Dept. of Biochemistry,

Animal Health Research Institute, Dokki.

Oxidative stress and some haematobiochemical changes in blood

of cattle during Theileriosis

(With 7 Tables and 6 Figures)

By

Amal M. Hosny, Sahar A. Aly and

Omima I. Ahmed*

* Dept. of Parasitology, Animal Health Research Institute, Dokki.

(Received at 20/2/2010)

الأجهاد التأکسدي والتغيرات الهيماتولوجية والبيوکيميائية للدم

في الأبقار المصابة بالثيليريا

أمل محمد حسنى ، سحر أحمد أحمد على ، أميمه ابراهيم أحمد

شملت هذه الدراسة الاستقصاء عن الاصابة بالثيلريا في عدد 30 بقره من احد المزارع  بمحافظة الفيوم وقد ظهرت عليها اعراض الأصابة بالمرض (ارتفاع في درجة الحرارة, تضخم العقد اللمفية السطحية وبخاصة امام اللوح, الهزال شحوب الاغشية المخاطية, تدمع العينين, ضعف الشهية, خشونة الجلد وزيادة معدلات التنفس وضربات القلب کما تم ملاحظة وجود القراد متطفلا علي مناطق مختلفة من جسم الحيوان المصاب) بالأضافة الي ذلک فقد تم تجمع عدد 96 عينة من ابقار سليمة ظاهريا قبل الذبح بمجزر البساتين. وقد وجد ان نسبة الأصابة بالثيلريا في المزرعة26(86.67%). أما في الحيوانات السليمة ظاهريا بالمجزر فکانت 14(14.58 %). اظهرت الفحوصات المعملية بالدم زيادة معنوية في مستوي المالون داي الدهايد مصحوبة بنقصا معنويا في مستوي الجلوتاثيون المختزل وانزيم الجلوکوز فوسفات دي هايدروحينيز. کما أظهر الفحص الخلوي للدم نقصا معنويا في کل من عدد الکريات الحمراء, نسبة الهيموجلوبين وحجم خلايا الدم المضغوطة مصحوبا بزيادة معنوية في متوسط حجم الکرية الحمراء هذا بالاضافة الي نقص معنوي في العدد الکلي لکرات الدم البيضاء وقد اظهر العد النوعي لها نقصا معنويا في کل من الخلايا اللمفاوية, النيتروفيل والأيزونوفيل. اما التحليل البيوکميائي فقد أظهر نقصا معنويا في مستويات کل من البروتين الکلي الألبيومين والجلوبيولين, الجلوکوزبالأضافة الي الزيادة المعنوية في کل من مستوى نشاط الأمينوترانسفيرازو کذلک البيلوربين الکلي, وکوليستيرول الدم الکلى والنوعى کما لوحظ انخفاضا معنويا في کل من مستويات الکالسيوم ,الفوسفور, الصوديوم ,البوتاسيوم والحديد.

SUMMARY

Investigation of the oxidative stress and some heamato- biochemical changes associated with Theileriosis in cows: The prevalence of Theileria infection was investigated in 30 cows from a private farm in Fayoum Governorate with suspected symptoms of theileriosis such as rise in temperature, enlargement of superficial lymph nodes, inappetance, tachycardia, weakness. In addition, a total of 96 blood samples were collected from apparently healthy cattle before slaughtering at EL-Bassatine abattoirs. Further more, 20 positive samples for theileriosis were subjected to investigation of the oxidative stress and some heamato- biochemical changes. In addition, samples from 10 microscopically negative healthy cows were subjected to the same examinations as a control Parasitological examination revealed that the incidence of Theileria infection in the farm was 26(86.67%) and in the apparently healthy cows at EL-Bassatine abattoir was 14(14.58%) with a total incidence of 40(31.58%). Blood analysis of positive samples for theileriosis revealed a significant increase in erythrocytic malondialdehyde level (MDA) accompanied by a significant decrease of reduced glutathione (R.GSH) as well as glucose-6- phosphate dehydrogenase (G6PD). Heamatological examination showed a significant decrease in total red blood corpuscles (T.RBCs) count,  Hemoglobin (Hb%), PCV%, (packed cell volume) accompanied by a significant increase in MCV(mean corpuscular volume) in addition to significant decrease in T.L.C, (total leucocytic count) neutrophils, eosinophils and lymphocytes numbers. Biochemical analysis of positive cases revealed significant decrease of glucose, total proteins (T.P), albumin, globulin levels as well as albumin/globulin ratio (A/G), in addition to a significant increased levels of aminotranseferases, T. Bilirubin, and cholesterol, whereas a significant decrease in iron, sodium, potassium, calcium and inorganic phosphorus, were recorded compared to the control group.

