Detection of a putative novel genotype of Ehrlichia sp . from opossums ( Didelphis aurita ) from Brazil

Erlichiosis affects humans and animals worldwide. Its distribution and prevalence depends on the presence of tick vectors and hosts in one geographic area. The aim of the present study was to investigate the occurrence of Ehrlichia spp. and Anaplasma spp. in opossums (Didelphis sp.) from the State of Rio de Janeiro, southeast Brazil. Blood samples from 37 animals were tested for these two pathogens using molecular methods. One animal (2.7%) was positive for Ehrlichia sp. by 16S rRNA-based nested PCR. In a phylogenetic analysis based on the 16S rRNA gene using the maximum likelihood method and the GTRGAMMA+I evolutionary model, we detected a novel Ehrlichia sp. genotype closely related to genotypes of E. canis previously reported in dogs from Brazil. To the authors’ knowledge, this is the first molecular detection of Ehrlichia sp. in opossums from this State in the southeastern region of the country.

However, to date there are few published studies on the occurrence of Anaplasmataceae in opossums.For instance, Lockhart et al. (1997) found 3 of 38 (8%) opossums (Didelphis virginianus) seropositive for E. chaffeensis in the State of Georgia, USA, with titres ranging between 64 and 512.Similarly, antibodies to E. chaffeensis were detected in serum samples from 3 of 19 (15.8%) opossums in the State of Mississipi, USA (CASTELLAW et al., 2011).Recently, antibodies against Ehrlichia canis were detected in blood specimens of 16 of 109 (14.67%) opossums from the State of São Paulo, southeast Brazil (MELO et al., 2016), and Ehrlichia and Anaplasma DNA were found in marsupials from the Brazilian Pantanal in the central-west region of the country (SOUSA et al., 2017).
The aim of the present study was to investigate the occurrence of Anaplasmataceae in opossums from the State of Rio de Janeiro, southeast Brazil, using molecular techniques including PCR.
Between January 2014 and November 2014, blood samples from 37 opossums (Didelphis sp.), selected by non-probability convenience collected at the Centro de Triagem de Animais Silvestres (CETAS) and at the Centro de Recuperação de Animais Silvestres from the Universidade Estácio de Sá (CRAS) located in the State of Rio de Janeiro, southeast Brazil.
This study was approved by the Committee on Animal Research and Ethics (Sisbio, n. 47791) in Brazil.
Blood samples were collected with anticoagulant by venipuncture of the ventral tail vein (JURGELSKI, 1974).DNA was extracted from 200 µL of each EDTA-whole blood sample using the ReliaPrep™ Blood gDNA Miniprep System (Promega™, Madison, Wisconsin, United States) according to the manufacturer's instructions.Ultra-pure sterile water (Invitrogen™, Carlsbad, California, United States) was used as negative controls in each batch of samples to assess DNA contamination during extraction of total DNA.
The concentration of extracted DNA samples was determined using a spectrophotometer NanoDrop 2000 (Thermo Scientific™).DNA samples were divided into aliquots and stored at -80°C for subsequent molecular analysis.
A DNA sample used as a positive control was obtained from dogs experimentally infected with the Jaboticabal strain of E. canis (CASTRO et al., 2004).A DNA sample used as a positive control was obtained from a dog naturally infected with A. platys from the  The reaction products were purified using a Silica Bead DNA Gel Extraction Kit (Fermentas®, São Paulo, SP, Brazil).Purified amplified DNA fragments were submitted for sequencing in an automatic sequencer (ABI Prism 310 Genetic Analyser -Applied Biosystem/ Perkin Elmer) at the Centro de Recursos Biológicos e Biologia Genômica (FCAV/ UNESP, Jaboticabal, SP, Brazil) and used for subsequent phylogenetic analysis.Consensus sequences were obtained through the analysis of the sense and antisense sequences using the CAP3 program (HUANG & MADAN, 1999).
We compared our sequences with those deposited in GenBank using the basic local alignment search tool (BLAST).The sequences were aligned with sequences published in GenBank using Clustal/W (THOMPSON et al., 1994) and manually adjusted in Bioedit v. 7.0.5.3 (HALL, 1999).Phylogenetic inference based on maximum likelihood criterion (ML) was inferred with RAxML-HPC BlackBox 7.6.3(STAMATAKIS et al., 2008) through the CIPRES Science Gateway.
Akaike information criterion was used with the software JModelTest on CIPRES Science Gateway in order to identify the most appropriate model of nucleotide substitution.The GTRGAMMA+I model was chosen as the most appropriate for the Maximum Likelihood analysis of the 16S rDNA alignment.
