THE EFFECT OF TROPICAL THEILERIOSIS ON SOME BLOOD CHEMISTRY AND HAEMATOLOGY OF CROSS BRED CALVES.

BY:

Omer Elfaroug Ibrahim Sid Ahmed B.Sc. of Veterinary Medicine

University of Khartoum 2001

A THESIS SUBMITTED FOR PARTIAL FULFILMENT FOR REQUIREMENTS OF THE DEGREE OF MASTER OF

BIOCHEMISTERY

SUPERVISOR: Prof. Omer Fadl Idris

FACULTY OF SCIENCE

DEPARTMENT OF BIOCHEMISTRY & BIOTECHNOLOGY ALNEELIN UNIVERSITY

November 2005

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To: My father's soul.

My great mother for her encouragement & intimate contact

during my all school days.

My elder brother A.Nasir, for his great sacrifices among all of

the family members.

My niece Aseel with ever lasting love.

My sisters, Amel, Bint Almoona, Zukaa.

My grandmother, Saadia, for her extreme kindness.

My cousin Abdalla Mukhtar Ahmed Elsheikh for his generousness.

My sister husband, Mohamed Abdel Azim Bashir.

All those who made this work possible.

Acknowledgements

To build up this thesis to its final form, I had considerable inspiration,

encouragement and support from several people who are worth thanking.

First, gratitude is expressed to my supervisor prof. Omer Fadl Idris for

his keen and priceless guidance. I'm also indebted to Dr. Barakat Elhusein, the

head of the Department of Biochemistry-University of Khartoum, Faculty of

Veterinary Medicine, and Dr. Nabiela Musa El bagir, the coordinator of

master program – Department of Biochemistry, U .of. K, Faculty of

Veterinary Medicine for their great efforts among me when I was a master

course student.

My scholarship was fully sponsored by the ministry of higher education

& scientific research. Deep thanks are due to the general manager of inside

training administration Dr. Azhari Omer Abd Elbagi who facilitates my

scholarship. Thanks also extended to Dr. Job Akuei Alith, the academic

secretary-Upper Nile University, for his extreme help.

Additional support was provided by Dr. Bashir Taha Mohamed, the

Director General of the Pan African Campaign for the Control of Epizootics

(PACE) - Sudan, for his technical support & facilitation.

The laboratory work of this study was done in the Department of

Biochemistry of the Central Vet. Research laboratory (Soba), Khartoum

educational hospital, and the Department of Preventive Medicine faculty of

veterinary medicine-University of Khartoum. My thanks are presented to

professional and assistant staff of these departments.

My thanks are also to my sincere friends, Dr. Mustafa Abd Al khalig,

Dr. khalid Ahmed Abd Alhafiez (Bahith), Dr. Salih osman, Dr. Omer ahmed,

Dr. Omer abashar, Dr. Husham Seri, Dr.Isam El din Abd Elbagi, Dr. Ayman

Kamal, Mohamed Madani, and Mamoun Elsamani Idris, for their invaluable

support in different forms. I would also like to express my appreciation to my

uncle Prof. Babiker Omer El Tayeb for his frequent asking about my study

and unlimited support.

My great appreciations to Dr. Ali Siddig & Dr. Mohamed Toum, of

Tick Research Institute - Ministry of Science & Technology, Soba. For their

unlimited support and supply with all required data concerning this study.

Eventually, I'm very greatly appreciated to all scientists whose

published papers were reviewed in the course of preparation of this thesis.

Contents

Dedication…………………………………………………………………………………... І Acknowledgement ………………………………………………………………………… П Contents………………………………………………………………………...................... ІV List of table …………………………………………………………………………………

VП

List of figure………………………………………………………………………………... VПІAbstract ……………………………………………………………………………………. ІХ Arabic abstract………………………………………………………………........................ ХІ Introduction …………………………………………………………………....................... ХП Objectives of the study …………………………………………………………………….. ХПІ

CHAPTER ONE: LITERATURE REVIEW

1.1: History ………………………………………………………………………………… 1 1.2: Etiology ……………………………………………………………………………….. 1 1.3: Epidemiology ……………………………………………………………..................... 1 1.3.1: Prevalence of Tropical Theileriosis …………………………………………............. 1 1.3.2: Transmission ………………………………………………………………………… 2 1.4: Pathogenesis …………………………………………………………………………… 2 1.5: life cycle and morphology …………………………………………………………….. 3 1.6: Classification of Theileria …………………………………………………………….. 7 1.7: Clinical features ……………………………………………………………………….. 7 1.8: Post mortem findings ………………………………………………………….............. 7 .9.1: Plasma proteins ……………………………………………………………………….. 8 1.9.2: General characteristics of plasma proteins ………………………………………….. 8 1.9.3: Albumin ……………………………………………………………………………... 8 1.9.4: Serum Calcium and phosphorus ……………………………………………………. 9 1.9.4.1: Distribution and functions of Ca and P……………………………………………. 9 1.9.4.2: Serum Calcium and phosphorus levels……………………………………………. 10 1.10: Serum Sodium ……………………………………………………………………….. 12 1.11: Serum Potassium …………………………………………………………………….. 12 1.11.1: Disturbances of potassium metabolism…………………………………………….. 12 1.11.2: Hypokalemia………………………………………………………………………... 13 1.11.3:Hyperkalemia……………………………………………………………………....... 13 1.12: Serum Magnesium …………………………………………………………………… 14 1.13: Hematology…………………………………………………………………………… 16 1.13.1: Red Blood Cells………………………………………………………….................. 16 1.13.2: Functions of the red cell………………………………………………….................. 16

1.13.3: Red cell metabolism………………………………………………………………… 17 1.14: White Blood Cells ……………………………………………………......................... 17 1.14.1: Granulocytes……………………………………………………………................... 18 1.14.2: Non granulocytes…………………………………………………………………… 19 1.14.2.1: Lymphocytes………………………………………………………….................... 19 1.14.2.2: Monocytes………………………………………………………………………… 20 1.14.2.3: Plasma cells……………………………………………………………………….. 20 1.15: Packed Cell Volume …………………………………………………………………. 21 1.16: Hemoglobin ……………………………………………………………….................. 21

CHAPTER TWO: MATERIALS AND METHODS 2.1: Area of investigation…………………………………………………………………… 23 2.2: Experimental animals……………………...…………………………………………... 23 2.3: Clinical examination…………………………………………………………………… 23 2.4: Collection and preparation of samples…………………………………………………. 23 2.5: Microscopical examination…………………………………………………………….. 24 2.6: Lymph node biopsy smears……………………………………………………………. 24 2.7: Staining………………………………………………………………………………… 24 2.8: Faecal samples…………………………………………………………………………. 24 2.8.1: Sedimentation ……………………………………………………………………….. 24 2.8.2: Flotation………………………………………………………………….................... 25 2.9: Biochemical investigation……………………………………………………………… 25 2.9.1: Serum albumin……………………………………………………………………….. 25 2.9.2: Serum total Proteins……………………………………………………...................... 26 2.9.3: Serum calcium concentration…………………………………………….................... 28 2.9.4: Serum phosphorus concentration…………………………………………………….. 29 2.9.5: Serum sodium and potassium concentration………………………………………… 30 2.9.6: Serum magnesium concentration……………………………………….……………. 32 2.10: Hematological investigation……………………………………………….................. 33 3.1 Serum chemistry findings……………………………………………………................. 34 3.1.1 Serum total protein……………………………………………………………………. 34 3.1.2 Serum albumin……………………………………………………………................... 34 3.1.3 Serum calcium …………………………………………………………….................. 34 3.1.4 Serum phosphorus………………………………………………………...................... 34 3.1.5 Serum magnesium…………………………………………………………………….. 34 3.1.6 Serum potassium……………………………………………………………………… 35 3.1.7 Serum sodium………………………………………………………………………… 35 3.2 Haematological findings……………………………………………………………….. 35 3.2.1 Red Blood Cell Count (RBC)………………………………………………………… 35 3.2.2 Hemoglobin (HGB)…………………………………………………………………... 35 3.2.3 Haematocrit (HCT)…………………………………………………………………… 35 3.2.4 Mean Corpuscular Volume (MCV)……………………………………....................... 35 3.2.5 Mean Corpuscular Hemoglobin (MCH)……………………………………………… 35 3.2.6 Mean Corpuscular Hemoglobin Concentration (MCHC)……………………………. 36

33

2.11: Statistical method…………………………………………………………..................

3.2.7 White Blood Cell total Count (WBC)…………………………………....................... 36 3.2.8 Lymphocytes…………………………………………………………………………. 36 3.2.9 Neutrophils…………………………………………………………………………… 36 3.2.10 Monocytes………………………………………………………………………….. 36

CHAPTER THREE: RESULTS

CHAPTER FOUR: DISCUSSION

4.1 Effect of tropical theileriosis on some serum proteins and minerals of cross-bred calves………………………………………………………………………………………...

43

4.1.1 Plasma total proteins and albumin……………………………………………………. 43 4.1.2 Serum calcium…………………………………………………………....................... 43 4.1.3 Serum phosphorus……………………………………………………………………. 43 4.1.4 Serum sodium and potassium………………………………………………………… 44 4.1.5 Serum magnesium……………………………………………………………………. 44 4.2 Effect of tropical theileriosis on hematological parameters of cross-bred calves………………………………………………………………………………………...

