1

Study of the efficacy of Nitazoxanide, Myrrh Total Oil and

Mirazid in comparison with Praziquantel in experimental

Schistosomiasis mansoni

Thesis

Submitted to the Medical Research Institute

University of Alexandria

In Partial Fulfillments of the

Requirements for the degree of

Master of Science

In

Applied & Molecular Parasitology

By

Mohammad Aziz Nawar Al-Kazzaz

Bachelor of Veterinary Medical Sciences

Faculty of Veterinary Medicine, University of Cairo, 1997

2014

2

Study of the efficacy of Nitazoxanide, Myrrh Total Oil and Mirazid in

comparison with Praziquantel in experimental Schistosomiasis mansoni

Prepared by

Mohammad Aziz Nawar Al-Kazzaz

Bachelor of Veterinary Medical Sciences

Faculty of Veterinary Medicine, University of Cairo, 1997

For the degree of

Master of Science in Applied & Molecular Parasitology

Examiner’s Committee Approved

Prof. Dr. Mona Hassan El-Sayad

Professor, Department of Parasitology

Medical Research Institute

University of Alexandria

Prof. Dr. Sanaa Ahmed El-Masry

Professor, Department of Tropical Health

High Institute of Public Health

University of Alexandria

Prof. Dr. Mostafa Abo El-hoda Mohamed

Professor, Department of Parasitology

Medical Research Institute

University of Alexandria

Assist. Prof. Dr. Hend Ali El-Taweel

Assistant Professor, Department of Parasitology

Medical Research Institute

University of Alexandria

Date 15 / 10 / 2014

3

SUPERVISORS

Prof. Dr. Mona Hassan El-Sayad

Professor, Department of Parasitology

Medical Research Institute

University of Alexandria

Assist. Prof. Dr. Hend Ali El-Taweel

Assistant Professor, Department of Parasitology

Medical Research Institute

University of Alexandria

Assist. Prof. Dr. Sahar Ahmed Abu-Helw

Assistant Professor, Department of Parasitology

Medical Research Institute

University of Alexandria

4

انرحيى انرح هللا بسى :قال هللا تبارك وتعايل

} رفع درجبت ي شبء وفىق كم ري عهى عهيى{ ( ٦٧قران كرمي )سورة يوسف اية

5

Dedication

TO

The spirit of my father

My mother

My brothers & sisters

My wife

6

Acknowledgement

First of all, I wish to thank God, the most gracious, the most merciful for helping me to complete this work. I would like to express my sincere thanks to Professor Dr. Mona Hassan El-Sayad, professor of Parasitology, Medical Research Institute (MRI), University of Alexandria for her precious advice, valuable guidance and great help to complete this work. My deepest gratitude to Dr.Hend Ali El-Taweel, assistant professor of Parasitology, Medical Research Institute, University of Alexandria for her keen supervision , constructive guidance and unlimited cooperation to complete this work. Many thanks to Dr.Sahar Ahmed Abou-Helw , assistant professor of Parasitology, Medical Research Institute ,University of Alexandria for her continuous advice and encouragement throughout this work. I want to express my great sympathy to Dr.Mostafa Yakoot, the Medical Director of Pharco Corporation for his support in the practical part of this work. Also I want to express my great appreciation to all members in the Department of Parasitology, Medical Research Institute,University of Alexandria who paid a lot of efforts in the practical part of this work. Last but not least, I warmly thank with sincere gratitude my family for their endless support, care and continuous encouragement throughout this work.

7

LIST OF CONTENTS

Chapter Page

LIST OF CONTENTS……………………………………….…………..........................................I

LIST OF TABLES………………………………………………………………………………....II

LIST OF FIGURES………………...………………………………..……………………...….....III

LIST OF ABBREVIATIONS……………….……………………………………………...........VII

I. INTRODUCTION……………………………………………………......................….…....1

II. AIM OF THE WORK………………………………………...…………...............................19

III. MATERIALS AND METHODS……………………………...………....……………….....20

IV. RESULTS…………………………………………………..……………………………......32

V. DISCUSSION………………………………………………….……………………..….......71

VI. SUMMARY AND CONCLUSION………………………………………………..……......89

VII. RECOMMENDATIONS…………………………………....................................................93

VIII. REFERENCES……………………………………………….………..................................94

IX. PROTOCOL

X. ARABIC SUMMARY

8

LIST OF TABLES

Table (I) Egg counts in the stool of S. mansoni-infected mice under different

treatments compared to non-treated mice.

33

Table (II) Egg counts in stool of different groups of S. mansoni-infected mice under

different treatments compared to non-treated infected mice.

34

Table (III) Worm burden in S.mansoni-infected mice treated and non-treated groups

by time (weeks).

35

Table (IV) Percentage of change in male and female worm distribution in different

S.mansoni-treated mice groups in different periods of follow up.

37

Table (V) Body length of S.mansoni worms recovered from different treatments

compared to non-treated mice at different follow up periods.

39

Table (VI) The tissue egg count in the liver and intestine of S. mansoni-infected

mice under different treatments at different periods of follow up.

41

Table (VII) The oogram pattern (percentage egg developmental stages) in the

intestine of S. mansoni-infected mice under different treatments in

different follow up periods.

44

Table (VIII) Erythrocytes and their related red blood cell indices in S. mansoni-

infected mice under different treatments at different follow up periods.

53

Table (IX) Total and Differential Leucocytic Counts in S. mansoni-infected mice

under different treatments at different follow up periods.

59

Table (X) Platelet counts in S.mansoni-infected mice under different treatments at

different follow up periods.

63

Table (XI) Liver function tests in S. mansoni-infected mice treated with different

drugs at different times.

65

Table (XII) Kidney function tests in S. mansoni-infected mice under different

treatments at different follow up periods.

68

Table (XIII) Blood Acetylcholinesterase level in S.mansoni-infected mice under

different treatments at different periods of follow up.

70

9

LIST OF FIGURES

Figure.1 Structural formula of praziquantel 11

Figure.2 Structural formula of Nitazoxanide 16

Figure.3 Steps of mice infection with S.mansoni cercariae 22

Figure.4 Perfusion pump machine 25

Figure.5 Mice perfusion 25

Figure.6 Measurement of female S. mansoni body length under dissecting

microscope with ordinary ruller.

26

Figure.7 Egg developmental stages 28

Figure.8 Blood collection from a mouse 30

Figure.9 Egg counts in stool of different groups of S. mansoni-infected mice

under different treatments compared to non-treated infected mice.

33

Figure.10 Percentage faecal egg count reduction in different groups of S. mansoni-

infected mice under different treatments at different periods of follow

up.

34

Figure.11 Percentage reduction in the mean total worm burden in different groups

under different treatments at different periods of follow up.

36

Figure.12 Percentage reductions in female worm burden in S.mansoni-infected

mice under different treatments at 1, 2 and 4 WPT.

38

Figure.13 Percentage reductions in male worm burden in S.mansoni-infected mice

under different treatments at 1, 2 and 4 WPT.

38

Figure.14 Percentage reductions of the body length of male S. mansoni worms

recovered from different treated groups at 1, 2 and 4 WPT.

40

Figure.15 Percentage reduction of the body length of female S.mansoni worms

recovered from different treated groups at 1, 2 and 4 WPT.

40

Figure.16 Percentage reduction in the mean hepatic egg counts in S. mansoni-

infected mice under different treatments at 1, 2 and 4 WPT.

42

Figure.17 Percentage reduction in the mean intestinal egg counts in S.mansoni-

infected mice under different treatments at 1, 2 and 4 WPT.

42

Figure.18 Percentage egg developmental changes in S.mansoni-infected mice

under different treatments at different periods of follow up.

45

Figure.19 Scanning electron micrographs of S.mansoni worms recovered from 47

10

infected non-treated mice showing normal tegument of male (A) and

female worms (B). Normal ventral sucker of male worms (C) and oral

sucker of female worms (D).The inner surface of the gynecophoric

canal of male (E) worms.

Figure.20 Effect of Praziquantel on the dorsal surface of female schistosoma

worms (F) and the male worms (G) recovered at 2 WPT.

48

Figure.21 Effect of Mirazid on dorsal aspects of the tegument of female (H), male

(I) S.mansoni worms and ventral sucker of male worms (J) recovered 2

WPT.

49

Figure.22 Scanning electron micrographs of S.mansoni worms recovered from

NTZ-treated mice showing normal tegument of female worms (K), the

tegument of male worms (L) ,the oral sucker of male worms (M), the

worm couple (N) and the gynecophoric canal of the male (O).

50

Figure.23 Scanning electron micrographs of the dorsal surface (P), oral and ventral

suckers (Q) of male S.msnsoni worms recovered from MTO-treated

mice at 2 WPT.

51

Figure.24 Mean RBCs counts in S.mansoni-infected mice under different

treatments at different follow up periods.

54

Figure.25 Mean Haemoglobin levels in the blood of S. mansoni-infected mice

under different treatments at different follow up periods.

54

Figure.26 Mean Packed cell volumes in the blood of S.mansoni-infected mice

under different treatments at different follow up periods.

55

Figure.27 Mean MCV of the RBCs in S.mansoni-infected mice under different

treatments at different follow up periods.

55

Figure.28 Mean MCH in S. mansoni-infected mice under different treatments at

different follow up periods.

56

Figure.29 Mean MCHC in S.mansoni-infected mice under different treatments at

different follow up periods.

56

Figure.30 Mean total leucocytic counts (TLC) in S.mansoni-infected mice under

different treatments at different follow up periods.

60

Figure.31 Mean Lymphocyte counts in S.mansoni-infected mice under different

treatments at different follow up periods.

60

Figure.32 Mean Neutrophils counts in S.mansoni-infected mice under different 61

11

treatments at different follow up periods.

Figure.33 . Mean Esinophils counts in S.mansoni-infected mice under different

treatments at different follow up periods.

61

Figure.34 Mean Monocytes counts in S.mansoni-infected mice under different

treatments at different follow up periods.

62

Figure.35 Mean Basophils counts in S.mansoni-infected mice under different

treatments at different follow up periods.

62

Figure.36 Mean Platelet counts in S.mansoni-infected mice under different

treatments at different follow up periods.

63

Figure.37 Liver functions tests {ALT (A), AST (B), ALP(C)} activity in

S.mansoni-infected mice under different treatments at different follow

up periods.

66

Figure.38 Kidney functions {Blood urea (A) and Serum creatinine (B)} in

S.mansoni-infected mice under different treatments at different follow

up periods.

69

Figure.39 Mean blood acetylcholinesterase levels in S.mansoni-infected mice

under different treatments at different periods of follow up.

70

12

LIST OF ABBREVIATIONS

AChE:

ALP:

ALT:

AST:

CBC:

CNS:

DALYs:

DLC:

ECG:

EDA:

ELISA:

EMR:

EPG:

FDA:

GMEC:

HB:

HCT:

HCV:

IC:

IHA:

KOH:

LC:

LD50:

MCH:

MCHC:

MCV:

MEO:

MOHP:

MRI:

MTO:

MVO:

Acetylcholinesterase Activity

Alkaline Phosphatase

Alanine Aminotransaminase

Aspartate Aminotransaminase

Complete Blood Count

Central Nervous System

Disability Adjusted Life Years

Differential Leucocytic Count

Electrocardiography

Egyptian Drug Authority

Enzyme-Linked Immunosorbent-Assay

Eastern Mediterranean Region

Egg per gram

Food And Drug Administration

Geometric Mean Egg Count

Haemoglobin

Haematocrit Value

Hepatitis C Virus

Inhibition Concentration

Indirect Haemagglutination

Potassium Hydroxide

Lethal Concentration

Lethal Dose 50

Mean Corpuscular Haemoglobin

Mean Corpuscular Haemoglobin Concentration

Mean Corpuscular Volume

Myrrh Essential Oil

Ministry of Health and Population

Medical Research Institute

Myrrh Total Oil

Myrrh Volatile Oil

13

MZD:

NTZ:

PCR:

PPM:

PT:

PZQ:

RBCs:

S/C :

SBSC:

SCE

SD:

SEAs:

SEM:

TB:

TBRI:

TGR:

US:

WHO:

WPI :

WPT:

µl:

μg :

Mirazid

Nitazoxanide

Polymerase Chain Reaction

Part Per Million

Post-Treatment

Praziquantel

Red Blood Cells

Subcutaneous

Schistosome Biologic Supply Center

Serum cholinesterase

Standard Deviation

Soluble Egg Antigens

Scanning Electron Microscopy

Tuberculosis

Theodore Bilharz Research Institute

Thioredoxin-Glutathione Reductase

United States

World Health Organization

Weeks Post-Infection

Weeks Post-Treatment

Microliter

Microgram

14

INTRODUCTION

15

INTRODUCTION

Schistosomiasis is a parasitic disease caused by the digenetic trematodes of the genus

Schistosoma (commonly known as blood flukes) (1)

. The disease is one of ten tropical diseases

especially targeted for prevention and control by the special programs for research and training in

tropical diseases of the United Nations development program, the World Bank and the World

Health Organization (WHO). It also represents one of the major communicable diseases of public

health and socio-economic importance in the Eastern Mediterranean Region (EMR) (2)

.

Schistosomiasis ranked second only to malaria and is the most important parasitic disease in terms

of prevalence, morbidity and mortality rates especially in rural areas of developing countries (3)

.

TAXONOMY OF SCHISTOSOMES:

Kingdom: Animalia

Phylum: Platyhelminthes

Class: Trematoda

Subclass: Digenea

Order: Strigeidida

Family: Schistosomatidae

Subfamily: Schistosomatinae

Genus: Schistosoma {Schisto= cleft & soma = body} (Weinland, 1858) (4)

.

Genus Schistosoma: There are 23 identified species of Schistosoma infecting man, mammals and

birds (5)

.

Human Schistosomes :

S. mansoni, S. hematobium and S. japonicum are the most important species from the

medical point of view that can infect humans (6)

. S. mansoni is found in Africa, South America,

Caribbean and Middle-East. Fresh water snails of the Biomphalaria are an important intermediate

host for this trematode. Among final hosts, humans are most important. S. haematobium,

commonly referred to as the bladder fluke, originally found in Africa, the Near East, and the

Mediterranean basin, Freshwater snails of the Bulinus are an important intermediate host for this

parasite. S. japonicum is found widely spread in Eastern Asia and the Southwestern Pacific

region. Fresh water snails of the Oncomelania are an important intermediate host for S.

japonicum. S. mekongi and S. intercalatum are considered human blood flukes of minor

importance from the medical point of view (7)

.

16

LIFE CYCLE AND BIOLOGY OF SCHISTOSOMA MANSONI:

Schistosomes are characterized by a complex life cycle involving two phases; (1) Sexual

phase in which sexual reproduction by adult worms occur in humans (definitive host), (2)

Asexual phase in specific aquatic snails (intermediate host, Biomphalaria species). Schistosomes

develop through successive stages: egg, miracidium, sporocyst, cercaria, schistosomula and adult.

S. mansoni eggs are oval with lateral spine. Each fertilized female worm releases many eggs each

day. The eggs of S. mansoni are released singly and may remain alive up to 3 weeks after

oviposition. It contains a single miracidium. Hatching and survival of the miracidia are dependent

on fresh water contact at a temperature between 200C-30

0C. In optimal conditions; miracidia will

survive for 5-6 hours (7)

.

When S. mansoni eggs reach fresh water, usually with faeces, they hatch and release tiny

miracidia. Although miracidia of schistosome do not have eye spots, they apparently have

photoreceptors and they are positively phototropic, they also display negative geotaxis and

possess chemotactic factors. At water flow rate of about 700 cm/ minute, they are stimulated to

swim more rapidly and change direction much more frequently, thus increasing their chances of

encountering the specific snail (Biomphalaria alexandrina) and attach to its soft part. Lytic

substances secreted from miracidial glands aid penetration. After penetration of a snail, the

miracidia lose their cilia, and become non-motile sac which metamorphoses into two generations

of sporocysts. The latter migrate to the digestive gland of the snail after about two weeks. The

mother sporocyt continues producing daughter sporocysts for up to 6-7 weeks. The daughter

sporocysts migrate to and grow in the hepatic and gonadal tissue of the snail. Sporocysts mature

into hundreds of infective larval forms of the parasite (cercariae) (7)

.

Cercariae start leaving the snail 4 to 6 weeks post-infection. They migrate through the

vascular sinuses and exit from the edge of the snail’s mantle. Cercariae are unisexual, fork-tailed,

free swimming and measure 400-600 µm in length. Cercariae may survive in fresh water up to 48-

72 hours but gradually begin to lose infectivity after 12 hours. Their activity in water alternates

between active movement towards the surface and slow sinking towards the bottom. A snail

infected by one miracidium can shed thousands of cercariae every day for months. Infection

occurs when humans come into contact with fresh water containing cercariae. Cercariae attach

themselves to the skin by their ventral and oral suckers assisted by mucoid secretions from the

postacetabular glands, they penetrate the skin. Following penetration, cercariae transform into

schistosomulae and develop a double-lipid bilayer tegument that helps in protecting the worm

from immune attack. Schistosomula secrete lytic enzymes and migrate through the dermis in

search for a vein, then travel through the blood stream within several days (7, 8)

.

17

The worms migrate along the pulmonary capillaries to enter the left side of the heart and

systemic circulation. Schistosomules are carried with the arterial blood flow through the aorta to

the mesenteric arteries, splanchnic capillaries and portal veins to reach the liver. The

schistosomules transversing the skin and pulmonary capillaries are the parasite stage most

susceptible to immune attack by the host. Only about 40 % of cercariae that penetrate the skin

eventually become viable adult worms. Survival is inversely related to the host-acquired

immunity to schistosomes (7)

. The worms mature within 4-6 weeks in the portal circulation .They

differentiate into male and female worms, mate in the small vasculature of the liver and migrate to

the inferior mesenteric veins of large intestine (draining intestines) against the blood flow.

Oviposition commences 4-7 weeks post infection and female worms produce 100-300 eggs per

day (8)

.

EPIDEMIOLOGY OF SCHISTOSOMIASIS MANSONI:

The epidemiology of schistosome parasites is based on their complex life cycle. The elegant

adaptational skill that allows these organisms to parasitize snails and humans also restricts their

geographic distribution.

Geographic distribution and global burden:

Schistosomiasis transmission has been documented in 78 countries. However those

requiring treatment targeted at most at-risk population groups live in 52 countries (9)

. S.mansoni is

present in 8 Eastern Mediterrean Region (EMR) countries including Egypt, Libya, Sudan, KSA,

Oman, Yemen, Djibouti and Somalia. During the past 20 years, schistosomiasis was eliminated in

Iran, Morocco, Lebanon and Tunisia (2)

.The geographical distribution of the different schistosome

species depends mainly on the ecology of their snail intermediate hosts (7)

. It was revealed that the

global burden of schistosomiasis and its consequences had been underestimated (10)

. This

underestimation of burden is attributed to multiple factors, including the chronic and

asymptomatic nature of most infections, non-specificity of some signs and symptoms, and low

sensitivity of parasitological diagnosis (10,11)

.

Impact of Schistosomiasis on Human Health:

In 2005, the weight of evidence from a meta-analysis of 135 interventional and

observational studies indicated that human schistosomiasis is significantly associated with chronic

symptoms of pain, diarrhea, fatigue, anaemia, impaired growth and exercise intolerance. These

frequently unacknowledged disease outcomes were substantially more prevalent than the

advanced ‘classic’ schistosomiasis-related disease outcomes, such as liver fibrosis, portal

hypertension, hepatosplenomegaly, or urinary tract obstruction. Although the former, more subtle

18

outcomes are less visible in their clinical presentation, they may actually represent the greatest

part of chronic disease burden associated with schistosomiasis (10,11)

.

Schistosomiasis negatively impacts on school performance in children due to long-term

developmental and cognitive effects as well as social and economic developments in heavily

affected areas (3, 8)

.

The concept of Disability Adjusted Life Years (DALYs) was introduced by Murray and

Lopez (1996) "to assess and refine estimates of the global burden of diseases". DALY is a

population health metric that combines the years lost from premature death and the years of life

lived with disability.It can be thought of as one lost year of healthy life. This index is calculated

from disease-specific prevalence, mortality, and disability weights of a certain disease (12)

. The

report of the WHO Expert Committee (2002) (13)

on the prevention and control of schistosomiasis

estimated 1.7 million DALYs were present. The burden of disease assigned to schistosomiasis-

associated disability was estimated to be 0.5%. However, a subsequent meta-analysis re-assessing

the chronic disease with a more robust measure of morbidity determined a schistosomiasis-

associated disability of about 2-15%(11)

.

There is a consensus that schistosomiasis-specific mortality occurs only in a small

percentage of individuals who develop a chronic disease. However, on revising the global burden

of schistosomiasis ; there were about 280,000 deaths per year was estimated in sub-Saharan

Africa alone where 150,000 per year due to non-functioning kidney from S.hematobium and

130,000 per year due to hematemesis from S.mansoni were detected(10)

.

Prevalence of Schistosomiasis and Human Host Factors:

Although schistosomiasis is highly prevalent, the associated morbidity is often variable

according to :

1-Age: No age is exempted from bilharziasis but higher disease rates among age groups from 15-

70 years(22.7%) followed by children in those between 5-14 years old(19.6%) (14)

.

2-Occupation: Schistosomiasis is considered an occupational disease related to water contact of

farmers or fishermen and also an environmental hazard. Agricultural workers and their families in

endemic areas that have continuous exposure to schistosoma-infested water through farming

,washing, bathing, and water recreation have great difficulty and perhaps no practical means of

remaining free of recurrent infection(8)

.

3-Socioeconomic level: Watts (2005) (15)

reported that the majority of schistosomiasis cases are

prevalent among poor people in Sub-Saharan Africa who lack access to health services, safe

water, sanitation, and education. Furthermore, the disease helps keep them poor by lessening their

ability to work, learn, and contribute to their communities. In Egypt ,the same findings were

19

noticed for the first time by Farooq et al (1966) (16)

and also by EL-Koby et al.,(2000)(17)

as lower

socioeconomic status among those who live in rural areas, who are more likely to be employed in

agriculture and have less convenient access to medical care (and treatment for schistosomiasis).

4-Sex: Males are more infected than females with schistosomiasis .This may be due to

occupational exposure to infected water canals in agriculture or by swimming (14, 17)

.

5-Education: It has been found to impact health-seeking behaviour, which may have an effect

on prevalence of infection (18)

. It also provides the impetus behind the success of deworming

programmes, preventing the contamination of the environment, and hence transmission (19)

.

Health education implies a long-term commitment and should ideally be integrated in the general

education system (20)

.

6-Hygienic measures or sanitation: The fundamental reason for the transmission of

schistosomiasis is the low level of sanitation in endemic areas, with the result that fecal material

containing viable schistosome eggs reach natural water bodies infested with fresh water snails

susceptible to infection (21)

. So provision of clean water supplies reduces exposure to cercariae and

sanitary disposal of excreta reduces the succession of the life cycle by supplying indoor water and

toilets. Communities with improved living standards were more likely to have satisfactory results

in eradication of schistosomiasis (7)

.

Relation of schistosomiasis to the environment:

Perennial irrigation is the modern system for irrigation in Egypt which ensures a water

supply all the year round, an abundant and an unbroken succession of crops but conversion from

basin to perennial irrigation resulted in an increase in the prevelance and intensity of schistosomal

infection due to flushing of snails. Both environmental changes that result from the development

of water resources and the growth or migration of populations can facilitate the spread of

schistosomiasis .The presence of Aswan high Dam in Egypt has led to the virtual elimination of S.

haematobium from the Nile Delta but has brought about the establishment of S. mansoni in upper-

Egypt (17)

.

Reservoir Hosts of Schistosomiasis mansoni:

S.mansoni infections have been found in rodents, baboons and insectivores in Africa and

South America which may constitute a health hazard as they may act as carriers after elimination

of human infection (7)

.

Intermediate Hosts of Schistosoma mansoni:

Endemic human schistosomiasis is ecologically most dependent on the presence of the snail

intermediate host and the deposition of human and reservoir host excreta into warm fresh water

habitat. Biomphalaria snails belong to the family Planorbidae, class Gastropoda. In Africa and the

20

Middle East; are divided into four species groups: B. alexandrina, B. pfeifferi, B. choanomphala,

and B.sudanica which act as the intermediate host for S.mansoni (7)

. B. alexandrina has

historically been implicated in the transmission of S. mansoni in Egypt (21)

. These fresh water

snails are characterized by their disk or lens-shaped shells, non-operculated, hermaphrodite,

vascularized mantle and haemocael. These snails live in lightly shaded, slow-flowing (15

m/minutes), shallow (less than 2m) waters (7)

.

Current Status of Schistosomiasis in Egypt:

According to the report of Schistosomiasis Working Group (2005) (22)

on schistosomiasis in

Egypt, it has been reported that S. hematobium is prevalent in the Upper Egypt governorates while

S.mansoni is prevalent in the Nile Delta governorates; By the end of 2004, both infections had

been greatly reduced to rates below 2 .WHO report (2007)(2)

cleared that the prevalence of S.

hematobium decreased to 1.2 % and S. mansoni to 1.5 % in Egypt. Fenwick (2011) reported

much decline in the prevalence for both S.mansoni and S. hematobium allover Egypt to less than

0.5 % in the year 2010(23)

. With the concept that schistosomiasis is present in high prevalence

rates in hot spots ; Khalil (2013)(24)

found among 100 school children in a village in Kafr El-

Sheikh Governorate; that the overall prevalence was 16% by percoll or 12 % by kato-katz

technique. Taman et al., (2014) (25)

found 26.6% overall prevalence rate among fishermen in Al-

Manzala lake.

Prevalence of Schistosomiasis mansoni and its Status in Alexandria, Egypt:

In the study of Abou-Basha et al., (2000) (14)

in Abis I village, the overall prevalence rate of

S.mansoni was 19.1 %. Hussein et al.,(2000)(26)

found that the prevalence of S. mansoni infection

in Abis 7 and 8 was 24.2 %, 37.8 % respectively and concluded that drinking water supply,

sanitary sewage disposal and proper disposal of animal wastes are still deficient in some houses of

the two villages.The prevalence of S.mansoni in a surveyed community (El-prince Village, EL-

Montazaa district, Alexandria Governorate) was found to be 15.4 % in the year 2002 while it was

78.4 % in 1985 and decreased to 24 % in the following year after chemotherapy with

praziquantel(27)

. Zaki et al., (2003) carried out a study in Abis 4 villages where the prevalence of

S.mansoni was 20.5 % being lower among females and children below 5 years. S.haematobium

was absent from urine samples (28)

.

Allam et al., (2009) (29)

examined stool samples of school-children in Abis 4 and Abis 8

villages and reported that the overall prevalence of S.mansoni in the 2 villages was 5.72%.The

Health Administrative Authority of the Egyptian Ministry of Health and population (MOHP) in

Alexandria Governorate performed a survey on 3782 school-children in the period from March to

April 2009 in different districts of the governorate by examining random stool samples, the study

21

revealed that overall prevalence of S.mansoni infection in Alexandria governorate was 1.3 % in

spite of the very low or nill infection rate in Biomphalaria snails in this period of the year .Hot

spots in some rural areas of Alexandria are present ,with prevalence of schistosomiasis mansoni

ranged from 1.5-7%. Even after mass treatment with PZQ, e.g.in Abis 8 villages, the prevalence

was still about 3.5 % (30)

. Hassan (2013) surveyed 420 children in Abis 8 village and reported that

the overall prevalence of S. mansoni by the kato-katz was 2.13% with GMEC of 16 epg among

infected persons with more light infection and none of them showed heavy intensity, while by

serologic tests ; prevalence was 5.7% by IHA and 21.9% by ELISA test (31)

.

