J. Vet. Med. A 38, 530-533 (1991) 0 1991 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 0931 - 184X

Department of Pathology', Department of Internal Diseases2. Faculty of Veterinary Medicine, Warsaw Agricultural University, Poland

Serum Lysozyme Activity and Nitroblue Tetrazolium Reduction Test in Dogs With Diabetes Mellitus:$ R. LECHOWSKI~, M. LENARCIK~, A, D E G ~ R Sand K I 'A. WINNICKA~ Address of authors: R. LECHOWSKI, Department of Internal Diseases, Veterinary Faculty, Agricultural University of Warsaw, Grochowska 272, 03-849 Warszawa, Poland

With 2 tables (Received for publication November 12, 1990)

Summary Serum lysozyme activity and nitroblue tetrazolium (NBT) reduction by blood phagocytes were studied in 24 dogs with controlled insulin-dependent diabetes mellitus and in 16 healthy controls aged from 6 to 12 years. In dogs with diabetes mellitus serum lysozyme activity was significantly (p < 0.001) lowered. The NBT reducing value of circulating phagocytes in diabetic dogs was increased (p < 0.05), whereas these cells increase in NBT reduction following stimulation (stimulation index) was decreased (p < 0.05). It is suggested that in diabetic dogs changes in activity of circulating and tissue phagocytes may occur.

Introduction

It is generally stated that dogs with diabetes mellitus are more susceptible to infection than non-diabetic animals. The results of some reports suggest that the mechanisms of specific immunity are not markedly impaired during diabetes mellitus in man (3), whereas certain parameters of nonspecific immunity are strongly altered (1,4). The most important mechanism leading to the increased predisposition of diabetic patients to infection seems to be impairement of function of neutrophils (14). The importance of non-specific immune mechanism in the defence system against an infectious agent is well recognized. Lysozyme activity is taken as a measure of intensity of phagocytosis in tissues and indirectly also as a measure of the size of tissue pool of neutrophils. O n the other hand, the phagocytic cell function may be examined by the nitroblue tetrazolium (NBT) reduction test. The serum lysozyme activity and N B T reduction values show several similarities. Both parameters are increased during bacterial infections (7) and are suppressed after high doses of antiinflammatory drugs in man (2). With this in mind, the purpose of the present study was to investigate the lysozyme activity in blood serum and to perform the N B T in dogs with diabetes mellitus. ':. Much of information contained in this report was presented at the 4th Congress of ICACB, July 18-22, 1990, Davis, California, USA. U.S.Copyright Clearance Center Code Statement:

0931 - 184X/91/3807- 530$02.50/0

Serum Lysozyme Activity and Nitroblue Tetrazolium Reduction Test in Dogs

531

Material Bnd Methods Twenty-four dogs with insulin dependent diabetes mellitus aged from 6 to 12 years were selected for the study. All were receiving insulin lente. The fasting plasma glucose levels were less than 6.11 mmol/l. None of them showed any other disease such as infection, renal faillure or ketoacidosis. The blood samples were obtained in the morning after insulin administration. Sixteen clinically healthy dogs aged from 6 to 10 years were used as a control sample. Serum lysozyme activity was determined by the modified lysoplate method (6) utilizing a dried strain of Micrococcus Iysodeikticus (Sigma, USA). For plotting the standard curve, hen egg-white lysozyme of an approximate activity of 10.000 sugar units per mg of protein (Reanal, Hungary) was used. The results are expressed as the serum lysozyme level in units per 1 ml of serum (U/ml). NBT reduction assay: EDTA-anticoagulated whole blood was used. The NBT reduction test was carried out according to the modified (11) microquantitative method (8), utilizing dextran sulphate potassium (Meito Sanguo Co., Japan) for blood phagocyte stimulation, nitroblue tetrazolium (Chemipan, Poland) as a substrate, and N,N-dimethyl formamide (Reachin, USSR) for the formazan precipitate extraction. The optical density of the NBT sample versus its blank (without NBT dye) was measured photometrically (Specol, Carl Zeiss, Jena) at 525 nm. The results of spontaneous and stimulated reduction were expressed as the increase in optical density per 106 phagocytes ( A OD/106 cells). The stimulation index (SI) was calculated from the following formula: St’imulated NBT reduction Stimulation index = Spontaneous NBT reduction Huernatology. Venous- blood was anticoagulated with EDTA. Total and differential leukocyte counts were determined by a Coulter Counter (Coulter Electronic Ltd., England) and by examination of May-Grunwafd-Giemsa stained smears respectively. The absolute differential and circulating phagocyte (neutrophils and monocytes) counts were calculated in 109 cells per litre (10911). The results were expressed as mean f standard deviations. The significance of the differences between groups was determined by Students t-test. P < 0.05 was taken as statistically significant (12).

