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PubMed1990

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Anaplasma centrale and Anaplasma marginale.

An enzyme-linked immunoassay (ELISA) was applied to detect antibodies to A. centrale and A. marginale using homologous and heterologous antigens. The assay was compared with the indirect fluorescent antibody (IFA) test, and although a similar degree of sensitivity was obtained, the ELISA test had several advantages. Partially purified Anaplasma initial bodies used for antigen preparations contained negligible amounts of residual erythrocytic material, and did not interfere with the specificity of the ELISA. The antigenic similarity between A. marginale and A. centrale was further substantiated by cross-reactivity obtained with heterologous antigens in both ELISA and IFA tests, and antibodies produced during natural infection with A. marginale were indistinguishable in both tests from those produced following vaccination with A. centrale.

Anaplasma↗
PubMed1998

Persistence of Anaplasma ovis infection and conservation of the msp-2 and msp-3 multigene families within the genus Anaplasma.

Goats which have recovered from acute Anaplasma ovis infection remain seropositive, although infected erythrocytes cannot be detected by microscopic examination. Persistence of A. ovis 17 to 21 months following experimental infection was demonstrated by PCR detection of the msp-5 gene. Quantitative analysis of persistent rickettsemia over time showed that all levels were below the limit of microscopic detection and ranged from a low of 10(2) organisms/ml to peaks of 10(6) organisms/ml. Two patterns of persistent rickettsemia were observed: the first was characterized by cyclic fluctuations at 6- to 9-week intervals, similar to the pattern described for A. marginale-infected cattle, while in the second pattern, repetitive cycles did not occur and the rickettsemia levels were relatively constant. The msp-2 and msp-3 multigene families, which provide the genetic capacity for outer membrane protein antigenic variation during persistent A. marginale rickettsemia, were identified in the A. ovis genome by Southern blot analysis, and expression of an MSP-2 homologue was confirmed by using immunoblots.

Anaplasma↗
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↗
PubMed1991

Analysis of protein compositions and surface protein epitopes of Anaplasma centrale and Anaplasma marginale.

Protein composition was compared and epitopes were analyzed among the isolates of Anaplasma centrale and A. marginale by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting using bovine antisera and monoclonal antibodies, and enzyme-linked immunosorbent assay. Common and unique proteins were found among the isolates. All isolates tested had a major surface protein with an apparent molecular weight of 38 to 40 kilodalton which had slight molecular size variations between species. This protein was also a dominant immunogen to the host. At least two species-common epitopes, one of which might contain carbohydrate(s), were present on the major surface protein. One species-specific epitope was identified on the major surface protein of A. marginale isolates.

Anaplasma↗
PubMed1994

Protein analysis of Anaplasma marginale and Anaplasma centrale by two-dimensional polyacrylamide gel electrophoresis.

Protein compositions of Anaplasma marginale and A. centrale were analyzed by two-dimensional gel electrophoresis. Both species had a major protein which was composed of 3-4 spots. The molecular weights of these two proteins were approximately 39 kDa. However, the position of these proteins in the gels were slightly different when 2 gel maps were superimposed. Five other protein spots were shared by A. marginale and A. centrale, whereas all the other protein spots were appeared to be unique to each of the species.

Anaplasma↗
PubMed1990

Frozen and fresh Anaplasma centrale vaccines in the protection of cattle against Anaplasma marginale infection.

The immunity induced by frozen and fresh Anaplasma centrale vaccines against anaplasmosis caused by A. marginale was tested in 12-month old Friesian steers. A. centrale parasitaemia occurred in all cattle inoculated with both types of vaccine. The average maximal decrease in PCV for the frozen and fresh vaccines was 41.0 and 40.3% respectively. All cattle recovered spontaneously. Vaccinated and control steers of the same age were challenged six months later with doses of 10(6), 10(7) or 10(8) A. marginale organisms. Vaccinated cattle showed average maximal A. marginale parasitemia of 1.2-4.0 versus 10.3-12.0% in control cattle. The average maximal decrease in packed cell volume (PCV) was 33.1 and 30.0% for steers vaccinated with frozen or fresh vaccine, respectively, and 57.4% for the non-vaccinated steers. All vaccinated cattle recovered spontaneously from the A. marginale infection while 7 out of 8 control steers required specific treatment. It thus appears that both frozen and fresh A. centrale vaccines are equally capable of inducing partial protection against infection with A. marginale and of preventing severe red blood cell destruction.

