Abstract
Samples of muscle tissue from the diaphragm, oesophagus and/or heart of eight adult red deer (Cervus elaphus hispanicus) from the Quintos de Mora Park in Toledo, Central Spain, were screened for sarcocysts by means of the compression method. From positive samples, individual sarcocysts were excised and examined in wet mounts under a light microscope (LM) in order to study their basic morphology before being preserved for molecular studies. In all red deer examined, only microscopic sarcocysts were found. Those in the diaphragm and oesophagus were spindle-shaped and about 1 × 0.1 mm in size, while those in cardiac muscle were sac-like and 500–800 × 80–180 μm. By LM, the sarcocysts either had densely packed, about 8-μm-long, hair-like protrusions (type 1), sparsely distributed indistinct projections (fuzzy outline; type 2) or no visible protrusions (smooth surface; type 3). In cardiac muscle, only sarcocysts without visible protrusions were found. One of the latter sarcocysts was examined by scanning electron microscopy (SEM) and found to possess thin ribbon-like protrusions. Forty-eight sarcocysts isolated from the diaphragm, oesophagus and heart of one red deer, as well as 55 sarcocysts from the heart of three other red deer, 103 sarcocysts in total, were characterized molecularly through PCR amplification and sequencing of the partial cytochrome c oxidase subunit I gene (cox1) of the mitochondrial genome, revealing the presence of six major cox1 sequence types. Each type comprised either a single sequence (three types) or a collection of several identical or nearly identical sequences. From selected isolates possessing each of these cox1 sequence types, the complete 18S ribosomal RNA (rRNA) gene was amplified and sequenced directly and/or after cloning of the 5′ end half. Supported by the sequence data from the latter gene, as well as the morphology of the sarcocysts from which the sequences originated, the six cox1 sequence types were considered to represent six separate Sarcocystis spp. Two cox1 sequence types were identified as belonging to the previously characterized species Sarcocystis hjorti (one sequence/sarcocyst) and Sarcocystis linearis (38 sequences/sarcocysts), respectively, whereas four sequence types were new. One of the latter types was assigned to the previously named species Sarcocystis cervicanis from red deer, since this sequence type was obtained from 52 sarcocysts from cardiac muscle, which matched the original morphological description (smooth surface) and habitat of this species. The remaining three sequence types were assigned to the three new species Sarcocystis iberica (one sequence/sarcocyst) Sarcocystis venatoria (10 sequences/sarcocysts) and Sarcocystis morae (one sequence/sarcocyst), respectively. The two species S. iberica and S. venatoria shared the same sarcocyst morphology (type 1) and habitat (diaphragm) and had virtually identical 18S rRNA gene sequences, but differed by 4% at cox1, which was considered sufficient to regard them as separate species. The single sarcocyst of S. morae (from the oesophagus) examined by LM had a smooth wall and this species was therefore believed to have the same type of ribbon-like protrusions (ultrastructurally) as sarcocysts of S. cervicanis and S. linearis, which were also the species most closely related to S. morae at cox1. Thus, there seems to be three species with similar ribbon-like cyst wall protrusions in red deer (S. cervicanis, S. linearis, S. morae), as well as three species with similar hair-like protrusions (S. hjorti, S. iberica, S. venatoria). Sarcocysts of S. cervicanis were only identified in cardiac muscle, whereas sarcocysts of S. linearis were found mainly in the diaphragm and oesophagus and rarely in the heart. The relative number of cox1 haplotypes was greater among sequences/isolates of S. linearis (17/38) than among isolates of S. cervicanis (7/52). Four of the species examined (S. cervicanis, S. linearis, S. iberica, S. venatoria) possessed considerable intra-isolate (intra-genomic) sequence variation (insertions/deletions, substitutions) in the 18S rRNA gene.
