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PubMed1983

Demonstration of Anaplasma marginale in hemolymph of Dermacentor andersoni by animal inoculation and by fluorescent-antibody technique.

Hemolymph was collected from adult Dermacentor andersoni Stiles that had been infected with Anaplasma marginale Theiler as nymphs. Before hemolymph was collected, the adult ticks were either incubated and unfed at 37 C for 2.5 days or fed for 6 days on sheep. Hemolymph collected from groups of 100 ticks was inoculated into susceptible splenectomized calves. Smears of hemolymph from the same groups of ticks were prepared for examination by fluorescent antibody technique. Hemolymph from incubated ticks caused anaplasmosis in 2 of 4 trials, and hemolymph from feeding ticks caused anaplasmosis in 4 of 4 trials. Moderately fluorescing bodies were demonstrated in some hemocytes from incubated ticks, whereas hemocytes from feeding ticks contained numerous clusters of brightly fluorescing bodies. Fluorescing bodies were not observed in hemocytes from control ticks.

Anaplasma↗
PubMed2002

Vaccination of cattle with Anaplasma marginale derived from tick cell culture and bovine erythrocytes followed by challenge-exposure with infected ticks.

Anaplasmosis, a hemolytic disease of cattle caused by the tick-borne pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) has been controlled using killed vaccines made with antigen harvested from infected bovine erythrocytes. We recently developed a cell culture system for propagation of A. marginale in a continuous tick cell line. In this study, we performed a cattle trial to compare the bovine response to vaccination with A. marginale harvested from tick cell culture or bovine erythrocytes. All immunized and control cattle were then challenge-exposed by allowing male Dermacentor variabilis infected with A. marginale to feed and transmit the pathogen. Nine yearling cattle (three per group) were used for this study and were immunized with cell culture-derived A. marginale, erythrocyte-derived A. marginale or received adjuvant only to serve as controls. Each vaccine dose contained approximately 2 x 10(10) A. marginale and three immunizations were administered at weeks 1, 4 and 6. At week 8, cattle were challenge-exposed by allowing 60 D. variabilis male that were infected with A. marginale as adults to feed on the cattle. Antibody responses of cattle against major surface proteins (MSP) 1a, 1b and 5, as determined by ELISAs, peaked 2 weeks after the last immunization. Cattle immunized with infected IDE8 cell-derived antigens had a preferential recognition for MSP1b while cattle immunized with erythrocyte-derived antigens had a preferential recognition for MSP1a. Protection efficacy was evaluated using the percent infected erythrocytes (PPE), the packed cell volume (PCV), and the prepatent period. A. marginale-immunized cattle showed lower PPE and higher PCV values when compared to control animals and did not display clinical anaplasmosis. The cell culture-derived A. marginale shows promise for use as antigen in development of a new killed vaccine for anaplasmosis.

Anaplasma↗
PubMed1987

Attempted transmission to cattle of Anaplasma marginale from overwintered Dermacentor andersoni ticks.

Since the 1983 summer outbreak of anaplasmosis in southern Saskatchewan, the role of the tick, Dermacentor andersoni as an overwintering reservoir for Anaplasma marginale has been questioned. The purpose of this study was to determine if spring-collected ticks carried virulent A. marginale. Sixteen splenectomized calves were assigned randomly to two groups of 14 principals and two controls. Adult D. andersoni, collected in April from areas having high transmission rates of A. marginale, were confined to the ears of the principals by special bags and allowed to feed for eight days. The two control calves were subsequently challenged intravenously with blood from a calf infected with the Virginia strain of A. marginale. Principals and controls were monitored for 60 and 50 days postexposure respectively for signs of infection by clinical, hematological and serological procedures. None of the principals developed anaplasmosis but both control calves developed signs of disease.

Anaplasmosis↗
PubMed1976

Cutaneous hypersensitivity and isoantibody production in cattle injected with live or inactivated Anaplasma marginale in bovine and ovine erythrocytes.

Delayed cutaneous hypersensitivity and the production of isoagglutinins against normal bovine erythrocytes were measured in cattle inoculated with erythrocytic preparations containing live or inactivated Anaplasma marginale. The effect of blood group substances versus parasitic components on induction of delayed cutaneous hypersensitivity and isoimmunity was examined by using test antigens derived from normal and parasitized erythrocytes, virulent A marginale in bovine erythrocytes, and attenuated A marginale in ovine erythrocytes. The use of inactivated vaccine of bovine origin induced production of isoantibodies and cutaneous hypersensitivity to both bovine and ovine blood group factors, whereas a similar vaccine preparation of ovine origin stimulated antibodies and cutaneous reactivity only against ovine blood group factors.

Anaplasma↗
PubMed1986

Prevalence of Anaplasma marginale, Babesia bigemina and B. bovis in Nigerian cattle using serological methods.

Serum samples collected randomly from 500 cattle from the 10 northern states of Nigeria were tested for antibodies against Anaplasma marginale by indirect fluorescent antibody (IFA), card agglutination (CT) and capillary tube-agglutination (CA) tests. The serum samples were also examined for antibodies to Babesia bigemina and B. bovis by the IFA test only. Of the serum samples tested, 79.4% had antibodies against A. marginale by the IFA test, 40 and 25% in the CT and CA tests, respectively. The IFA test results for B. bigemina and B. bovis were 29.4 and 14.1%, respectively.

Agglutination Tests↗
PubMed1996

Effect on intraerythrocytic Anaplasma marginale of soluble factors from infected calf blood mononuclear cells.

Blood mononuclear cells (lymphocytes and monocytes) were isolated from infected calves during in vivo control of acute anaplasmosis and cultured with Anaplasma marginale organisms. Supernatants from the cultures reduced the proportion of erythrocytes containing viable A. marginale in vitro, indicating that an antibody-independent mechanism of rickettsemia control might occur during acute anaplasmosis.

Acute Disease↗
PubMed1995

Detection of Anaplasma marginale DNA in bovine erythrocytes by slot-blot and in situ hybridization with a PCR-mediated digoxigenin-labeled DNA probe.

A 409-base pair (bp) DNA fragment derived from the msp-1 beta gene of Anaplasma marginale was amplified and simultaneously labeled with digoxigenin-11-dUTP by a polymerase chain reaction (PCR) assay. The resulting digoxigenin-labeled 409-bp PCR product was used as a probe for slot-blot and in situ hybridization to detect A. marginale DNA from experimentally infected bovine erythrocytes. The hybrid formation was detected with alkaline phosphatase-conjugated anti-digoxigenin antibody and substrates 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium salt. In slot-blot hybridizations, the probe detected A. marginale DNA from approximately 1,000-10,000 infected erythrocytes in 1.25 ml of whole blood, which is equivalent to a parasitemia level of 0.00001%. The probe proved to be a A. marginale-specific when tested with 17 species of microorganisms. The applicability of the probe for diagnosis was tested by screening A. marginale infections in 2 experimentally infected splenectomized cattle before microscopically detectable parasitemias and after acute infection. After inoculation of infected blood, A. marginale infections were detected with the probe 14 days prior to detection in stained smears. Microscopically inapparent parasitemias were also detected with the probe for 2 months after acute disease. When the probe was used for in situ hybridization on methanol-fixed blood smears, probe reaction could be visualized with light microscopy on A. marginale inclusions within infected erythrocytes. The probe reaction was not observed on leukocytes and uninfected erythrocytes from infected blood smears, on erythrocytes from uninfected blood samples, or on samples infected with A. ovis, Babesia bovis, or B. bigemina. This PCR-mediated nonradioactive probe appears to be a sensitive diagnostic test for A. marginale.

Anaplasma↗
PubMed1993

Opsonization of Anaplasma marginale mediated by bovine antibody against surface protein MSP-1.

Antibody from cattle immunized with purified major surface protein-1 (MSP-1) was demonstrated to significantly enhance phagocytosis of Florida strain Anaplasma marginale by bovine macrophages in vitro. Serum immunoglobulin from individual MSP-1 immunized, protected cattle varied in ability to promote phagocytosis, however all sera were significantly opsonic as compared with sera from sham immunized control cattle.

Anaplasma↗
PubMed1997

Increased resistance to Anaplasma marginale infection in cattle chronically infected with Theileria buffeli (syn. T. orientalis).

