To the best of our knowledge, this is the first worldwide report of S. lutrae parasitizing N. procyonoides and P. lotor, thus increasing its host spectrum to nine species. Sarcocysts of S. lutrae were previously reported and molecularly characterized in different species of mustelids and canids from the Czech Republic, Latvia, Lithuania, Norway and Scotland [2,3,4,5,6]. This parasite is the most common species among mustelids, with prevalence ranging between 67–82% in the European badger (M. meles) from Scotland and the Czech Republic [3, 5]. In the present survey its prevalence in N. procyonoides was low (7%), although this host probably acts as natural intermediate host, since other canids, such as red foxes (V. vulpes) from Latvia (0.2%) and Lithuania (2%) , also showed low prevalence. A similar pattern occurs in a congeneric species, S. arctica, in red fox (V. vulpes) (prevalence 3.8%) from the Czech Republic . On the other hand, the single P. lotor examined was positive to S. lutrae, thus suggesting that the parasite occurs in various hosts, although the sample size should be increased. More data about prevalence and molecular analysis of S. lutrae in procyonids are needed to determinate its host spectrum and the role of these hosts in the life-cycle of this parasite.
Our finding of S. lutrae in hind-limb differs from that of Kirillova et al. , who found no sarcocysts of this parasite in 294 N. procyonoides from Latvia, even though they examined the same kind of muscle. These differences could be related to the availability of potential hosts and their interactions, regional parasite occurrence, low susceptibility of raccoon dogs to this parasite or to the quality of samples examined (fresh vs autolytic).
The present nucleotide sequences were identical to those of S. lutrae in the European badger and European otter from the Czech Republic, thus indicating that the parasite is spread across the country in various carnivore hosts, at the same time, with some mustelid and canid hosts from other European countries. In order to elucidate the real host spectrum of this parasite, it is important to molecularly characterize those records of Sarcocystis sp. in several intermediate  and other possible definitive hosts, since the morphology of sarcocysts and bradyzoites is rather similar among species. All published sequences of S. lutrae using the 18S rRNA gene share high identity (> 99%) with those of S. halieti (GenBank: MF946587) and S. lari (GenBank: MF946588) in the white-tailed sea eagle (H. albicilla), S. corvusi (GenBank: JN256117) in the jackdaw (Corvus monedula) and S. columbae (GenBank: HM125054) in the wood pigeon (Columba palumbus), cox1 gene sequences were 100% identical to S. lari (GenBank: MF946584) and > 99% to Sarcocystis sp. in Accipiter cooperii (GenBank: KY348756), see also . Therefore, other loci are recommended to distinguish closely related Sarcocystis species where carnivores are intermediate hosts, such as the 28S rRNA gene and ITS1. 28S rRNA gene sequences of S. lutrae were 98% or less similar to other closely related Sarcocystis species (e.g. S. lari), whose intermediate and definitive hosts are the great black-backed gull (Larus marinus) (GenBank: JQ733509) and white-tailed sea eagle (GenBank: MF946611), respectively. ITS1 showed strong support for the species delimitation , since our sequence was 86–92% similar to S. lari (e.g. GenBank: MF946597). Thus, S. lutrae appears most closely related to species that use medium-to-large size raptorial birds as definitive hosts. Differences in the known sequences of S. lutrae are a result of the intraspecific variability, especially within ITS1 [2, 4] and 28S rRNA gene , as observed in the present loci from P. lotor (GenBank: MT036249 and MT036253). Despite some limitations (unsuccessful ITS1 sequencing), the data allowed to clearly determine that both mammal species are hosts of this parasite.
Thus, P. lotor is the first reported procyonid host for S. lutrae; this host and N. procyonoides act as intermediate hosts of this parasite.