Craig K. Thompson

Trypanosomes genetic diversity, polyparasitism and the population decline of the critically endangered Australian marsupial, the brush tailed bettong or woylie (Bettongia penicillata)

Graphical abstract

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))

BackgroundThe 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.MethodsWoylies 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.ResultsThe 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.ConclusionsHere 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.

Trypanosomes of Australian mammals: A review

Approximately 306 species of terrestrial and arboreal mammals are known to have inhabited the mainland and coastal islands of Australia at the time of European settlement in 1788. The exotic Trypanosoma lewisi was the first mammalian trypanosome identified in Australia in 1888, while the first native species, Trypanosoma pteropi, was taxonomically described in 1913. Since these discoveries, about 22% of the indigenous mammalian fauna have been examined during the surveillance of trypanosome biodiversity in Australia, including 46 species of marsupials, 9 rodents, 9 bats and both monotremes. Of those mammals examined, trypanosomes have been identified from 28 host species, with eight native species of Trypanosoma taxonomically described. These native trypanosomes include T. pteropi, Trypanosoma thylacis, Trypanosoma hipposideri, Trypanosoma binneyi, Trypanosoma irwini, Trypanosoma copemani, Trypanosoma gilletti and Trypanosoma vegrandis. Exotic trypanosomes have also been identified from the introduced mammalian fauna of Australia, and include T. lewisi, Trypanosoma melophagium, Trypanosoma theileri, Trypanosoma nabiasi and Trypanosoma evansi. Fortunately, T. evansi was eradicated soon after its introduction and did not establish in Australia. Of these exotic trypanosomes, T. lewisi is the sole representative that has been reported from indigenous Australian mammals; morphological forms were recorded from two indigenous species of rodents (Hydromys chrysogaster and Rattus fuscipes). Numerous Australian marsupial species are potentially at risk from the native T. copemani, which may be chronically pathogenic, while marsupials, rodents and monotremes appear at risk from exotic species, including T. lewisi, Trypanosoma cruzi and T. evansi. This comprehensive review of trypanosome biodiversity in Australia highlights the negative impact of these parasites upon their mammalian hosts, as well as the threatening biosecurity concerns.

Temporal and spatial dynamics of trypanosomes infecting the brush-tailed bettong (Bettongia penicillata): a cautionary note of disease-induced population decline

BackgroundThe brush-tailed bettong or woylie (Bettongia penicillata) is on the brink of extinction. Its numbers have declined by 90% since 1999, with their current distribution occupying less than 1% of their former Australian range. Woylies are known to be infected with three different trypanosomes (Trypanosoma vegrandis, Trypanosoma copemani and Trypanosoma sp. H25) and two different strains of T. copemani that vary in virulence. However, the role that these haemoparasites have played during the recent decline of their host is unclear and is part of ongoing investigation.MethodsWoylies were sampled from five locations in southern Western Australia, including two neighbouring indigenous populations, two enclosed (fenced) populations and a captive colony. PCR was used to individually identify the three different trypanosomes from blood and tissues of the host, and to investigate the temporal and spatial dynamics of trypanosome infections.ResultsThe spatial pattern of trypanosome infection varied among the five study sites, with a greater proportion of woylies from the Perup indigenous population being infected with T. copemani than from the neighbouring Kingston indigenous population. For an established infection, T. copemani detection was temporally inconsistent. The more virulent strain of T. copemani appeared to regress at a faster rate than the less virulent strain, with the infection possibly transitioning from the acute to chronic phase. Interspecific competition may also exist between T. copemani and T. vegrandis, where an existing T. vegrandis infection may moderate the sequential establishment of the more virulent T. copemani.ConclusionIn this study, we provide a possible temporal connection implicating T. copemani as the disease agent linked with the recent decline of the Kingston indigenous woylie population within the Upper Warren region of Western Australia. The chronic association of trypanosomes with the internal organs of its host may be potentially pathogenic and adversely affect their long term fitness and coordination, making the woylie more susceptible to predation.

Morphological and phylogenetic description of Trypanosoma noyesi sp. nov.: An Australian wildlife trypanosome within the T. cruzi clade

A number of trypanosome isolates from Australian marsupials are within the clade containing the human pathogen Trypanosoma cruzi. Trypanosomes within this clade are thought to have diverged from a common ancestral bat trypanosome. Here, we characterise Trypanosoma noyesi sp. nov. isolated from the critically endangered woylie (Bettongia pencillata) using phylogenetic inferences from three gene regions (18S rDNA, gGAPDH, and CytB) coupled with morphological and behavioural observations in vitro. We also investigated potential vectors and the presence of T. noyesi in the grey-headed flying fox (Pteropus poliocephalus). Phylogenetic analysis revealed T. noyesi and similar genotypes grouped at the periphery of the T. cruzi clade. T. noyesi is morphologically distinct both from other species of Australian trypanosomes and those within the T. cruzi clade. Although trypanosomes were not observed in the digestive tract of ectoparasites and biting flies collected from T. noyesi infected marsupials, tabanid and biting midges tested positive for T. noyesi DNA, indicating they are vector candidates. Tissues from flying foxes were negative for T. noyesi. This study provides novel information on the morphology and genetic variability of an Australian trypanosome within the T. cruzi clade.

