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PubMed2005

Variation among geographically separated populations of Dermacentor andersoni (Acari: Ixodidae) in midgut susceptibility to Anaplasma marginale (Rickettsiales: Anaplasmataceae).

Anaplasma marginale is a tick-borne rickettsial pathogen of cattle that is endemic throughout large areas of the United States. Cattle that survive acute infection become life-long persistently infected carriers. In the intermountain west the Rocky Mountain wood tick, Dermacentor andersoni Stiles, is the most common vector of A. marginale. Male D. andersoni acquire A. marginale when feeding on persistently infected cattle and biologically transmit it when they transfer from infected to susceptible hosts. Host-seeking adult D. andersoni were collected from four widely separated natural populations and tested for susceptibility to midgut colonization with A. marginale. Male ticks were fed on calves persistently infected with a strain of A. marginale naturally transmitted by D. andersoni. Gut infection rates ranged from 12.5% of ticks collected from a mountain site near Hamilton, MT, to 62.5% of ticks from a rangeland site near Riley, OR. Sites near Miles City, MT, and Kamloops, British Columbia, Canada, had intermediate levels of susceptibility. The infection rates differed significantly among populations, and the same populations sampled in two consecutive years were not significantly different from one year to the next. Although there was variation among the populations in the size of ticks, size was unrelated to acquisition of gut infection. Quantitative polymerase chain reaction (PCR) demonstrated that there was no significant difference between populations in the mean number of genome copies in the guts of infected ticks. A. marginale from infected ticks was genotyped to confirm that they were all infected with the laboratory strain, and a sample of 682 field-collected D. andersoni was surveyed for A. marginale by nested PCR; none were found to be naturally infected. Infection of the gut is an essential constituent of vector competence for A. marginale; in this study, we have demonstrated significant variation among populations in this key component of vector competence.

Anaplasma marginale↗

PubMed1984

Serologic and clinical responses of premunized, vaccinated, and previously infected cattle to challenge exposure by two different Anaplasma marginale isolates.

Two Anaplasma marginale isolates, one originating in Florida (FAM) and the other from Virginia (VAM), were compared immunologically by cross-challenge exposure of 14 Anaplasma carrier cattle, 8 previously infected cattle, and 6 splenectomized carrier calves. In addition, 28 cattle vaccinated with a commercially available adjuvant killed vaccine and 22 nonvaccinated cattle were challenge exposed with either FAM or VAM. A detectable clinical response was not produced by either FAM or VAM challenge exposure in carrier and previously infected cattle; however, evidence of A marginale growth as characterized by low percentages of parasitemia and increased serum complement-fixation titers was seen in carrier cattle given a heterologous challenge organism and in previously infected cattle inoculated with either homologous or heterologous organisms. Among splenectomized calves, there was virtually no cross protection to the heterologous challenge exposure, whereas a homologous challenge failed to elicit any detectable response. Vaccinated cattle were resistant to VAM exposure, but the clinical response to FAM exposure was severe with a 47% mortality. Most of these cattle displayed typical acute anaplasmosis that was only marginally less severe than that encountered in nonvaccinated cattle.

Anaplasma↗

PubMed2004

Infection of endothelial cells with Anaplasma marginale and A. phagocytophilum.

Anaplasma marginale and A. phagocytophilum are obligate intracellular, tick-borne pathogens that target erythrocytes and neutrophil granulocytes, respectively. Because ticks do not directly tap blood vessels, an intermediate tissue may mediate infection of blood cells. We considered that vascular endothelium interacts with circulating blood cells in vivo, and could be involved in pathogenesis and dissemination of the organisms. We used light and electron microscopy and immune labeling to show that A. phagocytophilum invaded rhesus (RF/6A), human (HMEC-1, MVEC), as well as bovine (BCE C/D-1b) endothelial cell lines, whereas A. marginale infected rhesus and bovine endothelial cells. A. marginale formed large intracellular inclusions that appeared smooth and solid at first, and subsequently coalesced into discrete granules. A. phagocytophilum formed numerous smaller inclusions in each cell. Within 1-3 weeks, the monolayers were destroyed, and lysed cultures were diluted onto fresh monolayers. Electron microscopy demonstrated uneven distribution of A. marginale inside large inclusions, with reticulated forms grouped more tightly than denser cells, whereas in A. phagocytophilum individual organisms appeared more evenly spaced. Specific polyclonal and monoclonal antibodies both labeled A. marginale and A. phagocytophilum in endothelial cells, and oligonucleotide primers complimentary to either A. marginale or A. phagocytophilum amplified their expected target from these cultures. In conclusion, we demonstrate that relevant microvascular endothelium is susceptible to anaplasmas in vitro and may present a link that could explain development of the immune response and persistent infection.

