Ashlie Hartigan

A Suspected Parasite Spill-Back of Two Novel Myxidium spp. (Myxosporea) Causing Disease in Australian Endemic Frogs Found in the Invasive Cane Toad

Infectious diseases are contributing to the decline of endangered amphibians. We identified myxosporean parasites, Myxidium spp. (Myxosporea: Myxozoa), in the brain and liver of declining native frogs, the Green and Golden Bell frog (Litoria aurea) and the Southern Bell frog (Litoria raniformis). We unequivocally identified two Myxidium spp. (both generalist) affecting Australian native frogs and the invasive Cane toad (Bufo marinus, syn. Rhinella marina) and demonstrated their association with disease. Our study tested the identity of Myxidium spp. within native frogs and the invasive Cane toad (brought to Australia in 1935, via Hawaii) to resolve the question whether the Cane toad introduced them to Australia. We showed that the Australian brain and liver Myxidium spp. differed 9%, 7%, 34% and 37% at the small subunit rDNA, large subunit rDNA, internal transcribed spacers 1 and 2, but were distinct from Myxidium cf. immersum from Cane toads in Brazil. Plotting minimum within-group distance against maximum intra-group distance confirmed their independent evolutionary trajectory. Transmission electron microscopy revealed that the brain stages localize inside axons. Myxospores were morphologically indistinguishable, therefore genetic characterisation was necessary to recognise these cryptic species. It is unlikely that the Cane toad brought the myxosporean parasites to Australia, because the parasites were not found in 261 Hawaiian Cane toads. Instead, these data support the enemy-release hypothesis predicting that not all parasites are translocated with their hosts and suggest that the Cane toad may have played an important spill-back role in their emergence and facilitated their dissemination. This work emphasizes the importance of accurate species identification of pathogens relevant to wildlife management and disease control. In our case it is paving the road for the spill-back role of the Cane toad and the parasite emergence.

Museum material reveals a frog parasite emergence after the invasion of the cane toad in Australia

BackgroundA parasite morphologically indistinguishable from Myxidium immersum (Myxozoa: Myxosporea) found in gallbladders of the invasive cane toad (Bufo marinus) was identified in Australian frogs. Because no written record exists for such a parasite in Australian endemic frogs in 19th and early 20th century, it was assumed that the cane toad introduced this parasite. While we cannot go back in time ourselves, we investigated whether material at the museum of natural history could be used to retrieve parasites, and whether they were infected at the time of their collection (specifically prior to and after the cane toad translocation to Australia in 1935).ResultsUsing the herpetological collection at the Australian Museum we showed that no myxospores were found in any animals (n = 115) prior to the cane toad invasion (1879-1935). The green and golden bell frog (Litoria aurea), the Perons tree frog (Litoria peronii), the green tree frog (Litoria caerulea) and the striped marsh frog (Limnodynastes peronii) were all negative for the presence of the parasite using microscopy of the gallbladder content and its histology. These results were sufficient to conclude that the population was free from this disease (at the expected minimum prevalence of 5%) at 99.7% confidence level using the 115 voucher specimens in the Australian Museum. Similarly, museum specimens (n = 29) of the green and golden bell frog from New Caledonia, where it was introduced in 19th century, did not show the presence of myxospores. The earliest specimen positive for myxospores in a gallbladder was a green tree frog from 1966. Myxospores were found in eight (7.1%, n = 112) frogs in the post cane toad introduction period.ConclusionAustralian wildlife is increasingly under threat, and amphibian decline is one of the most dramatic examples. The museum material proved essential to directly support the evidence of parasite emergence in Australian native frogs. This parasite can be considered one of the luckiest parasites, because it has found an empty niche in Australia. It now flourishes in > 20 endemic and exotic frog species, but its consequences are yet to be fully understood.

