Development and field evaluation of a nested polymerase chain reaction-restriction fragment length polymorphism (nPCR-RFLP) analysis to identify A. marginale-infected and A. centrale-vaccinated cattle.

A nested polymerase chain reaction-restriction fragment length polymorphism (nPCR-RFLP) assay based on the amplification of the Anaplasma spp. highly conserved msp5 gene and posterior digestion with HindIII endonuclease was developed and evaluated in field samples. Results were compared using an nPCR specific for Anaplasma marginale (nPCR-Am) based on the msp1ß gene and an nPCR specific for A. centrale (nPCR-Ac) based on the msp2 operon (msp2-o) gene. Amplicons dilutions of msp1ß and msp5 of A. marginale and msp2-o and msp5 of A. centrale and dilutions of parasited erythrocytes (PE) with A. marginale and A. centrale were used to determine the detection limits. The results were 20 DNA copies/reaction and 30 PE for A. marginale and A. centrale by nPCR-RLFP and nPCR-Am/Ac. A mix of msp5-Am and msp5-Ac was used to evaluate the interference of msp5 from one species for the detection of the other. Co-amplification of the DNA from both species was observed up to a 1:7 ratio of one species to the other. Field samples positive for Anaplasma spp. antibodies (n = 260) from 32 herds were evaluated. Strength of agreement between results by nPCR-RFLP and nPCR-Am or nPCR-Ac was 78% (κ = 0.44) and 94% (κ = 0.85), respectively. Thirty-four samples were positive for A. marginale by nPCR-RFLP but negative by nPCR-Am. msp1ß amplicons of 10 samples from 5 herds with discrepancies between nPCR-Am and nPCR-RFLP results were cloned and sequenced. The analysis of the msp1ß sequence showed several mutations in the target region of the internal forward primer that would explain the failure in the amplification. Only 10 of the 20 coinfections identified by nPCR-Ac/nPCR-Am were detected by nPCR-RFLP. nPCR-RFLP is a sensitive, low-cost and accessible molecular method for low-complexity laboratories. More studies are needed to establish in which circumstances coinfections can be underestimated.

Molecular detection and phylogenetic analysis of Anaplasma marginale and Anaplasma centrale amongst transhumant cattle in north-eastern Uganda.

There is little molecular data from Anaplasma marginale and Anaplasma centrale isolates from cattle in Uganda. Between November 2013 and January 2014, blood was collected from 240 cattle in 20 randomly-selected herds in two districts of the Karamoja Region in north-eastern Uganda. A duplex quantitative real-time polymerase chain reaction (qPCR) assay was used to detect and determine the prevalence of A. marginale (targeting the msp1ß gene) and A. centrale (targeting the groEL gene). The qPCR assay revealed that most cattle (82.9%; 95% confidence interval [CI] 78.2-87.7%) were positive for A. marginale DNA, while fewer cattle (12.1%; 95% CI 7.9-16.2%) were positive for A. centrale DNA. A mixed effects logistic regression model showed that the age of cattle was significantly associated with A. centrale infection, while the prevalence of A. marginale varied significantly according to locality. The near full-length 16S ribosomal RNA (16S rRNA) gene and the heat shock protein gene, groEL, for both Anaplasma species were amplified from a selection of samples. The amplicons were cloned and the resulting recombinants sequenced. We found three novel A. marginale 16S rRNA variants, seven A. marginale groEL gene sequence variants and two A. centrale groEL gene sequence variants. Phylogenetic trees were inferred from sequence alignments of the 16S rRNA gene and GroEL amino acid sequences determined here and published sequences using maximum likelihood, Bayesian inference and parsimony methods Phylogenetic analyses classified the 16S rRNA gene and GroEL amino acid sequences into one clade for A. marginale and a separate clade for A. centrale. This study reveals a high prevalence and sequence variability of A. marginale and A. centrale, and is the first report on the phylogenetic characterisation of A. marginale and A. centrale from cattle in Uganda using molecular markers. Sequence variation can be attributed to mobile pastoralism, communal grazing and grazing with wildlife. These data support future epidemiological investigations for bovine anaplasmosis in Uganda.

Co-infections with multiple genotypes of Anaplasma marginale in cattle indicate pathogen diversity.

