Veterinary Clinical Pathology ISSN 0275-6382
ORIGINAL ARTICLE
Further investigation of the blood characteristics of Australian quoll (Dasyurus spp.) species Hayley J. Stannard, Lauren J. Young, Julie M. Old Native and Pest Animal Unit, School of Science and Health, University of Western Sydney, Penrith, NSW, Australia
Key Words Blood chemistry, dasyurid, hematology, leukocyte, marsupial Correspondence Hayley Stannard, Native and Pest Animal Unit, School of Science and Health, University of Western Sydney, Hawkesbury Campus Bldg K8, Locked Bag 1797, Penrith NSW 2751, Australia E-mail: [email protected] DOI:10.1111/vcp.12094
Background: The Eastern and Spotted-Tailed Quolls are “near threatened” Australian dasyurid marsupials that have undergone significant reduction in their geographic ranges in the past 200 years. Captive breeding and research colonies now exist, allowing further efforts to more fully understand the physiology of these carnivorous species. Objectives: The aims of the study were to provide a more detailed study of blood chemistry and differential WBC counts for Eastern and SpottedTailed Quolls, and to determine the influence by 3 biologic factors. Methods: Blood samples were taken from conscious, captive, healthy Eastern Quolls. A small number of samples from Spotted-Tailed Quolls were also available and were included in the study for comparison. Blood chemistry and differential WBC counts were compared to determine season-, age-, and sex-related differences. Results: For many of the analytes, blood chemistry results were comparable to other marsupial ranges, and no significant differences between sexes were detected (P > .05). Seasonal differences were determined for total bilirubin, glucose, creatinine, and potassium concentrations in the Eastern Quoll. Generally, higher concentrations of these analytes were observed in the summer; however, amylase activity was significantly higher in autumn (southern hemisphere). Eastern Quolls one year of age and younger had significantly (P < .05) higher ALP activities than older animals. Conclusions: The normal ranges determined in this study can be used to assess clinical health of quolls and will assist with captive management and future reintroduction programs to the wild.
Introduction Quolls are carnivorous marsupials that inhabit Papua New Guinea and Australia, with 4 species inhabiting Australia: the Eastern Quoll (Dasyurus viverrinus), Spotted-Tailed Quoll (D maculatus), Western Quoll or Chuditch (D geoffroii), and Northern Quoll (D hallucatus). Eastern Quolls are either black or fawn, with white spots on the body and no spots on the tail. They weigh between 600 and 2000 g and exhibit sexual dimorphism.1,2 The Eastern Quoll occupies forests, woodlands, and open heaths in Tasmania.2 Its geographic range has been reduced by 50–90% since European settlement, and the species is now extinct on mainland Australia.3 Pressures such as hunting, agriculture,
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predation, and competition from exotic pest species have contributed to the reduced range and loss of Eastern Quoll.2,4 Resembling the Eastern Quoll, the Spotted-Tailed Quoll is larger (up to 4 kg) and has a thick brown to reddish coat, and a long spotted tail.5–7 The SpottedTailed Quoll is the largest carnivorous marsupial on mainland Australia, since the extinction of the Thylacine (Thylacinus cynocephalus) and Tasmanian Devil (Sarcophilus harrisii).5,7 It inhabits forests, rainforests, and heathland along the eastern coast of Australia, including Tasmania.5,7 The Spotted-Tailed Quoll has also undergone a significant geographic range reduction,3 with much of its current distribution relying heavily on the abundance of prey.8 Both the Eastern
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and Spotted-Tailed Quolls are listed as “near threatened” on the 2008 International Union for the Conservation of Nature red list of threatened species.9,10 Studies of hematology and blood chemistry profiles have shown differences between marsupial and eutherian mammals, albeit minor ones. Generally, marsupials have no or very low numbers of basophils, and serum enzyme activities are higher than in eutherians.11,12 Determining reference intervals (RI) for hematologic variables within each species cohort can be more informative than reference to generic hematologic data at the class level, but these data can be difficult to obtain for threatened, native animals. Data for different populations within a species can provide a baseline to detect changes in homeostasis and immune status, as in the case of an infection.13,14 For example, analysis of hematology variables was used to determine the occurrence of anemia, polycythemia, lymphocytosis, and neutrophilia in ill Western Quolls from a captive population.15 A number of biologic factors influence hematology variables and blood chemistry analytes, such as age, sex, reproductive status, habitat, season, and nutrition. In some cases, age influences susceptibility to disease, and hydration status and gender can influence RBC counts.16 For example, differences between sexes have been found in dunnarts (Sminthopsis spp.) for neutrophil and lymphocyte counts.17 Hemoglobin concentration and HCT values have also shown a decline due to season in some dasyurid species.18–20 Therefore, it is essential to measure the influence of different physiologic factors on hematology and blood chemistry variables of different species to understand what is “normal” for a particular species. Hematology RIs have been reported for a few dasyurid species, including the Fat-Tailed Dunnart (Sminthopsis crassicaudata), Stripe-Faced Dunnart (S macroura), Western Quoll, Tasmanian Devil, and Eastern Quoll.16–24 Previous studies have determined RIs for Eastern Quoll hematology variables in males and females, and blood chemistry variables for only 2 animals.11,12,23 One study reported blood chemistry variables of 2 male Spotted-Tailed Quolls.22 However, the influence of season and age on hematology and chemistry variables has not been determined in Eastern and Spotted-Tailed Quolls. The influence of sex has been determined in Eastern Quoll hematology, but not in blood chemistry.23 In contrast, detailed descriptions of WBC morphology have been reported for all 4 Australian quoll species.16 With the limited blood chemistry data available for the Eastern and Spotted-Tailed Quolls, and no hematology data available for the Spotted-Tailed
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Quoll, this paper aims to provide a more detailed study of blood chemistry variables and differential WBC counts for Eastern and Spotted-Tailed Quolls. In addition, differences correlating with biologic factors such as age, sex, and season are presented.
