Epidemiological and Biomolecular Studies on Echinococcus granulosus in Sudan

By

Rihab Ali Omer Abdalla Hamid

B.V.SC., M.V.SC

A thesis submitted in partial fulfillment of the requirements

of the degree of PhD University of Khartoum

Supervisor

Prof. Imad Eldain Elamin Eltahir Aradaib

April, 2006

Dedication

To the soul of my father who has dreamed a lot

to see the day I get this title

To my mother who has supported me and gave me

her blessing to overcome all the difficulties I

faced during my work

To my sisters and brother

To my husband Ayman, the one who was always

behind me providing all the support encouragemen,

power and strength

To my son Monzir, the beautiful smile which shone and enlightened my life during the last period of my work

List of contents

i Dedication

ii List of contents

iii List of Tables

iv List of Figures

v Acknowledgement

vi Summary

vii Arabic summary

1 Chapter One: General Introduction and Literature Review

1 Introduction 1.1

2 Objectives of the study

3 Classification 1.2

4 Life Cycle (Fig 1) 1.3

7 Distribution of cystic echinococcosis (Fig 2) 1.4

9 Cystic echinococcosis in Africa 1.5

10 Cystic echinococcosis in Sudan 1.6

12 Molecular epidemiology of cystic echinococcosis 1.7

13 Techniques of strain characterization 1.8

14 General morphology 1.8.1

15 Biochemical Methods 1.8.2

15 Developmental method in vivo 1.8.3

16 Developmental method in vitro 1.8.4

16 Genetic Characterization 1.8.5

17 Sequencing of partial mitochondrial cytochrome coxidase subunit 1 (cox1). (Bowles et al., 1992).

1.8.5.a

17 Analysis of ribosomal DNA regions ITS1 by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) (Bowles and McManus, 1993)

1.8.5.b

18 Analysis of ribosomal DNA region ITS2 by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) (Gasser and Chilton, 1995)

1.8.5.c

18 A PCR system for detection of species and genotypes of Echinococcus granulosus complex (Dinkel et al, 2004)

1.8.5.d

19 Diagnosis of Echinococcus in definitive and intermediate host 1.9 19 Diagnosis of Echinococcus in definitive host 1.9.1

19 Detection of serum antibodies 1.9.1.a

20 Detection of coproantigens 1.9.1.b

21 Detection of copro-DNA 1.9.1.c

21 Diagnosis of cystic hydatidosis in intermediate host 1.9.2

22 Serodiagnosis of hydatid disease in animals 1.9.2.a

23 Diagnosis of cystic hydatidosis in humans 1.9.3

23 Imaging Techniques 1.9.3.a

23 Immunodiagnosis of human hydatidosis 1.9.3.b

27 Chapter Two: General Material and Methods

27 Epidemiological survey 2.1

27 Hydatid cysts 2.2

30 Collection of hydatid cysts or cyst material from human patients

2.3

30 Parasitological studies 2.4

30 Preservation of hydatid and adult E.granulosus samples 2.5

30 Preservation of the faecal samples 2.6

31 Preparation of hydatid cyst material from intact cysts for DNA extraction

2.7

31 DNA extraction 2.8

32 DNA extraction from the faecal samples 2.9

33 Determination of the DNA concentration in samples 2.10

33 Polymerase Chain Reactions 2.11

33 Cestode specific PCR (cs PCR) 2.11.1

33 PCR assay specific for E.granulosus G1 (g1 PCR) 2.11.2

34 PCR assay specific for E.granulosus G6/7 and E. ortleppi (g5/6/7 PCR, g6/7 PCR, g5 PCR

2.11.3

34 Semi nested PCRs specific for E. ortleppi and E. granulosus (G6/7)

2.11.4

35 Detection of the PCR products (Agarose gel electrophoresis) 2.12 35 Preparation of the gel 2.12.1 35 Preparation of the sample and running of the gel 2.12.2 35 Visualization of the PCR products 2.12.3 34 Mitochondrial gene sequencing 2.13

36 Used Chemicals 2.14

36 Puffers and solutions 2.15

37 Enzymes 2.16

37 Oligonucleotides 2.17

38 2/7 Disposables 2.18

38 2/8 Instruments 2.19

40 Chapter Three : Epidemiological and Biomolecular Study of Cystic Echinococcosis in Humans and Animals in Sudan

40 Abstract

41 Introduction

43 Materials and Methods

43 1. Epidemiological survey Epidemiological survey

43 2. Parasitological studies

44 3. DNA extraction

44 4. Polymerase chain reaction (PCR ) and molecular characterization

45 5. Visualization of the PCR products

45 6. Mitochondrial gene sequencing

46 Results

46 1. Epidemiological Survey

47 2. Strain characterization

60 Discussion

63 Chapter Four: Strain Characterization and Coprodiagnostic PCR of E.granulosus in Central Sudan

63 Abstract

64 Introduction

66 Materials and Methods

66 1. Stray dogs

66 2. Scraping

66 3. Preservation of the faecal samples and harvested worms

67 4. DNA extraction from E.granulosus adult worms

67 5. DNA extraction from the faecal samples

68 6. Polymerase Chain Reactions (PCRs)

69 7. Visualization of the PCR products

69 8. Control for inhibition

69 9. Mitochondrial gene sequencing

71 Results

71 1. Necropsy

71 2. PCR of the E.granulosus worms

71 3. Copro PCR

79 Discussion

81 Chapter Five: Experimental Infection of Dogs with Protoscoleces of Camel Origin and Molecular Characterization of the Maturing Adult Worms Using PCR

81 Abstract

83 Introduction

85 Materials and Methods

85 1. Experimental Animals

85 2. Infective Materials

85 3. Parasitological Parameters

86 4. Necropsy

86 5. Molecular characterization of the adult worms

88 Results

88 Parasitological findings

88 Necropsy

88 Molecular Characterization of the adult worms.

92 Discussion

94 Chapter Six : General Discussion

102 References

List of Tables

Chapter one

26 Table 1 : Species and strains of Echinococcosis complex

Chapter two

28 Table 2 Numbers of examined camels, cattle, sheep and goats in different study areas

Chapter three

48 Table 1: Prevalence of cystic echinococcosis in livestock in Central Sudan

50 Table 2: Prevalence of cystic echinococcosis in livestock in Western Sudan

52 Table 3 Prevalence of cystic echinococcosis in livestock in Southern Sudan

55 Table 4 Strain characterization of hydatid cyst samples from camel, cattle, sheep, and goats from different parts of Sudan

Chapter four

72 Table 1 Necropsy and Copro-PCR results of the 42 dogs shot in central Sudan

Chapter five

89 Table 1

Results of the experimental infection of dogs in groups A and B with protoscolices of camel origin

List of Figures

Chapter one

6 Figure 1: Life cycle of Echinococcus granulosus

8 Figure 2: World Distribution of cystic echinococcosis

Chapter two

29 Figure 1: Study area

Chapter three

49 Figures 1 and 2: Prevalence and fertility of hydatid cysts from camel, cattle, sheep and goats in central Sudan

51 Figures 3 and 4: Prevalence and fertility of hydatid cysts from camel, cattle, sheep and goats in Western Sudan

53 Figures 5 and 6: Prevalence and fertility of hydatid cysts from camel, cattle, sheep and goats in southern Sudan

54 Figure 7: Predilection sites of hydatid cysts in camel, cattle, sheep and goats in different parts of Sudan

56 Figure 8: Visualization of the first amplified 254 bp PCR product from differnt cyst samples.

57 Figure 9: Semicamel PCR amplification of the 171 bp specific PCR product of the camel (G6) strain from the first 254 bp PCR product for the above gel.

58 Figure 10: Semicattle PCR amplification of the 171 bp specific PCR product of the cattle (E.ortleppi) strain from the first 254 bp PCR product for the G5/6/7 PCR.

59 Figure 11: Visualization of the 254 bp G1 (sheep strain) PCR product from differnt cyst samples .

Chapter four

73 Figure 1: Visualization of the first amplified 254 bp PCR product from E.granulosus worms.

74 Figure 2: Semicamel PCR amplification of the 171 bp specific PCR products of the camel (G6) strain from the first 254bp

75 Figure 3: Visualization of the first amplified 373bp PCR product from copro DNA

76 Figure 4: Visualization of the 254bp G5/6/7 nested PCR from the first cestode specific PCR

77 Figure 5: Semicamel PCR amplification of the 171bp specific PCR products of the camel (G6) strain from the G5/6/7 PCR

78 Figure 6: Visualization of the amplified 254 bp G1 (sheep strain) PCR product from copro DNA.

Chapter five

90 Figure 1: Visualization of the first amplified 254 bp PCR products from differnt worm samples.

87 Figure 2, Seminested PCR amplification of the 171 bp specific PCR product of the camel (G6) strain from the first 254 bp PCR product for the above gel.

1 3 4

5 6 7 8 9 10 11 12 NC PC

Acknowledgement

First, many thanks to Allah, my God who insert the desire of

work in my sides, and give me the support to work and power to

continue.

Many thanks to Prof. Imad Aradaib, my supervisor who helped

in designing the research protocol and looked after me with their kind

supervision and advices.

Thanks to the members of the Department of Parasitology,

University of Hohenehim, Germany for helping me during the course

of the study and for making my stay in Germany easy and enjoyable.

Special thanks are due to Prof Ute Mackenstedt for her valuable

supervision as well as official and personal helps. Many thank Dr.

Thomas Romig for helping me in planning my work, and for his

valuable advice and suggestions and for revising the manuscript.

Special thanks to Dr. Anke Dinkel for her supervision and for helping

me in performing the molecular biology techniques. May thanks are

also due to Dr. Michael Merli for his useful guidance during my work

in Hohenheim.

May thanks are also extended to Dr. Mohamed Elamin and

International Red Cross Commission (ICRC), Khartoum for providing

me with the human samples

My thanks are also due the family of the Central Veterinary

Research laboratories, Soba, specially Prof. Mohamed Salih

Algablabi, and Prof. Salah Mukhtar. Special thanks to the staff of

Parasitology, Soba, specially Mr. Nasir, Khalil, Ahmed Elshik for

technical help.

Thanks to my colleges, Dr. Osman Mukhtar, Dr. Yahia Hassan Beik,

Dr. Wegdan Hassan Ali and Dr. Selma Osman. and all other friends in

the Central Veterinary Research Laboratories (CVRL), Soba.

Thanks are also extended to the German Academic Exchange

Service (DAAD) for financial support of this work and for being a real

family during my stay in Germany.

I also acknowledge the important support offered from my mother,

sisters and brother.

My thanks are also due to my husband Dr. Ayman Elnahas for

being very helpful during the work and also for support and

encouragement. Thanks Monzir for sharing this time with me.

Summary This study represents an epidemiological and biomolecular study

about Echinococcus spp. and hydatid disease in definitive and

intermediate hosts including humans in Sudan. In this study, a survey

of cystic echinococcosis was conducted during the period from May

2001 to July 2003 in different parts of the Sudan. The prevalence rates

in camels, cattle, sheep and goats examined in different states of the

Sudan was found to be 59.8% (466/779), 6.1% (299/4893), 11.3%

(1180/10422) and 1.9% (106/5565) respectively. The encountered

number of cysts was 2387 in camels, 333 in cattle, 1514 in sheep and

108 in goats. Fertility rates were found to be 73.7%, 77%, 19% and

31.5% in camel, cattle, sheep and goat respectively. The favorite site

for cysts in camels was the lung (1627/2387). The liver was found to

be the preferred site in cattle (206/333) whereas the peritoneum was

the predilection site in sheep (1242/1514) and goats (53/108).

Strain characterization of the E. granulosus complex in human and

livestock population was described for the first time by using

polymerase chain reaction amplification and sequencing technology.

Even though we were able to detect E. ortleppi and sheep strain (G1)

in some samples, camel strain (G6) appears to be the predominant

strain causing cystic echinococcosis in humans and animals in Sudan.

533 of a total of 542 of all isolates were characterized as belonging to

this strain. In this study, the sheep strain of E.granulosus was reported

for the first time in Sudan in two samples of human origin and five

samples of sheep origin.

Secondly, 42 dogs shot as a part of the rabies control program in

Tamboul and Rofa, central Sudan, were autopsied and their intestinal

contents were examined for the presence of Echinococcus worms.

Faecal samples were taken for coprodiagnosis. Worm burden in

positive dogs was determined using dilution method and the harvested

worms were characterized using G5/6/7 and G1 PCRs. From the 42

euthanized dogs, 12 (28.5) were harboring E.granulosus worms The

worm burden was 22-80*103 in the positive dogs. All the DNA

samples extracted from the worm suspension were characterized as

camel (G6) strain of E.granulosus. 83.3% (10/12) of the DNA

extracted from the faecal samples collected from the 12 dogs which

were found to be positive at necropsy were also found positive with

copro PCR and the strain was characterized as camel (G6) strain of

E.granulosus. Two samples were considered inconclusive as there was

no signal in the inhibition test. 93.3% (28/30) copro DNA samples

from the 30 samples collected from the dogs which were reported

negative at necropsy were also negative using copro-diagnostic PCR.

The other two samples were positive and characterized as sheep (G1)

strain of E.granulosus. This copro PCR method was used for the first

time in such a survey. Disregarding the inhibited samples, the overall

sensitivity of the test was found to be 100%.

For the purpose of this study, hydatid cysts were obtained from

the lungs of naturally infected camels (Camelus dromedarius) in

Tamboul slaughterhouse in central Sudan. Viable protoscolices were

collected from these cysts and used for experimental infection of dogs

at different doses. Ten dogs were divided into two groups (A and B)

of five dogs each. Dogs in group A received a dose of 4×103

protoscolices each whereas dogs in group B received a dose of

8×103.protoscolices each. Fecal samples were examined for patent

infection during the study period. Dogs were necropsied at 45 dpi

(group A) and 54 dpi (group B). No eggs were detected in fecal

samples from group A throughout the experimental period (45 days).

However, eggs were first demonstrated in faeces 52 dpi in group B.

The experimental animals in both groups did not show any adverse

clinical signs during the experimental study. Echinococcus granulosus

worms were recovered from both groups at the time of necropsy.

Molecular characterization of the adult worms was made

possible using the polymerase chain reaction (PCR)-based detection

assay. The worms were identified as G6 (camel) strain of E.

granulosus. It was found that the prepatency period in dogs after

experimental infection with protoscolices of camel origin is longer

than the reported for other strains of E.granulosus. These are the first

data on prepatency periods of the camel strain G6 in dogs confirmed

by molecular characterization.

