Parasitology Research

, Volume 117, Issue 10, pp 3109–3118 | Cite as

First molecular evidence of Babesia caballi and Theileria equi infections in horses in Cuba

  • Adrian Alberto Díaz-Sánchez
  • Marcus Sandes Pires
  • Carlos Yrurzun Estrada
  • Ernesto Vega Cañizares
  • Sergio Luis del Castillo Domínguez
  • Alejandro Cabezas-Cruz
  • Evelyn Lobo Rivero
  • Adivaldo Henrique da Fonseca
  • Carlos Luiz Massard
  • Belkis Corona-GonzálezEmail author
Original Paper


Equine piroplasmosis is a disease of Equidae, including horses, donkeys, mules, and zebras, caused by either Theileria equi or Babesia caballi. This disease represents a serious problem for the horse industry and its control is critical for the international trade of horses. The objective of the present study was to detect B. caballi and T. equi infections in horses reared in western Cuba. Blood samples from 100 horses were tested for the presence of piroplasms by using Giemsa-stained blood smears and nested PCR (nPCR) assays targeting merozoite antigen genes of B. caballi (bc48) and T. equi (ema-1). All animals were inspected for the detection of tick infestation and tick specimens were collected for species identification. Erythrocyte inclusions were observed in 13 (13%) of the analyzed samples. nPCR analysis showed that 25 (25%) samples were positive for B. caballi, 73 (73%) for T. equi, and 20 (20%) showed dual infections. Only one tick species was found infesting horses, Dermacentor nitens. In addition, three nearly full-length sequences of T. equi 18S rRNA gene were obtained and subjected to phylogenetic analyses. This study reports a high prevalence of T. equi and B. caballi single and coinfections in horses in western Cuba. Molecular analysis of the 18S rRNA gene of T. equi suggested that different genotypes of this hemoparasite circulate in Cuba. To the best of our knowledge, this is the first report describing the molecular detection of B. caballi and T. equi in horses in Cuba.


Equine piroplasmosis Babesia caballi Theileria equi nPCR 18S rRNA 



The authors would like to thank Dr. MVZ Osvaldo Fonseca Rodríguez for his kindness in preparing the map included in this study, as well as Nelson Albelo Chávez and Eleuterio Hernández Hernández for their technical assistance, and Dr. nat. sc. Marina L. Meli at University of Zurich (UZH) for her very helpful comments on the manuscript.

Funding sources

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Compliance with ethical standards

Conflict of interests

The authors declare that they have no conflict of interest.


