First Detection and Circulation of RHDV2 in New Zealand.

Rabbit haemorrhage disease virus 2 (RHDV2) is a highly pathogenic lagovirus that causes lethal disease in rabbits and hares (lagomorphs). Since its first detection in Europe in 2010, RHDV2 has spread worldwide and has been detected in over 35 countries so far. Here, we provide the first detailed report of the detection and subsequent circulation of RHDV2 in New Zealand. RHDV2 was first detected in New Zealand in 2018, with positive samples retrospectively identified in December 2017. Subsequent time-resolved phylogenetic analysis suggested a single introduction into the North Island between March and November 2016. Genetic analysis identified a GI.3P-GI.2 variant supporting a non-Australian origin for the incursion; however, more accurate identification of the source of the incursion remains challenging due to the wide global distribution of the GI.3P-GI.2 variant. Furthermore, our analysis suggests the spread of the virus between the North and South Islands of New Zealand at least twice, dated to mid-2017 and around 2018. Further phylogenetic analysis also revealed a strong phylogeographic pattern. So far, no recombination events with endemic benign New Zealand rabbit caliciviruses have been identified. This study highlights the need for further research and surveillance to monitor the distribution and diversity of lagoviruses in New Zealand and to detect incursions of novel variants.

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.

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.

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.

The mixed liver and heart transcriptome dataset of the New Zealand brushtail possum, Trichosurus vulpecula.

New Zealand suffers greatly from invasive mammal predators including rats, stoats, feral cats and possums all of which not only damage or prey on New Zealand's unique terrestrial biodiversity, but also have huge impact on NZ's economy as many of these pests act as vectors of disease to farm and game animals. As such, the NZ government has invested nearly $90 m to support an ambitious plan to make the country predator free by 2050. Although there are adequate means to control invasive predator populations, it is widely agreed that current technologies are not sufficient for total eradication and that improved technologies are required. The Achilles Heel approach is one such developmental technology that attempts to exploit variation in the genes of target species that are vital to key physiological or cellular pathways within the body, such that interference with these genes will cause a species-specific death without the harmful effects on the environment and non-targets species that the current suite of control agents engender. Interference could either be through species-specific gene knock-down using such agents as siRNA and/or the use of species-selective chemical toxicants specifically developed against these targets. To assist with identifying species-specific gene targets in the New Zealand brushtail possum (Trichosurus vulpecula) we have assembled and annotated a possum mixed heart and liver transcriptome.

Assessment of endocrine disruption and oxidative potential of bisphenol-A, triclosan, nonylphenol, diethylhexyl phthalate, galaxolide, and carbamazepine, common contaminants of municipal biosolids.

The use of biosolids as a soil conditioner and fertiliser is hindered by the limited knowledge on the risks of micro-contaminants they contain. This study investigated the binding of six organic contaminants commonly found in biosolids, to the estrogen (ER), androgen (AR), aryl hydrocarbon (AhR), and transthyretin (TTR) receptors and their redox activity. Triclosan (TCS), bisphenol-A (BPA), and technical nonylphenol (TNP) had affinity for the TTR with relative potencies of 0.3, 0.03, and 0.076 respectively. Further, binding to TTR was the only toxicological response observed for carbamazepine, which induced sub-maximal response and relative potency of 0.0017. Estrogenic activity was induced by BPA, galaxolide (HHCB), diethylhexyl phthalate (DEHP) and TNP with BPA having the strongest potency of 5.1 × 10-6 relative to estradiol. Only BPA showed androgenic activity but it was not quantifiable. BPA also showed anti-androgenic activity along with TCS, HHCB, and TNP in the order of TNP > HHCB > TCS ~ BPA (relative potencies 0.126, 0.042, 0.032, 0.03). No compounds exhibited anti-estrogenic or AhR activity, or were redox-active in the dithiothreitol assay. The results highlight the multiple modes of action through which these compounds may impact exposed organisms, and the concentrations at which effects may occur. This allows assessment of the likelihood of effects being observed at environmental concentrations, and the potential contribution of these compounds.

Exploratory investigation of the chemical characteristics and relative toxicity of ambient air particulates from two New Zealand cities.

