Abstract
This study was conducted to identify the Sarcocystis species that infect the opossum Didelphis aurita in order to determine which sporocysts they are excreating in to the environment and help determine the role of D. aurita in the epidemiology of Sarcocystis. Sporocysts were obtained from intestinal tracts of 8 of 13 D. aurita trapped in Rio de Janeiro state, Brazil, and were orally inoculated into Melopsittacus undulatus and Balb/c nude Mus musculus. Portions of organs and muscles were processed for histology, immunohistochemistry, transmission electron microscopy (TEM), and PCR using primers JNB 33/54, and ITS. Amplification products were subjected to RFLP using DraI and HinfI. Some birds were euthanized 6, 7, 13, 16, and 24 days after inoculation (DAI). All other birds and all mice were euthanized 60 DAI. Schizonts were observed in the lungs using histology and immunostaining in birds examined prior to 60 DAI. Sarcocysts with a ~ 1.5-μm-thick wall were found in the breast, thigh, and tongue of some birds. Sarcocystis asexual stages were isolated in cell cultures inoculated with sporozoites. Parasite DNA isolated from bird tissues and cell cultures demonstrated that S. falcatula-like parasites were present in all samples derived from positive opossums. Asexual stages molecularly characterized as S. lindsayi-like were isolated in cell culture from one opossum with an apparent multiple infection. This study demonstrated that D. aurita is a definitive host for S. falcatula-like parasites and indicates that S. lindsayi-like parasites can be found in coinfections of this opossum species.
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References
Altschul SF, Madden TL, Schaffer AA, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389–3402. https://doi.org/10.1093/nar/25.17.3389
Box ED, Duszynski DW (1978) Experimental transmission of Sarcocystis from icterid birds to sparrows and canaries by sporocysts from the opossum. J Parasitol 64(4):682–688. https://doi.org/10.2307/3279960
Box ED, Meier JL, Smith JH (1984) Description of Sarcocystis falcatula Stiles, 1893, a parasite of birds and opossums. J Protozool 31(4):521–524. https://doi.org/10.1111/j.1550-7408.1984.tb05495.x
Brown BE (2004) Atlas of new world marsupials. Fieldiana Zoology, Chigago. https://doi.org/10.5962/bhl.title.3410
Casagrande RA, Cesar MO, Pena HFJ, Zwarg T, Teixeira RHF, Nunes ALV, Neves DVDA, Gomes M, Quagglia Neto F, Milanello L, Fontenelle JH, Matushima ER (2009) Occurrence of Sarcocystis spp. in opossums (Didelphis aurita and Didelphis albiventris) in regions of the state of São Paulo, Brazil. Braz J Vet Res An Sci 96:101–106
Cerqueira R, Lemos B (2000) Morphometric differentiation between Neotropical blackeared opossums, Didelphis marsupialis and Didelphis aurita (Didelphimorphia, Didelphidae). Mammalia 64:319–327
Cheadle MA, Dame JB, Greiner EC (2001) Sporocyst size of isolates of Sarcocystis shed by the Virginia opossum (Didelphis virginiana). Vet Parasitol 95(2-4):305–311. https://doi.org/10.1016/S0304-4017(00)00396-4
Dubey JP, Lindsay DS (1999) Sarcocystis speeri n. sp. (Protozoa: Sarcocystidae) from the opossum (Didelphis virginiana). J Parasitol 85(5):903–909. https://doi.org/10.2307/3285830
Dubey JP, Speer CA, Fayer R (1989) Sarcocytosis of animals and man. CRC Press Inc, Boca Raton, Florida
Dubey JP, Venturini L, Venturini C, Basso W, Unzaga J (1999) Isolation of Sarcocystis falcatula from the south American opossum (Didelphis albiventris) from Argentina. Vet Parasitol 86(4):239–244. https://doi.org/10.1016/S0304-4017(99)00145-4