BACKGROUND: Only a few studies have examined the presence of Anaplasma marginale and Anaplasma centrale in South Africa, and no studies have comprehensively examined these species across the whole country. To undertake this country-wide study we adapted a duplex quantitative real-time PCR (qPCR) assay for use in South Africa but found that one of the genes on which the assay was based was variable. Therefore, we sequenced a variety of field samples and tested the assay on the variants detected. We used the assay to screen 517 cattle samples sourced from all nine provinces of South Africa, and subsequently examined A. marginale positive samples for msp1α genotype to gauge strain diversity. RESULTS: Although the A. marginale msp1ß gene is variable, the qPCR functions at an acceptable efficiency. The A. centrale groEL gene was not variable within the qPCR assay region. Of the cattle samples screened using the assay, 57% and 17% were found to be positive for A. marginale and A. centrale, respectively. Approximately 15% of the cattle were co-infected. Msp1α genotyping revealed 36 novel repeat sequences. Together with data from previous studies, we analysed the Msp1a repeats from South Africa where a total of 99 repeats have been described that can be attributed to 190 msp1α genotypes. While 22% of these repeats are also found in other countries, only two South African genotypes are also found in other countries; otherwise, the genotypes are unique to South Africa. CONCLUSIONS: Anaplasma marginale was prevalent in the Western Cape, KwaZulu-Natal and Mpumalanga and absent in the Northern Cape. Anaplasma centrale was prevalent in the Western Cape and KwaZulu-Natal and absent in the Northern Cape and Eastern Cape. None of the cattle in the study were known to be vaccinated with A. centrale, so finding positive cattle indicates that this organism appears to be naturally circulating in cattle. A diverse population of A. marginale strains are found in South Africa, with some msp1α genotypes widely distributed across the country, and others appearing only once in one province. This diversity should be taken into account in future vaccine development studies.

Molecular survey and genetic characterization of Anaplasma centrale, A. marginale and A. bovis in cattle from Algeria.

Bovine anaplasmosis could be caused by several Anaplasma species. The causative agents are transmitted by ticks and haematophagous arthropods with a high impact on both human and animal health. This study was conducted to estimate the infection rate and to characterize Anaplasma spp. in cattle from Algeria. A molecular survey was performed in Setif district (Northeast Algeria) where a total number of 180 cattle blood samples were collected and tested for the presence of Anaplasma spp. by PCR. Positive samples were genetically characterized based on the 16S rRNA and msp4 genes. PCRs revealed that the infection rates of Anaplasma spp., Anaplasma centrale, Anaplasma marginale and Anaplasma bovis were 42.2%; 39.4%; 11.1% and 4.4%, respectively. All tested animals were negative for A. phagocytophilum. Co-infection occurred in 10% (18/180) of the tested animals, and the most common co-infection pattern was an association between A. centrale and A. marginale (5.5%). Five cattle (2.7%) were co-infected by the three Anaplasma species. Holstein animals (58.1%) were more infected by A. centrale than the other breeds (p = .01). The molecular prevalence of A. centrale was significantly higher in males (54.2%) than in females (34.1%) (p = .001). A. marginale msp4 genetic analysis indicated a high sequence diversity of Algerian strains, suggesting the importation of live cattle from different origins. Phylogenetic analysis of the 16S rRNA gene of A. bovis and A. centrale revealed a low degree of genetic diversity. Our study suggests that different species of Anaplasma are simultaneously present in the Algerian cattle. To the best of our knowledge, this is the first molecular study and genetic characterization of Anaplasma spp. in Algerian cattle.

Comparison of three nucleic acid-based tests for detecting Anaplasma marginale and Anaplasma centrale in cattle.

Several nucleic acid-based assays have been developed for detecting Anaplasma marginale and Anaplasma centrale in vectors and hosts, making the choice of method to use in endemic areas difficult. We evaluated the ability of the reverse line blot (RLB) hybridisation assay, two nested polymerase chain reaction (nPCR) assays and a duplex real-time quantitative polymerase chain reaction (qPCR) assay to detect A. marginale and A. centrale infections in cattle (n = 66) in South Africa. The lowest detection limits for A. marginale plasmid DNA were 2500 copies by the RLB assay, 250 copies by the nPCR and qPCR assays and 2500, 250 and 25 copies of A. centrale plasmid DNA by the RLB, nPCR and qPCR assays respectively. The qPCR assay detected more A. marginale- and A. centrale-positive samples than the other assays, either as single or mixed infections. Although the results of the qPCR and nPCR tests were in agreement for the majority (38) of A. marginale-positive samples, 13 samples tested negative for A. marginale using nPCR but positive using qPCR. To explain this discrepancy, the target sequence region of the nPCR assay was evaluated by cloning and sequencing the msp1ß gene from selected field samples. The results indicated sequence variation in the internal forward primer (AM100) area amongst the South African A. marginale msp1ß sequences, resulting in false negatives. We propose the use of the duplex qPCR assay in future studies as it is more sensitive and offers the benefits of quantification and multiplex detection of both Anaplasma spp.

Propagation of the Israeli vaccine strain of Anaplasma centrale in tick cell lines.

Anaplasma centrale has been used in cattle as a live blood vaccine against the more pathogenic Anaplasma marginale for over 100 years. While A. marginale can be propagated in vitro in tick cell lines, facilitating studies on antigen production, immunisation and vector-pathogen interaction, to date there has been no in vitro culture system for A. centrale. In the present study, 25 cell lines derived from 13 ixodid tick species were inoculated with the Israeli vaccine strain of A. centrale and monitored for at least 12 weeks by microscopic examination of Giemsa-stained cytocentrifuge smears. Infection of 19 tick cell lines was subsequently attempted by transfer of cell-free supernate from vaccine-inoculated tick cells. In two separate experiments, rickettsial inclusions were detected in cultures of the Rhipicephalus appendiculatus cell line RAE25 28-32 days following inoculation with the vaccine. Presence of A. centrale in the RAE25 cells was confirmed by PCR assays targeting the 16S rRNA, groEL and msp4 genes; sequenced PCR products were 100% identical to published sequences of the respective genes in the Israeli vaccine strain of A. centrale. A. centrale was taken through three subcultures in RAE25 cells over a 30 week period. In a single experiment, the Dermacentor variabilis cell line DVE1 was also detectably infected with A. centrale 11 weeks after inoculation with the vaccine. Availability of an in vitro culture system for A. centrale in tick cells opens up the possibility of generating a safer and more ethical vaccine for bovine anaplasmosis.

First molecular survey and novel genetic variants' identification of Anaplasma marginale, A. centrale and A. bovis in cattle from Tunisia.

Few data are available about the presence and distribution of Anaplasma species in cattle in North African countries. In this study prevalence, co-infections, risk factors and genetic diversity of Anaplasma species were evaluated in bovines from Northern Tunisia. A total of 232 cattle from 36 randomly selected farms in three Tunisian localities were investigated for the presence of Anaplasma species in blood by Real-time PCR and/or nested PCR. Overall infection rates of Anaplasma spp., Anaplasma marginale, Anaplasma centrale and Anaplasma bovis were 34.9%, 25.4%, 15.1%, and 3.9%, respectively. Anaplasma phagocytophilum was not detected in cattle. The most common co-infection pattern was an association of A. marginale and A. centrale (11.2%). Five cattle (2.1%) all reared in the sub-humid bioclimatic area, were co-infected by the three Anaplasma species. Molecular prevalence of Anaplasma infection varied significantly according to locality, bioclimatic area, tick infestation and type of breeding. Animals of the Holstein breed were less infected by A. marginale and A. centrale than other breeds. Genetic analysis of A. marginale msp4 gene indicated a high sequence diversity of Tunisian strains, suggesting a multiple introduction of infected cattle from different origins. Phylogenetic studies based on the 16S rRNA gene showed that the most prevalent A. centrale strains were closely related to the A. centrale vaccine strain. Moreover, all A. bovis variants clustered with other A. bovis sequences obtained from domestic and wild ruminant strains. This is the first molecular investigation on Anaplasma species in Tunisian cattle providing pivotal background for designing epidemiological studies and to develop control strategies in the country.

[Detection of Anaplasma / Ehrlichia Species of Cattle and Ticks in Aydin Region]. / Aydin Yöresinde Sigirlarda ve Kenelerde Anaplasma / Ehrlichia Türlerinin Belirlenmesi.

OBJECTIVE: The aim of this study is to detect the Anaplasma/Ehrlichia species of cattle and ticks and to provide knowledge on the prevalence of these species during sampling periods. METHODS: A total of 679 blood and 186 tick samples were collected from the Osmanbükü, Akçaova, Dalama, and Söke districts of Aydin. The samples were screened with genus polymerase chain reaction (PCR) for Anaplasma/Ehrlichia spp., species-specific polymerase chain reaction for Anaplasma marginale and A. centrale, and nested PCR for A. bovis and A. phagocytophilum. RESULTS: A. centrale was detected in Söke during September and in Dalama and Akçaova during March, June, September, and December. A. marginale was detected in Osmanbükü during June; in Söke during March and December; in Akçaova during June, September, and March; and in Dalama during the entire sampling period. A. phagocytophilum was detected in all regions during the entire sampling period. None of the samples were positive for A. bovis. Mixed infections were detected in 50 blood samples. A. marginale and A. phagocytophilum were detected in the tick samples. CONCLUSION: In this study, A. phagocytophilum was abundantly detected compared with A. marginale and A. centrale. A. phagocytophilum and A. centrale were extensively found in Akçaova and A. marginale was mostly seen in Dalama. Parasites were extensively detected in September and March. The analysis indicated that collected ticks were infected with different Anaplasma/Ehrlichia species.

