Plasma Biochemistry and Hematology Values in Juvenile Hawksbill Turtles (Eretmochelys imbricata) Undergoing Rehabilitation

Transcription

1 Plasma Biochemistry and Hematology Values in Juvenile Hawksbill Turtles (Eretmochelys imbricata) Undergoing Rehabilitation Valentina Caliendo 1, DVM, MRCVS, Peter McKinney 1, MVB, Cert Zoo Med, David Robinson 2, BSc, MSc, Warren Baverstock 2, Kevin Hyland 3, BSc 1. Al Wasl Veterinary Clinic, PO Box 75565, Dubai, United Arab Emirates 2. Jumeirah Group, Dubai, United Arab Emirates 3. Wildlife Protection Office, Dubai, United Arab Emirates ABSTRACT: The hawksbill turtle (Eretmochelys imbricata) is a regular inhabitant of the Arabian Gulf. Sea turtles are included in the IUCN red list, and the hawksbill is listed as critically endangered. From March 2004 to September 2010, the Dubai Turtle Rehabilitation Project received 150 sick and injured juvenile hawksbill turtles, rescued in the Arabian Gulf. Blood samples taken from the animals during convalescence and prior to release were analyzed, establishing a hematology and biochemistry reference interval for clinically healthy rehabilitated juvenile hawksbill turtles from the Arabian Gulf. Hemoglobin, packed cell volume (PCV), white blood cell count (WBC), aspartate aminotransferase (AST), creatine kinase (CK), uric acid, glucose, calcium, phosphorus, total protein, albumin, globulin, potassium, and sodium were evaluated. When compared to values at first presentation, clinically healthy rehabilitated turtles had significantly higher PCV (P = 0.006) and significantly lower WBC (P = ), heterophils (P = 0.005), monocytes (P = 0.04), AST (P = 0.03), and CK (P = ). There was no significant change in hemoglobi n, eosinophils, basophils, uric acid, glucose, calcium, phosphorus, total protein, albumin, globulin, potassiu m, or sodium levels between turtles at first presentation and post-rehabilitation. KEY WORDS: Arabian Gulf, Eretmochelys imbricata, Hawksbill turtle, juvenile, reference interval, rehabilitation. INTRODUCTION The hawksbill turtle (Eretmochelys imbricata) is one of the species of sea turtle that lives and reproduces in Arabian Gulf waters. The Gulf is a unique, semi-enclosed shallow sea that possesses characteristic ecological and biological properties (Samour et al., 1998). Its high level of salinity (36 parts per thousand during the nonmonsoon season), high temperature (up to 28 C, 82.4 F), and strong upsurge of phosphate represent natural challenges for the flora and fauna. In addition, oil spills, coastal development, landfill, pollution, and fishing practices are among human threats that menace marine life of the region. Hawksbill turtles have been classified as critically endangered by the World Conservation Union (IUCN red list). Populations of nesting and foraging hawksbills reside in the Gulf, making it an ideal area to pursue scientific studies of the species to better understand its physiology. The aim of this study was to investigate the blood parameters of clinically healthy rehabilitated juvenile hawksbill turtles, prior to release back to the wild, to establish a reference useful for future conservation and rehabilitation programs. MATERIALS AND METHODS From March 2004 to September 2010, 150 juvenile hawksbill turtles were rescued from around the United Arab Emirates (UAE) coastline and treated by the Dubai Turtle Rehabilitation Project. Ninety-two percent presented for malnutrition, 2% for foreign body ingestion, 1% for trauma caused by boat strike, and 5% for unidentified causes. The bodyweights ranged from 2.6 kg to 19.2 kg (mean: 10.9 ± 7.2 kg) and the mean straight carapace length (SCL) ranged from 22.7 cm to 49.6 cm (8.9 in to 19.5 in) (mean: 36.6 cm ± 9.8 cm [14.4 in ± 3.9 in]). The turtles were treated for two to nine months (median: four months), and during rehabilitation gained 0.9 ± 0.6 kg in weight and increased their SCL by 