Abstract

Introduction

During fall and early winter of 1999, a major sea turtle stranding occurred on the banks of Cape Cod Bay, Massachusetts. Loggerhead sea turtles (Caretta caretta) washed up alive in a state of severe hypothermia, commonly known as cold-stunned.² Turtles were collected by the Massachusetts Audubon Society and transported to the New England Aquarium for rehabilitation. Each animal was examined upon arrival, and venous blood samples were collected for plasma biochemistry profiles and complete blood counts. Throughout the period of rehabilitation, blood samples were collected to closely monitor the progress of each animal.

Little is known about hematologic parameters for sea turtles, particularly loggerheads. Whitaker and Krum (1999) reported white blood cell (WBC) counts in captive loggerheads weighing less than 2 kg to be 14.67 ± 5.80 X 10³/µ (Natt-Herrick method).⁶ However, to our knowledge, there are no published data for WBC counts in cold-stunned loggerhead sea turtles, nor reports for various leukocyte parameters in these turtles. Therefore, the present study focused on hematologic data collected from loggerheads of the 1999 stranding event. The primary objective of this project was to record and analyze total white blood cell counts and differential values of juvenile loggerhead sea turtles at the time of cold-stunning and at the time of release.

Methods

Animals

Of sixteen loggerhead sea turtles that survived the cold-stunning event, ten have been successfully rehabilitated to date and were therefore included in the study. These animals were classified as juvenile loggerheads based on their weights (ranging from 10.7-25.0 kg) and straight carapace lengths (ranging from 42.2-63.1 cm).⁴ Animals presented with severe hypothermia, and initial body temperatures ranged between 40-58°F. Complete blood counts were performed within 10 days of arrival, with body temperatures ranging from 61-73°F at the time of sampling. All of the animals were at 76°F for the final blood samples, which were collected after 12-18 wk of medical treatment and rehabilitation.

Blood Collection

Blood was taken from the dorsal cervical sinus (0.5 cc) using a 1.5-in, 22-ga needle and a 3-cc syringe.⁵ Blood was transferred into a pediatric lithium heparinized tube, and blood smears were made immediately and allowed to air dry. Next, the samples were refrigerated at 40°F and transported to Tufts Veterinary Diagnostic Laboratory (North Grafton, MA), where complete blood counts were performed within 24 hr of sample collection.

Identification of WBC Types

Individual WBC types were identified on Wright-Giemsa stained blood smears by the Tufts Veterinary Diagnostic Laboratory. Heterophils, lymphocytes, monocytes, eosinophils, basophils, and azurophils were classified and then photographically documented based on morphologic differences.³ Cytologic images were acquired with an Olympus BX40 microscope (Olympus America Incorporated, Melville, NY) linked to a Javelin camera (Smart Cam, JE3762DSP; Fusion Electronics, Miami, FL).

Complete Blood Counts

The Wright-Giemsa stained blood smears were used to determine the WBC differential values. To quantitate the total WBC counts, the Eosinophil Unopette method was utilized. Heparinized whole blood (< 0.3 cc) was drawn into an Eosinophil Unopette (#5877), and then incubated at room temperature for 10 min. The Unopette contained a substance, Phloxine B, that specifically stained heterophils and eosinophils during the incubation period.¹ The sample was then transferred to a Neubauer hemacytometer and incubated for an additional 10 min. Heterophils and eosinophils, which were stained bright orange-red, were manually counted within the nine square grids of the hemacytometer, and the average number per square was quantitated. The following equation was used to calculate the total WBC count.

A = average number of heterophils and eosinophils (Eosinophil Unopette) x 35.2
B = % of heterophils and eosinophils (WBC differential)
A/B = total WBC count

This total WBC number was then used to determine the absolute values for each cell type. Therefore, relative white blood cell counts were established from the WBC differential values, and the absolute counts were derived from the total WBC number.

Statistics

A paired, two-sample student's t-test was used to compare the arrival and release mean hematology values. The significance level was set at 0.05, therefore P values (two-tailed) < 0.05 were considered statistically significant.

