Babesia canis canis and Babesia canis vogeli infections in dogs from northern Portugal Luı ´s Cardoso a,b, * ,A ´ lvaro Costa c , Joana Tuna c , Lisete Vieira c , Osnat Eyal d , Yael Yisaschar-Mekuzas d , Gad Baneth d a Department of Veterinary Sciences and CECAV, University of Tra ´s-os-Montes e Alto Douro, P.O. Box 1013, 5001-801 Vila Real, Portugal b Parasite Disease Group, Instituto de Biologia Moleculare Celular (IBMC), Universidade do Porto, Portugal c Os Bichos–Veterinary Clinic, 5400-266 Chaves, Portugal d School of Veterinary Medicine, Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel Received 8 February 2008; received in revised form 7 May 2008; accepted 15 May 2008 Abstract Canine babesiosis represents an important veterinary medical problem. This study describes the molecular characterization of babesial parasites detected in eight clinically suspected dogs from northern Portugal, affected by lethargy, muscle tremors, weight loss, pale mucous membranes, hyperthermia or red-coloured urine. Microscopic examination of peripheral blood smears showed large intraerythrocytic piroplasms morphologically compatible with Babesia canis in all eight animals. DNA was extracted from blood on filter paper, and a Babesia spp. infection confirmed by polymerase chain reaction (PCR) amplification of a 408 bp fragment of the 18S rRNA gene. Analysis of PCR-derived sequences revealed that seven dogs were infected with B. canis canis and one with B. canis vogeli. This is the first molecular identification report of both the species B. canis and the subspecies B. canis canis and B. canis vogeli in dogs from Portugal. # 2008 Elsevier B.V. All rights reserved. Keywords: Babesia canis canis; Babesia canis vogeli; Babesiosis; Dog; Portugal; 18S rRNA gene 1. Introduction Canine babesiosis (or piroplasmosis), caused by tick- borne protozoa, represents an important veterinary medical problem worldwide (Lobetti, 1998). Two species have traditionally been identified as the aetiological agents of the disease in dogs: Babesia canis and Babesia gibsoni, which correspond to large (3–5 mm) and small (0.5–2.5 mm) intraerythrocytic parasitic forms (or piroplasms), respectively (Boozer and Macintire, 2003). The clinical features of canine babesiosis often include hyperthermia, anaemia, hae- moglobinuria, lethargy and anorexia (Bourdoiseau, 2006), but clinical signs in dogs may vary depending on the pathogen (Schetters et al., 1997) and host immunity (Branda ˜o et al., 2003). The species B. canis has been subdivided into three subspecies – B. canis canis, B. canis vogeli and B. canis rossi – on the basis of differences in vector specificity, geographical distribution, pathogenicity and antigenic properties (Uilenberg et al., 1989). Characterization with molecular methods confirmed the existence of three distinct genotypes of B. canis, and it has additionally been suggested that each of the subspecies www.elsevier.com/locate/vetpar Available online at www.sciencedirect.com Veterinary Parasitology 156 (2008) 199–204 * Corresponding author at: Department of Veterinary Sciences, University of Tra ´s-os-Montes e Alto Douro, P.O. Box 1013, 5001- 801 Vila Real, Portugal. Tel.: +351 259 350 458; fax: +351 259 350 629. E-mail address: lcardoso@utad.pt (L. Cardoso). 0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.05.027