The impact of integrating rabbit haemorrhagic disease virus (K5) release with pindone baiting on wild rabbit populations.

Several conventional and recently available tools are available for an integrated control of European rabbits in Australia. We quantified the impact of the release of rabbit haemorrhagic disease virus K5 (RHDV K5, hereafter K5) and pindone (2-pivalyl-1,3-indandione) baiting at 13 sites within Cudlee Creek fire scar in the Adelaide Hills, South Australia. K5 release was followed by pindone baiting between December 2021 and March 2022; the application of both control methods followed industry best practice. We counted rabbits using spotlights before and after the application of both control methods. Fly samples and livers from dead rabbits were collected to track K5 transmission within and between sites, and to detect the natural circulation of rabbit haemorrhagic disease virus 2 (RHDV2). K5 release had minimal impact on rabbit populations, with treated populations increasing by a mean of 65.5% at 14 days post-release and 27.9% at 77 days post-K5 release across all sites, comparable to the changes at control sites. K5 detection in flies up to 77 days post its release, and its detection in rabbit livers, demonstrates that it can survive and transmit in the environment for prolonged periods and that it can lethally infect some rabbits. This limited impact of K5 is consistent with previous studies and may be explained by pre-existing RHDV/RHDV2 immunity in the target populations or the presence of young rabbits with natural innate RHDV immunity. The detection of K5 in flies from control sites demonstrates that it was vectored beyond its release location. A reduction in rabbit counts post-pindone baiting was observed at most treatment sites, with a mean population reduction of 36.6% across all sites. Landholders need to carefully and strategically plan their integrated rabbit control programmes. Not all combinations of controls, even if theoretically logical, achieve meaningful outcomes for rabbit management.

Bayesian evaluation of temporal changes in sensitivity and specificity of three serological tests for multiple circulating strains of rabbit haemorrhagic disease virus.

Competition and indirect ELISAs are currently being used to monitor rabbit haemorrhagic disease viruses (RHDV1 and RHDV2) in rabbits worldwide. Temporal changes in the sensitivity (Se) and specificity (Sp) of RHDV1 competition-ELISA (cELISA1), RHDV2 competition-ELISA (cELISA2), and RHDV1 Immunoglobulin G (IgG1) ELISA, were investigated using Bayesian Latent Class models (BCLM) in the Australian wild rabbit population where both viruses circulate simultaneously and a long-term serological dataset exists. When cELISA1 was compared to IgG1 ELISA, the Se of cELISA1 improved while the Sp of IgG1 ELISA declined over the 2011-21. This corresponded with a decline in the true RHDV1 prevalence in 2018-21, suggesting that a large proportion of RHDV1 exposed rabbits survived the introduction and dominance of RHDV2 up to approximately 2017/2018, after which they died and were not replaced. The Se and Sp estimates for 2014-15 for both cELISA1 and IgG1 ELISA, and the true prevalence when analysing all three tests together were similar to those obtained from the analysis of cELISA1/IgG1 ELISA. The same was also true for the Se and Sp of cELISA2 and IgG1 ELISA estimates from 2018 onwards. This suggests that RHDV1 was the dominant infection type in 2014-15, but RHDV2 was the dominant infection type in 2018-21. Further, the increase in Se of cELISA2 and the low Sp of IgG1 ELISA in the cELISA2/IgG1 ELISA analysis, compared to the Se of cELISA2 and Sp of IgG1 ELISA when analysing all three tests together suggests that the underlying infection status was more influenced by RHDV2 and that the higher Se of IgG1 ELISA is due to cross-reaction of RHDV2 antibodies on IgG1 ELISA. The true prevalence data suggest that RHDV2 exposure peaked in 2017. Our findings show that test characteristics changed in response to the changing virus prevalences over time. IgG1 ELISA, currently having a high Se, should be used to monitor both viruses and will perform better than both cELISAs.

Utilizing Molecular Epidemiology and Citizen Science for the Surveillance of Lagoviruses in Australia.