Dubey JP, Lindsay DS, Rezende PC, Costa AJ (2000a) Characterization of an unidentified Sarcocystis falcatula-like parasite from the south American opossum, Didelphis albiventris from Brazil. J Eukaryot Microbiol 47(6):538–544. https://doi.org/10.1111/j.1550-7408.2000.tb00087.x
Dubey JP, Lindsay DS, Venturini L, Venturini C (2000b) Characterization of Sarcocystis falcatula isolates from the Argentinian opossum, Didelphis albiventris. J Eukaryot Microbiol 47(3):260–263. https://doi.org/10.1111/j.1550-7408.2000.tb00045.x
Dubey JP, Lindsay DS, Rosenthal BM, Kerber CE, Kasai N, Pena HFJ, Kwok OCH, Shen SK, Gennari SM (2001a) Isolates of Sarcocystis falcatula-like organisms from south American opossums Didelphis marsupialis and Didelphis albiventris from São Paulo, Brazil. J Parasitol 87(6):1449–1453. https://doi.org/10.1645/0022-3395(2001)087[1449:IOSFLO]2.0.CO;2
Dubey JP, Garner MM, Stetter MD, Marsh AE, Barr BC (2001b) Acute Sarcocystis falcatula-like infection in a carmine bee-eater (Merops nubicus) and immunohistochemical cross reactivity between Sarcocystis falcatula and Sarcocystis neurona. J Parasitol 87(4):824–832. https://doi.org/10.1645/0022-3395(2001)087[0824:ASFLII]2.0.CO;2
Dubey JP, Rosenthal BM, Speer CA (2001c) Sarcocystis lindsayi n. sp. (Protozoa: Sarcocystidae) from the south American opossum, Didelphis albiventris from Brazil. J Eukaryot Microbiol 48(5):595–603. https://doi.org/10.1111/j.1550-7408.2001.tb00196.x
Dubey JP, Johnson GC, Bermudez A, Suedmeyert KW, Fritz DL (2001d) Neural sarcocystosis in a straw-necked ibis (Carphibis spinicollis) associated with a Sarcocystis neurona-like organism and description of muscular sarcocysts of an unidentified Sarcocystis species. J Parasitol 87(6):1317–1322. https://doi.org/10.1645/0022-3395(2001)087[1317:NSIASN]2.0.CO;2
Dubey JP, Calero-Bernal R, Rosenthal BM, Speer CA, Fayer R (2015) Sarcocystosis of animals and humans. CRC Press, Boca Raton. https://doi.org/10.1201/b19184
Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32(5):1792–1797. https://doi.org/10.1093/nar/gkh340
Emmons L (1997) Neotropical rainforest mammals: a field guide. University of Chicago Press, Chicago
Finnie EP (1986) Monotremes and marsupials (anatomy) in: fowler ME (ed.) zoo and wild animal medicine. Saunders Company, Estados Unidos, pp 558-560
Gardner AL (2005) Order Didelphimorphia. In: Wilson DE, Reeder DM (eds) Mammal species of the world: a taxonomic and geographic. Johns Hopkins University Press, Baltimore, pp 3–18
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(7):579–591. https://doi.org/10.1016/j.ijpara.2013.02.004
Hershkovitz P (1992) The south American gracile mouse opossums, genus Gracilinanus Gardner and Creighton, 1989 (Marmosidae, Marsupialia): a taxonomic review with notes on general morphology and relationships. Fieldiana Zool 70:1–56
Holmdahl OJM, Mattsson JG, Uggla A, Johansson KE (1994) The phylogeny of Neospora caninum and Toxoplasma gondii based on ribosomal RNA sequences. FEMS Microbiol Lett 119(1-2):187–192. https://doi.org/10.1111/j.1574-6968.1994.tb06887.x
Kumar S, Stecher G, Tamura K (2016) MEGA 7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33(7):1870–1874. https://doi.org/10.1093/molbev/msw054
Kutkienè L, Prakas P, Sruoga A, Butkauskas D (2010) The mallard duck (Anas platyrhynchos) as intermediate host for Sarcocystis wobeseri sp. nov. from the barnacle goose (Branta leucopsis). Parasitol Res 107(4):879–888. https://doi.org/10.1007/s00436-010-1945-4