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References
Dahlgren SS, Gjerde B (2010a) Molecular characterization of five Sarcocystis species in red deer (Cervus elaphus), including Sarcocystis hjorti n. sp., reveals that these species are not intermediate host specific. Parasitology 137:815–840. https://doi.org/10.1017/S0031182009991569
Dahlgren SS, Gjerde B (2010b) The red fox (Vulpes vulpes) and the arctic fox (Vulpes lagopus) are definitive hosts of Sarcocystis alces and Sarcocystis hjorti from moose (Alces alces). Parasitology 137:1547–1557. https://doi.org/10.1017/S0031182010000399
Dubey JP, Jolley WR, Thorne ET (1983) Sarcocystis sybillensis sp. nov. from the north American elk (Cervus elaphus). Can J Zool 61:737–742. https://doi.org/10.1139/z83-098
Entzeroth R, Neméseri L, Scholtyseck E (1983) Prevalence and ultrastructure of Sarcocystis sp. from the red deer (Cervus elaphus L.) in Hungary. Parasitol Hung 16:47–52
Entzeroth R, Chobotar B, Scholtyseck E, Nemeseri L (1985) Light and electron microscope study of Sarcocystis sp. from the fallow deer (Cervus dama). Z Parasitenkd 71:33–39. https://doi.org/10.1007/bf00932916
Gjerde B (1986) Scanning electron microscopy of the sarcocysts of six species of Sarcocystis from reindeer (Rangifer tarandus tarandus). Acta Pathol Microbiol Immunol Scand [B] 94:309–317. https://doi.org/10.1111/j.1699-0463.1986.tb03058.x
Gjerde B (2013) Phylogenetic relationships among Sarcocystis species in cervids, cattle and sheep inferred from the mitochondrial cytochrome c oxidase subunit I gene. Int J Parasitol 43:579–591. https://doi.org/10.1016/j.ijpara.2013.02.004
Gjerde B (2014a) Sarcocystis species in red deer revisited: with a re-description of two known species as Sarcocystis elongata n. sp. and Sarcocystis truncata n. sp. based on mitochondrial cox1 sequences. Parasitology 141:441–452. https://doi.org/10.1017/S0031182013001819
Gjerde B (2014b) Morphological and molecular characteristics of four Sarcocystis spp. in Canadian moose (Alces alces), including Sarcocystis taeniata n. sp. Parasitol Res 113:1591–1604. https://doi.org/10.1007/s00436-014-3806-z
Gjerde B (2016) Molecular characterisation of Sarcocystis bovifelis, Sarcocystis bovini n. sp., Sarcocystis hirsuta and Sarcocystis cruzi from cattle (Bos taurus) and Sarcocystis sinensis from water buffaloes (Bubalus bubalis). Parasitol Res 115:1473–1492. https://doi.org/10.1007/s00436-015-4881-5
Gjerde B, Giacomelli S, Bianchi A, Bertoletti I, Mondani H, Gibelli LR (2017) Morphological and molecular characterization of four Sarcocystis spp., including Sarcocystis linearis n. sp., from roe deer (Capreolus capreolus) in Italy. Parasitol Res 116:1317–1338. https://doi.org/10.1007/s00436-017-5410-5
Hernández-Rodríguez S, Navarrete I, Martínez-Gómez F (1981a) Sarcocystis cervicanis, nueva especie parásita del ciervo (Cervus elaphus). Rev Ibér Parasitol 41:43–51
Hernández-Rodríguez S, Martínez-Gómez F, Navarrete I, Acosta-García I (1981b) Estudio al microscopio óptico y electrónico del quiste de Sarcocystis cervicanis. Rev Ibér Parasitol 41:351–361
Hu JJ, Liu TT, Liu Q, Esch GW, Chen JQ, Huang S, Wen T (2016) Prevalence, morphology, and molecular characteristics of Sarcocystis spp. in domestic goats (Capra hircus) from Kunming, China. Parasitol Res 115:3973–3981. https://doi.org/10.1007/s00436-016-5163-6
Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874. https://doi.org/10.1093/molbev/msw054
Kutkienė L (2003) Investigations of red deer (Cervus elaphus) Sarcocystis species composition in Lithuania. Acta Zool Lituanica 13:390–395. https://doi.org/10.1080/13921657.2003.10512311
Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451–1452. https://doi.org/10.1093/bioinformatics/btp187
