Experimental Parasitology 120 (2008) 255–260 0014-4894/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2008.07.011 Contents lists available at ScienceDirect Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr 1. Introduction Neospora caninum is an obligate intracellular protozoan that causes abortion and economic loss in the cattle industry. There are still many aspects of the lifecycle of N. caninum that remain unknown. At present only the dog and coyote are proven definitive hosts (McAllister et al., 1998; Gondim et al., 2004) but the recent finding of oocysts in the faeces of the red fox suggests this may also be a definitive host (Wapenaar et al., 2006). There is currently much speculation about the role wildlife may play as a reservoir of infection (e.g. Gondim, 2006). Recent findings indicate that small mammals such as mice (Mus domesticus and Mus musculus), rats (Rattus norvegicus) and wood mice (Apodemus sylvaticus) may be natural intermediate hosts (Huang et al., 2004; Hughes et al., 2006; Jenkins et al., 2007; Ferroglio et al., 2007; Barratt et al., 2008). Inves- tigation of risk factors associated with bovine neosporosis in Nor- mandy (France) found the presence of rabbits and/or ducks were a risk factor for seropositivity in dairy cattle (Ould-Amrouche et al., 1999). To our knowledge there has been no investigation of the prevalence of N. caninum in wild rabbits in the UK, and only one previous serological study in rabbits in Spain (Almería et al., 2007). In this latter study no evidence of exposure to the parasite was detected in rabbits. The closely related parasite Toxoplasma gondii has been more extensively studied and has been found to be a natu- rally occurring infection of wild rabbits in Europe (e.g. Hejlicek and Literak, 1994; Almería et al., 2004; Figueroa-Castillo et al., 2006). The objectives of this study were to investigate the prevalence and co-infection of N. caninum and T. gondii, using PCR techniques, in a naturally infected population of wild rabbits from Northern England; the tissue distribution of N. caninum in rabbits and the range of strain types of T. gondii present in this sample of rabbits. 2. Materials and methods 2.1. Collection of samples and DNA extraction Rabbits (Oryctolagus cuniculus) n = 57 were collected from within 2 km of Malham Tarn in the Yorkshire Dales over a three Neospora caninum: Detection in wild rabbits and investigation of co-infection with Toxoplasma gondii by PCR analysis J.M. Hughes a , D. Thomasson a , P.S. Craig a , S. Georgin a , A. Pickles b , G. Hide a, * a Centre for Parasitology and Infectious diseases, Biomedical Sciences Research Institute, University of Salford, Salford M5 4WT, UK b Field Studies Council at Malham Tarn, Settle, North Yorkshire BD24 9PU, UK article info abstract Article history: Received 9 May 2008 Received in revised form 22 July 2008 Accepted 23 July 2008 Available online 30 July 2008 Neospora caninum is an important pathogen of cattle causing significant economic loss. There is much cur- rent interest in wild animal reservoirs for this parasite. The role of the rabbit in this is currently unknown. DNA samples from the brains of wild rabbits (Oryctolagus cuniculus) collected from the Malham area of the Yorkshire dales were investigated by species-specific PCR for the presence of N. caninum and Toxoplasma gondii. We found prevalences of N. caninum of 10.5% (6/57) and T. gondii of 68.4% (39/57) with 8.8% (5/57) co-infected. Strain typing of T. gondii positive rabbits revealed strain types I–III were pres- ent in this population. Investigation of tissue distribution determined N. caninum DNA was most often detected in the brain and heart, less often in the tongue and not in the liver. To our knowledge this is the first report of N. caninum detection in naturally infected wild rabbits. © 2008 Elsevier Inc. All rights reserved. Keywords: Apicomlexa Neospora caninum Toxoplasma gondii PCR Co-infection Rabbit Wildlife DNA, deoxyribonucleic acid ELISA, enzyme linked immunosorbent assay IFAT, indirect florescent test PCR, polymerase chain reaction SAG, surface antigen gene TE, Tris–EDTA buffer * Corresponding author. Fax: +44 (0)161 2955015. E-mail address: g.hide@salford.ac.uk (G. Hide).