Calves chronically infected with the benign haemoprotozoan parasite Theileria buffeli (syn. T. orientalis) and T. buffeli-free calves were experimentally infected with virulent Anaplasma marginale. The daily mean maximum parasitaemia in the T. buffeli-carrier calves was lower and delayed relative to that of the Theileria-free calves. Anaemia was less marked in the Theileria infected calves, although this difference was not statistically significant. The susceptibility of Theileria-carrier and Theileria-free older cattle to virulent A. marginale infection was also investigated. The mean maximum parasitaemia observed in the Theileria-infected cattle was significantly lower than that of the Theileria-free cattle and the time to maximum parasitaemia was increased significantly in the Theileria-infected relative to the Theileria-free cattle. Of the Theileria-carrier cattle, 33% exhibited maximum parasitaemias of less than 0.1% infected erythrocytes and no clinical anaemia as a result of A. marginale infection. In contrast, the lowest maximum parasitaemia observed in the Theileria-free cattle was 7%. The percentage of cattle requiring treatment to prevent mortality due to anaemia was 50% and 91% in the Theileria-infected and Theileria-free cattle respectively. For the duration of increasing A. marginale parasitaemia, the level of Theileria in carrier cattle was significantly depressed or undetectable. Following the resolution of peak A. marginale parasitaemia, the level of Theileria parasites increased rapidly to become significantly higher than that prior to infection and then decreased gradually to a level similar to that prior to infection. The mechanism of the increased resistance to A. marginale infection conferred by T. buffeli-carrier state is unknown, but is likely to involve non-specific cell-mediated immunity, as no serological cross-reactivity exists between these two highly divergent parasite species. The susceptibility of relatively mature cattle to clinical anaplasmosis under field conditions is likely to be significantly affected by the widespread distribution and common occurrence of T. buffeli throughout the range of A. marginale in Australia, Africa and southeast Asia.

Anaplasma↗
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↗
PubMed1982

Serologic evidence of Anaplasma marginale infection in Illinois white-tailed deer.

A total of 175 deer and 54 cattle from Crab Orchard National Wildlife Refuge in southern Illinois were tested for antibody to Anaplasma marginale, using the capillary tube agglutination test. A total of 12 (6.9%) deer and 7 (13%) cattle were serologically positive. Serologic prevalence in deer increased from 0 at 6 months of age to 14% at 2 1/2 or more years of age. Cattle less than approximately 1 year of age were not seropositive.

Anaplasmosis↗
PubMed2001

Highly conserved regions of the immunodominant major surface protein 2 of the genogroup II ehrlichial pathogen Anaplasma marginale are rich in naturally derived CD4+ T lymphocyte epitopes that elicit strong recall responses.

Genogroup II ehrlichia, including the agent of human granulocytic ehrlichiosis, Ehrlichia phagocytophila, and the bovine pathogen Anaplasma marginale, express a markedly immunodominant outer membrane protein designated major surface protein 2 (MSP2). MSP2 is encoded by a multigene family, resulting in the expression of variant B cell epitopes. MSP2 variants are sequentially expressed in the repeated cycles of rickettsemia that characterize persistent A. marginale infection and control of each rickettsemic cycle is associated with development of a variant-specific IgG response. Importantly, these persistent rickettsemic cycles are controlled at levels 100-1000 times lower than those responsible for clinical disease during acute infection. Control of rickettsemia during persistence could result from an anamnestic Th lymphocyte response to conserved regions of MSP2 that enhances the primary Ab response against newly emergent variants. Comparison of MSP2 variants reveals conserved N and C termini flanking the central, surface-exposed hypervariable region that represents the variant B lymphocyte epitopes. We demonstrate MSP2-specific CD4(+) T lymphocyte recognition of epitopes common to several strains of A. marginale and the related pathogen A. ovis. Furthermore, T lymphocyte lines from three individuals identified six to nine overlapping peptides representing a minimum of four to seven dominant or subdominant epitopes in these conserved N and C termini. Immunodominant peptides induced high levels of IFN-gamma, a cytokine associated with protection against ehrlichia and needed for rapid generation of variant-specific IgG2. The presented data support the potential importance of a strong Th lymphocyte response to invariant MSP2 epitopes in controlling rickettsemia during persistent infection to subclinical levels.

Amino Acid Sequence↗
PubMed1994

Heterologous strain challenge of cattle immunized with Anaplasma marginale outer membranes.

