Morphologic and cytochemical characteristics of blood cells of juvenile loggerhead sea turtles (Caretta caretta)

Morphologic and cytochemical characteristics of blood cells of juvenile loggerhead sea turtles (Caretta caretta)

Research in Veterinary Science 82 (2007) 158–165 www.elsevier.com/locate/rvsc Morphologic and cytochemical characteristics of blood cells of juvenile...

1MB Sizes 0 Downloads 31 Views

Research in Veterinary Science 82 (2007) 158–165 www.elsevier.com/locate/rvsc

Morphologic and cytochemical characteristics of blood cells of juvenile loggerhead sea turtles (Caretta caretta) A.B. Casal, J. Oro´s

*

Department of Morphology, Unit of Histology and Pathology, Veterinary Faculty, University of Las Palmas de Gran Canaria (ULPGC), Trasmontan˜a s/n, 35416 Arucas, Las Palmas, Spain Accepted 30 July 2006

Abstract A morphologic classification based on the cytochemical characteristics of blood cells of 35 juvenile loggerhead sea turtles (Caretta caretta) is described. Cytochemical stains included benzidine peroxidase, chloroacetate esterase, alpha-naphthyl butyrate esterase (with and without sodium fluoride), acid phosphatase (with and without tartaric acid), Sudan black B, periodic acid-Schiff, and toluidine blue. The morphologic characteristics of erythrocytes were similar to those reported in green turtles. Six types of white blood cells were identified: heterophils, eosinophils, basophils, lymphocytes, monocytes and thrombocytes. Except for the basophils, the rest of the white blood cells from loggerhead turtles had different cytochemical characteristics compared to blood cells from other sea turtle species. The leukocyte differential count was different from that reported for other sea turtle species. Heterophils were the most numerous leukocytes from these loggerhead turtles, followed by lymphocytes, eosinophils, monocytes and basophils. This paper provides a morphologic classification of blood cells of loggerhead sea turtles that is useful for veterinary surgeons involved in sea turtle conservation. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Blood cells; Caretta caretta; Cytochemistry; Haematology; Sea turtle

1. Introduction Currently, two families and seven species of sea turtles are recognised (Pritchard, 1997). The family Cheloniidae includes the green turtle (Chelonia mydas), loggerhead (Caretta caretta), hawksbill (Eretmochelys imbricata), Kemp’s ridley (Lepidochelys kempi), olive ridley (Lepidochelys olivacea), and flatback turtle (Natator depressus). The family Dermochelyidae includes only the leatherback (Dermochelys coriacea). Only five species have been reported in the Canary Islands: loggerhead, green turtle, leatherback, hawksbill, and Kemp’s ridley sea turtle (Mateo et al., 1997; Barbadillo et al., 1999). All species of sea turtles are included on the Red List of the World Conservation Union (IUCN/SCC, 2004). *

Corresponding author. Tel.: +34 928 454375; fax: +34 928 451130. E-mail address: [email protected] (J. Oro´s).

0034-5288/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2006.07.017

The efforts to conserve sea turtles, the advances in their medical management, and the studies on physiological parameters have increased in recent years. In addition, many veterinary surgeons are involved in sea turtle conservation in wildlife rehabilitation hospitals around the world. The classification of blood cells in reptiles is inconsistent because variable criteria are used to categorize cells or because cellular lineages are uncertain (Work et al., 1998). Descriptions of the morphologic characteristics of blood cells of sea turtles are limited (Wood and Ebanks, 1984; Cannon, 1992; Aguirre et al., 1995; Work et al., 1998; Gelli et al., 2004). However, the classification of blood cells from loggerhead turtles is important because this species is intensively scrutinized in captivity in clinical settings and in the wild during research projects. This paper provides a morphologic classification of blood cells of loggerhead sea turtles, as a reference for future haematological studies of this species.

