feeding ixodid ticks. The variable appearance of A. marginale in saliva, regardless of the method used to induce salivation, suggests that transmission of A. marginale may be affected by the physiological state of the tick.

Anaplasma↗

PubMed1988

Preliminary studies of the development of Anaplasma marginale in salivary glands of adult, feeding Dermacentor andersoni ticks.

On each day of feeding on susceptible calves, salivary glands obtained from groups of adult ticks that transmitted Anaplasma marginale were examined for A marginale colonies by use of light microscopy and transmission electron microscopy. On day 8 of feeding, salivary glands were examined, using fluorescein-labeled antibody and methyl green-pyronine stain. Use of fluorescein-labeled antibody consistently revealed small numbers of fluorescent foci in salivary gland acinar cells obtained from ticks that had fed for 8 days. Colonies of A marginale were seen by transmission electron microscopy only in salivary gland acini of male ticks; these colonies could not be identified, using light microscopy, in companion 1-micron plastic sections stained with Mallory stain. Methyl green-pyronine stain, used commonly to detect theilerial parasites in tick salivary glands, did not differentiate A marginale from cytoplasmic inclusions normally found in salivary gland acinar cells.

Anaplasma↗

PubMed1981

Comparative titration of Anaplasma marginale antibodies by card agglutination and complement-fixation tests.

The card agglutination test for anaplasmosis was improved so that bovine serum could be titrated for Anaplasma antibodies. The modified test was validated when its results on 1 set of serum samples were compared with the results furnished by the complement-fixation test on the same set. The samples tested were from cows inoculated with an Anaplasma vaccine or with either 1 of 2 virulent strains of Anaplasma marginale. A correlation coefficient of 0.60 was calculated between the values of the titers furnished by the 2 tests, and regression equations were calculated and used to estimate the serum titer in 1 test from a titer measured in the other. The results indicate that the simple card agglutination test can yield titers comparable with those of the complement-fixation test.

Agglutination Tests↗

PubMed1975

Exit of Anaplasma marginale from bovine red blood cells.

Incubation of Anaplasma marginale-infected bovine red blood cells in a flow culture system resulted in a decrease of observable marginal bodies. The decrease was twice the degree of hemolysis, indicating that marginal bodies may leave red blood cells without concomitant lysis of the host cell.

Anaplasma↗

PubMed1996

Anaplasma marginale: detection of carrier cattle by PCR-ELISA.

A highly sensitive and specific polymerase chain reaction (PCR) based assay for the detection of the minute levels of Anaplasma marginale present in the blood of long-term carrier cattle was developed. A simple lysis method was used to remove most of the haemoglobin from the blood to facilitate direct input of samples into the PCR reactions without prior purification of the DNA. PCR product was detected by enzyme-linked immunosorbent assay (ELISA) to simplify the processing of large numbers of samples. The sensitivity limit of the PCR-ELISA was 0.00015% parasitaemia (24 infected erythrocytes per microlitre of blood). No cross-reactivity of the assay was observed when A. marginale-negative blood infected with Babesia bovis or Theileria orientalis was tested. The PCR-ELISA was shown to be 92% efficient in the detection of long-term A. marginale carrier cattle. No false-positive results were obtained. These results compared favourably with 2 serological assays for detection of A. marginale carrier cattle (card agglutination test and ELISA) which were applied to the same experimental animals.

Agglutination Tests↗

PubMed1984

A rapid staining procedure for Anaplasma marginale in bovine erythrocytes.

A commercial product that stains cells in less than 1 minute was compared with the Giemsa and the Wright-Giemsa methods for detection of the intraerythrocytic parasite, Anaplasma marginale, in bovine blood smears. The total number of A marginale-infected erythrocytes detected in 5 microscope fields of 18 different samples was determined for each staining procedure. In samples where less than 2% to greater than 25% of the RBC were infected, statistical analysis of the data indicated that the rapid staining method was superior to the Giemsa and Wright-Giemsa methods with regard to determination of the percentage of bovine erythrocytes infected with A marginale.