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Results and Discussion Haematological values for clinically healthy as well as diabetic dogs are presented in Table 1. As compared to healthy animals, the dogs with diabetes mellitus showed a slightly increased absolute number of neutrophils and of total leukocyte count. In diabetic patients the peripheral PMN count is normal suggesting an adequate size of the circulating granulocyte pool, while the marrow reserve seems to be considerably reduced (13). Similar

Table 1. Total and differential leukocyte count in diabetic and clinically healthy dogs

Total leukocyte count

Healthy dogs n = 16 (mean k 1 SD)

Diabetic dogs n = 24 (mean k 1 SD)

Significance of differences

9.44 f 2.7

11.77 f 3.96

p >0.05

6.74 f 2.391

7.82 f2.93

p >0.05

2.65 f 1.04

3.72 f 1.74

p 0.05

70.5 f 11.3

66.4 f 8.74

p >0.05

28.2 k 11.OO

30.9 t 14.7

p >0.05

1.00 t 1.00

1.44 f 1.24

p >0.05

(10911)

Absolute neutrophil count ( I 09/11 Absolute lymphocyte count (1094

Absolute eosinophil count (10911) Neutrophils (Yo)

Lymphocytes (Yo ) Eosinophils (Yo)

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LECHOWSKI, LENARCIK, D E G ~ R Sand K IWINNICKA

Table 2. Lysozyme activity, NBT reduction and stimulation index in diabetic and clinically healthy dogs Parameter

Diabetic dogs

Lysozyme (Uiml) NBT (OD/lOb) Stimulation index

(n = 24) x f SD

Healthy dogs (n = 16) x f SD

55.2 ?c 14.3 1.21 f 0.44 0.88 f 0.17

96.4 ? 22.4a 0.77 k 0.42b 1.15 k 0.34b

x = mean value; SD = standard deviation; OD = optical density; 3 = p < 0.001;

b

=

p < 0.05

results were observed in the marginal pool (13). The authors suggest that in the course of diabetes mellitus serious kinetic disturbances exist in the P M N including an increased turnover of the cells with impairment of the possibility of their mobilisation (13). In the present study similar haematological results were obtained. The differential leukocyte count in diabetic dogs did not differ significantly from healthy animals. However the absolute lymphocyte number was markedly increased which might have been due to chronic subclinical infection probably existing in these dogs. The results of lysozyme activity in serum, N B T and stimulation index are presented in Table 2. The dogs with diabetes mellitus showed markedly lowered value of lysozyme activity. It is well known that the release of lysozyme into the serum is a result of the breakdown and/or stimulation of phagocytes (neutrophils and monocytes) and serum lysozyme activity is associated with the number of the cells entering the tissue (5). Serum lysozyme is taken as a measure of intensity of phagocytosis in tissue and indirectly also as a measure of the tissue pool of neutrophils. The obtained results of lysozyme activity in diabetic dogs are in accordance with the suggestions of other authors that the marginal pool of PMN is relatively reduced (14). The NBT values showed a significant increase in diabetic dogs (p