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Rapid identification of isometamidium-resistant stocks of Trypanosoma b. brucei by PCR–RFLP

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Abstract

Analyses were made on the adenosine transporter-1 gene in Trypanosoma brucei (TbAT1), encoding a P2-like nucleoside transporter, from T. brucei brucei field stocks to investigate a possible link between the presence of mutations in this gene and isometamidium resistance. We have analysed the gene from 11 isometamidium-sensitive field stocks isolated from cattle in Uganda, two sensitive reference clones and two resistant reference clones. A sequence alignment showed that the isometamidium-sensitive T. b. brucei contained the wild-type sequence patterns. In contrast, the isometamidium-resistant T. b. brucei stocks showed the mutant-type sequence patterns with six point mutations that had previously been reported in a laboratory-derived arsenical-resistant T. brucei strain. To analyse the restriction fragment length polymorphism pattern of a fragment of TbAT1 (nucleotides 430–1108), the 677-bp polymerase chain reaction products from eight of the isometamidium-sensitive and two of the isometamidium-resistant T. b. brucei were subjected to digestion with Sfa NI. The results revealed two different banding patterns: the digest resulted in fragment sizes of 566 and 111 bp in the case of TbAT1 from isometamidium-sensitive stocks, whereas it produced fragment sizes of 435 and 242 bp in the case of TbAT1 from isometamidium-resistant stocks. Thus, the isometamidium-sensitive and isometamidium-resistant T. b. brucei could be successfully distinguished by digestion with the restriction endonuclease Sfa NI.

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References

  • Afework Y (2005) Parasitological and molecular characterisation of isometamidium-sensitive and -resistant Trypanosoma congolense and T. brucei brucei isolates from cattle in East and West Africa. Ph.D. thesis, Faculty of Veterinary Medicine, Freie Universität Berlin, pp 149

  • Afework Y, Clausen P-H, Abebe G, Tilahun G, Mehlitz D (2000) Multiple-drug resistant Trypanosoma congolense populations in village cattle of Metekel district, northwest Ethiopia. Acta Trop 76:231–238

    Article  Google Scholar 

  • Assefa E, Abebe G (2001) Drug-resistant Trypanosoma congolense in naturally infected donkeys in north Omo Zone, southern Ethiopia. Vet Parasitol 99:261–271

    Article  PubMed  CAS  Google Scholar 

  • Brun R, Jenni L, Tanner M, Schonenberger M, Schell KF (1979) Cultivation of vertebrate infective forms derived from metacyclic forms of pleomorphic Trypanosoma brucei stocks. Short communication. Acta Trop 36:387–390

    PubMed  CAS  Google Scholar 

  • Carter NS, Fairlamb AH (1993) Arsenical-resistant trypanosomes lack an unusual adenosine transporter. Nature (Lond) 361:173–176

    Article  CAS  Google Scholar 

  • Carter NS, Berger BJ, Fairlamb AH (1995) Uptake of diamidine drugs by the P2 nucleoside transporter in melarsen-sensitive and -resistant Trypanosoma brucei brucei. J Biol Chem 270:28153–28157

    Article  PubMed  CAS  Google Scholar 

  • Chitambo H, Arakawa A (1991) Therapeutic effect of Berenil and Samorin in mice infected with four trypanosome populations isolated from Zambian cattle. Vet Parasitol 39:43–52

    Article  PubMed  CAS  Google Scholar 

  • Clausen P-H, Sidibe I, Kabore I, Bauer B (1992) Development of multiple drug resistance of Trypanosoma congolense in Zebu cattle under high natural tsetse fly challenge in the pastoral zone of Samorogouan, Burkina Faso. Acta Trop 51:229–236

    Article  PubMed  CAS  Google Scholar 

  • Codjia V, Mulatu W, Majiwa PA, Leak SG, Rowlands GJ, Authie E, d’Ieteren GD, Peregrine AS (1993) Epidemiology of bovine trypanosomiasis in the Ghibe valley, southwest Ethiopia. 3. Occurrence of populations of Trypanosoma congolense resistant to diminazene, isometamidium and homidium. Acta Trop 53:151–163

    Article  PubMed  CAS  Google Scholar 

  • De Koning HP (2001) Uptake of pentamidine in Trypanosoma brucei brucei is mediated by three distinct transporters: implications for cross-resistance with arsenicals. Mol Pharmacol 59:586–592

    PubMed  Google Scholar 

  • De Koning HP, Anderson LF, Stewart M, Burchmore RJ, Wallace LJ, Barrett MP (2004) The trypanocide diminazene aceturate is accumulated predominantly through the TbAT1 purine transporter: additional insight on diamidine resistance in African trypanosomes. Antimicrob Agents Chemother 48:1515–1519

    Article  PubMed  CAS  Google Scholar 

  • Delespaux V, Geysen D, Majiwa PA, Geerts S (2005) Identification of a genetic marker for isometamidium chloride resistance in Trypanosoma congolense. Int J Parasitol 35:235–243

