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PubMed1964

IMMUNOLOGY AND SEROLOGY OF ANAPLASMA MARGINALE. I. FRACTIONATION OF THE COMPLEMENT-FIXING ANTIGEN.

Rogers, Thomas E. (Louisiana State University, Baton Rouge), Richard J. Hidalgo, and George T. Dimopoullos. Immunology and serology of Anaplasma marginale. I. Fractionation of the complement-fixing antigen. J. Bacteriol. 88:81-86. 1964.-Studies were conducted to fractionate and purify the complement-fixation (CF) antigen of Anaplasma marginale in infected erythrocytes of cattle. Initial attempts were made to resolve the antigen from crude stromatal preparations by various chemical and physical methods. Fractionation procedures involving partial and total lipid extraction suggested that the CF antigen was lipoprotein in nature. Fluorocarbon deproteinization of stromatal antigens was also attempted. A method was developed for the preparation of a desirable Anaplasma CF antigen which involved disintegration of infected erythrocytes by sonic vibration and separation of the antigen by differential centrifugation. Antigens prepared by this method were highly specific, colorless, did not exhibit anticomplementary activity, and possessed higher titers than standard Anaplasma antigens. When density-gradient sedimentation was applied to sonic extracts of infected cells, it was demonstrated that the CF antigen could also be fractionated by this method.

Anaplasma↗
PubMedDate not supplied

Detection of Anaplasma phagocytophilum in animals by real-time polymerase chain reaction.

The aim of this study was to detect Anaplasma phagocytophilum in wild and domesticated animals and to identify the phylogenetic relationships of different strains of this bacterium. We adapted six published conventional methods targeting 16S fragments for real-time polymerase chain reaction. Initial screening of samples from 419 animals found 37 Anaplasma positives, later confirmed with several different primers and a TaqMan probe. We also performed DNA quantification and melting curve analysis. The nucleic acid of Anaplasma sp. was detected in a higher percentage of cases in members of the deer family, hares, bank voles and mice (12.5 approximately 15%) than in foxes, boars, cows, and horses (around 4 approximately 6%). We also performed blood analysis of cows, horses, mice, and ticks removed from animals, evaluating the presence of antibodies against granulocytic Anaplasma sp. Finally, we subjected 11 randomly selected PCR amplified products to direct sequencing and we constructed the corresponding phylogenetic tree with respect to the Ehrlichia equi sequence, homologous to the human granulocytic ehrlichiosis agent. Mutual identity of the sequencing ranged from 99% to 100%.

Anaplasma↗
PubMed2007

Characterization of Anaplasma phagocytophilum major surface protein 5 and the extent of its cross-reactivity with A. marginale.

Major surface protein 5 (Msp5) of Anaplasma marginale is highly conserved in the genus Anaplasma and the antigen used in a commercially available competitive enzyme-linked immunosorbent assay (cELISA) for serologic identification of cattle with anaplasmosis. This study analyzes the degrees of conservation of Msp5 among various isolates of Anaplasma phagocytophilum and the extent of serologic cross-reactivity between recombinant Msp5 (rMsp5) of Anaplasma marginale and A. phagocytophilum. The msp5 genes from various isolates of A. phagocytophilum were sequenced and compared. rMsp5 proteins of A. phagocytophilum and A. marginale were used separately in an indirect ELISA to detect cross-reactivity in serum samples from humans and dogs infected with A. phagocytophilum and cattle infected with A. marginale. Serum samples were also tested with a commercially available competitive ELISA that uses monoclonal antibody ANAF16C1. There were 100% sequence identities in the msp5 genes among all of the A. phagocytophilum isolates from the United States and a horse isolate from Sweden. Sheep isolates from Norway and dog isolates from Sweden were 99% identical to one another but differed in 17 base pairs from the United States isolates and the horse isolate. Serologic cross-reactivity was identified when serum samples from cattle infected with A. marginale were reacted with rMsp5 of A. phagocytophilum and when serum samples from humans and dogs infected with A. phagocytophilum were reacted with rMsp5 of A. marginale in an indirect-ELISA format. Serum samples from dogs or humans infected with A. phagocytophilum did not cross-react with rMsp5 of A. marginale when tested with the commercially available cELISA. These results suggest that rMsp5 of A. phagocytophilum is highly conserved among United States and European isolates and that serologic distinction between A. phagocytophilum and A. marginale infections cannot be accomplished if rMsp5 from either organism is used in an indirect ELISA.

