Piroplasms

Piroplasms are tick-borne protozoan parasites that infect mammalian and avian erythrocytes. These organisms can cause significant economic losses because of their high pathogenicity in domestic production animals and horses worldwide and severe clinical disease in compan­ion animals (Colwell et al.

The Order Piroplasmida includes three genera: Babe­sia, Theileria and Cytauxzoon. Of these, Babesia and Theileria are known to infect Australian mammals. Cur­rently, there are 14 formally named species of piroplasms recorded in Australian mammals and genetic data from six unnamed marsupial-derived piroplasms. The species’ names, known vertebrate hosts, geographical distribu­tion and overall clinical significance are presented in Table 26.2.

2.1 Epidemiology

The true prevalence and geographic distribution of piro- plasms in native mammals is unknown, although there have been studies of limited populations in geographi­cally disparate regions. Piroplasms parasitising native mammal hosts have been recorded in most states and territories of Australia (Table 26.2). High prevalence of T. gilberti in free-ranging populations of the Gilbert’s potoroo (Potorous gilbertii) (100%, 16/16) and T. penicil- lata in brush-tailed bettongs (80.4%, 123/153) from WA (Lee et al. 2009; Rong et al. 2012) have been reported. In another study of managed care and free-ranging marsu­pial populations in WA, the overall detection rate of piroplasm-infected animals was 7.1% (8/113). Of these, a novel Babesia was identified in seven free-ranging brush-tailed bettongs and a single Theileria positive sample was identified from one of three managed bur­rowing bettongs (B.

A recent haemoprotozoan molecular investigation identified, for the first time, Babesia sp. in the Tasmanian devil (Sarcophilus harrisii), albeit at low prevalence (1.05%, 1/95) (Egan et al. 2020). Babesia sp. were also described for the first time, and at a high prevalence (72.2%, 95% CI: 49.4-88.5%), in common brush-tailed possums inhabiting urban and peri-urban areas in NSW and WA (Egan et al. 2021). These novel genotypes were genetically most closely related to Babesia lohae (previ­ously identified from Australian Ixodes ticks (Greay et al. 2018; Loh et al. 2018a)). B. lohae and B. lohae-like genotypes were also reported in common brush-tailed possums from Sydney (100%, 3/3) and in the township of Kioloa, on the mid-south coast of NSW (33.3%, 1/3) (Gofton et al. 2022). High prevalence (75%-90.9%) of Theileria sp. (presumably Theileria peramelis) was reported in long-nose bandicoots from NSW (Egan et al. 2021; Gofton et al. 2022).

Ticks are believed to be the main vectors of piroplasms, as they are ubiquitous and parasitise all vertebrate hosts of Babesia and Theileria species recorded to date. Ticks collected from infected animals have also been positive for piroplasms using molecular methods. In this context, native Haemaphysalis spp. and Ixodes spp. seem likely vector candidates (Dawood et al. 2013; Loh et al. 2018a; Loh et al. 2018b; Gofton et al. 2022). Under natural condi­tions, piroplasms are transmitted from the invertebrate vector to the definitive host via the ticks’ saliva during feeding.

Small Babesia spp. found in Australian ticks exhibit transovarial transmission, in which the parasites migrate to the ovaries and are transmitted vertically from the gravid female to larvae. In contrast, Theileria spp. undergo only transstadial transmission, in which the parasite is passed on from one life stage (‘stadium’) to the next. Furthermore, Babesia spp. only multiply in red blood cells, but Theileria spp. undergo extra- erythrocytic schizogony in lymphocytes or macrophages before invading erythrocytes (Uilenberg 2006; Sivakumar et al. 2014).

2.2 Pathogenesis and clinical significance

Although most piroplasms reported in Australian mam­mals appear to be non-pathogenic, there are some exam­ples of morbidity and mortality associated with infection. Haemolytic anaemia and thrombocytopenia are recognised as the most typical pathology of piroplas- mosis. These may be the result of physical effects of the parasites on cells or the activation of an immune response that leads to cell destruction by macrophages (Clark et al. 2004). Clinical signs of piroplasmosis may include lethargy, reduced mentation, diarrhoea and neu­rological signs and may vary with the species of parasite and host.

Interestingly, the pathogenesis of B. macropus in macropods appears to have similarities with that of B. bovis in cattle, characterised by sequestering of infected erythrocytes in the capillaries of organs, leading to low-level peripheral parasitaemia, disseminated

Table 26.2. Piroplasms (Babesia and Theileria) OfAustraIian native mammals

396 CurrentTherapyin MedicineofAustraIian Mammals

¹PriestIey 1915; ²Backhouse and Bolliger 1959; ³Mackerras 1959; ⁴Barkerefa/.

