<sup>1. Department of Anatomy, Faculty of Veterinary Science, Chulalongkorn University,
Bangkok, Thailand. Fax no. 6622189657
E-mail:ksumolya@chula.ac.th
2. Samutprakan Farm and Zoo, Samutprakan 10280, Thailand.
3. Sixth year students, Faculty of Veterinary Science, Chulalongkon University</sup>

^Abstract

^{Blood smear from 54 crocodiles of three breeds (Crocodylus siamensis, C.porosus and crossbred) are examined after Wright-Giemsa staining. Detection for alkaline phosphatase and peroxidase activities are performed. Four type of granulocytes are distinguished : heterophil, eosinophil, basophil and azurophill. Heterophil with the size of 9-10 um has oval eccentric nucleus with mostly spiculate granules and a few tear drop-shaped granules. Heterophil contains both alkaline phosphatase and peroxidase. Eosinophil with the size of 8-9 µm has round or oval eccentric nucleus. The round , rod and few tear drop-shaped and spiculate granules are packed in the cytoplasm. Eosinophil is strongly peroxidase positive but devoided of alkaline phosphatase. Basophil is 9 - 11 µm with centrally located round nucleus. Its’ cytoplasm is full of round various sizes of granules and contains neither alkaline phosphatase nor peroxidase activity. Another granulocyte (8-10 µm) has centrally located round, oval or kidney shaped nucleus with small amount of azurophilic granules. The cytoplasm of this cell gives very strong alkaline phosphatase activity but no peroxidase is detected. Morphological and physiological charactristics of the questionable leukocyte, suggest the possibility that this cell is azurophil.}

^{All granulocytes (monocyte and lymphocyte) and thrombocyte are devoided of both alkaline phosphatase and peroxidase activities.}

^{Key words : Crocodiles, granulocyte, alkaline phosphatase, peroxidase}

Successful in disease control and prevention depend largely on a precise and rapid diagnosis. This is a key to success in animal production industry. Among several parameters, leukocyte differential count is definitely needed in assisting scientists to proximate the status of the animal body. Owing to the fact that every fragment of the crocodile can be made valuable and also the endurance of crocodile to various diseases, quite a few farmers have turn their interest into crocodile farming. In the present time, development in diagnosis of the diseases of the crocodile has not gone far enough to a satisfy level. Additional information concerning the morphological and physiological characteristics of crocodilian blood leukocytes are needed to make a sucessful differential diagnosis and disease monitoring. Apart from differences in form and size of nucleus, cytoplasm and staining characteristics of cell constituent of each leukocytes, specific enzymes content in leukoctyes can be an integral indicator to identify the specific leukocyte. In crocodile, heterophil is used in place of neutrophil as its granules have strong affinity to eosin. They become acidophilic and some time very much resemble the eosinophil. Moreover, there has beeen confusion between azurophil and monocyte or lymphocyte (Hawkey and Dunnette,1989). Frye (1991) reports that azurophil has the appearance of mammal neutrophil mixed with monocyte. Alkaline phosphatase and peroxidase activities will assist in differentiation of heterophil, eosinophil and azurophil.

Materials and methods

Animal: A combination of 38 Crocodylus siamensis (C.siamensis), 8 C. porosus and 8
crossbreds, 6 - 7 years of age are used in the experiment (Table 1).
Diagnostic kits: 1. Sigma alkaline phosphatase (Al-P), leukocyte diagnostic kit*
2. Sigma Peroxidase (P), leukocyte diagnostic kit*
Staining: Wright’s stain with Giemsa (W-G stain)⁺

2. Centrifuged at 2,500-3,000 rpm to obtain the buffy coat.

3. Six buffy coat smears are processed using W-G stain and techniques for alkaline phosphatase and peroxidase activities.

4. Examine under light microscope (x100). A total of 200 leukocytes will be observed and differentiate into granulocytes (heterophil, eosinophil, azurophil and basophil), agranulocytes (monocyte and lymphocyte) and thrombocyte.

Scoring: Sites of alkaline phosphatase activity will appear as red granules and brown- black granulation will indicate peroxidase activity. Rate from 0 to 3⁺ on the basis of quantity and intensity of precipitated dye within the cytoplasm of the cell.

