TRYPANOSOMES

Trypanosomes are flagellated protozoan parasites that generally have a diphasic life-cycle occurring between a vertebrate host and an invertebrate vector. Although some trypanosome species can cause serious human dis­eases, such as African and American trypanosomiasis (sleeping sickness and Chagas’ disease, respectively), the majority of wildlife trypanosomes have historically been considered benign in their vertebrate hosts (Hoare 1972; Thompson et al.

The basic morphology of mammalian blood-borne trypomastigotes is somewhat lanceolate and oval in transverse section, containing a kinetoplast (an extra- nuclear mass of DNA near the posterior end) and a single undulating flagellum at the anterior end (Figs

26.1 and 26.2). Recent studies have demonstrated great morphological diversity within and between trypano­some species in Australia, with reported average lengths ranging from 8.3 μm to 38 μm and average widths rang­ing from 1.3 μm to 15 μm (Austen et al. 2009; McInnes et al. 2009; Thompson et al. 2013; Austen et al. 2015a; Cooper et al. 2017).

Australian trypanosomes also exhibit high intra- and interspecies genetic diversity. Interestingly, phylogenetic studies have demonstrated that some indigenous species are more closely related to species outside Australia. For instance, Trypanosoma noyesi and T. teixeirae are geneti­cally more similar to T. cruzi and bat-derived trypanosomes from South America and Africa than to other Australian native marsupial-derived trypanosomes such as T. irwini, T. copemani, T. gilletti and T. vegrandis (Hamilton et al. 2004;

Fig. 26.1. Trypanosoma copemani isolated from the blood of a Gilbert's potoroo (Potorousgilbertii). (a) Light micrograph, scale bar = 10 μm. (b) Scanning electron micrograph of T.

Hamilton et al. 2012; Thompson et al. 2014a; Barbosa et al. 2016a; Cooper et al. 2017).

In total, 76 native Australian mammal species have been screened for trypanosomes to date, including 48 of the 162 marsupial species (30%), 10 of the 66 rodent spe­cies (15%), 16 of the 76 bat species (21%) and both mono- treme species (100%) (Thompson et al. 2014a; Austen et al.

Fig. 26.2. Light micrograph of Trypanosoma teixeirae isolated from the blood of a little red flying-fox (Pteropus scapulatus). Scale bar =

10 μm.

2015b; Barbosa et al. 2017a; Austen et al. 2020; Egan et al. 2020; Hall et al. 2021). From these, 10 native Trypanosoma spp. have been taxonomically described and 13 genotypes reported (Table 26.1).

1.1 Epidemiology

There is relatively limited knowledge on the prevalence of trypanosomes in Australian mammals. Molecular studies have detected trypanosomes in the blood of up to 74% of koalas (Phascolarctos cinereus) from Qld and NSW, and brush-tailed bettongs (Bettongia penicillata) from WA (McInnes et al. 2011b; Botero et al. 2013; Bar­bosa et al. 2017b). A recent survey conducted in the NT found prevalence of 27% and 24% in northern brown bandicoots (Isoodon macrourus) and common brush­tailed possums (Trichosurus vulpecula), respectively (Barbosa et al. 2017a). High prevalence has also been reported in platypuses (Ornithorhynchus anatinus) (86%) and various species of bats (82%) (Paparini et al. 2014; Austen et al. 2015b). Trypanosoma was identified in 55% of brush-tailed bettongs in south-western Australia (Northover et al. 2019), while polyparasitism with Tryp­anosoma spp. was reported in 50% of samples from brush-tailed bettongs, common brush-tailed possums, and Western quolls (Dasyurus geoffroii) in WA (Cooper et al. 2018). A recent study identified the presence of Trypanosoma spp.

However, studies to date have indicated that Austral­ian trypanosomes appear to have a widespread geograph­ical distribution, having been recorded in all states (Thompson et al. 2014a; Barbosa et al. 2017a; Portas et al. 2020), except SA where limited investigations have been conducted. Additionally, there are many vertebrate popu­lations across the Australian mainland and islands that have not yet been examined, meaning that current knowl­edge on the biogeography of Australian trypanosomes is far from complete.