A.B. Casal, J. Oro´s / Research in Veterinary Science 82 (2007) 158–165

2. Materials and methods Thirty-five juvenile loggerhead sea turtles were used in this study. The turtles had been rehabilitated in outdoor facilities with seawater belonging to the Tafira Wildlife Rehabilitation Centre (TWRC) (Las Palmas de Gran Canaria, Canary Islands, Spain) during 2003 and 2004. Original stranding locations of these turtles included five islands: Gran Canaria Island (n = 17), Tenerife Island (n = 10), Fuerteventura Island (n = 4), La Gomera Island (n = 3), and La Palma Island (n = 1). The mean and standard deviation of the straight carapace length of juvenile turtles was 37.3 ± 4.6 cm (range, 18–52 cm). The minimum and maximum time in rehabilitation were respectively 10 and 184 days. Blood samples were obtained just before the turtles were set free, when they were clinically normal and in good physical condition. Two millilitres of blood were collected from the cervical sinus (Owens and Ruiz, 1980) in tubes without anticoagulant, using sterile syringes and needles. Twenty blood smears for each turtle were prepared immediately and air-dried (Campbell, 1996; Bradley et al., 1998). Two blood smears of each turtle were stained with a quick Romanowsky-type stain, Diff Quick (DQ) (Everest, Barcelona, Spain), according to the manufacturer’s instructions for differential leukocyte count. Two hundred leukocytes were counted and classified as lymphocytes, monocytes, eosinophils, heterophils, or basophils. Twenty erythrocytes and fifteen leukocytes were measured (area, perimeter, maximum length, minimum length) using an image analysis program, Image-Pro Plus, Version 4.1 (Media Cybernetics). Haematocrit was determined using the manual microhaematocrit method. The remaining 18 blood smears from each turtle were stained as follows: two blood smears from each turtle were stained with each stain, except for the alpha-naphthyl butyrate esterase and acid phosphatase stains in which four blood smears were stained. Stains included benzidine peroxidase (PER), chloroacetate esterase (CAE), alpha-naph-

159

thyl butyrate esterase (NBE) (with and without sodium fluoride), acid phosphatase (ACP) (with and without tartaric acid), Sudan black B (SBB), periodic acid-Schiff (PAS), and toluidine blue (TB) (Jain, 1986) using commercial kits (Sigma Diagnostics, Sigma Aldrich Quı´mica S.A., Spain) following the protocols given by the manufacturer. Normal human blood smears were used as controls to ensure correct staining procedure. A statistical analysis of the cell dimensions based on the Student’s t test was made using the program SPSS 11.0 for Windows, obtaining the following data: mean, standard deviation and range. 3. Results Dimensions of the blood cells of juvenile loggerhead turtles are shown in Table 1. Erythrocytes stained with DQ were oval with a pale pink cytoplasm and a violet–blue oval or round, some times indented, nucleus. Some erythrocytes had small basophilic intracytoplasmic inclusions. Erythrocytes did not take up any cytochemical stain (Table 2). Heterophils were big and round cells with a dense, oval, strongly purple, often eccentric nucleus, which contained clumped chromatin. When the nucleus was centrally located, it acquired a round form. The abundant cytoplasm showed weak eosinophilia, containing numerous fusiform granules with the same coloration as that of the cytoplasm, as a result of which, in most cases, heterophil granules were not seen easily (Fig. 1). Heterophils were stained with ACP (with and without tartrate) (Fig. 2), PER (Fig. 3), CAE and SBB; and they were moderately stained with PAS (Table 2). Eosinophils were round, with a generally eccentric, oval or round, strongly purple nucleus, which contained clumped chromatin. The abundant cytoplasm was weakly basophilic, and contained scarce or a moderate number of round, approximately 1 lm in diameter, well-defined eosinophilic granules (Fig. 4). These granules were stained with ACP (with and without tartrate), NBE (with sodium

Table 1 Mean, standard deviations, and ranges of the dimensions of the blood cells of loggerhead turtles on blood smears Blood cell

Area

Perimeter

Maximum length diameter

Minimum length diameter

Erythrocyte

194.28 ± 27.54 126.43–288.80

51.19 ± 3.53 41.89–61.40

19.05 ± 1.35 16.22–22.76

12.85 ± 1.25 9.69–16.53

Heterophil

228.94 ± 47.21 133.20–333.36

54.16 ± 5.61 41.56–64.68

17.83 ± 1.87 13.94–21.06

16.30 ± 1.81 11.75–20.26

Eosinophil

259.60 ± 50.12 160.40–375.80

58.67 ± 5.72 46.43–70.32

20.02 ± 2.23 15.65–25.31

16.87 ± 1.92 13.17–21.26

Lymphocyte

106.82 ± 24.56 66.72–177.70

37.09 ± 4.30 29.87–49.60

12.31 ± 1.48 9.70–16.66

11.10 ± 1.33 8.06–14.55

Monocyte

187.81 ± 40.55 108.96–301.91

49.69 ± 5.53 38.44–64.73

16.41 ± 1.91 12.97–20.60

14.77 ± 1.65 10.34–19.34

Thrombocyte

64.52 ± 16.38 35.58–109.40

32.88 ± 5.06 23.71–42.75

13.50 ± 2.45 9.15–18.35

6.19 ± 0.99 4.01–9.36

All dimensions are shown in lm, except area, in lm2.

