أ

 

The Effect of Theileria lestoquardi on the Reproductive Performance of Experimentally Infected Desert Sheep By Aisha Abbass Elsadig B.Sc. (1983), Faculty of Science, U of K.

M.Sc. (1993) Faculty of Veterinary Medicine U of K

A thesis submitted in partial fulfillment of the requirements for

the Degree of Doctor of Philosophy (Ph,D).

Supervisor Professor Hamid Suliman Abdalla

Department of Parasitology

Faculty of Veterinary Medicine

University of Khartoum

July 2009

 

 

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Dedication This thesis is dedicated with respect and love to:- - My deceased parents.

- My deceased brother Mustafa.

- My husband Baha.

- My sons Ahmad and Ayman.

-My daughters Amal and Israa.

- All friends and colleagues.

- All those who made this work possible

 

 

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List of content

Title Page

Dedication 11

List of content 111

List of tables V111

List of figures X

Acknowledgement X1

Abstract X111

ملخص االطروحة XV

Chapter one: Introduction and Literature review

1

1.1 Introduction……………………………………………………… 2

1.2 Theileriosis………………………………………………………... 5

1-2-1 Classification of Theileria lestoquardi………………………….... 5

1-2-2 Life cycle of the parasite…………………………………………... 6

1-2-2-1 Life cycle in ticks………………………………………………….. 6

1-2-2-2 Life cycle in mammals…………………………………………… 6

1. 2.3 Life cycle of Hyalomma anatolicum anatolicum…………………... 7

1.2.3.1 Parasitic stages……………………………………………………. 7

1.2.3.2 Non-Parasitic Stages………………………………………………. 8

1.2.3.2.1 Eggs Production…………………………………………………… 8

1.2.3.2.2 Pre- eclosion period………………………………………………... 9

1.2.3.2.3 Hatching…………………………………………………………... 9

1.2.3.2.4 Larval moulting period…………………………………………… 9

1.2.3.2.5 Nymphal moulting period………………………………………… 9

1.3 Host range………………………………………………………… 10

1.4 Geographical Distribution of Theileria…………………………… 11

1.5 Epidemiology of the disease…………………………………….. 12

1.5.1 Theileriosis in sheep……………………………………………… 12

 

 

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1.5.2 Theileriosis in goats……………………………………………… 13

1.5.3 Theilerial carrier stage……………………………………………. 13

1.5.4 Vertor and transmission…………………………………………... 14

1.6 Infectivity………………………………………………………… 15

1.7 Theilerial infection rates in ticks…………………………………. 17

1.8 Pathology and Pathogenesis……………………………………… 18

1.8.1 Clinical Signs…………………………………………………… 18

1.8.2 Post mortem Findings…………………………………………….. 19

1.9 Diagnosis…………………………………………………………. 20

1.9.1 Parasitological findings…………………………………………… 20

1.9.2 Lymphocytic stages……………………………………………….. 20

1.9.3 Erythrocytic forms………………………………………………… 20

1.9.4 Serological findings……………………………………………….. 21

1.10 Immunity and immunization……………………………………… 23

1.11 Effect of Theileria on reproduction……………………………….. 25

1.12 Haematological and Biochemical changes due to theileriosis…... 26

1.13 Molecular biology studies on Theileria……………………………… 30

1.14 Sheep in Sudan……………………………………………………. 35

1.14.1 Sheep Reproduction……………………………………………….. 35

1.14.2 Estrus Synchronization………………………………………… 36

1.14.3 Progesterone……………………………………………………. 37

1.14.4 Gestation length…………………………………………………… 38

1.14.5 Conception rate……………………………………………………. 39

1.14.6 Lambs survival and mortality rates………………………………. 40

1.14.7 Lamb birth weight…………………………………………………. 42

1.14.8 Puberty………………………………………………………….. 43

 

 

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2 Chapter Tow: Materials and methods 44

2.1 Study area…………………………………………………………. 45

2.2 Study duration……………………………………………………… 45

2.3 Study population…………………………………………………... 45

2.4 Housing……………………………………………………………. 45

2.5 Management and health control…………………………………… 45

2.6 Feeding……………………………………………………………. 46

2.7 Experimental design……………………………………………… 46

2.7.1 Experiment one…………………………………………………… 46

2.7.2 Experiment two…………………………………………………… 46

2.8 Tick collection and identification…………………………………. 46

2.8.1 Rearing of ticks……………………………………………………. 47

2.8.2 Laboratory maintenance of ticks…………………………………... 47

2.8.3 Handling and counting of tick stages……………………………… 47

2.9 Source of infective material……………………………………….. 48

2.9.1 Experimental infection of ticks……………………………………. 48

2.9.2 Experimental infection of animals ………………… 49

2.10 Detection of infection in experimental animals throughout the experiment 49

2.10.1 Physiological examination-temperature 49

2.10.2 Parasitological examination blood smear…………………………………. 49

2.10.3 Serological examination- indirect fluorescent antibody test(IFAT) 49

2.11 Oestrous synchronization………………………………………….. 50

2.12 Ram reproductive evaluation……………………………………… 50

2.13 Heat detection and maiting……………………………………….. 50

2.14 Collection of samples……………………………………………… 50

2.14.1 Sampling protocol…………………………………………………. 50

2.15 Heamatological investigation………………………………………. 50

2.15.1. Packed cell volume (PCV)………………………………………… 51

2.15.2 Haemoglobin concentration……………………………………… 51

 

 

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2.15.3 White blood cell count………………………………………... 51

2.16 Analysis of serum…………………………………...................... 51

2.16.1 Reagents and equipments………………………………………... 52

2.16.2 Estimation of serum total protein………………………… 52

2..16.3 Estimation of serum albumin………………………………. 53

2.16.4 Estimation serum globulin……………………………………… 53

2.16.5 Estimation of creatinine……………………………………… 53

2.16.6 Estimation of serum urea……………………………………… 54

2.16.7 Estimation of serum progesterone By Radio Immuno Assay (RIA) 55

2.16.8 Estimation of serum progesterone by Enzyme Immuno Assay (EIA) 58

2. 17 Statistical analysis………………………………………………… 59

Chapter Three: Results

60

3 Experiment one (studies on experimentally infected ewes) 61

3.1 Detection of the infection with Thieleria lestoquardi 61

3.1.1 Parasitological examination- Blood smears 61

3.1.2 Physical examination- Temperatures detection 61

3.1.3 Serological examination - Indirect fluorescent antibody test (IFAT)  61 3.2 Reproductive performance of experimentally infected and control ewes 65

3.2.1 Conception rate…………………………………………………. 65

3.2.2 Gestation length…………………………………………………. 65

3.2.3 Body weights of born lambs 65

3 .2.4 Number of delivered lambs, twining, abortions and survival rates: 66

3 .2.5 Progesterone rise post delivery…………………………………….. 66

3. 3 Haematological parameters…………………………………………. 72

3. 3. 1 Haemoglobin concentration (Hb)……………………………………. 72

3. 3. 2 Packed cell volumes (P CV)…………………………………………. 72

3. 3. 3 White blood cells (WBCs)…………………………………………… 79

3. 4 Biochemical parameters……………………………. 70

3. 4. 1 Serum total proteins………………………………………………… 79

 

 

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3.4. 2 Serum albumin…………………………………………………….. 86

3.4. 3 Serum globulins………………………………………………… 86

3.4.4 Serum creatinine……………………………………………………….. 94

3. 4. 5 Serum urea……………………………………………………………… 94

3.5 Experiment two(studies on experimentally infected lambs) 102

3.5.1 Detection of the infection with Thieleria lestoquardi 102

3.5.1.1 Parasitological examination - blood smears 102

3.5.1.2 Physical examination -temperature ………………… 102

3.5.1.3 Serological examination Indirect fluorescent antibody test (IFAT)... 102

3.5.2 Puberty…………………………………………………………. 105

3. 5.3 Biochemical parameters of lambs………………………………….. 110

3. 5.3.1 Serum total proteins……………………………………… 110

3. 5.3.2 Serum albumin…………………………………………… 110

3. 5.3.3 Serum globulins:-…………………………………………………… 110

3. 5.3. 4 Serum urea………………………………………………………… 110

3. 5.3. 5 Serum creatinine…………………………………………………… 111

4 Chapter Four: Discussion 123

5 References………………………………………… 135

6 Appendix…………………………………………. 163

 

 

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List of Table

Title Page

1 Indirect fluorescent antibody test (IFAT) for T. lestoquardi in the

infected ewes through out the experimental period in month…………

64

2 The number and percentage of conception rates of ewes in GC and GA 67

3 Gestation length (mean days ±SD) in GC and GA……………… ……. 68

4 Mean body weight kg of lambs in GA, GB and GC …………………  69

5 Number of delivered ewes, abortions and lamb survival rates in GC

and GA……………………………………………………………….

70

6 Intervals to progesterone rise(days) post delivery GA, GB and GC…  71

7 Weekly haemoglobin Hb concentration (mean±SD) in GC and GA  73

8 Mean ± SD values of HB (g/dL) concentrations in different pregnancy

periods in GC and GA…………………………………………………

75

9 Weekly packed cell volume (mean ± SD) in GC and GA post tick

application………………………………………………………………

76

10 PCV %( mean ± SD) during pregnancy in GC and GA………………. 78

11 Weekly white blood cell count X103/ (mean ± SD) in GC and GA post

tick application………………………………………………………….

80

12 WBCs counts per mm3 (X103) (mean ± SD) during pregnancy in GC

and GA………………………………………………………………….

82

13 Weekly values of total protein g/l (mean ± SD) in GC and GA post tick

application infection…..........................................................................

83

14 Total Protein values (g/dl) (mean ± SD) during pregnancy in GC and

GA…………………………………………………………………….

85

15

Weekly values of albumin g/l (mean± SD) in GC and GA post tick

application……………………………………………………………..

88

16 Albumin concentrations (g/dl) (mean ± SD) during pregnancy in GC

and GA………………………………………………………………..

90

 

 

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17 Weekly values of globulin g/l (mean± SD) in GC and GA post tick

application……………………………………………………………

91

18 Globulin concentration (g/dl) (Mean ± SD) during pregnancy in GC

and GA………………………………………………………………….

93

19 Weekly values of creatinine mg/dl(Mean ± SD) In GC and GA post

tick application………………………………………………………….

96

20 Creatinine concentrations (mg/dl) (Mean ± SD) during pregnancy in

GC and GA……………………………………………………………

98

21 Weekly values of urea mg/dl (Mean ± SD) in GC and GA post tick

application………………………………………………………

99

22 Urea concentrations (mg/dl) (mean ± SD) during pregnancy in GC and

GA………………………………………………………………………

101

23 Indirect fluorescent antibody test (IFAT) for T. lestoquardi in the

infected lambs through out the experimental period in month…………

104

24 Age at puberty in control ewe lambs G1 and infected lamb ewes G2 …

106

25 Weekly values of total protein g/l(mean ± SD) in G1 and G2 post tick

application…………………………………………………………….

112

26 Weekly values of albumin g/l (mean ± SD) in G1 and G2 post tick

application…………………………………………………………….

114

27 Weekly values of globulin mg/dl (Mean ± SD) in G1 and G2 post tick

application………………………………………………………………

116

28 Weekly values of urea mg/dl(mean ± SD) in G1 and G2 post tick

application……………………………………………………………..

118

29 Weekly values of creatinine mg/dl (mean ± SD) in G1 and G2 post tick

application………………………………………………………………

120

 

 

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page

List of figures

Title

1 Piroplasm of Thieleria lestoquardi in red blood cell………………… 62

2 Schizont of Thieleria lestoquardi in white blood cell……………….. 62

3 Daily body temperature(oC) in ewes 10 days after tick application 63

4 Haemoglobin concentration g/dl in GC and GA 10 weeks postinfection 74

5 Packed cell volume (%) in GC and GA post tick application 77

6 White blood cells in GC and GA post tick application……… 81

7 Total protein concentration g/dl in GC and GA post tick application 84

8 Albumin concentration g/dl in GC and GA post tick application 89

9 Globulin concentrationg/dl in GC and GA GA post tick application 92

10 Creatinine concentration mg/d in GC and GA post tick application.. 97

11 Urea concentration mg/dl in GC and GA post tick application ……... 100

12 Daily body temperature(oC) in lambs 10 days after tick application 103

13 Progesterone profile in lambs G1………………… 107

14 Progesterone profile in lambs G1………………… 107

15 Progesterone profile in lambs G2………………….. 108

16 Progesterone profile in lambs G2………………….. 108

17 Progesterone profile in lambs G2………………….. 109

18 Progesterone profile in lambs G2………………. 109

19 Total Protein concentration in G2 and G1 9 post tick application 113

20 Albumin concentration g/dl in G2 and G1 post tick application … 115

21 Globulin concentration g/dl in G2 and G1 post tick application …. 117

22 Urea concentration mg/dl in G2 and G1 post tick application ……… 119

23 Creatinine concentration mg/dl in G2 and G1 post tick application 121

 

 

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Acknowledgement

I would like to express my gratitude and appreciation to my

supervisor professor Hamid Suliman Abdalla, Department 0f

Parasitology, Faculty of Veterinary Medicine for his help, valuable

advices and suggestions throughout the period of study.

My special thanks and appreciation are also extended to Dr.

Yousif Hussein Elmansory, Head, Department, Radioisotopes,

Central Veterinary Research Laboratories, who inspired and

encouraged me to achieve my goals. I also thank him for reading

and revising the manuscript.

I am also indebted to Professor Abdelrahim Hussein. General

Director of the Central Laboratory and the staff in the Immunology

Department for their assistance to do part of the bench work in the

laboratory under his guidance.

My gratitude is also extended for my colleague Dr. Amna Eltaib

Babikir for her great support.

My thanks are also extended to Dr. Husna Mohamed Albasheer, who

help me in the statistical analysis

Also, I am greatly indebted to Dr. Ali Sideeg, Abdelazeez

Babikir Abdalla and Dr. Limya Mobarak in Entomology and Tick

department.

I am also indebted to Dr. Ahmad Algali and Dr. Kalid at

Atbara Research Station who sent me the infected sheep for the

study.

I would like also to extend my deepest gratitude to my all

 

 

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colleagues at the Isotope Department (Doctors, technicians. assistant

technicians and laborers).

I would finally like to show my appreciation to my husband

Dr. M. Baha Eldin Saad for his patience and for also reading and

revising the manuscript.

 

 

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Abstract

This study was aimed to investigate the effect of Theileria

lestoquardi on the reproductive performance of Sudanese desert

ewes. Thirty sudanese desert ewes were divided into three groups of

10 each. Group A (GA) was infected with Theileria lestoquardi

before mating; group B (GB) was infected with Theileria lestoquardi

2-3 months after conception and group C (GC) remained as the

uninfected control. GA was subjected to estrus synchronization and

hand mating. Conception rate was found to be11.1% in the first

mating, and increased to 33.3% and 44.4% in the second and third

mating. On the other hand, GC conception rate reached 90%. In GB,

three ewes aborted 20 days post infection. The gestation period was

two weaks shorter in GA. The effect of infection on body weight of

the newly born lambs was shown by 30% reduction in GA and by

50% mortality rate which was statistically significant at p < 0.05.

The study revealed that there was a statistically significance delay

(p<0.05) in the first progesterone rise after delivery.

The study was also concerned with the effect of Theileria

lestoquardi on the time of reaching puberty on the lambs. Fifteen

sudanese desert lamb ewes (4-5 months) were divided into two

groups; group 1 (G1) consisted of 7 lamb ewes infected with

Theileria lestoquardi, and group 2 (G2) remained as uninfected

control. Puberty was reached 12-15 and 8-9 months in G1 and G2

respectively

The study also revealed the effect of Theileria. lestoquardi on

 

 

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the haematological and biochemical picture in GA compared to GC.

There was a significant decrease in haemoglobin concentration,

packed cell volume and white blood cells counts (p<0.05). There

was also a significant decrease in serum total protein (p<0.05) but no

significant difference in serum albumin concentration. Serum

globulin values were also decreased significantly (P< 0.05). There

was a statistically significance (p<0.05) increase in serum urea

concentration and serum creatinine concentration.

The study also reflected the changes in the heamatological and

the biochemical parameters with the progress of pregnancy. The

results showed that there was a decrease in the haemoglobin

concentration and packed cell volume in GA and GC. White blood

cells count in GA only showed a significant decrease with the

progress of pregnancy. Serum total protein and albumin had

significantly decreased in GA and GC, while serum globulin and

serum urea showed significant decrease in GC only. Creatinine

concentrations decreased significantly in GA only.

 

 

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بسم اللة الرحمن الرحيم

األطروحةملخص

األداءبطفيل الثيليريا لستوكاردى على اإلصابة تأثيرمن هدفت هذة الدراسة للتحقيق

تمت الدراسة على ثالثين نعجة. االنتاجى للنعاج الصحراوية السودانية التناسلي

تحتوي كل مجموعه,ثالث مجموعات إلىبدورها قسمت والتي سودانيةصحراوية

, التزاوجبطفيل الثيليريا لسيوكاردى قبل أ المجموعة عدوىتمت , نعاج) 10(ى عل

فقد بقيت جالمجموعة أما. اإلخصابشهور بعد 3- 2 المجموعة ب عدوىوتمت

وجد . تم السماح لها بالتزاوج أ ثم تم تنسيق الشبق للمجموعة. للمقارنة إصابةبدون

فى % 44.4و % 33.3ثم ازداد الى % 11.1يمثل المجموعة في اإلخصابمعدل أن

فى % 90و من ناحية اخرى فان معدل االخصاب وصل الى , التزاوج الثانى و الثالث

العدوى يوم من 20لثالث نعاج بعد إجهاضحدث بالمجموعة في .جالمجموعة

تأثير العدوى على وجد أن . أ المجموعة في أسبوعين نقصت فقد ملالحفترة أما

وأن نسبة الموت قد وصلت % 30تدنى بنسبة قد متوسط أوزان الحمالن المولودة حديثا

.فى المجموعة أ% 50

في بعد الوالدةلبروجستيرون أفراز هرمون ا في تأخرهناك نأعلى الدراسةآشفت

.أالمجموعة

.عكست الدراسة تأثير الثايليربا ليستوآاردى على وقت البلوغ في الحمالن المصابة

نعاج تم 7وتتكون من 1 مجموعتين المجموعةالى ) أشهر 5-4( نعجة 15قسمت

9-8شهرا و 15- 12سجل وقت البلوغ , لم تعدى 2عدوتها بطفيل الثيليريا والمجموعة

 

 

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.على التوالى 2و 1أشهر للمجموعتين

فيطفيل الثيليريا لستوآاردى على التغيرات بالعدوى تأثير أيضاآشفت الدراسة

ترآيزات فينقص تمثلت فى جمقارنة بالمجموعة أ المجموعة في آيمياء الدمو الدم

نقص و هناك ايضا ,الدم البيضاءآريات نخفاض عددوا حجم خاليا الدم, الهيموقلوبين

تأثير علىبينما لم يكن هناك و في قيم القلبيولين المصلى آمية البروتين المصلى في

ترآيزات اليوريا والكرياتينين فى الوقت الذى ارتفعت فية .ترآيزات االلبيومين المصلى

.المصل في

أن فقد وجد , م و آيمياء الدم مع تقدم الحملأيضا التغيرات فى الد الدراسة أوضحت

أظهرت بينما جوالمجموعة أالهيموقلوبين وحجم المجموعة ترآيزي فيهناك نقص

اظهر البروتين وأيضا . أالمجموعة في فقط نقصا هاما الكريات الدموية البيضاء عدادأ

هر القلوبيولين واليوريا بينما اظ. جوالمجموعة أالمجموعة فيوااللبيومين نقصا هاما

ترآيزات الكرياتينين أظهرتبينما جالمجموعة فيالمصل نقص ملحوظ فقط في

.فقط أالمجموعة فينقصا ملحوظا

1 

 

CHAPTER ONE

2 

 

Introduction and Literature Review 1.1 Introduction

Tick and tick-borne diseases (TBDs) are widespread in the Sudan

(FAO, 1983 a, b and c). Theileriosis is a tick-borne disease of cattle,

sheep, goats, buffalo and occasionally wild ruminants caused by a species

of protozoa belonging to the genus Theileria (Losos, 1986). The parasite

is transmitted by a species of tick known as Hyalomma anatolicum

anatolicum. Clinical manifestations are shown as rise in temperature,

abortion and death may occur in severe cases, therefore, one of the ways

in which livestock production can be increased is by reduction of losses

due to tick-borne diseases.

