TY - JOUR
T1 - Rapid identification of isometamidium-resistant stocks of Trypanosoma b. brucei by PCR-RFLP
AU - Afework, Yohannes
AU - Mäser, Pascal
AU - Etschmann, Benjamin
AU - Von Samson-Himmelstjerna, Georg
AU - Zessin, Karl Hans
AU - Clausen, Peter Henning
N1 - Funding Information:
Acknowledgements We acknowledge the Katholischer Akade-mischer 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.
PY - 2006/8
Y1 - 2006/8
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=33745748047&partnerID=8YFLogxK
U2 - 10.1007/s00436-006-0141-z
DO - 10.1007/s00436-006-0141-z
M3 - Article
C2 - 16541260
AN - SCOPUS:33745748047
SN - 0932-0113
VL - 99
SP - 253
EP - 261
JO - Parasitology Research
JF - Parasitology Research
IS - 3
ER -