ANIMAL MODELS OF SCHISTOSOMIASIS MANSONI:

Experimental S.mansoni infection of laboratory animals has frequently been used to study

the anatomical, pathological and physiological features of the infection in humans as well as for

the study of immunity and chemotherapy (32)

. The complex nature of the schistosome parasite and

its interaction with the mammalian host necessitate the continued use of live intact animal models

in schistosomiasis research (33)

. Schistosome infections in experimental animals are less complex,

or at least more readily studied, than infections in humans (34)

.

A variety of animal models have been used in schistosomiasis research as mice, rat,

hamster, rabbit, chimpanzee and baboons. These hosts may be classified into two types according

to their susceptibility to schistosomal infection into permissive or non- permissive. Permissive

hosts are those animal hosts in which schistosome parasites can reach maturity as mice and

hamsters which are among the most susceptible host species while rats are known to be non-

permissive hosts (35)

.

Although the rat is a non-permissive host for schistosomiasis, it has been extensively

studied as it provides an immunological model for successful parasite immune-mediated rejection

studies (36)

. Schistosomes do not reach sexual maturity in the rat, being spontaneously eliminated

in the third week following infection. This schistosome attrition is immune-mediated and the

antibody dependent cell-mediated cytotoxicity mechanism plays a major role (37)

.The laboratory

rat (Rattus norvegicus) is considered a semipermissive host in that the majority of worms are

removed before reaching maturity in the portal tract in a self-cure around day 28. Although the

schistosome larvae are faster in this host than in the mouse, only 25% to 30% reach the liver, and

IgE has been directly implicated in this phenomenon (37, 38)

. On the other hand, the black rat

(Rattus rattus), which is a natural host for S. mansoni, is considered a fully permissive host (39)

.

Cioli et al. (1977) studied the survival, growth, and egg laying capacity of S.mansoni worms

surgically transplanted from mice into rats or from rats into hamsters. They found that in the rat,

22

worms were stunted, localized in the liver, and laying nonfertile eggs in small numbers. When

transferred to the hamster, they increased in size approaching normal hamster-grown worms

within 3 weeks following transplantation, were localized in the mesenteric veins, and produced

large numbers of eggs. Conversely, when adult mouse worms were injected into rats, they

regressed in size, remained in the liver, and produced small numbers of incompletely developed

eggs (40)

.

The baboon is the most frequently used non-human primate in schistosomiasis research

because of a multiplicity of qualities that make them more relevant models than rodents (41)

.

Baboons maintain natural infections in the wild (7)

and are highly susceptible to experimental

infections (42)

. They are a good model for vaccine efficacy studies but constraints limiting the use

of baboons in schistosomiasis research include the high costs involved in trapping and

maintaining monkeys in captivity. There may also be variation in data obtained from wild-caught

baboons due to their heterogeneous genetic background. In addition, some specific immunological

reagents suitable for baboon work may not be currently available (33)

.

Murine schistosomiasis has been the most studied experimental model in many aspects of

the disease as the progress of schistosomiasis in mice is approximately similar to that in humans.

Mice have tended to be the animals of choice because of their easy availability, high fertility and

susceptibility to experimental infection (43)

. Female mice are more susceptible to infection with

S.mansoni cercariae with higher mortality rate (80%) than male as fewer worms develop in male

than in female when exposed to the same number of cercariae indicating that schistosomula are

more successful in developing into adult worms in female mice (43,44)

.

PATHOLOGICAL ASPECTS OF SCHISTOSOMIASIS MANSONI :

The main immunopathlogy of the disease is the granulomatous inflammatory and

fibrosing reaction against tissue-trapped parasite eggs in the liver and intestine or other tissues (45)

.

Granuloma formation is a manifestation of cell-mediated, delayed-type hypersensitivity to soluble

egg antigens (SEAs) released by eggs that peak at the eighth week post-infection (46)

. Granuloma

formation is beneficial for the host because it blocks the hepatotoxic effects of the antigens

released from parasite eggs. However, this process may lead to fibrosis with excessive

accumulation of collagen and other extracellular matrix proteins in the periportal space (46, 47)

. Egg

granulomas activate antigen-specific CD4+T-helper cells, i.e.,Th-1 and Th-2, inducing the release

of specific immunomodulating antifibrogenic and fibrogenic cytokines (46, 48)

.

Acute pathology: Acute schistosomiasis occurs in immunologically naive, previously uninfected

people, such as immigrants.

It is a toxemic disease characterized by hyper-reactivity to

23

schistosome worm and egg antigens. It is usually seen as an acute febrile illness three to four

weeks after exposure coincident with the oviposition onset. The intestinal mucosa becomes

edematous and hyperemic with small hemorrhages, early granulomas as well as shallow ulcers.

The anatomic features of the acute disease in humans include a massive dissemination of

granulomas around the eggs, especially in the liver, lung, pancreas and lymph nodes (49)

.

Chronic pathology: Infection with S. mansoni results in a relatively tolerable chronic disease

(most chronically infected individuals have few or no symptoms), however, 5-10% of patients

suffer from a severe form that leads to severe hepatic fibrosis, portal hypertension, ascites, portal

systemic shunting, gastrointestinal haemorrhage and death(45,48)

.Chronic schistosomiasis mainly

affects people born and residing in endemic areas (45)

. In heavy infections, about 50% of the eggs

are trapped in the mucosa and submucosa of the colon, resulting in colonic polyposis with the

formation of pseudotubercles, granulomas and pseudopapillomas. Although chronic liver disease

develops in only 4-8% of individuals with schistosomiasis; hepatic schistosomiasis is one of the

leading causes of liver disease internationally (12)

. The granulomatous inflammation due to the

sustained chronic infection and ongoing immune responses may cause anemias of chronic

inflammation and iron-deficiency, caloric undernutrition, growth stunting (11)

.

Several studies

have suggested that chronic S.mansoni infection can increase the susceptibility to and progression

of many diseases (50-53)

. Chronic infection can increase the susceptibility to frequent falciparum

malaria attacks among children. The incidence rate of malaria has been found to be higher in those

with concomitant S. mansoni infection (51)

. On the other hand, there has been found an association

between the progression of active TB among those infected with HIV-1 and co-infected with S.

mansoni (52)

. Similarly, a significant increase has been reported in the progression rate of HCV-

mediated fibrosis in patients co-infected with schistosomiasis (52, 53)

.

Complications: Hepatosplenic complications are the most serious and life-threatening

consequences of schistosomiasis mansoni (54)

. Typically, schistosomiasis mansoni is the most

common cause of portal hypertension worldwide (55)

. Esophageal varices subsequent to portal

hypertension as a consequence of extended periportal fibrosis is the major cause of morbidity and

mortality associated with the disease (13,56)

. In schistosomiasis, fibrosis is restricted to the portal

area, with preservation of the lobular architecture of the liver where the macroscopic appearance

may show large fibrous septa, referred to as the Symmers' clay "pipestem" fibrosis (57)

. In S.

mansoni infection, fibrosis develops over five to fifteen years in comparison to S. japonicum

infection, in which it may progress more rapidly. Besides being developed in a small percentage

of infected people, the incidence of periportal fibrosis has been correlated with the age and gender

24

of the patient as well as the intensity of infection. It is more common among males than females,

and increases with age (58)

.

CHEMOTHERAPY OF SCHISTOSOMIASIS:

General overview:

Schistosomiasis control can be achieved through health education, sanitation, snail control,

immunization and chemotherapy. The chemotherapy of schistosomiasis is considered the most

effective tool for control of schistosomal morbidity in human (59)

. These chemotherapeutic drugs

had been developed and categorized into old drugs and new ones ,The old drugs could be

classified into two groups, Antimonial (Tarter emetic, Fouadin, Astiban, Anthiomaline) and Non-

antimonial compounds (Leucanthone, Hycanthone, Niridazole, Oltipraz)(60)

. The toxicity and

repeated intravenous injections of antimonials were a major limitation for considering them as a

treatment option, especially for mass therapy (61)

. The non-antimonials were abandoned because of

their toxicity to the liver, kidney, heart and carcinogenicity(62)

.

The new antischistosomal drugs include Metrifonate, Oxamniquine and Praziquantel .With

the advent of these drugs, they could be administered in a single oral dose, with good therapeutic

activity and less intense side effects than old antischistosomal drugs, it was possible to initiate

control programs in various endemic areas(61)

. Metrifonate, an organophosphorous drug which

was used firstly as an insecticide in the early 1960s, exhibits activity against S.haematobium only

by inhibition of cholinesterase of the worm (63)

. Oxamniquine is effective only against S. mansoni.

The drug was used on a large scale only in Brazil (64, 65)

. Following a half-century search for an

effective antischistosomal drug, the development of PZQ in the mid-1970s and its wide use since

the 1980s was essential feature for the great reductions in morbidity and mortality due to

schistosomiasis(63,65)

. A lot of novel therapeutic approaches under research performed to discover

new schistosomicidal agents either chemically designed (e.g., praziquantel derivatives) or

naturally (e.g., artemisinin and myrrh derivatives) (65-67)

.

The Current Antischistosomal Therapy of Schistosomiasis Mansoni:

Praziquantel is available all-over the world since 1980 in the form of 600 mg tablets (65)

and

Mirazid is present only in Egypt since 2002 as an alternative to PZQ in the form soft gelatin

capsules containing 300 mg oleoresin extract of Commiphora molmol or myrrh .

25

1-Praziquantel (PZQ):

Figure.1. Structural formula of praziquantel (C19H24N2O2) (63)

.

The antiparasitic activity of PZQ was observed in the early 1970s at the laboratories of Bayer and

E.Merck, Germany, when a large series of pyrazino-isoquinoline compounds were synthesized as

potential tranquilizers (68)

.

Antischistosomal Properties of PZQ:

In experimental animals, the therapeutic dose of PZQ depends mainly on the host species.

The dose ranges from 200 to 1000 mg/kg body weight for mice and from 100 to 500 mg/kg body

weight for hamsters (69-71)

. Experimental studies have shown that the activity of PZQ is stage-

dependent. Immature (2-4 weeks old) worms are less susceptible to PZQ than larval stage (1-2

weeks old) or adult (5 weeks old or older) worms. Hence, doses of drug that are curative against

larval or mature adult infections are sub-curative against developing worms (72)

. In man, several

regimens of PZQ treatment have been reported for the different species of schistosomes. The

standard dose of PZQ safely used for mass treatment leading to a decrease in the prevalence of

schistosomiasis mansoni is a single oral dose of 40 mg/kg body weight of PZQ (73)

. Higher doses

(60 mg/kg) (74,75)

but without significant efficacy advantage over the standard dose (75)

.

By 1989, the distribution of PZQ doses, free of charge, to all diagnosed schistosomiasis

cases was implemented through different health facilities including the network of rural health

units. In 2007, the MOHP has decided to move the control programme forward to achieve

elimination of schistosomiasis from Egypt. To accomplish the goal of elimination , the programme

plans to implement several rounds effective mass chemotherapy (1-2 rounds/ year) in '' hot spot"

areas using PZQ (2)

.

Mechanism of action of PZQ as anti-schistosomal drug: Despite the high success of PZQ in

treatment of schistosomiasis and reduction of its prevalence all-over the world, the mechanism of

action of PZQ is not known precisely and remains unresolved three decades following its

introduction.The detailed molecular mechanism of action has not been elucidated (72)

.A number of

researchers have been studied the mechanism of schistosomicidal action of PZQ (65,73,76-80)

, some

26

observations were noticed; it may induce violent muscle contraction that is linked to calcium

influx and results in shortening of the worm(76)

. But Pica-Mattoccia et al., (2008) (77)

observed that

“calcium accumulation by itself, at least as measured by whole parasites maintained in vitro.The

drug appears to damage the tegumental membrane disrupting the active immune evasion and

exposing surface antigens that were previously masked. This exposes the worm to the host

humoral immune attack which leads to worm death by host immune-mediated mechanisms. It has

also been suggested that PZQ exerts its effect by reducing schistosomal glutathione

concentrations (78)

. PZQ may bind to schistosomal Actin leading to disruption of the tegumentof

the worm (79)

or inhibiting adenosine receptors uptake (80)

. A number of metabolic alterations have

been observed in schistosomes exposed to PZQ; glucose uptake, lactate excretion and glycogen

content are all decreased (78)

.

Advantages of praziquantel in treatment of schistosomiasis:

PZQ is characterized by high efficacy, excellent tolerability, few and transient side effects,

simple administration, and competitive cost. The drug is equally suited for individual or large

scale treatment (65)

. So PZQ deserves to be included in the WHO model list of essential drugs (81)

.

Drawbacks of praziquantel in treatment of schistosomiasis:

1-Schistosomal Resistance: Even though PZQ efficacy is generally high, reported cure rates are

variable ranging from 60 to 95% (82)

. There are increasing concerns about the development of

resistance to the drug, but most published discussions of this topic conclude that convincing

evidence for the clinically relevant emergence of PZQ resistance in the field is still lacking (83-92)

.

2-PZQ is not used for prophylaxis: as PZQ is not active against immature stages of schistosomes

(schistosomula) (93)

.

3-PZQ is not ovicidal: it was reported that PZQ has no ovicidal properties (63)

.

Pharmacovigilance of praziquantel:

Abdominal discomfort, diarrhea, malaise, headache and dizziness are common side effects

of PZQ observed in a relatively large percentage of patient (30-60%), but also these are usually

mild and transient disappearing within 24 h (92,94)

. Some recipients of PZQ manifest allergic

symptoms with fever, rash, pruritis and eosinophilia in response to released worm antigens (95)

.

2-Mirazid (MZD):

Mirazid is a pharmaceutical natural preparation introduced to the Egyptian market by

Pharco pharmaceuticals (Alexandria, Egypt). The Egyptian drug authority (EDA) of MOHP has

registered this product for treatment of schistosomiasis (Reg.No.21655/2002).

27

Antischistosomal Properties of Mirazid:

MZD has been investigated, both experimentally and clinically against schistosomiasis with

controversy regarding its efficacy (96)

. Regarding the effect of MZD in vitro on S. mansoni adult

worms, Hassan et al., (2003) exposed the worms to various concentration of MZD from 100-400

μg/ml. It elicited maximal somatic muscle contraction at the highest concentration (400μg/ml) by

muscle tension method (97)

. Also Sharaf (2004) (98)

and Bakr et al., (2007) (99)

showed strong lethal

effect of MZD at both concentrations (100 and 200μg/ml) after 24 hrs of exposure of S. mansoni

adult males .Karamustafa et al., (2011) (100)

showed that MZD had antischistosomal activity

against S.mansoni larvae in vitro (IC50=7.18-32.69 µg/ml).

In experimental animals, Massoud et al., (1996) (101)

and Massoud (1999) (102)

started the

evaluation of myrrh (crude, fractions of oil or resins or combination of oil and resins) on S.

mansoni-infected hamster. They found that a combination of volatile oil and resins in special

formula was more effective as antischistosomal than the crude myrrh or separate volatile oil or

resins. Massoud et al., (2004) (103)

, Hamed and Hetta (2005) (104)

, Bakr et al. (2009)(99)

used MZD

in dose ranged from 250-600mg/kg for 3-5 days in S.mansoni infected-mice; the drug had a

valuable schistosomicidal effect against different maturation stages of S. mansoni worms (the rate

of worm reduction ranged from 81.1%-98.4%). The results of the previous experimental studies

were greatly conflicting with the following disappointing studies as Badria et al.,(2001)(105)

,

Guirguis and Mahmoud (2003) (106)

, Botros et al., (2004) (107)

, Ebeid et al .(2005) (108)

, Emam et

al., (2009) (109)

, El-Gamal et al., (2009) (110)

, Ramzy et al., (2010) (111)

, Abdul-Ghani et al., (2010)

(112), Lotfy et al., (2013)

(113) and EL-Malky et al., (2013)

(114) found low worm burden reduction

rate varied from (0% to 75%) either in S.mansoni or S. heamatobium or S. japonicum–treated

mice or hamster with MZD oral doses from 250-500 mg/kg for 2-5 days .

Efficacy of MZD as human anti-schistosomiasis drug was evaluated by Massoud et al.,

(1998) (115)

, Sheir et al. (2001) (116)

, Gaballah et al. (2001) (117)

, Abo-Madyan et al., (2004) (118)

,

Soliman et al.,(2004) (119)

and Massoud et al., (2010) (120)

. They enrolled 365 schistosomiasis-

infected patients (adults or children) treated with MZD in a dose of 10-11.5 mg/kg for 3-6 days.

At 2-3 months post-treatment, the drug effectiveness was assessed either parasitologically (faecal

egg count), clinically, biopsy or sigmoidoscopically. MZD achieved parasitological cure rate

varied from 80.7-100% with non-significant side effects on the liver and kidney functions .On the

other hand, many clinical studies showed that MZD has little or no beneficial activity in treatment

of schistosomiasis; as Botros et al.,(2005) (121)

, Barakat et al.,(2005) (122)

and Osman et al.,(2010)

(123) enrolled 206 patients (adults or children) orally administered the drug

in a dose of 300mg for

28

3 days, 600 mg for 3 days and 6oo mg for 6 days, respectively. MZD resulted in parasitological

cure ranged from 3.7%-15.6% at 4-8 weeks after treatments.

Mechanism of action of MZD as anti-schistosomal:

Although the exact mechanism of the schistosomicidal action of MZD has not been known.

It has been attributed to the permanent musculature loss of worms leading to unpairing of male

and female couples and their shift to the liver where subsequent destruction takes place

(97), this

may be related to the ability of some constituents to block the inward sodium current in

membranes leading to smooth muscle relaxing action and loss of attachment between male

worms and the inner linning of the blood vessels (117)

or to the increase of intra-parasite calcium

level (98)

. MZD caused destruction, deformity and blunting of spines on the tubercles of male

worm tegument, including the lateral margin of the gynecophoric canal (99)

. It was attributed that

the change in oogram pattern produced by MZD to an early interruption of egg laying capacity in

the intestinal wall or most probably blocking the development of reproductive organs (105)

.

Pharmacovigilance of Mirazid:

MZD possesses high safety margins in human application as it has no significant effects on

liver and kidney functions in healthy volunteers. It can be given for patients with

hepatosplenomegaly as the liver enzymes nearly returned to the normal level 8 weeks after

treatment (115,116)

. MZD has no arrhythmogenic activity as it had no siginificant effect on the ECG

parameters.Side effects reported to MZD administration were transient and mild and occurred in

only 11.8% of the treated cases and in none of the healthy volunteers. The most frequently

reported side effects were giddiness, somnolence, mild fatigue, abdominal pain or discomfort (116)

.

Drug Discovery and Development for Novel Treatments of Schistosomiasis

The fear for possible emergence of drug tolerance or appearance of new resistant strains to

PZQ especially with reinfection and re-treatment makes the search for new antischistosomal drugs

an essential target either from synthetic or natural origins.

A-Synthetic compounds:

1-Praziquantel derivatives and its combinations: Commercially produced PZQ is a racemic

mixture of levo (-) and dextro (+) enantiomers, only the levo enantiomer showed schistosomicidal

activity (124)

. Adoption of an enantioselective method of synthesis should therefore theoretically

provide drug that can be administered at higher dose without any increase in toxicity or adverse

events (125)

. Intense efforts are now directed to have a single drug for such a dreadful infection via

synthesizing derivatives of PZQ (68)

. Many variants of PZQ but were less active than the parent

compound (73)

.Combination of PZQ with other substances has been attempted for the treatment of

29

schistosomiasis mansoni aiming to reduce the PZQ dose, potentiate its schistosomicidal action,

and alleviate side effects (126)

. A lot of compounds either anthelmintics as Albendazole (127)

,

Artesunate (128)

, Artemether (129)

, Oxamniquine(130)

, or non-anthelmintics as Coenzyme-Q10(131)

,

Zinc (132)

, N-Acetyl-L-Cysteine (133)

, DDB (134)

, Dexamethasone (134)

, Pentoxifylline (135)

and

Silymarin (136)

were used.

2-Oxadiazoles (Furoxan derivatives): Oxadiazoles have been found to possess inhibitory

activity against S. mansoni and S. japonicum redox protein thioredoxin-glutathione reductase

(TGR).Oxadiazole-2-oxide surpassed criteria established by the WHO for potential lead

compounds for schistosomiasis in its effectiveness in experimental studies (137,138)

.

3-Cysteine Protease Inhibitors: Abdulla et al., (2007) introduced a novel chemotherapy of

human schistosomiasis through targeting cysteine proteases by phenyl vinyl sulfone or

(K11777).The inhibition of these schistosome specific enzymes resulted in a significant reduction

in parasite burden and pathology (139)

.

4-Trioxaquines: They were initially developed against malaria and exhibit a dual mode of action:

alkylation of heme with its trioxane entity, and stacking with heme due to its aminoquinoline

moiety, thus explaining their potent anti-S.mansoni activity in vitro and in vivo (140)

.

5-Trioxolanes (secondary ozonides): Trioxolanes isomers (OZ-78, OZ-209 and OZ-288) showed

significant schistosomicidal activity in vitro and in vivo against S. mansoni and S. japonicum.

High worm burden reductions (71.7 to 86.5%) were observed after administration of single 200-

mg/kg doses of OZ-78 and OZ-288 to hamsters infected with either juvenile or adult S.mansoni

and 94.2 to 100% in S.japonicum (141)

.

6- Imidazolidines: They had broad biological anti-microbial and anti-fungal activities, were also

used for treatment of schistosomiasis because of their potent in vitro schistosomicidal effects (142)

.

7-Benzimidazole derivatives: Triclabendazole (143)

, Flubendazole (144)

, Albendazole (145)

and

Mebendazole (146)

showed some promising anti-schistosomal activity in vitro and/or in vivo.

8- Thiazoles e.g., Nitazoxanide (NTZ):

Figure. 2. Structural formula of Nitazoxanide (C12H9N3O) (147)

30

Antiparasitic activity of Nitazoxanide:

NTZ was originally discovered in the 1980s at the Pasteur Institute. NTZ is a broad-

spectrum antiparasitic drug with activity against protozoa, nematodes and trematodes.The US

Food and Drug Administration (FDA) approved oral suspension of NTZ at December 2002 for the

treatment of diarrhea caused by Cryptosporidium species and Giardia intestinalis in pediatric

patients 1-11 years of age, and in July 2004, NTZ was approved for treatment of diarrhea caused

by G. intestinalis in adults (148)

. Two reports assessed the antischistosomal activity of NTZ against

S.mansoni in experimentally infected mice with controversy results as Abdel-Rahman et al.,

(1997) (149)

proved that the drug succeeded to reduce 59.91 % of worm load but Abdulla et al.,

(2009)(150)

proved that NTZ failed to affect worm burden .

Mechanism of Action of Nitazoxanide as anti-parasitic: The mechanism of Nitazoxanide’s

activity against helminths is unknown but it interfered with the pyruvate-ferredoxin

oxidoreductase enzyme-dependent electron transfer reaction which is essential to anaerobic

energy metabolism (151)

.

Pharmacovigilance of Nitazoxanide:

NTZ is generally well tolerated, and no significant adverse events have been noted in

human trials. Adverse events have been mild and transient and principally related to the

gastrointestinal tract, such as abdominal pain, diarrhea, and nausea. Adverse events occurring in

11% of more than 2000 patients participating in clinical trials included anorexia, flatulence,

increased appetite, enlarged salivary glands, fever, infection, malaise, elevated creatinine levels,

elevated serum ALT levels, pruritus, sweat, pale yellow sclerae, rhinitis, dizziness, and discolored

urine. In addition, there have been no significant changes in results of electrocardiography, vital

signs, or hematologic, clinical chemistry, or urinalysis parameters in patients treated with NTZ, it

has been well tolerated up to the maximum dose of 4 g when taken with or without food, but the

frequency of gastrointestinal side effects increases significantly with the dose level (152)

.

9-Miscellaneous synthetic drugs: A lot of synthetic compounds were examined experimentally

for anti-schistosomal activity eg; Oxamniquine derivatives (153)

, Ro 15-5458 (154)

,Antox(155)

,

pegylated tartar emetic (156)

, Adenine derivative(157)

, Thiazolo-Derivatives(158)

,Ro-354(159)

, Nano-

compounds(160)

, Tribendimidine(161)

, Clorsulon (162

, Benzothiazoles(163)

, Ozone(164)

, Nucleoside

phosphonates(165)

,Mefloquine(166)

,Substituted Pyrimidinedione derivatives (167)

, Anti-androgens(168)

,

Arachidonic acid (169)

,Interferon(170)

, Miltefosine(171)

, Thioxo-imidazolidine compounds(172)

,

endoperoxide N-89 (173)

, Licarin (174)

, Benzodiazepines (175)

, Aryl Ozonides (176)

, Imatinib (177)

and

Ivermectin (178)

.A considerable number of these compounds were tested and proved promising

31

anti-schistosomal activities, the majority of them were consigned to the museums of history, but

few succeeded in reaching more advanced developmental phases of clinical trials.

B-Natural products or naturally derived compounds:

As considerable efforts are ongoing to develop novel schistosomicidal agents, many natural

compounds with promising antischistosomal properties have been identified.

1-Artemisinin derivatives: Artemisinin is a sesquiterpene lactone with a peroxide group derived

from the leaves of the Chinese wormwood (Artemisia annua L.) which belongs to the family

Asteraceae, Artemether and artesunate are the most common Artemisinin derivatives (179)

. These

compounds are commonly used as antimalarial agents. In the early 1980s, it was discovered that

artemisinins exhibit antischistosomal properties .A comparative evaluation between artemether

and artesunate was performed by Utzinger et al., (2002), It revealed that artemether shows

consistently higher schistosomicidal activity than artesunate due to differences in the rates of

metabolism of the drugs (180)

.These artemisinin derivatives were found to be active against all

human schistosome species (181)

.

Artemether treatment in S.mansoni infected mice 4-6 week post-infection (WPI) with

doses ranging from (100 to 800 mg/kg for 2 to 4 days) resulted in worm load reduction varies

from 40 % to 61 %. Artemether shows its highest activity against the juvenile stages of the three

major human schistosome,so broadly defined as chemoprophylactic for schistosomiasis and exerts

ovicidal activity (180-185)

. S. mansoni immature worms exposed to artemether in vitro and

experimentally in mice resulted in high worm reduction (97-100%) between days 7 and 28 post-

infection (182)

. In fact, this is the period when praziquantel and other antischistosomal drugs are

less effective (73)

. Utzinger et al., (2000) (185)

reported the prophylactic activity of oral artemether

on S.mansoni in a randomized, double-blind placebo-controlled trial in western Côte d'Ivoire. The

incidence of infection was 50% lower in children who received artemether rather than placebo,

and the intensity of infection among those uncured was also reduced.

2-New Myrrh-Derivatives:

Myrrh oil can be prepared from the crude myrrh either by steam distillation or solvent

extraction (petroleum ether). It is named myrrh essential oil (MEO) or myrrh volatile oil (MVO)

or myrrh total oil (MTO). Allam and El-Sayad (2001) (186)

found molluscicidal activity in

B.alexandrina and the lethal concentration (LC 50) of the oil was 155 ppm in 24 hrs. While El-

Ashry et al., (2003) (187)

found (LC 50) was 6-7 ppm for 24 hrs. in the same snail. Oral lethal dose

(LD50) of MEO in rats was 1650 mg/Kg (188)

. Two reports assessed the antischistosomal activity

of MTO against experimental S.mansoni infection .The oil showed promising antischistosomal

32

activity by Massoud et al. (1999) (102)

and Abo-El-Maaty (2002) (189)

in hamster or mice without

hepatic hazard. In addition, MTO was tested as anticestodal drug and proved that 75 % of H.nana-

infected rats orally treated with 834 mg/kg were cured (190)

.