Key words: Theileria, cows, oxidative stress, hematology, serum biochemistry.

INTRODUCTION

Tropical theileriosis, a tick borne haemoprotozoan disease caused by protozoan parasite of the genus Theileria, and transmitted by Hyalomma sp., is one of the most devastating blood parasite-affecting cattle. (Sayin et al., 2003). The prevalence, morbidity, and mortality of tropical theileriosis are considerably high. It is estimated that 250 million cattle endemic are as from the Mediterranean basin to China, causing serious economic losses through bovine mortality and lost of productivity (Aktas et al., 2004). In their mammalian hosts, the parasites have a complex life cycle. Infection is initiated by the transformation of macroschizont-infected cells in the lymph nodes draining the site of inoculation of sporozoites by ticks. The infective form of the parasite is the sporozoite transforming into schizonts in WBCs of the mononuclear lineage. The schizont undergoes further differentiation to merozoites, which are released upon lysis of the infected cells. Once released from host cells, the merozoites enter erythrocytes. This is followed by the development of piroplasms in erythrocytes and the parasite becomes infective for the vector (Nalbantogiu, 2003). In the arthropod vector, the different stages of theileria species develop in gut epithelial cells and salivary glands till reaching the sporozoite which is the infective stage to the mammals (Shkap et al., 2003).

Weakness, weight loss, anorexia, high body temperature, petechia on the conjunctival mucosa, swollen lymph nodes, anemia, and cough are the most common clinical symptoms in theileriosis. On later stages of theileriosis, infected animals cannot stand up. Their body temperatures are under normal values and icterus, dehydration, and blood in feces are occasional clinical symptoms (Bakheit, et al., 2004). The main symptom of this disease is anemia; however, the pathogenesis of this anemia is not clear. It has been reported that abnormal osmotic fragility and morphological erythrocyte disorder were observed according to parasitemia, that the erythrocyte survival rate declines with the parasitemia, and that these phenomena occur in both parasitized and un parasitized erthrocytes (Yagi et al., 1998).

The occurrence of parasites produces lesions in the endothelial lining of blood vessels, tissue damage in organs such as liver, kidney, lung, and multiple petechial haemorrages (Forsyth et al., 1999).

There are some evidences that the oxidative stress and lipid peroxidation incorporate theileriosis in the pathogenesis of anaemia. Lipid peroxidation is a general mechanism, which, by free radicals, induces tissue damages and is involved under several pathological conditions (Halliwell and Gutteridge, 1999; Knight, 1995). Free radicals have been reported to act as cytotoxic agents (Gutteridge, 1995). The erythrocyte, due its role as O2 and CO2 transporter, is under constant exposure of free radicals (Harvey, 1997). However, red cells have a potent antioxidant protection that modifies free radicals into substantially less reactive intermediates (Cimen, 2008). Oxidative stress arises when there is an imbalance between radical-generating and radical-scavenging activity. It may therefore cause an increase in the formation of oxidation products (Gutteridge, 1995). Oxidation of the erythrocytes induces membrane injury, methemoglobin (MetHb) formation and eventually destruction of the cell (Harvey, 1997). Lipids especially polyunsaturated fatty acids are sensitive to oxidation, leading to the term lipid peroxidation, of which, malondialdehyde (MDA) is the most abundant (Halliwell and Chirico, 1993; Esterbauer, 1996). The accumulation of MDA in tissues or biological fluids is indicative of the extent of free radical generation, oxidative stress and tissue damage (Gutteridge, 1995; Yagi, 1998)                 

The present study aimed to focus light on prevalence of Theileriainfection in cows and detection of some stages of Theileria in hard ticks.    The present investigation also, was undertaken to ascertain the changes in hematological and biochemical constituents of blood as a result of Theileria infection in cows.

