SSCP analysis of the P2 purine transporter TcoAT1 gene of Trypanosoma congolense leads to a simple PCR-RFLP test allowing the rapid identification of diminazene resistant stocks.
Analyses were made on a Trypanosoma congolense contig coding a putative P2-like nucleoside transporter (the contig was named in this study TcoAT1). The sequence includes a start and stop codon and presents a high similarity with the gene TbAT1 of T. brucei (Smallest Sum Probability 2.8e-136). To investigate a possible link between point mutations and diminazene aceturate (DA) resistance in mice, the TcoAT1 putative genes of 26 T. congolense strains, characterised for DA sensitivity in the single dose mouse test, were screened by means of the Single Strand Conformation Polymorphism technique (SSCP). Results showed that the SSCP profiles of 23 out of 26 (88.5%) T. congolense strains were confirmed by the sensitivity test in mice with the commonly accepted criterion for sensitivity to diminazene being a CD80 of 20mg/kg in the mouse test. The remaining T. congolense strains showed a resistant SSCP profile and relapsed in mice after treatment at doses lower than 20mg/kg indicating that the SSCP is more sensitive than the single dose mouse test for the detection of resistance to diminazene. However, none of the strains used in this study showed a sensitive SSCP profile while they were resistant in the single dose mouse test. The sequencing of the TcoAT1 gene of two sensitive, two intermediate and two resistant strains allowed the set up of a PCR-RFLP test for the discrimination between sensitive and resistant strains confirming the SSCP results for the 26 strains of this study.
Delespaux
V
V
Animal Health Department, Institute of Tropical Medicine (Antwerp), Nationalestraat 155, B-2000 Antwerp, Belgium. vdelespaux@itg.be
Chitanga
S
S
Geysen
D
D
Goethals
A
A
van den Bossche
P
P
Geerts
S
S
D000818:Animals; D002417:Cattle; D004133:Diminazene / Q000031:analogs & derivatives / Q000494:pharmacology; D004351:Drug Resistance / Q000235:genetics; D051379:Mice; D033703:Nucleoside Transport Proteins / Q000235:genetics* / Q000378:metabolism; D021261:Parasitic Sensitivity Tests / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D018807:Polymorphism, Single-Stranded Conformational; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014344:Trypanocidal Agents / Q000494:pharmacology; D014348:Trypanosoma congolense / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism
D033703:Nucleoside Transport Proteins; C486446:P2 transporter, Trypanosoma brucei; D015800:Protozoan Proteins; D014344:Trypanocidal Agents; C003915:diminazene aceturate; D004133:Diminazene