Australia has multiple lagoviruses with differing pathogenicity. The circulation of these viruses was traditionally determined through opportunistic sampling events. In the lead up to the nationwide release of RHDVa-K5 (GI.1aP-GI.1a) in 2017, an existing citizen science program, RabbitScan, was augmented to allow members of the public to submit samples collected from dead leporids for lagovirus testing. This study describes the information obtained from the increased number of leporid samples received between 2015 and 2022 and focuses on the recent epidemiological interactions and evolutionary trajectory of circulating lagoviruses in Australia between October 2020 and December 2022. A total of 2771 samples were tested from January 2015 to December 2022, of which 1643 were lagovirus-positive. Notable changes in the distribution of lagovirus variants were observed, predominantly in Western Australia, where RHDV2-4c (GI.4cP-GI.2) was detected again in 2021 after initially being reported to be present in 2018. Interestingly, we found evidence that the deliberately released RHDVa-K5 was able to establish and circulate in wild rabbit populations in WA. Overall, the incorporation of citizen science approaches proved to be a cost-efficient method to increase the sampling area and enable an in-depth analysis of lagovirus distribution, genetic diversity, and interactions. The maintenance of such programs is essential to enable continued investigations of the critical parameters affecting the biocontrol of feral rabbit populations in Australia, as well as to enable the detection of any potential future incursions.

Optimising the Delivery of RHDV to Rabbits for Biocontrol: An Experimental Evaluation of Two Novel Methods of Virus Delivery.

Rabbit haemorrhagic disease virus (RHDV) is established as a landscape-scale biocontrol that assists the management of invasive European rabbits and their impacts in both Australia and New Zealand. In addition to this, it is also available to land managers to augment rabbit control efforts at a local scale. However, current methods of deploying RHDV to rabbits that rely on the consumption of virus-treated baits can be problematic as rabbits are reluctant to consume bait when there is abundant, green, protein-rich feed available. We ran a suite of interrupted time-series experiments to compare the duration of infectivity of two conventional (carrot and oat baits) and two novel (meat bait and soil burrow spray) methods of deploying RHDV to rabbits. All methods effectively killed exposed rabbits. Soil burrow spray and carrot baits resulted in infection and mortality out to 5 days post their deployment in the field, and meat baits caused infection out to 10 days post their deployment. In contrast, oat baits continued to infect and kill exposed rabbits out to 20 days post deployment. Molecular assays demonstrated high viral loads in deployed baits beyond the duration for which they were infectious or lethal to rabbits. Based on our results, we suggest that the drying of meat baits may create a barrier to effective transmission of RHDV by adult flies within 10 days. We therefore hypothesise that fly larvae production and development on infected tissues is critical to prolonged viral transmission from meat baits, and similarly from carcasses of RHDV mortalities, via mechanical fly vectors. Our study demonstrates that meat baits and soil spray could provide additional virus deployment options that remove the need for rabbits to consume baits at times when they are reluctant to do so.

Sustained Impact of RHDV2 on Wild Rabbit Populations across Australia Eight Years after Its Initial Detection.

Following the arrival of rabbit haemorrhagic disease virus 2 (RHDV2) in Australia, average rabbit population abundances were reduced by 60% between 2014 and 2018 based on monitoring data acquired from 18 sites across Australia. During this period, as the seropositivity to RHDV2 increased, concurrent decreases were observed in the seroprevalence of both the previously circulating RHDV1 and RCVA, a benign endemic rabbit calicivirus. However, the detection of substantial RHDV1 seropositivity in juvenile rabbits suggested that infections were continuing to occur, ruling out the rapid extinction of this variant. Here we investigate whether the co-circulation of two pathogenic RHDV variants was sustained after 2018 and whether the initially observed impact on rabbit abundance was still maintained. We monitored rabbit abundance and seropositivity to RHDV2, RHDV1 and RCVA at six of the initial eighteen sites until the summer of 2022. We observed sustained suppression of rabbit abundance at five of the six sites, with the average population reduction across all six sites being 64%. Across all sites, average RHDV2 seroprevalence remained high, reaching 60-70% in adult rabbits and 30-40% in juvenile rabbits. In contrast, average RHDV1 seroprevalence declined to <3% in adult rabbits and 5-6% in juvenile rabbits. Although seropositivity continued to be detected in a low number of juvenile rabbits, it is unlikely that RHDV1 strains now play a major role in the regulation of rabbit abundance. In contrast, RCVA seropositivity appears to be reaching an equilibrium with that of RHDV2, with RCVA seroprevalence in the preceding quarter having a strong negative effect on RHDV2 seroprevalence and vice versa, suggesting ongoing co-circulation of these variants. These findings highlight the complex interactions between different calicivirus variants in free-living rabbit populations and demonstrate the changes in interactions over the course of the RHDV2 epizootic as it has moved towards endemicity. While it is encouraging from an Australian perspective to see sustained suppression of rabbit populations in the eight years following the arrival of RHDV2, it is likely that rabbit populations will eventually recover, as has been observed with previous rabbit pathogens.

