Ribosomal DNA analysis of tsetse and non-tsetse transmitted Ethiopian Trypanosoma vivax strains in view of improved molecular diagnosis.

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.

Trypanosome infection in dromedary camels in Eastern Ethiopia: Prevalence, relative performance of diagnostic tools and host related risk factors.

A cross-sectional study was conducted in Chifra and Dewe districts of Afar region, Eastern Ethiopia, to determine the prevalence, agreement between diagnostic tests and host related risk factors of trypanosome infection in camel. An overall prevalence of 2%, 24.1%, 21.3%, 9.5% and 7.8% was recorded with respectively Giemsa stained thin blood smear, CATT/T. evansi, RoTat1.2 PCR, 18S PCR and ITS-1PCR in a cohort of 399 animals. Only one T. vivax infection was confirmed by TvPRAC PCR indicating T. evansi as the predominant species affecting camels in the study area. No single animal was positive when tested with T. evansi type B specific EVAB PCR. There was slight agreement between the CATT/T. evansi and the molecular tests. Among the PCR methods, RoTat 1.2 PCR yielded a significantly higher positivity rate compared to 18S PCR and ITS-1 PCR. There was no significant difference in the positivity rate observed in each gender of camels (p>0.05). The positivity rate was significantly higher in camels with poor body condition and in older animals when tested using the CATT/T.evansi or RoTat 1.2 PCR (p>0.05). Camels that tested positive with all tests had significantly lower PCV's (p<0.05). This study provides further evidence that T. evansi is endemic in the Afar region of Ethiopia. The latent class analysis indicated an estimate overall prevalence of 19% (95% CI: 13-28). Moreover, the model indicated low sensitivity of CATT/T. evansi (43%) and the PCR tests (39-53%) but higher specificity of the PCR tests (86-99%) and low specificity of CATT/T. evansi (80%). This study suggests that improved sensitivity and reliability of the tests would help diagnosis of trypanosomosis. Further studies are required to determine the prevalence of clinical disease and losses due to trypanosomosis.

A proline racemase based PCR for identification of Trypanosoma vivax in cattle blood.

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.