Trypanosomes of Australian Mammals: Knowledge Gaps Regarding Transmission and Biosecurity.

Trypanosomes infect humans, domestic animals, and wildlife, and are transmitted by haematophagous invertebrate vectors. Eight native trypanosome species have been described from Australian indigenous mammals, along with other unnamed isolates and genotypes. Associated difficulties relating to the confirmation of cyclical and mechanical vector candidates has hindered vector identification in Australia, with no successful experimental transmission documented for any of these native trypanosomes to indigenous mammals. We discuss pending biosecurity issues, with significant importance placed on the close phylogenetic and phenotypic relationship shared between Trypanosoma cruzi and some Australian trypanosomes. With such a dearth of information, we highlight the importance of keeping an open mind, which considers all possibilities during future investigations of vectors and their associated biosecurity issues in Australia.

Sensitivity testing of trypanosome detection by PCR from whole blood samples using manual and automated DNA extraction methods

Automated extraction of DNA for testing of laboratory samples is an attractive alternative to labour-intensive manual methods when higher throughput is required. However, it is important to maintain the maximum detection sensitivity possible to reduce the occurrence of type II errors (false negatives; failure to detect the target when it is present), especially in the biomedical field, where PCR is used for diagnosis. We used blood infected with known concentrations of Trypanosoma copemani to test the impact of analysis techniques on trypanosome detection sensitivity by PCR. We compared combinations of a manual and an automated DNA extraction method and two different PCR primer sets to investigate the impact of each on detection levels. Both extraction techniques and specificity of primer sets had a significant impact on detection sensitivity. Samples extracted using the same DNA extraction technique performed substantially differently for each of the separate primer sets. Type I errors (false positives; detection of the target when it is not present), produced by contaminants, were avoided with both extraction methods. This study highlights the importance of testing laboratory techniques with known samples to optimise accuracy of test results.

Survival, age estimation and sexual maturity of pouch young of the brush-tailed bettong (Bettongia penicillata) in captivity

The brush-tailed bettong or woylie (Bettongia penicillata) is a continuous and rapid breeder. However, research investigating the monthly survival and development of young woylies from parturition to parental independence is incomplete. The reproductive biology of eight female woylies was observed for 22 consecutive months within a purpose-built enclosure. Adult female woylies bred continuously and were observed caring for a dependant young 96% of the time. Pouch life of the young was ~102 days, with sexual maturity of female offspring reached as early as 122 days post partum. Crown–rump measurement was an accurate predictor of age for young restricted to the pouch, while skeletal morphometrics were a better predictor of age for ejected pouch young, young-at-foot and subadults. A four-month period between May and August of each study year accounted for 85% of pouch young mortality and 61% of pouch young births where the neonate went on to survive to subadult age. Here we discuss the possibility that pouch young born during the cooler, wetter months of May to August may have an increased chance of survival in the wild, resulting from an increased maternal investment being directed towards the rearing of ‘fitter’ progeny.

Trypanosome co-infections increase in a declining marsupial population

Understanding the impacts of parasites on wildlife is growing in importance as diseases pose a threat to wildlife populations. Woylie (syn. brush-tailed bettong, Bettongia penicillata) populations have undergone enigmatic declines in south-western Western Australia over the past decade. Trypanosomes have been suggested as a possible factor contributing towards these declines because of their high prevalence in the declining population. We asked whether temporal patterns of infection with Trypanosoma spp. were associated with the decline patterns of the host, or if other factors (host sex, body condition, co-infection or rainfall) were more influential in predicting infection patterns. Species-specific nested PCRs were used to detect the two most common trypanosomes (T. copemani and T. vegrandis) from 444 woylie blood samples collected between 2006 and 2012. Time relative to the decline (year) and an interaction with co-infection by the other trypanosome best explained patterns of infection for both trypanosomes. The prevalence of single species infections for both T. copemani and T. vegrandis was lower after the population crash, however, the occurrence of co-infections increased after the crash compared to before the crash. Our results suggest an interaction between the two parasites with the decline of their host, leading to a higher level of co-infection after the decline. We discuss the possible mechanisms that may have led to a higher level of co-infection after the population crash, and highlight the importance of considering co-infection when investigating the role of parasites in species declines.