# The glycoprotein TRP36 of Ehrlichia sp. UFMG-EV and related cattle pathogen Ehrlichia sp. UFMT-BV evolved from a highly variable clade of E. canis under adaptive diversifying selection.

Alejandro Cabezas Cruz, James J. Valdes, Jose de La Fuente

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Alejandro Cabezas Cruz, James J. Valdes, Jose de La Fuente. The glycoprotein TRP36 of Ehrlichia sp. UFMG-EV and related cattle pathogen Ehrlichia sp. UFMT-BV evolved from a highly variable clade of E. canis under adaptive diversifying selection. Parasites & Vectors, 2014, 7 (584), 10.1186/s13071-014-0584-5. doi: 10.13071-014-0584-5

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# The glycoprotein TRP36 of *Ehrlichia* sp. UFMG-EV and related cattle pathogen *Ehrlichia* sp. UFMT-BV evolved from a highly variable clade of *E. canis* under adaptive diversifying selection

Alejandro Cabezas-Cruz<sup>1,2*</sup>, James J Valdés<sup>3</sup> and José de la Fuente<sup>2,4*</sup>

**Abstract**

**Background:** A new species of *Ehrlichia*, phylogenetically distant from *E. ruminantium*, was found in 2010 infecting cattle in Canada. In 2012 and 2013, we reported the *in vitro* propagation, molecular and ultrastructural characterization of *Ehrlichia* sp. UFMG-EV (*E. mineirensis*), a new species of *Ehrlichia* isolated from the haemolymph of Brazilian *Rhipicephalus (Boophilus) microplus* ticks. A new organism, named *Ehrlichia* sp. UFMT-BV, closely related to *Ehrlichia* sp. UFMG-EV, was recently described in Brazil and after experimental infection it was shown to be pathogenic for cattle. This new emerging clade of cattle *Ehrlichia* pathogens is closely related to *E. canis*. The major immunogenic Tandem Repeat Protein (TRP36; also known as gp36) is extensively used to characterize the genetic diversity of *E. canis*. Homologs of TRP36 were found in both *Ehrlichia* sp. UFMG-EV and *Ehrlichia* sp. UFMT-BV.

**Findings:** Herein, we characterized the evolution of this new *Ehrlichia* clade using TRP36 sequences. Our working hypothesis is that *Ehrlichia* sp. UFMG-EV and related microorganisms evolved from a highly variable *E. canis* clade. In support of our hypothesis we found that *Ehrlichia* sp. UFMG-EV and *Ehrlichia* sp. UFMT-BV TRP36 evolved from a highly divergent and variable clade within *E. canis* and this clade evolved under episodic diversifying selection with a high proportion of sites under positive selection.

**Conclusion:** Our results suggest that *Ehrlichia* sp. UFMG-EV and *Ehrlichia* sp. UFMT-BV evolved from a variable clade within *E. canis*.

**Keywords:** *Ehrlichia* sp. UFMG-EV, *Ehrlichia* sp. UFMT-BV, *E. mineirensis*, Host-shift, Diversifying episodic selection

**Findings**

*Ehrlichia* sp. UFMG-EV and *Ehrlichia* sp. UFMT-BV belong to a new clade of cattle-related *Ehrlichia*

Anaplasmataceae is a family of α-proteobacteria that includes the genera *Anaplasma*, *Ehrlichia*, *Neorickettsia* and *Wolbachia*. From these genera, *Ehrlichia* and *Anaplasma* are important pathogens affecting animals and humans. *Ehrlichia* are obligate intracellular gram-negative, tick-borne bacteria that grow within membrane-bound vacuoles in human and animal leukocytes causing ehrlichiosis. With

a worldwide distribution ehrlichioses are considered emerging diseases that can cause serious illness in a variety of hosts, including humans, livestock and pets. Three news species of cattle-related *Ehrlichia* spp have been recently reported: (i) a new species that naturally infect cattle from British Columbia, Canada [1], (ii) *Ehrlichia* sp. UFMG-EV (referred as *E. mineirensis* in [2,3]) that was isolated from *R. microplus* hemolymph [2-4], and (iii) *Ehrlichia* sp. UFMT-BV that was found to be pathogenic for cattle in Brazil [5]. These three organisms are closely related to *E. canis* [1,2,5]. *Ehrlichia* sp. UFMG-EV and *Ehrlichia* sp. UFMT-BV, however, present new sequence of tandem repeats different to the one reported for *E. canis* TRP36 [2,5,6].

