Upload
andre-ngo
View
64
Download
1
Embed Size (px)
DESCRIPTION
Values reported for wild basilisk lizard hematology/blood chemistry
Citation preview
Journal of Zoo and Wildlife Medicine 42(2): 205–213, 2011
Copyright 2011 by American Association of Zoo Veterinarians
HEMATOLOGYAND CLINICAL CHEMISTRY VALUES OF FREE-
RANGING BASILISK LIZARDS (BASILISCUS PLUMIFRONS) IN
COSTA RICA
Rebecca K. Dallwig, D.V.M., Joanne Paul-Murphy, D.V.M., Dipl. A.C.Z.M., Chester Thomas, D.V.M.,
Scott Medlin, D.V.M., Christopher Vaughan, Ph.D., Linda Sullivan, D.V.M., Kurt K. Sladky, M.S.,
D.V.M., Dipl. A.C.Z.M., Oscar Ramirez, B.S., M.S., and Geovanny Herrera
Abstract: Twenty-three lizards were captured for this study, both males and females (12 males, 10 females, 1
undetermined), with a large range in body weights (40–286 g) appeared to be healthy based on activity level,
physical examinations, and body condition scores. Heparinized blood samples from 20 free-ranging basilisk
lizards (Basiliscus plumifrons) in Costa Rica were used for determining complete blood cell counts, plasma, and
heparinized whole blood biochemical analysis. This information will serve as baseline reference data for future
health assessment studies of free-ranging and captive basilisk lizards, as well as epidemiologic, conservation, and
captive-breeding studies. A point-of-care analyzer was useful for this field study, and clinical chemistry values
from heparinized whole blood samples were similar to values from plasma, which indicates that separation of
plasma may not be necessary to process blood samples on site in remote areas. To the authors’ knowledge, this is
the first report of hematologic and plasma biochemical data from free-ranging B. plumifrons.
Key words: Basiliscus plumifrons, clinical biochemistry, hematology, lizard, plasma biochemistry, reference
range.
INTRODUCTION
The green basilisk lizard, Basiliscus plumifrons,
also called the plumed or double-crested lizard; or
the ‘‘Jesus Christ Lizard,’’ because of its unique
ability to walk on water, is an integral part of the
species diversity of Costa Rica. This species is
abundant in the tropical rain forests of Central
America. To date, there is limited information
regarding the ecologic, biologic, clinical, and
pathologic conditions of captive and free-ranging
B. plumifrons, and published information is pri-
marily based on fortuitous observations.24,29 The
large basilisk population on an organic cacao
(Theobroma cacao) plantation and adjacent areas
in Limon Province, Costa Rica, provided the
opportunity to gather biologic information on
the health status of B. plumifrons.29 The goal of this
study was to document hematologic and biochem-
ical values, including differences between sexes
for B. plumifrons in a Costa Rican wet forest. In
addition, the study compared heparinized whole
blood with plasma clinical chemistry values in this
species to evaluate the usefulness of a point-of-
care analyzer under field conditions.
MATERIALS AND METHODS
Study site
This study was performed in a premontane wet
forest in the Guapiles region, La Rita District,
Limon Province, Costa Rica (10819"N, 83835"W).
From the School of Veterinary Medicine, University
of Wisconsin, 2126 Veterinary Medicine Building, 2015
Linden Drive, Madison, Wisconsin 53706, USA (Dall-
wig, Medlin); Department of Medicine and Epidemiol-
ogy, School of Veterinary Medicine, University of
California Davis, School of Veterinary Medicine, 2108
Tupper Hall, Davis, California 95616, USA (Paul-
Murphy); Department of Natural Resources–Wildlife
Ecology, College of Agriculture and Life Sciences,
University of Wisconsin, 233 Russell Laboratories,
1630 Linden Drive, Madison, Wisconsin 53706, USA
(Vaughan); International Institute for Wildlife Con-
servation and Management, Universidad Nacional,
Heredia, Costa Rica (Vaughan); Department of Patho-
biological Sciences, School of Veterinary Medicine,
University of Wisconsin, 2126 Veterinary Medicine
Building, 2015 Linden Drive, Madison, Wisconsin
53706, USA (Sullivan, Thomas); Department of Surgical
Sciences, School of Veterinary Medicine, University of
Wisconsin, 2015 Linden Drive, Madison, Wisconsin
53706, USA (Sladky); School of Biological Sciences,
Universidad Nacional, Heredia, Costa Rica (Ramirez);
and Milwaukee Public Museum, Milwaukee, Wisconsin
53233, USA (Herrera, Vaughan). Present addresses
(Dallwig): Chicago Zoological and Aquatic Animal
Residency Program, College of Veterinary Medicine,
University of Illinois, College of Veterinary Medicine,
3505 Veterinary Medicine Basic Sciences, 2001 South
Lincoln Avenue, Urbana, Illinois 61802, USA; (Medlin):
Stahl Exotic Animal Veterinary Services, 4105 Rust
Road, Fairfax, Virginia 22030, USA. Correspon-
dence should be directed to Dr. Paul-Murphy
205
The site was an active organic cacao plantation
situated in an agriculturally altered landscape,
with premontane wet forest nearby. The farm is
bordered by a banana plantation, a large pineap-
ple crop, grasslands, and pastures with scattered
trees. Shade on the cacao plantation is provided
by Eucaliptus globulus, Zygia longifolia, Leucaena
leucocephala, and Cocos nucifera. Several streams
and water channels cross the plantation, which
produce abundant leaf litter and undergrowth.29
Animals
Basiliscus plumifrons, a diurnal species, were
captured at nightfall by using nylon cords at-
tached to a 2-m metal pole, mesh nets, and leather
gloves. Global positioning system locations were
recorded and maintained in a data record system,
and the lizards were returned to the capture
location. Twenty-three lizards (12 males, 10
females, 1 undetermined) were evaluated. The
lizard body weights ranged from 40 to 255 g
(median body weight, 127 g) and the snout-to-
vent lengths ranged from 100 to 220 mm (median
snout-to-vent length, 167.5 mm). The lizards were
captured between 20 and 26 July 2006, transport-
ed to a field station in cloth drawstring bags, and
kept in bags overnight until processing, approxi-
mately 12 hours later. Basiliscus plumifrons are a
diurnal species and inactive at night, therefore,
this period without access to food and water was
considered within acceptable limits.28 The mean
ambient air temperature at the time of sampling
was 26.68C (808F).