Anaplasma↗
PubMed1983

Influence of a second Anaplasma exposure on the success of treatment to eliminate Anaplasma carrier infections in cattle.

Treatment of adult Anaplasma carrier cows, with long-acting oxytetracycline at dosage levels generally successful in eliminating infection, was unsuccessful when the treatment was preceded or accompanied by a 2nd exposure to A marginale on days 0, 7, or 14 before treatment. Noninfected calves exposed to A marginale 7 days before a similar treatment developed anaplasmosis and became carriers of infection.

Anaplasma↗
PubMed2005

Serologic and molecular characterization of Anaplasma species infection in farm animals and ticks from Sicily.

Although Anaplasma marginale was known to be endemic in Italy, the diversity of Anaplasma spp. from this area have not been characterized. In this study, the prevalence of Anaplasma spp. antibodies in randomly selected farm animals collected on the island of Sicily was determined by use of a MSP5 cELISA for Anaplasma spp. and an immunofluorescence test specific for Anaplasma phagocytophilum. Genetic variation among strains of Anaplasma spp. from animals and ticks was characterized using the A. marginale msp1alpha and the Anaplasma spp. msp4 genes. Eight species of ticks were collected and tested by PCR. Seropositivity for Anaplasma spp. and A. phagocytophilum was detected in bovine and ovine samples. All the donkeys were seropositive for A. phagocytophilum but not for Anaplasma spp. Four A. marginale genotypes were identified by msp4 sequences from bovine and tick samples. Two new genotypes of Anaplasma ovis were characterized in sheep. The sequences of A. phagocytophilum from three donkeys proved to be identical to the sequence of the MRK equine isolate from California. Six A. marginale genotypes were found in cattle and one tick using the A. marginale msp1alpha sequences. All genotypes had four repeated sequences in the N-terminal portion of the MSP1a, except for one that had five repeats. The Italian strains of A. marginale contained three repeat sequences that were not reported previously. Definition of the diversity of Anaplasma spp. in Sicily reported, herein is fundamental to development of control strategies for A. marginale, A. ovis and A. phagocytophilum in Sicily.

Amino Acid Sequence↗
PubMed1989

Cloned DNA probes identify Anaplasma ovis in goats and reveal a high prevalence of infection.

Anaplasma organisms are observed in erythrocytes from goats with anemia and weight loss in Kenya. Three anaplasmas have been isolated in nature, Anaplasma ovis, Anaplasma marginale, and Anaplasma centrale. The two recognized species, A. ovis and A. marginale, are known to infect goats. Since only A. ovis causes clinical disease in goats, the Anaplasma species in goats in Kenya were identified. To detect A. ovis, a 9.6-kilobase-pair section of genomic DNA was cloned into pBR322 (pAO12A) and was used in conjunction with an A. marginale DNA probe previously derived from a gene coding for a 105,000-molecular-weight surface protein (Am105L) of A. marginale. In Southern blots, pAO12A DNA hybridized to several at least partially homologous sequences that were present in A. ovis and A. marginale genomic DNAs. The pAO12A DNA did not hybridize to Babesia bovis genomic or goat leukocyte DNA. The Anaplasma species that infected goats was identified as A. ovis by (i) DNA hybridization with pAO12A, (ii) hybridization of the A. marginale DNA probe to A. centrale and A. marginale genomic DNAs and lack of hybridization to A. ovis genomic DNA from an isolate obtained in Idaho and Anaplasma DNA from infected goats in Kenya, (iii) the intraerythrocytic location of Anaplasma organisms in infected goat blood, and (iv) the host specificity of the Anaplasma organisms for goats but not for cattle. Also, by using the two Anaplasma DNA probes, the prevalence of A. ovis in goats from seven locations in Kenya was found to range from 22 to 87%. The pAO12A DNA probe detected a 0.0035% A. ovis parasitemia in infected blood, an improved sensitivity which is suitable for use in surveillance and epidemiological studies.

