A study was conducted to develop a Trypanosoma vivax (T. vivax) specific PCR based on the T. vivax proline racemase (TvPRAC) gene. Forward and reverse primers were designed that bind at 764–783 bp and 983–1002 bp of the gene. To assess its specificity, TvPRAC PCR was conducted on DNA extracted from different haemotropic pathogens: T. vivax from Nigeria, Ethiopia and Venezuela, T. congolense Savannah type, T. brucei brucei, T. evansi, T. equiperdum, T. theileri, Theileria parva, Anaplasma marginale, Babesia bovis and Babesia bigemina and from bovine, goat, mouse, camel and human blood. The analytical sensitivity of the TvPRAC PCR was compared with that of the ITS-1 PCR and the 18S PCR-RFLP on a dilution series of T. vivax DNA in water. The diagnostic performance of the three PCRs was compared on 411 Ethiopian bovine blood specimens collected in a former study. TvPRAC PCR proved to be fully specific for T. vivax, irrespective of its geographical origin. Its analytical sensitivity was lower than that of ITS-1 PCR. On these bovine specimens, TvPRAC PCR detected 8.3% T. vivax infections while ITS-1 PCR and 18S PCR-RFLP detected respectively 22.6 and 6.1% T. vivax infections. The study demonstrates that a proline racemase based PCR could be used, preferably in combination with ITS-1 PCR, as a species-specific diagnostic test for T. vivax infections worldwide.
References
[1]
Abebe G, Jobre Y (1996) Trypanosomiasis: a threat to cattle production in Ethiopia. Rev Méd Vét 147: 897–902.
[2]
Davila AM, Silva RA (2000) Animal trypanosomiasis in South America. Current status, partnership, and information technology. Ann N Y Acad Sci 916: 199–212.
[3]
Oluwafemi A, Ilemobade A, Laseined E (2007) The impact of African animal trypanosomosis and tsetse on the livelihood and wellbeing of cattle and their owners in the BICOT study area of Nigeria. Sc Res Essays 2: 380–383.
[4]
Desquesnes M, McLaughlin G, Zoungrana A, Davila AM (2001) Detection and identification of Trypanosoma of African livestock through a single PCR based on internal transcribed spacer 1 of rDNA. Int J Parasitol 31: 610–614.
[5]
Thumbi SM, Jung'a JO, Mosi RO, McOdimba FA (2010) Spatial distribution of African animal trypanosomiasis in Suba and Teso districts in Western Kenya. BMC Res Notes 3: 6.
[6]
Masiga DK, Smyth AJ, Hayes P, Bromidge TJ, Gibson WC (1992) Sensitive detection of trypanosomes in tsetse flies by DNA amplification. Int J Parasitol 22: 909–918.
[7]
Adams ER, Malele II, Msangi AR, Gibson WC (2006) Trypanosome identification in wild tsetse populations in Tanzania using generic primers to amplify the ribosomal RNA ITS-1 region. Acta Trop 100: 103–109.
[8]
Njiru ZK, Constantine CC, Guya S, Crowther J, Kiragu JM, et al. (2005) The use of ITS1 rDNA PCR in detecting pathogenic African trypanosomes. Parasitol Res 95: 186–192.
[9]
Geysen D, Delespaux V, Geerts S (2003) PCR-RFLP using Ssu-rDNA amplification as an easy method for species-specific diagnosis of Trypanosoma species in cattle. Vet Parasitol 110: 171–180.
[10]
Hamilton PB, Adams ER, Malele II, Gibson WC (2008) A novel, high-throughput technique for species identification reveals a new species of tsetse-transmitted trypanosome related to the Trypanosoma brucei subgenus, Trypanozoon. Infect Genet Evol 8: 26–33.
[11]
Ventura RM, Paiva F, Silva RAMS, Takeda GF, Buck GA, et al. (2001) Trypanosoma vivax: characterization of the spliced-leader gene of a Brazilian stock and species-specific detection by PCR amplification of an intergenic spacer sequence. Exp Parasitol 99: 37–48.
[12]
Cortez AP, Rodrigues AC, Garcia HA, Neves L, Batista JS, et al. (2009) Cathepsin L-like genes of Trypanosoma vivax from Africa and South America—characterization, relationships and diagnostic implications. Mol Cell Probes 23: 44–51.
