First report of zoonotic Cryptosporidium parvum GP60 subtypes IIaA15G2R1 and IIaA16G3R1 in wild ponies from the northern Iberian Peninsula

  • S. Couso-Pérez
  • F. Bárcena-Varela de Limia
  • E. Ares-Mazás
  • H. Gómez-CousoEmail author
Protozoology - Original Paper


Studies on the prevalence and molecular characterization of Cryptosporidium spp. affecting feral horses are scarce. The highland areas of the northern Iberian Peninsula are home to a large population of wild ponies which generally roam free in the ancient natural range and are subjected to a traditional exploitation regime. In the present study, a total of 79 non-diarrhoeal faecal samples from the wild ponies were collected from the ground immediately after defecation. Cryptosporidium was detected in 10 of the samples (12.6%) by a direct immunofluorescence antibody test and DNA amplification and sequencing. Analysis of partial sequences of the small subunit ribosomal RNA (SSU-rRNA) and heat shock protein (hsp70) loci revealed the presence of Cryptosporidium parvum. In addition, amplification and sequencing of a fragment of the 60-kDa glycoprotein (GP60) locus identified C. parvum subtypes IIaA15G2R1 and IIaA16G3R1. This study reports, for the first time, the occurrence of C. parvum in wild ponies in Europe, specifically in the northern Iberian Peninsula. Identification of the common subtype IIaA15G2R1 and also subtype IIaA16G3R1 (first description) indicates that these hosts may play a role in the sylvatic transmission of C. parvum and that they may act as a reservoir of zoonotic cryptosporidiosis.


Wild ponies Cryptosporidium parvum Immunofluorescence microscopy Molecular characterization Iberian Peninsula 



The authors thank María Dolores Fernández de Córdoba Castosa for collaboration in this research.

Funding information

This study was funded by the Autonomous Government of Galicia (grants GPC2014/069 and ED431C 2017/31).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.


