1. health and fitness
2. disease

Republic of Iraq
Ministry of Higher
Education
& Scientific Research
University of Baghdad
College of Science
Seroprevalence of Toxoplasmosis in a
Sample of Iraqi Schizophrenic Patients
A Thesis
Submitted to the counsil of the College of Science
University of Baghdad
In partial fulfillment of the Requirements for the Degree
of Master (M.Sc.)
In
Biology/Zoology
By
Suhair Dakhil Neamah AL-Maamuri
B.Sc. 2009
Under supervision of
Prof.Fawzia A. AL-Shinawy
June 2014
Prof. Dr. Alice K. Agop
Shaaban 1435
‫ﺑِ ْﺴ ِﻢ اﻟﻠﱠ ِﻪ اﻟ ﱠﺮ ْﺣ َﻤ ِﻦ اﻟ ﱠﺮِﺣﻴﻢ‬
‫ﺖ ﺑِﻨِ ْﻌﻤ ِ‬
‫ﻚ ﺑِﻤ ْﺠﻨُ ٍ‬
‫ﻮن *‬
‫ﺑ‬
‫ر‬
‫ﺔ‬
‫ﱢ‬
‫َ‬
‫ن َواﻟْ َﻘﻠَ ِﻢ َوَﻣﺎ ﻳَ ْﺴﻄُُﺮو َن * َﻣﺎ أَﻧْ َ َ َ َ‬
‫ﻚ َﻷَ ْﺟﺮا ﻏَْﻴـﺮ ﻣﻤﻨُ ٍ‬
‫ﻚ ﻟَ َﻌﻠﻰ ُﺧﻠُ ٍﻖ َﻋ ِﻈ ٍ‬
‫ﻴﻢ *‬
‫ﻮن * َوإِﻧﱠ َ‬
‫َوإِ ﱠن ﻟَ َ ً َ َ ْ‬
‫صدق ﷲ العظيم‬
‫القلم ‪4 - 1‬‬
Certification of Supervisors
We certify that this thesis title (Seroprevalence of Toxoplasmosis in
a sample of Iraqi Schizophrenic Patients) was prepared under
supervision at the College of Science , University of Baghdad, as partial
fulfillment of requirement for degree of Master of Science in BiologyZoology .
Signature:
Prof. Fawzia A. AL-Shinawy
Supervisor
/ /2014
Department of Biology
College of Science
University of Baghdad
Prof. Dr. Alice K. Agop
Supervisor
/ /2014
Department of Biotechnology
College of Science
University of Baghdad
In view of the availablem recommendation , Iforward this thesis for debate by
the examining committee .
Prof. Dr. Sabah N. Alwachi
Head of department Committee
of Graduate Studies in Biology
University of Baghdad
Date:
/ / 2014
Examination Committee Certification
We, the examining committee, certify that we have read this thesis title
(Seroprevalence
of Toxoplasmosis in a sample of Iraqi Schizophrenic
Patients) and have examined that student in its contents and that in our opinion it
is adequate for awarding Degree of Master of Science in Biology- Zoology.
Signature
Signature
Dr. Khawla H. Zghair
Dr. Talib A. Hussein
Member (Assist. Prof.)
Member (Assist. Prof.)
Signature
Dr. Abdul Rasul K. Saeed
Chairman (Assist. Prof.)
Signature
Prof. Fawzia A.AL-Shinawy
Assist. Prof. Dr. Alice K. Agop
Supervisor
Supervisor
/ / 2014
/ / 2014
Prof. Dr. Mohammed A. Atiya
Dean of College of Science
University of Baghdad
Date: /
/ 2014
Declaration
This is to certify that the dissertation / thesis entitled:
“Seroprevelance of Toxoplasma gondii in schizophrenic
patients in Iraq "
.
Submitted by: Suhair Dakhil Neamah AL-Maamuri
Department: Biology
College:
Science
has been linguistically corrected and its language in its present
form is acceptable.
Name: Enas T. Inssaif
Address:
Signature
Acknowledgement
At the prime praise be to (Allah), lord of the Universe, blessing and peace on
Muhammad (Allah’s peace and pray be upon him). There are quite a few I would like
to thank for their participations, directly or indirectly, in the completion of this
thesis:
Above all I would like to express my sincere and deep gratitude to both my
supervisors, ( Prof. Fawzia A. Al-shinawy) and ( Prof. Dr. Alice K. Agop), for their
indescribable help, advice and encouragement throughout all steps of my research.
My profound thanks to the Dean of the College of Science – University of Baghdad ,
the head of Biology department Assist. Prof. Dr. Sabah N. Alwachi .
Gratitude is expressed to all staff members of Department of Laboratories in AlRashad Teaching Hospitals for their help during sample collection.
My profound thanks owe to all patients and volunteers for their kindly cooperation.
My deep respect to both Dr. Nahlah G. Abdul-Majeed and Dr. Leen K. Mustafa in
Medical city Teaching Laboratories for helping me .
I express my deep sense of gratitude to my family members especially my father for
the encouragement and support they offered to me.
Finally, I wish to express my thanks to all individuals who cooperated with me, and
without their help, this work would not have been accomplished.
Suhair
The Dedication
To all patients who have
schizophrenia
I dedicate this work
I
SUMMARY
Toxoplasma gondii is a widespread parasite of great importance, and that
the disease toxoplasmosis does not show any clinical specific signs .The
present study was performed on 200 patient with schizophrenia and 100
healthy individuals, considered as control group. Blood samples were
collected from all study groups ( patients and healthy, males and femals)
during the period begining from December 2012 until the end of February
2013. The parasite T. gondii antibodies in the sera were detected using Latex
test (LAT) and Modified Latex test (MLAT) . Also they were detected using
Enzyme Linked Immunosorbent Assay (ELISA) compared with the control
healthy serum individuals. The concentration of interleukin-12 in the serum
was also measured in 40 samples (schizophrenic patients and control) to
determine
the
possible
association
between
schizophrenia
and
toxoplasmosis infection and one of the immunological markers . The results
are summarized as follows :
 The study revealed that 71.5 % of the schizophrenic patients were positive to
T.gondii antibodies detected using LAT test compared to control group
(45%) with significant differences between them . whereas during using
MLAT test the percentage of schizophrenic patients were 93.7 % for IgG and
6.3% for IgM , while the percentage of healthy individuals were 100% IgG
with significant differences between the percentages of infection .
 Using ELISA test sera of the schizophrenic patients showed 79.7% antiToxoplasma IgG antibodies compared to 73.3% of the healthy individuals
with no significant differences. Also detection of IgM in the sera of the
schizophrenic patients showed 4.19% anti-Toxoplasma IgM antibodies
compared to 0 % of healthy individuals with significant differences between
them .
II
 The study indicated significant differences between the percentages of T.
gondii infection individuals detected by using ELISA test in relation to level
of education , occupation , type of drinking water, presence of cats and other
pets , consuming milk , type of consuming meat , age and gender, type of
locality .
 No significant differences were found between the concentration of
interleukin -12 (IL-12) in sera of schizophrenic patients with toxoplasmosis
infection and healthy individuals (control) with the mean concentrations of
(5.79 ± 0.61 pg\ml) and(5.01 ± 0.89 pg\ml) respectively. But when
comparing IL-12 level according to gender , a significant differences were
found between the two groups. The mean concentration in sera of male
patients was (5.626 ± 0.93 pg\ml) compared to healthy control (3.777 ±
0.423 pg\ml), and in female patients (5.976 ± 0.80 pg\ml) compared to
healthy control (6.260 ± 1.62 pg\ml) .
III
LIST OF CONTENTS
SUBJECT
Page
Summary
List of Contents
I
III
List of Abbreviations
List of Figures
List of Tables
X
VII
IX
CHAPTER ONE
INTRODUCTION
AND
LITERATURES REVIEW
1-1: Introduction
1-2: Literatures review
1
5
1-2-1:Toxoplasmosis
1-2-2: History of toxoplasmosis
1-2-3: Taxonomy of parasite
1-2-4: Morphology of Toxoplasma gondii
1-2-4-1:Oocyst
1-2-4-2: Tachyzoite
1-2-4-3: Bradyzoite
1-2-5 : Life cycle of Toxoplasma gondii
1-٢-6: Toxoplasma gondii and human toxoplasmosis
1-2-6-1:Epidemiology
1-2-6-2:Transmission of toxoplasmosis
1-2-7: Pathogenesis
1-2-7-1:Acute toxoplasmosis
1-2-7-2: Subacute Infection
1-2-7-3: Latent toxoplasmosis
1-2-8: Clinical syndromes
1-٢-8-1: Toxoplasmosis in the immunocompetent patient
1-2-8-2: Toxoplasmosis in the immunodeficient patient
1-2-8-3: Congenital toxoplasmosis
1-2-8-4 :Ocular toxoplasmosis
1-٢-8-5:Toxoplasmosis role in schizophrenia and behavioral
5
6
7
8
8
9
10
11
13
13
16
17
18
19
19
20
20
20
21
23
25
IV
changes
1-2-9:Immune Response
1-2-9-1:Innate Immune Response
1-2-9-2:Adaptive Immune Response
1-2-9-3:Humoral Immune Response
1-2- 10: Diagnosis
1-2-10-1:Direct methods
1-2-10-1-1:Isolation of the parasite
1-2-10-1-2:Histological diagnosis
1-2-10-2: Indirect methods (Serological tests)
1-2-10-2-1: Sabin-Feldman Dye Test (SFDT)
1-2-10-2-2:Indirect Fluorescent Antibody Test (IFAT)
1-2-10-2-3: Complement Fixation Test (C FT)
1-2-10-2-4:Indirect Haemagglotination Test (IHAT)
1-2-10-2-5: Latex Agglutination Test (LAT)
1-2-10-2-6: Modified Latex Agglutination Test (MLAT)
1-2-١٠-2-7: Enzyme Linked Immunosorbant Assay (ELISA)
1-2-10-2-8:VITEK Immunodiagnostic Assay System (miniVIDAS)
1-2-10-2-9:Enzyme Immunoassay (EIA)
26
26
28
30
31
32
32
32
33
33
34
34
34
35
35
35
36
36
1-2-10-2-10:Immunosorbent Agglutination Assay (ISAGA)
37
1-2-10-3: Others
37
1-2-10-3-1:Skin Test
1-2-10-3-2:Polymeras Chain Reaction (PCR)
1-2-10-3-3:Diagnosis by Computerized Tomography (CT and
Magnetic Resonance Imaging (MRI)
37
38
38
CHAPTER TWO
Materials and Methods
2: Subjects , Materials and Methods
2-1: Subjects collection
2-1-1: Blood Sample collection
2-2: Materials
2-2-1: Instruments
2-2-2: Equipments
2-2-3: Chemicals, Solutions and Kits
2-3: Methods
2-3-1:Latex Agglutination Test (LAT)
39
39
39
40
40
41
42
42
42
V
2-3-1-1:The principle of the method
2-3-1-2: Techniques
2-3-2:Toxoplasmosis latex agglutination test with 2-Mercaptoethanol (2-ME) (MLAT).
