First published in the Bulletin of the Association of Reptile and Amphibian Veterinarians,Volume 7, Number 4, 1997. Reproduced by permission of ARAV
NOTE - This article is provided for informational purposes only and is not intended to replace the advice or services of a qualified veterinarian. Always consult a qualified veterinarian before beginning any kind of treatment for a sick or injured Bearded Dragon.
Key Words - Hematology, Plasma chemistry, Inland bearded dragon, Pogona vitticeps.
Introduction
The inland bearded dragon, Pogona vitticeps, native to inland Australia, is a diurnal, oviparous,
omnivorous, agamid lizard (Fogel, 1993, De Vosjoli and Mailloux, 1993). Due to its placid nature
and relative hardiness, it is rapidly becoming popular in captive collections. Despite this
popularity, very little hematological data has been gathered for this species (Cranfield, et al,
1996). The purpose of this study was to determine normal hematological and plasma chemistry
values for a captive population of P. vitticeps.
Materials and Methods
Blood samples were gathered from what appeared to be 21 healthy adult captive bred inland bearded dragons from July to August, 1996. The animals consisted of one female, two males, and all of their progeny. The animals were maintained in four separate colonies at the Philadelphia Zoological Garden. They were maintained in an indoor enclosifte in an ambient temperature that ranged from 26-28ºC (79-82ºF), with a 35ºC (95ºF) basking area. The photoperiod was 12 hours dark, 12 hours light. The lighting in the enclosures consisted of a Cool White fluorescent light, a VitaLite (Duro Test) and a 100-watt incandescent bulb. The lights were 24-30" from the bottom of the enclosure and all lights were less than 18 months old. The animals were fed a salad consisting of mixed greens, vegetables, fruit, horse meat, and a vitamin-mineral supplement. Crickets or mealworms were available on the days the salad was not fed. All animals had access to food 24 hours prior to time the blood samples were taken.
Samples were obtained from manually restrained lizards by venipuncture using a lateral approach
to the coccygeal vein. A 26- ga needle and syringe pretreated with sodium heparin was used to
draw blood. Approximately 1 cc of blood was then transferred to a lithium heparin collection tube
(Microcontainer, Becton-Dickson, Rutherford, NJ, USA). A small aliquot of blood was
transferred from the collection tube into an nonheparinized microhematocrit tube and centrifuged.
Packed cell volume was measured and total solids were determined using a refractometer. Blood
smears were obtained using a push slide technique and stained with Wright-Giemsa. The WBC
technique was obtained using the eosinophil unopette (Becton-Dickson, Rutherford, NJ, USA)
technique. The rest of the blood was centrifuged, and the plasma was separated. The plasma was
frozen at -76ºC (-105ºF) and read within six months from the time it was frozen. Plasma
chemistries were determined on a dry slide chemistry analyzer (Kodak Ektachem DT6O Analyzer,
Eastman Kodak Co., Rochester, NY).
Results
Erythrocytes: The erythrocytes were similar in morphology to those reported for other reptilian species (Frye, 1991). Immature erythrocytes averaged <1/HPF in all samples.
Thrombocytes: The thrombocytes contained a round, densely staining nucleus. The nuclear to cytoplasmic ratio was greater than one. Most of the thrombocytes were ellipsoidal. Thecytoplasm was clear and the thrombocytes tended to clump together. These latter two identifying characteristics helped distinguish the thrombocytes from the lymphocytes.
Lymphocytes: The lymphocyte contained a small amount of blue staining cytoplasm and a round nucleus with a fine reticular pattern. The lymphocyte was more spheroid than the thrombocyte, with a nuclear to cytoplasmic ratio of greater than one.
Basophils: The basophil was easily identified by its deeply staining purple, large, round granules that remained tightly adhered to the centrally located nucleus. In some instances the granules completely occluded the nucleus.
Heterophils: The heterophils contained a reniform to lobulated nucleus, often eccentrically located. The cytoplasm contained numerous rod shaped granules, which stained bright pink to orange on the Wright-Giemsa stain.
Monocytes: The monocyte was the largest leukocyte in the series, containing a lightly staining azurophilic, vacuolated cytoplasm. The nucleus was reniform in shape.
Azurophils: The azurophils were equal to slightly smaller in shape than the monocytes. The distinguishing feature of the azurophil was its heterochromatic cytoplasm, with both pink and blue staining circular granules. The nucleus was eccentrically located and was round to reniform in shape.
WBC and Differential - The results of the white blood cell count and differential are listed in Table 1.
The average WBC in
this study was 12,053 cells/microliter, which is slightly higher than the average WBC reported
in this and other agamid lizards (Pienarr, 1962, Efrati, et al, 1970, Cranfield, et al, 1996).
The leukocyte with the highest percentage of the differential was the lymphocyte, with an average of 59% and a range of 47 to 69% of the total white blood cell count. No eosinophils were found in the differential. This finding was expected, since none to a rare few eosinophils are found in the differential of squamates (Montali, 1988).
Plasma Chemistries - The results of the plasma chemistry values are listed in Table 2.
