FACTORS AFFECTING HEMATOLOGY AND PLASMA BIOCHEMISTRY IN THE SOUTHWEST CARPET PYTHON (MORELIA SPILOTA IMBRICATA) Gillian L. Bryant, 1,3 Patricia A. Fleming, 1 Leanne Twomey, 2 and Kristin A. Warren 1 1 School of Veterinary & Biomedical Sciences, Murdoch University, South Street, Murdoch, Western Australia 6150, Australia 2 Vetpath Laboratory Services, 39 Epsom Avenue, Ascot, Western Australia 6104, Australia 3 Corresponding author (email: [email protected]) ABSTRACT: Despite increased worldwide popularity of keeping reptiles as pets, we know little about hematologic and biochemical parameters of most reptile species, or how these measures may be influenced by intrinsic and extrinsic factors. Blood samples from 43 wild-caught pythons (Morelia spilota imbricata) were collected at various stages of a 3-yr ecological study in Western Australia. Reference intervals are reported for 35 individuals sampled at the commencement of the study. As pythons were radiotracked for varying lengths of time (radiotransmitters were surgically implanted), repeated sampling was undertaken from some individuals. However, because of our ad hoc sampling design we cannot be definitive about temporal factors that were most important or that exclusively influenced blood parameters. There was no significant effect of sex or the presence of a hemogregarine parasite on blood parameters. Erythrocyte measures were highest for pythons captured in the jarrah forest and at the stage of radiotransmitter implantation, which was also linked with shorter time in captivity. Basophil count, the only leukocyte influenced by the factors tested, was highest when the python was anesthetized, as was globulin concentration. Albumin and the albumin:globulin ratio were more concentrated in summer (as was phosphorous) and at the initial stage of radiotransmitter placement (as was calcium). No intrinsic or extrinsic factors influenced creatinine kinase, aspartate aminotransferase, uric acid, or total protein. This study demonstrates that factors including season, location, surgical radiotransmitter placement, and anesthetic state can influence blood parameters of M. s. imbricata. For accurate diagnosis, veterinarians should be aware that the current reference intervals used to identify the health status of individuals for this species are outdated and the interpretation and an understanding of the influence of intrinsic and extrinsic factors are limited. Key words: Anesthetic, captivity, hemoparasite, radiotransmitter, reference intervals, reptile, season, snake. INTRODUCTION Monitoring hematologic measures and serum or plasma biochemical analytes is important for evaluating the health status of reptiles kept in captivity for research, as pets, or in zoos (Nordøy and Thoresen, 2002). Evaluating hematologic and bio- chemical responses can facilitate the diagnosis of stress and disease states in reptile species (Christopher et al., 1999). Blood analysis can be used to detect conditions such as anemia, inflammatory disease, parasitemia, hematopoietic disor- ders, and hemostatic alterations by com- paring individual samples with a clinically normal sample population (Campbell and Ellis, 2007). Reptilian blood analysis has not been evaluated for clinical application to the same extent as in mammals, although various blood values are known to be influenced by factors such as age, sex, and nutritional status (Campbell, 2004). Reference intervals have typically not accounted for these variations in intrinsic and environmental factors, mak- ing interpretation difficult (Campbell, 2004, 2006). The International Species Information System (ISIS) reports hematologic mea- sures and plasma biochemical analytes for samples collected from 25 pythons (Mor- elia spilota) from 10 institutions. However, no details are provided on source of the animals, nor is any information given to indicate sex, season, or length of time in captivity before blood sampling (Teare, 2002). The Near-Threatened southwest Journal of Wildlife Diseases, 48(2), 2012, pp. 282–294 # Wildlife Disease Association 2012 282
Transcript
FACTORS AFFECTING HEMATOLOGY AND PLASMA BIOCHEMISTRY
IN THE SOUTHWEST CARPET PYTHON (MORELIA
SPILOTA IMBRICATA)
Gillian L. Bryant,1,3 Patricia A. Fleming,1 Leanne Twomey,2 and Kristin A. Warren1
1 School of Veterinary & Biomedical Sciences, Murdoch University, South Street, Murdoch, Western Australia 6150,Australia2 Vetpath Laboratory Services, 39 Epsom Avenue, Ascot, Western Australia 6104, Australia3 Corresponding author (email: [email protected])
ABSTRACT: Despite increased worldwide popularity of keeping reptiles as pets, we know littleabout hematologic and biochemical parameters of most reptile species, or how these measuresmay be influenced by intrinsic and extrinsic factors. Blood samples from 43 wild-caught pythons(Morelia spilota imbricata) were collected at various stages of a 3-yr ecological study in WesternAustralia. Reference intervals are reported for 35 individuals sampled at the commencement of thestudy. As pythons were radiotracked for varying lengths of time (radiotransmitters were surgicallyimplanted), repeated sampling was undertaken from some individuals. However, because of ourad hoc sampling design we cannot be definitive about temporal factors that were most important orthat exclusively influenced blood parameters. There was no significant effect of sex or the presenceof a hemogregarine parasite on blood parameters. Erythrocyte measures were highest for pythonscaptured in the jarrah forest and at the stage of radiotransmitter implantation, which was alsolinked with shorter time in captivity. Basophil count, the only leukocyte influenced by the factorstested, was highest when the python was anesthetized, as was globulin concentration. Albumin andthe albumin:globulin ratio were more concentrated in summer (as was phosphorous) and at theinitial stage of radiotransmitter placement (as was calcium). No intrinsic or extrinsic factorsinfluenced creatinine kinase, aspartate aminotransferase, uric acid, or total protein. This studydemonstrates that factors including season, location, surgical radiotransmitter placement, andanesthetic state can influence blood parameters of M. s. imbricata. For accurate diagnosis,veterinarians should be aware that the current reference intervals used to identify the health statusof individuals for this species are outdated and the interpretation and an understanding of theinfluence of intrinsic and extrinsic factors are limited.