45

References…………………………………………………………………………………… 47

LIST OF TABLES

Table (1) page The effect of infection with T.annulata on hematological Indices in crossbred calves in Khartoum state region………………………37 Table (2) The effect of infection with T.annulata on serum total proteins, albumin, calcium, phosphorus, magnesium, sodium, and potassium. In crossbred calves in Khartoum state region...........................................................................................…….38 Table (3) The effect of infection with T.annulata on the mean of Total WBC count and differential WBC count in crossbred calves in Khartoum state region……………………………………………………………………….39

LIST of FIGURES

Figure (1) page Theileria annulata piroplasms, inside bovine red blood cells………..….5

Figure (2) Bovine leukocytes immortalized by Theileria annulata………………....6 Figure (3) The Effect of Theileria infection on Blood chemistry of crossbred calves…………………………………………………………………….40 Figure (4) The Effect of theileria infection on hematological indices of crossbred calves…………………………………………………………………….41 Figure (5) The effect of theileria infection on total white blood cells count and differential W.B.Cs count of crossbred calves……………………….…...................42

ABSTRACT

This work was conducted at the Department of Biochemistry Faculty of

Veterinary Medicine, University of Khartoum to study the effect of tropical

theileriosis on some serum chemistry and hematology of cross-bred calves

(Friesian - Kenana), in Khartoum state .

Out of 74 cross calves their age vary between two to six months were

examined clinically and parasitologically, 30 were found as positive cases for

T. annulata. Animals without overt clinical signs and carriers with very low

parasitaemia were also considered. An average of 1–5 piroplasm forms in the

RBC were observed in all cases with a range of 10–45% parasitaemia.

Schizont parasitosis was generally high (more than 10%).

Determination of the theilerial parasites either the piroplasms in the

blood smears or schizonts in the lymph nodes biopsies, and the faecal samples

for the exclusion of the internal parasites of the infected calves was done. The

Haematology of both infected and healthy calves was also done. Serum

albumin, total proteins, calcium, phosphorus, sodium and potassium,

magnesium, of both infected and healthy calves were determined.

The results obtained showed that, the total proteins, albumin, and

calcium were significantly low in all theileria infected calves compared to the

control calves. Whereas phosphorus, magnesium, and sodium were

significantly higher in theileria infected calves compared to the control calves.

There was no significant difference in potassium levels between

theileria infected calves and the control.

The hematological findings revealed that Red Blood Cells count

(RBCs), Hemoglobin (HGB), Hematocrit (HCT), Mean Corpuscular Volume

(MCV), and Mean Corpuscular Hemoglobin Concentration (MCHC), were

significantly lower in theileria infected calves compared to the control calves.

Whereas Mean Corpuscular Hemoglobin (MCH) was not statistically

different between theileria infected calves and the control.

White blood cells total Count (WBCs), lymphocytes, neutrophils, and

monocytes were found to be lower in theileria infected calves with very high

statistical difference compared to the control calves.

بسم االله الرحمن الرحيم

ملخص الدراسة

لدراسة اثر ,جامعة الخرطوم –اجرى هذا العمل بقسم الكيمياء الحيوية بكلية الطب البيطرى

ة على بعض المكونات الدموية والصور الدموية فى العجول الهجين الثايليريا المداري (آنانة -فريزيان )

.بولاية الخرطوم

,شهور تم اجراء الاختبارات الاآلنيكية والطفيلية 6-2عجل تتراوح اعمارهم مابين 74من خلال

ثايلرياعجل آحالات موجبة للثايلريا المدارية 30حيث وجد من بينهم لحيوانات التى لم ا .انيولاتا .

تمت .والحاملة للمرض بدرجات تطفلية دموية متدنية تم اعتبارها ,تظهر عليها علامات المرض

%45-10من الاشكال البيروبلازمية فى الكريات الحمراء لكل الحالات المصابة بمعدل 5-1ملاحظة

)وقد آان التطفل الشيزونتى بدرجات عالية ,من درجة التطفل الدموى آثر من ا 10%).

تم تحديد الطفيليات الثايليرية البيروبلازمية او الشيزونتية فى المسحات الدموية والخزعات

.آذلك تم اخذ عينات برازية لاستبعاد الطفيليات الداخلية للعجول المصابة .الماخوذة من العقد الليمفاوية

ايضا تم تحديد الالببيومين ,تم اجراء القياسات للصور الدموية للعجول المصابة والسليمة

والماغنزيوم للعجول المصابة والسليمة ,البوتاسيوم ,الصوديوم ,الفسفور ,الكالسيوم ,البروتين الكلى

.على السواء

وديوموالص ,الالبيومين ,النتائج المتحصل عليها اظهرت بان البروتين الكلى والكالسيوم فى ,

بينما آانت .العجول المصابة بالثايلريا المدارية آانت متدنية بدرجة معنوية مقارنة بالعجول السليمة

مستويات الفسفور والماغنزيوم والبوتاسيوم آانت مرتفعة بدرجة معنوية عالية فى العجول المصابة

.مقارنة بالعجول السليمة

فى مستويات البوتاسيوم للعجول المصابة مقارنة بالعجول لم يكن هناك فرق معنوى واضح

.السليمة

,الهيماتوآريت ,الهيموغلوبين ,اظهرت النتائج الدموية بان العدد الكلى للكريات الحمراء

ومتوسط ترآيز الهيموغلوبين الكروى آانت متدنية بدرجة معنوية فى ,والحجم المتوسط الكروى

جول السليمةالعجول المصابة مقارنة بالع بينما لم يظهر متوسط الهيموغلوبين الكروى اى فروقات .

.معنوية ما بين العجول المصابة والسليمة

العدد الكلى للكريات البيضاء والليمفاويات والمتعادلة والاحادية اظهرت مستويات متدنية

.بدرجات معنوية عالية فى العجول المصابة مقارنة بالعجول السليمة

Introduction

Tropical theileriosis is a disease caused by theileria annulata, protozoan

parasite of cattle common in some part of the world (Uilenbeng, 1981;

Robinson, 1982). The parasite is transmitted from cattle to cattle by ticks of

the genus hyalomma (Robinson, 1982; Gautam and Dhar, 1983; Dumanli,

1987; Karaer, 1987). Several hyalomma species has been implicated in the

transmission of the parasite. The main vectors of Theileria annulata are

H.a.anatolicum, (Dhar, et al. 1982, Sangwan, et al. 1989) and H.detritum

(Samish and Pipono, 1978).H.a.exacavatum, H.m.marginatum, and

H.dromadarii also transmit T.annulata (Samish and pipano, 1983;

Bhattacharyulu, et al. 1975).

In Sudan T.annulata is considered to be major threat to the cattle

industry since the causative agent causes economical losses particularly in

imported and cross bred cattle (Mohammed, A.S, 1992).

Recently Khartoum state became an important business center with

dramatic human population growth. Therefore, demand for animal protein

became more than the supply provided by the traditional animal production

capacities. As far as milk is concerned, intensive milk farming therefore

required importation of high producing foreign breeds and establishment of

breed improvement programs, as a result, large numbers of foreign and cross

breed cattle were raised. Such animals are highly susceptible to tropical

theileriosis and high mortalities were reported among them (Mohammed, A.S,

1992).

For the previously mentioned aspects of the disease importance of the

disease is realized. Animal owners became aware enough of the economical

impact of the disease since great consideration is now given to the individual

animal as a unit of production instead of the traditional looks that consider the

size of the herd. However tropical theileriosis is considered as one of the

greatest threats to the breed improvement programs, milk production industry

all over the country.

Unfortunately, little had been done on tropical theileriosis in the Sudan

with the exception of the work done by the FAO 1979-1983(FAO, 1983) and

few researches were done by some practitioners. Hence no enough data on the

biochemical and hematological investigations affected by the disease.

Therefore, further researches are required to avail such data which are

important for the control and therapeutic strategy.

Objectives of the study:

1 To determine the effect of tropical theileriosis on some serum

chemistry (total serum protein-albumin-magnesium-calcium-

phosphorus-sodium-potassium).

2 To investigate the effect of tropical theileriosis on some

hematological parameters (Hemoglobin-total red blood cells-

total white blood cells-packed cell volume).

CHAPTER ONE LITERATURE REVIEW

1.1: History:

The pirosomic cattle diseases were not known to occur in the Sudan till

1905 when Balfour found a small piroplasm in blood cells of Sudanese cattle.

In the following three years piroplasmosis was reported in several districts in

the Sudan .all reported cases were attributed in error to east coast fever till

1923. In 1929 the name Thieleria dispar was given. Since 1939 it was

confirmed as T.annulata (Anon, 1914 - 1939). Tropical theileriosis was then

realized and became of common occurrence. It was however, of a lesser

significance than the current disease epidemics because of the enzootic

stability.

1.2: Etiology:

Theileria annulata (synonym T.dispar) is a member of apicoplex

group, like T.parva. It is highly virulent for European dairy cattle. infection in

local zebu cattle is often subclinical.

1.3: Epidemiology:

1.3.1: Prevalence of Tropical Theileriosis:

Tutshin, (1981) stated that in epizootic foci in Kazakhstan clinical

theileriosis was observed to affect young and adult cattle almost equally,

while in latent foci 91.7 - 96.6% of adult and 60 – 70% of 1 – 2 years old

cattle are theileria carrier and the disease is least prevalent among calves less

than 6 month old. Bansal et al (1987) found that 47 – 90% of randomly

selected animals from different age groups in India were showing

seroprevalence through the immuno fluorescent assay (IFA) test while direct

blood smears revealed low grade parasitaemia. They concluded that animals

of all age groups were carriers and the overall picture was indicative of

subclinical theileriosis rather than clinical one. Sisodia and Mandial, (1986)

showed that high incidence reported to be more in the organized farms as

compared to those private dairies. This may be attributed to the greater

susceptibility of high yielders usually maintained in organized farms.