Anaplasma marginale↗

PubMed2001

Molecular phylogeny and biogeography of North American isolates of Anaplasma marginale (Rickettsiaceae: Ehrlichieae).

Anaplasma marginale (A. marginale) is a tick-borne ehrlichial pathogen of cattle that causes the disease anaplasmosis. Six major surface proteins (MSPs) have been identified on A. marginale from cattle and ticks of which three, MSP1a, MSP4 and MSP5, are from single genes and do not vary within isolates. The other three, MSP1b, MSP2 and MSP3, are from multigene families and may vary antigenically in persistently infected cattle. Several geographic isolates have been identified in the United States which differ in morphology, protein sequence and antigenic properties. An identifying characteristic of A. marginale isolates is the molecular weight of MSP1a which varies in size among isolates due to different numbers of tandemly repeated 28-29 amino acid peptides. For these studies, genes coding for A. marginale MSP1a and MSP4, msp1alpha and msp4, respectively, from nine North American isolates were sequenced for phylogenetic analysis. The phylogenetic analysis strongly supports the existence of a south-eastern clade of A. marginale comprised of Virginia and Florida isolates. Analysis of 16S rDNA fragment sequences from the A. marginale tick vector, Dermacentor variabilis, from various areas of the United States was used to evaluate possible vector-parasite co-evolution. Our phylogenetic analysis supports identity between the most parsimonious tree from the A. marginale MSP gene data and the tree that reflected the western and eastern clades of D. variabilis. These phylogenetic analyses provide information that may be important to consider when developing control strategies for anaplasmosis in the United States.

Amino Acid Sequence↗

PubMed1989

Immunization of cattle with the MSP-1 surface protein complex induces protection against a structurally variant Anaplasma marginale isolate.

The Anaplasma marginale surface protein complex MSP-1 of the Florida isolate is composed of a 105-kilodalton (kDa) polypeptide, which bears a neutralization-sensitive epitope, and a 100-kDa polypeptide. Antigenically similar polypeptides in the Okanogan, Wash. (Washington-O), isolate MSP-1 are 86 and 100 kDa, respectively. Immunization of cattle with Florida isolate MSP-1 induced antibody titers to both MSP-1 polypeptides and protected cattle against homologous and heterologous challenge.

Anaplasma↗

PubMed1994

Intermolecular relationships of major surface proteins of Anaplasma marginale.

Immunization with Anaplasma marginale membranes containing major surface proteins (MSPs) induces protective immunity against clinical disease (N. Tebele, T. C. McGuire, and G. H. Palmer, Infect. Immun. 59:3199-3204, 1991). For use in design of a recombinant antigen subunit vaccine for A. marginale, intermolecular relationships of known A. marginale MSPs were analyzed. Under nonreducing conditions, MSP-2 and MSP-5 occur as multimers. A large (> 300-kDa-molecular-mass), nonreduced protein complex contained MSP-1a linked by disulfide bonds to MSP-1b and by noncovalent bonds to MSP-5. MSP-2 was also noncovalently bound to this complex. The nearest neighbor membrane proteins were identified by cross-linking reactions followed by immunoblotting with anti-MSP antibodies. A cross-linked aggregate retained in the stacking gel contained MSP-1a, MSP-1b, MSP-2, MSP-3, MSP-4, and MSP-5. Collectively, the data indicate that MSP-2 and MSP-5 occur as monomers and disulfide-bonded multimers. The MSP-1 complex occurs as both disulfide-bonded and noncovalently associated MSP-1 and MSP-1b, and MSP-2 and MSP-5 are noncovalently associated with MSP-1. Also, MSP-1, MSP-2, MSP-3, and MSP-4 are nearest neighbors, and MSP-5 is noncovalently associated with this cross-linked complex.