Results and Discussion

The range and mean ± standard deviation of arrival and release hematology values for the ten juvenile loggerhead sea turtles are listed in Table 1. The total WBC counts and all of the absolute differential WBC counts decreased throughout the rehabilitation period. Although these changes were determined to be clinically significant, only the decreases seen in the total WBC count (P = 0.004) and the heterophil absolute count (P = 0.004) were statistically significant. On arrival, the animals were hypothermic and had secondary medical conditions including pneumonia, sepsis, gastrointestinal disease, and traumatic shell wounds. These conditions resolved with medical management, including antibiotic and antifungal therapy. Since the animals were clinically healthy prior to release, the higher blood values seen at arrival were considered to be abnormal. Therefore, the decreased values in the total WBC counts and absolute differential WBC counts seen at release were considered to reflect normal values for rehabilitated loggerhead sea turtles.

Upon release, the total WBC count had decreased by an average of 70%. Although all cell types including lymphocytes and monocytes contributed to the total WBC count decrease, an increase in the percentage of lymphocytes (P = 0.049) and monocytes (P = 0.041) in the WBC differential was seen. These increases represented a shift in the leukogram distribution of the two cell types at arrival versus release. As previously stated, the animals were clinically healthy prior to release, therefore the distribution of lymphocytes and monocytes in the release leukogram were considered to be normal.

These data represent a distinct population of hypothermic juvenile loggerhead sea turtles, therefore caution should be taken when utilizing this information for the evaluation of turtles under different environmental and/or medical circumstances. In addition, it is important to note that these complete blood counts were determined using the Eosinophil Unopette method. Arnold (1994) documented discrepancies in white blood cell counts of loggerhead sea turtles when comparing this method to the Natt-Herrick method.¹ Therefore, the data presented here should not be compared with white blood cell counts determined with the Natt-Herrick method. However, the results of this study should prove to be a useful guide when medically evaluating cold-stunned juvenile loggerhead sea turtles.

Table 1. Range and mean ± standard deviation of hematology values for cold-stunned juvenile loggerhead sea turtles (Caretta caretta) (n = 10) at arrival and prior to release after rehabilitation.

a. Statistically significant difference in arrival and release values (P < 0.05).

Acknowledgments

We thank Dr. Andrew Stamper, Connie Merigo, Belinda Rubinstein, Jim Rice, Kristen Patchett, Melissa Hoge, Kristen Dubé, Deana Edmunds, Casey Sugarman, Robert Cooper, Susan Goodridge, Katarina Peterson, Dr. Sonia Mumford, John Dayton, and the volunteers of the New England Aquarium for their support of this project. We also thank Dr. Joyce Knoll for assistance with white blood cell identification.

References

1.  Arnold J. 1994. White Blood Cell Discrepancies in Atlantic Loggerhead Sea Turtles: Natt-Herrick Vs. Eosinophil Unopette. AZVT 14th Annual Proceedings: 15 B 22.

2.  George RH. 1997. Health Problems and Diseases of Sea Turtles. In: The Biology of Sea Turtles. CRC Press, Boca Raton, FL. Pp. 377-378.

3.  Hawkey CM, TB Dennett. 1989. Color Atlas of Comparative Veterinary Hematology. Iowa State University Press, Ames, Iowa.

4.  Musick JA, CJ Limpus. 1997. Habitat Utilization and Migration in Juvenile Sea Turtles. In: The Biology of Sea Turtles. CRC Press, Boca Raton, FL. Pp. 146-148.

5.  Owens DW, GJ Ruiz. 1980. New Methods of Obtaining Blood and Cerebrospinal Fluid from Marine Turtles. Herpetologica 36: 17.

6.  Whitaker BR, H Krum. 1999. Medical Management of Sea Turtles in Aquaria. In: M.E. Fowler and R.E. Miller, (eds). Zoo & Wildlife Animal Medicine. W.B. Saunders Company, Philadelphia, PA. Pp. 224-225.