  1. Alhassan A, Pumidonming W, Okamura M, Hirata H, Battsetseg B, Fujisaki K, Yokoyama N, Igarashi I (2005) Development of a single-round and multiplex PCR method for the simultaneous detection of Babesia caballi and Babesia equi in horse blood. Vet Parasitol 129:43–49. CrossRefPubMedGoogle Scholar
  2. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Asgarali Z, Coombs DK, Mohammed F, Campbell MD, Caesar E (2007) A serological study of Babesia caballi and Theileria equi in Thoroughbreds in Trinidad. Vet Parasitol 144:167–171. CrossRefPubMedGoogle Scholar
  4. Barros-Battesti DM, Arzua M, Bechara GH (2006) Carrapatos de importância médico-veterinária da região neotropical: um guia ilustrado para idenfiticação de espécies. ICTTD-3/Instituto Butantan.Google Scholar
  5. Battsetseg B, Lucero S, Xuan X, Claveria FG, Inoue N, Alhassan A, Kanno T, Igarashi I, Nagasawa H, Mikami T, Fujisaki K (2002) Detection of natural infection of Boophilus microplus with Babesia equi and Babesia caballi in Brazilian horses using nested polymerase chain reaction. Vet Parasitol 107:351–357. CrossRefPubMedGoogle Scholar
  6. Bhoora R, Franssen L, Oosthuizen MC, Guthrie AJ, Zweygarth E, Penzhorn BL, Jongejan F, Collins NE (2009) Sequence heterogeneity in the 18S rRNA gene within Theileria equi and Babesia caballi from horses in South Africa. Vet Parasitol 159:112–120. CrossRefPubMedGoogle Scholar
  7. Bhoora R, Quan M, Franssen L, Butler CM, van der Kolk JH, Guthrie AJ, Zweygarth E, Jongejan F, Collins NE (2010) Development and evaluation of real-time PCR assays for the quantitative detection of Babesia caballi and Theileria equi infections in horses from South Africa. Vet Parasitol 168:201–211. CrossRefPubMedGoogle Scholar
  8. Butler C (2013) Can Theileria equi be eliminated from carrier horses? Vet J 196:279. CrossRefPubMedGoogle Scholar
  9. Castresana J (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 17:540–552CrossRefPubMedGoogle Scholar
  10. de Waal DT (1992) Equine piroplasmosis: a review. Br Vet J 148:6–14. CrossRefPubMedGoogle Scholar
  11. Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet F, Dufayard JF, Guindon S, Lefort V, Lescot M, Claverie JM, Gascuel O (2008) robust phylogenetic analysis for the non-specialist. Nucleic Acids Res 36:W465–W469. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Hall CM, Busch JD, Scoles GA, Palma-Cagle KA, Ueti MW, Kappmeyer LS, Wagner DM (2013) Genetic characterization of Theileria equi infecting horses in North America: evidence for a limited source of U.S. introductions. Parasit Vectors 6:35. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Heim A, Passos LMF, Ribeiro MFB, Costa-Júnior LM, Bastos CV, Cabral DD, Hirzmann J, Pfister K (2007) Detection and molecular characterization of Babesia caballi and Theileria equi isolates from endemic areas of Brazil. Parasitol Res 102:63–68CrossRefPubMedGoogle Scholar
  14. Ikadai H, Xuan X, Igarashi I, Tanaka S, Kanemaru T, Nagasawa H, Fujisaki K, Suzuki N, Mikami T (1999) Cloning and Expression of a 48-Kilodalton Babesia caballi Merozoite Rhoptry Protein and Potential Use of the Recombinant Antigen in an Enzyme-Linked Immunosorbent Assay. J Clin Microbiol 37:3475–3480PubMedPubMedCentralGoogle Scholar
  15. Kerber CE, Labruna MB, Ferreira F, De Waal DT, Knowles DP, Gennari SM (2009) Prevalence of equine Piroplasmosis and its association with tick infestation in the state of Sao Paulo, Brazil. Rev Bras Parasitol Vet 18:1–8CrossRefPubMedGoogle Scholar
  16. Kizilarslan F, Yildirim A, Duzlu O, Inci A, Onder Z, Ciloglu A (2015) Molecular detection and characterization of Theileria equi and Babesia caballi in horses (Equus ferus caballus) in Turkey. J Equine Vet Sci 35:830–835. CrossRefGoogle Scholar
  17. Knowles D Jr (1996) Equine babesiosis(piroplasmosis): a problem in the international movement of horses. Br Vet J 152:123–126CrossRefPubMedGoogle Scholar
  18. Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874. CrossRefGoogle Scholar
  19. Liu Q, Zhao JL, Zhou YQ, Liu EY, Yao BA, Fu Y (2005) Study on some molecular characterization of Babesia orientalis. In: Vet Parasitol, vol 130, pp 191–198. CrossRefGoogle Scholar
  20. Liu Q, Meli ML, Zhang Y, Meili T, Stirn M, Riond B, Weibel B, Hofmann-Lehmann R (2016) Sequence heterogeneity in the 18S rRNA gene in Theileria equi from horses presented in Switzerland. In: Veterinary parasitology, vol 221, pp 24–29. CrossRefGoogle Scholar
  21. Machado RZ, Toledo CZ, Teixeira MC, Andre MR, Freschi CR, Sampaio PH (2012) Molecular and serological detection of Theileria equi and Babesia caballi in donkeys (Equus asinus) in Brazil. Vet Parasitol 186:461–465. CrossRefPubMedGoogle Scholar
  22. Malekifard F, Tavassoli M, Yakhchali M, Darvishzadeh R (2014) Detection of Theileria equi and Babesia caballi using microscopic and molecular methods in horses in suburb of Urmia, Iran. Vet Res Forum 5:129–133 PubMedPubMedCentralGoogle Scholar
  23. Mehlhorn H, Schein E (1998) Redescription of Babesia equi Laveran, 1901 as Theileria equi Mehlhorn, Schein 1998. Parasitol Res 84:467–475CrossRefPubMedGoogle Scholar
  24. Munkhjargal T, Sivakumar T, Battsetseg B, Nyamjargal T, Aboulaila M, Purevtseren B, Bayarsaikhan D, Byambaa B, Terkawi MA, Yokoyama N, Igarashi I (2013) Prevalence and genetic diversity of equine piroplasms in Tov province, Mongolia. Infect Genet Evol 16:178–185. CrossRefPubMedGoogle Scholar