3-Miscellaneous Natural products:

Likewise, research on other natural products and natural product-derived compounds

against schistosomes has been performed by many groups. Accordingly, several natural products

with antischistosomal properties have been described in the literature; Citrus reticulata(104)

,

Curcumin (Curcuma longa)(191,192)

,Ginger (Zingiber officinale)(193)

,Nigella sativa(194)

, Garlic

(Allium sativum)(194)

, piplartine (Piper) (195)

,Holothuria polii (196)

, propolis (197)

, Ailanthus altissima

(198), Ziziphus spina christi

(198), Camel milk

(199), Ferula assafoetida

(200), Cleome droserifolia

(201),

Chenopodium ambrosioides(202)

,Conyza dioscorides(202)

, Sesbania sesban(202)

, Balanites

aegyptiaca(203)

, Euphorbia schimperiana (204)

, Carica Papaya (205)

, Pomegranate (Punica

granatum) (206)

and Baccharis trimera (207)

.

In Egypt, large scale surveys were done on hundreds of natural products and found strong

in vitro antischistosomal activity against Schistosoma mansoni for the extracts of 30 species

which are (Agave Americana var. marginata and A. lophantha) , Furcraea selloa, Calotropis

procera, Pergularia tomentosa , Asclepias sinaica, Alkanna orientalis , Khaya grandifoliola,

Swietenia mahogany, Pimenta racemosa, Pinus canariensis, Verbascum sinaiticum ,(Solanum

elaeagnifolium, Solanum nigrum), Brachychiton rupestris, (Callistemon viminalis, C. rigidus , C.

speciosus , C. citrinus) , (Eucalyptus citriodora , E. rostrata, Eugenia edulis , E. javanica) ,

(Melaleuca leucadendron, M. stypheloides), Cryptostegia grandiflora , Zilla spinosa , Ficus

trijuja , Fagonia mollis and Nerium oleander (208-210)

. Most of the extracts or natural compounds

were only evaluated in vitro studies; it is expected that they will be evaluated using in vivo

experimental models and finally various phases of clinical trials should be followed to find the full

data of their effectiveness.

33

AIM OF THE WORK

34

AIM OF THE WORK

The aim of the study is to assess efficacy of Nitazoxanide, Myrrh Total Oil and the

commercially available product of Myrrh (Mirazid) in comparison with Praziquantel in treatment

of schistosoma mansoni infected mice.

35

MATERIALS

AND

METHODS

36

MATERIALS AND METHODS

MATERIALS

I.Experimental animals: The study included 120 Eight-week-old female Swiss albino mice (Mus

musculus) of the CD-1 strain weighing 18-25 gm. The animal groups were bred in separate

stainless steel wire-mesh cages under controlled conditions (Temperature 18-25°C, humidity 30-

70%, 12 hours light and 12 hours dark cycles). Animals were fed a standard pellet diet and water

ad libitum.

II.Parasite strain: Laboratory-bred B.alexandrina snails infected with miracidiae of Egyptian

(CD) strain of Schistosma mansoni were obtained from the Schistosome Biologic Supply Center

(SBSC), Theodore Bilharz Research Institute (TBRI). Cercariae shedding out of infected snails

were used to infect the experimental mice.

III.Drugs: The drugs under investigation were:

Nitazoxanide was purchased from a pharmacy in Alexandria as Nitazode powder for oral

suspension produced by Sigma pharmaceutical company for Al-Andalus Medical

Company, Batch No: 21581.

Mirazid capsules were obtained as free medical samples from Pharco Pharmaceuticals,

Batch No: 296.

Myrrh total oil was obtained from Safepharma.

Praziquantel was purchased as Biltricide tablets manufactured in Alexandria Company

for pharmaceutical and chemical industries, Batch No: 9118014.

IV. Chemicals:

Iodine solution Cremophore EL Distilled water

KOH (potassium hydroxide Petroleum ether Saline

V. Equipement:

Animal house Oesophageal syringe Markers

Sensitive balance Electricity supply Aquarium

Black plastic Glass conical flasks Electric pump

White fluorescent light lamp Magnetic rod Magnetic plate

Stereobinocular microscope Dissecting microscope Glass test tubes

Plastic pippetes (1ml) Glass slides Beakers

37

Volumetric flasks A pair of scissors Dissecting board

Ordinary Ruller Plastic small tubes Vortex mixer

Incubator Tray Forceps

Epindorf Micropipette Centrifuge

Vacutainers Diagnostic kits Capillary tubes

Haematology automated cell counter Haemocytometer

Chemistry analyser Critical Point Dryer Fine coater

Scanning electron microscope Statistical programme Computer

METHODS:

І-Mice infection with S.mansoni (figure 3):

*Cercarial shedding and counting of cercariae in the suspension (211)

:

Infected Biomphalaria alexandrina snails were washed with dechlorinated water and kept at

an aquarium in an aerated (by using electric pump), dark place (covering the glass bath with black

plastic bag). Before use, snails were rinsed gently with small volume of water to remove faeces

and other debris, then resuspended in water (1 ml /1 snail) and left uncovered in a glass test tube

under white fluorescent light for a period of 30-60 min to release cercariae .After shaking gently

to ensure homogenous distribution of cercariae, 1 ml of cercarial suspension was pipetted and

placed on glass slides, a drop of iodine was added to each slide to kill and stain the cercariae.

With the aid of a stereobinocular microscope, the number of cercariae was counted in each slide.

Generally 3 counts were made in 3 ml cercarial suspension and the average number per 1 ml was

calculated.

* Infection of mice (212)

:

Mice were allowed to urinate and defecate by its exposure to fresh water in a glass bath.

Mice were then infected using paddling technique .Each mouse was exposed separately to about

100 S.mansoni cercariae for one hour at room temperature (22-28OC) in a glass conical flask

containing 10 ml dechlorinated water mixed with the cercarial suspension .Infected mice were

then segregated in groups of 10 in separate stainless steel wire-mesh cages.The date of infection

was recorded. Mice received a standard well balanced diet and water. Stool examination was

performed 50 days post-cercarial infection to investigate the presence of S.mansoni eggs.

38

Figure.3. Steps of mice infection with S.mansoni cercariae (1-cercarial shedding,

2- cercarial counting, 3- cercarial inoculation)

3

2

1

39

II-Preparation of Drugs Suspensions:

1- Preparation of Praziquantel Suspension (99)

:

Fresh suspension was prepared by dissolving the tablet (600mg) in 6 ml of 4% Cremophore

EL (4 ml cremophore EL+ 96 ml sterile distilled water). Each mouse (20g) requires 0.1 ml

solution. A magnetic rod was placed into the flask, and then the flask was put on a magnetic plate.

The mixture was stirred for 30 minutes to ensure complete homogeneity of the drug suspension.

The suspensions were dispensed into sterile labeled tubes with tight stoppers.

2- Preparation of Mirazid suspension (103)

:

Each capsule of the drug (300 mg) was evacuated in a flask containing 3 ml of 4 %

cremophore EL. Each mouse requires 0.1 ml solution.

3- Preparation of Nitazoxanide suspension (213)

:

Nitazoxanide After reconstitution with distilled water, each 5 ml suspension contains 100

mg NTZ. Each mouse (20g) requires 0.1 ml solution.

4- Preparation of Myrrh Total Oil Suspension (214)

:

0.3ml (100 mg by weight) of the oil was mixed with 27 ml Cremophor EL 4 %. Each mouse

(20g) requires 0.1 ml solution.

N.B. Cremophor EL is a castor oil derivative used as an emulsifying and solubilising agent for the

production of aqueous preparations containing volatile oils and other hydrophobic substance.

N.B. Drug suspensions were freshly prepared within the week of the performance of experiments,

and put in the refrigerator until use. Doses equivalent to those predetermined in the dosing

regimen were then calculated as mentioned and orally administered to each mouse using

eosophageal syringe.

III. Study Grouping:

The study was carried out on six groups of 20 mice each .The mice were housed in a room

with a controlled adequate environmental temperature .Groups of 10 mice in each cage were

allowed free access to water and food. They were acclimatized for 1 week before test and only

healthy mice were assigned to the present study. Mice of all groups were randomly allocated

through treatment and control groups, just prior to drug administration. In these groups, treatment

started 50 days post infection. The drugs were administered after overnight fasting and eating was

allowed after one hour as shown in the following:

Group1: infected and treated orally with MZD 500 mg/kg bw/day for 5 consecutive days (103,107)

.

Group 2: infected and treated orally with MTO 18 mg /kg bw/day for 3 days (102,214)

.

Group 3: infected and treated orally with NTZ 100 mg/kg bw/day for 7 consecutive days (213)

.

40

Group 4: infected and treated orally with PZQ 500 mg/kg bw/day for 2 consecutive days (99)

.

Group 5: infected and non-treated (+ control G).

Group 6: normal non-infected and non-treated (- control G).

N.B. Infected non-treated control and normal non-infected non-treated mice were given only the

vehicle (4% Cremophor EL).

IV- Drug Evaluation:

Evaluation of efficacy was based on the following parameters:

I.Parasitological Studies:

They were performed to assess the efficacy of the different treatments on fecal egg counts,

worm burdens, sexes and lengths, tissue egg loads and oogram patterns.

a-Feacal Egg Count : Eggs of S.mansoni were counted in mice stool {each pellet was weighed ,

thoroughly mixed with saline and spread on glass slide then eggs were counted every other day

starting two days post-treatment and continued till mice sacrifice (215)

.

b-Recovery of adult worms: Perfusion technique was done in experimentally infected mice (G1-

G5).After mice sacrifice 1, 2 and 4 weeks post-treatment as follow:

Perfusion technique

1. Mice were sacrificed by cervical dislocation (99)

.

2. Blood samples were collected immediately.

3. Their bodies were skinned, washed with tap water to remove any adherent hair.

4. Mice were fixed to an inclined dissecting board, laid on a stainless steel pan in which the

perfusate was collected. The abdominal muscles and peritoneum were opened to expose the

internal organs. The portal vein was quickly ligated and closed to its entrance to the liver to

prevent shift of the parasites.

5. perfusion was done according to the technique of Smithers and Terry (1965) (212)

, using

perfusion pump machine (figure 4), A twenty liter glass beaker with outlet of rubber tubing and

20 gauge needle, containing citrated solution, was put in the perfusion machine. The pressure

required for the perfusion was provided by a rotator peristaltic pump. The needle connected to

automatic machine was inserted into inferior vena cava for pumping citrated saline into the liver.

The portal vein was then cut and the perfusate flowing from it was collected .Perfusion was

continued until the fluid coming from the animal was free of blood. The needle was then removed

while the pump was still operating and inserted into the thoracic aorta downward to perfuse the

mesenteric vessels. The perfusate flew out of the portal vein. Coils of the intestine were lift and

washed down in order to dislodge any worms adhering to them. The viscera with surrounding fat

deposits,were searched thoroughly for worms.

41

Figure.4. Perfusion pump machine

Figure.5. Mice perfusion

Worm Burden :

-The worms coming out with the perfusates of the liver and mesenteries were collected.Then, the

sediment was transferred into a Petri dish using a Pasteur pipette for worms to be counted and

sexed under a stereoscopic microscope using low-power magnification (x10).

-The percentage reduction in the total worm burden were calculated by comparing the number of

worms recovered from the treated mice with those recovered from the corresponding control

according to Tendler et al.,(1986)(216)

with the following formula:

42

% Worm reduction(R) =

Mean worm count non-treated group(c)-Mean worm count treated group(t) x100

Mean worm count non-treated group(c)

Or % R= C-T/C X100

Worm length:

Random samples of collected worms from each group were classified and their length were

measured using ordinary ruller and dissecting binocular microscope (143)

(figure 6).

Figure.6. Measurement of female S. mansoni body length under dissecting microscope with

ordinary ruller.

c-Tissue egg count :

To evaluate the number of eggs present in the tissues of the liver and small intestine

according to Cheever et al., (1968) (217)

.Tissues were frozen but not formalized till examination.

KOH digestion technique:

This technique was performed in the following steps:

- Fragments of the small intestine were slit-opened and washed with saline to remove any faecal

matter present in the lumen. Also fragments of the liver were taken and washed with saline.

- The specimens of the hepatic and the intestinal tissues were weighed separately (about 1g)

before digestion.

- These weighted fragments were placed in a test-tube containing 5 ml KOH solution (5%) at

37°C overnight in an incubator till complete digestion of tissues.

43

-After overnight incubation, the test tubes containing the tissue emulsion were mixed thoroughly

in a vortex mixer.

-The digest was well shaken and three samples, (each = 0.1ml or 100 µL) were pipetted out from

each tube by micropipette and placed on slides.

-The samples were then examined microscopically with low magnification (10 X) and the number

of ova in the 3 samples was recorded.

Calculation of tissue egg loads:

* Average number of ova in 0.1ml = Total number of eggs in the three samples/3

* Total number of ova in the examined tissue (in 5 ml) = Average number of ova in 0.1ml × 5(5

ml KOH solution) / 0.1

* Number of ova/g tissue (epg) = Total number of ova in 5ml /weight of liver or intestine in grams

recorded before digestion.

Calculation of tissue egg load reduction:

Reduction in tissue egg loads was calculated by comparing the mean tissue egg loads in the

treated groups with those of the corresponding control group using the Following formula:

% Tissue epg reduction=

Mean epg in non-treated(c) - Mean epg in the treated (t) x100

Mean epg in non-treated group(c)

d. Oogram pattern (percentage of egg developmental stages) (218)

:

For studying the oogram (figure 7); eggs were classified according to their viability (dead

or viable). Generally, dead eggs appear semitransparent, granular or darkened with retracted

embryo. If death takes place at immature stages, whereas those dying after maturation appear

roughly granulated, disintegrated, calcified or as egg-shells. The viable eggs were then divided

according to their stages of maturation (immature or mature).

Oogram technique was performed in the following steps:

1-The small intestine of the perfused mouse was separated and transferred into a Petri dish

containing normal saline. The intestinal contents were removed by squeezing the intestine gently.

2- About 10 centimeters of the middle part of the small intestine were opened longitudinally with

scissors and rinsed with saline after removing excess mucus.

3- Three fragments (1-cm pieces) were cut off, slightly dried with filter paper and placed between

two slides to be examined under microscope using low-power magnification (x10).

4- One hundred S. mansoni ova were counted in each fragment and subsequently classified

according to their developmental stages into: dead or viable (mature or immature).

44

5- The mean numbers and percentages of various egg developmental stages as well as dead eggs

were calculated for each group of mice.

Figure.7. Egg developmental stages (218)

[Dead eggs of S. mansoni (immature) (A, darkened eggs, B, granular egg, C and D, semi -

transparent eggs, E, egg with retracted embryo) and Viable eggs of S. mansoni (F) immature egg,

(G) mature egg].

C D E

45

2. Scanning Electron Microscpic Studies:

Scanning electron microscopy (SEM) was utilized to examine the effect of the drug on the

tegument or external surface of the worm. This technique was applied according to Anderson

1951(219)

with some modifications according to Bricker et al. (1983) (220)

.

Technique:

1- Fixation: Two worms (one male and one female) were removed from each group then

fixed with 3% glutaraldehyde buffered with 0.1 M phosphate buffer pH 7.4 at 40C.

2- Dehydration (to remove water): Washing of worms with phosphate buffer to remove the

fixative then worms were dehydrated in graded concentration series of Acetone

30%,40%,50% each for 15 min, then the worms were kept in 70% acetone until the time of

examination. Before examination, worms were washed for three times, the first and second

were for 30 min in 80% and 90% acetone respectively, while the last wash was for 1 hour

in 100% acetone.

3- Drying: worms were transferred to liquid CO2 at the critical point for drying in critical

point dryer.

4- Coating: The dried specimens were mounted on metal stabs then coated with gold (to make

the surface more conductive for electrons).

5- Imaging: Specimens were examined in Joel JSM-5300 scanning electron microscope in the

electron micrcoscpy unit, Faculty of science, Alexandria University, Egypt).

3. Hematological Studies:

Blood samples obtained before sacrification of mice using capillary tubes introduced into the

medial retro-orbital venous plexus (figure 8) , a part of blood (about 300 ul) was collected into

vacutainer tubes containing an anticoagulant [ethylene diamine tetra-acetic acid (EDTA)] for

determination of Complete Blood Count (CBC) (221)

by using haematology fully automated cell

counter (Mindray BC-3200) .

46

Figure.8. Blood collection from a mouse

4. Biochemical studies:

Another part of the blood (about 1ml) was collected in tubes without anticoagulant,

centrifuged at 3000 rpm for 5 minutes for collection of serum then estimation of biochemical

parameters using commercial kits. The liver function tests were assessed using alanine

aminotransaminase (ALT), aspartate aminotransaminase (AST,Diasys diagnostics) according to

Reitman and Frankel (1957) (222)

and alkaline phosphatase (ALP,Tecno diagnostics) according to

Kind and King(1954)(223)

. Blood urea and serum creatinine were used to assess kidney functions

using urea and creatinine kits (Diamond Diagnostics) according to Fawcett and Scott (1960) (224)

,

Husdon and Rapoport (1964)

(225) respectively. The previous tested parameters were counted by

photometer 5010 (fully-automated chemistry analyser, India), Cholinesterase (ChE) level was

selected to assess the neurotoxic potential in mice blood using Spinreact chemistry analyser

/Spinlab(Spain) according to the colorimetric method of Ellman et al.(1961)(226)

.

ETHICAL CONSIDERATIONS:

The study protocol was reviewed and approved by the ethics committee of the medical

research institute (MRI), University of Alexandria.

47

STATISTICAL ANALYSIS

The data were coded , collected, tabulated and analysed using one way analysis of variance

(ANOVA) followed by independent two-sample t-test or Student’s t test for comparison of means

of two corresponding groups using Minitab statistical software version 14. Descriptive statistics

were expressed as arithmetic mean ± Standard Deviation (SD) as measures of central tendency

and dispersion respectively. As regards the level of significance (P > 0.05 was considered

statistically non-significant while p<0.05 and p<0.01were considered statistically significant and

highly significant, respectively). Data were presented using Microsoft excel sheet for graphical

presentation.

The % of change between non-treated, non-infected and treated groups was calculated as follow:

% change in infected =

Mean values in non-infected (c) - Mean values in infected non-treated (n) × 100

Mean values in non-infected(c)

% change in treated =

Mean values in non-treated(c) - Mean values in treated (t) × 100

Mean values in non-treated (c)

48

RESULTS

49

RESULTS

1. PARASITOLOGICAL STUDIES

a. Egg count in stool

In S.mansoni-infected mice, it was noticed that the prepatent period lasted 49 days, as the

first patch of eggs was found in the stool of infected mice on day 50 post-infection. Non-

significant change in faecal egg count was found either in infected non-treated mice or treated

with different regimens before the 7th

day of follow up. There was gradual rise in egg count of

infected non-treated mice starting from the 8th

WPI till the 10th

week with fluctuation from day to

day in the follow up period (table I and figure 9, 10). Treatment of infected mice with PZQ caused

highly significant % reduction in the faecal egg counts, 1WPT (63%, p<0.01) as compared to the

infected non-treated group. Eggs were not detected in the faeces of infected treated mice at 2

WPT (egg reduction 100%, p<0.01) and this continued throughout the rest of the follow up period

till 4 WPT.

The results of MZD-treated S.mansoni -infected group showed non-significant % reduction

in the number of eggs 1WPT (4.5%, p>0.05), but MZD caused highly significant reduction

(39.5%, 69.6%, p< 0.01) at 2 and 4 WPT. As regards the effect of NTZ on egg count in stool of

S.mansoni -infected mice, it caused non-significant reduction (4.9%, p>0.05) at 1WPT but it

resulted in significant % reduction (22.5%, p< 0.05) at 2 WPT and highly significant reduction

(50.6 %, p< 0.01) at 4 WPT. MTO treatment resulted in insignificant change in faecal egg count

% reduction (1% and 9.8%, p>0.05) at the 1st and 2

nd PT. The oil significantly reduced (19.4%,

p< 0.05) at the 4th

weeks of treatment (table I, II and figures 9, 10).

50

Table (I): Mean egg counts per gram stool of S. mansoni-infected mice under different

treatments compared to infected non-treated mice.

NTZ = Nitazoxanide, MTO= Myrrh total oil, MZD=Mirazid, PZQ= praziquantel , PT: post-treatment,

epg= egg count per gram , the statistical test was done by independent two-sample t-test ,Values were

expressed as mean ± SD,

a: Statistically significant at P.value < 0.05.

A : Statistically highly significant at P.value < 0.01.

Figure.9. Egg counts in stool of different groups of S. mansoni-infected mice under different

treatments compared to non-treated infected mice.

Days

(PT)

Groups

Egg counts (epg)

3 5 7 9 11 14 16 18 20 22 24 26 28

NTZ

184.00

±30.50

272.00

±67.97

538.25

±39.47

638.00

±119.6

639.00

±135a

592.50

±90.69a

632.50

±126.0a

550.00

±111.80a

426.67

±118.4A

452.50

±113.2A

395.00

±70.4A

350.00

±84.85A

355.00

±28.07A

MTO

202.50

±47.87

292.50

±93.59

560.00

±66.58

655.67

±83.27

733.33

±85.05

690.00

±57.00

696.67

±65.06

583.33

±61.10

597.33

±15.53

511.00

±205.0a

580.08

±95 a

509.00

±155 a

580.0

±77.5a

MZD

177.33

±18.62

226.67

±50.86

540.00

±42.74

558.33

±108.70

556.00

±26.17A

462.50

±39.09A

533.80

±70.05A

494.33

±127.1A

392.50

±94.54A

320.00

±113.5A

255.00

±77.2A

225.00

±63.64A

218.75

±12.97A

PZQ

178.33

±66.76

279.17

±76.97

204.0

±15.7A

156.00

±81.12A

70.00

±18.71A

0.00

±0.00 A

0.00

±0.00 A

0.00

±0.00 A

0.00

±0.00 A

0.00

±0.00 A

0.00

±0.00A

0.00

±0.00A

0.00

±0.00 A

Infected

non-

treated

178.00

±20.49

220.90

±78.81

566.00

±55.05

624.00

±79.25

745.00

±113.28

765.00

±70.83

735.00

±54.47

657.50

±80.98

657.50

±83.42

810.00

±127.67

821.00

±83.86

770.00

±81.85

720.00

±62.11

51

Table (II): Effect of different types of treatments on mean egg counts among different

groups of S. mansoni-infected mice compared to non-treated infected mice according to

WPT.

WPT Mice groups (infected and treated)

Infected

non-treated NTZ MTO MZD PZQ

1 538.25 ±39.47

(-4.9%)

560.00 ±66.58

(-1%)

540.0±42.74

(-4.5%)

204.0 ±15.7A

(-63.9%)

566.00 ±55.05

2 592.50 ±90.69a

(-22.5%)

690.00 ±57.00

(-9.8%)

462.50±39.09A

(-39.5%)

0.00±0.00A

(-100%) 765.00 ±70.83

4 355.00 ±28.07A

(-50.6%)

580.0 ±77.5a

(-19.4%)

218.75±12.97A

(-69.6%)

0.00±0.00A

(-100%) 720.00±62.11

NTZ = Nitazoxanide,MTO= Myrrh total oil, MZD=Mirazid, PZQ= praziquantel, WPT=weeks post-

treatment, the statistical test was done by independent two-sample t-test ,Values were expressed as

mean ± SD, Numbers in parentheses indicate the percentage of change compared to the infected non-

treated group .

a: Statistically significant at P.value < 0.05.

A : Statistically highly significant at P.value < 0.01.

Figure. 10. Percentage faecal egg count reduction in different groups of S. mansoni-infected

mice under different treatments at different periods of follow up.

52

b. Worm Burden, Sex and Length:

Treatment of S. mansoni-infectbed mice with PZQ caused pronounced and a highly

significant reduction (83%,94 %,97%, P<0.01) in the mean number of total worms at 1,2 and 4

WPT, respectively as compared with the infected control group. MZD caused significant reduction

in the total worm burden 34% (P<0.05), 50% and 71% (P<0.01) at 1, 2 and 4 WPT, respectively.

NTZ-treated group showed insignificant reduction in total number of worms (26%, P>0.05) at 1

WPT. There was significant reduction 45% and 65% (P<0.01) at 2 and 4 WPT, respectively. MTO

gained non-significant rate of total worm reduction 9% and 27% (P>0.05) at 1 and 2 WPT but the

oil possessed significant rate of worm reduction 29 % (P<0.05) at 4 WPT (table III and figure 11).

Table (III): Worm burden in S.mansoni-infected mice treated and non-treated groups by

time (weeks).

Mice groups WPT Total worm burden

(Mean ± SD)

TWR

%

NTZ

1 14.60±1.81 26

2 15.75±0.95 A 45

4 10.00±1.82 A

65

MTO

1 18.00±4.83

8.5

2 20.67±3.21

27

4 20.33±2.08a

29

MZD

1 13.00±2.53 a

34

2 14.25±2.63 A

50

4 8.25±1.50 A

71

PZQ

1 3.33±1.75 A

83

2 1.6±0.54 A

94

4 1.00±0.70 A

97

Infected

non-treated

1 19.70±2.86 -

2 28.34±5.51 -

4 28.67±4.73

WPT: weeks post-treatment, TWR: Total worm reduction, NTZ = Nitazoxanide, MTO= Myrrh total oil,

MZD=Mirazid, PZQ= praziquantel, the statistical test was done by independent two-sample t-test. Values were expressed as mean ± SD.

a: Statistically significant at P.value < 0.05.

A : Statistically highly significant at P.value < 0.01.

53

Figure.11. Percentage reduction in the mean total worm burden in different groups under different

treatments at different periods of follow up.

In this study, PZQ showed equal sensitivity on the numbers of both sexes as there was

significant reduction 83.3%, 93.5% and 95.7% (P<0.01) in male worms and 82.6%, 95.8% and

98% (P<0.01) of female worms respectively at 1, 2 or 4 WPT. MZD affected males more than

females at 1, 2 and 4 WPT as the drug killed 38.4%, 57.1% and 77.2% (P<0.01) of the male

worms and 25.3%, 35.3% (P<0.05) and 60% (P<0.01) of females. NTZ affected male worms

more than females as there was significant reduction 27.6% (P<0.05), 47.7% and 67.8% (P<0.01)

of male worms and 22.3% (P>0.05), 37.9 % (P<0.05) and 60% (P<0.01) of females at 1, 2 or 4

WPT. MTO resulted in significantly reduced males at 2 and 4 WPT 35.7% (P<0.05) and 35.7%

(P<0.01) more than females (10.3%, and 16.7%, P>0.05) (table IV, fig.12, 13).

54

Table (IV): Percentage of change in male and female worm distribution in different

S.mansoni-treated mice groups in different periods of follow up.

WPT: weeks post-treatment, NTZ =Nitazoxanide, MTO= Myrrh total oil, MZD=Mirazid, PZQ=

praziquantel, the statistical test was done by independent two-sample t-test. Values were expressed as

mean ± SD.

a: Statistically significant at P.value < 0.05,

A: Statistically highly significant at P.value < 0.01.

Mice groups WPT Total male

Total female

NTZ

1 9.40±1.14a (27.6%)

5.20±0.83 (22.3%)

2 9.75±0.95A (47.7%) 6.00±0.81a (37.9%)

4 6.00±1.41A (67.8%) 4.00±0.81A (60%)

MTO

1 11.30±3.37 (13%) 6.70±1.82 (0%)

2 12.00±1.00a (35.7%) 8.67±2.31 (10.3%)

4 12.0±1.00A (35.7%) 8.33±1.15 (16.7%)

MZD

1 8.00±1.41A (38.4%) 5.00±1.89a (25.3%)

2 8.00±1.63A (57.1%) 6.25±1.25a (35.3%)

4 4.25±1.25A (77.2%) 4.0±1.41A (60%)

PZQ

1 2.16±1.60A (83.3%)

1.16±0.4A (82.6%)

2 1.20±0.44A (93.5%)

0.40±0.54A (95.8%)

4 0.80±0.44A (95.7%)

0.20±0.44A (98%)

Infected

Non-treated

1 13.00±2.24

-

6.70±2.59

-

2 18.67±3.21

-

9.67±3.06

-

4 18.67±2.08

-

10.00±2.65

-

55

Figure.12. Percentage reductions in female worm burden in S.mansoni-infected mice under

different treatments at 1, 2 and 4 WPT.