New Species of Myxosporea from Frogs and resurrection of the genus Cystodiscus Lutz, 1889 for species with myxospores in gallbladders of Amphibians

Two new myxosporean species in the gallbladders of frogs have recently spread across eastern Australia and cause disease. Cystodiscus axonis sp. n. and Cystodiscus australis sp. n. are species of Myxosporea (Myxozoa) identified from a range of Australian frogs and tadpoles including the introduced Cane toad (Rhinella marina). The new species are defined by their distinct genetic lineage, myxospore morphology and ultrastructure of the pre-sporogonic development. Spores of both species are produced in the gallbladder. Spores of C. axonis sp. n. possess distinct filiform polar appendages (FPA). The pre-sporogonic development of C. axonis sp. n. is within myelinated axons in the central nervous system of hosts, as well as bile ducts of tadpoles. Pre-sporogonic and sporogonic development of C. australis sp. n. is confined to tadpole bile ducts and myxospores of C. australis sp. n. are devoid of FPA. The genus Cystodiscus Lutz, 1889 introduced for Cystodiscus immersus Lutz, 1889 is emended to accompany myxosporean parasites affecting amphibians previously classified in the genus Myxidium sensu lato. A synopsis of described species within Cystodiscus is provided.

Extensive production of Neospora caninum tissue cysts in a carnivorous marsupial succumbing to experimental neosporosis

Experimental infections of Sminthopsis crassicaudata, the fat-tailed dunnart, a carnivorous marsupial widely distributed throughout the arid and semi-arid zones of Australia, show that this species can act as an intermediate host for Neospora caninum. In contrast to existing models that develop relatively few N. caninum tissue cysts, dunnarts offer a new animal model in which active neosporosis is dominated by tissue cyst production. The results provide evidence for a sylvatic life cycle of N. caninum in Australia between marsupials and wild dogs. It establishes the foundation for an investigation of the impact and costs of neosporosis to wildlife.

Comparative Pathology and Ecological Implications of Two Myxosporean Parasites in Native Australian Frogs and the Invasive Cane Toad

Myxosporean parasites Cystodiscus axonis and C. australis are pathogens of native and exotic Australian frog species. The pathology and ecological outcomes of infection with these parasites were investigated in this study. Gliosis was correlated to Cystodiscus axonis plasmodia in the brains of (9/60) tadpoles and (3/9) adult endangered Green and golden bell frogs using ordinal regression. Severe host reactions to C. axonis (haemorrhage, necrosis, and vasulitis) were observed in the brains of threatened Southern bell frogs (8/8), critically endangered Booroolong frogs (15/44) and Yellow spotted bell frogs (3/3). Severe brain lesions were associated with behavioural changes, neurological dysfunction, and spontaneous death. Both C. axonis and C. australis develop in the bile ducts of tadpoles, the plasmodia were significantly associated with biliary hyperplasia, inflammation and the loss of hepatocytes in (34/72) Green and golden bell frog tadpoles using ordinal regression. These lesions were so severe that in some cases 70% of the total liver was diseased. Normal liver function in tadpoles is necessary for metamorphosis, metabolism, and immune function. We postulate that this extensive liver damage would have significant host health impacts. Severe hepatic myxosporidiosis was more prevalent in tadpoles examined in autumn and winter (overwintered), suggestive of delayed metamorphosis in infected tadpoles, which would have serious flow-on effects in small populations. We compared the sensitivity of histopathology and species-specific PCR in the detection of C. australis and C. axonis. PCR was determined to be the most sensitive method (detection limit 1 myxospore equivalent of ribosomal DNA). Histology, however, had the advantage of assessing the impact of the parasite on the host. It was concluded that these parasites have the potential for significant ecological impacts, because of their high prevalence of infection and their ability to cause disease in some frogs.

Looks can deceive: Molecular identity of an intraerythrocytic apicomplexan parasite in Australian gliders

Two yellow-bellied gliders (Petaurus australis) had an intraerythrocytic parasite closely related to the cyst-forming coccidia (Apicomplexa: Sarcocystidae). The parasitaemia persisted for 3 months or more but was observed to clear within 3 years in captivity. The parasite appears not to significantly debilitate its infected host. Traditionally, using morphological identification, the intraerythrocytic parasite would have been classified within the Hepatozoon species typically found in red blood cells. However, molecular diagnostic techniques targeting the parasites SSU rDNA and LSU rDNA demonstrated the unusual identity of this blood parasite and disputed its identity as a haemogregarine parasite of the genus Hepatozoon. The sequence was compared with available sequences from diverse mammalian and non-mammalian blood parasites (malaria, piroplasms, hemosporidia and sarcosporidia). The intraerythrocytic blood parasite was found to be most closely related to the cyst-forming coccidia including Besnoitia spp., Cystoisospora spp., Hammondia spp., Hyaloklossia lieberkuehni, Neospora caninum, Sarcocystis spp. and Toxoplasma gondii. The life cycle of this intraerythrocytic parasite remains unknown. The presented DNA identification demonstrates its suitability for an improved identification of blood parasites.