The lizards were weighed in their bags by using
a digital postage scale (Pelouze Model SP5,
Sandford Corporation, Oak Brook, Illinois
60523, USA) and manually restrained by using
disposable gloves for physical examination and
morphometric measurements. Physical examina-
tion included body condition score based on a
scale of 1–5, and determined by two authors
(RKD and SM) for standardization, weight,
snout-to-vent length (millimeters), respiratory
rate (breaths per minute), and heart rate (beats
per minute) by using a Doppler ultrasonic flow
detector (model 811-B, Parks Medical Electron-
ics, Inc., Aloha, Oregon 97007, USA) by placing a
pediatric transducer over the ventral thoracic
area. The method for body condition scoring used
in this study was similar to that described in a
study with leopard geckos (Eublepharis macular-
ius).6 Each lizard was temporarily marked (Shar-
piet, Sandford Corporation) with an
identification number bilaterally on the skin of
the thoracic region. In addition, each lizard was
subjectively sexed based on the presence or
absence of a hemipene bulge, and the presence
or absence of distinctive crests. If sex could not
confidently be determined, then the animal was
classified as an unknown.
Sample collection and processing
Blood samples were collected by using mini-
mally coated preheparinized (1000 USP units/ml,
Baxter Health Care Cooperation, Deerfield, Illi-
nois 60015-4625, USA) 1.0-ml syringes, with 25-
or 23-gauge needles (3/4 inch) to prevent coagu-
lation during venipuncture. The ventral tail was
prepped for coccygeal venipuncture by using a
square gauze pad and 70% alcohol. Collected
blood volumes were less than 1% of the total body
weight of the animal (range, 0.16–1.1 ml). Each
blood sample was divided into multiple aliquots.
Two heparinized microhematocrit tubes were
filled to approximately 75% the length of the tube
and were sealed. The packed cell volume was
measured from the microhematocrit tubes after
centrifuging for 5 min in a ZIPocrit microhemat-
ocrit centrifuge (WARD’S Natural Science, Ro-
chester, New York 14692-9012, USA). Plasma
total solids were measured by using a refractom-
eter (Schuco Clinical Refractometer, Model 5711–
2020, Schuco International Ltd., Challenge
House, London, N12 ONE, England) from the
same microhematocrit samples. The remainder of
the whole blood samples were immediately placed
into a heparinized Microtainer (Becton Dickinson
and Company, Franklin Lakes, New Jersey 07417,
USA), capped, and mixed to ensure that no clots
were present. All the samples were clear-to-mildly
hemolyzed and considered acceptable for analy-
sis. Samples were processed within 15 min of
collection by using a point-of-care analyzer
(VetScant Avian Reptilian Profile Plus, PN: 500-
7131, Rev: C, � 2003, Abaxis, Inc., Union City,
California 94587, USA). Reptile and avian rotors
were filled with 0.1 ml heparinized blood for
measurement of 12 blood analytes: aspartate
aminotransferase (AST), bile acids, creatine ki-
nase (CK), uric acid, glucose, total calcium,
phosphorus, total protein (TP), albumin (Alb),
globulin (Glob), potassium, and sodium. The
remaining blood was centrifuged within 27–190
min (median, 70 min), and the plasma was
transferred to cryovials and refrigerated at 48C
for temporary storage. Plasma samples were
analyzed as a batch within 3–72 hr (median, 11.5
hr) of initial sample collection by using the same
point-of-care analyzer and rotor combination.
206 JOURNAL OF ZOO AND WILDLIFE MEDICINE
A minimum of 3 blood smears were made from
each heparinized whole blood sample. Slides were
fixed on site with methanol, stored for transpor-
tation, and stained with Wright-Giemsa at the
University of Wisconsin–School of Veterinary
Medicine. Cell morphology and differential leu-
kocyte counts were determined. The leukocyte
differential was based on examination of 100
leukocytes, and cells were classified into 1 of 5
groups: heterophils, monocytes–azurophils, lym-
phocytes, eosinophils, and basophils. Red blood
cells were also evaluated for hemoparasites. A
total white blood cell count (WBC) was obtained
by using eosinophil Unopettes (Becton Dickinson
Vacutainer Systems, Becton Dickinson and Com-
pany, Franklin Lanes, New Jersey 07417-1885,
USA) within 15–30 min of sample collection, and
the calculations were made after completion of
the blood cell differentials (total HET/EO¼ (total
heterophil/eosinophil count)/18) 3 32 3 10, with
the total WBC per ml¼ ([total HET/EO]/%HET/
EO)3 100). Basophils were found in low numbers
and were not included in the total count. Throm-
bocyte numbers were defined as decreased, ade-
quate, or increased. Decreased thrombocytes by
definition are less than 2–5 per high power field
(hpf ), with no visible thrombocyte clumps, ade-
quate numbers are 2–5 thrombocytes per hpf with
an average of only 2 clumps of thrombocytes per
field, increased numbers of thrombocytes are
defined as greater than 5 per hpf or numerous
clumps present on the slide. (K. Harr, Avian
Hematology SOP, Phoenix Central Laboratory
for Veterinarians, Everett, Washington 98204,
USA, pers. comm.)
