Histopathological, morphological, and molecular characterization of Sarcocystis species in elk (Cervus elaphus) from Pennsylvania, USA

Abstract

Sarcocystis sarcocysts are common in many species of domestic and wild animals. Here, we report sarcocystosis in muscles from 91 free range elk (Cervus elaphus) from Pennsylvania, USA, tested by histopathology, transmission electron microscopy (TEM), and DNA sequencing. Sarcocysts were detected in hematoxylin and eosin (HE)-stained sections from 83 of 91 (91.2%) elk, including 83/91 (91.2%) tongues and 15/17 (88.2%) hearts. With respect to age, sarcocysts were found in 0/5 calves, 8/9 (88.8%) yearlings, and 75/77 (97.4%) adults. Sarcocysts were identified in 62/69 (89.4%) females and 21/22 (91.2%) males. Associated lesions were mild and consisted of inflammatory foci around degenerate sarcocysts. There were two morphologically distinct sarcocysts based on wall thickness, thin (< 0.5 μm) and thick-walled (> 4.0 μm). Thin-walled sarcocysts had a TEM "type 2" and villar protrusions (vps), identical to Sarcocystis wapiti previously described from elk in western USA. This species was present both in tongue and heart samples and was detected in all infected elk. Thick-walled sarcocysts consisted of three morphologic variants, referred to herein as subkinds A, B, C. Subkind A sarcocysts were rare; only four sarcocysts were found in three elk. Histologically, they had a 5-8-μm thick wall with tufted vp. By TEM, the sarcocyst wall was "type 12" and appeared similar to Sarcocystis sybillensis, previously described from elk in USA. Subkind B, Sarcocystis sp.1 sarcocysts were also rare, found in only 1 elk. These sarcocysts had 6.7-7.3-μm-thick wall with TEM "type 15b" vp. Subkind C Sarcocystis sp.2 sarcocysts were more common (22/91). Morphologically, the sarcocyst wall was 6.1-6.8 μm thick and contained "type 10b" vp. Comparisons of ribosomal DNA loci with published sequences indicated all sarcocysts were similar to what has previously been isolated from cervid hosts across the northern hemisphere. Phylogenetic analysis placed the thin-walled S. wapiti within a strongly supported clade with S. linearis and S. taeniata, while the thick-walled cysts were very closely related to S. truncata, S. elongata, S. silva, and S. tarandi. Further sequencing is needed to produce molecular diagnostics to distinguish among these species. North American elk are hosts to multiple Sarcocystis species with diverse morphology, deriving from two separate evolutionary lineages.

Identification of a new Sarcocystis sp. in marsh deer (Blastocerus dichotomus) from wetlands of Argentina

“…Regarding the ITS1 fragment, a low level of amplification was also observed, and no consensus sequence was achieved. Similar difficulties in amplifying and sequencing the 18S rRNA and ITS1 fragments from S. wapiti from North American C. elaphus have been mentioned by Cerqueira-Cézar et al (2018) . Probably, the priming regions from S. wapiti and the species detected in marsh deer are not conserved, as previously assumed from most Sarcocystis spp.…”

“…detected in cervids in Argentina ( Chang Reissig et al, 2016 , 2020 ). The cyst wall ultrastructure from four cysts obtained from two marsh deer from Iberá Wetlands showing ribbon-like protrusions resembled other species in cervids muscles like S. cervicanis S. grueneri, S. linearis, S. morae, S. taeniata and S. wapiti ( Gjerde, 2014a ; Dubey et al, 2016 ; Gjerde et al, 2017a , b ; Cerqueira-Cézar et al, 2018 ; Delgado de Las Cuevas et al, 2019 ; Rudaitytė-Lukošienė et al, 2021 ). A similar image was obtained previously from cysts in pudu muscles from Argentina ( Chang Reissig et al, 2020 ).…”

“…in marsh deer showed a low homology (93.7–96.7%) with sequences from S. cervicanis in red deer in Spain ( Gjerde et al, 2017b ) S. grueneri from reindeer in Norway ( Dahlgren and Gjerde, 2007 ), S. linearis in roe deer from Spain and Lithuania ( Rudaityté- Lukošienė et al, 2020b ), S. morae in fallow deer in Lithuania ( Rudaityté- Lukošienė et al, 2020a ); Sarcocystis sp. from pudu from Argentina ( Chang Reissig et al, 2020 ), S. taeniata in sika deer in Lithuania ( Prakas et al, 2016 ), S. tarandivulpes from reindeer from Iceland ( Dahlgren et al, 2007 ) and S. wapiti from elk ( Cerqueira-Cézar et al, 2018 ). The differences at this target suggested that the Sarcocystis sp.…”

“…As far as we know, there are no reports of COI sequences from S. wapiti ( Cerqueira-Cézar et al, 2018 ), so proper comparisons are not possible. Despite morphological similarities, the high sequence divergence at 18S rRNA and COI allowed the assumption that Sarcocystis sp.…”

Sarcocystis infection in red deer (Cervus elaphus) with eosinophilic myositis/fasciitis in Switzerland and involvement of red foxes (Vulpes vulpes) and hunting dogs in the transmission

“…Molecular studies are necessary because different Sarcocystis species affecting cervids have a highly similar cyst morphology by light and electron microscopy ( Dahlgren and Gjerde, 2010a ; Gjerde et al, 2017b ; Abe et al, 2019a ), and oocysts and sporocysts in the DH cannot be discriminated by microscopic techniques ( Dubey et al, 2016 ). Several studies based the identification of Sarcocystis at the species level on the 18S ribosomal RNA gene sequence as it was performed in this study, but also the 28S rRNA gene, the ITS1 region, the mitochondrial cytochrome c oxidase subunit I gene ( cox 1) or different combinations of these targets have been used for this purpose, enhancing the diagnostic possibilities ( Gjerde 2013 , 2014b ; Gjerde et al, 2017b ; Moré et al, 2016 ; Dahlgren and Gjerde, 2010a ; Cerqueira-Cézar et al, 2018 ). The methodology based on the 18S rRNA gene amplification with subsequent cloning and sequencing has been successfully used to identify co-infections with several Sarcocystis spp.…”