title: Development and evaluation of an ITS1 "Touchdown" PCR for assessment of drug efficacy against animal African trypanosomosis.
abstract: Animal African trypanosomoses (AAT) are caused by flagellated protozoa of the Trypanosoma genus and contribute to considerable losses in animal production in Africa, Latin America and South East Asia. Trypanosoma congolense is considered the economically most important species. Drug resistant T. congolense strains present a threat to the control of AAT and have triggered research into discovery of novel trypanocides. In vivo assessment of trypanocidal efficacy relies on monitoring of treated animals with microscopic parasite detection methods. Since these methods have poor sensitivity, follow-up for up to 100 days after treatment is recommended to increase the chance of detecting recurrent parasitaemia waves. Molecular techniques are more amendable to high throughput processing and are generally more sensitive than microscopic detection, thus bearing the potential of shortening the 100-day follow up period. The study presents a "Touchdown" PCR targeting the internal transcribed spacer 1 of the ribosomal DNA (ITS1 TD PCR) that enables detection and discrimination of different Trypanosoma taxa in a single run due to variations in PCR product sizes. The assay achieves analytical sensitivity of 10 parasites per ml of blood for detection of T. congolense savannah type and T. brucei, and 100 parasites per ml of blood for detection of T. vivax in infected mouse blood. The ITS1 TD PCR was evaluated on cattle experimentally infected with T. congolense during an investigational new veterinary trypanocide drug efficacy study. ITS1 TD PCR demonstrated comparable performance to microscopy in verifying trypanocide treatment success, in which parasite DNA became undetectable in cured animals within two days post-treatment. ITS1 TD PCR detected parasite recrudescence three days earlier than microscopy and had a higher positivity rate than microscopy (84.85% versus 57.58%) in 66 specimens of relapsing animals collected after treatments. Therefore, ITS1 TD PCR provides a useful tool in assessment of drug efficacy against T. congolense infection in cattle. As the assay bears the potential for detection of mixed infections, it may be applicable for drug efficacy studies and diagnostic discrimination of T. vivax and T. congolense against other pathogenic trypanosomes, including T. brucei, T. evansi and T. equiperdum.
authors.affiliation: Global Alliance for Livestock Veterinary Medicines (GALVmed), Doherty Building, Pentlands Science Park, Bush Loan, Edinburgh EH26 0PZ, UK; Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium.
authors.forename: Thao
authors.initials: T
authors.lastname: Tran
authors.affiliation: Global Alliance for Livestock Veterinary Medicines (GALVmed), Doherty Building, Pentlands Science Park, Bush Loan, Edinburgh EH26 0PZ, UK.
authors.forename: Grant
authors.initials: G
authors.lastname: Napier
authors.affiliation: Global Alliance for Livestock Veterinary Medicines (GALVmed), Doherty Building, Pentlands Science Park, Bush Loan, Edinburgh EH26 0PZ, UK.
authors.forename: Tim
authors.initials: T
authors.lastname: Rowan
authors.affiliation: ClinVet International (Pty) Ltd., Uitzigweg, Bainsvlei, Bloemfontein, South Africa.
authors.forename: Claudia
authors.initials: C
authors.lastname: Cordel
authors.affiliation: ClinVet International (Pty) Ltd., Uitzigweg, Bainsvlei, Bloemfontein, South Africa.
authors.forename: Michel
authors.initials: M
authors.lastname: Labuschagne
authors.affiliation: Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium.
authors.forename: Vincent
authors.initials: V
authors.lastname: Delespaux
authors.affiliation: Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium.
authors.forename: Nick
authors.initials: N
authors.lastname: Van Reet
authors.affiliation: ClinVet International (Pty) Ltd., Uitzigweg, Bainsvlei, Bloemfontein, South Africa.
authors.forename: Heidi
authors.initials: H
authors.lastname: Erasmus
authors.affiliation: ClinVet International (Pty) Ltd., Uitzigweg, Bainsvlei, Bloemfontein, South Africa.
authors.forename: Annesca
authors.initials: A
authors.lastname: Joubert
authors.affiliation: Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium. Electronic address: pbuscher@itg.be.
authors.forename: Philippe
authors.initials: P
authors.lastname: Büscher
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000188:drug therapy; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D004351:Drug Resistance; D051379:Mice; D016133:Polymerase Chain Reaction / Q000592:standards / Q000662:veterinary*; D012680:Sensitivity and Specificity; D014344:Trypanocidal Agents / Q000592:standards* / Q000627:therapeutic use; D014353:Trypanosomiasis, African / Q000175:diagnosis / Q000188:drug therapy / Q000662:veterinary*
chemical_list: D021903:DNA, Ribosomal Spacer; D014344:Trypanocidal Agents
keywords: Cattle; ITS1; Touchdown PCR; Trypanosoma congolense; Trypanosoma vivax; Trypanozoon