Malignant ovine theileriosis (MOT) is an endemic disease which

causes massive losses among sheep. It appears as a rise in temperature,

anemia, emaciation and low activity, abortion and death may occur in

severe cases. The production is reduced and affected by the inefficient

conversion of food which adversely affects weight gain and milk

production.

In the Sudan, animal diseases which cause epidemics and outbreaks

were studied extensively and vaccines are available to most of the serious

bacterial and viral diseases. On the other hand, parasitic diseases in

general and specially protozoan blood parasites are not studied widely at

the herd level because their clinical effects are overlooked when

secondary infections are more apparent. This is why little work is done

on these diseases. Many previous studies reflected the economical impact

of these parasites (Mac Hattie et al, 1962 and Yousif, 1969).

The continuing increase in the human population in Khartoum state

3 

 

resulted in an increases demand for animal proteins and milk. The need

to increase agricultural production in the developing world will

necessarily involve an increase in livestock production. Major constrains

to livestock development are related to poor animal health, nutrition and

genetic potential. Poor animal health is often a consequence of infectious

diseases. This attracted the attention of workers to the importance of

piroplasmosis. The animal owners are now aware of the economical

consequences of the diseases and their great threat to their herds, hence,

affects their prospects in increasing animal production.

Sudan is the largest country in Africa and has the largest livestock

populations. Sheep comprised about 36% of the total livestock population

of 45 million animals in (Watson et. al.1977).

Livestock industry is of great importance to Sudanese economy as

it is one of the main sources of food, employment and foreign currency.

Sheep population is estimated at 48.136 million head (Ministry of

Animal Resources, 2002). In recent years, Sudanese sheep namely Sudan

desert type, has received great interest as export commodity to the Arab

countries. In 2000-2001 for example, sheep exports has contributed

$261.34 million to the national exchange earning at annual off take rate

of 21.778 million heads (Ministry of Animal Resources, 2002).

Little work is done on the reproductive performance of sheep that carry

piroplasms in their blood.

4 

 

General objective:

To investigate the effect of Theileria lestoquardi on the

reproductive potential of experimentally infected desert sheep.

Specific objectives:

1 To study the conception rate of experimentally infected ewes.

2 To estimate the first progesterone rise, the kidding interval and the

gestation period of experimentally infected ewes.

3 To investigate the general condition of the newborns delivered by

the experimentally infected ewes.

4 To study the effect of Thieleria lestoquardi on the age of puberty of

the Sudanese desert ewes.

5 To investigate some changes in the hematological and biochemical

parameters of the experimentally infected ewes.

5 

 

1.2 Theileriosis:

Theileriosis is an infectious, non-contagious protozoan disease

transmitted by some species of hard ticks (Acari: Ixodidae). The disease

affects mainly cattle and to a lesser extent sheep, goats, camels and wild

ungulates (Soulsby, 1982, Mossalam et al, 1984). Sheep and goats are

affected by three species of Theileria, of which Theileria hirci

(Dzhunkovskii and Urodshevich, 1924), named Theileria lestoquardi

nomen novum (Morel and Uilenberg, 1981) is the most pathogenic one

.The parasite causes malignant ovine theilerioses which assumes acute,

sub acute or chronic forms causing high mortality rates among sheep.

Theileria ovis and Theileri seperata, on the other hand, are less

pathogenic and of low importance than Theileria lestoquardi (Soulsby,

1982; Arnold and Dias, 1983; Uilenberg, 1983).

1-2-1 Classification of Theileria lestoquardi:-.

According to the scheme set by Levine et al (1980), Theileria

lestoquardi is classified as follows:

Sub-Kingdom: protozoa

Phylum : Apicomplexa

Class : Sporozoa.

Sub-class : Piroplasmina.

Order : Piroplasmida

Family : Theileriidae

Genus : Theileria.

Species : Theileria lestoquardi nomen novum.

6 

 

1-2-2 Life cycle of the parasite:

As in other species of Theileria, lestoquardi completes its cycle in

two hosts, these are ticks and mammals.

1-2-2-1 Life cycle in ticks:

During feeding of ticks on an infected animal, they ingest

piroplasm infected erythrocytes which start to differentiate into

macrogametes and microgametes. The gametes unite to from zygote in

the lumen of the tick’s gut. The zygote invades the gut epithelium to

develop a kinete that invades type III acini of the salivary gland (Schein,

1975). After moulting to the next stage, the parasite begins to develop

and the acini undergo marked hypertrophy (Irvin et. al., 1981).

Development and hypertrophy vary from tick to tick and from acinus to

another in the same tick. With the onset of tick attachment and feeding on

a new host, there is a rapid increase in acinus hypertrophy and the

parasite undergoes multiple divisions to form sporont giving rise to active

sporozoites (Fawcett et. al., 1985).

1-2-2-2- Life cycle in mammals:

As infected ticks feed on a susceptible mammalian host, mature

sporozoites get into the blood stream and invade the lymphocytes.

Macroschizont is the first stage reported to be seen in the lymphoid cells

of regional lymph nodes (Uilenberg, 1983); Macroschizonts usually

contain about eight large irregular reddish –purple nuclei (Stag et.

al.1981) with pale surrounding cytoplasm when stained with Geimsa

stain (Losos,1986). Presence of macroschizonts inside the lymphoid cells

stimulates their mitosis (Uilenberg, 1981). During this division,

macroschizonts undergo successive multiplication forming numerous

7 

 

microschizonts, each containing many small dense nuclei. These

disintegrate liberating merozoites which are released into the blood

stream by rupture of the lymphoid cells and enter the erythrocytes giving

rise to piroplasms (Uilenberg, 1981). Piroplasms are round, rod, ring or

comma-like in shape (Fischer and Say, 1989).

1. 2.3 Life cycle of Hyalomma anatolicum anatolicum:

1.2.3.1 Parasitic stages:

Feeding periods of different stages of Hyalomma anatolicum

anatolicum on host may slightly be affected by environmental

temperature. However, the tick feeding period on guinea pigs within an

ambient temperature range of 20-35 0C showed significant variation in

length (Snow, 1969). In cyclic development, the tick behaves as a three

host tick (Serdyukova, 1946; Feldman-Mohsam, 1948; Daubney and

Said, 1951 and Deply, 1952). Experimentally, especially when fed on an

abnormal host, Hyalomma anatolicum anatolicum may take two-host and

three host behavior (Serdyukova, 1946). That was illustrated by feeding

on guinea pigs (Snow, 1969) and rabbits (ElGhali, 1992; Ahmad, 1999

and Osman, 1999).

Larvae feeding period on rabbits were reported to be 2-3 days

(ElGhali, 1992) or 4-5 days (Yassir et al, 1992 and Ahmed, 1999). In

sheep, it has been reported that the larval feeding period was 4-5 days

(Ahmad, 1999). Nymphal feeding period as estimated by Ahmad (1999)

on rabbits and sheep were 6-8 days and 4-5 days respectively. Female

adults of Hyalomma anatolicum anatolicum complete feeding on rabbits

within 5-11 days (Snow, 1969; ElGhali, 1992; Yassir et al, 1992 and

Ahmad, 1999). However, Ahmad (1999) reported a period of 5-7 days

8 

 

when Hyalomma anatolicum anatolicum adult fed on sheep.

1.2.3.2. Non-parasitic stages:

1.2.3.2.1 Eggs production:

Capability of laying any numbers of eggs is affected by ambient

temperature. Hyalomma analoticum anatolicum females failed to oviposit

or layed small number of eggs at low temperature (Snow and Arthur,

1966). ElGhali (1992) recorded failure of laying at 150C. Snow and

Arthur (1966), Dipeolu (1983), Dipeolu (1989), ElGhali (1992) and

Ahmed (1999) reported a linear relationship (positive correlation)

between the engorgement weight of Hyalomma anatolicum anatolicum

female and number of their laid eggs. Dipeolu (1983) reported an average

of 0.97 gms engorgement weight of different Hyalomma species ticks

and maximum number of 2010 eggs laid by an individual tick. ElGhali

(1992) found that the hightest egg conversion ratio for Hyalomma

anatolicum anatolicum was 57.1 at 270C and 65.5-61.5% relative

humidity.

Type of host has a pronounced effect on the fecundity by the

source of feeding of ticks. Dipeolu and Adeyefa (1984) showed that ticks

fed on sheep and horses laid significantly less number of eggs and were

generally less biologically viable than those fed on cattle. They

concluded that the latter are better hosts. Dipeolu and Akinboade (1984)

subsequently compared the fecundity of some ticks which had been fed

on the red flanked duiker and cattle. Ticks fed on duiker produced more

eggs even though their engorgement weights were similar to those fed on

cattle.

9 

 

1.2.3.2.2 Pre- eclosion period:

The pre-eclosion period includes pre-oviposition and pre-hatching

periods. At 27±10C temperature, mean pre- ovipsoition and oviposition

of Hyalomma anatolicum anatolicum ticks were reported to be 4.6 and 15

days respectively (Yassir et al, 1992). ElGhali (1992) showed that both

periods decreased by increase of temperature, but they increased again at

40 0C. Pre-eclosion period is reported to range between 20 and 24 days

(Ahmed, 1999).

1.2.3.2.3 Hatching:

Eggs of Hyalomma anatolicum anatolicum failed to hatch at 150C,

while hatched within 22-26 days at 270C and at 350C where hatchability

reached 92.6 ± 0.18% (ElGhali, 1992). Ahmed (1999) reported

hatchability in eggs of Hyalomma anatolicum ticks which fed on sheep

and rabbits as 85-97% and 67-94% respectively.

1.2.3.2.4 Larval moulting period:

Ahmed (1999) studied the life cycle of Hyalomma anatolicum

anatolicum in both sheep and rabbits. He found that a period of 16 to 18

days was required for moulting of larvae fed on both animal species

without significant difference. At room temperature (10-033C) and

relative humidity 75-85%, larval moulting periods were found to range

between 9 and 12 days (Osman, 1999). This period was estimated under

optimum conditions (35oC temperature and 6.5-99% relative humidity) as

7.7± 0.5 days (ElGhali, 1992).

1.2.3.2.5 Nymphal moulting period:

Engorged nymphs of Hyalomma anatolium anatolium ticks

detached from sheep and rabbits moulted within 11-14 days (Ahmed,

10 

 

1999). ElGhali (1992) reported a range of eight to nine days for moulitng

of nymbhs of the tick fed on rabbits under optimum laboratory

conditions.

Generally, in all stages of Hyalomma anatolicum anatolium ticks, the

survival and developmental periods have been affected by the

surrounding atmospheric temperature (Snow, 1969; Dipeolu and Ogungi,

1980; Dipeolu, 1989; ElGhali, 1992 and Yassir et al, 1992), while the

relative humidity (Rh) does not affect the rates of development of ticks. It

does, however, critically affect survival of eggs and larvae. Nymphs and

adults are more resistant, and natural tick populations probably survive

hot and dry season in these stages (Tukahirwa, 1976). Ouhelli and Pandy

(1984) showed that at 35 0C, relative humidity has a significant influence

on the oviposition period but not on the number of laid eggs and the pre-

ovipositional period of Hyalomma lusitanicum, while at 25 0C, the pre

ovipositional period was shorter at 93% (9 days) than at 62% and 25%

(50-51 days).

1.3 Host range:

Bumann (1939), Soulsby (1982) and Mossalam et al, (1984)

mentioned that Theileria lestoquardi affects both sheep and goats.

Littlewood (1915), Lestoquard (1926) and Khayat and Gilder (1947)

reported the occurrence of the disease in sheep, while Dzhunkovskii and

Urodshevich (1924), Pabs-Gamon and Foley (1974) described Theileria

lestoquardi infection in goats in Serbia and Sierra Leone respectively. In

the Sudan, Nagwa (1986), Tageldin et al (1992), El Hussein et al (1993)

reported the disease in sheep. Moreover, El Hussein et al (1998) reported

the occurrence of theilerial piroplasms in the blood of apparently healthy

11 

 

goats in Nahr Elnil state. After an experimental infection, a single goat

showed neither clinical symptoms nor parasitic stages of Theileria inspite

of affection of the 39 Awassi sheep used in the same trials (Hooshmand-

Rad and Hawa, 1973a.).

Recently, the disease was shown to be widely distributed in main

sheep grazing area of the country.

1.4 Geographical distribution of Theileria:

Salih et. al. (2003) mentioned that “the prevalence of Theileria

lestoqurdi antibodies in Sudanese sheep from nine geographical areas in

Sudan was determined using indirect fluorescent antibody (IFA) test. Out

of 315 samples examined, 51(16.2%) were found positive and ranged

between 23.4% in River Nile state and 10% in Kasala and Darfour

provinces with an overall prevalence of 16.2% indicating widespread

distribution of the infection. They also reported the presence of

antibodies reactive to Theileria annulata in sheep sera.

El-Azazy et al (2001) collected blood, lymph nodes and tick

samples from animals being treated or necropsied at the veterinary

diagnostic laboratory in Jeddah and Makkah (Western region), and

Burieda ( Al-Qasim Central Region). Blood and lymph node smears were

prepared and examined for Theileria species. Theileria hirci = (T.

lestoquardi) was found in lymph node smears of one out of 36 sheep

(2.8%) in Jeddah and six of 25 sheep (24%) in Bureida. The erythrocytic

forms were detected in 5-8% of RBCs. Ticks were found in relatively

less number of sheep in Bureida and Jeddah (17/180 and 26/125

respectively). All Theileria-infected sheep were infested with Hyaloma

impeltatum . They suggested that H. impeltatum is a potential vector of

12 

 

malignant theileriosis in Saudia Arabia.

Luo et. al., (1997) mentioned that theileriosis is an important disease

of sheep and goats in west China. They reported that the outbreak period

is from late March to July with April-May being the peak months. This is

the period of most intensive tick attaching by Haemaphysalis

ginghaiensis (77.2-99.24%) during the year. They also found that the

disease was more serious in lambs and exotic adult animals than native

adult animals. The prevalence rate of lambs, exotic and native adult

animals were 78-85%, 41- 62.5% and 65% respectively.

Friedhoff (1997) mentioned that malignant theileriosis caused by

Theileria lestoquardi is an important disease in Iran and India. He

reported that it is transmitted by H. a. anatolicm but occurs outside the

distribution area of this tick. He also reported that an attenuated

macroschizont is successfully being used for vaccination in Iran.

1.5 Epidemiology of the disease:

1.5.1 Theileriosis in sheep:

Malignant ovine theileriosis has been reported in various countries

throughout the world. It is prevalent in Eastern Europe, Middle East,

North Africa, Iran, Iraq and Sudan (Dzhunkovskii and Urodshevich,

1924; Hooshmand- Rad and Hawa, 1973; Soulsby, 1982; Tageldin et al,

1992; Latif et al, 1994). Its economic importance is gradually and

increasingly becoming evident (Hawa et al, 1981), and this may be due to

high mortality rates which may reach 100% among infected animals

(Soulsby, 1982; Mossalam et al, 1984).

In Sudan, the disease was firstly described by Mason (1915), then

reported in Khartoum state and Western parts of the country (Nagwa,

13 

 

1986; Tageldin et al, 1992; Latif et al, 1994). In Northern Sudan, it was

reported by Elhussein et al (1993), ElGhali and EIhussein (1995) and

Ahmed (1999) as causing serious losses among sheep. The disease flares

up in summer, in a pattern of seasonal outbreaks, where 63.5% of sheep

admitted to Atbara veterinary hospital and Atbara veterinary research

laboratory were diagnosed as suffering from theileriosis during the

summer season (ElGhali and Elhuussein, 1995).

1.5.2 Theileriosis in goats:

Although many authors described theileriosis in small animals as a

disease of sheep and goats, no detailed studies were mentioned about the

infection in goats. Dzhunkovskii and Urodshevich (1924) reported the

disease in goats in Serbia. In Sierra Leone, theileriosis has been reported

in Sahelian goats with marked clinical signs (Pabs-Garnon and Foley,

1974).

In Northern Sudan, (Edamer province), clinical signs of caprine

theileriosis were not detected, but in a survey conducted by ElHussein et

al (1998) among apparently healthy goats, 10 out of 82 (12.2%) goats

showed piroplasms in their blood.

1.5.3 Theilerial carrier stage:

In carrier animals, the disease flares up post exposure to stress

factors (Shommein, 1979). Flach et al (1995) studied the factors

influencing the transmission and incidence of tropical theileriosis in an

endemic region of Morocce during 1991 season. They concluded that the

prevalence of subclinically infected carriers of Theileria annulata, the

number of adult Hyalomma species tick, and the probability of becoming

newly infected with Theileria annulata increased significantly with the

14 

 

age of cattle, although the age effect on new infections may be a result of

increased tick number on older animals. The probability of clinical

disease in newly infected cattle was not significantly influenced by age or

number of ticks, but was positively associated with cattle population. In

Spain, 92 field samples of apparently `healthy cattle blood were collected

and tested. 22% were positive by PCR for Theileria annulata infection

(D,Oliveira, et al, 1995). In Kenya, Theileria parva infection was

transmitted experimentally from carrier to susceptible cattle (Kariuki et

al, 1995).

1.5.4 Verctor and transmission:

Rhipicephalus bursa was suggested to be the main vector

responsible of transmition of Theileria lestoquardi (Dzhunkovskii and

Urodshevich, 1924; Soulsby, 1982). Mazlum (1982) suspected

Hyalomma species to be the vector for transmission of the disease in

South and South East Iran where sheep and goat malignant theileriosis

occurs in Iraq. Hooshmad-Rad and Hawa (1973b) transmitted the disease

via Hyalomma anatolicum anatolicum. It was also suggested that this tick

may be the main vector of the disease in the Sudan (Nagwa, 1986; latif et

al, 1994; Elhussein et al, 1993; Ahmed, 1999 and Osman, 1999).

Tageldin et. al. (1992) reported that all ticks collected from sheep

suffering from theileriosis during an outbreak in Khartoum state were

identified as Hyalomma anatolicum anatolicum.

Hyalomma anatolicum anatolicum as reported to be prevalent along the

North and North East parts of Africa, Middle East, South Russia and

Turkey in areas of lower humidity where rainfall is less than 250mm,

while in the Sudan, it has been reported in Northern Kassala, Kordofan

15 

 

and Gezira provinces (Hoogstraal, 1956). It is most abundant in the

desert scrub zone and it’s distribution in the South is limited to the North

of latitude 14 N (Jongejan et. at.1987).

Adults of Hyalomma anatolicum anatolicum infest sheep, goats,

cattle, camels, horses, donkeys and pigs ( Hoogstraal, 1956).

Transovarian transmission has not been reported in theilerosis (Losos,

1986), while transstadial transmission, is well known (Hooshmand-Rad

and Hawa, 1973b; Losos, 1986). (Hooshmand-Rad and Hawa, 1973b)

show that infected larvae failed to transmit the disease after moulting to

nymphs, but nymphs generated from infected larvae can transmit

malignant ovine theileriosis after moulting to adult.

Malignant ovine theileriosis has been experimentally transmitted to

susceptible sheep and goats via parasitaemic blood injected intravenously

and subcutaneously (Sasmal et al, 1983 and Elhussein et. al. 1998).

Intrauterine infection has also been reported (Robertson, 1976). Friedhoff

(1997) reported that Theileria hirci is transmitted by H .a .anatolicum but

occurs outside the distribution area of this tick. Malignant theileriosis of

sheep and goats is an important disease in Iraq, Iran and India. An

attenuated macroschizont vaccine is successfully being used in Iran.

1.6 Infectivity:

Leemans et al (1999) reported that in a series of experiments,

sporozoite stabilates of a Theileria lestoquardi (lahr) and a T.annulata

(Ankara) stocks prepared from Hyalomma anatolicum anatolicum ticks

were used to examine the infectivity of both parasite species for sheep

and cattle and to study the development of cross-immunity between these

species. In the first experiment, sheep and cattle were inoculated with T.