Statistical analyses
Descriptive statistics and distributions for each
data variable were examined by using Reference
Value Advisor v1.4 (RefValAdv) (National Veter-
inary School, Toulouse, France). Reference value
Advisor v1.4 is a set of Excelt (Microsoft
Corporation, Redmond, Washington 98052,
USA) macros that compute reference intervals
from data contained in spreadsheets by using
methods that closely adhere to Clinical and
Laboratory Standards Institute guidelines.5 Un-
transformed data as well as Box–Cox transformed
data were evaluated. Data were tested for good-
ness-of-fit to the Gaussian distribution by using
the Anderson–Darling statistic. Upper and lower
limits of the reference interval were computed by
parametric (standard) and iterative (robust) meth-
ods for transformed and untransformed data and,
when samples sizes were large enough, by a
nonparametric method. For each method of
reference interval estimation, a 90% confidence
interval about the upper and lower bound was
calculated. Statistical outlier detection was eval-
uated in RefValAdv by both the methods of
Dixon10 and that of Tukey.27 Data identified as
outliers were excluded from the analysis of that
analyte. Data identified as suspect outliers were
not excluded. The analyte values obtained from
blood and plasma were compared by using a
paired t-test. Differences in mean values, by sex,
of whole blood biochemistry and hematology
were compared by using a 2-sample t-test. These
tests of hypotheses were performed by using
SYSTATt version 12, (SYSTAT Software, Inc.,
Richmond, California 94804-3559, USA). Signif-
icance was defined as P , 0.05.
RESULTS
Other than minor resolved skin wounds, lizards
appeared to be active and healthy, based on
normal body scores and physical examinations.
Physical values for both males and females
included respirations per minute (median, 60;
range, 22–90) and heart beats per minute (median,
108; range, 60–180) at an ambient temperature of
26.68C (808F). The females had a median heart
rate of 108 beats per minute (range, 60–180) and a
median respiratory rate of 54 breaths per minute
(range, 22–72), whereas male lizards had a median
heart rate of 120 beats per minute (range, 84–156)
and a median respiratory rate of 61 breathes per
minute (range, 50–90).
Statistical analysis for hematology and bio-
chemistry is reported on analyte values from 20
animals. Data were collected on 23 animals,
however, the data set from 3 lizards was not
included in hematology or biochemistry analysis
because they were identified as outliers because of
WBC elevations.
Hematology
Hematology results are provided in Table 1. All
data from 3 lizards with total WBCs of approxi-
mately 64,000, 50,000, and 45,000 cells/ll of
blood were excluded from the entire data set. No
eosinophils and only 1–2 basophils per slide were
identified in the 20 leukocyte counts. Thrombo-
cytes were not statistically evaluated but were
found to be present in adequate numbers. In
addition, no hemoparasites or detectable abnor-
malities in blood cell morphology were present.
There were no significant differences in any
measured hematologic analytes between sexes.
DALLWIG ET AL.—BASILISK LIZARDS HEMATOLOGYAND CLINICAL CHEMISTRY 207
Table
1.
Hemato
logyvaluesfor20free-rangingadult
basilisk
liza
rds(B
asiliscusplumifrons)
from
theGuapilesregionofCostaRica.a
Analyte
NM
ean
SD
Median
Min
Max
RIb
Lowerbound
/90
%CIc
Upperbound
/90
%CIc
Dist.
WBC
(10
3/ll)
20
18.7
8.4
17.2
3.9
35.5
0.7;36.7
S�4.2;6.2
30.9;41.9
Norm
al
PCV
( %)
19
31.4
8.0
30
20
52
19;55.8
R12.3;20.8
39.5;48.6
Norm
al
TS(g/dL)
19
4.2
1.0
4.2
2.4
6.0
2.0;6.4
S1.4;2.7
5.7;7.1
Norm
al
Hetero
phil(10
3/ll)
20
13.2
5.9
11.7
3.1
24.1
0.5;25.9
S�3.0;4.4
21.9;29.6
Norm
al
Monocy
te–a
zuro
phil(10
3/ll)
18
1.4
1.2
1.3
0.08
4.2
0.04;5.3
R0.001;0.2
3.4;7.1
Box–C
oxnorm
al
Lymphocytes(10
3/ll)
20
3.6
2.2
2.9
0.77
7.4
0.3;11.5
R0.2;0.9
7.8;15.3
Box–C
oxnorm
al
Eosinophils(10
3/ll)
20
00
00
00
00
Nonparametric
Baso
phils(10
3/ll)
20
0.28
00.28
0.19
0.39
ND
ND
ND
Nonparametric
aM
in,minim
um;M
ax,maxim
um;RI,
referenceinterval;CI,
confidence
interval;Dist.,distribution;W
BC,whitebloodce
llco
unt;PCV,packedcellvolume;TS,totalso
lids;
ND,not
determ
ined.
bRI,
reference
interval:Numericvaluesare
thelowerandupperestim
atesoftheRI.
UpperandlowerlimitsoftheRIwere
computedbyparametric
(standard
)anditerative(robust)
methodsfortransform
ed
and
untransform
ed
data
and,when
samplessize
swere
largeenough,byanonparametric
method.S
indicatesstandard
method,and
Rindicatesthero
bust
method.
cForeach
methodofRIestim
ation,a90
%CIabouttheupperandlowerboundwasca
lculated.Numericvaluesare
the90
%co
nfidence
interval(C
I)forthelowerestim
ate
(lower
bound)andtheupperestim
ate
(upperbound)oftheRI.
