Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Development of a recombinant indirect ELISA for the diagnosis of Theileria sp. (China) infection in small ruminants

Abstract

Theileria sp. (China) causes severe limitations on the development of the livestock industry in the north-west of China. An enzyme-linked immunosorbent assay (ELISA) based on merozoite homogenate of the parasite for diagnosis of infection has been established; however, cross-reactivity with other small ruminant-infecting piroplasms could not be excluded. Thus, a prerequisite for epidemiological surveys and diagnosis was the establishment of a recombinant protein-based ELISA. To this end, serum from Theileria sp. (China)-infected sheep was used to screen a Theileria lestoquardi expression library, resulting in the identification of a specifically reacting clone with a high identity to the heat shock protein 70 (HSP70) of Theileria parva and Theileria annulata and thus named TlHSP70. An HSP70 homologue was also confirmed to be expressed by Theileria sp. (China) merozoites (TcHSP70). A part of the TlHSP70 protein, found to be conserved in TcHSP70, was recombinantly expressed and used to establish an ELISA. A total of 260 field serum samples tested resulted in a sensitivity and specificity of 94.3 and 89.5%, respectively, in comparison with the merozoite homogenate ELISA. The potentials of the application of the test in epidemiological surveys to map out the prevalence of the disease and for routine diagnostics are described.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. Ahmed JS, Yin H, Schnittger L, Jongejan F (2002) Ticks and tick-borne diseases in Asia with special emphasis on China. Parasitol Res 88:S51–S55

  2. Bai Q, Liu D, Ren J, Li X (2002) Isolation and preliminary characterization of a large Babesia sp. from sheep and goats in the eastern part of the Gansu Province, China. Parasitol Res 88:S16–S21

  3. Bakheit MA (2004) Identification and characterization of a Theileria lestoquardi immunogenic protein and the application in a diagnostic ELISA. Dissertation, Freie Universität, Köster, Berlin

  4. Bakheit MA, Schnittger L, Salih DA, Boguslawski K, Beyer D, Fadl M, Ahmed JS (2004) Application of the recombinant Theileria annulata surface protein in an indirect ELISA for the diagnosis of tropical theilerosis. Parasitol Res 92:299–302

  5. Bakheit MA, Seitzer U, Ahmed JS (2005) A new recombinant protein-based ELISA for the diagnosis of malignant theileriosis of sheep and goats. Parasitol Res (in press). DOI 10.1007/s0043600500346

  6. Gao YL, Yin H, Luo JX, Ouyang WQ, Bao HM, Guan GQ, Zhang QC, Lu WS, Ma ML (2002) Development of an enzyme-linked immunosorbent assay for the diagnosis of Theileria sp. infection in sheep. Parasitol Res 88:S8–S10

  7. Kolaskar AS, Tongaonkar PC (1990) A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett 276:172–174

  8. Kubota S, Matsuba T, Tanaka M, Sugimoto C, Onuma M (1993) Molecular cloning and sequencing of hsp70 cDNA of Theileria sergenti. Mol Biochem Parasitol 62:317–318

  9. Luo J, Yin H (1997) Theileriosis of sheep and goats in China. Trop Anim Health Prod 29:8S–10S

  10. Miranda J, Stumme B, Beyer D, Cruz H, Oliva A, Bakheit M, Wicklein D, Yin H, Lou J, Ahmed J, Seitzer U (2004) Identification of antigenic proteins of a Theileria species pathogenic for small ruminants in China recognized by antiserum of infected animals. Ann N Y Acad Sci 1026:161–164

  11. Nagore D, Garcia-Sanmartin J, Garcia-Perez AL, Juste RA, Hurtado A (2004) Identification, genetic diversity and prevalence of Theileria and Babesia species in a sheep population from Northern Spain. Int J Parasitol 34:1059–1067

  12. Schnittger L, Yin H, Jianxun L, Ludwig W, Shayan P, Rahbari S, Voss-Holtmann A, Ahmed JS (2000a) Ribosomal small-subunit RNA gene-sequence analysis of Theileria lestoquardi and a Theileria species highly pathogenic for small ruminants in China. Parasitol Res 86:352–358

  13. Schnittger L, Yin H, Jianxun L, Ludwig W, Shayan P, Rahbari S, Voss-Holtmann A, Ahmed JS (2000b) Phylogenetic analysis by rRNA comparison of the highly pathogenic sheep-infecting parasites Theileria lestoquardi and a Theileria species identified in China. Ann N Y Acad Sci 916:271–275

  14. Schnittger L, Shayan P, Biermann R, Gerdes J, Ahmed JS (2000c) Molecular genetic characterization and subcellular localization of Theileria annulata mitochondrial heat-shock protein 70. Parasitol Res 86:444–452

  15. Schnittger L, Yin H, Luo J, Katzer F, Beyer D, Shayan P, Ahmed JS (2002) Characterization of a polymorphic gene of T. lestoquardi and of a recently identified Theileria species pathogenic for small ruminants in China. Parasitol Res 88:553–556

  16. Schnittger L, Yin H, Gubbells MJ, Beyer D, Niemann S, Jongejan F, Ahmed JS (2003) Phylogeny of sheep and goat Theileria and Babesia parasites. Parasitol Res 91:398–406

  17. Schnittger L, Yin H, Bai Q, Gubbells MJ, Beyer D, Niemann S, Jongejan F, Ahmed JS (2004) Simultaneous detection and differentiation of Theileria and Babesia parasites infecting small ruminants by reverse line blotting. Parasitol Res 92:189–196

  18. Yin H, Luo J, Guan G, Lu B, Ma M, Zhang Q, Lu W, Lu C, Ahmed JS (2002a) Experiments on transmission of an unidentified Theileria sp. to small ruminants with Haemaphysalis qinghaiensis and Hyalomma anatolicum anatolicum. Vet Parasitol 108:21–30

  19. Yin H, Luo J, Guan G, Gao Y, Lu B, Zhang Q, Ma M, Lu W, Lu C, Yuan Z, Guo S, Wang B, Du H, Schnittger L, Ahmed JS, Jongejan F (2002b) Transmission of an unidentified Theileria species to small ruminants by Haemaphysalis qinghaiensis ticks collected in the field. Parasitol Res 88:S25–S27

  20. Yin H, Liu G, Luo J, Guan G, Ma M, Ahmed JS, Bai Q (2003) Observation on the schizont stage of an unidentified Theileria sp. in experimentally infected sheep. Parasitol Res 91(1):34–39

  21. Yin H, Luo J, Schnittger L, Lu B, Beyer D, Ma M, Guan G, Bai Q, Lu C, Ahmed J (2004) Phylogenetic analysis of Theileria species transmitted by Haemaphysalis qinghaiensis. Parasitol Res 92:36–42

Download references

Acknowledgements

This work was supported in part by the European Union (ICA4-1999-30151) and the Portuguese Foundation for Science and Technology (SFRH/BD/6494/2001). Thanks are due to Prof. F. Jongejan for providing sera from T. annulata-infected cattle. We declare that the experiments performed and presented in this report comply with the current laws of the Federal Republic of Germany.

Author information

Correspondence to U. Seitzer.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Miranda, J., Bakheit, M.A., Liu, Z. et al. Development of a recombinant indirect ELISA for the diagnosis of Theileria sp. (China) infection in small ruminants. Parasitol Res 98, 561–567 (2006). https://doi.org/10.1007/s00436-005-0105-8

Download citation

Keywords

  • Small Ruminant
  • Babesia
  • Indirect Fluorescent Antibody Test
  • Infected Sheep
  • Fish Gelatin