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(6):1259–1261. https://doi.org/10.1645/0022-3395(2002)088[1259:APBRAO]2.0.CO;2
Lindsay DS, Dubey JP (1989) Immunohistochemical diagnosis of Neospora caninum in tissue sections. Am J Vet Res 50(11):1981–1983
Lindsay DS, Verma SK, Scott D, Dubey JP, Von Dohlen AR (2016) Isolation, molecular characterization, and in vitro schizogonic development of Sarcocystis sp. ex A. cooperii from a naturally infected Cooper’s hawk (Accipiter cooperii). Parasitol Int 66(2):106–111. https://doi.org/10.1016/j.parint.2016.12.002
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(1-2):85–94. https://doi.org/10.1016/j.vetpar.2013.04.024
Morrison DA, Bornstein S, Thebo P, Wernery U, Kinne J, Mattsson JG (2004) The current status of the small subunit rRNA phylogeny of the coccidia (Sporozoa). Int J Parasitol 34(4):501–514. https://doi.org/10.1016/j.ijpara.2003.11.006
Neill PJG, Smith JH, Box ED (1989) Pathogenesis of Sarcocystis falcatula (Apicomplexa: Sarcocystidae) in the budgerigar (Melopsittacus undulatus). IV. Ultraestructure of developing, mature and degenerating sarcocysts. J Protozool 36(4):430–437
Porter RA, Ginn PE, Dame JB, Greiner EC (2001) Evaluation of the shedding of Sarcocystis falcatula sporocysts in experimentally infected Virginia opossums (Didelphis virginiana). Vet Parasitol 95(2-4):313–319. https://doi.org/10.1016/S0304-4017(00)00397-6
Price MN, Dehal PS, Arkin AP (2010) FastTree 2—approximately maximum-likelihood trees for large alignments. PLoS ONE 5, 1–10 e9490 https://doi.org/10.1371/journalpone0009490 Acessed 14 January 2017, 5, e9490
Rambaut A (2017). FigTree-version 1.4.3, a graphical viewer of phylogenetic trees. Available from <http://tree.bio.ed.ac.uk/software/figtree/>. Acessed 16 February 2017
Smith JH, Meier JL, Neill PJG, Box ED (1987) Pathogenesis of Sarcocystis falcatula in the budgerigar. I. Early pulmonary schizogony. Lab Investig 56(1):60–71
Spalding MG, Yowell CA, Lindsay DS, Greiner EC, Dame JB (2002) Sarcocystis meningoencephalitis in a northern Gannet (Morus bassanus). J Wildl Dis 38(2):432–437. https://doi.org/10.7589/0090-3558-38.2.432
Stabenow CS, Oliveira FCR, Albuquerque GR, Lopes CWG (2008) Sarcocystis lindsayi-like (Apicomplexa: Sarcocystinae) of the opossum (Didelphis aurita) from southeastern Brazil. Rev Bras Parasitol Vet 17:342–344
Stabenow CS, Ederli NB, Lopes CWG, Oliveira FCR (2012) Didelphis aurita (Marsupialia: Didelphidae): a new host for Sarcocystis lindsayi (Apicomplexa: Sarcocystidae). J Parasitol 98(6):1262–1265. https://doi.org/10.1645/GE-3140.1
Tanhauser SM, Yowell CA, Cutler TJ, Greiner EC, Mackay RJ, Dame JB (1999) Multiple DNA markers differentiate Sarcocystis neurona and Sarcocystis falcatula. J Parasitol 85(2):221–228. https://doi.org/10.2307/3285623
Zajac AM, Conboy GA (2011) Veterinary clinical parasitology. Wiley-Blackwell, United Kingdom
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The authors would like to thank the research funding agency CAPES (Coordenação de perfeiçoamento de Pessoal de Nível Superior) for financial support (Case: BEX 2755/15-7).
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Gallo, S.S.M., Lindsay, D.S., Ederli, N.B. et al. Identification of opossums Didelphis aurita (Wied-Neuweid, 1826) as a definitive host of Sarcocystis falcatula-like sporocysts. Parasitol Res 117, 213–223 (2018). https://doi.org/10.1007/s00436-017-5695-4
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DOI: https://doi.org/10.1007/s00436-017-5695-4