2.4 cm ± 0.6 cm (0.9 in ± 0.2 in). Turtles were deemed healthy when their physiological functions were restored to normality, and the clinical examination, fecal examination, and whole body radiographs did not show any abnormalities. Blood samples were obtained at first presentation and before release. Two milliliters of blood were collected from the cervical sinus using 3 ml disposable syringes. Blood was placed in collection tubes (MiniCollect Tubes, Greiner bio-one, Maybachstrasse 2, D Frickenhausen, Germany) containing lithium heparin (Bolton et al., 1992; Casal and Oros, 2009; Casal et al., 2009). A blood smear of each sample was prepared immediately and stained with a quick Romanowsky type stain (Diff-Quick Solution, Astral Diagnostics, West Depford, NJ) for differential leukocyte counts. The samples were processed within one hour of sampling for hematology, including packed cell volume (PCV), hemoglobin value (Hb), white blood cell (WBC) total and differential count, and plasma biochemistries. Only samples with no significant interference levels of lipemia, icterus, and hemolysis were included in the study. Volume 20, No. 4, 2010 Journal of Herpetological Medicine and Surgery 117

2 Packed cell volume was determined using microhematocrit capillary tubes (Glass Microhematocrit tube, Cardinal Health, Dublin, Ireland) centrifuged (Biofuge Haemo, Heraeus Instruments, Sepatech, Osterode, Germany) at 9,000 g for five minutes. Hemoglobin concentration was measured with a Haemocue B-Haemoglobin analyzer (Haemocue Ltd., Derbeyshire, UK). White blood cell counts were performed with a Neubauer hemocytometer (Blau Brand, Wertheim, Germany), using the 1% ammonium oxalate technique (Ammoniumoxalat-1-Hydrate, Riedel De Haen, Seelze, Germany). For WBC differential counts, 200 leukocytes were counted and classified as lymphocytes, monocytes, heterophils, eosinophils or basophils. Biochemical values were measured with an automated biochemical analyzer (Vetscan, Abaxis Inc., Darmstadt, Germany), using an Avian-Reptile rotor (Abaxis Inc.) and 100 µl of whole blood, according to the manufacturer s instructions (Wolf et al., 2008; Atkins et al., 2010). The biochemical parameters investigated were aspartate aminotransferase (AST), creatine kinase (CK), uric acid (UA), glucose (glu), total calcium (Ca), phosphorus (phos), total protein (TP), albumin (alb), globulin (glob), potassium (K), and sodium (Na). Statistical analysis was performed with MedCalc (Mariakerke, Belgium). The Kolmogorov-Smirnov test was used to determine the distribution of the data. To determine normal parameter values, the two sided tolerance interval was calculated for normally distributed data, and the distribution-free two-sided tolerance interval was used for other values (Geffre et al., 2009). Statistical significance was set at P The 95% reference interval was calculated by 2.5 and 97.5 percentiles. Comparison between values at first presentation and values of clinically healthy rehabilitated juvenile hawksbill turtles was performed using the paired t-test for normally distributed data and the Wilcoxon matched pairs test for nonparametric data. RESULTS Tables 1 and 2 show the mean hematological (Table 1) and biochemical (Table 2) blood parameters of juvenile hawksbill turtles at first presentation, immediately after Table 1. Hematological values of 150 juvenile hawksbill turtles at first presentation. PCV (%) Hemoglobin (mg/dl) WBC count (10 9 /L) Heterophils (10 9 /L) Eosinophils (10 9 /L) Basophils (10 9 /L) Lymphocytes (10 9 /L) Monocytes (10 9 /L) Table 2. Plasma chemistry values of 150 juvenile hawksbill turtles at first presentation. AST (U/L) 56 1, ,374.6 CK (U/L) 606 7,561 1, ,453 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) Journal of Herpetological Medicine and Surgery Volume 20, No. 4, 2010