روحةملخص االط

هذه الدراسة تشمل دراسة عن وبائية و التصنيف الجزئي لجنس

Echinococcus) و داء الحويصالت المائية في العائل الرئيسي و الوسيط ) الدودة الكلبية

تم اجراء مسح عن داء الحويصالت المائية في الفترة من . بما فيها االنسان في السودان

و قد وجد معدل حدوث المرض . اطق بالسودان في عدة من2003 الى يوليو 2001مايو

% 6.1, ) 779/466% (59.8في االبل و االبقار و الضان و الماعز التي فحصت

. يــالتوال علي) 5565/106 ( 1.9%, ) %10422/1180(11.3, ) 4893/299(

في 1514 في االبقار و 333 في االبل و 2387وكانت الحويصالت التي تم حصرها

في % 31.5, %19, % 77, % 73.7نسبة الخصوبة وجدت . في الماعز 108الضأن و

و وجد أن االعضاء التي يكثر تواجد . الضأن و الماعز على التوالي, البقار, االبل

الغشاء ) 333/206(الكبد في االبقار , )2387/1627(الحويصالت بهاهي الرئة في االبل

تم ). 108/53(روتيني في الماعز و الغشاء الب) 1514/1242(البروتيني في الضأن

تصنيف عترات الدودة الكلبية في االنسان و الحيوانات االليفة الول مرة في هذه الدراسة

و Ortleppi كما تم ايضا تحديد عترة sequencingباستخدام تفاعل البلمرة المتسلسل و

تسبيبا لداء و قد وضح أن عترة االبل هي األكثر . في بعض العتراتG1)(عترة الضأن

الحويصالت المائية في االنسان و الحيوانات المختلفة في السودان حيث كانت نسبتها

في عينتان (G1)و قد تم تسجيل عترة الضأن . من كل العينات الموجبة) 542/533(

.من اإلنسان و خمس عينات من الضأن في السودان الول مرة في هذه الدراسة

كلب تم اعدامهم ضمن حملة مكافحة السعر 42عدد تم فحص محتويات االمعاء ل

بمنطقة تمبول و رفاعة بوسط السودان للبحث عن الدودة الكلبية و كذلك فحصت عينات

و قد تم تحديد مدى وجود الديدان في الكالب الموجبة باستخدام طريقة . من البراز

من جملة الكالب المعدمة التخفيف كما تم تصنيفها باستخدام تفاعل البلمرة المتسلسلز وجد

* 80- 22به ديدان الدودة الكلبية و تراوح عدد الديدان بين %) 28.5 (12عدد ) 42(

كما تم تصنيف الحمض النووي بجميع العينات المستخلصة من . في الكالب الموجبة103

المستخلص من عينات النووي من الحمض )12/10% (83.3وجد أن والديدان و الذي

% 93.3ووجد ان . عينتان غير محددتانال بينما )G6 ( نتمى لعترة اإلبلي البراز

عينة من الحمض النووي من عينات الكالب التي وجدت سلبية عند التشريح ) 30/28(

وجدت ايضا سلبية باستخدام تفاعل البلمرة المتسلسل بينما العينتان المتبقيتان كانتا موجبنان

هذا النوع من تفاعل البلمرة G1). ( عترة الضأن و تم تصنيفهما ضمن دودة الكلبية

في هذه .%100المتسلسل استخدم الول مرة في هذه الدراسة وجدت نسبة حساسيته

ط السودان ـلخ تمبول في وسسالدراسة تم جمع الحويصالت المائية من رئات االبل في م

في الكالب ية منها و استخدامها الحداث عدوــو تم جمع االطوار المعدية الحي

لكل منها خمسة كالب ) و بأ( كالب الى مجموعتين10قسمت . بجرعات مختلفة

. لكل منها103*8 بينما المجموعة ب اعطيت جرعة 103*4المجموعة أ أعطيت جرعة

لم يتم . يوما54 و المجموعة ب بعد يدوــ يوم من الع45د ـالمجموعة أ شرحت بع

أ طوال فترة التجربة بينما وجد بيض في براز راز المجموعة ـالعثور على بيض في ب

لم يشاهد اي اعراض جانبية سالبة خالل فترة . ي يوم من العدو52المجموعة ب بعد

تم العثور على الدودة الكلبية في المجموعتين عند التشريح كما تم اجراء . التجربة

دودة الكلبية عترة التصنيف الجزئي للديدان باستخدام تفاعل البلمرة المتسلسل ووجدت ال

بالطور يلوحظ أن الفترة السابقة لظهور الدودة في الكالب عند العدو . (G6)االبل

المعدي المستخلص من االبل كانت اطول من تلك المسجلة عند االصابة بالعترات االخرى

بعترة االبل في ويو تعد هذه اول مرة تسجل فيها فترة ما قبل ظهور الدودة بعد العد

.ب و تاكيدها بواسطة التصنيف الجزئيالكال

Chapter One

General Introduction and Literature Review

1.1 Introduction Echinococcosis and hydatidosis are terms used to describe

infection of animals and humans with the adult tapeworm or larval

metacestode stage of cestode species belonging to the genus

Echinococcus. Members of this genus within the family of Taeniidae

are small tapeworms at 1.2-7mm in length, possessing only a

maximum of 7 segments (proglottids). The parasite is of pathogenic

and economic significance in intermediate and aberrant intermediate

hosts, where the larval parasite develops into a hydatid cyst, a fluid -

filled cystic or vesicular structure composed of two main layers or

membranes. The laminated layer is a carbohydrate –rich, acellular

structure which is unique for the genus Echinococcus. It is both

supportive and also physically encloses the asexually produced

protoscolices which bud off from the living germinal layer and are the

infective stage for the definitive host. Production of protoscolices by

Echinococcus spp. is prolific ensuring high worm burdens in the

carnivore definitive host. Infection with E.granulosus metacestodes

results in the development of one or several unilocular hydatid cysts

that may grow for the life of the host. Echinococcus species are of

medical and veterinary importance because infection with

metacestodes may cause severe illness and death in the intermediate

host (Jenkins et al, 2005). Tremendous research was conducted in

echinococcosis. In Sudan, in spite the sizeable work appeared on the

subject, molecular biology of the parasite is not fully investigated

which enhance the establishment of this work.

Objectives of the Study • Epidemiological and biomolecular study of cystic

echinococcosis in humans and animals in the Sudan.

• Strain characterization and Copro diagnosis of

E.granulosus in central Sudan.

• Determination of the prepatent period of the camel strain,

G6 of E.granulosus.

1.2 Classification Echinococcus was classified as follows:

Kingdom Protisa

Sub/Kingdom Metazoa

Phylum Platyhelminthes

Class Eucestoda

Orde Cyclophyllidea

Family Taeniidae

Genus Echinococcus

Currently there are six species recognized within the genus

Echinococcus (Jenkins et al., 2005), these are E.granulosus,

E.multilocularis, E.vogeli, E.oligarthus, E.ortleppi and E.equinus.

Seventh species, Echinococcus shiquicus, was recently described

(Xiao et al., 2005). Based on morphology, host specificity and

molecular characteristics (Pearson et al., 2002, McManus and

Thompson, 2003), the E.granulosus complex is divided into three

species and eight defined strains (Table 1). The present recognition of

Echinococcus species reflects a series of largely host adapted species

that are maintained in distinct cycles of transmission (Thompson,

2001., Thompson and McManus, 2002). These are characterized by

the principal intermediate hosts which are; sheep, horses, cattle,

camels and different species of rodents (Table 1). Although these

cycles of transmission may overlap in some geographical areas, the

parasites involved have been shown to maintain their genetic identity

(Thompson et al, 1995, Thompson and McManus 2001., 2002.,

McManus and Thompson, 2003, Haag et al, 2004).

Echinococcus granulosus is the most widely distributed species

which exists as a series of genetically distinct strains/genotypes, some

of these genotypes are likely to warrant species status in the future,

particularly those in pigs, camels and cervids (Harandi et al., 2002,

Thompson and McManus, 2002, Lavikainen et al, 2003). However,

more research is required to determine their host and geographic

ranges and whether their genetic characteristics are conservd between

different endemic regions (Jenkins et al, 2005).

Although the most frequent strain associated with human cystic

echinococcosis (CE) appears to be the common sheep strain (G1),

other strains such as the Tasmanian sheep strain (G2), camel strain

(G6), pig strain (G7/G9) and cervid strain (G8) occur in a significant

number of cases in some locations. E.orttlepi and E.equinus were

previously characterized as cattle (G5) and horse (G4) strains of

E.granulosus respectively (Le et al., 2002., McManus, 2002.,

Thompson and McManus, 2002) E.multilocularis species recognition

occurred as late as 1953 when the parasite and its life cycle in foxes

and rodents were described by Rausch (1954) and Vogel (1957).

Although a number of E.multilocularis isolates have been described

from different geographical areas, their genetic variability remains

undetermined (Jenkins et al, 2005).

The description of E.vogeli and E.oligarthus was relatively

recent (Thatcher and Sousa, 1966., Rausch and D,Alessandro, 1978).

To date, there is no evidence of significant intraspecific variation in E.

equinus, E. ortleppi, E. oligarthus and E. vogeli (Jenkins et al, 2005).

1.3 Life Cycle (Fig 1) Like other cestodes, species of Echinococcus require two

different host species to complete their life cycles. Definitive hosts

harbouring the adult tapeworm in the small intestine are exclusively

carnivores and intermediate hosts harbouring the larval stage

(metacestodes) are herbivorous or omnivorous. The adult worms are

less than 7mm in length. They feed on the intestinal contents of the

host without causing any symptoms as they do not invade tissues.

When mature, Echinococcus worms shed the terminal proglottids

containing eggs which pass with the faeces to the environment. The

intermediate hosts acquire the infection by accidental ingestion of the

eggs with contaminated food or water. Larvae contained in the eggs

(oncospheres) emerge from the eggs in the small intestine, invade

blood vessels and may migrate into almost every part of the body.

There, the metacestodes grow for months or years forming fluid-filled

cysts or vesicles. Protoscolices are produced within the metacestode in

a phase of non-sexual reproduction. Once the metacestode is eaten by

a suitable definitive host, these protoscolices will grow into adult

tapeworms. The occurrence of a parasite in a particular host

assemblage like dog/sheep or dog/horse reflects a variable degree of

host parasite adaptation (Torgerson and Budke, 2003).

Figure 1

Life cycle of Echinococcus granulosus

1.4 Distribution of cystic echinococcosis (Fig 2) Cystic echinococcosis is the most wide spread disease caused

by Echinococcus species. The adaptation of Echinococcus to a wide

variety of host species and the repeated introduction and movement of

domestic animals throughout the world had made possible its broad

geographical distribution (Schantz et al, 1995).

In Europe, zoonotic members of the E.granulosus complex have

been reported in every country with the exception of Ireland, Iceland

and Denmark. They are most intensely endemic in the Mediterranean

areas and parts of Eastern Europe such as Bulgaria (Torgerson and

Budke, 2003). In the UK, the parasite has a restricted distribution,

being found mainly in mid and southern Wales. In Asia, the parasite is

endemic in large parts of China and is an important reemerging

zoonosis in the former Soviet Republics in Central Asia (Torgerson et

al., 2002a, b). Parasites are also found throughout the Indian

Subcontinent and the Middle East. In North America they are found in

Canada and Alaska, mainly in a sylvatic cycle. In USA, the parasite is

very sporadic with just a few foci such as certain communities in Utah

and California. In South America the parasite is extensive, particularly

in Argentina, Uruguay and Peruvian Andes. In Australia, the parasite

is common due to a sylvatic cycle between dingoes and wallabies with

over 25% of dingoes and up to 65% of macropod marsupials infected

(Jenkins and Morris, 1995., Jenkins, 2002).

Figure 2

World Distribution of cystic echinococcosis

Approximate geographic distribution of Echinococcus granulosus (1999) Institute of Parasitology, University of Zurich (J. Eckert, F. Grimm & H. Bucklar) Note: exact identification of endemic and higly endemic areas in all region is not possible because of incomplete or lacking data.

1.5 Cystic echinococcosis in Africa Echinococcus infections have been reported in 20% of dogs in

Algeria (Senevet, 1951), 20–51% of dogs in Morocco (Sirol and

Lefevre, 1972., Pandey et al.,1988), 22–43% of dogs in Tunisia

(Jaiem, 1984., Bchir et al., 1985), 8–38% in Libya (Packer and Ali,

1986., Gusbi, 1987., Awan et al., 1990) and 1–10% in Egypt (Moch et

al., 1974., Abo–Shady, 1980., Hegazi et al., 1986). In some of these

countries dogs become infected from offal discarded during home

slaughtering of sheep (Bchir et al., 1987).

The cystic hydatid disease focus in North Africa include

Morocco (Pandey et al., 1988., Quhelli and Dakkak, 1992), Algeria

(Cherid and Nosny, 1972., Abada et el., 1977., Larbaoui and Allyulya,

1979), Tunisia (Ben Rachid et al., 1984., Bchir et al., 1985., Gharbi et

al., 1985), Libya (Gebreel et al., 1983., Aboudaya, 1985., Shambesh

et al., 1992) and to a lesser extent Egypt (Hegazi et al., 1986). The

human populations of the first four of these countries total

approximately 60 million. The annual incidence of hydatidosis was

between 3.4 –4.6, 6.5–7.8 per 100,000 inhabitants in Algeria and

Morocco respectively (Larbaoui and Allyulya, 1979., Quhelli and

Dakkak,1992). In Tunisia, between 800 and 1,200 new case of

hydatidosis are diagnosed every year for an annual average incidence

of 16.5 per 100,000 (Gharbi et al., 1985., Achour et al., 1989).

In East Africa, camels are important intermediate hosts. Sheep

and goats are frequently infected in Kenya (Macpherson, 1985) and in

Ethiopia (Bekele et al., 1988., Wosene, 1991). Many pastoral peoples

do not bury their dead, allowing dogs and wild carnivores access to

human hydatid cysts. Echinococcus infection has been found in 39–

70% of dogs in Turkana, Kenya (Nelson and Rausch, 1963.,

complexEchinococcus Species and Strains of : 1Table

Species Strain/genoty

pe

Known intermediate hosts Infective to

humans

Disease in humans Known definitive

hosts

Echinococcus

granulosus

Sheep/G1 Sheep (cattle, pigs,

camels,goats,macropods

Yes Cystic (Unilocular) Dog, fox, dingo,

jackal and hyena

Tasamanian

sheep/G2

Sheep (cattle?) Yes Cystic (Unilocular) Dog, fox

Buffalo/G3 Buffalo (cattle?) ? ? Dog, fox?

Camel/G6 Camels (sheep) Yes Cystic (Unilocular) Dog

Pig/G7 Pigs Yes Cystic (Unilocular) Dog

Cervid/G8 and

G10

Cervids Yes Cystic (Unilocular) Wolf, dog

?/G9 Yes ? Lion

Lion/? Zebra, wildbeest, warthog, bushpig,

buffalo, various antelope, giraffe?

Hippopotamus?

? ?

E. equinesHorse/G4 Horses and other equines No - Dog

E. ortleppi Cattle/G5 Cattle Yes Cystic (Unilocular) Dog

SpeciesStrain/genotype

Known intermediate host Infective to

human

Disease in human Known definitive

hosts

Echinococcus

multilocularis

Some isolate

variation

Rodents, domestic and wild pigs,

dog, monkey, (horse)

Yes Alveolar

(multivesicular`)

Fox, dog, cat, wolf,

raccoon-dog,

coyote

Echinococcus

shiquicus

? Lagomorphs (pika) ? ? Tibetan fox

Echinococcus

vogeli

None reported Rodents Yes Polycystic Bush dog

Echinococcus

oligarthus

None reported Rodents Yes Polycystic Wild felids

Macpherson, 1985) and 23% in stray dogs in Somalia (Macchioni et

al., 1985). The initial surgical incidence rate for Turkana, was

estimated to be 40 per 100 000 per year (Schwabe, 1964).

1.6 Cystic echinococcosis in Sudan In Sudan, the disease was first mentioned at the beginning of

the 20th century (Christopherson, 1909). Abel Malek (1959), made a

list of the parasites of the domesticated animals in Sudan, including a

single case of liver hydatidosis in cattle, 3 cases in camels as well as 4

cases of Echinococcus in dogs. The work done by Eisa and Mustafa

(1962) drew attention to this serious zoonosis in southern Sudan,

specially in Kapoeta district in Equatoria where they reported 86.4%

prevalence rate of Echinococcus in dogs and 21% in cattle in the

Upper Nile Province. About 33% and 9.4% infection rates amongst

sheep and goats were reported respectively (Eisa et al, 1962). Another

survey carried out by Eisa (1982) in Malakal and Rank district

revealed over all infection of 2.7% among cattle. Elkhawad et al

(1977) encountered an infection rate of 25%, 12% and 10% in cattle,

sheep and goats respectively in Western Province. In another survey

in the central region of Sudan, Elkhawad et al, (1979) reported an

infection rate of 4.3%, 8.1%, 3.2% and 53.3% in cattle, sheep, goats

and camel respectively. In Elobied (Kordofan region), the prevalence

of hydatid cysts among camels was 67% (Saad et al., 1989). 3.84%

cattle and 48.69% camels were found to harbour the parasite in central

Sudan (Saad and Magzoub, 1989). Recently, Elmahdi et al (2004)

recorded a prevalence rate of 44.6%, 6.9% and 3.0% in camels, sheep

and cattle respectively. Given the high rate of non-fertile or calcified

cysts in sheep, these animals appear to play an unimportant role in the

transmission cycle of echinococcosis in the studied areas(Saad and

Magzoub 1989a; Saad and Magzoub 1989b; Elmahdi, 1998; Omer et

al., 2002; Elmahdi et al, 2004 and Omer et al, 2004).

The prevalence rate of the disease in animals is high, thus

providing high risk of the disease for human populations in different

parts of the Sudan. However, no hospital records are currently

available. The disease seems to be endemic in rural communities

where people live in close contact with dogs (definitive host) and

large numbers of livestock (intermediate hosts). The disease was

prevalent in 0.33% (1 from 300) villagers in a village in central Sudan

(Elmahdi et al, 2004). A special situation may exist among the Taposa

and neighbouring tribes in Equatoria where the hyper endemic focus

of the disease among the Turkana extends into Sudan (Eisa et al.,

1962). Cahill et al (1965) conducted a serological study on 152

individuals in Southern Sudan. The prevalence rate among these

individuals was estimated by 13.3%. Cystic echinococcosis was found

in 2% (132/6728) of the human population in selected areas of

southern Sudan (Njoroge, 2001). Mann (1974) stated that 50% of the

human cases of hydatidosis in Uganda were immigrants from

Southern Sudan

Concerning the presence of Echinococcus species and strains,

so far there is only one study available (Dinkel et al, 2004), that 44 of

46 samples characterized from different intermediate host in central

Sudan were identified as G6 (camel strain), the two exceptions being

cattle samples identified as E.ortleppi (previously the cattle strain,

G5).

1.7 Molecular epidemiology of cystic echinococcosis The value of DNA analysis in molecular epidemiological

studies of cystic echinococcosis, as well as in clarifying the complex

issue of strain variation of E.granulosus, is well recognized. Some

molecular epidemiological surveys are summarized below.

In China, examination by a combination of DNA techniques has

indicated that the common sheep strain is the most predominant in the

northwest region of China (McManus et al, 1994).

In Argentina, several distinct genotypes including the common

sheep strain (G1) and Tasmanian sheep strain (G2) in sheep and

humans, the pig strain (G7) in pigs and the camel strain (G6) in

humans where identified (Rozenzvit et al., 1999). Furthermore, that

was the first report about the presence of the Tasmanian sheep strain

(G2) and the camel strain (G6) in humans. In Nepal, three strains of E.

granulosus, namely the sheep strain (G1), cattle strain (E.ortleppi) and

camel strain (G6) were identified in buffalo, sheep, goat and human

hosts, of which two human isolates were identified as G6 (Zhang et

al., 2000). In Iran, the sheep strain was found to be the most common

genotype of E.granulosus affecting sheep, cattle, goat and

occasionally camels. The majority of camels were infected with the

camel strain (G6), as were 3 of 33 human cases (Harandi et al., 2002).

Biochemical and genetic studies indicated that there may be at least

three strains of Echinococcus in Africa, the sheep strain involving

sheep, goats and humans, the camel strain in camels and goats and the

cattle strain (McManus and Macpherson, 1984., McManus et al,

1987). The existence of predator –prey parasite transmission between

lion and its main food animal suggests that a distinct wildlife form of

the parasite may exist in some parts of sub–Saharan Africa. Evidence

for this is that the parasite found in the lion is morphologically distinct

from E.granulosus (Ortlepp, 1937). In North Africa E.granulosus is

propagated primarily by a domestic cycle involving domestic dogs as

the definitive host and many species of livestock including camels,

cattle, sheep, goats, pigs, horses, donkeys and buffaloes as

intermediate hosts (El–Kordy, 1946., Dar and Taguri, 1978., Larbaoui

et al., 1980., Jaiem, 1984., Gusbi et al., 1987., Pandey et al., 1988.,

Ahmed, 1991). The local importance of each intermediate host species

in maintaining the cycle varies in different regions. In Kenya,

molecular analysis showed the presence of sheep (G1), camel (G6)

and cattle (E.ortleppi) strains. The range of intermediate hosts for

these strains appeared to be similar sheep, cattle, camel and humans

except that the camel strain was detected in one case of human and

E.ortleppi was detected in a single case from pig (Dinkel et al, 2004).

Additionally, the camel strain (G6) was characterized in two samples

of human origin in Mauritania (Bardonnet et al., 2001).

18 Techniques of strain characterization Several techniques that investigated the morphology, chemical

composition, metabolism, and developmental stages of Echinococcus

had been implemented to characterize different strains and species.

Epidemiological studies on hydatid disease reported the occurrence of

different strains in various countries (Kumaratilake and Thompson.,

1982., McManus and Macpherson, 1984., McManus and Thompson,

1985). These studies emphasized the importance of strain

characterization for the reason that strains may differ in their

infectivity to various intermediate and final hosts (Thompson, 1977).

The biochemical composition of various developmental stages of

different strains demonstrated considerable discrepancies (McManus,

1981., Kumaratilake and Thompson., 1984). This may cause a

variation in the susceptibility of the parasite to different

chemotherapeutic agents (Schantz et al., 1982., McManus et al.,

1985). Strain variation may have practical implication in vaccinations

attempts and in control programs.

1.8.1 General morphology The study of rostellar hook morphology was established by

InDialy and Sweatman (1965). Protoscolex rostellum hooks as well as

strobilar hooks of adult worms were used successfully in

morphological characterization. Total lengths, width, blade length of

hooks were all used to identify Echinococcus strains in various parts

of the world (William and Sweatman., 1963., Kumaratilake and

Thompson, 1984). Echinococcus of camel origin is distinct from the

sheep strain with regard to the shape of the hooks and hook length

whereas differences in hook measurement characteristics between

camel, cattle and horse material were less marked (Eckert et al. 1989).

Characteristics of the strobilar morphology such as total worm length,

length of the terminal segment, maximal number of segments, position

of the sexually mature segment, position of the genital pore, number

and distribution of testes, shape and size of the cirrus sac, shape of the

ovary and the female reproductive system are all considered as

important tools for strain characterization. The sexually mature

segment is always terminal in worms of camel origin and always

penultimate in worms of cattle origin. Testes are found throughout the

segment in case of worms of camel, sheep and horse origin and

confined to the centre of the segment in worms of cattle origin

(Thompson et al, 1984., Kumaratilake et al, 1986, Eckert et al, 1989).

Morphological characterization enabled the identification of two

morphologically different strains that specifically affected sheep and

horse. A virulent goat/dog strain was morphologically identified in

India by Pandey (1972).

1.8.2 Biochemical Methods Biochemical techniques using Sodium Dodycyl Sulphate Poly-

acrylamide gel electrophoresis (SDS-PAGE), isolectric focusing and

isoenzyme analysis were reported to be highly useful in strain

characterization of helminthes parasites in general and E.granulosus

in particular. Hydatid cyst fluids collected from pigs and sheep

showed different SDS-PAGE protein patterns as reported by

Zvolinskene et al., 1977. Similar DNA contents collected from

hydatid cysts in sheep and horse showed different composition of

poly-saccharides and lipids (McManus and Smyth, 1982). In addition,

protoscolices of the horse strain were found to produce more lactate

than that of the sheep strain. Isoelectric focusing was also used to

differentiate between strains in UK and Australia (Kumaratilake and

Thompson, 1984).

1.8.3 Developmental method in vivo Different strains of Echinococcus require different periods to

reach maturity in dogs. In worms of cattle origin, thin-shelled eggs

appeared in the uterus as early as 30 days post infection (PI)., with the

majority of worms being in this condition at 35 days and certain to be

infective by about 35-37 days (Thompson et al., 1984). In Australia,

different prepatent periods have been recorded in isolates from

different regions (Kumaratilake et al, 1893). A prepatent period of 35

days has also been reported in a dog infected with hydatid material

from giraffe of South West African origin (Tscherner, 1978).

Maturation of a particular strain can vary with the host in which

it develops (Thompson and Kumaratilake, 1985). Thus, although in

Australia the domestic sheep strain of E.granulosus matures in both

dogs and dingoes, the growth of the sylvatic form in dogs was found

to be substantially retarded compared with that in dingoes.

1.8.4 Developmental method in vitro Culturing of protoscolices using a special media of Parker 858

and 20% fetal serum was successfully implemented in differentiation

between horse and sheep strains (Smyth and Davis, 1974). Horse

protoscolices failed to segment in the culture system while those from

sheep segmented and matured. This variation is presumably based on

the nutritional requirements of different strains.

1.8.5 Genetic characterization Methods used for strain typing include sequencing of partial

mitochondrial cytochrome c oxidase subunit 1 (cox1) and of NADH

dehydrogenase 1 (nad1) genes (Bowles et al., 1992., Bowles and

McManus, 1993), analysis of ribosomal DNA regions (ITS1, ITS2) by

polymerase chain reaction-restriction fragment length polymorphism

(PCR-RFLP., Bowles and McManus, 1993., Gasser and Chilton,

1995) and random amplification of polymorphic DNA (RAPD)-PCR

(Scott and McManus, 1994., Siles-Lucas et al., 1994).

1.8.5. a Sequencing of partial mitochondrial cytochrome c oxidase subunit 1 (cox1). (Bowles et al., 1992).

Different isolates of Echinococcus were analyzed for sequence

variation within a region of the mitochondrial cytochrome c oxidase

subunit 1 (CO1) gene. For each Echinococcus isolate examined, a

single double-stranded PCR product that has a size of 446bp was

visualized on an ethidium-bromide stained agarose gel. Eleven distinct

partial CO1 sequences were detected amongst the examined

Echinococcus isolates. This method enabled the division of 7 discrete

groups (G1-G7) of E.granulosus. The CO1 sequences of some of

these groups were very similar. The sequence obtained with the

Tasmanian sheep sample (G2) differed from the standard sheep

sequence at 3 of the 366 nucleotide sites examined. A common

sequence (G4) was found in isolates of horse and donkey origin. A

unique CO1 sequence (G5) was found with a cattle isolate from

Holland, whereas camel isolates from Somalia and Sudan and a goat

isolate from Turkana region shared the same sequence G6. This was

found to be different from the sequence found in pig isolates from

Poland (G7) at only one nucleotide position.

1.8.5 /b Analysis of ribosomal DNA regions ITS1 by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) (Bowles and McManus, 1993)

Due to the fact that most of the polymorphism detected in

Echinococcus using pSM889 as a probe (McManus and Rishi, 1989)

results from sequence dissimilarity in the ITS1 and ITS2 spacer

regions and it is also likely that the non-transcribed spacers are too

variable for the purpose of identification. This method focused on the

ITS region of the rDNA repeat. The PCR-RFLP allows Echinococcus

isolates to be easily and rapidly distinguished using size and sequence

of the nuclear genomic rDNA ITS1 region as a genetic marker. This

method supports the existence and pattern of intraspecific genetic

variation in the genus. The sharp delineation between the

E.granulosus sheep, horse, cattle and camel/pig isolate groups

indicated that various strains are not interbreeding and are following

separate evolutionary paths.

1.8.5.c Analysis of ribosomal DNA region ITS2 by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) (Gasser and Chilton, 1995)

The PCR-RFLP of ITS2 rDNA provides a method of

identifying some taeniid cestodes including different isolates of

E.granulosus. This method enabled the differentiation between

isolates from camel and horse and a representative of the sheep strain.

In spite of this variation, it appears that digestion of ITS2 with the

digestion enzyme Rsa1 provides a reliable specific marker for

E.granulosus, since the restriction of isolates representing different

strains shared a common restriction fragment of 310 bp.

1.8.5.d A PCR system for detection of species and genotypes of E granulosus complex (Dinkel et al, 2004)

With this method those Echinococcus species and genotypes,

which are presently assumed to have the greatest public health impact,

can be easily and rapidly distinguished. It is a PCR system which uses

a part of the mitochondrial 12S rRNA gene as a target sequence. This

PCR system identifies g1 (sheep strain) and G5/6/7 (cattle, camel and

pig strain P) each with a different pair of primers. To discriminate

between E.ortleppi and E.granulosus G6/7, seminested PCRs are used

in a second step.

1.9 Diagnosis of Echinococcus in definitive and intermediate hosts

1.9.1 Diagnosis of Echinococcus in definitive host Diagnosis of Echinococcus in definitive hosts is difficult,

because the eggs of all Echinococcus and Taenia species are

morphologically indistinguishable from one another and the

characteristic small segments of Echinococcus species may be absent

from the faeces or can be easily overlooked. The two major

parasitological methods are purgation with arecoline salts and

examination of the small intestine at necropsy (Deplazes and Eckert,

1996). Both these techniques are time consuming, laborious and

require special safety precautions.

Therefore, indirect methods such as detection of serum

antibodies, coproantigens and parasite DNA in faecal sample are

described to simplify and improve epidemiological investigations.

1.9.1.a Detection of serum antibodies Taeniid cetsode antigens derived from adult worms, juvenile

intestinal stages or onchospheres interact with the immune system of

the host and may lead to the production of specific antibodies (Jenkins

and Rickard, 1985). Enzyme Linked immunoassays (ELISA) based on

detection of these antibodies have been developed for diagnosis of

Echinococcus spp infection in final hosts (Jenkins and Rickard, 1986.,

Gasser et al, 1988). A problem encountered with this method is the

persistence of antibodies after elimination of the cestodes (Gottstein et

al, 1991., Gasser et al. 1993). Usage of an ELISA based on the

detection of antibodies directed against Em2 antigen revealed a

sensitivity of 60% (Gottstein et al, 1991). Even though no cross

reactions were shown with sera from dogs with various helminthes

when using this ELISA, animals without a detectable intestinal

infection were also positive. Serological diagnosis of canine

echinococcosis due to E.granulosus has been based on the detection of

antibodies (IgG, IgA and IgE) directed against a protoscolex antigen

preparation (Gasser et al. 1993). Overall specificity ranged from 97-

100% but sensitivity varied (53-84%) according to geographic region,

though it could be increased by combining the evaluation of various

immunoglobulins classes (Gasser et al. 1993., Craig et al. 1995).

1.9.1.b Detection of coproantigens Coproantigen ELISAs were developed for both E.multilocularis and

E.granulosus. The most important advantage of these methods over

the conventional serum antibody assays is that they indicate current

infection (Allan et al. 1992., Deplazes et al. 1992, 1999). The

sensitivity of coproantigen ELISAs developed for E.multilocularis

was reported to be 83.6% whereas the overall sensitivity of the

ELISAs against E.granulosus has shown different levels of sensitivity

ranging between 50 and 87.5% (Allan et al. 1992., Craig et al. 1995.,

Moro et al. 1999). The sensitivity of the ELISAs developed for both

species of Echinococcus depends on the worm burden (Craig et

al.2003)

1.9.1.c Detection of copro-DNA The advantage of detecting Echinococcus DNA by polymerase

chain reaction (PCR) in faecal samples is that it has the potential to

provide absolute species specificity assays with high sensitivity.

Additionally, as the test is aimed to mainly detect eggs in faecal

samples, it provides a more accurate estimation of the potential risk

for human infection than ELISA-based methods. Problems

encountered when extracting DNA from faecal samples are that these

samples often contain PCR inhibiting substances that create false

negatives. Different kits have been utilized to purify DNA,

eliminating inhibitory substances, but this method is time consuming

and it may enhance the possibility of losing the DNA present in the

faecal samples. Sieving of the faecal samples in several steps

concentrates eggs from the sample to improve sensitivity (Monnier et

al., 1996), but may make the protocol complicated and time

consuming and may not be suitable for large epidemiological studies

(Craig, 2003). Usage of a commercial kit, the Qiaamp Ministool kit

(Qiagen, UK) combined with a single sieving step provided good

results (Abbasi et al. 2003).

A PCR was described by Bretagne et al (1993) for the detection

of E. multilocularis DNA in faecal samples of foxes by using the U1

snRNA. However, more specific results for the same parasite were

obtained by E.multilocularis specific PCR (Dinkel et al, 2004). This

method can also be applied to E.granulosus

1.9.2 Diagnosis of cystic hydatidosis in intermediate host The most frequent approach for the detection of cystic

echinococcosis in domestic livestock is by examination at necropsy.

Radiological (X-ray) diagnosis of ovine hydatidosis has been applied

successfully on a very small scale (Wyn-Jones and Clarkson, 1984),

but it is not a suitable system for epidemiological surveys. Use of

sonography for hydatidosis screening in some animals such as sheep

could be useful in the establishment of an epidemiological control

system as well as in the analysis and assessment of risks that the

disease poses for humans and animals (Eduardo et al. 2001).

Prevalence of hydatid cysts in goats in northewestern Turkana, Kenya,

and Taposa land, southern Sudan was determined using

ultrasonography (Njoroge et al.2000). Unlike with abattoir surveys,

the author considered the results of this survey as unbiased because it

covers the entire flock.

1.9.2 Serodiagnosis of hydatid disease in animals Even though serological diagnosis of Echinococcus infection in

domestic livestock had not been successful, few studies had been

undertaken to characterize the problems with sensitivity and

specificity of immunodiagnosis in animals (Craig, 1993). The

principal problem is that animals with fertile cysts have undetectable

levels of specific antibodies and cross-reactivity occurs against other

parasites (Verastegui et al, 1992). However, identification of exposure

to E.granulosus at the flock or herd level by use of mean values for

serum antibody activity is possible using hydatid cyst fluid antigens in

ELISA (Lightowlers and Gottstein, 1995). Three ELISA tests using a

purified Antigen B subunit, a recombinant oncospheral antigen

(EG95) and a crude protoscolex antigen were evaluated in naturally or

experimentally infected sheep. The highest diagnostic sensitivity was

63% with the protoscolex antigen (Kittelberger et al. 2002).

1.9.3 Diagnosis of cystic hydatidosis in humans

1.9.3.a Imaging Techniques Imaging methods for the detection of space occupying lesions

are used for the diagnosis of cystic and alveolar echinococcosis.

Computerized tomography (CT), magnetic resonance imaging (MRI),

ultrasound (US) and radiography (X-ray) are currently the most useful

imaging techniques. (Eckert et al, 2001). CT scans provide the best

overall resolution for both diseases but are limited to well equipped

hospitals and not applicable for field studies (Craig. et al, 2003)

Introduction of ultrasound imaging (US) had improved both the

diagnosis of the disease and the understanding of the natural history of

the disease (Ammann and Eckert, 1995., Caremani et al, 1997., Von

Sinner, 1997), but is not applicable when cysts are located in lungs,

bone or brain. The US examination is a suitable technique for

population studies aimed at detecting cases and determining the

prevalence of the disease.

All imaging techniques rely on the expertise of the operator in

differentiating Echinococcus infection from other space occupying

lesions. The image can be confused with other cystic lesions such as

tumors, and hepatic abscesses, but the identification is easier when

some pathognomonic lesions such as a visible laminated layer or

daughter cysts are present (Craig. et al, 2003).

1.9.3.b Immunodiagnosis of human hydatidosis Immunodiagnosis has been practiced for the detection of both

cystic and alveolar echinococcosis. The source of the antigen used for

the detection of cystic echinococcosis has been hydatid fluid removed

from cysts in livestock. The crude cyst fluid can be used for

procedures such as immunoelectrophoresis (detection of arc 5) and the

indirect haemagglutination assay with good results, but now

components such as Antigen B, a heat stable lipoprotein, are

frequently purified from the cyst fluid (Wen and Craig, 1994).

Recombinant Antigen B proteins have also been used but they are less

sensitive than the native Antigen B (Rott et al. 2000).

For alveolar echinococcosis, the antigens used are derived either from

cyst masses or protoscolices obtained from experimentally infected

rodents. The carbohydrate rich Em2, which is affinity purified, or the

commercially available preparation Em2 plus belong to the most useful

antigens. (Siles-Lucas and Gottstein, 2002). Whole protoscolex

homogenate has been used in ELISA with some success but it is better

when used in immunoblotting as a band with relative mass of 18 kDa

has been reported to be highly specific for alveolar echinococcosis

infections (Ito, Schantz and Wilson, 1995).

Antigens available for the diagnosis of alveolar echinococcosis

are usually more specific than those used for cystic echinococcosis

(Craig et al.2003). The major problems regarding immunodiagnostic

tests are due to the existence of significant numbers of both false

negative and false positive cases. The average sensitivity of ELISA-

based diagnostics is around 80%. Specificities with reference to other,

non cestode infections is usually high (95-100%) but significant levels

of cross reactivity exist within the main larval taeniid infections.

Serology is more problematic in geographical areas where E.

granulosus, E. multilocularis and Taenia solium occur together.

Individuals with previous exposure to the parasite without the

development of further infection or significant pathology (Cohen et al.

1998, Bartholomot et al. 2002) are often antibody positive when

serologically examined.

Benefits such as confirmation of the imaging or clinical

evidence, identification of asymptomatic individuals and providing

information on the state of the infection and the immune response

against the parasite can be gained when serology is combined with

other diagnostic procedures such as imaging techniques (Craig.et al.

2003).

Chapter Two

Material and Methods

2.1 Epidemiological survey During the period of May 2001 to July 2003 frequent visits

were made to slaughterhouses in different representative areas of the

Sudan (central, eastern, western and southern Sudan). A total number

of 21659 animals (779 camels, 4893 cattle, 10422 sheep and 5565

goats) was examined (Table 2 and Fig.2). Different organs including

lungs, liver, heart, spleen and kidneys of the slaughtered animals were

thoroughly inspected. Hydatidosis positive animals and the number of

cysts encountered were recorded.

2.2 Hydatid cysts DNA was extracted from 527 ethanol preserved hydatid cyst

samples (317 none calcified and 210 calcified) collected from camel

(157, 50), cattle (62, 45), sheep (81, 60), goats (10, 55) and humans

(7, 0) from different parts of central, western and southern Sudan

during the epidemiological survey. Representative samples from cattle

(4, 3) and camel (3, 5) were obtained from northern and eastern Sudan

respectively for the purpose of DNA characterization. Additional 26

samples of sheep origin were also obtained from central Sudan for

DNA characterization. DNA was also extracted from adult

E.granulosus worms obtained from 15 dogs in central Sudan.

Table 2: Numbers of the examined camels, cattle, sheep and goats in different

study areas

Location (Sudan) Camels Cattle Sheep Goats Central

(Khartoum, Medani, Tamboul, Rofa)

214 250 400 0

Western

(Darfur and Kordofan Staes)

565 4318 9727 5523

Southern

(Juba and Malakal).

0 325 295 42

Total 779 4893 10422 5565

Figure 2 Study area

2.2.2 Collection of hydatid cysts or cyst material from human patients

Visits were made to hospitals in the same areas to estimate the

numbers of human cystic echinococcosis cases. Hydatid cysts or cyst-

Only representative samples obtained

Epidemiological survey and representative samples

derived materials were obtained from 7 patients, admitted to

Khartoum and Juba medical teaching hospitals, for diagnosis of the

disease. Human samples were used for further molecular

characterization.

2.2.3 Parasitological studies Cysts or cyst material were examined microscopically to

confirm whether or not they are hydatid cysts. Fertility of the collected

hydatid cysts was assured by detection of protoscolices in aspirated

fluid samples. Sterile or calcified cysts were considered infertile.

2.2.4 Preservation of hydatid cysts and adult E. granulosus samples

Representative intact cysts or cyst material as well as

E.granulosus worms were preserved in 70% alcohol for further DNA

characterization.

2.2.5 Preservation of the faecal samples Collected faecal samples were preserved in -20˚C and later

upon arrival in Germany at -80 ˚C for at least three days for the

inactivation of eggs.

2.2.6 Preparation of hydatid cyst material from intact cysts for DNA extraction

Each individual cyst was placed in a Petri dish and opened

using sterilized scissors and scalpel. Cyst fluid, if present was

obtained using a pipette. If the cyst was calcified, part of the cyst wall

was taken using a scissors. Scissors and scalpels were sterilized after

the opening of each individual cyst using ethanol, flame, and water

subsequently.

2.2.7 DNA extraction DNA was isolated from ethanol preserved or frozen cysts as

described by Dinkel et al, 1998, that up to 0.5 g protoscolices or cyst

wall (germinal and laminar layer) or adult worm was cut into small

pieces using sterilized scalpel or scissor and transferred to 1.5 ml

Eppendorf reaction tube. 500 µl digestion buffer, 10 µl 1 M

dithiothreitol and 60 µl proteinase K were added to the sample and

incubated for 3-12 hours at 56◦C with frequent agitation to allow the

process of digestion until suspension was clear.

DNA was then extracted with phenol chloroform isoamyl

(25:24:1), that 570 µl of the phenol chloroform isoamyl preparation

was added to the sample which was then centrifuged at 13.200 rpm for

10 min. The upper watery phase (~500 µl) containing the DNA was

transferred in a new 1, 5 ml reaction tube and subsequently 50 µl 3 M

Na-acetate (0.1 volume of the aqueous volume) and 1000 µl (double

the volume of the DNA containing phase) cold absolute ethanol

(-20˚C) were added and then incubated at -20˚C for 2-12 hours. The

DNA was precipitated by centrifugation at 14.000 g for 30 minutes at

4˚C. The pellet was then washed with 70% cold (-20˚C) ethanol and

Examined

animals

Total

number

examined

Prevalence

%

Total

number of

cysts

Fertility

%

Predilection

site

Cysts examined for

genotype n (n Non

calcified-n calcified)

Genotype

(n)

Camel 214 55.6%

(119/214)

360 75%

(270/360)

Lung

(304/360)

87 (57-30) G6 (87)

Cattle 250 20%

(50/250)

63 100%

(63/63)

Liver

(49/63)

27 (27-0) G6 (27)

Sheep 400 2.5%

(10/400)

12 0%

(0/10)

Liver

(6/10)

12 (0-12) G6 (10)

Goat 0 - 0 - 0 0 0

Table 1: Prevalence of cystic echinococcosis in livestock in Central Sudan

subsequent centrifugation at 14.000 g for 10 minutes. Evaporation of

the ethanol was allowed at 35-40 ˚C in an oven. The DNA was

resuspended by the addition of 100-200 µl nuclease free water

depending on the amount of the precipitated DNA and then left

overnight at 4 ˚C.

2.2.8 DNA extraction from the faecal samples 0.5 gr of each faecal sample was diluted 1:2 (vol/vol) with

distilled water and then 1500 µl of this suspension was transferred in a

reaction tube (12 ml). Under the hood, 108 µl of 1 M KOH and 30 µl

1M Dithiothreitol were added, thoroughly mixed and left for 30 min in

a water bath at 65 ˚C. For neutralization, 270 µl 2 M Tris-HCl (pH

8.3) and 40.5 µl HCl were added to each sample. 1950 µl phenol-

chloroform-isoamylalcohol was then mixed with each sample and

centrifuged at 10.000 g for 10 minutes. From the upper watery phase,

which contains the DNA, 1800 µl was transferred into a new 12 ml

reaction tube. After the addition of 5400 µl binding buffer and 30 µl

of a silica matrix the samples were incubated at 37 ˚C for 60 minutes

with frequent agitation in a hybridization oven. After that samples

were centrifuged for 30 seconds at 10.000 g, and 1000 µl binding

buffer was added to the pellet after discarding the supernatant. This

was then transferred into a new 12 ml reaction tube and centrifuged at

13.000 g for 30 seconds. The pellet was then washed 3 times with

washing buffer. After drying at 35-40 ˚C in an oven to eliminate

ethanol, 100 µl nucleic acid free water was added to the pellet and

then incubated in a water bath at 50˚C for 15 minutes to resuspend

DNA. After centrifugation at 13.000 g for 60 seconds, 85 µl of this

solution were transferred in a new 1.5 reaction tube.

2.2.9 Determination of the DNA concentration in samples 4 µl of the sample was diluted with 200 µl nuclease free water.

The concentration of the DNA in the samples was measured

photometrically at 260 nm (nucleic acids) and also at 280 nm

(proteins) to calculate the purity of the DNA.

2.2.10 Polymerase Chain Reactions

2.2.10.1 Cestode specific PCR (cs PCR) A PCR with the primer pairs P60for and P375rev. was

performed as described by Dinkel et al (1998, 2004). The 50 or 100 µl

reaction mixture consisted of 5 or 10 µl of the PCR buffer (10 mM

Tris-HCl (pH 8.3), 50 mM KCl), 5 or 10µl of MgCl2 (2.5 mM) , 1 or

2µl of deoxynucleoside triphosophate mixture each at a concentration

of 200 µM, 2 or 4µl of each primer (20 or 40 pmol) and 0.25 µl of

Ampli-Taq Polymerase (1.25 units). The amount of the added DNA

was calculated at 200ng. The reaction volume was then completed to

50 or 100µl using nucleic acid free water. PCR was running for 40

cycles (denaturation for 30 s at 94 ˚C, annealing for 1 min at 55 ˚C

and elongation for 30 s at 72 ˚C). Positive and negative controls were

used to assess the quality of the PCR.

2.2.10.2 PCR assay specific for E. granulosus G1 (g1 PCR) As described by Dinkel et al, (2004), PCR was performed in a

50 µl volume containing 5µl PCR buffer consisted of 5 µl of the PCR

buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl), 4µl of MgCl2

(2.mM), 1µl of deoxynucleoside triphosophate mixture each at a

concentration of 200 µM, 2.5µl of each of the primer pair ss1for and

ss1rev. (each 25 pmol) and 0.25 µl of Ampli-Taq Polymerase (1.25

units.) The amount of the DNA was calculated at 200 ng. The reaction

volume was then completed to 50µl using nucleic acid free water.

PCR was performed with 40 cycles (denaturation for 30 s at 94 ˚C,

annealing for 1 min at 57 ˚C and elongation for 40 s at 72 ˚C). Positive

and negative controls are used to assess the quality of the PCR.

2.2.10.3 PCR assay specific for E.granulosus G6/7 and E. ortleppi (g5/6/7 PCR, g6/7 PCR, g5 PCR)

For the first PCR (g5/6/7), the primer pair E.gcs1for and

E.gcs1rev. was used (Dinkel et al, 2004). 50 µl volume containing 5µl

PCR buffer consisted of 5 µl of the PCR buffer (10 mM Tris-HCl (pH

8.3), 50 mM KCl), 4µl of MgCl2 (2.mM), 1µl of deoxynucleoside

triphosophate mixture each at a concentration of 200 µM, 2.5µl of

each of the primer pair ss1 and ss1 rev. (each 25 pmol) and 0.25 µl of

Ampli-Taq Polymerase (1.25 units) The amount of the DNA was

calculated at 200ng. The reaction volume was then completed to 50µl

using nucleic acid free water. PCR was running for 40 cycles

(denaturation for 30 s at 94 ˚C, annealing for 1 min at 57 ˚C and

elongation for 40 s at 72 ˚C). Positive and negative controls are used

to assess the quality of the PCR.

2.2.10.4 Semi nested PCRs specific for E. ortleppi and E. granulosus (G6/7)

Seminested PCR was performed to discriminate between

E.ortleppi and E. granulosus G6/7. The reaction mixture of 50 µl

contained 1.5 µl of the positive amplification product from the g5/6/7

PCR, 5µl PCR buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl), 4µl of

MgCl2 (2.mM), 1µl of deoxynucloside triphosopate mixture each at a

concentration of 200 µM, 2.5µl of each of the primer pair Eg camel or

cattle for and cs1 rev. (each 25 pmol) and 0.25 µl of Ampli-Taq

Polymerase (1.25 units.). PCR was performed for 3o cycles

(denaturation for 30 s at 94 ˚C, annealing for 1 min at 60 ˚C and

elongation for 30 s at 72 ˚C).

2.2.11 Detection of the PCR products (Agarose gel electrophoresis)

2.2.11.1 Preparation of the gel Agarose gel was prepared by melting 0.75 g of the Agarose

powder in 50 ml 1ҳ TBE buffer until solution is completely clear.

After cooling the Agarose solution to about 50 ˚C it was poured into

electrophoresis plate and mixed with 1µl ethidium bromide, comb was

placed and left to solidify. The chamber was then filled with 1 ҳ TBE

buffer.

2.2.11.2 Preparation of the sample and running of the gel 10 µl of each of the PCR products was mixed with 2 µl loading buffer

and loaded onto the Agarose gel. 100bp ladder as a marker was also

loaded with the samples to determine the different band sizes.

Samples were then allowed to be separated using a power of 5v/cm.

2.2.12 Visualization of the PCR products The gel was photographed under ultraviolet light using a

computerized documentation system (Bio-Profil®, Image Analysis

Software).

2.2.13 Mitochondrial gene sequencing As a reference method, the sequence of a part of the

mitochondrial cox1 with primer pair2575 (′5 TTT TTT GGG CAT

CCT GAG GTT TAT 3′) and 3021 (′5 TAA AGA AAG AAC ATA

ATG AAA ATG 3′; Bowles et al., 1992) and nad1 using primer pair

JB11 (5 AGA TTC GTA AGG GGC CTA ATA 3) and JB12 (5 ACC

ACT AAC TAA TTC ACT TTC 3; Bowles and McManus, 1993b)

were obtained. Both PCRs were performed as described previously

(Bowles et al., 1992; Bowles and McManus, 1993). After purification

of the PCR products over QIAquick TM columns, cycle sequencing

was performed with AB1 Prism Big Dye Terminator Cycle

Sequencing Ready Reaction Kit using the corresponding PCR primers

(forward and reverse) as sequencing primers. Cycling conditions (25

cycles) were: denaturation for 10 s at 94˚C and annealing for 4 min at

60˚C. Electrophoresis was carried out on the AB1 Prism 310 Genetic

Analyzer. Nucleotide sequence analysis was made using the National

Centre for Biotechnology Information BLAST programs and

databases.

2.3 Used Chemicals Agarose, Roth, Seakem, AGS

Dithiothreitol (DTT), Roth

DNA-Molecular weight marker (100bp ladder), Fermentas

EDTA (Ethylendiamentetra Acetic Acid), Merck

Ethidium bromide (10mg/ml), Roth

Ficol 400, Pharmacia

Phenol-Chloroform-Isoamylalcohol (25:24:1), Roth

Proteinase K, Fermentas

Potassiumhydroxide, Merck

Tris pure (Tris-(hydroxylmethyl)amino methane), Biomol

Tris-HCl (Tris-( hydroxylmethyl)amino methane. HCl), Biomol

2.4 Puffers and solutions

Digestion Buffer 10mM Tris-HCl pH 7.5

10mM EDTA pH 8.0

50mM NaCl

2% SDS pH 7.5

DNA-Loading Buffer (6 ҳ)

0.25% Bromphenolblue

0.25% Xylencyanol FF

15% Ficoll 400

in 1ҳ TBE

TBE Buffer (5 ҳ)

108 g Tris

55 g Boric acid

40 ml 0.5 M EDTA

2L H2o

2.5 Enzymes Taq-Polymerase, Ampli-Taq, Applied Biosystem.

2/6 Oligonucleotides E.g ss1for. 5' GTA TTT TGT AAA GTT GTT CTA '3

E.g ss1rev. 5' CTA AAT CAC ATC ATC TTA CAA T '3

E.g cs1for 5' ATT TTT AAA ATG TTC GTC CTG '3

E.g cs1rev 5' CTA AAT AAT ATC ATA TTA CAA C '3

E.g camelfor 5' ATG GTC CAC CTA TTA TTT CA '3

E.g cattlefor 5' ATG GTC CAC CTA TTA TTT TG '3

P60 for5' TT AAG ATA TAT GTG GTA CAG GAT TAG

ATA CCC '3

P375 rev.5' GGT ACA CAC CGC CCG TCA CCC TCG GTT

'3

All primers were synthesized by Roth.

2.7 Disposables Pipette tips; Sarstedt and Eurolab companies

Petri dishes; Greiner and Sarstedt

Reaction tubes; 0.2 ml; Applied Biosystem

0.5 ml; and 1,5 ml Sarstedt

2.0 ml Eppendorf

12 ml Greiner

15 and 50 ml Greiner

2.8 Instruments Centrifuge: Eppendorf centrifuge 5415D

Cold Centrifuge: Eppendorf Centrifuge 5417 R

Gel electrophoresis: Bio Rad Mini-Sub ® cell

Geldocumentation system: Ltf- Labortechnik GmbH and Co.Kg

(Camera: CCD video camera module, Computer: Belina,

Prinetr: Mitsubishi ).

Hybridization oven: Selutec hyp 10-S, Steuer Electrolabor and

Umwelttechnik.

Incubator: Memmert, Germany

Laminar flow: Heraeus Instrument, Germany

PCR thermocycler: Gene Amp PCR system 2400 Applied

Biosystem.

Pipettes: Eppendorf, autoclavables

Power Supply: Bio Rad Power PAC 300

Printer: DPU-414 thermal printer

Refrigerators: Bosch electronics

Spectrophotometer: Eppendorf Biophotometer

Votrex: Genie 2, Bender and Hober AZ, Zuerich, Switzerland

Waterbath: Julabo P

Chapter Three

Epidemiological and Biomolecular Study of

Cystic Echinococcosis in Humans and Animals in Sudan

Abstract A survey of cystic echinococcosis was conducted during the

period from May 2001 to July 2003 in different parts of the Sudan.

The prevalence rates in camel, cattle, sheep and goats examined in

different states of the Sudan was found to be 59.8% (466/779), 6.1%

(299/4893), 11.3% (1180/10422) and 1.9% (106/5565) respectively.

The encountered number of cysts was 2387 in camel, 333 in cattle,

1514 in sheep and 108 in goats. Fertility rates were found to be 73.7%,

77%, 19% and 31.5% in camel, cattle, sheep and goat respectively.

The favorite site for cysts in camels was the lung (1627/2387). The

liver was found to be preferred site in cattle (206/333) whereas the

peritoneum was the predilection site in sheep (1242/1514) and goats

(53/108).

Strain characterization of E. granulosus in human and livestock

population was described for the first time by using polymerase chain

reaction amplification and sequencing technology. Even though we

were able to detect E. ortleppi and sheep strain (G1) of E. granulosus

in some samples, the camel strain (G6) of E. granulosus appears to be

the predominant strain causing cystic echinococcosis in humans and

animals in Sudan as 533 of the 542 examined DNA samples were

characterized as belonging to this strain. In this study, the sheep strain

of E.granulosus was reported for the first time in Sudan in two

samples of human origin and five samples of sheep origin.

Introduction

Echinococcosis is a cyclozoonotic infection of a world wide

distribution and a variable geographic incidence. The disease is caused

by the larval cystic stage of the E. granulosus complex and it

constitutes a serious health hazard with increasing incidence rates in

many endemic areas of the world. Parasite transmission may occur in

domestic, sylvatic or semi-domestic animal host cycles depending on

the Echinococcus species (and strains) (Craig et al. 2003). The E.

granulosus complex is known to exist as biological distinct taxa,

which may vary in their infectivity to domestic animals and possibly

to man (Thompson, 1986., McManus and Smyth, 1986). These species

or strains vary in features such as morphology, biochemistry,

physiology, pathogenicity, developmental patterns and infectivity to

human and domestic animals (Eckert et al. 1989) with important

implications for the epidemiology of hydatid disease.

The disease has been extensively studied in a number of

different geographical areas and is now present in Asia, Africa, South

and Central America and the Mediterranean region (McManus and

Smyth, 1986). It is found to be common in parts of the United

Kingdom, Europe and Australia (Cook, 1989; Schantz, 1990).

In the Sudan, studies on animal echinococcosis were conducted

by several workers (Saad and Magzoub 1989a; Saad and Magzoub

1989b; Elmahdi, 1998; Omer et al., 2002; Elmahdi et al, 2004 and

Omer et al, 2004). While the disease is most abundant in camel and

cattle, sheep appear to play an unimportant role in the transmission

cycle of echinococcosis. The prevalence rate of the disease in animals

is high, thus providing high risk of the disease for human populations

in different parts of the Sudan. However, no hospital records are

currently available. The disease seems to be endemic in rural

communities where people live in close contact with dogs (definitive

host) and large numbers of livestock (intermediate host). Cystic

echinococcosis was found in 2% (132/6728) of the human population

in selected areas of southern Sudan (Njoroge, 2001) and in 0.33% (1

from 300) of villagers in central Sudan (Elmahdi et al, 2004). Forty

cases of human hydatidosis were recorded in the National Health

Institute, Khartoum, Sudan in 2001 (Personal communication).

Currently, there is no information available about the dominant

strain of the parasite and its infectivity to human. The objective of the

present investigation is to provide information about the epidemiology

and the molecular characterization of the prevalent taxa of E.

granulosus complex in human and animals in the Sudan.

Materials and Methods

1. Epidemiological survey Frequent visits were made to slaughterhouses in different

representative areas of the Sudan (Central: Khartoum, Tamboul and

Medani, Eastern: Kassala and Gedarif, Western: Darfur state and

Southern: Juba, Malakal) during the period of May 2001 to July 2003.

A total number of 21659 animals (779 camels, 4893 cattle, 10422

sheep and 5565 goats) was examined. Different organs including

lungs, liver, heart, spleen and kidneys of the slaughtered animals were

thoroughly inspected and hydatidosis positive animals and the number

of cysts encountered were recorded. Visits were also made to hospitals

in the same vicinity to estimate the number of human cystic

echinococcosis cases. Hydatid cysts or cyst-derived materials were

obtained from 7 patients, admitted to Khartoum (5) and Juba medical

teaching hospitals (2), for diagnosis of the disease. Theses samples

were used for the molecular characterization.

Epidemiological surveys about the disease were not conducted

in Eastern and Northern Sudan but some hydatid cyst samples from

camels (8) and cattle (7) were obtained for DNA characterization.

2. Parasitological studies Cysts or cyst materials were examined macro and microscopically.

Fertility of the collected hydatid cysts was determined by detection of

protoscolices in aspirated fluid samples. Sterile or calcified cysts were

considered infertile.

Exemplary samples of intact cysts or cyst materials were

preserved in 70% alcohol for DNA characterization.

3. DNA extraction DNA was extracted from 527 ethanol preserved hydatid cyst

samples (317 none calcified and 210 calcified) collected from camel

(157, 50), cattle (62, 45), sheep (81, 60), goats (10, 55) and humans

(7, 0) from different parts of central, western and southern Sudan

during the epidemiological survey. Some samples from cattle (4, 3)

and camel (3, 5) were obtained from northern and eastern Sudan

respectively for the purpose of DNA characterization.

DNA extraction was done as by Dinkel et al. (1998). Briefly,

500ul of hydatid fluid containing protoscolices were digested by

lysing buffer containing 500 µl digestion buffer, 10 µl 1 M

dithiothreitol and 60 µl proteinase K. The total nucleic acid was then

extracted using the standard phenol-chloroform-isoamylalcohol

(25:24:1) and precipitated with absolute alcohol. The DNA

concentration was quantified using spectrophotometer at 260 nm wave

length.

4. Polymerase chain reaction (PCR) and molecular characterization

Strain characterization was done using a previously described

G5/6/7 polymerase chain reaction (G5/6/7 PCR) and G1 (g1 PCR) for

the samples which were negative with the G5/6/7PCR (Dinkel et a.,l

2004), that the PCR was performed in a 50 µl volume containing 5 µl

PCR buffer consisted of 5 µl of the PCR buffer (10 mM Tris-HCl (pH

8.3), 50 mM KCl), 4 µl of MgCl2 (2 mM), 1 µl of deoxynucleoside

triphosophate mixture each at a concentration of 200 µM, 2.5 µl of

each of the primer pairs cs1forward and cs1reverse (each 25 pmol) for

the first PCR and ss1for and ss1rev for the G1 PCR and 0.25 µl of

Ampli-Taq Polymerase (1.25 units) The amount of the DNA was

calculated at 200 ng. Positive and negative controls were used to

assess the quality of the PCR. Samples which were positive with the

G5/6/7 PCR when visualized on ethidium bromide gel were then

subjected to a second semi-nested PCR amplification using the primer

pair E.g.camel or E.g.cattle for and cs1rev to determine if the sample

is camel/pig or cattle strain of E.granulosus. Some calcified cysts

were negative when tested with G5/6/7 PCR or G1 PCR, these

samples were subjected to cestode specific PCR using the primer pair

P60.for and P375.rev as described by Dinkel,. et al. (1998).When

positive, 0.5-1 µl of this PCR product was used with the G5/6/7 or G1

PCRs.

5. Visualization of the PCR products The etidium bromide stained agarose gel was photographed

under ultraviolet light using a computerized documentation system

(Bio-Profil®, Image Analysis Software).

6. Mitochondrial gene sequencing For confirmation of the PCR results, sequencing of a part of the

mitochondrial cox1 with primer pair 2575 and 3021 (Bowles et al.

1992) and nad1 using primer pair JB11 and JB12 (Bowles and

McManus, 1993b) was performed

Results

1. Eipdemiolgical survey

In Central Sudan, the prevalence rate of cystic echinococcosis

in camels was found to be 55.6% (119/214). The total number of cysts

was 360. These were primarily located in the lungs 84% (304/360)

followed by the liver and spleen. No cysts were found in the heart and

kidneys. In cattle and sheep, the prevalence rates were 20% (50/250)

and 2.5% (10/400), respectively. The predilection site in cattle and

sheep was the liver with a percentage of 77% and 60% respectively.

Fertility was found to be 100% in cattle and 75% in camels whereas

none of the cysts was found to be fertile in sheep (Table 1, Figures 1

and 2).

In Western Sudan, the highest prevalence rate was recorded in

camels as 61.4% (347/565). In contrast, the prevalence rate in cattle,

sheep and goats was found to be 5.2% (226/4318), 11.9% (1162/9727)

and 1.9% (103/5523), respectively.

Predilection site was the lung in camel (1323/2018) and the

liver in cattle (138/247), whereas mesentery was the predilection site

in sheep (1242/1494) and goats (51/76). The fertility rate was 73.8%

(1490/2018) in camel, 82.3 %( 186/226) in cattle, 19% (289/1494) in

sheep and 32.4% (34/105) in goats (Table 2, Figures 3 and 4).

In Southern Sudan, the prevalence rate was 7% (23/325) in

cattle, 3% (8/295) in sheep and 7% (3/42) in goat. Camels were not

examined. Predilection site was the liver in cattle (82.6%), sheep

(100%) and goats (100%). The fertility rate was 3% (8/23) in cattle,

whereas in sheep and goats the fertility was found to be 0% (Table 3,

Figures 5 and 6).

2. Strain characterization The camel strain of E granulosus was detected by polymerase

chain reaction and sequencing of DNA samples extracted from

hydatid cysts of camel, cattle and goat origin. The cattle strain was

found in two samples of cattle from Eastern and Southern Sudan. The

sheep strain (G1) was found in five DNA samples extracted from

hydatid cysts of sheep origin in central Sudan.

The camel (G6) strain was found in five DNA samples

extracted from hydatid cysts of humans whereas the common sheep

strain (G1) was found in two samples

Two calcified cysts of sheep origin were negative (Table 4,

Figures 8, 9, 10 and 11).

Figures 1 and 2: Prevalence and fertility of hydatid cysts from

camel, cattle, sheep and goats in central Sudan

Prevalence of hydatid disease in camel, cattle, sheep and goats in central Sudan

-200

-100

0

100

200

300

400

500

Camel Cattle Sheep Goats

Animal Species

Num

ber

Total Examined Number Number of Positives

Fertilty Percentage of hydatid cysts collected from camel, cattle and sheeps in central Sudan

0% 20% 40% 60% 80% 100% 120%

Camel

Cat t le

Sheep

Fert ilty Percentage

50

Table 2: Prevalence of cystic echinococcosis in

livestock in Western Sudan

Examine

d animals

Total

number

examined

Prevalence

%

Total

number

of cysts

Fertility

%

Predilection

site

Cysts

examined for

genotype n (n

fertile-n

infertile)

Genotype

(n)

Camel 565 61.4%

(347/565)

2018 (73.8%)

1490/2018

Lung

1323/2018

120 (100-20) G6 (120)

Cattle 4318 5.2%

(226/4318)

247 82.3%

186/247

Liver

138/247

60 (30-30) G6(60)

Sheep 9727 12%

(1162/9727)

1494 19%

289/1494

Peritoneum

1242/1494

95 (55-40) G6 (93)

Goats 5523 1.9%

(103/5523)

105 34/105

32.4%

Peritoneum

51/76

62(10-52) G6 (62)

51

Figures 3 and 4: Prevalence and fertility of hydatid cysts from camel, cattle,

sheep and goats in western Sudan

P r e v a l e n c e o f h y d a t i d d i s e a s e i n c a m e l , c a t t l e , s h e e p a n d g o a t s i n w e s t e r n S u d a n

- 4 0 0 0

- 2 0 0 0

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

1 2 0 0 0

C a m e l C a t t le S h e e p G o a t s

A n i m a l S p e c i e s

Num

ber

T o t a l E x a m in e d N u m b e r N u m b e r o f P o s it iv e s

Fert ilt y Percentage

0 % 10 % 2 0 % 3 0 % 4 0 % 5 0 % 6 0 % 7 0 % 8 0 % 9 0 %

Camel

Cat t le

S heep

Goat s

Fer t ilit y Pecr cen t age

Fert ilt y Percentage

52

Table 3: Prevalence of cystic echinococcosis in

livestock in Southern Sudan

Examined

animals

Total

number

examined

Prevalence

%

Total

number

of cysts

Fertility

%

Predilection site Cysts

examined

for

genotype n

(n fertile-n

infertile)

Genotype

(n)

Camel 0 - 0 - 0 0 0

Cattle 325 7%

(23/325)

23 34.7%

8/23

Liver

19/23

20 (5-15) E.ortleppi

(1)

G6 (19)

Sheep 295 2.7%

(8/295)

8 0% Liver

8/8

8 (0-8) G6 (8)

Goat 42 7%

(3/42)

3 0% Liver

3/3

3 (0-3) G6 (3)

53

Figures 5 and 6: Prevalence and fertility of hydatid cysts from camel,

cattle, sheep and goats in southern Sudan

Prevalence of hydatid disease in camel, cattle, sheep and goats in southern Sudan

-100

-50

0

50

100

150

200

250

300

350

Camel Cat t le Sheep Goat s

A ni ma l S pe c i e s

Tot al Examined Number Number of Posit ives

F e r t i l t y P e r c e n t a g e o f h y d a t i d c y st s c o l l e c t e d f r o m c a t t l e , sh e e p a n d g o a t s i n so u t h e r n S u d a n

0 % 5 % 1 0 % 1 5 % 2 0 % 2 5 % 3 0 % 3 5 % 4 0 %

C a t t l e

Sh e e p

G o a t s

Fer t i l ty Per centage

54

Figure 7: Predilection sites of hydatid cysts in camel, cattle, sheep

and goats in different parts of Sudan

Predilection Sites of hydatid cysts in camel, cattle, sheep and goats in different parts of Sudan

68%

61%

83%

65%

Lung

Liver

Peritonium

Peritonium

Cam

elC

attle

Shee

pG

oats

Percentage

55

Table 4: Strain characterization of hydatid cyst samples from camel,

cattle, sheep, and goats from different parts of Sudan

ANIMALS

LOCATION CAMEL CATTLE SHEEP GOATS

Central Sudan 87 (G6) 27 (G6) 38, 33 (G6)

5 (G1)

0

Eastern Sudan 5 (G6) 3, 2 (G6)

1 (E.ortleppi)

0 0

Western Sudan 120 (G6) 60 (G6) 95 (G6) 62 (G6)

Northern

Sudan

3 (G6) 4 (G6)

Southern

Sudan

0 20, 19 (G6)

1 (E.ortleppi)

8 (G6) 3 (G6)

56

Figure 8: Visualization of the first amplified 254 bp PCR

products from different cyst samples .

Lane MW: molecular weight marker, Lane 1: G5/6/7 DNA (positive

control), Lane 2: negative control, Lane 3-11: DNA extracted from

hydatid cyst samples from different representative animals.

MW 1 2 3 4 5 6 7 8 9 10 11

254 bp

57

Figure 9: Semi-nested PCR amplification of the 171 bp specific

PCR product of the camel (G6) strain from the

first 254 bp PCR product for the above gel.

Lane MW: molecular weight marker, Lane 1: E. granulosus (G6) strain

DNA (positive control), Lane 2: negative control, Lane 3-11: DNA

extracted from hydatid cyst samples from different representative

animals.

MW 1 2 3 4 5 6 7 8 9 10 11

254 bp 171 bp

58

Figure 10: Semi-nested PCR amplification of the

171 bp specific PCR product of the cattle (E.ortleppi) strain

from the first 254 bp PCR product for the G5/6/7 PCR.

Lane MW: molecular weight marker, Lane PC: E. ortleppi DNA (positive

control), Lane NC: negative control, Lane 1-2: DNA extracted from

hydatid cyst samplesfrom cattle from eastern and southern Sudan.

254bp

171bp

59

Figure 11: Visualization of the 254 bp G1 (sheep strain)

PCR products from different cyst samples.

Lane MW: molecular weight marker, Lane PC: G1 DNA (positive

control), Lane NC: negative control, Lane 1-6: DNA extracted from

hydatid cyst samples from sheep and humans.

254bp

60

Discussion Hydatidosis (cystic echinococcosis) is a highly endemic zoonotic

disease in most of the Mediterranean countries, North Africa and Middle

East (Matossian et al., 1977). The disease is of public health importance

and requires both surgical and chemotherapeutic interventions (Ibrahim,

1990; El-Mufti et al., 1993).

In Sudan, several studies were conducted to investigate the

epidemiology of this disease. Tola (1987) reported prevalence rates of

56.4%, 2.1% and 2.0% in camels, cattle and sheep respectively, whereas

Saad and Magzoub (1989), reported prevalence rates of 48.6% and 38.4%

in camel and cattle, respectively.

In the present study, hydatidosis was investigated in a total number of

21659 animals (779 camels, 4893 cattle, 10422 sheep and 5565 goats) in

the Sudan. The highest rate of infection in the total examined number was

encountered in camel (60%, 466/779) followed by sheep (11.3%,

1180/10422). The prevalence rate of cystic echinococcosis in camel was

the highest compared with other examined animals in central and western

Sudan (55.6% and 61.4% respectively). The number of cysts encountered

in camels were high comparing with the other animals (2387).The high

prevalence of infection in camels may be due to the fact that, in nomadic

environments, camels are usually found in close contact with dogs

(definitive host), thus providing infection to more susceptible livestock or

human populations. The prevalence rate of the disease in sheep in

Western Sudan was the highest comparing to its prevalence in the same

animal in other investigated areas Sudan (19%).

61

Fertility rates of the cysts in camel and cattle in central Sudan was

75% and 100% respectively (Table 1) whereas in western Sudan it was

found to be 73.8% and 75% respectively. However, the fertility rate in

sheep and goats in western Sudan was 19% and 32.4% respectively. This

fertility was reduced to 0% in both animals in central and southern Sudan.

It is well documented that sheep and goats are unimportant hosts in the

transmission cycle of cystic echinococcosis in the Sudan (Saad and

Magzoub, 1989, Omer et al., 2002., Elmahdi et al., 2004). This could be

attributed to the fact that sheep and goats are usually slaughtered at

younger age before development of mature cysts. However, in the present

study the animals (3-5years) developed calcified or infertile cysts. This

may be due to immunoresponce of sheep to hydatidosis infection (Saad

and Magzoub, 1989a; Omer et al, 2002), or due to genetic characteristics

of the parasite strain or species (Elmahdi et al, 2004).

Postmortem inspection of all examined animals indicated that the

predilection sites of hydatid cysts appeared to be the lung for camels,

liver for cattle and peritoneum for sheep and goats.

Elkhawad (1978) reported the liver to be the predilection site of

hydatidosis in sheep whereas camels were mostly affected at the lungs.

This result is in disagreement with Dada et al (1981) who reported the

pulmonary cysts to be common in sheep and goats.

It is not known yet to what extent epidemiological significant

differences (such as host range, infectivity, virulence and drug

susceptibility) reflect genetic heterogeneity within Echinococcus

populations (Bowles et al, 1992). However, in Sudan, with the exception

of the report on strain characterization of E. granulosus described by

Dinkel et al, 2004, no information is currently available in this regard.

The author reported the presence of camel strain in central Sudan, and

62

cattle strain of E. granulosus in 25% (2/8) of the samples obtained from

cattle origin. In our study we detected by PCR and DNA sequencing

twice the cattle strain in cattle in Eastern and Southern Sudan. The

hydatid cyst samples obtained from camels and cattle in eastern and

central Sudan and sheep in central Sudan were identified as camel (G6)

strain. Five of the DNA samples extracted from human cysts were

characterized as camel (G6) strain. The other two samples as well as five

of the samples obtained from sheep in central Sudan were found to be G1

(sheep strain). It is obvious that the high prevalence and fertility rates of

hydatid cysts in camel indicate the important role of camel for

maintenance of hydatidosis in Sudan. This is in contrast to many other

countries where the sheep strain of E. granulosus is the responsible strain

for most of cystic echinococcosis. In the human population, the camel

strain of E. granulosus was reported in Argentina, Iran, Kenya and Nepal

(Dinkel et al., 2004). In the present study, we report the first records of

camel strain of E. granulosus in the Sudanese population as well as the

presence of the sheep strain in human and sheep in Sudan. The link

between genetically homogenous strains and features of epidemiological

importance can be useful in implementation of control programs.

In conclusion, prevalence and strain characterization of E.

granulosus for livestock and human population was described for the first

time in Sudan using polymerase chain reaction and DNA sequencing

technologies.

63

Chapter Four

Strain Characterization and Coprodiagnostic PCR of Echinococcus. granulosus in Central Sudan

Abstract 42 dogs shot as a part of the rabies control program in Tamboul and

Rofa, central Sudan were autopsied and their intestinal contents were

examined for the presence of E. granulosus worms. Faecal samples were

taken as well for coprodiagnosis. The worm burden in positive dogs was

determined using a dilution method and the harvested worms were

characterized using G5/6/7 and G1 PCRs. From the 42 euthanized dogs,

12 (28.5%) were harboring E. granulosus worms. The worm burden was

22-80*103 in the positive dogs. All the DNA samples extracted from the

worms were characterized as camel (G6) strain of E. granulosus. 83.3%

(10/12) of the DNA extracted from the faecal samples collected from the

12 dogs which were found to be positive at necropsy were found positive

with copro- PCR and the strain was characterized as the camel (G6) strain

of E. granulosus. Two samples were considered inconclusive as there was

no signal in the inhibition test. 93.3% (28/30) copro- DNA samples from

the 30 samples collected from the dogs which were reported negative at

necropsy were negative using copro-diagnostic PCR. The other two

samples were positive and characterized as sheep (G1) strain of

E.granulosus. This copro-PCR method is used for the first time in such a

survey. The overall sensitivity of the test was found to be 100% as all the

necropsy positive dogs were found to be positive using copro-PCR.

Introduction

64

Hydatidosis is a disease caused by cestodes of the E. granulosus

complex which possesses a two host life cycle involving dogs as

definitive hosts and different animals and humans as intermediate hosts.

Hydatidosis in intermediate hosts results from accidental ingestion of

tapeworm eggs passed into the environment with faeces from definitive

hosts. The disease is of an economic importance in intermediate and

aberrant intermediate hosts (Torgerson and Budke, 2003). Diagnosis of

the disease in the definitive host is an important tool in establishing a

successful control program especially in areas where there are a lot of

stray dogs and uncontrolled slaughtering. Diagnosis of E. granulosus in

dogs is difficult, because the eggs of all Echinococcus and Taenia species

are morphologically indistinguishable from one another and the

characteristic small segments of Echinococcus species may be absent

from the faeces or can be easily overlooked. Methods like purgation with

arecoline salts and examination of the small intestine at necropsy

(Deplazes and Eckert, 1996) are not suitable for large scale

epidemiological studies as they are time consuming, laborious and require

special safety precautions. Serum antibody detection and copro-ELISA

have been successfully employed for the diagnosis of Echinococcus spp.

in dogs but the sensitivity of the former varied according to geographic

region and the sensitivity of the latter depends on the worm burden (Craig

et al. 2003). CoproPCR have been used for the detection of E

.multilocularis in foxes (Bretagne et al. 1993, Dinkel et al., 1998) and

also for the detection of E.granulosus (Abbasi et al. 2003)

In this study we used for the first time the copro-PCR assay which

was developed for the detection of E. multilocularis in foxes (Dinkel et

al., 1998) in a survey for the prevalence and strain characterization of E.

granulosus in stray dogs in central Sudan.

65

Materials and Methods

1. Stray dogs As a part of rabies control program, a campaign was done in

Central state (Tamboul and Rofa), central Sudan, for the purpose of

controlling the increasing population of stray dogs at that area. The whole

intestines (small and large intestines) of all the shot dogs (42) were

securely removed avoiding hazards of contamination to the environment

and workers.

2. Scraping Within 2-4 hours, the whole intestine of each individual animal was

opened after securing its content by making tight double adjacent

ligatures at both ends of the intestine. The mucosa was scraped with a

scalpel. The intestinal contents and the mucosal scrapings were washed

through an 80-mesh-per-inch brass sieve. The material retained by the

sieve was then washed into a glass container and examined for worms

with a hand lens. The number of worms recovered from each individual

material was estimated using a dilution method. Faecal samples were

collected from the rectum of each intestine (42 samples) and labeled.

3. Preservation of the faecal samples and harvested worms

Harvested E. granulosus adult worms were preserved in 70%

alcohol for further molecular characterization. Collected fecal samples

were frozen at -20˚C and later upon arrival in Germany at -80 ˚C for 3

days for the inactivation of eggs.

66

4. DNA extraction from E. granulosus adult worms DNA was extracted form worm suspensions obtained from 12

dogs. After washing the worms three times from the alcohol using nucleic

acid free water, DNA was extracted as previously described by Dinkel et

al (2004). Briefly, 500 ul of hydatid fluid containing protoscolices were

digested by lysing buffer containing 500 µl digestion buffer, 10 µl 1 M

dithiothreitol and 60 µl proteinase K. The total nucleic acid was then

extracted using the standard phenol-chloroform-isoamylalcohol (25:24:1)

and precipitated with absolute alcohol.

5. DNA extraction from the faecal samples DNA was extracted from the faecal samples as previously

described by Dinkel et al (1989). Briefly, 0.5 g of the sample were diluted

1:2 (vol/vol) with distilled water. A total of 1500 of the resulting solution

was used for the DNA extraction starting with a stage of alkaline

hydrolysis using 108 of 1M KOH and 30 of 1 M dithiothreitol. After

vortexing and incubation at 65° C for 30 min, the samples were

neutralized with 270 µl of 2 M Tris-HCl (pH 8.3) and 40.5 µl of 25%

HCl. 1950 µl phenol-chloroform-isoamylalcohol was then mixed with

each sample. The aqueous phase was transferred to a new tube and the

DNA was precipitated by the addition of 5400µl binding buffer and 30 µl

of a silica matrix. The samples were then incubated at 37 ˚C for 60

minutes with frequent agitation centrifuged for 30 seconds at 10.000 g,

and 1000 µl binding buffer was added to the pellet after discarding the

supernatant. This was then transferred into a new 12 ml reaction tube and

centrifuged at 13.000 g for 30 seconds. The pellet was washed 3 times

with washing buffer. After drying at 35-40 ˚C in an oven to eliminate

ethanol, 100 µl nucleic acid free water was added to the pellet and

incubated in a water bath at 50˚C for 15 minutes to re-suspend DNA.

67

After centrifugation at 13.000g for 60 seconds, 85 µl of this solution were

transferred in a new 1.5 µl reaction tube.

6. Polymerase Chain Reactions (PCRs) For the DNA extracted from the E. granulosus adult worms, strain

characterization was determined using our previously described G5/6/7

polymerase chain reaction (G5/6/7 PCR) (Dinkel et al 2004). The PCR

was performed in a 50 µl volume containing 5µl PCR buffer consisting of

5 µl of the PCR buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl), 4 µl of

MgCl2 (2mM), 1 µl of deoxynucleoside triphosphate mixture each at a

concentration of 200 µM, 2.5 µl of each of the primer pair cs1forward

and cs1reverse (each 25 pmol) and 0.25µl of Ampli-Taq Polymerase

(1.25 units) The amount of the DNA was calculated at 200 ng. Positive

and negative controls were used to assess the quality of the PCR.

After visualization on ethidium bromide gel, samples were

subjected to a second semi-nested PCR amplification using the primer

pair E.g. camel or E.g. cattle for and cs1rev to determine if the sample is

camel/pig or cattle strain of E. granulosus. For the DNA obtained from

the faecal samples, first a cestode specific PCR was performed as

described by Dinkel et al (1998, 2004). The 100 µl reaction mixture

consisted of 10 µl of the PCR buffer (10 mM Tris-HCl (pH 8.3), 50 mM

KCl), 10µl of MgCl2 (2.5 mM) , 2µl of deoxynucleoside triphosphate

mixture each at a concentration of 200 µM, 4 µl of each primer (20 or 40

pmol) and 0.25 µl of Ampli-Taq Polymerase (1.25 units). 10 µl of the

DNA was added and the volume was completed to 100 using nucleic acid

free water. PCR was running for 40 cycles (denaturation for 30 s at 94 ˚C,

annealing for 1 min at 55 ˚C and elongation for 30 s at 72 ˚C). Positive

and negative controls are used to assess the quality of the PCR.

68

In a second step, a nested G5/6/7 PCR was performed using 1,5 of

the PCR products of the first PCR and consequently a seminested PCR

were performed as previously described.

7. Visualization of the PCR products The ethidium bromide stained agarose gel was photographed under

ultraviolet light using a computerized documentation system (Bio-Profil®,

Image Analysis Software).

8. Control for inhibition Due to unknown factors present in some faecal samples, PCRs may

occasionally be inhibited and may therefore give false negative results

(Wilde et al., 1990). To control such inhibitions 10 µl of E. granulosus

DNA was added to each negative sample and the first PCR was repeated

(Dinkel et al.1998). The test sample was recorded as negative only if a

signal was obtained, if not, the result was considered inconclusive.

9. Mitochondrial gene sequencing The sequence of a part of the mitochondrial cox1 gene with primer

pair2575 and 3021 (Bowles et al. 1992) and nad1 gene using primer pair

JB11 and JB12 (Bowles and McManus, 1993b) was performed.

69

Results

1. Necropsy From the 42 euthanized dogs, 12 (28.5%) were harboring E.

granulosus worms (table 1).The worm burden was 22-80*103 in the

positive dogs.

2. PCR of the E. granulosus worms All the DNA samples extracted from the worm suspension were

characterized as camel (G6) strain of E. granulosus Fig (1, 2).

3. Copro-PCR 83.3% (10/12) of the Copro DNA extracted from the faecal

samples collected from the 12 dogs which were found to be positive at

necropsy were found positive with copro PCR and the strain was

characterized as camel (G6) strain of E.granulosus samples (Table 1 and

Fig 3,4,5). The result of the other two samples was considered

inconclusive as there was no signal in the inhibition test. 93.3% (28/30)

copro-DNA samples from the 30 samples collected from the dogs which

were reported negative at necropsy were negative using copro-diagnostic

PCR. The other two samples were positive and characterized as the sheep

(G1) strain of E. granulosus (Table 1 and Fig 6).

70

Table 1: Necropsy and Copro-PCR results of

the 42 dogs shot in central Sudan Necropsy CoproPCR and strain characterization

Positive: n=12 Positiv: n=10 (G6)

PCR inhibited n=2

Negatives: n= 30 Negative: n=28

Positive: n= 2(G1)

71

Figure 1: Visualization of the first amplified 254 bp

PCR products from E. granulosus worms.

Lane MW: Molecular weight marker, Lane PC: E. granulosus positive

DNA, Lane NC: DNA negative control, Lanes 1-12, DNA from E.

granulosus worms

1 2 3 4

5 6 7 8 9 10 11 12 NC PC M

254bp

72

Figure 2: Semi-nested PCR amplification of the

171 bp specific PCR products of the camel (G6)

strain from the first 254bp

Lane MW: Molecular weight marker, Lane PC: E. granulosus positive

DNA, Lane NC: DNA negative control, Lanes 1-12, DNA from E.

granulosus worms

254bp 171b

73

Figure 3: Visualization of the first amplified

373bp PCR product from copro DNA.

Lane MW: Molecular weight marker, Lane PC: E. granulosus positive

DNA, Lane NC: DNA negative control, Lanes 1-12, Copro-DNA.

Figure 4: Visualization of the 254bp G5/6/7 nested

PCR from the first cestode specific PCR

373bp

74

Lane M: Molecular weight marker, Lane PC: E. granulosus positive

DNA, Lane NC: DNA negative control, Lanes 1-12: CoproDNA.

Figure 5: Semi-nested PCR amplification of the 171bp specific

PCR products of the camel (G6) strain from the G5/6/7 PCR

254bp

75

Lane MW: Molecular weight marker, Lane PC: E. granulosus positive

DNA, Lane NC: DNA negative control, Lanes 1-12|: CoproDNA.

Figure 6: Visualization of the amplified 254 bp G1

(sheep strain) PCR products from copro DNA.

171bp 254bp

76

Lane MW: Molecular weight marker, Lane PC: E. granulosus positive

DNA, Lane NC: DNA negative control, Lanes 1-2: CoproDNA

Discussion

Sudan is the largest country in Africa and of the Arab world and it

has a large livestock population. Preliminary data about the prevalence of

hydatidosis in different intermediate hosts in Sudan revealed that the

254bp

77

disease occurs in all domesticated intermediate host species in Sudan

(camel, cattle, sheep and goats) with the highest infection rates in camels

(Elmahdi et al 2004, Omer et al., 2002 and 2004). Infection rates in

humans are considerable. The study area is located in Central Sudan and

considered the largest market for camel meat in the country. This area is

inhabited by villagers who own dogs for the purpose of guarding. In most

of the cases these dogs are fed on the offal obtained from the

slaughterhouse. These are mostly hydatidosis infected camel lungs. This

in turn ensures a continuous focus of echinococcosis in that area. In our

survey we examined a number of 42 dogs, 12 of these (28.5%) were

found to be positive at necropsy. From the data encountered about the

prevalence of hydatidosis in camels in that area (55.6%) and the high rate

of cyst fertility (Saad and Magzoub, 1988., Omer et al., 2002., Elmahdie

et al., 2004), it was suggested that the infection rate in dogs should be

high, however the relatively low prevalence rate reported here may be

due to particular risk behaviours in dogs like having access to offal which

can markedly differ according to the area and season (Macpherson et al.

1985., Wachira et al. 1994). The worm burden in these dogs was very

high (22-80*103) and the worms were mostly mature. This may explain

the high prevalence of the disease in intermediate hosts, because most of

the animals are reared under open grazing conditions and dogs are always

accompanying the herd. CoproPCR is used for the first time in such

studies in Sudan. 10 of the faecal samples obtained from the necropsy of

positive dogs were found positive using PCR and the strain was

characterized as the camel strain. Thus, the sensitivity of this test is

100%. The result of the other two positive necropsy samples was

considered inconclusive as the DNA was inhibited. From the 30 samples

obtained from the dogs which were negative at necropsy, 2 samples were

found to be positive using copro-PCR and they were characterized as

78

sheep strain (G1) of E.granulosus. This strain of E.granulosus has always

thought to be absent from Sudan since it was never detected in the

previous surveys about strain identification in Sudan (Omer et al., 2004.,

Dinkel et al., 2004). Nevertheless, we found the sheep strain in 1.3%

(7/542) of the samples that we have characterized from different

intermediate hosts in Sudan (Omer et al. 2006). The reason for this

apparent rarity of the G1 strain is not clear from the available

epidemiological data, and there is an urgent need for additional studies.

In conclusion, 100% of the DNA samples extracted from the fecal

samples obtained from the positive dogs at necropsy were also found

positive at PCR. Sensitivity of this coproPCR is 100%. Thus, integration

of such method in future surveys of the disease is very useful comparing

with the traditional methods concerning specificity, sensitivity, cost and

the processing time needed (Dinkel et al., 1998).

Chapter Five Experimental Infection of Dogs with Protoscolices of Camel Origin and Molecular Characterization of the Maturing Adult Worms Using PCR

ABSTRACT

For the purpose of this study, hydatid cysts were obtained from the

lungs of naturally infected camels (Camelus dromedarius) in Tamboul

79

slaughterhouse in central Sudan. Viable protoscolices were collected

from these cysts and used for experimental infection of dogs at different

doses. Ten dogs were divided into two groups (A and B) of five dogs

each. Dogs in group A received a dose of 4×103 each protoscolices

whereas dogs in group B received a dose of 8×103.protoscolices each.

Fecal samples were examined for patent infection during the study

period. Dogs were necropsied at 45 dpi (group A) and 54 dpi (group B).

No eggs were detected in fecal samples from group A throughout the

experimental period (45 days). However, eggs were first demonstrated in

faeces 52 dpi in group B. The experimental animals in both groups did

not show any adverse clinical signs during the experimental study.

Echinococcus granulosus worms were recovered from both groups at the

time of necropsy.

Molecular characterization of the adult worms was made possible

using the Polymerase chain reaction (PCR)-based detection assay. The

worms were identified as G6 (camel) strain of E. granulosus. It was

found that the prepatency period in dogs after experimental infection with

protoscolices of camel origin is longer than the reported for other strains

of E. granulosus. These are the first data on prepatency periods of the

camel strain G6 in dogs confirmed by molecular characterization.

80

INTRODUCTION

Cystic echinococcosis or hydatidosis is an important cyclo-

zoonotic parasitic disease caused by a specific tapeworm, E. granulosus

which cycles in a predator/prey relationship between a carnivore

(definitive host) and a herbivore (intermediate host). Humans can

accidentally become infected by ingestion of eggs of the adult worm. The

disease is common in open range livestock raising areas. In addition,

uncontrolled slaughtering of meat-animals and the presence of large stray

dog population also contribute towards the endemicity of the disease

(Anderson et al., 1997). The parasite causes serious public health

problems in certain parts of the world including the Sudan (Saad and

Magzoub, 1988). The camel is an important host of E. granulosus and is

commonly infected throughout Africa and the Middle East (Schwabe,

1986). The camel has attracted much interest as an intermediate host of E.

granulosus, particularly in its role as a reservoir of infection in humans

(Eckert et al.1989). In addition, it has been proposed that the camel form

of Echinococcus may be a different subspecies or strain (Pandey et

al.1986). However, very little comparative work has been carried out to

characterize isolates from E. granulosus from camels (Thompson and

Lymbery, 1988).

The prepatent period of E. granulosus in camels was reported to be

as early as 5 weeks in the definitive host (Eckert et al., 1989). However,

some workers reported the prepatent period to be as long as 12 weeks

post infection (Saad and Magzoub., 1988). Currently, no information is

available about the prepatent period in regard to the strain of the parasite.

In a previous report, it has been suggested that different strains of the

parasite may have different prepatent period in the final host (Eckert et

al., 1989). The morphological and biological characteristics of the

81

intestinal stages of E. granulosus of camel origin were previously studied

by Eckert et al., 1989 without performing the molecular characterization.

The objectives of the present study were to experimentally transmit

infection to dogs using different doses of protoscolices of camel origin,

and to determine the parasitological data of the infection using

conventional methods as well as the strain characterization using

polymerase chain reaction (PCR).

82

Materials and Methods

1. Experimental Animals Ten stray dogs (four males and six females) about 1-2 weeks –old

were selected. Young dogs are usually easy to handle and are more

susceptible to infection. The dogs were divided randomly into two groups

(five dogs each). Dogs in each group were housed together and received

milk, cooked meat, bread and water. The animals were subjected to

parasitological examination to eliminate the possibility of helminth

infection. The experimental animals received a total dose of 4×103 and

8×103 protoscolices for dogs in group A and B, respectively.

2. Infective Materials Hydatid cysts were obtained from camels in Tamboul

slaughterhouse (Central Sudan). The cysts were collected from the lungs

of the infected camels. Hydatid fluid containing protoscolices was

aspirated with sterile needles. The protoscolices were then kept in clean

sterile jars. The viability of the protoscolices was determined by eosin

exclusion and observation for flame cell movement basically as described

by Smyth et al., 1980. Viable protoscolices were counted individually

with dilution method, mixed with milk and fed orally to the dogs at the

prescribed dose.

3. Parasitological Parameters Fecal samples were collected from both groups and were examined

every two days from day 28-35 post infection and daily from day 36 to 54

post infection for the presence of eggs of the parasite in feces.

Conventional parasitological methods including both direct faecal

83

examination and centrifugal flotation methods (Soulsby, 1968) were used

to examine the faeces for presence of proglottids or eggs.

4. Necropsy Dogs in groups A and B were necropsied after euthanasia at days

45 and 54 (pi), respectively. The whole small intestine was removed.

Tight double adjacent ligatures were made at both ends of the intestine to

secure the contents. The intestine was then opened and the mucosa was

scraped with a scalpel. The intestinal contents and the mucosal scrapings

were washed through 80-mesh-per-inch brass sieve. The material retained

by the sieve was then washed into a glass container and examined for

worms with a hand lens. Adult worms were examined microscopically to

determine the extent of development. Approximate worm counts were

obtained by a dilution technique. Worms from each dog were preserved

in 70% alcohol for molecular characterization.

5. Molecular characterization of the adult worms Adult worms recovered from each dog in each group were washed

thoroughly with nucleic acid free water by centrifugation to remove the

70% alcohol. DNA was then extracted from the washed worms according

to the method described by Dinkel et al (1998). Briefly, the worms were

homogenized and the homogenate was lysed using lysing buffer (500 µl

digestion buffer, 10 µl 1 M dithiothreitol and 60 µl proteinase K). The

total nucleic acid was then extracted using the standard phenol-

chloroform-isoamylalcohol (25:24:1) and precipitated with absolute

alcohol. The DNA concentration was quantified using spectrophotometer

at 260 nm wave length. Strain characterization was determined using

G5/6/7 polymerase chain reaction (G5/6/7 PCR) (Dinkel et al., 2004),

that the PCR was performed in a 50 µl volume containing 5 µl PCR

84

buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl), 4 µl of MgCl2 (2.mM),

1µl of deoxynucleoside triphosophate mixture each at a concentration of

200 µM, 2.5 µl of each of the primer pair cs1forward and cs1reverse

(each 25 pmol) and 0.25 µl of Ampli-Taq Polymerase (1.25 units) The

amount of the DNA was calculated at 200 ng. Positive and negative

controls were used to assess the quality of the PCR.

After visualization on ethidium bromide gel, samples were then subjected

to a second semi-nested PCR amplification using the primer pair

Eg.camel or E.g.cattle forward and cs1reverse to determine if the sample

is camel/pig or cattle strain of E. granulosus.

85

RESULTS

1. Parasitological findings Microscopic examination of the faecal samples collected from dogs

in group A revealed that there were no eggs detected throughout the

course of the study. The examination of the faecal samples from dogs in

group B revealed the first detection of the eggs at day 52 d.p.i.

2. Necropsy Dogs in group A were necropsied at day 45 d.p.i. The recovered

number of worms was estimated to be 15-35×102 / animal (37.5%-

87.5%). The infection was considered prepatent as none of the recovered

worms were gravid. However, in group B the number of the recovered

worms was estimated to be 50-75×102 (62.5%-93.8%). The infection in

this group was considered patent as all harvested worms had gravid

segments. (Table1).

3. Molecular Characterization of the adult worms. Application of PCR to DNA extracted from adult worm resulted in

amplification of a 254 bp PCR product. The semi-nested amplification

produced a 171 bp PCR which is specific for G6 strain (camel strain) of

E. granulosus.

86

Table 1: Results of the experimental infection of dogs in groups A and B with

protoscolices of camel origin.

Group Number Infection dose

(Protoscolices)

Duration of the

study period

The first

appearance of

eggs

No of

recovered

worms

1 (n=5) 4×103 45 - 15-35×102

2 (n=5) 8×103 56 52 50-75×102

87

Figure 1: Visualization of the first amplified 254 bp

PCR products from different worm samples.

Lane MW: molecular weight marker, Lane 1: G5/6/7 DNA (positive control), Lane 2:

negative control, Lane 3-11: DNA extracted from E. granulosus worm samples from

different animals

MW 1 2 3 4 5 6 7 8 9 10 11

254 bp

88

Figure 2: Seminested PCR amplification of the

171 bp specific PCR product of the camel (G6) strain from the first

254 bp PCR product for the above gel.

Lane MW: molecular weight marker, Lane 1: E. granulosus (G6) strain DNA

(positive control), Lane 2: negative control, Lane 3-11: DNA extracted from E.

granulosus worms from dogs in group A and B.

MW 1 2 3 4 5 6 7 8 9 10 11

254 bp 171 bp

89

DISCUSSION

The camel population in the Sudan exceeds three million and is

raised mainly in a belt north of 12°N latitude (Majid et al., 2001). The

most densely populated areas are Kordofan, Eastern and Darfur states,

followed by other regions in the Central and Northern provinces. The

camels are owned by migratory pastoralists as a source of milk and meat

as well as pack and riding animals. Extensive research has been

conducted to evaluate the role played by Sudanese camels for

transmission of parasitic diseases with special emphasis on diseases of

public health importance. A few epidemiological studies were carried out

to determine the prevalence of cystic echinococcosis in camels in Sudan.

The prevalence rate of the disease in camel was found to be 48.69%

(Saad and Magzoub, 1989), 56.6% (Omer et al, 2004) and (Elmahdi et al,

2004). In addition, studies were also conducted to determine the prepatent

period of the parasite in dogs using protoscolices of camel origin. Saad

and Magzoub (1988) found all of four puppies tested to be readily

susceptible to infection with E. granulosus from camel in Sudan. In

Egypt, El-Hafez Ahmed (1977) infected five 1.5-month-old puppies, of

which only three harboured intestinal stages 6 days after infection. In the

same country, three dogs were successfully infected with protoscolices of

camel origin (Aba-Yazed, 1988).The prepatent period was reported to be

ranging from 35-97 days in the definitive host ( Slepnev et al, 1977; Saad

and Magzoub, 1988). Eckert et al. (1989) reported the prepatent period of

E. granulosus in puppies infected with protoscolices of camel origin to be

40 days. However, in the present study the experimentally infected dogs

have successfully developed adult worms and the prepatent period was

found to be 52 days. This is in agreement to the study conducted in

Nyala, Western Sudan by Adam (2004) who reported the prepatent period

90

to be 54 d.p.i. This result indicated the important role played by camel for

maintenance of the life cycle of the parasite in Sudan. Despite the fact

that all experimental dogs developed adult worm, only dogs in group B

developed patent infection. In addition, very little information is available

in regard to influence of the strain of the parasite on the prepatent period.

The number of the harvested worms was extremely high compared with

the infection dose in both groups (37.5%-87.5% in group A and 62.5%-

93.8% in group B). It was suggested that protoscolices of camel strain

would probably take longer time to produce patent infection in dogs. The

molecular characterization study showed that the recovered worms were

belonging to the G6 strain (camel strain) of E. granulosus. This should be

the reason for the prolongation of the prepatent period in the experimental

dogs. To advance beyond the current knowledge of the epidemiology of

the disease, we propose that attempts should be made to conduct

comparative experimental infection with echinococcosis in dog using

protoscolices of different strains. We recognize that we had a small

sample size (n=5 for each group), but we believe that the experiment

supports the important role of the camel in the transmission cycle of

echinococcosis in Sudan.

In conclusion, the scientific data presented in this study indicated

that camel can serve as intermediate host for maintenance of the adult

worm of the parasite in dogs. In addition protoscolices of camel origin

take longer time to develop in dogs compared to other strains.

Chapter Six

91

General Discussion

Sudan is the largest country in Africa, with an area of 2.44 million

square kilometers, extending from 4˚N to 22˚N. It has a population of

about 25 million, mostly living in rural areas. Climatic condition is

diverse, with average rain fall varying from less than 25 mm in the north

to 1500 mm in the south. Sudan has the second largest animal population

in Africa. In 1995 the livestock was estimated by 103 million head, of

which 30 million were cattle, 37 million were sheep, 33 million goats and

about 3 million camels. Western Sudan has the most livestock (40%),

followed by southern Sudan (27%) and central Sudan (23%). The

majority of breeds are well adapted to harsh environment and often trek

long distances in search of feed and water. This trip is mostly

accompanied by dogs as guard animals.

In this study we tried to examine the general feature of

echinococcosis in both definitive and intermediate hosts. To determine

the epidemiological status of the disease in different intermediate host, a

survey was conducted in the period from May 2001 to July 2003 in parts

of central, western and southern Sudan. The prevalence rates in camel,

cattle, sheep and goats examined in different states of the Sudan was

found to be 59.8% (466/779), 6.1% (299/4893), 11.3% (1180/10422) and

1.9% (106/5565) respectively. The encountered number of cysts was

2387 in camel, 333 in cattle, 1514 in sheep and 108 in goats. Fertility

rates were found to be 73.7%, 77%, 19% and 31.5% in camel, cattle,

sheep and goat respectively. The favorite site for cysts in camels was the

lung (1627/2387). The liver was found to be prediliction site for cyst in

cattle (206/333) whereas the peritoneum was the predilection site in sheep

(1242/1514) and goats (53/108). Other sites of infection were the liver

and spleen.

92

From this result it is obvious that the higher prevalence rate of the

disease was encountered in camels followed by cattle and this was also

true regarding the fertility of cysts. Elkhawad et al. (1979) reported a

prevalence rate of 35.3% in camels in central Sudan. Among the 141

camels examined at Tamboul area, central Sudan, 64 (45.4%) were

harbouring hydatid cysts (Saad et al. 1983). Tola (1987) mentioned the

prevalence rates of hydatid disease in camel and cattle as 56.4% and 2.1%

respectively which were higher than that in sheep and goats. Even hydatid

cysts were reported in sheep and goats in this study, but the fertility of the

encountered cysts was very low. The prevalence rates of cystic

hydatidosis in camel and cattle in western Sudan was found to be 79.5%

and 6.4% in Nyala slaughterhouse in western Sudan (Mohamed and

Elmalik, 2000). Abattoir survey in areas in central Sudan revealed the

highest infection rate and fertility rate of hydatid cysts in camel and

cattle. The prevalence rates and fertility percentages in sheep and goats

was mentioned to be of no significance by all authors in Sudan that cysts

encountered in sheep and goats were calcified or semicalcified (Elkhawad

et al.,1979., Idris et al., 1985., Tola., 1987,Saad and Magzoub., 1989a

and 1989b., Omer et al.,. 2002 and 2004 and Elmahdi et al., 2004).

These results may lead to the suggestion that in contrast to many

parts of the Africa including parts of southern Sudan, Kenya and the

countries of Maghreb, where sheep play the most important role in the

cycle of hydatid disease (Macpherson and Wachira, 1997), camels are the

most important animals in the life cycle of E. granulosus in Sudan.

Although sheep in Kenya may be no more likely to be infected than those

in central Sudan, with prevalence of 8.1 % recorded in the sheep of

Masailand (Macpherson, 1985) and 3.6% in the sheep of Turkana district

(Njoroge et al, 2002), they are far more likely to harbour fertile cysts that

-50%-88% of Kenyan sheep cysts are fertile. The high level of fertility

93

seen in the cysts of Kenyan sheep is comparable with that seen in regions

with much higher prevalences of sheep infection, such as Tunisia

(Lahmar et al., 1999). Peru (Dueger and Gilman, 2001) and Kazakhstan

(Torgerson et al., 2003)..

The camel (Camelus dromedarius) is an important livestock

species uniquely adapted to hot and arid environments. It produces milk,

meat, wool, hair and hides, serves for riding and as a draft animal for

agriculture and short distance transport. The camel is considered a

browsing animal, but they acquire diseases which are transmitted by

contaminated food or water such as fascoliasis and schistomosis beside

echinococcosis. This may be due to the fact that camels are changing

their habitat (Majid et al. 2002).

In this study it was assumed that the prevalence rate in the human

population should be high in areas with high prevalence rates in livestock

and dogs, the disease should be particularly prevalent in rural

communities where there is close contact between dogs and livestock,

and the unhygienic conditions in some slaughterhouses should lead to

higher infection rates. Human hydatidosis in Sudan was first reported by

Christopherson (1909). Eisa et al (1962) reported the occurrence of

hydatidosis as an endemic disease amongst Taposa tribe in Kapoeta

district of the Equatoria Province in southern Sudan. It was reported that

about half of the hydatidosis patients in Uganda were immigrants from

southern Sudan (Ower and Bitakarmine , 1975). Studies about

hydatidosis conducted in southern Sudan reported a prevalence ranging

from 0.5%-3.5% in humans (Magambo et al. 1996). Data obtained about

the prevalence of cystic echinococcosis in humans considered

insufficient. This is because estimation of such data depends primarily on

the evaluation of hospital records. Unfortunately, no such records exist, in

any reliable form, in Sudan.

94

Strain characterization was conducted in a number of 542 hydatid

cyst samples obtained from different intermediate hosts (camel, cattle,

sheep, goats and humans) in different parts of the Sudan. The camel strain

of E. granulosus was detected by polymerase chain reaction and DNA

sequencing of all DNA samples extracted from hydatid cysts of camel,

cattle and goat origin. The cattle strain (E. ortleppi) was found in two

samples of cattle origin from eastern and southern Sudan. Sheep strain

(G1) was found in five DNA samples extracted from hydatid cysts of

sheep origin in central Sudan.

Camel (G6) strain was found in five DNA samples extracted from

hydatid cysts of humans whereas as the common sheep strain (G1) was

found in two samples. There is only one previous study about strain

characterization of E. granulosus in Sudan (Dinkel et al., 2004). From 46

samples obtained from camels, cattle, sheep and goats in central Sudan,

two samples of cattle origin were characterized as E. ortleppi whereas the

other samples were characterized as camel strain (G6) of E. granulosus

Hydatid cysts samples of human origin are characterized for the first time

in Sudan in this study. Camel strain (G6) was found in 71.4% (5/7) of the

surgical obtained cysts. The infectivity of this strain to human was

previously reported in sporadic cases from one case in Argentina

(Rozenzwit et al.,1999), two cases from Nepal (Zhang et al. 2000), two

samples from Mauritania (Bardonnet et al., 2001) 3 of 33 samples from

Iran (Harandi et al., 2002) and in 1 of 178 in Kenya (Dinkel et al.2004).

It is obvious that the percentage of human infection due to the camel

strain of E.granulosus in Sudan is higher than in other countries. This is

may be due to the fact that this strain is more prevalent and more fertile in

Sudanese livestock ensuring the source of infection to the definitive host.

95

The sheep strain (G1) of E. granulosus was reported for the first

time in Sudan in this study in both animals and humans. However, the

fact that 7 of 542 from Sudanese samples of human and sheep origin

belonged to this strain indicates that this strain may be one of the

genotypes which are not predominant in the country. The prevalence of

echinococcosis in stray dogs in selected area in central Sudan was

determined by examining the intestinal contents of shot dogs at necropsy

as well as by means of coprodiagnostic PCR. From the 42 dogs examined

12 (28.5%) were harboring E. granulosus worms. The worm burden was

(22-80*103) in the positive dogs. This rate was lower than that reported

by Saad and Magzoub (1986) in the same study area (central Sudan) as

they stated that 51% of the dogs were found to be infected The

prevalence rate of E. granulosus in dogs was found to be 86.5% in

Kapoeta district in southern Sudan and 6% of 33 dogs in Khartoum (Eisa

et al. 1962 and 1977). In 1985, the prevalence rate of E.granulosus in

dogs in Khartoum was reported to be 17.5% (Idris, 1985). These variable

figures about the infection rate of E.granulosus are certainly due to

particular risk behaviours in dogs like access to offal which can markedly

differ according to the area and season (Macpherson et al., 1985.,

Wachira et al., 1994).

CoproPCR was involved for the first time in Sudan in this survey,

from the 12 dogs found positive at necropsy, 10 were found positive by

PCR and the strain was characterized as camel (G6) strain. The result of

the other two samples was considered inconclusive as the DNA extracted

from the faecal samples obtained from these animals was inhibited.

Hence the sensitivity of the test is considered 100%.

From the 30 dogs which where found negative at necropsy, two

were positive using copro-PCR and the strain was characterized as sheep

(G1) strain. This result may be considered normal when compared with

96

identified strains in the intermediate host. That as mentioned before, the

camel strain was found to be the common strain in intermediate host and

hence the available strain for dog infection. Characterization of sheep

strain (G1) supported the suggestion that the sheep strain (G1) is present

but to a lesser extent in Sudan.

For the determination of the prepatent period of E. granulosus of

camel origin in experimentaly dogs, ten dogs were divided into two

groups, five dogs each. The first group (A) received a dose of

protoscolices 4×103 whereas the second one (B) received a total dose of

8×103 protoscolices. Fecal samples were examined for patent infection

during the study period. No eggs were detected in fecal samples from

group A through out the experimental period (45 days). However, eggs

were first demonstrated in faces at day 52 pi in group B which was

necropsied at day 54 pi. None of the groups showed any adverse reactions

throughout the course of the infection. Previous studies to determine the

prepatent period required by protoscolices of camel origin in

experimentally infected dogs indicated that protoscolices of camel origin

are infective to dogs. Saad and Magzoub (1988) found all of four puppies

tested to be readily susceptible to infection with E. granulosus from

camel in Sudan. In Egypt, El-Hafez Ahmed (1977) infected five 1.5-

month-old puppies, of which only three harboured intestinal stages 66

days after infection. In the same country, three dogs were successfully

infected with protoscolices of camel origin (Aba-Yazed, 1988). The

production of fully developed egg in E. granulosus of camel origin was

mentioned to be 54 days (Adam, 2004) which agree with our findings.

Some strains of E.granulosus mature earlier like E.ortleppi which

requires 35 days. E.granulosus of camel origin is distinct in various

morphological features and does not conform to any described subspecies

of E. granulosus (Eckert et al., 1989) and hence the different and

97

relatively long prepatent period mentioned in E. granulosus of camel

origin may be attributed to this difference.

E. granulosus worms harvested during this study were

characterized by means of PCR and were reported to be camel (G6). This

match well with our previous mentioned results that camel strain is the

most abundant strain of E. granulosus in definitive and intermediate hosts

in Sudan.

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