Theileria annulata was found to be highly pathogenic to susceptible

Friesian and Friesian crossed with Kenana in Sudan causing mortality over

80%. Serological surveys showed the parasite to be endemic in the northern,

Khartoum state and Gezira area extending as far as south to Senar district

(FAO, 1983).

1.3.2: Transmission:

Transmission by ticks takes place only after a period of feeding which is

necessary for the development of the infective form of the parasite

(Robertson, 1976). Sergent, et al. (1945) referred to this period to contain the

moulting process of the tick which is necessary for the development of the

parasite to the stage which is infective to the bovine host.

Most of the ticks which are considered as vector for theileriosis undergo

two host tick (Hyalomma .detritum) in the mediterranean basin or three host

life cycle(Hyalomma. anatolicum) in central – western Asia and north-eastern

Africa. Upon feeding on a vertebrate host they either engorge as larvae and

nymphs on the first host before attaching to a second one for the adult feed or

can utilize a fresh host for each blood meal. Normal ticks are infected when

feeding as larvae or nymphs and transmit then parasite in the following

instars. This is known as trans-stadial Transmission (Bhattcharyulu, et al.

1975).

1.4: Pathogenesis:

The pathogenesis is similar to that of East Coast Fever (ECF), but

damage is caused by both schizonts in lymphocytes and piroplasms in

erythrocytes. Consequently, there is lymphadenopathy and panluckopenia on

one hand, and hemolytic anemia with icterus on the other. Over 90% of

erythrocytes may be parasitized by one or more merozoites.

Immunosupression may occur in the acute phase, but is less marked than ECF

(Sharpe, et al. 1983), probably because leukocytes numbers return to normal

soon after the acute phase.

1.5: Life cycle and morphology:

In the bovine host following the bite of an infected tick the parasite

multiplies as schizont in the lymphocytes (figure 2). From these stages

merozoites develop which localize as piroplasms in the erythrocytes(figure 1).

The piroplasms are then ingested by the vector tick when it feeds and sexual

reproduction of the parasite occurs in the gut of the tick (Schein,

1975).Ookinetes develop which migrate through the haemolymph and appear

as sporonts in the salivary glands of the next instar where they develop into

infective sporozoites (Schein,1975). Indications of sexual cycle have been

demonstrated in all studied species T.annulata, T.parva, T.mutans,

T.taurotragi and T.velifera (Irvin and Boarer, 1980). Karaer (1987) describe

the development of T.annulata in the intestine of engorged nymph of

Hyalomma dromedarii (Koch, 1844) and he mentioned that three phases were

recognized:

Phase One: - lasts for about 4 days after feeding in which thread like micro

gametes develop from erythrocytic merozoites.

Phase Two: - Occurs during day 5-12 of feeding where rounded zygotes

appear with characteristic kinetes.

Phase Three: - Being in the 13th day with liberation of kinetes and lasts for

about 3 days.

The development of theileria species in ticks follows a uniform pattern

but there are differences in the morphology rate of development of different

species (Schein, et al. 1977 and Young, et al. 1980).however , according to

Schein, et al. (1978) merozoites of T.parva, T.lawrenceii, T.mutans, and

T.annulata are so similar that they can not identified on their morphology

alone. Not only that but also the morphology of stages of theileria is in many

cases not distinct enough to give an accurate identification for them, young, et

al. (1980) and Schein, et al. (1977) demonstrated two forms of erythrocytic

stages which probably represent gamonts from which gamete stages are

formed when ingested by the tick. In the tick gamete are formed and zygote

formation is deduced. Zygote increase in size to reach a diameter of

approximately 10µm at 12 days after repletion.

After 13 days invagination occurs and club shaped form is produced

which is considered to be Ookinetes. These Ookinetes pass to the acinar cells

of the salivary glands type � & � and rounded up into sporonts and

commence sporogeny.

Figure (1):

Theileria annulata piroplasms, inside bovine red blood cells.

Webbed by: www.cvm.okstate.edu/.../ disk6/images/img0041.jpg

1536 x 1024 pixels - 319k

Figure (2):

Bovine leukocytes immortalized by Theileria annulata. Note the infected cell Webbed by: www.gla.ac.uk

1.6: Classification of Theileria:

The classification of the genus Theileria according to the revision of the

Committee on Systematic and evolution of the Society of Protozoologists

(CSESP) which was published by Levine, et al. (1980).

Phylum Apicomplexa

Class Sporozoea

Subclass Piroplasmia

Family Theileridae

Genus Theileria

1.7: Clinical features:

In a stable endemic situation, there may be no clinical disease in local

zebu cattle (Jacquiet, 1994).Clinical signs in exotic cattle are so longer and

may last for weeks before death. Anemia develops within a few days and

mucous membranes may become icteric.Other signs include pyremia,

anorexia, depressed milk yield, respiratory distress, diarrhea, and weight loss

(Preston, 1992). Rarely, there may be small subcutaneous nodules containing

schizonts (Manickam, 1984).

1.8: Post mortem findings:

Losos, (1986a) cited that the pathological picture is dominated by

changes which occur in the three components of the lymphoid system, (the

major lymphoid organs, the peripheral lymphoid tissues and the lymphocytes

in the circulation). He described the emaciation of the carcass with viable

amounts of froth escaping from the nostrils, enlargement of the peripheral

lymph nodes and evidence of diarrhea, which may contain mucous and blood,

as major external changes Losos (1986b). While hydrothorax, hydro-

pericardium, distended lungs with discoloration, solid texture, exudation and

ecchymotic hemorrhages are the main internal findings. Marked enlargement

of spleen and liver, renal infarctions, edematous lungs, swelling of the lymph

nodes, icteric mucous membrane, often with petichae and inflammation

especially of the mucous membranes of the intestines and abomasums with

the occurrence of characteristic ulcers (2-12mm) in diameter are the common

findings. Necrotic infarction of the brain may occur in cases with cerebral

involvement (Soulsby, 1982).

1.9: Blood chemistry:

1.9.1: Plasma proteins:

1.9.1.1: General characteristics of plasma proteins:

Most plasma proteins are synthesized in the liver; however the gamma

globulins are synthesized in other sites, such as endothelial cells. Plasma

proteins are generally synthesized on membrane bound poly ribosomes. All

most of plasma proteins are glycoprotein, many plasma proteins exhibit poly

orphism, each plasma protein has a characteristic half life in circulation, the

level of certain proteins in plasma increases during acute inflammatory state

or secondary to certain types of tissue damage (Henry, et al. 1974).

Elhag and Hamid, (2003) stated that the total proteins during theileria

infection in crossbred calves are significantly decreased when compared with

control calves.

1.9.1.2: Albumin:

Albumin is the most prominent of the serum proteins and in animals

constitutes between 35 and 50% of the total serum proteins, in contrast to

human beings and nonhuman primates, in which albumin accounts for 60-

67% of the total. Its tertiary structure is globoid or ellipsoid, and it is the most

homogenous fraction discernible on Serum Protein Electrophoresis (SPE). It

is often described as the only discreet protein species which can be detected

by SPE. Cornelius, et al. (1962) stated that the turn over of albumin in cows is

about 16.5 days.

Albumin is initially secreted as preprotein. The only known function of

prealbumin is thyroxin binding and transport (Oppenheimer, et al. 1965), it is

responsible for 75 – 80 % of the osmotic pressure of plasma, so the major

function of albumin is determination of plasma osmotic pressure, another

important function of albumin is acting as general carrier in plasma because

of its ability to bind to various ligands including bilirubin, free fatty acids,

ions, metals, hormones, and a variety of drugs (Murray, 1988).True over

production of albumin alone has not been known to occur in any animal. Any

rise in albumin can be interpreted as dehydration (Jiro, 1980). Thyroxin

binding function of postalbumin fraction has been observed in horses

(Blackmore, 1978).

Decreases in albumin are a common form of dysproteinemia and,

depending on the stage of the disease, can be associated with either slight

hyper-, normo-, or, in its advanced stages, hypoproteinaemia. Due to its small

size and osmotic sensitivity to fluid movements, albumin is selectively lost in

renal (Osborne and Vernier, 1973), gut disease (Meuten, et al. 1978; Kaneko,

et al. 1965), and intestinal parasitism (Dobson, 1965). The hypoalbuminemia

of intestinal parasitism is aggravated by increased albumin catabolism

(Cornelius et al. 1962; Holiday et al. 1968; Holmes, et al. 1968). Furthermore,

due to the sensitivity of albumin to nutritional influences, albumin loss

impairs albumin synthesis. Usually, decreased albumin precedes the

development of generalized hypoproteinaemia in dietary protein deficiencies.

Albumin means values have been found to be significantly decreased

during theileria infection (Dhar and Gautam, 1979).

1.9.2: Serum Calcium and phosphorus:

1.9.2.1: Distribution and functions of Ca and P:

Over 70% of the ash of the body consists of calcium and phosphorus.