Materials and Methods Study animals Captive Eastern Quolls (n = 35; 16 males and 19 females) at the Australian Ecosystems Foundation Inc. (Lithgow, NSW, Australia) and Spotted-Tailed Quolls (n = 3, 2 males and one female) at Featherdale Wildlife Park (Doonside, NSW, Australia) were used for this study. Both quoll species were housed in outdoor wire enclosures, in an environment that provided a natural photoperiod and temperature fluctuations consistent with the local area. Mean seasonal temperatures at Lithgow during the collection period were summer, 24.5°C; autumn, 18.5°C; winter, 7.5°C; spring, 12.3°C, and at Doonside, summer, 29.4°C; autumn, 24.5°C; winter, 17.3°C; and spring, 23.8°C.25 The quolls were maintained on a diet of kangaroo mince, chicken necks, insects, day-old chicks, and rats.
Blood collection and analysis Sample collection and analysis protocols were approved by the University of Western Sydney’s Animal Care and Ethics Committee, A6087. Blood samples were collected in the early morning hours to reduce the possibility of heat-related stress. Each animal was removed from the nest box and placed into a hessian bag. The individual was restrained inside the bag with the tail exposed to allow access to the lateral caudal vein. Sufficient blood volumes for blood chemistry analysis were obtained from 26 of the 35 Eastern Quolls available for this study. Up to 150 lL of blood were collected from the lateral tail vein using a 25-gauge winged infusion needle and syringe, as reported for collection from large dasyurids.16 Animals were conscious throughout the procedure. Once the blood sample was taken, the animals were returned to their enclosures, fed and monitored for signs of stress for the following hour. Eastern Quoll blood samples were collected each February, April, and October over a period of 2 years, to obtain seasonal values. As a cautionary measure and to reduce inadvertent influence on breeding cycles, samples were not collected during the winter season to avoid blood sampling-related stress and disturbed breeding behavior during the main breeding season of Eastern Quoll.
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Spotted-Tailed Quoll samples were obtained at random from 3 animals: 2 9-month-old males, and one 3-year-old female. They were included here for comparison with the Eastern Quoll. After collection, a 100 lL aliquot of whole blood without anticoagulant was immediately transferred into an analysis plate and analyzed in the VetScan Chemical Analyzer (Abaxis, CA, USA). Blood chemistry values were obtained for albumin (ALB), amylase (AMY), total bilirubin (TBIL), ALP, ALT, globulin, glucose (GLU), sodium, potassium, urea, creatinine (CRE), calcium, phosphorus, and total protein (TP) using a comprehensive rotor (Abaxis, CA, USA). The remainder (< 50 lL) of the blood was used to make a blood smear. Differential WBC counts were determined on blood smears stained with Diff Quik (SigmaAldrich, St. Louis, MO, USA). Further hematologic tests were not performed as only small sample volumes could be collected. The VetScan Analyzer was kept upto-date with new software as it was released by Abaxis, and each year it was serviced to ensure quality results. Statistical differences between seasons and age groups were determined using one-way ANOVA, and differences between sexes were determined using an unpaired t-test (Statistical Package for the Social Sciences). RIs were determined using the freeware program Reference Value Advisor.26 As the data represented a small sample size of a wildlife species (n < 40), Box-Cox transformation and a robust
method with 90% confident intervals (CIs) were used. Due to the small sample size of Spotted-Tailed Quolls, only the raw data obtained for each analyte are presented.
Results Eastern Quoll RBCs were anucleated biconcave discs ranging in diameter from 6 to 9 lm. Lymphocytes were proportionally the most abundant WBCs and ranged from 10 to 19 lm in diameter. Lymphocyte nuclei were round or bean-shaped with dark-stained chromatin and surrounded by a fine rim of basophilic cytoplasm (Figure 1A). Neutrophils were 15 to 19 lm in diameter and generally had a 3- to 5-lobed nucleus, and fine pink-staining granules in the cytoplasm. A number of neutrophils were ring neutrophils, as they had an annular nucleus (Figure 1B). Monocytes were large ranging from 15 to 20 lm in diameter and had an indented to “horseshoe” -shaped nucleus and pale cytoplasm. Eosinophils ranged from 16 to 20 lm in diameter and had a 2- to 3-lobed nucleus. The cytoplasm contained coarse round granules that stained a reddish purple color (Figure 1C). Basophils were not observed in the blood samples taken from either species. Morphology of Spotted-Tailed Quoll blood cells was similar to that of the Eastern Quoll; however,
A
B
C
D
Figure 1. WBCs in peripheral blood smears from quolls. Diff Quik. Bar = 25 lm (A) Neutrophil with an annular nucleus (left) and lymphocyte (right) from an Eastern Quoll. (B) Neutrophil with an annular nucleus from an Eastern Quoll. (C) Eosinophil (left) with coarse granules stained a reddish purple color and lymphocyte (right) from an Eastern Quoll. (D) Neutrophil with a segmented nucleus from a Spotted-Tailed Quoll.
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Table 1. Reference intervals for WBC differential counts and blood chemistry variables of Eastern Quoll.
