Hematology and Clinical Chem Values of Free Ranging Basilisk Lizards in Costa Rica

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

([email protected]).

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


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


atesoftheRI.

UpperandlowerlimitsoftheRIwere


(standard

)anditerative(robust)

methodsfortransform

ed

and

untransform

ed

data

and,when

samplessize

swere


method.S

indicatesstandard

method,and

Rindicatesthero

bust

method.

cForeach

methodofRIestim

ation,a90



the90

%co

nfidence

interval(C

I)forthelowerestim

ate

(lower


ate



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


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


(standard

)anditerative(robust)methodsfortransform

edanduntransform

eddata

and,when

samplessize

swere


method.Numericvaluesare


atesoftheRI.

Sindicatesstandard

method,andR

indicatesthero

bust

method.

cForeach

methodofRIestim

ationa90



the90

%co

nfidence

interval(C

I)forthelowerestim

ate

(lower


ate



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


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


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.

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Received for publication 26 January 2009


Documents

Hematology and Clinical Chem Values of Free Ranging Basilisk Lizards in Costa Rica