16 

 

lestoquardi sporozoites. Surviving animals and naïve sheep and cattle

were, in the second experiment, inoculated with T. annulata. In the third

experiment, naïve sheep and sheep previously infected with T. annulata,

were inoculated with T. lestoquardi. The following responses to

inoculations were monitored: clinical and haematological signs of

infection, appearance of parasitic stages of the parasites in lymph node

biopsies and in peripheral blood and serological response to T.

lestoquardi and T.annulata schizont antigens. While T. lestoquardi

readily infected sheep and caused severe disease, it did not infect cattle.

On the other hand, T. annulata infected both cattle and sheep. However,

whereas cattle became severely affected, infected sheep showed mild

clinical sypmtoms only and piroplasms did not develop. Despite their

different behavior in the host species examined, cross-immunity studies

suggested that the parasite species are very closely related. Experiments

in sheep indicated that T. lestoquardi infection gave protection against

subsequent T. annulata infection. On the other hand, recovery from

T.annulata infection did not prevent infection by sporozoites of

T.lestoquardi, resulting in the establishment of schizonts and their

subsequent development into piroplasms, although it was protected

against the major clinical effects of T. lestoquardi infection.

The maturation and quantification of Theileria lestoquardi parasites in

unfed and partially fed adult Hyalomma anatolicum anatolicum ticks was

studied using: 1- methyl green pyronin (MGP) staining of salivary

glands, 2- in vitro infection of peripheral blood mononuclear cell (PBM)

with parasites harvested from infected ticks and 3- a semi quantitative

polymerase chain reaction (PCR) with MGP staining. The greatest

17 

 

infection rate was seen in unfed ticks. Feeding resulted in a gradual

reduction in the number of infected acini with a concomitant increase in

the maturity of the parasites. In vitro infection of sheep PBM with titrated

group-up tick supernate (GUTS) demonstrated that infectivity peaked

between 2 and 4 days of tick feeding whereas GUTS prepared from unfed

ticks was not infective. The polymerase chain reaction (PCR) was both

sensitive and specific; detecting T. lestoquardi DNA in unfed, partially

fed and 2-days fed ticks, though it gave no indication of the infectivity of

the parasite (Kirvar et.al., 1998).

Theileria species specific primers were used in a PCR to determine

the identity of the parasites in the cell lines. These invitro studies

confirmed earlier observations that T. lestoquardi was unable to infect

cattle, whereas infection of all sheep with T annulata was proven.

Moreover, earlier indications of the development of partial cross-

immunity in sheep of T. annulata to T .lestoquardi and vice versa were

strengthened. These findings may thus have consequences on the

understanding of the epidemiology of T .lestoquardi infections of sheep.

(Leemans et al).

1.7 Theilerial infection rates in ticks:

Determination of the theilerial infection rate in the vector’s

salivary gland is an important component in the study of the

epidemiology of the disease (Sangwan et. al. 1994). Infection rate of

Theileria annulata was estimated by Walker and Mckellar (1993),

Sangwan et al (1986), Flach et al (1993), Sangwan et al (1994) and Flach

et al (1995), while Theileria parva infection rate in the salivary gland of

ticks was studied by Purnell et. al., (1971), Blewett and Branagan (1973),

18 

 

Walker et, al (1979), Irvin et. al., (1981) and Yassir et. al. (1992).

In the Sudan, Theileria annulata infection rate in Hyalomma marginatum

rufipes has been reported under laboratory conditions to reach 100%

(Jongejan et al, 1983), while Walker et al (1983) estimated the same

theilerial species natural infection rate in Hyalomma anatolicum

anatolicum as 38-86% in Khartoum state. Infection rate of

Rhipicephalus appendiculatus ticks with theileria parva was reported as

1-2% in enzootic areas Leitch and Young, (1981). Little attention has so

far been paid to such studies in Theileria Iestoquardi. However, Ahmed

(1999) reported the infection rate of Theileria Iestoquardi in Hyalomma

anatolicum anatolicum ticks naturally fed on sheep at Alakad area in

Edamer province to be 6.19%. The infection was higher 7.86% in male

ticks than 4.95% in female ones. The infection rate with Theileria

Iestoquardi was low (2%) in the salivary glands of Hyalomma

anatolicum anatolicum ticks fed on carrier sheep in Atbara, Northern

Sudan, while it was much higher (14%) when ticks were fed on an

experimentally infected sheep (Osman, 1999). No parasites were detected

in the salivary glands of Hyalomma anatolicum anatolicum ticks

collected from sheep in Khartoum state during an outbreak of malignant

ovine theileriosis (Nagwa, 1986).

1.8 Pathology and pathogenesis:

1.8.1 Clinical signs:

Malignant ovine theileriosis has three forms: acute, sub acute and

chronic. Acute form may lead to death after a short course of fever and

one or more of the following clinical symptoms that may appear:

depression, inappetance, fever, swelling of superficial lymph nodes,

19 

 

respiratory manifestations, appearance of pale visibie mucous

membranes, jaundice and oedema of the throat (Soulsby, 1982; Losos,

1986; Tageldin et al, 1992; ElHussein et al, 1993; Latif et al, 1994 and

Osman, 1999). Similar symptoms are noticed in the subacute and chronic

forms but they are less marked (Losos, 1986).

In goats, symptoms observed were inappetance, slight fever,

respiratory distress with nasal discharge, lacrymation, enlargement of

superficial lymph nodes and paleness of conjunctiva (Pabs-Garnon and

Foley, 1974; Sasmal et al, 1983 and Elhussien et al, 1998). However,

goats seem to be resistant to the development of clinical disease

(Elhussein et.al. 1998).

1.8.2 Post mortem findings:

Posmortem changes reported in carcasses of infected sheep are

gelatinization of intermuscular and subcutaneous adipose tissue,

congestion and oedema of the lungs with frothy ductules and alveoli,

flabby heart and hydropericardium, enlarged friable yellow to yellowish

brown liver, distended gall bladder, jaundice, enlargement of lymph

nodes and spleen, congestion and ulceration of abomasal mucosa,

congestion of the mucous membrane of the last part of the intestine may

be accompanied with the presence of haemorrhagic batches (Hooshmand-

Rad and Hawa, 1973a; Tageldin et al, 1992; Elhussein et al 1993; Latif et

al, 1994 and Osman, 1999).

Pabs-Garnon and Foley (1974) described postmortem findings in

two goats that died after suffering from malignant caprine theileriosis as

petichial haemorrhages and whitish foci on the liver and kidneys,

distended gall bladder with dark green to black fluid and consolidation of

20 

 

apical lobe of the lung.

1.9 Diagnosis:

1.9.1 Parasitological findings:

As with the other theileria species, Theileria lestoquardi develops

lymphocytic and erythrocytic stages.

1.9.2 Lymphocytic stages:

Schizonts are usually detected in smears of lymph node biopsies

ten to twelve days after infected ticks start feeding on sheep (Osman,

1999). Macroschizont contains up to eight large irregular nuclei (Stagg et

al, 1981), while microschizont contains up to 80 nuclei that appear as

numerous small dense dots. Schizonts can also be seen in the impression

smears from the liver, lung, brain and spleen (Hooshmand-Rad and

Hawa, 1973 a, Osman, 1999).

In goats, schizont had been detected with difficulty in peripheral lymph

nodes biopsies 13-18 days post infection (Sasmal et al, 1983; Elhussein

et al, 1998). However, impression smears from spleen and liver showed

intercellular schizonts (Pab-Garnon and Foley, 1974).

1.9.3 Erythrocytic forms:

Piroplasms appear in the red blood cells (erythrocytes). as round,

ring rod or comma-like shaped (Fischer and Say, 1989) with reddish

purple nuclei and faint blue surrounding cytoplasm when stained with

Geimsa stain (Hooshman-Rad and Hawa, 1973 a). Piroplasms seen in

goats erthrocytes were similar to those of sheep in size and shape (Sasmal

et al, 1983). They had been detected in goats blood 10-32 days post

infection (Sasmal et al, 1983). Experimental inoculation of Indian goats

with infected sheep blood revealed 18.25 - 31.86% parasitaemia (Sasmal

21 

 

et al, 1983), while Elhussein et al, (1998) detected a comparatively very

low parasitaemia which rarely reached 1/1000 in experimentally infected

indigenous Sudanese goats.

1.9.4 Serological findings:

Many serological tests were used for detection of antibodies

produced by Theileria species, such as complement fixation, capillary

agglutination and indirect haemagglutination tests, but their reliability is

not always proven (Uilenberg, 1981). Indirect fiuorescent antibody test

(IFAT) is commonly used for detection of antibodies produced by

Theileria lestoquardi in sheep, and the peak titer is manifested shortly

before death or at early recovery from the acute stage of infection (Hawa

et al, 1976; Uilenberg, 1981). IFAT was applied by leemans et al (1997)

using a schizont culture. The antibodies were firstly detected 15 days and

disappeared 90 days post inoculation. The above authors reported an

antigenic relationship between ovine Theileria lestoquardi and Theileria

annulata of cattle. Similar results were reported by Hooshmand-Rad et

al, (1993).

Gao et. a.l (2002) reported that a rapid, sensitive and specific

diagnostic method (an enzyme-linked immunosorbent assay (ELISA))

was developed for the diagnosis of Theileria sp. Infection in sheep; and

optimal conditions were established, such as antigen concentration,

serum dilution, coating time, Tween-20 concentration and conjugate. The

results were analyzed by measuring the coefficient of variation (CV).

Three sera titers (high, middle, and low) were analyzed over the

measurement range, resulting in a CV of around 10% whereas a 30%

variation is the maximum acceptable. The cut-off value was determined

22 

 

by the mean of a negative control plus three standard deviations. Cross-

reactions were found only with Babesia ovis. However, this result may be

questionable, because it cannot exclude that these sheep were already

infected with both Theileria sp. and B. ovis. The ELISA described in the

resent studies proved to be a useful tool for studying the epidemiology of

Theileria sp.

Leemans et al (1997) reported that an indirect fluorescent antibody

test (IFAT) based on schizont-infected lymphoblastoid cells, was applied

to study the course of antibody production in adult sheep inoculated with

attenuated, in vitro grown , Theileria lestoquardi infected cells. Bright

fluorescence of the intracellular schizonts could first be demonstrated 15

days after inoculation. A 32-64- fold rise in antibody titres was recorded

one month after infection and substantial titres were still observed 90

days after inoculation. Fluorescence was absent with negative control

sera and background staining was minimal. No serological cross-

reactions were detected with sheep sera positive for Babesia motasi,

Babesia ovis or Toxoplasma gondii. Results obtained did not differ when

antigens prepared form three different strains of T. lestoquardi infected

lymphoid cells were compared. Testing for reactivity to non-pathogenic

Theileria species of sheep revealed a low degree of cross-reaction of

Theileria ovis and Theileria separata antiserum to Thieleria antigen.

Cross-reaction was also observed with bovine sera positive for Theileria

annulata and Theileria parva. Moreover, T. lestoquardi positive sera

reacted almost equally strongly with bovine T. annulata antigen as with

their homologous antigen, whereas cross-reaction with bovine T. parva

antigen was less pronounced. These results indicate a close antigenic

23 

 

relationship between T. lestoquardi of sheep and T. annulata of cattle.

In molecular studies, Bakheit (2004) reported that several clones

were selected, purified and sequenced either at random or on the basis of

the immuno-reactivity. One of these clones, against which sheep sera

samples were noticeably reactive, was chosen for further characterization

and was given the name Clone-5. The antigenicity of clone-5 protein was

repeatedly confirmed during this study. The result showed that Clone-5

could be a suitable tool for diagnosis of malignant theileriosis of sheep

and goats and for testing larger numbers of sera.

1.10 Immunity and immunization:

It is well known that there is a variable resistance to theileriosis

within the host species. European cattle imported to the Sudan and

crossbreeds being particularly, more susceptible to tropical theileriosis

than indigenous breeds (Shommein, 1979). In India, indigenous Zebu

cattle (Bos indicus) are claimed to be resistant to Theileria annulata

infection, whereas in North Africa, the indigenous cattle (Bos Taurus) are

also thought to be almost equally resistant to the same organism. In both

regions, tropical theileriosis is almost exclusively a disease of imported

cattle, predominantly but not exclusively of European taurine origin

(Edwards, 1927). This fact is not always evident, as Seigent et. al. (1945)

reported a lower mortality rate (12%) among French imported cattle than

23% among indigenous Algerian cattle infected with Theileria annulata.

Hooshmand-Rad et al, (1993) reported that, specific enzyme linked

immunosorbent assay (ELISA) and molecular DNA, RNA detection

techniques have been developed. These could help in giving accurate

diagnosis for detailed studies on the epidemiology and transmission of

24 

 

the disease (Uilenberg, 1981).

Indian cattle derived from East Coast Fever (ECF) endemic areas

do, however, show evidence of ECF resistance eventhough they may

have been maintained free from tick or disease challenge for a number of

generations (Guilbride and Opwata, 1963).What is evident, however, is

that resistance to ixodid ticks, particularly Hyalomma species and

Rhipicephalus appendiculatus plays a major role in determining death or

survival of cattle in an endemic area (Young et al, 1988).

Thielerial antibodies arise after treatment and / or recovery from

infection. Cattle which recover from theilerial infection and / or treated

animals are solidly resistant to homologous challenge with a

heterologous strain of Theileria, as for example, when cattle are

transported to a new area can result in a complete breakdown of

immunity (Irvin, 1985). Therefore, in case of East Cost Fever,

immunization with a combination of different Theileria strains (cocktail)

gives a broad infection that could be controlled by a single treatment with

long acting tetracycline (Radely et al, 1975a; Radely et al, 1975b).

Shommein (1979) mentioned that tropical theileriosis usually follows

stress factors e.g. long travel, vaccination with hot vaccines, starvation

and thirst.

Efforts for immunization against thileriosis are continuing.

Immunization by infection and treatment using stabilates of unfed ticks

and simultaneous tetracycline administration has been done for Thieleria

parva (Brown et al, 1977a; Brown et al, 1977b,) and Theileria annulata

(Pipano et al, 1981) with successful results. Immunization using schizont

attenuated by serial passage in tissue culture has also been carried out for

25 

 

Theileria annulata (Pipano and Tsur, 1966; Pipano et al, 1977) and for

Theileria lestoquardi (Hawa et al, 1981).

Lawrence (1997) reported that a line vaccine for Theileria hirci

(lestoquardi) consisting of schizonts propagated in a lymphoid cell

culture and attenuated by passage has also been used successfully in the

Middle East.

1.11 Effect of Theileria on reproduction:

Rumberia et. al. (1993) found that five of eight recovered animals

in the Theileria parva low infection group cycled regularly post

infection, while three others had extended periods of ovarian dysfunction

post infection. Both infected and recovered animals in the Theileria

parva high infection -group similarly had extended periods of ovarian

dysfunction post infection. All controls in both experiments cycled

regularly throughout the study period. Differences in cyclical status were

not related to the febrile response, parasitological or hematological

differences. The animals that continued to cycle throughout lost the least

amount of weight. Animals that stopped cycling lost between 16 and 29%

of their initial body weights post infection. They concluded that their

study confirmed previous anecdotal evidence that theileriosis has an

adverse effect on reproduction. Kumar and Sharma (1991) found that

anoestrus cows and repeat-breeder animals had significantly lower

haemoglobin concentration values than normally cycling cows. It also

came in line with Hossain et. al. (2003) observations in which he

reported that the number of pregnant ewes were higher in high energy

supplemented ewes compared to low energy supplemented ewes.

Similarly, birth weight of lambs was also higher on high energy diet

26 

 

compared with that on low energy diet. They also reported that, size,

weight and health status of ewes may be an important factor which

affects birth weight of lambs.

1.12 Haematological and biochemical changes due to theileriosis:

Concerning changes in blood chemical components .Ydav and

Sharma (1986) who experimentally infected calves with Theileria

annulata showed that the level of glucose, calcium, proteins,

phosphorous, magnesium and potassium decreased and that of bilirubin,

cholesterol and alkaline phosphatase increased with no changes in

sodium and acid phosphatase levels. They attributed these biochemical

alterations to the injury of the liver and other organs. In another study

aimed to evaluate serum chemistry and haematological picture of 80

cross-bred calves infected naturally with Theileria annulata compared

with healthy controlled calves, El Haj (2003); revealed low values of the

haematological findings compared with that of the control calves. He

reported that the packed cell values (PCV) averaged to 15.88% ,

erythrocytes count (RBCs) to 4.65 million cell/ ml, total leucocytes count

(WBCs) to 2.8810 cell/ml, hemoglobin concentration (Hb) to 5.84 g/dl,

mean corpuscular volume (MCV) to 33.85 Fl, mean corpuscular

hemoglobin (MCH) to 12.6 pg, mean corpuscular hemoglobin

concentration (MCH+C) to 37.54 g/dl. For the control calves, the average

values were 28.07%, 11.01, 8.37.10 cell/ml, 33.54 Fl, 39.32 g/dl, 5.3110

cell/ml for PCV, Hb, RBC, MCV, MCHC and WBC respectively.

Ramazan and Ugur (2007) reported that changes in selected serum

components were investigated to elucidate metabolic profile in cattle

naturally infected with T. annulata. Statistically significant increases

27 

 

were observed in the mean serum activity of aspartate aminotransferase,

alanine aminotransferase, gama-glutamyl transferase, bilirubin,

creatinine, urea and creatinine kinase and statistically significant

decreases were seen in the mean serum contents of glucose, total protein,

albumin, triglycerides, cholesterol, calcium and phosphorus along with a

non-significant decrease in iron level in infected animals when compared

with controls (uninfected). The study has shown that T. annulata

infection in cattle is associated with profound biochemical changes.

ömer Kizil et. al. (2007) conducted a study to detect the differences of

biochemical parameters related with the degree of anemia in cattle with

theileriosis. In their study, 30 cattle with tropical theileriosis were divided

into 3 groups according to their haematocrit values, and 10 healthy cattle

were used as control. They observed significant differences in

biochemical parameters such as glucose, urea, triglisrides, total protein,

albumin, total bilirubin, and AST, ALT, CK, Na and K level in cattle

with tropical theileriosis related with the degree of anemia.

Singh, et al. (2001) observed that there was a progressive decrease

(P<0.05) in the haemoglobin and packed cell volume. Also, serum

analysis revealed a decrease (P< 0.05) in the concentration of calcium,

cholesterol and triglycerides, while there was an increase (p< 0.05) in the

concentrations of blood urea nitrogen as compared to day 0 values. The

total serum proteins, albumin and serum immunoglobulin concentrations

and the albumin-to immuno-globulin ratio showed marked decreases (p<

0.05) .Omer et. al., (2003), studied biochemical parameters in adult and

young Friesian cattle naturally infected with Theileria annulata. They

found that cattle infected with T. annulata had significantly lower serum

28 

 

total protein , albumin, globulin, creatinine, calcium, phosphorus,

magnesium, potassium, iron and copper concentrations and significantly

higher AST activity and bilirubin concentration than the healthy cattle.

The heamatological profiles were studied. Changes in blood parameters

in T. annulata infected cattle indicated severe macrocytic hypochronic

anemia, panleucopenia, lymphocytopenia, eosinopenia, neutropenia and

thrombocytopenia but no reticulocytosis. Ahmed and Yassir (2004)

observed that calves infected with T. annulata showed low levels of

packed cell volume, haemoglobin concentration and glucose

concentration compared to healthy calves. Infected calves showed low

levels of total protein and albumin compared to healthy calves. Serum

urea and triglycerides of infected calves showed high levels compared to

healthy calves. Serum alanine transaminase and aspartate transaminase

were increased in the infected calves compared to healthy calves. Serum

sodium and potassium showed slightly decrease in the infected calves

compared to healthy calves.

Getnet (2005) found that variations in blood values due to late

pregnancy were observed. WBCs, RBCs, PCV and Hb were slightly higher

(P>0.05) for late pregnant camels compared with the values obtained for

non-pregnant ones. Similar findings were seen in cattle (Jain, 1986). An

increase in the leukocyte picture was observed for pregnant camels

comparable with the results reported by Coles (1986) for cattle. This could

be due to the physiological increase of neutrophil count at late stage of

pregnancy. Increased WBC count particularly neutrophil count was noted at

the period near to parturition day in cattle (Doxey, 1977), which was in

agreement with the present study. Amina (2002) concluded that there was a

29 

 

decrease in total protein, albumin and globulin in late pregnancy in cross

bred goats, but pregnancy did not affect the level of urea. Ahmad et. al.