Anaplasma marginale↗
PubMed2003

Prevalence of four species of Borrelia burgdorferi sensu lato and coinfection with Anaplasma phagocytophila in Ixodes ricinus ticks in central Germany.

A total of 305 Ixodes ricinus ticks collected from three areas of Thuringia in central Germany were investigated for infection with Borrelia burgdorferi sensu lato species and Anaplasma phagocytophila. Overall, 11.1% were infected with Borrelia burgdorferi and 2.3% with Anaplasma phagocytophila. Adult ticks showed a significantly higher rate of infection with both borreliae and Anaplasma phagocytophila. Borrelia garinii (55.9%) was detected most frequently, followed by Borrelia burgdorferi sensu stricto (32.4%), Borrelia afzelii (17.6%), and Borrelia valaisiana (5.9%). Four ticks had dual infection with Borrelia garinii and Borrelia burgdorferi sensu stricto. Two of the Borrelia-positive ticks were coinfected with Anaplasma phagocytophila.

Adult↗
PubMed1998

Characterization of Anaplasma isolates from eland (Taurotragus oryx). Pathogenicity in cattle and sheep and DNA profiles analysis.

Two eland Anaplasma isolates, AnapE1, from Kenya, and AnapE2, from South Africa were characterised. Their characterization was based on their pathogenicity to intact and splenectomized cattle and sheep and also their DNA profiles. Their DNA profiles were analysed and compared to Anaplasma marginale, A. ovis and A. centrale after endonuclease restrictions and probing with Anaplasma DNA probes, AC5-12 and AC-1. The results of the pathogenicity trials showed AnapE1 to be similar to A. ovis and AnapE2 an isolate of A. marginale. On DNA profiles, AnapE1 was close to A. ovis, with differences that occur even in same Anaplasma species isolates from different locations. On the other hand, AnapE2, resembled one of the A. marginale isolates known to occur in South Africa. The DNA profiles correlated well with the pathogenicity results. It is concluded that elands are carriers of both A. marginale and A. ovis parasites and are therefore important reservoirs that need attention in epidemiology of anaplasmosis.

Anaplasma↗
PubMed2000

Establishment and characterization of an Oklahoma isolate of Anaplasma marginale in cultured Ixodes scapularis cells.

Anaplasma marginale is a tick-borne hemoparasite of cattle worldwide. The Virginia isolate of A. marginale was propagated previously in a cell line derived from embryos of the tick, Ixodes scapularis. The cultured Anaplasma (VA-tc) was passaged continuously for over 4 years and retained its infectivity for cattle and antigenic stability. We report herein the continuous in vitro cultivation of a second isolate of A. marginale derived from a naturally infected cow in Oklahoma (OK-tc). Blood from the infected cow was subinoculated into a splenectomized calf and blood collected at peak parasitemia was frozen, thawed and used as inoculum on confluent tick cell monolayers. Colonies of Anaplasma were apparent in low numbers at 9 days post exposure (PE) and infection in monolayers reached 100% by 4-5 weeks PE. Cultures were passaged by placing supernatant onto fresh tick cell monolayers at a dilution of 1:5 or 1:10. By the third passage development of the OK-tc was similar to that of the VA-tc and a 1:5 dilution resulted in 100% infection in 10-12 days. Inoculation of OK-tc into a splenectomized calf caused clinical anaplasmosis and Dermacentor ticks that fed on this calf transmitted the organism to a second susceptible calf. Major surface proteins (MSPs) 1-5 of the OK-tc were compared with homologous proteins present on VA-tc and the erythrocytic stage of the Oklahoma isolate. The MSPs 1, 2, 4, 5 were conserved on the OK-tc but there was evidence for structural variation in MSP3 between the cultured and erythrocytic stage of Anaplasma. MSP2 and MSP3 were the major proteins recognized by serum from infected cattle. Two-dimensional gels also identified positional differences between VA-tc and OK-tc in MSP2 and MSP3. The OK-tc may have potential to be used as antigen for development of an improved vaccine for anaplasmosis in the South Central United States.