Anaplasma↗

PubMed1986

Experimental anaplasmosis in American bison: persistence of infections of Anaplasma marginale and non-susceptibility to A. ovis.

Blood collected 314 and 496 days after experimentally infecting splenectomized and spleen-intact American bison (Bison bison) with Anaplasma marginale was infective for splenectomized bovine steers. The pathogenesis was identical to that seen in bovine studies using bovine blood inoculations. A splenectomized bison remained normal clinically, hematologically and serologically for 10 mo after repeated inoculation of ovine blood infected with A. ovis.

Anaplasma↗

PubMed1997

Effect of breed of cattle on innate resistance to infection with Babesia bovis, B bigemina and Anaplasma marginale.

OBJECTIVE: To assess the innate resistance of naive Bos taurus, Bos taurus cross Bos indicus and Bos indicus cattle to virulent Babesia bovis, B bigemina and Anaplasma marginale parasites. DESIGN: Groups of 10, pure B indicus, 1/2 B indicus cross, 1/4 B indicus cross and pure B taurus steers were infected with virulent B bovis, B bigemina and A marginale parasites [corrected]. PROCEDURE: Sequential infections were carried out by intravenous inoculation of infected blood containing 1 x 10(8) parasites of B bovis, followed by B bigemina and then A marginale. To assess resistance, measurements were made of parasitaemia, rectal temperature, packed cell volume and the number within a group requiring chemotherapy to control infection. There was a recovery period between each infection. RESULTS: Infection with B bovis showed that pure B indicus steers were significantly more resistant to B bovis infection than the other groups, with none of this group requiring treatment. There was no significant difference between 1/2 B indicus cross and 1/4 B indicus cross with 30% and 20%, respectively, of steers in these groups requiring treatment [corrected]. The pure B taurus steers were significantly more affected then those in the other three groups with 80% requiring treatment. Infections of B bigemina produced a mild response in comparison to that of B bovis and none of the steers required treatment. However, the pure B taurus group was significantly more affected than the other three groups for all other measurements. After the A marginale infection, B indicus steers were moderately affected with 50% requiring treatment, whereas 70% of the 1/2 B indicus group, 80% of the 1/4 B indicus cross group and 100% of the pure B taurus group required treatment [corrected]. CONCLUSIONS: All breeds of cattle, ranging from pure B indicus to pure B taurus may be at risk of severe disease if exposed to virulent A marginale. The results confirm that pure B indicus cattle are relatively resistant to B bovis, but there could be a significant risk of severe mortalities if cross-bred herds are exposed to virulent infection.

Anaplasma↗

PubMed1988

Isolation and restriction endonuclease cleavage of Anaplasma marginale DNA in situ in agarose.

Bacterial restriction endonucleases were used to produce DNA cleavage patterns that could be useful as tools to study the relatedness among Anaplasma marginale isolates. Bovine erythrocytes infected with A. marginale were lysed, washed, and embedded in agarose. The embedded erythrocytes and bacterial pathogens were partially digested by sequential infiltration of the agarose with acetone, lysozyme, sodium dodecyl sulfate, and proteinase K. The unfragmented genomic DNA was left supported and protected in a porous matrix. The DNA was digested in situ in agarose under the following conditions: (i) brief treatment with phenol, (ii) brief washing with distilled water, and (iii) adjustment of restriction enzyme digestion mixture to compensate for the volume of the agarose. The cleaved DNA was electrophoresed horizontally to produce a DNA cleavage pattern. Of 19 restriction enzymes screened, 12 produced distinct DNA bands from the genomes of each of the five A. marginale isolates examined. The DNA cleavage pattern produced from each isolate with a given restriction enzyme was reproducible. However, the DNA cleavage patterns produced from different isolates with a given restriction enzyme were not necessarily identical. This procedure could be modified for general bacterial DNA isolation, in situ agarose digestion, and manipulations.