2-3-3: Enzyme linked Immunosorbent Assay (ELISA-IgG)
2-3-3-1: The principle of the method
2-3-3-2: Enzyme Linked Immunosorbent Assay Reagent
2-3-3-3: Method
2-3-3-4: Calculation and interpretation of results
2-3-4: Enzyme linked Immunosorbent Assay (ELISA-IgM)
2-3-5: Determination of Human Interleukin 12 ELISA Kit ( ELISA IL12)
2-3-5-1: Reagent
2-3-5-2: Assay procedure
2-3-5-3: Calculation of results
2-3-6 : Statistical analysis
CHAPTER THREE
RESULTS AND DISCUSSION
3: Results and Discussion
3-1: Demographic presentation of the studied groups
3-2: Serological diagnosis of T. gondii Abs in sera of schizophrenic
patients and healthy individuals by using LAT\MLAT Abs
tests
3-3: Serological diagnosis of anti-T. gondii Abs in sera of
schizophrenic patients and healthy individuals by using ELISA
IgG\IgM test
3-4:Frequencies of T.gondii antibodies in sera of Schizophrenic
patients and healthy individuals(control) according to
educational level as measured by ELISA-IgG (Abs.) test
3-5:Frequencies of anti-T.gondii antibodies in sera of Schizophrenic
patients and healthy individuals(control) according to
occuption as measured by ELISA-IgG (Abs.) test
3-6:Frequencies of anti-T.gondii antibodies in sera of Schizophrenic
patients according to type of drinking water as measured by
ELISA-IgG( Abs.) test
3-7:Frequencies of anti-T.gondii antibodies in sera of Schizophrenic
patients and healthy individuals according to presence of
cats and pets as measured by ELISA-IgG test
42
43
43
44
44
44
45
45
46
47
47
48
49
49
50
50
50
53
57
58
60
62
VI
3-8: Frequencies of anti-T.gondii antibodies in sera of Schizophrenic
patients and healthy individuals according to consuming
milk as measured by ELISA-IgG test
3-9:Frequencies of anti- T.gondii antibodies in sera of Schizophrenic
patients and healthy individuals according to type of
meat (fresh or canned) consumed as measured by
ELISA-IgG test
3-10: Frequencies of anti- T.gondii antibodies in sera of
Schizophrenic patients and healthy individuals according to
age and gender groups as measured by ELISA-IgG test
3-11:Frequencies of T.gondii antibodies in sera of Schizophrenic
patients and healthy individuals according to type of
locality as measured by ELISA-IgG test
3-12: Presence of IL-12 in Schizophrenic patients and healthy
individuals as measured by ELISA-IL 12 kit
69
Conclusions and Recommendations
References
77
80
64
66
72
74
VII
Figure
No.
LIST OF FIGURES
TITLE
Page
1-1
Endodyogeny of T. gondii
10
1-2
Schematic drawings of a tachyzoite (left) and a bradyzoite (right)
of T. gondii. The drawings are composites of electron
micrographs
11
1-3
Diagram summarising the complete life cycle of
Toxoplasma gondii
Showing Congenital toxoplasmosis (hydrocephalitis)
Active retinitis area adjacent to previous cicatricial foci
12
Development of a model of immunity to T. gondii
Enzyme linked Immunosorbent Assay (ELISA-IgG) kit
use in this study.
Enzyme linked Immunosorbent Assay (ELISA-IgM) kit
use in this study
Human Interleukin 12 ELISA (IL-12) kit use in this
study
The percentage distribution of T.gondii antibodies in ٢٠٠
sample sera of Schizophrenic patients and 100 sample of
healthy individuals (control) in Iraq as measured by LAT
(Abs) test.
The percentage distribution of T.gondii antibodies in sera
of Schizophrenic patients and healthy individuals (control)
in Iraq as measured by MLAT(Abs) test
The percentage distribution of anti-T.gondii antibodies
in sera of Schizophrenic patients and healthy
individuals (control) in Iraq as measured by ELISA-IgG
(Abs.) test.
28
46
3-4
The percentage distribution of anti-T.gondii antibodies
in sera of Schizophrenic patients and healthy
individuals (control) in Iraq as measured by ELISA-IgM
(Abs.) test.
54
3-5
Frequencies of anti-T.gondii antibodies in sera of
Schizophrenic patients and healthy individuals(control)
in Iraq according to educational level as measured by
ELISA-IgG (Abs.) test.
57
1-4
1-5
1-6
2-1
2-2
2-3
3-1
3-2
3-3
23
24
47
49
51
52
53
VIII
3-6
3-7
3-8
3-9
3-10
3-11
3-12
3-13
3-14
3-15
3-16
Frequencies of anti-T.gondii antibodies in sera of
Schizophrenic patients and healthy individuals in Iraq
according to occuption as measured by ELISA-IgG (Abs.)
test
Frequencies of anti-T.gondii antibodies in sera of
Schizophrenic patients in Iraq according to drinking
water type as measured by ELISA-IgG (Abs.) test.
Frequencies of anti-T.gondii antibodies in sera of
Schizophrenic patients in Iraq according to presence of
cats as measured by ELISA-IgG test.
Frequencies of anti-T.gondii antibodies in sera of
Schizophrenic patients and healthy individuals in Iraq
according to presence of pets animals as measured by
ELISA-IgG test.
Frequencies of anti-T.gondii antibodies in sera of
Schizophrenic patients and healthy individuals in Iraq
according to consuming milk as measured by ELISA-IgG
test.
Frequencies of anti- T.gondii antibodies in sera of
Schizophrenic patients and healthy individuals in Iraq
according to type of meat consumed as measured by
ELISA-IgG test
Frequencies of anti-T.gondii antibodies in sera of
Schizophrenic patients and healthy individuals
according to consumed canned meat as measured by
ELISA-IgG test.
Frequencies of anti-T.gondii antibodies in sera of
Schizophrenic patients and healthy individuals in Iraq
according to age group as measured by ELISA-IgG test.
Frequency of anti-T. gondii antibodies in sera o
Schizophrenic patients and healthy individual in Iraq
according to the sex as measured by ELISA –IgG test.
Frequencies of anti-T.gondii antibodies in sera of
Schizophrenic patients in Iraq according to type of locality
as measured by ELISA-IgG test.
Concentration of IL-12 in schizophrenic patients and
healthy individuals
59
61
62
63
65
66
67
70
70
73
75
IX
No.
LIST OF TABLES
TITLE
Page
2-1
2-2
2-3
Instruments used in this study
Equipments used in this study
Chemical materials and solutions used in this study.
40
41
42
2-4
Reagents
47
X
List of Abbreviations
Abs
Antibodies
Ag
Antigen
AIDS
Acquired Immunodeficiency syndrome
APC
Antigenic Presenting Cell
CD4
Cluster of Differentiation 4
CD8
Cluster of Differentiation 8
CFT
Complement Fixation Test
DAT
Direct Agglutination test
DCs
Dendritic Cells
DHT
Delayed Hypersensitivity Test
DT
Dye Test
ELISA
Enzyme-Iinked Immunosorbent Assay
FR
Free-Range Chicken
HRP
Horse Radish Peroxidase
IFAT
Indirect Fluorescent Antibody Test
IFN- γ
Interferon gamma
IgG
Immunoglobulin G
IgM
Immunoglobulin M
IHAT
Indirect Haemagglutination Test
XI
IL-2
Interleukin-2
IL-4
Interleukin-4
IL-5
Interleukin-5
IL-6
Interleukin-6
IL-12
Interleukin-12
IQ
Intelligence Questions
LAT
Latex Agglutination Test
MLAT
Modified Latex Agglutination Test
MØ
Macrophages
NK
Natural Killer
NO
Nitrogen monoxide
PAS
Periodic Acid Schiff
PO
Peroxidase
PV
Parasitophorous Vacuole
SAG1
Surface Antigen 1
SAS
Statistical Analysis System
TL
T lymphocyte
Th1
T helper one
Th2
T helper two
TMB
Tetra Methyl Benzidine
TNF- α
Tumour Necrosis Factor- alpha
TMN
Tubulovesicular Membranous Network
CHAPTER ONE
INTRODUCTION
AND
LITERATURES REVIEW
Chapter One
Introduction and Literatures review
1
1-1:Introduction :Schizophrenia is a serious neuropsychiatric disease of uncertain etiology.
