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Parasitology Research

, Volume 95, Issue 3, pp 186–192 | Cite as

The use of ITS1 rDNA PCR in detecting pathogenic African trypanosomes

  • Z. K. Njiru
  • C. C. Constantine
  • S. Guya
  • J. Crowther
  • J. M. Kiragu
  • R. C. A. Thompson
  • A. M. R. Dávila
Original Paper

Abstract

There are 11 different pathogenic trypanosomes in trypanosomiasis endemic regions of Africa. Their detection and characterisation by molecular methods relies on species-specific primers; consequently several PCR tests have to be made on each sample. Primers ITS1 CF and ITS1 BR, previously designed to amplify the internal transcribed spacer (ITS1) of rDNA, have been evaluated for use in a universal diagnostic test for all pathogenic trypanosomes. Blood was collected from 373 cattle and 185 camels. The primers gave constant PCR products with the stocks of each taxon tested. Members of subgenus Trypanozoon (T. brucei brucei, T. evansi, T. b. rhodesiense and T. b. gambiense) gave a constant product of approximately 480 bp; T. congolense, savannah 700 bp, T. congolense kilifi 620 bp and T. congolense forest 710 bp: T. simiae 400 bp, T. simiae tsavo 370 bp, T. godfreyi 300 bp and T. vivax 250 bp. The sensitivity of the test ranged from 10 pg for Trypanozoon, T. congolense clade and T. vivax to 100 pg for T. simiae and T. godfreyi. The primers detected cases of multi-taxa samples, although the sensitivity was reduced with an increase in the combinations. A better detection rate of trypanosome DNA was recorded with buffy coats than from direct blood. With the field samples, the diagnostic sensitivity was close to the sensitivity obtained using single reactions with species-specific primers for Trypanozoon 38/40 (95%) and T. congolense savannah 30/33 (90.9%) but was lower with T. vivax 25/31 (77.4%). The primers offer promise as a routine diagnostic tool through the use of a single PCR; however, further evaluation is recommended.

Keywords

Internal Transcribe Spacer Sterile Insect Technique Trypanosomiasis Trypanosome Infection Improve Detection Rate 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgements

This work received financial support from the International Atomic Energy Agency (IAEA) under grant no. KEN 11414 to Z.K. Njiru and supplementary funds from the Kenyan Government. Z.K. also thanks Dr. Wendy Gibson for supplying some reference DNA and the staff of Biochemistry Division, KETRI for technical support. The work reported here complies with the current laws of Kenya where the work was done. Nucleotide sequence data reported in this paper is available in the GenBank and DDBJ data bases under accession number AY661891.

