ORIGINAL ARTICLE In vitro Culture of a Novel Genotype of Ehrlichia sp. from Brazil E. Zweygarth 1 , H. Sch € ol 1 , K. Lis 1 , A. Cabezas Cruz 2 , C. Thiel 1 , C. Silaghi 1 , M. F. B. Ribeiro 3 and L. M. F. Passos 1,4 1 Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians-Universit€ at M€ unchen, Munich, Germany 2 Faculty of Science and Biology Centre of the ASCR, Institute of Parasitology, University of South Bohemia,  Cesk e Bud ejovice, Czech Republic 3 Departamento de Parasitologia, ICB-UFMG, Belo Horizonte, Brazil 4 Departamento de Medicina Veterin aria Preventiva, INCT-Pecu aria, Escola de Veterin aria- UFMG, Belo Horizonte, Brazil Keywords: Ehrlichia; Rhipicephalus (Boophilus) microplus; in vitro culture; tick cell; DH82; endothelial cell; cattle; 16S rRNA; Brazil Correspondence: E. Zweygarth. Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians- Universit€ at M€ unchen, Leopoldstrasse 5, Munich D-80802, Germany. Tel.: +49 89 21806837; Fax: +49 89 21803623; E-mail: ZweygarthE@gmail.com Received for publication November 15, 2012 doi:10.1111/tbed.12122 Summary Ehrlichiae are obligate intracytoplasmic Gram-negative, tick-borne bacteria belonging to the Anaplasmataceae family. Ehrlichioses are considered emerging diseases in both humans and animals. Several members of the genus Ehrlichia have been isolated and propagated in vitro. This study describes the continuous propagation of a Brazilian Ehrlichia sp. isolate in IDE8 tick cells, canine DH82 cells and bovine aorta cells. Initially, the organisms were isolated from the hae- molymph of a Rhipicephalus (Boophilus) microplus tick into IDE8 cells. Infected IDE8 cells were brought from Brazil to Germany, where the organisms were con- tinuously propagated in IDE8, DH82 and bovine aorta cells. Bovine aorta cells were infected and propagated for 3 months, corresponding to six subcultures, whereas the other two infected cell lines were kept for more than 1 year. During the cultivation period, 36 and 14 subcultures were carried out in IDE8 and DH82 cell cultures, respectively. Reinfection of IDE8 cells with organisms grown in DH82 cells was achieved. Sequence analysis made with a fragment of the 16S rRNA gene showed that this Ehrlicha sp. is closely related to Ehrlichia canis. How- ever, the maximum likelihood phylogenetic tree shows that it falls in a separate phylogenetic clade from E. canis. Introduction Ehrlichiae are obligate intracytoplasmic Gram-negative, tick-borne bacteria belonging to the Anaplasmataceae fam- ily. Ehrlichioses are considered emerging diseases in both humans and animals. At present, the genus Ehrlichia con- sists of five recognized species: Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia muris and Ehrlichia ruminantium (Dumler et al., 2001). Four of these species have been propagated in vitro, namely E. ruminantium, the causative agent for heartwater in ruminants (Bezuidenhout et al., 1985), E. canis, which causes tropical canine pancy- topenia (Dawson et al., 1991), E. chaffeensis, which causes moderate to severe disease in humans, and E. muris (Mun- derloh et al., 2009), isolated from a wild mouse and not yet attributed to a human disease. Recently, an Ehrlichia-like agent (Munderloh et al., 2009) and a new pathogenic Ehrli- chia species from the United States (Pritt et al., 2011) were isolated using in vitro culture techniques. New Ehrlichia spp. have been isolated from Rhipicephalus (Boophilus) microplus ticks in Asia and characterized molec- ularly (Wen et al., 2002; Parola et al., 2003), but these have not yet been propagated in vitro. In Brazil, three Ehrlichia spp. have been reported: E. canis (Costa et al., 1973), E. ewingi (Oliveira et al., 2009) and E. chaffeensis (Machado et al., 2006), of which E. canis was the only species estab- lished in cell culture (Torres et al., 2002). Ehrlichia chaffensis was found in wild herbivores, namely the marsh deer (Blast- ocerus dichotomus) (Machado et al., 2006) and the brown brocket deer (Mazama gouazoubira) (Silveira et al., 2012). In this study, we describe the continuous propagation of a Brazilian Ehrlichia sp. isolate in IDE8 tick cells, canine © 2013 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 60 (2013) (Suppl. 2) 86–92 86 Transboundary and Emerging Diseases