Epidemiological and neuropathological studies have indicated that some cases of
schizophrenia may be associated with environmental factors, such as exposure to
infectious
agents. However, specific infectious agents associated
with the
development of schizophrenia have not been identified (Torrey and Yolken, 2000 ;
Tanyuksel et al., 2010). In humans, acute infection with Toxoplasma gondii can
produce psychotic symptoms similar to those displayed by persons with
schizophrenia (Yolken and Torrey, 2008 ). Consequently, T. gondii may be a
candidate infectious agent related to schizophrenia (Tanyuksel et al., 2010). A study
by Mortensen et al. (2007) revealed that newborns who have antibodies to T. gondii
have an increased risk of later being diagnosed with schizophrenia. Toxoplasma
gondii is a protozoan parasite found worldwide (Alvarado-Esquivel et al., 2006)
that infects all kinds of mammals, including cats, livestock, and human beings. In its
life cycle, cats and other felids are the definitive hosts and the other warmblooded
vertebrates are intermediate hosts (Daryani et al., 2010). Human response to T.
gondii is related to immune status of the infected person, strain of T. gondii and
course of infection (Suzuki, 2002). T. gondii invades any type of nucleated cells
(Carruthers and
Blackman, 2005) and persists intracellularly in brain cells
including glia and neurons ( Luder et al., 1999 ; Daryani et al., 2010 ).In
immunocompetent
subject , primary infection is usually asymptomatic or
associated with self
limited symptoms such as fever , malaise , and cervical
lymphodenopathy (Daryani et al., 2010). Infection acquired during pregnancy is
frequently associated with transmission of T.gondii to their fetus, resulting in
congenital disease. In
immunocompromised
women ,reactivation of latent
infection can cause life threatening complications ( Montoya and Liesenfeld ,
2004). If a pregnant woman contracts toxoplasmosis, it may be passed through the
placenta to the fetus, resulting in congenital toxoplasmosis, which is a cause of
Chapter One
Introduction and Literatures review
2
mortality and malformation (Lambert, 2009). Infection in human generally occurs
through consuming food or drink contaminated with oocysts and tissue cysts from
undercooked meat. Congenital transmission and organ transplantation are also
other routes of infection (Dalimi and Abdoli,2012). Most of Toxoplasma infections
are asymptomatic to mild and in some infected persons cervical lymphadenopathy,
ocular disease (Alvarado-Esquivel et al., 2006); central nervous system
manifestation (Bossi et al., 1998) and brain abscess may occur (Silva et al., 2001). In
congenital toxoplasmosis the organisms may cross the placenta and infect the fetus.
The symptoms of congenital toxoplasmosis include hydrocephaly, microcephaly,
intracranial calcifications, damage to the retina, and mental retardation (Torrey and
Yolken, 2003). With regard to neurotropism of T. gondii, psychiatric manifestations
such as disorientation, anxiety, depression and even psychoses with schizophernia
form characters are seen in 60% of immunocompromised individuals with AIDS in
whom latent infections are reactivated (Arendt et al., 1999). Similar psychiatric
complications and meningoencephalitis can also occur in T. gondii-infected
immunocompetent human hosts ( Kaushik et al., 2005), and human studies revealed
that latent toxoplasmosis may cause personality changes (Flegr et al., 1996) and
decreased IQ (Flegr et al.,2003). However, more recent studies on model animals
have suggested that behavioral changes are manifest following infection. Moreover,
recent associations have been made between parasite infection and neurological
disorders, such as schizophrenia (Kamerkar and Davis, 2012). Studies showed that
people with schizophrenia and other mental health problems have a higher
incidence of exposure to T. gondii than healthy control, measured by the presence of
antibody to the parasite (Alipour et al.,2011). Different methods can be used for the
diagnosis of T. gondii including isolation of the parasite and several serological tests
that are available for the detection of T. gondii antibodies. In one type, the observer
judges the given color of tachyzoites under a microscope, such as with the Dye Test
(DT) and Indirect Fluorescent Antibody Test (IFAT), Another depends on the
principle of agglutination of Toxoplasma tachyzoites, red blood cells or latex
Chapter One
Introduction and Literatures review
particles,
such
as
with
the
3
Direct
Agglutination
Test
(DAT),Indirect
Haemagglutination Test (IHAT) and latex Agglutination Test (LAT), respectively.
With the Enzyme-linked Immunosorbent Assay (ELISA), the degree of color change
defines the quantity of specific antibody in a given solution (Juma and Salman,
2011). Interleukin (IL)-12 consistently proved crucial for both the initiation and
maintenance of host-protective immunity against T. gondii .The IL-12 molecule is a
heterodimer of 70 kDa consisting of two disulphide-bonded subunits, IL-12 p40 and
IL-12 p35, which must be expressed in the same cell to generate bioactive IL-12.
Production of IL-12 is restricted to a subset of hematopoietic cells including
macrophages, granulocytes, B cells and dendritic cells and is elicited by microbial
stimuli or during interaction with T cells via CD40 ligation (Schade and
Fischer,2001). Multiple evidence indicates that T. gondii can trigger production of
IL-12 in a T cell-independent manner as shown in vivo ( Schulz et al., 2000) and on
isolated macrophages, neutrophils or dendritic cells ( Fischer et al., 1999, 2000).
Such pathway has been implicated to be pivotal for the establishment of IFNdependent innate resistance (Schade and Fischer,2001).
Chapter One
Introduction and Literatures review
4
Aims of the study
This study was designated to determine the prevalence rate of antiToxoplasma antibodies (IgG and IgM ) and IL-12 among local schizophrenic
patients compared with healthy individuals. Accordingly the following
assessments were achieved :
1- Collection of blood serum from patients with schizophrenia and from
healthy individuals used as control.
2 - Detection of the specific T. gondii antibodies in the serum of patients and
control individuals using different detecting methods: LAT , MLAT and ELISA.
3 - Estimating the concentration of IL-12 in the serum of patients and in
control individuals.
Chapter One
Introduction and Literatures review
5
1-2: Literatures review:
1-2-1: Toxoplasmosis:Toxoplasmosis is considered one of the most important worldwide zoonotic
diseases caused by the protozoan T. gondii. Besides vertical infection during
pregnancy, humans can get infected post-natally either by peroral uptake of
sporulated Toxoplasma oocysts or by ingestion of tissue cysts upon consumption of
raw or undercooked meat (Berger-Schoch et al., 2011). T. gondii infection can cause
severe neurological ocular disease in the fetus during human pregnancy. Humans
acquire their infections from ingestion of oocyst-contaminated soil and water, from
tissue cysts in undercooked meat, by through placenta, blood transfusion,
laboratory accidents (Schaffner, 2001; Dubey and Jones, 2008).Milk may serve as a
potential source for human toxoplasmosis (Asgari et al., 2011).
Beside that,
tachyzoites of T. gondii were found in the milk of several species, including sheep,
goats, cows, mice and cats (Maksimov et al., 2011). Acquisition of direct oral
transmission by the parasite was an evolutionary change that has led to wide spread
expansion of Toxoplasma (Su et al., 2003 ).T. gondii infection in free-range chickens
(FR) is considered important as one of the best indicators for soil contamination
with T. gondii oocysts because they feed from the ground, and tissues of infected
chickens are considered a good source of infection for cats. Additionally, ingestion of
infected chicken meat can be a source of infection for T. gondii infection in humans
and other animals. Rarely, toxoplasmosis can cause clinical disease in chickens,
direct detection of T. gondii oocysts in soil is difficult. Therefore, chickens have been
used as one of the indicators of soil contamination by oocysts. In 2002, initiated a
worldwide survey of T. gondii infection in (FR) chickens with the ultimate objective
of studying the genetic diversity of T. gondii on a worldwide basis (Lehmann et al.,
2006; Dubey and Su, 2009). T. gondii consists of three main genotypes, designated
type I, II, and III which differ in virulence and epidemiological pattern. Type I strain
is associated with high-level virulence in mice. This type has been recorded in
Chapter One
Introduction and Literatures review
6
patients with ocular toxoplasmosis. Type II is nonvirulent for mice but generate
chronic infection with persistence of tissue cysts. This strain is also most commonly
associated with human infections in Europe and North America.
Type III is
nonvirulent for mice and less frequent than type II in Europe and North America.
The last types regarded as the most frequent strain from animals. Moreover, type I
and II strains have been recorded in patients with congenital disease and AIDS
patients (Dalimi and Abdoli, 2012).
1-2-2: History of Toxoplasmosis :Toxoplasmosis was discovered almost a century ago, in 1908 by Nicolle and
Manceaux, and the pathogen itself was identified in the North African rodent
(Ctenodactylus gundii) (Nicolle and Manceaux,1908), as it was being used for
research in the Pasteur Institute in Tunis. In the same year 1908 Splendore
published his description of Toxoplasma from a laboratory rabbit at the Italian
hospital in Sao Paulo, Brazil, then in (1909) differentiated the disease from
Leishmania and named it T. gondii(Sukthana,2006) .Castellani (1913), described the
organism in spleen smear from 14 year old boy with protracted, fever and
splenomegaly. Junku (1923) observed the cyst in the retina of a child with
hydrocephalus and chorioretinitis in a case of congenital toxoplasmosis but he failed
to isolate the organism. The development of serological tests by Sabin and Feldman
(1948) led to the recognition of asymptomatic infection in human and animal's
populations all over the world. Frenkle(1948) introduced the toxoplasmine skin
test. Ocular toxoplasmosis was reported and proved by Wilder (1953).
Goldman(1957)used immunofluorescence microscope to study the human immune
response . Remington and Kaufman (1960), isolated the parasite from the women
uterus in one of nine necrosis and in the three of 34 cases of hysterectomies.
Moreover they suggested that the chronic infection with Toxoplasma during
pregnancy may facilitate transporting the parasite to the fetus to form congenital
toxoplasmosis. The complete life cycle of T. gondii was not examined in its entirety
Chapter One
Introduction and Literatures review
7
until when Frenkle et al. (1970)discovered the sexual stages of life cycle of parasite.
It was shown that cats are definitive hosts to the pathogen, and are excreted as
oocyst in their feces and they recognized coccidian parasite, while humans and
other mammals, as well as birds, are intermediate hosts .Blewett et al. (1983)
reported a method of isolation of Toxoplasma tissue cyst (bradyzoite) from foetal
ovine brain by simple direct isolation technique . In (1957) Goldman used the
immunofluorescent microscope to study the human immune response (Alcamo ,
1997) . In Iraq , Machattie (1938) was the first who recorded the parasite by a
smear from spleen and lungs of two stray dogs in Baghdad . During the
years 1980 and 1990 methods were developed to recognize genetic differences
among T. gondii isolates from humans and animals, (Dubey and Jones , 2008).
