Journal of Zoo and Wildlife Medicine 42(2): 205213, 2011 Copyright 2011 by American Association of Zoo Veterinarians

HEMATOLOGY AND CLINICAL CHEMISTRY VALUES OF FREERANGING 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 (40286 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 eld 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 rst 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
From the School of Veterinary Medicine, University of Wisconsin, 2126 Veterinary Medicine Building, 2015 Linden Drive, Madison, Wisconsin 53706, USA (Dallwig, Medlin); Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California Davis, School of Veterinary Medicine, 2108 Tupper Hall, Davis, California 95616, USA (PaulMurphy); Department of Natural ResourcesWildlife 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 Conservation and Management, Universidad Nacional, Heredia, Costa Rica (Vaughan); Department of Pathobiological 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. Correspondence should be directed to Dr. Paul-Murphy (paulmurphy@ucdavis.edu).

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 primarily 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 biochemical 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-ofcare analyzer under eld 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).

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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 pineapple 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 attached 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-tovent 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, transported to a eld station in cloth drawstring bags, and kept in bags overnight until processing, approximately 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 examination included body condition score based on a scale of 15, 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 ow detector (model 811-B, Parks Medical Electronics, 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 macularius).6 Each lizard was temporarily marked (Sharpiet, Sandford Corporation) with an identication 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 condently be determined, then the animal was classied as an unknown. Sample collection and processing Blood samples were collected by using minimally coated preheparinized (1000 USP units/ml, Baxter Health Care Cooperation, Deereld, Illinois 60015-4625, USA) 1.0-ml syringes, with 25or 23-gauge needles (3/4 inch) to prevent coagulation 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, 0.161.1 ml). Each blood sample was divided into multiple aliquots. Two heparinized microhematocrit tubes were lled 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 microhematocrit centrifuge (WARDS Natural Science, Rochester, New York 14692-9012, USA). Plasma total solids were measured by using a refractometer (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 analysis. Samples were processed within 15 min of collection by using a point-of-care analyzer (VetScant Avian Reptilian Prole Plus, PN: 5007131, Rev: C, 2003, Abaxis, Inc., Union City, California 94587, USA). Reptile and avian rotors were lled with 0.1 ml heparinized blood for measurement of 12 blood analytes: aspartate aminotransferase (AST), bile acids, creatine kinase (CK), uric acid, glucose, total calcium, phosphorus, total protein (TP), albumin (Alb), globulin (Glob), potassium, and sodium. The remaining blood was centrifuged within 27190 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 372 hr (median, 11.5 hr) of initial sample collection by using the same point-of-care analyzer and rotor combination.

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A minimum of 3 blood smears were made from each heparinized whole blood sample. Slides were xed on site with methanol, stored for transportation, and stained with Wright-Giemsa at the University of WisconsinSchool of Veterinary Medicine. Cell morphology and differential leukocyte counts were determined. The leukocyte differential was based on examination of 100 leukocytes, and cells were classied into 1 of 5 groups: heterophils, monocytesazurophils, lymphocytes, 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 Company, Franklin Lanes, New Jersey 07417-1885, USA) within 1530 min of sample collection, and the calculations were made after completion of the blood cell differentials (total HET/EO (total heterophil/eosinophil count)/18) 3 32 3 10, with the total WBC per ml ([total HET/EO]/%HET/ EO) 3 100). Basophils were found in low numbers and were not included in the total count. Thrombocyte numbers were dened as decreased, adequate, or increased. Decreased thrombocytes by denition are less than 25 per high power eld (hpf ), with no visible thrombocyte clumps, adequate numbers are 25 thrombocytes per hpf with an average of only 2 clumps of thrombocytes per eld, increased numbers of thrombocytes are dened 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 Veterinary 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 Untransformed data as well as BoxCox transformed data were evaluated. Data were tested for goodness-of-t to the Gaussian distribution by using the AndersonDarling statistic. Upper and lower limits of the reference interval were computed by parametric (standard) and iterative (robust) methods 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% condence interval about the upper and lower bound was calculated. Statistical outlier detection was evaluated in RefValAdv by both the methods of Dixon10 and that of Tukey.27 Data identied as outliers were excluded from the analysis of that analyte. Data identied 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). Significance was dened 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, 2290) and heart beats per minute (median, 108; range, 60180) at an ambient temperature of 26.68C (808F). The females had a median heart rate of 108 beats per minute (range, 60180) and a median respiratory rate of 54 breaths per minute (range, 2272), whereas male lizards had a median heart rate of 120 beats per minute (range, 84156) and a median respiratory rate of 61 breathes per minute (range, 5090). Statistical analysis for hematology and biochemistry 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 identied as outliers because of WBC elevations. Hematology Hematology results are provided in Table 1. All data from 3 lizards with total WBCs of approximately 64,000, 50,000, and 45,000 cells/ll of blood were excluded from the entire data set. No eosinophils and only 12 basophils per slide were identied in the 20 leukocyte counts. Thrombocytes were not statistically evaluated but were found to be present in adequate numbers. In addition, no hemoparasites or detectable abnormalities in blood cell morphology were present. There were no signicant differences in any measured hematologic analytes between sexes.

