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Further investigation of the blood parameters of Australian quoll (Dasyurus spp.) species

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Further investigation of the blood parameters of Australian quoll (Dasyurus spp.) species
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  O R I G I N A L A R T I C L E FurtherinvestigationofthebloodcharacteristicsofAustralianquoll( Dasyurus spp.)species HayleyJ.Stannard,LaurenJ.Young,JulieM.Old NativeandPestAnimalUnit,SchoolofScienceandHealth,UniversityofWesternSydney,Penrith,NSW,Australia KeyWords Bloodchemistry,dasyurid,hematology,leukocyte,marsupial Correspondence HayleyStannard,NativeandPestAnimalUnit,SchoolofScienceandHealth,UniversityofWesternSydney,Hawkesbury CampusBldgK8,LockedBag1797,PenrithNSW2751, AustraliaE-mail:h.stannard@uws.edu.auDOI:10.1111/vcp.12094 Background:  TheEasternandSpotted-TailedQuollsare“nearthreatened”Australian dasyurid marsupials that have undergone significant reductionin their geographic ranges in the past 200 years. Captive breeding andresearch colonies now exist, allowing further efforts to more fully under-standthephysiologyofthesecarnivorousspecies. Objectives:  The aims of the study were to provide a more detailed study of blood chemistry and differential WBC counts for Eastern and Spotted-TailedQuolls,andtodeterminetheinfluenceby3biologicfactors. Methods:  Blood samples were taken from conscious, captive, healthyEastern Quolls. A small number of samples from Spotted-Tailed Quollswere also available and were included in the study for comparison. Bloodchemistry and differential WBC counts were compared to determineseason-,age-,andsex-relateddifferences. Results:  For many of the analytes, blood chemistry results were compara- ble to other marsupial ranges, and no significant differences between sexesweredetected( P >  .05).Seasonaldifferencesweredeterminedfortotalbil-irubin, glucose, creatinine, and potassium concentrations in the EasternQuoll. Generally, higher concentrations of these analytes were observed inthesummer;however,amylaseactivitywassignificantlyhigherinautumn(southern hemisphere). Eastern Quolls one year of age and younger hadsignificantly( P   <  .05)higherALPactivitiesthanolderanimals. Conclusions:  The normal ranges determined in this study can be used toassess clinical health of quolls and will assist with captive management andfuturereintroductionprogramstothewild. Introduction Quolls are carnivorous marsupials that inhabit PapuaNew Guinea and Australia, with 4 species inhabitingAustralia:theEasternQuoll (Dasyurusviverrinus ),Spot-ted-Tailed Quoll ( D maculatus ), Western Quoll or Chu-ditch ( D geoffroii  ), and Northern Quoll ( D hallucatus ).Eastern Quolls are either black or fawn, with whitespots on the body and no spots on the tail. They weigh between 600 and 2000 g and exhibit sexual dimor-phism. 1,2 The Eastern Quoll occupies forests, wood-lands, and open heaths in Tasmania. 2 Its geographicrange has been reduced by 50  –  90% since Europeansettlement,andthespeciesisnowextinct onmainlandAustralia. 3 Pressures such as hunting, agriculture,predation, and competition from exotic pest specieshavecontributedtothereducedrangeandlossofEast-ernQuoll. 2,4 Resembling the Eastern Quoll, the Spotted-TailedQuoll is larger (up to 4 kg) and has a thick brown toreddish coat, and a long spotted tail. 5  –  7 The Spotted-Tailed Quoll is the largest carnivorous marsupial onmainland Australia, since the extinction of the Thyla-cine ( 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 hasalso undergone a significant geographic range reduc-tion, 3 with much of its current distribution relyingheavily on the abundance of prey. 8 Both the Eastern VetClinPathol 42/4(2013)476–482 ©  2013AmericanSocietyforVeterinaryClinicalPathologyandEuropeanSocietyforVeterinaryClinicalPathology 476 VeterinaryClinicalPathology ISSN0275-6382  and Spotted-Tailed Quolls are listed as “near threa-tened” on the 2008 International Union for the Con-servationofNatureredlistofthreatenedspecies. 