(2004) also, found that, infected calves showed low levels of pakced cell

volume, haemoglobin concentration and glucose concentration compared to

healthy calves. Also, they found that, infected calves showed low levels of

total protein and albumin, serum urea and triglycerides compared to healthy

calves.

Differences were seen between young and mature animals as Otto et.

al. (2000) reported. Among the enzymes measured, only, ALP showed a

significant difference, being much higher in young animals. This difference

is seen in other species and is the result of the faster growth rate in young

animals, and leakage of the enzyme from the growing bones and intestines

into the blood The significant difference between young and mature Angoni

cattle in serum proteins is due to longer exposure to the various antigens and

pathogens and production of antibodies. The higher globulin concentrations

led to higher total serum proteins and lower albumin to globulin ratio.

Among the metabolites measured, the only significant difference seen was in

serum urea which was higher in the mature animals. This could be a result of

the greater efficiency of converting nitrogenous substances to amino acids

and proteins, leading to a faster growth rate in young animals. The reason for

the significantly lower levels of calcium and potassium seen in mature

Angoni was not clear. As for calcium, it may be a result of continuous

suckling by the young cattle, calcium-rich milk and lactating by mature

females losing calcium in the milk. Otto e. al. (2000), has also found that The

physiological status of the cows significantly affected the serum levels of

some blood constituents. Differences were seen in serum LDH, ALP,

30 

 

glucose and calcium, which were highest in the non-pregnant, non-lactating

cows and total proteins which were the lowest in the 3 groups. Highest

globulin and cholesterol levels were seen in the lactating cows. All these

differences could be related to the differences in the animals’ metabolism,

needs for milk production and metabolic changes related to the development

of the fetus.

1.13 Molecular biology studies on Theileria:

A 398-bp DNA fragment was specifically amplified from blood

samples obtained from sheep naturally infected with T. ovis. No PCR

products resulted from T. lestoquardi, T. annulata, T. parva, T. buffeli and

Babesia spp. on using these specific primers. The sensitivity of the nested

PCR for T .ovis which was assessed showed that one infected cell in 107

sheep erythrocytes, equivalent to a blood parasitemia of 0.00001% could be

detected. This is more sensitive than examining 200 fields under light

microscopy (Altay et al. 2005). They reported that the primer pairs described

in their study will be useful for epidemiological studies on ovine theileriosis

and for discrimination between T. lestoquardi and T. ovis infections in

sheep.

Spitalska et. al. (2004) found that Theileria lestoquardi and

T.annulata can occur in similar vectors, and current available probes based

on the 18th rRNA gene showed cross-reaction between the two species.

However, they developed a species-specific RFLP test based on the Mspl

restriction enzyme, able to cut amplified products from T. lestoquardi only

and to discriminate the two species in both tick and blood samples.

Schnittger et al. (2004) found that characteristic sequence signature was

31 

 

identified within the hyper variable region 4 (v4 region) of the small

ribosomal RNA gene of ovine/caprine piroplasm species including

Theileria lestoquardi, T.ovis, T. separate, Babesia ovis, B. motasi, B.

crass (comprising strains B. crassa (Iran) and B.crassa (turkey))and

several novel species: Theileria sp. 1 (China), Theileria sp. 2 (China) and

Babesia sp. (China) [comprising strain Babesia sp. (Lintan), and Babesia

sp. (Ningxian)] as defined previously. Based on the ascertained gene

variations, a reverse line blotting (RLB) assay was developed enabling

direct, concurrent, highly specific and sensitive identification of virtually

all presently known ovine/caprine piroplasm species. All probes are

bound to their respective target sequence only; therefore, no cross-

reaction was observed resulting in clear recognition of individual strains,

species or groups. No signal was observed when ovine and caprine

genomic DNA was used as the control, demonstrating that the signals are

due to the presence of parasite DNA in investigated samples.

Furthermore, the sensitivity of RLB could be considerably to detect a

parasitemia level of at least 10 -12 % by reamplification of PCR products

(nested PCR) thereby, substantially increasing the possibility of

identifying carrier animals.

Schnittger et. al. (2002) identified a gene from Theileria

lestoquardi and from a recently described Theileria species which is

highly pathogenic for small ruminants in China. Since the taxonomic

position of the latter parasite is still not clear, they refered to it as

Theileria (China) species. The gene described in their study comprises an

open reading frame of about 948 bp which prospectively encodes a 35-

kda protein. Its sequence is most closely related to the polymorphic

32 

 

immunodominant membrane protein of T. parva (36% identity). A search

for sequence patterns and motifs within the predicted amino acid

sequence revealed that this gene possesses three membrane-spanning

regions at its C-terminal part, suggesting that it is a membrane protein.

Several allelic variants of this gene were found in each parasite species

demonstrating interspecies and interspecies variation. The predicted

amino acid sequence variants display a substantial size and sequence

polymorphism in the central part of its presumed extra cellular region,

while the N-terminal and the trans membrane / intracellular regions are

highly conserved.

Need has arisen for sensitive and specific diagnostic tests that will

distinguish between the two species in the vector tick, allowing the

epidemiology of both diseases to be clearly defined. A contribution to

this has been the development of a polymerase chain reaction using

specific primers which amplify, only in T. lestoquardi infected ticks, a

785 bp fragment of the gene that codes for a 30 KD merozoites surface

protein. The sensitivity of this test and its application to the detection of

T. lestoquardi in infected H. anatolicum anatolicum tick, in the blood of

three species of domestic ruminants and in cell cultures established in

mononuclear cells of sheep and goats is also discussed (Kirvar et

al.1998). Leemans et. al. (2001) studied phenotypes of sheep cell lines

infected with Theileria lestoquardi or T.annulata by flow cytometric

analysis, following immunolabelling with a panel of monoclonal

antibodies reacting to leukocyte differentiation antigens cell lines from

cattle undergoing acute T. annulata infection. Besides the non-lineage,

specific markers CD45, MHC class I and MHC class II, myeloid lineage-

33 

 

associated antigens and B cell-specific markers were expressed in all five

different types of lines, suggesting that both T. lestoquardi and T.

annulata had infected the same cell types in sheep as T. annulata in

cattle, notably monocytes, macrophages and B cells. Lineage- specific

markers were generally expressed at low frequency and intensity. Any

differences between the five types of cell lines were quantitative, rather

than qualitative. Thus, relative rather than absolute differences in cell

preference of sporozoites of T. lestoquadi and T. annulata may contribute

to the differences observed in previous studies in the course of the

infection of sheep with each of these two parasites and in the infection of

cattle with T. annulata.

Schnittger et. al. (2000) reported that a fatal disease of sheep and goats in

the northwestern part of China has been reported to be due to Theileria

lestoquardi (syn. T. hirci). However, some characteristics of the causative

agent are not in accordance with attributes ascribed to this parasite. They,

therefore, determined the nucleotide sequence of the small-subunit

ribosomal RNA (srRNA) gene of T. lestoquardi and the parasite

identified in China and compared it with that of other Theileria and

Babesia species. In the inferred phylogenetic tree, the srRNA sequence of

the Chinese parasite in the northwestern part of China has been reported

to be due to Theileria lestoquardi (syn. T. hirci). However, some

characteristics of the causative agent are not in accordance with attributes

ascribed found to be most closely related to T. buffeli and clearly

divergent from T. lestoquardi, suggesting that it is as yet unrecognized

Theileria species. Extensive structural similarities were observed

between the srRNA sequences of T. lestoquardi and T.annulata,

34 

 

revealing a close phylogenetic relationship between these two Theileria

species. On the basis of the srRNA nucleotide sequence, polymerase

chain reaction (PCR) primers were designed that specifically amplified

genomic DNA of the Chinese Theileria species. These primers may be

valuable tools in future epidemiology studies.

Bakheit (2006) reported that a PCR strategy was used to identify

potential antigenic proteins of T. lestoquardi suitable for development of

an ELISA by searching for homologous proteins previously identified in

other theilerial parasites to be antigenic. Bakheit et. al; (2006) Applied a

newly discovered surface protein of T. lestoquardi (Clone-5) in an

enzyme-linked immunosorbent assay (ELISA) and the potentials of the

application of the test in epidemiological surveys and diagnosis were

described. ELISA based on this recombinant protein, demonstrated a

satisfactory performance with a calculated sensitivity and specificity of

94.6 and 88%, respectively, when counter tested with a standard indirect

fluorescent antibody test. Moreover, no cross-reactions could be

demonstrated against Theileria species (China). This test is

recommended for further field validation experiments. Also, Bakheit et.

al. (2006) isolated , established and characterized a Theileria lestoquardi-

infected cell culture (line) as a source of biological material and

generated a schizont cDNA library for further studies aiming at the

identification of antigenic protein.

Awadia et.al. (2008), studied potential molecular markers for

identification of attenuation in a T. lestoquardi-infected cell line to be

used vaccination trials. Two markers associated with attenuation in T.

annulata vaccine strains were analyzed. The result showed a decreased

35 

 

activity of MMP9 and decreased mRNA expression of TNFα with

increasing passage number.

1.14 Sheep in Sudan:

Sheep belong to the tribe Caprini of the family Bovidae in the sub-

order Ruminantia of the order Artiodactyla of the genus Ovis (Mason

1951).

Four main sudanese groups of sheep have been identified (Tothill,

1948; Mcleroy, 1961a; 1961b; Wilson and Clarke, 1975) in relation to

physical features and to ecological distribution. These groups are: sudan

desert; Sudan nilotic; Sudan arid upland; and Sudan equatorial upland. In

addition, fused ecotype groups have been recognized, resulting from non-

systematic crossbreeding of the pure type. The Sudan desert comprises

more than 65% of Sudanese sheep. It is more productive than the other

local sheep groups which gives it priority in research and development

programmes.

Tribal subtypes of Sudan desert sheep include, Shugor, Dubasi,

Watish, Kababish and Baggara. Other classifications include Gezira

1.14.1 Sheep reproduction:

Reproductive performance may be measured in many ways .

Because the emphasis is on selection for reproductive performance, the

measures used have to be made on the individual ewe under near normal

condition of folk management. For this purpose, the usual measure is

reproductive rate which is defined as the number of lambs weaned per

ewe per year. It is a function of fertility, fecundity and survival.

Reproduction efficiency can be measured and expressed by the kidding

rate, weaning rate, kidding interval, live weight of kids and the length of

36 

 

reproductive cycle. (Greyling, 1988; 2000).

1.14.2 Estrus synchronization:

Estrus synchronization (ES) has been considered as a main and

valuable method to manage reproduction in farm animals (Lumeng et. al.

2008). Estrus synchronization in live stock focuses on the manipulation

of either the luteal or the follicular phase of the estrus cycle. In does and

ewes, the opportunity for control is greater during the luteal phase, which

is of longer duration and more responsive to manipulation (Wildeus,

1999).

Several methods of estrus synchronization are known to exist of

which intravaginal sponges have been the traditional treatments of choice

in small ruminants. They are impregnated with progestagens that are

effective at lower dose levels than the natural progesterone. Two types of

intravaginal sponges are commonly used for synchronization and or

induction of oestrus: Medroxyprogesterone acetate (MAP) marketed as

veramix intravaginal sponges and Flurogestrone acetate (FGA) marketed

as chronogest (Baril et. al., 1993; Romano 1996; AK at. al., 1998;

Romano 1998; Motlomelo et. al. 2002).

Sponges are usually inserted over a period of 9 to 19 days and used

with conjunction with pregnant mare serum gonadotrophin (PMSG).

PMSG administration with follicle stimulating hormone (FSH) and

luteinizing hormone (LH) activity 48 hours before or at progestagen

withdrawal, have been shown to stimulate follicular growth in both

anoestrous and cycling goats and after the time of ovulation (Bretzlaff,

1997; Leboeuf et. al., 1998).(Usually, PMSG administered at the time of

proestagen removal during the non breeding season (Motlomelo et.

37 

 

al.,2002) whereas during the non breeding season administered 48h prior

to progestagen removal (Baril et.al., 1993).) At the same time, the use of

the synthetic prostaglandin F2α analogue, cloptostenol, cases luteolysis

in does having a functional corpus luteum at the end of the treatment

(Baril et.al. 1993, Bretzlaff, 1997; Leboeuf et. al. 1998; Motlomelo et. al.

2002).

Another form of progesterone method is what is known as the

Controlled Internal Drug Release (CIDR) device which is made of

progesterone – impregnated medical silicone elastomers and were

developed in New Zealand. However, the use of progestagensin

improving fertility, pregnancy and kidding rates will continue to be

available option in reproductive management of goats (Chemineau et. al.,

1999; Sahlu and Goetsch, 2005).

1.14.3 Progesterone:

Hormones are chemical substances secreted directly into the blood

stream by ductless glands to aid in the integration of body processes by

their stimulatory or inhibitory effects on target organs.

Progesterone is a steroid hormone of molecular weight about 314.5KDa.

It is usually secreted by the corpus luteum in woman during menstrual

cycle and in a much higher amount by the placenta during pregnancy. It

is also secreted in a minor quantity by the adrenal cortex in both male and

female. The majority of circulating progesterone is bound to albumin and

corticosteroid globulin (CBG). The bioactive free hormone represents

only 2.5-3% of the total progesterone (IAEA, 1984).

In both goats and sheep, pregnancy is maintained by progesterone

(P4). P4 in the ewe is produced frist by the corpus luteum and then by the

38 

 

placenta in the last-third of pregnancy. However, in goats the sole source

of P4 throughout pregnancy is the corpus luteum (Thornburn and

Schneider, 1972).

Progesterone is critical for maintenance of pregnancy (Vasconcelos,

et. al., 2003). Plane of nutrition combined with high clearance rate of P4

in the liver has been found to have an inverse relationship with

circulating p4 in ewes and gilts (Parr et. al., 1993; Prime, 1993).

Measurement of serum progesterone is of diagnostic value for

reproduction disorder and infertility. Plasma P4, fat milk P4 and fat free

milk P4 have been examined and high correlation were found between

their values (Heap and Holdsworth, 1981).

1.14.4 Gestation length:

It could be defined as the period from successful fertilization of the

ova to parturition. Gestation length is influenced by several factors including

sire breed (Fogarty et al., 2005), dam age, litter size and litter weight at

birth. Older dams have significantly longer gestation lengths (Vatankhah et

al., 2000; Koyuncu et al., 2001), as do ewes carrying single lambs compared

to those carrying multiples (Osinowo et al., 1994; Vatankhah et al., 2000;

Koyuncu et al., 2001; Dwyer, 2003; Fogarty et al., 2005). Gestation length

increases with total litter birth weight (Knight et al., 1988; Osinowo et al.,

1994; Vatankhah et al., 2000; Fogarty et al., 2005) and may be longer when

the dam is carrying a male lamb compared to a female (Koyuncu et al.,

2001; Vatankhah et al., 2000; Fogarty et al., 2005).

The gestation length for most breeds ranges from 143-153 (ELNaim,

1979). Musa et.al, (2005) revealed that there are differences between

breeds and with in the same breed in gestation length. It increases with

39 

 

increasing age of ewe to 4 years of age, recorded that the mean gestation

length for experimental ewes was 150.44 ± 2.64 days ranging between

145-156 days. The effect of ewe age on gestation length was not

significant (p>0.05).The average gestation length for male-born lambs

was 150.95 ± 2.48 days, while for female born lamb it was 149.50 ± 2.59

days. In both sexes, single born lamb had longer gestation period than

twin born lambs (Musa et.al. (2005). Mohamed et. al. (2008) mentioned

that dietary supplementation decreased the gestation period significantly

(p<0.05); the mean values for the non-supplemented and the

supplemented were 155.7 ± 0.3 and 152.8 ± 0.4 days respectively.

1.14.5 Conception rate:

Conception rate is defined as the percentage of ewes

present and percentage of ewes pregnant Older ewes had a better

conception rate lower barrenness rate and higher litter size than

the young 2teeth ewes, as reported by Musa et.al, (2005).

Mohamed et. al. (2008) found that the pregnancy diagnosis test of

Sudanese desert sheep indicated that the conception rate was

higher in the supplemented ewes compared to thenon-

supplemented ewes.

Sudanese sheep are not seasonal breeders and may have lambs

throughout the year. Many local breeders, however, have

attempted to establish two main breeding seasons, one during the

wheat and cotton harvest in the winter and the other during the

rainy season (two lambing seasons) some breeders are now

turning to more intensified rearing so that lambs feed and allow

40 

 

rams to run freely with recently lambed ewes.

1.14.6 Lambs survival and mortality rates:

Lamb birth weight was the single greatest factor influencing lamb survival.

Lambs born light had low lamb survival as compared to lambs born with

medium or higher birth weights. Several authors (e.g. Dalton et al., 1980;

Scales et al., 1986; Ahmad et al., 2000) have also demonstrated that

increasing birth weight result in a decrease in mortalities but with an

inverted U-shaped distribution of birth weight with survival. however, birth

weights on the higher end of the range are not detrimental to survival. Birth

weight is influenced by ewe prenatal nutrition, litter size, placental size and

foetal genotype (Haughey, 1993). Factors which contribute to low birth

weight also tend to reduce foetal lipid reserves, limit neonatal vigour, impair

colostrum production and restrict ewe milk production (Mellor & Murray,

1985a, b). Data on birth weight consistent with optimal lamb survival in

tropical breeds are scarce.

Results of preliminary analysis on lamb survival indicated that the overall

flock pre- and post-weaning mortality averaged 20 and 24%, respectively,

leading to a flock mortality rate of 44% (including 1.6% stillbirths) up to 1

year of age. Mortality rate was influenced by many factors whose

importance varied with lamb age. The pre- and post-weaning death rates

were 25.3 and 34.2%, respectively, for Horro in contrast to 8.8 and 19.3%

for Menz sheep. As a consequence, mortality up to 1 year of age was about

twice as high for Horro than Menz lambs (59 vs. 28%, respectively). Markos

(2006)

Birth weight was also a major risk factor for pre-weaning Mortality In

comparison to heavy weight lambs, this was in agreement with the

41 

 

observation that lambs born multiple and/or lambs born lighter were also

more likely to die of any cause than single-born lambs

Female lambs had a better chance of survival during all periods compared

with males. Twin born lambs had about 50% greater hazard of mortality than

singles for the early postnatal period (1-120 days) and the hazard of

mortality for triplets were about twice as large as that for twin born lambs

for the same period. The effect of type of birth on hazard of mortality

decreased with lamb age (Judith Collins and Joanne Conington (2005).

Lamb mortality is the single most important constraint limiting productivity.

Studies indicate that up to 50% of the lambs born can die mainly due to

diseases and other causes such as adaptation failure, dystocia, cold stress,

starvation and mismothering Hinch et al., 1986). Information is required on

pattern and causes of mortality to improve survival.

Mazumdar et.al. (1980) reported mortality of 100% for kids weighing 1.0

to 1.5 kg and 32.2% for kids weighing from 2-2.5%kg. Whereas Mtenga

et. al., (2006) reported a mortality rate of 57.9% for kids less than 1.5kg.

The effect of nutrition on embryo survival is limited as recently reviewed

by Abecia and Rhind (1994) and by Hanrahan (1994). According to the

later review, only 10% of the probability of embryo survival can be

explained by changes in feeding levels within the range of 0.5-1.5 of

maintenance requirements

Birth weight has a quadratic relationship with hazard of mortality with both

lighter and heavier lambs at greater risk (eg of hypothermia and dystocia

respectively, among other factors).However, as mentioned above, higher

birth weight lambs have a better chance of survival during the early

postnatal period. Selection for optimal rather that maximum birth weight

42 

 

should therefore be practiced when viability lamb at birth and birth weight

are to be improved simultaneously (Sawalha et. al., 2006).

Mukasa (2002) reported that young mortality rates in sheep and goats

from birth to weaning range from5 to more than 50% and represent a

serious reduction in biological efficiency

1.14.7 Lambs birth weight:

Musa et.al. (2005) revealed that lamb birth weight is an important

factor in sheep production. It is influenced by genetic and environmental

factors. Age of dam had an effect on lamb birth weight. Males and

single born lambs were heavier than females and multiple born lambs.

