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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)


About 200 individual sarcocysts were excised from 12 samples of cattle beef from five countries (Argentina, Brazil, Germany, New Zealand, Uruguay) and tentatively identified to species or cyst type on the basis of their size and shape and cyst wall morphology. Genomic DNA was extracted from 147 of these sarcocysts and used initially for PCR amplification and sequencing of the partial mitochondrial cytochrome c oxidase subunit I gene (cox1) in order to identify the sarcocysts to species and/or sequence type. In addition, seven Sarcocystis sinensis-like sarcocysts collected from the oesophagus of water buffaloes in Egypt were examined at cox1 for comparative purposes. Based on the results from the cox1 marker, selected sarcocyst isolates from both hosts were further characterised at one to three regions of the nuclear ribosomal (r) DNA unit, i.e. the complete 18S rRNA gene, the complete internal transcribed spacer 1 (ITS1) region and the partial 28S rRNA gene. This was done in order to compare the results with previous molecular identifications based on 18S rRNA gene sequences and to evaluate the utility of these regions for species delimitations and phylogenetic inferences. On the basis of sarcocyst morphology and molecular data, primarily the cox1 sequences, four Sarcocystis spp. were identified in the samples of cattle beef. Twenty-two microscopic sarcocysts (1 × 0.1 mm) with hair-like protrusions were assigned to Sarcocystis cruzi, 56 macroscopic sarcocysts (3–8 × 0.5 mm) with finger-like protrusions were assigned to Sarcocystis hirsuta and 45 and 24 microscopic sarcocysts (1–3 × 0.1–0.2 mm) with finger-like protrusions were assigned to Sarcocystis bovifelis and Sarcocystis bovini n. sp., respectively. Sarcocysts of S. cruzi were identified in samples of beef from Argentina and Uruguay; sarcocysts of S. hirsuta in samples from Argentina, Brazil, Germany and New Zealand; sarcocysts of S. bovifelis in samples from Argentina and Germany; and sarcocysts of S. bovini in samples from Argentina and New Zealand. The microscopic sarcocysts from water buffaloes were confirmed to belong to S. sinensis. The cox1 sequences of S. bovifelis and S. bovini, respectively, shared an identity of 93–94 % with each other, and these sequences shared an identity of 89–90 % with cox1 of S. sinensis. In contrast, the intraspecific sequence identity was 98.4–100 % (n = 45), 99.3–100 % (n = 24) and 99.5–100 % (n = 7) for sequences of S. bovifelis, S. bovini and S. sinensis, respectively. In each of the latter three species, an aberrant type of cox1 sequences was also identified, which was only 91–92 % identical with the predominant cox1 type of the same species and about 98 % identical with the aberrant types of the two other species. These aberrant cox1 sequences are believed to represent non-functional nuclear copies of the mitochondrial genes (numts or pseudogenes). They might be used as additional markers to separate the three species from each other. Sequencing of a considerable number of clones of S. bovifelis, S. bovini and S. sinensis from each of the three regions of the rDNA unit revealed intraspecific sequence variation in all loci in all species and particularly in the ITS1 locus (78–100 % identity). As regards the 18S rRNA gene, it was possible to separate the three species from each other on the basis of a few consistent nucleotide differences in the less variable 3′ end half of the gene. A comparison of the new sequences with GenBank sequences obtained from S. sinensis-like sarcocysts in cattle in other studies indicated that previous sequences derived from cattle in Germany and Austria belonged to S. bovifelis, whereas those derived from cattle in China belonged to S. bovini. On the basis of the new 28S rRNA sequences, it was possible to separate S. sinensis from S. bovifelis and S. bovini, whereas the latter two species could not be separated from each other. Based on ITS1 sequences, the three species were indistinguishable. Phylogenetic analysis using maximum parsimony placed with fairly high support cox1 sequences of S. bovifelis, S. bovini and S. sinensis, respectively, into three monophyletic clusters, with S. bovifelis and S. bovini being a sister group to S. sinensis. In contrast, phylogenies based on each of the three regions of the rDNA unit did not separate sequences of the three species completely from each other. Characterisation of cox1 of 56 isolates of S. hirsuta from four countries revealed only 13 haplotypes and an intraspecific sequence identity of 99.3–100 %. In the three regions of the rDNA unit, there was more extensive sequence variation, particularly in the ITS1 region. The 22 cox1 sequences of S. cruzi displayed a moderate intraspecific variation (98.6–100 %), whereas there was no variation at the 18S rRNA gene among 10 sequenced isolates. Sequencing of 16 clones of the partial 28S rRNA gene of S. cruzi yielded two markedly different sequence types, having an overall sequence identity of 95–100 %.