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Table 1. Hematology values for 20 free-ranging adult basilisk lizards (Basiliscus plumifrons) from the Guapiles region of Costa Rica.a

Analyte

Mean

SD

Median

Min

Max

RIb

Lower bound /90% CIc Dist.

S

Upper bound /90% CIc

WBC (10 /ll) PCV (%) TS (g/dL) Heterophil (103/ll) Monocyteazurophil (103/ll) Lymphocytes (103/ll) Eosinophils (103/ll) Basophils (103/ll)

20 19 19 20 18 20 20 20

18.7 31.4 4.2 13.2 1.4 3.6 0 0.28

8.4 8.0 1.0 5.9 1.2 2.2 0 0

17.2 30 4.2 11.7 1.3 2.9 0 0.28

3.9 20 2.4 3.1 0.08 0.77 0 0.19

35.5 52 6.0 24.1 4.2 7.4 0 0.39

0.7; 36.7 19; 55.8R 2.0; 6.4S 0.5; 25.9S 0.04; 5.3R 0.3; 11.5R 0 ND

4.2; 6.2 12.3; 20.8 1.4; 2.7 3.0; 4.4 0.001; 0.2 0.2; 0.9 0 ND

30.9; 41.9 39.5; 48.6 5.7; 7.1 21.9; 29.6 3.4; 7.1 7.8; 15.3 0 ND

Normal Normal Normal Normal BoxCox normal BoxCox normal Nonparametric Nonparametric

a Min, minimum; Max, maximum; RI, reference interval; CI, condence interval; Dist., distribution; WBC, white blood cell count; PCV, packed cell volume; TS, total solids; ND, not determined. b RI, reference interval: Numeric values are the lower and upper estimates of the RI. Upper and lower limits of the RI were computed by parametric (standard) and iterative (robust) methods for transformed and untransformed data and, when samples sizes were large enough, by a nonparametric method. S indicates standard method, and R indicates the robust method. c For each method of RI estimation, a 90% CI about the upper and lower bound was calculated. Numeric values are the 90% condence interval (CI) for the lower estimate (lower bound) and the upper estimate (upper bound) of the RI.

Table 2. Heparinized whole blood biochemical analytes and descriptive statistics for free-ranging adult basilisk lizards (Basiliscus plumifrons) collected in the Guapiles region of Costa Rica by using a Vetscan point-of-care analyzer.a
Min
R

Analyte

Mean

SD

Median

Max

RIb

Lower bound 90% CIc

Upper bound 90% CIc

Dist.

