title: Evaluation of cytochrome b as a sensitive target for PCR based detection of T. annulata carrier animals.
abstract: Bovine tropical theileriosis, caused by the tick-borne protozoan Theileria annulata, imposes a serious constraint upon breed improvement programmes and livestock production in tropical and sub-tropical regions of the world. Animals that recover from primary infection serve as carriers and play a critical role in the epidemiology of the disease, acting as reservoirs of infection. However, conclusive identification of carrier animals can be problematic. This study describes assessment of candidate target genes for PCR assay-based detection of T. annulata infected carrier animals. Following in silico screening and rejection of three major multi-copy gene families, an assay based on PCR amplification of a 312 bp segment of the T. annulata gene for cytochrome b (Cytob1 assay) was established. Sensitivity was evaluated using serial dilutions of blood obtained from experimentally infected calves, while specificity was confirmed by testing DNA representing twelve different T. annulata stocks and other Theileria and Babesia species. Direct comparison with other target genes and published data indicated that Cytob1 PCR-based assays provide the greatest level of sensitivity, combined with a high level of specificity and the ability to detect different T. annulata genotypes. It can be concluded that the cytochrome b gene is the optimal target for PCR amplification and its incorporation in a Reverse Line Blot Assay offers the most sensitive method yet devised to detect the parasite in carrier animals. The use of this assay will increase the accuracy of epidemiological studies aimed at improving disease control in endemically unstable regions.
authors.affiliation: Faculty of Veterinary Medicine, Department of Parasitology, Adnan Menderes University, Işikli Mevki, 09016 Aydin, Turkey. huseyin bilgic@yahoo.com
authors.forename: Huseyin B
authors.initials: HB
authors.lastname: Bilgic
authors.forename: Tülin
authors.initials: T
authors.lastname: Karagenç
authors.forename: Brian
authors.initials: B
authors.lastname: Shiels
authors.forename: Andy
authors.initials: A
authors.lastname: Tait
authors.forename: Hasan
authors.initials: H
authors.lastname: Eren
authors.forename: William
authors.initials: W
authors.lastname: Weir
mesh_terms: D000818:Animals; D015415:Biomarkers; D002353:Carrier State / Q000175:diagnosis*; D002417:Cattle; D003001:Cloning, Molecular; D045303:Cytochromes b / Q000235:genetics / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D008297:Male; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D016796:Theileria annulata / Q000302:isolation & purification*; D013801:Theileriasis / Q000175:diagnosis* / Q000453:epidemiology
chemical_list: D015415:Biomarkers; D016054:DNA, Protozoan; D045303:Cytochromes b