We examined the chemical composition and biological response associated with particulate emissions from the two largest cities in New Zealand, Auckland and Christchurch. The organic and water-soluble fractions were isolated from the particulate matter (PM). The organic fraction was examined for PAH content, direct mutagenicity, CYP1A1 induction, and cytotoxicity and TNF-alpha release in RAW264.7 macrophages. The water-soluble fraction was examined for metal content, and cytotoxicity and TNF-alpha release in RAW264.7 macrophages. Particulate, PAH and water-soluble metal concentrations were all higher in PM collected from Christchurch, being highest in May-July when woodburners for home heating are widely in use. In contrast, PM from Auckland showed the highest concentrations in March, but PAH and metal concentrations were highest in July. We found marked differences in the biological response elicited by ambient air PM: the organic extracts of Christchurch PM(2.5) and PM(10) showed higher mutagenicity and CYP1A1 induction compared with PM(10) from Auckland. In contrast, water-soluble extracts of Auckland PM were more cytotoxic and resulted in greater TNF-alpha release than those from Christchurch PM, although they had a lower metal content. The organic fraction of PM from both cities did not induce any cytokine release, and the organic extract from Auckland samples showed no cytotoxicity; smaller PM mass was available for testing for these samples. Biological responses typically occurred at lower doses of the organic extract, indicating that organic components may be more important in eliciting effects than water-soluble components. Preliminary apportionment of the biological responses to the dominant sources of PM in both cities-woodburners and vehicles-was undertaken. This indicated that for both cities, vehicles have a greater contribution to the direct mutagenic activity of ambient PM than woodsmoke, despite a lower contribution to ambient PM. In contrast, woodsmoke is estimated to have a greater contribution to CYP1A1 induction of ambient PM. The calculated activity forms only a small proportion of the activity observed in extracts of ambient PM from Christchurch, particularly for mutagenicity, and may indicate a significant influence of atmospheric transformation processes on biological response. Only data for mutagenicity and CYP1A1 activity could be used for apportionment as low and/or variable cytotoxicity or TNF-alpha release response were obtained for either the individual source or ambient PM at the doses tested. Further, in the case of the water-soluble extracts from Auckland, additional components are suggested to have a role in the observed activity.

The effect of ambient air pollution on respiratory health of school children: a panel study.

BACKGROUND: Adverse respiratory effects of particulate air pollution have been identified by epidemiological studies. We aimed to examine the health effects of ambient particulate air pollution from wood burning on school-age students in Christchurch, New Zealand, and to explore the utility of urine and exhaled breath condensate biomarkers of exposure in this population. METHODS: A panel study of 93 male students (26 with asthma) living in the boarding house of a metropolitan school was undertaken in the winter of 2004. Indoor and outdoor pollution data was continuously monitored. Longitudinal assessment of lung function (FEV1 and peak flow) and symptoms were undertaken, with event studies of high pollution on biomarkers of exposure (urinary 1-hydroxypyrene) and effect (exhaled breath condensate (EBC) pH and hydrogen peroxide concentration). RESULTS: Peak levels of air pollution were associated with small but statistically significant effects on lung function in the asthmatic students, but not healthy students. No significant effect of pollution could be seen either on airway inflammation and oxidative stress either in healthy students or students with asthma. Minor increases in respiratory symptoms were associated with high pollution exposure. Urinary 1-hydroxypyrene levels were raised in association with pollution events by comparison with low pollution control days. CONCLUSION: There is no significant effect of ambient wood-smoke particulate air pollution on lung function of healthy school-aged students, but a small effect on respiratory symptoms. Asthmatic students show small effects of peak pollution levels on lung function. Urinary 1-hydroxypyrene shows potential as a biomarker of exposure to wood smoke in this population; however measurement of EBC pH and hydrogen peroxide appears not to be useful for assessment of population health effects of air pollution.Some of the data presented in this paper has previously been published in Kingham and co-workers Atmospheric Environment, 2006 Jan; 40: 338-347 (details of pollution exposure), and Cavanagh and co-workers Sci Total Environ. 2007 Mar 1;374(1):51-9 (urine hydroxypyrene data).

Elevated concentrations of 1-hydroxypyrene in schoolchildren during winter in Christchurch, New Zealand.

Particulate air pollution is significantly elevated during the winter in Christchurch, New Zealand, largely attributable to use of wood burners for domestic home heating, topography, and meteorological conditions. Polycyclic aromatic hydrocarbons (PAHs) are a key component of airborne particulate matter (PM) and urinary 1-hydroxypyrene (1-OHP) has previously been used to assess exposure of people to PAHs. We examined urinary 1-OHP in Christchurch male non-smoking schoolchildren (12-18 yr) on two occasions after high pollution events (48 and 72 microg PM(10)/m(3) 24-h average) and two occasions during periods of low pollution (19 and 12 microg PM(10)/m(3)). Concentrations of urinary 1-OHP were significantly elevated in the students during high pollution events (median (mean+/-SD) 0.043 (0.051+/-0.032) and 0.042 (0.060+/-0.092) micromol OHP/mol creatinine respectively) compared with low pollution periods (median (mean+/-SD) 0.019 (0.026+/-0.032) and 0.025 (0.028+/-0.018) micromol/mol creatinine respectively). The observed 1-OHP concentrations are at the lower end of those determined in children and non-occupationally exposed adults in international studies and suggest a generally low exposure to PAHs. The increased urinary 1-OHP concentrations following nights of elevated particulate concentrations in ambient air suggest increased exposure to ambient air pollution during winter time, and could potentially be used as a biomarker of exposure in this population.