Table
2.
Heparinizedwhole
blood
bioch
emicalanalytesand
descriptivestatisticsforfree-rangingadult
basilisk
liza
rds(B
asiliscusplumifrons)
collected
inthe
GuapilesregionofCostaRicabyusingaVetsca
npoint-of-ca
reanalyze
r.a
Analyte
NM
ean
SD
Median
Min
Max
RIb
Lowerbound90
%CIc
Upperbound90
%CIc
Dist.
AST
(U/L)
16
48.3
26.2
39
19.5
115
16;47.4
R12.6;21.8
88.7;236.8
Box–C
oxnorm
al
CK
(U/L)
12
6,323
2,074
6,313
2,497
8,893
1,571;11,075
S�66.5;3,440
9,120;12,950
Norm
al
UA
(mg/dl)
15
1.7
0.8
1.5
0.6
2.9
�0.03;3.4
S�0.6;0.6
2.8;4.0
Norm
al
Glu
(mg/dl)
19
193
48.1
184
108
279
88.8;296
S59.3;121
262;327
Norm
al
Ca(m
g/dl)
19
10.6
1.3
10.5
8.3
12.4
7.8;13.4
S7.0;8.6
12.5;14.2
Norm
al
Phos(m
g/dl)
19
5.6
1.6
5.0
4.1
9.3
3.5;13.3
R3.3;3.9
8.3;32.4
Box–C
oxnorm
al
TP
(g/dl)
19
4.4
1.6
53.1
6.6
3.0–6
.7R
2.7;3.3
5.8;7.9
Box–C
oxnorm
al
Alb
(g/dl)
19
1.8
0.3
1.8
1.3
2.6
1.3;2.7
R1.1;1.4
2.3;3.0
Box–C
oxnorm
al
Glob(g/dl)
19
2.6
0.7
2.6
1.6
4.7
1.6;4.5
R1.3;1.8
6.3;5.4
Box–C
oxnorm
al
K(m
mol/L)
16
5.4
1.7
5.2
2.3
7.9
1.7;9.1
S0.0;2.9
7.7;10.1
Norm
al
Na(m
mol/L)
19
153.5
7.0
152
142
167
139;169
S134;143
163;173
Norm
al
aM
in,minim
um;M
ax,maxim
um;CI,
confidenceinterval;Dist.,distribution;AST,asp
artate
aminotransferase;CK,creatininekinase;UA,uricacid;Glu,glucose;Ca,calcium;Phos,
phosp
horu
s;TP,totalpro
tein;Alb,albumin;Glob,globulin;K,potassium;Na,so
dium.
bRI,
reference
interval:Numericvaluesare
thelowerandupperestim
atesoftheRI.
UpperandlowerlimitsoftheRIwere
computedbyparametric
(standard
)anditerative(robust)
methodsfortransform
ed
and
untransform
ed
data
and,when
samplessize
swere
largeenough,byanonparametric
method.S
indicatesstandard
method,and
Rindicatesthero
bust
method.
cForeach
methodofRIestim
ation,a90
%CIabouttheupperandlowerboundwasca
lculated.Numericvaluesare
the90
%co
nfidence
interval(C
I)forthelowerestim
ate
(lower
bound)andtheupperestim
ate
(upperbound)oftheRI.
208 JOURNAL OF ZOO AND WILDLIFE MEDICINE
Whole blood and plasma biochemistry
Heparinized whole blood biochemical analytes
are provided in Table 2, and plasma biochemical
analytes are presented in Table 3. All values were
normally distributed. Bile acid concentrations
were not obtained because all values were less
than the detection range of the point-of-care
analyzer (i.e., ,35 lmol/L). Because only 1% of
the lizard’s body weight was acceptable for blood
volume sampling, smaller volumes obtained
(,0.25 ml) were insufficient for both heparinized
whole blood and plasma analysis. For this reason,
the sample sizes in Tables 2 and 3 do not match.
Creatine kinase was not reported for every lizard
because some values were above the range of the
analyzer (i.e., .14,000 U/L). There were no
statistical differences in whole blood versus
plasma biochemical analytes or between sexes
for plasma biochemical analytes. However, when
comparing males to females for whole blood
biochemical analytes, there was a statistical
difference in TP (P ¼ 0.013), with a mean of 4.93
for males and 3.97 for females; Alb (P ¼ 0.047),
with a mean of 1.97 for males and 1.68 for
females; and Glob (P ¼ 0.036), with a mean of
2.95 for males and 2.29 for females.
DISCUSSION
Cacao plantations in Costa Rica’s lowland
tropics provide habitat and refuge for healthy
free-ranging B. plumifrons populations. Blood was
sampled in the field from a presumed adult subset
of this population for the purpose of this study by
using a point-of-care analyzer. The results are
similar to reported values in other reptilian
species. As previously stated, the complete blood
cell count and biochemistry data sets from 3
animals with WBC of 64,000, 50,000, and 45,000
cells/ll were excluded from analysis. There were
no statistical differences for biochemistry analy-
tes between heparinized whole blood and plasma.
TP, Alb, and Glob were found to be statistically
different between males and females for whole
blood but not for plasma. The elevated CKvalues
and variations in WBCs can be explained by
physiologic changes.
This study included lizards of differing weights
and lengths, and presumably of differing ages. A
complete ecologic reference to differentiate juve-
niles from adult B. plumifrons lizards was not
available; therefore, the data were not compared
based on weight or length of the lizards. A recent
study of B. plumifrons in the same geographic area
of this current study reported a snout-to-vent
Table
3.