Anaplasma↗
PubMed2003

Phylogenetic analysis of the erythrocytic Anaplasma species based on 16S rDNA and GroEL (HSP60) sequences of A. marginale, A. centrale, and A. ovis and the specific detection of A. centrale vaccine strain.

Phenotypic criteria for the identification of erythrocytic ruminant Anaplasma species has relied on subjective identification methods such as host pathogenicity (virulence for cattle or sheep) and/or the location of Anaplasma inclusion bodies within the host's red cells. Sequence comparisons of new and available GenBank Accessions were investigated to elucidate the relationships among these closely related Anaplasma species. Twenty-one 16S rDNA and GroEL (HSP60) sequences from 13 Anaplasma marginale (South Africa, Namibia, Zimbabwe, Israel, USA, Australia and Uruguay), three A. centrale (South Africa and Japan), two A. ovis (USA and South Africa), and two unknown Anaplasma species isolated from wild ruminants (South Africa), were compared. 16S rDNA maximum-likelihood and distance trees separated all A. marginale (and the two wild ruminant isolates) from the two South African A. centrale (including original vaccine strain, Theiler, 1911). The Japanese A. centrale (Aomori) demonstrated the lowest sequence identity to the remaining erythrocytic Anaplasma species. A. ovis inter-species relationships could not be resolved through the 16S rDNA analyses, whereas strong bootstrap branch support is demonstrated in the GroEL distance tree using A. ovis OVI strain. All erythrocytic Anaplasma species and isolates were confirmed to belong to the same cluster showing strong branch support to Anaplasma (Ehrlichia) phagocytophilum with Ehrlichia (Cowdria) ruminantium and Rickettsia rickettsii serving as appropriate out-groups. Based on groEL sequences, a specific PCR method was developed which amplified A. centrale vaccine (Theiler, 1911) specifically. This study confirms the suitability of 16S rDNA sequences to define genera and demonstrates the usefulness of GroEL sequences for defining species of erythrocytic Anaplasma.

Anaplasma↗
PubMed1965

CATALASE ACTIVITY IN ANAPLASMA MARGINALE.

Wallace, W. R. (Louisiana State University, Baton Rouge), and G. T. Dimopoullos. Catalase activity in Anaplasma marginale. J. Bacteriol. 91:309-311. 1965.-Extracts of erythrocytes infected with Anaplasma marginale were found to contain more catalase activity than normal erythrocytic preparations. The increase in catalase activity appeared concurrently with increases in the number of erythrocytes containing Anaplasma bodies. Antisera against normal bovine erythrocytes and Anaplasma-infected erythrocytes were prepared in rabbits to test the source of increased catalase activity during anaplasmosis. Antiserum against normal erythrocytes decreased the catalase activity of extracts of normal erythrocytes by 271 units per ml and of partially purified Anaplasma bodies by only 120 units. Rabbit antiserum against Anaplasma-infected bovine erythrocytes removed only 73 units of activity from normal erythrocytes, but decreased the activity of the partially purified Anaplasma bodies by 260 units, indicating the association of catalase with the marginal body.

Anaplasma↗
PubMed2004

Pathogens and symbionts in ticks: prevalence of Anaplasma phagocytophilum (Ehrlichia sp.), Wolbachia sp., Rickettsia sp., and Babesia sp. in Southern Germany.

