Veterinary Parasitology, 27 (1988) 245-249 245 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

The Oxidant Defence System in Water-Buffaloes (Bubalus bubalis) Experimental ly Infected with Anaplasma marginale

G.R. REDDY 1, T. MORE', S.P. SHARMA 2 and L.N. SINGH 1

1Division of Biochemistry and Food Science and 2Division of Parasitology, Indian Veterinary Research Institute, Izatnagar-243122, U.P. (India)

(Accepted for publication 31 March 1987)

ABSTRACT

Reddy, G.R., More, T., Sharma, S.P. and Singh, L.N., 1988. The oxidant defence system in water- buffaloes (Bubalus bubalis) experimentally infected with Anaplasma marginale. Vet. Parasi- tol., 27: 245-249.

The glutathione (GSH) -oxidant defence system protects the erythrocytes and leucocytes from oxidative damage. Leucocyte -superoxide dismutase (SOD), GSH-peroxidase (GSH-px), GSH- reductase (GR), GSH-S-transferase (GSH-S-t) and arginase were examined in samples from buffaloes infected with Anaplasma marginale. All the enzymes, except arginase, were also studied in the red cell haemolysates from these animals. GSH-S-t, GSH- and glutathione-reductase (GR) levels in leucocytes decreased in infected animals suggesting a decline in the efficiency of the GSH- oxidant defence system. SOD levels increased but there was no change in leucocyte-arginase ac- tivity due to infection. Infection caused no significant changes in red cell SOD, GSH-px, GR and GSH. However, GSH- S-t significantly decreased (P < 0.05).

INTRODUCTION

The erythrocytes of 80% of Murrah buffaloes contain reduced glutathione ( GSH ), a free-radical scavenger, in the range 50-100 mg dl - ' red cells ( More and Nivasarker, in press). The free radicals are produced by monocytes, neu- trophils and macrophages, when activated in the presence of micro organims, parasites and other foreign elements ( Goldstein et al., 1975; Root and Metcalf, 1977). However, this response is found to differ in some of the haemoproto- zoan infections ( Seth et al., 1985; Remalay et al., 1985). In the present study, buffalo calves experimentally infected with a haemorickettsial agent, Ana- plasma marginale, have been used as a model for monitoring the oxidant def- ence systems in both red and white cells on which no information has hitherto been available. The related parameters e.g., reduced GSH, GSH-peroxidase

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(GSH-px) (EC 1.11.1.9), GSH-S-transferase (GHH-S-t) (E.C.2.5.1.18) and glutathione-reductase (GR) (E.C.1.6.4.2) were also studied.

MATERIALS AND METHODS

Anaplasma infection was established in four male 7-8-month-old buffalo calves by intraveneously inoculating 50 ml. of blood with 70% parasitaemia. Three uninfected calves of a similar age were used as controls. Blood samples were collected in an EDTA s.olution ( Sodium EDTA, 1 mg ml - 1 ) during pat- ent periods of infection. Reduced GSH in whole blood was estimated according to Prins and Loos (1969). Leucocytes were separated (Woldhehiwet et al., 1981) and the cell pellets suspended in phosphate-buffered saline (pH 7.3). They were then sonicated and the homogenates used for enzyme assays. Red cells were washed twice with normal saline solution and haemolysates were prepared. In both red cell haemolysates and leucocyte homogenates, the su- peroxide dismutase ( SOD ) was determined according to Andrewartha and Ca- ple (1980), GSH-px and GR were determined according to Beutler (1976) and GSH-S-t was estimated according to the method of Habig et al., (1976). Total protein content and arginase activity were also determined in leucocyte ho- mogenates by the methods described by Lowry et al. (1951) and Schwartz (1971), respectively. Haemoglobin was estimated according to Beutler (1976). The data were analysed by using Student 's t-test.

RESULTS

Anaplasma-infected buffalo calves developed 8.5% mean parasitaemia on 29 + 2.5 days post infection ( d.p.i. ). The clinical manifestations included rise in body temperature (mean 39.8 °C), anorexia, rough hair coat, pale mucous membranes, nasal and ocular discharge and debility. Different enzyme levels measured both in the control and infected groups are presented in Table I. The activity of the GSH-dependent oxidant-defence system, in terms of GSH-px and GR in leucocytes, declined during infection (P < 0.05 ) whereas SOD tended to increase. There was no influence of infection on leucocyte arginase activity. No significant deviation in the parameters studied was observed in erythro- cytes, except that GSH-S-t declined significantly (P < 0.05 ) (Table II).