[13]
Nakayima J, Nakao R, Alhassan A, Mahama C, Afakye K, et al. (2012) Molecular epidemiological studies on animal trypanosomiases in Ghana. Parasit Vectors 5: e217.
[14]
Dickin SK, Gibson WC (1989) Hybridisation with a repetitive DNA probe reveals the presence of small chromosomes in Trypanosoma vivax. Mol Biochem Parasitol 33: 135–142.
[15]
González LE, Garcia JA, Nú？ez C, Perrone TM, González-Baradat B, et al. (2005) Trypanosoma vivax: a novel method for purification from experimentally infected sheep blood. Exp Parasitol 111: 126–129.
[16]
Silva RAMS, Egüez A, Morales G, Eulert E, Montenegro A, et al. (1998) Bovine trypanosomiasis in Bolivian and Brazilian lowlands. Mem Inst Oswaldo Cruz 93: 29–32.
[17]
Hoare CA (1972) The trypanosomes of mammals. Oxford: Blackwell Scientific Publications. 749 p.
[18]
Masake RA, Majiwa PAO, Moloo SK, Makau JM, Njuguna JT, et al. (1997) Sensitive and specific detection of Trypanosoma vivax using the polymerase chain reaction. Exp Parasitol 85: 193–205.
[19]
Gibson W (2007) Resolution of the species problem in African trypanosomes. Int J Parasitol 37: 829–838.
[20]
Steverding D (2008) The history of African trypanosomiasis. Parasit Vectors 1: 3.
[21]
Fikru R, Goddeeris BM, Delespaux V, Moti Y, Tadesse A, et al. (2012) Widespread occurrence of Trypanosoma vivax in bovines of tsetse- as well as non-tsetse-infested regions of Ethiopia: a reason for concern? Vet Parasitol 190: 355–361.
[22]
Deborggraeve S, Lejon V, Ali Ekangu R, Mumba Ngoyi D, Pyana PP, et al. (2011) Diagnostic accuracy of PCR in gambiense sleeping sickness diagnosis, staging and post-treatment follow-up: a 2-year longitudinal study. PLoS Negl Trop Dis 5: e972.
[23]
Morlais I, Ravel S, Grébaut P, Dumas V, Cuny G (2001) New molecular marker for Trypanosoma (Duttonella) vivax identification. Acta Trop 80: 207–213.
[24]
Chamond N, Cosson A, Coatnoan N, Minoprio P (2009) Proline racemases are conserved mitogens: characterization of a Trypanosoma vivax proline racemase. Mol Biochem Parasitol 165: 170–179.
[25]
Viera AJ, Garrett JM (2005) Understanding interobserver agreement: the kappa statistic. Family Medicine 37: 360–363.
[26]
Leeflang P, Buys J, Blotkamp C (1976) Studies on Trypanosoma vivax: infectivity and serial maintenance of natural bovine isolates in mice. J Parasitol 6: 413–417.
[27]
Delespaux V, Geysen D, Van den Bossche P, Geerts S (2008) Molecular tools for the rapid detection of drug resistance in animal trypanosomes. Trends Parasitology 24: 236–242.
[28]
Van Meirvenne N, Janssens PG, Magnus E (1975) Antigenic variation in syringe passaged populations of Trypanosoma (Trypanozoon) brucei. I. Rationalization of the experimental approach. Ann Soc Belg Méd Trop 55: 1–23.
[29]
Bajyana Songa E, Hamers R (1988) A card agglutination test (CATT) for veterinary use based on an early VAT RoTat 1/2 of Trypanosoma evansi. Ann Soc Belg Méd Trop 68: 233–240.
[30]
Barrowman PR (1976) Observations in the transmission, immunology, clinical signs and chemotherapy of dourine (Trypanosoma equiperdum infection) in horses, with special reference to cerebrospinal fluid. J Vet Res 43: 55–66.
[31]
Verloo D, Brandt J, Van Meirvenne N, Büscher P (2000) Comparative in vitro isolation of Trypanosoma theileri from cattle in Belgium. Vet Parasitol 89: 129–132.
[32]
Geysen D (2000) The application of molecular techniques to analyse diversity in Theileria parva populations in Zambia [dissertation]. Brunel University, UK. 268 p.