Materials and Methods

A total number of thirty cows from a private farm in Fayoum Governorate of both sexes with suspected symptoms of theileriosis were bled and examined for Theileria infection. The animals were clinically examined for signs of health and disease with special care to body temperature, nasal discharge, enlargement of superficial lymph nodes, corneal opacity and wasting (Radostits et al., 2000).In addition a total of ninety-six blood samples were collected from apparently healthy cows   before slaughtering at EL-Bassatine abattoir. Blood samples were collected as two groups: the first, on silicone coated vacutainer tubes with EDTA (ethylene- diamine tetra acetate) for parasitological, oxidative and haematological examination. The second group on sterile Silicone- coated vacutainer tubes centrifuged at 3000 rpm for 5 minutes to collect the serum for biochemical analysis. Thin blood smears were made from each blood sample, fixed and stained with Giemsa stain for detection of blood parasite microscopically according to Coles (1986). Collection of ticks from the body of animals. Ticks were prepared for dissection according to Rosell- Davis and Coons (1989). Both the salivary glands and the gut of each tick were taken separately and spread on a slide then stained with Giemsa stain. Developmental stages of these protozoa in blood films and salivary or gut smears were measured then illustrated.

After parasitological examination, 20 samples proved to be highly positive for theilaria were subjected to haematological and biochemical analysis. In addition, samples from 10 microscopically negative healthy cows were subjected to the same examination as control.

The blood collected on (EDTA) for both groups was used for the determination of the following parameters represented in Table (1).

Table 1:

Blood film from each blood sample was stained with Leishman stain and observed microscopically to study WBCs differential count.

The collected serum samples were used for the determination of the following parameters represented in Table (2).

Table 2:

The mean values obtained from hemograms and biochemical assays of positive samples were compared with data of negative samples using the T- test(Petrie and Watson, 1999) Differences were considered to be statistically significant with values of P<0.05

RESULTS

Table 3: The Prevalence of tropical theileriosis among cattle.

Table 4: Effect of tropical theileriosis on oxidative stress indicators in cows

  The mean difference is significant at the P<0.05.

Table 5: Effect of tropical theileriosis on blood hematology in cows

The mean difference is significant at the P<0.05.

Table 6: Effect of tropical theileriosis on some blood serum biochemical parameters in cows    

The mean difference is significant at the P<0.05.

Table 7: Effect of tropical theileriosis on some mineral           concentration in cows 

  The mean difference is significant at the P<0.05.

Fig. 1: Macroshizont in lymphocyte of blood smear of infected cattle

Fig. 2: Microshizont in lymphocyte of blood smear of infected cattle

Fig. 3: Ring form of theileria species within infected erthrocytes

Fig. 4: Elongated form in gut smears from tick.

Fig. 5: Ring form in gut smears from tick.

Fig. 6: Ookinete in salivary gland smears from tick.

DISCUSSION

1- Parasitological examination

The clinical signs on Theileria infection in cattle were rise in body temperature, enlargement of superficial lymph nodes, inappetance, tachycardia and weakness.

In the present study, the parasite was found to occur in different morphological forms within the thin blood smears. The schizonts were detected in the lymphocytes with two forms; macroschizonts and microschizonts. Macroschizonts measured 8.5× 3.5 μm (Fig.1).  Microschizonts measured 9.75× 3.75 μm (Fig.2.). Ring form was found in infected erthrocytes with transparent central part and nucleus located at the margin was found in most cases and measured 1.3 μm (Fig.3).

Regarding tick examination, various developmental stages of different shapes and forms were observed in the gut and salivary smears. Elongated structure was found in the gut smear, measured 3 μm long with 2 nuclei were seen (Fig.4). and ring form was detected also in the gut smear measured 1.85 μm (Fig.5) Concerning salivary gland smears examination revealed numerous sporonts (ookinete). Each one was round and measured 3-4 μm in diameter with centrally located nucleus a hallow was seen around the nucleus (Fig.6).