Differential WBC Count (n = 35) Lymphocytes (%) Neutrophils (%) Ring-Neutrophils (%) Monocytes (%) Eosinophils (%) Basophils (%) Blood Chemistry (n = 26) Albumin (g/L) ALP (U/L) ALT (U/L) Amylase (U/L) Total Bilirubin (lmol/L) Urea (lmol/L) Calcium (lmol/L) Phosphorus (mmol/L) Creatinine (lmol/L) Glucose (mmol/L) Sodium (mmol/L) Potassium (mmol/L) Total Protein (g/L) Globulin (g/L)
Reference Interval*
Median (Min–Max)
Mean SD 66.9 14.5 22.0 13.4 7.1 3.9 3.0 2.1 1.0 1.7 0
66 (28–95) 20 (3–63) 6.5 (0–17) 3 (0–8) 0 (0–8) 0 (0)
11.5–44.8 44.0–503.7 19.6–169.3 21.0–1216.5 3.2–9.0 6.5–26.5 1.5–3.5 0.7–3.9 11.5–70.3 2.3–9.5 112.5–181.0 2.1–8.2 13.2–67.8 16.2–43.1
28 (8–34) 171 (55–529) 44 (19–153) 518 (219–1261) 5 (4–8) 19 (5.5–27.1) 2.4 (1.1–2.7) 2.1 (0.8–3.8) 31 (18–61) 5.5 (2.3–9.9) 146 (100–155) 5.2 (2.1–8.5) 57 (20–66) 25 (19–37)
90% Confidence Interval Lower Limit
90% Confidence Interval Upper Limit
5.4–17.9 31.7–71.4 18.7–21.8 116.8–193.3 3.0–3.8 5.2–11.0 1.0–1.8 0.4–1.1 8.9–16.5 1.2–3.1 102.7–126.8 0.8–3.2 19.0–41.7 14.4–19.5
38.9–48.5 369.2–637.5 108.5–250.8 1035.8–1382.2 7.7–9.3 24.5–28.2 3.1–3.7 3.4–4.3 56.8–78.8 8.3–10.6 164.5–192.0 7.3–9.0 69.2–80.6 36.3–47.7
*Reference intervals were determined using Reference Value Advisor.26
WBCs with annular nuclei were only occasionally observed. Lymphocytes measured 10 to 16 lm in diameter. Neutrophils were 16 to 20 lm in diameter with a finely granulated cytoplasm and segmented nuclei with 3- to 6-lobes (Figure 1D). Monocytes were 18 to 22 lm in diameter with “horseshoe” -shaped nuclei. Eosinophils were 16 to 18 lm diameter. There was no significant difference (P > .05) between male and female Eastern Quolls for differential WBC counts and blood chemistry analytes (Table 1). Significantly higher concentrations were determined for TBIL (F2,25 = 7.128; P = .004), CRE (F2,25 = 3.333; P = .054), GLU (F2,25 = 5.644; P = .010), and potassium (F2,25 = 5.644; P = .016) in summer compared with autumn. Amylase activity was significantly higher (F2,25 = 3.262; P = .05; Table 2) in autumn compared with spring. There was no significant difference in the concentrations of blood chemistry analytes between spring and summer (Table 2). Twelve of the 26 Eastern Quolls were one year old, 10 were 2 years old, and 4 were 3 years old. A significant difference (F2,25 = 5.911; P = .008) was found for ALP activities between the ages of one and 2 years (P < .05), and one and 3 years (P < .05). ALP activities in older Eastern Quolls were around 50% the activities of the one-year-old animals (ALP: one year: 328 131 U/L; 2 year: 176 72 U/L; 3 year: 136 65 U/L).
Table 2. Blood chemistry variables in Eastern Quolls in summer, autumn, and spring.
Albumin (g/L) ALP (U/L) ALT (U/L) Amylase (U/L) Total Bilirubin (lmol/L) Urea (mmol/L) Calcium (mmol/L) Phosphorus (mmol/L) Creatinine (lmol/L) Glucose (mmol/L) Sodium (mmol/L) Potassium (mmol/L) Total Protein (g/L) Globulin (g/L)
Summer n=9
Autumn n = 12
Spring n=5
27.5 9.2 248.4 137 64.8 31.4 588.1 254 6.6 1.2
26 5 217.7 115.9 49.4 37.9 710.5 320.2 4.8 0.75
20.8 9.7 141 70.4 60.2 48.4 447.4 157* 5.6 1.1**
19.5 5.9 2.4 0.49 2.4 0.75 44.3 12 7 1.5 139.4 16.1 6.4 1.2 53.7 13 24.8 3.4
16.8 3.1 2.2 0.32 2.3 0.76 29.8 10.3 5 1.3 147.9 3.6 4.8 0.69 53.3 6.2 27.1 5.5
16.6 5.4 2.1 0.68 1.7 0.71 36 17.1** 4.8 1.8** 128.4 26.1 4.5 2** 50.8 17.7 31.5 8.4
Data are means standard deviation. *P < .05; **P < .01.
In comparison, the Spotted-Tailed Quolls generally showed similar concentrations and activities of blood chemistry analytes. The Spotted-Tailed Quolls had lower activity of ALP, ALT, and AMY, and globulin concentration was also lower (Table 3). It should be noted that one male Spotted-Tailed Quoll had much
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Table 3. Differential WBC counts and blood chemistry variables of 3 Spotted-Tailed Quolls.
Differential WBC Count Lymphocytes (%) Neutrophils (%) Ring-Neutrophil (%) Monocytes (%) Eosinophils (%) Basophils (%) Blood Chemistry Albumin (g/L) ALP (U/L) ALT (U/L) Amylase (U/L) Total Bilirubin (lmol/L) Urea (mmol/L) Calcium (mmol/L) Phosphorus (mmol/L) Creatinine (lmol/L) Glucose (mmol/L) Sodium (mmol/L) Potassium (mmol/L) Total Protein (g/L) Globulin (g/L)
Male 1
Male 2*
Female
65 25 5 4 1 0
67 26 7 0 0 0
46 45 3 5 1 0
46 115 22 148 9 13.2 2.47 2.06 94 7.9 144 6.2 67 21
2 15 8 16 6 2 0.85 0.47 43 2.6 100 – 10 –
40 65 31 202 7 18.2 2.37 1.64 93 7.6 143 5.3 69 29
*Although the values from male 2 are much lower than those from the other 2 quolls, male 2 appeared healthy and the blood was not clotted.
lower values for each analyte than the other 2 animals, but there was no obvious rationale to exclude its data from this report, as the animal appeared to be in good health at the time of sampling, and the collected blood was not clotted.
Discussion Although hematology and blood chemistry profiles have been studied previously in Eastern and SpottedTailed Quolls, sample numbers were often small and thus not representative for a population.12,22 In the present study, correlations between sexes, age, and season for hematology and chemistry variables were calculated in a captive population of Eastern Quoll. Specifically, it appears that serum enzyme activities in Eastern Quolls were influenced by season, and ALP activity was influenced by age. Generally, the morphology of RBCs and WBCs in the Eastern and Spotted-Tailed Quolls was consistent with previous descriptions for both species, and similar to that of other dasyurid species, although some minor differences in the diameter and number and proportion of granulocytes were recorded. As reported for dunnarts,17 ringed neutrophils were common in quoll blood.
