New species of Ehrlichia isolated from Rhipicephalus (Boophilus) microplus shows an ortholog of the E. canis major immunogenic glycoprotein gp36 with a new sequence of tandem repeats

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al.[Background]: Ehrlichia species are the etiological agents of emerging and life-threatening tick-borne human zoonoses that inflict serious and fatal infections in companion animals and livestock. The aim of this paper was to phylogeneticaly characterise a new species of Ehrlichia isolated from Rhipicephalus (Boophilus) microplus from Minas Gerais, Brazil. [Methods]: The agent was isolated from the hemolymph of Rhipicephalus (B.) microplus engorged females that had been collected from naturally infested cattle in a farm in the state of Minas Gerais, Brazil. This agent was then established and cultured in IDE8 tick cells. The molecular and phylogenetic analysis was based on 16S rRNA, groEL, dsb, gltA and gp36 genes. We used the maximum likelihood method to construct the phylogenetic trees. [Results]: The phylogenetic trees based on 16S rRNA, groEL, dsb and gltA showed that the Ehrlichia spp isolated in this study falls in a clade separated from any previously reported Ehrlichia spp. The molecular analysis of the ortholog of gp36, the major immunoreactive glycoproteins in E. canis and ortholog of the E. chaffeensis gp47, showed a unique tandem repeat of 9 amino acids (VPAASGDAQ) when compared with those reported for E. canis, E. chaffeensis and the related mucin-like protein in E. ruminantium. [Conclusions]: Based on the molecular and phylogenetic analysis of the 16S rRNA, groEL, dsb and gltA genes we concluded that this tick-derived microorganism isolated in Brazil is a new species, named E. mineirensis (UFMG-EV), with predicted novel antigenic properties in the gp36 ortholog glycoprotein. Further studies on this new Ehrlichia spp should address questions about its transmissibility by ticks and its pathogenicity for mammalian hosts.A. Cabezas Cruz is a Marie Curie Early Stage Researcher (ESR) supported by the POSTICK ITN (Post-graduate training network for capacity building to control ticks and tick-borne diseases) within the FP7- PEOPLE – ITN programme (EU Grant No. 238511).Peer Reviewe

Functional and Immunological Relevance of Anaplasma marginale Major Surface Protein 1a Sequence and Structural Analysis.

Bovine anaplasmosis is caused by cattle infection with the tick-borne bacterium, Anaplasma marginale. The major surface protein 1a (MSP1a) has been used as a genetic marker for identifying A. marginale strains based on N-terminal tandem repeats and a 5'-UTR microsatellite located in the msp1a gene. The MSP1a tandem repeats contain immune relevant elements and functional domains that bind to bovine erythrocytes and tick cells, thus providing information about the evolution of host-pathogen and vector-pathogen interactions. Here we propose one nomenclature for A. marginale strain classification based on MSP1a. All tandem repeats among A. marginale strains were classified and the amino acid variability/frequency in each position was determined. The sequence variation at immunodominant B cell epitopes was determined and the secondary (2D) structure of the tandem repeats was modeled. A total of 224 different strains of A. marginale were classified, showing 11 genotypes based on the 5'-UTR microsatellite and 193 different tandem repeats with high amino acid variability per position. Our results showed phylogenetic correlation between MSP1a sequence, secondary structure, B-cell epitope composition and tick transmissibility of A. marginale strains. The analysis of MSP1a sequences provides relevant information about the biology of A. marginale to design vaccines with a cross-protective capacity based on MSP1a B-cell epitopes

Protection in the absence of exclusion between two Brazilian isolates of Anaplasma marginale in experimentally infected calves

This study investigated whether a low pathogenicity isolate of Anaplasma marginale with an appendage (UFMG1) could protect calves from infection with a pathogenic A. marginale isolate (UFMG2). Two groups of five Friesian calves were each inoculated with UFMG1 by intravenous injections of either A. marginale-infected tick cell cultures (group 1) or blood stabilates (group 2); a third (control) group was injected with saline. All animals were inoculated with a blood stabilate containing a high pathogenicity A. marginale isolate (UFMG2) 75. days after the UFMG1 inoculation. After infection with UFMG2, animals in groups 1 and 2 presented low rickettsaemia, but no clinical signs and no reduction in packed cell volume (PCV). Control animals became sick, with high rickettsaemia (16% infected erythrocytes) and a reduction in PCV (71%), resulting in 60% deaths. Up to 2. weeks after the UFMG2 inoculation, msp1α UFMG1 sequences were detected in groups 1 and 2. Four weeks after UFMG2 inoculation, UFMG2 sequences were detected in these animals, along with a new msp1α genotype sequence, closely related to that of the UFMG2 isolate. Control animals had UFMG2 msp1α sequences up to 4. weeks after inoculation with UFMG2 and the new msp1α genotype sequence could be detected on the sixth week. The origin of the new A. marginale genotype was unknown, but may represent the first example of MSP1a antigenic variation in infected cattle. The results confirmed the low pathogenicity of the UFMG1 isolate, which provided clinical protection against the highly pathogenic A. marginale UFMG2. Infection with UFMG1 did not prevent the establishment of a second isolate, suggesting protection without infection-exclusion among A. marginale isolates.This research was supported by FAPEMIG (Brazil), the Ministerio de Ciencia e Innovación, Spain (project BFU2008-01244/BMC) and scholarships provided by CAPES and CNPq.Peer Reviewe

Use of Percoll gradients to purify Anaplasma marginale (Rickettsiales: Anaplasmataceae) from tick cell cultures

