CHAIN-BREAKING ANTIOXIDANTS, ASCORBIC ACID AND VITAMIN E, IN RESEPONSE

Assiut Vet. Med. J. Vol. 52 No. 108 January 2006

Dept. of Animal Medicine, Clinical Laboratory Diagnosis,

Fac. Vet. Med., Assiut University.

CHAIN-BREAKING ANTIOXIDANTS, ASCORBIC ACID AND VITAMIN E, IN RESEPONSE

TO CATTLE THEILERIOSIS

(With 2 Tables and 2 Figures)

By M.R. ABDELLAH; A.M. ABD EL-FATAH; GHADA A. ABOU EL-ELLA* and M.F. RAGHIB * Dept. Biochemistry, Fac. of Vet. Med., South Valley University.

(Received at 19/12/2005)

العلاقة بين مضادات الأکسدة المتمثلة في حامض الأسکوربيک

وفيتامين ه ومرض الثيليريا في العجول محمود رشدي عبد اللاه،عمرو محمد عبد الفتاح،غادة أحمد أبو العلا

محمد فاروق راغب

استهدفت الدراسة فحص تأثير مرض الثيليريا في العجول على مستوى مضادات الأکسدة مثل حامض الأسکوربيک وفيتامين ه في مصل الحيوانات المصابة. تم اجراء الدراسة على عدد ۱۸ بقرة هوليشتاين من أحد المزارع الخاصة بمحافظة المنيا حيث تم فحص

کل الحيونات إکلينيکية وتم أخذ عينات دم وذلک لتشخيص الإصابة بطفيليات الدم وعمل اختبارات صورة الدم وکذلک قياس مستوى حامض الأسکوربيک وفيتامين ه في الحيونات محل الدراسة. أظهرت الدراسة وجود ثمانية حيوانات مصابة بالقراد وتظهر عليها أعرض الإصابة بطفيليات الدم والتي تم تأکيد الإصابة بها وذلک بتشخيص طفيل الدم الثيليريا في أفلام الدم الخاصة بها، وعلى الجانب الأخر أظهرت الدراسة خلو باقي الحيوانات من

طفيليات الدم وأي أعراض مرضية أخرى وتم استخدامها کمجموعة ضابطة في الدراسة. أظهرت النتائج وجود انخفاض معنوي في مستوى مضادات الأکسدة حامض الأسکوربيک ( 0.01 >P) وفيتامين ه

( 0.01

>P) في الحيوانات المصابة بالمقارنة بالحيوانات السليمة.

هذا وقد أظهرت الدراسة زيادة التأثير السلبي للعوامل المؤکسدة التي انعکست على الحيوانات المصابة في صورة الانخفاض المعنوي في مضادات الأکسدة وعلى ذلک توصي الدراسة بإستخدام مصادر حامض الأسکوربيک وفيتامين ه کإضافات علائق للحيوانات المصابة وذلک لتلافي التأثير السلبي للعوامل المؤکسدة على الحيوانات المصابة.

SUMMARY

The goal of the present study was to investigate the effect of cattle theileriosis on blood serum ascorbic acid and vitamin E levels as a

203

Assiut Vet. Med. J. Vol. 52 No. 108 January 2006

model for studying the blood oxidative status in parasitized cattle. A total number of 18 Holstein cows were subjected to the study. All animals were examined clinically and blood samples were collected for evaluation of blood parasite, hemogram picture and vitamin E and ascorbic acid levels. Out of the examined number, 8 cattle were found infested with ticks and showed clinical manifestation of blood parasite infection, which were confirmed by the positive blood film for Theileria infection. The rest of the animals were proved healthy and used as control group for the study. The results of the evaluation study of the chain-breaking antioxidants, vitamin E and Ascorbic acid, in the diseased group as compared to the control group revealed significant decrease in blood serum ascorbic acid (p< 0.01) and vitamin E (p< 0.01) levels. The hematological picture revealed significant decrease in total erythrocytes count, hemoglobin concentration and packed cell volume %. The over all results suggest the presence of an increased oxidative stress in Theileria-infected cattle which was manifested by the reduction of the chain-breaking antioxidants levels, ascorbic acid and vitamin E in the blood of parasitized animals. The results of the study recommend the supplementation of Theileria-infected cattle with sources of vitamin C and vitamin E in addition to traditional treatment to overcome the oxidative stress associating the infection and to avoid the possible complications of secondary vitamin deficiency.