Alkaline phosphatase positive control: Segmented neutrophil of normal rat is used as
positive control for alkaline phosphatase activity (Moloney, 1958).

Peroxidase negative control : Heat and catalase destroy peroxidase (Humason, 1979).

Our pilot study indicates that fixed blood smear incubated at 100^ºC with 70-80% humidity for 30 minutes can destroy peroxidase activity. Crocodile buffy coat smear incubated to destroy peroxidase activity is used as negative control.
 

Results

Granulocytes [heterophil (Figs.1 and 2), eosinophil (Fig.3 and 4), basophil (Figs.5 and 6) and azurophil (Fig.7)], agranulocytes [monocyte and lymphocyte (Fig. 7) and thrombocyte (Fig. 7) are studied. Their morphologies and characteristics of alkaline phosphatase and peroxidase are in table 2.

Alkaline phosphatase activity (Table 2, 3)

Rat buffy coat smear for positive control has red cytoplasm (Fig.8). Heterophil(Fig. 9) has Al-P⁺ with red granules in cytoplasm. The light blue nucleus is oval and eccentric. Heterophil comprises 52.5% (40-64) of the granulocytes. Azurophil (Fig. 10), is round with Al-P⁺⁺⁺ cytoplasm . Centrally located nucleus is blue and can be round, oval or kidney shaped. Azurophil is 13%(5-21). Eosinophil (Figs.11,12) is 6.5%(2-10) with no alkaline phosphatase activity. Nucleus is eccentric and round or oval. Basophil (Fig.13) is Al-P^- with round, centrally located nucleus.

Peroxidase activity (Tables 2,3)

Heterophil (Fig.15) is peroxidase positive (P⁺⁺) with brown-black granules dispersed all over the cytoplasm. spiculate granules are observed. Heterophil comprises 49.5% (41-59). Eosinophil (Fig.16,17) has P⁺⁺⁺ with round, rod and tear drop-shaped brown-black granules densely packed in the cell. Eosinophil shares 7.1% (2-11) of the granulocytes. Basophil (Fig.18), is devoided of peroxidase activity (P^-). It comprises 29.3%(21-39). Azurophil (Fig.19) has P^- and shares 14.1% of the granulocytes

Neither monocyte nor lymphocyte has peroxidase activity (Figs. 20). The thrombocyte (Fig. 15) is also devoided of peroxidase. Heterophil (Fig. 21) and eosinophil (Fig. 22) on the heated buffy coat film (negative control) display no peroxidase activity.

Disscussion

Wright-Giemsa stain displays 4 types granulocytes(heterophil, eosinophil, basophil and azurophil), agranulocytes (monocyte and lymphocyte) and thrombocyte. Morphology of these cells are the same as described by Frye (1973,1977 and1991), Hawkey and Dennette (1989) and Kanchanapangka and Youngprapakorn (1994). However, confusion between heterophil and eosinophil happens. Also azurophil can be mixed up with neutrophil and monocyte, especially when azurophil nucleus is eccentric (Hawkey and Dennette,1989 and Frye,1991). The study of alkaline phosphatase and peroxidase activities can help differentiate the granuloctyes from one anothers and monoctye (Table 2). The agranulocytes and thrombocyte are all alkaline phosphatase and peroxidase negative.

Granulocyte differential count when W-G stain, alkaline phosphatase and peroxidase techniques are applied gives approximately the same percentage (Table 3).

This is in concert with the report by Hawkey and Dennette (1989) and Kanchanapangka and Youngprapakorn (1994) that heterophil is the most abundant (51.2% in the present study). Frye (1977) suggests that reptile has a high number of basophil (31.7% in the present study). Azurophil shares 10.5% and eosinophil is the least (6.6%). Sex, season,

Lymphocyte of the 3 crocodilian species are similar to American alligator (Mateo et al.,1984). In contrast, thrombocyte of the American alligator has fusiform shape with long prominent fold on the nucleus.