Anaplasma marginale (Rickettsiales: Anaplasmataceae) is an obligate intracellular bacterium that multiplies exclusively within membrane-bound vacuoles in the cytoplasm of host cells. A number of A. marginale isolates can be propagated in the Ixodes scapularis IDE8 tick cell line, which provides a reliable source of antigens for a wide variety of studies. However, because of its intracellular nature, separation of bacteria from host cell materials remains an important constraint for researchers. In the present study, we evaluated the use of Percoll gradients for purification of two Brazilian strains of A. marginale grown in IDE8 tick cells. The purified A. marginale monitored in Giemsa-stained smears contained only minimal amounts of IDE8 cell stroma. The total protein yields were 1.2mg and 1.7mg, while the DNA titers quantified with real-time PCR were 6.4×109 for UFMG1 and 4.87×109 for UFMG2 copies in the purified material, respectively. Additionally, we confirmed the viability of purified bacteria by infecting tick cells after being freshly purified and after retrieval from long-term storage. Importantly, the viability of the organisms is preserved after use of this separation method, and therefore the purified organisms can be used in enzymatic assays and other research approaches where live organisms would be preferred. © 2014 Elsevier GmbH.This research was supported by POSTICK ITN (Post-graduate training network for capacity building to control ticks and tick-borne diseases) within the FP7-PEOPLE–ITN Programme (EU Grant no. 238511).Peer Reviewe

Functional and immunological relevance of Anaplasma marginale major surface protein 1a sequence and structural analysis

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.Bovine anaplasmosis is caused by cattle infection with the tick-borne bacterium, Anaplasma marginale. The major surface protein 1a (MSP1a) has been used as a genetic marker for identifying A. marginale strains based on N-terminal tandem repeats and a 5′-UTR microsatellite located in the msp1a gene. The MSP1a tandem repeats contain immune relevant elements and functional domains that bind to bovine erythrocytes and tick cells, thus providing information about the evolution of host-pathogen and vector-pathogen interactions. Here we propose one nomenclature for A. marginale strain classification based on MSP1a. All tandem repeats among A. marginale strains were classified and the amino acid variability/frequency in each position was determined. The sequence variation at immunodominant B cell epitopes was determined and the secondary (2D) structure of the tandem repeats was modeled. A total of 224 different strains of A. marginale were classified, showing 11 genotypes based on the 5′-UTR microsatellite and 193 different tandem repeats with high amino acid variability per position. Our results showed phylogenetic correlation between MSP1a sequence, secondary structure, B-cell epitope composition and tick transmissibility of A. marginale strains. The analysis of MSP1a sequences provides relevant information about the biology of A. marginale to design vaccines with a cross-protective capacity based on MSP1a B-cell epitopes.This research was supported by POSTICK ITN (Post-graduate training network for capacity building to control ticks and tick-borne diseases) within the FP7-PEOPLE-ITN programme (EU Grant No. 238511) and BFU2011-23896 grant to JF. JJV was sponsored by project CZ.1.07/2.3.00/30.0032, co-financed by the European Social Fund and the state budget of the Czech Republic.Peer reviewe

Complete genome sequence of ehrlichia mineirensis, a novel organism closely related to ehrlichia canis with a new host association

We report here the complete genome sequencing of Ehrlichia mineirensis, an Ehrlichia organism that was isolated from the hemolymph of Rhipicephalus microplus–engorged females. E. mineirensis is the best characterized Ehrlichia isolate from a novel cattle-related clade closely related to the monocytotropic pathogen E. canis

B-cell epitope analysis in A. marginale MSP1a tandem repeats.

<p>The B-cell epitopes were predicted using BCPRED server. The type 1 B-cell epitope was used as reference (Model) for comparisons. (A) Clustalw alignment and amino acid changes in the 5 more represented MSP1a tandem repeat B cell epitopes. B-cell epitope types model (light violet), 1 (blue), 10 (yellow), 11 (dark violet) and 17 (red) are shown. (B) Percent of tandem repeats containing each type of B cell epitopes. (C) Neighbor joining phylogenetic tree based on B cell epitope amino acid sequences showing the two clusters formed by the 5 more represented B cell epitopes. Cluster-1: Types 1 and 11 and Cluster-2: Types Model, 10 and 17. Correlations between VaxiJen/Blastp (D), BCPRED/Blastp scores (E) and VaxiJen/BCPRED (F) scores are shown. These correlations suggest that the epitopes with higher homology (Blastp score) share in common the immunogenic properties represented by VaxiJen/BCPRED.</p

Association of <i>A. marginale</i> MSP1a R1 sequences with world ecoregions.

<p>World ecoregions were built upon temporal series of NDVI values.</p>(a)<p>R1 sequences recorded in one ecoregion only.</p>(b)<p>R1 sequences that have been reported in other ecoregions.</p

Phylogenetic tree based on MSP1a tandem repeat amino acid sequences.

<p>The MSP1a sequences from tick-transmissible and non-transmissible strains (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065243#pone-0065243-t002" target="_blank">Table 2</a>) were included in the phylogenetic analysis. The phylogenetic tree was reconstructed using the neighbor joining and maximum likelihood methods. Reliability for internal branch was assessed using the bootstrapping method with 1000 bootstrap replicates. Bootstrap values are shown as % in the internal branch. The tree shows four phylogenetic clusters containing different patterns of MSP1a tandem repeat 2D structures. Cluster β-α-c (blue), cluster α-1 and cluster α-2 (beige) contain tick-transmissible A. marginale strains while in cluster β (red) fall the non-tick-transmissible strains.</p