Physiological biomarkers of Hendra virus infection in Australian Flying-foxes (Pteropus spp.)

Abstract

Since 1994 when the disease was first described, there have been over 50 Hendra virus incidents involving more than 90 confirmed or possible equine cases, and 7 associated human cases, with case fatality rates approaching 90% and 60% respectively. Epidemiological studies suggest that flying-foxes, in particular the Black flying-fox (Pteropus alecto) and the closely related Spectacled flying-fox (P. conspicillatus) are the primary natural reservoir hosts. This research project investigates a hypothesised causal association between ecological and/or physiological stress and Hendra virus infection in Pteropus species. During a 12-month study of flying-foxes in Southeast Queensland (SEQ), samples were collected from 446 captured wild P. alecto for analysis of 18 hematologic, 22 biochemical and 8 urinary biomarkers, establishing normal reference ranges and temporal changes for this suite of biomarkers. The population demonstrated statistically significant temporal variation in some biomarkers consistent with life cycle events. In Hendra virus RNA-positive animals all biomarker mean values were within established normal ranges, but some were significantly different compared to Hendra virus RNA-negative animals, notably, increased lymphocyte percent, decreased neutrophil percent, decreased plasma triglyceride levels, increased plasma alkaline phosphatase levels and increased urinary protein levels. The study demonstrated that Hendra virus infection in P. alecto was sub-clinical, and that there was no apparent relationship with nutritional stress, reproductive stress or extreme metabolic demand, which would have caused substantial changes to biomarker values. The relationship between increased urinary protein levels, decreased triglyceride levels and Hendra virus infection is of interest, and may support the premise of urinary tract association and physiological demand with Hendra virus infection of P. alecto. In a second study, a method of collection and assay of urinary cortisol to measure physiological stress in roosting flying-foxes was validated. Differences between the four mainland flying-fox species (P. alecto, P. poliocephalus, P. scapulatus, P. conspicillatus) were established through collection of urine samples from a wide geographic range throughout Queensland and New South Wales from single species roosts. Then, over a three-year study, population urinary cortisol and Hendra virus urinary excretion prevalence was measured from a total of 2208 pooled urine samples from mixed species roosts of flying-foxes from two geographically distinct regions, subtropical SEQ and tropical Far North Queensland (FNQ). An autumn elevation in urinary cortisol in the FNQ population was detected, but no significant seasonal fluctuations in Hendra virus excretion prevalence were found. Urinary cortisol was significantly elevated in both autumn and winter in the SEQ population, the latter temporally associated with a significant winter elevation in Hendra virus excretion prevalence. There was also a strong correlation between urinary cortisol concentration and minimum temperature on the day of sampling, with urinary cortisol increasing exponentially as temperature decreased. As mixed species population urinary cortisol measurements have the inherent difficulty of an inability to interpret the effect of species and cohort, a novel urinary collection and molecular analysis method was employed on 464 individual animal urine samples collected from the SEQ roost site over a 12-month study to assess the effects of sex and species on cortisol levels and Hendra virus excretion status. P. alecto was found to be the only species present at the site excreting Hendra virus, and there were no significant differences between sex for either Hendra virus excretion, or urinary cortisol excretion levels. The autumn peak in urinary cortisol is most plausibly primarily driven by the physiological demand of peak mating; the winter peak in urinary cortisol measurements, which is temporally correlated with a pulse in Hendra virus excretion, is not associated with reproductive stressors and may be driven by the physiological demand of thermoregulation of the historically tropical species P. alecto in a subtropical/temperate niche during winter. The research addresses significant knowledge gaps in relation to baseline physiological biomarkers in flying-foxes, tests hypotheses on risk factors for Hendra virus infection in P. alecto, and provides a platform for further investigation of the disease ecology of Hendra virus in flying-foxes. More broadly, it provides a valuable template for the investigation of the disease ecology of various wildlife-associated emerging zoonoses of public health significance.