    Article  PubMed  CAS  Google Scholar 

  • Donald AD (1994) Parasite, animal production and sustainable development. Vet Parasitol 54:27–47

    Article  PubMed  CAS  Google Scholar 

  • Geerts S, Holmes PH (1998) Drug management and parasite resistance in bovine trypanosomiasis in Africa. PAAT technical and scientific series 1. Food and Agriculture Organization of the United Nations, Rome

    Google Scholar 

  • Geerts S, Holmes PH, Eisler MC, Diall O (2001) African bovine trypanosomiasis: the problem of drug resistance. Trends Parasitol 17:25–28

    Article  PubMed  CAS  Google Scholar 

  • Hassan HF, Coombs GH (1988) Purine and pyramidine metabolism in parasitic protozoa. FEMS Microbiol Rev 4:47–83

    PubMed  CAS  Google Scholar 

  • Hawking F (1976a) The resistance to human plasma of Trypanosoma brucei, T. rhodesiense and T. gambiense. I. Analysis of composition of trypanosome strains. Trans R Soc Trop Med Hyg 70:504–512

    Article  PubMed  CAS  Google Scholar 

  • Hawking F (1976b) The resistance to human plasma of Trypanosoma brucei, T. rhodesiense and T. gambiense. II. Survey of strains from East Africa and Nigeria. Trans R Soc Trop Med Hyg 70:513–520

    Article  PubMed  CAS  Google Scholar 

  • Hide G, Tilley A, Welburn SC, Maudlin I, Tait A (2000) Trypanosoma brucei: identification of trypanosomes with genotypic similarity to human infective isolates in tsetse isolated from a region free of human sleeping sickness. Exp Parasitol 96:67–74

    Article  PubMed  CAS  Google Scholar 

  • Higuchi R (1989) Simple and rapid preparation of samples for PCR. In: Ehrlich HA (ed) PCR technology. Stockton, New York, pp 31–38

    Google Scholar 

  • Joshua RA (1988) Drug resistance in recent isolates of Trypanosoma brucei and Trypanosoma congolense. Rev Élev Méd Vét Pays Trop 41:359–364

    PubMed  CAS  Google Scholar 

  • Kalu AU (1995) Sensitivity of animal-derived Trypanozoon stocks from sleeping sickness endemic foci of Nigeria to trypanocides and human plasma. Rev Élev Méd Vét Pays Trop 48:139–144

    PubMed  CAS  Google Scholar 

  • Kinabo LD, Bogan JA (1988) The pharmacology of isometamidium. J Vet Pharmacol Ther 11:233–245

    Article  PubMed  CAS  Google Scholar 

  • Lanham SM, Godfrey DG (1970) Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Exp Parasitol 28:521–534

    Article  PubMed  CAS  Google Scholar 

  • Leach TM, Roberts CJ (1981) Present status of chemotherapy and chemoprophylaxis of animal trypanosomiasis in the Eastern hemisphere. Pharmacol Ther 13:91–147

    Article  PubMed  CAS  Google Scholar 

  • Mäser P, Sütterlin C, Kralli A, Kaminsky R (1999) A nucleoside transporter from Trypanosoma brucei involved in drug resistance. Science 285(5425):242–244

    Article  PubMed  Google Scholar 

  • Matovu E, Iten M, Enyaru JC, Schmid C, Lubega GW, Brun R, Kaminsky R (1997) Susceptibility of Ugandan Trypanosoma brucei rhodesiense isolated from man and animal reservoirs to diminazene, isometamidium and melarsoprol. Trop Med Int Health 2:13–18

    Article  PubMed  CAS  Google Scholar 

  • Matovu E, Geiser F, Schneider V, Mäser P, Enyaru JCK, Kaminsky R, Gallati S, Seebeck T (2001a) Genetic variants of the TbAT1 adenosine transporter from African trypanosomes in relapse infections following melarsoprol therapy. Mol Biochem Parasitol 117:73–81

    Article  PubMed  CAS  Google Scholar 

  • Matovu E, Seebeck T, Enyaru JC, Kaminsky R (2001b) Drug resistance in Trypanosoma brucei ssp., the causative agents of sleeping sickness in man and nagana in cattle. Microbes Infect 3:763–770

    Article  PubMed  CAS  Google Scholar 

  • Matovu E, Stewart ML, Geiser F, Brun R, Mäser P, Wallace LJM, Burchmore RJ, Enyaru JCK, Barrett MP, Kaminsky R, Seebeck T, de Koning HP (2003) Mechanisms of arsenical and diamidine uptake and resistance in Trypanosoma brucei. Eukaryot Cell 2:1003–1008