  25. OIE (2014) Chapter 2.5.8: piroplasmosis equina. Manual of diagnostic tests and vaccines for terrestrial animals, Volume 1:963–972Google Scholar
  26. Oliver A, Hernandez J, Roman EI (1985) Concomitant haemoparasitosis with Piroplasma caballi and Nuttallia equi in a horse from the province of Ciego de Avila. Ciencia y Técnica en la Agricultura, Veterinaria 7:79–82Google Scholar
  27. Posada-Guzman MF, Dolz G, Romero-Zuniga JJ, Jimenez-Rocha AE (2015) Detection of Babesia caballi and Theileria equi in blood from equines from four indigenous communities in Costa Rica. Vet Med Int 2015:236278. CrossRefPubMedPubMedCentralGoogle Scholar
  28. Qablan MA, Obornik M, Petrzelkova KJ, Sloboda M, Shudiefat MF, Horin P, Lukes J, Modry D (2013) Infections by Babesia caballi and Theileria equi in Jordanian equids: epidemiology and genetic diversity. Parasitology 140:1096–1103. CrossRefPubMedGoogle Scholar
  29. Rosales R, Rangel-Rivas A, Escalona A, Jordan LS, Gonzatti MI, Aso PM, Perrone T, Silva-Iturriza A, Mijares A (2013) Detection of Theileria equi and Babesia caballi infections in Venezuelan horses using competitive-inhibition ELISA and PCR. Vet Parasitol 196:37–43. CrossRefPubMedGoogle Scholar
  30. Rothschild CM (2013) Equine piroplasmosis. J Equine Vet Sci 33:497–508. CrossRefGoogle Scholar
  31. Salabarria FF, Gonzalez M, Jimenez T (1982) Complement fixation in the serological diagnosis of Nuttalia equi. Revista Cubana de Ciencias Veterinarias 13:81–84Google Scholar
  32. Salim B, Bakheit MA, Kamau J, Nakamura I, Sugimoto C (2010) Nucleotide sequence heterogeneity in the small subunit ribosomal RNA gene within Theileria equi from horses in Sudan. Parasitol Res 106:493–498. CrossRefPubMedGoogle Scholar
  33. Sant C, d'Abadie R, Pargass I, Basu AK, Asgarali Z, Charles RA, Georges KC (2016) Prospective study investigating transplacental transmission of equine piroplasmosis in thoroughbred foals in Trinidad. Vet Parasitol 226:132–137. CrossRefPubMedGoogle Scholar
  34. Schwint ON, Knowles DP, Ueti MW, Kappmeyer LS, Scoles GA (2008) Transmission of Babesia caballi by Dermacentor nitens (Acari: Ixodidae) is restricted to one generation in the absence of alimentary reinfection on a susceptible equine host. J Med Entomol 45:1152–1155CrossRefPubMedGoogle Scholar
  35. Scoles GA, Ueti MW (2015) Vector ecology of equine piroplasmosis. Annu Rev Entomol 60:561–580. CrossRefPubMedGoogle Scholar
  36. Short MA, Clark CK, Harvey JW, Wenzlow N, Hawkins IK, Allred DR, Knowles DP, Corn JL, Grause JF, Hennager SG, Kitchen DL, Traub-Dargatz JL (2012) Outbreak of equine piroplasmosis in Florida. J Am Vet Med Assoc 240:588–595CrossRefPubMedGoogle Scholar
  37. Tamura K, Nei M (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol 10:512–526. CrossRefPubMedGoogle Scholar
  38. Teglas M, Matern E, Lein S, Foley P, Mahan SM, Foley J (2005) Ticks and tick-borne disease in Guatemalan cattle and horses. In: Vet Parasitol, vol 131, pp 119–127. CrossRefGoogle Scholar
  39. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Wise L, Kappmeyer L, Mealey R, Knowles D (2013) Review of equine piroplasmosis. J Vet Intern Med 27:1334–1346CrossRefPubMedGoogle Scholar
  41. Wise LN, Kappmeyer LS, Silva MG, White SN, Grause JF, Knowles DP (2017) Verification of post-chemotherapeutic clearance of Theileria equi through concordance of nested PCR and immunoblot. Ticks Tick-Borne Dis 9:135–140. CrossRefPubMedGoogle Scholar
  42. Xuan X, Nagai A, Battsetseg B, Fukumoto S, Makala LH, Inoue N, Igarashi I, Mikami T, Fujisaki K (2001) Diagnosis of equine piroplasmosis in Brazil by serodiagnostic methods with recombinant antigens. J Vet Med Sci 63:1159–1160CrossRefPubMedGoogle Scholar
  43. Zhang J, Kelly P, Li J, Xu C, Wang C (2015) Molecular detection of Theileria spp. in livestock on five Caribbean islands. Biomed Res Int 2015:624728. CrossRefPubMedPubMedCentralGoogle Scholar
  44. Zobba R, Ardu M, Niccolini S, Chessa B, Manna L, Cocco R, Pinna Parpaglia ML (2008) Clinical and laboratory findings in equine piroplasmosis. J Equine Vet Sci 28:301–308. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Adrian Alberto Díaz-Sánchez
    • 1
  • Marcus Sandes Pires
    • 2
  • Carlos Yrurzun Estrada
    • 3
  • Ernesto Vega Cañizares
    • 1
  • Sergio Luis del Castillo Domínguez
    • 1
  • Alejandro Cabezas-Cruz
    • 4
  • Evelyn Lobo Rivero
    • 1
  • Adivaldo Henrique da Fonseca
    • 2
  • Carlos Luiz Massard
    • 2
  • Belkis Corona-González
    • 1
    • 5
    Email author
  1. 1.National Center for Animal and Plant Health (CENSA)San José de las LajasCuba
  2. 2.Department of Epidemiology and Public HealthFederal Rural University of Rio de Janeiro (UFRRJ)Rio de JaneiroBrazil
  3. 3.Department of Animal Prevention, Veterinary Medicine CollegeAgrarian University of Habana (UNAH)San José de las LajasCuba
  4. 4.UMR BIPAR, INRA, ANSES, Ecole Nationale Vétérinaire d’AlfortUniversité Paris-EstMaisons-AlfortFrance
  5. 5.Animal Health DivisionNational Center for Animal and Plant Health (CENSA)San José de las LajasCuba

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