Tick-transmitted diseases like tick-borne encephalitis and Lyme borreliosis have been well known in Germany for decades. Ongoing research now gives an additional focus to a broad range of other bacteria and parasites in ticks like Anaplasma phagocytophilum, former Ehrlichia sp., Rickettsia sp. and Babesia sp. Knowledge about the prevalence of these infectious agents in ticks is an important prerequisite for risk assessment of human diseases. Therefore nymphs and adult Ixodes ricinus ticks were collected and examined for Anaplasma phagocytophilum (n = 5424 ticks), Rickettsia sp. (n = 1187), and Babesia sp. (n = 3113). For the detection of Anaplasma phagocytophilum, DNA from the 16S rDNA gene was amplified by nested PCR and hybridized with a DIG-labeled oligonucleotide probe. The examination of Rickettsia sp. was performed by single PCR. A partial sequence of the citrate synthase gene was amplified. As a target for the detection of Babesia sp., DNA from the 18S rDNA gene was amplified, also by single PCR. All positive PCR products were sequenced to control specificity. Anaplasma phagocytophilum was detected by PCR in n = 103 (1.9%) out of 5,424 examined ticks from 11 investigation areas. However, not all positive PCR products hybridized using DIG-labeled oligonucleotide probe. Thus, the result of sequencing indicated that only 1.0% (n = 54) belonged to Anaplasma phagocytophilum and nearly half of these PCR products (0.9%) were identified as Wolbachia sp. Rickettsia sp. in Ixodes ricinus ticks from 3 areas were found in n = 105 (8.9%) out of 1,187 ticks examined (range from 13.3% to 5.6%). Sequencing showed Rickettsia helvetica exclusively. In about 2.6% of Rickettsia-positive ticks, double infection with Anaplasma phagocytophilum was found. Babesia sp. was detected in n= 31 (1.0%) out of 3,113 ticks examined, which originated from 4 different areas. By sequencing, n = 28 (90.0%) were identified as Babesia divergens. Three of all Babesia-positive ticks were identified as harboring Babesia microti. The detection of Anaplasma phagocytophilum, Rickettsia sp. and Babesia sp. demonstrates their possible role as a source of human infection in Germany.

Anaplasma phagocytophilum↗
PubMed2001

Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila.

The genera Anaplasma, Ehrlichia, Cowdria, Neorickettsia and Wolbachia encompass a group of obligate intracellular bacteria that reside in vacuoles of eukaryotic cells and were previously placed in taxa based upon morphological, ecological, epidemiological and clinical characteristics. Recent genetic analyses of 16S rRNA genes, groESL and surface protein genes have indicated that the existing taxa designations are flawed. All 16S rRNA gene and groESL sequences deposited in GenBank prior to 2000 and selected sequences deposited thereafter were aligned and phylogenetic trees and bootstrap values were calculated using the neighbour-joining method and compared with trees generated with maximum-probability, maximum-likelihood, majority-rule consensus and parsimony methods. Supported by bootstrap probabilities of at least 54%, 16S rRNA gene comparisons consistently clustered to yield four distinct clades characterized roughly as Anaplasma (including the Ehrlichia phagocytophila group, Ehrlichia platys and Ehrlichia bovis) with a minimum of 96.1% similarity, Ehrlichia (including Cowdria ruminantium) with a minimum of 97.7% similarity, Wolbachia with a minimum of 95.6% similarity and Neorickettsia (including Ehrlichia sennetsu and Ehrlichia risticii) with a minimum of 94.9% similarity. Maximum similarity between clades ranged from 87.1 to 94.9%. Insufficient differences existed among E. phagocytophila, Ehrlichia equi and the human granulocytic ehrlichiosis (HGE) agent to support separate species designations, and this group was at least 98.2% similar to any Anaplasma species. These 16S rRNA gene analyses are strongly supported by similar groESL clades, as well as biological and antigenic characteristics. It is proposed that all members of the tribes Ehrlichieae and Wolbachieae be transferred to the family Anaplasmataceae and that the tribe structure of the family Rickettsiaceae be eliminated. The genus Anaplasma should be emended to include Anaplasma (Ehrlichia) phagocytophila comb. nov. (which also encompasses the former E. equi and the HGE agent), Anaplasma (Ehrlichia) bovis comb. nov. and Anaplasma (Ehrlichia) platys comb. nov., the genus Ehrlichia should be emended to include Ehrlichia (Cowdria) ruminantium comb. nov. and the genus Neorickettsia should be emended to include Neorickettsia (Ehrlichia) risticii comb. nov. and Neorickettsia (Ehrlichia) sennetsu comb. nov.

Anaplasma↗