2,3,7,8-TCDD equivalence and mutagenic activity associated with PM10 from three urban locations in New Zealand.

Ambient particulate matter (PM(10)) in urban centres varies depending on emission sources, geography, demography, and meteorology. Hence physical (PM(10), wind speed, rainfall, temperature), chemical (polycyclic aromatic hydrocarbons, PAH), and toxicological (Ames Test, H4IIE EROD Assay) analyses were done on daily PM(10) (approximately 1640 m(3)/day) collected from three New Zealand urban sites where winter emissions were predominantly due to domestic home heating. Daily PM(10) levels ranged between 9.7 and 20.8 in summer and between 21.8 and 61.0 microg/m(3) in winter. Daily PAH concentrations were 0.5, 0.45, and 1.5 ng/m(3) in summer and 52.1, 128.9, and 5.8 ng/m(3) in winter at sites Christchurch, Alexandra and Dunedin, respectively. During winter, 74% of PM(10) extracts from all three sites showed significant mutagenicity in the Ames Test (TA 98, -S9), whereas approximately 25% of the daily PM(10) was mutagenic in summer. Benzo[a]pyrene and BaP carcinogenic equivalence concentrations during winter were strongly correlated to both mutagenicity and TCDD-like activity at two sites. Daily levels of TCDD toxicity equivalence concentrations ranged from 0.5 to 3.6 pg TCDD/m(3) air in summer and from 0.3 to 4009 pg TCDD/m(3) air in winter. Chemically and biologically derived TCDD toxicity equivalent concentrations were significantly correlated in all study locations indicating that PAH may represent most of the TCDD-like activity present in the PM(10).

Molecular adsorption at particle surfaces: a PM toxicity mediation mechanism.

Fine atmospheric particles depositing in the lung present a large adsorbent surface for the adsorption of bronchoalveolar lining fluid (BALF) components, including lung surfactant and its associated proteins. Such adsorption at invading particle surfaces is known to be important in biological particle clearance, and the immunological and toxicological fate of these particles. In the experiments conducted here, it was hypothesized that this is also true for particles of nonbiological origin, and that fine particles with large surface areas would selectively adsorb the opsonizing components of BALF. This work quantifies the adsorption rates (adsorption of compound per unit surface area) of isolated BALF components. Elemental carbon (EC) is a ubiquitous component of fine urban particulate matter (PM2.5), and particular forms of EC are extremely surface active (e.g., activated carbon). EC originates largely from fossil fuel combustion, and vehicles in particular contribute a significant proportion of PM(2.5) EC mass in urban areas. Since the size distribution of EC is submicrometer, industrially produced carbon blacks in the 25-100 nm size range can be used as a surrogate for urban EC, in terms of surface area and chemistry. Three types of carbon black (CB) particles were used. Two were identical in size (25 nm) but different in surface treatment; R330, a CB with a nonoxidized surface, and R400, a CB produced with an oxidized surface. The third particle type, M120, was 75 nm, different in size from R330 and R400, but similar to R330 in surface chemistry, that is, nonoxidized. Particles were first washed and resuspended in phosphate-buffered-saline (PBS, pH 7.0) three times to remove surfactant coatings added during their manufacture. Colloidal suspensions of M120, R330, and R400 particles with decreasing surface areas were then generated and separated into reaction vials. BALF proteins were added spanning physiological concentrations while the dominant phospholipid in surfactant was added at a fixed concentration lower than physiological lung lavage concentrations to ensure the lipid remained in suspension during experimentation ex situ. For dipalmitoylphosphatidylcholine (DPPC) combinations with particles, visible particle agglomeration occurred within 1 h. Marked changes in the size distribution of the immersed particles were observed, compared to a phosphate buffer control. Differences in particle agglomeration and particle settling were observed between M120, R330, and R400. Reduction of DPPC occurred in a surface- and size-dependent manner. This indicates that surface adsorption was responsible for the observed agglomeration and the gross reductions in phospholipid concentrations. Combination of particles with fibrinogen and albumin revealed little agglomeration/precipitation at the protein concentrations chosen. However, surfactant protein (SP-D) was completely eliminated from suspension upon combination with all three-particle types. This reaction between SP-D particles was therefore concluded to be independent of surface chemistry. Further investigation as to whether this is size- or surface-area-dependent is recommended. The biological implication is that molecular adsorption at nonbiological particulate matter (PM) surfaces in BALF may mediate the toxicity of PM via one or both of these mechanisms, as in the case of biological particles.