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Abstract Background Six Sarcocystis species are known to use cattle (Bos taurus) as the intermediate host, two of which, S. hominis and S. heydorni, are zoonotic. There is a need for a method that will enable rapid identification of the Sarcocystis species in cattle. Methods The diaphragm muscles of 102 cattle from Lithuania were examined for the presence of Sarcocystis spp., using two different methods for species identification. Individual sarcocysts were isolated from squash preparations of the diaphragm muscle under the light microscope, followed by genetic characterisation of excised cysts using sequence analysis of the 18S rRNA (18S rRNA) and cytochrome c oxidase subunit I (cox1) genes. The same cattle muscle samples were digested and species-specific PCR analyses targeting cox1 were developed to identify the Sarcocystis isolates to the species level. Results Under the light microscope, sarcocysts were detected in 87.3% of animals, and Sarcocystis infection was verified in all digested samples. Three species, namely S. cruzi (n = 20), S. bovifelis (n = 23) and S. hirsuta (n = 6), were identified by DNA sequence analysis of isolated sarcocysts. Based on sequence analysis of cox1, the level of genetic variability depended on Sarcocystis species and geographical location. Four Sarcocystis species, S. cruzi (96.1%), S. bovifelis (71.6%), S. hirsuta (30.4%) and S. hominis (13.7%), were confirmed in the digested samples. In individual samples, the most common finding was two species of Sarcocystis (44.1%), followed by three species (26.5%), a single species (24.5%) and four species (4.9%). Conclusions Although examination of tissue preparations under the light microscrope did not detect any sarcocysts belonging to S. hominis, this species was identified in the digested samples subjected to a cox1-specific PCR analysis. These results demonstrate the need for effective molecular diagnosis techniques to detect Sarcocystis spp., which may be present at a lower prevalence and not detectable among the limited number of sarcocysts identified individually under the light microscope.

Abstract Background Typically, carnivores serve as definitive hosts for Sarcocystis spp. parasites; currently, their role as intermediate hosts is being elucidated. The present study aimed to identify and molecularly characterize Sarcocystis cysts detected in striated muscle of red foxes from different populations in Latvia, Lithuania and Spain. Methods Muscle samples from 411 red foxes (Vulpes vulpes) and 269 racoon dogs (Nyctereutes procyonoides) from Latvia, 41 red foxes from Lithuania and 22 red foxes from Spain were examined for the presence of Sarcocystis sarcocysts by light microscopy (LM). Sarcocystis spp. were identified by transmission electron microscopy (TEM) and molecular biology techniques. Results Sarcocystis cysts were detected in 11/411 (2.7%) Latvian, 3/41 (7.3%) Lithuanian, and 6/22 (27.3%) Spanish red foxes, however, cysts were not observed in the muscles of racoon dogs. Based on LM, TEM, 18S rDNA, 28S rDNA, ITS1, cox1 and rpoB sequences, Sarcocystis arctica and Sarcocystis lutrae cysts were identified in red fox muscles from Latvia and Lithuania, whereas only S. arctica was detected in Spain. The 18S rDNA, 28S rDNA and ITS1 sequences from the 21 isolates of S. arctica from Latvia, Lithuania and Spain were identical. By contrast, two and four haplotypes were determined based on mtDNA cox1 and apicoplast rpoB sequences, respectively. Polymorphisms were not detected between the two isolates of S. lutrae from Latvia and Lithuania. Based on phylogenetic results, S. arctica and S. lutrae were most closely related to Sarcocystis spp. using predatory mammals as intermediate hosts and to Sarcocystis species with a bird-bird life-cycle. Conclusions Based on current knowledge, the red fox and Arctic fox (Vulpes lagopus) could act as intermediate host for the same two Sarcocystis species. Molecular results suggest the existence of two genetic lineages of S. arctica, and such divergence relies on its geographical distribution but not on their intermediate host species.