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normal

AST (U/L) CK (U/L) UA (mg/dl) Glu (mg/dl) Ca (mg/dl) Phos (mg/dl) TP (g/dl) Alb (g/dl) Glob (g/dl) K (mmol/L) Na (mmol/L)

16 12 15 19 19 19 19 19 19 16 19

48.3 6,323 1.7 193 10.6 5.6 4.4 1.8 2.6 5.4 153.5

26.2 2,074 0.8 48.1 1.3 1.6 1.6 0.3 0.7 1.7 7.0

39 6,313 1.5 184 10.5 5.0 5 1.8 2.6 5.2 152

19.5 2,497 0.6 108 8.3 4.1 3.1 1.3 1.6 2.3 142

115 8,893 2.9 279 12.4 9.3 6.6 2.6 4.7 7.9 167

16; 47.4 1,571; 11,075S 0.03; 3.4S 88.8; 296S 7.8; 13.4S 3.5; 13.3R 3.06.7R 1.3; 2.7R 1.6; 4.5R 1.7; 9.1S 139; 169S

12.6; 66.5; 0.6; 59.3; 7.0; 3.3; 2.7; 1.1; 1.3; 0.0; 134;

21.8 3,440 0.6 121 8.6 3.9 3.3 1.4 1.8 2.9 143

88.7; 9,120; 2.8; 262; 12.5; 8.3; 5.8; 2.3; 6.3; 7.7; 163;

236.8 12,950 4.0 327 14.2 32.4 7.9 3.0 5.4 10.1 173

BoxCox Normal Normal Normal Normal BoxCox BoxCox BoxCox BoxCox Normal Normal

normal normal normal normal

a Min, minimum; Max, maximum; CI, condence interval; Dist., distribution; AST, aspartate aminotransferase; CK, creatinine kinase; UA, uric acid; Glu, glucose; Ca, calcium; Phos, phosphorus; TP, total protein; Alb, albumin; Glob, globulin; K, potassium; Na, sodium. b RI, reference interval: Numeric values are the lower and upper estimates of the RI. Upper and lower limits of the RI were computed by parametric (standard) and iterative (robust) methods for transformed and untransformed data and, when samples sizes were large enough, by a nonparametric method. S indicates standard method, and R indicates the robust method. c For each method of RI estimation, a 90% CI about the upper and lower bound was calculated. Numeric values are the 90% condence interval (CI) for the lower estimate (lower bound) and the upper estimate (upper bound) of the RI.

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Table 3. Plasma biochemical analytes and descriptive statistics for free-ranging adult basilisk lizards (Basiliscus plumifrons) collected in the Guapiles region of Costa Rica by using a VetScan point-of-care analyzer.a

normal normal

DISCUSSION
Cacao plantations in Costa Ricas lowland tropics provide habitat and refuge for healthy free-ranging B. plumifrons populations. Blood was sampled in the eld 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 analytes 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 CK values 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 juveniles 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

AST (U/L) CK (U/L) UA (mg/dl) Glu (mg/dl) Ca (mg/dl) Phos (mg/dl) TP (g/dl) Alb (g/dl) Glob (g/dl) K (mmol/L) Na (mmol/L)

Analyte

11 12 12 13 12 13 12 12 11 11 12

33.8 4,441 2.8 161 10.7 6.1 4.7 1.9 2.6 4.7 153.4

Mean

16.4 2,778 2.5 52.4 1.1 2.6 0.9 0.3 0.4 1.5 5.6

SD

28.5 4,300 1.7 174 10.5 5.5 4.2 1.8 2.6 4.8 152

Median

13 330 0.6 16 8.9 1.1 3.5 1.4 2.1 1.8 144

Min

53 8,381 7.6 213 12.6 11.8 6.6 2.6 3.2 7.0 162

Max

4.3; 85.2R 1,923; 10,804S 0; 21.8R 18.6; 243R 8.3; 13.2S 0; 12.4R 3.2; 8.2R 1.33.0R 1.8; 3.5S 1.3; 8.1S 141; 166S

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 lizards body weight was acceptable for blood volume sampling, smaller volumes obtained (,0.25 ml) were insufcient 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 0.013), with a mean of 4.93 for males and 3.97 for females; Alb (P 0.047), with a mean of 1.97 for males and 1.68 for females; and Glob (P 0.036), with a mean of 2.95 for males and 2.29 for females.

Dist.