Anaplasma↗
PubMed2002

A msp1alpha polymerase chain reaction assay for specific detection and differentiation of Anaplasma marginale isolates.

Anaplasma marginale is the causative agent of bovine anaplasmosis, a disease which can be protected by vaccination with the less pathogenic Anaplasma species, A. centrale. Currently, there is no polymerase chain reaction (PCR) assay available which differentiates between different species of Anaplasma or which can differentiate isolates of A. marginale within outbreaks and between different countries. A molecular test specific for A. marginale would be ideal for the identification of Anaplasma species in wild ruminants, as possible reservoirs of anaplasmosis, and to differentiate between A. marginale from A. centrale. A PCR assay was designed to amplify the major surface protein 1alpha gene of the rickettsial bovine pathogen, A. marginale both as an inter- and intra-specific test. The test did not amplify A. centrale or A. ovis, and discriminated A. marginale by amplifying repeat regions within the msp1alpha gene which vary in number between many isolates. The nested A. marginale amplicons varied in size from 630 to 1190bp representing one to eight internal repeats. All 22 Australian isolates tested amplified a 630bp product (one repeat) in contrast to all 19 non-Australian isolates tested. Eight sequences from Australian isolates from different geographical regions confirmed the conserved nature of the Australian A. marginale msp1alpha genes. The Australian 'repeat unit' MSP1a deduced amino acid sequence has been designated as Australian type 1. The msp1alpha PCR method developed here enabled the amplification and comparison of A. marginale isolates originating from North and South America, Africa, Israel and Australia. The method is sensitive and specific for A. marginale. Although additional msp1alpha products were amplified from at least two Australian isolates, the results suggest limited introduction of A. marginale into Australia.

Amino Acid Sequence↗
PubMed2003

Identification of Ehrlichia chaffeensis, Anaplasma phagocytophilum, and A. bovis in Haemaphysalis longicornis and Ixodes persulcatus ticks from Korea.

A total of 1,467 tick (1,463 of Haemaphysalis longicornis, three of Ixodes persulcatus and one of I. turdus) collected from nine provinces of Korea were examined by TaqMan real-time PCR for the presence of Ehrlichia and Anaplasma species. One set of primers and a probe were designed for detection of all of the Ehrlichia and Anaplasma species. Template DNAs (total 803) were prepared either from pools of larvae, nymphs, adult males and females, or from the salivary gland and midgut of adult ticks. Only DNAs positive in TaqMan PCR were examined for A. phagocytophilum with nested PCR and for E. chaffeensis with PCR. Four A. phagocytophilum 16S rRNA gene PCR products were sequenced for comparison with sequences previously reported. Amplification of a 16S rRNA gene fragment of Ehrlichia and Anaplasma species was observed in 364 tick DNAs (45.3% of the total). Of these 364 positive ticks, species-specific PCRs confirmed that 35 H. longicornis and one I. persulcatus were positive for A. phagocytophilum and one I. persulcatus was positive in E. chaffeensis. Except for one (AB-GGHL, GenBank accession number [GAN] AF470698), three of the four 16S rRNA gene fragment sequences of the A. phagocytophilum-positive samples were similar or identical to the sequences of variants of A. phagocytophilum deposited in GenBank. The 16S rRNA gene fragment sequence of AB-GGHL was similar to that of Anaplasma (Ehrlichia) bovis 16S rRNA (GAN U03775). The identities of the Anaplasmataceae genus and species DNA in the 327 ticks that could not be confirmed infected with either E. chaffeensis, A. phagocytophilum, or A. bovis are not known. This study is the first to demonstrate the presence of E. chaffeensis, A. phagocytophilum and A. bovis in Korean ticks.