Monitoring hematologic measures andserum or plasma biochemical analytes isimportant for evaluating the health statusof reptiles kept in captivity for research, aspets, or in zoos (Nordøy and Thoresen,2002). Evaluating hematologic and bio-chemical responses can facilitate thediagnosis of stress and disease states inreptile species (Christopher et al., 1999).Blood analysis can be used to detectconditions such as anemia, inflammatorydisease, parasitemia, hematopoietic disor-ders, and hemostatic alterations by com-paring individual samples with a clinicallynormal sample population (Campbell andEllis, 2007). Reptilian blood analysis hasnot been evaluated for clinical application
to the same extent as in mammals,although various blood values are knownto be influenced by factors such as age,sex, and nutritional status (Campbell,2004). Reference intervals have typicallynot accounted for these variations inintrinsic and environmental factors, mak-ing interpretation difficult (Campbell,2004, 2006).
The International Species InformationSystem (ISIS) reports hematologic mea-sures and plasma biochemical analytes forsamples collected from 25 pythons (Mor-elia spilota) from 10 institutions. However,no details are provided on source of theanimals, nor is any information given toindicate sex, season, or length of time incaptivity before blood sampling (Teare,2002). The Near-Threatened southwest
Journal of Wildlife Diseases, 48(2), 2012, pp. 282–294# Wildlife Disease Association 2012
282
carpet python (Morelia spilota imbricata)that inhabits southwest Western Australiameasures up to 2.4 m snout-to-vent length(SVL) and can weigh up to 6 kg (IUCN,1998; Pearson et al., 2002). Since 2003it has been legal to obtain a license andkeep reptiles as pets in Western Australia,including the southwest carpet python(Edwards, 2003). As part of a widerecology study on wild-caught M. s. im-bricata, blood samples were collected toassess whether the health of pythons wasadversely affected by having a radiotrans-mitter surgically implanted into theircoelomic cavity, which enabled in situmonitoring of individuals. This studytherefore presented a unique opportunityto carry out repeated sampling for thispopulation of wild pythons over time,before and after surgery. We presenthematologic and biochemical data col-lected from 43 individuals and analyzethe effects of season, sex, study site,anesthesia, duration of time in captivity,radiotransmitter placement over time, andthe presence of parasites (Haemogregari-na sp.) within red blood cells.
MATERIALS AND METHODS
Study animals and sample collection
Forty-three southwest carpet pythons werecollected through opportunistic hand capturefor an ecology and thermal biology researchproject. Individuals were captured in thesouthwest of Western Australia from twohabitat types (referred to elsewhere as studysite): 1) coastal woodland: included animalscaptured and monitored at Martin’s Tank(32u519S, 115u409E) and Leschenault Penin-sula Conservation Park (33u269S, 115u419E)and 2) jarrah forest: areas surrounding Dwell-ingup Township (32u439S, 116u49E). Thisproject was approved by the Animal EthicsCommittees of Murdoch University (W2028/07) and Department of Environment andConservation, Western Australia (DEC AEC/55/2006 and DEC AEC54/2006).
Body mass (Mb) was determined with acalibrated spring balance (60.2 kg), and SVLwas measured with a tape measure in a straightline along the ventral surface of the pythonfrom the tip of the mouth to the cloaca. Thesex of each python was determined by eversion
of hemipenes or by insertion of a lubricatedblunt probe into the cloaca and then directedtoward the tail to determine the presence orabsence of hemipenes. Sex was determined bydepth of the probe insertion, as measured bythe number of overlying subcaudal scales.Females probed to between one and fivescales, whereas males probed to depthsequivalent to 7–20 scales. Adult male pythonsweighed an average 6946(SD)285 g (range298–1,500 g) and measured SVL 134617 cm(range 97–170 cm). Adult female pythonsweighed 1,4206554 g (range 103–3,731 g)and measured SVL 1796119 (range 90–200cm).
Upon initial capture, pythons were broughtinto a holding facility at the DEC ResearchCenter, Dwellingup, Western Australia be-tween December 2006 and November 2008 toundergo surgical implantation of radiotrans-mitters (for surgical details see Bryant et al.,2010). When held at the research center, theanimals were housed in a 25 C temperature-controlled room in purpose-built ventilatedenclosures with flooring lined to approximately2-cm depth with recycled newspaper kittylitter (Old News Cat Litter, Ciber Cycles PtyLtd., Toowoomba, Queensland, Australia).The enclosures contained a hide box as acardboard box with an entrance hole cut intoone side, cage furniture including a branchand rocks, ad libitum water, and an externalheat pad positioned under the plywoodflooring that heated that portion of the cagefloor above the heat pad to approximately 30 C.All cages were kept in a room under naturallighting provided by a large window. If theywere held for .3 wk during warmer seasons,pythons were fed dead laboratory mice andrats that were stored frozen and thawed beforefeeding. Pythons were not fed during winter.Animals were held for varying lengths of timebecause of logistic reasons, including thenecessity of customizing a radiotransmitterfor surgical implantation. Length of time eachpython spent in captivity before samplecollection was recorded and scored for statis-tical analyses as 0–30 days (n533), 30–60 days(n518), and .60 days (n57).