9,10 Studies of hematology and blood chemistry pro-files have shown differences between marsupial andeutherian mammals, albeit minor ones. Generally,marsupials have no or very low numbers of basophils,and serum enzyme activities are higher than in euthe-rians. 11,12 Determining reference intervals (RI) for he-matologic variables within each species cohort can bemore informative than reference to generic hemato-logic data at the class level, but these data can be diffi-cult to obtain for threatened, native animals. Data fordifferent populations within a species can provide a baselinetodetect changesinhomeostasisandimmunestatus, as in the case of an infection. 13,14 For example,analysis of hematology variables was used to deter-mine the occurrence of anemia, polycythemia, lym-phocytosis, and neutrophilia in ill Western Quollsfromacaptivepopulation. 15 Anumberofbiologicfactorsinfluencehematologyvariables and blood chemistry analytes, such as age,sex,reproductivestatus,habitat,season,andnutrition.In some cases, age influences susceptibility to disease,and hydration status and gender can influence RBCcounts. 16 Forexample,differencesbetweensexeshave been found in dunnarts ( Sminthopsis  spp.) for neutro-philandlymphocytecounts. 17 Hemoglobinconcentra-tion and HCT values have also shown a decline due toseason in some dasyurid species. 18  –  20 Therefore, it isessential to measure the influence of different physio-logic factors on hematology and blood chemistry vari-ables of different species to understand what is“normal”foraparticularspecies.Hematology RIs have been reported for a fewdasyurid species, including the Fat-Tailed Dunnart( Sminthopsis crassicaudata ), Stripe-Faced Dunnart( S macroura ), Western Quoll, Tasmanian Devil, andEastern Quoll. 16  –  24 Previous studies have determinedRIs for Eastern Quoll hematology variables in malesand females, and blood chemistry variables for only 2animals. 11,12,23 One study reported blood chemistryvariables of 2 male Spotted-Tailed Quolls. 22 However,the influence of season and age on hematology andchemistry variables has not been determined in East-ern and Spotted-Tailed Quolls. The influence of sexhasbeendeterminedinEasternQuollhematology,butnot in blood chemistry. 23 In contrast, detailed descrip-tions of WBC morphology have been reported for all 4Australianquollspecies. 16 With the limited blood chemistry data availablefor the Eastern and Spotted-Tailed Quolls, and nohematology data available for the Spotted-TailedQuoll, this paper aims to provide a more detailed studyof blood chemistry variables and differential WBCcounts for Eastern and Spotted-Tailed Quolls. In addi-tion, differences correlating with biologic factors suchasage,sex,andseasonarepresented. MaterialsandMethods Studyanimals Captive Eastern Quolls ( n  =  35; 16 males and 19females) at the Australian Ecosystems Foundation Inc.(Lithgow, NSW, Australia) and Spotted-Tailed Quolls( n  =  3,2malesandonefemale)atFeatherdaleWildlifePark (Doonside, NSW, Australia) were used for thisstudy. Both quoll species were housed in outdoor wireenclosures, in an environment that provided a naturalphotoperiod and temperature fluctuations consistentwith the local area. Mean seasonal temperatures atLithgow 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 weremaintained on a diet of kangaroo mince, chickennecks,insects,day-oldchicks,andrats. Bloodcollectionandanalysis Sample collection and analysis protocols wereapproved by the University of Western Sydney’s Ani-malCareandEthicsCommittee,A6087.Bloodsampleswere collected in the early morning hours to reducethe possibility of heat-related stress. Each animal wasremoved from the nest box and placed into a hessian bag. The individual was restrained inside the bag withthe tail exposed to allow access to the lateral caudalvein. Sufficient blood volumes for blood chemistryanalysis were obtained from 26 of the 35 Eastern Quo-lls available for this study. Up to 150  l L of blood werecollected from the lateral tail vein using a 25-gaugewinged infusion needle and syringe, as reported forcollection from large dasyurids. 