PARASITOLOGICAL AND BIOCHEMICAL STUDIES ON CATTLE THEILERIOSIS AT DAKAHLIA GOVERNORATE WITH SPECIAL REFERENCE TO ITS CONTROL

Assiut Vet. Med. J. Vol. 52 No. 109 April 2006

Dept. of Parasitology, Animal Health Research Institute, El-Mansoura Provincial Laboratory.

PARASITOLOGICAL AND BIOCHEMICAL STUDIES

ON CATTLE THEILERIOSIS AT DAKAHLIA GOVERNORATE WITH SPECIAL REFERENCE

TO ITS CONTROL (With 6 Tables and 2 Figures)

By NABILA M. EL-MASRY; S.A. EL-DESSOUKY*

and S.A. ABO-ELKHEIR *Dept. of Biochemistry, Animal Health Research Institute,

El-Mansoura Provincial Laboratory

(Received at 2/2/2006)

دراسات طفيلية وبيوکيميائية على مرض ثيلريا الأبقار في محافظة الدقهلية

مع الإشارة إلي طرق المقاومة نبيلة محمود المصري،صبري عبده الدسوقي،شعبان عبد ربه أبو الخير تم إجراء هذه الدراسة على عدد ۱۰۰ بقرة تتراوح أعمارها من 6 شهور - أکثر من

5 سنوات تنتمي إلي بعض المزارع الخاصة في المناطق المختلفة لمحافظة الدقهلية في الفترة من سبتمبر ۲۰۰4 إلى نهاية أغسطس ۲۰۰۵. وقد أظهر الفحص الإکلينيکي (الظاهري) الاشتباه في عدد 46 حيوان عليها أعراض الإصابة بطفيل الثيٹريا وبإجراء الفحص

الطفیلی

لهذه الحيوانات معمليا تأکد إصابة عدد 4 حيوان بطفيل الثيثريا أنيولاتا (

۲۹,۳۳ %) بالنسبة العدد الکلي حيث وجد ارتفاع في درجة الحرارة (40-41 °م)، تضخم في بعض الغدد الليمفاوية، فقدان الشهية، احتقان الأغشية المخاطية، زيادة في معدلات التنفس وفي الحالات المتقدمة من المرض ظهرت أعراض الأنيميا والضعف العام بدرجات متفاوتة مع تواجد القراد علي جسم الحيوان. وقد أظهرت الدراسة أن أعلي نسبة إصابة کانت في فصل الخريف (

47,61

%) يليها فصل الصيف (۳۹٫۶۷%) ثم الربيع والشتاء بنسبة (۲۰%)،

( %۰٫۷۱) علي الترتيب. أوضحت الدراسة أن الحيوانات عند عمر 6 شهور إلي سنة کانت نسبة الإصابة (

۳۷,۵۰ %) بلها العمر من ۱-۳ سنوات (۳۲,۰

%) فالعمر من

5-3 سنوات (

۲۷,۷۷

%) وفوق خمس سنوات (۰٫۸۸%). وبالنسبة للتغيرات في صورة ومصل الدم للحيوانات المصابة بالثيلريا أنيو لاتا عند مقارنتها بحيوانات المجموعة الضابطة کانت علي النحو التالي: أظهرت الفحوص الدموية وجود أنيميا تمثلت في وجود نقص معنوي عال في کل من العد الکلي لکرات الدم الحمراء وترکيز الهيموجلوبين وحجم الخلايا المضغوطة وأيضا الخلايا الليمفوسيت ومعنوي لخلايا المونوسیت وفي الجانب الأخر وجدت زيادة معنوية عالية في الخلايا المتعادلة والحمضية ومعنوي في الخلايا القاعدية وفيما يخص

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معنوي عال في مستوي کل

الفحوص البيوکيميائية لمصل الدم للحيوانات المصابة وجد نقص من الألبيومين وسکر الدم وعلى النقيض وجدت زيادة معنوية عالية في مستوي کل من