We recorded repeated samples from py-thons at three time points; however, thenumber of samples collected from pythonsvaried between these points. At the initialstage of presurgical implantation of the radio-transmitter 38 blood samples were collectedin the laboratory (pre-TM, n538 individuals).Twenty four of these 38 pythons wereanesthetized via inhalation of 1.5% isofluranegas. The remaining 14 pythons were manuallyrestrained by holding carefully within calico
BRYANT ET AL.—HEMATOLOGY AND BIOCHEMISTRY OF SOUTHWEST CARPET PYTHON 283
bags, with the tail exposed for blood collectionfrom the ventral coccygeal vein. Animals wereopportunistically allocated to these two treat-ments on the basis of logistics around thetransportation of blood samples to the clinicallaboratory for analysis. The reference intervalswere calculated from the samples collectedfrom these 38 animals. Pythons were releasedapproximately 2 wk after surgical implantationand were radiotracked weekly to monitorfeeding behavior and habitat use. Whenpythons could be captured by hand from theirretreat site, 3–12 mo (average 6.8564.86 mo)postimplantation of radiotransmitters (post-TM, n522), a second blood sample wascollected from manually restrained consciousanimals in the field. One python was movedback into temporary captivity for surgicalreplacement of a radiotransmitter due tobattery failure, and the posttransmitter (sec-ond) blood sample was collected underanesthesia. Pythons were anesthetized at thetime of radiotransmitter removal, allowing athird (removal-TM, n520) blood sample.Twelve individuals lost their transmittersduring the period of tracking and 13 diedover the 3-yr study (postmortem examinationswere performed when possible and the resultsare presented elsewhere; Bryant, 2012). Intotal, we collected 77 blood samples from 43animals. Sixteen pythons were sampled once,17 were sampled twice, and 10 were sampledthree times.
A range of needle sizes (depending on thesize of the python; 25 G for SVL,100-cmpythons, 25 G, 23 G for intermediate sizes,and 21 G for SVL.150 cm) and a 2-ml syringe(BD PrecisionGlideTM Needle; BD Slip TipSyringe; Becton Dickinson, Singapore) wereused to collect blood samples from the ventralcoccygeal vein. Fresh blood smears withoutanticoagulate were made immediately aftercollection; the remainder of the sample wascarefully transferred immediately into 2-ml(13375 ml) lithium heparin vacutainers (BDVacutainerH, Becton Dickinson, Plymouth,UK) and kept refrigerated at approximately4 C during transportation to a commerciallaboratory. Analysis was completed by VetpathLaboratory Services (Ascot, Western Australia,Australia) within 48 hr of collection. Samplescollected in the field were kept on ice in aninsulated container or refrigerated whenpossible before transport to the laboratory.Whenever possible, a complete set of hema-tologic and plasma biochemical data wascollected from each blood sample. Incompleteblood analysis was common, generally dueto insufficient volumes of plasma for the fullbiochemistry panel.
Hematology analysis
Blood samples were analyzed using aCELL-DYNTM (Cell Dyn 3700, Abbott Diag-nostics, North Ryde, New South Wales,Australia) analyzer using the reptilian/aviansetting to take nucleated erythrocytes intoaccount. The blood variables analyzed includ-ed hemoglobin (Hb), packed cell volume(PCV), red blood cell count (RBC), meancell hemoglobin (MCH), mean cell volume(MCV), mean cell hemoglobin concentration(MCHC), and total white blood cell count(WBC) using the automated system. Manualcalculations for PCV were made to correcthemoglobin values (Hb, MCH, MCV, andMCHC) and if these measures did notcorrespond (8/77 samples), they were removedfrom further analysis and only PCV wasincluded.
Blood smears were air-dried and stainedwith Wright’s–Giemsa stain by an automatedslide stainer (Hematek, Siemens, OsbornePark, Western Australia). The proportions ofheterophils (including potential eosinophils,which could not be definitively identified bymorphology alone; Stirk et al., 2007), lympho-cytes, basophils, and combined monocytes/azurophils (Fig. 1a–d) were classified throughmanual counts of blood smears. Parasites inred blood cells that were morphologicallyconsistent with a Hemogregarina species werefound in some samples (Fig. 1e). Followingdescriptions provided by Mackerras (1961) ofthe seven Hemogregarina species found inAustralian Boidae (O’Donoghue and Adlard,2000), the species in this study appears to beHemogregarina moreliea. Samples were cate-gorized according to the presence or absenceof the hemoparasites in smears.
Plasma biochemical analysis
A minimum of 150 ml of plasma fromheparinized blood samples was used for bio-chemical analysis. Plasma was analyzed forcreatinine kinase (CK), aspartate aminotrans-ferase (AST), uric acid, total protein, albumin,globulin, albumin-to-globulin ratio (A/G),calcium, phosphorous, and glucose using anautomated chemical analyzer (Olympus AU400, Integrated Science, Tokyo, Japan). Be-cause of varying lengths of time beforelaboratory analysis (up to 48 hr) and incompletedata collection, glucose results are variable andtherefore, although range of data is indicated,caution should be taken when using the valuespresented here. For these reasons, multipleregression analyses were not performed onglucose.
284 JOURNAL OF WILDLIFE DISEASES, VOL. 48, NO. 2, APRIL 2012
Statistical analysis
Reference intervals for M. s. imbricata wereinvestigated for the first time period samplingpoint only (pre-TM implantation) for 38individuals. Blood samples from three femalepythons were removed as they were consid-ered outliers using Dixon’s range test withinthe program Reference Value Advisor V 1.4(Anonymous, 2010); reference intervals weretherefore calculated using blood samples for35 individuals. Standard descriptive statisticsinclude sample size, mean, standard deviation,minimum, median, and the maximum values.Normality tests were performed and if re-quired, data were transformed with Box-Coxtransformation. The lower and upper limit ofthe reference interval as well as the 2.5, 5, 90,and 97.5% confidence intervals are givenwhere possible using the program ReferenceValue Advisor V 1.4 (Anonymous, 2010).
Data from our study (M. s. imbricata) areexpressed as a proportion of reported ISISaverage values (M. spilota) following theformula:
Proportional difference~
Average M: s: imbricata{average M: spilotað Þ
=average M: spilota
Multiple regression analysis (Statistica 9.0,Statsoft Inc.) was carried out separately foreach blood measure (dependent variable)against the same independent factors: sex(male or female), study site (coastal woodlandor jarrah forest), season (summer: December–February; autumn: March–May; winter: June–August; spring: September–November), anes-thesia (anesthetized or conscious), the exper-imental phase of sample collection (threecategories: pre-TM, post-TM, and removal-TM), the time in captivity from capture untilsample collection (three categories: 0–30 days,31–60 days, and .60 days; animals sampledin the field were ascribed as 0 days), andhemoparasite presence (positive or negative).The blood variables examined included: Hb,PCV, RBC, MCH, MCV, MCHC, WBC,heterophil, lymphocyte, basophil, and com-bined monocyte and azurophil counts, CK,AST, uric acid, total protein, albumin, globu-lin, A/G ratio, calcium, and phosphorus.