A.B. Casal, J. Oro´s / Research in Veterinary Science 82 (2007) 158–165

160

Table 2 Cytochemical staining characterization of blood cells of juvenile loggerhead turtles Blood cell

Cytochemical stains ACPa

Erythrocyte Heterophil Eosinophil Basophil Lymphocyte Monocyte Thrombocyte

PER

T

NT

+ +

+ +

NBEb F

CAE

SBB

PAS

+ +

+ ±

± +

TB

NF

+ +

+ ± ± +

ACP: acid phosphatase; PER: benzidine peroxidase; NBE: alpha-naphthyl butyrate esterase; CAE: chloroacetate esterase; SBB: Sudan black B; PAS: periodic acid-Schiff; TB: toluidine blue. (+) positive; ( ) negative; (±) moderately positive. a Two ACP stains were made with tartaric acid (T) and without tartaric acid (NT). b Two NBE stains were made with sodium fluoride (F) and without sodium fluoride (NF).

Fig. 1. A loggerhead turtle heterophil (arrowhead) stained with Diff Quick. Note also the typical morphology of an erythrocyte (*). Bar = 3.6 lm.

fluoride), CAE (Fig. 5) and PAS. They were also moderately stained with SBB (Table 2). Basophils were difficult to find in the blood smears of loggerhead turtles. These cells were round, and contained a dense, violet–blue, generally eccentric nucleus, which presented a clumped chromatin pattern. The cytoplasm contained numerous big basophilic granules that often masked the nucleus (Fig. 6). Due to the scarcity of basophils in the blood of loggerhead turtles, it was not possible to determine their dimensions. Basophil granules stained only with TB (Table 2). Lymphocytes were small and round with a well-defined round purple–blue nucleus that contained prominently clumped chromatin. The nucleus was surrounded by a rim of moderately granular basophilic cytoplasm (Fig. 7); the visual estimate nucleus:cytoplasm area ratio was typically 0.6. Lymphocytes from loggerhead turtles stained

Fig. 2. Heterophil (arrowhead) of a loggerhead turtle showing positive acid phosphatase (without tartaric acid) stain. Bar = 4.1 lm.

only moderately with NBE (without sodium fluoride) (Table 2). Monocytes had a diameter that was 33% greater than that of lymphocytes. These cells were round or amoeboid. The nucleus was purple–blue, generally oval, kidney-like form or fusiform, eccentric location, and had a chromatin pattern slightly less clumped than that of the lymphocyte. The cytoplasm was weak to moderately basophilic, sometimes containing variably sized intracytoplasmic vacuoles (Fig. 8). The visual estimate nucleus:cytoplasm area ratio was typically 0.5, less than lymphocytes. Azurophilic granules were not seen. Some monocytes had pseudopods. Monocytes were only moderately stained with NBE without fluoride (Table 2).

A.B. Casal, J. Oro´s / Research in Veterinary Science 82 (2007) 158–165

161

Fig. 3. Two heterophils (arrowheads) of a loggerhead turtle showing positive benzidine peroxidase stain. Bar = 4.3 lm. Fig. 5. Eosinophil (arrowhead) of a loggerhead turtle showing positive chloroacetate esterase stain. Bar = 3.2 lm.

Fig. 6. A loggerhead turtle basophil (arrowhead) stained with Diff Quick. Note the big basophilic granules. Bar = 4.3 lm.

Fig. 4. A loggerhead turtle eosinophil (arrowhead) stained with Diff Quick. Note the well-defined eosinophilic granules. Bar = 3.3 lm.

