Morphological polymorphism of Trypanosoma copemani and description of the genetically diverse T. vegrandis sp. nov. from the critically endangered Australian potoroid, the brush-tailed bettong (Bettongia penicillata (Gray, 1837))

Abstract

Background: The trypanosome diversity of the Brush-tailed Bettong (Bettongia penicillata), known locally as the woylie, has been further investigated. At a species level, woylies are critically endangered and have declined by 90% since 1999. The predation of individuals made more vulnerable by disease is thought to be the primary cause of this decline, but remains to be proven.

Methods: Woylies were sampled from three locations in southern Western Australia. Blood samples were collected and analysed using fluorescence in situ hybridization, conventional staining techniques and microscopy. Molecular techniques were also used to confirm morphological observations.

Results: The trypanosomes in the blood of woylies were grouped into three morphologically distinct trypomastigote forms, encompassing two separate species. The larger of the two species, Trypanosoma copemani exhibited polymorphic trypomastigote forms, with morphological phenotypes being distinguishable, primarily by the distance between the kinetoplast and nucleus. The second trypanosome species was only 20% of the length of T. copemani and is believed to be one of the smallest recorded trypanosome species from mammals. No morphological polymorphism was identified for this genetically diverse second species. We described the trypomastigote morphology of this new, smaller species from the peripheral blood of the woylie and proposed the name T. vegrandis sp. nov. Temporal results indicate that during T. copemani Phenotype 1 infections, the blood forms remain numerous and are continuously detectable by molecular methodology. In contrast, the trypomastigote forms of T. copemani Phenotype 2 appear to decrease in prevalence in the blood to below molecular detectable levels.

Conclusions: Here we report for the first time on the morphological diversity of trypanosomes infecting the woylie and provide the first visual evidence of a mixed infection of both T. vegrandis sp. nov and T. copemani. We also provide supporting evidence that over time, the intracellular T. copemani Phenotype 2 may become localised in the tissues of woylies as the infection progresses from the active acute to chronic phase. As evidence grows, further research will be necessary to investigate whether the morphologically diverse trypanosomes of woylies have impacted on the health of their hosts during recent population declines.

Figure 1

Remaining natural populations of woylies and the sample locations for this study in Western Australia.

Figure 2

The different morphological forms of T. copemani identified from the same host. A - Broad trypomastigote form. B - Slender trypomastigote form. K= Kinetoplast, N= Nucleus, FF= Free Flagellum.

Figure 3

The different morphological phenotypes of T. copemani identified from different hosts. A - T. copemani Phenotype 1 (P1). B - T. copemani Phenotype 2 (P2). K= Kinetoplast, N= Nucleus, FF= Free Flagellum.

Figure 4

The morphological form of T. vegrandis sp. nov. A and B - Trypomastigote forms. K= Kinetoplast, N= Nucleus, FF= Free Flagellum.

Figure 5

Fluorescence in situ hybridisation of T. vegrandis sp. nov. A and B - Fluorescent trypomastigote forms. K= Kinetoplast, N= Nucleus, FF= Free Flagellum.

Figure 6

Trypomastigotes of T. copemani P1 (arrow) and T. vegrandis (circle) from NAR.

Figure 7

Smallest reported length and average smallest length of the species within each subgenus of Trypanosoma[21], T. copemani and T. vergrandis.