ORIGINAL PAPER Erich Zweygarth ® Laura M. Lopez-Rebollar Jane Nurton ® Alan J. Guthrie Culture, isolation and propagation of Babesia caballi from naturally infected horses Received: 24 September 2001 /Accepted: 11 December 2001 / Published online: 2 March 2002 Ó Springer-Verlag 2002 Abstract Thirteen blood samples of horses from South Africa, five of which were seropositive for Babesia cab- alli and eight for both B. caballi and Theileria equi, were subjected to in vitro culture to identify carrier animals. None of the animals had a detectable parasitaemia on Giemsa-stained blood smears before culture initiation. Cultures were initiated in L-cysteine-enriched medium, either in an oxygen-reduced gas mixture or in a 5% CO 2 - in-air atmosphere. All five animals seropositive for B. caballi were identified as carrier animals using an oxygen-reduced atmosphere, whereas only four samples became culture positive under normal atmospheric conditions. Among the eight samples seropositive for both B. caballi and T. equi, two were identified as car- riers for both. The remaining six samples were identified as carrying only T. equi. Introduction Equine piroplasmosis is a tick-borne disease of equidae (horses, mules, donkeys and zebras) caused by two intra- erythrocytic protozoan parasites, Babesia caballi and Theileria equi. The latter, previously known as Babesia equi, multiplies in lymphoid cells before invading ery- throcytes (Schein et al. 1981) and has therefore recently been reclassified as a Theileria species (Mehlhorn and Schein 1998). Equine piroplasmosis is widespread in South Africa. A serological survey revealed that, of 6,350 serum samples collected from all over South Africa, nearly 80% were positive for T. equi and approximately 50% were positive for B. caballi (De Waal 1995). Despite this, during culture diagnostic procedures designed to detect T. equi parasites in carrier animals (Zweygarth et al. 1997, 1999), B. caballi was only incidentally demon- strated. A definite diagnosis of equine piroplasmosis relies on the demonstration of parasites in a stained blood smear; a demonstrable parasitaemia is rarely observed. In vitro culture techniques may therefore be considered for diagnosis, either alone or to supplement other procedures such as serology or DNA-based methods. In the case of B. caballi the carrier status of four out of nine horses previously experimentally infected has been successfully confirmed by culture (Holman et al. 1993). The aim of this study was to demonstrate the pres- ence of, and to isolate, B. caballi parasites found in naturally infected horses by using a culture technique originally developed for the in vitro diagnosis of T. equi. Materials and methods Blood samples Blood samples were collected from horses at the National Yearlings Sale in March 2000. Serum was prepared and examined for anti- B. caballi and anti-T. equi antibodies using the indirect fluorescent antibody (IFA) test as described for B. caballi by Madden and Holbrook (1968). A titre of ‡1:80 was considered to be positive, and 13 out of 515 samples were found to be positive for B. caballi, and of these 13, 8 were also positive for T. equi. Blood samples were collected from the B. caballi seropositive horses by venipuncture into sterile vacuum tubes containing EDTA as anticoagulant (Vacutainer, Becton Dickinson, Meylan, France). The samples were brought to the Onderstepoort Veterinary Insti- tute (OVI) on ice in a polystyrene container and processed as below. Thin blood smears, prepared from all blood samples, were stained with Giemsa stain and examined microscopically. No par- asites were detected during these examinations. None of the horses that were serologically positive for B. caballi had a history of previous treatment with antiprotozoal agents. Culture medium A modified HL-1 medium (BioWhittaker, Walkersville, Md.) as described recently for the culture initiation of T. equi (B. equi) Parasitol Res (2002) 88: 460–462 DOI 10.1007/s00436-002-0609-4 E. Zweygarth (&) ® L.M. Lopez-Rebollar Onderstepoort Veterinary Institute, Parasitology Division, Private Bag X5, Onderstepoort 0110, South Africa E-mail: erich@moon.ovi.ac.za J. Nurton ® A.J. Guthrie Equine Research Centre, Faculty of Veterinary Science, University of Pretoria, Private Bag X4, Onderstepoort 0110, South Africa