In this study, we tested the hypothesis that outer membrane immunization would induce protection against an antigenically variant strain. Outer membranes were isolated from the Virginia strain of Anaplasma marginale using density gradient centrifugation, combined with saponin adjuvant, and used to immunize Friesian cattle in Zimbabwe. Immunized cattle developed high antibody titers (80,000-160,000) against outer membrane polypeptides including MSP-2 and MSP-5 in both the homologous Virginia and heterologous Zimbabwe strains. Outer membrane immunized cattle were protected significantly following challenge with 10(4) Zimbabwe strain parasitized erythrocytes, demonstrated by significant differences in prepatent period and peak rickettsemia compared with adjuvant immunized control cattle. One outer membrane immunized animal was completely protected against infection. However, there were no overall significant differences in severity of anemia between cattle immunized with outer membrane and the control group, indicating that a significant reduction in rickettsemia does not necessarily result in less severe anemia.

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↗
PubMed1993

Antigen recognition on Anaplasma marginale and bovine erythrocytes: an electron microscopy study.

The reactivity of sera from Anaplasma marginale-infected bovine with red blood cells and with purified anaplasma bodies was analyzed by electron immunomicroscopy. Red blood cells from non-infected and from anaplasma-infected cows and A. marginale bodies separated from parasitized erythrocytes, were incubated with control, pre-immune and immune sera followed by anti-bovine IgG-Peroxidase. Immune sera from cows infected with the venezuelan and Florida isolate reacted with red blood cell membranes from normal and infected bovines, while sera from non-infected cows did not. The immune sera also recognized epitopes localized on the cell wall, membrane and on unidentified intracellular structures of the purified anaplasma bodies. Thus, we propose that A. marginale infection may cause structural and biochemical modifications of the plasma membrane of the bovine red blood cells during its intraerythrocytic cycle. This in turn could elicit an autoimmune type of response against its own cells that would stimulate erythrophagocitosis. The strong reactivity of the immune sera with the Anaplasma bodies suggests that the bovine immune system also recognizes epitopes located on the parasite.

Anaplasma↗
PubMed2001

CD4(+) T lymphocytes from calves immunized with Anaplasma marginale major surface protein 1 (MSP1), a heteromeric complex of MSP1a and MSP1b, preferentially recognize the MSP1a carboxyl terminus that is conserved among strains.

Native major surface protein 1 (MSP1) of the ehrlichial pathogen Anaplasma marginale induces protective immunity in calves challenged with homologous and heterologous strains. MSP1 is a heteromeric complex of a single MSP1a protein covalently associated with MSP1b polypeptides, of which at least two (designated MSP1F1 and MSP1F3) in the Florida strain are expressed. Immunization with recombinant MSP1a and MSP1b alone or in combination fails to provide protection. The protective immunity in calves immunized with native MSP1 is associated with the development of opsonizing and neutralizing antibodies, but CD4(+) T-lymphocyte responses have not been evaluated. CD4(+) T lymphocytes participate in protective immunity to ehrlichial pathogens through production of gamma interferon (IFN-gamma), which promotes switching to high-affinity immunoglobulin G (IgG) and activation of phagocytic cells to produce nitric oxide. Thus, an effective vaccine for A. marginale and related organisms should contain both T- and B-lymphocyte epitopes that induce a strong memory response that can be recalled upon challenge with homologous and heterologous strains. This study was designed to determine the relative contributions of MSP1a and MSP1b proteins, which contain both variant and conserved amino acid sequences, in stimulating memory CD4(+) T-lymphocyte responses in calves immunized with native MSP1. Peripheral blood mononuclear cells and CD4(+) T-cell lines from MSP1-immunized calves proliferated vigorously in response to the immunizing strain (Florida) and heterologous strains of A. marginale. The conserved MSP1-specific response was preferentially directed to the carboxyl-terminal region of MSP1a, which stimulated high levels of IFN-gamma production by CD4(+) T cells. In contrast, there was either weak or no recognition of MSP1b proteins. Paradoxically, all calves developed high titers of IgG antibodies to both MSP1a and MSP1b polypeptides. These findings suggest that in calves immunized with MSP1 heteromeric complex, MSP1a-specific T lymphocytes may provide help to MSP1b-specific B lymphocytes. The data provide a basis for determining whether selected MSP1a CD4(+) T-lymphocyte epitopes and selected MSP1a and MSP1b B-lymphocyte epitopes presented on the same molecule can stimulate a protective immune response.

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↗