Although there is no conclusive evidence on the iden­tity of invertebrate vectors of trypanosomes in Australian native species, various ectoparasites, including fleas, tab- anid flies and several tick species (Ixodes spp., Haema- physalis spp., Amblyomma triguttatum), have been reported as potential vectors (Mackerras 1959; Noyes et al. 1999; Hamilton et al. 2005; Austen et al. 2011; Paparini et al. 2014; Botero et al. 2016a; Barbosa et al. 2017b; Krige et al. 2021). However, no experimental transmission stud­ies to confirm their vectorial competence have been con­ducted to date (Krige et al. 2019).

In terms of transmission dynamics, trypanosomes are divided in two groups: Salivaria and Stercoraria. The former undergo morphological and physiological trans­formation in the salivary glands of the vectors and are transmitted by inoculation during a blood meal; whereas the latter reside in the invertebrate vector’s gut and are released in the faeces and deposited on the skin of the host.

1.2 Pathogenesis and clinical significance

Although subclinical trypanosome infections are common in wildlife, disease can result in morbidity and mortality, particularly in immunocompromised hosts. Haemolytic anaemia is common and may be a conse­quence of physical effects of the parasites (e.g. mechani­cal injury to erythrocytes and release of proteases) or of extravascular haemolysis arising from antibody-medi­ated erythrocyte destruction (Clark et al. 2004). In addi­tion to effects on erythrocytes, trypanosomes can invade host cells (e.g. T. copemani), causing tissue inflammation, reducing fitness of the host and thereby potentially increasing susceptibility to predation (Botero et al. 2013).

Trypanosomes can also potentiate the effects of con­current infections by compromising the immune system of their hosts (Khan and Lacey 1986; Goossens et al. 1997; Carrera et al. 2009). Trypanosome-induced immunosup­pression involves various mechanisms of depression of cellular and humoral immune responses, including quan­titative, biochemical and functional changes of T-cells, B-cells and macrophages (Zuniga et al. 2000; Vincendeau and Bouteille 2006).

Although most of the endemic trypanosomes identi­fied to date are thought to be non-pathogenic (Thompson et al. 2014a; Cooper et al. 2017), there are several docu­mented cases suggesting that some of these species have potential to be pathogenic. Additionally, exotic trypano­some species may also have a negative impacton the health of naive mammalian hosts in Australia (Abbott 2006; Thompson et al.

1.2.1 Trypanosoma lewisi and extinction of endemic rats of Christmas Island

Following the unintentional introduction of black rats (Rattus rattus) infected with T. lewisi and their flea vectors to Christmas Is. in the early 1900s, two endemic rodent species, Maclear’s (R. macleari) and bulldog (R. nativitatis) rats became extinct. Reports at the time described Maclear’s rats being frequently found sick or moribund and heavily infected with trypanosomes and demonstrat­ing pathological changes consistent with trypanosome infection at necropsy (Andrews 1909). A century after their extinction, molecular analysis of ancient DNA sug­gested that native trypanosomes were absent from the

Table 26.1. Trypanosomes of native Australian mammals

26 - Haemoprotozoan parasites 389

Table 26.1. (continued)

390 CurrentTherapyin MedicineofAustraIian Mammals

26 - Haemoprotozoan parasites

endemic rodents on Christmas Island before introduction of the black rat (Wyatt et al. 2008), lending weight to the hypothesis that T. lewisi infection contributed to the extinctions.