<footnote>* Correspondence: cabezasalejandrocruz@gmail.com; jose_delafuente@yahoo.com</footnote>

<footnote n="1">¹Center for Infection and Immunity of Lille (CIIL), INSERM U1019 – CNRS UMR 8204, Université Lille Nord de France, Institut Pasteur de Lille, Lille, France</footnote>

<footnote n="2">²SaBio, Instituto de Investigación de Recursos Cinegéticos</footnote>

<footnote>IREC-CSIC-UCLM-JCCM, Ronda de Toledo s/n, 13005 Ciudad Real, Spain</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
The results of this work expand on our previous find-
ings regarding the evolution and differentiation of TRP36
in *Ehrlichia* sp. UFMG-EV [2]. Herein, we showed that
the gene *trp36* presents episodic bursts of selection,
unequally distributed across sites and that diversifying

selection occurs only in few branches of the *trp36* phylo-
genetic tree. Our results showed that *Ehrlichia* sp.
UFMG-EV and the new *Ehrlichia* sp. UFMT-BV affecting
cattle evolved from a highly divergent and variable clade
within *E. canis*.

<figure><img src="image_2.png" /><figcaption>**Figure 1** *Ehrlichia* sp. UFMG-EV and *Ehrlichia* sp. UFMT-BV strain belong to a variable clade within *E. canis*. The *trp36* (*E. canis*), *gp47* (*E. chaffeensis*) and mucin like protein (*E. ruminantium*) nucleotides sequences were aligned and gap regions removed. Phylogenetic analyses were conducted using ML and NJ. The figure shows that *Ehrlichia* sp. UFMG-EV and *Ehrlichia* sp. UFMT-BV fall in a divergent clade of *E. canis* *trp36* having low homology (less than 80%: red and pink boxes) compared to the isolate *E. canis* USA Jake 2. The amino acid sequence of the different TRP36 tandem repeats variants are shown (Coloured circles). The positions of the sequons are shown (red sticks on the boxes). The position of TRP36 ancestor clades I, II and III at internal branches (white circles) and position of sequons on ancestors (red sticks on white boxes) are also shown. The topologies obtained with the two methods were similar. The numbers above the internal branches represent bootstrap values. Only bootstrap values higher that 70 are shown.</figcaption></figure>
**Ehrlichia** sp. UFMG-EV *trp36* gene evolved from a highly
divergent clade within *E. canis*

To study the evolution of *trp36* gene we used a com-
bination of phylogenetic and evolutionary analysis (see
Additional file 1 for detailed description of materials
and methods). The gene *trp36* has been widely used to
study the genetic diversity of *E. canis* strains [7-10].
We performed maximum likelihood and neighbor join-
ing phylogenetic analyses with *trp36* nucleotide se-
quences available in GenBank (Additional file 1) to
study the evolution of *Ehrlichia* sp. UFMG-EV and
*Ehrlichia* sp. UFMT-BV *trp36* in relation to *E. canis*
*trp36*. The phylogenetic analysis showed that *Ehrlichia*

sp. UFMG-EV and *Ehrlichia* sp. UFMT-BV *trp36* are
separated but clustered together with *E. canis* strains
from South Africa, Taiwan and Brazil (Figure 1). Using
the *E. canis* strain USA Jake-2 as a reference, the
TRP36 amino acid sequences from the Taiwanese and
South African *E. canis* strains, together with *Ehrlichia*
sp. UFMG-EV and *Ehrlichia* sp. UFMT-BV, presented
the lowest percent (<86%) of homology (Figure 1, red
and pink boxes). The results demonstrated that *E.
canis* strain USA Jake-2 belongs to a conservative
TRP36 clade within *E. canis* (Figure 1). Members of
this clade have a high percent (>90%) of amino acid
homology in TRP36 (Figure 1, black boxes).