Chi-square analysis was used to determinevariation in hemoparasite prevalence acrossseasons with the expected values calculatedassuming an equal proportion of the popula-tion was positive each season. A similaranalysis was carried out for H. morelieahemoparasite prevalence at each samplingperiod, assuming an equal proportion ofsamples collected pre-TM, post-TM, or re-moval-TM were positive (i.e., expected valuescalculated assuming an equal distributionacross these three time points). Chi-squareanalysis was also used to test for a significantdifference in H. moreliea prevalence betweenstudy sites.
RESULTS
Reference intervals
Reference intervals are shown for 35pythons for blood collected before surgicalimplantation of radiotransmitters (pre-TM) only (Table 1). Raw, untransformeddata are shown for reference intervals.Most average values found in this studywere similar (,20% difference) to pub-lished ISIS values for M. spilota (Teare,2002). However, MCV were twice those ofpublished ISIS values, WBC counts were
FIGURE 1. Peripheral blood films of a southwestcarpet python (Morelia spilota imbricata) showing: a)heterophil (black arrow); b) lymphocytes (blackarrows) and thrombocytes (open arrow); c) basophil(double black arrow); a lymphocyte (single blackarrow) and thrombocyte (open arrow) are alsovisible; d) azurophil (black arrow); and e) erythrocytecontaining a Hemogregarina moreliea hemoparasite(black arrow). Images (a–e) stained with Wright–Giemsa stain, 1,0003, bar 5 15 mm.
BRYANT ET AL.—HEMATOLOGY AND BIOCHEMISTRY OF SOUTHWEST CARPET PYTHON 285
TA
BL
E1.
Hem
atolo
gy
and
pla
sma
bio
chem
ical
valu
es
for
35
app
aren
tly
heal
thy,
wil
d-c
augh
tso
uth
west
carp
et
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hon
s(M
orel
iasp
ilot
aim
bri
cata
)sa
mp
led
befo
rera
dio
tran
smit
ter
imp
lan
tati
on
.S
amp
lesi
zeva
ries
amon
gm
eas
ure
sb
eca
use
of
dif
fere
nce
sin
the
volu
me
of
blo
od
that
cou
ldb
eco
llect
ed
(som
ete
sts
not
con
du
cted
beca
use
of
smal
lsa
mp
lesi
ze),
du
eto
vari
atio
nin
tim
eto
anal
ysis
(not
all
sam
ple
sw
ere
anal
yzed
for
glu
cose
beca
use
of
tran
sport
dela
ys),
or
du
eto
dis
crep
anci
es
inery
thro
cyte
cou
nts
(see
text
for
furt
her
deta
il).
Val
ues
were
calc
ula
ted
usi
ng
Refe
ren
ceV
alu
eA
dvi
sor
v1.4
pro
gra
man
dgiv
en
toth
ree
sign
ific
ant
dig
its,
or
were
inte
rpre
ted
asze
rofo
rn
egat
ive
resu
lts.
Val
ue
nM
ean
Med
ian
SD
Min
imu
mM
axim
um
Co
nfi
den
cein
terv
als
Tra
nsf
orm
atio
na
Lo
wer
lim
itU
pp
er
lim
it2
.5%
5%
90
%9
7.5
%
Hem
oglo
bin
(g/l
)26
76.5
79.0
17.1
28.0
105
BoxC
ox
30.4
104
048.7
97.4
109
Pac
ked
cell
volu
me
30
0.2
32
0.2
40
0.0
51
0.0
80
0.3
0B
oxC
ox
0.0
90
0.3
07
00.1
55
0.2
93
0.3
19
Red
blo
od
cou
nt
(310
12/l
)26
0.6
69
0.7
00
0.1
74
0.2
00
0.9
00
BoxC
ox
0.0
27
0.9
05
Mean
cell
hem
oglo
bin
con
cen
trat
ion
(g/l
)26
343
342
17.7
315
374
UT
305
380
297
314
369
389
Mean
cell
hem
oglo
bin
(pg)
26
117
111
19.8
96.0
178
UT
67.2
152
51.0
86.1
133
170
Mean
cell
volu
me(f
l)26
339
327
49.5
279
484
UT
219
430
188
260
386
465
Wh
ite
blo
od
cell
cou
nt
(310
9/l
)35
14.9
13.4
6.9
44.9
029.3
UT
029.4
02.7
024.8
32.2
Hete
rop
hil
s(3
10
9/l
)31
7.7
27.1
35.1
70.4
10
24.8
BoxC
ox
0.6
20
21.7
0.1
41
1.7
916.8
27.1
Lym
ph
ocy
tes
(310
9/l
)35
3.0
92.5
92.1
90.4
00
10.3
BoxC
ox
0.3
67
9.0
70.2
75
0.6
03
6.9
111.3
Bas
op
hil
s(3
10
9/l
)28
0.2
32
0.1
55
0.2
46
0.0
00.9
40
BoxC
ox
01.2
17
00
0.7
78
1.8
3M
onoc
ytes
&az
uro
ph
ils(3
109/l)
32
4.5
73.9
42.7
61.0
812.3
BoxC
ox
1.0
812.9
0.7
68
1.5
09.7
717.0
Cre
atin
ine
kin
ase
(U/l
)32
1,8
80
1,6
51
1,1
15
365
5,1
47
BoxC
ox
379
4,7
67
293
542
3,7
26
5,7
46
Asp
arta
team
inot
ran
sfer
ase
(U/l)
32
84.8
63.0
63.8
17.0
259
BoxC
ox
17.8
296
13.9
23.6
187
445
Uri
cac
id(m
mol/
l)28
0.1
86
0.1
84
0.0
61
0.0
81
0.3
33
UT
0.0
47
0.3
01
0.0
19
0.0
88
0.2
67
0.3
42
Tota
lp
rote
in(g
/l)
32
73.0
71.5
11.7
56.0
105
UT
47.0
96.0
41.0
53.6
88.2
103
Alb
um
in(g
/l)
32
20.9
21.0
3.0
316.0
27.0
UT
14.5
27.1
13.0
15.7
25.3
28.8
Glo
bu
lin
(g/l
)32
52.0
50.5
9.2
539.0
78.0
UT
31.5
70.4
26.3
37.0
64.2
76.0
Alb
um
in-t
o-g
lob
uli
nra
tio
32
0.4
07
0.4
20
0.0
45
0.2
80
0.4
90
BoxC
ox
0.2
96
0.4
85
0.2
51
0.3
43
0.4
69
0.4
99
Cal
ciu
m(m
mol/
l)32
3.4
33.4
60.3
02.9
04.4
1U
T2.7
94.0
22.6
03.0
03.8
24.1
9P
hosp
horu
s(m
mol/
l)32
1.1
41.1
40.3
16
0.4
30
1.8
6U
T0.4
68
1.7
80.3
37
0.6
43
1.5
71.9
4G
luco
se(m
mol/
l)28
2.8
42.1
01.9
40.1
00
8.3
0B
oxC
ox
0.1
89
8.2
10.0
51
0.4
91
5.7
510.3
aU
T5
un
tran
sfo
rmed
rob
ust
dat
a;B
oxC
ox
5B
ox
Co
xro
bu
sttr
ansf
orm
atio
n.