Upper bound 90% CIc

Lower bound 90% CIc

RIb

0; 4,116; 0; 0; 7.4; 0; 3.0; 1.2; 1.5; 0.1; 136;

13.5 580 0 102 9.2 2.6 3.7 1.5 2.1 2.7 146

55.6; 8,187; 6.9; 214; 12.2; 9.8; 6.0; 2.4; 3.1; 6.6; 161;

86.5 13,316 66.7 259 14.1 15.3 10.1 3.8 3.8 9.4 171

BoxCox Normal BoxCox BoxCox Normal BoxCox BoxCox BoxCox Normal Normal Normal

normal normal normal

normal

Whole blood and plasma biochemistry

a Min, minimum; Max, maximum; CI, condence interval; Dist., distribution; AST, aspartate aminotransferase; CK, creatinine kinase; UA, uric acid; Glu, glucose; Ca, calcium; Phos, phosphorus; TP, total protein; Alb, albumin; Glob, globulin; K, potassium; Na, sodium. b RI, reference interval: Upper and lower limits of the RI were computed by parametric (standard) and iterative (robust) methods for transformed and untransformed data and, when samples sizes were large enough, by a nonparametric method. Numeric values are the lower and upper estimates of the RI. S indicates standard method, and R indicates the robust method. c For each method of RI estimation a 90% CI about the upper and lower bound was calculated. Numeric values are the 90% condence interval (CI) for the lower estimate (lower bound) and the upper estimate (upper bound) of the RI.

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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 100220 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 species 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 dimorphic 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 identied 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, 3.8735.5). Considerations for the discrepancy in the WBC include stress, infection, inammation, or elevated body temperature. 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 temperature could not be evaluated in this study. Previous publications reported signicantly decreased total thrombocyte counts and WBCs, including individual leukocytes, when heparinized whole blood was used compared with nonanticoagulated whole blood in green iguanas and Chinese water dragons.14,22 Heparin also has been reported to cause an increase in pyknotic leukocytes and lysed cells.22 A study in Hermanns

tortoises (Testudo hermanii), however, concluded heparin to be the choice anticoagulant for hematology when compared with ethylenediaminetetraacidic acid (EDTA).23 Studies in other reptile species, such as Burmese pythons (Python molurus bivittatus), found no signicant 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 subjectively correlated to the lizards with the lowest absolute WBCs. Although comparing anticoagulants 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 (azurophils) 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 compared 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 conrmed because endoparasite and ectoparasitic sampling was not the goal of this study. Alternatively, eosinophil numbers can be inuenced 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 Kemps 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

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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 signicant 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 (Geochelone gigantean) and Burmese mountain tortoises (Manouria emys) concluded that samples of serum and plasma stored at 48C signicantly improved the stability of potassium and sodium concentrations, when compared with samples stored at 258C.1 No signicant differences were found in analytes tested between heparinized whole blood and plasma. Because of this congruity, 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 biochemical 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 addition, the plasma biochemical values in B. plumifrons fell within published references ranges for bearded dragons (Pogona vitticeps), with the exception of TP, Alb, Glob, and CK, which were not reported.12 Calcium values were not signicantly 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 American alligators (Alligator mississippiensis) are documented to have increased plasma calcium levels that correspond to ovarian response.13 In addition, plasma concentration of ionized calcium in healthy iguanas was not found to be signicantly different among adult males, adult females, or juveniles, and were concluded to be tightly regulated.8 These ndings 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 signicantly lower reproductive activity during February and March, with more gravid females identied 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, thereby allowing the possibility of misidentication of males and females. A statistically signicant 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 signicant differences in any plasma biochemical analytes between males and females, nor were there any signicant differences in analytes between whole blood and plasma biochemical 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 signicant nding. 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 included. The elevated CK results were conrmed by the codes indicated on the VetScan troubleshooting report that corresponds to each CK value. As in other species, CK in reptiles is considered an enzyme specic to muscular cell damage. The

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elevated CK results may be secondary to the animals activities during the 1214-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 Ricords iguanas (Cyclura ricordii) also caused elevations 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 eld. 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, transport, 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 Prole Plus chemistry rotors, and the WARDS Natural Science company for the donation 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 collaborates with a mission of the Conservation Health Consortium and the Milwaukee Public Museum/University of WisconsinMadison/ United States Department of Agriculture (581275-2-026 funding Theobroma cacao: Biodiversity in Full and Partial Canopies), a project to study Costa Rican biodiversity in cocoa plantations.

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