Thrombocytes were typically oval-shaped cells, although sometimes we observed round thrombocytes. The nucleus was generally oval, strong violet–blue and with a clumped chromatin. The scant cytoplasm was seen accumulated in the two poles of the cell when the thrombocyte presented an oval morphology; or a slim cytoplasmic halo around the nucleus was seen when the cell acquired a round form. In all the cases, the cytoplasm presented a very pale coloration, almost transparent, a characteristic that helped to distinguish this cell from the lymphocytes. Other important characteristics of thrombocytes were their tendency to aggregate in the blood smears. Thrombocytes stained only with PAS (Table 2, Fig. 9).

Table 3 lists the results of the leukocyte differential count and the haematocrit values from the loggerhead turtles of our study. 4. Discussion The morphologic characteristics of erythrocytes from juvenile loggerhead sea turtles in this study were similar to those reported in green turtles (Wood and Ebanks, 1984; Work et al., 1998), young loggerhead sea turtles (Bradley et al., 1998) and in other terrestrial chelonians (Sypek and Borysenko, 1988; Alleman et al., 1992). The erythrocytes identified in our study were 19 lm long. The erythrocytes of immature green turtles studied by Work et al. (1998) were 17–20 lm long. However, there are no descriptions of the size of the erythrocytes in the haemato-

162

A.B. Casal, J. Oro´s / Research in Veterinary Science 82 (2007) 158–165

Fig. 7. A loggerhead turtle lymphocyte (arrowhead) stained with Diff Quick. Bar = 4.8 lm.

Fig. 9. Two loggerhead turtle thrombocytes (arrowheads) showing intracytoplasmic positive cytochemical periodic acid-Schiff stain. Bar = 4.4 lm.

Table 3 Mean, standard deviations, and ranges of the haematocrit and leukocyte differential count values of juvenile loggerheads

Haematocrit (%) Heterophils (%) Eosinophils (%) Basophils (%) Lymphocytes (%) Monocytes (%)

Fig. 8. A loggerhead turtle monocyte (arrowhead) stained with Diff Quick. Bar = 3.2 lm.

logical studies on black turtles (Grumbles et al., 1990), Kemp’s ridley sea turtles (Cannon, 1992), or young loggerhead turtles (Bradley et al., 1998). Work et al. (1998) observed small amorphous intracytoplasmic inclusions in numerous erythrocytes of green turtles. Ultrastructurally those inclusions were identified as degenerating organelles and were similar to those found in erythrocytes of desert tortoises (Gopherus agassizii) (Alleman et al., 1992). Basophilic inclusions are common in the erythrocytes of healthy chelonians (Heard et al., 2004). We observed similar basophilic inclusions but no ultrastructural study was carried out. The negative cytochemical stain of erythrocytes from loggerhead turtles was similar to the results obtained from studies on erythrocytes of green turtles (Work et al., 1998).

Mean ± standard deviation

Range

28 ± 5.77 75.80 ± 9.08 4.51 ± 6.33 0±0 18.41 ± 7.11 1.20 ± 1.36

19–43 51.61–88.61 0–29.40 – 4.40–30.92 0–5.31

Based on the results obtained during this study we classified six types of white blood cells in loggerhead turtles: heterophil, eosinophil, basophil, lymphocyte, monocyte and thrombocyte, similar to those described in reptiles by Sypek and Borysenko (1988), and described in green turtles (Work et al., 1998). Light microscopy showed that heterophils from loggerhead turtles were similar to those reported for other reptiles (Sypek and Borysenko, 1988; Bounous et al., 1996). In our study, heterophils were stained with ACP (with and without tartrate), PER, CAE and SBB; and they were moderately stained with PAS. There are no previous references on cytochemical characterization of heterophils from loggerhead turtles. In a similar study, heterophils from green turtles stained only with NBE and PAS (Work et al., 1998). Heterophils from other terrestrial reptiles stained with ACP and alkaline phosphatase (ALP) (Sypek and Borysenko, 1988; Alleman et al., 1992). The differences in the cytochemical characteristics of heterophils from different species of sea turtles show that the heterophils of these species have different enzymes. Heterophils in reptiles (Montali, 1988) and birds (Brooks et al., 1996) have a similar function to that performed by the neutrophils in mammals. We did not identify neutrophils in loggerhead turtles. Neutrophils are rare in reptiles, but have been described in the tuatara (Sphenodon punctatus) (Desser, 1978). There are several descriptions of