286 JOURNAL OF WILDLIFE DISEASES, VOL. 48, NO. 2, APRIL 2012
40%, and heterophil counts were 172%
greater than ISIS values. All other leuko-cyte values were lower than those report-ed in ISIS for M. spilota. Creatinine kinase(+292%) and AST (+239%) were higherthan reference values and uric acid(257%), albumin (230%), calcium(246%), and phosphorous (259%) con-centrations were lower than average val-ues reported by ISIS.
Pythons from the jarrah forest hadgreater RBC (t5652.04, P,0.05) andlower concentrations of MCH (t565
22.41, P,0.05) and MCV (t56522.18,P,0.05) compared with pythons fromcoastal woodland. There was no statisticaldifference in any hematologic variable orplasma biochemical analytes betweenmales and females (all P.0.05, Tables 2and 3).
Season, anesthesia, surgical implanta-tion, and time in captivity significantlyinfluenced some hematologic and bio-chemical analytes; however, due to thelack of independence of the factors tested,the effects of season and radiotransmitterplacement cannot be differentiated. Sea-son significantly influenced values forMCH (t5653.33, P,0.01), MCV (t565
2.55, P,0.01), albumin (t6552.07,P,0.05), A/G ratio (t6552.42, P,0.05),and phosphorous (t6553.23, P,0.01). ForMCH, MCV, albumin, and phosphorous,the lowest average values were recordedin winter (Tables 2 and 3), whereas theA/G ratio peaked in summer (Table 3).Blood values were apparently influencedby anesthetic state, with anesthetizedanimals having higher average basophilcounts (t5552.10, P,0.05) and globulinconcentrations (t6552.01, P,0.05; Ta-bles 2 and 3). The A/G ratio was alsolower for anesthetized animals (t655
22.09, P,0.05; Table 3). The time ofblood collection (categorized by threestages of radiotransmitter implantation)influenced several hematologic parame-
ters (Hb: t5752.16, P,0.04; PCV:t5652.78, P,0.01; RBC: t5653.03,P,0.01; and basophil count: t55522.44,P,0.05) and plasma biochemical analytes(albumin: t6553.54, P,0.001; A/G ratio:t6554.24, P,0.001; and calcium: t6553.50,P,0.001). All these measures were high-est at the pre-TM implantation samplingperiod. The length of time in captivitybefore blood sample collection (0–30 days,30–60 days, or .60 days) significantlyinfluenced only MCH (t56522.09,P,0.05) and MCV measures (t56522.78,P,0.05; Table 2), where values werehigher for animals that had spent lesstime in captivity.
The presence of H. moreliea was onlymarginally associated with reduced totalprotein (t6551.88, P.0.06), albumin (t655
1.85, P.0.07), and globulin (t6551.88,P.0.06; Table 3) measures. There wasno significant difference in the occurrenceof the intracellular hemoparasite betweenpythons captured from coastal woodlandor jarrah forest (x2
152.43, P.0.05). Agreater proportion of blood samples col-lected from pythons during spring (Sep-tember–November) were positive forH. moreliea (x2
158.44, P,0.01; Fig. 2).Significantly more pythons sampled atremoval-TM were positive for hemogreg-arines compared with pre-TM and post-TM (x2
158.30, P,0.01; Fig. 2).
DISCUSSION
We examined the influence of intrinsicand extrinsic factors on hematologic pa-rameters and plasma biochemical analytesin a Western Australian python species.Health screening provides useful baselinedata for conservation management pro-grams of wild populations of threatenedspecies and for species held in captivity forzoologic collections and as pets (Espinosa-Aviles et al., 2009). We provide referenceintervals for wild-caught southwest carpetpythons that could contribute to healthscreening of these animals for conservationmanagement. There was no significant
BRYANT ET AL.—HEMATOLOGY AND BIOCHEMISTRY OF SOUTHWEST CARPET PYTHON 287
effect of sex or the presence of a hemo-gregarine parasite, but season, time incaptivity, anesthesia, and the stage ofradiotransmitter implantation did influenceblood parameters.