*Species classification based on morphological characterisation only.

26 - Haemoprotozoan parasites

intravascular coagulation and severe cerebral babesio­sis (Dawood et al. 2013).

2.2.1 Babesia/Theileria tachyglossi and short-beaked echidna mortality

Piroplasms (B. tachyglossi and T. tachyglossi) found in short-beaked echidnas were originally described based solely on microscopy (Priestley 1915; Backhouse and Bol- liger 1957, 1959; Mackerras 1959). At the time of the origi­nal reports, the genera Babesia and Theileria had not yet been defined, with regard to the existence of schizonts and/or transstadial versus transovarial transmission. Molecular data suggests that only a single piroplasm spe­cies, believed to be T. tachyglossi, infects echidnas (Slapeta et al. 2017).

Infection with piroplasms was implicated in the deaths of 12 zoo-housed short-beaked echidnas (Back­house and Bolliger 1957). Subsequently, there have been no further reports of pathological effects of piroplasms in echidnas, despite these parasites being frequently observed in blood films (L Vogelnest pers. comm.). Monotreme populations in Australia remain relatively understudied with respect to the prevalence and clinical effects of piroplasm infection.

2.2.2 Anaemia in bandicoots associated with Theileria peramelis

Although natural infections with T. peramelis have been recorded in southern brown (I. obesulus) and long-nosed bandicoots (Perameles nasuta), there is only one report associating experimental infection with T. peramelis and anaemia in these species. The anaemia observed was strongly regenerative, characterised by increased aniso­cytosis and immature erythrocytes (Mackerras 1959).

2.2.3 Babesia infection and fatal anaemia in antechinuses

Infection with a Babesia sp. has been associated with clinical piroplasmosis in the brown antechinus (Antechi­nus stuartii) and male agile antechinuses (A. agilis) undergoing physiological stress in the post-mating period. These animals presented with decreased PCV, haemoglobinuria and haemosiderosis of the lung and spleen (Cheal et al. 1976; Barker et al. 1978).

2.2.4 Theileria Ornithorhynchi and fatal anaemia in platypuses Although T. ornithorhynchi is generally considered non- pathogenic, this parasite has been associated with fatal haemolytic anaemia in an orphaned juvenile platypus. The animal had concurrent fungal dermatitis and a heavy tick burden (Kessell et al. 2014). Association of T. ornitho- rhynchi with disease is further supported by a recent report of high (10-15%) parasitaemia in three diseased male wild platypuses (two juveniles and one sub-adult). One of them was also infected with Eimeria sp. The ani­mals were dehydrated and anorexic. Histopathology revealed severe suppurative enteritis and active spleen with increased erythrophagocytosis (Slapeta et al. 2017).

2.2.5 Babesia macropus and severe babesiosis in macropods

Although subclinical infection with Babesia spp. appears to be relatively common in macropods, B. macropus infec­tion has been associated with a syndrome of haemolytic anaemia and debility in hand-reared and free-ranging juvenile eastern grey kangaroos, agile wallabies (Nota- macropus agilis), red-necked wallabies (N. rufogriseus) and swamp wallabies (Wallabia bicolor). Clinical signs included severe pallor, lethargy, polydipsia, polyuria, neu­rological signs and death (Vogelnest and Portas 2008; Dawood et al. 2013; Donahoe et al. 2015). PCV of less than 10% are commonly observed in affected animals, which may also be hypoproteinaemic with total protein levels as low as 30 g/L (Vogelnest and Portas 2008).

Other variable clinical pathological findings associated with this disease included thrombocytopenia, neutrope­nia, hyperamylasaemia, azotaemia and bilirubinaemia. The neurological signs observed in some kangaroos may be related to intravascular sequestration of parasitised erythrocytes within the CNS. Potential pathogenic mech­anisms include hypoxia, inflammatory cytokine release following obstructive sequestration of erythrocytes and endothelial damage (Donahoe et al. 2015). Necropsy find­ings included diffuse pallor of the carcass and visceral organs; thin, watery blood; widespread petechiae; ecchy- moses, tissue oedema; splenomegaly; and generalised lymphadenomegaly (Donahoe et al. 2015).

As most of the infected kangaroos were in managed care or hand-reared, it is likely that the stress of han­dling, transportation and confinement contributed to the development of disease. Furthermore, kangaroos hand-reared by carers lack the relevant maternal anti­bodies and also have less exposure to naturally occur­ring parasites compared with free-ranging kangaroos, therefore rendering them more susceptible to infection (Donahoe et al. 2015). Alternatively, it is possible that the disease was more likely to be detected in managed animals than in free-ranging animals.