More than 99% of the total Ca and 80-85% of the P are contained in the

skeleton and the teeth. The minor portions present in the body fluids, although

negligible in amount, play an extremely important role in the maintenance of

normal body functions. The Ca in the extracellular fluid is critical for (1)

normal neuromuscular excitability, (2) capillary and cell membrane

permeability, (3) normal muscle contraction, (4) normal transmission of

impulses, and (5) normal blood concentration (Jiro, 1980).

Phosphorus outside the skeleton plays an even more fascinating role. It

is involved in vital cellular structures and serves in the degradation and

synthesis of numerous carbon compounds. High-energy phosphate bonds play

a fundamental role in storage, liberation, and transfer of energy. Finally, the

ability of P to be excreted either as H2PO4 2- or HPO4

- gives a broad margin

for the acid-base metabolism in the body (Jiro, 1980).

Calcium and phosphorus always occur in an approximately 2:1 ratio,

and this is not altered even in conditions of partial demineralization of the

bones. Therefore, without ash percentage, the percentage of Ca and P is a

poor indication of animal's mineral reserve (Duncan, 1958). Cancelllous bone

is more readily resorbed then the compact bone, and accordingly changes are

demonstrated in vertebrae or ribs.

1.9.2.2: Serum Calcium and phosphorus levels:

Calcium is one of the more precisely regulated constituents of plasma.

Calcium homeostasis is achieved by balancing the absorption of Ca from the

alimentary tract with the urinary and endogenous fecal excretion of Ca, as

well as fetal or lactational drain of Ca. if the intake of dietary Ca falls below

that required, plasma Ca concentration is maintained by a shift of bone

calcium turnover in favor of net resorption.

The serum concentration of calcium in normal adult animals averages

10 mg/dl (or 2.5mmole/liter). The normal range is usually stated to be 9-12

mg/dl. The serum Ca level of normal young animals is of the same order as

that of adults. The quantity of Ca contained in red blood cells (RBC) is

negligible (Mylera and Bayfield, 1968).

Extracellular Ca is present in at least three forms: free Ca ions, Ca ions

that are ionically bound but ultrafiltrable, and Ca bound to plasma protein.

The serum contains several chelators of Ca, including citrate, and there is also

probably some interaction between Ca and P or carbonate, which decreases

the Ca ion activity. The ionized Ca is diffusible, the complexed Ca is

diffusible but not ionized, whereas the protein-bound Ca is neither ionized nor

diffusible. Although all these forms of Ca are in equilibrium with one another,

in body fluids only the ionized fraction is under direct control (Copp, 1969).

Ultrafiltrable serum Ca in cattle has been found to constitute from 40%

to about 60% of total serum Ca (Hallgren, 1959).

Most of the P of the blood is present as organic esters of P within the

RBC; these contain small amounts of Pi at any given moment. Serum contains

about 14-15mg of total P per deciliter, but of these 5-8 mg are lipid P. a trace

of the rest is ester P, the most significant portion of the reminder being Pi.

Serum Pi concentration in normal adult animals is 4-7mg/dl (1.3-2.3

mmole/liter), young animals usually have a higher and more variable

concentration [5-9mg/dl (1.6-2.9 mmole/liter)]. The Pi level appears to be

intimately related to carbohydrate metabolism. During the increased

carbohydrate utilization the level tends to decrease and during fasting an

increase usually is observed [1-2mg/dl (0.3-0.6 mmole/liter)]. A principal

rule, to which there are many exceptions, is that increasing serum P is

accompanied by decreasing serum Ca. Generally the actual concentrations

depend upon the following conditions:

(1) Intestinal absorption.

(2) Uptake from the Skelton.

(3) Amount of PTH and calcitonin.

(4) Urinary execration.

Serum Ca concentration is precisely regulated by three major

regulatory hormones – PTH, calcitonin, and the active metabolites of vitamin

D – there does not appear to be any precise homeostatic regulation of serum P

concentration (Mylera and Bayfield, 1968).

1.10: Serum Sodium:

Physiological Functions Sodium is the primary electrolyte that regulates the extracellular fluid

levels in the body. Sodium is essential for hydration because this mineral

pumps water into the cell. In turn, potassium pumps the by-products of

cellular processes out of the cell, eventually eliminating these “wastes” from

the body. In addition to maintaining water balance, sodium is necessary for

osmotic equilibrium, acid-base balance and regulation of plasma volume,

nerve impulses and muscle contractions (McDowell, 1993).

1.11: Serum Potassium:

Potassium serves an important role in the maintenance of intracellular

osmotic pressure. In addition, this element is essential to many physical and

chemical processes in the body. Potassium concentration in the ECF

influences the development of membrane potentials.

The potassium concentration in the ECF is very low, approximately

one-thirtieth of the sodium concentration. The reverse is true of most cells of

the body: the potassium concentration is much greater than the sodium

concentration. This is presumably the result of the active extrusion of sodium

from the cell by the so called sodium pump.

1.11.1: Disturbances of potassium metabolism:

While the sodium concentration in the plasma is a reliable index of the

status of the body with respect to sodium, plasma potassium is not always a

meaningful indication of the status of the body with respect to potassium.

This is because most of the potassium is intracellular, and there is no

consistently reliable relationship between intra- and extracellular potassium

concentration (Jiro, 1980).

1.11.2: Hypokalemia:

Decreased concentration of potassium in the serum is called

hypokalemia or hypopotassemia. This may be the result of decreased intake of

potassium. Potassium depletion occurs even if the serum concentration is not

recognizably altered. When anorectic animals continue to drink water or when

they are kept hydrated by parenteral administration of potassium-free fluids

for many days, frank Hypokalemia may recognized. Potassium depletion and

Hypokalemia are also seen when patient has lost large quantities of fluids by

vomiting and diarrhea. Prolonged administration of adrenal steroids that have

some mineralocorticoid activity may promote potassium excretion and result

in Hypokalemia. Certain diuretics may cause potassium depletion (Abbrecht,

1969).

1.11.3: Hyperkalemia:

Increased potassium within the cell is apparently not harmful; the toxic

effects of excess potassium in the body are the direct result of increased levels

in the ECF. Hyperkalemia is most frequently the result of acidosis causing a

redistribution of body potassium, i.e., the movement of intracellular

potassium into the ECF.

The principal danger in hyperkalemia is cardiac arrest. Ward (1966) has

reported the death of a cow following oral administration of potassium

chloride. Infected calves with theileria have shown high levels of serum

potassium (Elhag, and Hamid, 2003).

Potassium is the most abundant cation in the intracellular fluid (ICF) of

both plants and animals. For this reason, this element is present in high

concentration in almost all normal foods. Therefore a deficiency of potassium

in the normal diet of any animal is rare (Tasker, 1967).

Because of the large quantities of potassium consumed by normal

animals, large amounts must be excreted to prevent potassium intoxication of

the organism. The kidney is principal organ for potassium excretion. It does

this by glomerular filtration as well as by tubular secretion (Pickering, 1965).

1.12: Serum Magnesium:

In term of amount, (Mg) is fourth among cations of the body, being

surpassed only by calcium, sodium, and potassium. In calves the Mg content

of the body is associated with the body weight according to the following

equation (Blaxter and McGill, 1956):

Mg (gm) = 0.655 weight (Kg) – 3.5

Mg is present in all tissues. About 70% of the total body Mg is located

in the skeleton, and approximately one-third of this is available for

mobilization to soft tissues when dietary intake is inadequate. Taylor, (1959)

demonstrated that Mg present in bone exists at least two different forms.

About 70% of the bone Mg could be removed relatively easily with dilute

acid, while the remaining 30% was intimately bound. The Mg of the skeleton

acts as a labile source of Mg, but there is striking difference between the

reactions of the young and those of the adult bovine (Blaxter and McGill,

1956).

Stevenson, and Wilson, (1963) suggested that the labile, exchangeable

form is present in a concentration which is practically identical with the Mg2+

concentration in the extracellular fluid. A simple diffusion equilibrium

probably exists between these two, while a more complex relation prevails

between the two intracellular forms.

The physiological functions of Mg may conveniently be divided into

two categories:

1. Intracellular function: Mg is an activator of numerous enzymes,

such as phosphatases and the enzymes catalyzing reactions

involving ATP. It may be inferred that the action of Mg extends

to all the major anabolic and catabolic processes involving the

main metabolites. Magnesium is an activator for enzymes that

require thiamine pyrophosphate as a cofactor. One of them is

active in the conversion of pyruvic acid to acetyl-CoA, an

example of oxidative decarboxylation, (Aikawa, 1963, and

1971).

2. Extracellular function: in the extracellular fluid Mg has an

important role in the production and decomposition of

acetylcholine, the substance necessary for the transmission of

impulses at the neuromuscular junction. A low concentration of

Mg, or more particularly a low Mg2+/Ca2+ ratio, potentiates the

release of acetylcholine (Del Castillo and Engbak, 1953).

Depending on the species, blood serum normally contains 2-5 mg Mg

per deciliter (0.8-2.1mmole/litre). In animals other than the ox, Mg occurs at a

higher concentration in the red cells than in plasma Salt, (1950) and Aikawa,

(1963) has found there is no apparent exchange of Mg between plasma and

RBC in the peripheral circulation. About 79% of the serum Mg has been

found to be an ultrafiltrable fraction independent of the total amount present

(Smith, 1957).

There is in some respects a reciprocal relationship between Mg and Ca

in the serum [the decrease in serum calcium is accompanied by an increase in

serum magnesium (Hallgren, 1959).