Key words: Chain-breaking antioxidants, Vitamin C, Vitamin E, Theileriosis.

INTRODUCTION

Important changes have been recorded in the biochemistry of hosts suffering from parasitic invasions that were based on the species of the parasites and the sites of the hosts they invade (Aksakal 1987).

Bovine theileriosis, is one of the most serious diseases in grazing cattle, the main symptoms of this disease is anemia (Abdel Rahman et al, 1989). Although the pathogenesis of this anemia is not clear, reports have showed that erythrocyte survival rate declines with the parasitemia and that these phenomena occurs in both parasitized and non parasitized erythrocytes (Yagi and Kunugiyama, 1991).

Antioxidant vitamins such as E, C, and A protect the cells from damage by free oxygen radicals generated as a result of parasitism (Medzyavichyus et al, 1989 and Smith 1989). Vitamin C status of ruminants may be important in relation to their health and disease. Ascorbic acid plays an important role as an antioxidant (Kanter 1998). It

204

Assiut Vet. Med. J. Vol. 52 No. 108 January 2006

has also been suggested to be of value in alleviating both physiological and pathological stress (Hemingway 1991). Moreover, the interaction between antioxidant vitamin C and E was investigated and it was reported that there is a sparing effects of vitamin C on vitamin E (Miller et al, 1993).

In the present study it is hypothesized that the oxidative free radicals released from parasitized blood cells are responsible for the consumption of the antioxidant agents and hence the reduction of the chain-breaking antioxidant levels in parasitized cattle. To test this hypothesis, the current study were conducted to investigate the alteration of the antioxidant agents, vitamin E and ascorbic acid, in response to cattle theileriosis as an indicator of the blood oxidative status of parasitized cattle.

MATERIALS and METHODS

ons.

1 - Animals: A total number of 18 Holstein cows (5-7 years old) from a private farm in Samalot district, El-Menya governorate, Egypt were subjected to the study. The animals were divided into two groups; the first group used as a diseased group and composed of 8 animals that showed signs of blood parasite infection upon clinical and laboratory examinations. The second group was the control group and composed of 10 that were proved to be healthy after careful clinical and laboratory examinations. 2 - Samples and adopted methods A. Whole blood samples: 5 ml whole blood samples were collected from the jugular vein in a clean, dry tubes containing disodium salt of EDTA as anticoagulant. Blood smear were done directly after collection, fixed in absolute methanol and kept in dry container. The remained blood samples were kept in cold container containing ice and used for hematological analysis (Coles 1986). Total red blood cell counts (T/l), hemoglobin concentration (Hb g/dl), packed cell volume (PCV, %), mean corpuscular volume (MCV fl), mean corpuscular hemoglobin (MCH pg) and mean corpuscular hemoglobin concentration (MCHC % were determined by using blood cell counter (Cell-Dyn 1700). Blood smears stained with Giemsa stain and differential leucocytic count was carried out using Four Field-Meander method according to Coles (1986). B. Blood serum samples: 10 ml whole blood samples without anticoagulant were collected in clean and dry tubes. These samples used for separation of serum according to Coles (1986). Blood serum levels of

205

Assiut Vet. Med. J. Vol. 52 No. 108 January 2006

ascorbic acid and vitamin E were determined according to Omaye et al, 1979 and Martinek 1964 respectively. C. Statistical analysis: Statistical analysis of the obtained data was done by means of software computer program SPSSWin 1997.