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Mean concentrations for blood chemistry analytes determined for the 2 quoll species were within the range observed for other dasyurids.11,12,21,22,24 Mean activity levels for ALP and AMY were higher and ALB concentration was lower when compared with the ranges for the Tasmanian devil.21 Enzyme activities in the Eastern and Spotted-Tailed Quolls were similar to those determined in the Western Quoll.24 Results for TBIL concentrations were higher and TP concentrations lower in both quoll species, and CRE concentrations lower in the Eastern Quoll compared with general eutherian ranges. In Eastern Quolls, the mean CRE activity varied significantly across seasons. Reduced CRE activity has been associated with poor nutrition and, consequently, loss of muscle mass in dolphins (Tursiops truncates), harbor seals (Phoca vitulina), and Eurasian badgers (Meles meles).27–29 In the Eastern Quolls, these reduced activity levels should not be related to nutrition as feeding was managed by human caretakers. Food items were rotated daily, possibly accounting for change in CRE between sampling periods. Elevated CRE in spring and summer could also suggest a temporary increase in muscle mass, which could be due to increased physical activity during that time of the year. Low urea concentrations have also been attributed to poor nutrition in the agile wallaby (Macropus agilis);30 however, urea activity in the quolls was higher than in the wallaby and did not differ significantly across the seasons, thus suggesting no seasonal change in nutritional status in the Eastern Quolls. Previously, GLU and urea values were determined to be higher in marsupials than in people;12 however, the results for GLU and urea from quolls in this study were comparable to a normal human range.31 Seasonal changes in GLU in Eastern Quolls could relate to hydration, although water was available ad libitum. Such GLU variations have also been reported in the Brushtail Possum (Trichosurus vulpecula), where they were associated with stress, and possibly water availability.32,33 In Bobcats (Felis rufus), capture stress was believed to be the reason for elevated GLU concentrations.34 These and other factors, such as capturerelated stress, changes in housing and animal groups or pairs, or hormonal changes related to breeding, could have influenced GLU concentrations in this study. The determined range of TBIL concentrations for Eastern Quolls fell within the range observed for the Western Quoll,24 but this level was significantly higher in summer compared with autumn. Opposing results were found in free-living Mountain Brushtail Possums (Trichosurus caninus),35 where TBIL concentrations were significantly lower in summer and autumn
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compared with winter and spring.35 TBIL can be related to liver function and, although quolls appeared to be healthy at the time of sampling, change in TBIL concentrations may suggest a current or recent change in liver function. Eastern Quolls reach sexual maturity at one year of age,2 and as ALP concentration is associated with skeletal growth activity(osteogenesis), it is reasonable to assume that Eastern Quolls under one year of age have higher ALP activities in the blood. Higher ALP values in juveniles have been determined for most domestic animals, and also for marsupials and other mammals, both in captivity and in the wild, including Western Quoll, Brush-Tailed Rock Wallaby (Petrogale penicillata), Tammar Wallaby (Macropus eugenii), Northern Hairy-Nosed Wombat (Lasiorhinus krefftii), Japanese Macaques (Macaca fuscata), wolves (Canis lupus), and Rock Hyrax (Procavia capensis).24,36–41 ALP activity in Eastern Quolls showed large variations between individuals; similarly large variations in ALP activity have been observed previously in 3 captive murid species.42 The data in the present study were generated from a captive population where the animals were exposed to natural environmental conditions rather than controlled housing; consequently, identified trends may be related to seasonal cues and climatic conditions of the local area. Although based on a relatively small sample size and despite the absence of validation data, the RI determined in the current study enable useful comparisons with Eastern Quolls translocated into the surrounding area in the future. Both quoll species have become important to conserve due to significant geographic range contractions and the extinction of the Eastern Quoll on mainland Australia. Spotted-Tailed Quoll blood chemistry should be based on a larger sample size of animals to determine whether the data obtained here are typical for this species. Nevertheless, the data presented here can now be used in combination with other indicators of health such as body condition, appetite, and mobility to provide a valuable resource for maintenance of captive animals and in the monitoring of translocated animals postrelease.
Acknowledgments We would like to thank the staff at AEFI and FWP for allowing access to their animals. Thanks to Dr Michelle Bingley for assistance with sample collection. The study was funded by a UWS Postgraduate Research Award and an Australian Geographic grant given to HJS. Disclosure: The authors have indicated that they have no affiliations or financial involvement with any organiza-
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tion or entity with a financial interest in, or in financial competition with, the subject matter or materials discussed in this article.