About 49 years ago Blaxter and McGill (1956) pointed out that there

was no evidence from critical experimentation indicating that the endocrine

glands had any specific effect on Mg metabolism. They therefore, suggested

that the Mg metabolism was regulated by a dynamic equilibrium in which the

skeleton acted as a labile source of Mg.

1.13: Hematology:

1.13.1: Red Blood Cells:

The mature red cell appears as a biconcave disc with no nucleus. It is

filled to saturation point with hemoglobin. The membrane of the red cell is

composed of a complex layer of lipids and proteins and the hemoglobin

within is in free solution. In addition to hemoglobin the cell contains sodium

and potassium in concentrations similar to those found in other cells, i.e. in

reverse concentrations to those found in the plasma. It also contains the

various enzymes necessary for its metabolism (which is almost entirely glycol

tic) and the various metabolic intermediates.

The concentration of red cells in the peripheral blood is maintained in a

steady state by a balance between their production in the bone marrow and

their destruction. Destruction in health is a result of ageing, each cell

disappearing after it has circulated for about 120 days. Alteration in the steady

state system may result from diminished production or increased destruction

when a deficiency of cells may result or from increased production where an

excess of cells occurs. These changes result in an anemia or polycythaemia

respectively. Anemia signifies a deficiency of circulating hemoglobin and

may result either from a lack of red cells which contain a normal amount of

hemoglobin, or lack of hemoglobin in red cells which are present in relatively

normal numbers. Theileria annulata piroplasms have been found to cause red

blood cells destruction (Losos, 1986).

1.13.2: Functions of the red cell:

Oxygen transport is the primary function of the red cell. It is

accomplished through the remarkable property of hemoglobin in combining

reversibly with oxygen in accordance with the external oxygen tension. The

full range of this property is apparent from an inspection of the oxygen

dissociation curve of hemoglobin. The electronic state of the iron in the haem

molecule controls the capacity of hemoglobin to take up oxygen, a process

which is accompanied by the loss of an electron (H+) from the globin portion

of the molecule.

Hemoglobin also plays an important part in the carriage of carbon

dioxide from the tissues, the globin moiety forming a reversible carbamino

compound in situations where the concentration of CO2 is high. However,

hemoglobin is not the only factor concerned in the carriage of CO2 in the

blood.

1.13.3: Red cell metabolism:

Red cells use glucose to perform a number of functions of which the

most obvious are:

1. Maintenance of the high concentration of potassium and low

concentration of sodium within the cell compared with outside.

2. Maintenance of a high concentration of reduced sulphydryl (- SH)

groups within the cell.

3. Maintenance of hemoglobin in the unoxidized state.

Two metabolic pathways exist in the red cell, and 90% of the glucose

utilized is broken down in the Embden-Myerhof glycolytic pathway. The

remaining 10% undergoes oxidation, but not through the respiratory

tricarboxylic acid cycle, which does not exist in the cell but through the

pentose phosphate pathway (London, 1961).

1.14: White Blood Cells:

The white blood cells may be broadly subdivided into (a) the

granulocytes, which have numerous granules in its cytoplasm and a

multilobed nucleus, and (b) the non granulocytes whose cytoplasm contains

few or no granules and whose nucleus is round or bean-shape (Lawrence,

1955).

1.14.1 Granulocytes:

The life-span of all granulocytes is thought to be the same and to

average about 10 days. To maintain normal equilibrium in the peripheral

blood about 11Х 1010 cells must be produced daily, but all these cells do not

reach the peripheral blood immediately.

The majorities is stored in the bone marrow and reach the blood after a

delay of about five days. These constitute the granulocyte reserves and may

amount to about 100 times the total in the peripheral blood; but in response to

a suitable stimulus, such as infection, they may be mobilized and discharged

into the blood producing a rapid increase in the number of circulating cells.

The destruction of granulocytes is probably due partly to natural ageing

and partly to their random removal in action against foreign invasion.

An increase in the peripheral blood content of granulocytes may result from a

sudden mobilization of the stores or a sustained increase in production by the

marrow. Increased production of granulocytes is usually accompanied by the

appearance of some primitive cells in the blood such as metamyelocytes, a

change sometimes designated as (a shift to the left).

A fall in the granulocyte may result from a decrease in the rate of

production or an increase in their rate of destruction. In the first instance there

will be a delay of about a week before the counts fall if the bone marrow

ceases to produce cells altogether, this period representing the time during

which the marrow stores are utilized. In the presence of increased destruction

however the count may fall rapidly. Little is known about abnormal

mechanisms of white cell destruction, although often immune processes are

involved.

Granulocytes are difficult to isolate from the other leucocytes in the

blood so that it is not easy to investigate their chemical activities, but

information about them can be obtained by special staining procedures. They

contain a wide variety of different enzymes, many of which are clearly not

concerned with cell metabolism; amongst these are peptidases, phosphatases,

lipases and esterases. Their granules contain phospholipids, and the cells also

contain glycogen.

The principal function of granulocytes is to protect the body against

infection and to help localize it. To this end the cells are phagocytic of

bacteria, and also contain various digestive enzymes which are liberated when

the cell lyses, and which break down tissue and bacterial components. The

combination of dead, dying, and decomposed granulocytes with partly

digested tissue and bacteria constitute pus. Polymorphonuclear cells in the

blood are in transit from the marrow to the tissues where they appear to be

attracted by certain chemicals, a phenomenon known as chemotaxis. In the

presence of bacteria the cell granules disappear, the cells become phagocytic

and they eventually disintegrate. In the process of lysis they release pyrogenic

material which often raises the body temperature.

Eosinophils have no known specific function but increase in the blood

in response to allergy and certain parasitic infections. Basophils appear to be

involved in the interaction of certain antigens and antibodies. Eosinophils

contain a high content arginine, whilst basophils are rich in histamine, a

heparin-like substance, and possibly serotonin. (Fredrick's, and Maloney,

1959).

1.14.2: Non granulocytes:

1.14.2.1: Lymphocytes:

Lymphocytes of two different sizes are found in the peripheral blood.

Large ones are about the size of polymorphs (diameter 10-14µ) and have a

pale staining cytoplasm with a single round nucleus containing irregular

clumps of chromatin. Small ones (diameter 7-10µ) consist almost entirely of

nucleus with only a thin surround of cytoplasm. Lymphocytes are produced in

lymph glands, the spleen, and in the thymus in young animals, but probably

only to a small extent in the bone marrow.

There is evidence that lymphocytes in lymph nodes and in the spleen

are the main source of antibody production in the body, and are therefore,

very active in globulin synthesis. They have no phagocytic activity. The

actual relation between the large and small lymphocytes is not clear but many

would accept that large lymphocytes are a resting stage of the small cell,

whilst the small cells may be actively mitotic.

Lymphocytes were originally thought to have a life-span of about 7-10

days but it appears that at least two varieties of small lymphocytes are

identifiable, one having a life-span of only days and the second having one of

perhaps years(Gowans and Knight, 1964).

1.14.2.2: Monocytes:

Monocytes are larger than other white cells (diameter 12-18µ).the cells

are motile, show amoeboid movements and are phagocytic. There are

relatively few Monocytes in the bone marrow and the origin of these cells is

not certain. It is probable that they develop from other free cells of the

reticulo-endothelial system. Their relationship to tissue macrophages is also

unclear but it seems likely that macrophages migrate into the tissues and

remain there as part of their life-cycle.

An increase in monocytes occurs in the peripheral blood in some viral

infections, especially infective hepatitis and glandular fever. Monocytosis

may also occur during recovery from pyrogenic infections. Chronic infections

such as tuberculosis and brucellosis may be associated with a monocytosis, as

may certain protozoal infections such as malaria and kala-azar (Valantine,

1960).

1.14.2.3: Plasma cells:

These cells are usually regarded as a form of lymphocyte and are

thought to be derived from them; they are 10-15µ in diameter, have a more

deeply basophilic cytoplasm often containing vacuoles, and eccentrically

placed nucleus. The nucleolus has irregular clumps of nucleoprotein which

are often arranged radially and have been described rather fancifully as of

(cart wheel) appearance. Plasma cells are responsible for the production of

certain anti bodies and globulins in the plasma. Plasma cells are not seen

normally in the peripheral blood and are present very sparsely in connective

tissue, lymph glands and bone marrow.

1.15: Packed Cell Volume:

The packed cell volume is a function of erythrocyte size and number of

cells per unit volume of blood. The hemoglobin content of the erythrocyte on

a volume basis is between 30-35 per cent with a mean of 33 per cent, except

in disease in which the utilization of iron is defective.

Anemia exist when the PCV falls below the minimum normal range

for the species in question, and hemoconcentration may be present when the

PCV exceeds the maximum of the normal range (Kaneko, 1959).

Laiblin (1978) has stated that in calves infected with theileria annulata

PCV is significantly decreased, as a result of the destruction of myeloid tissue

by lymphoblastoid toxins.

1.16: Hemoglobin:

Hemoglobin is a protein containing four haem molecules linked to two

pairs of protein chains which constitute globin. The two chains are α and β

polypeptides, the α chain contains 141 and the β chain 146 amino acids

residues arranged in known sequence. Each haem molecule consists of a ring

linkage of four pyrrole molecules with one iron atom forming a common bond

between haem and globin molecules. The iron atom in the haem molecules is

responsible for the reversible oxygen linkage of hemoglobin and it achieves

this linkage without a change in valency it can form a more stable linkage

with oxygen, when the iron changes into the ferric form, methemoglobin, and

it can also form stable linkages with carbon monoxide and cyanide.