Anaplasma spp. in wild mammals and Ixodes ricinus from the north of Spain.

Our objectives were to investigate the presence of Anaplasma spp. infection in red deer, wild boars, and Ixodes ricinus removed from deer surveyed in La Rioja, as well as to analyze the presence of Anaplasma spp. in I. ricinus from different Spanish regions--ours included. A total of 21 deer and 13 wild boar blood samples as well as 295 I. ricinus removed from deer, vegetation, or asymptomatic people were tested by polymerase chain reaction targeting Anaplasma spp. 16S rRNA gene and groESL heat shock operon. Twelve deer blood samples were found to be infected with Anaplasma centrale (n = 7) or Anaplasma phagocytophilum (n = 5). No wild boar blood samples gave positive polymerase chain reaction results. Further, A. phagocytophilum was detected in 12 out of 89 I. ricinus removed from deer and in 18 out of 168 I. ricinus collected over vegetation in the North of Spain. Anaplasma spp. was not detected in any of the 38 I. ricinus removed from people. Nucleotide sequences for 16S rRNA gene showed substancial heterogeneity. The etiological agent of human anaplasmosis was found in two deer blood samples, an adult tick from deer, and a nymph from vegetation. The 16S rRNA sequences for 12 out of 35 samples matched the sequence of the Ap-variant 1 strain previously described in the United States, and the remaining 19 positive samples (deer blood and I. ricinus) showed variations with unknown significance. Although the groEL DNA sequences varied among analyzed strains, the deduced amino acid sequences did not change for any of them. This study suggests that deer population from La Rioja harbors strains of A. phagocytophilum similar to that pathogen for humans and other of unknown pathogenicity. Further, it seems that the Ap-variant 1 is circulating among I. ricinus ticks from the North of Spain more frequently than the A. phagocytophilum strain associated to human anaplasmosis.

Identification of Anaplasma marginale outer membrane protein antigens conserved between A. marginale sensu stricto strains and the live A. marginale subsp. centrale vaccine.

Live vaccination with Anaplasma marginale subsp. centrale (synonym for Anaplasma centrale) induces protection against severe disease upon challenge with A. marginale sensu stricto strains. Despite over a century of field use, the targets of protective immunity remained unknown. Using a broad proteomic approach, we identified the proteins in a challenge sensu stricto strain that were bound by the relevant antibody isotype induced by live vaccination with Anaplasma marginale subsp. centrale. A core of 15 proteins was identified in vaccinated animals across multiple major histocompatibility complex (MHC) haplotypes. This core separated into two structural/functional classes: "housekeeping" proteins involved in replication and metabolism and outer membrane proteins (OMPs). Orthologous proteins of both classes were identified within the vaccine strain and among sensu stricto strains. In contrast to the broad conservation among strains in the sequences of the housekeeping proteins, there was significantly greater divergence in the OMPs and greater divergence in both OMP sequences and the encoding locus structure between the vaccine strain and the sensu stricto strains than among the sensu stricto strains. The OMPs bound by live vaccine-induced antibody overlapped with OMPs that were immunogenic in animals vaccinated with inactivated vaccines and subsequently protected against bacteremia and disease. The identification of this core set of OMPs is consistent with the hypothesis that "subdominant" immunogens are required for vaccine-induced protection against A. marginale and provides clear direction for development of a safer, more effective vaccine.

Identification of Anaplasma centrale major surface protein-2 pseudogenes.

The present study was aimed to identify msp2 pseudogenes and MSP2 variants in the vaccine Anaplama centrale strain. Five msp2 pseudogenes were identified in the A. centrale genome, and multiple MSP2 variants that emerged during both acute and persistent infection were detected. The pseudogene copies of msp2 were truncated; they contained a central hypervariable region flanked by short portions of the 5' and 3' conserved regions. Alignment of the hypervariable region sequence of the expression site of MSP2 variants with msp2 pseudogenes showed that MSP2 variants are generated by two mechanisms, previously described in Anaplasma marginale: (i) recombination of the whole pseudogene into the single msp2 expression site, and (ii) recombination of small segments of pseudogenes into the expression site by segmental gene conversion. The present study showed that the A. centrale MSP2 variants and the msp2 pseudogene repertoire were different from those reported for A. marginale. Unique MSP2 variants and pseudogenes identified in the vaccine strain allow the A. centrale-vaccinated cattle to be superinfected with the field strains of A. marginale. The knowledge gained in the present study on the mechanisms of antigenic variations in the vaccine strain of A. centrale is a further step in the development of a new generation vaccine against anaplasmosis.