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22. Parsons R, Guiler ER. Observations on the blood of the marsupial tiger cat, Dasyurops maculatus (Kerr) (Dasyuridae). Comp Biochem Physiol A. 1972;43:935–939. 23. Melrose WD, Pearse AM, Jupe DMD, Baikie MJ, Twin JE, Bryant SL. Haematology of the Australian eastern quoll, Dasyurus viverrinus. Comp Biochem Physiol A. 1987;88:239–241. 24. Svensson A, Mills JN, Boardman WSJ, Huntress S. Haematology and serum biochemistry reference values for anesthetized chuditch (Dasyurus Geoffroii). J Zoo and Wildl Med. 1998;29:311–314. 25. Bureau of Meteorology. Climate Online Data. Available at: www.bom.gov.au/climate/data/. Accessed October 18, 2011. 26. Geffre A, Concordet D, Braun JP, Trumel C. Reference Value Advisor: a new freeware set of macroinstructions to calculate reference intervals with Microsoft Excel. Vet Clin Pathol. 2011;40:107–112. 27. Domingo-Roura X, Newman C, Calafell F, Macdonald DW. Blood biochemistry reflects seasonal nutritional and reproductive constraints in the Eurasian badger (Meles meles). Physiol Biochem Zool. 2001;74:450–460. 28. Trumble SJ, Castellini MA, Mau TL, Castellini JM. Dietary and seasonal influences on blood chemistry and hematology in captive harbour seals. Mar Mammal Sci. 2006;22:104–123. 29. Hall AJ, Wells RS, Sweeney JC, et al. Annual, seasonal and individual variation in hematology and clinical blood chemistry profiles in bottlenose dolphins (Tursi-
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36. McKenzie S, Deane EM, Burnett L. Haematology and serum biochemistry of the tammar wallaby, Macropus eugenii. Comp Clin Pathol. 2002;11:229–237. 37. Barnes TS, Goldizen AW, Coleman GT. Haematology and serum biochemistry of the brush-tailed rock-wallaby (Petrogale penicillata). J Wildl Dis. 2008;44:295–303. 38. Reiss A, Portas T, Horsup A. Hematologic and serum biochemical reference values for free-ranging northern hairy-nosed wombats. J Wildl Dis. 2008;44:65–70. 39. Takenaka A, Gotoh S, Watanabe T, Takenaka O. Developmental changes of plasma alkaline phosphatase, calcium, and inorganic phosphorus in relation to the growth of bones in the Japanese macaque (Macaca fuscata). Primates. 1988;29:395–404. 40. Seal US, Mech LD, Van Ballenberghe V. Blood analyses of wolf pups and their ecological and metabolic interpretation. J Mammal. 1975;56:64–75. 41. Aroch I, King R, Baneth G. Hematology and serum biochemistry values of trapped, healthy, free-ranging rock hyraxes (Procavia capensis) and their association with age, sex, and gestational status. Vet Clin Pathol. 2007;36:40–48. 42. Old JM, Connelly L, Francis J, Branch K, Fry G, Deane EM. Haematology and serum biochemistry of three Australian desert murids: the plains rat (Pseudomys austrlis), the Spinifex hopping-mouse (Notomys alexis) and the central rock-rat (Zyzomys pedunculatus). Comp Clin Pathol. 2005;14:130–137.
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ORIGINAL ARTICLE
Further investigation of the blood characteristics of Australian quoll (Dasyurus spp.) species Hayley J. Stannard, Lauren J. Young, Julie M. Old Native and Pest Animal Unit, School of Science and Health, University of Western Sydney, Penrith, NSW, Australia
Key Words Blood chemistry, dasyurid, hematology, leukocyte, marsupial Correspondence Hayley Stannard, Native and Pest Animal Unit, School of Science and Health, University of Western Sydney, Hawkesbury Campus Bldg K8, Locked Bag 1797, Penrith NSW 2751, Australia E-mail: [email protected] DOI:10.1111/vcp.12094
Background: The Eastern and Spotted-Tailed Quolls are “near threatened” Australian dasyurid marsupials that have undergone significant reduction in their geographic ranges in the past 200 years. Captive breeding and research colonies now exist, allowing further efforts to more fully understand the physiology of these carnivorous species. Objectives: The aims of the study were to provide a more detailed study of blood chemistry and differential WBC counts for Eastern and SpottedTailed Quolls, and to determine the influence by 3 biologic factors. Methods: Blood samples were taken from conscious, captive, healthy Eastern Quolls. A small number of samples from Spotted-Tailed Quolls were also available and were included in the study for comparison. Blood chemistry and differential WBC counts were compared to determine season-, age-, and sex-related differences. Results: For many of the analytes, blood chemistry results were comparable to other marsupial ranges, and no significant differences between sexes were detected (P > .05). Seasonal differences were determined for total bilirubin, glucose, creatinine, and potassium concentrations in the Eastern Quoll. Generally, higher concentrations of these analytes were observed in the summer; however, amylase activity was significantly higher in autumn (southern hemisphere). Eastern Quolls one year of age and younger had significantly (P < .05) higher ALP activities than older animals. Conclusions: The normal ranges determined in this study can be used to assess clinical health of quolls and will assist with captive management and future reintroduction programs to the wild.
Introduction Quolls are carnivorous marsupials that inhabit Papua New Guinea and Australia, with 4 species inhabiting Australia: the Eastern Quoll (Dasyurus viverrinus), Spotted-Tailed Quoll (D maculatus), Western Quoll or Chuditch (D geoffroii), and Northern Quoll (D hallucatus). Eastern Quolls are either black or fawn, with white spots on the body and no spots on the tail. They weigh between 600 and 2000 g and exhibit sexual dimorphism.1,2 The Eastern Quoll occupies forests, woodlands, and open heaths in Tasmania.2 Its geographic range has been reduced by 50–90% since European settlement, and the species is now extinct on mainland Australia.3 Pressures such as hunting, agriculture,
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predation, and competition from exotic pest species have contributed to the reduced range and loss of Eastern Quoll.2,4 Resembling the Eastern Quoll, the Spotted-Tailed Quoll is larger (up to 4 kg) and has a thick brown to reddish coat, and a long spotted tail.5–7 The SpottedTailed Quoll is the largest carnivorous marsupial on mainland Australia, since the extinction of the Thylacine (Thylacinus cynocephalus) and Tasmanian Devil (Sarcophilus harrisii).5,7 It inhabits forests, rainforests, and heathland along the eastern coast of Australia, including Tasmania.5,7 The Spotted-Tailed Quoll has also undergone a significant geographic range reduction,3 with much of its current distribution relying heavily on the abundance of prey.8 Both the Eastern
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and Spotted-Tailed Quolls are listed as “near threatened” on the 2008 International Union for the Conservation of Nature red list of threatened species.9,10 Studies of hematology and blood chemistry profiles have shown differences between marsupial and eutherian mammals, albeit minor ones. Generally, marsupials have no or very low numbers of basophils, and serum enzyme activities are higher than in eutherians.11,12 Determining reference intervals (RI) for hematologic variables within each species cohort can be more informative than reference to generic hematologic data at the class level, but these data can be difficult to obtain for threatened, native animals. Data for different populations within a species can provide a baseline to detect changes in homeostasis and immune status, as in the case of an infection.13,14 For example, analysis of hematology variables was used to determine the occurrence of anemia, polycythemia, lymphocytosis, and neutrophilia in ill Western Quolls from a captive population.15 A number of biologic factors influence hematology variables and blood chemistry analytes, such as age, sex, reproductive status, habitat, season, and nutrition. In some cases, age influences susceptibility to disease, and hydration status and gender can influence RBC counts.16 For example, differences between sexes have been found in dunnarts (Sminthopsis spp.) for neutrophil and lymphocyte counts.17 Hemoglobin concentration and HCT values have also shown a decline due to season in some dasyurid species.18–20 Therefore, it is essential to measure the influence of different physiologic factors on hematology and blood chemistry variables of different species to understand what is “normal” for a particular species. Hematology RIs have been reported for a few dasyurid species, including the Fat-Tailed Dunnart (Sminthopsis crassicaudata), Stripe-Faced Dunnart (S macroura), Western Quoll, Tasmanian Devil, and Eastern Quoll.16–24 Previous studies have determined RIs for Eastern Quoll hematology variables in males and females, and blood chemistry variables for only 2 animals.11,12,23 One study reported blood chemistry variables of 2 male Spotted-Tailed Quolls.22 However, the influence of season and age on hematology and chemistry variables has not been determined in Eastern and Spotted-Tailed Quolls. The influence of sex has been determined in Eastern Quoll hematology, but not in blood chemistry.23 In contrast, detailed descriptions of WBC morphology have been reported for all 4 Australian quoll species.16 With the limited blood chemistry data available for the Eastern and Spotted-Tailed Quolls, and no hematology data available for the Spotted-Tailed
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Quoll, this paper aims to provide a more detailed study of blood chemistry variables and differential WBC counts for Eastern and Spotted-Tailed Quolls. In addition, differences correlating with biologic factors such as age, sex, and season are presented.