There is a tendency for hemoglobin in the natural state to undergo auto-

oxidation to the ferric form which of course operates against the efficiency of

oxygen transport. within the cell any excessive oxidation is reversed by a

system which continuously reduces methemoglobin, so that in the normal cell

it remains at less than 2%.certain chemical agents, particularly aromatic

amino compounds, will oxidized hemoglobin beyond the methemoglobin

state; this results in a denaturaion of the globin moiety and the formation of

compounds, known as choleglobins, which can often be demonstrated as

Heinz bodies in specially stained cells. The presence of a high concentration

of reduced glutathione in the cell tends to prevent this sequence of reactions,

glutathione being more readily oxidized than hemoglobin. A further metabolic

system reduces any oxidized glutathione which accumulate in the cell,

through the enzyme glutathione reductase. The principal value of the pentose

phosphate pathway in the red cell appears to be that it provides a system for

main- tainig a high concentration of reduced glutathione, and thus prevents

the oxidative denaturation of hemoglobin, whilst glycolysis provides energy

and maintains methemoglobin reduction.

Different types of hemoglobin have been discovered which differ in the

structure of their globin polypeptide chains. The haem molecule is identical in

all animals but the globin molecule (although of fairly constant molecular

weight – about 60.000kd) is species specific (Perutz, et al, 1990).

CHAPTER TWO MATERIALS AND METHODS

Determination of the theilerial parasites either the piroplasms in the

blood smears or schizonts in the lymph nodes biopsies, and the faecal samples

for the exclusion of the internal parasites of the infected calves was done. The

haematology of both infected and healthy calves was also done. Serum

albumin, total proteins, calcium, phosphorus, sodium potassium, and

magnesium, of both infected and healthy calves were determined.

2.1. Area of Investigation:

Field samples of naturally infected calves were collected from the farm

of University of Khartoum in Shambat, and other farms situated at Hilat Kuku

in Khartoum north province. Poor health measurements were observed in all

farms which facilitate the abundance of many clinical cases.

2.2. Experimental Animals

Forty crossbred calves (Friesian with Kenana) with their age range

between 2-6 months were used. Thirty of them were theileria positive,

infected with Theileria. annulata. The other ten calves were healthy and used

as a control group.

2.3. Clinical examination

The following observations were carefully recorded:

• Rectal body temperature.

• Pulse, heart, and respiratory rate.

• Lymph node palpation.

• Examination of mucous membranes.

2.4 Collection and preparation of samples

Five ml blood was collected by venepuncture from jugular vein into

plain vacutainer and allowed to clot (overnight), and then the serum was

separated and stored at -20°C to be analyzed later for biochemical Parameters.

Two ml blood was taken from calves into EDTA blood containers, which

later was used for blood films and examined microscopically for the presence

of Theileria. annulata and haematological investigations.

2.5. Microscopical examination

Thin blood smears were made by putting a drop of blood onto a slide

margin and drawn by the aid of another slide, allowed to dry and fixed with

absolute methanol for 5 minutes. Parasitosis was recorded as the percentage

of schizont-infected cells and merozoite-infected cells in 200 cells; piroplasm

parasitaemia was recorded as the number of piroplasm-infected erythrocytes

in 100cells.

2.6. Lymph node biopsy smears:

A sterile disposable syringe was inserted into prescapular lymph node

and lymph node materials were drown out by negative pressure. A drop from

these materials was dropped onto a slide margin and drawn by the aid of

another slide, allowed to dry and fixed with absolute methanol for 5 minutes.

Then kept until examined.

2.7. Staining:

10% of Giemsa stain was used to examine the blood smears and lymph

node biopsy smear slides. These slides were put in an absolute methanol for

5-minutes, and then allowed to dry. Then were put into 10 % giemsa stain for

30 minutes, washed with distilled water for 30 seconds. Dried and examined

under the light microscope at (100Х). Whenever examining a smear, a drop of

emersion oil was dropped into it.

2.8. Faecal samples:

2.8.1. Sedimentation:

Small amount of faeces was placed in a centrifuge tube. Water was

added and mixed thoroughly with a glass rod. The tube was filled with water

and centrifuged at 1500 rpm for 5-minutes. Then the supernatant was

decanted. By using the pipette a drop from the top layer of the sediment was

placed onto the slide, another from the middle layer and a third from the

bottom layer. The three slides were covered with cover slides and examined

systematically under the lower power (100Х).

2.8.2. Flotation:

Small amount of faeces was taken in a carteny bottle. The faeces was

covered with saturated salt solution, mixed well, the bottle was filled to the

rim with saturated salt solution and few drops of salt solution were added in

excess. The bottle was covered with cover slide. The cover slide was removed

after one hour and examined systematically using the lower magnification

(40Х).

2.9. Biochemical analysis:

2.9.1. Serum Albumin:

Determination of serum albumin was performed according to Spencer

and Prince (1977), modified method.

Principle:

This method depends on the binding of Bromo Cresol Green (BCG)

with albumin at pH 4.1 using succinate buffer.

Reagents:

Stock succinate buffer, pH 4.1, 0.5 mol/L:

10g of sodium hydroxide and 56g succinic acid were dissolved in 800

ml of distilled water. The pH was adjusted to 4.1 at 20°C with molar solution

of sodium hydroxide. The volume was made up to one liter with distilled

water and stored at 4°C.

Stock BCG dye solution, 10 mmol/L:

1.75g BCG was dissolved in 5ml of one mole/L of sodium hydroxide

and was made up to 250ml with distilled water. When diluted 1/1000 with

succinic buffer, pH 5.3, 0.2 mole/L, and reading was against distilled water at

632 nm.

Stock sodium azide:

40g of sodium azide were dissolved in one liter of distilled water.

Stock Brij 35: 250g/L:

25g of solid Brij 35 were warmed in distilled water to dissolve and

made up to 100 ml with distilled water.

Working dye solution:

500 ml of the stock succinate buffer, 40 ml of the stock dye solution,

12.5 ml of the stock sodium azide, and 12.5 ml of the stock Brij 35 were

mixed in 5 liter volumetric flask and kept at 20 °C.

Stock standard albumin solution, 100g/L:

10g of bovine serum albumin and 50 mg sodium azide were dissolved

in distilled water and the mixture was made up to 100 ml with distilled water.

Working albumin standards:

20, 30, 40, 50, and 60g/L were prepared by diluting the stock standard

with a 500mg/L solution of sodium azide in distilled water, and was kept at

4°C.

Procedure:

20µL of the serum were added to 4 ml of the working dye solution at

25°C, then after 10 minutes, in a cuvette the test was read in the

spectrophotometer at 632nm against the blank of the working dye solution.

For standard 20µL of the stock standard albumin solution was added to 4ml of

the working dye solution.

Calculations:

Serum albumin (g/L) =

Optical density of unknown Х Concentration of the standard Optical density of standard 2.9.2 Serum Total Proteins:

Determination of total protein was done according to Reinhold method

(1953).

Principle:

The determination of total protein by Biuret method depends on the

precipitation of globulins by a mixed sodium sulphate and sulphite solution in

proportions of 3:1.

Reagents:

Sulphate – sulphite solution (278g/L):

208g of anhydrous sodium sulphate and 70g of anhydrous sodium

sulphite were dissolved in 900 ml of distilled water to which 2 ml

concentrated sulphuric acid was added in 2-liter beaker. Then the pH was set

above 7.0, and the solution was kept at 37°C in the incubator.

Stock Biuret reagent:

45g of Rochelle salt was dissolved in 400 ml of 200mmol/L sodium

hydroxide, and then 15 g of copper sulphate was added and followed by 5g of

potassium iodide. Thereafter, the solution was made up to a liter with 200

mmol/L of sodium hydroxide.

Biuret reagent for use:

200 ml of the stock reagent was diluted to a liter with 200 mmol/L of sodium

hydroxide containing 5g of potassium iodide per liter.

Tartarate-iodide solution:

9 g of Rochelle salt was dissolved in 400ml of 200 mmol/L sodium

hydroxide containing 5g potassium iodide/L.

Standard serum:

Bovine serum g/100 ml.

procedure:

6.0 ml of sulphate-sulphite solution were pipetted into centrifuge tube,

and to it 0.4 ml of the serum were added to 5 ml of Biuret reagent in test tube.

Serum blank: 2 ml serum sulphate-sulphite mixture was added to

5 ml tartarate-iodide solution and mixed well.

Biuret blank: 2ml of sulphate-sulphite solution was added to 5ml of the

Biuret reagent.

Standard: 0.4 ml of the standard serum was pipetted into 6.0 ml of the

sulphate-sulphite solution as above, and 2ml of the mixture was transferred

into 5ml of Biuret reagent in a test tube.

Standard serum blank: it was prepared as described for test serum

blank.

After shaking, all tubes were placed in a water bath at 37°C for 10

minutes. Then they were allowed to cool for 5 minutes at room temperature.

Reading by (jenway 6105, u.v/Vis. spectrophotometer) was at 540-560nm

wave length. Serum blank was read against the tartarate-iodide solution, test

and standard were read against the Biuret blank.

Calculations:

Serum total protein (g/L) =

Reading of unknown – Reading of unknown serum blank Х Concentration Reading of standard - Reading of standard serum blank of standard 2.9.3 Serum Calcium concentration:

Serum calcium was evaluated using Flamephotometer according to

Gindler and King method (1972).