Because of the physiologic flexibility ofreptiles (e.g., poikilothermy, long-termfasting, and rapid up-regulation of thedigestive system upon feeding; Bedfordand Christian, 2001; Secor and Ott, 2007),reference intervals are difficult to establish(Campbell and Ellis, 2007). The overallaverage leukocyte counts in this study forM. s. imbricata varied substantially fromISIS reports for the species (all valuespresented as M. spilota). This discrepancymay reflect differences between subspe-cies of Morelia that include varying dietand potential climatic influences such asrainfall, temperature, and humidity, butalso reflects that ISIS values do not followthe current Clinical and Laboratory Stan-dards Institute (CLSI) guidelines for
calculation and presentation (C28-A3)and should be updated (CLSI, 2008).Leukocyte counts for M. s. imbricatadiffered from ISIS values in a mannerconsistent with heterophilia and lympho-penia, which can occur with a stressresponse to capture and handling inwild-caught pythons (Campbell, 2004,2006; Campbell and Ellis, 2007). Alterna-tively, there may be differences in leuko-cyte identification between individualpathologists. The high heterophil numbersby comparison with low lymphocytes,basophils, and monocytes/azurophils maysuggest the latter. However, we alsorecorded higher CK and AST in M. s.imbricata compared with ISIS averagevalues, which may suggest muscle damageassociated with handling of wild-caughtindividuals (assuming that the majority ofsamples used for the ISIS values are fromcaptive animals that may be habituated tohuman presence and handling). The
TABLE 2. Average6SD values for each hematologic measure analyzed by multiple regression for all factors.Bold values indicate a significant (* P,0.05, ** P,0.005, *** P,0.001) difference between the categories inthe analysis. Values to three significant digits.
Factor
Category, n5numberof pythons (N5number
of samples)Hb (g/l),
N565PCV (l/l),
N564RBC (31012/l)a,
N564MCH (pg)b,
N564
Study site Coastal, n528 (54) 69.6616.3 0.20960.050 *0.600±0.155 *118±17.5Jarrah forest, n513 (23) 74.369.62 0.22760.038 0.672±0.102 113±14.7
mean cell volume; MCHC 5 mean cell hemoglobin concentration; WBC 5 total white blood cell count.a Nonnormal distribution for Box-Cox transformed data.b Box-Cox transformed data with normal distribution.
288 JOURNAL OF WILDLIFE DISEASES, VOL. 48, NO. 2, APRIL 2012
differences between values presented inthis study and reported ISIS values maytherefore reflect real differences for wild-caught animals.
Although multiple regression analysisshould simultaneously take multiple fac-tors into consideration in its computation,we nevertheless urge caution in interpre-tation of data where there are clearlyunequal samples collected for each factorand where multiple factors could influ-ence the results. Multiple regressionanalysis did not distinguish between sam-ples collected from male and female M. s.imbricata for either hematologic or plasmabiochemical analytes tested. Morelia spi-lota imbricata are capital breeders and willbreed only every second year, at best(Pearson, 2002). Over the 3-yr study, onlyseven females were identified as gravidonce over this time; only two of these weresampled while gravid (none were sampledpostoviposition) and they did not stand outas outliers. Therefore, to our knowledge,the majority of female M. s. imbricatasampled were nonreproductive. Similarly,serum biochemistry does not vary between
male and female black diamond watersnakes (Nerodia rhombifera rhombifera;McDaniel et al., 1984), a viviparousspecies. By contrast, other studies haveidentified sex differences in plasma ana-lytes for various species of snakes, withelevations of plasma calcium, phosphorus,and protein concentrations in femalesduring estrus and egg production (Des-sauer, 1970; Campbell, 2004). It is there-fore likely that blood measures only differbetween the sexes under specific repro-ductive states.
We found significant seasonal differencesfor MCH, MCV, albumin, A/G ratio, andphosphorous concentration. Wojtaszek (1992)noted a significant decrease in RBC, PCV,and Hb concentration in spring, the matingperiod for the grass snake (Natrix natrixnatrix), and attributed the hematologicchanges to a hormonal influence producedby decreased erythropoietic activity andfrom some RBC breakdown during winter.The reduction in albumin and phosphorousconcentrations in winter for M. s. imbricatais most likely associated with fasting(Campbell, 2004).
BRYANT ET AL.—HEMATOLOGY AND BIOCHEMISTRY OF SOUTHWEST CARPET PYTHON 289
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290 JOURNAL OF WILDLIFE DISEASES, VOL. 48, NO. 2, APRIL 2012
The state of pythons during sampling(anesthetized or conscious) appeared toinfluence some blood measures. Basophilcount and globulin concentration wereelevated for anesthetized M. s. imbricata(and the reverse for the A/G ratio). Wehave little understanding of how anesthet-ics influence hematologic and biochemistryvalues in snakes (McDaniel et al., 1984).Further testing including perioperative,intraoperative, and postoperative stages ofanesthesia and surgery would be beneficialin deciphering the interpretation of anes-thetic effects on specific hematologic andplasma biochemical values.
The time individuals were sampled inrelation to radiotransmitter placementappeared to affect several hematologicand plasma biochemical values. However,given that we opportunistically collectedblood samples, it was not possible tocontrol for the seasonal spread of samplescollected at each radiotransmitter implan-tation stage. As pythons are most activeduring spring and summer, they were
opportunistically captured at higher ratesduring that time (20 of 35 samples werecollected in summer; Fig. 2), and thestudy concluded in spring of 2008 whenmost radiotransmitters were removed (19of 20 removal-TM samples were collectedin spring; Fig. 2). Hemoglobin, PCV,RBC, calcium, albumin, and the A/G ratiowere all highest at the pre-TM stage ofimplantation. The majority of pre-TMsamples were collected during summer,and although season did not statisticallyinfluence Hb, PCV, RBC, and calcium,the seasonal effect was significant foralbumin and A/G ratio. These parametersmay reflect hydration or nutritional status.A study by Lentini et al. (2011), specifi-cally designed to test the inflammatoryresponse of implanting radiotransmittersin rattlesnakes (Sistrurus catenatus cate-natus), found that 33% of implantedsnakes had grade 3 or higher reactions(on the basis of histopathologic tissueexamination) with extensive and activeinflammation. The reaction to the implant
FIGURE 2. Of 77 blood samples collected from 43 southwest carpet pythons Morelia spilota imbricata, agreater proportion of blood samples collected from pythons during spring was positive for intracellularhemoparasites (September–November; x2
158.44, P,0.01), whereas a greater proportion of samples collectedat the time of radiotransmitter removal was positive (x2
158.30, P,0.01) compared with preradiotransmitterand postradiotransmitter placement. The morphology of the hemoparasites seen in blood smears wasconsistent with Haemogregarina moreliea. The numbers shown in the table indicate sample size for eachsample group.