3 hospitalization, whereas tables 3 and 4 show the mean hematological (Table 3) and biochemical (Table 4) blood parameters of clinically healthy juvenile hawksbill turtles after medical treatment, before their release in their natural environment. When compared to values at first presentation, clinically healthy rehabilitated turtles had significantly higher PCV (P = 0.006) and significantly lower WBC (P = ), heterophils (P = 0.005), monocytes (P = 0.04), AST (P = 0.03) and CK (P = ). There was no significant change (all P > 0.05) in Hb, eosinophils, basophils, UA, glu, Ca, phos, TP, alb, glob, K, or Na levels between turtles at first presentation and post-rehabilitation. Reptiles are ectothermic animals and, as such, it is important to consider the influence of extrinsic factors, such as environmental conditions, as well as intrinsic factors, such as age and sex, when interpreting blood values (Campbell, 1996, 2006). Furthermore, sample techniques and the stress of the animal during the procedure can complicate the establishment of reference values in reptiles (Lutz and Dunbar-Cooper, 1987; Campbell, 1996; Gelli et al., 2008). This study attempted to minimize these influences by limiting the region of study to the Arabian Gulf population, by only analyzing data from blood samples collected in winter, and by studying only juvenile turtles undergoing rehabilitation (using size as an indirect indicator of age). Because all turtles studied were juveniles, no separation by sex was made, in accordance with similar studies (Casal and Oros, 2009; Casal et al., 2009). DISCUSSION When compared to values at first presentation, clinically healthy rehabilitated turtles had significantly higher PCV values. Normal PCV of most reptiles range from approximately 20% to 40% (Campbell, 1996, 2006). Values less than 20% are suggestive of anemia, whereas values higher than 40% suggest hemoconcentration or erythrocytosis. In this study, the PCV in clinically healthy rehabilitated turtles ranged from 10.5% to 24.0%, markedly lower than other published references, particularly when compared to Table 3. Hematological values of 150 clinically healthy juvenile hawksbill turtles after medical treatment. PCV (%) Hemoglobin (mg/dl) WBC count (10 9 /L) Heterophils (10 9 /L) Eosinophils (10 9 /L) Basophils (10 9 /L) Lymphocytes (10 9 /L) Monocytes (10 9 /L) Table 4. Plasma chemistry values of 150 clinically healthy juvenile hawksbill turtles after medical treatment. AST (U/L) CK (U/L) 516 2,338 1, , ,028 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) Volume 20, No. 4, 2010 Journal of Herpetological Medicine and Surgery 119

4 Atlantic animals (Bolten et al., 1992; Casal and Oros, 2009; Casal et al., 2009). Previous studies in free living juvenile green turtles have reported slightly lower PCV values in the turtles from the Arabic Gulf compared with those from the Atlantic Ocean (Samour et al., 1998). Furthermore, studies in other sea turtle species have shown that the age and size of the animal, along with season sampled, can contribute to significant variations in PCV values within individuals (Frair, 1977; Wood and Gwynne, 1984; Casal et al., 2009; Fong et al. 2010). It has been demonstrated in green turtles that the PCV reference interval is lower in subadults than in adults and that this gradually increases with age (Casal et al., 2009). In this study, samples were taken in winter, and studies in other reptile species and amphibians have correlated low temperatures with a lower PCV (Wesley, 1976; Hasbun et al., 1998; Denis et al., 2004). Studies comparing PCV of juveniles with healthy adults in the same region, and during different seasons, would provide additional data for individuals rehabilitating these animals. Normal hemoglobin concentration in other species ranges from 6 g/dl to 10 g/dl, whereas plasma total protein ranges from 3 g/dl to 7 g/dl (Campbell, 2006; Wyneken, 2006). In this study, the majority of animals presented