Amoah and Gelaye (1990), in their study in the reproductive

performance of goats in south pacific coutries, found that the period of

breeding had a positive relationship with birth weight of kids. Musa et.

al,(2005), investigated the production performance of West African

Sheep in Sudan found that the overall birth weight of lambs was 2.90 ±

0.50kg, the average for males and females were 3.18±0.49 and 2.70 ±

0.41kg respectively. They also found that male lambs were similar to

females during all periods of the first year from birth. On the other hand,

body weight for adult rams and ewes was recorded. For rams, it ranged

between 35.35-49.53kg with an average of 43.83kg. While for females,

it ranged between 29.20-37.58 kg with an average of 29.79kg. Mohamed

et. al, (2008) reported that the birth weight was significantly higher for

lambs born to supplemented ewes compared to those born to non-

supplemented ewes.

43 

 

1.14.8 Puberty :

Puberty in the female sheep occurs in the first or second year of

life .It is the age at which females show interest to the male, associated with

many physiological (hormonal) and morphological changes. Puberty, the

time of first sexual activity, it is a function of both age and body weight

Lambs reared on a high plane of nutrition reached puberty earlier than

those on a low plane. This is partly a reflection of the tendency to reach

puberty only above a certain body weight and partly a direct effect of

nutrition on the reproductive system. Suffolk lambs and Merino ewes

reach puberty at 5 to 9 months of age. In a small group of ewe lambs

measurement of progesterone concentration in peripheral blood showed

that there was no ovulation until the time of the first estrus detected by a

ram. In another small group, ewe lambs ovulated once or twice before

demonstrating estrus. Spontaneous estrus and ovulation, however, appear

to depend on the initiation of hypothalamic activity by estrogens. (Hare,

et. al. 1982). Ewes typically reach puberty at 5 to 12 months, depending

on breed, nutrition, and date of birth. Well grown Merino ram lambs

reach puberty at around 6 months of age and by 12 months can produce

Disclaimer, (2008).

In Sudan studies , at ElHuda research station, on three sub-types of

Sudan desert ewes showed that first lambing for Shugor type was 428 ±

3.0 days (14.26 monthes), 429 ± 5.6 (14.3) for Dubasi , 406 ± 9.0 (13.5)

for Watish, and 13-15 months for Southern Darfur traditional system. By

subtracting months of gestation, puberty for Shugor type was

approximately 6-9, Dubasi was approximately 6-9, Watsish was

approximately 5-9, and 8-10 for Southern Darfur traditional system.

44 

 

CHAPTER TWO

45 

 

Materials and methods

2.1 Study area:

The study was carried out at the Department of Radioisotopes, Central

Veterinary Research Laboratories, Soba West, which is located 25

kilometers south east of Khartoum.

2.2 Study population:

The study was conducted on Sudanese desert ewes purchased from

Abuzeid market which is located west of Omdurman. First purchase was

30 adult ewes aged 2-3 years and the second purchase was 15 female

lambs approximately 4-5 months old. They were bought after confirming

that they were free of ticks, parasitic infections and Brucella.

Three healthy adult rams (2-4 years) were also purchased from the same

market for the purpose of natural mating.

2.4 Housing:

Each group was housed separately at the Department of Isotopes barns of

20 square meters each. They were provided with metal feeding troughs

and plastic containers for water.

2.5 Management and health control:

Before the commencement of the experiments, the animals were kept for

five weeks to adapt themselves to the new environment and to be

clinically examined for any clinical detectable diseases such as

brucellosis, trichomniasis and any reproductive disorders or

abnormalities.

All animals were dosed with broad spectrum antihelmenthics and anti

coccidial drugs.

46 

 

2.6 Feeding:

The animals were fed on green forage and supplemented with a

commercial pelleted diet

2.7 Experimental design:

Two major experiments were conducted. They comprised the

following:

2.7.1 Experiment one:

This experiment was conducted on 30 Sudanese desert ewes. They

were randomly divided into three groups (A,B and C) of ten each.. Ewes

of group A were experimentally infected with Theileria lestoquardi

before mating and conception. Ewes in Group B were also

experimentally infected with the same strain of Theileria lestoquardi but

after confirmation of being pregnant (1-3 months), whereas, ewes in

group C were subjected to mating and remained as the uninfected control.

2.7.2 Experiment two:

This experiment was conducted on 15 female lambs  aged from 5-6

months at the start of the experiment. They were divided into two groups.

Group 1 (7 ewe lambs) were experimentally infected with Theileria

lestoquardi and group 2 (8 ewes lambs) were kept as the uninfected

control.

2.8 Tick collection and identification:

Engorged adult ticks were collected from sheep raised in the farm of

the University of Khartoum which is located at Shambat using a pair of

blunt forceps .Ticks collected were identified at the department of ticks at

Soba with the aid of the key of Hoogstral and Kaiser (1959). After

identification, only Hyalomma anatolicum anatolicum was collected.

47 

 

2.8.1 Rearing of ticks:

Ticks reared for the pick up trials were first allowed to feed on

clean rabbits. The ticks were reared to the fifth generation in order to

ensure their freedom of infection, and also to harvest as many ticks as

possible.

2.8.2 Laboratory maintenance of ticks:

Engorged females of Hyalomma anatolicum anatolicum were

kept individually in 10 x1.5cm round bottomed plastic tubes covered

with a piece of cotton and gauze. Flat larvae were allowed to feed on

sheep or goat till moulting to the next nymphal stage in 7x1.5 cm round

bottomed plastic tube covered the same way as above. Flat nymphs were

also allowed to feed on a sheep or goat by applying the same method

described before till moulting to flat adults which is also fed till they get

engorged. These ticks were kept in 10 x1.5cm round bottomed plastic

tubes. Each tube was then covered as previously mentioned, labeled

indicating the starting date of the developmental stage and the number of

generation. All tubes were placed in a desiccator containing saturated

solution of sodium chloride (Nacl) to maintain a 75% relative humidity

according to Buxton and Mellanby (1934). Petroleum wax was smeared

on to the rim of the desiccator cover to ensure tight sealing. The

desiccator was incubated at 280C. Ventilation was provided during daily

inspection.

2.8.3 Handling and counting of tick stages:

A fine hair water- color brush was used for handling and

counting of eggs, larvae and nymphs in a petri-dish placed on a white

48 

 

paper on ice to minimize the movement of ticks.

2.9 Source of infective material:

A naturally infected sheep was diagnosed at Atbra Regional

Veterinary Research Laboratory as having 10% infection with T.

lestoquardi was brought to Khartoum. The infection was confirmed by

The Department of Entomology and Ticks at Soba. The sheep was then

kept and used as the source of infection.

2.9.1 Experimental infection of ticks:

Flat nymphs of Hyalomma anatolicum anatolicum were applied

on the infected sheep to pick up the infection using ear bags according to

the method described by Bailey (1960). Ear bag is made of white cotton

sleeves 6-8 inches long and 2-3 inches wide which is open from both

sides. Sheep was prepared by clipping the hair around the ear base. One

end of the ear bag was fixed to the base of the ear by shoe glue secured

by zinc oxide adhesive tape. Ticks were placed into plastic tubes, 30 ticks

each (15 males and 15 females) and plugged with a cotton wool. The tube

was inserted into the ear bag through the open free end which is extended

well beyond the ear tip. The cotton plugs were removed and the tube was

palpated by a brush allowing ticks to get out and spread inside the ear

bag. The open end was sealed using a piece of adhesive tape. The ear bag

was inspected every other day by opening of the free end and rolling

back the sleeve to expose the ear. After 5-7 days, engorged nymphs

inside the ear bag were collected in a plastic bag, counted and placed in

plastic tubes as described before. The ticks were left in the desicator to

molt to the next stage (flat adult stage) which was considered the source

of infection to the experimental ewes.

49 

 

2.9.2 Experimental infection of animals:

Infected flat adults were allowed to feed on the experimental ewes

and experimental lambs by the same method of the ear bag described

above.

2.10 Detection of infection in experimental ewes and lambs

throughout the period of study:

2.10.1 Physiological examination- temperature detection:

Seven to 21 days post tick application, establishment of infection

was monitored daily by measuring rectal temperature.

2.10.2 Parasitological examination - blood smear:

Thin blood smears from the ear vein were prepared daily, fixed with

absolute methanol and then stained with fresh Giemsa stain { prepared

according to the method described by Kolmer et al (1951)}. until the

appearance of the stages of Theileria lestoquardi.

2.10.3Serologically examination – indirect fluorescent antibody test

(IFAT):

The indirect fluorescent antibody test as described by Morzaria et,

al. (1977) was carried out to show the presence of the parasite (T.

lestoquardy) through out the experiment. Antigen was prepared from

cultures made from parasitized blood collected from infected sheep.

These cultures were washed 3 times in phosphate buffered saline (PBS)

at 7.2 PH (KH2PO4 83.3 mM, Na2 HPO4 66 mM, Na Cl 14.5 mM) then

smeared on a slide which was circled with a polish. These slides were

smeared with the prepared antigen then, dried and fixed with acetone 20

minutes at room temperature, dried and stored at normal refrigerator.

Standardization was done with sera collected from the experimental

50 

 

animal in the discriminating dilution 1:80. An amount of 10 µ of the

diluted serum was added to each circle on the slide and incubated for 30

minutes at 370C. following three washes with PBS, the smears were

incubated for 30 minutes with rabbit anti-bovine IgG fluorescin

isothiocyanate conjugate (Sigma) diluted 1:400 in PBS. Finally, the

smears were washed three times, dried and one drop of 50% glycerin/

PBS solution was placed on each slide and covered with a cover-slip

then examined by epifluorescent microscopy. Negative sera showed no

background fluorescence, while positive sera showed strong fluorescence

2.11 Oestrous synchronization:

Synchronization was performed so as to bring about oestrus

cycle for experimental ewes at the same time. Synchronization was

conducted by insertion of progesterone releasing intravaginal device

(CIDR) that contains 0.3 gm slow release progesterone (Inter Ag,

Hamilton, Netherlands). The CIDR remained in situ for 14 days (Ritar,

et. al. 1984). At time of CIDR withdrawal, the ewes received an

intramuscular injection of 400-500 IU of pregnant mare serum

gonadotrophin (PMSG -intervrt, UK).

2.12 Ram reproductive evaluation:

The rams were selected on the basis of their sexual desire (libido)

and they were also tested for their semen quality in the animal

production research center at kuku

2. 13 Heat detection and mating:

Heat was detected at 24 - 72 hours following the removal of CIDR

and injection of PMSG. ewes that showed heat signs which appear in

rapid tail movement or raised tail, nervousness and reddened swollen

51 

 

vulva, were mated, but due to the difficulty of signs detection in ewes ,

the 3 rams were introduced to each group 24 hours post PMSG, and

ensuring that breeding occurred at least twice per mating.

2.14 Collection of samples:

After the appearance of Theileria in the blood of infected ewes,

whole blood samples were taken for heamatological investigations.

Serum was collected for the indirect fluorescent antibody test,

biochemical investigations and for detection of progesterone level.

2.14.1 Sampling protocol:

Weekly blood samples of 5ml were collected into plain and

heparinized vacutainer tubes from each ewe through out the study.

Heparanized blood samples were used for haematological analysis,

whereas, the blood in plain vacuatiners was allowed to clot and then

centrifuged at 3000rpm for 5 minutes and serum was then removed into

sealed plastic tubes and kept at -20 0C until analyzed. All the

haematological parameters were investigated using the techniques

described by Schalm (1965).

2.15 Haematological investigation:

2.15.1 Packed cell volume (PCV):

The packed cell volume values were determined by the

microhematocrit method, capillary tube for at least two-third of their total

length, sealed firmly at one end with cristaseal, and were then centrifuged

in Hawksley microhaematocrit centrifuge at 12000 rpm for 5 minutes

(Hawksly and Sons LTD, England). They were read using the

haematocrit scale described by Daci and lewis (1975).

52 

 

2.15.2 Haemoglobin concentration (Hb):

Drabkin colorimetric test:

The technique was principally based on that haemoglobin is

oxidized by potassium ferricyanide into methahaemoglbin which is

converted into cyanomethhaemoglobin by potassium cyanide. The

intensity of absorbance of the cyanomthaemoglobin is proportional to

haemoglobin concentration.

Procedure:

20µ of serum sample and standard was added to 5.0 ml of working

reagent (Drabkin) in a test tube, then mixed and incubated for 3 minutes at

room temperature; this is measured at absorbance (wavelength) 540 nm

against blank reagent. The concentration of haemoglobin was then read

from a table to change the absorbance to g/dl (Van Kempen, (1961).

2.15.3 Total white blood cells (WBCS):

Leucocytes (WBCs) were counted in an improved Neubauer

haemocytometer (Hawksley and Sons LTD, England) using Tuerk,s

solution as a diluent consisting of 1% glacial acetic acid colored with

gention violet as described by Jain (1986).

2.16 Analysis of serum:

2.16.1 Reagents and equipments:

Commercial Randox kits, (Diamond Road, Crumlin, Co. Antrim,

United Kingdom) BT29 4Qy were used for serum metabolic profile

analysis.

Spectrophotometer, Jenway, 6105 U/V spectrophotometer, U.K.,

Colorimeter, Jenway 6030 colorimeter (Jenway Ltd. Felsted Dunmow,

Essex m6-3LB U. K.), were used for the measurement of the serum

53 

 

metabolic profile.

2.16.2 Estimation of serum total proteins:

Protein in serum forms blue violet complex when mixed with copper

ions in alkaline solution (Biuret reaction). Tartarate is added as a stabilizer

and iodine is used to prevent auto reaction of the alkaline complex. The

absorbance of this complex at 546 nm is proportional to the protein

concentration, so the buffer reagent contains a mixture of sodium

hydroxide 200 m mol/l, potassium sodium tartarate 32 m mol/l, copper

sulphate and potassium iodide 30 m mol/l. 0.02 ml of the serum sample

was pipetted in a test tube. 1 ml from the mixture of the buffer reagent was

added, mixed and incubated at room temperature for 5 minutes and the

absorbance (wavelength) of the sample was then measured against a

reagent blank (0.02 distilled water in 1 ml buffer reagent) within 30

minutes, then calculated using the equation:

Total protein concentration ═ Sample X Standard concentration

Standard

2.16.3 Estimation of serum albumin:-

Principally, the measurement of serum albumin is based on its

quantitative binding to the indicator 3, 3', 5, 5'- tetrabromo-m cresol

sulphonephthalien (bromocresol green BCG). The albumin-BCG-

complex absorbs maximally at 578 nm, the absorbance being directly

proportional to the concentration of albumin in the sample.

The samples and standards were mixed according to the Randox

protocol and then incubated for 5 minutes at +20 to +25 0C. The

absorbance of the sample and the standard were measured against the

54 

 

blank reagent. The albumin concentrations of the samples were

calculated using the following formula:

Albumin concentration ═ Sample X conc. of standards (g/l or g/dl)

Standard

2.16.4 Estimation 0f serum globulins:

The serum globulins were determined by subtracting the obtained

serum albumin from that of the total protein values.

2.16.5 Estimation of creatinine:

Creatinine in alkaline solution reacts with picric acid to form a

colored complex. The amount of complex formed is directly proportional

to the creatinine concentration. The reagent composition are standard

177µmol/l (2mg/dl), picric acid 35mmol and sodium hydroxide 0.32

mol/l. The procedure requires adjustment of spectrophotometer

wavelength to 492 (490-510nm). The reagents were added according to

the randox protocol then they were mixed and the absorbance A1 of the

standard and sample was read after 30 seconds exactly. 2 minutes later,

the absorbance A2 of the standard and sample was read again.

The result was then calculated by substracting A1 from A2 to give

∆ A standard.

Concentration of creatinine in serum ═ ∆ A sample X 177 µmol/l

∆A standard

or ═ ∆A sample X2 mg/dl

∆A standard

2. 16.6 Estimation of serum urea:

Modified urease –Berthelot method:

The method is based on that the urea in serum is hydrolyzed to

55 

 

ammonia in the presence of urease; the ammonia is then measured

photometrically by Berthelot´s reaction.

Urea + water Urease ═ 2NH3 + C O2

Salicylate and hypochloride in the reagents react with the ammonium

ions to form a green complex (2.2 dicarboxylindophenol). The

absorbance of the samples and standard were read against the blank with

the wave length adjusted at 546 nm, the color of the reaction is stable for

8 hours.

Urea concentration in mmol/l ═ Sample X 13.3

Standard

In mg/dl ═ Sample X 80

Standard

2.16.7 Estimation of serum progesterone by radioimmuno assay

(RIA) method:-

Progesterone [I125] RIA –kit Ref: Rk-460.

The progesterone [I125] assay system provides the quantitative invitro

estimation of progesterone in serum. Progesterone can be assayed in the range

of 0-120 nmol/l using 50 µ serum samples. Each kit contains materials

sufficient for 100 assay tubes, permitting the concentration of standard curve

and assay of 80 unknowns (serum samples) and two controls in duplicate.

Principle of the test:

This assay is based on the competition between unlabeled

progesterone and a fixed quantity of I125 labeled progesterone for a

limited number of binding sites on progesterone specific antibody.

Allowing a fixed amount of tracer and antibody to react with different

amounts of unlabeled ligands, the amount of tracer bound by the

56 

 

antibody will be inversely proportional to the concentration of

unlabeled ligand. Upon addition of magnetizable immunosorbent, the

antigen-antibody complex is bound on solid particles which will then be

separated by magnetic sedimentation. Counting the radioactivity of

solid phase enables a standard curve to be constructed and samples to

be quantitated.

Contents of the kit:

-One vial of I125 tracer ready to use (11 ml per vial) containing about

130Kbq progesterone-11-hemisuccinate-[I125]TME in buffer with

0.1%NaN3.

* Six vials of standards ready to use. Their concentrations as 0, 1.5,

4, 12, 40, 120 nmol/l in serum with 0.1% NaN3 as a preservative.

* One vial of antiserum, ready to use (11 ml per vial) containing

polyclonal anti-progesterone (IgG) from rabbit in buffer with 0.1%

NaN3

* One vial containing control serum, ready to use.

* One bottle containing magnetic immunosorbent (MIS) ready to use,

consist of 55 ml per bottle containing paramagnetic particles in buffer

with 0.1% NaN3.

Assay procedure:

All reagents were equilibrated to room temperature. Duplicate

tubes were labeled for total count (T) non- specific binding (NSB), zero

standard (standard 1=Bo), standards (S 2-6), control (C) and samples

(Sx). Reagents and samples were thoroughly mixed before use.

Excessive foaming was avoided. 50µl each of the standards, control,

and samples were pipetted into the properly labeled tubes. 100µl of

57 

 

tracer solution was pipetted into all tubes. 100µl of antiserum was

pipetted into all tubes except T and NSB. All tubes were thoroughly

mixed except T, and then incubated for 2 hours at room temperature.

The bottle containing magnetic immunosorbent was gently shaken and

swirled until homogeneity. 500µl was then added to each tube. All

tubes were thoroughly vortexed, and were then incubated for 15

minutes at room temperature. The bound fraction was separated by

using magnetic separation.

Magnetic separation: The rack was attached to the magnetic separator

base and was ensured that every tube was in contact with the base plate.

The MIS particles were left to settle for 5 minutes. The rack was not

removed from the separator base. After the separation of solid and

liquid phase, the supernatant was poured off and discarded. The

separator was kept inverted, and the tubes were placed on a pad of

absorbent tissue and were allowed to drain for 2 minutes. The

radioactivity of all tubes was counted in a time not less than 60 seconds.

The concentration was calculated as described below.

Calculation of results:

The average counts per minute (CPM) for each pair of assay tubes

were calculated and the percent B for zero standard (S1) was calculated

by using the following equation: B/T% = 100 (S1-NSB)/ T.

The normalized percent binding for each standard, control and sample

were calculated by using the following equation:

B/B% = 100 (S2-6; Mx-NSB) / (S1-NSB)

Semi-logarithmic graph paper plot B/B% for each standard versus the

corresponding concentration of progesterone was used.

58 

 

2.16.8 Estimation of serum progesterone by enzyme immuno assay

(EIA) method:

Displacement of progesterone from serum binding proteins:-

This EIA system was developed for the measurement of

progesterone in serum or plasma, which is a direct 2-step serum EIA with

no pre-extraction of samples. The concentration range covered by the

standards is approximately 0-100 nmol/l.