أميناز، الحديد،

الجلوبيولين الکلي والألفا جلوبيولين، والجاما جولبيولين، أنزيمات الترانس النحاس ومعنوية في المستوي الکلي للاصباغ المرارية، بولينا الدم، الکرياتينين، السيلينيوم، المنجنيز، الزنک عند مقارنتها بالمجموعة الضابطة. وقد تم عزل الحيوانات المصابة حيث عولجت بعقار بيوتالکس وتيراميسين طويل المفعول وأنتوبلکس کمقوي عام. وقد تم رش الحظائر والحيوانات للقضاء علي القراد بعقار البوتکس مما أدي إلي تمام الشفاء وعودة الحيوانات لحالتها الطبيعية واختفاء الأعراض وتحسن ملحوظ في صورة الدم والقياسات البيوکيميائية وعودتها إلى معدلاتها الطبيعية.

SUMMARY

This study was carried out on 150 cow, their ages ranged from 6 months - over 5 years belonged to some private farms in different localities at El-Dakahlia governorate in the period from September 2004 to August 2005. The clinical examination showed 46 suspected cases which have the signs of theileria infection, laboratory parasitological examination confirmed that infection of 44 animals infected with Theileria annulata (29.33%) to total animal number where high temperature (40°-41°C), enlargment of some superficial lymph nodes, anorexia, congestion of mucus membranes, increased respiratory rates were noticed. In advanced cases of the disease signs of anaemia, general weakness were present with different degrees, in addition to presence of ticks on animal body. The study revealed that the higher rate of infection was in Autumn season (47.61%) followed by Summer season (39.47%), Spring and Winter (20%) & (5.71%) respectively. The study cleared that animals at the age of 6 months to one year have the higher rate of infection (37.50%) followed by the age of 1-3 years (32.50%), 3-5 years (27.77%) and finally over 5 years (5.88%). Concerning the changes in the blood and serum of Theileria annulate infected animals when compared with control group were as follow: Blood picture investigations proved presence of anaemia represented by high significant decrease for total erythrocytic count, haemoglobin concentration and packed cell volume and lymphocyte cells, also significant decrease for monocyte cells. On contrary presence of high significant increase in neutrophil and oesinophil cells and significant in basophil cells. Serum biochemical investigation of infected animals, showed that high significant decrease in Albumin and glucose level, while on the other side there was high significant increase in the level of both total globulin, alfa-globulin, gamma-globulin, transamenase enzymes, iron and copper, while the level of total bilirubin, blood urea, creatinine, selenium, manganese and

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zinc showed significant increase. All infected animals were isolated and treated with Butalex®, Terramycin L.A and Antoplex as general haematinic. Animals and their houses were sprayed for ticks using Butox, who lead to complete recovery for diseased animals and marked improvement in haematological and biochemical parameters towards its normal levels.

Key words : Theileria annulata, prevalence, serum, biochemical changes.

INTRODUCTION

Tropical theileriosis is a tick born disease of cattle caused by Theileria annulata, transmitted by hard tick (Hyalomma anatolicum) (Shkap et al., 2003 and Akats et al., 2004). The disease is a major constraint to livestock improvement programes in many parts of the middle east and Asia, where about 200 million cattle are said to be at risk of infection (Bakheit and Latif, 2002). The disease is clinically manifested by marked fever, swelling of superficial lymph nodes, inappetence, tachycardia, dyspnoea and weakness (Omer et al., 2003). Theileria annulata infection rate, reach its maximum in both autumn and summer seasons (El-Metenawy, 2000). Studies of Forsyth et al., (1999) had presented a new view on the destructive effect of the parasite on the host organs and tissues. As the number of cattle in Dakahlia governorate reaches 172099 head (GOVS, 2005). So the present study aimed to facus light on disease situation at Dakahlia governorate, its clinical signs, seasonal prevalence, age susceptibility, the changes of some blood biochemical components associated with Theileria annulta infection in addition to treatment trails of diseased animals.