The plasma chemistry
values for BUN, the electrolytes and AST were within normal range for reptile species (Campbell, 1996). All values for ALT were <3 IU/l, with the exception of one lizard whose ALT was 5.
Additionally, most of the calcium values fell within the normal range expected for calcium, which is
between 1.99-4.99 mmol/l, 8-20 mg/dl (Campbell, 1996). This study found most calcium levels within
that range, with one outlying value of 6.79 mmol/l, 27.2 mg/dl.
The phosphorus values in this sample were elevated compared to those values reported in the
literature but the overall calcium to phosphorus ratio averaged at 2.2:1 (Campbell, 1996).
Glucose was higher than values normally reported in reptilian species (Campbell, 1996), with a
range of 7.72-16.15 mmol/l, 139-291 mg/dl and an average of 11.7mmol/l (210 mg/dl).
Cholesterol values were high in this study (8.07-31.7µmol/l, 312-1224 mg/dl) compared to
cholesterol values that have been reported previously in reptiles (Holcolmb, et al, 1972, Marks and Citino, 1990, Wright and Skeba, 1993, Raphael, et al, 1994, Anderson, 1996,). The uric acid levels were within range to slightly elevated compared to uric acid values found in the literature (Campbell, 1996). The total solids were mostly within normal limits, with one value that was
slightly high (9.Sg/dl) (Frye, 1991, Raphael, et al, 1994, Cranfield, et al, 1996). Packed cell
volume values were also mostly in range (17-33%) (Pienaar, 1962, Sodeinde and Ogunjobi,
1994, Campbell, 1996), with one outlying value of 50%.
Discussion
The average white blood cell count is close to average white blood cell counts that have been reported for this and other agamid lizards (Pienaar, 1962, Efrati, et al, 1970, Cranfield, et al, 1996). The WBC ranged from 6,736-19,946 cells/microliter, and all of the lizards, even the ones on the higher end of the range, were clinically normal and had unremarkable plasma chemistries. White blood cell counts in reptiles have been documented to fluctuate according to a variety of factors, including season and temperature (Duguy, 1970, Frye, 1991, Anderson, 1996), and it is generally agreed that reptilian leukograms can vary widely (Duguy, 1970, Hawkey, 1989, Campbell, 1996). A more accurate assessment of disease in a reptile patient may be obtained by looking at the abnormal morphology of the leukocytes (i.e., toxic cells, phagocytozed particles, and enlarged leukocytes) (Hawkey, 1989). Though no eosinophils were found in this study, additional cytochemical work on Pogona vitticeps leukocytes has revealed that the heterophils stain positive for Sudan black B (author, yet unpublished work). This stain is characterized as an eosinophil marker (Montali, 1988). These findings perhaps further support the suggestion that squamate combine the function of eosinophil and heterophil into one cell (Montali, 1988). The azurophil in this study took up both azurophilic and eosinophilic (blue and red) dyes, but pure azurophilia has been reported in other studies (Saint Girons, 1970).
Most of the plasma chemistry values were within the range of those reported for other reptile species. The elevated glucose values have been associated with factors such as increased temperature as well as stress and activity level prior to sampling (Anderson, 1996). Elevated cholesterol levels may be due to a diet that includes a high percentage of mammalian prey or a fat difference between domestic crickets and the insects the bearded dragon normally eats in the wild. High uric acid level may reflect post-prandial collection or renal dysfunction and blood sampling should be repeated when high uric acid levels (above 892 µmol/l,15mg/dl) are obtained (Rapheal, 1995, Campbell, 1996.). The high uric acid levels in this study may have been due to the high protein diet or the fact that food was not restricted prior to sampling. The one high calcium level that was found was taken from a female who laid a clutch of eggs one month after this study was done. It has been documented that reptiles undergoing active folliculogenesis may have significantly elevated calcium levels (Raphael, 1995). The average phosphorus level in this study was high. Elevated phosphorus has also been linked to egg production in reptiles (Raphael, 1995), but hyperphosphatemia may be a reflection of the age of the animal, hypervitaminosis D, high dietary phosphorus, or renal disease (Campbell, 1996). The one high PCV may have been due to dehydration or another transient physiological state since the animal appears to be normal one year after sampling.
A few concerns need to be addressed concerning the material and methods used in this experiment. Due to time constraints, the plasma was frozen at -76ºC (-105ºF) and read at a later time. The analytes that were chosen for this study have been shown to remain stable over extended periods of time at this temperature (Thoresen, 1995). In addition, the animals used in this study were the progency of two females and one male. Further studies of hematological and plasma chemistry values are therefore needed for bearded dragons of disparate lineage.
Acknowledgments: The author would like to thank Kevin M. Wright, DVM for providing faculty assistance in this project and Sandra Skeba A.H.T. for her technical assistance in analyzing the blood and the Reptile House keepers for their help in restraining animals.
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Further, this article is provided for informational purposes only and is not intended to replace the
advice or services of a qualified veterinarian. Always consult a qualified veterinarian before
beginning any kind of treatment for a sick or injured Bearded Dragon.