A.B. Casal, J. Oro´s / Research in Veterinary Science 82 (2007) 158–165

neutrophils in loggerhead turtles (George, 1997) and green turtles (Wood and Ebanks, 1984; Aguirre et al., 1995), being the leukocyte least numerous in the differential leukocyte count. However cytochemical stains were not used by these authors to confirm the identification of the different leukocytes. Work et al. (1998) suspected the neutrophils described by Wood and Ebanks (1984) in green turtles to be large degranulated eosinophils. Eosinophils from loggerhead turtles were homogeneous in size, 20 lm in diameter, unlike eosinophils from green turtles, which are large as well as small (Work et al., 1998). Large and small eosinophils have also been described in Kemp’s ridley turtle (Cannon, 1992). Large and small eosinophils are uncommonly reported in reptiles. Work et al. (1998) suspected that large eosinophils in green turtles represent activated cells that contain degranulated or coalescent granular material in response to a parasitic infection or other inflammatory stimulus. In our study, granules from the eosinophils were stained with ACP (with and without tartrate), NBE (with sodium fluoride), CAE and PAS. They were also moderately stained with SBB. Large and small eosinophils from green turtles stained strongly only with CAE, and marginally with NBE and PAS (Work et al., 1998). Small eosinophils from Kemp’s ridley turtles stained strongly with ACP whereas large eosinophils stained moderately with ALP, PAS, and SBB (Cannon, 1992). Basophils were scarce in loggerhead turtles, similar to those described by Work et al. (1998) in green turtles. Cannon (1992) did not identify basophils in Kemp’s ridley turtles. In our study, basophils were slightly smaller than heterophils, and basophil granules stained only with TB. This cytochemical pattern is similar to that described by Work et al. (1998) in green turtles. However, basophils of some snakes stain with PAS, but not with TB (Alleman et al., 1992). Lymphocytes from loggerhead turtles were morphologically similar to lymphocytes reported for green turtles (Work et al., 1998) and Kemp’s ridley turtles (Cannon, 1992). Lymphocytes from loggerhead turtles were not difficult to distinguish from thrombocytes using DQ stain because freshly prepared smears were used. However, lymphocytes from other reptilian species can be difficult to distinguish from thrombocytes (Alleman et al., 1992; Bounous et al., 1996), specially if blood smears are prepared from blood that has been chilled (Work et al., 1998). Lymphocytes from loggerhead turtles stained only lightly with NBE (without sodium fluoride) whereas lymphocytes from green turtles did not stain with any cytochemical stain used by Work et al. (1998). However, Cannon (1992) found that lymphocytes from Kemp’s ridley turtles stained with nonspecific esterase. Monocytes from loggerhead turtles were similar to the monocytes from green turtles described by Work et al. (1998). Other authors did not identify monocytes from green turtles (Wood and Ebanks, 1984; Aguirre et al., 1995) or Kemp’s ridley turtles (Cannon, 1992). According