References

  1. Desquesnes M, Davila AM (2002) Applications of PCR-based tools for detection and identification of animal trypanosomes: a review and perspectives. Vet Parasitol 11:213–231CrossRefGoogle Scholar
  2. Desquesnes M, McLaughlin G, Zoungrana A, Davila AM (2001) Detection and identification of Trypanosoma of African livestock through a single PCR based on internal transcribed spacer 1 of rDNA. Int J Parasitol 31:610–614Google Scholar
  3. Gibson WC, Stevens JR, Mwendia CMT, Makumi JM, Ngotho JM, Ndung’u JM (2001) Unraveling the phylogenetic relationships of African trypanosomes of suids. Parasitology 122:625–631CrossRefGoogle Scholar
  4. Hernandez P, Martin-Parras L, Martinez-Robles ML, Schvartzman JB (1993) Conserved features in the mode of replication of eukaryotic ribosomal RNA genes. EMBO J 12:1475–1485Google Scholar
  5. Lehane MJ, Msangi AR, Whitaker CJ, Lehane SM (2000) Grouping of trypanosomes species in mixed infections in Glossina pallidipes. Parasitology 120:583–592CrossRefGoogle Scholar
  6. Majiwa PAO, Thatti R, Moloo SK, Nyeko JHP, Otieno LH, Moloo S (1994) Detection of trypanosome infections in the saliva of tsetse flies and buffy coat samples from antigenic but aparasitaemic cattle. Parasitology 108:313–322Google Scholar
  7. Malele I, Craske L, Knight C, Ferris V, Njiru ZK, Hamilton P, Stella L, Lehane M, Gibson WC (2003) The use of specific and generic primers to identify trypanosome infections of wild tsetse flies in Tanzania by PCR. Infect Genet Evol 3:271–279CrossRefGoogle Scholar
  8. Masake RA, Majiwa PAO, Moloo SK, Makau JM, Njuguna JT, Maina M, Kabata J, Ole-MoiYoi, OK, Nantulya VM (1997) Sensitive and specific detection of Trypanosoma vivax using the polymerase chain reaction. Exp Parasitol 85:193–205CrossRefGoogle Scholar
  9. Masiga DK, Smyth AJ, Hayes P, Bromidge TJ, Gibson WC (1992) Sensitive detection of trypanosomes in tsetse flies by DNA amplification. Int J Parasitol 22:909–918CrossRefGoogle Scholar
  10. McLaughlin Gl, Ssenyonga SS, Nanteza E, Rubaire-Akiki, Wafula O, Hansen RD, Vodkin MH, Novak RJ, Gordon VR, Montenegro-James S, James M, Aviles H, Armijos R, Santrich C, Weigle K, Saravia N, Wozniak E, Gaye O, Mdachi R, Shapiro SZ, Chang KP, Kakoma I (1996) PCR based detection and typing of parasites. In: Zcel MA, Alkan MZ (eds) Parasitology for the 20th century. CAB International, Wallingford, pp 261–287Google Scholar
  11. Morlais I, Ravel S, Grebaut P, Dumas V, Cuny G (2001) New molecular marker for Trypanosoma (Duttonella) vivax identification. Acta Trop 80:207–213CrossRefGoogle Scholar
  12. Mugittu KN, Silayo RS, Majiwa PAO, Kimbita EK, Mutayoba BM, Maselle R (2000) Application of PCR and DNA probes in the characterization of trypanosomes in the blood of cattle in farms in Morogoro Tanzania. Vet Parasitol 94:177–189CrossRefGoogle Scholar
  13. Murray M, Murray PK, McIntyre IM (1977) An improved parasitological technique for the diagnosis of African trypanosomiasis. Trans R Soc Trop Med Hyg 71:325–326CrossRefGoogle Scholar
  14. Njiru ZK, Makumi JN, Okoth S, Ndungu JM, Gibson WC (2004) Identification of trypanosomes in Glossina pallidipes and G. longipennis in Kenya. Infect Genet Evol 4:29–35CrossRefGoogle Scholar
  15. Stevens J, Rambaut A (2001) Evolutionary rate differences in trypanosomes. Infect Genet Evol 1:143–150CrossRefGoogle Scholar
  16. Solano P, Michel JF, Lefrancois T, De La Rocque S, Sidibe I, Zoungrana A, Cuisance D (1999) Polymerase chain reaction as a diagnosis tool for detecting trypanosomes in naturally infected cattle in Burkina Faso. Vet Parasitol 30:95–103CrossRefGoogle Scholar
  17. Woo PTR (1970) The haematocrit centrifuge technique for the diagnosis of African trypanosomes. Acta Trop 27:384–386Google Scholar
  18. Wuyts N, Chokesajjawatee N, Panyim S (1994) A simplified and highly sensitive detection of Trypanosoma evansi by DNA amplification. S E Asian J Trop Med Public Health 25:266–271Google Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Z. K. Njiru
    • 1
  • C. C. Constantine
    • 1
  • S. Guya
    • 2
  • J. Crowther
    • 3
  • J. M. Kiragu
    • 2
  • R. C. A. Thompson
    • 1
  • A. M. R. Dávila
    • 4
  1. 1.Division of Veterinary and Biomedical Sciences, Western Australian Biomedical Research Institute (WABRI)Murdoch UniversityMurdochAustralia
  2. 2.Kenya Trypanosomiasis Research Institute (KETRI)KikuyuKenya
  3. 3.Joint FAO/IAEA DivisionInternational Atomic Energy Agency ViennaAustria
  4. 4.Departmento de Bioquimica e Biologia MolecularInstituto Oswaldo CruzFioCruzBrazil

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