Plasm
abiochemicalanalytesanddescriptivestatisticsforfree-rangingadult
basilisk
liza
rds(B
asiliscusplumifrons)
collectedin
theGuapilesregionof
CostaRicabyusingaVetS
canpoint-of-care
analyze
r.a
Analyte
NM
ean
SD
Median
Min
Max
RIb
Lowerbound90
%CIc
Upperbound90
%CIc
Dist.
AST
(U/L)
11
33.8
16.4
28.5
13
53
4.3;85.2
R0;13.5
55.6;86.5
Box–C
oxnorm
al
CK
(U/L)
12
4,441
2,778
4,300
330
8,381
�1,923;10,804
S�4,116;580
8,187;13,316
Norm
al
UA
(mg/dl)
12
2.8
2.5
1.7
0.6
7.6
0;21.8
R0;0
6.9;66.7
Box–C
oxnorm
al
Glu
(mg/dl)
13
161
52.4
174
16
213
18.6;243
R0;102
214;259
Box–C
oxnorm
al
Ca(m
g/dl)
12
10.7
1.1
10.5
8.9
12.6
8.3;13.2
S7.4;9.2
12.2;14.1
Norm
al
Phos(m
g/dl)
13
6.1
2.6
5.5
1.1
11.8
0;12.4
R0;2.6
9.8;15.3
Box–C
oxnorm
al
TP
(g/dl)
12
4.7
0.9
4.2
3.5
6.6
3.2;8.2
R3.0;3.7
6.0;10.1
Box–C
oxnorm
al
Alb
(g/dl)
12
1.9
0.3
1.8
1.4
2.6
1.3–3
.0R
1.2;1.5
2.4;3.8
Box–C
oxnorm
al
Glob(g/dl)
11
2.6
0.4
2.6
2.1
3.2
1.8;3.5
S1.5;2.1
3.1;3.8
Norm
al
K(m
mol/L)
11
4.7
1.5
4.8
1.8
7.0
1.3;8.1
S0.1;2.7
6.6;9.4
Norm
al
Na(m
mol/L)
12
153.4
5.6
152
144
162
141;166
S136;146
161;171
Norm
al
aM
in,minim
um;M
ax,maxim
um;CI,
confidence
interval;Dist.,distribution;AST,asp
artate
aminotransferase;CK,creatininekinase;UA,uricacid;Glu,glucose;Ca,calcium;Phos,
phosp
horu
s;TP,totalpro
tein;Alb,albumin;Glob,globulin;K,potassium;Na,so
dium.
bRI,
reference
interval:UpperandlowerlimitsoftheRIwere
computedbyparametric
(standard
)anditerative(robust)methodsfortransform
edanduntransform
eddata
and,when
samplessize
swere
largeenough,byanonparametric
method.Numericvaluesare
thelowerandupperestim
atesoftheRI.
Sindicatesstandard
method,andR
indicatesthero
bust
method.
cForeach
methodofRIestim
ationa90
%CIabouttheupperandlowerboundwasca
lculated.Numericvaluesare
the90
%co
nfidence
interval(C
I)forthelowerestim
ate
(lower
bound)andtheupperestim
ate
(upperbound)oftheRI.
DALLWIG ET AL.—BASILISK LIZARDS HEMATOLOGYAND CLINICAL CHEMISTRY 209
length of 250 mm in males and 174 mm in
females.29 In this current study, the median
snout-to-vent length was 167.5 mm, with a range
of 100–220 mm, which corresponds with the
aforementioned study and indicates the majority
of animals reported in this study are adults that
represent both sexes. The closely related Basilicus
basiliscus were reported to reach maturity at about
20 and 16 mo of age for females and males,
respectively. They are a sexually dimorphic spe-
cies with males having enlarged crests on the
head, tail, and body, and to be larger in size than
females.24 In this current study, sex was easily
determined by physical characteristics in all but
the smallest lizard, by using the sexually dimor-
phic characteristics of B. basiliscus as a model,
which allows comparison of male to female
hematology and clinical chemistry values.
For this data set, all statistical outliers were
evaluated in RefValAdv by both methods of
Dixon and Tukey. However, when more than one
outlier is present, either method may fail to detect
extreme values because of a phenomenon termed
‘‘masking.’’26 All data from 3 lizards with a total
WBC of approximately 64,000, 50,000, and
45,000 cells/ll of blood that were not identified
as outliers by RefValAdv were excluded from the
entire data set. This was an empirical decision by
using the rationale that such values were unlikely
to have come from individuals representative of a
healthy reference population.
The WBC in this study has a wide range (mean,
18.71 103/ll range, 3.87–35.5). Considerations for
the discrepancy in the WBC include stress,
infection, inflammation, or elevated body temper-
ature. It has been noted that reptiles can have a
wide range of WBCs with certain leukocyte
numbers changing with environmental, seasonal,
and temperature factors, in addition to diseases.2
All of the lizards sampled appeared healthy on
physical examination, all were collected from the
same approximate site, and animals with elevated
WBCs determined to be outliers were excluded
from the data set, therefore, infectious etiology is
unlikely. Because the body temperatures of the
lizards were not measured, the effect of temper-
ature could not be evaluated in this study.
