The taxonomy of ruminant Trypanosoma theileri and its relatives (Kinetoplastida: Trypanosomatidae) is controversial, with recent phylogenetic studies segregating T. theileri in cattle and other ruminants worldwide into two major genetic lineages (the TthI and TthII clades) based on genetic markers. In the present study, T. theileri-like trypanosomes isolated from Honshu sika deer (Cervus nippon) in the western Japan (YMG isolate) were genetically characterized using a number of genetic markers. Sika deer trypanosomes of the YMG isolate were genetically different from the Trypanosoma sp. TSD1 isolate previously recorded from Hokkaido sika deer in northern Japan, with the former trypanosome isolate being genetically closer to European cervid trypanosomes and the bovine T. theileri TthII lineage. In contrast, the latter isolate exhibited greater relatedness to North American cervid trypanosomes and the bovine T. theileri TthI lineage, although a clear genetic distinction between these was apparent. Furthermore, trypanosomes in Honshu sika deer from the central part of Japan harboured additional genetic diversity and were closer to either TSD1 or YMG isolates, while distinct from known T. theileri-related genotypes. Importantly, cervids and wild ruminants worldwide might harbour divergent descendants of a T. theileri ancestor, which exhibit rigid host specificity to either bovines or cervid species.

We propose a taxonomic revision of the dixenous trypanosomatids currently classified as Endotrypanum and Leishmania, including parasites that do not fall within the subgenera L. (Leishmania) and L. (Viannia) related to human leishmaniasis or L. (Sauroleishmania) formed by leishmanias of lizards: L. colombiensis, L. equatorensis, L. herreri, L. hertigi, L. deanei, L. enriettii and L. martiniquensis. The comparison of these species with newly characterized isolates from sloths, porcupines and phlebotomines from central and South America unveiled new genera and subgenera supported by past (RNA PolII gene) and present (V7V8 SSU rRNA, Hsp70 and gGAPDH) phylogenetic analyses of the organisms. The genus Endotrypanum is restricted to Central and South America, comprising isolates from sloths and transmitted by phlebotomines that sporadically infect humans. This genus is the closest to the new genus Porcisia proposed to accommodate the Neotropical porcupine parasites originally described as L. hertigi and L. deanei. A new subgenus Leishmania (Mundinia) is created for the L. enriettii complex that includes L. martiniquensis. The new genus Zelonia harbours trypanosomatids from Neotropical hemipterans placed at the edge of the Leishmania–Endotrypanum-Porcisia clade. Finally, attention is drawn to the status of L. siamensis and L. australiensis as nomem nudums.

Trypanosoma irwini was previously described from koalas and we now report the finding of a second novel species, T. gilletti, as well as the extension of the host range of Trypanosoma copemani to include koalas. Phylogenetic analysis at the 18S rDNA and gGAPDH loci demonstrated that T. gilletti was genetically distinct with a genetic distance (±s.e.) at the 18S rDNA locus of 2·7±0·5% from T. copemani (wombat). At the gGAPDH locus, the genetic distance (±s.e.) of T. gilletti was 8·7±1·1% from T. copemani (wombat). Trypanosoma gilletti was detected using a nested trypanosome 18S rDNA PCR in 3/139 (∼2%) blood samples and in 2/29 (∼7%) spleen tissue samples from koalas whilst T. irwini was detected in 72/139 (∼52%) blood samples and T. copemani in 4/139 (∼3%) blood samples from koalas. In addition, naturally occurring mixed infections were noted in 2/139 (∼1·5%) of the koalas tested.

The morphology and genetic characterization of a new species of trypanosome infecting koalas (Phascolarctos cinereus) are described. Morphological analysis of bloodstream forms and phylogenetic analysis at the 18S rDNA and gGAPDH loci demonstrated this trypanosome species to be genetically distinct and most similar to Trypanosoma bennetti, an avian trypanosome with a genetic distance of 0·9% at the 18S rDNA and 10·7% at the gGAPDH locus. The trypanosome was detected by 18S rDNA PCR in the blood samples of 26 out of 68 (38·2%) koalas studied. The aetiological role of trypanosomes in koala disease is currently poorly defined, although infection with these parasites has been associated with severe clinical signs in a number of koalas. Based on biological and genetic characterization data, this trypanosome species infecting koalas is proposed to be a new species Trypanosome irwini n. sp.