EFFECT OF SOME DAILY MANAGEMENTAL FACTORS ON APPEARANCE OF ABNORMAL BEHAVIOR AND HEALTH STATUS OF DONKEYS

Assiut Vet. Med. J. Vol 51 No. 107 October 2005

Department of Animal Hygiene Faculty of Veterinary Medicine, Assiut University

EFFECT OF SOME DAILY MANAGEMENTAL FACTORS ON APPEARANCE OF ABNORMAL BEHAVIOR AND HEALTH STATUS OF DONKEYS

(With 4 Tables and 2 Figures)

By MADEHA H.A. DARWISH and M.A.ABDEL-RAHMAN

(Received at 20/9/2005)

تأثير بعض

المعاملات اليومية على ظهور السلوک الغير طبيعي

وعلى الحالة الصحية للحمير

مديحه حسنى أحمد درويش،معتز أحمد محمد عبد الرحمن

تم إجراء هذه التجربة على عدد 5 من إناث الحمير والتي کان متوسط أعمارها 5 سنوات وأوزانها حوالى ۲۰۰ کيلوجرام. سکنت هذه الحيوانات بالمزرعة التجريبية الخاصة بکلية الطب البيطري بجامعة أسيوط داخل حوش مفتوح ذو أرضية خرسانية دون وجود فراش وتحت الظروف البيئية السائدة. تم تغذية هذه الحيوانات بحرية على عليقة حافظة مکونة من مخلوط مرکزات الأعلاف والتبن مع تقديم الماء لها بصورة مستمرة لمدة أسبوعين وذلک کفترة ض ابطة. تلت هذه الفترة الفترات التجريبية والتي کانت مدة کل منها أسبوعين کالآتي:- 1- وضع فرشة من قش الأرز تحت الحيوانات. ۲- زيادة نسبة الطاقة والبروتين بتقديم البرسيم للحيوانات مع العليقة الحافظة. 3- تقديم ماء الشرب للحيوانات کل يومين بدلا من السقي اليوم. 4- تسکين الحيوانات داخل غرفة مغلقة بدلا من الحوش المفتوح. تم تسجيل سلوکيات هذه الحيوانات وکذلک تم فحصها وذلک لتحديد حالتها الصحية مع أخذ عينات من دم هذه الحيوانات التقدير نسب بعض مکونات الدم ومستوى هرمون الکورتيزول بها. أثبتت النتائج التي تم الحصول عليها أن المعاملات اليومية التي ترتبط ببقاء الحيوانات داخل المساکن کان لها التأثير الأقوى في ظهور السلوک الغير طبيعي بين الحمير وأن بعض من هذه العوامل کانت مسببة للإجهاد وإنعکست بصورة واضحة على الحالة الصحية وبعض مکونات الدم لهذه الحيوانات. وقد أوصت النتائج بملاحظة عدم حدوث أي تغييرات على برنامج الرعاية الخاص بالفصيلة الخيلية المرباه داخل المزارع وذلک من أجل الحصول على الحد الأقصى من الإراحة وبالتالي الإنتاج.

SUMMARY

Five non pregnant, non lactating she-donkeys of about 5 years in age and 250 kg in weight were used in this investigation. Animals were tied under shed in an open yard with a concrete floor belonging to the

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experimental farm of faculty of veterinary medicine, Assiut University. They were ad libitum fed a maintenance ration. Drinking water was freely available allover the experiment. Donkeys were subjected to four experimental trials, two weeks each (bedding the floor, offering Barseem hay with the ration, watering each two days and housing inside closed pens). Behavior of the experimented animals was recorded. Moreover, their health status and some of blood parameters were estimated. The obtained results indicated that, managemental factors related to the time spent in the stable showed the strongest associations with stereotypic and abnormal behavior. Some of these conditions were considered as a stressful factor and reflected prominently on the animal's health status and blood parameters. Therefore, changes in the management program within the equine farms should be avoided to provide the animals with a comfortable situation.

Key words: Management, behavior, health, donkeys

INTRODUCTION

I

A great knowledge on the effect of management factors is required to investigate the ontogney of abnormal behavior in the stabled equines. Stabled horses often exhibit behaviors which owners regard as unwelcomed. These include stereotypies, defined as repetitive, relatively invariant and apparently functionless activities (Mason, 1991) and redirected behaviors, which are directed towards an inappropriate target (Fraser and Broom, 1990). Cage design, isolation rearing and food deprivation have been implicated as proximate causes of stereotypic behaviors (Morgan, 1973; Odberg, 1986 and Appleby & Lawrence, 1987). Arousal generated by frustrated motivation is a possible shared cause (Duncan et al., 1993). Heterogeneity also emphasized as a cause of different stereotypies (Mason, 1991).

Despite much work on farm and laboratory species, the cause of stereotypic and redirected behavior in equines remain unclear Heritability plays some role but little is known about the relative importance of management factors that might frustrate motivation in equines. Feeding practice have a greater effect than housing practices on the incidence of abnormal behaviors (Marsden, 1993). Other possible causes of abnormal behaviors in equines include factors associated with weaning, social contact, crowding and training practices (Kiley Worthington, 1983 & 1987). Exposure to stereotypic neighbor may also increase the likelihood of stereotypy development or performance.

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The aim of the present study was to establish the relative influence of some management factors on the development and performance of abnormal behaviors in donkeys.