Gut microbiota composition does not associate with toxoplasma infection in rats.

Toxoplasma infection in intermediate host species closely associates with inflammation. This association has led to suggestions that the behavioural changes associated with infection may be indirectly driven by the resulting sustained inflammation rather than a direct behavioural manipulation by the parasite. If this is correct, sustained inflammation in chronically infected rodents should present as widespread differences in the gastrointestinal microbiota due to the dependency between the composition of these microbiota and sustained inflammation. We conducted a randomized controlled experiment in rats that were assigned to a Toxoplasma-treatment, placebo-treatment or negative control group. We euthanised rats during the chronic phase of infection, collected their caecal stool samples and sequenced the V3-V4 region of the 16S rRNA gene to characterize the bacterial community in these samples. Toxoplasma infection did not induce widespread differences in the bacterial community composition of the gastrointestinal tract of rats. Rather, we found sex differences in the bacterial community composition of rats. We conclude that it is unlikely that sustained inflammation is the mechanism driving the highly specific behavioural changes observed in Toxoplasma-positive rats.

Cross-protection, infection and case fatality rates in wild European rabbits experimentally challenged with different rabbit haemorrhagic disease viruses.

Rabbit haemorrhagic disease virus 2 (RHDV2) is now the dominant calicivirus circulating in wild rabbit populations in Australia. This study compared the infection and case fatality rates of RHDV2 and two RHDVs in wild rabbits, as well as their ability to overcome immunity to the respective other strains. Wild rabbits were allocated to groups either blindly or based on pre-screening for RHDV/RHDV2 antibodies at capture. Rabbits were monitored regularly until their death or humane killing at 7 days post infection. Liver and eyeball samples were collected for lagovirus testing and aging rabbits, respectively. At capture, rabbits showed high seroprevalence to RHDV2 but not to RHDV. In RHDV/RHDV2 seronegative rabbits at capture, infection rates were highest in those inoculated with RHDV2 (81.8%, 18 out of 22), followed by K5 (53.8%, seven out of 13) and CZECH (40.0%, two out of five), but these differences were not statistically significant. In rabbits with previous exposure to RHDV2 at capture, infection rates were highest when inoculated with K5 (59.6%, 31 out of 52) followed by CZECH (46.0%, 23 out of 50), with infection rates higher in younger rabbits for both viruses. In RHDV/RHDV2 seronegative rabbits at capture, case fatality rates were highest for those inoculated with K5 (71.4%), followed by RHDV2 (50.0%) and CZECH (50.0%). In rabbits with previous exposure to RHDV2 at capture, case fatality rates were highest in rabbits inoculated with K5 (12.9%) followed by CZECH (8.7%), with no case fatalities following RHDV2 inoculation. Case fatality rates did not differ significantly between inoculums in either serostatus group at capture. Based on multivariable modelling, time to death post RHDV inoculation increased in rabbits with recent RHDV2 exposure compared with seronegative rabbits and with age. The results suggest that RHDV2 may cause higher mortalities than other variants in seronegative rabbit populations but that K5 may be more effective in reducing rabbit populations in an RHDV2-dominant landscape.

Changes in virus transmission dynamics following the emergence of RHDV2 shed light on its competitive advantage over previously circulating variants.

Rabbit haemorrhagic disease virus (RHDV) is highly pathogenic to European rabbits. Until recently, only one serotype of RHDV was known, GI.1/RHDV. RHDV2/GI.2 is a novel virus that has rapidly spread and become the dominant pathogenic calicivirus in wild rabbits worldwide. It is speculated that RHDV2 has three competitive advantages over RHDV: (a) the ability to partially overcome immunity to other variants; (b) the ability to clinically infect young rabbits; and (c) a wider host range. These differences would be expected to influence virus transmission dynamics. We used markers of recent infection (IgM/IgA antibodies) to investigate virus transmission dynamics pre and post the arrival of RHDV2. Our data set contained over 3,900 rabbits sampled across a 7-year period at 12 Australian sites. Following the arrival of RHDV2, seasonal peaks in IgM and IgA seropositivity shifted forward one season, from winter to autumn and spring to winter, respectively. Contrary to predictions, we found only weak effects of rabbit age, seropositivity to non-pathogenic calicivirus RCV-A1 and population abundance on IgM/IgA seropositivity. Our results demonstrate that RHDV2 enters rabbit populations shortly after the commencement of annual breeding cycles. Upon entering, the population RHDV2 undergoes extensive replication in young rabbits, causing clinical disease, high virus shedding, mortality and the creation of virus-laden carcasses. This results in high virus contamination in the environment, furthering the transmission of RHDV2 and initiating outbreaks, whilst simultaneously removing the susceptible cohort required for the effective transmission of RHDV. Although RHDV may enter the population at the same time point, it is sub-clinical in young rabbits, causing minimal virus shedding and low environmental contamination. Our results demonstrate a major shift in epidemiological patterns in virus transmission, providing the first evidence that RHDV2's ability to clinically infect young rabbits is a key competitive advantage in the field.