Reagents:

Chloranilic acid (0.5%):

0.5g of Chloranilic acid was dissolved in 100ml distilled water

containing 0.4ml ethanolamine. Then 50 mg sodium cyanide were added. The

solution was allowed to stand overnight and the clear layer was stored in the

refrigerator.

Ferric nitrate:

10 g of ferric nitrate were dissolved in 200 ml distilled water and 30 ml

of NH3SO4 and the solution was made up to 250 ml with water.

Standard stock solution:

0.25g of calcium carbonate was dissolved in 0.1 HCL and made up to

100 ml with acid.

Working standard solution:

4.0ml of stock solution was dissolved in 100 ml distilled water.

Calcium chloride stock solution:

1.0ml of stock solution was dissolved in 10 ml distilled water.

Procedure:

1- In three test tubes marked: test, blank, standard. 0.5 ml

serum+1.0 of 0.5% Chloranilic acid were added to the test tube. Into standard

tube 0.5 ml of working standard+1.0 ml of Chloranilic acid were added and

stand for 15minutes. Ferric nitrate was used as blank.

2- the Standard tube and the test were centrifuged at 3000 (rpm) for 5

minutes. The precipitate was washed with 0.5ml distilled water and

centrifuged, decanted, and drained. The precipitate was dissolved in 4 ml of

4% ferric nitrate. Then stand for 5miutes.

3- The three tubes (test, blank, standard) were read at 500nm.

Calculations:

Calcium (mg/100ml). = Test - Blank Х 10 STD - Blank 2.9.4 Serum phosphorus concentration:

Serum phosphorus was determined according to Varley (1967).

Reagents:

10% Trichloroacetic acid:

10 g of trichloroacetic acid were dissolved into 100 ml distilled water.

Ammonium Molybdate:

15 g of ammonium molybdate were dissolved in about 400 ml distilled

water, 100 ml of 10 n sulphuric acid was added, and the solution was made up

to 800 ml with distilled water.

Metol:

1g of P-methyl amino-phenol was dissolved in 100ml of 3% solution of

sodium bisulphate.

Standard:

0.2197g of potassium dihydrogen phosphate was dissolved and made

up to 1liter of distilled water. Few drops of chloroform were added.

Procedure:

1- In three test tubes marked: test, blank, standard. 1ml of plasma was

put into test tube labeled test, and 9 ml of 10% trichloroacetic acid, then

filtered. 5 ml was taken from the supernatant.

2- 0.5ml of working standard+4.5ml of 10% trichloroacetic acid were

put into the standard tube.

3- 5ml of 10% trichloroacetic acid was put into blank tube. To all tubes

1ml of ammonium molybdate solution was added and mixed. 1 ml of Metol

was added and mixed. Then allowed to stand for 30 minutes at room

temperature. Then the three tubes were read at 680nm.

Calculations:

Phosphorus (mg/100ml) = Test Х 5 STD 2.9.5 Serum Sodium and Potassium concentration:

Serum sodium and potassium were determined using Flamephotometer

according to Varley et al, (1967 and 1980).

Principle:

In flamephotometer, a solution containing the substance to be

determined was passed under carefully controlled conditions as a very fine

spray into the air supply o the burner. In the flame the solution was

evaporated and the substance was first converted to the atomic state. As the

temperature was raised the thermal energy of the flame made the electrons

able to be absorbed. Light of characteristic wave length is emitted and passes

through specific filter for sodium or potassium onto a selenium cell, and the

amount was read on a galvanometer.

Reagents:

Stock standard of sodium:

58.5g of sodium chloride were dissolved in one liter of distilled water.

Working sodium standard:

High standard: 8 ml of the stock standard in one liter distilled water.

Low standard: 7 ml of stock standard in one liter distilled water.

Stock standard of potassium:

7.5 g of potassium chloride was dissolved in one liter distilled water.

Working potassium standard:

High standard: 7ml of stock potassium chloride in one liter distilled

water.

Low standard: 5ml of stock potassium chloride in one liter distilled

water.

High standard of sodium and potassium:

8ml of high sodium standard and 7ml of high potassium standard then

made up to one liter with distilled water.

Low standard of sodium and potassium:

7 ml of low sodium standard and 5 ml of low potassium standard were mixed,

the volume was made up to one liter with distilled water.

Procedure:

The flamephotometer was used as follow: the butane, burner, air

compressor were adjusted. The zero point of the galvanometer was adjusted

against distilled water, and the 100 point of sodium or potassium was adjusted

against the high standard of each, after selecting the wave length key of

sodium or potassium. For either sodium or potassium 0.1ml serum sample

was diluted in 9.9ml of distilled water. The concentration of sodium was 140

mEqu/L and of potassium was 5 mEqu/L. the diluted sample was read against

the low standard of both sodium and potassium.

Calculations:

* Serum sodium (mEq/L) =

Reading of the unknownХ140mEqu/L Reading of the low standard of sodium * Serum potassium (mEq/L) = Reading of the unknownХ5mEqu/L Reading of the low standard of potassium

2.9.6. Serum Magnesium concentration:

Determination of serum magnesium was performed according to

method described by Norbert, (1982).

Reagents:

Stock standard of magnesium:

8.8 g of magnesium acetate was dissolved in distilled water and the

solution was made up to 1liter of distilled water.

Working Stock standard of magnesium:

1ml of stock standard solution was diluted up to 200 ml of distilled

water (fresh standard).

Polyvinyl alcohol reagent:

1g of polyvinyl alcohol was dissolved in about 400 distilled water using

gently heat. The solution was cooled and made up to 1liter with distilled

water. This solution was stable.

Titan yellow (stock):

0.5g of titan yellow was dissolved and made up to 100 ml with distilled

water.

Titan yellow working reagent:

2.0ml of the stock solution was diluted up to 100 ml with distilled

water (fresh for each test).

Procedure:

1- In four test tubes marked: test, blank, low standard, high standard.

0.2ml serum were added to test. Into blank tube 3.0ml of distilled water were

pippeted. Into low standard solution tube 1.0ml of working standard+2.0ml

distilled water were pippeted. Into high standard solution tube 2.0ml of

working standard+1.0ml distilled water were pippeted.

2- To all tubes in the stated order, mixing after each addition: 0.5ml

polyvinyl alcohol reagent, 0.5ml titan yellow working reagent , and 1.0 ml

7.5% w/v sodium hydroxide were added

3- All tubes were allowed to stand for 5 minutes.

4- Absorbance's of test and standard were read at 540nm after setting zero

absorbance with the blank.

Calculations:

Mg conc. (mg/100ml) = Test Х 2.5 Low STD 2.10 Hematological investigation:

Haemoglobin (Hb) concentration, haematocrit (HCT), red blood cell

(RBC), white blood cell (WBC), differential WBC counts, mean corpuscular

volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular

haemoglobin concentration (MCHC), were determined by using the Sysmex

KX-21 machine which performs speedy and accurate analysis of 18

parameters in blood and detects the abnormal samples. To assure easy sorting

of abnormal analysis data with abnormal marks attached on the screen. Thus

displayed analysis data allows detecting those samples which are outside the

tolerance and need further analysis and reconsideration.

The Sysmex KX-21 employs three detector blocks and two kinds of

reagents for blood analysis. The WBC count is measured by the WBC

detector block using the direct count (DC) detection method. The RBC and

platelets are taken by the RBC detector block, also using the DC detection

method. The HGB detector block measures the hemoglobin concentration

using the non-cyanide hemoglobin method. (Sysmex, 1998).

2.11 Statistical method:

A two-way analysis of variance procedure was performed separately for

each variable using paired-samples T-test by SPSS PC version 12 Apache

system Foundation (ASF, 2003), USA.

CHAPTER THREE RESULTS

Out of 74 cross calves their age vary between two to six months were

examined clinically and parasitologically, 30 were found as positive cases for

T. annulata. Animals without overt clinical signs and carriers with very low

parasitaemia were also considered. An average of 1–5 piroplasm forms in the

RBC were observed in all cases with a range of 10–45% parasitaemia.

Schizont parasitosis was generally high (more than 10%).

3.1 Serum chemistry findings:

3.1.1 Serum total protein:

The effect of Theileria annulata on serum total protein in crossbred

calves is presented in Table (2) and figure (3). Thus total proteins studied

showed significantly (p<0.05) lower values in theileria infected calves

compared to control calves.

3.1.2 Serum albumin:

The level of albumin in theileria infected calves is significantly

(p<0.05) lower compared to the control, as seen in table (2) and (fig.3)

3.1.3 Serum calcium:

The effect of Theileria annulata on serum calcium in crossbred calves

is presented in Table (2) and figure (3). Thus calcium studied showed

significantly (p<0.05) lower values in theileria infected calves compared to

control calves.

3.1.4 Serum phosphorus:

As seen in Table (2) and figure (3), theileria infected calves have

significantly (p<0.05) higher serum phosphorus values compared to control

calves.

3.1.5 Serum magnesium:

Table (2) and figure (3) showed that thieleria infected calves have

significantly higher values of serum magnesium (p<0.01) compared to the

control calves.

3.1.6 Serum potassium:

As seen in Table (2) and figure (3), theileria infected calves have non

significantly higher serum potassium values compared to control calves.

3.1.7 Serum sodium:

The effect of Theileria annulata on serum sodium in crossbred calves is

presented in Table (2) and figure (3). Thus sodium studied showed

significantly (p<0.01) lower values in the theileria infected calves compared

to the control calves.