1.2.2 Trypanosoma spp. and koala morbidity

Although trypanosome infections in koalas may be asymptomatic, clinical signs of trypanosomiasis have been reported and include poor body condition and regenerative anaemia caused by extravascular haemoly­sis. Trypanosomes were identified on blood films, with a regenerative erythroid response evident in blood and bone marrow. Some cases progressed to developing neurological signs such as nystagmus, tremors and sei­zures; however, it is unclear if these signs could be attributed to the trypanosomes. Histopathology revealed small organisms in the liver and CNS, sugges­tive of trypanosome intracellular amastigotes; however, their identity could not be confirmed. Other findings included lymphocytic/plasmacytic choroiditis in the animals with neurological signs, with putative trypano­somes in some of the choroid vessels (A Gillett pers. comm.).

A recent study provided further evidence that trypa­nosome infections may impact koala health and sur­vival and may be contributing to the decline of koala populations in eastern Australia (McInnes et al. 2011b). A significant association was reported between infec­tion with T. gilletti and low PCV and body condition scores in koalas with signs of concurrent diseases (chlamydiosis, bone marrow dysplasia or koala retrovi­rus (KoRV)-associated immune deficiency). This sug­gests that normally benign trypanosome infections may become pathogenic in koalas that are immunosup­pressed or concurrently infected with other pathogens, particularly KoRV. Alternatively, the trypanosome infection may be inducing immunosuppression and therefore potentiating the effects of concomitant dis­eases (McInnes et al. 2011b).

Mixed infections with up to six Trypanosoma spp., including T. irwini, T. gilletti, T. copemani, T. vegrandis and T. noyesi, have been reported in koalas (McInnes et al. 2011a, McInnes et al. 2011b, Barbosa et al. 2016b, Barbosa et al. 2017b). Further research is required to unravel the biological interactions involved in mixed trypanosome infections and in mixed chlamydial/KoRV and trypanosome infections.

1.2.3 Trypanosoma infection and the brush-tailed bettong population decline

The brush-tailed bettong (woylie) is critically endan­gered and has undergone a rapid population decline of 90% (Thompson et al. 2014b). It has been suggested that trypanosomes may have played a role in this decline. Studies have provided evidence that T. copemani amas- tigote stages can migrate to and replicate in a range of brush-tailed bettong tissues, including heart, skeletal muscle, oesophagus and tongue, resulting in inflamma­tion and tissue degeneration (Botero et al. 2013; Botero et al. 2016b). These effects may reduce fitness and coor­dination, making them more susceptible to predation (Botero et al. 2013). This observation has been further supported by a temporal association between T. copem­ani prevalence and declines of the Kingston indigenous brush-tailed bettong population in the Upper Warren region in WA (Thompson et al. 2014b).

A study conducted by Hing et al. (2016) revealed a relationship between T. copemani infection and the functional efficiency of innate immunity (phagocytosis) in brush-tailed bettongs. The T. copemani-infected bet­tongs had higher faecal cortisol metabolites (FCM) associated with a lower phagocytosis index. However, when the bettongs were trypanosome-negative, higher FCM was associated with higher phagocytosis index. This suggests that during periods of Trypanosoma para- sitaemia, the animals are more vulnerable to the immu­nosuppressive effects of glucocorticoids. Alternatively, a combination of host stress physiology and infection status may affect the efficiency of leucocyte function (Hing et al. 2016).

Pathological changes in brush-tailed bettongs may also occur with mixed infections involving T. vegran- dis and T. noyesi, suggesting a greater immunosup­pressive effect or enhanced pathogenicity with mixed infections (Botero et al. 2013). The finding of a higher prevalence of mixed infections involving T. copemani, T. vegrandis and T. noyesi in a declining brush-tailed bettong population compared with a stable one seems to support this theory (Botero et al. 2013). Conversely, it has been hypothesised that interspecific competi­tion may exist between T. copemani and T. vegrandis, where an existing T. vegrandis infection may moderate the sequential establishment of T. copemani (Thompson et al. 2014b; Godfrey et al. 2018). As with koalas, the clinical impact of trypanosome co-infections on brush-tailed bettong health warrants further investigation.

