Abstract
Characteristic sequence signatures were identified within the hypervariable region 4 (V4 region) of the small ribosomal RNA gene of ovine/caprine piroplasm species including Theileria lestoquardi, T. ovis , T. separata , Babesia ovis , B. motasi , B. crassa [comprising strains B. crassa (Iran) and B. crassa (Turkey)] and several novel species: Theileria sp. 1 (China), Theileria sp. 2 (China) and Babesia sp. (China), [comprising strain Babesia sp. (Lintan), and Babesia sp. (Ningxian)] as defined previously. Based on the ascertained gene variations a reverse line blotting (RLB) assay was developed enabling direct, concurrent, highly specific and sensitive identification of virtually all presently known ovine/caprine piroplasm species. All probes bound to their respective target sequence only, therefore, no cross-reaction was observed resulting in clear recognition of either individual strains, species or groups. No signal was observed when ovine and caprine genomic DNA was used as the control, demonstrating that the signals are due to the presence of parasite DNA in investigated samples. Furthermore, the sensitivity of RLB could be considerably enhanced to detect a parasitemia level of at least 10-12% by reamplification of PCR products (nested PCR) thereby substantially increasing the possibility of identifying carrier animals.
Similar content being viewed by others
References
Alani AJ, Herbert IV (1988) Morphology and transmission of Theileria recondita (Theileriidae: Sporozoa) isolated from Haemaphysalis punctata from North Wales. Vet Parasitol 28:283–291
Bai Q, Liu G, 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 Gansu Province, China. Parasitol Res 88: S16–21
Calder JA, Reddy GR, Chieves L, Courtney CH, Littell R, Livengood JR, Norval RA, Smith C, Dame JB (1996) Monitoring Babesia bovis infections in cattle by using PCR-based tests. J Clin Microbiol 34:2748–2555.
d’Oliveira C, van der Weide M, Habela MA, Jacquiet P, Jongejan F (1995) Detection of Theileria annulata in blood samples of carrier cattle by PCR. J Clin Microbiol 33:2665–2669
Gubbels MJ, de Vos AP, van der Weide M, Viseras J, Schouls LM, de Vries E, Jongejan F (1999) Simultaneous detection of bovine Theileria and Babesia species by reverse line blot hybridization. J Clin Microbiol 37:1782–1789
Gubbels MJ, Yin H, van der Weide M, Qi B, Nijman IJ, Guangyuan L, Jongejan F (2000) Molecular characterisation of the Theileria buffeli / orientalis group. Int J Parasitol 30:943–952
Gubbels MJ, Yin H, Bai Q, Liu G, Nijman IJ, Jongejan F (2002) The phylogenetic position of the Theileria buffeli group in relation to other Theileria species. Parasitol Res 88: S28–32
Herwaldt BL, Kjemtrup AM, Conrad PA, Barnes RC, Wilson M, McCarthy MG, Sayers MH, Eberhard ML (1997) Transfusion-transmitted babesiosis in Washington State: first reported case caused by a WA1-type parasite. J Infect Dis 175:1259–1262
Hooshmand-Rad P, Hawa NJ (1973a) Malignant theileriosis of sheep and goats. Trop Anim Health Prod 5:97–102
Katzer F, McKellar S, Kirvar E, Shiels B (1998) Phylogenetic analysis of Theileria and Babesia equi in relation to the establishment of parasite populations within novel host species and the development of diagnostic tests. Mol Biochem Parasitol 95:33–44
Kirvar E, Ilhan T, Katzer F, Wilkie G, Hooshmand-Rad P, Brown D (1998) Detection of Theileria lestoquardi (hirci) in ticks, sheep, and goats using the polymerase chain reaction. Ann N Y Acad Sci 849:52–62
Leemans I, Hooshmand-Rad P, Uggla A (1997) The indirect fluorescent antibody test based on schizont antigen for study of the sheep parasite Theileria lestoquardi. Vet Parasitol 69:9-18
Luo J, Yin H (1997) Theileriosis of sheep and goats in China. Trop Anim Health Prod 29:8S-10S
Medlin L, Elwood HJ, Stickel S, Sogin ML (1988) The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene 71:491–499
Mehlhorn H, Schein E (1984) The piroplasms: life cycle and sexual stages. Adv Parasitol 23:37–103
Mehlhorn H, Schein E, Ahmed JS (1994) Theileria. In: Kreier JP (eds) Parasitic Protozoa, part 2, vol 7. Academic Press, San Diego, Calif., pp 217–304
Persing DH, Herwaldt BL, Glaser C, Lane RS, Thomford JW, Mathiesen D, Krause PJ, Phillip DF, Conrad PA (1995) Infection with a babesia-like organism in northern California. N Engl J Med 332:298–303
Quick RE, Herwaldt BL, Thomford JW, Garnett ME, Eberhard ML, Wilson M, Spach DH, Dickerson JW, Telford SR, Steingart KR, Pollock R, Persing DH, Kobayashi JM, Juranek DD, Conrad PA (1993) Babesiosis in Washington State: a new species of Babesia? Ann Intern Med 119:284–290
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
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
Thomford JW, Conrad PA, Telford SR, Mathiesen D, Bowman BH, Spielmann A, Eberhard ML, Herwaldt B, Quick RE, Persing DH (1994) Cultivation and phylogenetic characterization of a newly recognized human pathogenic protozoan. J Infect Dis 169:1050–1056
Uilenberg G, Andreasen MP (1974) Haematoxenus separatus sp.n. (Sporozoa, Theileriidae) a new blood parasite of domestic sheep in Tansania. Rev Elev Med Vet Pays Trop 27:459–465
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 (2002) Transmission of an unidentified Theileria species to small ruminants by Haemaphysalis qinghaiensis ticks collected in the field. Parasitol Res 88:S25-S27
Acknowledgements
This work was supported by the following European Union INCO-DEV Program contracts: contract no. ICA4-CT2000–300028 (Molecular and immunological characterisation of merozoite antigens and their encoding genes of a Theileria species highly pathogenic for small ruminants in China: application for the development of diagnostics and vaccine); contract no. ICB1CT-2000–80004 ( Theileria China antigens: application for the development of diagnostics and vaccines for small ruminants in China); contract no. ICA4-CT-2000–30006 [International Consortium on Ticks and Tick-Borne Diseases (ICTTS-2)]; a bursary was awarded to Y. H.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Schnittger, L., Yin, H., Qi, B. et al. Simultaneous detection and differentiation of Theileria and Babesia parasites infecting small ruminants by reverse line blotting. Parasitol Res 92, 189–196 (2004). https://doi.org/10.1007/s00436-003-0980-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00436-003-0980-9