RESULTS

Clinical findings: Diseased animals were found infested with ticks in the preneal, flank and neck regions. Upon clinical examination, they showed different degrees of emaciation, enlarged prescapular and/or prefemoral lymph nodes, anemic mucous membranes and lacrimation. Laboratory findings: Theileria infected cattle were identified by examination of blood smears, which revealed the presence of intracellular signet ring Theileria species parasites. The hematological analysis, as shown in Table 1, revealed significant decreases in total erythrocytes count (p <0.01), hemoglobin concentration and in PCV % (p < 0.01). Analysis of antioxidant vitamins as shown in Table 2 revealed significant decrease in blood serum vitamin C level (p<0.01) as well as blood serum vitamin E level (p< 0.01).

Table 1: Values (Mean = standard deviation (X + SD) of erythrocytes

picture in control and Theileria-infected cattle.

PCV

MCV

MCH

Total RBCs count (1/1)

MCHC

Hb Conc. (g/dl)

(%)

(pg)

Clinically healthy (Control)

cattle

| 6.85 +0.50

6.18 -7.63

11.40 + 0.65 10.60 - 12.60

32.00 +2.00 28.00 - 34,00

47.18 3.80 44.40 - 55.50

| 16.64 +0.64

15.63 - 17.50

35.27 +2.51 31.70 - 38.70

Theileria infected cattle

5.06 $1.23** 3.30 -6.50

6.88 0.89** 5.20 - 8.20

25.75 +5.14** 20.00 - 35.00

53.01 – 7.35* 43.70 - 85.30

14.21 +3.32* 9.07- 19.40

27.37 +5.38** 21.07 - 35.60

*. Significant (p <0.05).

**. Highly significant (p <0.01).

Table 2: Values (Mean + standard deviation (X + SD) of serum vitamin

C and E levels in control and theileria infected cattle.

Vitamin C (mg/dl)

Vitamin E (ug/ml)

Clinically healthy cattle (Control)

0.17 + 0.067 0.12 -0.31

6.02 +0.93 4.27 - 7.46

Theileria infected cattle

0.064 + 0.031** 0.02 -0.11

3.63 + 0.65** 2.95 -5.01

**: Highly significant (p <0.01).

206

Assiut Vet. Med. J. Vol. 52 No. 108 January 2006

Figure 1: Mean values of serum vitamin C level in control and

theileria infested cattle

ip/հա

Control

Theileria infected cattle

Figure 2: Mean values of serum vitamin E level in control and

diseased cattle.

0

w/bn

Control

Theileria infected cattle

DISCUSSION

The tick-born protozoan disease, theileriosis, poses important problem for the health and management of domestic cattle in the tropics and subtropics. In Egypt, bovine theileriosis represents an important obstacle for the development of the livestock industry through the losses of milk and meat production (Abdel Rahman 1989).

207

Assiut Vet. Med. J. Vol. 52 No. 108 January 2006

The significant decrease in total erythrocytic counts and hemoglobin concentration can be attributed to the intracellular parasitism. Intra-erythrocytic parasites metabolize hemoglobin and produce O2, which causes oxidative stress, as a result, the free O2 and H2O2 increase lipid peroxidation and causes oxidation of the sulfhydryl groups of the globin chains and damage of the erythrocyte cell membrane with the subsequent removal of the affected erythrocytes by active macrophages (Mishra et al, 1994 and Stocker et al, 1986). This was emphasized by a number of studies, which demonstrated that the amount of reactive oxygen radicals which cause lipid peroxidation are increased in the cells of hosts infected with different species of parasites, thereby causing cell and tissue damages (Smith 1989 and Sarin et al, 1993). The decreased PCV % (p< 0.01) occurred secondary to reduced total erythrocytic counts.

The interactions between antioxidant vitamin C and E was investigated and a sparing effects of vitamin C on vitamin E as well of vitamin E on vitamin C were recorded (Tanaka et al, 1997). The significant decrease in serum vitamin levels in the present study is attributed to the assimilation of these vitamins by the continuous release of reactive oxygen species. This was in accordance with previous study (Miller et al, 1993), which declared that stress, disease and induction of immune response increase requirement for nutrients, including vitamin E, A and C and essential trace elements. Vitamin C and vitamin E are chain-breaking antioxidants that protect the cells from oxidative damage. Vitamin E is very important for the synthesis of vitamin C (potent antioxidant vitamin) and synthesis of coenzyme Q-a compound that participates in the respiratory mechanism of the cell.

