Molecular characterization of the Babesia caballi rap-1 gene and epidemiological survey in horses in Israel Adi Rapoport, Karin Aharonson-Raz, Dalia Berlin, Saar Tal, Yuval Gottlieb, Eyal Klement, Amir Steinman ⇑ Koret School of Veterinary Medicine, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, POB 12, Rehovot 76100, Israel article info Article history: Received 8 October 2013 Received in revised form 28 January 2014 Accepted 30 January 2014 Available online 10 February 2014 Keywords: Piroplasmosis Babesia caballi cELISA RAP-1 Horses abstract Equine piroplasmosis imposes great concerns for the equine industry regarding international horse movement, and therefore requires reliable diagnostic tools. Recent studies from South Africa and Jordan, including a preliminary study in Israel, reported extremely low seroprevalence to Babesia caballi (B. cab- alli) (0–1%) using the acceptable rhoptry-associated protein-1 (RAP-1) cELISA. In accordance with the study from South Africa demonstrating a significant heterogeneity in the rap-1 gene sequence of South African B. caballi isolates, the objectives of this study were to phylogenetically characterize the rap-1 gene of the Israeli isolates and determine the prevalence of B. caballi in horses in Israel. Out of 273 horses tested using the RAP-1 cELISA, only one was sero-positive, while 9.3% were positive on PCR performed on the rap-1 gene. Phylogenetic analysis of the rap-1 gene grouped the Israeli isolates in a cluster together with the South African strains (99% nt identity), but in a separate cluster from the American/ Caribbean strains (81–82% nt identity). These findings support the existence of heterogeneity in the RAP-1 amino-acid sequences of the Israeli and South African isolates as compared to that used in the cEL- ISA commercial kit and raise doubts as to the ability of this assay to serve as a sole regulatory test for international horse movement. Risk factor analysis found management and age to significantly associate with prevalence of B. caballi, as higher prevalence was noted in horses held out on pasture and a negative association was recorded with age. In addition, B. caballi was not detected in horses in the steppe-arid and extreme-arid climatic regions as compared to the wetter regions. Findings of this study emphasize the need to combine several detection methods to ameliorate the control and spread of the disease. Ó 2014 Elsevier B.V. All rights reserved. 1. Introduction Located at the junction of 3 continents: Europe, Asia and Africa, Israel has a Mediterranean climate ranging from temperate to trop- ical, and is known to be endemic to many arthropod borne dis- eases. Equine piroplasmosis (EP) is a tick-borne disease caused by two haemoprotozoan parasites, Babesia caballi (B. caballi) and Theileria equi (T. equi). Previous studies conducted in Israel, using serological and molecular assays, have shown that T. equi is highly prevalent in Israel with a 33.7% seroprevalence and 26.4% preva- lence tested by PCR in 1998 and 2002, respectively (Shkap et al., 1998; Steinman et al., 2012). B. caballi is reported annually by the Israeli Veterinary Services and Animal Health, with few sero- logically positive diagnosed cases a year (Bellaiche, 2013). How- ever, its prevalence in horses in Israel has not been adequately addressed. Identification of the specific EP agent based on clinical signs alone is not probable due to the similar clinical signs manifested by horses infected with both parasites, including fever, anaemia, jaundice, edema, depression and even death (Knowles, 1996). Mixed infection with both parasites have been documented (Ros- García et al., 2013) as well as a subclinical carrier state identified for T. equi (de Waal, 1992). Identification of the parasite by means of blood smear examination can provide, in some cases, differenti- ation between the parasites. Paired merozoites joined at their pos- terior ends are a diagnostic feature of B. caballi infection (Mehlhorn and Shein, 1984), while a typically smaller four pear-shaped mer- ozoites forming a tetrad known as a ‘Maltese cross’ is a character- istic feature of T. equi (Brüning, 1996). However, the differentiation is difficult, particularly when parasitaemia is low and when mixed infections are encountered. Molecular methods for parasite detec- tion in blood are both sensitive and specific in the clinical stage but may fail to detect the parasite in carrier animals (Ribeiro et al., 2013). Therefore, serological tests are currently the most recom- mended detection method by the World Organization for Animal Health (OIE) (OIE, 2008). Among those methods is the http://dx.doi.org/10.1016/j.meegid.2014.01.033 1567-1348/Ó 2014 Elsevier B.V. All rights reserved. ⇑ Corresponding author. Tel.: +972 3 9688534; fax: +972 3 9604079. E-mail address: Steinman@agri.huji.ac.il (A. Steinman). Infection, Genetics and Evolution 23 (2014) 115–120 Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid