Deep-sequencing to resolve complex diversity of apicomplexan parasites in platypuses and echidnas: Proof of principle for wildlife disease investigation.

The short-beaked echidna (Tachyglossus aculeatus) and the platypus (Ornithorhynchus anatinus) are iconic egg-laying monotremes (Mammalia: Monotremata) from Australasia. The aim of this study was to demonstrate the utility of diversity profiles in disease investigations of monotremes. Using small subunit (18S) rDNA amplicon deep-sequencing we demonstrated the presence of apicomplexan parasites and confirmed by direct and cloned amplicon gene sequencing Theileria ornithorhynchi, Theileria tachyglossi, Eimeria echidnae and Cryptosporidium fayeri. Using a combination of samples from healthy and diseased animals, we show a close evolutionary relationship between species of coccidia (Eimeria) and piroplasms (Theileria) from the echidna and platypus. The presence of E. echidnae was demonstrated in faeces and tissues affected by disseminated coccidiosis. Moreover, the presence of E. echidnae DNA in the blood of echidnas was associated with atoxoplasma-like stages in white blood cells, suggesting Hepatozoon tachyglossi blood stages are disseminated E. echidnae stages. These next-generation DNA sequencing technologies are suited to material and organisms that have not been previously characterised and for which the material is scarce. The deep sequencing approach supports traditional diagnostic methods, including microscopy, clinical pathology and histopathology, to better define the status quo. This approach is particularly suitable for wildlife disease investigation.

A new coccidian parasite of the boodie, Bettongia lesueur (Mammalia: Marsupialia: Potoroidae), from Australia.

Four of 28 wild boodies or burrowing bettongs, Bettongia lesueur (Quoy et Gaimard) passed oocysts of species of Eimeria Schneider, 1875. The boodies are surviving on off-shore islands and in large predator-proof sanctuaries on the mainland where they were reintroduced. The boodie is a potoroid marsupial extinct from the mainland of Australia due to predation from red foxes and feral cats. Comparison with other species of the genus Eimeria indicates that the coccidium found represents a new species. Sporulated oocyst of Eimeria burdi sp. n. are pyriform, 21.0-24.0 µm (mean 22.6 µm) by 14.0-16.0 µm (14.9 µm), with a length/width ratio 1.31-1.71 (1.52) and 1-µm-thick yellowish bilayered wall. Micropyle is present at the thinner apex end filled with hyaline body. Polar granules are absent. Sporocysts are ellipsoidal, 10.0-13.5 µm (11.8 µm) by 7.0-8.5 µm (7.4 µm), shape index is 1.42-1.89 (1.63) and a very thin, poorly defined unilayered sporocyst wall is 0.2 µm thick with a domelike almost indistinct Stieda body. Substieda body is indistinct.

Congenital vestibular disease in captive Sumatran tigers (Panthera tigris ssp. sumatrae) in Australasia.

The Sumatran tiger (Panthera tigris ssp. sumatrae) is a critically endangered species in the wild. To ensure that demographic and genetic integrity are maintained in the longer term, those Sumatran tigers held in captivity are managed as a global population under a World Association of Zoos and Aquariums Global Species Management Plan (GSMP). A retrospective study, including segregation and pedigree analysis, was conducted to investigate potential cases of congenital vestibular disease (CVD) in captive Sumatran tigers in Australasian zoos using medical and husbandry records, as well as video footage obtained from 50 tigers between 1975 and 2013. Data from the GSMP Sumatran tiger studbook were made available for pedigree and segregation analysis. Fourteen cases of CVD in 13 Sumatran tiger cubs and one hybrid cub (Panthera tigris ssp. sumatrae × Panthera tigris) were identified. Vestibular signs including head tilt, circling, ataxia, strabismus and nystagmus were observed between birth and 2 months of age. These clinical signs persisted for a median of 237 days and had resolved by 2 years of age in all cases. Pedigree analysis revealed that all affected tigers were closely related and shared a single common ancestor in the last four generations. A genetic cause for the disease is suspected and, based on pedigree and segregation analysis, an autosomal dominant mode of inheritance is likely. Further investigations to determine the world-wide prevalence and underlying pathology of this disorder are warranted.

Diagnosis and management of tuberculosis (Mycobacterium tuberculosis) in an Asian elephant (Elephas maximus) with a newborn calf.

In 2006, five Asian elephants (Elephas maximus) were imported to Taronga Zoo, Australia, from Thailand. Pre-import and initial postarrival tuberculosis screening was performed by trunk wash (TW) culture and was negative for Mycobacterium tuberculosis. In April 2009, the ElephantTB STAT-PAK (SP) assay was used to test the elephants. A 15.5-yr-old pregnant cow was reactive. TW frequency for this cow was increased from annually to quarterly. TW cultures remained negative on all other elephants. In February 2010, the Dual Path Platform (DPP) VetTB assay was used for the first time, and the SP-reactive cow also reacted on the DPP. A SP was run concurrently and was reactive. All other elephants were nonreactive on both assays. Treatment was not initiated due to concern about the effect of antituberculous drugs on the fetus. Quarterly TW cultures continued. The cow gave birth on 2 November 2010. A routine TW on 24 November 2010 was culture positive for M. tuberculosis. Although previous shedding could not be ruled out, reactivation of latent infection or exacerbation of subclinical disease due to parturition was suspected. Treatment with isoniazid, pyrazinamide, rifampicin, and ethambutol commenced. A 12-mo treatment course was completed within a 15-mo period. The isolate was susceptible to these drugs and genotyped as a Beijing strain. Stored serum samples from 2004 and 2006 were tested retrospectively and were reactive on SP and DPP. TW, SP, and DPP screening frequency increased to monthly for the positive cow on commencement of treatment in January 2011. Monthly serum biochemistry indicated drug-induced hepatitis. Therapeutic drug monitoring was conducted to ensure therapeutic levels were achieved. The infant calf was reactive on DPP, but TW culture negative, and was not treated. Serial DPP results for the cow and calf during and after treatment indicated that the antibody levels were declining, suggesting a favorable response to therapy in the dam, and that the origin of the antibodies in the calf were maternal, rather than a response to infection.