Figure.13. Percentage reductions in male worm burden in S.mansoni-infected mice under different

treatments at 1, 2 and 4 WPT.

The effect of the studied medications on the body length of both male and female worms

after recovery indicated that PZQ caused equal sensitivity in shortening of worm length as it

caused significant reduction in the body length of female worms 26.1%, 45.2% (P<0.05), 65.6%,

(P<0.01) and 25%, 50% and 61% (P<0.01) of male worms at 1, 2 and 4 WPT, respectively. MZD

decreased body length of male worms 11.8% (P>0.05), 40.4% and 41% (P<0.01) than in female

worms 3% (P>0.05), 20.5% (P<0.05) and 33.6% (P<0.01) respectively at 1, 2 and 4 weeks after

56

treatment. NTZ and MTO had negligible effect on worm length of both sexes as there was non-

significant decrease in the body length of worms recovered at different times 1,2 or 4 weeks post-

treatment (6.5%, 9.5% and 5.2% for males and 0%,6.8% and 5.6% for female respectively in

NTZ-treated worms, also in MTO (2.6%,4.7% and 0% for males and 0% ,5.9% and 12% for

females at 1,2 and 4 WPT) (table V and figure 14,15).

Table (V): Body length of S.mansoni worms recovered from different treatments compared

to non-treated mice at different follow up periods.

Worms

sex

WPT

The body length of S.mansoni worms recovered (mm)

Infected Treated Mice Infected

Non-treated NTZ MTO MZD PZQ

Male

1 7.10±0.58

(-6.5%)

7.40±1.15

(-2.6%)

6.7±1.53

(-11.8%)

5.70±0.10a

(-25%)

7.60±0.55

2 7.60±1.15

(-9.5%)

8.00±1.00

(-4.7%)

5.00±1.00a

(-40.4%)

4.20±0.58a

(-50%)

8.40±1.50

4 9.00±1.41

(-5.2%)

9.50±0.71

(0%)

5.60±1.53a

(-41%)

3.70±1.15A

(-61%)

9.50±0.70

Female

1 13.00±1.73

(0%)

13.0±0.58

(0%)

12.60±0.58

(-3%)

9.60±0.58a

(-26.1%)

13.00±1.00

2 10.9±0.58

(-6.8%)

11.00±1.73

(-5.9%)

9.30±0.58a

(-20.5%)

6.40±0.58a

(-45.2%)

11.70±1.50

4 11.80±0.71

(-5.6%)

11.00±1.41

(-12%)

8.30±1.1A

(-33.6%)

4.30±0.58A

(-65.6%)

12.50±0.71

NTZ=Nitazoxanide,MTO=Myrrh total oil, MZD=Mirazid, PZQ= praziquantel, WPT=weeks post-

treatment , the statistical test was done by independent two-sample t-test ,Values were expressed as

mean ± SD, Numbers in parentheses indicate the percentage of reduction compared to the infected group.

a : Statistically significant at P value < 0.05.

A : Statistically highly significant at P value < 0.01.

57

Figure.14. Percentage reductions of the body length of male S. mansoni worms recovered from

different treated groups at 1, 2 and 4 WPT.

Figure.15. Percentage reduction of the body length of female S.mansoni worms recovered from

different treated groups at 1, 2 and 4 WPT.

c. Tissue egg count

The infected non-treated mice were loaded with higher number of eggs in the intestinal

tissues than the hepatic ones. PZQ-treated mice showed significant reduction in tissue egg load in

both liver and intestine as it was able to reduce the intestinal egg load (69.9%, 79.1% and 88%,

P<0.01) more than the reduction in the hepatic tissues (64.4%,69.2 % and 85.8%, P<0.01) at 1,2

and 4 WPT, respectively.

58

MZD produced significant reduction (28.9% ,49.3% and 66%,P<0.01) at 1,2 and 4 WPT in

the intestinal egg loads and non-significant reduction in the hepatic tissue egg load at

1WPT(22.2%,P>0.05) but it was able significantly to reduce the egg count later on in the 2nd

and

4th

week after therapy (42.8% and 65.3%,P<0.01). NTZ reduced the intestinal egg count (22.1%,

45.2% and 46.6%, p<0.01) at 1, 2 and 4 WPT. It was unable to reduce hepatic tissue egg load at 1

WPT (20%, p>0.05) but significant reduction was achieved (23.8%, p< 0.05 and 30.7%, p<0.01)

at 2 and 4 WPT, respectively. On the other hand, MTO resulted in significant reduction in the

intestinal egg count (12.2%, p<0.05), (23.2% and 31.5%, p< 0.01) at 1, 2 or 4 WPT .The oil

insignificantly reduced the hepatic tissue egg count (7.7% and 26%, p<0.05) at 1 and 2 WPT but

significant reduction was achieved (42.3%, p<0.01) at 4 WPT (table VI and figure 16, 17).

Table (VI): The tissue egg count in the liver and intestine of S. mansoni-infected mice under

different treatments at different periods of follow up.

Mice

groups

Tissue egg count(epg) x103 (% R)

Intestine (% Reduction) Liver (% Reduction)

1st week 2

nd week 4

th week 1

st week 2

nd week 4

th week

NTZ

8.00±1.1A

(-22.1%)

8.4±1.6A

(-45.2%)

8.8±0.7A

(-46.6 %)

3.6±1.4

(-20%)

4.8±0.8a

(-23.8%)

5.4±0.5A

(-30.7%)

MTO

9.02±1.06a

(-12.2%)

11.2±0.7A

(-23.2%)

11.3±1.7A

(-31.5%)

4.15±1.18

(-7.7%)

4.66±1.4

(-26%)

4.5±1.9a

(-42.3%)

MZD

7.30±1.3A

(-28.9%)

7.4±0.5A

(-49.3%)

5.6±1.00A

(-66%)

3.5±1.6

(-22.2%)

3.6±0.8A

(-42.8%)

2.7±1.0A

(-65.3%)

PZQ

3.09±0.16A

(-69.9%)

3.05±0.9A

(-79.1%)

1.96±0.4A

(-88%)

1.6±0.7A

(-64.4%)

1.94±0.8A

(-69.2%)

1.1±0.19A

(-85.8%)

Infected

Non-

treated

10.28±0.4

14.6±0.3

16.5±0.6

4.5±0.7

6.3±0.6

7.8±1.3

WPT:weeks post-treatment, NTZ=Nitazoxanide, MTO=Myrrh total oil, MZD=Mirazid, PZQ=

praziquantel, the statistical test was done by independent two-sample t-test ,Values were expressed

as mean ± SD.

a: Statistically significant at P value < 0.05.

A : Statistically highly significant at P value < 0.01.

59

Figure.16. Percentage reduction in the mean hepatic egg counts in S. mansoni-infected mice under

different treatments at 1, 2 and 4 WPT.

Figure.17. Percentage reduction in the mean intestinal egg counts in S.mansoni-infected mice

under different treatments at 1, 2 and 4 WPT.

d. Oogram pattern (percentage of egg developmental stages):

In the present work, the oogram pattern showed that about 60% of eggs in the infected non-

treated group were immature whereas dead eggs constituted only 7-12% and the mature eggs

formed 24-32% of the total eggs at different follow up periods. PZQ induced marked changes in

the oogram pattern in comparison to the non-treated infected group, it produced a highly

significant increase in the percentage of dead eggs to 79.87 % at 1 WPT, 83.2% at 2 WPT and

60

85.75% at 4 WPT as well as highly significant reduction in immature eggs where they constituted

only 3.67% at 1 WPT, 2.6% at 2 WPT, and 1.5% at 4 WPT, respectively. As regards mature eggs,

PZQ induced significant reduction in their percentage as 16-13% of the eggs in the oogram were

mature.

MZD resulted in significant increase in the percentage of dead eggs. However, the changes

were less marked than that induced by PZQ (24.83 %, at 1 WPT), (37% and 37.33%, p<0.01 at 2

and 4 WPT). MZD also induced significant reduction in immature eggs starting from the 2nd

WPT

(23.25 %) and continued to the 4th

WPT (10.77%). MZD produced a significant increase in the

percentage of mature eggs compared to the non-treated infected mice and at 4 WPT, more than

50% of the eggs were mature. NTZ treatment resulted in significant increase in the percentage of

dead eggs to 19.50%, 22.25% and 30.5% at 1 ,2 and 4 WPT , respectively with progressive

increase in the mean percentage of total mature eggs (27.25% , p<0.01) at 1 WPT ,(34.25%,

p<0.01) at 2 WPT and (51%, p<0.01) at 4 WPT, respectively as well as progressive reduction in

immature eggs (56%, p<0.05), (43.25% and 18.50%, p<0.01) at 1,2 and 4 WPT , respectively. In

comparison to the non-treated infected mice, MTO resulted in initial and significant increase in

immature eggs to 63.25%, p<0.05 at 1WPT followed by highly significant reduction to 47% at 2

WPT and 24.5%, p<0.01) at 4 WPT. MTO also induced an initial and significant reduction in

mature eggs to 20.25% at 1 WPT followed by significant increase to (33% and 49% p<0.01) at 2

and 4 WPT, respectively. The mean percentage of the dead eggs increased significantly (16.50%,

20%, 26.5%, p<0.01) at 1, 2 and 4 WPT, respectively (table VII, figure 18).

61

Table (VII): The oogram pattern (percentage egg developmental stages) in the intestine of S.

mansoni-infected mice under different treatments in different follow up periods.

Type of egg

WPT

Mice Groups

NTZ MTO MZD PZQ Infected

Non-treated

Immature

1

56.00±0.35a 63.25±1.99a 55.50±4.42 3.67±0.03A 59.60±2.28

2

43.25±2.50A 47.00±0.16A 23.25±0.99A 2.60±0.05A 61.50±3.00

4

18.50±0.71A 24.50±0.54A 10.77±0.52A 1.50±0.09A 62.50±0.71

Mature

1

27.25±0.50a 20.25±0.99A 19.37±2.16A 16.67±0.84A 32.60±1.99

2

34.25±1.02A 33.00±1.72A 39.75±1.71A 14.20±0.76A 27.00±0.16

4

51.00±1.41A 49.00±4.24A 52.00±3.00A 13.00±1.16A 24.50±0.12

Dead

1

19.50±0.89A 16.50±1.08A 24.83±2.62A 79.87±5.20A 7.80±0.59

2

22.25±0.99A 20.00±0.16A 37.00±2.55A 83.20±5.54A 11.50±0.38

4

30.50±0.71A 26.50±0.71A 37.33±2.52A 85.75±3.30A 12.00±0.41

WPT=weeks post-treatment, NTZ = Nitazoxanide, MTO= Myrrh total oil, MZD=Mirazid, PZQ=

praziquantel. The statistical test was done by independent two-sample t-test. Values expressed as

mean percentage ± SD.

a: Statistically significant at P.value < 0.05.

A : Statistically highly significant at P.value < 0.01.

62

Figure.18. Percentage egg developmental changes in S.mansoni-infected mice under different

treatments at different periods of follow up.

63

II. Scanning electron microscopic studies:

In non-treated infected mice, there is a difference between male and female teguments as

male tegument shows hill-shaped tubercles covered with pointed spines and convoluted surface of

the tegumental membranes between tubercles but female tegument does not have tubercles

(smooth) with fines spines on the surface especially on the dorsal aspect.Oral and ventral suckers

showed intact features and organization. The gynecophoric canal of male worms showed fine

spines in its inner aspect (figure 19).

PZQ showed a pronounced tegumental damage by scanning electron microscopic

examination of S.mansoni worms recovered from treated mice 2 WPT in the form of extensive

tegumental damage with rupture of tubercles and loss of spines in wide areas in male worms.

Moreover, a marked ulceration in the tegument was detected in the outer surface of female worms.

Some teguments showed severe erosion or even sloughing of tegumental membranes exposing the

underlying muscle layers. The male tegument was more affected than females (figure 20).

MZD showed mild tegumental damage in female and male S. manoni worms without any

obvious deeper effects as the changes were topically confined to the outer surface. There was

focal erosion and ulceration with shrinkage of the outer surface in the female tegument. The

tegumental damage in male worms manifested by rupture of tubercles with marked loss of spines

and if present, lost their sharpness .There was higher sensitivity in the tegumental damages in

males than females.The oral sucker still intact (figure 21). NTZ resulted in mild tegumental

damaging effect manifested by focal lesions in the inter-tubercular ridges, disorganization of the

oral sucker of male worm and loss of spines in the gynecophoric canal (figure 22). MTO resulted

only in oedematous swelling of both oral and ventral suckers without detectable alteration in the

tegument (figure 23).

64

Figure.19.Scanning electron micrographs of

S.mansoni worms recovered from infected non-

treated mice showing normal tegument of male

(A) and female worms (B). Normal ventral

sucker of male worms (C) and oral sucker of

female worms (D).The inner surface of the

gynecophoric canal of male (E) worms.

(T = Tubercles, S= Spines, ITR= Inter-Tubercular

Ridge, VS= Ventral Sucker, OS= Oral Sucker).

T

S

ITR

VS

VS

S

S

S X

OS

S

65

Figure.20. Effect of Praziquantel on the dorsal surface of female schistosoma worms (F) and the

male worms (G) recovered at 2 WPT.

(T = Tubercles, S= Spines, ITR= Inter-Tubercular Ridge, X= Ulceration and erosion).

G

66

Figure.21.Effect of Mirazid on dorsal

aspects of the tegument of female (H), male

(I) S.mansoni worms and ventral sucker of

male worms (J) recovered 2 WPT.

(T=Tubercles,S= Spines, ITR= Inter-Tubercular

Ridge,X=Ulceration and Erosion , VS= Ventral

Sucker).

T S

ITR

X

VS

67

Figure.22. Scanning electron micrographs of

S.mansoni worms recovered from NTZ-treated

mice showing normal tegument of female

worms (K), the tegument of male worms (L)

,the oral sucker of male worms (M), the worm

couple (N) and the gynecophoric canal of the

male (O).

(T = Tubercles, S= Spines, ITR= Inter-Tubercular

Ridge, OS= Oral Sucker).

S

OS

OS

S

ITR ITR

S T

68

Figure.23.Scanning electron micrographs of the dorsal surface (P), oral and ventral suckers (Q) of

male S.msnsoni worms recovered from MTO-treated mice at 2 WPT.

(T=Tubercles, S= Spines, ITR= Inter-Tubercular Ridge, VS= Ventral Sucker, OS= Oral Sucker)

III.Haematological Studies

1-Erythrocytes and their related paramters

Data presented in (table VIII and figure 24-29) showed highly significant and progressive

decrease in RBCs count (13%, 26.8% and 30.8%, p<0.01), haemoglobin level (27.5%, 46.6%,

59.4%, p<0.01) and the haematocrit value (HCT) 21.6%,35.6% and 41.7%, p<0.01 in infected

non-treated mice at 8,9 and 11 WPI respectively as compared to the non-treated mice. There was

progressive decrease in red blood cell indices, MCV (9.7%, 12.8%, 25%, p<0.01), MCH (16.8%,

27.14%, 47.88%, p<0.01) and MCHC (7.5%, 17.2%, 30.33%, p<0.01).

Treatment of infected mice with PZQ resulted in progressive increase in the RBCs count

(4%,16.9% and 12.9%), HB level (11.9%,57% and 107.6%), HCT% (12.5%,40.1% and 42.5%),

red blood cell indices [MCV (3.2%,20% and 26.2%),MCH (7.3%,34.12% and 83.9%) at 1,2 and 4

WPT and MCHC (12% and 45.7%) at 2 and 4 WPT, respectively as compared to the infected

non-treated mice (table VIII and figure 24-29).

Treatment with MZD in a dose of 500 mg/kg for 5 days in infected mice resulted in

insignificant increase in RBCs count (1.4%) , HB level (3.6%) and HCT value (0%), red blood

cell indices; MCV (1.5%), MCH (1.9%) and MCHC (3.6%) at 1 WPT but after the 2nd

week of

treatment , the drug was able significantly to elevate RBCs count (15%) ,HB (44.9%), and HCT

value (13.5%), red blood cell indices ;MCHC (27.4%) but not the MCV and MCH . After the 4th

O

69

week of treatment, the drug elevated RBCs count (11%), HB (98.4%), HCT (35%), MCV (10%),

MCH (61.2%) and MCHC (46.7%) (Table VIII and figure 24-29). NTZ 100 mg/kg orally for 7

days to S. mansoni-infected mice at 7 WPI was unable to induce elevation in HB level (5.4%),

PCV (3.1%) and red blood cell indices (MCV,3.1%, MCH 5% and MCHC 2.2%) in comparison

to non-treated group, but at 2 WPT, significant elevation was reported in the HB level

(28.7%),PCV% (28.2%) and red blood cell indices (MCV,20%, MCH 20.5%) in comparison to

the infected non-treated group but there was non-significant effect on MCHC. At 4 weeks after

treatment, significant elevation in the HB level (64%), PCV (32.9%) and red blood cell indices

(MCV,22.9%, MCH 51.5% and MCHC 22.8%) were observed in comparison to non-treated

group but RBCs counts had insignificant change (table VIII and figure 24-29). With MTO

treatment, the only significant changes were the elevation in HB (16.9%) at 4 WPT and in some

red blood cell indices (MCH,18.8% at 2 WPT and 17.9% at 4 WPT), MCHC, 16.1% at 4 WPT

(table VIII and figure 24-29).

70

Table (VIII): Erythrocytes and their related red blood cell indices in S. mansoni-infected

mice under different treatments at different follow up periods.

Parameter

/Group WPT NTZ MTO MZD PZQ

Infected

Non-treated

Non-

infected

Non-

infected

RBCs

(106 Cell/ul)

1 7.00±0.46

(0%)

6.27±0.90

(-10.4%)

7.10±0.07

(+1.4%)

7.28±0.60

(+4%)

7.00±0.23A

[-13%]

8.05±0.14

2 6.40±0.57

(+6.6%)

5.70±0.71

(-5%)

6.90±0.73b

(+15%)

7.00±0.67b

(+16.9%)

6.00±0.27A

[-26.8%]

8.20±0.48

4 6.70±0.13

(+8%)

6.10±0.06

(-1.6%)

6.90±0.22b

(+11%)

7.00±0.01b

(+12.9%)

6.20±0.53A

[-30.8%]

8.96±0.63

HB

(g/dl)

1 9.70±0.95

(+5.4%)

8.98±0.61

(-2.3%)

9.54±0.35

(+3.6%)

10.30±0.44b

(+11.9%)

9.20±0.77A

[-27.5%]

12.70±0.57

2 9.10±0.28B

(+28.7%)

8.00±0.57

(+13.1%)

10.25±1.0B

(+44.9%)

11.10±0.61B

(+57%)

7.07±0.90A

[-46.6%]

13.26±1.00

4 9.40±0.56B

(+64%)

6.70±0.73b

(+16.9%)

11.37±1.11B

(+98.4%)

11.90±0.61B

(+107.6%)

5.73±0.36A

[-59.4%]

14.12±1.69

PCV

(%)

1 33.00±1.51

(+3.1%)

29.00±1.00

(-9.3%)

32.00±1.15

(0%)

36.00±1.81b

(+12.5%)

32.00±3.21A

[-21.6%]

40.82±1.30

2 35.00±1.56B

(+28.2%)

28.5±1.48

(+4.3%)

31.00±2.55b

(+13.5%)

38.27±1.12B

(+40.1%)

27.30±2.60A

[-35.6%]

42.40±1.95

4 39.50±0.71B

(+32.9%)

29.90±1.41

(+0.6%)

40.10±2.03B

(+35%)

42.33±2.52B

(+42.5%)

29.70±2.03A

[-41.7%]

50.98±3.04

MCV

(fl)

1 47.14±2.57

(+3.1%)

46.20±2.85

(+1.2%)

45.00±2.55

(-1.5%)

47.20±.02

(+3.2%)

45.70±3.66a

[-9.7%]

50.83±3.59

2 54.60±1.70B

(+20%)

50.00±3.14

(+9.8%)

44.90±2.12

(-1.3%)

54.60±2.53B

(+20%)

45.50±4.06a

[-12.85%]

52.21±3.00

4 58.90±4.27B

(+22.9%)

49.00±3.51

(+2.2%)

52.70±2.57b

(+10%)

60.47±2.03B

(+26.2%)

47.90±4.10A

[-25%]

64.04±3.68

MCH

(pg)

1 13.80±0.30

(+5%)

14.30±0.90

(+8.8%)

13.40±0.81

(+1.9%)

14.10±1.05

(+7.3%)

13.14±1.02A

[-16.8%]

15.81±0.61

2 14.20±0.35B

(+20.5%)

14.00±0.41B

(+18.8%)

14.80±0.57B

(+25.6%)

15.80±1.46B

(+34.12%)

11.78±0.70A

[27.14%]

16.17±0.56

4 14.00±1.03B

(+51.5%)

10.90±0.24B

(+17.9%)

16.40±0.84B

(+61.2%)

17.00±1.00B

(+83.9%)

9.24±0.55A

[-47.88%]

17.73±0.70

MCHC

(%)

1 29.39±1.40

(+2.2%)

30.90±1.38

(+7.4%)

29.80±1.73

(+3.6%)

28.60±1.98

(-0.5%)

28.75±3.32

[-7.5%]

31.11±2.57

2 26.00±1.28

(+0.4%)

28.00±1.35

(+8.1%)

33.00±1.58B

(+27.4%)

29.00±1.86b

(+12%)

25.89±2.07A

[-17.2%]

31.27±1.85

4 23.70±1.87B

(+22.8%)

22.40±1.47B

(+16.1%)

28.30±1.40B

(+46.7%)

28.11±1.42B

(+45.7%)

19.29±1.56A

[-30.33%]

27.69±1.84

NTZ=Nitazoxanide, MTO= Myrrh total oil, MZD=Mirazid, PZQ= Praziquantel, WPT: weeks post-

treatment , MCV=mean cell volume, fl= femtolitre (10-15

) g/dl , MCH=mean corpuscular haemoglobin,

pg = picogram (10-12

)g/dl, MCHC=mean corpuscular haemoglobin concentration. RBCs: Red blood cells,

HB: Haemoglobin, PCV: packed cell volume. The statistical test was done by independent two-

sample t-test. Values were expressed as mean ± SD. Numbers in parentheses indicate the percentage

change, Numbers in parentheses [ ] indicate the percentage of change in relation to non- infected non-

treated mice and Numbers in parentheses ( ) indicate the percentage of change in relation to infected non-

treated.

a: Statistically significant at P < 0.05 compared to non-infected,

A: highly significant at P < 0.01compared to non-infected,

b : Statistically significant at P < 0.05 compared to non-treated ,

B: highly significant at P < 0.01 compared to non-treated.

71

Figure.24. Mean RBCs counts in S.mansoni-infected mice under different treatments at different

follow up periods.

Figure.25. Mean Haemoglobin levels in the blood of S. mansoni-infected mice under different

treatments at different follow up periods.

72

Figure.26. Mean Packed cell volumes in the blood of S.mansoni-infected mice under different

treatments at different follow up periods.

Figure. 27. Mean MCV of the RBCs in S.mansoni-infected mice under different treatments at

different follow up periods.

73

Figure.28. Mean MCH in S. mansoni-infected mice under different treatments at different follow

up periods.

Figure.29. Mean MCHC in S.mansoni-infected mice under different treatments at different follow

up periods.

2. Leucocytes and differential leucocytic count:

Infection with S. mansoni was found to cause a significant elevation in the total leucocytic

counts (TLC) or Leukocytosis at 9 and 11 WPI as noticed in the CBC findings of non-treated

infected group compared to the non-infected mice).This study revealed progressive lymphocytosis

in relation to the time of infection as there was significant increased count (5.1%, p<0.05) at 8

WPI. At 9 and 11 WPI, The level of lymphocytosis was increased in high statistical significance

74

(17% and 38.8%, p<0.01) respectively, compared to non-treated non-infected mice). Neutropenia

was evident in an ascending manner in response to infection in non-treated infected mice in a rate

of (24%, 33.3% and 62.1%) at 8, 9 and 11 WPI). Eosinophilia was reported in non-treated

infected mice in progress to infection time as there was increased rate (51.9%, 83.3% and 90.4%)

at 8, 9 and 11 WPI. Monocytes and basophils were insignificantly changed in their count in

response to infection (p>0.05) (table IX, figure 30-35).

Treatment of infected mice with PZQ resulted in non-significant decrease in TLC count

(10.8%,p>0.05) 1 WPT as compared to non-treated infected group, but the drug was able in high

statistical significance to reduce the TLC 17.8% and 48.8% at 2 and 4 WPT, respectively. Also

resulted in significant decrease in lymphocytes (3%, P<0.05) at 1WPT as compared to non-treated

group, the drug was able in high statistical significance to reduce the lymphocytes (31.7% and

57.9 %, P<0.01) at 2 and 4 WPT, respectively. PZQ showed increase in the neutrophils count in

high statistical significance (25.6%, 87.6% and 287.4%, P<0.01) at 1, 2 and 4 WPT, respectively

compared to non-treated infected mice. Also it resulted in significant decrease in esinophils count

(10.1% and 15.1%, P<0.05), (40.2%, P<0.01) at 1, 2 and 4 WPT, respectively.There was no

significant change in the monocytes and basophils in the blood picture of PZQ-treated mice in any

time post-treatment (table IX,figure 30-35).

MZD caused non-significant change in TLC at 1WPT (0.1%, p>0.05). It reduced

leukocytosis significantly at 2 WPT (14.2%, P<0.05) as well as at 4 WPT, the reduction rate was

highly significant (35.1%, P<0.01). The drug caused non-significant change in lymphocytes count

1WPT (1.4%, p>0.05). It reduced lymphocytosis in high statistical significance at 2 or 4 WPT

(18.2% and 42%, P<0.01). MZD caused non-significant increase in neutrophils count at 1WPT

(10.1%, p>0.05), also increased neutrophils in high statistical significance at 2 or 4WPT (53.5%

and 215.7%, P<0.01). Esinophils decreased significantly at 1WPT (10.1%, P<0.05).The

eosinophilia was highly significantly reduced at 2 or 4 WPT (9% and 34.5%, P<0.01),

respectively. There was no change in the monocytes and basophils counts in the MZD-treated

mice in any time post-treatment (table IX, figure 30-35).

In NTZ treatment, the TLC was changed insignificantly (+1.2% and -6.6%, P<0.05) at 1

and 2 WPT compared to the non-treated infected mice, but the drug significantly reduced

leukocytosis (22.5%, P<0.01) at 4 WPT when compared to the non-treated infected mice. The

differential leucocytic counts (DLC) in the blood of NTZ-treated infected mice showed

insignificant increased level of lymphocytes (1.2%, p>0.05, at 1WPT but the level was highly

decreased significantly at 2 and 4 WPT in rates of (7.7% and 8.6%, P<0.01). Decreament of

neutrophils count was insignificantly encountered in a rate of (5.7%, p>0.05) compared to the

75

non-treated mice at 1 WPT, but the drug increased neutrophils significantly in high statistical

level at 2 or 4WPT (21.4% and 137.1%, P<0.01) compared to the non-treated mice. Esinophils

increased in NTZ-treated mice (2.5 %, p>0.05) insignificantly at 1 WPT but significant elevation

was observed at 2 WPT (25.7%, P<0.01) in high statistical significance. After 4 WPT, there was

marked reduction in esinophils count (and 42.4%, P<0.01). Monocytes and basophils were

insignificantly changed in any time post-treatment with NTZ (table IX, figure 30-35).

