Trypanosome diversity in wildlife species from the serengeti and Luangwa Valley ecosystems

Abstract

Background: The importance of wildlife as reservoirs of African trypanosomes pathogenic to man and livestock is well recognised. While new species of trypanosomes and their variants have been identified in tsetse populations, our knowledge of trypanosome species that are circulating in wildlife populations and their genetic diversity is limited.

Methodology/principal findings: molecular phylogenetic methods were used to examine the genetic diversity and species composition of trypanosomes circulating in wildlife from two ecosystems that exhibit high host species diversity: the Serengeti in Tanzania and the Luangwa Valley in Zambia. Phylogenetic relationships were assessed by alignment of partial 18S, 5.8S and 28S trypanosomal nuclear ribosomal DNA array sequences within the Trypanosomatidae and using ITS1, 5.8S and ITS2 for more detailed analysis of the T. vivax clade. In addition to Trypanosoma brucei, T. congolense, T. simiae, T. simiae (Tsavo), T. godfreyi and T. theileri, three variants of T. vivax were identified from three different wildlife species within one ecosystem, including sequences from trypanosomes from a giraffe and a waterbuck that differed from all published sequences and from each other, and did not amplify with conventional primers for T. vivax.

Conclusions/significance: Wildlife carries a wide range of trypanosome species. The failure of the diverse T. vivax in this study to amplify with conventional primers suggests that T. vivax may have been under-diagnosed in Tanzania. Since conventional species-specific primers may not amplify all trypanosomes of interest, the use of ITS PCR primers followed by sequencing is a valuable approach to investigate diversity of trypanosome infections in wildlife; amplification of sequences outside the T. brucei clade raises concerns regarding ITS primer specificity for wildlife samples if sequence confirmation is not also undertaken.

Figure 1. Neighbour-joining tree based on partial 18S, 5.8S and partial 28S trypanosomatid sequences.

Bodo caudatus was included as an outgroup. Bootstrap values are shown where support is >70%. Sequences generated in this study are shown in blue (identified sequences in dark blue, unidentified sequences in light blue), and labelled with sample identity; pathogen species (UnK if unknown); host species; Genbank ID. Other sequences were retrieved from Genbank and are shown in black, and are labelled with pathogen species and Genbank ID. T. brucei clade indicated in grey box.

Figure 2. Unrooted neighbour-joining dendrogram of ITS1, 5.8S and ITS2 sequences for Trypanosoma vivax.

Bootstrap values are shown at nodes with >70% support. Sequences generated in this study shown in black; other sequences retrieved from Genbank and listed in Table 3. Sequence locations are shown by colour: Tanzania (this study, black); Kenya (blue); Mozambique (green); Nigeria (purple); South America (red). Host species from which sequence was amplified are indicated. Accession numbers for reference sequences are: IL3905 cl8, Genbank ID:DQ316040; IL3905 cl4Ro, DQ316043; IL3905 cl4, DQ316039; IL3905 cl3Ro, DQ316042; IL3905 cl2Ro, DQ316041; IL3905 cl5Ro, DQ316044; IL3905 cl2 DQ316037; IL3905 cl3, DQ316038; TviBrMi cl4, DQ316048; TviBrPo cl13, DQ316049; TviBrCa cl2, DQ316045; Y485, U22316; TviBrMi cl2, DQ316047; TviVeMe cl1, DQ316051; TviVeMe cl12, DQ316052; TviBrCa cl13, DQ316046; TviBrPo cl6, DQ316050; TviMzNy cl5, EU482080; TviMzNy cl2, EU482079; TviMzNy cl8, EU482082; TviMzNy cl1, EU482078; TviMzNy cl6, EU482081.