Mediterranean cost fever or tropical theileriosis is a tick born disease of cattle caused by Theileria infection, extending from Morocco to China and consider one of the major health problems of live stock (Devendra, 1995). Clinical examination of animals infected with Theileria revealed rise in body temperature, enlargement of superficial lymph nodes, loss of weight, anorexia. These findings were previously recorded by Abd EL-Raouf et al. (2000); Salah and Mahran (2003); Omer et al. (2003b) and El-Masry et al. (2006).

Examination of blood films (Table 3) revealed that 40 (31.58%) out of 126 animals were infected with theileriosis. This result was similar to that of Loria et al. (1999); Abd EL-Raouf et al. (2000); Inoue   et al. (2001) and El-Masry et al. (2006).They recorded that the incidence of theileriosis among cattle were 29.4%, 33.66% 27.5% and 29.33% respectively. However higher prevalence rates were reported by        EL-Metenawy (2000); Inoue et al. (2001) and Song and Sang (2003) who detected that theileriosis among cattle were 76.5%, 50.4% and 67.8% respectively. These differences may be attributed to the difference in localities and species of examined animals. Also blood films revealed the presence of different forms of the intra erythrocytic trophoites, schizonts (Koch's blue bodies) in lymphocytes. This finding is completely typical to those previously described by Radostits et al.(2000); Salah and Mahran, (2003) and El-Masry et al. (2006).

The identification of the collected ticks revealed that they were Hyalomma anatolicum anatolicum. This result coincide with Bakhiet and Latif(2002); Shkap et al. (2003) and Aktas et al. (2004) who recorded that Theileria annulata, the causative agent of Tropical theileriosis in cattle, is transmitted by ticks of genus Hyalomma.

Tick examination revealed different developmental stages of Theileria, which were present in gut and salivary glands. Present result coincides with Schein (1975), who studied the life cycle of T. annulata in mid gut of Hyalomma anatolicum excavation, and found elongated structure measuring 3 μm long with 2-4 nuclei, describing it as mature microgamont, whereas Magda et al. (1998)found elongated structure with 3 nuclei. Also a ring form measuring 1.85 μm was observed. The latter stage may be the stage seen just after replication of ticks due to the similarity of its measurement with that of the ring form detected in the erythrocytes, which was 1.3 μm, this result was agreed withMagda et al. (1998).

Examination of the salivary glands revealed a round shape measuring 3-4 μm in diameter with centrally located nucleus and a hallow around it. This result is similar to those of Schein (1975) and El-Refai et al. (1998)whostated that the ookinete penetrated the alveoles of the salivary glands and commenced rounding up into sporonts.

2- The Oxidative stress of Theileriosis:

Since erythrocytes membrane is rich in polyunsaturated fatty acids,it is considered as the primary target for reactions involving free radicals, and is very susceptible to lipid peroxidation (Devasena et al., 2001).   

Malondialdehyde (MDA), a product of polyunsaturated fatty acid oxygenation, is a reliable and a commonly used biomarker for assessing lipid peroxidation (Sheu et al., 2003). In present study, there was a significant increases (p < 0.05) in the levels of MDA in Theileria infected cows, compared with healthy ones, In contrast, there was a significant reduction (p < 0.05) in the level of R.GSH Table (4). These results come in accordance with Asri-Rezaei and Dalir-Naghadeh (2006) and Yoldas and Ertekin (2009). El- Deeb and Younis (2009) recorded that, increased MDA concentration with decreased levels of R.GSH, in affected cattle. It seems that such decreased level may be an indicator of high oxidative stress in theileriosis. The oxidative stress appears when the production of free radicals and reactive metabolites of oxygen exceeds their safe disposal by antioxidant mechanisms, causing lipid peroxidation and gave MDA as the finished product. Shiono et al. (2003) and Asri-Rezaei and Dalir-Naghadeh (2006) showed that the levels of MDA began to increase remarkably in proportion to the decrease of packed cell volume and the increase of parasitemia in T. sergenti infected cattle during the onset of anaemia. They added that, during the serious stage of anaemia, this oxidative index reached its maximum value, concluding that the oxidative damage to the RBCs might play an important role in the pathogenesis of anaemia in bovine theileriosis.

Morever, our results revealed a significant decrease (p < 0.05) in G-6-PDactivities in Theileria infected cattle, compared with healthy ones. This result come in accordance withEl- Deeb and Younis (2009). The significant decrease in the activity of G6PD in affected cattle suffering of severe anaemia is an indicator of a metabolic disturbance of erythrocytes. This enzyme has a key role in the pentose phosphate pathway, which has critical significance in the survival of erythrocytes (Beutler, 1984). G6PD enzyme is the principal source of NADPH, which helps in maintaining glutathione at reduced state, thus protecting erythrocytes from oxidative stress. So G6PD serves as an antioxidant enzyme and the low activity of G6PD has been associated with increased haemolysis in cattle affected by theileriosis (Singari et al., 1991) and increased oxidative stress in endothelial cells (Leopold et al., 2003). Thus the, low G6PD activity can be followed by reduced activities of GSH, because of the dependence of the activity of this enzymes on NADPH + H levels in the cell.

3- Haematological examination

In the present investigation, total red blood cells count, haematocrit value, and haemoglobin concentration were significantly lower (p < 0.05) in infected cows compared to control group (Table 5): In addition, MCV, was significantly increased, where as MCHC was non significantly decreased indicating macrocytic hypochromic anaemia  in the infected group compared to the control group. These observation were compatible with those reported by Beniwal et al. (2000); Omer     et al. (2002); Ramazan and Ugur (2006); El-Masry et al. (2006); Abdellah et al. (2006); Hasanpour et al. (2008) and El-Deeb and Younis (2009). The cause of anemia during blood parasite infection may be multifactorial. The extent decrease in total RBC is correlated with the intensity of infection which indicated the destruction of erythrocytes and reduction in the Hb content as evidenced by anemia (Chengalva and Hafeez, 1995). The removal of the piroplasm- infected erythrocytes by macrophages in the organs of the reticuloendothelial system has been suggested as a cause of anemia. Beniwal et al. (2000) and Singh et al. (2001), recorded reticulocytosis to compensate increased RBCs destruction in theileriosis Also, this decrease could be due to increased levels of activated complement products (Stockham et al., 2000; Omer et al., 2002). In addition, pro-inflammatory cytokines, particularly TNF-α, have been implicated in mediating anemia associated with tropical theileriosis (Forsyth et al., 1999; Graham et al., 2001). Moreover, oxidized erythrocytes are easily destroyed by erythropagocytosis, and oxygen radicals may also be involved in the pathogenesis of the resultant anemia (Clark, et al., 1986; Mbassa, et al., 1994; Yagi, et al., 2002)

Some researchers have demonstrated an increased leucocytic number just after Theileria infection followed by a significant decrease within several days in calves,(Sandhu et al., 1998) andin camels(Al-Saad, et al., 2006). Present data showed a significant decrease (p < 0.05) in TLC, neutrophils, eosinophils and lymphocytes number accompanied by insignificant difference in absolute basophile and monocyte number in infected cows compared to the control ones (Table 5). Similar findings were reported by Omer et al. (2002); Ramazan and Ugur (2006); in cattle and with Osman and AL-Gaabary (2007); Hasanpour    et al. (2008) and EL-Deeb and Younis (2009), in buffaloes, and with Hala and Eman, (2006), in sheep. This decrease was related to the destruction of lymphocytes in lymphoid organs and infiltration of these cells into various organs (Sandhu et al., 1998; Omer et al., 2002). Forsyth et al. (1999)suggested that Theileria infection induced leucopoenia in cattle, mainly mediated by TNF- α. Graham et al. (2001) added that, cytokines (TNF-α, IL-1, and IL-6) produced by infected mononuclear cells are responsible for the diverse clinical symptoms of tropical theileriosis, such as depression, pyrexia, anorexia, cachexia, and disseminated hemorrhages.

4- Biochemical examination

Serum proteins, albumin, globulins levels and A/G ratio, were decreased significantly (P<0.05) in the infected cows in comparison with the control group (Table 6). This results coincided withFarah et al. (1999); Omer et al. (2003a); Ramazan and Ugur, (2007) and Yurtseven and Uysal (2009) who attributed this decrease in total serum proteins to hypoalbuinaemia and hypoglobulinaemia arising from decreased protein production as a result of tissue damage in the liver which occurs during Theileria infection in cattle.Stockham et al. (2000), suggested that such decrease could be explained by the lesser food intake due to anorexia, in addition to the extravascular accumulation of pertinacious fluids, resulting from diseased lymph nodes and thus explained the edema and body cavity effusions.   

In this investigation animals with theileriosis revealed a significantly higher level (P<0.05) of AST and ALT activity levels than the control group. (Table 5). These findings support the results of Sandhu  et al. (1998); Singh et al. (2001); Omer et al. (2003a); Dabak      et al. (2004); Ramazan and Ugur (2007), in cattle and with Hasanpour     et al. (2008), in buffaloes, and with Hala and Eman, (2006), in sheep. The occurrence of parasites in any tissue causes the parasitic tissue damage (Radostitis et al., 2000). Theileriainfection causes hepatic tissue damage that include coagulative necrosis, distortion of hepatic cord and heavy infiltration of lymphocytes in the peripheral areas, which indicate severe damage in the hepatobiliary system due to hypoxia resulting from anaemia and jaundice (Sandhu, 1998). FurthermoreRamazan and Ugur, (2007),recorded that, the significant rise in serum AST and creatine kinase activities in theileriosis were due to muscle trauma caused by prolonged recumbency.

In the same time a significant increase in bilirubin concentration in infected animals compared to control ones was observed (Table 6). This observation is in agreement with Sandhu et al. (1998); Farah et al. (1999); Omer et al. (2003a) and Ramazan and Ugur (2007), who related this rise in bilirubin concentration to the destruction of parasitized erythrocytes by erythropagocytosis in the spleen, lymph nodes and other organs of the reticuloendothelial system. In additionto the hepatic dysfunction and haemolytic anemia accompanying theileriosis.

Table (6) represents a significant increase (P<0.05) in the total cholesterol, HDL and LDL levels while triglyceride level showed no significant change, compared with the control ones. This result is in agreement with those of Yadav and Sharma, (1986) and Dabak et al. (2004)in cattle and with Hala and Eman (2006)who attributed these result to the liver damage.

In this study, theileriosis caused a significant decrease (P<0.05) in the concentration of serum glucose (Table 6). This result is in agreement with those of Dabak et al. (2004); El-Masry et al. (2006) and Ramazan and Ugur (2007) in cattle, and with EL-Deeb and Younis (2009) in buffaloes, who suggested that hypoglycemia could be due to the utilization of glucose by the parasite, in addition to the damaged liver. Also, Attia (2001) attributed hypoglycemia in blood parasite diseases to anorexia and depraved metabolic process of diseased animals.     

In present study, cows with theileriosis showed a significant decrease (P < 0.05) in calcium, inorganic phosphorus, sodium, potassium and iron levels, in comparison with healthy animals,      (Table 7). These results are coincided with Farah et al. (1999); Omer   et al. (2003a); Dabak et al. (2004); Ramazan and Ugur (2007), in cattleandwithHasanpour et al. (2008), in buffaloes, and withHala and Eman (2006) in sheep. In theileriosis, hypocalcaemia and hyponatremia were probably due to the hypoproteinemia, decreased dietary intake, intestinal malfunction, and kidney damage (Ramazan and Ugur, 2007). Whereas, the decreased serum phosphorus concentration in cattle with theileriosis may resulted from the diarrhea and renal tubular defect(Sandhu et al., 1998; Omer et al., 2003a). Sodium and potassium are required to maintain electrolyte balance and osmotic pressure of the tissues. During theileriosis, animals suffer from severe dehydration, diarrhea and those parameters are thus found to be disturbed (Farah et al., 1999). The significant decrease in iron level may be attributed to the inability of the damaged liver to synthesize transferring,(Kumar and Malik, 1999; Omer et al., 2003a).

              In conclusion, cattle theileriosis resulted in oxidative and heamato- biochemical changes, which may indicate of tissue damage and might form an indicative basis for subsequent studies, and it should be used as a useful tool for diagnosis, prognosis, and evaluation of the therapy applied.

              Theileriosis in cows resulted in oxidative and heamato- biochemical changes, which may indicate tissues damage and might form an indicative basis for subsequent studies.

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