1-2-3: Taxonomy of parasite :T.gondii is classified according to Keas (1999) as follows:Kingdom …………………………... Protista
Phylum ….………………………... Apicomplexa
Class ………………………………. Sporozoa
Subclass ……………………………Coccidiasina
Order ……………………………... Eucoccidiorida
Suborder …………………………..Eimeriorina
Family ……………………………..Sarcocystidae
Subfamily ………………………….Toxoplasmatinae
Genus ……………………………... Toxoplasma
Species …………………………….. gondii
Chapter One
Introduction and Literatures review
8
1-2-4: Morphology of T. gondii:1- Oocyst which produce sporozoites , are shed in cat feces .
2- Tachyzoite or trophozoite which is the active proliferation form found in any
type of host cells except red blood cells (RBCs) .
3- Bradyzoite which is the resting form found in the tissue cysts (Acha and
Szyfres, 2003 ; Jones and Dubey ,2010 ).
1-2-4-1:Oocyst :This form of the parasite develops in the epithelial cell of the intestine
of the definitive host (cat) by gametogony(sexual reproduction ). It is spherical ,
about 10-12 µ in diameter . The mature one is transparent , contains two
sporocysts , each has four sporozoites infective form . which are released
by ingestion in the intestine ( Paniker , 2002 ) . Oocysts of T. gondii are shed
during the first infection (in young cats) and the cat is then immune to reinfection . Older
cats whose immune system is compromised may also shed
oocysts (Thomas, 2005).Contamination of the environment with oocysts of parasite
may be due to infected domestic cats or wild felines. After primary infection , a
single cat may shed more than 100 million oocysts into the environment (Tenter et
al., 2000).Under environmental conditions with sufficient aeration, humidity , and
warm temperature , oocysts may sporulate and become infective in less than
one day. Depending on the Toxoplasma strain , ingestion of as few as 10
sporulated oocysts may cause an infection in intermediate hosts (Dubey et
al.,1996 ) . Sporulated oocysts of Toxoplasma are very resistant to environmental
conditions . They survive short periods of cold and dehydration , and remain
infective in moist soil or sand for up to 18 months (Frenkel, 2000). Under
laboratory conditions, sporulated oocysts survived storage at 4ºC for up to 54
Chapter One
Introduction and Literatures review
9
months and freezing at - 10ºC for 106 days. However , they are killed within 1-2
min by heating to 55- 60ºC ( Dubey , 1998 ) .
1-2-4-2: Tachyzoite:
The term ’tachyzoite‘‘(tachos= speed in Greek) was coined by Frenkel (1973) ,
to describe the stage that rapidly multiplied in any cell of the intermediate host
and in non intestinal epithelial cell of the definitive host . Tachyzoites have also
been termed endodyozoites or endozoites (Dubey et al.,1998).The tachyzoite is
often crescent shaped approximately 2 by 6 µ. with a pointed anterior (conoidal
) end and a rounded posterior end ( Gutierrez ,2000). Although tachyzoites can
move by gliding , flexing , undulating , and rotating , they have no visible means
of locomotion such as cilia , flagella , or
pseudopodia (Frenkel , 1973) .
Tachyzoites are obligate intracellular forms ,which can invade and replicate
and
multiply
in
the cytoplasm within
parasitophorous vacuole(pv) by
endodyogeny within all mammalian cells except a nuclear erythrocytes , they
appear rounded
enclosed by a host cell membrane called pseudocyst , and
intracellular multiplication continues until the host cell lyses or a tissue cyst is
formed (Kasper,2005).Tachyzoites enter host cells by actively penetrating through
the host cell plasmalemma or by phagocytosis (Bonhomme et al., 1992), after
entering the host cell, the tachyzoite becomes ovoid and is surrounded by a
parasitophorous vacuole , which appears to be derived from both the parasite and
the host cell. Soon after penetration ,a tubulovesicular membranous network (TMN)
develops within the PV. Some of the TMN membranes are connected to the
parasitophorous vacuolar membrane (Sibley et al., 1985) .Tachyzoites multiply
asexually within the host cell by repeated endodyogeny, a specialized form of
reproduction in which two progeny form within the parent parasite, consuming it
(Melton and Sheffield ,1968) (Figure 1-1).Rarely, tachyzoites of certain strains
divide by binary fission (Ferguson and Hutchison, 1981).
Chapter One
Introduction and Literatures review
10
Fig(1-1): Endodyogeny of T. gondii (Wikipedia, 2007).
1-2-4-3: Bradyzoite:The term "bradyzoite" (brady = slow in Greek) was also mentioned by Frenkel
(1973) to describe the organism multiplying slowly within a tissue cysts.
Bradyzoites are also called cystozoites.Tissue cysts grow and remain intracellular
as the bradyzoites divide by endodyogeny (Lambert, 2009). Bradyzoites differ
structurally only slightly from tachyzoites. They have a nucleus situated toward the
posterior end, whereas the nucleus in tachyzoites is more centrally located. The
contents of rhoptries in bradyzoites are usually electron dense, whereas those in
tachyzoites are labyrinthine (Saavedra, 2003).However, the contents of rhoptries in
bradyzoites vary with the age of the tissue cyst. Bradyzoites in younger tissue cysts
may have labyrinthine rhoptries, whereas those in older tissue cysts are electron
dense. Also, most bradyzoites have one to three rhoptries, which are looped back on
themselves. Bradyzoites contain several amylopectin granules which stain red with
Chapter One
Introduction and Literatures review
11
Periodic Acid Schiff (PAS) reagent; such material is either in discrete particles or
absent in tachyzoites also bradyzoites are more slender than are tachyzoites and
are less susceptible to destruction by proteolytic enzymes than are tachyzoites
(Dubey and Odening, 2008)(Figure 1-2).
Fig(1-2): Schematic drawings of a tachyzoite (left) and a bradyzoite (right) of
T. gondii. The drawings are composites of electron micrographs (Dubey et al.,
1998).
1-2-5 : Life cycle of T. gondii :The life cycle of T. gondii has two phases. The sexual part of the life cycle
(coccidia like) takes place only in cats, both domestic and wild (family Felidae),
which makes cats the parasite's primary host (Johnsen, 2009). The second phase,
the asexual part of the life cycle, can take place in other warm-blooded animals,
including cats, mice, humans, and birds (Aranda- Lozano, 2011). The hosts in which
asexual reproduction takes place is called the intermediate host (De souse, 2009).
Tissue cysts or mature oocysts are ingested by a cat. The cysts survive passage
through the stomach of the cat and the parasites infect epithelium of the small
Chapter One
Introduction and Literatures review
12
intestine where they undergo asexual reproduction. Asexual form of division occurs
first leading to formation of schizonts which contain merozoites. Some of the
merozoites are transferred into the sexual stages, initiating gametogony.A
macrogamete is fertilized by a motile microgamete resulting in the formation of a
zygote (Bruno et al.,2006). The zygote secretes a protective coat and is transformed
into oocyst. The oocysts are passed in the feces. Animals and humans that ingest
mature oocysts (e.g., by eating unwashed vegetables) or tissue cysts in improperly
cooked meat become infected. The parasite enters macrophages in the intestinal
lining and is distributed via the blood stream throughout the body (Blader and Saeij,
2009). In the first phase tachyzoites multiply rapidly in many different types of host
cells (Benavides et al., 2011).Tachyzoites of the last generation initiate the second
phase of development which results in the formation of tissue cysts containing
bradyzoites (Munoz et al.,2011), which are the slowly replicating version of the
parasite (De souse, 2009) (Figure 1-3).
Fig.(1-3):Diagram summarising the complete life cycle of Toxoplasma gondii
(Ferguson, 2002).
Chapter One
Introduction and Literatures review
13
1-2-6: T. gondii and human toxoplasmosis :1-2-6-1:Epidemiology
T. gondii is among the global major zoonotic diseases (Dubey, 2010). It has been
estimated that one third of the world ppopulation has been infected(Zhou et al.,
2011). Environmental conditions , differences in the type of food consumed ,
animal species used in the food industry,and the number of cats are examples of
factors that may influence the spread of parasite .Water-born transmission of T.