MTO treatment did not cause significant change in the TLC either at 1, 2 or 4 WPT. MTO-

treated mice showed insignificant increase in the lymphocytes count at 1or 2 WPT, but there was

a significant reduction at 4 WPT (7.8%, P<0.05). MTO insignificantly changed the neutrophils or

esinophils counts at 1or 2 WPT. The oil was able to elevate neutrophils 4 WPT (40.8%, P<0.01)

in high statistical significance and to reduce eosinophilia (7.1%, P<0.05). Monocytes and

basophils were insignificantly changed in the MTO-treated mice in any time post-treatment (table

IX, figure 30-35).

76

Table (IX): Total and Differential Leucocytic Counts in S. mansoni-infected mice under

different treatments at different follow up periods.

Parameters

/Group

WPT

Mice groups

NTZ MTO MZD PZQ Infected

Non-treated

Non-infected

Non-treated

Total

Leucocytes

(103 cell//ul)

1 12.50±1.80

(+1.2%)

12.67±2.52

(+2.6%)

12.36±1.83

(+0.1)

11.00±1.84

(-10.8%)

12.34±2.50

(+20.7%)

10.22±1.91

2 12.95±0.97

(-6.6%)

13.55±4.03

(-2.3%)

11.90±1.70b

(-14.2%)

11.40±0.78B

(-17.8%)

13.87±0.70A

(+46.9%)

9.44±1.29

4 10.95±0.07B

(-22.5%)

12.80±1.3

(-9.4%)

9.17±2.15B

(-35.1)

7.23±0.55B

(-48.8%)

14.13±0.85A

(+101.2%)

7.02±0.62

Lymphocytes

(%)

1 71.90±1.40

(+1.2%)

73.20±1.50

(+3%)

70.00±1.20

(-1.4%)

68.80±1.77b

(-3%)

71.00±3.02a

(+5.1%)

67.50±0.48

2 60.80±1.32B

(-7.7%)

63.70±2.70

(-3.3%)

53.90±1.52B

(-18.2%)

45.00±3.17B

(-31.7%)

65.90±1.51A

(+17%)

56.30±0.05

4 63.00±0.54B

(-8.6%)

63.60±4.95b

(-7.8%)

40.00±1.79B

(-42%)

29.00±1.13B

(-57.9%)

69.00±1.53A

(+38.8%)

49.70±0.71

Neutrophils

(%)

1 18.00±1.50

(-5.7%)

18.00±1.00

(-5.7%)

21.20±1.46

(+10.9%)

24.00±1.58B

(+25.6%)

19.10±1.98A

(-24.5%)

25.30±1.98

2 30.60±1.7B

(+21.4%)

26.00±1.41

(+3.1%)

38.70±1.90B

(+53.5%)

47.30±3.61B

(+87.6%)

25.20±1.15A

(-33.3%)

37.80±0.39

4 37.70±1.9 B

(+137.1%)

22.40±2.55B

(+40.8%)

50.20±1.04B

(+215.7%)

61.60±9.50B

(+287.4%)

15.90±1.74A

(-62.1%)

42.00±0.49

Esinophils

(%)

1 8.10±0.91

(+2.5%)

7.20±2.70

(-8.8%)

7.10±0.65b

(-10.1%)

7.10±0.11b

(-10.1%)

7.90±0.45A

(+51.9%)

5.20±0.37

2 8.30±0.69B

(+25.7%)

8.10±4.03

(22.7%)

6.00±0.32B

(-9%)

5.60±0.25b

(-15.1%)

6.60±0.68

(+83.3%)

3.60±0.28

4 8.00±1.41B

(-42.4%)

12.90±0.85b

(-7.1%)

9.10±0.08B

(-34.5%)

8.30±0.35B

(-40.2%)

13.90±0.14

(+90.4%)

7.30±0.31

Monocytes

(%)

1 1.30±0.05

(0%)

1.10±0.01

(+15.3%)

1.30±0.09

(0%)

1.40±0.03

(-7.6%)

1.30±0.20

(0%)

1.30±0.61

2 1.80±0.21

(0%)

1.80±0.21

(0%)

1.90±0.07

(+5.5%)

1.80±0.26

(-5.2%)

1.90±0.03

(0%)

1.90±0.09

4 1.10±0.08

(0%)

1.10±0.97

(0%)

1.10±0.06

(0%)

1.10±0.01

(-9%)

1.10±0.11

(+10%)

1.00±0.07

Basophils

(%)

1 0.70±0.01

(0%)

0.70±.006

(0%)

0.4±0.01

(-42.8%)

0.70±0.05

(-14.2%)

0.70±0.01

(0%)

0.70±0.01

2 0.30±0.02

(-25%)

0.40±0.05

(0%)

0.30±0.02

(-42.8%)

0.30±0.01

(-25%)

0.40±0.01

(0%)

0.40±0.01

4 0.00±0.00

(0%)

0.00±0.00

(0%)

0.00±0.02

(0%)

0.00±0.00

(0%)

0.00±0.00

(0%)

0.00±0.00

NTZ:Nitazoxanide, MTO=Myrrh total oil, MZD=Mirazid, PZQ= praziquantel, WPT: weeks post-

treatment . The statistical test was done by independent two-sample t-test .Values were expressed as

mean ± SD. Numbers in parentheses indicate the percentage change. a: Statistically significant at P < 0.05

compared to non-infected. A: highly significant at P < 0.01compared to non-infected. b : Statistically

significant at P < 0.05 compared to non-treated. B: highly significant at P < 0.01 compared to non-treated.

77

Figure.30. Mean total leucocytic counts (TLC) in S.mansoni-infected mice under different

treatments at different follow up periods.

Figure.31. Mean Lymphocyte counts in S.mansoni-infected mice under different treatments at

different follow up periods.

78

Figure.32. Mean Neutrophils counts in S.mansoni-infected mice under different treatments at

different follow up periods.

Figure.33. Mean Esinophils counts in S.mansoni-infected mice under different treatments at

different follow up periods.

79

Figure.34. Mean Monocytes counts in S.mansoni-infected mice under different treatments at

different follow up periods.

Figure.35. Mean Basophils counts in S.mansoni-infected mice under different treatments at

different follow up periods.

3. Total Platelet Counts

In the current study, it was noticed that non-treated infected mice showed progressive

thrombocytopenia (the platelet counts decreased in response to the duration of infection compared

to the infected non-treated mice 14.1% (P<0.05), 20.7% (P<0.01) and 33.6% (P<0.01) at 8, 9 and

11 WPI, respectively). With PZQ, MZD and NTZ treatment at 2 or 4 WPT ,the platelet counts

were highly significantly increased 31.9% and 72.1%, (P<0.01), 25.5% and 50.4% (P<0.01) and

80

18.8% and 40.1% (P<0.01), respectively compared to the infected non-treated mice. MTO

treatment decreased platelet counts significantly only at 1WPT (-14.2%, P<0.05) (table X, figure

36).

Table (X): Platelet counts in S.mansoni-infected mice under different treatments at different

follow up periods.

WPT

Mice groups

NTZ MTO MZD PZQ Infected

Non-

treated

Non-Infected

Non-treated

1 8.44±0.09

(+1.4%)

7.13±0.05b

(-14.2%)

8.93±0.11

(+7.3%)

9.13±1.04

(+9.7%)

8.32±0.86a

(-14.1%)

9.68±0.99

2 7.61±0.06B

(+18.8%)

6.13±0.02

(-6.6%)

8.04±0.10B

(+25.5%)

8.45±0.5B

(+31.9%)

6.40±0.46A

(-20.7%) 8.08±0.56

4 8.55±0.07B

(+40.1%)

7.05±0.13

(+15.5%)

9.18±0.04B

(+50.4%)

10.5±1.0B

(+72.1%)

6.10±0.51A

(-33.6%) 9.20±0.15

NTZ=Nitazoxanide, MTO=Myrrh total oil,MZD =Mirazid, PZQ= praziquantel,WPT: weeks post-

treatment , The statistical test was done by independent two-sample t-test .Values were expressed as

mean ± SD, Numbers in parentheses indicate the percentage change.

a: Statistically significant at P value < 0.05 compared to non-infected.

A : Statistically highly significant at P value < 0.01 compared to non-infected.

b : Statistically significant at P value < 0.05 compared to non-treated ,

B: Statistically highly significant at P value < 0.01 compared to non-treated.

Figure.36. Mean Platelet counts in S.mansoni-infected mice under different treatments at different

follow up periods.

81

IV. Biochemical Studies:

Regarding the activity of liver enzymes, S.mansoni-infected non-treated mice showed

high significant elevation of serum ALT (54.5%, 80.6% and 202.2%,), AST (45.8%, 49.2% and

79.5%) and ALP level (123.2%, 127.9% and 212.2%) as compared to the non-infected mice at 8,

9 and 11 WPI as seen in (table XI, figures 37). Under the effect of PZQ, serum ALT decreased

significantly 1 WPT (29.1%). At 2 and 4 WPT, there was more reduction (35.1% and 53.5%)

compared to the infected non-treated mice. Serum level of AST decreased insignificantly (7.6%)

at 1WPT but there was high statistically significant decrease (21.3% and 50.3%) at 2 and 4 WPT

as compared to the non-treated mice. The serum level of ALP decreased insignificantly (30%) at

the 1st week post-therapy but ALP activity was highly significantly decreased at 2 and 4 WPT

(43.2% and 65.5%) as compared to the non-treated mice (table XI, figures 37).

MZD treatment of infected mice resulted in high significant reduction in ALT activity at 1,

2 and 4 weeks after treatment (23.5%, 25.9 % and 40.1%) as compared to the infected non-treated

mice. One week after treatment with MZD; the serum level of AST decreased insignificantly

(8.3%). MZD reduced significantly AST activity at 2 weeks after treatment (9.7%) but the drug

highly significantly reduced AST level 4 WPT (40.2%). One week after treatment with MZD; the

serum level of ALP decreased insignificantly (20.6%). MZD reduced highly significantly ALP

activity at 2 and 4 WPT (32.2%, 49.3%).

NTZ treatment resulted in highly significant reduction in ALT activity at 1, 2 and 4 weeks

after treatment (16.6%, 26.4% and 30.7%) as compared to the non-treated mice. The serum AST

level decreased insignificantly (7.4% and 9%) at 1 or 2 WPT but decreased significantly at 4 WPT

(22.9%).The serum ALP level decreased insignificantly at 1 WPT (11.4%) but was significantly

reduced at 2 and 4 WPT (20.2% and 40.7%) (Table XI, figures 37).

MTO treatment of S.mansoni-infected mice resulted in significant increase in ALT activity

at 1 WPT (10.2%) as compared to the non-treated mice. There was insignificant change in ALT

after 2 weeks of treatment (0.5%) but the oil could reduce ALT significantly (15.2%) at 4 WPT.

AST activity increased (18.3%) at 1WPT but insignificant change occurred at 2 WPT (2.4%). At

4 WPT, the oil reduced the serum AST significantly (29.8%). MTO resulted in insignificant

reduction in serum ALP activity at 1WPT (1.7%) but significant decrease was achieved at 2 and 4

WPT (18% and 25.9%) (table XI, figures 37).

82

Table (XI): Liver function tests in S. mansoni-infected mice treated with different drugs at

different times.

Liver

Enzymes

WPT

Mice Groups

NTZ MTO MZD PZQ Infected

Non-treated

Non-infected

Non-treated

ALT

(U/L)

1 53.67±3.11B

(-16.6%)

71.00±5.49b

(+ 10.2%)

49.25±4.66B

(-23.5%)

45.60±1.40B

(-29.1%)

64.40±3.90A

(54.5%)

41.37±6.21

2 68.00±4.49B

(-26.4%)

93.00±6.66

(+0.5%)

68.50±8.61B

(-25.9%)

60.00±6.56B

(-35.1%)

92.50±3.54A

(80.6%)

51.20±7.96

4 69.50±7.02B

(-30.7%)

85.00±5.90B

(-15.2%)

60.00±6.79B

(-40.1%)

53.75±6.27B

(-53.5%)

100.33±6.51A

(202.2%)

33.20±5.89

AST

(U/L)

1 110.67±9.66

(-7.4%)

141.57±7.99B

(+18.3%)

109.67±4.93

(-8.3%)

110.50±10.66

(-7.6%)

119.60±11.00A

(45.8%)

82.00±6.96

2 131.00±12.83

(-9%)

140.50±11.85

(-2.7%)

130.00±12.7b

(-9.7%)

113.33±11.73B

(-21.3%)

144.00±7.13A

(49.2%)

96.50±7.92

4 122.50±9.61B

(-22.9%)

111.50±4.75B

(-29.8%)

95.00±9.08B

(-40.25%)

79.00±4.85B

(-50.3%)

159.00±8.17A

(79.52%)

88.57±9.24

ALP

(U/L)

1 99.33±29.70

(-11.4%)

110.25±22.60

(-1.7%)

89.00±6.24

(-20.6%)

78.50±26.41

(-30%)

112.20±37.60A

(123.2%)

50.25±17.71

2 139.50±8.00B

(-20.2%)

143.50±7.78B

(-18%)

118.50±8.33B

(-32.2%)

99.33±4.16B

(-43.2%)

175.00±9.40A

(127.9%)

76.77±2.01

4 92.00±5.56B

(-40.7%)

115.00±7.21B

(-25.9%)

78.67±3.32B

(-49.3%)

53.50±4.57B

(-65.5%)

155.33±6.01A

(212.2%)

49.75±2.41

WPT:weeks post-treatment, NTZ=Nitazoxanide, MTO= Myrrh total oil, MZD=Mirazid, PZQ=

praziquantel. The statistical test was done by independent two-sample t-test .Values were expressed as

mean ± SD, Numbers in parentheses indicate the percentage of change.

a: Statistically significant at P value < 0.05 compared to non-infected.

A : Statistically highly significant at P value < 0.01 compared to non-infected.

b : Statistically significant at P value < 0.05 compared to non-treated.

B: Statistically highly significant at P value < 0.01 compared to non-treated.

83

Figure.37. Liver functions tests {ALT(A), AST(B), ALP(C)} activity in S.mansoni-infected mice

under different treatments at different follow up periods.

B

A

C

84

As regards the kidney functions, the blood urea and serum creatinine in S.mansoni-

infected mice increased in response to the period of infection as they were progressively raised the

blood urea level (61.2%, 88.3%, 255.3%, p<0.01) and the serum creatinine level (17.1%, p>0.05,

78.5%, p<0.05 and 150%, p<0.01) at 8, 9 and 11 WPI. Treatment of infected mice with PZQ 500

mg/kg for 2 days at 7 weeks post-infection resulted in highly significantly reduced blood urea

(12.5%, 33.9% and 60.2%, p<0.01) at 1,2 and 4 WPT. The serum creatinine changed

insignificantly after 1 and 2 weeks of treatment (26.4% and 28%, p>0.05) respectively but the

drug reduced creatinine at 4 WPT, (48.2%, p<0.05) (table XII, figures 38).

Treatment of infected mice with MZD 500 mg/kg for 5 days at 7 weeks post-infection

resulted in highly significantly reduced blood urea (21.3%,26.8% and 45.7%, p<0.01) at 1,2 and

4 WPT. The serum creatinine was insignificantly changed after 1 and 2 weeks of treatment

(20.7% and 5.6%, p>0.05) respectively but the drug reduced creatinine at 4 WPT, (33.1%,

p<0.05). NTZ-treated mice after one week of treatment with 100 mg/kg for 7 days showed

insignificant increase in blood urea (7.5 %,p>0.05) but highly significantly decreased urea level

at 2 and 4 WPT(26.3% and 33.4%, p<0.01). Serum creatinine changed insignificantly at 1, 2 and

4 WPT (+22.6%, 0% and -24.1%, p>0.05). MTO-treated mice showed highly significant increase

in blood urea at 1WPT (25%,p<0.01). At 2 weeks after treatment, the oil insignificantly reduced

the blood urea (10%, p>0.05).The oil highly significantly reduced the blood urea at 4 weeks after

treatment (28%, p <0.01). The serum creatinine was highly significantly increased at 1 or 2

weeks after treatment (50% and 12%, p<0.01) but was insignificantly reduced at 4 WPT (10.3%,

p>0.05) (table XII, figures 38).

85

Table (XII): Kidney function tests in S. mansoni-infected mice under different treatments at

different follow up periods.

Kidney

functions

WPT

Mice groups

NTZ MTO MZD PZQ Infected

Non-treated

Non-infected

Non-treated

Blood

Urea

(mg/dl)

1 43.00±5.47

(+7.5%)

50.00±6.89B

(+25%)

31.45±2.58B

(-21.3%)

35.00±2.60B

(-12.5%)

40.00±2.92A

(+61.2%)

24.80±1.74

2 40.50±6.36B

(-26.3%)

49.50±4.95

(-10%)

40.25±3.89B

(-26.8%)

36.33±6.03B

(-33.9%)

55.00±4.14A

(+88.3%)

29.20±2.66

4 49.00±9.90B

(-33.4%)

53.00±4.24B

(-28%)

40.00±3.00B

(-45.7%)

29.25±3.30B

(-60.2%)

73.67±7.75A

(+255.3%)

20.73±3.84

Serum

Creatinine

(mg/dl)

1 1.30±0.06

(+22.6%)

1.59±0.22B

(+50%)

1.28±0.09

(+20.7%)

0.78±0.31

(-26.4%)

1.06±0.30

(+17.7%)

0.9±0.44

2 1.25±0.05

(0%)

1.40±0.02B

(+12%)

1.18±0.08

(-5.6%)

0.90±0.36

(-28%)

1.25±0.09a

(+78.5%)

0.70±0.43

4 1.10±0.02

(-24.1%)

1.30±0.07

(-10.3%)

0.90±0.01b

(-33.1%)

0.75±0.24b

(-48.2%)

1.45±0.54A

(+150%)

0.58±0.17

WPT: weeks post-treatment, NTZ = Nitazoxanide,MTO= Myrrh total oil, MZD=Mirazid, PZQ=

praziquantel , The statistical test was done by independent two-sample t-test .Values were expressed

as mean ± SD,

Numbers in parentheses indicate the percentage change.

a: Statistically significant at P value < 0.05 compared to non-infected.

A : Statistically highly significant at P value < 0.01 compared to non-infected.

b : Statistically significant at P value < 0.05 compared to non-treated ,

B: Statistically highly significant at P value < 0.01 compared to non-treated.

86

Figure.38. Kidney functions {Blood urea (A) and Serum creatinine (B)} in S.mansoni-infected

mice under different treatments at different follow up periods.

AChE activity in S.mansoni-infected mice 8 WPI with 100 cercariae showed progressive

decrease with the time of infection as there was significant decrease 11.4%,p<0.05 at 8WPI, and

at 9 and 11 WPI , there was highly significant decrease in blood AChE activity 19.8%, 23.5%,

p<0.01,respectively) which may be due hepatocellular damage and consequently low serum

protiens or may secretion of toxins by the adult schistosomes inhibiting the enzyme activity.

Treatment of infected mice with PZQ insignificantly raised the depressed level of blood

AChE at 1WPT (3.5%, p>0.05), while at 2 and 4 WPT, the drug increased highly significantly the

AChE level (22.5% and 31.8%, p<0.01) in comparison to the non-treated group.This effect may

be due higher reduction of adult worms and/or amelioration of hepatic damage. MZD-treated

mice showed progressive improvement in blood AChE (10.3%, p<0.05), (16.2% and 25.4%,

A

B

87

p<0.01) at 1, 2 and 4 WPT, respectively. NTZ decreased the blood AChE activity insignificantly

(4.4%, p>0.05) at 1 WPT. But it had the power to elevate the enzyme level at 2 and 4 WPT with

high significant difference (13.1 % and 16.2%, p<0.01 respectively). MTO significantly decreased

the blood AChE activity (23%, p<0.05) compared to the non-treated group at 1 WPT in spite of its

lower activity on adult worms; may be due inhibitory effects of the oil on ChE. The oil did not

have the power to elevate the enzyme level at 2 and 4 WPT in treated mice in comparison to the

non-treated group as there was insignificant reduction in enzyme level (16.2% and 4.2% ,p>0.05

respectively) in comparison to the non-treated group (table XIII and figure 39).

Table (XIII): Blood Acetylcholinesterase level in S.mansoni-infected mice under different

treatments at different periods of follow up.

AChE

activity

/ WPT

Mice groups

NTZ MTO MZD PZQ Infected

Non-treated

Non-infected

Non-treated

1 8.60±0.40

(-4.4%)

8.10±0.09b

(-23%)

9.91±1.5b

(+10.3%

9.32±0.19

(+3.5%)

9.00±0.8a

(-11.3%)

10.15±0.65

2 9.05±0.49B

(+13.1%)

7.50±0.55

(-16.2%)

9.30±0.40B

(+16.2%)

9.80±0.40B

(+22.5%)

8.00±0.17A

(-19.8%)

9.98±0.48

4 8.80±0.28B

(+16.2%)

7.25±0.49

(-4.2%)

9.50±0.92B

(+25.4%)

9.98±0.15B

(+31.8%)

7.57±0.66A

(-23.5%)

9.90±0.40

WPT: weeks post-treatment, NTZ=Nitazoxanide, MTO= Myrrh total oil, MZD=Mirazid, PZQ=

praziquantel .The statistical test was done by independent two-sample t-test .Values were expressed

as mean ± SD. Numbers in parentheses indicate the percentage change.

a: Statistically significant at P value < 0.05 compared to non-infected.

A : Statistically highly significant at P value < 0.01 compared to non-infected.

b : Statistically significant at P value < 0.05 compared to non-treated ,

B: Statistically highly significant at P value < 0.01 compared to non-treated.

88

Figure.39. Mean blood acetylcholinesterase levels in S.mansoni-infected mice under different

treatments at different periods of follow up.

89

DISCUSSION

90

DISCUSSION

Discovery of antiparasitic agents is a challenging process, requiring discovery of molecules

with the ability to kill parasites but not their hosts. Although efficacy is central to the success of a

drug, there are many other parameters that impact on the successful development of it as safety is

a vital component of delivering a new antiparasitic drug (227)

. All antiparasitic drugs have been

discovered by empirical screening in parasites (in vitro) or animal models (in vivo) (228,229)

.

Assessment of effectiveness of antischistosomal drugs in murine models of schistosomiasis

depends principally on the parasitological studies (fecal egg counts, worm burden, tissue egg

counts and oogram patterns) (99,103,113)

, and one or more of the following parameters

e.g.,histopathological(110)

,immunological(110)

,molecular(113)

,histochemical(143)

,biochemical(215)

,

haematological(215)

and scanning electron microscopic studies (99,111)

.

Changes in patterns of schistosome egg elimination are mostly used to determine the

drug’s effectiveness, in terms of a cure rate (disappearance of eggs) from stool and/or egg

reduction rate (73)

. In this study, examination of fecal samples of infected non-treated control mice

at 49 days (7 WPI) showed the presence of the characteristic eggs of S.mansoni. With day after

day of successive examination, there was an initial rise in the egg-output from 7-8 WPI with

maximum output at 8-10 WPI. There was non-significant change in faecal egg count either in

infected non-treated or treated mice before the day 7 of follow up .This may be due to the fact that

excreted eggs need about 6 days to be fully mature (218)

.

PZQ showed highly significant and sharp reduction in the faecal egg counts at 1WPT

(63%,p<0.01) and complete disappearance of eggs on the 2nd

and 4th

WPT. Similarly Khalil

(2000)(230)

investigated the effect of 600 mg/kg PZQ on murine infection with 120 S. mansoni

cercariae 8 WPI , and found 69.2% faecal egg count reduction at 1WPT and 100 % reduction at 2

or 4 WPT, This is also similar to that reported by Issa (2007) (197)

who found that PZQ (500mg/kg

for 2 days after 45 days of infection with 100 cercariae) was able to reduce faecal egg count

(98.3%) in S. mansoni infected mice at 2 WPT. Lower rates were reported by Abou El-Maatti et

al.,(2006)(231)

and Abdel-Hady et al.,(2013)(232)

who found 50.6 % and 73.5% reduction,

respectively in faecal egg count of S. mansoni- infected mice (with 100 cercariae at 6 WPI)

treated with PZQ (500 mg/kg bw single oral dose or for 2 days) 4 WPT. Botros et al. (2005) (121)

and Barakat et al.(2005) (122)

examined the efficacy of PZQ in patients infected with human

91

schistosomiasis mansoni in a dose of 40 mg/kg and the cure rate varied from 62.5-79.7% and egg

reduction rate (40-88%) at 3-6 WPT.

MZD in this experimental study, resulted in non-significant (4.5%) reduction in the number

of S.mansoni eggs 1WPT, but it caused highly significant reduction (39.5%, 69.6%,p<0.01) in

faecal egg count of treated mice when compared to the infected non-treated group at 2 and 4

WPT, nearly as previously reported by Abou El-Maatti et al., (2006) (231)

who found 6.6%,44 %

reduction at 2 and 4 WPT with (MZD 500mg/kg for 5 days),respectively. Lotfy et al.,(2013) (113)

found 97 % reduction in faecal egg count (236 epg at 45 days PI to 7 epg) at 4 weeks after therapy

in S.mansoni-infected mice treated with MZD (600mg/kg for 6 days). In a study carried out by

Massoud et al., (1998)(115)

who used myrrh in special formulation (consisting of 8.5 parts resins

and 3.5 parts volatile oil in a soft gelatin capsules) given to 62 S. mansoni -infected patients on an

empty stomach in a dose of 11.5 mg/kg for 3 days and patients were examined 1, 2 ,4 and 8 WPT.

Fecal egg count dropped from 181 epg to 4 epg at 8 WPT (98.8 % reduction). Sheir et

al.(2001)(116)

, in a study carried out on 171 patients with schistosomiasis mansoni, examined the

efficacy of MZD in a dose of 10 mg/kg/day for three days. They reported a cure rate of 91.2 % at

2 months after treatment. Gaballah et al. (2001) (117)

enrolled 364 diagnosed cases of S. mansoni

infection under MZD treatment in a dose 10 mg/kg/day for three consecutive days. The cure rates

at 3 months after a single course of treatment were 96.7% by examination of colonic and rectal

snips (with the absence of immature eggs in all snips), 95.9 % by hatching test and 91.8% by Kato

smear. Abo-Madyan et al.,(2004) (118)

stated that the parasitological cure rate by Kato-Katz

technique among 26 S. mansoni-infected patients was 88.5% and 96.2% at 2 and 3 months post-

treatment with MZD (600mg daily on an empty stomach before breakfast for 6 days) respectively

and the egg count dropped from 280 epg to 16 epg (egg reduction =94%) at 2 months PT and eggs

were dropped from 440 to 8 epg at 3 months after treatment (egg reduction = 98%).Soliman et al.

(2004)(119)

examined the effectiveness of MZD on 8 children infected with schistosomiasis

mansoni in a dose of 10 mg/kg/day an hour before breakfast for 3 consecutive days. They reported

that the parasitological cure (by kato-katz and rectal snip techniques) was 100 % 3 months post-

drug administration.

In contrary to these findings, Botros et al. (2005) (121)

used MZD in a dose of 300 mg daily

for three days for household members (n=79) and school children (n=55). At 5-6 weeks post-

treatment, MZD showed low parasitological cure rates of 8.9% and 9.1% in household members

and school children, respectively. Osman et al., (2010) (123)

and Yakoot (2010) (233)

explained that

the low cure rate observed by Botros et al. (2005) was due to using a sub-theraputic dose of MZD

and they had estimated the effectiveness at 4-6 weeks after treatment. Similarly, Barakat et al.,

92

(2005) (122)

conducted a study to assess the activity of MZD in 45 S.mansoni-infected patients;

MZD was given in a dose of 600 mg daily for three consecutive days. The drug was given twice

with a three-week interval. The cure rate was very low, 15.6% after the first treatment, and 8.9%

after the second treatment, respectively. Osman et al.(2010) (123)

in a field study in Abis area

,Alexandria ; found low cure rate (14.8%) at 1 month after treatment of 31 S.mansoni-infected

patients with full dose of Mirazid capsules (600 mg for 6 consecutive days) on empty stomach 1

hour before breakfast. The cure rate dropped to 3.7% after 2 months with non-significant egg

count reduction.

As regards the effect of NTZ on egg count in the stool of S.mansoni -infected mice, it

caused non-significant reduction (4.9%) in faecal eggs one week after treatment but it resulted in

highly significant reduction (22.5%) 2 WPT and this value was elevated with high statistical

significance at 4WPT to (50.6 %). On the other hand ,MTO in a dose of 18 mg/kg for 3 days to

S.mansoni-infected mice resulted in insignificant change in faecal egg count (+12.1% and

9.8%,p>0.05) after the 1st and 2

nd WPT and caused very modest reducible effect at 4 WPT

(12.5%). These results do not agree with the findings of Abou-El Maaty (2002)(189)

who used the

oil in doses 250 mg/kg single oral dose in PZQ-resistant S.mansoni-infected mice .It resulted in

73.3% reduction in faecal egg count at 2 WPT and 85% reduction in faecal egg counts at 4 WPT.

Reduction in worm burden is an important parameter for the assessment of

antischistosomal activity of drugs in laboratory animals but not human (107)

. The activity of a

compound surpasses criteria established by the World Health Organization for potential lead

compounds for schistosomiasis: active `hits' should result in 100% inhibition of motility of adult

parasites at 5 μg/ml in vitro and highly active lead compounds are defined as those with 80%

reduction in worm burdens in vivo experimental models to be considered as a promising potential

for further human clinical evaluation (234)

.

Treatment of S.mansoni-infected mice at 7 WPI with PZQ in a dose of 500mg/kg for two

successive days caused pronounced curative effects, where the mean number of total worms

showed a highly significant reduction (83%, 94% and 97%) at 1, 2 and 4 WPT, respectively.

Botros et al. (2007) (235)

used the same dose of the drug ,they reported that the total worm

reduction was 93% at 1 WPT. Khalil (2000)(230)

found 69.2%,92.3% and 100% reduction in worm

load in PZQ-treated mice (600mg/kg single oral dose at 7 WPI with 100 S. mansoni cercariae by

paddling technique) at 1,2 and 4 WPT, respectively. Also, Emam et al., (2009) (109)

found 100 %

worm reduction in PZQ-treated mice 2 WPT (500mg/kg for 2 days 8 WPI). Bakr et al. (2009) (99)

and Helmy et al.(2009) (131)

used PZQ 500 mg/kg for two successive days in S.mansoni-infected

93

mice, the drug caused 91.47% and 97.2 % total worm reduction at 2 and 4 weeks after cessation

of therapy, respectively. On the contrary , Sharaf (2004)(98)

found that a lower reduction in total

worm recovery (58.9%) in infected female mice 2 WPT with 400 mg/kg PZQ single oral dose 7

WPI with 150 Puerto Rican strain of S.mansoni S/C.

In this study, MZD reduced significantly the total worm burden 34%, 50% and 71% at 1, 2

and 4 WPT, respectively. Massoud et al., (2004) (103)

reported a strong antischistosomal activity of

MZD (500 mg/kg b w for 5 days 45 days post-infection) against S. mansoni worms as it resulted

in lethality of 98.46% of the worms 5 WPT. Bakr et al. (2009)(99)

examined the activity of MZD

in doses of 500 mg/kg for 3 days in S.mansoni-infected mice.There was 82.5% reduction in the

total worm load 1 month after treatment . Hamed and Hetta (2005) (104)

reported that MZD in a

dose of 600 mg/kg for 3 days in S.mansoni-infected mice at 8 WPI leads to 81.1 % worm

reduction at 27 days PT. On contrary to these results, Badria et al. (2001) (105)

used the drug in a

dose of 500 mg/kg twice a day for 3 days in S.mansoni-infected mice. MZD recorded 75% worm

burden reduction at five days PT. Lotfy et al., (2013) (113)

used MZD (600 mg/kg for 5 days) in

murine model of schistosomiasis mansoni .There was significant worm load reduction (69.3%) at

4 WPT. El-Gamal et al., (2009) (110)

studied single dose of MZD 250 mg/kg for S.mansoni

infected mice at 6 weeks after infection.The drug killed 58.6% of total worms at 2 WPT. In the

same way,Guirguis and Mahmoud(2003) (106)

,Botros et al. (2004)(107)

,Ebeid et al. (2005)(108)

,

Ramzy et al. (2010) (111)

, Abdul-Ghani et al. (2010) (112)

and EL-Malky et al. (2013) (114)

found

low worm burden reduction rates not more than 10% either in S.mansoni , S.haematobium or

S.japonicum-infected mice or hamster treated with MZD in oral doses varied from 250-500 mg/kg

for 2-5 days. This may be due to low dose of the drug, less days of administration or infection

with lower numbers of cercariae.

In the current study, NTZ-treated group showed significant reduction in total number of

worms (45% and 65%) at 2 and 4 WPT, respectively). These results agree with Abdel-Rahman et

al., (1997) (149)

who reported 59.91% reduction in the hepatic worm load in S.mansoni-infected

mice treated with NTZ in a dose of 65 mg/kg twice daily for 7 days and contradict with Abdulla et

al. (2009)(150)

who found that NTZ (100 mg/kg once or twice daily for 4 days) had no effect on

worm burden in S.mansoni-infected mice .

MTO in doses of 18mg/kg for 3 days in infected mice gained non-significant rate of total

worm reduction at 1 and 2 WPT but the oil possessed significant rate of worm reduction (29%) at

4 WPT. On the contrary, Massoud (1999) (102)

reported that the volatile oil of myrrh (in doses of

15 and 30 mg/kg bw for 3 days) in S. mansoni-infected hamster reduced total worm load 55.1% to

79.9%) at 1, 2 and 4 WPT, respectively. Abo-El-Maaty (2002)(189)

found 74.6%,80.3% total worm

94

reduction when the myrrh oil was used at a dose of 250 mg/kg in mice infected with 100 PZQ-

resistant S.mansoni cercariae at 2 and 4 WPT. This is due to use of different animal model or dose

or different type of myrrh oil.

Regarding the effect of various drugs on worm sex, Our findings showed equal sensitivity of

female and male worms after treatment with PZQ at 1,2 or 4 WPT as there was significant

reduction (83%,94% and 96%) in male worms while (83%,96% and 98%) of female worms were

reduced, respectively. Similar results were reported by Nessim and Demerdash (2000) (236)

, Botros

et al., (2004) (107)

and Sewify (2009) (215)

. Other studies by Xiao and Catto (1989) (237)

in vitro or

Gonnert and Andrews (1977) (238)

, Khalil (2000) (230)

and Seif-El-Din et al. (2013) (239)

in vivo,

demonstrated that PZQ was more killer to adult male worms than female ones but Bakr et al.,

(2009) (99)

and Soliman et al., (2012) (240)

demonstrated that female worms were more affected than

males.

In the present work, MZD decreased the numbers of both male and female worms, yet males

were more affected than females at 1 and 2 WPT as there was significant reduction (38.4% and

57.1%) of the male worms and (25.3% and 35.3%) of female worms .At 4 WPT, there was

significant reduction of both male and female worms (77% and 60%).Botros et al., (2004) (107)

found that MZD resulted in non-significant reduction in male and female worms 2 WPT(12.4%

and 24.5%), respectively. Bakr et al. (2009)(99)

found 75.1% and 83.8% reduction in male and

female worms recovered from MZD-treated mice (500mg/kg for 3 days) and sacrificed 4 WPT.

The difference in rates may be due to the number of cercariae used for infection of mice or the

dose of drug.

NTZ decreased the the number of recovered males during the period of the study (28%,48%

and 68%) of male worms more than the number of females (24%,38% and 60%) . These results

contradict with that reported byAbdulla et al. (2009) (150)

who found no different effect of NTZ on

both sex of recovered worms.

MTO affected male worms more than females at study time of recovery (1,2 or 4 WPT) as

there was significant reduction in male worms (14%,36% and 36%) but the changes in female

worm count was not-significant at any time of the follow up period (0%,10% and 17% reduction).

These results were contradictory to that mentioned by Massoud (1999) (102)

who reported lethal

activity of myrrh oil at 1,2 and 4 weeks of treatment with 15 mg/kg for 3 days with equal

sensitivity on both sexes .

In this study, The effect of the studied medications on the body length of both male and

female worms indicated that PZQ had equal sensitivity in shortening of worm length as it caused

reduction in the body length of female worms 26.1%, 45.2% and 65.6% and in male worms 25%,

95

50% and 61% at 1, 2 and 4 WPT, respectively. This result agree with Eissa et al., (2004)(145)

who

examined S.mansoni worms recovered from PZQ-treated mice and reported that the mean length

of worms decreased 2 days after treatment . At 1, 2 and 4 WPT, MZD reduced body length of male

worms 11.8%, 40.4% and 41% more than in female worms 3%, 20.5% and 33.6% respectively.

Nearly similar result was reported by Hassan et al., (2003) (97)

who observed that MZD caused 48

% shortening in the length of worms after in vitro exposure.

The number of schistosome eggs found in the tissues of infected animals is affected by the

number of eggs laid by the worm (about 300 eggs /day), the number passed in the excreta (about

50%) and the number destroyed in the tissues of the host. It was found that the eggs per worm pair

increase in the tissues with time in a nearly linear fashion .The use of different antischistosomal

drugs for the treatment of S.mansoni infection seemed to account for the apparent destruction of

eggs in tissues (34)

.

In the current work, the infected non-treated mice were loaded with higher number of eggs

in the intestinal tissues than the hepatic tissue as reported by Botros et al.(2004) (107)

, Sewify

(2009) (215)

and Seif El-Din (2010) (132)

but against Khalil(2000) (230)

who reported that the hepatic

tissue was more loaded with eggs than the intestinal one. PZQ-treated mice showed significant

reduction in tissue egg load in both liver and intestine as it was able to reduce the intestinal egg

load (69.9%-88%) more than the reduction in the hepatic tissues (64.4%-85.8%). These results

agree with El-Shafei et al. (2002) (241)

who reported that PZQ reduced 98.9% of the intestinal eggs

and 87.9% of the hepatic eggs at 2 WPT. Also, Botros et al., (2004) (107)

found 90% reduction of

the intestinal eggs and 60% of the hepatic eggs. Khalil (2000) (230)

found that PZQ reduced eggs in

the intestines (77.5%- 93%) more than that present in the liver (69%-89%) at 1,2 and 4 WPT.

Regarding the effect of MZD, it produced significant reduction in the intestinal egg loads

(28.9%-66%) and non-significant reduction in the hepatic tissue egg load at 1WPT (22.2%) but it

was able significantly to reduce the egg count later on in the 2nd

and 4th

week after therapy (42.8%

and 65.3%). Higher tissue egg count reduction was reported by Massoud et al., (2004) (103)

who

found 98.2% and 97% reduction in the intestinal and hepatic tissue egg count, respectively at 5

WPT but Bakr et al., (2009) (99)

found lower percentage reduction (41% and 28.9%) in hepatic and

intestinal tissue egg count 4 WPT (MZD 500 mg/kg for 3 days). On the contrary, Guirguis and

Mahmoud (2003)(106)

and Botros et al., (2004) (107)

found non-significant change in both intestinal

and hepatic tissue egg load 2 WPT (MZD 300 or 500 mg/kg for 3 or 5 days at 7 WPI).

NTZ was able in high statistical significance to reduce the rate of intestinal egg load

22.1%, 45.2% and 46.4% at 1, 2 and 4 WPT, respectively. It was unable to reduce hepatic tissue

egg load at 1 WPT but significant reduction was achieved (23.8% and 30.7%) at 2 and 4 WPT,

96

respectively. On the other hand, MTO resulted in significant reduction in the rate of intestinal egg

count (12.2%, 23.2% and 31.5%) at 1, 2 and 4 WPT. The oil was able to reduce tissue egg count

in the liver (42.3%) at 4 WPT. The current rates of intestinal and hepatic egg counts are less than

rates reported by Massoud (1999)(102)

who found 60.9%-73.1% reduction in intestinal eggs and

72.9-96.4% reduction in the hepatic eggs after treatment of S. mansoni-infected hamster with the

volatile oil of myrrh in dose of 30mg/kg. Abou-El Maaty (2002) (189)

reported that, the percentage

of ova reduction per gram of liver tissue was 78.7% and 85.5% at 2 and 4 WPT (myrrh oil 250

mg/kg). This may be due to using of different routes of S. mansoni cercariae infection, different

doses or different animals.

Changes in the number and character of eggs (oogram) provide a simple, sensitive, and

reliable criterion for the screening of drugs activity against S. mansoni. It assesses the effects of a

drug on oviposition as well as on the maturation and survival of eggs trapped in the intestinal

mucosa (242,243)

. This can be achieved by studying the alteration in the percentages of the various

stages of viable eggs (mature or immature) as well as of the increase in the percentage of dead

eggs in the mucosa of the small intestine.A drug is considered with an antischistosomal activity

when the corresponding oogram shows 50% or more mature or dead eggs and absence of

immature eggs of one or more developmental stages (218)

.

Farag et al. (1978) reported that the changes in the oogram after drug administration is due

to 3 factors, loss of muscle tone with shift of worms, action of the drug on the reproductive organs

of the parasite and death of the worms (244)

.Viable eggs play an important role in immunologically

mediated pathogenesis of schistosomiasis as miracidia in mature eggs secrete antigens which

trigger the host granulomatous reaction. Thus killing eggs in situ might lessen the host reaction

and no response is observed around dead eggs (243)

. Also, an increase of dead eggs can be

considered as hallmark effect for effective antischistosomal role (107)

.

In the present work, the oogram pattern showed that about 60% of eggs in the infected non-

treated group were immature whereas dead eggs constituted only 7-12% at different follow up

periods and the mature eggs formed 24-32% of the total eggs. PZQ induced marked changes in

the oogram pattern as it produced a highly significant increase in the percentage of dead eggs

(79.87 % at 1 WPT, 83.2% at 2 WPT and 85.75% at 4 WPT) as well as significant reduction in

immature eggs where they constituted only 3.67% at 1 WPT, 2.6% at 2 WPT, and 1.5% at 4

WPT, respectively. As regards mature eggs, PZQ induced significant reduction in their percentage

as 13-16% of the eggs in the oogram were mature.These results are similar to that reported by

97

Khalil (2000)(230)

who found that after PZQ therapy, immature ova stage was present as 18% at 1

WPT and complete disappearance occurred in the 2nd

and 4th

week after treatment with

progressive reduction of mature ova stage (28%,12% and 2 %) and marked increase in dead ova

stage (54%,88% and 98%. Similar results were obtained by Nessim and Demerdash (2000) (236)

who reported that in S.mansoni-infected mice 2 weeks post-PZQ administration, all eggs were

dead. This is an important issue because only mature eggs can cross gut tissue to be excreted with

the host’s feces. The possible explanation is that PZQ could be acting on the reproductive system

of female worms. In this situation, eggs could display low development capacity or the dosages

used here were enough to act on the mature eggs. These results were confirmed by morphological

studies through scanning confocal laser microscopy for determination of egg viability (245)

and

functional criteria such as labeling the eggs with the Hoechst 33258 fluorescent probe (246)

and

intestine histopathology (247)

.

MZD resulted in significant increase in the percentage of dead eggs. However, the

reduction was less than that induced by PZQ. MZD produced a significant increase in the mean

percentage of mature eggs compared to the non-treated infected mice. At 4 WPT, more than 50%

of the eggs were mature. Massoud et al. (2004) (103)

attributed the changes in the oogram pattern

produced by MZD to an early interruption of egg laying in the intestinal wall or blocking the

development of oviposition. Also, Badria et al. (2001) (105)

reported that MZD caused separation

of male and female-coupled worms and shifting of female worms from their normal habitat to the

liver resulting in a progressive reduction in the immature eggs laid in the wall of the small

intestine with an increase in mature eggs (93%) but they did not report on dead ova. These results

do not coincide with the results reported by Guirguis and Mahmoud (2003) (106)

, Botros et al.,

(2004) (107)

and Ebeid et al., (2005) (108)

who found absence of oogram alterations after MZD

treatment of mice or hamster.

NTZ resulted in increase in the percentage of dead eggs significantly (19.50%, 22.25%

and 30.5%) with progressive increase in % of total mature eggs (27.25%-51%) as well as

progressive reduction in immature eggs (56%, 43.25% and 18.51%) at 1, 2 and 4 WPT,

respectively. MTO resulted in significant increase in immature eggs 63.25% (p<0.05), 47% and

24.5% (p<0.01) at different times of follow up at 1, 2 or 4 WPT, respectively. Also, there was

progressive decrease in mature eggs 20.25%, 33% and 49% (p<0.01) at 1, 2 and 4 WPT. The

mean % of the dead eggs increased significantly 16.50%, 20% and 26.5% (p<0.01) at 1, 2 and 4

WPT, respectively. Massoud (1999) (102)

reported that the oil administered in doses of 15 mg/kg

for 3 days for S.mansoni-infected hamster resulted in highly significant decrease in the immature

98

eggs (3.3%) compared to (51.2%) in the control non-treated group with increase in the mature

eggs (60.7%) in treated group versus (47.3%) in the non-treated group and increase in the dead

eggs in treated hamster (36%) versus (1.5%) in the non-treated group. Abou-El Maaty (2002)(189)

used the oil in treatment of mice infected with 100 S.mansoni cercariae. 7 WPI, mice were orally

adminstered with 250 mg/kg single oral dose. It was noticed at 2 WPT that the immature eggs

were not detected, the mature eggs decreased from (23.9%) in the non-treated mice to (19.5%) in

the treated group as well as the dead eggs showed marked increase to (80.5%).

The tegument of schistosomes has been described as a living, anucleate, and cytoplasmic

structure covering the outer surface of the worm (248)

. The tegument has secretory functions, is

involved in nutrient absorption and shields schistosomes from the immune response by the

infected host (249,250)

. Scanning electron microscopy (SEM) has become a useful tool for the study

of the ultrastructural changes of the surface of the Schistosoma worms in response to

chemotherapy by showing the effect on the tegumental structures (tubercles, spines, inter-

tubercular ridges), oral and ventral suckers (251)

.The ultrastructural changes are directly

proportional with the potency of antischistosomal drugs (252)

and may clarify the procedure of

killing these worms (97)

. As it was suggested that focal tegumental damage induced by an anti-

schistosomal drug might be repaired effectively over the course of 7-14 days after cessation of the

drug while in case of severe tegumental damage, the host immune response might impact this

repair process (253)

. These morphological alterations are accompanied by an increased exposure of

schistosome antigens (epitopes) at the parasite surface (254)

, leading to disappearance of the

immunological 'disguise' of the worm and also inability to engulf food by oral and ventral suckers.

This is believed to be of prime importance in causing death of the worms (255,256)

.

In this study, PZQ caused a pronounced tegumental damage with rupture of tubercles and

loss of spines in wide areas in male worms and marked tegumental ulceration in female worms

exposing the underlying muscle layers. The male tegument was more affected than females. These

results were in accordance with several studies that detected extensive tegumental damage in adult

S. mansoni worms in vitro (98, 99, 220,256- 261)

or in vivo (98, 99, 111, 145, 252, 262-264)

.

MZD resulted only in superficial tegumental damages in female S.mansoni worms with

shrinkage of the outer surface and rupture of male tubercles with marked loss of spines and if

present, lost their sharpness. These results agreed with those reported by Hassan et al. (2003) (97)

,

Sharaf (2004) (98)

and Bakr et al. (2009) (99)

but did not agreed with the results of Ramzy et al.,

(2010) (111)

who reported that the dorsal and ventral surfaces of S.haematobium–recovered from

MZD-treated hamster (500mg/kg for 3 days) were intact 3 months after treatment time. On the

other hand, NTZ resulted in mild tegumental damaging effect manifested by focal lesions in the

99

inter-tubercular ridges without effect on tubercles and spines, disorganization of the oral suckers

and loss of spines in the gynecophoric canal of male worms. MTO resulted only in oedematous

swelling of both oral and ventral suckers without detectable alteration in the tegument.

Pointing to the haematological findings, data presented in this study showed that anaemia

was a characteristic feature in S.mansoni-infected non-treated mice as there was highly significant

and progressive decrease in haemoglobin level, RBCs count and the haematocrit value (HCT)

respectively at 8, 9 and 11 WPI as compared to the non-treated mice. There was progressive

decrease in MCV especially at 11 WPI indicating microcytosis .The decreased levels of MCH and

MCHC indicate hypochromacia as reported by several authors(265,266)

.So there was microcytic

hypochromic anaemia which directs the etiology to iron deficiency anaemia as reported

previously in human(267)

or experimentally infected animals (268,269)

.

The exact mechanisms involved in schistosomiasis-associated anemia are unclear (270)

.

Many investigators tried to explain the cause of anemia; due to blood loss, increased hemolysis

and subsequent iron deficiency (271,272)

.The direct loss of erythrocytes may arise from the

extrusion of schistosomal ova by piercing the submucosa of intestine by the lateral spines during

escape of eggs (267)

or because of the consumption of blood by schistosomes (each worm

consumes about 0.88 µl blood per day) (273,274)

. Gaafar et al., (1992) concluded that the plasma

lipoprotein abnormalities in patients with hepatic schistosomiasis cause changes in lipid

composition of erythrocytes membranes that in turn may be responsible in part for haemolysis and

shortened life span of RBCs (275)

.

The implication of a haemolytic element is confirmed by the finding that the haemoglobin

level gradually decreases with disease progress accompanied by a gradual rise in reticulocyte

count and a parallel decrease in serum haptoglobin (276,277)

. Further evidence is provided by the

rise in serum bilirubin, increase in the median corpuscular fragility of RBCs and the detection of

elevated HbF levels in Hb-electrophoresis (277,278)

. Chemical damage to erythrocyte membranes

via an oxidative stress pathway may be implicated (279)

. Splenomegaly leading to RBCs

sequestration and anaemia of inflammation are other possible causes of anaemia in

schistosomiasis(280)

. Alternatively, schistosomiasis may cause anemia by inducing pro-

inflammatory cytokine-mediated dyserythropoiesis, as seen in anemia associated with

inflammation (281,282)

. Dyserythropoietic changes demonstrated in bilharzial patients are correlated

to both the serum iron and the percentage iron saturation (283)

.

PZQ resulted in progressive increase in the HB level, RBCs count, HCT% and red blood

cell indices [MCV, MCH and MCHC] at 1, 2 and 4 WPT .These findings indicate that PZQ had

100

no damaging effect on blood parameters of erythrocytes and their indices in S.mansoni infected

mice as previously reported by Ahmed (1993) (270)

.

MZD resulted in insignificant increase in HB level, RBCs count, HCT and red blood cell

indices at 1 WPT but there was significant improvement in these parameters in the 4th

week of

treatment. MZD proved to be safe on erythrocytic parameters in this study as previously reported

by El-Ashmawy et al.(2000)(284)

who used the drug in doses 50,100 and 200 mg/kg for 2 months

in normal rats ,and reported that HB,HCT and RBCs counts did not significantly changed .

Soliman et al., (2004) (119)

and Waheeb and Abdel Hafeez (2001) (285)

found improvement in HB

level (8%-14%) at 3 months after MZD treatment (15 mg/kg for 3 days) in 49 patients with

intestinal schistosomiasis.

NTZ 100 mg/kg orally given for 7 days to S.mansoni-infected mice at 7 WPI was unable

to induce elevation in the RBCs, HB, HCT and red blood cell indices. Two WPT, significant

elevation was reported in the HB level, HCT and red blood cell indices (except MCHC). At 4

weeks after treatment, significant elevation in the HB, HCT and red blood cell indices (MCV,

22.9%, MCH 51.5% and MCHC 22.8%) were observed in comparison to non-treated group but

RBCs counts had insignificant change at all the study duration. NTZ proved to be safe on

erythrocytic parameters with mild improvement according to the period follow up. With MTO

treatment, the only significant changes were the elevation in red blood cell indices at 4 weeks

after treatment. This denotes that MTO had mild deleterious effects on erythrocytic parameters

without improvements.

In this study, Infection with S.mansoni was found to cause a significant elevation in WBC's

counts (Leukocytosis) (46.9% and 101.2% at 9 and 11 WPI respectively). This was nearly similar

to the results previously reported by Shaker et al., (1980) (286)

, Ahmed (1993) (270)

, Soliman et al.,

(2003) (287)

, Freudenstien-Dan et al., (2003) (288)

, and Allan et al., (2014) (289)

. The increase in total

leukocytes counts was attributed to the stimulation of the cellular production as a powerful

defense reaction against the schistosomes and/or their ova. Freudenstien-Dan et al. (2003)(288)

reported that activated leucocytes are known to participate in immunity to S.mansoni ,where they

attach to the parasite surface and secrete schistosomicidal substances as cationic protiens

,hydrolytic enzymes,and oxidants implicated in the damage of the schistosomes. Other studies

indicated presence of leucopenia (low WBCs) in S. mansoni-infected mice at 8 WPI as reported

by Allam (2009) (192)

and Mahmoud &El-Bessoumy (2013) (290)

. However, Abdel-Mottaleb et al.

(2008) (291)

reported non-significant changes in the total leucocytic count in S. mansoni-infected

mice at 11WPI.

101

In this work, the differential leucocytic counts of infected non-treated group revealed

progressive lymphocytosis in relation to the time of infection reaching 38.8% at 4 WPI .These

results were in agreement with the results of Allan et al.(2014) (289)

and Soliman et al.,(2003)(287)

who reported lymphocytosis at 6 or 9 WPI but not agreed with Thabet et al.(2007)(164)

, Allam

(2009) (192)

, Abdel-Mottaleb et al.(2008) (291)

, Mahmoud and El-Bessoumy (2013) (290)

who

reported lymphopenia (low lymphocytes) ranging from 20.2 to 59.2% at 8 or 11 WPI.

Neutropenia (low neutophils) was evident in an ascending manner in response to infection in

non-treated mice in a rate of (24%,33.3% and 62.1%) at 8,9 and 11 WPI as reported by

Mahmoud and El-Bessoumy (2013)(290)

, Abdel-Mottaleb et al.(2008) (291)

who reported decreased

rate of neutophils (38.1% and 59.5%) ,respectively in infected non-treated mice at 8 or 11 WPI .

On the contrary, Soliman et al., (2003)(287)

, Thabet et al.(2007)(164)

and Allam (2009)(192)

reported

variable rates of neutrophilia ranging from 16.4% to 231% in S.mansoni–infected mice at 8,9

and 11 WPI. Eosinophils flourished in non-treated infected mice in progress to infection time as

there was increased rate of 51.9%, 83.3% and 90.4% at 8, 9 and 11 WPI. Allam (2009) (192)

and

Mahmoud and El-Bessoumy (2013) (290)

reported eosinophilia (high eosinophils) at 8 WPI as the

rate of increase was 238.4% and 158.3% respectively. While Soliman et al., (2003) (287)

observed

70% increase in the mean value of esinophils in infected non-treated animals at 9 WPI. Thabet et

al.,(2007) (164)

and Abdel-Mottaleb et al.,(2008) (291)

reported 175.4% and 415.3% increase in the

level of esinophilic count at 11 WPI , respectively. Eosinophils are known to be involved in

destruction of schistosome eggs and modulation of the granuloma (292)

as they are able to generate

superoxides (293)

. Eosinophils activation was suggested to occur by the enhanced release of the

haematopoietic cytokines such as IFN-γ (169)

.Generally, it's not uncommon to find an increase in

the total counts of eosinophils in helminthic infection (294)

. It has been reported that eosinophilia is

associated with schistosomiasis where the living ova and worms produce specific stimuli leading

to bone marrow, blood and local tissue eosinophilia (295)

. Eosinophilia may be observed in most of

the patients with or without increase of leukocyte counts (296)

. Monocytes and basophils were not

significantly changed in infected non-treated mice at both 1,2 or 4 weeks post-infection as

previously reported by Allam (2009) (192)

and Abdel-Mottaleb et al.,(2008) (291)

at 8 or 11 WPI.