Duplex real-time polymerase chain reaction for simultaneous detection and quantification of Anaplasma marginale and Anaplasma centrale.

Anaplasma marginale and Anaplasma centrale are rickettsial pathogens responsible for acute disease and mild infections, respectively, in cattle herds. A duplex real-time polymerase chain reaction (PCR) assay with probes labeled with different fluorophores was developed for simultaneous detection and quantification of A. marginale and A. centrale DNA in bovine blood samples. The assay was able to detect as few as 10(1) and 10(2) DNA copies for A. marginale and A. centrale, respectively, with optimal specificity and reproducibility. Analysis by real-time and nested PCR carried out on 54 samples previously tested by reverse line blot hybridization showed that the established duplex real-time PCR assay can detect and quantify the 2 Anaplasma spp., even if present simultaneously in the same blood samples. Such an assay could be used in pathogenesis studies on bovine acute anaplasmosis.

Novel genetic variants of Anaplasma phagocytophilum, Anaplasma bovis, Anaplasma centrale, and a novel Ehrlichia sp. in wild deer and ticks on two major islands in Japan.

Wild deer are one of the important natural reservoir hosts of several species of Ehrlichia and Anaplasma that cause human ehrlichiosis or anaplasmosis in the United States and Europe. The primary aim of the present study was to determine whether and what species of Ehrlichia and Anaplasma naturally infect deer in Japan. Blood samples obtained from wild deer on two major Japanese islands, Hokkaido and Honshu, were tested for the presence of Ehrlichia and Anaplasma by PCR assays and sequencing of the 16S rRNA genes, major outer membrane protein p44 genes, and groESL. DNA representing four species and two genera of Ehrlichia and Anaplasma was identified in 33 of 126 wild deer (26%). DNA sequence analysis revealed novel strains of Anaplasma phagocytophilum, a novel Ehrlichia sp., Anaplasma centrale, and Anaplasma bovis in the blood samples from deer. None of these have been found previously in deer. The new Ehrlichia sp., A. bovis, and A. centrale were also detected in Hemaphysalis longicornis ticks from Honshu Island. These results suggest that enzootic cycles of Ehrlichia and Anaplasma species distinct from those found in the United States or Europe have been established in wild deer and ticks in Japan.

Molecular and serological detection of A. centrale- and A. marginale-infected cattle grazing within an endemic area.

A reverse line blot hybridization (RLB) one-stage nested PCR (nPCR) for Anaplasma centrale and a nested PCR for Anaplasma marginale were used to detect infected cattle grazing within an endemic region in Israel. A novel set of PCR primers and oligonucleotide probes based on a 16S ribosomal RNA gene was designed for RLB detection of both Anaplasma species, and the performance of the molecular assays compared. The immunofluorescent antibody test (IFA) was used to detect antibodies to both Anaplasma species, whereas, a highly sensitive and specific competitive enzyme-linked immunosorbent assay (cELISA) was used to detect antibodies in A. centrale-vaccinated cattle. The RLB and the nested PCR procedures showed bacteremia with sensitivity of 50 infected erythrocytes per milliliter. Up to 93% of the A. centrale vaccinates carried specific antibodies that were detected by cELISA, and up to 71% of the vaccinated cattle were found to be naturally infected with A. marginale according to the PCR and the RLB assays. Nevertheless, no severe outbreaks of A. marginale infection occurred among vaccinated herds in this endemic region. It appears that both, molecular tools and serology are useful for evaluation of the vaccine efficacy. In the light of wide natural field infection with A. marginale, strong recommendations to continue the A. centrale vaccination program regime will continue until a new generation of non-blood-based vaccine will be developed.

Amplification of 16S rRNA genes of Anaplasma species in China for phylogenetic analysis.

In this study, a phylogenetic tree was inferred through comparing five 16S rRNA gene sequences of four isolates of Anaplasma ovis and one of Anaplasma marginale in China with all nineteen 16S rRNA gene sequences deposited in GenBank (12 A. marginale, 3 A. ovis and 4 Anaplasma centrale derived from America, Uruguay, South Africa, Zimbabwe, Australia, Isreal and Japan). The analysis showed that all A. ovis isolated in China were separated into an A. ovis cluster, while the A. marginale in China was separated into an A. marginale cluster (see Fig. 1). This analysis demonstrated that there are at least two different Anaplasma species widespread among ruminants in North China.

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.