<figure><img src="image_3.png" /><figcaption>**Figure 2 Branches under episodic diversifying selection in the trp36 tree.** The tree of *trp36* orthologs is shown. Branch-site REL model (Additional file 1) was used to determine branches under episodic diversifying selection (highlighted in red). Branches were considered under episodic diversifying selection when corrected *p-value* < 0.05 (see Additional file 1 for methods). For the rest of the tree (branches in black) there is no evidence of episodic diversifying selection (Additional file 2).</figcaption></figure>
## The new TRP36 tandem repeat variants evolved from the typical *E. canis* tandem repeat

The tandem repeat composition of the divergent clade was highly variable, encoding the typical *E. canis* TRP36 tandem repeat (TEDSVSAPA), but also other variants – AQVSADSGA (*Ehrlichia* sp. UFMT-BV), EASVVPEA (New Brazilian variant of *E. canis*) and VPAASGDAQ (*Ehrlichia* sp. UFMG-EV) (Figure 1, coloured circles). The conservative TRP36 clade, however, only presented the tandem repeat variant TEDSVSAPA amongst all members. Ancestral sequence reconstruction (see Additional file 1 for detailed description of ancestral sequence reconstruction methods) showed that all the new TRP36 variants evolved from the typical TRP36 tandem repeat, TEDSVSAPA (Figure 1, white circles and roman numerals).

There is currently no experimental evidence that TRP36 has N-linked glycans. The evolution of highly divergent variants of TRP36, however, was associated with an increase in the number of sequons of N-glycosylation in TRP36 (Figure 1, red sticks on colored boxes). In agreement with this finding, the evolution of TRP36 ancestors from clades I to III was associated with the gain of one sequon of N-glycosylation for each evolutionary step (from I to II and from II to III – Figure 1, red sticks on white boxes). One of three sequons present in the ancestor of TRP36 clade III was lost in *Ehrlichia* sp. UFMG-EV and in the South African strains, but it is present in *Ehrlichia* sp. UFMT-BV and the Taiwanese strains. The second sequon in TRP36 ancestor clade I and the strains from USA, Spain, Israel, Central Africa and Brazil possess a proline (P) residue in the second position making it improbable that the asparagine (N) will be glycosylated (Figure 1, asterisks on red sticks). The relevancy of whether these sequons are glycosylated or not is that changes in glycosylation patterns may contribute to evade host immune system [11] and antigenic drift [12].

## *Ehrlichia* sp. UFMG-EV trp36 evolved under episodic diversifying selection

Our next step was to test whether different branches or codon sites of the trp36 phylogenetic tree evolved under episodic diversifying selection. Results showed that the diversifying selection events among the branches were scarce along the phylogenetic tree (Figure 2). Only 8 (A1, A3, A5, 1, 2, 7, 9 and 10) out of 51 (15.6%) branches were found to be under episodic diversifying selection (Corrected *p*-value ≤ 0.05 – Figure 2 and Additional file 2). Episodic diversifying selection was detected only in branches belonging to the highly divergent clade of TRP36 described above (Figure 1). The patterns of episodic diversifying selection were complex, with differences in extent and strength of selection along the diversifying branches. The branches can be separated into four groups: (i) 2, 9, A1, A3 and A5 that experienced strong selective

force (ω+ > 3333.56) in a small proportion of sites (Proportion < 0.07), (ii) 1 that experienced low selective force (ω+ = 7.86) in a high proportion of sites (Proportion = 0.17), (iii) 7 that experienced low selective force (ω+ = 46.08) in a low proportion of sites (Proportion = 0.05), and (iv) 10 that experienced middle selective force (ω+ = 166.14) in a high proportion of sites (Proportion = 0.15). Among the branches experiencing episodic selection, 11 out of 171 (6.4%) codon sites were under episodic diversifying selection (Table 1, Additional file 3). Most of these sites were concentrated in branches 7 and 1.

Searching the sequences for evidence of positive and negative selection using SLAC, FEL, REL and MEME (see materials and methods) showed that many sites experienced positive or negative selection (Table 2). The higher proportion of sites inferred to be evolving under positive selection was found in the ancestral branches 1, 7 and 10. The branches A1, A2, B2-B6, which are associated to deep branches 1 and 10 (Figure 2), were related to the occurrence of new forms of TRP36 tandem repeats (Figure 1). This relation suggests that early, strong selective events on lineages 1 and 10 may have been related to the occurrence of new tandem repeats. The sites under negative selection were concentrated in ancestral lineage 2.

Codon 77 evolved under diversifying (positive) and codon 116 evolved under negative selection. These two codons code for amino acids involved in the formation of sequons among TRP36 homologs (Additional file 4).