Anaplasma↗

PubMed1988

Radioimmunoassay for Anaplasma marginale antibodies in cattle.

A radioimmunoassay is described for use in the detection of Anaplasma marginale antibodies in cattle sera. Optimal sensitivity and specificity were obtained by using 2 antigens, an A marginale antigen and a RBC antigen (obtained before infection was established) from the same calf. In addition, sera were preabsorbed with RBC from healthy cattle and with sonicated Babesia bovis. Of 86 sera obtained from cattle with A marginale infection (as determined by blood smear examination or by results of subinoculation of blood from such infected cattle into splenectomized calves), 85 had positive results by use of this test. Of 100 sera obtained from cattle raised in an anaplasmosis-free area, 98 yielded negative results, and sera obtained from 35 cattle (97 sera) infected with B bigemina and from 18 cattle infected with Theileria orientalis yielded negative results. By use of this test, 99 of 100 sera obtained from cattle with B bovis infection were negative for A marginale. Anaplasma marginale antibodies were detected in 18 cattle that had been pastured in a Boophilus microplus-free area for 2 years after natural infection. After 3 years, 16 of these cattle were still seropositive for A marginale. Sixteen cattle pastured in a Bo microplus-infested area had detectable antibody against A marginale 27 months after initial infection with A marginale. Sensitivity and specificity of the test were assessed as 98.8% for each.

Anaplasma↗

PubMed1990

Development of a recombinant Anaplasma marginale DNA probe.

An Anaplasma marginale DNA probe has been developed by using an improved method for the isolation of genomic DNA. Purified genomic A. marginale DNA from the St. Croix isolate was partially digested with Sau 3A1 into fragments (greater than or equal to 5.0 kb). The restriction fragments were cloned using standard techniques in the pBR322 vector and used to transform E. coli (DH5) host cells. The recombinant A. marginale DNA library was screened by the colony lifting procedure. Colonies containing plasmids with A. marginale DNA inserts were identified by hybridization with a genomic A. marginale DNA radiolabeled probe (32P). Seven recombinant A. marginale DNA probes were evaluated by dot-blot in vitro hybridization assays to identify candidates as diagnostic tools in bovine anaplasmosis studies. Specificity and sensitivity experiments were carried out by using heterologous and homologous DNAs. The heterologous panel contained bovine DNA (WBC) and blood parasites DNA from Babesia bovis (Bb), Babesia bigemina (Bbi), Eperythrozoon suis (Es) and Eperythrozoon wenyoni (Ew). The homologous DNA panel included A. marginale DNAs of 12 different isolates which were isolated in the Caribbean, Mexico, and the U.S.A. The selected diagnostic probe was identified as pSt. Croix A1, and labeled with 32P by using in vitro nick translation and random primer techniques. The pSt. Croix A1 probe demonstrated 100% specificity and high sensitivity by hybridization in dot blotting and Southern blotting. The probe can detect 500-1000 infected erythrocytes per microliters which corresponds to a parasitemia of less than 0.01%. The A. marginale DNA insert was approximately 6.4 kb in size and a partial restriction map has been constructed.

Anaplasma↗

PubMed1991

Induction of protective immunity by using Anaplasma marginale initial body membranes.

Anaplasma marginale initial bodies of the Norton Zimbabwe strain were disrupted and separated into two membrane fractions banding at 1.15 and 1.22 g/cm3 by sucrose density centrifugation. The membrane fractions differed in their morphology and polypeptide composition. Membranes banding at 1.22 g/cm3 shared epitopes with surface-exposed polypeptides of the Florida strain of A. marginale, confirming the outer membrane location of these polypeptides. Immunization of cattle with either membrane fraction induced protection against homologous challenge, as demonstrated by significantly less anemia and lower peak rickettsemia values compared with those of adjuvant-immunized and nonimmunized calves. Protection correlated with antibody titer to membrane polypeptides. Although both membrane fractions induced protection, a 31-kDa polypeptide was the only common antigen to both fractions, as shown by reactivity of immune sera. Identification of membrane antigens capable of inducing protective immunity should facilitate development of vaccines against anaplasmosis suitable for use in Zimbabwe.