gondii has earlier been considered uncommon ,human outbreaks connected to
water reservoirs have been reported(Hill and Dubey , 2002).Toxoplasmosis is a
disease affecting 500 million people worldwide. The seroprevalence varies from 5%
to 90% depending on geographical location, age, eating habit, raw meat or
unwashed fruit and vegetables, and generallevel of hygiene(Al-jebouri et al.,
2013).The incidence of infection in humans
and
animals
may vary
among
countries , within different areas of a country and within the same city (
Dubey,2010 ).Infection is more common in warm climates and at lower altitudes
than in cold climates and mountainous (Barbosa et al.,2009).In United States
between 2004-2005 national probability sample found that 33.1% of U.S. persons
above 12 years of age haveToxoplasma –specific IgG antibodies. This prevalence has
significantly increased from the 1999-2000 data (Chatterton et al., 2011). However,
there is a large variation among countries, in France, around 88% of the population
are carriers probably due to a high consumption of raw and slightly cooked meat. In
Germany, Netherlands and Brazil also have high prevalence of toxoplasmosis
around 80%, over 80% and 67% respectively (Dabritz and Conrad, 2011). In Britain
about 22% are carriers, and south Korea's rate is only 4.3% (De souse,
2009).Individuals in countries such as France, Ethiopia, and Brazil have a high
prevalence of antibodies to T. gondii. In France and Ethiopia, the high infection rates
are thought to be attributable to the cultural custom of eating undercooked or
uncooked meat; in Brazil, the high rate has been attributed to water supplies
Chapter One
Introduction and Literatures review
contaminated
with
feline
oocysts
14
as
well
as
to
undercooked
meat
consumption(Bahia-Oliveira et al., 2003). Though a recent serological survey carried
out in 3281 individuals from the northeast and the south of China showed a 12.3%
anti-T. gondii IgG positive rate, which indicated the increasing growth in prevalence
(Xiao et al.,2010 ).The prevalence of T. gondii in China was still relatively low,
comparing with 50-75% seropositive in France, and 20% in UK(Darrel, 2009 ;Zhou
et al.,2011 ). In Turkey rate of infection was 39.4 % among women by using
Solid-phase enzyme immunoassay (Durmaz et al., 1995).In Iran, Assmar et al.,
(2000) reported aseroposetive prevalence of 51.8 % using a polymerase chain
reaction (PCR) method. In Arabe countries the prevalence rate varied among
people from different Arab countries using different techniques. In Somalia it was
53% (Zardi et al., 1980) . In Libya the rate of infection in pregnant women was
47.4% by using IHAT test (Kassem and Morsy , 1991) . In Tunisia was 58.4%
(Bouratbine et al., 2001) . In Egypt was 81.4% (Soliman et al ., 2001) .In Saudi
Arabia it was 31.2% of pregnant women attending Abdul -Aziz University Hospital
in Jaddah (Basalamach and serbour , 1981), and in Kuwait was 58.2% (AL-Nakib
et al .,1983) . In Jordan it was 26% ( Abdel Hafez et al.,1986 ).Other study in
Saudi
Arabia on
women was recorded 25% by using LAT, IHAT, IFAT and
ELISA(AL-Meshari et al.,1989).In the United Arab Emirates (UAE) the correlation
between toxoplasmosis seropositivity rate and different occupation groups
was studied .It was declared that the highest prevalence was among the
office workers 33.5% , field farmers 33.3%, domestic workers13% and laborers
22.4% (Mohammad et al .,1997). Other study in UAE in blood donors was 34%
(Uduman et al. ,1998). Pappas and Wordrop(2004)commented on high prevalence
of foci in the Middle East including Turkey, Iran, Iraq and Kuwait. A general
population study in Eastern Saudi Arabia showed a lower prevalence, reaching 25%,
similar to pregnant women in Bahrain and to the general population prevalence
observed in Qatar. In Asia, high rates were found in Indonesia >60% and Malaysia
49%, but significantly lower rates in China and Vietnam 10–11%. In India, most
Chapter One
Introduction and Literatures review
15
studies showed a rate in excess of 40%.In Iraq, Niazi et al. (1988)and AL-Kalaby
(2008) showed that, 39% of the examined women were IgG positive and 60% of
Iraqi women of child bearing age were susceptible and might acquire the infection
during pregnancy. Mohammed and Al- Nasiry (1996) reported a prevalence rate of
20.4% toxoplasmosis in Iraqi women. Al- Hamdani and Mahdi (1997) reported that,
infection had been much more frequently 18.5% in women who are with habitual
abortion than in the normal pregnancy group 5.9% by using LAT. Al-Dageli (1998)
reported that, the Toxoplasma infection rate in age 16-20 years was 9.4% . Hasson
(2004) recorded that, the Toxoplasma infection rate of positive cases in Najaf
governorate, was 133 (40.4%) out of the total number of 325 pregnant women, 64
(19.7%) were positive by LAT , 40 (12.3%) were positive by IgM-ELISA and IgG–
IFAT respectively. In Duhok, North of Iraq, Razzak et al. (2005) found low
Toxoplasma infections of about 0.97%. In Sulaimania, Karem (2007) found out that
by using ELISA, the seropositivity was 32.6% in women. In a study carried out in
Basrah by Yacoub et al. ,(2006) the prevalence of Toxoplasmosis had been shown to
be 41.1 to 52.1%.In Baghdad, Juma and Salman (2011) found that the infection of T.
gondii in women was 19.17%. In Tikrit, Al-Doori (2010) showed that the prevalence
rate was infection of about 49 to 95% and higher rate of infection lies among those
of 25 to 31 years old in the women and their husbands. Al-Jebouri et al. (2013)
found that the prevalence of toxoplasmosis among female patients in Al-Hawija was
53% and was 30% in Al-Baiji.The seroepidemiological study of toxoplasmosis
among pregnant women in Salah –Aldden where 226 pregnant women that had
single or multiple fetal loss were examined and found 29.2% of them had
toxoplasmosis (ADdory, 2011).
For schizophrenic patients in Iran 80 schizophrenia patients and 99 healthy people
were examined for the presence of IgG and IgM antibodies to T. gondii by (ELISA)
the highest 10th percentile of IgG titers in schizophrenia individuals was 18.8%
which was significantly higher than control group 6.1% (Daryani et al.,2010).The
sero-positivity rate among patients with schizophrenia was 67.7% which was
Chapter One
Introduction and Literatures review
16
significantly higher than control group 37.1% ( Alipour et al.,2011).In Iraq
Mahmoud and Hasan (2009) investigated the Seroprevalence rate for anti
Toxoplasma IgG and IgM antibodies by ELISA in 96 schizophrenic and 96 healthy
Individuals(control). The seropositivity rate for anti-Toxoplasma IgG antibodies
among schizophrenic patients was 53% significantly higher than in healthy
individuals 23% . Jassam (2010) showed significantly higher positive IgG Ab among
schizophrenic group (49%) compared to control group 16% .
1-2-6-2:Transmission of Toxoplasmosis:Transmission of T. gondii may occur through the following :1. Ingestion of raw or partly cooked meat , especially pork , lamb ,or venison
containing Toxoplasma tissue cysts .Infection prevalence
in countries where
undercooked meat is traditionally eaten has been related to this transmission
method , tissue cysts may also be ingested during hand-to-mouth contact after
handling undercooked meat , or from using knives , utensils , or cutting boards
contaminated by raw meat. (Jones and Dubey , 2010).Humans may become
infected with this parasite after eating undercooked infected chicken meat (Dubey ,
2009).
2. Ingestion fruits and vegetables contaminated by cat feces . This can
through hand-to-mouth
occur
contact following gardening , cleaning a cat's litter
box , contact with children's sand pits , or touching any- thing that has come
into contact with cat feces (Jones and Dubey , 2010).
3. Drinking water contaminated with Toxoplasma oocyst .
4. Through
congenital
tachyzoites from
transplacental
transmission
of
rapidly replicated
mothers who become infected during pregnancy and pass
the infection to the fetus .
5. Receiving an infected organ transplant or blood transfusion, although this
is extremely rare (Andreoletti et al. ,2007).
Chapter One
Introduction and Literatures review
17
1-2-7: Pathogenesis
T. gondii is an obligate intracellular parasite existing in three forms: the
oocyst, shed only in cat feces,the tachyzoite (a rapidly dividing form observed
in the acute phase of infection ) , and the bradyzoite (a slow growing form
observed within tissue cysts) .During a primary infection a cat can shed millions of
immature oocysts daily for a period of one to three weeks . These oocysts may
remain infectious for over a year (Montoya and Liesenfeld, 2004) . T. gondii is
released from either tissue cyst or oocyst by digestive process in the gastrointestinal
tract, invading the intestinal enterocyst. Tachyzoites then disseminate via the blood
stream or lymphatic to a variety of organs, particularly lymphatic tissue, skeletal
muscle, myocardium, inside monocyte and granulocyte (Markall et al., 1999).
Necrosis in intestinal and mesenteric lymph nodes may occur before other organ
becomes severely damaged. Focal areas of necrosis may develop in many organs.
T.gondii does not produce a toxin, necrosis is caused by intracellular multiplication
of tachyzoites(Gazzinelli et al.,1993). An unusual feature of toxoplasmosis is that the
organisms persist in cyst in various organs for the remainder of the infected
person's life (Larry and John ,2002).Toxoplasamosis
is generally a self-limited
,asymptomatic disease in immunocompetent patients , although infection can
reactivate at a later time if the patient becomes immunosuppressed (Montoya et
al.,2004).Toxoplasmosis is considered harmless for a non-pregnant woman ,but
it is potentially harmful
during
pregnancy , especially at first trimester .
Primary maternal infection during pregnancy can be transmitted to the fetus
and result in serious sequel (Giannoulis et al., 2008 ).Acute infection in adult
humans goes unrecognized in as many as 90% of cases, because either it
is
subclinical or symptoms are nonspecific and it is falsely takenas a viral illness
(Montoya and Rosso ,2005). During acute infection, the tachyzoites invade every
kind of host cell except non-nucleated red blood cells. The host cell invasion is a
major step in its biological cycle and in pathogenesis. Tachyzoites enter host cells by
actively penetrating through the host cells plasmalemma or by phagocytosis. They
Chapter One
Introduction and Literatures review
18
multiply intracellular causing host cell disruption, the liberated parasites invade and
destroy adjacent cells, producing progressively larger focal lesions. If the initial
infection occurs when the host is pregnant, the tachyzoites associated with the acute
phase can cross the placenta and infect the fetus, which can result severe birth
defects,
including
hydrocephaly,
calcification,
neurological
defects
and
chorioretinitis, which may be recurrent. However, with the onset of the host
immune response, a subpopulation of tachyzoites in the brain, undergo stage
conversion to bradyzoites which multiply slowly to form large tissue cysts with
numerous bradyzoites that are capable of persisting for the life of the host (Montoya
and Liesenfeld ,2004;Waree, 2008). Even though subclinical disease is the rule ,
signs present at birth may include fever,a maculopapular rash, hepatosplenomegaly,
microcephaly
,seizures,jaundice
,thrombocytopenia
,and
rarely
generalized
lymphadenopathy. The so-called classic triad of congenital toxoplasmosis consists
of chorioretinitis, hydrocephalus and intracranial calcifications (Berrebi et al.,
2007).Toxoplasma is a frequent cause of intracerebral focal lesions resulting in
toxoplasmic encephalitis (Kappagoda et al., 2011). If left untreated, toxoplasmic
encephalitis can be fatal (El-Beshbishi et al., 2011).