Further research on the impact of co-infections involv­ing trypanosomes and other parasites or pathogens is also essential for informing brush-tailed bettong conservation efforts. In their 2018 study, Northover et al. conducted a diagnostic investigation on a polyparasitised brush-tailed bettong infected with T. copemani, Sarcocystis sp., Potorostrongylus woyliei, and Paraustrostrongylus sp. Clinical manifestations included poor body condition, diffuse alopecia, debilitating skin lesions and severe ectoparasite infestation. Blood tests indicated moderate hypomagnesemia, mild hypokalemia, mild hyperglobu- linemia, and mild hypoalbuminemia. The authors pro­posed a hypothesis based on pathology findings and the generally low pathogenicity associated with other para­sites, suggesting that T. copemani may influence co­infecting parasites, immune function, and host health. However, additional research is needed to explore this hypothesis further.

1.2.4 Trypanosomiasis in a little red flying-fox

The first case report of a trypanosome infection associ­ated with clinical disease in bats involved an adult female little red flying-fox (Pteropus scapulatus), found on the ground in Redcliffe, Qld. The animal presented with icterus and severe anaemia. Necropsy and histo­logical findings were consistent with trypanosome infection of lymphoid tissue and intravascular haemol­ysis (Mackie et al. 2017). Examination of a blood smear by light microscopy revealed the presence of numerous trypanosomes and molecular analysis revealed the parasite was a novel species (Mackie et al. 2017). More extensive phylogenetic analysis confirmed the species status of this novel trypanosome, which was named T. teixeirae. It was shown to be closely related to the pathogenic T. cruzi of South America (Barbosa et al. 2016a).

1.2.5 Trypanosoma vegrandis and anaemia in northern brown bandicoots

A recent investigation of haemoprotozoan parasites in native mammals from northern Australia demonstrated that northern brown bandicoots positive for T. vegrandis were more likely to have lower PCVs than negative indi­viduals. No other clinical signs were associated with infection (Barbosa et al. 2017a).

1.2.6 Trypanosoma sp. and morbidity in eastern bettongs and mortality of eastern grey kangaroos

During an investigation of a recent mortality event of eastern grey kangaroos (Macropus giganteus) and weight loss in eastern bettongs (B. gaimardi) in the ACT, a novel Trypanosoma sp. was detected in 5/10 kangaroos and 6/9 bettongs. The novel species was genetically distinct from, but most closely related to T. copemani (genetic distance = 14%) (Barbosa et al. unpublished). Haematol­ogy revealed regenerative anaemia suggestive of chronic blood loss. The role of Trypanosoma sp. in mortality and morbidity events is unclear and its potential involvement should be considered with caution, in the context of a broader investigation of these events. For instance, infec­tions with Theileria sp. (see section 2.2.6) and moderate endoparasitism were also observed, as well as unusual climatic conditions (i.e. a prolonged period of sub-zero temperatures) and low pasture biomass during the winter period (Barbosa et al. unpublished; T Portas pers. comm.).

1.3 Diagnosis

Giemsa or Wright’s stained blood smears are used for detection of trypanosomes by light microscopy (Figs 26.1a and 26.2). Although convenient and readily available, microscopic detection of trypanosomes is rela­tively insensitive, particularly in the chronic stages of infection and in cases of low-level parasitaemia (e.g. T. noyesi) (Botero et al. 2016a). Various molecular tech­niques are now available for detection of trypanosomes. For research purposes, electron microscopy is particu­larly useful for ultrastructural characterisation of the parasites (Fig. 26.1b).

PCR assays are able to amplify very small amounts of trypanosome DNA in blood. Additionally, PCR com­bined with Sanger sequencing can be used for species identification. Morphological characterisation is insuf­ficient for species identification, because of the overlap­ping morphometry between species and polymorphic life-cycle stages in vertebrate hosts (Thompson et al. 2013; Austen et al. 2015a). PCR and sequence analysis of partial fragments of the 18S rRNA gene are the most widely used diagnostic method for Trypanosoma iden­tification. However, amplification at additional loci, such as the nuclear glyceraldehyde 3-phosphate dehy­drogenase gene and the mitochondrial cytochrome b gene, can be adopted for a more robust genetic characterisation (Hamilton et al. 2004; Botero et al. 2016a; Cooper et al. 2017).

