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. 2012 Dec 11:5:291.
doi: 10.1186/1756-3305-5-291.

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

Alejandro Cabezas Cruz  1 , Erich Zweygarth, Mucio Flavio Barbosa Ribeiro, Julia Angelica Gonçalves da Silveira, Jose de la Fuente, Libor Grubhoffer, James J Valdés, Lygia Maria Friche Passos
Affiliations

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

Alejandro Cabezas Cruz et al. Parasit Vectors. .

Abstract

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.

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Figures

Figure 1
Figure 1
AB Phylogenetic trees based on the 16S rRNA (A) and groEL (B) genes sequences from members of the family Anaplasmataceae. The tree shows that E. mineirensis (UFMG-EV) falls in a clade separated from all the previous reported sequences. Bootstrap values are shown as % in the internal branch. Only bootstrap values equal or higher than 50% are shown. Rickettsia prowazekii 16S rRNA sequence was used to root the 16S rRNA tree and E.coli groEL gene was used to root the groEL tree. The GenBank accession numbers of the sequences used to build the 16S rRNA tree are: E. muris, AB013008; E. chaffeensis, AF147752; E. ruminantium, AF069758; E. ewingii, U96436; A. marginale, M60313; A. phagocytophilum, M73224; A. platys, M82801; N. helminthoeca, U12457; N. sennetsu, M73225; N. risticii, AF036649; E. canis, GU810149; R. prowazekii, NR044656. The GenBank accession numbers of the sequences used to build the groEL tree are: E. muris, AF210459; E. chaffeensis, L10917; E. ruminantium, U13638 ; E. ewingii, AF195273; A. marginale, AF165812; A. phagocytophilum, U96729; A. platys, AY008300; N. sennetsu, U88092; N. risticii, U96732; E. canis, U96731; E. coli, X07850.
Figure 2
Figure 2
A highly variable region of sequence located at 5’ end of the 16S rRNA gene revealed by multiple alignments of 16S rRNA gene sequences of Ehrlichia genus. Underlined are the nucleotide differences found between E. canis and E. mineirensis (UFMG-EV). The GenBank accession numbers of the sequences show in the alignment are: E. muris, AB013008; E. chaffeensis, AF147752; E. ruminantium, AF069758; E. ewingii, U96436 and E. canis, GU810149.
Figure 3
Figure 3
Phylogenetic unrooted tree based on the dsb gene sequences from members of the family Anaplasmataceae. The tree shows that E. mineirensis (UFMG-EV) falls in a clade separated from all the previous reported sequences and the previously reported E. canis dsb sequences. Bootstrap values are show as% in the internal branch. Only bootstrap values equal or higher than 50% are shown. The GenBank accession numbers of the dsb sequences used to build the tree are: E. canis, AF403710; E. canis Uberlandia, GU586135; E. canis Jaboticabal, DQ460716; E. canis Sao Paulo, DQ460715; E. muris, AY236484; E. chaffeensis, AF403711; E. ruminantium, AF308669, clon 18hw; E. ewingii, AY428950.
Figure 4
Figure 4
Phylogenetic tree based on the citrate synthase (gltA) gene sequences from members of the family Anaplasmataceae. The tree shows that E. mineirensis (UFMG-EV) falls in a clade separated from all the previously reported sequences. Bootstrap values are show as % in the internal branch. Only are showed bootstrap values equal or higher than 50%. N. risticii gltA sequence was used to root the tree. The GenBank accession numbers of the gltA sequences used to build the tree are as follow: E. canis, AF304143; E. muris, AF304144; E. chaffeensis, AF304142; E. ruminantium, AF304146; E. ewingii, DQ365879; A. marginale, AF304140; A. phagocytophilum, AF304138; A. platys, AY077620.
Figure 5
Figure 5
A-E Epitope identification. The modeled 3D structures for E. mineirensis (UFMG-EV) (A), E. canis (B; GenBank: EF560599), and E. chaffeensis (C and D; GenBank: DQ085430, DQ146157, respectively) depict the position of the predicted epitope (→). Protein structures are colored from blue (N-terminus) to red (C-terminus) according to the residue position. An epitope Cα superimposition (E) of E. mineirensis (UFMG-EV) (cyan), E. canis (brown), E. chaffeensis (GenBank: DQ085430; green) and E. chaffeensis (GenBank: DQ146157; yellow) depicting the differences in their overall structures, E. mineirensis (UFMG) having a 5-6 Å difference compared with the other epitopes).
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