Materials and Methods Study animals Captive Eastern Quolls (n = 35; 16 males and 19 females) at the Australian Ecosystems Foundation Inc. (Lithgow, NSW, Australia) and Spotted-Tailed Quolls (n = 3, 2 males and one female) at Featherdale Wildlife Park (Doonside, NSW, Australia) were used for this study. Both quoll species were housed in outdoor wire enclosures, in an environment that provided a natural photoperiod and temperature fluctuations consistent with the local area. Mean seasonal temperatures at Lithgow during the collection period were summer, 24.5°C; autumn, 18.5°C; winter, 7.5°C; spring, 12.3°C, and at Doonside, summer, 29.4°C; autumn, 24.5°C; winter, 17.3°C; and spring, 23.8°C.25 The quolls were maintained on a diet of kangaroo mince, chicken necks, insects, day-old chicks, and rats.
Blood collection and analysis Sample collection and analysis protocols were approved by the University of Western Sydney’s Animal Care and Ethics Committee, A6087. Blood samples were collected in the early morning hours to reduce the possibility of heat-related stress. Each animal was removed from the nest box and placed into a hessian bag. The individual was restrained inside the bag with the tail exposed to allow access to the lateral caudal vein. Sufficient blood volumes for blood chemistry analysis were obtained from 26 of the 35 Eastern Quolls available for this study. Up to 150 lL of blood were collected from the lateral tail vein using a 25-gauge winged infusion needle and syringe, as reported for collection from large dasyurids.16 Animals were conscious throughout the procedure. Once the blood sample was taken, the animals were returned to their enclosures, fed and monitored for signs of stress for the following hour. Eastern Quoll blood samples were collected each February, April, and October over a period of 2 years, to obtain seasonal values. As a cautionary measure and to reduce inadvertent influence on breeding cycles, samples were not collected during the winter season to avoid blood sampling-related stress and disturbed breeding behavior during the main breeding season of Eastern Quoll.
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Spotted-Tailed Quoll samples were obtained at random from 3 animals: 2 9-month-old males, and one 3-year-old female. They were included here for comparison with the Eastern Quoll. After collection, a 100 lL aliquot of whole blood without anticoagulant was immediately transferred into an analysis plate and analyzed in the VetScan Chemical Analyzer (Abaxis, CA, USA). Blood chemistry values were obtained for albumin (ALB), amylase (AMY), total bilirubin (TBIL), ALP, ALT, globulin, glucose (GLU), sodium, potassium, urea, creatinine (CRE), calcium, phosphorus, and total protein (TP) using a comprehensive rotor (Abaxis, CA, USA). The remainder (< 50 lL) of the blood was used to make a blood smear. Differential WBC counts were determined on blood smears stained with Diff Quik (SigmaAldrich, St. Louis, MO, USA). Further hematologic tests were not performed as only small sample volumes could be collected. The VetScan Analyzer was kept upto-date with new software as it was released by Abaxis, and each year it was serviced to ensure quality results. Statistical differences between seasons and age groups were determined using one-way ANOVA, and differences between sexes were determined using an unpaired t-test (Statistical Package for the Social Sciences). RIs were determined using the freeware program Reference Value Advisor.26 As the data represented a small sample size of a wildlife species (n < 40), Box-Cox transformation and a robust
method with 90% confident intervals (CIs) were used. Due to the small sample size of Spotted-Tailed Quolls, only the raw data obtained for each analyte are presented.
Results Eastern Quoll RBCs were anucleated biconcave discs ranging in diameter from 6 to 9 lm. Lymphocytes were proportionally the most abundant WBCs and ranged from 10 to 19 lm in diameter. Lymphocyte nuclei were round or bean-shaped with dark-stained chromatin and surrounded by a fine rim of basophilic cytoplasm (Figure 1A). Neutrophils were 15 to 19 lm in diameter and generally had a 3- to 5-lobed nucleus, and fine pink-staining granules in the cytoplasm. A number of neutrophils were ring neutrophils, as they had an annular nucleus (Figure 1B). Monocytes were large ranging from 15 to 20 lm in diameter and had an indented to “horseshoe” -shaped nucleus and pale cytoplasm. Eosinophils ranged from 16 to 20 lm in diameter and had a 2- to 3-lobed nucleus. The cytoplasm contained coarse round granules that stained a reddish purple color (Figure 1C). Basophils were not observed in the blood samples taken from either species. Morphology of Spotted-Tailed Quoll blood cells was similar to that of the Eastern Quoll; however,
A
B
C
D
Figure 1. WBCs in peripheral blood smears from quolls. Diff Quik. Bar = 25 lm (A) Neutrophil with an annular nucleus (left) and lymphocyte (right) from an Eastern Quoll. (B) Neutrophil with an annular nucleus from an Eastern Quoll. (C) Eosinophil (left) with coarse granules stained a reddish purple color and lymphocyte (right) from an Eastern Quoll. (D) Neutrophil with a segmented nucleus from a Spotted-Tailed Quoll.