163

to Work et al. (1998) monocytes are more difficult to differentiate from lymphocytes when smears are made from blood that has been chilled 8 h, partly because of cellular shrinkage. Monocytes from loggerhead turtles stained only moderately with NBE (without sodium fluoride). Monocytes from green turtles stained with ACP and PAS, and only some of them with CAE and NBE (Work et al., 1998). In our study, azurophils were not identified in the blood of loggerhead turtles. References on azurophils in the blood of sea turtles are rare; Arnold (1994) found azurophils only in one of the blood samples collected from loggerhead turtles. Smith et al. (2000) identified azurophils in loggerhead turtles. Keller et al. (2004) also found azurophils to be approximately 5% of the total white blood cell count for juvenile loggerhead turtles. However, most other authors did not identify azurophils in sea turtles (Wood and Ebanks, 1984; Cannon, 1992; Aguirre et al., 1995; Bradley et al., 1998; Work et al., 1998; Work and Balazs, 1999; Harms et al., 2000). Light microscopy showed thrombocytes from loggerhead turtles to be similar to those reported for green turtles (Work et al., 1998). Cannon (1992) did not report thrombocytes from Kemp’s ridley turtles. Wood and Ebanks (1984) found it difficult to differentiate between thrombocytes and basophils in green turtles. Thrombocytes from other reptilian species can be difficult to distinguish from lymphocytes (Alleman et al., 1992; Bounous et al., 1996); according to Work et al. (1998), thrombocytes usually retain their morphologic characteristics on freshly prepared smears. However, if blood smears are prepared from blood that has been chilled, cellular shrinkage makes differentiation more difficult. In our study, thrombocytes were differentiated from lymphocytes cytochemically by staining only with PAS. Thrombocytes from green turtles stain with NBE and PAS (Work et al., 1998). Thrombocytes from other terrestrial reptiles stain also with ACP (Sypek and Borysenko, 1988; Bounous et al., 1996). Mean ± standard deviation of haematocrit from loggerhead turtles was 28 ± 5.77% (range, 19–43%). This value is similar to that reported for loggerhead turtles by Gelli et al. (2004). However it is slightly lower than the mean observed in other studies on loggerhead turtles (31.5–32%) (Dessauer, 1970; Keller et al., 2004). The difference can be attributed to the age of the turtles. An increase in the erythrocyte total count was observed in adult green turtles compared with juvenile turtles (Wood and Ebanks, 1984). In addition, a significant correlation between haematocrit and corporal size was also observed in captive green turtles (Wood and Ebanks, 1984). However, this correlation was not observed in juvenile wild green turtles (Bolten and Bjorndal, 1992). It has been proven that diet, health status, exercise and stress can affect haematocrit in captive sea turtles (Frair, 1977). The leukocyte differential count of juvenile loggerhead turtles is different from that reported for green turtles by Work et al. (1998). In our study, heterophils were the most numerous leukocytes in the leukocyte differential count of

164

A.B. Casal, J. Oro´s / Research in Veterinary Science 82 (2007) 158–165

juvenile loggerhead turtles, followed by lymphocytes, which is similar to that described for loggerhead turtles by other authors (Arnold, 1994; Bradley et al., 1998; Smith et al., 2000; Gelli et al., 2004). However, in a survey on wild-caught juvenile loggerheads from inshore waters of North Carolina, lymphocytes were the most numerous circulating leukocytes, followed by heterophils, eosinophils, azurophils and basophils (Keller et al., 2004). In another study on wild-caught juvenile to adult loggerheads captured in offshore waters of South Carolina, Georgia and northeastern Florida, lymphocytes were found to be 61.7% of the total leukocyte count (Maier et al., 2004). Differences in criteria used to identify blood cells, differences in wild-caught versus rehabilitated turtles, age range differences, or geographical differences could explain these different results. Lymphocytes were also the most numerous circulating leukocytes from green turtles, followed by eosinophils, heterophils, and monocytes (Work et al., 1998). In our study, eosinophils were the third leukocyte in abundance in the leukocyte differential count; in other studies on loggerhead turtles, eosinophils represent the fourth leukocyte (Bradley et al., 1998; Smith et al., 2000) or the third leukocyte (Arnold, 1994; Bradley et al., 1998; Keller et al., 2004). Monocytes were the fourth leukocyte in abundance in the leukocyte differential count; other authors count the monocytes from loggerhead turtles as the third cell (Smith et al., 2000; Harms et al., 2002), or the third or fourth cell (Bradley et al., 1998) in the leukocyte differential count, or they are not even described (Arnold, 1994; Keller et al., 2004). The difficulties in differentiating monocytes from lymphocytes, described above, can explain these results. Basophils are very rare in blood smears from juvenile loggerhead turtles, which are similar to that described in other reptiles, including sea turtles (Sypek and Borysenko, 1988; Cannon, 1992; Arnold, 1994; Work et al., 1998; Raskin, 2000; Keller et al., 2004). Owing to the absence of previous cytochemical studies on blood cells of loggerhead turtles, this study provides a morphologic classification of blood cells in this species, useful for the veterinary surgeons involved in sea turtle conservation. Acknowledgments The authors would like to thank Pedro Castro, Department of Morphology, University of Las Palmas de Gran Canaria, for technical assistance. They are grateful to Dr. Pascual Calabuig (TWRC) and members of Consejerı´a de Medio Ambiente, Cabildo Insular de Gran Canaria, for providing us the turtles. This study was partially supported by the national project I + D CGL2004-01111. References Aguirre, A.A., Balazs, G.H., Spraker, T.R., Gross, T.S., 1995. Adrenal and hematological responses to stress in juvenile green turtles (Chelonia mydas) with and without fibropapillomas. Physiological Zoology 68, 831–854.

