Abstract
A total of 33 macroscopically visible (3–11 × 1–5 mm) sarcocysts of Sarcocystis fusiformis were excised from the oesophagus of 12 freshly slaughtered water buffalos in Giza, Egypt. Genomic DNA was extracted from the sarcocysts, and all isolates were characterised at the mitochondrial cytochrome c oxidase subunit I (cox1) gene through PCR amplification and direct sequencing, whereas a few selected isolates were characterised at the 18S and 28S ribosomal (r) RNA genes and the internal transcribed spacer 1 (ITS1) region of the nuclear rDNA unit following cloning. Among the 33 cox1 sequences (1,038-bp long), there was a total of 13 haplotypes, differing from each other by one to seven substitutions and sharing an identity of 99.3–99.9 %. In comparison, the sequence identity was 98.8–99.0 % among eight complete 18S rRNA gene sequences (1,873–1,879-bp long), 98.1–100 % among 28 complete ITS1 sequences (853–864-bp long) and 97.4–99.6 % among five partial 28S rRNA gene sequences (1,607–1,622 bp). At the three nuclear loci, the intraspecific (and intra-isolate) sequence variation was due to both substitutions and indels, which necessitated cloning of the PCR products before sequencing. Some additional clones of the 18S and 28S rRNA genes were highly divergent from the more typical clones, but the true nature of these aberrant clones could not be determined. Sequence comparisons and phylogenetic analyses based on either 18S rRNA gene or cox1 nucleotide sequences, placed S. fusiformis closest to Sarcocystis cafferi from the African buffalo, but only the analyses based on cox1 data separated the two taxa clearly from each other and showed that they were separate species (monophyletic clusters and 93 % sequence identity at cox1 versus interleaved sequences and 98.7–99.1 % sequence identity at the 18S rRNA gene). Two cats experimentally infected with sarcocysts of S. fusiformis started shedding small numbers of sporocysts 8–10 days post-infection (dpi) and were euthanized 15 dpi. Sporocysts isolated from the intestinal mucosa of both cats were identified molecularly as belonging to S. fusiformis through PCR amplification and sequencing of the partial cox1. The two sporocyst-derived cox1 sequences were identical with the most common sarcocyst-derived cox1 haplotype.
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References
Abu-Elwafa SA, Al-Araby MA, Abbas IEA (2015) Sarcocystis fusiformis (Railliet, 1897) infecting water buffaloes (Bubalus bubalis) in Dakahlia Province, Egypt. Int J Adv Res 3:116–120
Achuthan HN (1983) Sarcocystis and sarcocystosis in buffalo (Bubalus bubalis) calves. Indian Vet J 60:344–346
Bhoora R, Franssen L, Oosthuizen M, Guthrie A, Zweygarth E, Penzhorn B, Jongejan F, Collins N (2009) Sequence heterogeneity in the 18S rRNA gene within Theileria equi and Babesia caballi from horses in South Africa. Vet Parasitol 159:112–120. doi:10.1016/j.vetpar.2008.10.004
Chen XW, Zuo YX, Hu JJ (2003) Experimental Sarcocystis hominis infection in a water buffalo (Bubalus bubalis). J Parasitol 89:393–394. doi:10.1645/0022-3395(2003)089[0393:ESHIIA]2.0.CO;2
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
Dahlgren SS, Gjerde B (2008) Sarcocystis in moose (Alces alces): molecular identification and phylogeny of six Sarcocystis species in moose, and a morphological description of three new species. Parasitol Res 103:93–110. doi:10.1007/s00436-008-0936-1
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
Dessouky MI, Mohamed AH, Nassar AM, Hilali M (1984) Haematological and biochemical changes in buffalo calves inoculated with Sarcocystis fusiformis from cats. Vet Parasitol 14:1–6. doi:10.1016/0304-4017(84)90127-4
Dissanaike AS, Kan SP (1978) Studies on Sarcocystis in Malaysia. I. Sarcocystis levinei n. sp. from the water buffalo Bubalus bubalis. Z Parasitenkd [Parasitol Res] 55:127–138
Dissanaike AS, Kan SP, Retnasabapathy A, Baskarna G (1977a) Demonstration of the sexual phases of Sarcocystis fusiformis (Railliet, 1897) and Sarcocystis sp. of the water buffalo Bubalus bubalis in the small intestine of cats and dogs. Trans R Soc Trop Med Hyg 71:271. doi:10.1016/0035-9203(77)90028-1
