Geographical segregation of Cryptosporidium parvum multilocus genotypes in Europe Simone M. Cacciò a,⇑ , Valerie de Waele b , Giovanni Widmer c a Istituto Superiore di Sanità, Department of Infectious, Parasitic and Immunomediated Diseases, Viale Regina Elena 299, Rome 00161, Italy b Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerpen, Belgium c Cummings School of Veterinary Medicine at Tufts University, Department of Infectious Disease and Global Health, North Grafton, MA 01536, United States article info Article history: Received 14 October 2014 Received in revised form 22 December 2014 Accepted 6 February 2015 Available online 14 February 2015 Keywords: Cryptosporidium parvum Multilocus genotypes Population structure Principal coordinate analysis Linkage disequilibrium abstract Cryptosporidium parvum is a common enteric protozoan pathogen of humans and livestock. Multilocus genotyping based on simple sequence repeat polymorphisms has been used extensively to identify transmission cycles and to investigate the structure of C. parvum populations and of the related pathogen Cryptosporidium hominis. Using such methods, the zoonotic transmission of C. parvum has been shown to be epidemiologically important. Because different genetic markers have been used in different surveys, the comparison of Cryptosporidium genotypes across different laboratories is often not feasible. Therefore, few comparisons of Cryptosporidium populations across wide geographical areas have been published and our understanding of the epidemiology of cryptosporidiosis is fragmented. Here we report on the genotypic analysis of a large collection of 692 C. parvum isolates originating primarily from cattle and other ruminants from Italy, Ireland and Scotland. Because the same genotypic markers were used in these surveys, it was possible to merge the data. We found significant geographical segregation and a correla- tion between genetic and geographic distance, consistent with a model of isolation by distance. The pres- ence of strong LD and positive I A S values in the combined MLG dataset suggest departure from panmixia, with different population structures of the parasite prevailing in each country. Ó 2015 Elsevier B.V. All rights reserved. 1. Introduction Species within the genus Cryptosporidium are obligate intracel- lular parasites that multiply in the epithelial cells of the gastro- intestinal tract of vertebrates. These pathogens have a complex life cycle that comprises two generations of asexual multiplication followed by a sexual cycle (Tzipori, 1988). In humans, Cryptosporidium hominis and Cryptosporidium parvum account for the vast majority of cases worldwide (Cacciò and Putignani, 2014; Widmer and Sullivan, 2012; Xiao, 2010). C. hominis is consid- ered a human parasite, albeit experimental and natural infection of animals with this species have occasionally been reported (Connelly et al., 2013; Giles et al., 2009; Ryan et al., 2005; Smith et al., 2005; Tanriverdi et al., 2003). C. parvum is a common parasite of ruminants, particularly of young animals, and has an established zoonotic potential (Learmonth et al., 2004; Sopwith et al., 2005). Infection is initiated by the ingestion of oocysts. Direct human- to-human or animal-to-human contact, or ingestion of con- taminated water and food are common routes of transmission. The oocysts are remarkably resistant to environmental stress and can withstand chlorination of drinking water. These properties, coupled with a low infectious dose (Chappell et al., 2006; Chappell et al., 1996), explain the large number of waterborne out- breaks, including the largest ever recorded outbreak in Milwaukee in the United States (Mac Kenzie et al., 1994). Understanding how natural parasite populations are structured and how population structures can vary in relation to ecological and epidemiological conditions has attracted a considerable inter- est, due to the implication such studies can have on the develop- ment of control measures. Indeed, as highly polymorphic genetic markers (mini- and micro-satellites) were identified (Cacciò et al., 2000; Cacciò et al., 2001; Feng et al., 2000; Mallon et al., 2003a), population genetics studies were initiated in different countries to evaluate genetic diversity among parasite isolates, to estimate the occurrence of mixed infections (Tanriverdi et al., 2003; Widmer et al., 2014) and to identify host-associated sub- populations (Drumo et al., 2012) and other population structures (Feng et al., 2014). These studies have shown the existence of human-adapted C. parvum multi-locus genotypes (MLGs) (Leav et al., 2002; Mallon et al., 2003b), or have demonstrated the influ- ence of different husbandry practices on the structure of bovine http://dx.doi.org/10.1016/j.meegid.2015.02.008 1567-1348/Ó 2015 Elsevier B.V. All rights reserved. ⇑ Corresponding author. Tel.: +39 06 4990 3016; fax: +39 06 4990 3561. E-mail address: simone.caccio@iss.it (S.M. Cacciò). Infection, Genetics and Evolution 31 (2015) 245–249 Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid