The use of specific and generic primers to identify trypanosome infections of wild tsetse flies in Tanzania by PCR

Abstract

The accurate identification of trypanosome species and subspecies remains a challenging task in the epidemiology of human and animal trypanosomiasis in tropical Africa. Currently, there are specific PCR tests to identify about 10 different species, subspecies or subgroups of African tsetse-transmitted trypanosomes. These PCR tests have been used here to identify trypanosomes in four species of tsetse (Glossina brevipalpis, G. pallidipes, G. swynnertoni, G. morsitans morsitans) from two areas of Tanzania. PCR using species-specific primers was performed on 1041 dissection-positive proboscides, giving an overall positive identification in 254 (24%). Of these, 61 proboscides (24%) contained two or more trypanosomes. The trypanosome with the greatest overall prevalence at both field sites was Trypanosoma simiae Tsavo, which was identified in a total of 118 infected tsetse proboscides (46%). At Pangani, T. godfreyi was found in G. pallidipes but not in G. brevipalpis, suggesting that these flies might have different susceptibility to this trypanosome or might have fed on a different range of hosts. A high proportion (about 75%) of trypanosome infections remained unidentified. To investigate the identity of these unidentified samples, we used primers complementary to the conserved regions of trypanosomal small subunit ribosomal RNA (ssu rRNA) genes to amplify variable segments of the gene. Amplified DNA fragments were cloned, sequenced and compared with ssu rRNA genes on database of known trypanosome species. In this way, we have tentatively identified two new trypanosomes: a trypanosome related to Trypanosoma vivax and a trypanosome related to T. godfreyi. The T. godfreyi-related trypanosome occurred frequently in the Tanzanian field samples and appears to be widespread. Molecular identification of these two new trypanosomes should now facilitate their isolation and full biological characterisation.