1-2-7-1:Acute Toxoplasmosis :During acute toxoplasmosis, symptoms are often influenza-like: swollen lymph
nodes, or muscle aches and pains that last for a month or more. Rarely, a patient
with a fully functioning immune system may develop eye damage or nasal lesions
from toxoplasmosis (Paul, 1999). Symptomatic infection is usually characterized by
lymphadenopathy and reticular cell hyperplasia(Montoya and Liesenfeld ,
2004;Dalimi and Abdoli, 2012). Young children and immunocompromised patients,
such as those with HIV/AIDS, those taking certain types of chemotherapy, or those
who have recently received an organ transplant, may develop severe toxoplasmosis.
This can cause damage to the brain (encephalitis) or the eyes (necrotizing
retinochoroiditis). Only a small percentage of infected newborn babies have serious
Chapter One
Introduction and Literatures review
19
eye and brain damage or nasal malformations at birth (Paul, 1999). Enlarged lymph
nodes will resolve within one to two months in 60% of patients. However, a quarter
of patients take 2–4 months to return to normal and 8% take 4–6 months. A
substantial number of patients (6%) do not return to normal until much later
(Remington and Desmonts, 1976).
1-2-7-2: Subacute Infection:During (1-3) weeks the antibodies start to appear after infection leading to
cessation of parasitaemia and decreased in number of organism in viscera
characterized by general lymphadenopathy without fever, irregular fever is lasting
for a few days, low blood pressure, leucopenia with lymphocytosis and sometime
spleno and hepatomegaly (Slutaker et al., 1999). Damaged may be more extensive
in the central nervous system(CNS) than in unrelated organs because of lower
immunocompetence in these tissues (Remington and Desmonts, 1976).
1-2-7-3: Latent toxoplasmosis
Most patients who become infected with T. gondii and develop toxoplasmosis do
not know it. In most immunocompetent patients, the infection enters a latent phase,
during which only bradyzoites are present, forming cysts in nervous , brain, retina,
skeletal and cardiac muscles. Most infants who are infected while in the womb have
no symptoms at birth but may develop symptoms later in life (Parker, 2003). In
immunocompromised patients such as AIDS, toxoplasmosis almost always happens
as a result of reactivation of chronic infection. In these patients, clinical symptoms
consist of mental status changes, seizures, sensory abnormalities, cerebellar signs,
movement disorders, and neuropsychiatric findings (Montoya and Liesenfeld , 2004
;Dalimi and Abdoli, 2012).
Chapter One
Introduction and Literatures review
20
1-2-8: Clinical syndromes:1-٢-8-1: Toxoplasmosis in the immunocompetent patient:Infection with Toxoplasma rarely causes severe disease in an immunocompetent
individual . Acquired Toxoplasma infection is asymptomatic in approximately
80 % of individuals . A considerable number of cases with mild symptoms
are either ignored by the patients or the nonspecific symptoms are not attributed
to a Toxoplasma infection. Usually, clinical symptoms occur 10-14 days after
infection and consist
primarily of
mild , local to generalized , self limiting
lymphadenopathy. Chronic active toxoplasmosis is rare and suspected when
clinical symptoms and high antibody concentrations persist for several months or
even years . Life-threatening cases of pneumonia due to Toxoplasma infection ,
have
recently been described in immunocompetent individuals from South
America and may be associated with
genetically atypical and
highly virulent
strains of the parasite ( Alapatt et al.,2009).
1-2-8-2: Toxoplasmosis in the immunodeficient patient:In contrast to the relatively favorable course of toxoplasmosis in almost all
immunocompetent individuals, immunologically impaired patients usually develop
a dreadful and often life-threatening disease. Immunocompromised patients at
higher risk for toxoplasmosis include those with hematologic malignancies
(particularly patients with lymphoma), bone marrow transplant, solid organ
transplant (including heart, lung, liver, or kidney), or AIDS. Toxoplasmic encephalitis is the most common presentation of toxoplasmosis in immunocompromised
patients (Israelski and Remington,1993). The most frequent cause of focal CNS
lesions in AIDS patients . It is unclear whether T. gondii penetrates the brain more
easily than other organs or whether it is more difficult for the brain, as an
immunologically privileged site, to eradicate the organism during the initial acute
Chapter One
Introduction and Literatures review
21
infection and once residual infection has been established (Montoya and
Remington,1997). A wide range of clinical findings, including altered mental state,
seizures, weakness, cranial nerve disturbances, sensory abnormalities, cerebellar
signs, meningismus, movement disorders, and neuropsychiatric manifestations are
observed in patients with toxoplasmic encephalitis (Liesenfeld et al., 1999). Other
organs commonly involved in immunocompromised patients with toxoplasmosis
are the lungs, eyes, and heart. In the vast majority of immunocompromised patients,
Toxoplasmosis results from reactivation of a latent infection. In contrast, in heart
transplant patients and in a small number of other immunocompromised patients,
the highest risk of developing disease is in the setting of primary infection (i.e., a
seronegative recipient who acquires the parasite from a seropositive donor via a
graft(Deroun et al., 1986) . Contrary to the situation in adults , toxoplasmosis
of the central
nervous system is not a common opportunistic infection in
children infected with HIV. Rather , severe congenital toxoplasmosis seems to be
more frequent among such children( Abrams , 2000) .
1-2-8-3: Congenital toxoplasmosis:Congenital toxoplasmosis occurs whenT. gondii , an obligatory intracellular
protozoan , reaches the
fetus transplacental. More than 90% of
pregnant
women who acquire a primary infection during gestation are asymptomatic
(Montoya and Rosso , 2005) .Acute primary maternal toxoplasmosis if acquired
during
the first trimester of pregnancy can cause significant morbidity and
mortality in developing fetuses (Singh , 2003 ; Thiebaut et al ., 2007). Congenital
infection of the fetus in women infected just before conception is extremely
rare , and even during the first few weeks of pregnancy, the maternal-fetal
transmission rate is low ( Press et al., 2005; Emna et al., 2006). Mother-to-child
transmission rates have been found to be around 20-50% (Jones et al., 2001) . In
several studies there are reports that point towards a more serious disease
when the mother is infected during the first trimester ( Montoya and Liesenfeld
Chapter One
Introduction and Literatures review
, 2004) . The presentation
22
of congenital
toxoplasmosis varies widely from
subclinical to severe cases , including fetal or neonatal death. Recent findings
indicate
that type
II strains of Toxoplasma can cause
both
benign and
complicated whereas the severity of infection is primarily related
period
of
maternal
infection
and
to
the
to the parasite concentrations in the
amniotic fluid (Romand et al., 2004). Infection during early pregnancy may cause
fetal death and abortion or severe damage like chorioretinitis, intracranial
calcification, hydrocephalus or microcephalus (Figur 1٤-) . Infection during late
pregnancy is often subclinical in the newborn with manifestations such as
retinochoroiditis or , rarely, neurological disorders. Manifestations are found in
approximately 5% of infected newborns (Syrocot , 2007) . The prevalence of T.
gondii risk factors and of previous infections varies from country-to-country
(Noorbakhsh et al., 2012).Congenital toxoplasmosis occurs almost exclusively as a
result of primary maternal infection during pregnancy. Rarely reactivation of
infection in immune compromised woman during pregnancy can result in
congenital toxoplasmosis. Most maternal infections are asymptomatic or result in
mild illnesses (Kravetz and Federman,2005; Noorbakhsh et al., 2012). The ELISA is
capable of detecting 85% of cases of congenital toxoplasmosis infections in the first
few days of life. Most important fact for the clinician is that patients with a positive
IgG titre and a positive IgM IFA or ELISA titre must be presumed to have a recently
acquired infection with T. gondii(Del Castillo ,2004; Noorbakhsh et al., 2012).
Prevention of congenital toxoplasmosis is needed by treatment of active T. gondii
infection in pregnant women .In congenital infection; treatment in the first year of
life is associated with diminished occurrence of this complication (Wallon et al.,
2004 ; Noorbakhsh et al., 2012). Prenatal diagnosis of congenital toxoplasmosis
validated by PCR in amniotic fluid against Indirect Fluorescent Antibody Assay in
mothers. Analysis of amniotic fluid by polymerase chain reaction for prediction of
congenital toxoplasmosis is useful (Neto et al.,2004).
Chapter One
Introduction and Literatures review
23
Fig (1-٤):- Congenital toxoplasmosis (hydrocephalitis) (Dubey and Beattie
, 1988).
1-2-8-4 :Ocular toxoplasmosis:Ocular toxoplasmosis leads to permanent loss of vision in affected eyes in
nearly 25 % of patients (Bosch-Driessen et al., 2002). Ocular disease occurs in a
large percentage of congenitally infected patients , but the classical explanation
that most cases were due to congenital infection has been challenged ( Gilbert
and Stanford , 2000) .At birth , infants
infected in utero may have ocular
disease broadly defined as retinochoroiditis or inflammation of the retina and
choroid with associated vitritis ( Mcleod et al ., 2006). New ocular lesions can
occur at any age after birth
in untreated and some treated children . Whilst
severity of disease is influenced by trimester in which infection is acquired by the
mother(Remingtone et al.,2005) . Recurrent posterior uveitis is the typical form of
this disease, characterized by unilateral, necrotizing retinitis with secondary
choroiditis, occurring adjacent to a pigmented retinochoroidal scar and associated
Chapter One
Introduction and Literatures review
24
with retinal vasculitis and vitritis. Multiple atypical presentations are also described,
and
severe
inflammation
is
observed
in
immunocompromised
patients.
Histopathological correlations demonstrate focal coagulative retinal necrosis, and
early in the course of the disease, this inflammation is based in the inner retina. For
typical ocular toxoplasmosis, a diagnosis is easily made on clinical examination. In
atypical cases, ocular fluid testing to detect parasite DNA by polymerase chain
reaction or to determine intraocular production of specific antibody may be
extremely helpful for establishing aetiology. Given the high seroprevalence of
toxoplasmosis in most communities, serological testing for T. gondii antibodies is
generally not useful. Despite a lack of published evidence for effectiveness of current
therapies, most ophthalmologists elect to treat patients with ocular toxoplasmosis
that reduces or threatens to impact vision (Butler et al., 2013 ).Ocular
toxoplasmosis can heal spontaneously after two to three months even in the
absence of therapy (Silveira et al., 2002) (Fig. 1-٥ ).