General description of the progesterone EIA:

Progesterone in serum occurs largely bound to corticosteroid

binding protein (CBG) and albumin (I). This protein bound progesterone

is unavailable for antibody binding, as antibodies can only bind to

unbound or free progesterone. In direct progesterone assay a blocking

reagent (which binds to serum binding proteins, but not to the antibody)

is used to displace progesterone from serum binding proteins, thus

making it available for antibody binding.

Elimination of serum protein binding of progesterone tracer by two –

step assay design:

It has been reported that tracer binding by serum proteins is a

source of error in conventional (one-step) direct progesterone assay (2).

Two-steps assay designs prevent contact between the tracer and serum

binding proteins and lead to patient results which agree well with those

of extraction RIA (2).

Direct 2-step progesterone EIA design:

The progesterone EIA is a direct 2- steps assay of a limited reagent

‘competitive’ design. The assay consists of the following main stages:

59 

 

Reaction of antibody with serum progesterone:

Anti-progesterone antibody, blocking reagent and magnetic anti-

mouse IgG antibody are incubated with the sample for 2 hours at 37oC in

a water-bath. Any progesterone which binds to the solid phase antibody

is isolated by means of a magnetic wash step. Other serum components,

including binding proteins, are thus removed ensuring that they do not

bind progesterone tracer in the next step.

Reaction of antibody with labeled progesterone:

The solid phase is incubated for 15 minutes at 37oC in a water-bath

alkaline phosphatase labeled progesterone tracer. Any progesterone tracer

which binds to the solid phase is isolated by two magnetic wash steps.

Colors development:

The solid phase is incubated with a color enzyme substrate for 1

hour at 37oC. The reaction is terminated by the addition of a stop buffer

and the optical density of all tubes is measured. The progesterone

concentration of test samples is interpolated from a calibration curve.

2. 17 Statistical analysis:

The obtained data were subjected to statistical analysis using the

computer program SPSS 16 for windows. The results obtained were

expressed as mean ± SD. Student’s t-test, ANOVA test or chi square test

and the level was considered significant at p < 0.05.

60 

 

CHAPTER THREE

61 

 

RESULTS 3. Experiment one

3.1 Detection of the infection with Thieleria lestoquardi

3.1.1 Parasitological examination:

-Blood smears:

Following tick application, daily examination of blood smears of

experimentally infected ewes showed the presence of Thieleria

lestoquardi parasite on day 10th – 21st of tick application. The observed

stages were schizonts within the lymphocytes and /or a piroplasm stage

within the red blood cells (Figures 1 and 2)

3.1.2 Physical examination:

-Temperatures detection:

The mean daily rectal temperatures of experimentally infected

ewes from day 7 are shown in Figure 3. The maximum mean daily

temperature was found to be 40.4 0C. This was recorded within the

second week of tick application.

3.1.3 Serological examination:

-Indirect fluorescent antibody test (IFAT):

The IFAT was carried out for the serum taken throughout the

experiment is shown in Table (1), The Ab level was detected in blood of

the experimental animals through out the experiment.

62 

 

Figure 1:Piroplasm of Theileria lestoquardy in red blood cell

Figure 2: Schizont of Theileria lestoquardy in white blood cell 

  Figure 2: Schizont of Theileria lestoquardy in white blood cell

 

63 

 

64 

 

Table 1: Indirect fluorescent antibody test (IFAT) for T. lestoquardi in ewes infected before

conception throughout the experimental period in month

Animal

No.

1st

month

2nd

month

3rd

month

4th

month

5th

month

6th

month

7th

month

8th

month

9th

month

         1  ‐ ‐  ‐  ‐      ‐ ‐ + + + D e a d        

         2  ‐ ‐  ‐  +  +  +  ‐ ‐ D e a d        

         3  ‐ ‐  ‐  +  +  +  ‐ ‐ + + + + + + + ‐ + + +  +  + + + + + + + + + + + + + + +  + 

         4  ‐ ‐    +  ‐  + ‐ + ‐ + + + + ‐ ‐ + + +  +  + + + + + ‐ + ‐ +  + 

          5  ‐  +    +  ‐  + + + + + ‐ ‐ + + + + +    + ‐ ‐ ‐ + + + + + + + + +  + 

          6  ‐ ‐  +  ‐  ‐  ‐  + + + + + ‐ + + + ‐ ‐ +  +  + + + + + + + + + + + + + + +  + 

       7  +    +  ‐  +  + + + + + + + + + + + ‐    + + + + + + + ‐ + + + + + +  + 

       8  +  +  ‐    +  + + + + + + + ‐ +  +  + + + + + + + + + + + + + + +  + 

       9  ‐ ‐  +  +  +  +  + ‐ + + + + + + + + +  +  + + + + + + + + + + + + + + +  + 

65 

 

3.2 Reproductive performance

3.2.1 Conception rate:

Table (2) shows the conception rate in ewes infected before mating

(GA) and non infected ewes (GC). Nine ewes in GC conceived from the

first mating. This constituted about 90% conception rate, while in GA,

only one ewe conceived from the first mating which constituted about

11.11% conception rate. In the second mating, 3 ewes conceived, and

this increased the conception rate a 33.33% whereas in the third mating,

4 ewes conceived making a 44.44% conception rate. However, the ewe

that did not conceive after the 4th mating got pregnant 5 months later.

The one that did not conceive at all in GC showed a persistent rise in

progesterone concentration.

3.2.2 Gestation length:

The gestation length of the control group ewes GC was found to be

159 ±7.15 days ranging between 151-168 day which is significantly

higher (p< 0.05) than that in infected first group GA and infected after

conception group GB, which where found to be 144.2 ± 4.76 and 153 ±

4.5 days ranging from 138-150 and 148-157 days respectively.(Table 3)

3.2.3 Body weights of born lambs:

The newly born lambs in GC were apparently healthy and their

mean body weight was found to be 2.62 ± 0.233 Kg, whereas in GA,

most of the newly born lambs were born weak and their mean body

weight was 1.78 ± 0.795, while GB were heavier than that of GA with

66 

 

mean body weight of 2.4 ± 0.38 kg (Table 4).The difference in body

weights was found to be statistically significant (P < 0.05).

3 .2 .4 Numbers of delivered lambs, twining, abortions and lambs

survival rates:

The numbers of delivered lambs, twining, abortions and lambs

survival rates are represented in table (5). Ewes in GC, gave birth to 14

healthy lambs, five of them gave twins with a survival rate amounted

for 100%. Two ewes in GA died during the execution of the experiment

and the remaining ones gave birth to 9 lambs, one of them delivered

triple lambs. However, most of the newly born lambs in GA were born

weak, died either immediately after birth or shortly after with a

mortality rate of 45%. On the other hand in GB, 3 ewes aborted, one

died and one delivered twins that gave birth to 5 lambs only 60% of

them survived

3 .1.5 Progesterone rise post delivery:

Mean first progesterone (P4) rise after delivery was found to be 21.33

± 8.06 days in GC, 59.67 ± 42.79, and 40.25 ± 28.63 days in GA and GB

respectively (Table 6). This difference in intervals to progesterone rise

was found to be statistically significant (P<0.05).

 

67 

 

Table 2: The number and percentage of conception rate of ewes in

Control group (GC) and infection before conception group (GA)

Animal Group

No. of animals

Conception rate

Not conceived

1st service

2nd

service

3rd service

GA

9

11.11%

33.33%

44.44%

11.11%

GC

10

90%

_

_

10%

68 

 

Table 3: Gestation length (mean days ±SD.) in ewes infected before

conception group (GA), ewes infected after conception

group (GB) and uninfected control group (GC)

Animal group

Gestation length in days

(mean ± SD)

Range

GA

144.2 ± 4.76 b

138-150

GB

153.25± 4.50 a

148-157

GC

159.00 ± 7.15a

151-168

P- value

0.003

------

Values with different superscript letters within same column were significantly different at P<0.05

69 

 

Table 4: Mean body weight (kg) of lambs born to ewes infected before mating (GA-lambs), lambs born to ewes infected after conception (GB-lambs) and control group (GC-lambs)

Animal group

Body weight in Kg

(mean± SD)

Range

GA-lambs

1.78 ± 0.795 b

1 - 3

GB-lambs

2.4 ± 0.380 a

2 - 3

GC-lambs

2.62 ± 0.233 a

2.25 -3

P-value

0.004

____

Values with different superscript letters within same column were significantly different at P<0.05

70 

 

Table 5: Number of delivered ewes, abortions, and lamb survival rates control group G C,

infected before conception G A and Infected after conception GB

 

 

Animal groups

No. of animals

No. of delivered animals

No. of abortions

No. of lambs

Twining

lamb survival

rate

GC 

 

   9   90% 

 

 0 

    

14  5  100% 

GA 

 

7  77% 

 

 0 

   

 9  1  55% 

GB 

 

4   50%  3 

   

 5      1  60% 

71 

 

Table 6: Days from lambing to first progesterone rise (P4) in ewes

infected before conception group (GA), ewes infected after conception group (GB) and uninfected control group (GC)

Values with different superscript letters within same column

  were significantly different at P<0.05

 

 

 

 

Animal group

Days from lambing to 1stprogesterone rise (M ± SD)

Range

GA

59.67 ± 42.79a

21-90

GB

40.25±28.63a

12-73

GC

21.33 ±8.06b

9-32

P-value

0.032

------

72 

 

3.3 Haematological parameters

3. 3. 1 Haemoglobin Concentration:

The mean haemoglobin concentrations (Hb) in mg/dL are presented in

Table 7 and Figure 4. The weekly mean Hb concentration was monitored

for 10 weeks post tick application. The concentrations of haemoglobin in

GA were slightly lower than in GC, however, the difference was

significant in weeks 2, 7, 8, and 9 (P<0.05). While in week 10, the mean

Hb concentration showed insignificant difference between the two groups

In pregnant ewes, the mean Hb concentrations in GC and GA showed

significant decrease (P<0.05) with pregnancy (table 8). The lowest

concentrations of Hb were noticed during late pregnancy period in GC while

in GA the lowest value was observed in the mid pregnancy. In early

pregnancy, the Hb value was significantly high in GC than in GA (P < 0.05).

3. 3. 2 Packed cell volumes (P CV):

The mean values of PCV% are represented in Table 9 and Figure 5 for

the first 10 weeks post tick application. However, the weekly means PCV

percent values in GA were found slightly lower than that of GC.

Whereas, the difference was significant in weeks 6, 8, and 9 (P < 0.05).

But in week 10, the mean PCV% showed insignificant difference

between the two groups (P < 0.05).

In pregnant ewes, it was noticed that the PCV% values in GC and GA

decreased significantly (P < 0.05) with pregnancy and the lowest values

were obtained during late pregnancy period (Table 10). However the mean

PCV% values were found to be higher in GA than in GC

73 

 

Table 7: Weekly haemoglobin concentrations (g/dl) (mean ± SD)

in control group(GC) and infected group

(GA) post tick application

Period in weeks

GC

GA

P- value

Week (1) 8.27±1.2a 8.10± 1.35a 0.604

Week (2) 7.65±1.12a 6.91±1.49 b 0.034

Week (3) 8.4 0±0.95 a 7.44±1.04 a 0.149

Week (4) 8.00±0.61a 7.88±0.79 a 0.567

Week (5) 7.97±0.78 a 7.91±1.04 a 0.811

Week (6) 8.38±0 .64 a 8.26±0.64 a 0.582

Week (7) 7.67±1.13 a 6.78± 1.10 b 0.012

Week (8) 8.35±1.82 a 4.20±0.01 b 0.001

Week (9) 6.92 ±1.7 a 6.98±1.27 b 0.000

Week (10) 8.90±1.13 a 8.55±0.33 a 0.750

Values with different small superscripts within same rows were

significantly different at P<0.05

74 

 

Time in weeks

1097654321

Mea

n va

lue

in (g

m/d

L)

9.5

9.0

8.5

8.0

7.5

7.0

Control Group

Infected groupo

Figure 4: Haemoglobin concentration (g/dl) in control group

(GC) and infected group (GA) post tick application

75 

 

Table 8: Mean ± SD values of Hb concentrations (g/dl) in different pregnancy periods

in control group GC and infected first group GA

 

 

                                                       

Values with different capital superscripts within same columns were significantly different at P<0.05 

                      

Animal groups

Pre pregnancy

Early

pregnany

Mid pregnancy

Late pregnancy

P-

value

 

GC  9.30 ±1.41aA  7.65±1.61bA 

 

6.87±1.52bA  6.58 ±1.61bA  0.027 

GA  7.51±1.49aA  7.52 ±1.69aA 

 

6.94 ±1.36bA  7.28 ±1.65aA  0.003 

 

P ‐Value  0.045  0.145 

 

0.115  0.766 

…...

76 

 

 

Table 9: Weekly packed cell volume (PCV %) (mean ± SD

in control group(GC) and infected group (GA)

post tick application 

Period in weeks

GC

GA

P- value

Week (1) 25.11±4.7a 24.11±5.62a 0.383

Week (2) 28.72±8.3a 25.56±6.44a 0.461

Week (3) 21.34±5.16a 23.00±5.17a 0.328

Week (4) 29.47±4.71a 27.88±4.58a 0.196

Week (5) 28.79±4.9 a 28.11±5.25 a 0.586

Week (6) 25.87±4.73a 23.00±4.44b 0.009

Week (7) 26.00±5.04a 25.43±4.65a 0.689

Week (8) 22.93±5.16a 21.89±6.39b 0.024

Week (9) 22.82±5.56a 23.75±6.41b 0.037

Week (10) 22.64±5.39a 22.33±4.00 a 0.523

Values with different small superscripts within same rows were

significantly different at P<0.05

77 

 

Time in weeks

10987654321

Mea

n %

32

30

28

26

24

22

20

Control Group

Infected Group

Figure 5: Packed cell volume (%) in control group (GC)

and infected group (GA) post tick application

78 

 

Table 10: PCV % (mean ±SD) during pregnancy in control group GC

and infected first group GA

   

Animal groups 

 

Pre­ 

Pregnancy 

 

Early 

pregnancy 

 

Mid 

pregnancy 

 

Late 

pregnancy 

 

P­ value 

 

GC  26.00±2.8a,A  24.67±3.27a,A 

 

19.31±3.56b,A  18.11±3.98b,A  0.001 

 

GA  25.58±6.81a,A  21.03±5.2b,B 

 

20.98±4.45bA  19.30±4.46b,A  0.000 

P – 

Value  0.871  0.050 

 

0.061  0.548  ‐‐‐‐‐‐‐ 

Values with different small superscripts within same rows were significantly different at P<0.0 

                                                        Values with different capital superscripts within same columns were significantly different at P<0.05 

 

79 

 

3. 3 White blood cells (WBCs):

The mean values for the white blood corpuscles in thousands per

cubic milliliter are represented in table 11 and figure 6. The weekly

WBCs in GA were generally found to be slightly lower than that of the

GC. However, the difference was significant only in week 5 and 6 post

tick application (P < 0.05). On the other hand, in weeks 8-10, the mean

WBCs showed insignificant difference between the two groups.

The WBCs in pregnant ewes decreased in late pregnancy in both

groups (table 12) however, the decrease was significant in GA (P<0.05),

whereas in GC, the decrease was not significant (P>0.05).

3. 4. Biochemical parameters:

3. 4. 1 Serum total proteins:

The mean values of serum total proteins in g/dL are given in table 13,

figure 7 whereas table 14 shows the mean serum total protein in pregnant

ewes. The weekly mean total protein concentrations for serum samples

collected for up to 10 weeks post tick application in GA were found to be

lower than that of GC However, the difference was significant (P < 0.05) in

weeks 3 - 7. Whereas, in week 8- 10, the mean values of serum total

proteins showed insignificant difference (P > 0.05) between the two groups.

In pregnant ewes, the mean values of serum total proteins in both GC and

GA showed significant decrease (P < 0.05) with pregnancy and the lowest

values were noticed in late pregnancy (table 14). However, in early

pregnancy the decrease in total proteins was significant in GA than in CG

(P<0.05).

80 

 

Table 11: Weekly white blood cell count (X103/mm3) (mean ± SD)

in control group(GC) and infected group

(GA) post tick application

Period in weeks

GC

GA

P- value

Week (1) 8.12±2.12a 8.36±2.24a 0.518

Week (2) 7.96±3.23a 7.52±3.25a 0.439

Week (3) 7.02±1.82a 6.69±1.66a 0.302

Week (4) 6.20±2.56a 6.10±1.93a 0.879

Week (5) 6.20±2.84a 4.96±1.41b 0.002

Week (6) 7.20±2.25a 6.31±4.65b 0.010

Week (7) 6.92±2.11a 6.78±2.23a 0.621

Week (8) 9.13±2.16a 9.33±2.29a 0.587

Week (9) 11.0±3.24a 12.0±2.59a 0.163

Week (10) 8.45±1.75a 8.92±1.09a 0.151

Values with different small superscripts within same rows were

significantly different at P<0.05

81 

 

          

 

 

   

 

Time in weeks

109654321

Mea

n va

lue

(x10

/L)

14000

12000

10000

8000

6000

4000

Control Group

Infected Group

 

Figure 6: White blood cells count (X103/mm3) in control group (GC) and infected group (GA) post tick applicatio

82 

 

                Table 12: WBCs counts per mm3 (X103) (Mean ± SD) during pregnancy

in control group GC and infected first group GA

Animal groups

Pre-

Pregnancy

Early

Pregnancy

Mid

Pregnancy

Late

Pregnancy

P- value

GC

8.82±2.29a,A

9.19±3.92a,A

8.70±3.22a,A

7.50±2.57a,A

0.203

GA

7.94±2.74a,A

6.52±2.03b,A

8.59±3.52a,A

5.08±1.86b,A

0.000

P –

Value

0.189

0.205

0.933

0.328

------

Values with different small superscripts within same rows were significantly different at P<0.05 

                                            Values with different capital superscripts within same columns were significantly different at P<0.05  

83 

 

                 

Table 13: Weekly values of total proteins (g/dl) (mean ± SD)

in control group(GC) and infected group

(GA) post tick application

Period in weeks

GC

GA

P- value

Week (1) 7.31±0.32 a 7.34±2.05a 0.875

Week (2) 7.45 ±0.49 a 7.73±2.62a 0.863

Week (3) 7.35 ±0.78 a 5.56±1.11b 0.003

Week (4) 7.27 ±0.37 a 6.11±0.15b 0.008

Week (5) 7.19 ±0.51 a 5.73±.69 b 0.005

Week (6) 7.24 ±0.40 a 5.98±1.36a 0.108

week (7) 7.37 ±0.44 a 5.83±1.25b 0.006

Week (8) 7.19 ±0.38 a 7.15±1.96a 0.862

Week (9) 7.17 ±0.36 a 7.63±1.69a 0.157

Week (10) 7.19 ±0.59 a 7.54±0.82a 0.148

Values with different small superscripts within same rows were

significantly different at P<0.05

84 

 

Time in weeks

987654321

Mea

n va

lue

in (g

/dl)

8.0

7.5

7.0

6.5

6.0

5.5

5.0

Infected Group

Control Group

Figure 7: Total proteins concentration (g/dl) in control group

(GC) and infected group (GA) post tick application

85 

 

                               Table 14: Total Proteins values (g/dl) (Mean ± SD) during pregnancy

In control group GC and infected first group GA

Animal groups

Pre-

Pregnancy

Early

pregnancy

Mid

pregnancy

Late

pregnancy

P- value

GC

7.38±0.40 a,A

7.27±0.48 a,A

6.15±1.35 b,A

5.14±1.74 c,A

0.00

GA

7.46±1.33 a,A

6.84±1.08 a,B

6.62±1.78 a,A

5.56±1.29 b,A

0.014

P –

Value

0.71

0.00

0.11

0.87

----

                    Values with different small superscripts within same rows were significantly different at P<0.05 

                                  Values with different capital superscripts within same columns were significantly different at P <0.05  

                       

86 

 

3.4. 2 Serum albumin:

Table 15 and figure 8 represent the mean concentrations of serum

albumin in g/dl in GA and GC ewes for 10 weeks post tick application

while table 16 represents the mean concentrations of serum albumin

during pregnancy. However, the values of the mean serum albumin

concentrations during the 8 weeks post tick application, showed slightly

lower values in GA compared to GC. While in week 9 and 10, the

values are comparable between the two groups.