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Table 1. Reference intervals for WBC differential counts and blood chemistry variables of Eastern Quoll.
Differential WBC Count (n = 35) Lymphocytes (%) Neutrophils (%) Ring-Neutrophils (%) Monocytes (%) Eosinophils (%) Basophils (%) Blood Chemistry (n = 26) Albumin (g/L) ALP (U/L) ALT (U/L) Amylase (U/L) Total Bilirubin (lmol/L) Urea (lmol/L) Calcium (lmol/L) Phosphorus (mmol/L) Creatinine (lmol/L) Glucose (mmol/L) Sodium (mmol/L) Potassium (mmol/L) Total Protein (g/L) Globulin (g/L)
Reference Interval*
Median (Min–Max)
Mean SD 66.9 14.5 22.0 13.4 7.1 3.9 3.0 2.1 1.0 1.7 0
66 (28–95) 20 (3–63) 6.5 (0–17) 3 (0–8) 0 (0–8) 0 (0)
11.5–44.8 44.0–503.7 19.6–169.3 21.0–1216.5 3.2–9.0 6.5–26.5 1.5–3.5 0.7–3.9 11.5–70.3 2.3–9.5 112.5–181.0 2.1–8.2 13.2–67.8 16.2–43.1
28 (8–34) 171 (55–529) 44 (19–153) 518 (219–1261) 5 (4–8) 19 (5.5–27.1) 2.4 (1.1–2.7) 2.1 (0.8–3.8) 31 (18–61) 5.5 (2.3–9.9) 146 (100–155) 5.2 (2.1–8.5) 57 (20–66) 25 (19–37)
90% Confidence Interval Lower Limit
90% Confidence Interval Upper Limit
5.4–17.9 31.7–71.4 18.7–21.8 116.8–193.3 3.0–3.8 5.2–11.0 1.0–1.8 0.4–1.1 8.9–16.5 1.2–3.1 102.7–126.8 0.8–3.2 19.0–41.7 14.4–19.5
38.9–48.5 369.2–637.5 108.5–250.8 1035.8–1382.2 7.7–9.3 24.5–28.2 3.1–3.7 3.4–4.3 56.8–78.8 8.3–10.6 164.5–192.0 7.3–9.0 69.2–80.6 36.3–47.7
*Reference intervals were determined using Reference Value Advisor.26
WBCs with annular nuclei were only occasionally observed. Lymphocytes measured 10 to 16 lm in diameter. Neutrophils were 16 to 20 lm in diameter with a finely granulated cytoplasm and segmented nuclei with 3- to 6-lobes (Figure 1D). Monocytes were 18 to 22 lm in diameter with “horseshoe” -shaped nuclei. Eosinophils were 16 to 18 lm diameter. There was no significant difference (P > .05) between male and female Eastern Quolls for differential WBC counts and blood chemistry analytes (Table 1). Significantly higher concentrations were determined for TBIL (F2,25 = 7.128; P = .004), CRE (F2,25 = 3.333; P = .054), GLU (F2,25 = 5.644; P = .010), and potassium (F2,25 = 5.644; P = .016) in summer compared with autumn. Amylase activity was significantly higher (F2,25 = 3.262; P = .05; Table 2) in autumn compared with spring. There was no significant difference in the concentrations of blood chemistry analytes between spring and summer (Table 2). Twelve of the 26 Eastern Quolls were one year old, 10 were 2 years old, and 4 were 3 years old. A significant difference (F2,25 = 5.911; P = .008) was found for ALP activities between the ages of one and 2 years (P < .05), and one and 3 years (P < .05). ALP activities in older Eastern Quolls were around 50% the activities of the one-year-old animals (ALP: one year: 328 131 U/L; 2 year: 176 72 U/L; 3 year: 136 65 U/L).
Table 2. Blood chemistry variables in Eastern Quolls in summer, autumn, and spring.
Albumin (g/L) ALP (U/L) ALT (U/L) Amylase (U/L) Total Bilirubin (lmol/L) Urea (mmol/L) Calcium (mmol/L) Phosphorus (mmol/L) Creatinine (lmol/L) Glucose (mmol/L) Sodium (mmol/L) Potassium (mmol/L) Total Protein (g/L) Globulin (g/L)
Summer n=9
Autumn n = 12
Spring n=5
27.5 9.2 248.4 137 64.8 31.4 588.1 254 6.6 1.2
26 5 217.7 115.9 49.4 37.9 710.5 320.2 4.8 0.75
20.8 9.7 141 70.4 60.2 48.4 447.4 157* 5.6 1.1**
19.5 5.9 2.4 0.49 2.4 0.75 44.3 12 7 1.5 139.4 16.1 6.4 1.2 53.7 13 24.8 3.4
16.8 3.1 2.2 0.32 2.3 0.76 29.8 10.3 5 1.3 147.9 3.6 4.8 0.69 53.3 6.2 27.1 5.5
16.6 5.4 2.1 0.68 1.7 0.71 36 17.1** 4.8 1.8** 128.4 26.1 4.5 2** 50.8 17.7 31.5 8.4
Data are means standard deviation. *P < .05; **P < .01.
In comparison, the Spotted-Tailed Quolls generally showed similar concentrations and activities of blood chemistry analytes. The Spotted-Tailed Quolls had lower activity of ALP, ALT, and AMY, and globulin concentration was also lower (Table 3). It should be noted that one male Spotted-Tailed Quoll had much
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Table 3. Differential WBC counts and blood chemistry variables of 3 Spotted-Tailed Quolls.