with anemia and hypoproteinemia. The association of low PCV, low hemoglobin values, and hypoproteinemia was recurrent in animals presenting with starvation, malnutrition, and chronic hemorrhage caused by trauma or internal parasites. The erythrocyte regenerative response in reptiles is slow and can take up to four months (Campbell, 2006); thus, rehabilitation of these animals can be prolonged. Heterophilia is associated with inflammatory disease, tissue injury and stress. Heterophils with toxic changes (e.g., increases in cytoplasmic basophilia, abnormal granulation, cytoplasmic vacuolation) were observed in severely compromised animals. Their disappearance was evaluated as an improvement of the prognosis (Fig. 1). The reduction in WBC, heterophils, and monocytes seen in the rehabilitated turtles in this study suggested that the inflammatory and stress responses many of these animals presented for were corrected prior to release. In previous studies, the most useful biochemical tests in reptiles included glu, UA, AST, CK, Ca, and phos (Campbell, 2006). During this study, the authors found that glu, CK, and Na were particularly helpful biochemical parameters for assessing health status. Normal glu in reptiles ranges from 60 mg/dl to 100 mg/dl (Campbell, 2006). Animals presented for starvation, malnutrition, and septicemia were often hypoglycemic. Furthermore, glu levels were useful in assessing the duration of the illness, and chronically ill animals had a characteristic, marked hypoglycemia. When dealing with these latter cases, it was paramount to dispense immediate care to restore normal glu levels. Sea turtles renally excrete UA, ammonia, and urea, and it is recommended to test plasma urea nitrogen (BUN) and UA when evaluating kidney function. Unfortunately, UA was the only indicator of kidney function in this study s screening panel. Although all of the turtles had normal, low levels of UA, this result was not considered conclusive enough to exclude renal disease because UA levels are typically only raised in the terminal stage of renal disease when two thirds or more of the functional renal mass is compromised (Campbell, 2006). Sea turtles tend to have higher normal Na concentrations than do other reptiles. Their salt glands regulate Na, K, and chloride levels in the blood; thus disorders of the salt gland may affect electrolyte balance (Campbell, 2006). Hypernatremia is associated with dehydration, and critically debilitated animals in this study presented with very high Na levels. Such cases received emergency care to restore the physiological electrolyte balance. Hypocalcemia and hypophosphatemia occurred in animals presenting with starvation and malnutrition. Although these values were not statistically different between the two groups in this study, these parameters merit consideration when rehabilitating sea turtles. It is also important to note that the Ca:phos ratio was inverted in this population of turtles, which is common in sea turtles in rehabilitation facilities (Stringer et al., 2010). It is likely that diet and captive environmental factors play a role in this finding. AST is not considered an organ-specific enzyme in reptiles, despite its higher activity in liver tissue. Turtles with muscle injury and generalized diseases, such as septicemia, commonly presented with an increased AST, which was also often associated with an increased CK. When the level of AST was high, without an associated high CK level, hepatic disease was suspected. CK is a muscle-specific enzyme to test for muscle cell damage, trauma, and systemic infection, which can also increase during handling and after intramuscular injection (Campbell, 2006). Turtles with decreased AST and CK during rehabilitation had a better prognosis and were released when clinically healthy. CONCLUSION Figure 1. Heterophil exhibiting toxic changes (arrow) consisting of abnormal granulation (granules with abnormal shapes and staining) in a hawksbill turtle blood smear (Diff Quick stain). This study has produced working reference intervals useful for recently rehabilitated juvenile hawksbill turtles for future conservation and rehabilitation projects in the Arabian Gulf and may be of assistance in similar programs worldwide. The Arabian Gulf marine ecosystem faces extensive threats, and to implement a thorough conservation program to protect hawksbill turtles, further study and greater knowledge of the species is imperative. 