MATERIALS and METHODS I- Animals:

The present study was carried out on 150 cows aged from 6 months to over 5 years old collected from different private farms in different localities at Dakahlia governorate during the period extending from the beginning of September 2004 to end of August 2005. The animals were clinically examined for signs of health and disease, special care to body temperature, nasal discharge, enlargement of superficial lymph nodes, corneal opacity and wasting according to the method described by Radostits et al., (2000), the animal divided into two groups the first consisted of 104 apparently healthy animals (free from blood parasite after blood examination), the second consisted of 46 animals suffered from signs of the disease blood examination of these animals.

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Revealed that, 44 animals was infected with Theileria spp. The adult ticks were collected manually from the animal and was examined and identified according to Bovattour et al., (1996). II- Samples: 1- Thin blood smears were individually prepared from superficial ear vein, fixed and stained with Giemsa stain for detection of blood parasites microscopically according to Coles, (1986). 2- Aspirated smears were also taken from superficial enlarged lymph nodes and examined according to Marquardt et al., (2000). 3- Two blood samples were collected from jugular vein via venoject system, from each examined animal, in a dry sterile centrifuge tubes. The first blood samples was collected with anticoagulant (sodium salt of EDTA) and used for the evaluation of haemo and Icucograme parameters according to the method adopted by Jain, (2000). The second blood sample was collected without anticoagulant for preparation of serum needed for biochemical investigations, where both of serum total proteins, serum glucose, serum aspartate aminotransferase (S.AST), serum alanine aminotransferase (S.ALT), serum total bilirbin, serum blood urea and serum creatinine were estimated using commercial diagnostic test kits supplied by BioMerieux France. Serum protein electrophoresis was separated according to their respective electrical charges at pH 8.8 on a cellulose plate using both electrophoretic and electroendosmotic force present in the system according to Alper, (1974). The serum iron, copper, selenium, manganese and zinc were estimated by atomic absorption spectrophotometer (PYE, Unicum, SP 90, England) according to Khan et al., (1995). III- Treatment trails and control:

Butox® (intervet) was used for eradication of ticks vectors on animals and in the surrounding areas with concentration of 25 PPM with spray manner according to manufacture recommendation. Theileria infected cows were treated with Butalex® (schering plough animal health), a single injection of 2.5 mg/kg (1ml Butalex per 20 kg.b.w) was used via i/m route in the muscle of the neck. Supportive treatment was done (Terramycin L-A (phizer) as antibiotic for secondary infection at a dose of 200mg (1mg/10kg.b.w), repeated after 3 day, beside Antoplex® (Labortrios Tornel, Mexico) as haematinic was used at a dose of 10 ml twice a week, via i/m route. IV- Statistical analysis:

All data were subjected to statistical analysis according to Snedcor and Cochram, (1982).

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RESULTS

The present study was carried out to focus light on disease situation at Dakahlia governorate, its clinical signs, seasonal prevalence, age susceptibility, changes of some blood biochemical components in addition to treatment trials. I- Clinical findings:

Close observation concerning the clinical survey conducted on 150 cows revealed presence of lacrimation, nasal discharge, enlargement of superficial lymph nodes, inappetance, tachcardia, weakness and corneal opacity on 44 animals. Gradual improvement in the general health condition of the diseased cows was noticed during treatment course, complete recovery was obtained after relapse of 15 day manifested by disappearance of clinical signs of the disease.

II- Laboratory findings:

Parasitological examination of blood smears revealed that there is no presence of Theileria infection after treatment. The results of the study dealing with parasitological and blood biochemical parameters before and after treatment were illustrated in Tables (1-6) and Figs. (land 2). Table 1: The prevalence of T. annulata infection among cattles in Dakahlia

Governorate: Examined animals

No.

No. of infected Infection % Appearently healthy

104

0

0% Suspected diseased

46

44

95.65 Total

150

29.33

44

Table 2: Seasonal prevalence of Theileria annulata infection among

cattle in Dakahlia Governorate:

No. of examined animals

No. of infected

Season Summer Autumn Winter Spring

Total

% of infection

39.47 47.61 5.71

20 29.33

150

Table 3: The prevalence of Theileria annulata infection among cattle of

different age: Age group I No. of examined No. of infected Infection % 6M-1 year

40

37.50 13 years

32.50 3-5 years

27.77 Over 5 years

5.88

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Table 4: Haemo and leucogram of cattle infected with Theileria

annulate before and 15 day after treatment.

Item Control healthy

Diseased group (92) Parameters

group (gi) Before treatment After treatment RBCs (x10°/ul)

8.71+0.33

6.91+0.38

8.47+0.31NS Hb (g/dl)

11.37+0.44

8.57+0.49** 11.13-0.37N.S PCV %)

34.63+1.30 28.40–1.46**

33.87+1.44NS Total WBCs (x103/ul)

9.66+1.73 17.89+1.41** 10.98+0.97N.S Neutrophils (%)

31.45+1.85 44.24+2.95*

31.50+2.47N.S Eosinophils (%)

5.55+0.37

8.47+0.45**

5.81+0.36N.S Basophils (%)

0.75+0.10

1.34+0.17*

0.77–0.12N.S Lymphocytes (%)

56.75+2.09 41.75+2.98**

57.43+1.33N.S Monocytes (%)

5.50+0.29

4.20+0.37*

4.49+0.38N.S * Significant at (P<0.05)**: Highly significant at (P<0.01) N.S: Non significant.

Table 5: Biochemical changes in serum of cattle infected with

Theilerice annulalta before and 15 day after treatment.

Item Control healthy Parameters

group (g) Total proteins (g/dl) 7.66Ł0.16 Albumin (g/dl)

4.13+0.19 Total globulin (g/dl) 3.35+0.28 C-globulin (g/dl)

1.123+0.064 B-globulin (g/dl)

0.567+0.071 d-globulin (g/dl)

1.840=0.102 Glucose (mg/dl)

55.16+5.31 AST (IU/L)

56.43+2.05 ALT (IU/L)

17.51+1.41 Total bilirubin (mg/dl) 0.945–0.061 Blood urea (mg/dl) 27.43+1.41 Greatinine (mg/dl)

1.60+0.12 *. Significant at (P<0.05) *** Very highly significant at (P<0.001)

Diseased group (92) Before treatment | After treatment

8.17+0.25N.S

7.89+0.22N.S 2.87+0.29**

4.07+0.22N.S 5.30+0.36*

3.82+0.30N.S 1.501+0.068**

1.280+0.061 N.S 0.9790.077**

0.578-0.067N.S 2.820+0.136***

1.962£0.103N.S 31.745.43**

58.91+4.78N.S 68.86+2.16** 56.98+1.69N.S 25.89–1.57**

18.23+1.32N.S 1.190=0.055* 0.961+0.052N.S 33.71+1.94 28.61+1.73N.S 2.11+0.17*

1.66+0.14N.S **. Highly significant at (P<0.01) N.S: Non significant.

Table 6: Mean values of serum microelements in cattle infected with

Theileria annulalta before and 15 day after treatment.

Item Parameters Iron (ug/dl) Copper (ug/dl) Selenium (ug/dl) Manganese (ug/dl)

Zinc (ug/dl) * Significant at (P<0.05)

Control healthy

Diseased group (92) group (g) Before treatment | After treatment 227.81+3.50 246.37+3.45* 229.11+2.72.5 113.26+2.69 129.78=2.96** 114.51=1.91N.S 3.295+0.274 4.395=0.235 3.191+0.243.N.S 51.18+2.59 59.43+2.20*

52.76+2.27N.S 121.52-4.15 136.55=4.49*

123.87+3.78.N.S **: Highiy significant at (P < 0.01) N.S: Non significant.

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Fig. 1: Blood film showing theileria trophozoites (X

10 x 100)

Fig. 2: Koch's blue bodies stained by Gimsa stain (X10 x 100)

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DISCUSSION

Mediterranean cost fever or tropical theileriosis is a tick borne disease of cattle caused by Theileria annulata infection, extending from Morocco to China and consider one of the major health problems of livestock (Devendra, 1995).