BRYANT ET AL.—HEMATOLOGY AND BIOCHEMISTRY OF SOUTHWEST CARPET PYTHON 291
was reflected with increases in counts ofheterophils and monocytes and a decreasein globulin concentrations after 6 mo.Hemoglobin was significantly lower com-pared with snakes without implants (Len-tini et al., 2011). Increases in theseleukocyte counts were not as pronouncedin our study at the three transmittersampling stages; however, reduced Hbconcentration at the last two samplingstages may indicate an anemic response,similar to that found by Lentini et al.(2011).
Hemogregarine parasites (Phylum Api-complexa, Family Haemogregorinidae)require a vertebrate host (e.g., reptiles)and an intermediate invertebrate host(e.g., ticks, mites, mosquitoes, or leeches)to complete their life cycle (Diethelm,2006). Infection with hemoparasites isoften subclinical in reptiles; however,heavy burdens can result in anemia(Diethelm, 2006). Hemogregarines arereasonably common among snake species,including brown tree snakes (Boiga irre-gularis) and slatey-grey snakes (Stegonotuscucullatus) from Queensland (Caudellet al., 2002). In these two species, therewas no significant difference betweeninfected (less than 10% of RBCs) andnoninfected snakes for Hb, PCV, albumin,calcium, phosphorus, protein, uric acid,AST, and CK (Caudell et al., 2002).Similarly, we found no significant differ-ences in any hematologic or biochemicalanalytes for M. s. imbricata according tohemogregarine parasite status. The inci-dence of hemoparasites in M. s. imbricatadid, however, vary significantly seasonally,with a greater prevalence in blood smearscollected in spring. Changes in seasonalprevalence of hemoparasites have beenobserved for some lizards, where there isgreater hemoparasite prevalence towardthe end of the mating season in spring(Amo et al., 2005; Huyghe et al., 2010),particularly in females (Amo et al., 2005).In comparison, males often show consis-tent levels of hemoparasite prevalenceacross seasons, which has been attributed
to the immunosuppressive effects of tes-tosterone (e.g., Amo et al., 2005). Increasingnumbers of invertebrate intermediate hostvectors during spring would potentiallydrive an increase in the prevalence of thehemoparasites and hence the number ofaffected vertebrate (python) hosts (Huygheet al., 2010). Furthermore, M. s. imbricatashow greater movement and activity pat-terns during spring (Pearson et al., 2005),which may increase exposure to intermedi-ate invertebrate hosts at this time. About95% of samples were collected frompythons in spring at the time of radio-transmitter removal and there was a signif-icant difference in the presence of hemo-parasites during this sampling period.Although no conclusions can be drawnregarding the relative influences of diet,breeding activity, or stage of capture/samplecollection, this seasonal pattern in hemo-parasites warrants further investigation.
In conclusion, it is important to con-sider a variety of factors when interpret-ing hematologic and biochemical valuesfor diagnosing diseases or evaluatinghealth status of pythons. We foundcompounded seasonal differences inblood analytes and the time of sampling,which may be related to feeding andhydration status. Although our data sug-gest an effect of radiotransmitter place-ment, we recommend that additionalresearch be undertaken. We hope toprovide the impetus to further improvethe understanding and interpretation ofhematology and biochemical analytes inreptile species.
ACKNOWLEDGMENTS
We thank all staff at Vetpath LaboratoryServices involved in analyzing samples for thisstudy. Thank you to all volunteers involved inhelping in fieldwork and assisting GLB withsample collection and delivery of samples toVetpath Laboratory Services, in particular M.Connor, S. Dundas, J. Clarke, J. Bryant, and A.Bryant. This project was financially supportedby the Department of Environment andConservation, Murdoch University and theAustralian Research Council LP0562099.
292 JOURNAL OF WILDLIFE DISEASES, VOL. 48, NO. 2, APRIL 2012
LITERATURE CITED
AMO, L., P. LOPEZ, AND J. MARTIN. 2005. Prevalenceand intensity of haemogregarine blood parasites andtheir mite vectors in the common wall lizard,Podarcis muralis. Parasitology Research 96: 378–381.
ANONYMOUS. 2010. Reference guide for ReferenceValue Advisor v1.4, Rykkinn, Norway, 7 pp.
BEDFORD, G. S., AND K. A. CHRISTIAN. 2001.Metabolic response to feeding and fasting inthe water python (Liasis fuscus). AustralianJournal of Zoology 49: 379–387.
BRYANT, G. L. 2012. Biology of the south west carpetpython (Morelia spilota imbricata): Is thereevidence for mesopredator release in responseto fox baiting? PhD Thesis, School of Veterinaryand Biomedical Sciences, Murdoch University,Perth, Western Australia, 232 pp.
———, P. EDEN, P. J. DE TORES, AND K. S. WARREN.2010. Improved procedure for implanting radio-transmitters in the coelomic cavity of snakes.Australian Veterinary Journal 88: 443–448.
CAMPBELL, T. W. 2004. Clinical chemistry of reptiles.In Veterinary hematology and clinical chemistry,M. A. Thrall (ed.). Blackwell Publishing Ltd,Oxford, UK, pp. 493–498.
