Meat-borne parasites of the genus Trichinella are exotic to mainland Australia although the geographical proximity of reservoirs, particularly of the non-encapsulated species Trichinella papuae in Papua New Guinea (PNG) and Trichinella pseudospiralis in the south-eastern Australian island state of Tasmania, suggests there are potential risks of entry. To date, surveillance data for the presence of Trichinella in Australian wildlife has relied on game meat export certification results and to a lesser extent on data collected from limited surveys in wildlife that both use the artificial digestion (AD) method. A surveillance framework and capacity for diagnosis of animal infections could be improved to clarify or better safeguard Australia‘s Trichinella-free status. This thesis aimed to contribute data to the status of Trichinella infection in Australian wildlife, improve the understanding and performance of methods for Trichinella diagnosis in wildlife surveillance and assess the risk of introduction of non-encapsulated Trichinella species to the mainland.
A wildlife survey using an AD method was conducted in 599 wild pigs as well as a limited number of foxes, wild dogs and cats. Diaphragm samples (20 g) were tested from 450 animals from north-eastern Australia‘s Cape York Peninsula (CYP) and Torres Strait islands, areas that are at-risk for introduction of a range of diseases and are adjacent to the Western Province of PNG, a known foci of T. papuae. One hundred and forty nine wild pigs were also tested from south-western Australia and 51 carnivore (wild dog, cat and fox) samples were obtained from the mid-eastern coast. No Trichinella muscle larvae were detected in any carnivore or pig samples from the mainland but a T. papuae (Kikori strain) infection was identified in a single wild pig from Gabba Island, Torres Strait. The findings supported a zero or low prevalence of Trichinella parasites in mainland Australian wildlife but narrowed the geographical proximity of T. papuae foci to the mainland and underpinned the importance of continued surveillance, particularly in northern regions.
A molecular assay utilising real-time PCR technology to increase sensitivity for low-level infections was developed for Trichinella wildlife surveillance. A genus-specific SYBR green assay that detected a 195 bp fragment of the small subunit ribosomal RNA (SSU-rRNA) gene was evaluated for its ability to detect Trichinella larvae in 10 g muscle samples and its specificity against host and other pathogen DNAs. The test sensitivity was as low as 0.1 larvae per gram in muscle from wild pigs, cat, fox, saltwater crocodile and a carnivorous marsupial (Tasmanian devil) spiked with ethanol-preserved T. pseudospiralis or T. papuae larvae. This level of sensitivity represented a 5-fold increase compared to AD using 20 g muscle samples and a field evaluation using naturally infected muscle samples of wild pigs and Tasmanian devils showed complete agreement with the AD method (ĸ-value=1.00). Positive amplification of mouse tissue experimentally infected with T. spiralis indicated the assay could also be used on encapsulated Trichinella species in situ.
Serological methods were investigated for their utility as sensitive, rapid diagnostic methods in wildlife surveillance. The performances of a commercially available (PrioCHECK Trichinella Ab, Prionics Pty Ltd, Zurich, Switzerland) and 'in-house‘ indirect-ELISA using excretory-secretory (E/S) antigens were compared using sera from 673 wild pigs from the CYP, Torres Strait and south-western Australia, the majority of which were paired with muscle and tested by AD previously. Reactive sera were further investigated using either Western blot (WB) or the newly developed real-time PCR and an additional 10 g of muscle. The ELISAs correctly classified all positive controls as well as the naturally infected wild pig from the Torres Strait and showed substantial agreement (ĸ-value= 0.66) that increased to very good (ĸ-value= 0.82) when WB-positive only samples were compared. The seroprevalence in wild pigs from mainland Australia, excluding Torres Strait samples, was 3.5% (95% C.I. 0.0-8.0) and 2.3% (95% C.I. 0.0-5.6) using the in-house and commercial assay respectively, resulting in a significantly higher (P< 0.05) estimate than the AD estimate of 0.0% (95% C.I. 0.0-1.1). Real-time PCR testing in ELISA-positive sera did not detect any positive mainland wild pigs, but revealed the presence of a second larvae-positive animal on Gabba Island. The serology results suggest Australian wildlife may be exposed to Trichinella parasites, but because of the likelihood of non-specific reactions with other parasitic infections, more work using well-defined cohorts of positive and negative samples should be conducted.
The final study involved a qualitative risk assessment for the introduction and establishment of T. pseudospiralis and T. papuae into mainland Australia. Risk pathways for introductions of T. pseudospiralis via infected, carnivorous birds from Tasmania and T. papuae via infected, saltwater crocodiles from PNG and their risk of establishing in introduced or native Australian wildlife were developed. The overall risk estimation for both pathways was 'very low‘ which is a value ascribed to risks likely to be minor to directly affected parties and unlikely to be discernible at any other level. Although there were moderate level consequences of the presence of non-encapsulated Trichinella in mainland wildlife that could be recognised at a national level, the low probability of entry and establishment reduced the risk rating significantly. Directly, the most important impacts were public health impacts in at-risk consumers including recreational hunters and consumers of game meats. Indirect impacts were likely to affect government agencies responsible for animal health and food security as well as game meat industries including crocodile meat production and commercial harvesting of wild boar.