Sensitivity of High Conservation Value Birds to Para-Aminopropiophenone (PAPP) Determined by Sub-Lethal Dose-Response Assay.

Para-aminopropiophenone (PAPP) is a methaemoglobin (MetHb) forming compound used for the lethal control of invasive carnivores and mustelids. By measuring the dose-dependent inhibition of O2 transport arising from the oxidation of haemoglobin (HbFe2+) to MetHb (HbFe3+), we determined the sensitivity of nine bird species to PAPP. A methaemoglobinaemia absorbance index (MAI) was validated in five common bird species to determine thresholds associated with a 99% probability of survival (ST99) and a 50% probability of mortality (LT50). Dose-response trials in high conservation value birds sought MAI values below the ST99 threshold, projecting the LT50 value and avoiding the need for lethal outcomes. Black-backed gull (LT50 = 1784.7) and eastern rosella (LT50 = 1074 mg kg-1) were the most tolerant species, while brown kiwi (LT50 = 8.4 mg kg-1) and weka (LT50 = 9.3 mg kg-1) were the most sensitive. Takahe were of intermediate acute sensitivity (LT50 = 51 mg kg-1), although protracted impacts on haemoglobin were observed in takahe up to 72 h later and associated with PAPP doses as low as 25.6 mg kg-1. In pukeko (LT50 = 138.4 mg kg-1), protracted declines in haemoglobin 72 h later occurred at doses as low as 29.5 mg kg-1, while at higher doses (253 and 112 mg kg-1), deaths resulted after 4-6 days. Based upon PAPP doses that caused acute and protracted responses, we provide estimates for the lowest observable adverse effect level (LOAEL) and no observable effects level (NOEL) for nine bird species.

Monitoring methaemoglobinaemia in birds using 5 µL of whole blood.

Methaemoglobin (MetHb) forming compounds such as para-aminopropiophenone (PAPP) and sodium nitrite (NaNO2) have recently been adopted for the lethal control of a range of invasive carnivores and mustelids. Determining the relative hazard of these compounds to non-target bird species is an important component of ecological risks evaluation. Problematically, some potential non-target bird species may be as small as 10 g in body mass, thus placing limitations on blood volumes that can be routinely sampled. Accordingly, we developed methods to quantify markers of increasing methaemoglobinaemia at their point of collection that required only 5 µL of whole blood. A 3 µL blood aliquot is pipetted into a plastic micro-cuvette and placed in a custom made holder optically coupled to the Ocean Optics spectrometer, enabling absorbance for oxyhaemoglobin (HbO: 575 nm) and MetHb (630 nm) to be determined. Haemoglobin (HbFe2+), packed cell volume (PCV) and lactate (LAC) data were generated from the remaining 2 µL aliquot apportioned to biosensor strips for the Cera-Check® and Lactate Scout® point-of-care devices. After oral doses of PAPP, a methaemoglobinaemia absorbance index (MAI = absorbance 575 nm-absorbance 630 nm) was strongly and significantly associated with dose-dependent declines in HbFe2+ in 9 bird species. Quantifying dose-dependent responses to MetHb-forming agents at the point of sample collection avoids analytical and storage artifacts arising from sample degradation that appears to be a much greater problem in avian blood compared to mammalian blood.

Age and Infectious Dose Significantly Affect Disease Progression after RHDV2 Infection in Naïve Domestic Rabbits.