On the other hands, in pregnant ewes, the albumin concentrations

in GC and GA showed significant decrease (P<0.05) with pregnancy

and the lowest values were noticed as pregnancy progressed (table 16).

Whereas in mid pregnancy period, the albumin concentrations showed

significant difference between the two groups (P< 0.05).

3. 4. 3 Serum globulins:

The mean concentrations of serum globulins in g/dl of GA and GC

ewes for 10 weeks post tick application, are shown in table 17 and

figure 9 and table 18 shows the serum globulin concentration during

pregnancy. The weekly mean globulins concentrations values post tick

application for GA were found to be slightly lower than that of GC.

However, the difference was significant (P< 0.05) during week 3 and 7

post tick application, whereas in pregnant ewes, a significant decrease

(P< 0.05) of the serum globulins concentrations was observed in both

groups with pregnancy and the lowest values were obtained during

late pregnancy (table 18). It was noticed that the values in GA were

87 

 

slightly higher than GC and the difference was significant during early

pregnancy period (P < 0.05).

88 

 

Table 15: Weekly values of albumin ( g/dl) (mean± SD)

in control group(GC) and infected group

(GA) post tick application

Period in weeks

GC

GA

P -value

Week (1) 3.32 ±0.33 a 3.31±.82 a 0.940

Week (2) 3.38±0.32 a 3.41±0.57 a 0.228

Week (3) 3.00±0.27 a 2.84±0.97 a 0.501

Week (4) 3.12±0.32 a 2.99 ±0.61 a 0.578

Week (5) 3.26±0.58 a 3.14 ±0.27 a 0.922

Week (6) 3.24±0.24 a 3.09±0.43 a 0.257

Week (7) 3.21±0.17 a 3.07 ±0.22 a 0.489

Week (8) 3.31±0.33 a 3.12 ±0.35 a 0.294

Week (9) 3.13±0.29 a 3.12±0.52 a 0.743

Week (10) 3.15 ±0.31 a 3.11 ± 0.42 a 0.587

Values with different small superscripts within same rows were

significantly different at P<0.05

89 

 

Time in weeks

987654321

Mea

n va

lue

in (g

/dl)

3.6

3.4

3.2

3.0

2.8

2.6

Infected group

Control Group

Figure 8: Albumin concentrations (g/dl) in control group

(GC) and infected group (GA) post tick application

90 

 

Table 16: Albumin concentrations (g/dl) (Mean±SD) during pregnancy

in control group GC and infected first group GA

Animal groups

Pre-

Pregnancy

Early

pregnancy

Mid

pregnancy

Late

pregnancy

P- value

GC

3.35±0.32 a,A

3.19±0.34 a,A

3.05±0.52 a,A

2.90±0.48 b,A

0.008

GA

3.37±0.47 a,A

3.13±0.40 a,A

2.96±0.58 ab,B

2.72±0.49 b,A

0.001

P -Value

0.929

0.223

0.015

0.688

 

Values with different small superscripts within same rows were significantly different at P<0.0 

Values with different capital superscripts within same columns were significantly different at P<0.05

91 

 

Table 17: Weekly values of globulins (g/dl) (mean± SD)

in control group(GC) and infected group

(GA) post tick application

Period in weeks

GC

GA

P- value

Week (1) 3.98±0.54 a 4.03±1.37 a 0.806

Week (2) 4.07±0.76 a 4.32±2.01 a 0.836

Week (3) 4.34±0.88 a 2.72±1.03 b 0.009

Week (4) 4.15±0.51 a 2.87±0.78 a 0.110

Week (5) 3.93±0.81 a 2.83±0.68 a 0.120

Week (6) 3.97±0.51 a 3.34±1.12 a 0.147

Week (7) 4.16±0.54 a 2.98±1.21 b 0.028

Week (8) 3.89±0.60 a 3.79±1.59 a 0.736

Week (9) 4.04±0.49 a 4.25±0.91 a 0.307

Week(10)

4.15±0.52 a 4.16±0.29 a 0.412

Values with different small superscripts within same rows were

significantly different at P<0.05

92 

 

Time in weeks

987654321

Mea

n va

lue

in (d

/dl)

5.0

4.5

4.0

3.5

3.0

2.5

Infected Group

Control Group

Figure 9: Globulins concentration (g/dl) in control group

(GC) and infected group (GA) post tick application

93 

 

Table 18: Globulin concentrations (g/dl) (Mean±SD) during pregnancy

in control group GC and infected first group GA

Animal groups

Pre-

Pregnancy

Early

pregnancy

Mid

pregnancy

Late

pregnancy

P- value

GC  4.03±0.64 a,A  4.07±0.65 a,A 

 

3.21±1.19 b,A  2.32±1.73 c,A  0.000 

GA  4.10±1.16 a,A  3.73±0.98 a,B 

 

3.37±1.39 a,A  2.82±1.19 b,A  0.073 

P – 

Value 

0.745  0.001 

 

0.089  0.470 

 

‐‐‐‐ 

Values with different small superscripts within same rows were significantly different at P<0.05 

Values with different capital superscripts within same columns were significantly different at P<

94 

 

3.4.4 Serum Creatinine:

The mean serum creatinine concentrations in mg/dl are shown in table

19 and figure 9 for ewes in GA and GC for 10 weeks post tick

application and table 20 represents the mean creatinine concentrations in

mg/dl during pregnancy. It was found that the weekly mean

concentrations of creatinine in GA were significantly higher than in GC

(P<0.05) during the first 7 weeks, however, the difference was

insignificant during weeks 8, 9 and 10.

In pregnant ewes, the creatinine concentrations in mg/dl showed

insignificant changes in GC (P>0.05) throughout the pregnancy period

(table 20), whereas, in GA a significant increase was noticed in early

pregnancy (P<0.05). However, the values of the mean creatinine

concentrations in GA were significantly higher (P<0.05) than that of

GC especially during pre- and early pregnancy period.

3. 4. 5 Serum urea:

The mean serum urea concentrations in mg/dl for ewes in GC and

GA for 10 weeks post tick application are shown in table 21 and figure

10 and table 22 represents the serum urea concentrations values during

pregnancy. It was observed that the weekly serum urea concentrations

were higher in GA than in GC during the period 2-8 weeks, whereas, at

weeks 9-10, the urea concentration of GA decreased more than that of

GC.

In pregnant ewes the mean urea concentrations in mg/dl in GA were

significantly higher than in GC (P<0.05) especially in mid and late

95 

 

pregnancy (table 22). However, the values in GC showed significant

decrease (P < 0.05) with pregnancy with the lowest values at late

pregnancy. on the other hand , the decrease in serum urea

concentrations in GA was insignificant (P>0.05).

96 

 

Table 19: Weekly values of creatinine (mg/dl) (Mean ± SD)

in control group(GC) and infected group

(GA) post tick application

Period in weeks

GC

GA

P- value

Week (1) 5.32±5.86 a 7.59±5.03 b 0.000

Week (2) 5.46±0.87 a 10.14±3.85 b 0.000

Week (3) 4.26±3.74 a 8.99±5.99 b 0.000

Week (4) 6.83±3.59 a 11.34±2.55 b 0.000

Week (5) 7.00±3.07 a 22.05±14.68b 0.000

Week (6) 6.16±5.51 a 10.27±4.17 b 0.000

Week (7) 4.29±2.20 a 9.21±3.59 b 0.000

Week (8) 10.26±1.21 a 12.74±4.12a 0.111

Week (9) 7.38±3.79 a 7.12±4.38a 0.243

Week(10) 4.30±2.81 a 4.13±3.25 a 0.343

Values with different small superscripts within same rows were

significantly different at P<0.05

97 

 

Time in weeks

987654321

Mea

n va

lue

in (m

g/l)

30

20

10

0

Control Group

Infected Group

Figure 10: Creatinine concentration (mg/dl) in control group

(GC) and infected group (GA) post tick application

98 

 

Table 20: Creatinine concentrations (mg/dl) (Mean±SD) during pregnancy

incontrol group GC and infected first group GA

Animal

group

Pre-

Pregnancy

Early

pregnancy

Mid

pregnancy

Late

pregnancy

P- value

GC  5.36±4.80 a,A  6.49±5.51 a,A 

 

6.87±5.01 a,A  6.45±3.09 a,A  0.848 

GA  8.87±4.50 ab,B  10.07±4.38 b,B 

 

7.59±3.90 a,A  7.55±3.55 ab,A  0.039 

P‐value  0.00  0.003 

 

0.457  0.306  ‐‐‐‐‐‐ 

 

 

Values with different small superscripts within same rows were significantly different at P<0.05 

Values with different capital superscripts within same columns were significantly different at P<0.0

99 

 

Table 21: Weekly values of urea (mg/dl) (Mean ± SD)

in control group(GC) and infected group

(GA) post tick application

Period in weeks

GC

GA

P- value

Week (1) 34.76±5.58 a 33.86±3.19 b 0.000

Week (2) 32.84±3.26 a 34.24±2.45 b 0.013

Week (3) 31.38±2.89 a 32.89±7.19 b 0.000

Week (4) 28.51±4.10 a 30.02±3.19 b 0.000

Week (5) 30.71±3.62 a 33.71±6.63 b 0.000

Week (6) 31.52±4.92 a 32.88±6.24 b 0.000

Week (7) 31.39±2.39 a 34.78±3.90 b 0.000

Week (8) 31.70±4.06 a 33.36±3.32 b 0.001

Week (9) 32.40±3.24 a 32.95±11.34a 0.063

Week(10) 32.31±4.25 a 32.59±5.68 a 0.065

Values with different small superscripts within same rows were

significantly different at P<0.05

100 

 

Time in weeks

987654321

Mea

n va

lue

in (m

g/l)

38

36

34

32

30

28

26

Infected group

ontrol Group

Figure 11: Urea concentration (mg/dl) in control group

(GC) and infected group (GA) post tick application

101 

 

Table 22: Urea concentrations (mg/dl) (Mean±SD) during pregnancy

in control group GC and infected first group GA

Animal

group

Pre-

Pregnancy

Early

pregnancy

Mid

pregnancy

Late

pregnancy

P- value

GC  33.74±4.47a,A  30.87±3.75a,A 

 

33.95±19.53a,A  15.85±7.14b,A  .000 

GA  34.05±2.72a,B  32.91±5.20a,A 

 

30.70±15.08a,B  26.28±9.91a,B  0.170 

P‐

value  0.000  0.861 

 

0.000  0.004 

Values with different small superscripts within same rows were significantly different at P<0.05 

Values with different capital superscripts within same columns were significantly different at P<0.05 

102 

 

3. 5 Experiment two:

3.5.1 Detection of the infection with Thieleria lestoquardi:

3.5.1.1 Parasitological examination:

-Blood smears:

Blood smears were examined for the presence of Thieleria lestoquardi

parasite from day five post tick application and on for lambs of G2. The

daily examination revealed that the blood smears showed the presence of

the Thieleria lestoquardi parasite after day 10th – 21st of tick application

as a schizont stage within the lymphocytes and /or a piroplasm stage

within the red blood cells (figure 1 and 2).

3.5.1.2 Physical examination:

-Temperatures detection:

The mean daily rectal temperatures of experimentally infected lambs

from day 7 to day 17th are shown in figure 12. The maximum mean daily

temperature was found to be 41.3 0C during the second week of tick

application.

3.5.1.3 Serological examination:

-Indirect fluorescent antibody test (IFAT):

The results of the indirect fluorescent antibody test (IFAT) for

collected serum during the course of the study was represented in table

23. The positive signs confirm the presence of the parasite in the infected

lambs.

103 

 

Days after tick application

2019

1817

1615

1413

1211

109

87

Missing

Tem

pera

ture

Co

42

41

40

39

38

37

Figure12: Daily body temperature (0C) in lambs after 7 days

Post tick application

104 

 

Table 23: Indirect fluorescent antibody test (IFAT) for T. lestoquardi in the infected lamb

ewes through out the experimental period in months

 

 

   

Animal

No.

Parasitemia /months

1st

month

2nd

month

3rd

month

4th

month

5th

month

6th

month

7th

month

    1 ‐  ‐ + + + + + ‐ + + + + ‐ ‐ +  + + ‐ + + + + + ‐ ‐ +

    2 ‐  ‐ ‐ ‐ + + + + + + + + ‐ + +  + ‐ ‐ + + + ‐ + + ‐ + ‐ +

    3 ‐  ‐ ‐ ‐ ‐ D e a d    

    4 ‐  ‐ ‐ ‐ + + D e a d    

   5 ‐  ‐ + + + + + + ‐ ‐ ‐ + + +   ‐  + + + ‐ ‐ + + + ‐

    6   ‐  ‐ ‐ + + + + ‐ ‐ + + + + + ‐  + + + + + + +

  

+ ‐ +

105 

 

3. 5.2 Puberty

Detectable progesterone (P4) level in lambs in G1 was observed at

about 12-13 weeks from the start of the experiment. Figures 13 and 14

showed regular oestrus cycles every 3-4 weeks (P4 peaks) indicating

that puberty age of the lambs in G1 is estimated to be reached at 8-9

months old.

On the other hand, a detectable level of progesterone (P4) in lambs

in G2 was observed from week 25-34 post the commencement of the

experiment (figures 15-18). The concentrations of P4 profile of G2 also

represented in these figures indicate an irregular oestrus cycles and that

puberty was delayed and the estimated age of puberty is 12- 15 month

old.

106 

 

Table 24: Age at puberty in control ewe lambs G1

and infected lamb ewes G2

Animal group

Age at puberty (month)

(mean ±SD) range

G1

8.58 ±0.4a

8-9

G2

13.33 ±1.1b

12-15

P-value

0.00

____

Values with different superscript letters within same column were significantly different at P<0.05

107 

 

Age in weeks

514743393531272319

P4 v

alue

in n

mol

/l

6

5

4

3

2

1

0

Figure 13: Progesterone profile in lamb No.1 control group

Figure14: Progesterone profile in lamb No.2 control group

Age in weeks

514743393531272319

P4

valu

e in

nm

ol/l

16

14

12

10

8

6

4

2

0

108 

 

Age in w eeks

575349454137514743393531272319

P4 v

alue

s in

nm

ol/l

6

5

4

3

2

1

0

Figur15: Progesterone profile in lamb N0. 3 infected group

Age in weeks

575349454137514743393531272319

P4

valu

es in

nm

ol/l

6

5

4

3

2

1

0

Figure16: Progesterone profile in lamb No.4 infected group

109 

 

Age in w eeks

575349454137514743393531272319

P4 in

nm

o/l

7

6

5

4

3

2

1

0

Figure17: Progesterone profile in lamb No. 5 infected group

Age in w eeks

575349454137514743393531272319

P4 v

alue

s in

nm

ol/l

10

8

6

4

2

0

Figure18: Progesterone profile in lamb No.6 infected group

110 

 

3.5.3 Biochemical parameters of the lambs:

3. 5.3.1 Serum total proteins:

The weekly mean serum total protein concentrations in g/dl in G2

were slightly lower than that of G1 (P > 0.05). However, the difference

was significant in weeks 4, 5, and 6 of infection (P < 0.05), whereas, in

weeks 8, 9 and 10, the mean total protein showed insignificant

difference between the two groups (table 24 figure 19).

3. 5.3.2 Serum albumin:

The weekly mean of albumin concentrations in g/dl in G2 were

slightly lower than that of G1 (P > 0.05). The difference was significant

in weeks 5 and 6 (P<0.05). Insignificant difference of the mean serum

albumin concentrations between the two groups was observed in week

9 and 10 (table 25, figure 20).

3. 5.3.3 Serum globulins:

The lambs weekly mean serum globulin concentrations in g/dl are

shown in table 26, figure 21. However, the serum globulin values in G2

were slightly lower than that of G1 (P > 0.05). The difference was

significant in weeks 5, 6, and 8 of infection (P < 0.05), whereas, the

difference was insignificant (P > 0.05) between the two groups in week 9

and 10 of infection

3. 5.3.4 Serum urea:

The weekly mean serum urea concentrations (mg/dl) values in G2

were generally higher than that of the mean concentrations of G1 (P <

111 

 

0.05). However, the difference was significant in week 2 (P < 0.05). In

week 4 – 10, the mean urea concentration values were comparable

between the two groups (table 27, figure 22).

3. 5.3.5 Serum creatinine:

The weekly mean serum creatinine concentrations in mg/dl in G2

were slightly lower than that of G1 (P > 0.05). However, the difference

was significant on weeks 7, 8, and 9 of infection (P < 0.05). On week

10, the mean creatinine concentrations showed insignificant difference

between the two groups (table 28, figure 23).

112 

 

Table 25: Weekly values of total proteins ( g/l) (mean ± SD)

In infected lambs (G2) and control lambs (G1)

post tick application

Period in weeks

G1

C2

P- value

1st 5.92±.524 a 5.57±2.09 a 0.741

2nd 4.99±1.17 a 4.70±1.29 a 0.668

3rd 5.21±0.78 a 4.27±0.22 a 0.082

4th 6.50 ±1.67 a 4.47±1.05 b 0.033

5th 6.53±1.35 a 4.65±0.77 b 0.013

6th 5.76±0.39 a 4.25±0.04 b 0.002

7th 6.22±1.50 a 4.33±6.90 a 0.148

8th 5.44±0.43 a 4.89±0.29 a 0.094

9th 5.11±0.64 a 4.87±4.20 a 0.575

10th 5.20±0.56 4.89±2.40 0.625

Values with different small superscripts within same rows were

significantly different at P<0.05

113 

 

Time in weeks

10987654321

Mea

n va

lue in g/dl

7.0

6.5

6.0

5.5

5.0

4.5

4.0

Inected group

Control group

Figure 19: Total proteins concentrations (g/dl) in infected lambs

(G2) and control lambs (G1) post tick application

114 

 

Table 26: Weekly values of albumin (g/l) (mean ± SD)

In infected lambs (G2) and control lambs (G1)

post tick application

Period in weeks

G1

G2

P- value

1st 2.83±.0.20 a 2.83±0.32 0.976

2nd 2.63 ±.18 a 2.27±0.34 a 0.511

3rd 2.36±0.55 a 2.27±0.35 a 0.713

4th 2.67±0.16 a 2.24±0.55 a 0.166

5th 2.75±0.24 a 2.47±0.07 b 0.022

6th 2.70±0.27 a 2.09±0.29 b 0.044

7th 2.64±0.39 a 2.21±0.37 a 0.176

8th 2.41±0.433 a 2.38±2.90 a 0.938

9th 2.52±0.36 a 2.29±0.12 a 0.436

10th 2.51±0.35 2.31±0.22 0.326

Values with different small superscripts within same rows were

significantly different at P<0.05

115 

 

Time in weeks

10987654321

Mea

n va

lue

in g

/dl

3.0

2.8

2.6

2.4

2.2

2.0

Infected Group

Control Group

Figure 20: Albumin concentrations (g/l) in infected lambs

(G2) and control lambs (G1) post tick application

116 

 

Table 27: Weekly values of globulin (mg/dl) (Mean ± SD)

In infected lambs (G2) and control lambs (G1)

post tick application

Period in weeks

G1

G2

P- value

1st 2.46±.62 a 2.74±2.26 a 0.767

2nd 2.78±0.52 a 2.00±1.26 a 0.57

3rd 3.07±0.99 a 1.19±0.17 a 0.391

4th 2.84±0.37 a 2.28±1.71 a 0.150

5th 3.07±0.20 a 2.39±0.71 b 0.039

6th 2.95±0.22 a 2.28±0.01 a 0.087

7th 3.8±0.60 a 2.05±0.94 a 0.247

8th 2.80±0.58 a 2.51±.46 b 0.049

9th 2.29±0.41 a 2.63±0.72 a 0.566

10th 2.28±0.42 2.22±0.62 0.623

Values with different small superscripts within same rows were

significantly different at P<0.05

117 

 

Time in weeks

10987654321

Mea

n va

lue

in g

/dl

3.2

3.0

2.8

2.6

2.4

2.2

2.0

1.8

Infected group

Control group

Figure 21: Globulins concentrations (g/l) in infected lambs

(G2) and control lambs (G1) post tick application

118 

 

Table 28: Weekly values of urea (mg/dl) (mean ± SD)

In infected lambs (G2) and control lambs (G1)

post tick application

Period in weeks

G1

G2

P- value

1st 23.71±6.33 a 19.38±8.17 a 0.289

2nd 22.41±10.47 a 46.32±11.45 b 0.002

3rd 39.43±19.40 a 57.55±9.49 a 0.085

4th 28.74±10.6 a 26.79±9.36 a 0.760

5th 22.15±10.54 a 23.11±4.81 a 0.840

6th 31.80±9.18 a 36.93±1.06 a 0.233

7th 19.34±4.498 a 22.03±8.73 a 0.660

8th 57.03±16.15 a 57.84±10.56 a 0.938

9th 36.07±22.91 a 28.51±10.74 a 0.579

10th 33.23±21.32 30.35±12.39 0.503

Values with different small superscripts within same rows were

significantly different at P<0.05

119 

 

Time in weeks

10654321

Mea

n va

lue

in m

g/dl

70

60

50

40

30

20

10

Infected group

Control group

Figure 22: Urea concentration (mg/l) in infected lambs (G2)

and control lambs (G1) post tick application

120 

 

Table 29: Weekly values of creatinine (mg/dl) (mean ± SD)

In infected lambs (G2) and control lambs (G1)

post tick application

Period in weeks

G1

G2

P- value

1st 9.51±5.38 a 8.13±1.56 a 0.524

2nd 6.55±3.39 a 9.43±3.93 a 0.213

3rd 8.78±3.97 a 5.63±6.82 a 0.352

4th 10.08±3.98 a 8.11±3.88 a 0.447

5th 7.26±3.54 a 8.25±4.32 a 0.087

6th 6.03±2.09 a 4.69±3.17 a 0.506

7th 4.05±4.39 a 8.05±2.56 a 0.241

8th 9.52±3.33 a 7.08±3.70 a 0.443

9th 8.16±.08 a 3.66±4.36 a 0.129

10th 7.82±3.25 4.95±2.35 0.321

Values with different small superscripts within same rows were

significantly different at P<0.05

121 

 

Time in weeks

10654321

Mea

n va

lue

in m

g/dl

14

12

10

8

6

4

2

Infected group

Control group

Figure 23: Creatinine concentrations (mg/l) in infected lambs

(G2) and control lambs (G1) post tick application

122 

 

CHAPTER FOUR

123 

 

Discussion

Production is best estimated by the annual reproductive rate which

is defined as the number of lambs weaned per ewe of reproductive age

per year. This estimate is influenced by litter size, lamb mortality,

interval between parturitions and age at puberty (Mukasa, 2002).