———. 2006. Clinical pathology of reptiles. InReptile medicine and surgery, 2nd Edition, D.Mader (ed.). Saunders, Elsevier, Philadelphia,Pennsylvania, pp. 453–470.
———, AND C. ELLIS. 2007. Hematology of reptiles.In Avian and exotic animal hematology andcytology, T. W. Campbell and C. Ellis (eds.).Blackwell Publishing Ltd, Oxford, UK, pp. 51–81.
CAUDELL, J. N., J. WHITTIER, AND M. R. CONOVER.2002. The effects of haemogregarine-like para-sites on brown tree snakes (Boiga irregularis)and slatey-grey snakes (Stegonotus cucullatus) inQueensland, Australia. International Biodeterio-ration & Biodegradation 49: 113–119.
CHRISTOPHER, M., K. BERRY, I. WALLIS, K. NAGY, B.HENEN, AND C. PETERSON. 1999. Referenceintervals and physiologic alterations in hemato-logic and biochemical values of free-rangingdesert tortoises in the Mojave Desert. Journal ofWildlife Diseases 35: 212–238.
CLINICAL AND LABORATORY STANDARDS INSTITUTE
(CLSI). 2008. Defining, establishing, and veri-fying reference intervals in the clinical laborato-ry: Approved guideline, 3rd Edition. Clinical andLaboratory Standards Institute, Wayne, Penn-sylvania, 59 pp.
DESSAUER, H. C. 1970. Blood chemistry of reptiles:Physiological and evolutionary aspects. In Biol-ogy of the reptilia, C. Cans and T. S. Parsons(eds.). Academic Press, New York, New York,pp. 1–72.
DIETHELM, G. 2006. Haemoparasites. In Reptile med-icine and surgery, D. R. Mader (ed.). WB Saunders,Philadelphia, Pennsylvania, pp. 1103–1117.
EDWARDS, J. 2003. Wildlife conservation (reptilesand amphibians; pet herpetofauna) notice 2003.Government Gazette Wildlife Conservation Act1950 CO301, The Department of Environmentand Conservation, Perth, Western Australia,pp. 658–659.
ESPINOSA-AVILES, D., V. M. SALOMON-SOTO, AND S.MORALES-MARTINEZ. 2009. Hematology, bloodchemistry, and bacteriology of the free-rangingMexican beaded lizard (Heloderma horridum).Journal of Zoo and Wildlife Medicine 39: 21–27.
HUYGHE, K., A. VAN OYSTAEYEN, F. PASMANS, Z. TADIC,B. VANHOOYDONCK, AND R. VAN DAMME. 2010.Seasonal changes in parasite load and a cellularimmune response in a colour polymorphiclizard. Oecologia 163: 867–874.
INTERNATIONAL UNION FOR CONSERVATION OF NATURE
(IUCN). 1998. Guidelines for re-introductions.Prepared by the IUCN/SSC Re-introductionSpecialist Group. Gland, Switzerland and Cam-bridge, UK, 10 pp.
LENTINI, A. M., G. J. CRAWSHAW, L. E. LICHT, AND
D. J. MCLELLAND. 2011. Pathologic and hemato-logic responses to surgically implanted transmit-ters in eastern massasauga rattlesnakes (Sistruruscatenatus catenatus). Journal of Wildlife Diseases47: 107–125.
MACKERRAS, M. 1961. The haematozoa of Australianreptiles. Australian Journal of Zoology 9: 61–122.
MCDANIEL, R., W. GRUNOW, J. DALY, AND M.PLUMMER. 1984. Serum chemistry of the dia-mond-backed water snake (Nerodia rhombiferarhombifera) in Arkansas. Journal of WildlifeDiseases 20: 44–46.
NORDØY, E. S., AND S. I. THORESEN. 2002. Referencevalues for serum biochemical parameters in free-ranging harp seals. Veterinary Clinical Pathology31: 98–106.
O’DONOGHUE, P. J., AND R. D. ADLARD. 2000.Catalogue of protozoan parasites recorded inAustralia. Memoirs of the Queensland Museum45: 1–163.
PEARSON, D. 2002. The ecology and conservation ofthe south-western Australian carpet python,Morelia spilota imbricata. PhD Thesis, Schoolof Biological Sciences, University of Sydney,Sidney, New South Wales, Australia, 152 pp.
———, R. SHINE, AND A. WILLIAMS. 2002. Geograph-ic variation in sexual size dimorphism within asingle snake species (Morelia spilota, Pythoni-dae). Oecologia 13: 418–426.
———, ———, AND ———. 2005. Spatial ecology ofa threatened python (Morelia spilota imbricata)and the effects of anthropogenic habitat change.Austral Ecology 30: 261–274.
SECOR, S. M., AND B. D. OTT. 2007. Adaptivecorrelation between feeding habits and digestivephysiology for boas and pythons. In Biology ofthe boas and pythons, R. W. Henderson and R.
BRYANT ET AL.—HEMATOLOGY AND BIOCHEMISTRY OF SOUTHWEST CARPET PYTHON 293
Powell (eds.). Eagle Mountain Publishing, EagleMountain, Utah, pp. 257–268.
STIRK, N., I. R. ALLENMAN, AND K. E. HARR. 2007.Circulating and inflammatory cells. In Infectiousdiseases and pathology of reptiles, E. R.Jacobson (ed.). CRC Press: Taylor & FrancisGroup, Boca Raton, Florida, pp. 167–189.
TEARE, J. A. (ed.). 2002. Reference ranges forphysiological values in captive wildlife: Interna-tional Species Information System physiological
data reference values. ISIS, Eagan, Minnesota,on CD-ROM.
WOJTASZEK, J. S. 1992. Seasonal changes of circu-lating blood parameters in the grass snakeNatrix natrix natrix. Comparative Biochemistryand Physiology Part A: Physiology 103: 461–471.
Submitted for publication 4 August 2010.Accepted 1 October 2011.
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