Hematology and Plasma Biochemistry of Wild Spectacled Flying Foxes (Pteropus conspicillatus) in Australia

The spectacled flying fox is a large frugivorous bat with a restricted Australian mainland range in Far North Queensland (Churchill 2008), integral to the Wet Tropics World Heritage area ecosystem as an important rain forest pollinator and seed disperser (Fox 2005). The North Queensland population is in decline (Tait et al. 2014), putatively contributed to decreasing habitat, climatic extremes, anthropogenic activities, and mass mortality events associated with tick paralysis and neonatal cleft palate syndrome (Tait et al. 2014). The species was listed in 2002 as vulnerable in Australia, under the Commonwealth Environment Protection and Biodiversity and Conservation Act 1999, yet knowledge of fundamental physiologic parameters of the species is limited, as published reference ranges are based on small numbers of captive or wild-caught animals, including opportunistic sampling of injured flying foxes. This study presents ranges of a suite of hematologic and biochemical analytes for apparently healthy adult wild-caught spectacled flying foxes and compares values between sex cohorts, and with previously published findings for the paraphyletic black flying foxes (Almeida et al. 2014; McMichael et al. 2015). Our findings provide baseline information essential to understand and meaningfully interpret flying fox population health in ecologic and epidemiologic contexts.

Blood and urine samples were collected from 50 wild-caught (28 males and 22 females), apparently healthy (determined by veterinary physical examination and assessment of any injuries or conditions) adult spectacled flying foxes during a single capture event in June 2015 (winter in the Southern hemisphere) at a rural roost at Gordonvale (17°06′S, 145°47′E), in Far North Queensland, Australia. Fieldwork was conducted under the Queensland Department of Agriculture, Fisheries and Forestry Animal Ethics Committee Permit SA 2011/12/375, and Department of Environment, Heritage and Protection Scientific Purposes Permits WISP05810609 and WISP14100614. Bats were captured before dawn in mist nets (18 m wide×4 m deep) hoisted between two trees within the roost site. Animals were anesthetized (under veterinary supervision) using the inhalation agent isoflurane and medical oxygen by facemask (Jonsson et al. 2004). Bats were assessed, sampled, and released at the capture site as per McMichael et al. (2015). Urine was collected by gentle manual expression of the urinary bladder.

All blood samples were collected within 2 h of capture. Two milliliters of blood was collected from the propatagial (cephalic) vein and dispensed into a 1.3-mL lithium heparin blood tube (Sarstedt 41.1393.105, Sarstedt, Nümbrecht, Germany) and a 0.5-mL EDTA tube (Microtainer 5974, Becton, Dickinson, Franklin Lakes, New Jersey, USA). Blood glucose concentration was measured at the time of bleeding by using an ACCU-CHEK Performa^TM glucometer (Roche Diagnostics GmbH, Mannheim, Germany). Plasma was separated by centrifugation within 2 h of bleeding. Samples were kept chilled before daily shipment to a commercial pathology laboratory (Queensland Medical Laboratories, Brisbane, Australia) for manual blood smear examination, complete blood counts using an XE-2100^TM automated hematology system (Sysmex Corp., Kobe, Japan), and complete biochemical analysis (ADVIA 2400 chemistry system, Siemens AG, Berlin, Germany). All values are reported in standard international units. Urine biochemistry analytes were measured within 2 h of urine collection by using Urispec Plus reagent test strips (Henry Schein 900-3567, Henry Schein Animal Health, Dublin, Ohio, USA). Urine specific gravity was measured using a hand-held clinical refractometer.

Reference ranges for hematologic (Table 1) and plasma and urine (Table 2) analytes were calculated according to Friedrichs et al. (2012), whereby the reference ranges were calculated as ±2 SD following outlier elimination. In addition, the effects of sex and species on hematologic (Table 3) and plasma and urine (Table 4) analytes were calculated. The species comparative analysis used data collected for black flying foxes by McMichael et al. (2015). This analysis was based on the contention that spectacled flying foxes and black flying foxes are paraphyletic species (Almeida et al. 2014), follow synchronous life cycles (Churchill 2008), were sampled at the same time of year, and have similar physiologic values and responses (McMichael et al. 2015). Each hematologic or biochemical variable was subjected to a restricted maximum likelihood analysis using GenStat (VSN International 2015). Normal or log normal distributions were used as appropriate for continuous variables. Where values for analytes were lower than the detectable limit of the assay, we adopted the recommended practice of assigning half of the lowest detectable concentration for the comparative analyses (Wood et al. 2011). Predicted mean values, SEs of the means, and significance levels are reported for each variable.

The reference ranges for spectacled flying foxes are temporally comparable to black flying fox population and sex cohort analyte values (McMichael et al. 2015, 2016). Higher protein, albumin, and glucose values of spectacled flying foxes likely reflect a higher quality and quantity of food resources in a tropical rain forest habitat, whereas higher triglycerides may be consistent with black flying fox winter depletion of fatty acid reserves with putative thermoregulation demands of subtropical southeastern Queensland winters (McMichael et al. 2017). Intraerythrocytic protozoal parasites, subsequently characterised as Hepatocystis sp. (Schaer et al. 2018), were present in 56% (28 of 50) of spectacled flying foxes compared to only 4% (16 of 385) of black flying foxes, likely reflecting differing climatic conditions conducive to vector presence and transmission. For spectacled flying foxes, the higher eosinophil and platelet counts, higher aspartate transferase (AST), alanine transferase (ALT), and gamma-glutamyl transferase (GGT) values, and lower hemoglobin, mean corpuscular hemoglobin, and mean corpuscular volume values are putatively consistent with the higher prevalence of intraerythrocytic parasites (Opara and Fagbemi 2008). Liver enzyme (AST, ALT, and GGT) elevations are alternatively an interesting parallel to the emergence of neonatal cleft palate syndrome in spectacled flying foxes, as June corresponds to early pregnancy and likely embryonic palatogenesis in flying foxes (Bush and Jiang 2012). By contrast, toxin exposure, plausibly causing liver enzyme derangement, during early pregnancy is known to cause birth defects in mammals (Rogers and Kavloc 1996). Our findings provide baseline data essential to measure and meaningfully interpret flying fox population health. First, in an ecologic and conservation context where ongoing mass mortality events threaten the vulnerable spectacled flying fox population in Far North Queensland; and second, in an epidemiologic context due to the importance of Pteropus sp. as known reservoir hosts of zoonotic infections.