Dissanaike AS, Kan SP, Retnasabapathy A, Baskaran G (1977b) Developmental stages of Sarcocystis fusiformis (Railliet, 1897) and Sarcocystis sp., of the water buffalo, in the small intestines of cats and dogs respectively. Southeast Asian J Trop Med Public Health 8:417
Dubey JP, Speer CA, Fayer R (1989a) Sarcocystosis of animals and man. CRC Press, Boca Raton, pp 94–95
Dubey JP, Speer CA, Shah HL (1989b) Ultrastructure of sarcocysts from water buffalo in India. Vet Parasitol 34:149–152. doi:10.1016/0304-4017(89)90176-3
Dubey JP, Lane EP, Wilpe E, Suleman E, Reininghaus B, Verma S, Rosenthal B, Mtshali M (2014a) Sarcocystis cafferi, n. sp. (Protozoa: Apicomplexa) from the African buffalo (Syncerus caffer). J Parasitol 100:817–827. doi:10.1645/13-467.1
Dubey JP, Fayer R, Rosenthal BM, Calero-Bernal R, Uggla A (2014b) Identity of Sarcocystis species of the water buffalo (Bubalus bubalis) and cattle (Bos taurus) and the suppression of Sarcocystis sinensis as a nomen nudum. Vet Parasitol 205:1–6. doi:10.1016/j.vetpar.2014.06.020
Dubey JP, Hilali M, Van Wilpe E, Verma SK, Calero-Bernal R, Abdel-Wahab A (2015) Redescription of Sarcocystis fusiformis sarcocysts from the water buffalo (Bubalus bubalis). Parasitology 142:385–394. doi:10.1017/S003118201400122X
El-Kelesh EAM, Abdel-Mawgood SZ, Abdel-Wahab AM, Radwan IGH, Ibrahim O (2011) The effect of freezing and heating on the infectivity of Sarcocystis fusiformis to cats and evaluation of ELISA for its diagnosis in water buffaloes (Bubalus bubalis). Am J Anim Vet Sci 7:55–58
El-Seify M, El-Morsey A, Hilali M, Zayed A, El-Dakhly K, Haridy M, Sakai H, Yanai T (2014) Molecular characterization of Sarcocytis fusiformis and Sarcocystis buffalonis infecting water buffaloes (Bubalus bubalis) from Egypt. Am J Anim Vet Sci 9:95–104. doi:10.3844/ajavssp.2014.95.104
El-Sherry S, Ogedengbe ME, Hafeez MA, Barta JR (2013) Divergent nuclear 18S rDNA paralogs in a turkey coccidium, Eimeria meleagrimitis, complicate molecular systematics and identification. Int J Parasitol 43:679–685. doi:10.1016/j.ijpara.2013.03.005
Gajadhar AA, Marquardt WC, Hall R, Gunderson J, Ariztia-Carmona EV, Sogin ML (1991) Ribosomal RNA sequences of Sarcocystis muris, Theileria annulata and Crypthecodinium cohnii reveal evolutionary relationships among apicomplexans, dinoflagellates, and ciliates. Mol Biochem Parasitol 45:147–154
Ghaffar FA, Hilali M, Scholtyseck E (1978) Ultrastructure study of Sarcocystis fusiformis (Railliet, 1897) infecting the Indian water buffalo Bubalus bubalis of Egypt. Tropenmed Parasitol 29:289–294
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
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. doi: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. doi: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. doi:10.1007/s00436-014-3806-z
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
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
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
Hilali M, Nassar AM (1983) Development of Sarcocystis fusiformis (Railliet, 1897) in experimentally infected buffalo calves (Bubalus bubalis). In: Bylund G (ed) Proceedings of the XI Symposium of the Scandinavian Society for Parasitology, Stockholm, Sweden. Åbo Akademi Parasitol Inst Information 1983 17:1–99
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
Holmdahl OJ, Mattsson JG, Uggla A, Johansson KE (1994) The phylogeny of Neospora caninum and Toxoplasma gondii based on ribosomal RNA sequences. FEMS Microbiol Lett 119:187–192
Huong LTT, Uggla A (1999) Sarcocystis dubeyi n. sp. (Protozoa: Sarcocystidae) in the water buffalo (Bubalus bubalis). J Parasitol 85:102–104
Huong LTT, Dubey JP, Nikkilä T, Uggla A (1997) Sarcocystis buffalonis n. sp. (Protozoa: Sarcocystidae) from the water buffalo (Bubalus bubalis) in Vietnam. J Parasitol 83:471–474
Jain PC, Shah HL (1986) Gametogony and sporogony of Sarcocystis fusiformis of buffaloes in the small intestine of experimentally infected cats. Vet Parasitol 21:205–209. doi:10.1016/0304-4017(86)90066-X
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