Rabbit haemorrhagic disease virus 2 (RHDV2 or GI.2, referring to any virus with lagovirus GI.2 structural genes) is a recently emerged calicivirus that causes generalised hepatic necrosis and disseminated intravascular coagulation leading to death in susceptible lagomorphs (rabbits and hares). Previous studies investigating the virulence of RHDV2 have reported conflicting results, with case fatality rates ranging from 0% to 100% even within a single study. Lagoviruses are of particular importance in Australia and New Zealand where they are used as biocontrol agents to manage wild rabbit populations, which threaten over 300 native species and result in economic impacts in excess of $200 million AUD annually to Australian agricultural industries. It is critically important that any pest control method is both highly effective (i.e., virulent, in the context of viral biocontrols) and has minimal animal welfare impacts. To determine whether RHDV2 might be a suitable candidate biocontrol agent, we investigated the virulence and disease progression of a naturally occurring Australian recombinant RHDV2 in both 5-week-old and 11-week-old New Zealand White laboratory rabbits after either high or low dose oral infection. Objective measures of disease progression were recorded through continuous body temperature monitoring collars, continuous activity monitors, and twice daily observations. We observed a 100% case fatality rate in both infected kittens and adult rabbits after either high dose or low dose infection. Clinical signs of disease, such as pyrexia, weight loss, and reduced activity, were evident in the late stages of infection. Clinical disease, i.e., welfare impacts, were limited to the period after the onset of pyrexia, lasting on average 12 h and increasing in severity as disease progressed. These findings confirm the high virulence of this RHDV2 variant in naïve rabbits. While age and infectious dose significantly affected disease progression, the case fatality rate was consistently 100% under all conditions tested.

Welfare Impacts of Pindone Poisoning in Rabbits (Oryctolagus cuniculus).

Control methods used to manage unwanted impacts of the European rabbit in Australia and New Zealand include the use of toxic bait containing the anticoagulant pindone. Towards increased certainty in evaluating the animal welfare impacts of pindone poisoning in rabbits, we recorded behavioral and post-mortem data from rabbits which ingested lethal quantities of pindone bait in a laboratory trial. Pindone poisoning in rabbits resulted in welfare compromise, primarily through functional impairments related to internal haemorrhage over a maximum duration of 7 days. Applying this data to a formal assessment framework for ranking animal welfare impacts indicated that pindone had relatively high severity and also duration of welfare impacts in comparison to other rabbit control methods.

Combining aspirin with cholecalciferol (vitamin D3)--a potential new tool for controlling possum populations.

The introduced Australian brushtail possum is a major vertebrate pest in New Zealand, with impacts on conservation and agriculture being managed largely through poisoning operations. Cholecalciferol (vitamin D3) is registered for use in controlling possums and despite its many advantages it is expensive and relatively inhumane. Combination of a high proportion of aspirin with a low proportion of cholecalciferol was effective in killing high proportions of groups of acclimatised, caged possums: this is attributed to both an unexpectedly high toxicity of the type of cholecalciferol used, and a proposed synergistic mechanism between the two compounds. Death was caused by localised damage to heart ventricles by aspirin, and inhibition of tissue repair by both aspirin and cholecalciferol. The observed toxicosis had lower impact on the welfare of possums than either compound administered alone, particularly aspirin alone. Residue analyses of bait remains in the GI tract suggested a low risk of secondary poisoning by either compound. The combination of cholecalciferol and aspirin has the potential to meet key requirements of cost-effectiveness and humaneness in controlling possum populations, but the effect of the combination in non-target species has yet to be tested.

Bacterial ghosts as a delivery system for zona pellucida-2 fertility control vaccines for brushtail possums (Trichosurus vulpecula).

The introduced brushtail possum is a serious pest in New Zealand and there is much interest in the development of an immunocontraceptive vaccine for population control. Immunisation of female possums against recombinant possum zona pellucida protein-2 (ZP2) is known to reduce embryo production by 72-75% but successful development of fertility control will depend on a delivery system that is effective for field use. Bacterial ghost vaccine technology is a promising system to formulate a non-living vaccine for bait or aerosol delivery. The N-terminal (amino acid residues 41-316, ZP2N) and C-terminal (amino acid residues 308-636, ZP2C) regions of possum ZP2 were fused to maltose-binding protein and expressed in the periplasmic space of Escherichia coli NM522 bacterial ghosts. Female possums (n=20 per treatment group) were immunised with 20mg of either plain ghosts, ZP2N ghosts, or ZP2C ghosts in phosphate-buffered saline applied to the nostrils and eyes (nasal/conjunctival mucosa) at weeks 0, 2 and 4. Effects of immunisation on fertility were assessed following superovulation and artificial insemination. Both constructs evoked humoral (antibody) and cell-mediated immune responses in possums and significantly fewer eggs were fertilised in females immunised against ZP2C ghosts. Results in this study indicate that bacterial ghosts containing possum ZP antigens can reduce possum fertility when delivered by mucosal immunisation and offer a promising delivery system for fertility control of wild possum populations.