    Article  PubMed  CAS  Google Scholar 

  • Mehlitz D, Zillmann U, Scott CM, Godfrey DG (1982) Epidemiological studies on the animal reservoir of Gambiense sleeping sickness. Part III. Characterisation of Trypanozoon stocks by isoenzymes and sensitivity to human serum. Tropenmed Parasitol 33:113–118

    PubMed  CAS  Google Scholar 

  • Miller EN, Turner MJ (1981) Analysis of antigenic types appearing in first relapse populations of clones of Trypanosoma brucei. Parasitology 82:63–80

    PubMed  CAS  Google Scholar 

  • Nicholas KB, Nicholas HB Jr (1997) GeneDoc: a tool for editing and annotating multiple sequence alignments. Multiple sequence alignment editor and shading utility version 2.6.002 (copyright 2000)

  • Peregrine AS (1994) Chemotherapy and delivery systems: haemoparasites. Vet Parasitol 54:223–248

    Article  PubMed  CAS  Google Scholar 

  • Pötzsch CJ (1999) Krankheits- und Produktionsfaktoren in peri-urbanen Milchrinderherden Ugandas. Dissertation, Freie Universität Berlin, journal no. 2300, p 149

  • Radwanska M, Chamekh M, Vanhamme L, Claes F, Magez S, Magnus E, de Baetselier P, Buscher P, Pays E (2002) The serum resistance-associated gene as a diagnostic tool for the detection of Trypanosoma brucei rhodesiense. Am J Trop Med Hyg 67:684–690

    PubMed  CAS  Google Scholar 

  • Ross CA, Barns AM (1996) Alteration to one of three adenosine transporters is associated with resistance to Cymelarsan in Trypanosoma evansi. Parasitol Res 82:183–188

    Article  PubMed  CAS  Google Scholar 

  • Scheer A (2001) In vitro-Untersuchungen zur Medikamentenempfindlichkeit (Isometamidiumchlorid) von Trypanosomen (Trypanosoma brucei) aus Milchviehherden Ugandas. Dissertation, Freie Universität Berlin, journal no. 2506, p 132

  • Sutherland IA, Peregrine AS, Lonsdale-Eccles JD, Holmes PH (1991) Reduced accumulation of isometamidium by drug-resistant Trypanosoma congolense. Parasitology 103:245–251

    Article  PubMed  Google Scholar 

  • Sutherland IA, Mounsey A, Eisler M, Holmes PH (1992) Kinetic modelling of isometamidum chloride (Samorin) uptake by Trypanosoma congolense. Parasitology 105:91–95

    PubMed  CAS  Google Scholar 

  • Wellde BT, Reardon MJ, Chumo DA, Kovatch RM, Waema D, Wykoff DE, Mwangi J, Boyce WL, Williams JS (1989) Cerebral trypanosomiasis in naturally-infected cattle in the Lambwe Valley, South Nyanza, Kenya. Ann Trop Med Parasitol 83:151–160

    PubMed  Google Scholar 

  • Zweygarth E, Röttcher D (1989) Efficacy of experimental trypanocidal compounds against a multiple drug-resistant Trypanosoma brucei brucei stock in mice. Parasitol Res 75:178–182

    Article  PubMed  CAS  Google Scholar 

  • Zweygarth E, Kaminsky R (1991) Evaluation of d-alpha-difluoromethylornithin against susceptible and drug-resistant Trypanosoma brucei brucei. Acta Trop 48:223–232

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We acknowledge the Katholischer Akademischer Ausländer-Dienst (KAAD), Bonn for the financial support we received to undertake this study. We thank Delia Grace for constructive comments on the manuscript. We declare that the animal experiments of this study comply with the current laws in Germany.

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Authors and Affiliations

  1. Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Königsweg 67, 14163, Berlin, Germany

    Yohannes Afework, Karl-Hans Zessin & Peter-Henning Clausen

  2. Institute of Cell Biology, Baltzerstrasse 4, 3012, Bern, Switzerland

    Pascal Mäser

  3. Institute of Veterinary Physiology, Freie Universität Berlin, Oertzenweg 19 b, 14163, Berlin, Germany

    Benjamin Etschmann

  4. Hannover School of Veterinary Medicine, Institute for Parasitology, Bünteweg 17, 30559, Hannover, Germany

    Georg von Samson-Himmelstjerna

Authors
  1. Yohannes Afework
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  2. Pascal Mäser
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  3. Benjamin Etschmann
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  4. Georg von Samson-Himmelstjerna
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  5. Karl-Hans Zessin
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  6. Peter-Henning Clausen
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Correspondence to Peter-Henning Clausen.

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Afework, Y., Mäser, P., Etschmann, B. et al. Rapid identification of isometamidium-resistant stocks of Trypanosoma b. brucei by PCR–RFLP. Parasitol Res 99, 253–261 (2006). https://doi.org/10.1007/s00436-006-0141-z

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  • DOI: https://doi.org/10.1007/s00436-006-0141-z

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