Eimeria spp. in italian chamois: morphological, statistical and molecular analysis

Coccidia of the genus Eimeria are common intestinal parasites affecting domestic and wild animals. In chamois, five species have been described: Eimeria alpina, Eimeria suppereri, Eimeria yakimoffmatschoulskyi, Eimeria riedmuelleri, Eimeria rupicaprae (the latter two highly prevalent in Italy). Accurate identification of these protozoa to the species level is crucial, particularly in hostpathogen interactions studies and when the parasitic pressure is high. However, this is often a challenging task. In this study, a survey was conducted to investigate Eimeria species and to explore their genetic features in the Alpine chamois, Rupicapra rupicapra rupicapra, and the Apennine chamois, Rupicapra pyrenaica ornata. From September 2013 to November 2015, fresh fecal samples were collected from R. r. rupicapra in Alps (N=262) and from R. p. ornata in Apennines (N=90), and stored in 2.5% potassium dichromate until microscopy analysis. Oocysts were recovered by flotation, photographed, measured and identified. Based on oocysts morphometry (i.e. length, width) a hierarchical cluster analysis was performed. The morphological features of Eimeria oocysts were then correlated with their genetic variability after 18SrDNA fragment cloning. Up to 94% of chamois harbored Eimeria oocysts and four species were morphologically identified: E. yakimoffmatschoulskyi, E. riedmuelleri, and E. rupicaprae in both R. r. rupicapra and R. p ornata, whereas E. suppereri was only found in Alpine chamois. Cluster analysis based on 894 oocysts from R. r. rupicapra and 450 oocysts from R. p. ornata generated dendrograms mostly consistent with the results obtained by morphological identification. Phylogenetic preliminary analysis provided evidences of distinct clades: 27 Eimeria cloned sequences derived from E. yakimoffmatschoulskyi and E. rupicaprae formed two well-supported clades; the other clades included all isolates from other Eimeria species from ruminants available in the GenBank. This study represents a first-step approach towards insights on the differentiating criteria and genetic variations of the coccidia in wild animals.