Examination of blood films and lymph node smears revealed that 44 (29.33%) out of 150 animals were infected with theileriosis (Table 1). This result was similar to that of Loria et al., (1999) in Italy, Abd El Raof et al., (2000) in Egypt and Inoue et al., (2001) in Vietnam, they recorded that the incidence of theileriosis among cattle were (29.4%, 33.66% and 27.5%) respectively.

However, higher prevalence rates were reported by El-Metenawy, (2000), Inoue et al., (2001) in Cambodia and Song Sang (2003), they detected that theileriosis among cattle were (76.5, 50.4 and 67.8%) respectively. This differences may be attributed to the difference, in localities and species of examined animals.

Also blood films and lymph node smears revealed the presence of different forms of intra-erythrocytic trophozoites. Schizonts (Koch's blue bodies) were observed in the lymph nodes and the circulating lymphocytes. This finding completely typical to those described previously by Radostits et al., (2000) and Saleh & Mahram, (2003) (Figs. 1,2).

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

Clinical examination of animals infected with Theileria revealed gradual rise in body temperature, enlargement of superficial lymph nodes, loss of weight, anorexia, nasal discharge, bilateral lacrimation which may reach to corneal opacity. This findings as recorded by Abd El-Raof et al., (2000), Saleh & Mahran, (2003) and Omer et al., (2003).

Concerning seasonal prevalence (Table 2), the peak of infection was in Autumn (47.61%) followed by Summer and Spring (39.47% & 20%) and the lowest rate was in winter (5.71%) which may be due to the absence of the tick vector during the winter season. In this respect similar finding were recorded by Abd El-Raof et al., (2000) and

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El-Metenawy, (2000) with some variation in the ratio which may be attributed to the number of animals examined in each season.

The present data in Table (3) revealed that calves with age of 6 months to 1 year were highly susceptible to theileriosis (37.50%) which is nearly equal to that from 1-3 years (32.50%), they decreased at the age from 3-5 years (27.77%) to reach its lowest infection rate at animals over 5 years (5.88%) which may be attributed to age resistance or to previous infection and presence of a sort of immunity. Around this point Abd El Raof et al., (2000) reported that buffaloes with age of 6 months to 5 years were highly susceptible to Theileriosis (54.91%) followed by those over 5 years old (15.79%), while Song & Sang, (2003) found that T. sergent, infection among the over 3 years old group of cattle was (75%) had a higher prevalence than that among the less than 3 years old group (61.8%). This variation may be attributed to the species of the examined animals.

Concerning the changes in haemogram parameters in Theileria annulata infected cows, (Table 4) showed that there was a noticeable mirocytic hypchromic anaemia as a result of high significant decrease (P <0.01) in the levels of total red blood cells count (RBCs), haemoglobin content (Hb) and packed cell volume (PCV%) when compared with control group.

This result come in accordance with those of Omer et al., (2002) and Emam and Al-Kabbany, (2004). Such changes could be due to the destructive effect of the parasites on erthrocytes and failure of bone marrow to produce enough erthrocytes response to the toxic material to compensate such losses (Radostits et al., 2002).

Dealing with the changes in leucogram parameters in the present study (Table 4) revealed that there was a high significant leucocytosis (P <0.01) as a result of high significant increase (P <0.01) of neutrophils, eosisophils and basophils, also Table (4) clarified a high significant and significant decrease (P <0.01 & P <0.05) in lymphocytes and monocytes in comparison with control group respectively.

Guglielmone et al., (1996), Omran and El-Kholany, (2003) explained such alterations by the fact that the stress exerted on the animals lead to release of corticoids into circulations so both of lymphocyte and monocyte number became reduced, at the same time neutrophils were attracted to injury site to participate in the developing inflammation.

Table (5) indicated a high significant decrease (P < 0.01) in albumin level, on contrary globulin level showed high significant

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