Previous publications reported significantly
decreased total thrombocyte counts and WBCs,
including individual leukocytes, when heparinized
whole blood was used compared with nonanti-
coagulated whole blood in green iguanas and
Chinese water dragons.14,22 Heparin also has been
reported to cause an increase in pyknotic leuko-
cytes and lysed cells.22 A study in Hermann’s
tortoises (Testudo hermanii), however, concluded
heparin to be the choice anticoagulant for hema-
tology when compared with ethylenediaminete-
traacidic acid (EDTA).23 Studies in other reptile
species, such as Burmese pythons (Python molurus
bivittatus), found no significant differences in
hematology values between EDTA and heparin.16
Heparin was used in this study because it is
required by the point-of-care analyzer. Although
the heparin in the preheparinized syringes was
considered negligible and only low numbers of
pyknotic leukocytes and lysed cells were noted in
our samples, the authors acknowledge that this
could contribute to a potentially falsely lowered
WBC. No evidence of dilutional effects secondary
to heparin use was evident in this study because
the samples with small volumes were not subjec-
tively correlated to the lizards with the lowest
absolute WBCs. Although comparing anticoagu-
lants was not the goal of this study, the reader
should consider which anticoagulant was used
with blood collection when comparing studies.
Monocytes with azurophilic granules (azuro-
phils) were noted on the hematology differentials
in this study and were included in the total
monocyte count. Previous research with green
iguanas has shown no cytochemical difference
between monocytes with and without azurophilic
granules.15 Reports of green iguanas and Chinese
water dragons include azurophils in the absolute
monocyte count, and it was decided to include
both types of monocytes under the same category
during the differential leukocyte counts in our
study.14,22
The number of circulating eosinophils in a
healthy lizard can vary; however, lizards generally
have lower numbers of eosinophils when com-
pared with other species, as was documented in
this study. The absence of eosinophils in this
study may be attributed to a low parasitic load,
although this cannot be confirmed because endo-
parasite and ectoparasitic sampling was not the
goal of this study. Alternatively, eosinophil num-
bers can be influenced by seasonal changes with
lower numbers of eosinophils documented in
lizards during the summer months, which is
consistent with the sampling period of this
study.2,14,15
Point-of-care analyzers used in previous studies
with psittacines and Kemp’s Ridley (Lepidochelys
kempii) sea turtles have been determined to be
very useful tools.17,19 Whole blood samples were
analyzed immediately after collection to avoid
changes in analytes associated with prolonged
sample storage of heparinized whole blood as
210 JOURNAL OF ZOO AND WILDLIFE MEDICINE
reported for Burmese python (P. molurus bivittatus)
samples.16 The effect of plasma refrigeration and
storage on clinical chemistry values is expected to
be minimal because a published report for
loggerhead sea turtles (Caretta caretta) found no
significant changes in analytes of stored plasma
compared with whole blood when samples were
centrifuged and plasma stored 24 hours before
analysis.11 A study on Aldabara tortoises (Geo-
chelone gigantean) and Burmese mountain tortois-
es (Manouria emys) concluded that samples of
serum and plasma stored at 48C significantly
improved the stability of potassium and sodium
concentrations, when compared with samples
stored at 258C.1 No significant differences were
found in analytes tested between heparinized
whole blood and plasma. Because of this congru-
ity, heparinized whole blood sampled with a
point-of-care analyzer could be used as a sole
sampling method in remote sites and eliminate
the need for centrifugation and refrigeration of
plasma samples. Alternatively, a point-of-care
analyzer to determine plasma clinical chemistry
values from stored plasma samples would be
reliable.
Clinical biochemical reference intervals were
determined in low numbers of free-ranging B.
plumifrons lizards. The results reported in this
study were compared with published values for
several different lizard species. The plasma bio-
chemical concentrations we recorded were within
the reported ranges for plasma biochemical
values in the green iguana (Iguana iguana)8,15 and
the rock iguana (Cyclura cychlura inornata)18 and
for serum biochemistry values in the Chinese
water dragon (Physignathus oncincinus).22 In addi-
tion, the plasma biochemical values in B. plumi-
frons fell within published references ranges for
bearded dragons (Pogona vitticeps), with the ex-
ception of TP, Alb, Glob, and CK, which were not
reported.12
Calcium values were not significantly different
between males and females in this study. Females
undergoing vitellogenesis or gravid females would
be expected to have higher plasma calcium
concentrations than males.3,4,7,20,21,25 Female Amer-
ican alligators (Alligator mississippiensis) are docu-
mented to have increased plasma calcium levels
that correspond to ovarian response.13 In addi-
tion, plasma concentration of ionized calcium in
healthy iguanas was not found to be significantly
different among adult males, adult females, or
juveniles, and were concluded to be tightly
regulated.8 These findings suggest that it was not
the reproductive season for the free-ranging B.
plumifrons during the sampling period. Although
the breeding season of B. plumifrons has not been
documented, a related species (B. basiliscus) is
reported to have significantly lower reproductive
activity during February and March, with more
gravid females identified during October and
November.28 If the B. plumifrons species is similar
in its breeding season, then it is plausible that the
lizards in this study were neither gravid nor
entering vitellogenesis when sampled in July.
Alternatively, it is possible that a number of the
lizards sampled in this study population were not
of breeding age. The weights and lengths of lizards
in our study are within the range of documented
adult sizes, however, the measurements could also
indicate that they are young adults and not yet of
breeding maturity.28 Sex determination was based
on subjective morphologic characteristics and not
DNA sexing or laparoscopic examination, there-
by allowing the possibility of misidentification of
males and females.
A statistically significant difference between
male and female TP, Alb, and Glob heparinized
whole blood analytes was noted. In all cases, the
males had higher mean values than the females,
for TP (mean, 4.72 vs. 3.97), Alb (mean, 1.97 vs.
1.68), and Glob (mean, 2.95 vs. 2.29), respectively.