Anaplasma↗

PubMed2004

CD4+ T lymphocytes from Anaplasma marginale major surface protein 2 (MSP2) vaccinees recognize naturally processed epitopes conserved in MSP3.

Major surface protein 2 (MSP2) and MSP3 of the persistent bovine ehrlichial pathogen Anaplasma marginale are immunodominant proteins that undergo antigenic variation. The recently completed sequence of MSP3 revealed blocks of amino acids in the N and C termini that are conserved with MSP2. This study tested the hypothesis that CD4+ T cells specific for MSP2 recognize naturally processed epitopes conserved in MSP3. At least one epitope in the N terminus and two in the C terminus of MSP2 were also processed from MSP3 and presented to CD4+ T lymphocytes from MSP2-immunized cattle. This T-lymphocyte response to conserved and partially conserved epitopes may contribute to the immunodominance of MSP2 and MSP3.

Amino Acid Sequence↗

PubMed1993

Expression and immune recognition of the conserved MSP4 outer membrane protein of Anaplasma marginale.

Defining conserved, protective epitopes is essential to the design of an effective vaccine against bovine anaplasmosis. MSP4, one of six initial body proteins recognized by a neutralizing serum, is conserved among Anaplasma marginale isolates at both the protein and the DNA levels. Sera from cattle immunized with an outer membrane fraction of A. marginale and protected from a virulent challenge bind MSP4. The gene for MSP4 has been cloned, and the recombinant protein has been expressed, isolated, and demonstrated to share epitopes with the native protein expressed on initial bodies. MSP4 may have a greater potential to protect cattle from a challenge by heterologous isolates than other A. marginale surface proteins, which vary widely in size and structure.

Anaplasma↗

PubMed1998

The repertoire of Anaplasma marginale antigens recognized by CD4(+) T-lymphocyte clones from protectively immunized cattle is diverse and includes major surface protein 2 (MSP-2) and MSP-3.

Major surface proteins of Anaplasma marginale are vaccine candidates. We recently demonstrated that immunization of calves with outer membranes of the Florida strain of A. marginale resulted in protective immunity that correlated with a memory CD4(+) T-lymphocyte response specific for major surface protein 1 (MSP-1), MSP-2, and MSP-3 (W. C. Brown, V. Shkap, D. Zhu, T. C. McGuire, W. Tuo, T. F. McElwain, and G. H. Palmer, Infect. Immun. 66:5406-5413, 1998). As immunogens, these proteins have been shown to induce complete or partial protection against homologous challenge. To further define the T helper (Th) cell response to these and other A. marginale antigens and to determine conservation of Th cell epitopes among genetically distinct A. marginale strains, Th cell clones obtained prior to challenge from three immunized calves were characterized for antigen-specific responses. Nine distinct antigenic profiles were defined by 11 Th cell clones derived by stimulation with the Florida strain. Several clones responded to MSP-2, MSP-3, or both. All of these MSP-2- or MSP-3-specific clones and the majority of other clones that did not respond to MSPs recognized all bovine blood-passaged strains of A. marginale. These results demonstrate conservation of certain Th cell epitopes between MSP-2 and MSP-3 and show that Th cell epitopes in MSP-2, MSP-3, and undefined antigens are conserved among strains of A. marginale. Of seven clones that responded to the blood-passaged Virginia strain, two did not recognize antigen prepared from this strain cultured in tick cells, suggesting differences in the antigenic composition between these stages. Analysis of the cytokines expressed by the Th cells revealed that all clones expressed gamma interferon and tumor necrosis factor alpha, and most coexpressed interleukin-4. Our results provide a rationale for identifying Th cell epitopes conserved among different strains of A. marginale for inclusion in a nucleic acid or recombinant protein vaccine.

Anaplasma↗