<table><caption>Table 1 Codons under episodic diversifying selection in specific branches</caption><thead><tr><th>Codons</th><th>Branches<sup>a</sup></th><th>From</th><th>To</th><th>Type of substitution<sup>b</sup></th></tr></thead><tbody><tr><td>10</td><td>7</td><td>aac (N)</td><td>ggt (G)</td><td>dN and dS</td></tr><tr><td>21</td><td>7</td><td>caa (Q)</td><td>tca (S)</td><td>dN</td></tr><tr><td rowspan="2">28</td><td>7</td><td>tca (S)</td><td>gta (V)</td><td>dN</td></tr><tr><td>1</td><td>gta (V)</td><td>aca (T)</td><td>dN</td></tr><tr><td rowspan="2">39</td><td>7</td><td>cat (H)</td><td>agt (S)</td><td>dN</td></tr><tr><td>1</td><td>agt (S)</td><td>cat (H)</td><td>dN</td></tr><tr><td>40</td><td>7</td><td>cct (P)</td><td>ggt (G)</td><td>dN</td></tr><tr><td>51</td><td>6</td><td>aat (N)</td><td>ggt (G)</td><td>dN</td></tr><tr><td rowspan="2">77</td><td>15</td><td>gct (A)</td><td>gtt (V)</td><td>dN</td></tr><tr><td>B1</td><td>gct (A)</td><td>gtt (V)</td><td>dN</td></tr><tr><td>105</td><td>2</td><td>tat (Y)</td><td>gaa (E)</td><td>dN</td></tr><tr><td>124</td><td>5</td><td>aat (N)</td><td>tct (S)</td><td>dN</td></tr><tr><td rowspan="2">142</td><td>8</td><td>tct (S)</td><td>ggt (G)</td><td>dN</td></tr><tr><td>10</td><td>tct (S)</td><td>gaa (E)</td><td>dN and dS</td></tr><tr><td rowspan="2">145</td><td>1</td><td>gct (A)</td><td>gtt (V)</td><td>dN</td></tr><tr><td>A1</td><td>gtt (V)</td><td>caa (Q)</td><td>dN and dS</td></tr></tbody></table>

<footnote><sup>a</sup>Internal and external branches are identified by numbers and letters and numbers as in Figure 2.</footnote>