Elevations in TP, Alb, and/or Glob have been
noted in gravid females when compared with
nongravid females and males but our study
population was presumed to not include gravid
or vitellogenic females.15 The heparinized whole
blood and plasma biochemical values for TP, Alb,
and Glob reported here are still within, or very
close to, the reference intervals reported for other
lizard species.8,15,18,22 In addition, because there
were no significant differences in any plasma
biochemical analytes between males and females,
nor were there any significant differences in
analytes between whole blood and plasma bio-
chemical analysis, it is plausible that these
differences could be artifactual and attributed to
chance and the small sample size, and, therefore,
are not a biologically significant finding.
The CK values of 12 heparinized whole blood
samples and 12 plasma samples are reported. The
CK concentrations for 7 heparinized whole blood
and 3 plasma samples were greater than the
detectable range for the point-of-care analyzer to
accurately measure and, therefore, are not includ-
ed. The elevated CK results were confirmed by
the codes indicated on the VetScan troubleshoot-
ing report that corresponds to each CK value. As
in other species, CK in reptiles is considered an
enzyme specific to muscular cell damage. The
DALLWIG ET AL.—BASILISK LIZARDS HEMATOLOGYAND CLINICAL CHEMISTRY 211
elevated CK results may be secondary to the
animals’ activities during the 12–14-hr holding
period, the length of the capture, or venipuncture
and restraint.2 Although it was not statistically
evaluated, only three of the animals sampled in
this study had concurrent elevations in AST and
CK. Capture and restraint of free-ranging Ric-
ord’s iguanas (Cyclura ricordii) also caused eleva-
tions in both CK and AST levels.21 A published
health assessment for wild caught rock iguanas
(Cyclura cychlura inornata) found elevated CK
values in nonhabituated animals compared with
animals habituated to people and handling.18
CONCLUSIONS
Hematologic and blood biochemical data from
this study provide useful ranges for evaluating the
health status of free-ranging B. plumifrons. The
data reported here was found to be comparable
with previously published data for other lizard
species, including the green iguana, Chinese water
dragon, and bearded dragon.9,12,22
Because no statistical differences were found
between the plasma and the heparinized whole
blood biochemistry analytes in this study, it was
concluded that the point-of-care analyzer used in
this study (VetScan) was useful for collection of
data in the field. Separation of plasma from red
blood cells may not be necessary to obtain
accurate data for B. plumifrons when using this
point-of-care analyzer, which suggests that using a
point-of-care analyzer may enable researchers to
process blood samples on site in remote areas,
thus, eliminating the need for refrigeration, trans-
port, and centrifugation of samples, and thereby
improving the accuracy of data collected from
free-ranging species.
The clinical chemistry and hematology data
presented add to the limited data available to
veterinarians and conservation biologists for this
lizard species. The limited sample size in this
study, and the limited methodologic comparison,
has generated data that should only be considered
preliminary. A larger sample size would be
necessary to verify the reliability and repeatability
of these results in both captive and free-ranging B.
plumifrons.
Acknowledgments: The authors thank the
ABAXIS company for generously supporting this
study by the donation of VetScan and the Avian
Reptilian Profile Plus chemistry rotors, and the
WARD’S Natural Science company for the dona-
tion of a ZIPocrit microhematocrit machine. In
addition, the authors thank Pete MacWilliams,
D.V.M., Ph.D., Dipl. A.C.V.P., Julia Klauer, B.S.,
and Ann Stewart, C.V.T., for their invaluable
assistance throughout the course of this study.
Finally the authors thank Hugo Hemerlink for the
use of his plantation (Finmac). Funding for this
study was provided by Henry Vilas Zoological
Society in Madison, Wisconsin (USA) and col-
laborates with a mission of the Conservation
Health Consortium and the Milwaukee Public
Museum/University of Wisconsin–Madison/
United States Department of Agriculture (58-
1275-2-026 funding ‘‘Theobroma cacao: Biodiver-
sity in Full and Partial Canopies’’), a project to
study Costa Rican biodiversity in cocoa planta-
tions.
LITERATURE CITED
1. Abou-Madi, N., and E. R. Jacobson. 2003. Effects
of blood processing techniques on sodium and potas-
sium values: a comparison between aldabra tortoises
(Geochelone gigantean) and Burmese mountain tortoises
(Manouria emys). Vet. Clin. Pathol. 32: 61–66.
2. Campbell, T. W. 2006. Clinical pathology of
reptiles. In: Mader, D. R. (ed.). Reptile Medicine and
Surgery, 2nd ed. Elsevier Inc., St. Louis, Missouri. Pp.
458–465.
3. Christopher, M. M., K. Berry, I. Wallis, K. A.
Nagy, B. T. Henen, and C. C. Peterson. Reference
intervals and physiologic alterations in hematologic
and biochemical values of free-ranging Desert tortois-
es in the Mojave desert. 1999. J. Wildl. Dis. 35: 212–
238.
4. Cree, A., L. J. Guillette, M. A. Brown, G. K.
Chambers, J. F. Cockrem, and J. D. Newton. 1991.
Slow estradiol-induced vitellogenesis in the tuatara,
Sphenodon punctatus. Physiol. Zool. 64: 1234–1251.
5. CSLI. 2008. Defining, establishing, and verifying
reference intervals. In: The Clinical Laboratory: Ap-
proved Guidelines, 3rd ed. Clinical and Laboratory
Standards Institute: Wayne, Pennsylvania. CSLI docu-
ment C28–A3.
6. Deming, C., E. G. Greiner, and E. W. Uhl. 2008.
Prevalence of cryptosporidium infection and charac-
teristics of oocyst shedding in a breeding colony of
leopard geckos (Eublepharis macularius). J. Zoo Wildl.
Med. 39: 600–607.