Kan SP, Dissanaike AS (1978) Studies on Sarcocystis in Malaysia. II. Comparative ultrastructure of the cyst wall and zoites of Sarcocystis levinei and Sarcocystis fusiformis from the water buffalo Bubalus bubalis. Z Parasitenkd [Parasitol Res] 57:107–116
Li QQ, Yang ZQ, Zuo YX, Attwood SW, Chen XW, Zhang YP (2002) A PCR-based RFLP analysis of Sarcocystis cruzi (Protozoa: Sarcocystidae) in Yunnan Province, PR China, reveals the water buffalo (Bubalus bubalis) as a natural intermediate host. J Parasitol 88:1259–1261. doi:10.1645/0022-3395(2002)088[1259:APBRAO]2.0.CO;2
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
Manuel MF, Misa GA, Yoda T (1983) Histomorphological studies of bubaline Sarcocystis in the Philippines. Philos J Vet Med 22:24–36
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
Nassar AM (1982) Studies on sarcocysts infecting water buffaloes (Bubalus bubalis) in Egypt. Ph.D. Thesis, Faculty of Veterinary Medicine, Cairo University, Egypt, 89 pp
Oryan A, Sharifiyazdi H, Khordadmehr M, Larki S (2011) Characterization of Sarcocystis fusiformis based on sequencing and PCR-RFLP in water buffalo (Bubalus bubalis) in Iran. Parasitol Res 109:1563–1570. doi:10.1007/s00436-011-2412-6
Parairo JR, Manuel MF, Icatlo FC Jr (1988) Ultrastructural studies on the cyst wall of Sarcocystis spp. in Philippine carabaos (Bubalus bubalis). Philos J Vet Anim Sci 14:40–54
Prakas P, Oksanen A, Butkauskas D, Sruoga A, Kutkiene L, Švažas S, Isomursu M, Liaugaudaite S (2014) Identification and intraspecific genetic diversity of Sarcocystis rileyi from ducks, Anas spp., in Lithuania and Finland. J Parasitol 100:657–661. doi:10.1645/13-395.1
Quandt S, Bengis RG, Stolte M, Odening K, Bockhardt I (1997) Sarcocystis infection of the African buffalo (Syncerus caffer) in the Kruger National Park, South Africa. Acta Parasitol 42:68–73
Rosenthal BM, Dunams DB, Pritt B (2008) Restricted genetic diversity in the ubiquitous cattle parasite, Sarcocystis cruzi. Infect Genet Evol 8:588–592. doi:10.1016/j.meegid.2008.04.004
Scholtyseck E, Hilali M (1978) Ultrastructural study of the sexual stages of Sarcocystis fusiformis (Railliet, 1897) in domestic cats. Z Parasitenkd [Parasitol Res] 56:205–209
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
Tongson MS, Molina RM (1979) Light and electron microscope studies on Sarcocystis sp. of the Philippine buffaloes (Bubalus bubalis). Philipp J Vet Med 18:16–31
Vangeel L, Houf K, Geldhof P, De Preter K, Vercruysse J, Ducatelle R, Chiers K (2013) Different Sarcocystis spp. are present in bovine eosinophilic myositis. Vet Parasitol 197:543–548. doi:10.1016/j.vetpar.2013.06.001
Vrba V, Poplstein M, Pakandl M (2011) The discovery of the two types of small subunit ribosomal RNA gene in Eimeria mitis contests the existence of E. mivati as an independent species. Vet Parasitol 183:47–53. doi:10.1016/j.vetpar.2011.06.020
Xiang Z, Chen X, Yang L, He Y, Jiang R, Rosenthal BM, Luan P, Attwood SW, Zuo Y, Zhang YP, Yang Z (2009) Non-invasive methods for identifying oocysts of Sarcocystis spp. from definitive hosts. Parasitol Int 58:293–296. doi:10.1016/j.parint.2009.03.004
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
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
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
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
Zaman V, Robertson TA, Papadimitriou JM (1980) Scanning electron microscopy of Sarcocystis fusiformis from the water buffalo (Bubalus bubalis). Southeast Asian J Trop Med Public Health 11:205–211
Zuo YX, Yang ZQ (2015) The validity of Sarcocystis sinensis. Dongwuxue Yanjiu [Zoological Research] 36:109–111. http://www.zoores.ac.cn/EN/Y2015/V36/I2/109
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 Chinese]. In: Proceedings of the Tenth Anniversary of the Founding of Chinese Parasitological Society, Chinese Science and Technology Press, Beijing, PR China, pp. 20–24
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Gjerde, B., Hilali, M. & Mawgood, S.A. 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 (2015). https://doi.org/10.1007/s00436-015-4566-0
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DOI: https://doi.org/10.1007/s00436-015-4566-0