Luzón M, Domínguez-González J, Soto-Carrión AM, Alunda JM (2015) Sarcocystosis in Cervus elaphus: comparison of diagnostic methods. Int J Parasitol Parasites Wildl 4:396–400. https://doi.org/10.1016/j.ijppaw.2015.11.001
Poli A, Mancianti F, Marconcini A, Nigro M, Colagreco R (1988) Prevalence, ultrastructure of the cyst wall and infectivity for the dog and cat of Sarcocystis sp. from fallow deer (Cervus dama). J Wildl Dis 24:97–104. https://doi.org/10.7589/0090-3558-24.1.97
Prakas P (2011) Diversity and ecology of Sarcocystis in Lithuanian game fauna. PhD thesis, Vilnius University, Vilnius, Lithuania
Prakas P, Butkauskas D, Rudaityte E, Kutkiene L, Sruoga A, Puraite I (2016) Morphological and molecular characterization of Sarcocystis taeniata and Sarcocystis pilosa n. sp. from the sika deer (Cervus nippon) in Lithuania. Parasitol Res 115:3021–3032. https://doi.org/10.1007/s00436-016-5057-7
Prakas P, Rudaityte E, Butkauskas D, Kutkiene L (2017) Sarcocystis entzerothi n. sp. from the European roe deer (Capreolus capreolus). Parasitol Res 116:271–279. https://doi.org/10.1007/s00436-016-5288-7
Reissig EC, Moré G, Massone A, Uzal FA (2016) Sarcocystosis in wild red deer (Cervus elaphus) in Patagonia, Argentina. Parasitol Res 115:1773–1778. https://doi.org/10.1007/s00436-016-4915-7
Santín-Durán M, Alunda JM, Hoberg EP, de la Fuente C (2004) Abomasal parasites in wild sympatric cervids, red deer, Cervus elaphus and fallow deer, Dama dama, from three localities across central and western Spain: relationship to host density and park management. J Parasitol 90:1378–1386. https://doi.org/10.1645/GE-3376
Soltis PS, Soltis DE (2003) Applying the bootstrap in phylogeny reconstruction. Stat Sci 18:256–267. https://doi.org/10.1214/ss/1063994980
Son H-Y, Kim N-S, Ryu S-Y, Kim H-C, Rhee J-H, Cho J-G, Park B-K (2009) Ultrastructure of Sarcocystis grueneri-like sarcocysts from cardiac muscle of red deer (Cervus elaphus) in Korea. J Vet Clin 26:595–599
Speer CA, Dubey JP (1982) Sarcocystis wapiti sp. nov. from the north American wapiti (Cervus elaphus). Can J Zool 60:881–888. https://doi.org/10.1139/z82-120
Stephan R, Loretz M, Eggenberger E, Grest P, Basso W, Grimm F (2012) Erster Nachweis von Sarcocystis hjorti bei der Fleischuntersuchung von Rothirschen in der Schweiz. Schweiz Arch Tierheilkd 154:539–542. https://doi.org/10.1024/0036-7281/a000403
Wesemeier HH, Sedlaczek J (1995a) One known Sarcocystis species and two found for the first time in red deer and wapiti (Cervus elaphus) in Europe. Appl Parasitol 36:245–251
Wesemeier HH, Sedlaczek J (1995b) One known Sarcocystis species and one found for the first time in fallow deer (Dama dama). Appl Parasitol 36:299–302
Acknowledgements
The authors would like to thank Ms. Elena González Sánchez for her appreciated help in the collection of muscular samples from the animals selected for this study and Mr. Ángel Moreno Gómez, Director of Quintos de Mora, for allowing us access to the park in order to collect biological samples from hunted animals. We would also like to extend our gratitude to Ms. Michaela Salajkova at the EM lab, Department of Biosciences, University of Oslo, for her skilful operation of the scanning electron microscope.
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Gjerde, B., Luzón, M., Alunda, J.M. et al. Morphological and molecular characteristics of six Sarcocystis spp. from red deer (Cervus elaphus) in Spain, including Sarcocystis cervicanis and three new species. Parasitol Res 116, 2795–2811 (2017). https://doi.org/10.1007/s00436-017-5590-z
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DOI: https://doi.org/10.1007/s00436-017-5590-z