16 Animals were con-scious throughout the procedure. Once the blood sam-ple was taken, the animals were returned to theirenclosures,fed andmonitoredfor signsofstressforthefollowing hour. Eastern Quoll blood samples were col-lected each February, April, and October over a periodof 2 years, to obtain seasonal values. As a cautionarymeasure and to reduce inadvertent influence on breeding cycles, samples were not collected during thewinter season to avoid blood sampling-related stressand disturbed breeding behavior during the main breedingseasonofEasternQuoll. VetClinPathol 42/4(2013)476–482 ©  2013AmericanSocietyforVeterinaryClinicalPathologyandEuropeanSocietyforVeterinaryClinicalPathology  477 Stannardetal Quollbloodcharacteristics  Spotted-Tailed Quoll samples were obtained atrandomfrom3animals:29-month-oldmales,andone3-year-old female. They were included here for com-parisonwiththeEasternQuoll.After collection, a 100  l L aliquot of whole bloodwithout anticoagulant was immediately transferredinto an analysis plate and analyzed in the VetScanChemical Analyzer (Abaxis, CA, USA). Blood chemis-try values were obtained for albumin (ALB), amylase(AMY), total bilirubin (TBIL), ALP, ALT, globulin, glu-cose (GLU), sodium, potassium, urea, creatinine(CRE), calcium, phosphorus, and total protein (TP)using a comprehensive rotor (Abaxis, CA, USA). Theremainder ( <  50  l L) of the blood was used to make a blood smear. Differential WBC counts were deter-mined on blood smears stained with Diff Quik (Sigma-Aldrich, St. Louis, MO, USA). Further hematologictestswerenotperformedasonlysmallsamplevolumescould be collected. The VetScan Analyzer was kept up-to-datewithnewsoftwareasitwasreleasedbyAbaxis,andeachyearitwasservicedtoensurequalityresults.Statistical differences between seasons and agegroups were determined using one-way ANOVA, anddifferences between sexes were determined using anunpaired  t  -test (Statistical Package for the SocialSciences). RIs were determined using the freewareprogram Reference Value Advisor. 26 As the datarepresented a small sample size of a wildlife species( n  <  40), Box-Cox transformation and a robustmethod 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 arepresented. Results Eastern Quoll RBCs were anucleated biconcave discsranging in diameter from 6 to 9  l m. Lymphocyteswere proportionally the most abundant WBCs andranged from 10 to 19  l m in diameter. Lymphocytenuclei were round or bean-shaped with dark-stainedchromatin and surrounded by a fine rim of basophiliccytoplasm (Figure 1A). Neutrophils were 15 to 19  l min diameter and generally had a 3- to 5-lobed nucleus,and fine pink-staining granules in the cytoplasm. Anumber of neutrophils were ring neutrophils, as theyhad an annular nucleus (Figure 1B). Monocytes werelargerangingfrom15to20  l mindiameterandhadanindented to “horseshoe” -shaped nucleus and palecytoplasm. Eosinophils ranged from 16 to 20  l m indiameter and had a 2- to 3-lobed nucleus. The cyto-plasm contained coarse round granules that stained areddish purple color (Figure 1C). Basophils were notobserved in the blood samples taken from eitherspecies.Morphology of Spotted-Tailed Quoll blood cellswas similar to that of the Eastern Quoll; however, A BC D Figure 1.  WBCsinperipheralbloodsmearsfromquolls.DiffQuik.Bar  =  25  l m( A )Neutrophilwithanannularnucleus(left)andlymphocyte(right)froman Eastern Quoll. ( B ) Neutrophil with an annular nucleus from an Eastern Quoll. ( C ) Eosinophil (left) with coarse granules stained a reddish purple colorandlymphocyte(right) fromanEasternQuoll. ( D )Neutrophil withasegmented nucleusfromaSpotted-Tailed Quoll. VetClinPathol 42/4(2013)476–482 ©  2013AmericanSocietyforVeterinaryClinicalPathologyandEuropeanSocietyforVeterinaryClinicalPathology 478 Quollbloodcharacteristics Stannardetal  WBCs with annular nuclei were only occasionallyobserved. Lymphocytes measured 10 to 16  l m indiameter. Neutrophils were 16 to 20  l m in diameterwith a finely granulated cytoplasm and segmentednuclei with 3- to 6-lobes (Figure 1D). Monocytes were18 to 22  l m in diameter with “horseshoe” -shapednuclei.Eosinophilswere16to18  l mdiameter.There was no significant difference ( P   >  .05) between male and female Eastern Quolls for differen-tial WBC counts and blood chemistry analytes(Table 1). Significantly higher concentrations weredetermined for TBIL (F 2,25  =  7.128;  P   =  .004), CRE(F 2,25  =  3.333; P   =  .054),GLU(F 2,25  =  5.644; P   =  .010),and potassium (F 2,25  =  5.644;  P   =  .016) in summercompared with autumn. Amylase activity was sig-nificantly higher (F 2,25  =  3.262;  P   =  .05; Table 2) inautumn compared with spring. There was no signifi-cant difference in the concentrations of blood chemis-tryanalytesbetweenspringandsummer(Table 2).Twelveofthe26EasternQuollswereoneyearold,10 were 2 years old, and 4 were 3 years old. A signifi-cant difference (F 2,25  =  5.911;  P   =  .008) was found forALP activities between the ages of one and 2 years( P   <  .05),andoneand3 years( P   <  .05).ALPactivitiesinolderEastern Quollswerearound50%theactivitiesof the one-year-old animals (ALP: one year:328    131 U/L; 2 year: 176    72 U/L; 3 year:136    65 U/L).In comparison, the Spotted-Tailed Quolls gener-ally showed similar concentrations and activities of blood chemistry analytes. The Spotted-Tailed QuollshadloweractivityofALP,ALT,andAMY,andglobulinconcentration was also lower (Table 3). It should benoted that one male Spotted-Tailed Quoll had much Table 1.  Reference intervalsforWBCdifferentialcounts andbloodchemistryvariablesofEasternQuoll.Reference Interval* Median(Min  –  Max) 90%ConfidenceInterval LowerLimit 90%ConfidenceInterval UpperLimitDifferential WBCCount( n  =  35) Mean    SDLymphocytes(%) 66.9    14.5 66(28  –  95)Neutrophils(%) 22.0    13.4 20(3  –  63)Ring-Neutrophils(%) 7.1    3.9 6.5(0  –  17)Monocytes (%) 3.0    2.1 3 (0  –  8)Eosinophils (%) 1.0    1.7 0 (0  –  8)Basophils(%) 0 0(0)BloodChemistry( n  =  26)Albumin(g/L) 11.5  –  44.8 28(8  –  34) 5.4  –  17.9 38.9  –  48.5ALP(U/L) 44.0  –  503.7 171(55  –  529) 31.7  –  71.4 369.2  –  637.5ALT(U/L) 19.6  –  169.3 44(19  –  153) 18.7  –  21.8 108.5  –  250.8Amylase (U/L) 21.0  –  1216.5 518(219  –  1261) 116.8  –  193.3 1035.8  –  1382.2TotalBilirubin ( l mol/L) 3.2  –  9.0 5 (4  –  8) 3.0  –  3.8 7.7  –  9.3Urea( l mol/L) 6.5  –  26.5 19(5.5  –  27.1) 5.2  –  11.0 24.5  –  28.2Calcium ( l mol/L) 1.5  –  3.5 2.4(1.1  –  2.7) 1.0  –  1.8 3.1  –  3.7Phosphorus(mmol/L) 0.7  –  3.9 2.1(0.8  –  3.8) 0.4  –  1.1 3.4  –  4.3Creatinine ( l mol/L) 11.5  –  70.3 31(18  –  61) 8.9  –  16.5 56.8  –  78.8Glucose (mmol/L) 2.3  –  9.5 5.5(2.3  –  9.9) 1.2  –  3.1 8.3  –  10.6Sodium(mmol/L) 112.5  –  181.0 146(100  –  155) 102.7  –  126.8 164.5  –  192.0Potassium (mmol/L) 2.1  –  8.2 5.2(2.1  –  8.5) 0.8  –  3.2 7.3  –  9.0TotalProtein(g/L) 13.2  –  67.8 57(20  –  66) 19.0  –  41.7 69.2  –  80.6Globulin(g/L) 16.2  –  43.1 25(19  –  37) 14.4  –  19.5 36.3  –  47.7*Referenceintervals weredeterminedusingReference ValueAdvisor. 26 Table 2.  Blood chemistry variables in Eastern Quolls in summer,autumn,andspring.Summer n  =  9Autumn n  =  12Spring n  =  5Albumin(g/L) 27.5    9.2 26    5 20.8    9.7ALP(U/L) 248.4    137 217.7    115.9 141    70.4ALT(U/L) 64.8    31.4 49.4    37.9 60.2    48.4Amylase (U/L) 588.1    254 710.5    320.2 447.4    157*TotalBilirubin( l mol/L)6.6    1.2 4.8    0.75 5.6    1.1**Urea(mmol/L) 19.5    5.9 16.8    3.1 16.6    5.4Calcium (mmol/L) 2.4    0.49 2.2    0.32 2.1    0.68Phosphorus(mmol/L) 2.4    0.75 2.3    0.76 1.7    0.71Creatinine ( l mol/L) 44.3    12 29.8    10.3 36    17.1**Glucose (mmol/L) 7    1.5 5    1.3 4.8    1.8**Sodium (mmol/L) 139.4    16.1 147.9    3.6 128.4    26.1Potassium (mmol/L) 6.4    1.2 4.8    0.69 4.5    2**TotalProtein(g/L) 53.7    13 53.3    6.2 50.8    17.7Globulin (g/L) 24.8    3.4 27.1    5.5 31.5    8.4Dataaremeans    standarddeviation. * P  <  .05; ** P  <  .01. VetClinPathol 42/4(2013)476–482 ©  2013AmericanSocietyforVeterinaryClinicalPathologyandEuropeanSocietyforVeterinaryClinicalPathology  479 Stannardetal Quollbloodcharacteristics  lowervaluesforeachanalytethantheother2animals, but there was no obvious rationale to exclude its datafrom this report, as the animal appeared to be in goodhealth at the time of sampling, and the collected bloodwasnotclotted. Discussion Although hematology and blood chemistry profileshave been studied previously in Eastern and Spotted-Tailed Quolls, sample numbers were often small andthus not representative for a population. 