Alleman, A.R., Jacobson, E.R., Raskin, R.E., 1992. Morphologic and cytochemical characteristics of blood cells from the desert tortoise (Gopherus agassizii). American Journal of Veterinary Research 53, 1645–1651. Arnold, J., 1994. White blood cell count discrepancies in Atlantic loggerhead sea turtles: Natt-Herrick vs. Eosinophil Unopette. In: Proceedings of the 14th Annual Conference of the Association of Zoo Veterinary Technicians, Cleveland, OH, September 16–21, 1994, pp. 15–22. Barbadillo, L.J., Lacomba, J.I., Pe´rez-Mellado, V., Sancho, V., Lo´pezJurado, L.F., 1999. In: Barbadillo, J.L. (Ed.), Anfibios y Reptiles de la Penı´nsula Ibe´rica, Baleares y Canarias. Geoplaneta Editorial, Barcelona, p. 419. Bolten, A.B., Bjorndal, K.A., 1992. Blood profiles for a wild population of green turtles (Chelonia mydas) in the southern Bahamas: size-specific and sex-specific relationships. Journal of Wildlife Diseases 28, 407– 413. Bounous, D.I., Dotson, T.K., Brooks, R.L., Ramsay, E.C., 1996. Cytochemical staining and ultrastructural characteristics of peripheral blood leukocytes from the yellow rat snake (Elaphe obsoleta quadrivittata). Comparative Haematology International6 6, 86–91. Bradley, T.A., Norton, T.M., Latimer, K.S., 1998. Hemogram values, morphological characteristics of blood cells and morphometric study of loggerhead sea turtles, Caretta caretta, in the first year of life. Bulletin of the Association of Reptilian and Amphibian Veterinarians 8, 8–16. Brooks Jr., R.L., Bounous, D.I., Andreasen, C.B., 1996. Functional comparison of avian heterophils with human and canine neutrophils. Comparative Haematology International 6, 153–159. Campbell, T.W., 1996. Sea turtle rehabilitation. In: Mader, D.R. (Ed.), Reptile Medicine and Surgery. W.B. Saunders Company, Philadelphia, Pennsylvania, pp. 427–436. Cannon, M.S., 1992. The morphology and cytochemistry of the blood leukocytes of Kemp’s ridley sea turtle (Lepidochelys kempi). Canadian Journal of Zoology 70, 1336–1340. Dessauer, H.C., 1970. Blood chemistry of reptiles: physiological and evolutionary aspects. In: Gans, C., Parsons, T.S. (Eds.), Biology of the Reptilia. Academic Press, New York, pp. 1–72. Desser, S.S., 1978. Morphological, cytochemical, and biochemical observations on the blood of the tuatara, Sphenodon punctatus. New Zealand Journal of Zoology 5, 503–508. Frair, W., 1977. Turtle blood cells packed cell volume, sizes and numbers. Herpetologica 33, 167–190. Gelli, D., Morgante, M., Ferrari, V., Mollo, A., Freggi, D., Romagnoli, S., 2004. Hematologic, serum biochemical, and serum electrophoretic patterns in loggerhead sea turtles (Caretta caretta). In: Proceedings of 11th Annual Conference of the Association of Reptilian and Amphibian Veterinarians, FL, May 8–11, 2004, pp. 149–152. George, R.H., 1997. Health problems and diseases of sea turtles. In: Lutz, P.L., Musick, J.A. (Eds.), The Biology of Sea Turtles. CRC Press, Boca Raton, FL, pp. 363–385. Grumbles, J., Rostal, D., Alvarado, J., Owens, D., 1990. Hematology study on the black turtle, Chelonia agaassizi, at Playa Colola, Michoacan, Mexico. In: Richardson, T.H., Richardson, J.I., Donnelly, M. (Compilers), Proceedings of the 10th Annual Workshop on Sea Turtle Biology and Conservation, National Oceanographic and Atmospheric Administration Technical Memorandum NMFS-SEFC278, Miami, FL, pp. 235–239. Harms, C.A., Keller, J.M., Kennedy-Stoskopf, S., 2000. Use of a two-step PercollÒ gradient for separation of loggerhead sea turtle peripheral blood mononuclear cells. Journal of Wildlife Diseases 36, 535–540. Harms, C.A., Lewbart, G., Beasley, J., Stamper, A., Chittick, B., Trogdon, M., 2002. Clinical implications of haematology and plasma biochemistry values for loggerhead sea turtles undergoing rehabilitation. In: Proceedings of the 20th Annual Symposium on Sea Turtle Biology and Conservation, National Oceanographic and Atmospheric Administration Technical Memorandum NMFS-SEFSC-477, pp. 190–191. Heard, D., Harr, K., Wellehan, J., 2004. Diagnostic sampling and laboratory tests. In: Girling, S.J., Raiti, P. (Eds.), Manual of Reptiles,