Myxozoans on the Move: Dispersal Modes, Exotic Species and Emerging Diseases

Increased global connectivity and shorter transportation times translate to a higher probability and frequency that hosts and their parasites will be introduced to foreign locations and will arrive viable. However, myxozoans are heteroxenous with two obligatory hosts and two free-living life stages, so even if a parasite is introduced, a myriad of factors must align for establishment to occur. Associated with successful introductions are ecological and economic impacts. Unless there is an established monitoring program, it is difficult to detect the emergence of a myxozoan before clinical signs become overt. In this chapter we examine features of myxozoan parasites that promote or impede dissemination and establishment. We present examples of modes of myxozoan introduction and dispersal, both natural and human-mediated. We review known and novel species responsible for emerging diseases, and recently identified species that have the potential to cause disease. We conclude by outlining strategies to understand and limit myxozoan introductions.

Emerging myxosporean parasites of Australian frogs take a ride with fresh fruit transport

BackgroundThe spread of wildlife pathogens into new geographical ranges or populations is a conservation concern for endangered species. Cystodiscus australis and Cystodiscus axonis are two species of myxosporean parasites infecting Australian frogs and tadpoles that have been recently recognised as important disease agents impacting amphibian conservation. Yet despite their importance to wildlife health, the mechanism of emergence for these parasites is unknown. We hypothesise that these parasites are capable of being accidentally translocated with their amphibian hosts in fresh produce (agricultural, horticultural and industrial) shipments into naïve environments and host populations.MethodsWe surveyed 33 Australian “Banana box” frogs from Sydney fruit markets during 2011 using faecal smears and multiplex species specific PCR on DNA isolated from frog faeces or using histopathology to demonstrate the presence of both C. australis and C. axonis.ResultsOne of the “Banana box” frogs, the Dainty green tree frog (Litoria gracilenta) was positive for C. australis and C. axonis in its faeces and continuously shed the parasites for eight months.ConclusionsWe present a possible mechanism for the emergence of Cystodiscus parasites and a non-invasive screening method to be used as a diagnostic test. In the future, vigilance and communication between wildlife managers/researchers and veterinarians will provide valuable information about these parasites, their host range and true distribution. This will aid risk management assessments for threatened populations within the range of Cystodiscus parasites and ultimately enhance conservation efforts.

New cell motility model observed in parasitic cnidarian Sphaerospora molnari (Myxozoa:Myxosporea) blood stages in fish

Cellular motility is essential for microscopic parasites, it is used to reach the host, migrate through tissues, or evade host immune reactions. Many cells employ an evolutionary conserved motor protein– actin, to crawl or glide along a substrate. We describe the peculiar movement of Sphaerospora molnari, a myxozoan parasite with proliferating blood stages in its host, common carp. Myxozoa are highly adapted parasitic cnidarians alternately infecting vertebrates and invertebrates. S. molnari blood stages (SMBS) have developed a unique “dancing” behaviour, using the external membrane as a motility effector to rotate and move the cell. SMBS movement is exceptionally fast compared to other myxozoans, non-directional and constant. The movement is based on two cytoplasmic actins that are highly divergent from those of other metazoans. We produced a specific polyclonal actin antibody for the staining and immunolabelling of S. molnari’s microfilaments since we found that neither commercial antibodies nor phalloidin recognised the protein or microfilaments. We show the in situ localization of this actin in the parasite and discuss the importance of this motility for evasion from the cellular host immune response in vitro. This new type of motility holds key insights into the evolution of cellular motility and associated proteins.

Myxosporean parasites in Australian frogs: Importance, implications and future directions.

Graphical abstract Highlights ► Australian frog species have been shown to be affected by myxosporidiosis. ► It is suggested that Myxosporea threaten amphibian health and conservation. ► Information gained in Australia is transferable to amphibians around the world.