Fig (1-٥): Active retinitis area adjacent to previous cicatricial
foci (Guex-Crosier ,2009).
Chapter One
Introduction and Literatures review
25
1-2-8-5:Toxoplasmosis role in schizophrenia and behavioral changes:Studies that have been conducted showed the Toxoplasma
parasite may
affect behavior and may present as or be the causative or contributory factor in
various psychiatric disorders such as depression, anxiety and schizophrenia
(Henriquez et al ., 2009 ) . In prospective studies, an increase in IgG antibodies
against T. gondii has been found in mothers of infants who later develop
schizophrenia (Brown et al., 2005 ; Zhou et al., 2011) . Many reports revealed that
Toxoplasma might represent a major pathogen in some cases of psychosis. It has
been proven that the parasite infection could increase the dopamine level in brains
(Huber et al., 2007 ; Zhou et al., 2011) . Behavioral changes associated with T. gondii
infection may contribute to serious neurological disorders in humans. Several
studies have observed an association between T. gondii seroprevalence with
schizophrenia (Brown and Derkits, 2010). Since T. gondii infection has been found
to last throughout the lifetime of the host, seroprevalence is likely to reflect chronic
infection (Dubey, 2010). Dopamine dysregulation is proposed to play a central role
in schizophrenia, potentially in combination with glutamate metabolism. How
dopamine dysregulation plays a role in schizophrenia, however, is still unknown.
The principal antipsychotic drug that has been used to treat schizophrenia,
dopamine antagonist haloperidol, can also block the development of behavior
changes in T. gondii infected rodents. It is possible that the increased dopamine
accumulation and release observed during T. gondii infection may contribute to T.
gondii associated schizophrenia. Dopamine metabolite concentration have been
inversely
correlated
with
gray
matter
volume
in
schizophrenia
patients(Prandovszky et al., 2011 ). Dopamine plays a key role in psychosis cases
such as schizophrenia, and bipolar disorder (Zhou et al., 2011). The predilection of
T. gondii for the CNS places it in a privileged position to manipulate host behavior
(McConkey et al., 2013).Lately work at the University of Leeds has found that
the parasite produces an enzyme with tyrosine hydroxylase and phenylalanine
hydroxylase activity ,this enzyme may contribute to the behavioral changes
Chapter One
Introduction and Literatures review
observed
in toxoplasmosis
26
by altering
the
production
of
dopamine , a
neurotransmitter involved in mood , sociability, attention, motivation and sleep
patterns . Schizophrenia has long
been linked to dopamine deregulation
(Jaroslav , 2007 ). Correlations have been found between latent Toxoplasma
infections and various characteristics :
Decreased novelty-seeking behaviour .

Slower reactions .

Lower rule-consciousness and greater jealousy (in men) .

Promiscuity and greater conscientiousness (in women) ( Jaroslav ,2007).
1-2-9:Immune Response:1-2-9-1:Innate Immune Response:Toxoplasma gondii is able to trigger the nonspecific activation of macrophage,
natural killer (NK) cells and the other cells such as fibroblasts, epithelial or
endothelial cells during the earliest stages of infection ,this activation is for limiting
parasite proliferation because of its direct or indirect cytotoxic action and to
activate a specific immune response in order to the presentation of Toxoplasma
antigens.This non-specific immune response reacts immediately after the first
contact between the parasite and the host, its peak at the end of the first week, and
then slowly reduces until absent in the second week of the beginning of infection. In
mice, the activation of macrophages cytokine interferon gamma (IFN-γ) in the
presence of co-signals, such as tumour necrosis factor- α (TNF- α) is necessary to
trigger the cytotoxic activity of the macrophages against T. gondii (Filisetti and
Candolfi, 2004).Macrophage ,NK cell and cytokines are the main components
involved in the innate immune response against T.gondii . In the acute phase of
the infection , tachyzoites stimulate macrophage to produce interleukin 12 (IL12) , which acts on NK cells to start producing interferon gamma(IFN-γ).The
Chapter One
Introduction and Literatures review
27
latter stimulates macrophages killing the phagocytosed tachyzoites ( Bhopale ,
2003 ) . The standard view of neutrophils is that they rapidly home to sites of
infection, phagocytose pathogens, release anti-microbial granules, and undergo
apoptosis. However, neutrophils can also release immunoregulatory cytokines and
chemokines, suggesting that they may also participate in shaping immunity (Nathan,
2006). There is evidence that neutrophils are important in recruiting and activating
dendritic cells in response to microbial pathogens including Toxoplasma (Bennouna
et al., 2003).Recently, these cells have been found to release chromatin and granuleassociated Neutrophil Extracellular Traps (NET) that ensnare and kill microbes.
Originally described as a response to bacterial and fungal pathogens, new studies
indicate that NET release also occurs in response to protozoan pathogens,including
Toxoplasma (Abi-abdallah et al.,2012). Both human and mouse neutrophils undergo
a vigorous parasite strain-independent NET response during tachyzoite co-culture,
and entrapment within NET could in principle interfere with the ability of T. gondii
to find safe harbor within host cells. In addition , activated CD+4 T cell produce
IL-2 , an
important T cell mitogen. Together , these events result
in
the
convergence of large numbers of parasite-specific CD+4 and CD+8 cell that
produce IFN-γ at the site of parasite invasion (Fig. 1-٦) (Taylor et al.,2007; Elia
and Christopher, 2009).Thus, macrophages and NK cell function cooperatively to
destroy tachyzoites and to minimize the spread of the parasite ( Filisetti and
Candolfi,2004).
Chapter One
Introduction and Literatures review
Fig. (1-٦) : Development of a model of
28
immunity to T. gondii (Elia and
Christopher , 2009).
1-2-9-2:Adaptive Immune Response:The
non-specific
macrophages and
immune
response
B- Lymphocyte
has
led
to
differentiation
of
into antigenic presenting cell (APC) . The
effector cells are stimulated by dendrite cells presenting the antigen to TLymphocyte (TL) T-cell receptors . However , this mechanism
requires a
close interaction between the APC and the TL (Reichmann et al., 2000) . These
effector cells , which are involved in resistance to Toxoplasma
infection ,
then exert their function via a cytotoxic activity and / or the secretion of
cytokines involved in the regulation of immune response (Hunter et al. ,1994).
Recognition of infection elicits a rapid and strong Th1- polarized immune
response that is necessary
for host
survival and long- term parasite
persistence (Denkers and Gazzinelli ,1998 ). In the context of chronic infection ,
characterized by presence of parasite cysts in tissues of the central nervous system
Chapter One
Introduction and Literatures review
29
and skeletal muscle, IFN-γ-producing CD4+ and CD8+ T lymphocytes are required
to maintain quiescent infection ( Gazzinelli et al. ,1992). The most dramatic
evidence underlining this concept is found during Acquired Immune Deficiency
Syndrome (AIDS) progression , where cyst reactivation may occur as T cell
numbers decline, often with devastating consequences ( Montoya and Liesenfeld
,2004). The specific protective immunity is predominantly a cell-mediated immune
response. This is because the T. gondii is an intracellular parasite. The cytokine, IFNγ is mainly secreted by T-cells (Tan et al.,2011). In addition, many studies showed
that normally avirulent strains of T. gondii become highly virulent in T-lymphocytedeficient animals. The macrophages and NK cells are primary cells of defense
against the parasite during the early infectionus stage ( Sher and Sousa 1998;
Waree, 2008). Interleukin 12 (IL-12), which is major cytokine produced by the
macrophages(MØ) and the DC during antigen stimulation, appears to play a major
anti-Toxoplasma role during the acute phase of the infection (Butcher et al., 2011).
Indeed, it activates the production of IFN-γ by NK cells and T-lymphocyte (CD4+ and
CD8+) cells. The CD4+ and CD8+ T-lymphocyte are the main players involved in
resistance of the host to Toxoplasma infection. In mice, mature CD4+ TL are divided
into T helper 1 (Th1) and 2 (Th2). This distinction is based on the list of cytokines
secreted following stimulation, the Th1 cells produce IL-2 and IFN-γ while the Th2
cells produce IL-4, IL-5, IL-6 and IL-10 (Debierre-Grockiego and Schwarz, 2010).
Both the proliferation of CD8+ cells and the maturation of NK cells also induce the
pro-increasing of IFN-γ production. (Khan and Kasper,1996 ; Waree, 2008). Based
on information from various studies, the cytokines involve in the immune process
against T. gondii infection include IL-2, IL-12, IFN-γ and TNF- α and possibly IL-6. It
appears that IFN-γ and TNF α -are the critical mediators in the cell mediated
immune response against T. gondii infection (Fung and Kirschenbaum,1996). IL-10
is considered to be an inhibitor of Th1 , Th2 and immune responses (Moore et
al ., 2001; O’Garra and Vieira ,2007). It is produced by macrophages , monocytes,
dendritic cells , B cells , and CD4+ and CD8+ T cells , and
acts broadly on
Chapter One
Introduction and Literatures review
30
accessory cells to down regulate pro inflammatory cytokine production and Major
Histocompatibility Complex (MHC) and costimulatory molecule
expression .
The role of IFN-γ in T. gondii infection, the mechanisms of action of the cytokines is
as follows:
IFN-γ activates the macrophages by enhancing their oxidative metabolism,
releasing hydrogen peroxide that kills the parasites. Nonoxidative mechanisms,
represented mainly by the production of Nitrogen Monoxide (NO) by macrophages
activated by IFN-γ with NO also involved during the chronic phase in inhibition of
intracerebral parasite proliferation ( Schluter et al., 1991). IFN-γ also increases the
activity of indoleamine 2, 3-dioxygenase, resulting in the breakdown of tryptophan,
which is required for growth of the parasite (Flávio et al. ,2011 ; Nascimento et al.,
2011 ).