Distribution and Genetic Diversity of Hepatitis E Virus in Wild and Domestic Rabbits in Australia.

In 2020, Hepatitis E virus (HEV) was detected for the first time in Australian rabbits. To improve our understanding of the genetic diversity and distribution of the virus, 1635 rabbit liver samples from locations across Australia were screened via RT-qPCR for HEV. HEV genomes were amplified and sequenced from 48 positive samples. Furthermore, we tested 380 serum samples from 11 locations across Australia for antibodies against HEV. HEV was detected in rabbits from all states and territories, except the Northern Territory. Seroprevalence varied between locations (from 0% to 22%), demonstrating that HEV is widely distributed in rabbit populations across Australia. Phylogenetic analyses showed that Australian HEV sequences are genetically diverse and that HEV was likely introduced into Australia independently on several occasions. In summary, this study broadens our understanding of the genetic diversity of rabbit HEV globally and shows that the virus is endemic in both domestic and wild rabbit populations in Australia.

INFECTION PRESSURE IS NECESSARY, BUT NOT SUFFICIENT BY ITSELF, TO EXPLAIN TOXOPLASMA GONDII SEROPREVALENCE IN INTERMEDIATE HOST SPECIES.

Parasite infection pressure is suggested to be a strong driver of transmission within ecosystems. We tested if infection pressure drives seroprevalence in intermediate host species for Toxoplasma gondii. We defined Toxoplasma infection pressure to intermediate host species as the combined influence of cat abundance, environmental conditions, and its prevalence in the cat population. We sampled and tested 2 species of rodent and collated information on Toxoplasma seroprevalence in koalas, wallabies, kangaroos, and sheep. All species were sampled using equivalent methods, within a 2-yr period, and from adjacent regions of low and high Toxoplasma infection pressure. The seroprevalence of Toxoplasma in kangaroos scaled with infection pressure, but we observed no statistical difference in seroprevalence for any other species between these 2 regions. Within the region of low infection pressure, Toxoplasma seroprevalence did not differ between species. However, within the region of high Toxoplasma infection pressure, we observed large variation in seroprevalence between species. Our results demonstrate that infection pressure is not sufficient by itself, but merely necessary, to drive Toxoplasma seroprevalence in intermediate host species. Where Toxoplasma seroprevalence in an intermediate host species is already low, further reducing infection pressure will not necessarily further decrease seroprevalence in those species. This has important ramifications for the mitigation of parasite infections and suggests that reductions in Toxoplasma infection pressure, intended to reduce infections, may be most effective and applicable to species that are known to experience high rates of infection.

The impact of PIT tags on the growth and survival of pythons is insignificant in randomised controlled trial.

Individual identification is fundamental to the study of captive and wild animals but can have adverse impacts if the method of identification is inappropriate for the species or question of interest. We conducted a randomised controlled trial to test whether passive integrated transponder (PIT) tags reduced the growth or survival of pythons. We randomly allocated 200 captive-bred Burmese python (Python bivittatus) hatchlings into two groups, tagged versus untagged. Hatchlings were individually identified using a combination of PIT tags and unique colour patterns, and their mass, snout-vent length (SVL) and body condition measured at 9, 73, 134, 220, 292 and 385 days of age. We recorded the date of all mortalities. Python morphometrics and their rate of change increased or fluctuated non-linearly with age. The impact of PIT tagging on python body mass and body mass growth over the 376 day study period was insignificant. PIT tagging additionally had an insignificant impact on python survival. However, we found minor differences in SVL growth between tagged and untagged pythons. These differences peaked at approximately 0.5 mm/day and appeared to drive similar, but more pronounced, differences between tagged and untagged pythons in their rate of change in body condition; peaking at approximately 3-4 g/day. While we cannot be certain that these small differences are, or are not, biologically meaningful, they nonetheless appear to be short-term and readily resolved. Unsurprisingly, the strongest driver of python growth was their age, with growth rapidly increasing or highest amongst younger snakes for all measures of size. Python sex was associated with their body mass and survival, with higher mass but lower survival amongst females. Python size at hatching did not impact on their growth or survival. Our results confirm that PIT tags are a valuable and effective tool for the identification and tracking of captive pythons, and snakes generally, and meet high safety and animal welfare standards.