Anaplasma↗
PubMed2002

Expression of major surface protein 2 variants with conserved T-cell epitopes in Anaplasma centrale vaccinates.

Major surface protein 2 (MSP-2), identified as a protection-inducing immunogen against Anaplasma marginale challenge, is an immunodominant outer membrane protein with orthologues in all examined Anaplasma species. Although immunization with live Anaplasma centrale has long been used to induce protection against acute disease upon challenge with virulent A. marginale, its MSP-2 structure and whether MSP-2 variants are generated during persistence of the vaccine strain was unknown. In this study, we showed that the A. centrale vaccine strain persisted for a minimum of 4 years postvaccination and generated sequential MSP-2 variants. Comparison of amino acid sequences encoded by A. centrale msp-2 transcripts from the initial postimmunization period and from sequential time points during persistence of the vaccine strain revealed a central hypervariable domain flanked by conserved amino and carboxy-terminal regions. This structure corresponded to that shown in A. marginale MSP-2, where the central hypervariable region encodes variant B-cell epitopes in the extracellular domain and the flanking transmembrane domains are rich in CD4(+)-T-cell epitopes. Importantly, at least four CD4(+)-T-cell epitopes are conserved between the two species, a finding consistent with A. marginale challenge triggering a recall response of CD4(+) T cells induced by A. centrale vaccination. The genomic arrangement is conserved between A. centrale and A. marginale with multiple msp-2 pseudogenes and a single operon-linked expression site for the full-length msp-2. This conservation of both genomic structure for generating MSP-2 variants and the CD4(+)-T-cell epitopes between these two genetically distinct Anaplasma species indicates that they present a similar repertoire of MSP-2 epitopes to the immune system and that this similarity may be responsible for all or part of the A. centrale vaccine efficacy.

Amino Acid Sequence↗
PubMed2004

Incidence and prevalence of infection with Anaplasma phagocytophilum. Prospective study in healthy individuals exposed to ticks.

The seroprevalence of human granulocytic anaplasmosis (former human granulocytic ehrlichiosis, HGE) has been documented in several studies, but little data exists on incidence rates in healthy individuals. In a prospective study, we tested 125 healthy adults (mean age 43 years)--workers of the Bialowieza Primeval Forest National Park, north-eastern Poland--for Anaplasma phagocytophilum IgG antibodies using an indirect immunofluorescence antibody assay, and for Borrelia burgdorferi IgG with ELISA in a 12-month interval. The data concerning clinical symptoms consistent with human granulocytic anaplasmosis were collected using a standardized questionnaire. Of these 125 subjects, 9 were anti-A. phagocytophilum positive at the study entry. Four participants (3.2 %) seroconverted from IgG negative to positive during the observation period. Three subjects (2.4 %) converted from initially anti-A. phagocytophilum positive to negative. Specific IgG antibodies against Borrelia burgdorferi were detected in 27 (21.6 %) individuals. Concurrence of Borrelia burgdorferi and Anaplasma phagocytophilum was observed in 3.2 %, whereas 4 % were Anaplasma phagocytophilum IgG positive and Borrelia burgdorferi IgG negative (not significant). Clinical symptoms associated with human granulocytic anaplasmosis were not present in seroconverting individuals. The obtained results confirm the occurrence of Anaplasma phagocytophilum infection in north-eastern Poland with asymptomatic clinical course.

Adult↗
PubMed2004

The risk of exposure to Anaplasma phagocytophilum infection in Mid-Eastern Poland.