A.B. Casal, J. Oro´s / Research in Veterinary Science 82 (2007) 158–165 second ed. British Small Animal Veterinary Association, Quedgeley, UK, pp. 71–86. IUCN/SSC, 2004. 2004 IUCN Red List of Threatened Species. (accessed 07.12.05.). Jain, N.C., 1986. Cytochemistry of normal and leukemic leukocytes. Schalm’s Veterinary Hematology, fourth ed. Lea and Febiger, Philadelphia, pp. 909–934. Keller, J.M., Kucklick, J.R., Stamper, M.A., Harms, C.A., McClellanGreen, P.D., 2004. Associations between organochlorine contaminant concentrations and clinical health parameters in loggerhead sea turtles from North Carolina, USA. Environmental Health Perspectives 112, 1074–1079. Maier, P.P., Segars, A.L., Arendt, M.D., Whitaker, J.D., Stender, B.W., Parker, L., Vendetti, R., Owens, D.W., Quattro, J., Murphy, S.R., 2004. Development of an index of sea turtle abundance based upon inwater sampling with trawl gear. Final Project Report to The National Marine Fisheries Service, National Oceanic and Atmospheric Administration, pp. 86. Mateo, J.A., Andreu, A.C., Lo´pez-Jurado, L.F., 1997. Las tortugas marinas de la Penı´nsula Ibe´rica, Baleares, Azores, Madeira y Canarias: introduccio´n. In: Pleguezuelos, J.M. (Ed.), Distribucio´n y Biogeografı´a de los Anfibios y Reptiles en Espan˜a y Portugal. Universidad de Granada Editorial, Granada, pp. 433–434. Montali, R.J., 1988. Comparative pathology of inflammation in the higher vertebrates (reptiles, birds and mammals). Journal of Comparative Pathology 99, 1–26.

165

Owens, D.W., Ruiz, G.J., 1980. New methods of obtaining blood and cerebrospinal fluid from marine turtles. Herpetologica 36, 17–20. Pritchard, P.C.H., 1997. Evolution, phylogeny, and current status. In: Lutz, P.L., Musick, J.A. (Eds.), The Biology of Sea Turtles. CRC Press, Boca Raton, FL, pp. 1–28. Raskin, R.E., 2000. Reptilian complete blood count. In: Fudge, A.M. (Ed.), Laboratory Medicine (avian and exotic pets). W.B. Saunders Company, Philadelphia, Pennsylvania, pp. 193–197. Smith, C.R., Hancock, A.L., Turnbull, B.S., 2000. Comparison of white blood cell counts in cold-stunned and subsequently rehabilitated loggerhead sea turtles (Caretta caretta). In: Proceedings of the American Association of Zoo Veterinarians and International Association for Aquatic Animal Medicine Joint Conference, New Orleans, Lousiana, September 17–21, 2000, pp. 50–53. Sypek, J., Borysenko, M., 1988. Reptiles. In: Rowley, A.F., Ratcliffe, N.A. (Eds.), Vertebrate Blood Cells. Cambridge University Press, Cambridge, pp. 211–256. Wood, F.E., Ebanks, G.K., 1984. Blood cytology and haematology of the green sea turtle, Chelonia mydas. Herpetologica 40, 331–336. Work, T.M., Balazs, G.H., 1999. Relating tumor score to hematology in green turtles with fibropapillomatosis in Hawaii. Journal of Wildlife Diseases 35, 804–807. Work, T.M., Raskin, R.E., Balazs, G.H., Whittaker, S.D., 1998. Morphologic and cytochemical characteristics of blood cells from Hawaiian green turtles. American Journal of Veterinary Research 59, 1252–1257.