DISCUSSION

The limited increase in the leucocyte SOD concentration in the present study indicated superoxide formation and dismutation to H202 by the enzyme, the neutrophils being activated by various invading organisms (Goldstein et al., 1975; Root and Metcalf, 1977). The involvement of the GSH-oxidant defence system as well as the myeloperoxidase-halide system is also reported. How-

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TABLE I

Effect of 'Anaplasma marginale infection on certain leucocyte enzymes

Parameter Control Infected

GSH-reductase (GR) nmole min- 1 rag- 1 protein ) GSH-peroxidase (GSH-px) (nmole min- 1 mg- 1 protein) GSH-S-transferase (GSH-S-t) (nmole rain -~ mg -1 protein) Superoxide dismutase (SOD) (units min 1 mg-i protein) Arginase /~mole urea min-~ mg-1 protein

70.1± 4.61 47.5 _+ 3.41"

325.3_+ 8.37 157.8 _+30.96*

36.3__ 10.47 10.04 ± 1.56"

10.6± 1.44 15.9 ± 2.39

9.0+ 2.88 13.0 ± 3.13

* Significantly different from the control values at P < 0.05.

ever, the decline of the G S H - p x of the leucocytes in the p r e s e n t s tudy sugges ted an inadequa te func t ion ing of the f o r m e r s y s t e m in th is infect ion. Also, Ris t ic (1980) r epo r t ed t h a t the p ro t ec t i on in a n a p l a s m o s i s is m e d i a t e d by the el im- ina t ion of pa ra s i t i zed red cells by the i m m u n e sys tem. I n v o l v e m e n t of G S H - S- t in e lec t rophi le de tox i f i ca t ion as a resu l t of G S H a n d leucot r iene p r o d u c t i o n by neu t roph i l s has a l r eady been es t ab l i shed ( B o y l a n d a n d Charseaud , 1979; P leszczynki , 1985) .

Decl ine in the obse rved G S H - S - t ac t iv i ty in leucocytes in the p r e s e n t s tudy could poss ib ly be due to i nac t iva t ion of th is e n z y m e in the p re sence of ana- p l a s m a infection. Leucocytes exam i ned in the p re sen t s tudy possessed five t imes g rea te r ac t iv i ty of G S H - S - t t h a n t h a t obse rved in red cells. T h i s is in agree- m e n t wi th the r e po r t ed va lues for h u m a n be ings ( H a r v e y and Beu t l e r 1982 ). T h e role of leucocytes in de tox i f i ca t ion appea r s , there fore , to be m o r e effect ive t h a n t h a t of red cells.

TABLE II

Effect ofAnaplasma marginale infection on certain red cell enzymes related to the GSH-oxidant defence system

Parameters Control Infected

Haematocrit ( To ) Red blood cell GSH (mM l- 1 ) GSH-reductase (nmole min -~ g-1 Hb) GSH-peroxidase ( nmole min- 1 g- 1 Hb) SOD ( 104 units min- 1 g- 1 Hb ) GSH-S-transferase (nmole min ± 1 g- 1 Hb)

30.5 _+2.0 20.6 ±2.7" 1.73 ± 0.13 1.98_ 0.29

13.24 + 2.02 12.08 ___ 1.20 140.0 _+7.27 137.3 ±6.10

7.1 ±0.80 8.71±1.4 5.93 _+ 0.20 1.85 ± 0.39*

*Denotes significantly different from the control values at P < 0.05.

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Leucocyte arginase act iv i ty in our expe r imen t s was s tudied wi th reference to the observa t ions of Schne ider and Dy (1985), who repor ted t h a t when lym- phocy tes and macrophages were ac t iva ted in v i t ro they con ta ined or re leased arginase. However , our in vivo f indings could no t conf i rm the i r observat ions; the dif ferences be tween the cont ro l and infected values were no t significant . Never the less , the p resence of arginase in per iphera l leucocytes was previous ly observed in the red cells of buffaloes (More , 1986).

T h e s ignif icant decline in red cell G S H - S - t ac t iv i ty m ay indicate de ter iora- t ion in G S H - b a s e d haemoglob in p ro t ec t ion against oxidat ive radicals ( H a r v e y and Beut ler , 1982). T h e r e was no s ignif icant decline in the G S H co n t en t of the red cells. W h e n this value was expressed in t e rms of whole blood, the de- cline in G S H level app roached signif icance ( P < 0.05), since h a e m a t o c r i t lev- els are low in anap lasmos i s -based anaemia due to haemolysis . Also, such condi t ions are accompan ied by an increase in per iphera l re t iculocytes which are r ich in G S H (Agar, 1975). F u r t h e r s tudies to pa r t i t i on the enzymes in Anaplasma-infected cells are needed.

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