Seroprevalence and risk factors for infection with equine coronavirus in healthy horses in the USA L.J. Kooijman a, K. Jamesb, S.M. Mapesb, M.J.P. Theelena, N. Pusterlab,* aDepartment of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 114, Utrecht 3584CM, The Netherlands bDepartment of Veterinary Medicine and Epidemiology, School of Veterinary Medicine, University of California, One Shields Avenue, Davis, California 95616, USA A R T I C L EI N F O Article history: Accepted 10 January 2017 Keywords: Equine coronavirus ELISA Horses Risk factors Seroprevalence A B S T R A C T Equine coronavirus (ECoV) is considered an enteric pathogen of foals and has only recently been asso- ciated with infections in adult horses. Seroprevalence data is needed to better understand the epidemiology of ECoV in adult horses, evaluate diagnostic modalities and develop preventive measures. The objective of this study was to investigate the seroprevalence and selective risk factors for ECoV in 5247 healthy adult horses in the USA, using a recently established and validated IgG enzyme-linked immunosorbent assay. Prevalence factors analysed in this study included geographic region, age, breed, sex and use. A total of 504/5247 horses (9.6%) horses tested seropositive. Geographic region (Mid-West; P = 0.008), breed (Draft horses; P = 0.003) and specifi c uses of horses (ranch/farm, P = 0.034; breeding use, P = 0.016) were all statistically signifi cant risk factors for seropositivity. 2017 Published by Elsevier Ltd. Introduction Coronaviruses are part of the coronaviridae family, which are positive-strand RNA viruses. The coronaviridae subfamily is grouped into four genera, based on genetic differences and serological cross- reactivity (Woo et al., 2009, 2012). Equine coronavirus (ECoV) is a -coronavirus, as are human coronavirus OC43 and HKU1, murine hepatitis virus, bovine coronavirus (BCoV), porcine haemagglutinating encephalomyelitis virus, canine respiratory coronavirus and rat coronavirus (Zhang et al., 2007). ECoV is considered an enteric patho- gen of foals (Guy et al., 2000) and has only recently been associated with infections in adult horses in Japan, the USA and Europe (Oue et al., 2011, 2013; Pusterla et al., 2013; Miszczak et al., 2014). Re- ported morbidity rates during outbreaks range from 2083% (Pusterla et al., 2013; Fielding et al., 2015; Giannitti et al., 2015). Despite the sporadic occurrence of ECoV outbreaks in adult horses, the overall seroprevalence of this virus in horse popula- tions remains largely unknown. Seroprevalence data is needed to better understand the epidemiology of ECoV, evaluate diagnostic modalities and develop preventive measures. Therefore, the objec- tive of this study was to investigate the seroprevalence and selective risk factors to ECoV in 5247 healthy adult horses originating from 18 US states using a recently established and validated ELISA (Kooijman et al., 2016). Materials and methods Study population The study population consisted of 5247 healthy horses from 18 states (CA, CO, FL, KY, MD, MN, MS, MO, NY, NC, OH, PA, TN, TX, VA, WA, WI, WY) representing the four regions of the USA (North-East, South, Mid-West, West). States sampled from the four regions were as follows: North-East (NY, MD, PA); South (FL, MS, KY, TN, NC, VA, TX); Mid-West (MO, OH, WI, MN); West (WA, CA, CO, WY). Banked sera col- lected by Zoetis USA for the purposes of another study were used. Blood samples were collected by convenience sampling from healthy horses by equine veterinar- ians from participating veterinary clinics across the USA during the autumn of 2013. Clinics enrolled voluntarily and each clinic sampled approximately 110 horses; no more than 10 horses sampled from each clinic resided on the same farm. Jugular venipuncture was used to collect 10 mL of whole blood from each horse. Addition- ally, age, breed, primary use, and sex data were collected for each horse by questionnaire. Blood samples were centrifuged, serum was separated, shipped on ice, processed and stored at 20 C. Serological analysis All sera were tested for IgG to ECoV using an ELISA based on a recombinant protein containing two immunodominant areas of the spike protein of ECoV, including the area with the highest predicted antigenic sequence (Kooijman et al., 2016). Briefl y, microtiter plates were coated with 0.0156 g recombinant protein per well and sera were tested at a dilution of 1 L serum in 100 L of buffer. Each plate contained known negative and positive controls. Negative controls consisted of sera collected from healthy adult horses from the university herd with no history of systemic disease and qPCR-negative feces for ECoV. Positive controls consisted of sera collected from horses 30 days after they developed clinical signs compatible with ECoV infection and tested qPCR-positive for ECoV in the faeces. A secondary horseradish-peroxidase labelled IgG was used to bind with ECoV-specifi c antibo