Anaplasma↗

PubMed2003

Antigenic characterization of Brazilian isolates of Anaplasma marginale.

Antigenic characterization of Anaplasma marginale isolates, by identifying conserved and variable epitopes of major surface proteins (MSP), is an important tool for vaccine development against this rickettsia. The B cell epitopes of A. marginale isolates from three microregions of the State of Pernambuco and one from the State of Mato Grosso do Sul, Brazil, were characterized by indirect fluorescent antibody technique (IFAT) and Western blot (WB) with 15 monoclonal antibodies (MAbs). The epitope recognized by MAb ANA22B1 (MSP-1a) was conserved by IFAT and WB (73-81 kDa). MSP-2 epitopes recognized by MAbs ANAO58A2 and ANAO70A2 were conserved by IFAT, while ANAO50A2 and ANA66A2 epitopes were polymorphic; in the WB, the MAbs ANAO50A2 and ANAO70A2 identified bands of 45 kDa only in the Pernambuco-Mata isolate. None of the isolates reacted with MAb ANAR75C2 (MSP-3). The MSP-4 epitope recognized by MAb ANAR76A1 was conserved by IFAT, as well as the MSP-5 epitope recognized by MAb ANAF16C1 by IFAT and WB (16 kDa). The MAbs ANAR17A6, ANAR83B3, ANAR94C1, ANAO24D5 and ANAR19A6 identified conserved epitopes by IFAT. MSP-1, MSP-2 and MSP-4, which previously showed partial protection in experimental trials, are also potential immunogens to be employed in Brazil, due to the B cell epitope conservation.

Anaplasma↗

PubMed1979

Electron microscope studies of Anaplasma marginale in an Aedes albopictus culture system.

Anaplasma marginale was seen by electron microscopy within the cytoplasm of Aedes albopictus cells in culture. Anaplasma marginale was seen in free bovine RBC as well as in RBC phagocytized by Ae albopictus cells. Some A marginale organisms, both intracellular and extracellular, remained intact for as long as 60 days in culture, and Anaplasma inclusion appendages were seen in free-lysed RBC and in lysed RBC phagocytized by Ae albopictus cells. Although some A marginale organisms had deteriorated by day 60, the structure of most A marginale organisms did not change appreciably from that in its natural bovine host. Reproduction of A marginale was not seen.

Aedes↗

PubMed2004

Stochastic transmission of multiple genotypically distinct Anaplasma marginale strains in a herd with high prevalence of Anaplasma infection.

Multiple genotypically unique strains of the tick-borne pathogen Anaplasma marginale occur and are transmitted within regions where the organism is endemic. In this study, we tested the hypothesis that specific A. marginale strains are preferentially transmitted. The study herd of cattle (n = 261) had an infection prevalence of 29% as determined by competitive inhibition enzyme-linked immunosorbent assay and PCR, with complete concordance between results of the two assays. Genotyping revealed the presence of 11 unique strains within the herd. Although the majority of the individuals (70 of 75) were infected with only a single A. marginale strain, five animals each carried two strains with markedly distinct genotypes, indicating that superinfection does occur with distinct A. marginale strains, as has been reported with A. marginale and A. marginale subsp. centrale strains. Identification of strains in animals born into and infected within the herd during the period from 1998 to 2003 revealed no significant difference from the overall strain prevalence in the herd, results that do not support the occurrence of preferential strain transmission within a population of persistently infected animals and are most consistent with pathogen strain transmission being stochastic.

Anaplasma marginale↗

PubMed2026

Molecular epidemiology and phylogenetic analysis of Anaplasma ovis and Anaplasma marginale in Ixodidae infesting livestock in northwestern Iran.