One (2.7%) out of 37 marsupials was positive for both E. canis and E. chaffeensis by nested PCR assays based on the 16S rRNA gene.The sequence was 100% identical to Ehrlichia canis (GenBank access number KR920044.1)by BLAST analysis.
All the blood samples tested in this survey were negative for Anaplasma spp.by PCR assays based on the 16S rRNA.
The only sample that was positive for Ehrlichia spp.by 16S rRNA-based PCR was negative in the PCR assays based on the dsb and omp-1 genes which precluded additional molecular characterization.
In the phylogenetic analysis based on the 16S rRNA gene using the maximum likelihood method and GTRGAMMA+I evolutionary model, the Ehrlichia sp.genotypes detected in opossums (Didelphis sp.) was closely related to E. canis and to genotypes related to E. canis previously been detected in dogs from Peru and Brazil (Figure 1).
The positive animal was a young female Didelphis aurita which weighed 363g and was captured in Vargem Grande neighborhood, Rio de Janeiro, RJ, Southeast Brazil, on March 21, 2014.This animal was transported to CRAS for rehabilitation and subsequent reintroduction into her original habitat.At the time of blood sampling on March 31, 2014, this marsupial was not infested by ticks.During treatment, the animal was kept in a cage with water provided ad libitum and was fed with fruits.Treatment and management were performed by a practitioner at CRAS.Although opossums are infested with different species of ticks and can often act as amplifier hosts of a number of pathogens, there are few published studies about the detection of Ehrlichia spp. in these marsupials (HORTA et al., 2009).
In Brazil, antibodies against Ehrlichia spp.have been detected in opossums from the State of São Paulo.Authors have suggested the possibility that a unclassified Ehrlichia yet to be reported circulates among these animals as cross-reactivity occurs in serological assays (MELO et al., 2016;HARRUS & WANER, 2011).Recently, Sousa et al. (2017) detected Ehrlichia DNA in 23.3% of marsupials in the Pantanal, central-west region of the country.
Our results reinforces such findings from previous studies published elsewhere, and also highlight the fact that opossums may play a role in the epidemiology of Ehrlichia infection.In the present survey, the Ehrlichia sp.16S rRNA sequence that was found in one female opossum belongs to Ehrlichia genotype closely related to E. canis.Unfortunately, we did not test the extracted DNA samples to a housekeeping gene.Therefore, we could not rule out the occurrence of false negative results, precluding any assumption on the prevalence of this agent in the studied population.Future studied should be conducted in order to investigate the prevalence of this putative novel genotype in opossum population from different Brazilian regions.
Although further molecular characterization based on other genes is desirable to better assess the phylogeny of Ehrlichia sp. that occurs in marsupials in Brazil, the amount of DNA from animals of this study was insufficient for additional testing.In addition, attempts to make additional phylogenetic inferences based on genes other than 16S were unsuccessful.Our failed attempts corroborate the results published by Benevenute et al. (2017).
The identity at the species level of the genotypes of Ehrlichia that circulate in wild animals in Brazil needs to be further investigated.A larger number of marsupials and better molecular and antigenic characterization are needed in order to better assess the role of these mammals in the epidemiology of ehrlichiosis in South America.
Although the opossums tested in the present study were not infested by ectoparasites at the time of blood collection, there is a possibility that ectoparasites may have left the animal during the 10 day period of captivity.
Larvae, nymphs, and adults of Amblyomma spp.and Ixodes loricatus ticks have been reported in opossums from the State of São Paulo, Brazil (HORTA et al., 2007).
Besides, I. loricatus and A. aureolatum have been found in Didelphis albiventris in the Brazilian States of Mato Grosso do Sul and Rio Grande do Sul, respectively (MIZIARA et al., 2008;MULLER et al., 2005).
In conclusion, a Ehrlichia sp.genotype closely related to E. canis circulates in opossums in the state of Rio de Janeiro, southeast Brazil.

Figure 1 .
Figure 1.Phylogenetic position of Ehrlichia sp.isolated from a Brazilian opossum based on 16S rRNA DNA sequences (300 bp).The tree was constructed using the neighbor-joining method, and the numbers on the tree indicate bootstrap values for the branch points.Accession numbers are indicated.

Table 1 .
Description of primers, PCR product size and references used in PCR assays for E. canis, E. chaffeensis and Anaplasma spp.based on 16S rRNA, omp and dsb genes.