<footnote><sup>b</sup>Type of substitution: non-synonymous (dN) and synonymous (dS).</footnote>
<table><caption>Table 2 Codons under positive and negative selection</caption><thead><tr><th>Codons</th><th>Branches<sup>a</sup></th><th>SLAC ω<sup>b</sup></th><th>SLAC p-value</th><th>FEL ω<sup>b</sup></th><th>FEL p-value</th><th>REL ω<sup>b</sup></th><th>Bayes factor</th><th>MEME ω<sup>b</sup></th><th>MEME p-value</th><th>Selection type<sup>c</sup></th></tr></thead><tbody><tr><td>6</td><td>B5</td><td>-4.047</td><td>0.333</td><td>-3.159</td><td>0.081</td><td>-1.126</td><td>229.790</td><td>-</td><td>-</td><td>Negative</td></tr><tr><td>8</td><td>7</td><td>-4.604</td><td>0.293</td><td>-4.597</td><td>0.071</td><td>-1.096</td><td>92.479</td><td>-</td><td>-</td><td>Negative</td></tr><tr><td>10</td><td>7</td><td>-0.686</td><td>0.787</td><td>-2.465</td><td>0.583</td><td>-0.149</td><td>0.595</td><td>&gt;100</td><td>0.030</td><td>Positive</td></tr><tr><td>21</td><td>7</td><td>4.991</td><td>0.423</td><td>3.013</td><td>0.222</td><td>0.810</td><td>3.221</td><td>&gt;100</td><td>0.019</td><td>Positive</td></tr><tr><td>28</td><td>7</td><td>8.543</td><td>0.184</td><td>5.818</td><td>0.082</td><td>1.678</td><td>28.536</td><td>&gt;100</td><td>0.010</td><td>Positive</td></tr><tr><td>39</td><td>7</td><td>6.912</td><td>0.371</td><td>3.907</td><td>0.444</td><td>1.265</td><td>8.352</td><td>&gt;100</td><td>0.013</td><td>Positive</td></tr><tr><td>40</td><td>7</td><td>4.047</td><td>0.444</td><td>2.432</td><td>0.350</td><td>0.476</td><td>1.985</td><td>&gt;100</td><td>0.011</td><td>Positive</td></tr><tr><td>49</td><td>1</td><td>-16.858</td><td>0.031</td><td>-10.024</td><td>0.007</td><td>-1.120</td><td>178.481</td><td>-</td><td>-</td><td>Negative</td></tr><tr><td>51</td><td>6</td><td>3.473</td><td>0.603</td><td>1.923</td><td>0.586</td><td>0.096</td><td>1.136</td><td>&gt;100</td><td>0.028</td><td>Positive</td></tr><tr><td>52</td><td>1</td><td>-8.094</td><td>0.111</td><td>-3.234</td><td>0.062</td><td>-1.087</td><td>77.050</td><td>-</td><td>-</td><td>Negative</td></tr><tr><td>71</td><td>1</td><td>-18.415</td><td>0.026</td><td>-14.187</td><td>0.004</td><td>-1.121</td><td>187.132</td><td>-</td><td>-</td><td>Negative</td></tr><tr><td>77</td><td>B1</td><td>4.047</td><td>0.444</td><td>2.150</td><td>0.377</td><td>0.340</td><td>1.681</td><td>&gt;100</td><td>0.025</td><td>Positive</td></tr><tr><td>83</td><td>5</td><td>-4.047</td><td>0.333</td><td>-3.402</td><td>0.073</td><td>-1.127</td><td>243.194</td><td>-</td><td>-</td><td>Negative</td></tr><tr><td>89</td><td>2</td><td>-9.551</td><td>0.153</td><td>-7.402</td><td>0.042</td><td>-1.101</td><td>99.411</td><td>-</td><td>-</td><td>Negative</td></tr><tr><td>94</td><td>2</td><td>-6.708</td><td>0.201</td><td>-4.324</td><td>0.059</td><td>-1.124</td><td>189.451</td><td>-</td><td>-</td><td>Negative</td></tr><tr><td>96</td><td>2</td><td>-4.047</td><td>0.333</td><td>-4.119</td><td>0.063</td><td>-1.127</td><td>242.661</td><td>-</td><td>-</td><td>Negative</td></tr><tr><td>105</td><td>2</td><td>3.569</td><td>0.689</td><td>2.357</td><td>0.608</td><td>0.330</td><td>1.536</td><td>&gt;100</td><td>0.030</td><td>Positive</td></tr><tr><td>116</td><td>2</td><td>-7.488</td><td>0.180</td><td>-3.380</td><td>0.074</td><td>-1.126</td><td>199.960</td><td>-</td><td>-</td><td>Negative</td></tr><tr><td>124</td><td>5</td><td>3.663</td><td>0.543</td><td>2.278</td><td>0.488</td><td>0.329</td><td>1.569</td><td>&gt;100</td><td>0.004</td><td>Positive</td></tr><tr><td>139</td><td>10</td><td>-17.080</td><td>0.037</td><td>-7.190</td><td>0.074</td><td>-1.007</td><td>23.196</td><td>-</td><td>-</td><td>Negative</td></tr><tr><td>142</td><td>1</td><td>6.862</td><td>0.316</td><td>6.098</td><td>0.511</td><td>1.459</td><td>31.512</td><td>&gt;100</td><td>0.009</td><td>Positive</td></tr><tr><td>145</td><td>1</td><td>4.902</td><td>0.413</td><td>4.701</td><td>0.571</td><td>1.230</td><td>7.496</td><td>&gt;100</td><td>0.017</td><td>Positive</td></tr><tr><td>152</td><td>10</td><td>20.127</td><td>0.244</td><td>41.463</td><td>0.238</td><td>1.510</td><td>66.006</td><td>&gt;100</td><td>0.224</td><td>Positive</td></tr><tr><td>158</td><td>1</td><td>2.355</td><td>0.585</td><td>-1.195</td><td>0.886</td><td>1.162</td><td>161.941</td><td>1.219</td><td>0.612</td><td>Positive</td></tr><tr><td>162</td><td>1</td><td>16.368</td><td>0.227</td><td>8.312</td><td>0.354</td><td>1.574</td><td>87.208</td><td>&gt;100</td><td>0.343</td><td>Positive</td></tr></tbody></table>
<table><caption>Table 2 Codons under positive and negative selection (Continued)</caption><tr><td>166</td><td>1</td><td>25.338</td><td>0.042</td><td>5.483</td><td>0.239</td><td>1.510</td><td>20.855</td><td>&gt;100</td><td>0.198</td><td>Positive</td></tr><tr><td></td><td>10</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>A8</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>167</td><td>10</td><td>28.242</td><td>0.032</td><td>8.115</td><td>0.083</td><td>1.677</td><td>53.176</td><td>&gt;100</td><td>0.084</td><td>Positive</td></tr><tr><td></td><td>B1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>170</td><td>10</td><td>-8.379</td><td>0.406</td><td>-74.785</td><td>0.031</td><td>0.536</td><td>0.396</td><td>-</td><td>-</td><td>Negative</td></tr><tr><td></td><td>9</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></table>