12,22 In thepresent study, correlations between sexes, age, andseason for hematology and chemistry variables werecalculated in a captive population of Eastern Quoll.Specifically, it appears that serum enzyme activities inEastern Quolls were influenced by season, and ALPactivitywasinfluencedbyage.Generally, the morphology of RBCs and WBCs inthe Eastern and Spotted-Tailed Quolls was consistentwith previous descriptions forboth species, andsimilarto that of other dasyurid species, although some minordifferences in the diameter and number and propor-tion of granulocytes were recorded. As reported fordunnarts, 17 ringed neutrophils were common in quoll blood.Mean concentrations for blood chemistry analytesdetermined for the 2 quoll species were within therange observed for other dasyurids. 11,12,21,22,24 Meanactivity levels for ALP and AMY were higher and ALBconcentration was lower when compared with theranges for the Tasmanian devil. 21 Enzyme activities inthe Eastern and Spotted-Tailed Quolls were similar tothose determined in the Western Quoll. 24 Results forTBIL concentrations were higher and TP concen-trations lower in both quoll species, and CRE concen-trations lower in the Eastern Quoll compared withgeneral eutherian ranges. In Eastern Quolls, the meanCRE activity varied significantly across seasons.Reduced CRE activity has been associated with poornutrition and, consequently, loss of muscle mass indolphins ( Tursiops truncates ), harbor seals ( Phoca vituli-na ), and Eurasian badgers (  Meles meles ). 27  –  29 In theEastern Quolls, these reduced activity levels shouldnot be related to nutrition as feeding was managed byhuman caretakers. Food items were rotated daily, pos-sibly accounting for change in CRE between samplingperiods.ElevatedCREinspringandsummercouldalsosuggest a temporary increase in muscle mass, whichcould be due to increased physical activity during thattime 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 quollswas higher than in the wallaby and did not differsignificantly across the seasons, thus suggesting noseasonal change in nutritional status in the EasternQuolls.Previously, GLU and urea values were determinedto be higher in marsupials than in people; 12 however,the results for GLU and urea from quolls in this studywerecomparabletoanormalhumanrange. 31 Seasonalchanges in GLU in Eastern Quolls could relate tohydration, although water was available ad libitum . Such GLU variations have also been reported in theBrushtail Possum ( Trichosurus vulpecula ), where theywere associated with stress, and possibly water avail-ability. 32,33 In Bobcats ( Felis rufus ), capture stress was believed to be the reason for elevated GLU concentra-tions. 34 These and other factors, such as capture-relatedstress,changesinhousingandanimalgroupsorpairs, or hormonal changes related to breeding, couldhaveinfluencedGLUconcentrationsinthisstudy.The determined range of TBIL concentrations forEastern Quolls fell within the range observed for theWesternQuoll, 24  butthislevelwassignificantlyhigherin summer compared with autumn. Opposing resultswere found in free-living Mountain Brushtail Possums( Trichosurus caninus ), 35 where TBIL concentrationswere significantly lower in summer and autumn Table 3.  Differential WBC counts and blood chemistry variables of 3Spotted-TailedQuolls.Male1 Male2* FemaleDifferential WBCCountLymphocytes(%) 65 67 46Neutrophils(%) 25 26 45Ring-Neutrophil(%) 5 7 3Monocytes (%) 4 0 5Eosinophils (%) 1 0 1Basophils(%) 0 0 0BloodChemistryAlbumin(g/L) 46 2 40ALP(U/L) 115 15 65ALT(U/L) 22 8 31Amylase (U/L) 148 16 202TotalBilirubin ( l mol/L) 9 6 7Urea(mmol/L) 13.2 2 18.2Calcium (mmol/L) 2.47 0.85 2.37Phosphorus(mmol/L) 2.06 0.47 1.64Creatinine ( l mol/L) 94 43 93Glucose (mmol/L) 7.9 2.6 7.6Sodium(mmol/L) 144 100 143Potassium (mmol/L) 6.2  –   5.3TotalProtein(g/L) 67 10 69Globulin(g/L) 21  –   29*Although the values from male 2 are much lower than those from theother2quolls,male2appearedhealthyandthebloodwasnotclotted. VetClinPathol 42/4(2013)476–482 ©  2013AmericanSocietyforVeterinaryClinicalPathologyandEuropeanSocietyforVeterinaryClinicalPathology 480 Quollbloodcharacteristics Stannardetal
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