Although Sanger sequencing of PCR amplicons, gen­erated by universal primers for the genus Trypanosoma, is a commonly used method for species identification, it does not allow characterisation of multiple genotypes involved in mixed trypanosome infections, which are common in brush-tailed bettongs and koalas (McInnes et al. 2011a; McInnes et al. 2011b; Paparini et al. 2011; Botero et al. 2013; Barbosa et al. 2017b). In these cases, application of alternative methods such as species-spe­cific conventional and real-time quantitative PCR (qPCR), cloning or next-generation sequencing are nec­essary to characterise mixed infections (McInnes et al. 2011b; Paparini et al. 2011; Barbosa et al. 2017b; Cooper et al. 2018; Keatley et al. 2020). Additionally, total RNA sequencing (meta-transcriptomics) has been successfully used for surveillance and diagnosis of trypanosomes in native Australian wildlife (Ortiz-Baez et al. 2020; Gofton et al. 2022).

1.4 Treatment and control

There are no specific treatment recommendations for trypanosomiasis in Australian mammals. Supportive care, including blood transfusions, IV fluid therapy, nutritional and thermal support, is useful. Additional symptomatic treatment, such as the use of diazepam in koalas exhibiting neurological signs, is recommended (A Gillettpers. comm.).

Prevention and control of trypanosome transmission through limiting exposure to invertebrate vectors is gen­erally not feasible. However, monitoring and surveillance to gather baseline data on the prevalence and genetic diversity of trypanosomes in Australian mammal popu­lations and their vectors is crucial for conservation man­agement, particularly in translocated populations.

1.5 Zoonotic potential

There is currently no evidence that Australian native trypanosomes are zoonotic, although preliminary evi­dence shows that T. copemani is naturally resistant to human serum and therefore may be potentially zoonotic (Austen et al. 2015c). The first report of the cosmopolitan parasite T. dionisii in Australian bats is of importance considering it has been reported in human cardiac tissue together with T. cruzi (Dario et al. 2016; Austen et al. 2020).

1.6 Biosecurity concerns

The potential negative effect of exotic trypanosomes such as T. lewisi, T. dionisii, T. cruzi and T. evansi, if they established and spread within Australian native mam­mals, presents a biosecurity concern (Thompson 2013; Thompson et al. 2014a). Of these, T. lewisi and T. dionisii are the only exotic species already introduced and found in native mammals (Thompson et al. 2014a; Austen et al. 2020; Egan et al. 2021; Gofton et al. 2022).

In 1907, surra-infected camels were imported into WA; fortunately, this infection was diagnosed quickly and T. evansi was eradicated before establishment (Mack­erras 1959). If T. evansi had established in Australia, the consequences for Australian native mammals and domestic livestock could have been devastating (Reid et al. 2001; Reid 2002).

The zoonotic pathogen T. cruzi is genetically very similar to some Australian trypanosomes, particularly T. noyesi and T. teixeirae. It has been hypothesised, for example, that the vector of T. noyesi could potentially transmit T. cruzi from humans (infected immigrants and travellers) to indigenous mammals (Thompson et al. 2014a; Thompson and Thompson 2015). Experimental T. cruzi infection has been demonstrated in the short- beaked echidna (Tachyglossus aculeatus) and brush-tailed possum (Backhouse and Bolliger 1951), confirming the potential for disease and deaths of native mammals and the potential for native mammals to act as a reservoir for human infection (Thompson and Thompson 2015).

Continued surveillance and identification of vectors of Australian trypanosomes are required to ascertain the biosecurity risks of exotic trypanosomes establishing in Australia (Thompson and Thompson 2015).

2.