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Neospora caninum Natural Infection in Tunisian Rams: Serological Study and Molecular Identification of Infection in Semen



The aim of this study was to estimate the seroprevalence and molecular prevalence and perform a molecular identification of Neospora caninum in semen of Tunisian rams.


A total of 92 blood samples were collected from four farms located in four Tunisian governorates (Jendouba, Kairouan, Zaghouan and Ben Arous) and samples were screened with a commercial ELISA kit for N. caninum antibodies. For the same rams, semen samples were collected and tested for the presence of N. caninum ITS1 gene using PCR. Five amplicons were randomly selected for sequencing. A phylogenetic tree was constructed to compare the partial sequences of the ITS1 gene with sequences deposited in GenBank.


The seroprevalence of N. caninum infection was 25% (23/92) and PCR revealed that the molecular infection prevalence in semen was 11.95% (11/92). Kappa test showed an average agreement between seroprevalence and parasite prevalence in semen (κ = 0.44). The highest molecular prevalence was for rams that accomplished more than two mating seasons (21.0 ± 12.1%) compared to those performed less than two mating seasons and yearling individuals (4.0 ± 5.5%) (P = 0.01). There were no differences in N. caninum molecular prevalence according to either breed or locality. Comparison of the partial sequences of the ITS1 gene revealed 99–100% similarity with those deposited in GenBank.


To the best of our knowledge, this is the first detection and molecular identification of N. caninum in semen from rams in North Africa. Our findings indicate that N. caninum infection rate was high in rams.

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  1. 1.

    Almería S, López-gatius F (2013) Bovine neosporosis: clinical and practical aspects. Res Vet Sci 95(2):303–309.

    Article  PubMed  Google Scholar 

  2. 2.

    Amdouni Y, Rjeibi MR, Awadi S, Rekik M, Gharbi M (2018) First detection and molecular identification of Neospora caninum from naturally infected cattle and sheep in North Africa. Transbound Emerg Dis.

    Article  PubMed  Google Scholar 

  3. 3.

    Arbabi M, Abdoli A, Dalimi A, Pirestani M, Abdoli A (2014) Identification of latent neosporosis in sheep in Tehran, Iran by polymerase chain reaction using primers specific for the Nc-5 gene. Onderstepoort J Vet Res.

    Article  Google Scholar 

  4. 4.

    Ayouni T (2011) Defended a thesis: Contribution à l’étude de la néosporose caprine: enquête sérologique chez des chèvres ayant avorté en Tunisie. Ecole Nationale de Médecine Vétérinaire de Sidi Thabet

  5. 5.

    Bergeron N, Fecteau G, Paré J, Martineau R, Villeneuve, A (2000) Vertical and horizontal transmission of Neospora caninum in dairy herds in Québec. Can Vet J 41(6): 464–467.

  6. 6.

    Bishop S, King J, Windsor P, Reichel MP, Ellis J, Jan S (2010) The first report of ovine cerebral neosporosis and evaluation of Neospora caninum prevalence in sheep in New South Wales. Vet Parasitol 170:137–142.

    Article  PubMed  Google Scholar 

  7. 7.

    Buxton D, Maley SW, Wright S, Thomson KM, Rae AG, Innes EA (1998) The pathogenesis of experimental neosporosis in pregnant sheep. J Comp Pathol 118:267–279.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Caetano-Da-Silva A, Ferre I, Collantes-Fernández E, Navarro V, Aduriz G, Ugarte-Garagalza C, Ortega-Mora LM (2004) Occasional detection of Neospora caninum DNA in frozen extended semen from naturally infected bulls. Theriogenol J 62(7):1329–1336.

    Article  Google Scholar 

  9. 9.

    Cheah TS, Mattsson JG, Zaini M, Sani RA (2004) Isolation of Neospora caninum from a calf in Malaysia. Vet Parasitol 126:263–269.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Boughzalad H (2000) Defended a thesis:Néosporose abortive bovine: Résultats d’une enquête sérologique en Tunisie

  11. 11.

    Davison HC, Guy CS, McGarry JW, Guy F, Williams DJL, Kelly DF, Trees AJ (2001) Experimental studies on the transmission of Neospora caninum between cattle. Res Vet Sci 70(2):163–168.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Davison HC, Otter A, Trees AJ (1999) Estimation of vertical and horizontal transmission parameters of Neospora caninum infections in dairy cattle. Int J Parasitol 29(10):1683.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Dechicha A, Gharbi S, Kebbal S, Chatagnon G, Tainturier D, Ouzrout R, Guetarn D (2009) Serological survey of etiological agents associated with abortion in two Algerian dairy cattle breeding farms. J Vet Med Ani Health 2(1): 1–5.

  14. 14.

    Derdour SY, Hafsi F, Azzag N, Tennah S, Laamari A, China B, Ghalmi F (2017) Prevalence of the main infectious causes of abortion in dairy cattle in Algeria. J Vet Res 61(3):337–343.

    Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Dijkstra T, Barkema HW, Eysker M (2002) Natural transmission routes of Neospora caninum between farm dogs and cattle. Vet Parasitol 105:99–104.

    Article  PubMed  Google Scholar 

  16. 16.

    Du L, Yang D, Zhai T, Gong P, Zhang X, Li J (2015) Detection of Neospora caninum-DNA in brain tissues from pigeons in Changchun, Jilin (China). Vet Parasitol 214(1–2):171–173.

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Dubey JP, Hartley WJ, Lindsay DS, Topper MJ (1990) Fatal congenital Neospora caninum infection in a lamb. J Parasitol 76(1):127–130.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Dubey JP, Hemphill A, Calero-Bernal R, Schares G (2017) Neosporosis in animals. CRC Press, Boca Raton, pp 133–336

    Book  Google Scholar 

  19. 19.

    Dubey JP, Schares G (2011) Neosporosis in animals-the last five years. Vet Parasitol 180:90–108.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Dubey JP, Schares G, Ortega-Mora LM (2007) Epidemiology and control of neosporosis and Neospora caninum. Clin Microbiol Rev 20:323–367.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Ezatpour B, Alirezaei M, Hassanvand A (2013) The first report of Neospora caninum prevalence in aborted and healthy sheep from west of Iran. Comp Clin Pathol.

    Article  Google Scholar 

  22. 22.

    Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 783:71.

    Article  Google Scholar 

  23. 23.

    Ferre I, Aduriz G, Del-Pozo I, Regidor-Cerrillo J, Atxaerandi R, Collantes- Fernández E, Ana H, Carlos UG, Ortega-Mora LM (2005) Detection of Neospora caninum in the semen and blood of naturally infected bulls. Theriogenology 63(5):1504–1518.

    Article  PubMed  Google Scholar 

  24. 24.

    Ghalmi F, China B, Kaidi R, Losson B (2011) Neospora caninum is associated with abortion in Algerian cattle. J Parasitol 97(6):1121–1124.

    Article  PubMed  Google Scholar 

  25. 25.

    Ghattof HH, Faraj AA (2015) Seroprevalence of Neospora caninum in goats in Wasit Province Iraq. Int J Curr Microbiol Appl Sci 4(7): 182–191.

  26. 26.

    Gholamreza R, Naseri Z (2017) Molecular detection of Neospora caninum infection in ovine aborted foetuses in the Mashhad area, Iran. Ann Parasitol 63(1):45–47.

    Article  Google Scholar 

  27. 27.

    Gottstein B, Hentrich B, Wyss R, Thür B, Bruckner L, Müller N, Kaufmann H, Waldvogel A (1999) Molecular and immunodiagnostic studies of bovine neosporosis in Switzerland. Int J Parasitol 28(4):679–691.

    Article  Google Scholar 

  28. 28.

    Howe L, West D, Collett M, Tattersfield G, Pattison R, Pomroy WE, Kenyon PR, Morris ST, Williamson NB (2008) The role of Neospora caninum in three cases of unexplained ewe abortions in the Southern North Island of New Zealand. Small Rumin Res 75:115–122.

    Article  Google Scholar 

  29. 29.

    Ibrahim HM, Huang P, Salem TA, Talaat RM, Nasr MI, Xuan X (2009) Prevalence of Neospora caninum and Toxoplasma gondii antibodies in Northern Egypt. Am J Trop Med Hyg 80(2):263–267.

    Article  Google Scholar 

  30. 30.

    Iovu A, Györke A, Mircean V, Gavrea R, Cozma V (2012) Seroprevalence of Toxoplasma gondii and Neospora caninum in dairy goats from Romania. Vet Parasitol 186(3–4):470–474.

    Article  PubMed  Google Scholar 

  31. 31.

    Liu ZK, Li JY, Pan H (2015) Seroprevalence and risk factors of Toxoplasma gondii and Neospora caninum infections in small ruminants in China. Prev Vet Med 118(4):488–492.

    Article  PubMed  Google Scholar 

  32. 32.

    Lucchese L, Benkirane A, Hakimi I, Idrissi AE (2016) Seroprevalence study of the main causes of abortion in dairy cattle in Morocco. Vet Ital 52(1):13–19.

    Article  PubMed  Google Scholar 

  33. 33.

    Masuda T, Kobayashi Y, Maeda R, Omata Y (2007) Possibility of Neospora caninum infection by venereal transmission in CB-17SCID mice. Vet Parasitol 149(1–2):130–133.

    Article  PubMed  Google Scholar 

  34. 34.

    Mesquita LP, Nogueira CI, Costa RC, Orlando DR, Bruhn FRPP, Lopes PFRR, Nakagaki KYR, Peconick AP, Seixas JN, Júnior PSB, Raymundo DL, Varaschin MS (2013) Antibody kinetics in goats and conceptuses naturally infected with Neospora caninum. Vet Parasitol 196(3–4):327–333.