Both the presence of Anaplasma phagocytophilum in ticks and the seroprevalence of human granulocytic anaplasmosis have been reported in different parts of Europe. There are few reports concerning this problem in Poland. The aim of the study was to assess the prevalence of Anaplasma phagocytophilum in ticks, and to detect antibodies against the HGE agent in serum of forest workers in the region of Mid-Eastern Poland. In our opinion, this should reflect the real probability of infection of people exposed to Ixodes tick bites. Seroactivity against Anaplasma phagocytophilum was detected in 20.6% of persons in the study group. Coexistence of anti-Borrelia burgdorferi was present in 84.6 % of individuals seropositive to A. phagocytophilum. The PCR test identifying Anaplasma phagocytophilum was positive in 13.1% of overall tick samples. The highest prevalence of infection (45.7%) was found in female ticks. Anaplasmal DNA was detected in 4.5% of male ticks and only in 0.9% of nymphs. The results of our study confirmed the existence of A. phagocytophilum in the natural environment of Mid-Eastern Poland. As the risk for infection exists, it should call the attention of public health services to the possibility of an increasing number of patients with this disease.

Anaplasma phagocytophilum↗
PubMed1988

Cultivation of Anaplasma marginale from cattle in a Dermacentor cell line.

A tick cell line derived from Dermacentor variabilis (RML-15) was inoculated with bovine RBC infected with Anaplasma marginale. Two hours after inoculation, numerous RBC were phagocytized by the tick cells. After one passage of the cell culture, numerous groups of Anaplasma-like particles were seen in the tick cell cytoplasm. Increased numbers of Anaplasma-like particles also were present. Seemingly, Anaplasma can multiply in tick cells.

Anaplasma↗
PubMed1978

Immune responses of cows to a chemically modified anaplasma antigen.

Cows were vaccinated at 0 and 28 days with either a standard or a chemically modified anaplasma antigen. The humoral antibody response was measured by the anaplasma complement-fixation and card agglutination tests. The antibody response to bovine erythrocyte antigens was also evaluated. Peripheral blood leukocytes were collected for an in vitro lymphocyte-transformation test. Both groups of vaccinated cows developed a significant cell-mediated response as measured by lymphocyte-transformation test. However, only the cows given the standard anaplasma antigen developed anaplasma antibody and significant titer of bovine erythrocyte antibodies. Vaccinated and control cows were challenge exposed 1 month after the 2nd antigen injection. The vaccinated cows developed lower levels of parasitemia than did the control nonvaccinated cows.

Agglutination Tests↗
PubMed1982

Morphologic alteration of Anaplasma marginale in calves treated with a dithiosemicarbazone.

Blood and clotted blood samples from 3 calves with anaplasmosis were examined by light and electron microscopies before and after the calves were treated twice with alpha-ethoxyethylglyoxal dithiosemicarbazone (given IV). In the treated calves anaplasma bodies were swollen and irregularly shaped and had rough surfaces when viewed by light microscopy. Posttreatment electron microscopic examinations revealed swelling and vacuolation of initial bodies of anaplasma bodies, and ballooning of the vesicular membrane surrounding the anaplasma body. Other initial bodies were ameboid in shape this was or was not accompanied by rupture of the vesicular membrane of the anaplasma body. Necrotic, ameboid-shaped initial bodies also were seen. Some necrotic initial bodies were surrounded by complete vesicular membrane, and others were close to a broken, curled vesicular membrane.

Anaplasma↗
PubMed1981

Infection of splenectomized calves with Anaplasma ovis.

An Anaplasma was not recovered from 2 splenectomized calves at 17 days after A ovis inoculation, but was recovered from 1 of the calves at 177 days after inoculation. In a 3rd calf exposed to A ovis, an Anaplasma was recovered at 177 and 262 days after inoculation. the Anaplasma recovered from these calves was determined to be A ovis on the basis of cross-infectivity trials, using Anaplasma marginale.

Anaplasma↗
PubMed1977

Cell-mediated immune response to virulent and attenuated Anaplasma marginale administered to cattle in live and inactivated forms.

