Download - M.aziz master thesis 2014
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
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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
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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
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انرحيى انرح هللا بسى :قال هللا تبارك وتعايل
} رفع درجبت ي شبء وفىق كم ري عهى عهيى{ ( ٦٧قران كرمي )سورة يوسف اية
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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.
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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
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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
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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
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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).
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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
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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
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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
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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
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INTRODUCTION
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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)
.
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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)
.
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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
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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 .
154
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
155
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