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  1. Bensasson D, Zhang D, Hartl DL, Hewitt GM (2001) Mitochondrial pseudogenes: evolution’s misplaced witnesses. Trends Ecol Evol 16:314–321. doi:10.1016/S0169-5347(01)02151-6

  2. Böttner A (1984) Vorkommen sowie licht- und elektronenmikroskopische Differenzierung von Sarkosporidienarten bei Schlachtrindern auf der Nordinsel Neuseelands. Diss Tierärztl Hochsch, Hannover

  3. Böttner A, Charleston WA, Hopcroft D (1987a) The structure and identity of macroscopically visible Sarcocystis cysts in cattle. Vet Parasitol 24:35–45. doi:10.1016/0304-4017(87)90128-2

  4. Böttner A, Charleston WA, Pomroy WE, Rommel M (1987b) The prevalence and identity of Sarcocystis in beef cattle in New Zealand. Vet Parasitol 24:157–168. doi:10.1016/0304-4017(87)90036-7

  5. Chen X, Zuo Y, Rosenthal BM, He Y, Cui L, Yang Z (2011) Sarcocystis sinensis is an ultrastructurally distinct parasite of water buffalo that can cause foodborne illness but cannot complete its life-cycle in human beings. Vet Parasitol 178:35–39. doi:10.1016/j.vetpar.2010.12.026

  6. Dahlgren SS, Gjerde B (2007) Genetic characterisation of six Sarcocystis species from reindeer (Rangifer tarandus tarandus) in Norway based on the small subunit rRNA gene. Vet Parasitol 146:204–213. doi:10.1016/j.vetpar.2007.02.023

  7. Dahlgren SS, Gjerde B (2010) 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. doi:10.1017/S0031182009991569

  8. Dubey JP, Udtujan RM, Cannon L, Lindsay DS (1990) Condemnation of beef because of Sarcocystis hirsuta infection. J Am Vet Med Assoc 196:1095–1096

  9. Dubey JP, Moré G, van Wilpe E, Calero-Bernal R, Verma SK, Schares G (2015) Sarcocystis rommeli, n. sp. (Apicomplexa: Sarcocystidae) from cattle (Bos taurus) and its differentiation from Sarcocystis hominis. J Eukaryot Microbiol. doi:10.1111/jeu.12248

  10. Fischer S, Odening K (1998) Characterization of bovine Sarcocystis species by analysis of their 18S ribosomal DNA sequences. J Parasitol 84:50–54. doi:10.2307/3284529

  11. Gestrich R, Mehlhorn H, Heydorn AO (1975) Licht- und elektronenmikroskopische Untersuchungen an Cysten von Sarcocystis fusiformis in der Muskulatur von Kälbern nach experimenteller Infektion mit Oocysten und Sporocysten der großen Form von Isospora bigemina der Katze Zbl Bakt I. Abt Orig A 233:261–276

  12. Gjerde B (2012) Morphological and molecular characterization and phylogenetic placement of Sarcocystis capreolicanis and Sarcocystis silva n. sp. from roe deer (Capreolus capreolus) in Norway. Parasitol Res 110:1225–1237. doi:10.1007/s00436-011-2619-6

  13. Gjerde B (2013a) Characterisation of full-length mitochondrial copies and partial nuclear copies (numts) of the cytochrome b and cytochrome c oxidase subunit I genes of Toxoplasma gondii, Neospora caninum, Hammondia heydorni and Hammondia triffittae (Apicomplexa: Sarcocystidae). Parasitol Res 112:1493–1511. doi:10.1007/s00436-013-3296-4

  14. Gjerde B (2013b) 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. doi:10.1016/j.ijpara.2013.02.004

  15. 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. doi:10.1017/S0031182013001819

  16. 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. doi:10.1007/s00436-014-3806-z

  17. Gjerde B (2014c) Molecular characterisation of Sarcocystis rileyi from a common eider (Somateria mollissima) in Norway. Parasitol Res 113:3501–3509. doi:10.1007/s00436-014-4062-y

  18. Gjerde B (2015) The resurrection of a species: Sarcocystis bovifelis Heydorn, et al., 1975 is distinct from the current Sarcocystis hirsuta in cattle and morphologically indistinguishable from Sarcocystis sinensis in water buffaloes. Parasitol Res. doi:10.1007/s00436-015-4785-4

  19. Gjerde B, Josefsen TD (2015) Molecular characterisation of Sarcocystis lutrae n. sp. and Toxoplasma gondii from the musculature of two Eurasian otters (Lutra lutra) in Norway. Parasitol Res 114:873–886. doi:10.1007/s00436-014-4251-8

  20. Gjerde B, Schulze J (2014) Muscular sarcocystosis in two arctic foxes (Vulpes lagopus) due to Sarcocystis arctica n. sp.: sarcocyst morphology, molecular characteristics and phylogeny. Parasitol Res 113:811–821. doi:10.1007/s00436-013-3711-x

  21. Gjerde B, Hilali M, Mawgood SA (2015) Molecular characterisation of three regions of the nuclear ribosomal DNA unit and the mitochondrial cox1 gene of Sarcocystis fusiformis from water buffaloes (Bubalus bubalis) in Egypt. Parasitol Res 114:3401–3413. doi:10.1007/s00436-015-4566-0

