title: Ribosomal DNA analysis of tsetse and non-tsetse transmitted Ethiopian Trypanosoma vivax strains in view of improved molecular diagnosis.
abstract: Animal trypanosomosis caused by Trypanosoma vivax (T. vivax) is a devastating disease causing serious economic losses. Most molecular diagnostics for T. vivax infection target the ribosomal DNA locus (rDNA) but are challenged by the heterogeneity among T. vivax strains. In this study, we investigated the rDNA heterogeneity of Ethiopian T. vivax strains in relation to their presence in tsetse-infested and tsetse-free areas and its effect on molecular diagnosis. We sequenced the rDNA loci of six Ethiopian (three from tsetse-infested and three from tsetse-free areas) and one Nigerian T. vivax strain. We analysed the obtained sequences in silico for primer-mismatches of some commonly used diagnostic PCR assays and for GC content. With these data, we selected some rDNA diagnostic PCR assays for evaluation of their diagnostic accuracy. Furthermore we constructed two phylogenetic networks based on sequences within the smaller subunit (SSU) of 18S and within the 5.8S and internal transcribed spacer 2 (ITS2) to assess the relatedness of Ethiopian T. vivax strains to strains from other African countries and from South America. In silico analysis of the rDNA sequence showed important mismatches of some published diagnostic PCR primers and high GC content of T. vivax rDNA. The evaluation of selected diagnostic PCR assays with specimens from cattle under natural T. vivax challenge showed that this high GC content interferes with the diagnostic accuracy of PCR, especially in cases of mixed infections with T. congolense. Adding betain to the PCR reaction mixture can enhance the amplification of T. vivax rDNA but decreases the sensitivity for T. congolense and Trypanozoon. The networks illustrated that Ethiopian T. vivax strains are considerably heterogeneous and two strains (one from tsetse-infested and one from tsetse-free area) are more related to the West African and South American strains than to the East African strains. The rDNA locus sequence of six Ethiopian T. vivax strains showed important differences and higher GC content compared to other animal trypanosomes but could not be related to their origin from tsetse-infested or tsetse-free area. The high GC content of T. vivax DNA renders accurate diagnosis of all pathogenic animal trypanosomes with one single PCR problematic.
authors.affiliation: College of Veterinary Medicine and Agriculture, Addis Ababa University, P.O. Box 34, Bushoftu, Ethiopia; Department of Biomedical Sciences, Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerp, Belgium; Department of Biosystems, Faculty of Bioscience Engineering, KU Leuven, Kasteelpark Arenberg 30, B-3001 Leuven, Belgium. Electronic address: fikruregassa@yahoo.com.
authors.forename: Regassa
authors.initials: R
authors.lastname: Fikru
authors.affiliation: Department of Biomedical Sciences, Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerp, Belgium. Electronic address: imatetovici@itg.be.
authors.forename: Irina
authors.initials: I
authors.lastname: Matetovici
authors.affiliation: Department of Biomedical Sciences, Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerp, Belgium. Electronic address: stijnroge@gmail.com.
authors.forename: Stijn
authors.initials: S
authors.lastname: Rogé
authors.affiliation: College of Veterinary Medicine and Agriculture, Addis Ababa University, P.O. Box 34, Bushoftu, Ethiopia. Electronic address: merga.bekana@gmail.com.
authors.forename: Bekana
authors.initials: B
authors.lastname: Merga
authors.affiliation: Department of Biosystems, Faculty of Bioscience Engineering, KU Leuven, Kasteelpark Arenberg 30, B-3001 Leuven, Belgium. Electronic address: bruno.goddeeris@kuleuven.be.
authors.forename: Bruno Maria
authors.initials: BM
authors.lastname: Goddeeris
authors.affiliation: Department of Biomedical Sciences, Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerp, Belgium. Electronic address: pbuscher@itg.be.
authors.forename: Philippe
authors.initials: P
authors.lastname: Büscher
authors.affiliation: Department of Biomedical Sciences, Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerp, Belgium. Electronic address: nvanreet@itg.be.
authors.forename: Nick
authors.initials: N
authors.lastname: Van Reet
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000469:parasitology*; D004275:DNA, Ribosomal / Q000235:genetics; D005002:Ethiopia; D006239:Haplotypes; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D014345:Trypanosoma / Q000145:classification / Q000235:genetics*; D014352:Trypanosomiasis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D014370:Tsetse Flies / Q000469:parasitology
chemical_list: D004275:DNA, Ribosomal; D012337:RNA, Ribosomal, 18S
keywords: Diagnosis; Ethiopia; Non-tsetse transmitted; PCR; Ribosomal DNA; Sequencing; Trypanosoma vivax; Tsetse-transmitted