Differential WBC Count Lymphocytes (%) Neutrophils (%) Ring-Neutrophil (%) Monocytes (%) Eosinophils (%) Basophils (%) Blood Chemistry Albumin (g/L) ALP (U/L) ALT (U/L) Amylase (U/L) Total Bilirubin (lmol/L) Urea (mmol/L) Calcium (mmol/L) Phosphorus (mmol/L) Creatinine (lmol/L) Glucose (mmol/L) Sodium (mmol/L) Potassium (mmol/L) Total Protein (g/L) Globulin (g/L)
Male 1
Male 2*
Female
65 25 5 4 1 0
67 26 7 0 0 0
46 45 3 5 1 0
46 115 22 148 9 13.2 2.47 2.06 94 7.9 144 6.2 67 21
2 15 8 16 6 2 0.85 0.47 43 2.6 100 – 10 –
40 65 31 202 7 18.2 2.37 1.64 93 7.6 143 5.3 69 29
*Although the values from male 2 are much lower than those from the other 2 quolls, male 2 appeared healthy and the blood was not clotted.
lower values for each analyte than the other 2 animals, but there was no obvious rationale to exclude its data from this report, as the animal appeared to be in good health at the time of sampling, and the collected blood was not clotted.
Discussion Although hematology and blood chemistry profiles have been studied previously in Eastern and SpottedTailed Quolls, sample numbers were often small and thus not representative for a population.12,22 In the present study, correlations between sexes, age, and season for hematology and chemistry variables were calculated in a captive population of Eastern Quoll. Specifically, it appears that serum enzyme activities in Eastern Quolls were influenced by season, and ALP activity was influenced by age. Generally, the morphology of RBCs and WBCs in the Eastern and Spotted-Tailed Quolls was consistent with previous descriptions for both species, and similar to that of other dasyurid species, although some minor differences in the diameter and number and proportion of granulocytes were recorded. As reported for dunnarts,17 ringed neutrophils were common in quoll blood.
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Mean concentrations for blood chemistry analytes determined for the 2 quoll species were within the range observed for other dasyurids.11,12,21,22,24 Mean activity levels for ALP and AMY were higher and ALB concentration was lower when compared with the ranges for the Tasmanian devil.21 Enzyme activities in the Eastern and Spotted-Tailed Quolls were similar to those determined in the Western Quoll.24 Results for TBIL concentrations were higher and TP concentrations lower in both quoll species, and CRE concentrations lower in the Eastern Quoll compared with general eutherian ranges. In Eastern Quolls, the mean CRE activity varied significantly across seasons. Reduced CRE activity has been associated with poor nutrition and, consequently, loss of muscle mass in dolphins (Tursiops truncates), harbor seals (Phoca vitulina), and Eurasian badgers (Meles meles).27–29 In the Eastern Quolls, these reduced activity levels should not be related to nutrition as feeding was managed by human caretakers. Food items were rotated daily, possibly accounting for change in CRE between sampling periods. Elevated CRE in spring and summer could also suggest a temporary increase in muscle mass, which could be due to increased physical activity during that time of the year. Low urea concentrations have also been attributed to poor nutrition in the agile wallaby (Macropus agilis);30 however, urea activity in the quolls was higher than in the wallaby and did not differ significantly across the seasons, thus suggesting no seasonal change in nutritional status in the Eastern Quolls. Previously, GLU and urea values were determined to be higher in marsupials than in people;12 however, the results for GLU and urea from quolls in this study were comparable to a normal human range.31 Seasonal changes in GLU in Eastern Quolls could relate to hydration, although water was available ad libitum. Such GLU variations have also been reported in the Brushtail Possum (Trichosurus vulpecula), where they were associated with stress, and possibly water availability.32,33 In Bobcats (Felis rufus), capture stress was believed to be the reason for elevated GLU concentrations.34 These and other factors, such as capturerelated stress, changes in housing and animal groups or pairs, or hormonal changes related to breeding, could have influenced GLU concentrations in this study. The determined range of TBIL concentrations for Eastern Quolls fell within the range observed for the Western Quoll,24 but this level was significantly higher in summer compared with autumn. Opposing results were found in free-living Mountain Brushtail Possums (Trichosurus caninus),35 where TBIL concentrations were significantly lower in summer and autumn
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compared with winter and spring.35 TBIL can be related to liver function and, although quolls appeared to be healthy at the time of sampling, change in TBIL concentrations may suggest a current or recent change in liver function. Eastern Quolls reach sexual maturity at one year of age,2 and as ALP concentration is associated with skeletal growth activity(osteogenesis), it is reasonable to assume that Eastern Quolls under one year of age have higher ALP activities in the blood. Higher ALP values in juveniles have been determined for most domestic animals, and also for marsupials and other mammals, both in captivity and in the wild, including Western Quoll, Brush-Tailed Rock Wallaby (Petrogale penicillata), Tammar Wallaby (Macropus eugenii), Northern Hairy-Nosed Wombat (Lasiorhinus krefftii), Japanese Macaques (Macaca fuscata), wolves (Canis lupus), and Rock Hyrax (Procavia capensis).24,36–41 ALP activity in Eastern Quolls showed large variations between individuals; similarly large variations in ALP activity have been observed previously in 3 captive murid species.42 The data in the present study were generated from a captive population where the animals were exposed to natural environmental conditions rather than controlled housing; consequently, identified trends may be related to seasonal cues and climatic conditions of the local area. Although based on a relatively small sample size and despite the absence of validation data, the RI determined in the current study enable useful comparisons with Eastern Quolls translocated into the surrounding area in the future. Both quoll species have become important to conserve due to significant geographic range contractions and the extinction of the Eastern Quoll on mainland Australia. Spotted-Tailed Quoll blood chemistry should be based on a larger sample size of animals to determine whether the data obtained here are typical for this species. Nevertheless, the data presented here can now be used in combination with other indicators of health such as body condition, appetite, and mobility to provide a valuable resource for maintenance of captive animals and in the monitoring of translocated animals postrelease.
Acknowledgments We would like to thank the staff at AEFI and FWP for allowing access to their animals. Thanks to Dr Michelle Bingley for assistance with sample collection. The study was funded by a UWS Postgraduate Research Award and an Australian Geographic grant given to HJS. Disclosure: The authors have indicated that they have no affiliations or financial involvement with any organiza-
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tion or entity with a financial interest in, or in financial competition with, the subject matter or materials discussed in this article.
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