BACKGROUND: Anaplasma marginale and Anaplasma ovis are tick-borne obligate intracellular bacteria causing anaplasmosis in cattle and small ruminants, respectively, with considerable economic losses worldwide. Given the favorable ecological conditions for tick survival and the limitation of data on Anaplasma spp. in local tick populations in northwestern Iran, this study aimed to molecular and phylogenetic analysis of A. ovis and A. marginale in Ixodidae infesting livestock in northwestern Iran. METHODS: In this cross-sectional study, a total of 780 ixodid ticks were collected from livestock across 198 herds in 11 counties of Ardabil Province during 2025. Ticks were morphologically identified and grouped into pools based on species and host type. Genomic DNA was extracted using a commercial kit and molecular detection of A. ovis and A. marginale was performed using PCR assays targeting the 16 S rRNA and groEL genes. Positive samples were submitted for Sanger sequencing to confirm the identity of Anaplasma spp., phylogenetic analysis was conducted using reference sequences from GenBank (NCBI) using MEGA software (version 12). Statistical analyses were conducted using SPSS (version 25), and associations between categorical variables were assessed using Fisher's exact test (p < 0.05). RESULTS: Eight tick species belonging to three genera were identified. Among the hosts, sheep exhibited the highest infestation rate (49.3%). Hyalomma anatolicum anatolicum was the most prevalent species (25.3%) and was present in all sampled counties. Tick distribution varied significantly among host species (p = 0.003) and geographic locations (p = 0.002). PCR analysis detected A. ovis DNA in 16.6% (4/24) and A. marginale DNA in 8.3% (2/24) of tick pools. Positive pools were primarily associated with Rhipicephalus spp. and Dermacentor marginatus. According to the results of the statistical analysis a significant association was found between tick species and host type (χ² = 13.87, p = 0.0031), with Hyalomma anatolicum anatolicum more prevalent in sheep (p = 0.001). Tick abundance varied across counties (χ² = 16.42, p = 0.002), with highest densities in Nir, Khalkhal, and Kowsar (χ² = 14.21, p = 0.0028). No significant association was observed between Anaplasma positivity and tick species (p = 0.21) or host type (p = 0.09). CONCLUSIONS: The detection of A. ovis and A. marginale DNA in ixodid tick pools indicates their circulation in Ardabil Province. However, due to pooled sampling and the limited number of positive samples, the infection rate at the individual tick level could not be determined. These findings also highlight the importance of a One Health approach, considering the interconnected roles of animal health, tick vectors, and the environment in the transmission and control of tick-borne diseases.

Animals↗

PubMed1985

High-yield preparation of purified Anaplasma marginale from infected bovine red blood cells.

Purification of Anaplasma marginale from infected bovine RBC was achieved through enzyme treatment and density-gradient centrifugation. A relative yield of 41.6% was obtained by dividing the number of organisms in the final purified preparation by the number of A marginale-infected RBC. Purified parasites were verified as A marginale by light microscopy, electron microscopy, and immunologic tests. The purified parasites reacted positively with calf and rabbit anti-A marginale sera in interfacial and slide agglutination tests. Anti-bovine RBC serum did not agglutinate purified A marginale, indicating absence of any contaminating RBC stroma. Anaplasma marginale was antigenic, but did not cause infection when the preparation was inoculated into a susceptible calf. The density of A marginale was determined to be 1.19 g/ml and cell diameters ranged from 0.25 to 0.63 micron. This method provided procedures for obtaining A marginale free of bovine RBC antigens for accurate biochemical assays and vaccine production.

Agglutination Tests↗

PubMed2004

Genetic diversity and molecular phylogeny of Anaplasma marginale isolates from Minas Gerais, Brazil.