120 Journal of Herpetological Medicine and Surgery Volume 20, No. 4, 2010

5 Acknowledgments: The authors thank HH Sh Mohammed bin Rashid Al Maktoum and HH Sh Hamdan bin Mohammed bin Rashid Al Maktoum for their ongoing support of the Dubai Turtle Rehabilitation Project and are grateful to Andrew Bull for his valued contribution. LITERATURE CITED Atkins A, Jacobson E, Hernandez J, Bolten AB, Lu X Use of a portable point-of-care (Vetscan Vs2) biochemical analyzer for measuring plasma biochemical levels in freeliving loggerhead sea turtles (Caretta caretta). J Zoo Wildl Med, 41(4): Bolten AB, Jacobson ER, Bjorndal KA Effects of anticoagulant and autoanalyzer on blood biochemical values of loggerhead sea turtles (Caretta caretta). Am J Vet Res, 53: Campbell TW Sea turtle rehabilitation. In Mader DR (ed): Reptile Medicine and Surgery. 1st ed. Saunders, Philadelphia, PA: Campbell TW Clinical pathology of reptiles. In Mader DR (ed): Reptile Medicine and Surgery. 2nd ed. Saunders, Philadelphia, PA: Casal AB, Camacho M, Lopez-Jurado LF, Juste C, Oros J Comparative study of hematologic and plasma biochemical variables in eastern Atlantic juvenile and adult nesting loggerhead sea turtles (Caretta caretta). Vet Clin Pathol, 38: Casal AB, Oros J Plasma biochemistry and haematology values in juvenile loggerhead sea turtles undergoing rehabilitation. Vet Rec, 164: Denis V, Brito S, Toledo L, Abe A Seasonal changes in blood oxygen transport and acid-base status in the tegu lizard, Tupinambis merianae. Respir Physiol, 140: Fong C, Chen H, Cheng I Blood profiles from wild populations of green sea turtles in Taiwan. J Vet Med Anim Health, 2:8 10. Frair W Sea turtle red blood cell parameters correlated with carapace lengths. Comp Biochem Physiol, 56: Geffre A, Friedrichs K, Harr K, Concordnet D, Trumel C, Braun JP. Reference values: a review Vet Clin Pathol, 38: Gelli D, Ferrari V, Zanella A, Arena P, Pozzi L, Nannarelli S, Vaccaro C, Bernardini D, Romagnoli S Establishing physiological blood parameters in the loggerhead turtle (Caretta caretta). Eur J Wildl Res, 55: Hasbun CR, Lawrance AJ, Naldo J, Samour JH, Al Ghasis SM Normal blood chemistry of free living green turtles, Chelonia mydas, from the United Arab Emirates. Comp Haematol Int, 8: Lutz PL, Dunbar-Cooper A Variation in the blood chemistry of the loggerhead sea turtle, Caretta caretta. Fish Bull, 85: Samour JH, Howlett JC, Silvanose C, Hasbun CR, Al Ghais SM Normal haematology of free-living green sea turtles (Chelonia mydas) from the United Arab Emirates. Comp Haematol Int, 8: Stringer EM, Harms CA, Beasley JF, Anderson ET Comparison of ionized calcium, parathyroid hormone, and 25-hydroxyvitamin D in rehabilitating and healthy wild green sea turtles (Chelonia mydas). J Herp Med Surg, 20(4): Wesley W Influence of temperature on the optimal hematrocrit of the bull frog (Rana catesbeiana). J Comp Physiol, 105: Wolf KN, Harms CA, Beasley JF Evaluation of five clinical chemistry analyzers for use in health assessment in sea turtles. J Am Vet Med Assoc, 233: Wood E, Gwynne K Blood cytology and hematology of green sea turtle, Chelonian mydas. Herpetologica, 40: Wyneken J Medical care of sea turtles. In Mader DR (ed): Reptile Medicine and Surgery. 2nd ed., Saunders, Philadelphia, PA: Volume 20, No. 4, 2010 Journal of Herpetological Medicine and Surgery 121