3.2 Haematological findings:

3.2.1 Red Blood Cell Count (RBC):

Table (1) and figure (4) showed that thieleria infected calves have

significantly lower values of RBCs count (p<0.01) compared to the control

calves.

3.2.2 Hemoglobin (HGB):

As seen in Table (1) and figure (4), theileria infected calves have

significantly lower hemoglobin values (p<0.01) compared to control calves.

3.2.3 Haematocrit (HCT):

The effect of Theileria annulata on hematocrit in crossbred calves is

presented in Table (1) and figure (4). Thus hematocrit studied showed

significantly (p<0.01) lowered values in theileria infected calves compared to

the control calves.

3.2.4 Mean Corpuscular Volume (MCV):

As seen in Table (1) and figure (4), theileria infected calves have

significantly lower mean corpuscular volume values (p<0.01) compared to

control calves.

3.2.5 Mean Corpuscular Hemoglobin (MCH):

Table (1) and figure (4) showed that thieleria infected calves have non

significantly lower values of mean corpuscular hemoglobin levels compared

to the control calves.

3.2.6 Mean Corpuscular Hemoglobin Concentration (MCHC):

As seen in table (1) and figure (4), theileria infected calves have

significantly lower mean corpuscular hemoglobin concentration values

(p<0.01) compared to control calves.

3.2.7 White Blood Cell Total Count (WBC):

The effect of Theileria annulata on WBC count in crossbred calves is

presented in table (3) and figure (5). Thus WBC count studied showed

significantly (p<0.01) lower values in theileria infected calves compared to

the control calves.

3.2.8 Lymphocytes:

Table (3) and figure (5) showed that thieleria infected calves have

significantly (p<0.01) lower values of lymphocytes compared to the control

calves.

3.2.9 Neutrophils:

As seen in Table (3) and figure (5), theileria infected calves have

significantly lower Neutrophils values (p<0.01) compared to control calves.

3.2.10 Monocytes:

Table (3) and figure (5) showed that thieleria infected calves

have significantly (p<0.05) lower values of monocytes compared to the

control calves.

Table (1): The effect of infection with T.annulata on hematological Indices (mean ± S.E.M) in crossbred calves in Khartoum state region

Indices uninfected calves

infected calves

RBC 103/1 7.26±.286 3.70±.206 p<0.01

HGB g/dl 11.62±.619 4.06±.369 p<0.01

HCT 1/1 34.57±2.079 16.99±1.60 p<0.01

MCV Fl 42.09±.422 32.59±.929 p<0.01

MCH Pg 14.19±.162 14.160±.225 p>0.05

MCHC g/dl 33.77±.345 29.060±.658 p<0.01

Table (2): The effect of infection with T.annulata on serum total proteins, albumin, calcium, phosphorus, magnesium, sodium, and potassium. (Mean ± S.E.M) in crossbred calves in Khartoum state region

Parameters Uninfected calves

Infected calves

Total Proteins (g/dl)

6.83±.139 5.75±.299 p<0.05

Albumin (g/dl)

4.00±.437 2.62±2.2 p<0.05

Calcium (mg/100ml)

7.47±.366 2.88±.264 p<0.01

Phosphorus (mg/100ml)

4.26±.056 7.54±.398 p<0.01

Magnesium (mEq/L)

2.01±.037 6.10±.373 p<0.01

Sodium (mEq/L)

152.70±1.612 119.85±1.394 p<0.01

Potassium (mEq/L)

4.02±.216

4.53±.030 p<0.05

Table (3): The effect of infection with T.annulata on The mean of Total WBC count and Differential WBC count (mean ± S.E.M) in crossbred calves in Khartoum state region.

Uninfected calves

Indices

Infected calves

WBC/Total 103/1

7.06±.220 3.55±.280p<0.01

Neutrophils 103/1

2.45±.176 1.33±.091p<0.01

Lymphocytes 103/1

3.58±.171 1.76±.199p<0.01

Monocytes 103/1

.38±.031 .303±.020p<0.05

CHAPTER FOUR DISCUSSION

4.1 Effect of tropical theileriosis on some serum proteins and minerals of cross-

bred calves:

4.1.1 Plasma total proteins and albumin:

The level of plasma total proteins and albumin are the most important factors

which are known to be affected by Theileria annulata infection. Several studies

showed that the level of total proteins and albumin in serum decreased after the

infection with theileria annulata Yasir,(2002 ) and Mohamed,(2003), reported a

significant decrease in serum total proteins in theileria infected calves compared to

the control calves.

In the present study the plasma total proteins and albumin were found to be in

line with these findings. The decrease was suggested to be due to the fact that hepatic

damage and gastroenteritis due to theilerial schizonts cause hypoalbuminemia and

consequently hypoproteinaemia (Meyer et al. 1992). This hepatic damage can cause

inadequate synthesis of protein. Moreover, the gastroenteritis results in inadequate

absorption in the gastrointestinal tract.

4.1.2 Serum calcium:

Serum calcium values were decreased significantly in theileria infected calves

compared to the control calves. The hypocalcaemia was in agreement with the work

of Mohamed (2003), Dhar and Gautam (1977). The decrease of serum calcium was

due to the decrease of serum albumin, because 40% of serum calcium is bound to

serum albumin (Meyer et al, 1992).

4.1.3 Serum phosphorus:

Serum phosphorus values were increased significantly in theileria infected

calves compared to control calves. The increased values may be attributed to the fact

that serum phosphorus tends to increase during fasting. Since theilerial infection

causes anorexia and dehydration this consequently causes an increase in serum

phosphorus values. Moreover, decreasing serum calcium levels is accompanied by

increased serum phosphorus levels (Mylera and Bayfield, 1968).

4.1.4 Serum sodium and potassium:

The main function of sodium and potassium is to maintain the intracellular

gradient. The pump that maintains this gradient is ATPase which is activated by

sodium and potassium. The ATPase has catalytic centers for both ATP and sodium

on the cytoplasmic side of the membranes, but potassium binding side is located at

the extracellular side of the membrane.

Na+ - K+ ATPase pump moves sodium ions from inside the cell to the outside

and brings two potassium ions from the outside to the inside for every molecule of

ATP hydrolyzed to ADP by the membrane associated ATPase. Inhibition of the

extracellular domain due to the disease inhibits this ATPase. Moreover, during

theileria infection animals show severe dehydration and hypoglycemia (Yasir, 2002)

which affect the synthesis of ATPase, sodium, and potassium levels.

4.1.5 Serum magnesium:

Serum magnesium values were increased significantly in theileria infected

calves compared to the control calves. In general red blood cells have great amount of

magnesium into their cytoplasm (Salt, 1950; and Aikwa, 1963). Since theileria is

intraerthrocytic parasite and causes erythrolysis (Losos, 1986). Therefore, great

amount of magnesium will be released into peripheral circulation.

4.2 Effect of tropical theileriosis on hematological parameters of cross-bred

calves:

Changes in blood parameters indicated severe anemia, panleukopenia,

lymphocytopenia, eosinopenia and neutropenia but no reticulocytosis. The decrease

in MCHC, HGB, and MCV values in infected calves indicated macrocytic

hypochromic anemia. These results came at variance with that of Sandhu, et al.

(1998) who reported normocytic normochromic anemia in cross-bred calves

experimentally infected with T. annulata and in line with Omer, et al. (2002). It may

be of relevance to note that severe macrocytic hypochromic anemia was reported in

sheep experimentally infected with Theileria recondita (Alani and Herbert, 1988).

However, no reticulocytosis was observed in infected calves. This could be attributed

to the fact that cattle demonstrate reticulocytosis only on extreme provocation such as

severe acute haemorrhage(Kerr, 1989).

Despite the fact that anaemia is one of the main symptoms of tropical

theileriosis, the precise mechanism is still unknown. It has been claimed to be a result

of removal of erythrocytes by phagocytosis rather than parasite-induced lysis

(Boulter and Hall, 2000).

Hooshmand-Rad (1976) proposed the involvement of an auto-immune

reaction. Tumour necrosis factor-_ (TNF-_) has also been implicated in the

pathogenesis of anaemia by suppressing haematopoietic progenitors resulting in

decreased erythrocyte production and also decreased erythrocyte survival (Boulter

and Hall, 2000). The activation of complement as a cause of erythrocyte destruction

could also be suggested. Activation of complement is known to occur in malaria

infection (Adam, et al. 1981). The manifestation of anemia, leukopenia and

lymphocytopenia in acute T. annulata infection were reported by several authors

(Hooshmand-Rad, 1976; Gill, et al. 1977; Preston, et al. 1992; Sandhu, et al. 1998;

Forsyth, et al. 1999; Singh, et al. 2001). The present investigation has shown that

acute T. annulata infection in crossbred calves was also accompanied by significant

decreases in neutrophils.

Recent studies have shown that monocytes are the host cells invaded and

transformed by T. annulata in vivo (Forsyth, et al.1997, Forsyth, et al. 1999, Preston,

et al. 1999). The syndrome of anaemia, panleukopenia of extreme neutropenia and

lymphopenia and thrombocytopenia was also reported in field cases of East Coast

Fever in Tanzania (Mbassa, et al. 1994). The cause of leukopenia in acute T. annulata

infection is not quite clear. However, a role for cytokines, particularly TNF-_ has

been proposed. (Forsyth, et al. 1999).

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