Anaplasma marginale (Rickettsiales: Anaplasmataceae), a tick-borne pathogen of cattle, is endemic in tropical and subtropical regions of the world, and many isolates of A. marginale may occur in a given geographic area. Phylogenetic relationships have been reported for A. marginale isolates from the US using gene and protein sequences of MSP1a and msp4. These studies demonstrated that msp4 sequences, but not MSP1a DNA or protein sequences, provide phylogeographic information and also that MSP1a sequences are highly heterogeneous among A. marginale populations. However, little information is available on the genetic diversity of A. marginale isolates from other regions of the world. The present study was undertaken to examine genetic variation among 10 isolates of A. marginale obtained from infected cattle in the State of Minas Gerais, Brazil, where A. marginale is endemic. Neighbor-joining analysis of msp4 sequences of Brazilian and New World isolates of A. marginale from Argentina, Mexico and the US provided bootstrap support for a Latin American clade. The sequences of the MSP1a repeats of four Brazilian isolates of A. marginale were compared to sequences of Latin American and US isolates. The MSP1a repeated sequences of Latin American isolates of A. marginale had nine repeat forms, alpha-phi, which have not been reported previously in North American isolates of A. marginale. Furthermore, the repeated forms tau, sigma and mu were only present in the Brazilian isolates. The results demonstrated that the genetic heterogeneity observed among isolates of A. marginale is common in endemic areas, independent of the predominant tick vector and is consistent with previous studies in which msp4 provided phylogeographic information about A. marginale isolates, while MSP1a was found not to be a useful marker for phylogeographic characterization of A. marginale isolates.

Amino Acid Sequence↗

PubMed2004

Molecular conservation of MSP4 and MSP5 in Anaplasma marginale and A. centrale vaccine strain.

Anaplasma centrale msp4 and msp5 genes were cloned and sequenced, and the recombinant proteins were expressed. The identity between Anaplasma marginale and A. centrale MSP4 was 83% in the nucleotide sequences and 91.7% in the encoded protein sequences. A. centrale msp5 nucleotide sequences shared 86.8% identity with A. marginale msp5, and there was 92.9% homology between A. centrale and A. marginale encoded amino acids of the MSP5 protein. Southern blots hybridized with probes derived from the msp4 and msp5 central regions indicate that msp4 and msp5 of A. centrale are encoded by single copy genes. Recombinant MSP4 and MSP5 fusion proteins reacted with anti-A. marginale monoclonal antibodies ANAR76A1 and ANAF16C, respectively, demonstrating the conservation of conformation-sensitive B-cell epitopes between A. centrale and A. marginale. These data demonstrate the structural and antigenic conservation of MSP4 and MSP5 in A. centrale and A. marginale. This conservation is consistent with the cross-protective immunity between A. marginale and A. centrale and supports the development of improved vaccines based upon common outer membrane proteins.

Amino Acid Sequence↗

PubMed2004

Recent studies on the characterization of Anaplasma marginale isolated from North American bison.

Anaplasma marginale (Rickettsiales: Anaplasmataceae), a tick-borne pathogen of cattle, is endemic in tropical and subtropical regions of the world. Many geographic isolates of A. marginale occur worldwide that have been identified by major surface protein (MSP) 1a, which varies in sequence and molecular weight owing to different numbers of tandem 28-29 amino acid repeats. Although serologic tests have identified American bison, Bison bison, as being infected with A. marginale, the present studies were undertaken to confirm A. marginale infection in bison, to characterize bison isolates, and to compare the phylogenetic relationship of the bison isolates with other A. marginale isolates from North America. Nine A. marginale isolates derived from Canadian bison possessed identical msp4 sequences with one characteristic silent nucleotide change. The sequence of MSP1a was determined for one Canadian and two U.S. bison isolates of A. marginale, and these isolates contained 4 and 5 tandem repeats, respectively. One U.S. bison isolate tested for infectivity proved to be infective for cattle and transmitted by Dermacentor variabilis ticks. the results of this study demonstrated that these A. marginale isolates obtained from bison were similar to ones derived from naturally infected cattle.

Anaplasma marginale↗

PubMed2006

Selection for simple major surface protein 2 variants during Anaplasma marginale transmission to immunologically naïve animals.

Anaplasma marginale, a rickettsial pathogen, evades clearance in the animal host by antigenic variation. Under immune selection, A. marginale expresses complex major surface protein 2 mosaics, derived from multiple donor sequences. However, these mosaics have a selective advantage only in the presence of adaptive immunity and are rapidly replaced by simple variants following transmission.

Anaplasma marginale↗