  22. Heydorn AO, Gestrich R, Mehlhorn H, Rommel M (1975) Proposal for a new nomenclature of the Sarcosporidia. Z Parasitenkd [Parasitol Res] 48:73–82

  23. Hilali M, El-Seify M, Zayed A, El-Morsey A, Dubey JP (2011) Sarcocystis dubeyi (Huong and Uggla, 1999) infection in water buffaloes (Bubalus bubalis) from Egypt. J Parasitol 97:527–528. doi:10.1645/GE-2656.1

  24. Hornok S, Mester A, Takács N, Baska F, Majoros G, Fok É, Biksi I, Német Z, Hornyák Á, Jánosi S, Farkas R (2015) Sarcocystis-infection of cattle in Hungary. Parasit Vectors 8:69. doi:10.1186/s13071-015-0685-9

  25. Jehle C, Dinkel A, Sander A, Morent M, Romig T, Luc PV, De TV, Thai VV, Mackenstedt U (2009) Diagnosis of Sarcocystis spp. in cattle (Bos taurus) and water buffalo (Bubalus bubalis) in Northern Vietnam. Vet Parasitol 166:314–320. doi:10.1016/j.vetpar.2009.08.024

  26. Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451–1452. doi:10.1093/bioinformatics/btp187

  27. Moré G, Schares S, Maksimov A, Conraths FJ, Venturini MC, Schares G (2013) Development of a multiplex real time PCR to differentiate Sarcocystis spp. affecting cattle. Vet Parasitol 197:85–94. doi:10.1016/j.vetpar.2013.04.024

  28. Moré G, Pantchev A, Skuballa J, Langenmayer MC, Maksimov P, Conraths FJ, Venturini MC, Schares G (2014) Sarcocystis sinensis is the most prevalent thick-walled Sarcocystis in beef for consumers in Germany. Parasitol Res 113:2223–2230. doi:10.1007/s00436-014-3877-x

  29. Odening K, Wesemeier HH, Walter G, Bockhardt I (1995) On the morphological diagnostics and host specificity of the Sarcocystis species of some domesticated and wild bovini (cattle, banteng and bison). Appl Parasitol 36:161–178

  30. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739. doi:10.1093/molbev/msr121

  31. Xiang Z, He Y, Zhao H, Rosenthal BM, Dunams DB, Li X, Zuo Y, Feng G, Cui L, Yang Z (2011) Sarcocystis cruzi: comparative studies confirm natural infections of buffaloes. Exp Parasitol 127:460–466. doi:10.1016/j.exppara.2010.10.012

  32. Yang ZQ, Zuo YX, Ding B, Chen XW, Luo J, Zhang YP (2001a) Identification of Sarcocystis hominis-like (Protozoa: Sarcocystidae) cyst in water buffalo (Bubalus bubalis) based on 18S rRNA gene sequences. J Parasitol 87:934–937. doi:10.1645/0022-3395(2001)087[0934:IOSHLP]2.0.CO;2

  33. Yang ZQ, Zuo YX, Yao YG, Chen XW, Yang GC, Zhang YP (2001b) Analysis of the 18S rRNA genes of Sarcocystis species suggests that the morphologically similar organisms from cattle and water buffalo should be considered the same species. Mol Biochem Parasitol 115:283–288. doi:10.1016/S0166-6851(01)00283-3

  34. Yang ZQ, Li QQ, Zuo YX, Chen XW, Chen YJ, Nie L, Wei CG, Zen JS, Attwood SW, Zhang XZ, Zhang YP (2002) Characterization of Sarcocystis species in domestic animals using a PCR-RFLP analysis of variation in the 18S rRNA gene: a cost-effective and simple technique for routine species identification. Exp Parasitol 102:212–217. doi:10.1016/S0014-4894(03)00033-X

  35. Zuo YX, Yang ZQ (2015) The validity of Sarcocystis sinensis. Dongwuxue Yanjiu [Zoological Research] 36:109–111.

  36. Zuo YX, Chen XW, Li YJ, Ma TC, Tang DH, Fan LX, Zhao ML (1995) Studies on Sarcocystis species of cattle and water buffalo with description of a new species of Sarcocystis. In: Proceedings of the Tenth Anniversary of the Founding of Chinese Parasitological Society, Chinese Science and Technology Press, Beijing, PR China, pp. 20–24 [in Chinese]

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Thanks are due to the personnel from the Norwegian Food Safety Authority for providing samples of imported cattle beef and to Dr. Mosaad Hilali for providing sarcocysts from water buffaloes.

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Correspondence to Bjørn Gjerde.

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Gjerde, B. 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 (2016).

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  • Sarcocystis bovifelis
  • Sarcocystis bovini
  • Sarcocystis hirsuta
  • Sarcocystis cruzi
  • Sarcocystis sinensis
  • Cattle
  • Water buffalo
  • cox1
  • Numts
  • 18S rRNA gene
  • 28S rRNA gene
  • ITS1
  • Phylogeny
  • Sarcocyst morphology