Cutaneous Chromatophoromas in Four Species of Australian Elapid Snake.

This report documents the clinicopathological features of cutaneous chromatophoromas in four wild-caught, captive Australian elapid snakes: a strap-snouted brown snake (Pseudonaja aspidoryncha), a tiger snake (Notechis scutatus), an Eastern brown snake (Pseudonaja textilis) and a Mengden's brown snake (Pseudonaja mengdeni). All tumours were subclassified as melanophoromas, with three assessed as malignant on the basis of invasive growth or presence of intracoelomic metastases. The chromatophoromas were single or multiple, black or orange pigmented, cutaneous, sometimes ulcerated, plaques or nodules. Microscopically, the neoplastic cells were often spindle shaped with low or variable pigmentation. Neoplastic cells in one tumour were notable for their pleomorphic round cell morphology and high mitotic rate. One snake with late-stage metastasis survived for over 5 years. There are few reports of chromatophoromas in elapid snakes and, to our knowledge, this is the first report of these tumours in Australian elapid snakes.

Fractional seroprevalence rates in common prey species can cause more than half of feral cats to be exposed to Toxoplasma gondii annually.

Rodents comprise a major component of cat (Felis catus) diets in many ecosystems, and life cycle diagrams of Toxoplasma gondii typically depict small rodents as quintessential intermediate hosts. Counter-intuitively, small rodents often experience a lower T. gondii seroprevalence than do larger sympatric mammals. This observation has repeatedly caused confusion about the relative importance of small rodents to the ecology of T. gondii. To address this confusion, we modified the Reed-Frost epidemic model to develop a simple binomial equation to model T. gondii transmission from prey to feline predators. This equation takes into account variations in prey seroprevalence and the frequency with which they are consumed by felids. Even when T. gondii seroprevalence in prey is < 1%, computation reveals that the risk of feline exposure to T. gondii can easily exceed 50 % annually. For example, if cats eat an average of 1 mouse per day, a seroprevalence of 0.2 % (1/500) in mice will cause 51.9 % of cats to be exposed to T. gondii annually. Our simple equation demonstrates that both prey seroprevalence and the rate at which prey are consumed are of approximately equal importance to the ecology of T. gondii. When inferring the importance of various prey species to the ecology of T. gondii, researchers must consider the predation and dietary habits of felids from within their study system. Our simple binomial equation could also be used to predict T. gondii exposure rates of humans or other carnivorous animals from various dietary sources or be applied to other predator-prey parasite life cycles.

Spatial Analysis of a Cat-Borne Disease Reveals That Soil pH and Clay Content Are Risk Factors for Sarcocystosis in Sheep.

Cat-borne parasites and their associated diseases have substantial impacts on human, livestock, and wildlife health worldwide. Despite this, large and detailed datasets that allow researchers to study broad-scale trends in the ecology of cat-borne diseases are either difficult to obtain or non-existent. One condition that is easily detected at slaughter is macroscopic sarcocystosis, a cat-borne parasitosis of sheep (Ovis aries). We conducted a cross-sectional study to describe the geographic distribution of sarcocystosis in sheep throughout South Australia and investigate ecosystem characteristics associated with the presence of disease. Data were obtained from two slaughterhouses which processed 3,865,608 sheep from 4,204 farms across 385,468 km2 of South Australia's land mass for the period 2007-2017. A Poisson point process model was developed to quantify environmental characteristics associated with higher densities of sarcocystosis-positive farms. Sarcocystosis was highly clustered on a large island off of the Australian coast and the density of sarcocystosis-positive farms increased in areas of low soil pH (intensity ratio: 0.86, 95% CI: 0.78, 0.95) and high clay content. We hypothesize that region was confounded by, and predominately acted as a proxy for, cat density. Our results have broader implications regarding the health, welfare, economic, and conservation impacts of other cat-borne parasitosis, such as toxoplasmosis.