7. Denardo, D. 2006. Reproductive biology. In:
Mader, D. R. (ed.). Reptile Medicine and Surgery, 2nd
ed. Elsevier Inc., St. Louis, Missouri. Pp. 389–390.
8. Dennis, P. M., A. R. Bennett, K. E. Harr, and B. A.
Lock. 2001. Plasma concentration of ionized calcium in
healthy iguanas. J. Am. Vet. Med. Assoc. 219: 326–328.
9. Diethelm, G., and G. Stein. 2006. Hematologic
and blood chemistry values in reptiles. In:Mader, D. R.
(ed.). Reptile Medicine and Surgery, 2nd ed. Elsevier
Inc., St. Louis, Missouri. Pp. 1103–1118.
10. Dixon, W. J. 1950. Analysis of extreme values.
Ann. Math. Statist. 21: 488–493.
212 JOURNAL OF ZOO AND WILDLIFE MEDICINE
11. Eisenhawer, E., C. H. Courtney, R. E. Raskin,
and E. Jacobson. 2008. Relationship between separa-
tion time of plasma from heparinized whole blood on
plasma biochemical analytes of loggerhead sea turtles
(Caretta caretta). J. Zoo Wildl. Med. 39: 208–215.
12. Eliman, M. M. 1997. Hematology and plasma
chemistry of the inland bearded dragon, Pogona
vitticeps. Bull. Assoc. Rept. Amph. Vet. 7: 4.
13. Guillette, L. J., A. R. Woodward, A. D. Crain, G.
R. Masson, B. D. Palmer, C. M. Cox, Q. You-Xiang,
and E. F. Orlando. 1997. The reproductive cycle of the
female American alligator (Alligator mississippiensis).
Gen. Comp. Endocrinol. 108: 87–101.
14. Hanley, C. S., S. Hernandez-Divers, S. Bush, and
K. Latimer. 2004. Comparison of the effect of dipotas-
sium ethylenediaminetetraacetic acid in lithium hepa-
rin on hematologic values in the green iguana (Iguana
iguana). J. Zoo Wildl. Med. 35: 328–332.
15. Harr, K. E., R. Alleman, P. Dennis, L. Maxwell,
B. Lock, A. Bennett, and E. Jacobson. 2001. Morpho-
logic and cytochemical characteristics of blood cells
and hematologic and plasma biochemical reference
ranges in green iguanas. J. Am. Vet. Med. Assoc. 218:
915–921.
16. Harr, K. E., R. E. Raskin, and D. J. Heard. 2005.
Temporal effects of 3 commonly used anticoagulants
on hematologic and biochemical variables in blood
samples from macaws and Burmese pythons. Vet. Clin.
Pathol. 34: 383–388.
17. Innis, C. J., M. Tlusty, C. Merigo, and S. E.
Weber. 2007. Metabolic and respiratory status of cold-
stunned Kemp’s ridley sea turtles (Lepidochelys kempii).
J. Comp. Physiol. B. 177: 623–630.
18. James, S. B., J. Iverson, V. Greco, and B.
Raphael. 2006. Health assessment of Allen Cays rock
iguana, Cyclura cychlura inornata. J. Herpetol. Med.
Surg. 16: 93–97.
19. Johnston, M. S., K. L. Rosenthal, and F. S.
Shofer. 2007. Assessment of a point-of-care biochem-
ical analyzer and comparison with a commercial
laboratory for the measurement of total protein and
albumin concentrations in psittacines. Am. J. Vet. Res.
68: 1348–1353.
20. Lance, V. 1976. Studies on the annual reproduc-
tive cycle of the female cobra (Naja naja). Seasonal
variation in plasma inorganic ions. Comp. Biochem.
Physiol. 53: 285–289.
21. Maria, R., J. Ramer, T. Reichard, P. J. Tolson,
and M. J. Christopher. 2007. Biochemical reference
intervals and intestinal microflora of the free-ranging
Ricord’s iguana (Cyclura ricordii). J. Zoo Wildl. Med.
38: 414–419.
22. Mayer, J., J. Knoll, C. Innis, and M. Mitchell.
2005. Characterizing the hematologic and plasma
chemistry profiles of captive Chinese water dragons,
(Physignathus oncincinus). J. Herpetol. 15: 16–23.
23. Muro, J., R. Cuenca, J. Pastor, L. Vinas, and S.
Lavin. 1998. Effects of lithium heparin and tripotas-
sium EDTA on hematologic values of Hermann’s
tortoises (Testudo hermanni). J. Zoo Wildl. Med. 29:
40–44.
24. Rose, B. 1982. Lizard home ranges: methodology
and functions. J. Herpetol. 16: 253–269.
25. Rosthal, D. C., V. Lance, J. Grumbles, and A.
Alberts. 1994. Seasonal reproductive cycle of the
Desert tortoise (Gopherus agassizii). Herpetological
Monographs. 8: 72–82.
26. Tietien, G. L., and R. Moore. 1972. Some grubb-
type statistics for detection of several outliers. Tech-
nometrics. 14: 583–597.
27. Tukey J. W. 1997. Exploratory Data Analysis.
Addison-Wesley: Reading, Massachusetts.
28. Van Devender, R. W. 1982. Comparative demog-
raphy of the lizard Basiliscus basiliscus. Herpetologica.
38: 189–208.
29. Vaughan, C., O. Ramirez, G. Herrera, U. Fallas,
and R. Henderson. 2007. Home range and habitat use
of Basiliscus plumifrons (Squamata: Corytophanidae) in
an active Costa Rician cacao farm. Appl. Herpetol. 4:
217–226.
Received for publication 26 January 2009
DALLWIG ET AL.—BASILISK LIZARDS HEMATOLOGYAND CLINICAL CHEMISTRY 213