MATERIALS and METHODS

I- Animals, feeding and management: -

Five non pregnant, non lactating she-donkeys of about 5 years in age and 250 kg in weight were used in this investigation. Animals appeared to be clinically healthy and parasitological examination revealed no gastrointestinal affections. They were housed under the prevalent environmental conditions as they tied under shed in an open yard with a concrete floor belonging to the experimental farm of faculty of veterinary medicine, Assiut University. Animals were ad libitum fed a maintenance ration consists of commercial concentrate mixture and wheat straw. Drinking water was freely available allover the experiment. II- Experimental trials: -

Animals were housed under the previously mentioned conditions for two weeks as a control trial. After that, they were subjected to four experimental trials as follows: -

- Trial one: -

Where the animals housed under the previously mentioned control conditions except that the floor was bedded with rice straw. - Trial two: -

Where the animals housed under the previously mentioned control conditions except that the energy and protein contents of the diet increased by adding Barseem hay to the previously mentioned maintenance ration with a rate of 2 kg/head. - Trial three: -

Where the animals housed under the previously mentioned control conditions except that they were watered each two days instead of daily watering. - Trial four: -

Where the animals housed under the previously mentioned control conditions except that they were housed and tied inside closed pen rather than open yard.

Each experimental trial was consists of two weeks followed by one week as a preliminary period during which, animals were reposed the control conditions (McGreevy et al., 1995).

JU,

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- Behavioral observation: -

Behavior of the experimented animals was recorded following the method of Martin & Bateson (1988) using the scan sampling method where the observer can observe all the animals without being seen by them. Behavior was observed and analyzed according to McGreevy et al. (1995). During the control and experimental periods, animals were observed during the second week for 8hours / day (four hours in the morning and four hours in the afternoon). The behavioral observations were carried out in the morning between 9:00 and 13:00 and in the afternoon between 13:00 and 17:00.

Animals were observed for incidence of any of the following abnormal behavioral patterns: -

1. Kicking 2. Eating dung 3. Eating bedding 4. Throwing food out of manger 5. Licking the walls 6. Weaving

7. Crib-biting and wind-sucking - Health status measurements: -

On the 1st, 7th and last day of all trials, experimented donkeys were examined clinically according to Blood and Henderson (1974) and Blood & Radostits (1990) to determine their average pulse rate, respiratory rate and their body temperature as well as the condition of their mucous membranes, fecal matters and coats. - Blood parameters: -

During the last two days of all trials, three blood samples, 5 ml each were drawn from the jugular vein of each animal. The first one was drawn into glass test tubes contain EDTA for leucocytic count according to Franke and Reitman (1963). The second blood sample was drawn into centrifuge tubes and centrifuged for 30 minutes at 3000 r.p.m and the obtained sera were assayed within three hours for their glucose concentration according to Tinder (1969). The third blood sample was drawn into centrifuge tubes and centrifuged for 30 minutes at 3000 r.p.m and the obtained sera were freezed at -80 °C and kept for further analysis to determine their cortisol level using TDX FLx system according to Dandliker and Sassure (1973). III- Statistical analysis: -

Statistical analyses of the collected data were carried out according to procedures of completely random design (SAS, 1995).

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Assjut Vet. Med. J. Vol 51 No. 107 October 2005

RESULTS

The results of this study were illustrated in tables 1,2,3,4 as well as figures 1 and 2.

DISCUSSION

I- Behavioral observations of experimented animals: -

The data represented in table (1) and assimilated on figure (1) showed the effect of the studied management conditions on the incidence of abnormal behavior among donkeys. These data revealed that, incidence and performance of abnormal behaviors by donkeys was significantly affected with changing the daily management conditions than the control one (P<0.01).

Bedding the floor of the yard with rice straw was reflected on the experimented donkeys with incidence of eating bedding with a rate of 60% as shown in trial one. However, increasing the energy and protein content of the diet during trial two by adding Barseem hay to their maintenance ration was associated with the incidence of kicking, throwing food out of manger, weaving and crib-biting & wind-sucking with rates of 80, 40, 40 and 40%, respectively. Kownacki et al. (1978); Duncan et al. (1993) and McGreevy et al. (1995) indicated that, both oral-based (crib-biting and wind-sucking) and locomotor (kicking and weaning) stereotypies are more prevalent among stabled equines that daily fed forage. This finding may be related to the enforced free time of the stabled animals which accompanied by high protein content of the offered food. At the same time, increased incidence of throwing food out of manger may be related to searching for their favorable food, Barseem

hay.

Moreover, watering the animals every two days instead of every day during trial three was associated with the incidence of kicking, eating dung and licking the walls with rates of 40, 60 and 80%, respectively. This finding may be related to the stressful situation water restriction which kept the animal nervous and searching for water even by eating their dung or licking the walls (Waring, 1983 and Kiley Worthington, 1987). At the same time, housing and tying the experimented donkeys inside closed pens rather than open yards resulted in incidence of kicking and weaving with rates of 60 and 40%, respectively. This may be related to the fact that locomotor (kicking and weaning) stereotypies are more prevalent among stabled animals.

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Moreover, weaving is the most likely behavior to be learnt among equines by observation. Stabled horses and donkeys may be more likely to encounter weavers however, large yards was generally associated with a reduced risk of these abnormal behaviors (Houpt, 1986 and McGreevy et al., 1995). II- Health status of the experimented animals: -

The data represented in table (2) showed the effect of the studied management conditions on pulse rate, respiratory rate (No. / min.) and body temperature (°C) as well as the conditions of mucous membrane, faecal matter and coat of the experimented donkeys. The average data were 38, 10, 37.4, normal, normal, normal following the control conditions; 38, 10, 37.3, normal, normal, normal after bedding the floor of the yard with rice straw; 40, 12, 37.4, normal, normal, normal after adding Barseem hay to the maintenance ration; 48, 18, 37.4, normal, firm & dark, normal following watering each two days and 38, 10, 37.4, normal, normal, normal after housing the animals inside closed pens, respectively. These results revealed that, among all experimented management conditions, watering the animals every two days was the only factor that had a significant effect (p<0.01) on health status of donkeys. Watering the animals every two days was associated with a significant increase in their pulse and respiratory rates with changing in the constancy of their faecal matter. This finding may be related to the physiological and biological adjustments and changes in the animal body to meet this new stressful situation (Hafez, 1975; Banerjee, 1982 and Radostits et al., 1994). Moreover, it could be related to the fact that insufficient water intake affects significantly the blood water content and increases the blood viscosity which reflects on the animal with an obvious effect on their pulse rate (Blood and Radostitis, 1990). III- Blood parameters of the experimented animals: -

The data represented in table (3) showed the effect of the studied management conditions on the differential leucocytic count of donkeys. These data showed that, the counts (10 /ul) of total WBCs, Neutrophils, Lymphocytes, Monocytes, Eosinophils and Basophils during control conditions were 11.14, 6.36, 3.12, 1.12, 0.42, 0.120, respectively. However, it was 11.19, 6.19, 3.32, 1.10, 0.45, 0.130 after bedding the floor of the yard with rice straw; 10.94, 6.31, 2.97, 1.14, 0.40, 0.120 after increasing the energy and protein contents of the diet; 11.11, 6.22, 3.22, 1.13, 0.42, 0.120 following watering the animals each two days and 10.97, 6.16, 3.12, 1.16, 0.40, 0.130 after housing the animals inside closed pens, respectively. This result indicated that,

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leucocytic series of the experimented donkeys was not significantly affected by any of the studied management conditions.

At the same time, The data illustrated in table (4) and assimilated on figure (2) showed the effect of these studied management conditions on the blood levels of cortisol and glucose of donkeys. Serum cortisol level was 0.97, 0.97, 0.88, 1.91 and 1.43 ug / L while, serum glucose level was 5.87, 5.87, 5.34, 7.52 and 7.48 Mmol / L following control, bedding the floor, offering Barseem hay with the maintenance ration, watering each two days and housing inside closed pens, respectively. These findings indicated that, both watering the animals each two days and housing inside closed pens were significantly affected blood cortisol and glucose levels (P<0.01) however, other factors did not do so. The significant increase in the blood cortisol level indicated an occurrence of stress due to these conditions where acute stress causes an outpouring of ACTH which intern causes the adrenal cortex to increase its secretion of glucocorticoids including cortisol (McDonald, 1969; Burchfield et al., 1980 and Stephens, 1981). However, the increase in blood glucose level may related to the fact that glucocorticoids, including cortisol, act mainly on the hepatocytes which induced to produce gluconegenic enzymes which in turn increase the rate of gluconeogenesis and enhance the conversion of protein to glucose. Moreover, cortisol causes a moderate reduction in the rate of glucose utilization by the body cells, which leads to a rise in blood glucose level (Guyton and Hall, 1996).

CONCLUSION

In conclusion, management factors related to the time spent in the stable showed the strongest associations with stereotypic and abnormal behavior. Some of these conditions are considered as a stressful factor and reflected prominently on the animal health status and serum level of both cortisol and glucose which are likely to upset its body homeostasis and so, its behavior. Therefore, changes in the management program within the equine farms should be avoided to provide the animals with a comfortable situation.

REFERENCES

Appleby, M.C. and Lawrence, A.B. (1987): Food restriction as a cause of

stereotypic behavior in gilts. J. Anim. Prod., 45: 103-110. Banerjee (1982): A textbook of animal husbandry. 5th Ed., Oxford and

publishing company.

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Assiut Vet. Med. J. Vol 51 No. 107 October 2005

Blood, D.C. and Henderson, J.A. (1974): Veterinary medicine. 4th Ed.,

Bailliere-Tindall-London. Blood, D.C. and Radostits, O.M. (1990): Veterinary medicine. 7th Ed.,

Great Britian. Burchfield, S.R.; Wood, S.C. and Elich, M.S. (1980): Pituitary

adrenocortical response to chronic intermittent stress. Physiol.

And Behav., 24; 297-302. Dandliker, W.B. and Sassure, D.V. (1973): Review article: fluorescent

polarization immunoassay. Theory and experimental method.

Immunochemistry, 10: 219-227. Duncan, I.J.H.; Rushen, I. and Lawrence, A.B. (1993): Conclusions and

implications for animal welfare. In: Stereotypic animal

behavior. UK, pp: 193-206. Franke, S. and Reitman, S. (1963): Clinical laboratory methods and

diagnosis. 6" Ed., C.V. Mosby Company, USA. Fraser, A.F. and Broom, D.M. (1990): Farm animal behavior and

welfare. Balliere Tindall, London, p. 390. Guyton, A. and Hall, J.E. (1996): Textbook of medical physiology. 9th

Ed., W.B. Saunders, Philadelphia, USA. Hafez, E.S. (1975): The behavior of domestic animals. 3rd Ed., Bailliere

Tindall-London. Houpt, K.A. (1986): Stable vices and trailer problems. Equine Pract., 2:

623-633. Kiley-Worthington, M. (1983): Stereotypies in horses. Equine Pract, 5:

34-40. Kiley-Worthington, M. (1987): In: The behavior of horses. London, p.

257. Kownacki, M.; Sasimewski, E., Budzynski, M.; Jezieski, T. and Kapron,

M. (1978): Observations of the twenty-four hours rhythm of natural behavior of Polish primitive horse bred for the conservation genetic resources in a forest reserve. Genitica

Polowaca, 19: 61-77. Marsden, M.D. (1993): Feeding practices have greater effects than

housing practices on the behavior and welfare of the horse. 4th international symposium of the Americium Society of Agricultural Engineers, University of Warwick, Coventry, pp.

314-318. Marten, P. and Bateson, P. (1988): In Measuring behavior. Cambridge

University Press, Cambridge, pp. 48-69.

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Mason, J.I. (1991): Stereotypies, a critical review. Anim. Behav., 41:

1015-1032. McDonalds, L.E. (1969): Veterinary endocrinology and reproduction. 1st

Ed., Lea and Febiger, Philadelphia, USA. McGreevy, P.J., French, N.P.; Green, L.E. and Christine, J.N. (1995):

Management factors associated with stereotypic and redirected behavior in the thoroughbred horse. Equine Vet. J., 27 (2): 86

91.

Morgan, M.J. (1973): Effects of post-weaning environment on learning

in the rat. Anim. Behav., 21: 429-442. Odberg, E.O.(1986): The jumping stereotypy in the bank vole. Biol.

Behav., 11: 130-143. Radostits, O.M.; Leslie, K.E. and Fetrow, J. (1994): Herd health. 2dn

Ed., UK SAS (1995): Statistical analysis system. User's Guide : Statistics. Version

6, 2nd Ed., SAS Inst. Inc., Cary, NC. Stephens, D.B. (1981): Stress and its measurments in domestic animals.

Adv. Vet. Comp. Msd., 24: 179-210. Tinder, P. (1969): Determination of glucose in blood using glucose

oxidase with an alternative oxygen acceptor. Annals. Clin.

Biochem., 6:24-27. Waring, J.H. (1983): In: Horse behavior. Park Ridge, New Jersey, USA,

p. 293.

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Assiut Vet. Med. J. Vol 51 No. 107 October 2005 Table 1: Incidence of abnormal behavioral patterns (%) of the

experimented animals

400

60b

oa 09

Trials Behavior

Control

Three Four Kicking

0a 800 Eating dung

6061 Eating bedding

oa Throwing food

09 Licking the walls Weaving

405 Crib-biting and wind-sucking 0

Trial one = Bedding the floor

Trial two = Offering Barseem hay plus maintenance ration Trial three = watering each two days Trial four = Housing inside closed pens Figures in the same raw with different superscripts differs significantly (p<0.01)

oa o a 400 oa

oa

O a

806 oa 09

406

40b

WINNI

%

Control

One

Two

Three

Four

Trials s Kicking B Eating bedding

Licking the walls Crib-biting and wind-sucking

Eating dung

Throwing food Weaving

Fig.

1 :- Incedince of abnormal behavioral

patterns of the experimented animals

Assiut Vet. Med. J. Vol 51 No. 107 October 2005

Table 2: Health status measurements of the experimented animals

Trials

Item

Control

One

Two

Three

Four

Pulse rate (No./min)

38+2

38+1°

40+2

48+2b

38+2*

Respiratory rate (No./min)

1041

10+1

12+12

18+1b

10+1

Body temperature (°C)

37.4+0.

1

37.30.1 | 37.4+0.1

37.4+0.

1

1

37.4+0.14

Mucous membrane

Normal

| Normal

Normal

Normal

Normal

Fecal matter

Normal

Normal

Normal

Firm & Dark

Normal

Condition of the coat

Normal

Normal

Normal

Normal

Normal

Trial one = Bedding the floor

Trial two = Offering Barseem hay plus maintenance ration Trial three = watering each two days Trial four = Housing inside closed pens Figures in the same raw with different superscripts differs significantly (p<0.01)

Table 3:- Differential leucocytic count (10° / ull of the experimented

animals

Trials

Item

Control

One

Two

Three

Four

Total WBCs

11.14+0.12

11.19+0.10

10.94+0.16

| 11.11+0.12

10.97+0.10

Neutrophils

6.36+0.10

6.19+0.10

6.31+0.13

6.22:0.10

6.16+0.18

Lymphocytes

3.12+0.10

3.32+0.10

2.97+0.12

3.22:0.10

3.12£0.10

Monocytes

1.12+0.04

1.10+0.02

1.14+0.06

1.13+0.02

1.16+0.04

Eosinophils

0.42+0.03

0.45+0.03

0.40+0.01

0.42+0.02

0.40+0.03

Basophils

0.120+0.01

0.130+0.02

0.120+0.01

0.120+0.01

0.130£0.01

Trial one = Bedding the floor

Trial two = Offering Barseem hay plus maintenance ration Trial three = watering each two days Trial four = Housing inside closed pens

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Table 4: Serum cortisol (ug / L) and glucose (Mmol/L) concentrations

of the experimented animals

Trials

Item

Control

One

Two

Three

Four

Cortisol

0.97+0.02

0.97+0.02

| 0.88+0.01

0.88+0,012

1.91+0.015

1.43+0.02

Glucose

5.87+0.20

5.87+0.10

5.34+0.20

7.520.20b

7.48+0.15

Trial one = Bedding the floor

Trial two = Offering Barseem hay plus maintenance ration Trial three = watering each two days Trial four = Housing inside closed pens Figures in the same raw with different superscripts differs significantly (p <0.01)

W

L

ARRA

ONOMNO

ARNA

w

Cortisol

Glucose

Control Trial One Trial Two Trial Three Trial Fou

Fig.2: - Serum cortisol (ug/L) and glucose (Mmol/L) concentrations of the experim

animals

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Dept. of Animal Medicine, Fac. of Vet. Med., Assiut University, Egypt

MYXOBOLUS INFESTATION IN OVARIES OF SHARPTOOTH CATFISH, CLARIAS

GARIEPINUS (With 2 Tables and 5 Figures)

By A.A. ELKAMEL and A. TANTAWY* *Department of Pathology, Faculty of Veterinary Medicine,

Moshtohor, Banha University, Egypt

(Received at 26/9/2005)

الإصابة بطفیل ميکزوبولاس في مبايض الأسماک القطية النيلية(القراميط)

أحمد عبد الهادي الکامل،أحمد عبد الحافظ طنطاوی الهدف من هذه الدراسة هو اجراء دراسة ميدانية وملاحظة الأعراض الإکلينيکية ونسب الإصابة وکذلک التغيرات المرضية نتيجة اصابة مبايض الأسماک القطية النيلية (القراميط) بطفيل الميکروبولس. تم فحص

عدد ۱۲۰ سمکة على مدار عام ۲۰۰4 بمعدل ۱۰ سمکات ش هريا ووجد أن عدد 15 سمکة کانت تحمل حويصلات طفيل الميکزوبولس في المبايض. وکانت معدلات الإصابة في آخر فصل الخريف قليلة ثم ازدادت تدريجيا في فصل الشتاء حتى وصلت إلى أعلى معدلاتها في بداية فصل الربيع، ولم تسجل أي حالة من حالات الإصابة في فصل الصيف. کما لوحظ أن الإصابة کانت في مبيض واحد فقط في 6 أسماک (

%40) من الأسماک المصابة في حين کانت الإصابة في المبيضين معا في و أسماک

(

%60) من الأسماک المصابة. اما حدة الإصابة فقد ازدادت ايضا خلال فصل الشتاء حتى وصلت أعلى مستوى لها في بداية فصل الربيع ثم تضاءلت سريعا مع بداية فصل الصيف. اظهر الفحص الميکروسکوبي لحويصلات طفيل الميکروبولس عدد کبير جدا من أبواغ (جراثيم) الطفيل عند درجات مختلفة من التطور والنمو. کما اظهر الفحص الميکروسکوبی المبيض الأسماک المصابة أن حويصلات طفيل الميکزوبولس قد قامت بالضغط على الويضات القريبة مما تسسب في ضمورها و موتها في بعض الأحيان، کما تسببت الحويصلات في حدوث اضطرابات دموية في الأنسجة المجاورة لها. ونظرا لأن هذا الطفيل وجد في مبايض القراميط فقد يکون نوعا جديدا من الطفيليات والذي يحتاج لمزيد من الدراسة مستقبلا.

SUMMARY

of this study was to investigate the clinical and The main aim

findings, seasonal prevalence, and histopathological postmortem

با 5

Assiut Vet. Med. J. Vol. 51 No. 107 October 2005

alterations that are caused by probably a new species of Myxobolus in ovaries of sharptooth catfish, Clarias gariepinus, in Assiut, Egypt. Out

120 fish examined over one year (2004), ovaries of only 15 (12.5 % fish were infested with macroscopic Myxobolus cysts (plasmodia and host cyst) that were embedded in the connective tissue among ova. Prevalence of infestation started low in late autumn and increased over winter and reached maximum in early spring. Infestation was not recorded in summer. Six (40%) out of the infested fish had Myxobolus cysts in only one ovary, meanwhile, the reminder (60%) of infested fish had both ovaries infested. Also, intensity of infestation gradually increased over winter and was maximal in early spring, but abruptly declined in summer. Microscopic examination of plasmodia showed numerous typical Myxobolus spores at various developmental stages. Mature spores are oval in shape with two anteriorly located polar capsules that have 4-5 coils of polar filaments. Microscopic examination of infested ovaries revealed that Myxobolus plasmodia were encapsulated within a thin connective tissue layer of host reaction. Myxobolus cysts compress neighboring tissues causing atrophy of ova and local circulatory disturbances. Based on the tissue location of plasmodia and morphological character of the mature spores, the parasite in the present study might be a new species.

Key words: Clarias gariepinus, myxobolus, ovaries

INTRODUCTION

Commercial farming of sharptooth catfish, Clarias gariepinus, is a rapidly growing aquaculture industry in Upper Egypt. C. gariepinus, has recently gained a consolidate position in the food fish market as it is widely accepted by consumers in Upper Egypt.

Myxosporea are economically important fish parasites which form an abundant and diverse group. They cause heavy infections, extensive lesions, and mortalities in cultured fish (Lom and Dyková, 1995). In Africa, about 100 species are currently known from the continent (Fomena and Bouix, 1997). In Egypt, myxosporean parasites were examined in River Nile fish by Aziza (1980), Imam et al. (1987), Abdel Ghaffar et al. (1998), and Ali (1999, 2000). Currently, study of myxosporean infections focuses on pathogenicity and significance of the parasite in both aquaculture and captured fish (Lom and Dykova, 1995).

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Clarias gariepinus, a carnivorous bottom feeder, acts as a host for plenty of parasites that have tremendous effects on fish health and population throughout the River Nile. In Egypt, C. gariepinus had been found to be infested with Myxobolus lazeri (Aziza, 1980), and Myxobolus clarii (Mandour et al., 1993). Histozoic Myxobolidae cause great destruction of the host tissues and are of serious concern to fish culture (Kabata, 1985).

There are scanty data on Myxobolus infestations and the pathology they cause in fish ovaries (Lom and Dykova, 1995; Gbankoto et al., 1998; Reed et al., 2003). In the present study, prevalence and intensity of infestation of C. gariepinus ovaries with probably a new species of Myxobolus have been investigated over one year. In addition, clinical and postmortem findings and histopathological alterations of C. gariepinus infested ovaries were studied.

MATERIALS and METHODS

Fish:

A total of 120 live, apparently healthy, female specimens of sharptooth catfish, Clarias gariepinus, of 300-800 g were collected from January 2004 to December 2004 (10 fish /month) from El-Ibrahemia canal and its tributaries, Assiut city. Parasitological examination of samples A-Clinical examination:

Fish were externally examined after capture for any apparent clinical signs or lesions. Fish were incised according to (Stoskopf, 1993) to examine ovaries for macroscopic Myxobolus cysts to determine prevalence of infestation (number of infested fish divided by the number of examined fish per month). Longitudinal incision was made in both ovaries to determine total numbers of Myxobolus cysts to determine intensity (number of cysts per infested fish) of infestation. Cysts were examined for size, consistency, and contents. B-Microscopic examination:

Impression smears were made from cysts, air dried, and then fixed in 40% ethanol for 10 min. Fixed smears were stained with Methylene blue or Lugol's iodine solution. Histopathological examination

Infested ovaries were excised from infested fish, fixed in 10% neutral buffered formalin for 48 hours, and then processed for microscopic examination. Thin paraffin sections were stained with

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Haematoxylin and Eosin (H&E), Toluidine blue, and Periodic Acid Schiff's (PAS) stains.

RESULTS

Parasitological examination of samples

Longitudinal incision of infested ovaries showed whitish round to oval cysts (Myxobolus plasmodia and host cyst) that were randomly scattered and embedded in connective tissue among ova (Fig. 1). Cysts were seen by naked eyes and were 1.2-1.5 mm in diameter. Cysts were located in ovaries of immature females and mature females at off spawning seasons, while ovaries of mature females at spawning season needed more careful examination because cysts are of average size of mature ova but of different color. Interestingly, wall of cysts collected in spring, the primary spawning season, were fragile and readily ruptured releasing mature spores; in contrast, wall of cysts collected during late autumn and winter were relatively firm and resistant to rupture if compared to those collected in spring. Seasonal prevalence and intensity of infestation

Out of the 120 fish examined, ovaries of only 15 (12.5%) fish were found to be infested with Myxobolus cysts. Infestations were not seen during summer, but were recorded at a relatively low rate when temperature started to drop in late autumn (Table 1). Prevalence gradually increased over winter and reached maximum when temperature started to rise in early spring, and then declined again in late spring (Fig. 2).

Intensity of infestation was determined according to number of cysts per ovary and whether one or both ovaries were infested (Table 2). Six (40%) out of the infested fish had Myxobolus cysts in only one ovary, meanwhile, the reminder (60%) of infested fish had both ovaries infested. Infestation was considered severe when 10 or more cysts were seen in one or both ovaries, while was considered moderate when 6-9 cysts were seen in one or both ovaries. Females were considered lightly infested when had 1-5 cysts in one or both ovaries.

Generally, during winter, when temperature is lowest in season, most cases of infestation were light. When temperature starts to rise, intensity of infestation gradually increases where moderate cases were recorded. Furthermore, Intensity of infestation continues to increase in spring when severe cases of infestation were seen and then rapidly declined and even disappeared in summer. In addition, during late

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autumn, when temperature starts to decline, infestations re-emerge when light cases of infestation were seen again. Microscopic examination

Microscopically, plasmodia were encapsulated within a thin fibrous connective tissue capsule of host reaction and infiltrated with few lymphocytes with dilated blood capillaries (Fig.3). Plasmodia were filled with numerous typical Myxobolus spores where mature spores located centrally, while the developing ones were peripherally arranged. Furthermore, plasmodia collected in early spring had mainly mature spores, while plasmodia collected during autumn and winter had mainly developing spores.

Mature spores are oval in shape with slightly pointed anterior end and more rounded posterior end, and measuring 10.6 X 9.4 u (Fig. 4). Also, mature spores have at the anterior end two oval polar capsules with pointed anterior end and rounded posterior one. Polar capsules are of equal size, and measuring 4.8 X 3.5 u. Each polar capsule has 4-5 coils of polar filament. Sporoplasm contains an iodinophilus vacuole that stain positively with lugol's iodine solution. Furthermore, thin sections of infested ovaries stained with PAS showed positively stained dark red spores. Histopathological examination

Toluidine blue and H&E stained sections showed that Myxobolus cysts exert pressure atrophy over the adjacent ovarian tissues and cause disturbances in local circulation. Adjacent ova show degenerative changes in nuclei and cytoplasm and separation of the squamous cell layer that covers ova (Fig.5).

DISCUSSION

Present study revealed that Myxobolus infestation of ovaries of sharptooth catfish, C. gariepinus, is a mildly spread among wild population. Prevalence of infestation was 12.5% of all fish examined over one year. Water temperature has a great influence over seasonal prevalence of myxosporean infestations (Negm-Eldin et al., 1999). The prevalence of ovarian infestations with Myxobolus cysts increased in winter and early spring, while decreased in autumn. Interestingly, during summer, there was no record of Myxobolus cysts in the ovaries of fish examined. Similar annual cycles were reported with other myxosporean infestations (Negm-Eldin et al., 1999). In accordance with Clifton Hadley et al., (1986) who concluded that water temperature influences

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maturation of spores and development of myxosporean infestations in fish, in the present study, mature spores were seen in plasmodia collected in early spring, while developing spores were seen in plasmodia collected during autumn and winter.

Intensity of infestation has a cycle similar to that of prevalence. Intensity of infestation has gradually increased over winter and spring and then abruptly declined in late spring and summer. Sudden decline in prevalence and intensity of infestations during summer may be due to dispersing of intact cysts with eggs laid by infested mature females during spawning or, alternatively, rupture of cysts releasing mature spores in ovarian tissues. Ovarian contractions during egg lying might promote rupture of the cysts. This is supported by the fact that the cysts' walls are fragile and easily to be ruptured during egg laying season, but relatively harder during off-spawning seasons. Furthermore, it is supported by the fact that sporogenesis is completed during winter, and by spring plasmodia contain fully developed mature spores.

Dispersing of Myxobolus cysts or spores with laid eggs might be the primary route of spreading of infection and completing of the parasite's life cycle. It is not clear how this parasite reaches fish ovaries, its target organs. The exact mechanism of host invasion is unknown, but many freshwater myxosporeans have an alternate stage of development in oligochaetes (Oumouna et al., 2002) or ploychaetes (Bartholomew et al., 1997) which produces actinosporean spores that invade host. Oral route of transmission is also common route for myxosporean infestations (Lom and Dykova, 1995). In either case, the sporoplasms cross the epithelial barrier and are carried by the blood stream or lymphatic system to the target organ (Kabata, 1985).

Encapsulation of Myxobolus cysts within a thin connective tissue capsule of host reaction indicates that plasmodia severely irritate ovarian tissues stimulating a proliferative inflammatory response. This capsule is driven from the surrounding population of connective tissue cells and from compressed cells of the neighboring tissues (Lom and Dykova, 1995).

The extent of damage to tissues infested with Myxosporea depends on species of parasite and its life cycle stage, intensity of infestation and the host reaction (Lom and Dyková, 1995). Microscop examination of infested ovaries revealed that the myxobolus cysts replaced original ovarian tissues, compressed neighboring ova, and caused disturbances in circulation in neighboring tissues. Henneguya oviperda causes similar lesions in ovaries of pike, Esox lucius, in

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Europe, where atrophy of large number of ova was observed with local circulartory disorders (Lom and Dykova, 1995). Myxobolus dahomeyensis has been reported to hinder the successful breeding of several species of tilapia and their hybrids in Benin. M. dahomeyensis is found in ovaries of brooder tilapia where it penetrates inside the mature ova and liquefies the content causing total destruction of mature ova. In severe cases of infestation, ovaries become like sacs full of whitish fluid with spores and damaged ova (Gbankoto et al., 1998).

Myxosporea are host, organ and tissue specific (Molnar, 1994). Myxosporean infestations had been reported in C. gariepinus in Egypt. Aziza (1980) described Myxobolus lazeri from kidneys, while Mandour et al. (1993) reported Myxobolus clarii from testis of C. gariepinus. Mature spores morphology is the key feature in identification of Myxobolus (Kabata, 1985). Mature spores of M. lazeri (9.8 X 6.1 u) are smaller than those of the parasite of the present study (10.6 X 9.4 u). In addition, polar capsule of M. lazeri spores are smaller (5.1 X 2.3 u) than those of the parasite spores of the present study (4.8 X 3.5 u). Morphological characters of the parasite's spores in the present study are close, but not similar, to those of M. clarii that is found in testis of C. gariepinus (Mandour et al., 1993). Mature spores of the parasite in the present study are relatively larger but within the average size as mature spores of Myxobolus clarii. Size of the polar capsules of M. clarii spores (5.1 X 2.5 u), however, is smaller than those of spores of the present study.

Plasmodia of Myxobolus gariepinus reported by Reed et al. (2003) in ovaries of C. gariepinus in Botswana were 2-3 mm in diameter, while fully mature plasmodia of the parasite in the present study was 1.2-1.5 mm. Mature spores of M. gariepinus (13.9 X 10.8 u) are larger than those of the parasite the present study. Furthermore, polar capsules of M. gariepinus spores are measured (6.2 X 3.5 u) and contain 5-6 coils of polar filaments, while polar capsules of the parasite of the present study were smaller and contain 4-5 coils of polar filament.

Based on its host species, tissue location, and mature spores morphology and dimensions, the parasite in the present study might be a new species. Classification of the parasite in the present study, however, needs further investigations including comparative ultra structure study and molecular identification.

Sharptooth catfish is widely accepted by consumers in Upper Egypt as a relatively cheaper choice of fish protein. Commercial farming of sharptooth catfish has significantly increased in Upper Egypt over the

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past few years. With no obvious method of treatment or control, ovarian infestation of sharptooth catfish with Myxobolus may affect fecundity (Lom and Dykova, 1995) and thus populations of wild and cultured fish.

ACKNOWLEDGMENT

We would like to thank Dr. Shaban M. Ahmed, professor of fish diseases and management, Faculty of Veterinary Medicine. Assiut University for all the help and guidance he provided through this study. We would like, also, to thank Dr. Gamal Abed, Professor of Zoology, Faculty of Science, Assiut University for his input and help in identifying of the parasite studied.