However, Soliman et al., (2003) (287)

and Mahmoud & El-Bessoumy (2013) (290)

reported

monocytosis at 9 or 11 WPI.

Treatment of infected mice with PZQ 500 mg/kg for 2 days at 7 WPI resulted in non-

significant decrease in WBCs count (10.8%) 1WPT but the drug reduce significantly TLC at 2

and 4 WPT, respectively in rate of (17.8% and 48.8%, P<0.01).This was accompanied by

102

significant reduction of lymphocytes (31.7% and 57.9 %) at 2 and 4 WPT, respectively. PZQ

increased the neutrophils count in high statistical significance (25.6%, 87.6% and 287.4 %) at 1, 2

and 4 WPT. Significant reduction in esinophils (10.1%, 15% and 40.2%) was observed at 1 or 2

WPT, respectively. On the hand, neutrophils increased progressively throughout the follow up.

MZD caused significant reduction in TLC (14.2% and 35.1%), in lymphocytes (18.2%

and 42%) at 2 and 4 WPT and in esinophils (10.1%, 22.3% and 34.5%) but significant increase in

neutrophils at 2 or 4 WPT (53.5% and 215.7%). El-Ashmawy et al., (2000)(284)

found non-

significant change in total and differential leucocytic counts at 1,2,4 and 8 WPT with MZD in

normal healthy rats orally in 3 dose levels 50,100 and 200 mg/kg for 2 months.Waheeb and Abdel

Hafeez (2001) (285)

reported non-significant change in WBCs count and esinophils 2 months after

treatment with MZD (15mg/kg for 3 days) in cases of intestinal schistosomiasis.

NTZ resulted in significant reduction in TLC at 4 WPT (22.5%), in lymphocytes at 2 or

4WPT (7.7% and 8.6%) but significant elevation in neutrophils at 2 or 4 WPT (21.4% and

137.1%) and esinophils (25%) at 2 WPT. As regards MTO treatment, there were only significant

changes in differential counts at 4 WPT where there was significant reduction in lymphocytes and

eosinophils and elevation in neutrophils.

In the current study, S.mansoni-infected non-treated mice showed progressive degree of

thrombocytopenia (as the platelet count decreased in response to time of infection in rate of

(14.1%, 20.7% and 33.6%) at 8,9 and 11 WPI, respectively as compared to the non-infected non-

treated control mice .These results agreed with those of Stanley et al., (2003) (297)

at 12 WPI but

were against El-Shenawy et al., (2008) (194)

who found significant thrombocytosis (+107.1%) at 7

WPI while Thabet et al.,(2007)(164)

reported non-significant change in platelet count in S.mansoni-

infected mice at 11WPI. Omran et al., (1978) (283)

reported quantitative and qualitative platelet

defects in late stage of hepatosplenic schistosomiasis. Thrombocytes have been shown to act as an

effector mechanism in immune animals, a property mediated by IgE antibodies (298,299)

. Ngaiza

and Doenhoff (1990) (300)

reported that schistosome egg excretion is partially platelet-dependent.

Treatment of infected mice with PZQ,MZD or NTZ caused significant increase in the

platelet count at 2 or 4 WPT , the effect was higher for PZQ (31.9% and 72.1%) followed by

MZD (25.5% and 50.4%) and NTZ (18.8% and 40.1%). This elevation in platelet counts may be

due worm death and subsequently decrease in egg excretion depending on the potency of the drug

but MTO did not induce elevation in platelet count, Instead, it caused significant platelet

reduction at 1 WPT (14.2%) which may be due to its weak antischistosomal activity.

Liver damage can be detected by measuring the changes in liver enzymes (ALT, AST and

ALP) activities compared to the control.Where its hepatocytes show differences in the localization

103

and concentration of some enzyme systems. These enzymes served as markers for different cell

organelles and any defect of them will be reflected to the enzyme activity itself (301,302)

.ALT is a

liver specific enzyme only significantly elevated in hepatobiliary diseases. Increase in AST level

can occur in connection with damages of heart or skeletal muscle as well as liver parenchyma.

ALP levels are of interest in the diagnosis of hepatobiliary disorders and bone diseases. Parallel

measurement of ALT, AST and ALP is therefore applied to distinguish liver from heart or skeletal

muscle damages.So studying changes in these enzymatic activities could be helpful in evaluating

the damaging effects of S.mansoni infection on the liver of infected hosts and evaluating the

possible side effects of different treatments and the improvement occurring in such enzymes after

treatment (303)

.

In this work, mice in the infected non-treated group showed highly significant elevation of

serum ALT (54.5%, 80.6% and 202.2%), AST (45.8%, 49.2% and 79.5%) and ALP levels

(123.2%, 127.9% and 212.2%) compared to non-infected normal mice at 8, 9 and 11 WPI. Botros

et al.,(2007) (235)

found 112.1% increase in ALT level in S.mansoni-infected mice at 8 WPI .El-

Lakkany et al.,(2012) (135)

reported 80% increase in serum ALT at 9 WPI, Saba-El Rigal and

Hetta (2006)(304)

found 238.9% and 119.3% elevation in the serum AST and ALP levels

respectively at 8 WPI. Abdel-Mottaleb et al., (2008) (291)

found 88% increase in serum ALP at 11

WPI. El-Shenawy et al. (2008)(194)

and Mahmoud et al., (2002) (305)

attributed the elevated liver

enzyme level to the hepatic cell damage and increased cell membrane permeability or to heavy

Schistosoma egg deposition. Awadalla et al., (1975) (306)

and El-Aasar et al., (1989) (307)

attributed

the increase in enzyme activity to the irritation of the liver cells by toxins or metabolic products of

growing schistosomules, adult worms and eggs or to increased loss of intracellular enzyme by

diffusion through cell membranes which appears to act as a stimulus to the synthesis of more

enzyme protein. Higher rates of formation would, in turn, increase the rate of diffusion and hence

increase serum activity.

In the current study, under the effect of PZQ, the serum ALT decreased significantly

29.1%, 35.1% and 53.5% at 1, 2 and 4 weeks after treatment. PZQ also reduced AST (21.3% and

50.3%) and ALP activity (43.2% and 65.5%) at 2 and 4 WPT and this was constant with the

findings of Botros et al., (2007) (235)

who found 33.2% and 43.3% reduction in ALT level in PZQ-

treated mice at 1 or 2 WPT (500 mg/kg for 2 days at 6 WPI). However, Sewify (2009) (215)

and El-

Lakkany et al., (2012)(135)

found insignificant change in the AST level at 2 WPT in PZQ-treated

mice .

MZD treatment of infected mice caused significant changes in the activity of liver

enzymes. This was manifested by significant reduction in ALT activity at 1, 2 and 4 WPT (23.5%,

104

25.9% and 40.1%). The significant reduction in serum AST and ALP activity occurred only at 2

WPT (9.7% and 32.2%) and at 4 WPT (40.25%, and 49.3%). El-Ashmawy et al.,(2000)(284)

reported non-significant change in serum liver enzymes in healthy rats orally given MZD doses

ranging from 50-200 mg/kg for 2 months at 1 , 2 or 4 WPT. Saba-El Rigal and Hetta (2006) (304)

used MZD in dose of 600 mg/kg for 3 days in S.mansoni-infected mice (100 cercariae at 8 WPI).

The level of ALT and AST was decreased 48.5% and 52.7%, respectively at 3 WPT compared to

the non-treated mice. Omar et al., (2005) (308)

studied the effect of MZD 500 mg/kg or PZQ 1500

mg/kg daily for 6 weeks on normal rats. There was non-significant increase in the mean value of

ALT in MZD-treated rats as compared to the normal non-treated control; whereas PZQ induced

significant increase in the mean value of ALT compared to MZD. Also, PZQ induced significant

increase in the mean value of AST activity compared to MZD.

Significant improvement in the liver functions was also observed after NTZ treatment of

S.mansoni-infected mice. There were significant reduction in ALT activity at 1, 2 and 4 WPT

(16.6%, 26.4% and 30.7%).The decrease in AST activity was significant at 4 WPT (22.9%) while

ALP activity showed significant reduction at 2 and 4 WPT (20.2% and 40.7%). MTO treatment of

S.mansoni-infected mice resulted in further significant increase in ALT activity (10.2%) and AST

activity (18.3%) at 1 WPT. The oil could significantly reduce ALT (15.2%) and AST (29.8%) at 4

WPT. Also, ALP activity decreased significantly (18% and 25.9%) at 2 and 4 WPT. Massoud

(1999) (102)

studied the effect of the oil in S.mansoni-infected hamster (in dose of 30 mg/kg for 3

days), the author found significant decrease in the serum liver enzymes at 1, 2 and 4 weeks after

treatment. These findings related to the effect of the studied drugs on liver functions in mice

declare that PZQ, MZD as well as NTZ were safe with variable degrees of improvements

according to the potency. MTO was slightly hazardous especially in the 1st week after treatment.

Nephropathay or nephrotic syndrome was reported in human and experimental animals

infected with S. mansoni. The disease is reported to progress to end stage renal failure (309,310)

.

Schistosomal nephropathy is most likely produced by chronic deposition of circulating immune

complexes, antischistosome antibodies and schistosome antigens (311)

. It is likely that schistosomal

nephropathy results from accumulated injury due to prolonged exposure to immune complexes

inefficiently cleared by liver and splenic mononuclear phagocyte (312)

. Alternatively, many cases

attributed to schistosomal glomerulopathy may actually be caused by immune complex disease

from chronic coinfections, such as hepatitis B, malaria and salmonellosis (313)

.

Blood urea and serum creatinine are routinely used as biomarkers for assessment of renal

functions. Urea is the final result of the metabolism of protiens; it is formed in the liver from their

destruction. Elevated urea can appear in the blood (uremia) in; diets with excess of protiens, renal

105

diseases, heart failure, gastrointestinal hemorrhage, dehydration. Creatinine is the result of the

degradation of creatine (component of muscles), it can be transformed into ATP, that is a source

of high energy for cells. The creatinine production depends on the modification of the muscular

mass, and it varies little and the levels usually are very stable. Creatinine is excreted by the

kidneys. With progressive renal insufficiency, there is retention in blood urea and elevated

creatinine level (303)

. In this study, the blood urea and serum creatinine in S.mansoni-infected mice

increased in response to the period of infection as they were progressively raised. EL-Shenawy et

al., (2008) (194)

reported nearly similar results as the blood urea and serum creatinine of mice

infected by S.mansoni, showed that significant increase (300% and 166.6%) respectively as

compared to non-infected mice at 7 WPI (100 cercariae).

Treatment of infected mice with PZQ or MZD caused progressive significant reduction in

the elevated levels of urea (12.5%, 33.9% and 60.2%) for PZQ and (21.3%, 26.8% and 45.7%) for

MZD at 1, 2 and 4 WPT, respectively. Both drugs significantly reduced creatinine at 4 WPT.

Sheir et al., (2001) (116)

and El-Ashmawy et al., (2000) (284)

reported that MZD was safe on kidney

functions of normal healthy rats (orally given 50,100 and 200 mg/kg for two months) or healthy

volunteers (10 mg /kg for 3 days after 2 months) as well as infected treated patients. NTZ-treated

mice showed significant decrease in blood urea at 2 and 4 WPT (26.3% and 33.4%). Serum

creatinine showed non-significant change. MTO caused significant elevation in blood urea 1WPT

(25%) and in creatinine level at 1 and 2 WPT (50% and 12%).However, a significant reduction in

blood urea (28%) was observed at 4WPT .

Cholinesterases are enzymes (protein in nature) present in cholinergic and non-cholinergic

tissues as well as blood and other body fluids. There are two isoforms according to their catalytic

properties and specificity for substrates, sensitivity to inhibitors and tissue distribution

[butyrylcholinesterase (BChE) or pseudocholinesterase (PChE) and acetylcholinesterase (AChE)

or true cholinesterase (TChE)] (314)

. The two isforms differ genetically, structurally and for their

kinetics (315)

.

Acetylcholinesterase (AChE) is synthesized mainly in the hepatocytes then released into

the blood to its target sites. It is an enzyme participating in cholinergic neurotransmission as it

breaks down acetylcholine (causes the stimulation of neurons) while cholinesterase causes the

ending of stimulation by breaking down the acetylcholine into choline and acetic acid which

terminates the neurotransmission process post-synaptically or through the neurosynaptic cleft in

both the central and the peripheral nervous system (reaction necessary to allow a cholinergic

neuron to return to its resting state after activation or stimulation)(316)

. The acetylcholinesterase is

mainly found in the brain, muscles, erythrocytes, lymphocytes and cholinergic neurons(317,318)

.

106

Inhibition of the AChE will cause high concentration of acetylcholine then accumulated leading to

toxicity manifested by nicotinic, muscarinic or central signs and symptoms according to the level

of inhibition and consequently the receptors affected. The butyrylcholinesterase (BChE) is found

in the intestine, liver, kidney, heart, lung, plasma and serum. Butyrylcholine is not a physiological

substrate in mammalian brain which makes the function of BChE of difficult interpretation with

lower level than AChE. Pseudocholinesterase (PChE) has a broader range of esterase activity that

can hydrolyze butyrylcholine, acetylcholine and other aliphatic esters (319-322)

.

In this current work, S.mansoni-infected mice at 8 WPI showed significant reduction of

blood AChE activity (11.3%). Such decline in the enzyme activity present in infected mice was

aggrevated with time (19.8% and 23.5%) at 9 and 11 WPI respectively.Nearly similar to the

results obtained by Sewify (2009) (215)

and Saba El-rigal and Hetta (2006)(304)

who found 14% and

56.1% inhibition in serum cholinesterase (SCE) activity in S.mansoni-infected mice at 7 or 8 WPI

(with 100 cercariae either by paddling technique or tail immersion method), respectively. The

later said that the low SCE level is attributed to low serum total proteins.

Treatment of mice with PZQ, MZD or NTZ caused significant elevation in the depressed

level of AChE, This effect was greater detected earlier for MZD (10.3%, p<0.01, 16.2% and

25.4%).It was observed (22.5% and 31.8%) at 2 and 4 WPT for PZQ and (13.1% and 16.2%) for

NTZ. Badria et al.(2001) (105)

stated that MZD in a dose of 500 mg/kg for 3 days for S.mansoni-

infected mice resulted in death of adult worms ;may be due to loss of musculature (paralysis) .

Hassan et al. (2003) (97)

and Sharaf (2004) (98)

examined the muscle tension of S.mansoni worms

under the effect of MZD in rising concentrations 100,200,300 and 400 µg/ml .The drug elicited

somatic muscle contraction and reached highest response with the higher concentration. It was

found that exposure of isolated rabbit duodenum to MZD 150-300 µg/ml induced inhibitory effect

on motility. However, it failed to evoke the contractile effect of acetylcholine (2µg/ml), so MZD

is devoid of an effect on the muscarinic receptors (323)

.Saba-El rigal and Hetta (2006) (304)

found

that MZD proved to have highly significant stimulatory activity on SCE level (14%) in normal

mice. MTO significantly declined the blood AChE activity (23%) at 1 WPT. This may be due to

lower effects on adult worms and liver compared to other drugs. The oil may have potent

inhibitory effects on blood AChE activity. It did not change the enzyme level at 2 and 4 WPT in

treated mice in comparison to non-treated group in spite of significant reduction of total worm

burden .The oil proved to have mild anticholinergic activity in mice.

107

SUMMARY

AND

CONCLUSION

108

SUMMARY AND CONCLUSION

The chemotherapy of schistosomiasis is considered the most effective tool for control of

schistosomal morbidity in human. It is based on the control of adult worms in infected patients,

with praziquantel being the most widely used drug. However, PZQ does not prevent reinfection, is

inactive against juvenile schistosomes, and has only a limited effect on already developed liver

and spleen lesions. These limitations, in Combination with a considerable concern about the

development of PZQ resistance, have motivated the scientific community to search for novel and

inexpensive drugs against schistosomiasis.

The aim of the study is to assess efficacy of Nitazoxanide, Myrrh Total Oil and the

commercially available product of Myrrh (Mirazid) in comparison with Praziquantel in treatment

of schistosoma mansoni infected mice.

In this experimental study, 120 mice in (six groups) were randomly allocated through

treatment and control groups. 100 mice were infected with 100 Schistosoma mansoni cecariae

(examination and treatment started 50 days post infection).

G1: infected and treated orally with MZD 500 mg/kg bw/day for 5 consecutive days.

G2: infected and treated orally with MTO 18 mg/kg bw/day for 3 days.

G3: infected and treated orally with PZQ 500 mg/kg bw/day for 2 consecutive days. G4: infected

and treated orally with NTZ 100 mg/kg bw/day for 7 consecutive days.

G5: infected and non-treated control.

G6: normal non-infected and non-treated.

Mice were sacrificed at 1, 2 and 4 WPT, examination of efficacy was assessed

parasitologically (through egg count in stool, worm burden, sex and length, tissue egg count in the

liver and intestine, and oogram pattern), scanning electron microscopic, haematological and

biochemical studies.

Statistically analysed results reported in this study:

I.Parasitological results:

PZQ showed highly significant and sharp reduction in the faecal egg counts at 1WPT (63%)

and complete disappearance of eggs on the 2nd

and 4th

WPT (100% reduction). MZD caused

highly significant reduction (39.5% and 69.6%) in faecal egg count at 2 and 4 WPT. NTZ

resulted in significant reduction in the faecal egg counts (22.5% and 50.6 %) at 2 and 4 WPT.

MTO resulted in insignificant change in faecal egg count (1%, 9.8%) after the 1st, 2

nd weeks of

109

treatment and significant reduction in faecal egg count in a rate of 19.4% only after the 4th

weeks post-treatment.

PZQ-treated mice showed a highly significant reduction in the total worm burden (83%, 94%

and 97%) as well as MZD reduced significantly the total worm burden (34%, 50% and 71%) at

1, 2 and 4 WPT. NTZ-treated group showed significant reduction in total number of worms

(45% and 65%) at 2 and 4 WPT. MTO-treated group possessed significant rate of worm

reduction (29 %) only at 4 WPT.

PZQ had equal effect on male and female worms,while MZD, MTO and NTZ affected male

worms more than females at 1,2 and 4 WPT .

PZQ had equal sensitivity in shortening of female and male worm length and MZD decreased

worm length of male worms more than in female worms at 1, 2 and 4 WPT but NTZ and

MTO resulted in non-significant decrease in the mean body length of worms recovered at

different times at 1, 2 or 4 WPT.

PZQ was able to reduce the intestinal egg load (69.9%, 79.1% and 88%) more than the

reduction in the hepatic tissues (64.4%, 69.2 % and 85.8%) at 1, 2 and 4 WPT. MZD produced

significant reduction in the intestinal egg loads (28.9%, 49.3% and 66%) at 1,2 and 4 WPT

and (42.8% and 65.3%) in the hepatic tissue egg load at 2 and 4 WPT. NTZ reduced the

intestinal egg count (22.1%, 45.2% and 46.6%) at 1, 2 and 4 WPT and significant reduction

was achieved in the hepatic tissue egg load (23.8% and 30.7%) at 2 and 4 WPT. MTO resulted

in significant reduction in the intestinal egg count (12.2%, 23.2% and 31.5%) at 1, 2 or 4 WPT

and significant reduction was achieved in the hepatic tissue egg load (42.3%) only at 4 WPT.

PZQ produced a highly significant increase in the percentage of dead eggs as well as

significant reduction in mature and immature eggs at 1, 2 and 4 WPT. MZD resulted

significant increase in the percentage of dead eggs at 2, 4 WPT, mature eggs at 4 WPT and

significant reduction in immature eggs at 4 WPT. NTZ resulted in an increase in the

percentage of dead eggs and mature eggs with significant reduction in immature eggs at 1, 2

and 4 WPT. MTO showed significant increase in the dead and immature eggs with

progressive increase in mature eggs at 1, 2 or 4 WPT.

II. Scanning electron microscopic examination of the recovered worms from treated mice:

PZQ caused a pronounced tegumental damage with rupture of tubercles and loss of spines in

wide areas in male worms and marked tegumental ulceration in female worms exposing the

underlying muscle layers. The male tegument was more affected than females.

110

MZD treatment resulted only in mild tegumental damage of male and female S. manoni

worms with shrinkage of the outer surface with rupture of tubercles and marked loss of spines

and if present, lost their sharpness without any obvious deeper effects.

NTZ resulted in mild tegumental damaging effect manifested by focal lesions in the inter-

tubercular ridges, disorganization of the oral suckers of male worms and loss of spines in the

gynecophoric canal. MTO resulted only in oedematous swelling of both oral and ventral

suckers without detectable alteration in the tegument.

III.Haematological and Biochemical findings:

In comparison between infected non-treated mice and treated infected gps , PZQ resulted in

progressive increase in the HB level, RBCs count, HCT%, red blood cell indices [MCV,

MCH and MCHC] at 1, 2 and 4 WPT. MZD treatment resulted in significant improvement in

the previous parameters in the 2nd

week of treatment (except the MCV and MCH) and at 4th

week of treatment, there was significant improvement in all parameters. NTZ-treated gp

resulted in significant elevation in the HB level, HCT and red blood cell indices (MCV,

MCH) at 2 WPT. At 4 weeks after treatment, there was significant elevation in all studied

parameters (except RBCs counts). With MTO treatment, the only significant changes were

the elevation in HB at 4WPT and in some red blood cell indices (MCH) at 2 or 4 WPT,

(MCHC) at 4 WPT.

PZQ reduced significantly TLC accompanied by significant reduction in lymphocytes and

esinophils at 1 or 2 WPT and increase in the neutrophils at 1, 2 and 4 WPT. MZD caused

significant reduction in TLC, lymphocytes at 2 and 4 WPT and esinophils at 1, 2 and 4 WPT

with significant increase in neutrophils at 2 or 4WPT. NTZ resulted in significant reduction

in TLC at 4 WPT and in lymphocytes at 2 or 4 WPT with significant elevation in neutrophils

at 2 or 4 WPT and esinophils at 4 WPT. As regards MTO treatment, there were only

significant changes in differential counts at 4 WPT where there was significant reduction in

lymphocytes, eosinophils and elevation in neutrophils. Monocytes and basophils were

insignificantly changed in any time post-treatment.

Treatment of infected mice with PZQ, MZD or NTZ caused significant increase in the

platelet count at 2 or 4 WPT. The effect was higher for PZQ treatment followed by MZD and

NTZ. MTO did not induce elevation in platelet count, Instead of this, it caused significant

platelet reduction at 1 WPT.

PZQ decreased significantly ALT at 1, 2 and 4 WPT as well as AST and ALP activity at 2

and 4 WPT. MZD resulted in significant reduction in ALT activity at 1, 2 and 4 WPT as well

111

as serum AST and ALP activity only at 2 and 4 WPT. NTZ significant reduction in ALT

activity at 1, 2 and 4 WPT and AST activity at 4 WPT while in ALP activity at 2 and 4 WPT.

MTO resulted in significant increase in ALT and AST activity at 1WPT. The oil reduced

ALT and AST at 4 WPT. Also, ALP activity decreased significantly at 2 and 4 WPT.

PZQ and MZD caused progressive significant reduction in the elevated levels of urea and

creatinine at 1, 2 and 4 WPT, respectively. Both drugs significantly reduced creatinine at 4

WPT. NTZ-treated mice showed significant decrease in blood urea at 2 and 4 WPT. Serum

creatinine showed non-significant change. MTO caused significant elevation in blood urea

1 WPT and in creatinine level at 1 and 2 WPT. However, a significant reduction in blood

urea was observed at 4 WPT.

Treatment of mice with PZQ, MZD or NTZ caused significant elevation in the level of

AChE, This effect was greater detected earlier for MZD, at 2 and 4 WPT for PZQ and NTZ.

MTO significantly declined the blood AChE activity at 1 WPT. It did not change the enzyme

level at 2 and 4 WPT.

Conclusion:

This study declared that PZQ is still the most important drug in treatment of

schistosomiasis because of its high lethality to schistosome worms as early as possible after two

weeks of treatment with higher safety margins on blood cells, liver and kidney functions tests as

well as blood AChE activity. MZD showed moderate antischistosomal activity less effective than

Praziquantel but highly safe without adverse haemtological or biochemical effects on infected

treated mice. Also, NTZ was less effective than PZQ and MZD but with less adverse health

effects. MTO exerted little antischistosomal activity with lower safety profile at the selected dose.

112

RECOMMENDATIONS

113

RECOMMENDATIONS

1. Continuity of use of Praziquantel as a standard treatment of schistosomiasis as the drug is

still effective and safe drug until production of new antischistosomal agents or vaccines.

2. The haematological effects of PZQ should be re-evaluated as there is scarcely available

literature in this concern.

3. When Mirazid is used as alternative to PZQ for treatment of S.mansoni infection; adequate

doses should be used and thorough parasitological re-assessment is essential as egg excretion

may continue at a low level.

4. Mirazid and Myrrh total oil are very complex mixture of compounds so fractionation of them

into fine components may yeild very promising new antischistosomal agents than the very

simple preparation of MZD.

5. Short course of treatment in MZD application as in PZQ should be tested to offer maximum

patient compliance.

6. Re-evaluation of cholinesterase activity of MZD in vitro on adult schistosomes may explore

the mechanism of action of the drug.

7. Re-evaluation of Nitazoxanide safety in various healthy animal models with various doses

and courses as well as its efficacy in treatment of schistosomiasis using in vitro and animal

models alone or in combination with PZQ.

8. For experimental discovery of antischistosomal activity of a substance, adopt WHO criteria

to save time and costs, so many substances may be assessed.

114

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141

PROTOCOL

142

بنبرازيكىاتم عهي انبههبرسيب انعىية بيقبرة انيرازيذو دراسة فبعهية عقبر انيتبزوكسبيذ وانسيث انكهي نببت انر

انتجريبية

Study of the efficacy of Nitazoxanide , Myrrh Total Oil and Mirazid in comparison with

Praziquantel in experimental Schistosomiasis Mansoni

Protocol of a thesis submitted to the فطخ ثؾش ملخ ا

Medical Research Institute ؼل اجؾس اطجخ

University of Alexandria عبؼخ االىلهخ

In partial fulfillment of the افبءا عيئب شوغ

Requirements for the degree of اؾصي ػ كهعخ

Master of Science in Applied & Molecular Parasitology انبجستير في انطفيهيبت انتطبيقية وانجسيئية

By

Mohammad Aziz Nawar Al-Kazzaz دمحم ػيي اه امياى

Bachelor of Veterinary Medical Sciences ثىبه اؼ اطجخ اجطوخ

Faculty of Veterinary Medicine وخ اطت اجطوي

University of Cairo عبؼخ امبوح

1997 ٧٩٩١

Department of Parasitology ل اطفبد

Medical Research Institute ؼل اجؾس اطجخ

University of Alexandria عبؼخ االىلهخ

2011 ١١٧٧

143

Supervisors انشرفى

Dr.Mona Hassan El Sayad الوزهح / ؽ اصبك

Professor, Department of Parasitology ازبم ثم ػ اطفبد

Medical Research Institute ؼل اجؾس اطجخ

University of Alexandria عبؼخ االىلهخ

Dr. Hend Aly El Taweel الوزهح / ل ػ اط

Assisstant Professor, Department of Parasitology ازبم بػل ثم ػ اطفبد

Medical Research Institute س اطجخ ؼل اجؾ

University of Alexandria عبؼخ االىلهخ

Dr. Sahar Ahmed Abou-Helw الوزهح / ؾو اؽل اثؽ

Assisstant Professor, Department of Parasitology ازبم بػل ثم ػ اطفبد

Medical Research Institute ؼل اجؾس اطجخ

University of Alexandria عبؼخ االىلهخ

144

Background

Schistosomiasis remains one of the most prevalent parasitic infections in the world. (1)

In

Egypt, schistosomiasis is a great socio-economic problem and has deleterious effects on various

tissues and organs of the human body. (2)

The government efforts is successful in reducing both

the prevalence and morbidity of this disease.(3)

However, schistosomiasis is still endemic in many

rural areas and transmission still occurs. (1)

Many drugs used for the treatment of schistosomiasis , however Praziquantel (PZQ) is the

drug of choice.(4)

The extensive reliance on just one drug is of concern, due to the possible

development of drug-resistant parasites . (5)

This necessitates a search for new safe and effective

anti-schistosomal drug.

Mirazid, a new anti-schistosomal drug introduced in the Egyptian market in the form of

soft gelatin capsules produced by Pharco (Alexandria, Egypt) since 2001. Mirazid is an oleo-resin

extract of myrrh obtained from the stem of the plant Commiphora molmol . (5)

Safety and efficacy

of Mirazid as anti-schistosomiasis drug was reported in some laboratory and clinical studies. (6-9)

while others clinical and experimental studies reported its ineffectiveness (5,10,11)

.

Nitazoxanide, a Nitrothiazole benzamide, is an antiprotozoal agent. In humans, one study

had indicated that Nitazoxanide is effective in treating F. hepatica infection.(12)

Nitazoxanide has

been tested against Schistosoma mansoni and Schistosoma hematobium in experimentally infected

mice and proved to be schistosomicidal in a cure rate of 59.91 % & 82.85% respectively (13)

, but

studies are still lacking as regards its efficacy and safety in schistosomiasis.

145

Aim of the Study

The aim of the study is to assess efficacy of Nitazoxanide , Myrrh Total Oil and the

commercially available product of Myrrh (Mirazid) in comparison with Praziquantel in treatment

of schistosoma mansoni infected mice.

146

Materials and Methods

This study will be carried out on six groups of 20 mice each.

Group1: infected & treated orally with Mirazid 500 mg / kg. body weight /day on an empty

stomach for 5 consecutive days (5)

Group 2: infected & treated orally with Myrrh Total Oil 18 mg / kg. body weight/day for 3 days on

an empty stomach (6,14)

Group 3 infected & treated orally with Nitazoxanide 100 mg / kg.body weight / day for 7

consecutive days (13,15)

Group 4 infected & treated orally with Praziquantel 500 mg / kg.body weight / day for 2

consecutive days (5)

Group 5 infected & non-treated control.

Group 6 normal non-infected & non-treated.

Groups 1,2,3,4 and 5 will be infected with 100 schistosoma mansoni cercariae for 1 hour by

paddling technique (16)

, then separated and kept in labeled cages. Stool examination will be

performed 50 days after infection to investigate presence of eggs. On day 50 each group will

receive the corresponding drug. Mice of all groups will be sacrified 1, 2 & 4 weeks after drug

administration.

The following parameters will be studied:

І.Parasitological studies:

1. Egg Count in Stool :

Eggs of S.mansoni will be counted in mice stool every other day starting 2 days post-

treatment and will be continued till animal sacrifice.

2.Perfusion of Mice & Counting of Worms

147

One and two weeks after the last doses of treatment, the mice will be sacrified , Perfusion of

the liver and portal vein will be done first in a separate container followed by the mesenteric blood

vessels in another container . Worms recovered from hepatic and mesentric vessels will be counted

& the sex will be identified.(17)

Random samples of collected worms from each group will be

examined and their length will be measured using micrometer and dissecting binocular

microscope.(18)

3.Tissue egg count will be performed in the liver and intestine (19)

4.Oogram pattern will be studied in the last part of the small intestine (17)

ІІ. Scanning Electron Microscopic Studies

Two worms will be collected and will be embedded in 3% glutaraldehyde and will be

investigated by scanning electron microscopy according to Anderson 1951. (20)

ІІІ.Hematological Studies:

A blood sample will be obtained before sacrification of mice for determination of complete

blood count (CBC). (21)

ІV. Biochemical studies :

Liver enzymes tests: serum Aspartate Amino-Transferase (AST), Alanine Amino-

Transferase (ALT) (22)

, and Alkaline Phosphatase (ALP) (23)

, Kidney functions tests: blood urea (24)

and serum creatinine (25)

, blood acetylcholinesterase (AchE) activity will be determined as an

indication of Neurotoxicity. (26)

148

References

1. Magnussen P. Treatment and re-treatment strategies for schistosomiasis control in different

epidemiological settings: a review of 10 years’ experiences. Acta Trop 2003; 86: 243–54.

2. Feachen R, Graham W, Trimacus I. Identifying health problems and health research priorities in

developing countries. J Trop Med Hyg 1989; 92(3):133-8.

3. Engels D, Chitsulo L, Montresor A, Savioli L. The global epidemiological situation of

schistosomiasis and new approaches to control and research. Acta Trop 2002; 82: 139–46.

4. Doenhoff MJ, Kimani G, Cioli D. Praziquantel and the control of schistosomiasis. Parasitol

Today 2000; 16(9): 346-66.

5. Botros S, William S, Ebeid F, Cioli D, Katz N, Day T , et al. Lack of evidence for an

antischistosomal activity of myrrh in experimental animals. Am J Trop Med Hyg 2004; 71:

206–10.

6. Massoud A. Efficacy of myrrh as a new schistosomiasis: An experimental study. Ain Shams

Med J 1999;50 :1287-98.

7. Sheir Z, Nasr A, Massoud A, Salama O , Badra G, El Shennawy H , et al . Herbal safe effective

anti-schistomicidal therapy derived from myrrh. Am J Trop Med Hyg 2001; 65(6): 700-4.

8. Abo-Madyan A, Morsy T, Motawea S, Morsy A. Efficacy of myrrh in treatment of

schistosomiasis (hematobium and mansoni) in Ezbet El-Bakly (Tamyia center) , Al-Fayoum

Governorate,Egypt. J Egypt Soc Parasitol 2004; 34(2): 423-46.

9. Kilany Y, Abou Holw S, Abouel-Nour M, Morsy A. Early development of osteoporosis in

male smokers with hypoandrogenism due to fascioliasis with or without schistosomiasis

added by life style . J Egypt Soc Parasitol 2009;39 (3): 789 – 802.

10. Barakat R, El-morshedy H, Fenwick A. Efficacy of myrrh in the treatment of human

schistosomiasis mansoni. Am J Trop Med Hyg 2005; 73(2):365–7.

149

11. Osman M, El-Taweel H, Shehab A, Farag H. Ineffectiveness of myrrh-derivative Mirazid

against schistosomiasis and fascioliasis in humans. East Mediter Health J 2010: 16(9):932-6.

12. Rossignol JF, Abaza H, Friedman H. Successful treatment of human fascioliasis with

Nitazoxanide. Trans R Soc Trop Med Hyg 1998; 92: 103–4.

13. Abdel-Rahman MS, El-Bahy MM, El-Bahy NM.Testing the parasiticidal efficacy of

Nitazoxanide. Alex J Vet Sci 1997;13: 447-58.

14. Khamis M. Formulation and evaluation of various dosage forms of myrrh. Master Thesis

2006. Faculty of Pharmacy, Alexandria University.

15. Sanad M, Al-Malki JS. Immunochemotherapy for cryptosporidiosis in immunosuppressed

mouse model. J Egypt Soc Parasitol 2007; 37( 3): 945-56.

16. Smithers SR, Terry R J .The infection of laboratory hosts with cercariae of S.mansoni and

the recovery of adult worms. Parasitol 1965;55(4): 695-700.

17. Pellegrino J, Oliveira CA, Faria J, Cunha AS. New approach to the screening of drugs in

experimental schistosomiasis mansoni in mice. Am J Trop Med Hyg 1962;11(2): 201–15.

18. El Sayad MH, Yehia MA, Ali AM. Experimental studies on triclabendazole in treatment of

schistosomiasis. Bull Alex Fac Med 2001;2(1): 59-70.

19. Cheever AW. Conditions affecting the accuracy of potassium hydroxide digestion

techniques for counting S.mansoni eggs in tissues . Bull WHO 1968 ;39:328-31

20. Anderson TF. Techniques for preservation of three-dimensional structure in preparing

specimens for the electron microscope. Trans New York Acad Sc 1951; 13:130–4.

21. Weiss DJ, Wardrop KJ. Schalm’s veterinary hematology. 6th

ed. Wiley-Blackwell 2010,

pp.246–50.

150

22. Reitman S, Frankel S. A Colorimetric method for the determination of serum glutamic

oxaloacetic and glutamic pyruvic transaminases. Am J Clin Pathol 1957;28: 56-63.

23. Kind PR, King EJ. Estimation of plasma phosphatase by determination of hydrolysed phenol

with antipyrine.J Clin Pathol 1954;7:322-6.

24. Fawcett JK, Scott JE.A rapid and precise method for determination of urea. J Clin Pathol

1960;13: 156- 9.

25. Husdon H, Rapoport A. Estimation of creatinine by Jaffe reaction. A comparison of three

methods . Clin Chem 1968;14:222-38.

26. Ellman RC, Courtney K, Andres V, Featherstone RM. A new and rapid colorimetric

determination of acetylcholinesterase activity. Biochem Pharmacol 1961;7: 88-95.

151

ARABIC

SUMMARY

152

انهخص انعربي

اغؼ ابئ ف اطوح ػ اؾبخ اوظخ الب اصبة ؼزجو اؼالط اىبي وض اجبهب ا

نا .ػ غوك اطوح ػ اللا اجبغخ ػمبه اجواىىاز االوضو ازقلاب ػبب ثزصوؼ ظخ اصؾخ اؼبخ

اظبفخ فؼبي غ اللا غو اجبغخ ما رأصو ؾلك ف ؽبخ عك اصبثبد اىجل اطؾبي . واؼمبه ال غ اػبكح االصبثخ غ

اىبخ عك مبخ نا الاء .و ن اؼلبد شغؼذ اؼبء جؾش ػ اكخ عللح هفصخ ظل وض اجبهب.ا

ل ايذ اى جبد اواقالصخ اغبيح جبد اوازفوح ف رلف ن الهاخ زم وفبءح ػمبه زبىوب

اق )واىل( مبهخ ثؼمبه اجواىىاز ؼالط افئوا اصبثخ ثبجبهب اؼخ .

٧١١ي االفوي ظبثطخ . ر ػل ؼبغخ ػ غوك اف بدفأها م وغػ ٧١١ف ن ازغوثخ ر ازقلا

اؼلي ر اىشف ػ االصبثخ ػالط افئوا ٥١ثؼل ووبهب كلا ثبهب ازم ى فأه ٧١١فأه ثؼلك

فأها ف و غػخ( ١١ (غػبد ٦اصبثخ ثؼل فزوح ص غاي ا ؼ ثزبي اطؼب ثؼل مه ثبػخ ى بن

وبألر:

اب ززبخ . ٥غ / وغ وغوػخ اؽلح ب لح ٥١١ صبثخ ػغذ ثؼمبهاواىل ثغوػخ : فئوا٧اغػخ

أب ٣غ / وغ وغوػخ اؽلح ب لح ٧١: فئوا صبثخ ػغذ ثؼمبهايذ اى جبد او ثغوػخ ١اغػخ

ززبخ .

غ / وغ وغوػخ اؽلح ب لح ززب. ٥١١اىىاز ثغوػخ : فئوا صبثخ ػغذ ثؼمبه اجو٣اغػخ

أب ززبخ . ١غ / وغ وغوػخ اؽلح ب لح ٧١١: فئوا صبثخ ػغذ ثؼمبه زبىوبل ثغوػخ ٤اغػخ

: فئوا صبثخ غو ؼبغخ. ٥اغػخ

و صبثخ غو ؼبغخ. : فئوا غجؼخ غ٦اغػخ

ابثغ اؼالط .ر اوزشبف وفبءح االكخ ازقلخ ػ ٤ ٧١زػخ ثؼل فزواد ػىر مثؼ فئوا اغػبد

كهاخ ػلك اجعبد ف أغخ -ػلك اللا رؾلل ع اللا غب -اجط ف اجواى كػلكهابد غفخ ) غوك

كهاخ ازغواد از رؾلس ف واؽ اجعبد كهاخ ثبىىة االىزو ابؼ -اىجل األؼبء اللمخ

اخ ؽع ب : زبئظ ن اله اإلؽصبئى ثبزؾ كهابد ثوبئخ .كهابد ػل فالب ال

لهابد اطفخ:ف ا -٧

ػلك اجعبد ف اجواى ثلءا" زػ إى اقفبض شلل م كالخ اؽصبئ ف ثؼمبه اجواىىازأكي اؼالط

قفبض ٪( ر افزفبء و اجعبد ف االجع اضب اواثغ اؼالط )ثجخ ا٦٣االجع االي ثؼل اؼالط )ثجخ

ػلك اجعبد ف اجواى ثؼل زػ ٪ م كالخ إؽصبئ ف ٦.٦٩٪ ٥.٣٩جخ ثؽمك ػمبهواىل اقفبض .٪( ٧١١

االجع ثؼل االجع اضب ٪ م كالخ اؽصبئ٦.٥١٪ ٥.١١جخ اواثغ . ؽمك ػمبه زبىوبل االجع اضب

٪ ١.٩ ٪٧ثت مي كالخ اؽصبئ )اواثغ . ايذ اى جبد او قفط زػ ػلك اجعبد ف اجواى

اواثغ .٪( ثؼل االجع االي اضب ٥.٧١

٩٤٪ ١٣) ثجخ ػلك اللا اىخزػ إى اقفبض شلل م كالخ إؽصبئ ف ثؼمبه اجواىىازأكي اؼالط ٪

ثؼل٪( ١٧٪٥١٪٣٤ػلك اللا اىخ )زػ ف ؽ ؽمك ػمبهواىل جخ ما كالخ اؽصبئ ف رم ٪( ٩١

ثجخ مي كالخ إؽصبئ ػلك اللا اىخؽمك ػمبه زبىوبل رم ف زػ االجع االي اضب اواثغ.

153

ىب م فمػ ٪١٩ جخى ايذ اى جبد او ؽمك ع اضب اواثغ اؼالط.االجف ) ٪ ٦٥ ٪ ٤٥ (

. ثؼل االجع اواثغ اؼالط ػلك اللا اىخزػ رم ف كالخ إؽصبئ

افئوا اؼبغخ ى ػمبهواىل ازقوعخ موه إبس اللا ػلك اصبها" زبخ ػ ؽمك ػمبهاجواىىاز

األجع االي ػمبهزبىوبل ايذ اى جبد او وب رأصو اوضوػ ػلك اللا انوه االبس ثؼل

اضب اواثغ.

و وجوػ غي موه إبس اللا ى ػمبهواىل وب رأصغي ؽبخ زبخ ػ ؽمك ػمبهاجواىىاز

ايذ اى جبد او رأصوػ غي اللا انوها ػمبهزبىوبلاللا انوه ػ االبس ف ؽ ؾلس

االجع االي اضب اواثغ. االبس ثؼل

بد ف االؼبء ثجخ اكي اؼالط ثؼمبه اجواىىاز ا اقفبض م كالخ اؽصبئخ ف رم زػ ػلك اجع

( ثؼل االجع األي اضب اواثغ. أكي ٪١.١٥ ٪١.٦٩ ٪٤.٦٤( اوضو اغخ اىجل )٪١١٪٧.١٩ ٪٩.٦٩)

( ٪٦٦٪٣.٤٩ ٪٩.١١ا اقفبض م كالخ إؽصبئخ ف اجعبد ف اغخ االؼبء ثجخ )واىل اؼالط ثؼمبه

أكي اؼالط األجع اضب اواثغ. ( ثؼل٪٣.٦٥ ٪١.٤١اوضو اغخ اىجل )غ األجع األي اضب اواث ثؼل

٪٧.١١ثؼمبه زبىوبل ا اقفبض م كالخ اؽصبئخ ف رم زػ ػلك اجعبد ف اغخ االؼبء ثجخ )

االجع اضب ( ثؼل٪١.٣١ ٪١.١٣اوضو اغخ اىجل )االجع االي اضب اواثغ ( ثؼل٪٦.٤٦٪١.٤٥

أكي اؼالط ثبيذ اى جبد او ا اقفبض مي كالخ إؽصبئخ ف زػ ػلك اجعبد ف اغخ اواثغ.

( ثؼل٪٣.٤١اوضو اغخ اىجل )األجع األي اضب اواثغ ( ثؼل٪٥.٣٧ ٪١.١٣ ٪١.٧١االؼبء ثجخ )

األجع اواثغ.

اجواىىاز ا ىبكح مي كالخ اؽصبئخ ػبخ ف ػلك اجعبد ازخ وب أكي ا رم اجعبد أكي اؼالط ثؼمبه

األجع ثؼلواىل ىبكح م كالخ اؽصبئخ ف ػلك اجعبد ازخ ف ؽ أكي اؼالط ثؼمبه اغو بظغخ ابظغخ

.األجع اواثغ ثؼل ر رم اجعبد اغو ابظغخ األجع اواثغ ثؼلبد ابظغخ ونه اجع اضب اواثغ

ػمبه زبىوبل ىبكح غوكخ ما كالخ إؽصبئخ ف اجعبد ازخ اجعبد ابظغخ غ رم ف اجعبد ؽمك

اقفبض غوكي ف ػلك فمل ؽمك ى جبد اوايذ ااب .األجع األي اضب اواثغ ثؼلاغو بظغخ

اجعبد ابظغخ ى ثجخ لخ ػل مبهز اجعبد ازخ غ ىبكح طوكح ف ػلك اجعبد اغو بظغخ

ثبألكخ األفوي.

كهاخ ثبىىة االىزو ابؼ:-١

اقبهع للا اجبهب ؽش أكي ا ريك ازءاد غ فملا اجواىىاز ا رلو ف اطؼأكي اؼالط ثؼمبه

األشان اعكح أػ ازءاد ف بغك زؼلكح ف اللا انوه ونه رموػ شلل ف اطؼ اقبهع ف اللا

بس .اإلبس ب أكي ا إظبه األغخ رؾذ اطؼ اقبهع . ازف ف انوه وب أوضو اإل

ا رله طؾ ف غطبء اللا اإلبس غ اىب ف اطؼ اقبهع ريك واىل أكي اؼالط ثؼمبه

ازءاد غ فملا االشان اعكح أػ ازءاد إ علد غ فمل ؽلرب ف اللا انوه غ ػل ؽلس رفبد ف

األغخ اؼمخ رؾذ اغطبء .

وبل أكي ا رف ففف ف غطبء كلا اجبهب ػ ئخ اصبثبد ف أعياء اطجمبد بث ازؤاد ػمبه زبى

غ رله شى اصبد افخ ف موه اللا فملا األشان اعكح كاف لبح اإلؽزعب .

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غطبء خ غ ػل ظه رغواد ؾظخ ف ايذ اى جبد او ي ازفبؿ ف اصبد افخ اجط ؾمك

اللا.

كهابد صهح ال : -٣

عث ػلك فالب جواىىاز أكي ا ىبكح غوكخ ف جخ ااثبمبهخ ثبفئوا اصبثخ غو اؼبغخ فب ػمبه

د فالب ال اؾواء ض )زػ ؽغ )اابرووذ( ؤشوا فالب ال اؾواء ازواوخجخ ال اؾواء

ووبد ال اؾواء زػ ى عث ووبد ال اؾواء زػ رووي اعث ف ووبد ال

فأكي اؼالط ثؼمبه اواىل ا رؾ ؾظ ف اؼا ابثمخ .األجع األي اضب اواثغ ثؼلاؾواء(

اضب )بػلا زػ ؽغ ووبد ال اؾواء زػ ى عث ووبد ال اؾواء( ف األجع األجع

جخ اعث جخ ازبىوبل ا ىبكحأكي ػمبه اواثغ اؼالط ر رؾ ثشى ؾظ ف و اؼا .

اؾواء ض )زػ ؽغ ووبد ال اؾواء زػ ى فالب ال اؾواء ازواوخ ؤشواد فالب ال

االجع اواثغ ثؼل اؼالط فمل ر ىبكح و اؼا اطثخ األجع اضب . اب فف عث ووبد ال اؾواء(

جبد او وب ازغو اؽل ف بيذ اى ثبجخ ؼالط ث )بػلا ػل فالب ال اؾواء از ؾلس ثب رغو(.

األجع اواثغ ثؼل اؼالط ونه ثؼط ؤشواد فالب ال اؾواء ض زػ ى ىبكح جخ اعث ف

ف األجع اضب اواثغ زػ رووي اعث ف ووبد ال اؾواء ف عث ووبد ال اؾواء

ثغ.األجع اوا

ػلك فالب ال اجعبء اى صبؽج لخ اقالب افبخ ف ثجخ م كالخ اؽصبئخ رمجواىىاز اجت ػمبه

األجع األي اضب اواثغ.ف األجع األي اضب ثؼل اؼالط ىبكح ف اقالب ازؼبكخ ف اقالب اؾبعخ

االجع اضب اواثغ ف اقالب ف ػلك فالب ال اجعبء اى اقالب افبخ ل رم جت ػمبه اواى

األجع اضب اواثغ.ف االجع االي اضب اواثغ غ ىبكح ؾظخ ف اقالب ازؼبكخ ف اؾبعخ

االجع اواثغ ف اقالب ف بء اى ثجخ م كالخ اؽصبئخ ازبىوبل أكي ا رم ف ػلك فالب ال اجع

ثبجخ ؼالط االجع اواثغ.ف االجع اضب اواثغ غ ىبكح ف اقالب ازؼبكخ اقالب اؾبعخف افبخ

اواثغ ؽش عل اقفبض ؾظ ف االجعبيذ اى جبد او عل فمػ رغواد ف ػلك اقالب ازفوم ف ث

اقالب ؽلح ااح اقالب امبػلخ ؾلس ثب رغو اقالب افبخ اقالب اؾبعخ ىبكح ف اقالب ازؼبكخ .

ؾظ ف اي لذ ثؼل اؼالط .

ا ازبىوبل جت ىبكح ف ػلك اصفبئؼ الخ اواىل ا جواىىازاػالط افئوا اصبثخ ثبزقلا ػمبه

ايذ اى جبد ازبىوبل اب جواىىاز اواىل صثؼل األجع اضب اواثغ وب ازأصو لب ثبزقلا ا

ف ػلك اصفبئؼ الخ ى جت لخ شللح ثؼل األجع االي. جت ىبكح او

الهابد اجوبئخ: -٤

اي اال ا روافوي ايبد اىجل ف اص ض جواىىاز ا رم شبغاأكي اؼالط ثؼمبه (ALT) ثجخ

اي اىب (AST)ا روافوياألجع األي اضب اواثغ ونه اي اجبهرذ م كالخ اؽصبئ ف

ثجخ م كالخ ALTاي رم شبغأكي اؼالط ثؼمبه اواىل ا األجع اضب اواثغ.ف (ALP)ففبري

أكي اؼالط األجع اضب اواثغ.ف AST ALPاألجع األي اضب اواثغ ونه اؽصبئ ف

ASTاألجع األي اضب اواثغ ونيثجخ م كالخ اؽصبئ ف ALT رم شبغا ازبىوبلثؼمبه

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ALP شبغ ىبكحا بيذ اى جبد او أكي اؼالط ثاألجع اضب اواثغ.ف ALTAST ثجخ م كالخ

االجع اضب اواثغ.ف ALPشبغ األجع األي ازطبع ايذ رم ف األجع اواثغ.ونه ل اؽصبئ ف

ا رم ؾظ غوكي جخ اهب ف ال ثجخ م كالخ اؽصبئ ف اواىل جواىىاز ااكي اؼالط ثؼمبه

األجع األي اضب اواثغ ػ ازا. غؼ اؼمبه ف رم جخ اىوبر ف ال ثشى ؾظ ف األجع

األجع اضب اواثغ ا رم ؾظ جخ اهب ف ال ف ف افئوا ازبىوبلأكي اؼالط ثؼمبه اواثغ.

ا ىبكح ؾظخ جخ اهب ف بيذ اى جبد اوأكي اؼالط ث ى غؼ ف رم جخ اىوبر ف ال .

االجع االي اضب غؼ ايذ ف إؽلاس ف اؼالط ىبكح جخ اىوبر ف ال االجع االي ال ف

االجع اواثغ ى غؼ ف رم جخ اىوبر .رم ؾظ جخ اهب ف ال ف

شبغ االي جت ىبكح ؾظخ ف جخ ا ازبىوبل اواىل جواىىازاػالط افئوا ثبزقلا ػمبه

ثؼل االجع اضب ف االجع اواىل ػمبهاى ازوي ف ال وب ازأصو وجوا ى الؽظز جىوا غ

ثؼل االجع لخ شللح فمل ل شبغ االي ايذ اى جبد اواب . ازبىوبل جواىىاز اواثغ ثبزقلا ا

االي ؾلس رغو ثؼل االجع اضب اواثغ.

انخالصة:

ؽش أظود الهاخ أ ػمبه اجواىىاز الىاي الاء االوضو اخ ف ػالط وض اجبهب ؽش اىفبءح

ػل فالب ال ظبئف اىجل اى ف ؽبخ ػبخ أكي مز كلا اجبهب ف افئوا اؼبغخ ثؼل اجػ اؼالط

اشبغ اؼصج الي اى ازوي ف ال ا ػمبه اواىل ال ف اىفبءح ػ اجواىىاز ى آ جت

ا ػمبه زبىوبل ال وفبءح ػ .افئوا اؼبغخ ف ص صهح ال ا ازأصواد اجوبئخشبو صؾخ ػ

اجواىىاز اواىل ى جت رأصواد صؾخ فطوح صجذ ػل وفبءح ايذ اى جبد او ف ػالط وض

اغوػخ ازقلخ .اجبهب ف افئوا اؼبغخ لخ اب ف ؽلك

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انشرفى

د/يي حس انصيبدا.

اطفبد , لأزبم

ؼل اجؾس اطجخ

عبؼخ االىلهخ

د /هذ عهي انطىيم ا.

اطفبد , لبػل أزبم

ؼل اجؾس اطجخ

عبؼخ االىلهخ

د /سحر احذ ابىحهىا.

اطفبد , ل بػل أزبم

ؼل اجؾس اطجخ

عبؼخ االىلهخ

157

عهي بنبرازيكىاتمبيقبرة انيرازيذو دراسة فبعهية عقبر انيتبزوكسبيذ وانسيث انكهي نببت انر

انبههبرسيب انعىية انتجريبية

مل

دمحم عسيس ىار انقساز

ثىبه اؼ اطجخ اجطوخ

٧٩٩١- عبؼخ امبوح. وخ اطت اجطوي

هحصىل عهي درجةن

انبجستيرفي عهى انطفيهيبت انتطبيقية وانجسيئية

يىافقى نجة انبقشة و انحكى

د / يي حس انصيبد.ا

اطفبد , لأزبم

ؼل اجؾس اطجخ

عبؼخ االىلهخ ثبء احذ انصري د /ا.

صؾخ ابغك اؾبهح, ل أزبم

ؼب صؾخ اؼبخؼل اا

عبؼخ االىلهخ

دمحم ابىانهذييصطفي د /.ا

اطفبد , لأزبم

اجؾس اطجخؼل

عبؼخ االىلهخ

هذ عهي انطىيم / و.دا.

اطفبد , لبػلأزبم

ؼل اجؾس اطجخ

عبؼخ االىلهخ

٧٥/٧١/١١٧٤ازبهـ

158

دراسة فبعهية عقبر انيتبزوكسبيذ وانسيث انكهي نببت انر و انيرازيذ يقبرة ببنبرازيكىاتم عهي

انبههبرسيب انعىية انتجريبية

ػخهبخ

عبؼخ االىلهخ-ؼل اجؾس اطجخملخ ا

افبءا عيئب شوغ اؾصي ػ كهعخ

انبجستيرفي عهى انطفيهيبت انتطبيقية وانجسيئية

مل

دمحم عسيس ىار انقساز

ثىبه اؼ اطجخ اجطوخ

٧٩٩١-عبؼخ امبوح . وخ اطت اجطوي

٤١٠٢

159