While codon 77 was selected in branches 15 and B1 (*E. canis*), codon 116 was selected in branch 2 (*E. canis*, *Ehrlichia* sp. UFMT-BV and *Ehrlichia* sp. UFMG-EV). This data therefore suggests that putative N-glycosylation associated with this sequon might be important in the host shift (see below) observed in *Ehrlichia* sp. UFMT-BV and *Ehrlichia* sp. UFMG-EV.

### Model of emergence of *Ehrlichia* sp. UFMG-EV and *Ehrlichia* sp. UFMT-BV within *E. canis*

The emergence of new pathogens is frequently associated to mutations that confer the ability to infect novel hosts, known as “host shift” [13]. *Ehrlichia* sp. UFMG-EV and *Ehrlichia* sp. UFMT-BV are closely related to *E. canis*, however they were associated to new invertebrate and vertebrate hosts, respectively. First, while the common tick vector for *E. canis* is *R. sanguineus* [14], *Ehrlichia* sp. UFMG-EV was isolated from *R. microplus* hemolymph [2]. Secondly, while *E. canis* is mainly pathogenic for dog [10], *Ehrlichia* sp. UFMT-BV was found to be pathogenic for cattle [5]. How pathogens can colonize new hosts is a challenging question in evolutionary biology [13]. Recently, Aguiar and colleagues [9] suggested that *E. canis* may have a wider range of hosts in Brazil than currently recognized. The host shift in this context may have occurred in a scenario where dogs infected with a variable *E. canis* strain, as previously found in Brazil [9], were the source of infection for *R. microplus* or *R. sanguineus* ticks that later infested cattle. Both tick species are able to infect dogs [15,16] and cattle [17]. The scenario involving *R. microplus* is unlikely as this is a one-host tick species. However, *R. microplus* moves among hosts during their parasitic lifetime [18], thereby increasing the chances of horizontal pathogen transmission among different hosts. Changes in evolutionary pressures on *E. canis*, related to new host association, may have resulted in a completely new species.

Our evidence supports the idea of differential evolutionary pressures on the glycoprotein TRP36 along different strains of *E. canis*, resulting in highly divergent variants of

TRP36. In the habitual host of *E. canis*, TRP36 must possess amino acid positions beneficial or neutral that may be deleterious in new hosts – the opposite may also be true. Within variable strains of a given pathogen, novel genetic variants may eventually deliver beneficial mutations that promote successful emergence, thereby providing a source for adaptive genetic variation in new hosts [13]. In agreement with this, we found a large proportion of sites that evolved under purifying (negative) selection, positive and diversifying selection. It is worth noting that the selective events were more frequent and strong in the deepest branches of *trp36* phylogenetic tree. This supports the hypothesis that most mutations that originated in the new TRP36 amino acid variants of *Ehrlichia* sp. UFMG-EV and *Ehrlichia* sp. UFMT-BV occurred before the emergence of the clade formed by these two organisms. The fact that the most recent common ancestor (Figure 1, ancestor clade III) between *Ehrlichia* sp. UFMG-EV, *Ehrlichia* sp. UFMT-BV and *E. canis* had a typical TRP36 tandem repeat structure, supports the aforementioned hypothesis. The divergence found in TRP36 tandem repeats was consistent with a 1.7% sequence divergence between 16S rRNA of *Ehrlichia* sp. UFMG-EV and *E. canis* [2]. Taking into account the high identity of 16S rRNA among *E. canis* strains (maximum 0.6%) [7], and thus the conservative nature of this gene, *Ehrlichia* sp. UFMG-EV may have diverged a long time ago from *E. canis*.