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Modrý D, Václavek P, Koudela B, Šlapeta J (2001) Placentophagia—an alternative way for horizontal transmission of Neospora caninum in cattle? Trends Parasitol 17(12):4922.

    Article  Google Scholar 

  36. 36.

    Moore DP, Draghi MG, Campero CM, Cetrá B, Odeón AC, Alcaraz E, Späth EAJ (2003) Serological evidence of Neospora caninum infections in beef bulls in six counties of the Corrientes province, Argentina. Vet Parasitol 114(4):247–252.

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Omar B, Leila DA, Rachida A, Nedjoua L, Assia B, Omar B (2013) Seroprevalence of Neospora caninum in dairy cattle in Eastern Algeria. Annals Bio Res 4(11): 76–79.

  38. 38.

    Ortega-Mora LM, Ferre I, Del-Pozo I, Caetano-Da-Silva A, Collantes- Fernández E, Regidor-Cerrillo J, Ugarte-Garagalza C, Aduriz G (2003) Detection of Neospora caninum in semen of bulls. Vet Parasitol 117(4):301–308.

    Article  PubMed  Google Scholar 

  39. 39.

    Pena HFJ, Soares RM, Ragozo AMA, Monteiro RM, Yai LEO, Nishi SM, Gennari SM (2007) Isolation and molecular detection of Neospora caninum from naturally infected sheep from Brazil. Vet Parasitol 147(1–2):61–66.

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Ruiz-Fons F, González-Barrio D, Aguilar-Ríos F, Soler AJ, Garde JJ, Gortázar C, Fernández-Santos MR (2014) Infectious pathogens potentially transmitted by semen of the black variety of the Manchega sheep breed: health constraints for conservation purposes. Anim Reprod Sci 149(3–4):152–157.

    Article  PubMed  Google Scholar 

  41. 41.

    Saitou N, Nei M (1987) The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425.

    CAS  Article  Google Scholar 

  42. 42.

    Sasani F, Javanbakht J, Seifori P, Fathi S, Hassan MA (2013) Neospora caninum as causative agent of ovine encephalitis in Iran. Pathol Discov.

    Article  Google Scholar 

  43. 43.

    Schwartz D (1993) Méthodes statistiques à l’usage des médecins et des biologistes, 3rd edn. Flammarion, Paris, pp 17–81

    Google Scholar 

  44. 44.

    Syed-Hussain SS, Howe L, Pomroy WE, West DM, Smith SL, Williamson NB (2013) Detection of Neospora caninum DNA in semen of experimental infected rams with no evidence of horizontal transmission in ewes. Vet Parasitol 197(3–4):534–542.

    CAS  Article  PubMed  Google Scholar 

  45. 45.

    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 Evo 28:2731–2739.

    CAS  Article  Google Scholar 

  46. 46.

    Toma B, Dufour B, Sanaa M, Bénet JJ, Ellis P, Moutou F, Louzä A (2007) Epidémiologie appliquée à la lutte collective contre les maladies animales transmissibles majeures. 2nd edn

  47. 47.

    Uggla A, Stenlund S, Holmdahl OJM, Jakubek EB, Thebo P, Kindahl H, Björkman C (1998) Oral Neospora caninum inoculation of neonatal calves. Int J Parasitol 28(9):1467–1472.

    CAS  Article  PubMed  Google Scholar 

  48. 48.

    Wang Y, Tian RM, Gao ZM, Bougouffa S, Qian PY (2014) Optimal eukaryotic 18S and universal 16S/18S ribosomal RNA primers and their application in a study of symbiosis. PLoS One.

    Article  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Zhan W, Deng C, Liu Q, Liu J, Wang M, Tian KG, Yu XL, Hu DM (2007) First identification of Neospora caninum infection in aborted bovine foetuses in China. Vet Parasitol 149:72–76.

    Article  Google Scholar 

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The work was funded by the laboratory of “Laboratoire d’épidémiologie des infections enzootiques des herbivores en Tunisie: application à la lutte’’ (LR16AGR01) (Ministère de l’enseignement supérieure et de la recherche scientifique, Tunisie) and was partly supported by the CGIAR Research Program on Livestock. The authors thank Mr. Bechir Guesmi, Mr. Limam Sassi and Mr. Taoufik Lahmar for their technical support and the farm owners. We also acknowledge Mustapha Lahmer from the Department of Animal Breeding, Office de l’Elevage et des Pâturages, Sidi Thabet, Tunisia.

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Correspondence to Yosra Amdouni.

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Amdouni, Y., Rouatbi, M., Lassoued, N. et al. Neospora caninum Natural Infection in Tunisian Rams: Serological Study and Molecular Identification of Infection in Semen. Acta Parasit. 64, 821–828 (2019).

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  • Neospora caninum infection
  • Ram
  • Semen
  • PCR
  • Tunisia