The leukocyte migration-inhibition test (LMIT) and lymphocyte transformation of blood leukocytes from cattle exposed to virulent, attenuated, and inactivated preparations of Anaplasma marginale served as indices of cell-mediated immunity in bovine anaplasmosis. The LMIT response increased markedly in cattle vaccinated with attenuated A marginale or injected with virulent A marginale. Cattle inoculated with inactivated anaplasma developed a transient, low-level LMIT response. The response to antigenic stimulation recorded in the lymphocyte transformation test of leukocytes collected from cattle which were given live anaplasma was less than the response elicited from leukocytes collected from cattle vaccinated with inactivated anaplasma in adjuvant.

Anaplasma↗
PubMed2006

Differential expression and sequence conservation of the Anaplasma marginale msp2 gene superfamily outer membrane proteins.

Bacterial pathogens in the genera Anaplasma and Ehrlichia encode a protein superfamily, pfam01617, which includes the predominant outer membrane proteins (OMPs) of each species, major surface protein 2 (MSP2) and MSP3 of Anaplasma marginale and Anaplasma ovis, Anaplasma phagocytophilum MSP2 (p44), Ehrlichia chaffeensis p28-OMP, Ehrlichia canis p30, and Ehrlichia ruminantium MAP1, and has been shown to be involved in both antigenic variation within the mammalian host and differential expression between the mammalian and arthropod hosts. Recently, complete sequencing of the A. marginale genome has identified an expanded set of genes, designated omp1-14, encoding new members of this superfamily. Transcriptional analysis indicated that, with the exception of the three smallest open reading frames, omp2, omp3, and omp6, these superfamily genes are transcribed in A. marginale-infected erythrocytes, tick midgut and salivary glands, and the IDE8 tick cell line. OMPs 1, 4, 7 to 9, and 11 were confirmed to be expressed as proteins by A. marginale within infected erythrocytes, with expression being either markedly lower (OMPs 1, 4, and 7 to 9) or absent (OMP11) in infected tick cells, which reflected regulation at the transcript level. Although the pfam01617 superfamily includes the antigenically variable MSP2 and MSP3 surface proteins, analysis of the omp1-14 sequences throughout a cycle of acute and persistent infection in the mammalian host and tick transmission reveals a high degree of conservation, an observation supported by sequence comparisons between the St. Maries strain and Florida strain genomes.

Amino Acid Sequence↗
PubMed2005

Potential vertebrate reservoir hosts and invertebrate vectors of Anaplasma marginale and A. phagocytophilum in central Spain.

Organisms in the genus Anaplasma are obligate intracellular pathogens that multiply in both vertebrate and invertebrate hosts. The type species, A. marginale, causes bovine anaplasmosis and only infects ticks and ruminants. A. phagocytophilum causes human and animal granulocytic anaplasmosis, and genetically closely related strains show a wide host range, including ticks, ruminants, rodents, equids, canids, birds, and humans. Recent reports demonstrated that A. marginale and A. phagocytophilum co-exist in geographic areas and that concurrent infections occur in ruminants and ticks. In this study, we characterized A. marginale and A. phagocytophilum infections in wild and domestic animals, and ticks collected in central Spain by serology, PCR, and sequence of 16S rRNA genotypes. Species tested included humans, cattle, dogs, rodents, Iberian red deer, European wild boar, birds, and ticks. Species of hematophagous Diptera were analyzed as potential mechanical vectors of Anaplasma spp. A. marginale was detected in tabanids, ticks, cattle, and deer, while A. phagocytophilum was detected in ticks, deer, cattle, and birds. Concurrent infections of the two Anaplasma were found in cattle and deer. These results illustrate the complexity of the epizootiology of A. marginale and A. phagocytophilum in regions where both pathogens co-exist and share common reservoir hosts and vectors. The increasing contact between wildlife, domestic animals, and human populations increases the risk of outbreaks of human and bovine anaplasmosis, and the difficulty of implementing surveillance and control measures.

Anaplasma marginale↗