### Conclusion

Altogether, these results suggest that this new group of organisms evolved from *E. canis* sensu stricto and has become ecologically independent from the parental species. In agreement with the new hosts association of this group of microorganisms, it was found that *Ehrlichia* sp. UFMG-EV was able to propagate in bovine aorta BA886 cell line, while *E. canis* did not [4]. This *in vitro* observation supports the above conclusions regarding the new host specificity of this novel group of cattle related agents. At the ultrastructural level, *Ehrlichia* sp. UFMG-EV shares ultrastructural features with other members of

<footnote>ªInternal and external branches are identified by numbers and letters and numbers as in Figure 2.</footnote>

<footnote>ᵇThe ratio between non-synonymous (dN) and synonymous (dS) nucleotide substitution per site (ω) analyzed by Datamonkey via SLAC, FEL, REL and MEME.</footnote>

<footnote>ᶜSites were considered under positive selection (ω > 1) or negative selection (ω < 1) when at least one of the methods shows significant difference (p-value < 0.05 (SLAC, FEL and MEME) or Bayes Factor > 50 (REL)).</footnote>
the genus *Ehrlichia* (*E. muris*, *E. canis* and *E. chaffeensis*). We found cells, however, with unusual structures (invasion of the cellular membrane) for which we yet do not have an explanation [3]. Further studies should clarify the role of major immunogenic surface exposed proteins in the evolution of bacterial host shift. The full genome of *E. mineirensis* (*Ehrlichia* sp. UFMG-EV) might be an important contribution to these studies.

## Additional files

**Additional file 1: Detailed description of materials and methods.**

**Additional file 2: Likelihood ratio test statistics for branches under episodic diversifying selection.** Branch-site REL model (Additional file 1) was used to determine branches under episodic diversifying selection (highlighted in red). Branches were considered under episodic diversifying selection when corrected *p-value* < 0.05 (see Additional file 1 for methods).

**Additional file 3: Likelihood ratio test statistics for sites under episodic diversifying selection.** MEME (Additional file 1) was used to determine sites (codons) under episodic diversifying selection (highlighted in red). Sites were considered under episodic diversifying selection when *p-value* < 0.05 (see Additional file 1 for methods).

**Additional file 4: Sequons of N-glycosylation in the N-terminus of TRP36 variants.** Putative sequons of N-glycosilation of the N-terminus of TRP36 variant included in this study are shown.

## Competing interests

The authors declare that they have no competing interests.

## Authors' contributions

ACC developed the overall concept, drafted the manuscript, performed and interpreted the phylogenetic and evolutionary analysis. JJV drafted the manuscript and performed and helped in the interpretation of phylogenetic and evolutionary analysis. JF drafted the manuscript and made critical revisions to the manuscript and interpretation of the data. All authors read and approved the final version of the manuscript.

## Acknowledgments

This research was partially supported by the EU FP7 ANTIGONE project number 278976. 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. ACC was supported by a grant from the Ministère de l'Éducation Supérieure et de la Recherche de France.

## Author details

Received: 24 September 2014 Accepted: 30 November 2014
Published online: 10 December 2014

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doi:10.1186/s13071-014-0584-5

**Cite this article as:** Cabezas-Cruz et al.: The glycoprotein TRP36 of *Ehrlichia* sp. UFMG-EV and related cattle pathogen *Ehrlichia* sp. UFMT-BV evolved from a highly variable clade of *E. canis* under adaptive diversifying selection. *Parasites & Vectors* 2014 **7**:584.

<footnote n="1">¹Center for Infection and Immunity of Lille (CIIIL), INSERM U1019 – CNRS UMR 8204, Université Lille Nord de France, Institut Pasteur de Lille, Lille, France.</footnote>

<footnote n="2">²SaBio, Instituto de Investigación de Recursos Cinegéticos
IREC-CSIC-UCLM-JCCM, Ronda de Toledo s/n, 13005 Ciudad Real, Spain.</footnote>

<footnote n="3">³Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, České Budějovice, Czech Republic. ⁴Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078, USA.</footnote>