Reproduction efficiency can be measured and expressed as the kidding

rate, weaning rate, kidding intervals, birth weight of kids and length of

the reproductive cycle (Greyling, 1988; 2000). Flock productivity may,

however, be affected by a wide range of disease problems and

reproductive wastage. The impact of disease may be reflected as

mortality, abortion, morbidity or in other words loss of foetus and also

may be reflected as sub clinical effects like weight loss or reduced

gains.

This study revealed that as far as the reproductive efficiency of the

Sudanese desert ewes is concerned, the effect of T. lestoquardi appeared

to have an adverse effect on the reproductive potential of Sudanese

desert sheep

Las Cruces (2000) reported that the normal oestrus cycle for ewes

is approximately 17 days, and may vary from 14 to 19 days. The heat

period usually lasts for 30 to 35 hours, with a range of 20 to 42 hours.

That was correlated to our observations that experimental ewes came on

heat 24-48 hours post PMSG inoculation , and the next heat was

observed in non conceived ewes 15-21 days.

The results of this study showed that the conception rate was 90%

124 

 

in the control ewes, while it was only 11.11% in the infected ewes. This

finding was not far from that of Las Cruces (2000) who reported that

fertility of mature ewes is usually high and often over 90% of ewes

conceived during 5 weeks joining period, provided that no adverse

factors present, such as oestrogenic pastures. However, it does vary,

with breed, date of mating, duration of mating and other management

factors. Our results were also consistent with Hossain et al. (2003) who

reported in a nutritional point of view that the numbers of pregnant ewes

were higher in high energy supplemented ones compared to the low

energy supplemented ewes. Elmansoury (2008) demonstrated that high

protein fed goats had higher conception rate than low protein fed goats.

In this study, the mean gestation length of the infected ewes (144.2

±4.76) days seemed to have been a little bit shorter than that of the control

ones (159 ±7.15 days). This may be due to the effect of T. lestoquardi

infection that might not keep pregnancy to its normal end. However, the

gestation length for most breeds of Sudanese goats as reported by ELNaim,

(1979) ranges from 143-153 days. Musa et al. (2005) revealed that the mean

gestation length for West African ewes was 150.44 ± 2.64 days ranging

between 145-156 days, whereas, Las Cruces (2000) reported gestation length

of 147 to152 days which was consistent with our results.

Fthenakis et al. (2001) observed a statistically significant difference

in the total lambs born per ewe naturally infected with sarcoptic mange

and those in the uninfected control (1.2 and 1.6, respectively). Mukasa

(2002) found that litter size in tropical sheep ranges from 1.0 to 1.5

indicating that twinning rate in sheep ranges from 0 and 50%. The above

125 

 

findings agrees with our findings that the control ewes gave more than one

lamb per year, while the infected ewes gave only one lamb per year.

Concerning lambs birth weights, the results showed that the control

ewes gave lambs with birth weights ranging from 2.3 to 3.0 kg with a mean

weight of 2.59 kg which is significantly higher than that of the infected

ones which ranged from 1.0 to 3.0 kg with a mean body weight of 1.87 kg.

This loss in birth weight along with shorter gestation length may be

attributed to the stress of pregnancy and the recruitment of the infection.

Musa et. al.,(2005) in their study on the production performance of West

African sheep in Sudan, found that the overall birth weight of lambs was

2.90 ± 0.50kg.

Lamb survival rates vary markedly. For Merinos, approximately

80% to 90% of single and 60% to 80% of twin-born lambs survive, if

conditions are good. Survival rate of triple or higher parity lambs is very

low under paddock conditions (Disclaimer, 2008). This was in

agreement with our result that the survival rate was found to be 100% in

the control ewes and dropped in the infected ones to about 50-60%.

In this study, the newly born lambs from infected ewes (with mean

body weight of 1.78) died within 24 hours from birth. This finding was in

agreement with that of Disclaimer, (2008) who concluded that lamb birth

weight was the single greatest factor influencing lamb survival. Lambs

born light had low lamb survival as compared to lambs born with medium

or higher birth weights. However, Markos (2006) observed that there was a

positive relationship between body weight and survival rate within breed at

all ages (P<0.001). Survival for lambs born light (< 2 kg body weight) was

126 

 

lower at least by 13% in Horro and 17% in Menz lambs than those with

medium body weight (2–3 kg body weight). Likewise, survival in lambs

with medium body weight was lower by 10-20% in the Horro and 10% in

the Menz lambs than heavy lambs at birth (3 kg body weight or more).

Hinch et al., (1986) concluded that up to 50% of the lambs born may

die mainly due to diseases and other causes such as adaptation failure,

dystocia, cold stress, starvation and mismothering. This was in agreement

with our present study. However, research has shown that nearly 80% of

lamb mortality was related to ewes’ nutrition during the last weeks before

lambing and the first week after lambing (Seymour, 1998). On the other

hand, acute deficit of energy and over fattening of ewes reduce

progesterone concentration in the blood and hence embryo survival during

the first stage of pregnancy (Landau and Molle, 1979). At birth, the

probability of mortality was greater for triplets than for twin born lambs

and greater by 25% for males compared with females (Judith Collins et. al.,

2005). This was closely related to our finding that triple lambs died within

a few hours from birth. Mukasa (2002) reported that mortality rates in

sheep and goats from birth to weaning range from 5 to more than 50% and

represent a serious reduction in biological efficiency.

Mazumdar et.al. (1980) reported a mortality of 100% for kids weighing

1.0 to 1.5 kg and 32.2% for kids weighing from 2-2.5%kg, whereas,

Mtenga et. al., (2006) reported a mortality rate of 57.9 % for kids less than

1.5 kg. Best survival rate was among lambs with weights ranging 3-5 kg.

Hossain, et.al. (2003) reported that birth weight of lambs was higher on

high energy diet compared with that on low energy diet. They concluded

127 

 

that, size; weight and health status of ewes may play an important factor

which affect birth and weight of lambs

In this study, Thieleria lestoquardi caused abortions in early pregnant

ewes but mid or late pregnant ewes did not abort and gave lambs that

were clinically normal. Abortion is an uncommon cause of reproductive

wastage in sheep. It appears to be more important in countries where

more intensive system of lambing management is used. One would

particularly expect this to be the case with diseases such as

toxoplasmosis, campylobacteriosis, salmonellosis, listeriosis,

brucellosis and ovine pestivirus (Las Cruces 2000)

With regard to post-partum heat, our study concluded that control

ewes return to their ovarian activity after 9-32 days post delivery with a

mean of 21.33 ± 8.06 days, whereas in the infected ewes, the post-partum

heat delayed to 21-90 days with a mean of 59.67 ± 42.79. This delay is

probably attributed to the weakness of the ewes due to the infection.

However, Walkden-Brown and Bocquier (2000) have observed in

Burundian meat goats a delay in post-partum resumption of cyclicity in

dry season due to poor quality and low availability of food.

It is worthmentioning that two ewes from the infected group

(before mating) died at late pregnancy and three ewes from the group

infected after pregnancy aborted after infection. This result was in line

with that of Martinez et.al. (1999), who infected ewes with Cowdria and

found that two ewes from the infected group aborted and one died during

late pregnancy. On the other hand, their findings contradicted the current

128 

 

result that persistent infection with Cowdria did not affect the measured

productivity parameters of ewes.

Our results revealed that the infection with Theileria lestoquardi

has greatly influenced the time of puberty in lambs as young ewes in the

uninfected control group reached puberty in 8-9 months, while, the

infected ones reached puberty in about 12-15 months. However, with

trypanosomes, Oscaer (1999) showed that their effect on puberty and

age at first lambing was indirectly mediated through depression of

growth rates. He added that “there was a delaying effect of insufficient

feeding on onset of puberty and reproductive performance in young

Djallonke sheep.

Ewes, typically, reach puberty at 5 to 12 months, depending on breed,

nutrition, and date of birth. Well grown Merino ram lambs reach puberty at

around 6 months of age (Disclaimer, 2008).

However, the age at puberty of some Sudanese sheep as reported in Elhuda

research station that the Shugor and Dubasi types reached puberty in

approximately 6-9 months, Watsish in approximately 5-9 months, and 8-10

months for Southern Darfur traditional system. These findings were in line

with our result for the control group. while a delayed puberty was observed

in the infected ones.

This study revealed that the reproductive parameters such as ,

lambing rate, abortions, embryonic losses and first progesterone rise

seemed to have been strongly affected by Theileria lestoquardi These

findings were in agreement with Rumberia et. al. (1993) who reported

similar results on the effect of Theileria parva on the reproductive

129 

 

functions of the Boran-Freisian heifers using low and high doses of

infection. They came to the conclusion that theileriosis has an adverse

effect on the reproductive potential of animals. Also, Las Cruces (2000)

reported that heavy infection of internal parasites can reduce the body

condition of breeding ewes and may reduce their reproductive

performance.

Atempts to explain the haematological changes on pathophysiological

basis, it was obvious that in infection with T. lestoquardi, there was a

marked decrease in haemoglobin, packed cell volume and white cell

counts. This decrease fluctuated in weeks 9 and 10 following tick

application, after which, the values returned back to normal. Our

findings were similar to the findings of other workers (Mehta et. al.

1988, Singh et. al. 2001, Rayules and Hafeez 1995, Sandhu et. al. 1998,

and Ahmed e., al. 2004. The decline of the above mentioned values

might probably be attributed to the destruction of erythrocytes by

macrophages in the lymph nodes, spleen and other organs of the

reticuloendothelial system as previously suggested by Singh et al

(2001). Omer et. al. (2002) reported that these changes in blood

parameters pictured by the decrease in haemoglobin, packed cell

volume and white blood cell counts may finally lead to the occurrence

of severe anemia. Low haemoglobin concentration values were also

reported by Kumar and Sharma (1991) in anoestrus cow and repeat

breeder animals compared to normally cycling cows.

With respect to the biochemical changes as a result of T. lestoquardi

infection, the study demonstrated that there was an apparent decrease in

130 

 

the concentrations of serum total protein, serum albumin and serum

globulins. This decrease fluctuated in the 9-10 weeks following the tick

application, after which, these values returned back to normal. This

finding is in line with that of Ramazan and Uguruslu (2007), Ahmed et.

al. (2004), Yadav and Sharma (1986) and Singh et al (2001). However,

Sandhu et. al. (1998) reported an insignificant decrease in these

parameters. Stockham et, al. (2000) attributed the decrease in the

concentrations of the serum protein and the serum albumin to the extra

vascular accumulation of proteinaceous fluids resulting from affected

lymph nodes. On the other hand, Singh et. al. (2001) and Omer et. al.

(2003) attributed the decrease in serum proteins to hypoalbuminaemia

and hypoglobulinaemia arising from liver failure.

Our findings indicated that ewes infected with Theileria lestoquardi

had higher concentrations of serum creatinine and serum urea. This

increase fluctuated in the 9-10 weeks following tick application, after

which, they returned to normal. These findings were in agreement with the

findings of Ramazan and uguruslu (2007), Ahmed et, al. (2004) and

Yerham (1998) who attributed the increase in creatinine to a damage

observed in the liver and kidney in babesiosis in sheep. The increase in

urea level was similar to that reported by Singh et, al. (2001) and Sandhu

et, al. (1998). However, our observation contradicts that of Omer et al.

(2003) who showed significant decrease in urea and creatinine in cattle

naturally infected with Theileria annulata,. In our opinion, the rise in urea

levels might probably be attributed to some sort of kidney damage.

Our result concluded that throughout the gestation period, the values of

131 

 

heamoglobin, packed cell volume and white blood cells decrease gradually

with the advancement of pregnancy. The same decrease was also observed

for serum total proteins, serum albumin, serum globulins, serum urea and

serum creatinine throughout the gestation period. These findings were in

agreement with Amina (2002) who concluded that there was a decrease in

total proteins, albumin and globulins in late pregnancy in cross bred goats.

However, she showed that pregnancy did not affect the level of urea. On

the other hand, our findings were not in agreement with the findings of

Getnet, (2005) who found that white blood cells, red blood cells, pakced

cell volume and haemoglobin were slightly higher for late pregnant camels

compared with the values obtained for non-pregnant ones.

Similar findings were seen in cattle (Jain, 1986). An increase in the

leukocytes picture that was observed for pregnant camels was comparable

with the results reported by Coles (1986) for cattle. This could be attributed

as he concluded to the physiological increase of neutrophils count at late

stage of pregnancy. Increased white blood cells count particularly

neutrophils was noted just before parturition in cattle (Doxey, 1977).

132 

 

Conclusions

Thieleria lestoquardi infection has greatly influenced the

reproductive potential of Sudanese desert ewes as evidenenced by the

following:

Decreased conception rate. Increased number of abortions. Decreased

body weights of the newly born lambs. Decreased survival rate of the

newly born lambs. Delayed progesterone rise after delivery. Delayed the

time of reaching puberty.

Thieleria lestoquardi infection has also influenced the heamatological

and biochemical parameters of infected ewes as with the following

noticeable changes:

Decreased values of Hb, PCV and WBCs.Decreased values of total

proteins, albumin and globulins.

Increased values of urea and creatinine.

infection might probably indicate an extensive tissue damage caused

by the parasite.

133 

 

Recommendations

Subsequent studies under natural and experimental field conditions are

recomended and may be used as a useful tool for the diagnosis, prognosis

and evolution of the therapy applied.

1- There is a need for economic analysis to determine a more sustainable

Theileria control strategy.

2- The establishment of endemic stability in livestock population which

requires the administration of vaccines, the treatment of clinically affected

animals and adjustment of acaricide application frequencies to allow

boosting of immunity.

3- Series of investigations on individual sheep and flock health may be carried

out in collaboration with the concerned institutions to add more knowledge

on the effect of Theileria lestoquardi on the reproductive potential of ewes.

134 

 

References

135 

 

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Appendix Table 1: Control group data (GC)

Animal No.

Date of service

conception Abortion Date of delivery

Gestation period(days)

Progesterone rise

Days to progesterone

rise 5 4/4/2006 not

conceive _ _ _ _

_

16 4/4/2004\6 conceived

_ 5.9.2007 154 14.9.2007 9

28 “ “ conceived

_ dead _ _ _

30 “ “ conceived

_ 19/9/2006 168 12.10.2007 23

31 “ “ conceived

_ 15.9.2007 164 12.10.2007 27

42 4/8/2006 conceived

_ 15/1/2007 164 16/2/2007 32

41 “ “ conceived

_ 2/1/2007 151 24.1.2007 22

45 “ “ conceived

_ 15/10/06 160 26.1.2007 15

53 “ “ conceived

_ dead dead Dead _

60 “ “ conceived

_ 5/1/2007 153 20.1.2007 15                        

164 

 

                                Table 2: Infected first data (G A) 

 

                           

Animal No. 

Date of service 

Conception(first 

service) 

Conception date 

abortion  

Date of delivery 

Gestation period (days) 

Progesterone rise 

24  2/11/2006 not conceived 

12.6.2007 _ 8/11/2007 147

28  “  “  not conceived 

_ _

32  “  “  dead _37  “  “  not 

conceived _ _

39  “  “  not conceived 

14.122006 _ 8/5/2007 145 21.7.2007

40  “  “  not conceived 

dead _

316  “  “  conceived 2.11.2006 _ 20/3/2007 138 30.6.2007319  “  “  not 

conceived 23.12.2007 _ 14/5/2007 141 4.62007

324  “  “        not conceived 

14.12.2007 _ 13/5/2007 150

165 

 

                                    Table 3: Pregnant then Infected data (G B) 

 

 

 

Animal No. 

Tickapplication 

Gestation length at 

tick application 

Date of delivery 

  

Gestation period (days) 

Remarks

6  11/1/2006 

2_3 months 29 / 3/2007 

157

16 “     “ dead  Dead on delivery 

29 “     “ 2_3 months 20/3/2005 

148

31 “     “ 1_2 months  27/4/2007 

151

41 6/7/2006  1_2months 

Aborted  Aborted on 

18/7/200739 2/9/2006

 One month Aborted  Aborted 

on 6/9/2007 

816 6/7/2006 1_2 months 11/12/ 007 

157

60 

6/7/2006 One month Aborted  31/7/2007

166 

 

                                        Table 4: lamb survival and body weight (infected first group) 

  

Animal No. 

Delivery condition 

No. of lambs 

lambs survival 

lambs body weight (kgs) 

24  normal 

one survive 2.5 

28  not conceived 

32  dead 

37  normal 

one survive 2.5 

39  normal 

one dead  1 

40 dead 

‐‐‐‐‐‐ ‐‐‐‐‐‐‐‐

316  very weak three not survive 

1, 1 , 1.5 

319  normal 

one survive 2.5 

324  normal 

one survive 3 

167 

 

                                                Table 5: lambs survival and body weight in control group 

 

Animal No. 

Delivery condition 

No. of lambs 

Lambssurvived 

lamb body weight 

5  

_ _ _ _

16 normal 2 Survive 

2.4, 2.75

28 

normal 1 “ 2.8

30 normal 

1 “ 2.5

31 normal 

2 “ 2.5, 2.25

42 normal 

1 “ 3.0

41 

normal 2 “ 2.75, 2.6

25 

normal 2 “ 2.35, 2.6

26 

normal 1 “ 3.0

60 

normal 2 “ 2.5, 2.5

168 

 

                                     Table 6: lambs survival and body weight in concepted first group 

 

 

 

Animal No. 

Condition at delivery 

No. of lambs 

Lambsurvived 

lamb body weight (kgs) 

6  normal 

1 2.2 

16  dead  

_ _ _

29  normal 

1 survive 2.5

31  normal 

2 survive 2, 2.3 

41  abortion 

_ _ _

39  aborted 

_ _ _

816  

normal 1 survive 3.0 

60  

aborted _ _ _