2.2.6 Theileria sp. and morbidity in eastern bettongs and mortality of eastern grey kangaroos

A novel Theileria sp. has been detected in 4/9 eastern bettongs and 4/10 eastern grey kangaroos involved in recent morbidity and mortality events, respectively, in the ACT (Barbosa et al. unpublished). Amongst other clinical signs, the animals exhibited regenerative anae­mia, which may be associated with Trypanosoma (see section 1.2.6) and Theileria infections. However, the role of these parasites in the morbidity and mortality events is unclear because of the presence of concurrent infec­tions and potential environmental causes such as unu­sual climatic conditions and lower pasture biomass (T Portas pers. comm.).

2.3 Diagnosis

Light microscopy is the primary method used for detec­tion of piroplasms, but it lacks sensitivity, particularly in chronic or subclinical cases (Clark et al. 2004). Morpho­logical features within intra-erythrocytic piroplasms are also indistinguishable among different genera and species (Homer et al. 2000). Schizonts of Theileria spp., present in leucocytes, may also be very difficult to find. Addition­ally, the morphology of piroplasms in Australian mam­mals is highly pleomorphic, with intra-erythrocytic organisms characterised as ring-shaped trophozoites and/or pairs or tetrads of pyriform-shaped merozoites (Fig. 26.3). Even though the infection typically affects less than 1% of erythrocytes (Clark et al. 2004), piroplasms may exhibit high parasitaemia, as observed for T. gilberti in the Gilbert’s potoroo (Lee et al. 2009) (Fig. 26.3a).

Molecular and phylogenetic analyses are required for confirmation of genus and species (Schnittger et al. 2003). Molecular methods are significantly more sensitive than microscopy (Clark et al. 2004). PCR assays targeting the 18S rRNA gene are the most widely used molecular method for the detection and characterisation of piro- plasms in Australian mammals (Paparini et al. 2012; Dawood et al. 2013). However, in addition to the 18S rRNA locus, additional molecular analysis at the cox3 and cytB loci is recommended to achieve superior taxo­nomic resolution (Barbosa et al. 2019). With regards to high-throughput methods which are commonly used to characterise parasite co-infections, a next-generation sequencing assay was developed to study the diversity of

Fig. 26.3. Piroplasms in Australian mammal species. (a) Ring­shaped trophozoites in the blood of a Gilbert's potoroo (Potorous gilbertii) exhibiting a high Theileria gilberti parasitaemia. (b) Photomicrograph of a tetrad of pyriform-shaped piroplasms isolated from the blood of a platypus (Ornithorhynchus anatinus) and identified by PCR as Theileria ornithorhynchi. Scale bars = 10 μm.

apicomplexan parasites (including piroplasms) in platy­puses and echidnas (Slapeta et al. 2017).

2.4 Treatment and control

Although a wide range of antiprotozoal drugs has been used for the treatment of piroplasmosis in domestic ani­mals, there is currently no robust scientific evidence of the effectiveness of these drugs in Australian mammals, or awareness of potential toxic effects. There are occasional reports of parenteral treatment with imidocarb dipropion­ate; however, response to this treatment remains anecdotal and unevaluated (Vogelnest and Portas 2008; Dawood et al. 2013; Kessell et al. 2014; Donahoe et al. 2015).

Suggested treatment regimens include provision of supportive care, including blood transfusions, IV fluids, improved nutrition and warmth if the animal is hypo­thermic (Vogelnest and Portas 2008).

As with trypanosomes, prevention of piroplasm trans­mission by vector control is not generally feasible. Administration of prophylactic doses of antiprotozoal medication has been suggested for macropods before and after translocation to prevent development of clinical babesiosis (Donahoe et al. 2015). Awareness of the preva­lence and genetic diversity of piroplasms is crucial for appropriate conservation management and translocation strategies.

2.5 Zoonotic potential

The first autochthonous case of human babesiosis reported in Australia was diagnosed in 2010 and the aetiological agent was identified as B. microti (Senanay- ake et al. 2012). Even though several Babesia spp. have been isolated from Australian mammals, there is cur­rently no reservoir identified for this zoonotic Babesia in Australia. There is no scientific evidence that Theileria spp. infecting Australian mammals are zoonotic.

ACKNOWLEDGEMENTS

We thank Dr Andrea Paparini, Dr Jill Austen and Dr Amber Gillett for their valuable contributions to this chapter.