Heterophil is used in place of neutrophil in reptile (Hawkey and Bennette, 1989)

Alkaline phosphatase activity is detected in mammal neutrophil ( Molony, 1958, Frye, 1997 and Vergnes et al., 1990. Interestingly, poultry and pigeon heterophil does not contain alkaline phosphatase. Although the avians do have nucleated red blood cell that resemble the crocodile. Azurophil also has alkaline phosphatase activity. Kaplow (1968) reports alkaline phosphatase activity in frog red blood cell and various eosinophil.

Heterophil and eosinophil have peroxidase activity. Kaplow (1965) reveals the present of peroxidase in human neutrophil and eosinophil. Using 3-amino-9-ethyl carbasole as substrate, Mateo et al. (1984) also find peroxidase in heterophil granules. Mateo et al. (1984) and Frye (1991) elaborate that only the round eosinophil granules contain peroxidase. However, all eosinophil granules has peroxidase positive in the present study.

Acknowledgement

The authors wish to thank Dr. Chitraporn Chanrajkit and the people at the Samutprakarn Crocodile Farm and Zoo for their excellent assistance in blood collecting. Chulalongkorn University, Rachadapiseksompoch Research Fund for the financial support .

Fig.1 Heterophil : Nucleus(N) is round or oval and eccentric. Spiculate acidophilic
granules are densly packed in the cytoplasm.(100x).

Fig.2 Abundant spiculate granules (hollow arrow) dispersed from the ruptured heterophil.
A few tear drop-shaped granules (arrow) are also found.(100x).

Fig.3 Eosinophil : Nucleus(N) is round and eccentric with deep eosinophilic round and rod granules (arrow) closely packed in the cytoplasm.(100x).

Fig.4 Lots of round(arrow head) and some rod(rectangle) granules are scattered from the ruptured eosinophil. A few tear drop-shaped (arrow) and spiculate granules are present.(100x).

Fig.5 Basophil :Round nucleus(N) is in the center of the cell. Various size round basophilic
granules are in the cytoplasm.(100x).

Fig.6 Abundant different sizes basophilic granules from the ruptured basophil.(100x).

Fig.7 Aurophils with kidney shaped nucleus(N) centrally located in the cell have fine azurophilic granules. Monocyte (M) at the left hand conner has a large amount
cytoplasm with foamy appearance(arrow).(100x).

Table 2 : Morphology, alkaline phosphatase and peroxidase activities of crocodilian
granulocytes, agranulocytes and thrombocyte
 

and peroxidase activities
 

References

Frye L.F. 1973. Hematology. In : Husbandry, medicine and surgery in captive
reptiles. VM publishing. North Nettleton. pp. 49-51.

Frye L.F. 1977. Hematology of captive reptiles (with emphasis on normal
morphology). In : Current Veterinary Therapy VI.R.W. Kirk (ed.).

W.B. Saunders. Philadelphia. pp. 792-798.

Frye L.F. 1991. Hematology as applied to clinical reptile medicine. In : Biochemical and surgical aspects of captive reptile husbandry. 2^nd ed., Vol 1. Krieger Publish Company. Malabar, Florida. pp. 211-221.

Hawkey C.M. and Dennett T.B. 1989. Normal and abnormal granulocytes.In : A colour atlas of comparative veterinary hematology. Wolfe publication. Ipswich. pp. 58-59, 107-108, 133-137.

Humason G.L. 1979. Alkaline phosphatase, peroxidase method. In : Animal tissue techniques. 4^th ed., W.H. Freeman and company. San Francisco. pp. 420-422.

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Kaplow L.S. 1968. Leukocyte alkaline phosphatase cytochemistry : Applications and methods. Ann. N.Y. Acad. Sci. 155:911-947.

Mateo M.R., Roberts E.D., and Enright, F.M. 1984. Morphologic, cytochemical, and functional studies of peripheral blood cells of young healthy American alligators (Alligator mississippiensis). Am.J.Vet.Res. 45(5) : 1046-1053.

Moloney W.C. 1958. Leukocyte alkaline phosphatase activity in the rat. Ann. N.Y. Acad. Sci. 15 : 31-36.

Vergnes H.A., Courdoughji M.K., Guelfi J.F. and Grozdea J.G. 1990. Effect of zine deficiency in lamb on plasma and neutrophil alkaline phosphatade. Small Ruminant Research 3 : 167-177.