REFERENCES

Abdel Ghaffar, F.; Ibrahiem, E.A.; Bashtar, A. and Ali, M.A. (1998):

Myxosporidia infecting saline-and freshwater fishes of Qarun and Wadi El-Raiyan lakes, Egypt. J Egypt Ger Soc Zool., 26:

209-229. Ali, M.A. (1999): Henneguya ghaffari sp. n. (Myxozoa: Myxosporea),

infecting the Nile perch Lates niloticus (Teleostei:

Centropomidae). Dis Aquat Org., 38: 225-230 Ali, M.A. (2000): Ortholinea basma n. sp. (Myxozoa: Myxosporea) from

the agile klipfish Clinus agilis (Teleosti: Clinidae), light and

scanning electron microscopy. Eur J Protistol., 36:100-102 Aziza Marwan, (1980): Studies on The Blood and Kidney Parasites of

Some Nile Fishes in Assiut Governorate, A.R. Egypt. M.Sc.

thesis, Assiut University, Assiut, A.R. Egypt Bartholomew, J.L.; Whipple, M.J.; Stevens, D.G. and Fryer, J.L. (1997):

The life cycle of Ceratomyxa shasta, a myxosporean parasite of salmonids, requires a freshwater polychaete as an alternate

host. J. Parasitol., 83, 859-868 Clifton-Hadley, R.S.; Richards, R.H. and Pucke, D. (1986): Proliferative

Kidney Disease (PKD) in rainbow trout Salmo gairneri: Further observations on the effects of temperature.

Aquaculture, 55, 165-171 Fomena, A. and Bouix, G. (1997): Myxosporea (Protozoa: Myxozoa) of

freshwater fishes in Africa: key to genera and species. Syst Parasitol., 37:161-178

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Gbankoto, A.; Sakiti, N. and Marques, A. (1998): Groupment Des

Protistologues De Langue Franciase, 36th Annual Meeting,

May 1998. Imam, E.A.; Ramadan, E.I. and Derhalli, F.S. (1987): Studies on some

internal protozoa infecting some Nile fishes in Egypt. J Egypt

Vet Med Assoc., 74:55-61 Kabata, Z. (1985): Parasites and diseases of fish cultured in the tropics.

Taylor & Francis Ltd., London, UK. Lom, J. and Dyková, I. (1995): Myxosporea (Phylum Myxozoa) in Woo,

P.T.K. Editor. Fish Diseases and Disorders, Vol.1.: Protozoan and Metazoan infections, pp. 97-148. CAB International,

Wallingford, Oxon, UK. Mandour, A.M.; Galal, A.A. and Abed, G.H. (1993): Myxobolus clarii in

the testis of fish Clarias lazera from the River Nile of Assiut.

Assiut Vet. Med. J. 29 (58): 108-114 Molnar, K. (1994): Comments on the host, organ and tissue specificity

of fish myxosporeans and on the types of their intrapiscine

development. Parasitol. Hungarica, 27, 5-20 Negm-Eldin, M.M.; Govedich, F.R. and Davies, R.W. (1999): Gill

myxosporeans on some Egyptian freshwater fish. Deutsche

Tierärztliche Wochenschrift.106 (11): 457-496 Oumouna, M.; Hallett, S.L.; Hoffmann, RW. and El-Matbouli, M.

(2002): Seasonal occurrence of actinosporeans (Myxozoa) and oligochaetes (Annelida) at a trout hatchery in Bavaria,

Germany. Parasitology Research, 89 (3): 170-184 Reed, C.C.; Basson, L. and Van As, L.L. (2003): Myxozoans infecting

the sharptooth catfish, Clarias gariepinus in the Okavango Rivery and Delta, Botswana, including describtions of two new species, Henneguya samochimensis sp. n. and Myxobolus

gariepinus sp. n. Folia Parasitologica, 50: 183-189. Stoskopf, M.K. (1993): Fish Medicine. W. B. Saunders Co. Philadelphia,

Pennsylvania, 19106, USA

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Table 1: Seasonal prevalence of Myxobolus infestation in Clarias

gariepinus ovaries.

Month

Examined Fish

Prevalence No. of infested fish

10

10 10

% 20 30 30 20

10

January February March April May June July August September

October November December

Total

10 10 10

10 10

10 10

10 10 20 12.5

10

120

15

Table 2: Intensity of Myxobolus infestation in Clarias gariepinus

ovaries.

Infestation case

Month

Intensity Total number of cysts

Ovaries infested

Severity Light

January

Light

Moderate

February

Moderate

Severe

12

Severe

March

16

Severe

25

Severe

Severe

April

Severe

Moderate

May

June - September

October November

Light Light Light Light

December

2

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IN

Fig. 1: A photograph of Clarias gariepinus ovary incised longitudinally

and infested with Myxobolus plasmodia (P) that are embedded in connective tissue among ova.

Jan

Feb March April May

June

July

Aug

Sep

Oct

Nov

Dec

Clarias

Fig. 2: Seasonal prevalence of Myxobolus infestation in

gariepinus ovaries.

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GC

DS

Fig. 3: Light microscope photograph of Myxobolus plasmodium in Clarias

gariepinus ovary. Plasmodia are enclosed in fibrous connective tissue capsule (CT) infiltrated with few lymphocytes. Germinating cells (GC) are located peripherally, while developing spores (DS) are toward the center. Toluidine stain (400X).

PC

SP

Fig. 4: Light microscope photograph of Myxobolus mature spores stained with

methlyene blue. Spores are oval in shape with sporoplasm (SP) and two polar capsules (PC) (1000X).

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CC

CT

Fig. 5: Light microscope photograph of a plasmodium of Myxobolus full

of spores (SP) encapsulated within a connective tissue capsule (CT) and compressing neighboring ova (CO) of Clarias gariepinus ovary. H&E (400X).

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