  1. Alves M, Xiao L, Sulaiman I, Lal AA, Matos O, Antunes F (2003) Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal. J Clin Microbiol 41:2744–2747. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bárcena F (2012) Os póneis selvagens (Equus ferus sp.) do norte da Península Ibérica. In: I Congresso Internacional do Garrano, candidatura a Patrimonio Nacional. Vila Verde, pp 75-96Google Scholar
  3. Caffara M, Piva S, Pallaver F, Iacono E, Galuppi R (2013) Molecular characterization of Cryptosporidium spp. from foals in Italy. Vet J 198:531–533. CrossRefPubMedGoogle Scholar
  4. Cañon J, Checa ML, Carleos C, Vega-Pla JL, Vallejo M, Dunner S (2000) The genetic structure of Spanish Celtic horse breeds inferred from microsatellite data. Anim Genet 31:39–48CrossRefGoogle Scholar
  5. Couso-Pérez S, Ares-Mazás E, Gómez-Couso H (2019) First report of Cryptosporidium molnari-like genotype and Cryptosporidium parvum zoonotic subtypes (IIaA15G2R1 and IIaA18G3R1) in brown trout (Salmo trutta). J Parasitol 105:170–179CrossRefGoogle Scholar
  6. Deng L, Li W, Zhong Z, Gong C, Cao X, Song Y, Wang W, Huang X, Liu X, Hu Y, Fu H, He M, Wang Y, Zhang Y, Wu K, Peng G (2017) Occurrence and genetic characteristics of Cryptosporidium hominis and Cryptosporidium andersoni in horses from Southwestern China. J Eukaryot Microbiol 64:716–720. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Díaz P, Quílez J, Chalmers RM, Panadero R, López C, Sánchez-Acedo C, Morrondo P, Díez-Baños P (2010) Genotype and subtype analysis of Cryptosporidium isolates from calves and lambs in Galicia (NW Spain). Parasitology 137:1187–1193. CrossRefPubMedGoogle Scholar
  8. Díaz P, Quílez J, Prieto A, Navarro E, Pérez-Creo A, Fernández G, Panadero R, López C, Díez-Baños P, Morrondo P (2015) Cryptosporidium species and subtype analysis in diarrhoeic pre-weaned lambs and goat kids from north-western Spain. Parasitol Res 114:4099–4105. CrossRefPubMedGoogle Scholar
  9. Feng Y, Ryan UM, Xiao L (2018) Genetic diversity and population structure of Cryptosporidium. Trends Parasitol 34:997–1011. CrossRefPubMedGoogle Scholar
  10. Galuppi R, Piva S, Castagnetti C, Iacono E, Tanel S, Pallaver F, Fioravanti ML, Zanoni RG, Tampieri MP, Caffara M (2015) Epidemiological survey on Cryptosporidium in an equine perinatology unit. Vet Parasitol 210:10–18. CrossRefPubMedGoogle Scholar
  11. Grinberg A, Oliver L, Learmonth JJ, Leyland M, Roe W, Pomroy WE (2003) Identification of Cryptosporidium parvum «cattle» genotype from a severe outbreak of neonatal foal diarrhoea. Vet Rec 153:628–631CrossRefGoogle Scholar
  12. Hong S-H, Anu D, Jeong Y, Abmed D, Cho S-H, Lee W-J, Lee S-E (2014) Molecular characterization of Giardia duodenalis and Cryptosporidium parvum in fecal samples of individuals in Mongolia. Am J Trop Med Hyg 90:43–47. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Inácio SV, Widmer G, de Brito RLL, Zucatto AS, de Aquino MCC, Oliveira BCM, Nakamura AA, Neto LS, Carvalho JGB, Gomes JF, Meireles MV, Bresciani KDS (2017) First description of Cryptosporidium hominis GP60 genotype IkA20G1 and Cryptosporidium parvum GP60 genotypes IIaA18G3R1 and IIaA15G2R1 in foals in Brazil. Vet Parasitol 233:48–51. CrossRefPubMedGoogle Scholar
  14. Jian F, Liu A, Wang R, Zhang S, Qi M, Zhao W, Shi Y, Wang J, Wei J, Zhang L, Xiao L (2016) Common occurrence of Cryptosporidium hominis in horses and donkeys. Infect Genet Evol 43:261–266. CrossRefPubMedGoogle Scholar
  15. Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120CrossRefGoogle Scholar
  16. Kostopoulou D, Casaert S, Tzanidakis N, van Doorn D, Demeler J, von Samson-Himmelstjerna G, Saratsis A, Voutzourakis N, Ehsan A, Doornaert T, Looijen M, De Wilde N, Sotiraki S, Claerebout E, Geurden T (2015) The occurrence and genetic characterization of Cryptosporidium and Giardia species in foals in Belgium, The Netherlands, Germany and Greece. Vet Parasitol 211:170–174. CrossRefPubMedGoogle Scholar
  17. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Laatamna AE, Wagnerová P, Sak B, Květoňová D, Aissi M, Rost M, Kváč M (2013) Equine cryptosporidial infection associated with Cryptosporidium hedgehog genotype in Algeria. Vet Parasitol 197:350–353. CrossRefPubMedGoogle Scholar
  19. Lagos Abarzuza L (2013) Ecología del lobo (Canis lupus), del poni salvaje (Equus ferus atlanticus) y del ganado vacuno semiextensivo (Bos taurus) en Galicia: interacciones depredador - presa. University of Santiago de CompostelaGoogle Scholar
  20. Leite JAV., Portas MCP (2013) Cavalo garrano. In: Livro Raças Autóctones Portuguesas. Direcção Geral de Alimentação e Veterinaria, Lisboa, pp 25-31Google Scholar
  21. Piva S, Caffara M, Pasquali F, Castagnetti C, Iacono E, Massella E, Zanoni RG, Galuppi R (2016) Detection and quantification of Cryptosporidium oocysts in environmental surfaces of an equine perinatology unit. Prev Vet Med 131:67–74. CrossRefPubMedGoogle Scholar
  22. Qi M, Zhou H, Wang H, Wang R, Xiao L, Arrowood MJ, Li J, Zhang L (2015) Molecular identification of Cryptosporidium spp. and Giardia duodenalis in grazing horses from Xinjiang, China. Vet Parasitol 209:169–172. CrossRefPubMedGoogle Scholar
  23. Quílez J, Torres E, Chalmers RM, Robinson G, Del Cacho E, Sánchez-Acedo C (2008) Cryptosporidium species and subtype analysis from dairy calves in Spain. Parasitology 135:1613–1620. CrossRefPubMedGoogle Scholar
  24. Ryan U, Xiao L, Read C, Zhou L, Lal AA, Pavlasek I (2003) Identification of novel Cryptosporidium genotypes from the Czech Republic. Appl Environ Microbiol 69:4302–4307CrossRefGoogle Scholar
  25. Snyder SP, England J, McChesney AE (1978) Cryptosporidiosis in immunodeficient Arabian foals. Vet Pathol 15:12–17. CrossRefPubMedGoogle Scholar
  26. USEPA (2005) Method 1623: Cryptosporidium and Giardia in water by filtration/IMS/FA. United States Environmental Agency, USAGoogle Scholar
  27. Wagnerová P, Sak B, McEvoy J, Rost M, Perec Matysiak A, Ježková J, Kváč M (2015) Genetic diversity of Cryptosporidium spp. including novel identification of the Cryptosporidium muris and Cryptosporidium tyzzeri in horses in the Czech Republic and Poland. Parasitol Res 114:1619–1624. CrossRefPubMedGoogle Scholar
  28. Wagnerová P, Sak B, McEvoy J, Rost M, Sherwood D, Holcomb K, Kváč M (2016) Cryptosporidium parvum and Enterocytozoon bieneusi in American Mustangs and Chincoteague ponies. Exp Parasitol 162:24–27. CrossRefPubMedGoogle Scholar
  29. Warmuth V, Eriksson A, Bower MA, Cañon J, Cothran G, Distl O, Glowatzki-Mullis M-L, Hunt H, Luís C, do Mar Oom M, Yupanqui IT, Ząbek T, Manica A (2011) European domestic horses originated in two Holocene refugia. PLoS One 6:e18194. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Laboratory of Parasitology, Department of Microbiology and Parasitology, Faculty of PharmacyUniversity of Santiago de CompostelaA CoruñaSpain
  2. 2.Institute of Food Research and AnalysisUniversity of Santiago de CompostelaA CoruñaSpain

Personalised recommendations