Results of the present study clearly demonstrate the importance of the additional supplementation of Theileria-infected animals with sources of vitamins along with the traditional systemic treatment to overcome the oxidative stress and to avoid the possible complications of vitamin E and C deficiencies. This is because vitamin E and vitamin C play an important role in the immune response and the reproduction of the animals. Previous studies have shown that low level of a-tocopherol (chemical, active form of vitamin E) represent a significant risk factor for clinical mastitis and other types of infection of affected animals (Weimann 1991).

208

Assiut Vet. Med. J. Vol. 52 No. 108 January 2006

REFERENCES

Abdel Rahman MSF, M.M.A.K.; El-Nimr, M.M. and El-Bahy, M.M.

(1989): A review on the works done for the preparation of a vaccine against Theileria annulata Egyptian strain. J. Egypt

Vet. Med. Ass. 49: 529-540. Aksakal Mall, E. (1987): Investigations on hematological values and

vitamin E levels of sera before and after treatment with antihelmentic drugs in Akkarman sheep. A U Vet. Fak Derg

34: 72-84. Coles, E. (1986): Veterinary Clinical Pathology. 4th Ed Saunders Comp

Philadelphia, London, Toronto. Hemingway, D. (1991): "Vitamin C in the prevention of neonatal calf

diarrhea." Can Vet. J. 32: 184. Kanter, M. (1998): Free radicals, exercise and antioxidant

supplementation. Proc. Nutr. Soc. 57: 9-13. Martinek, R. (1964): Method for determination of vitamin E (total

tocopherol) in serum. Clin. Chem. 10: 1078-1086. Medzyavichyus AK, B.V.Y.; Drebitskene, G.A., Mozalene, E.E.,

Medzevicius, A.; Bebravicius, V.; Drebickiene, G. and Mozaliene, E. (1989): Effect of vitamin A and C on immunity to Trichuriasis in piglets under industrial farming conditions.

Acta Parasitol Lit 23: 115-129. Miller, J.; Brzezinska-Slebodzinska, E. and Madson, F.C. (1993):

Oxidative stress, antioxidants and animal function. J. of Dairy

Sci. 6(7): 2812-2823. Mishra, N.; Kabilan, L. and Sharma, A. (1994): Oxidative stress and

malaria-infected erythrocytes. Ind J. Malariol 31: 77-87. Omaye, S., Turnbul, J.D. and Savberligh, H.E. (1979): Ascorbic acid

analysis. II. Determination after derivatisation with 2.2. dinitrophenylhydrazine. Selected methods for determination of ascorbic acid in animal cells, tissues and fluids. Methods in

Enzymology London 62: p.7. Sarin, K.; Kumar, A.; Prakash, A. and Sharma, A. (1993): Oxidative

stress and antioxidant defence mechanism in Plasmodium vivax malaria before and after chloroquin treatment. Ind. J. Malariol.

30: 127-133. Smith NCB, C. (1989): Free radical generation during primary

infections with Nippostrongylus brasiliensis. Parasit Immunol 11: 147-160.

209

Assiut Vet. Med. J. Vol. 52 No. 108 January 2006

SPSSWin. (1997): Software program for statistical analysis under

windows, USA. Stocker, R.; Hunt, N.H. and Wiedemann, M.J. (1986): Antioxidants in

plasma from mice infected with Plasmodium vinckei. Biochem.

Biophys. Res. Commun. 134: 152-158. Tanaka, K.S., Igurchi, H., and Kojo, S.I. (1997): Interaction between

vitamin C and E are observed in tissue of inherently corbutic

rats. Nutr. 127: 2060-2064. Weimann, B. (1991): Functions of vitamin E in reproduction and in

prostacyclin and immunoglobulin synthesis in rats. American

Journal of Clinical Nutrition 53: 1056-1060. Yagi, Y.; Ito, N. and Kunugiyama, I. (1991): Decrease in erythrocyte

survival in Theileria sergenti-infected calves determined by non-radioactive chromium labelling method. J. Vet. Med. Sci. 53: 391 - 394.

210