1-2-9-3:-Humoral Immune Response
Humoral immune response which appears to play a minor role for the host
ability to control T. gondii infection , but is essential for the serological diagnosis
of toxoplasmosis in humans ( Lappalainen and Hedman , 2004 ) .IgM antibodies
may appear within the first week of infection and generally decline within a few
months , IgG antibodies appear within one to two weeks of infection, peak in six to
eight weeks and then decline over the next two years; they remain detectable for
life(Giannoulis et al., 2008).There are four
unequal
proportions during
subclasses
which
appear
in
toxoplasmosis . IgG1, IgG2, IgG3 and IgG4 are
thought to be predominant . They
also enable
cytotoxicity or opsonization , through
antibody dependent cell
binding to fragment creystallizable
receptors existing on the monocyte macrophages and the polynuclear cells or to
cytolysis mediated by the complement or by NK cells . They play a role in
protection of the fetus because they are capable of crossing the placenta . The main
target antigens of IgG are the surface antigens of the parasite (Pinon et al. 1996).
Until the early 1990 , the presence of IgM antibodies against toxoplasmosis was
Chapter One
Introduction and Literatures review
31
interpreted as diagnosis of the acute form of the disease.However, introduction of
highly sensitive immune enzymatic tests to detect IgM revealed that low levels are
able to persist for many months , even years, after acute infection. Such IgM
antibodies are called residuals and their presence does not indicate recent infection
( Marcel et al.,2008) .In general , anti- Toxoplasma IgM antibodies disappear at an
early period ( 3-5 week) after the onset of infection , but occasionally a low IgM
titer may persist for a year or even more ( Nazan-Dalgic ,2008) . Recent
improvements in sensitivity of IgM tests has made IgM detection an extremely
protracted
acute
phase
marker ,
and
IgG avidity evalution test became
necessary . Observations has shown that a correlation
can
be established
between IgM levels and avidity percentages , suggesting that frequently the
avidity test may be necessary ( Leite et al. ,2008). Since maternal IgM antibodies
usually do not cross the placental barrier and the fetus is able to develop IgM
antibody , the demonstration of Toxoplasma IgM antibody in the neonate reflects
acute congenital toxoplasmosis ( Correa et al.,2007).
1-2-10:- Diagnosis
Clinical
signs of
toxoplasmosis
are non-specific and
not sufficiently
characteristic for a definite diagnosis , and in fact mimic several other infectious
diseases ( Foulon et al. ,1999 ) . In general , diagnosis of toxoplasmosis can be
achieved by serological methods or by direct detection of the parasite , its
proteins or its nucleic
acid .
Different diagnostic strategies exist for the
detection of an acute or chronic Toxoplasma infection in the immunocompetent,
immunocompromised, pregnant or newborn organism (Remington et al .,2004).
Another, more direct form of diagnosis is through the staining of body fluid samples
such as blood or cerebrospinal fluid. This method is not used as frequently as
serological testing due to various technical difficulties. Various molecular
techniques are being developed to detect the presence of the parasite's DNA in the
Chapter One
Introduction and Literatures review
32
affected body fluid. These procedures can be useful in identifying the presence of T.
gondii in the fetus by testing the amniotic fluid(Linda et al.,2008).
1-2-10-1:-Direct methods
1-2-10-1-1:-Isolation of the parasite
Toxoplasma gondii can be isolated
from the placenta , umbilical cord, or
infant blood by inoculation into mice or cell culture (Boyer , 2001). Tachyzoites
may be detected in various tissues and body fluids by cytology during acute
illness .Tachyzoites are rarely found in blood , cerebrospinal fluid ( CSF ) , fine
needle aspirates, and transtracheal or bronchoalveolar washings, but are more
common in the peritoneal and thoracic fluids of animals developing thoracic
effusions or ascites (Dubey et al. , 2009). The isolation of T . gondii from blood
or body fluid establishes that the infection is acute . Attempts to isolate
the
parasite can be performed by inoculation of specimens secretions, excretions
body fluids , tissues taken by biopsy and tissues with macroscopic lesions taken
postmortem in the peritoneal cavities of
laboratory animals or tissue-cell
cultures of virtually any human tissue or body fluid . Using such specimens , one
may
not
only
attempt
the
isolation , but
may search
for T.gondii
microscopically using traditional histochemical stains or by immunohistochemical
staining of the parasite (Fleck,1989). After ( 1-2 week ) , drow samples from
inoculated laboratory animals from peritoneal cavities then tested to detection
tachyzoites . Left for two month then taken tissues section from brain , heart
,muscles ,and lung for histological diagnosis , to bradyzoites observation ( Dubey
and Thulliez , 1993) .
1-2-10-1-2:- Histological diagnosis
Tachyzoites in tissue sections or smears of body fluid (e.g., CSF, amniotic fluid)
establishes the diagnosis of acute infection (Nissapatorn et al, 2011). Multiple tissue
cysts near an inflammatory necrotic lesion probably establish the diagnosis. It is
Chapter One
Introduction and Literatures review
33
often difficult to show tachyzoites in stained tissue sections (Cenci-Goga et al ,
2011). Fluorescent antibody staining may be useful, but often yields nonspecific
results. The immunoperoxidase technique, which uses antisera to T. gondii, has
proved sensitive and specific result (Fauquenoy et al., 2011). Tachyzoites can also
be detected in impression smears from the same organs by Giemsa staining
or immunofluorescence . A rapid and
detection
of
T. gondii
technically simple used method is the
in air –dried , Wright or
Giemsa-stained
slides of
centerifuged sediment of CSF and a brain aspirate , or in impression smears of
biopsy tissues ( Press et al.,2005).
1-2-10-2:- Indirect methods (Serological tests)
Diagnosis of acute or chronic toxoplasmosis is generally performed by serological
assays , followed by confirmatory parasitological tests of fetal T. gondii
infection (
Carvalheiro et al., 2005). There are numerous serological procedures available for
the detection of humoral antibodies .
1-2-10-2-1:- Sabin-Feldman Dye test (SFDT)
Is the so-called ‘gold standard’ serological test for Toxoplasma antibodies in
humans (Cenci-Goga et al., 2011). Live Toxoplasma tachyzoites are incubated with a
complement-like accessory factor and the test serum at 37°C for 1 hour before
methylene blue is added. Specific antibody induces membrane permeability in the
parasite so that the cytoplasm is able to leak out and the tachyzoite does not
incorporate the dye and so appears colourless. Tachyzoites not exposed to specific
antibody (i.e. a negative serum sample) take up the dye and appear blue (Dixon,
2010). The DT is both specific and sensitive in humans, but may be unreliable in
other species ( Dubey, 2010).
Chapter One
Introduction and Literatures review
34
1-2-10-2-2:- Indirect Fluorescent Antibody Test (IFAT)
The test is based on the use of anti globulins labeled with fluorescent dyes, the
fluorochrome emit visible light after excitation by ultraviolet light (Gutierrez and
Little, 1991). The antibodies ( Abs) that are detected by IFAT act mainly against the
cell wall of the parasite similar to the Dye test Abs, but do not need live parasites.
The test can detect both IgM and IgG in serum. This test is simple and easy to apply
in comparison with the Dye test and the results are parallel to those of the Dye test
(Willis et al., 2002).
1-2-10-2-3:- Complement Fixation Test (C FT)
The test was employed for the first time in diagnosis of Toxoplasma by Warren and Russ
in 1948 (Warren and Russ,1948) . This test uses a soluble parasite antigen in contrast to
whole organisms used in the DT, the CFT becomes positive after the DT becomes positive
,reverts to negative within several years, and reach high titers only in patients who have
high titers, as determined by other procedures .Results may vary greatly and are dependent
on the antigen preparation. Because of technical difficulties associated with this test in
general and its lack of sensitivity in comparison with other tests, the CFT test is rarely used
today (Kistiah, 2009).
1-2-10-2-4:-Indirect Haemagglotination Test (IHAT)
This IHAT test was first used in 1958 for the serological diagnosis of T. gondii
by
Jacobs
and
Lunde. The test uses animal
red blood
cell sensitized with
Toxoplasma antigens of a cytoplasmic origin to react with serum specimen (Elliot et
al.,1985).These erythrocytes react with specific antibodies present in human or
animal serum , thus
forming a homogeneous network in the plate ( positive
reaction ). In case specific antibodies are not present , erythrocytes settle forming a
neat button in the
plate
( negative
reaction ) ( James , 1996) . The major
disadvantages should not be used for detection of congenital and newborn
Chapter One
Introduction and Literatures review
35
infection and also variation of RBC quality and variation in antigen (Wilson et al.,
1990).
1-2-10-2-5:- Latex Agglutination Test (LAT)
In this test patient’s serum is reacted without treatment with sensitized latex
particles. If Toxoplasma specific antibodies are present, agglutination takes place.
The LAT has been considered useful for IgG screening purposes. A small percentage
of false positive reactions do take place and have been attributed to non specific IgM
reactions. The LAT is also simple, easy to perform, inexpensive and can be used in
both human beings and animals (Antoniazzi et al., 2008).
1-2-10-2-6:-Modified Latex Agglutination Test (MLAT)
This test is used for detection of T. gondii IgG antibodies in patient
serum , in this test T . gondii IgM antibodies will distracted by using 2Mercaptoethanol
component , immunoglobulin IgM
antibodies
bond will
distracted and IgG antibodies remain without any effect . In this test the presence
of agglutination means that result is IgG positive , in the other hand if no
agglutination occure the result is positive for IgM ( Parker and Cubitt , 1992).
1-2-10-2-7:- Enzyme Linked ImmunoSorbant Assay (ELISA)
Firstly this technique was used in 1971 by Van Weaman and Shrurus. This assay
was applied to Toxoplasma, by Voller (Voller et al.,1976).
ELISA
automated
samples , and in
routine
and thus are suited to test large numbers of
screening . Some ELISA have been
shown
to be
can
both
be
highly
sensitive and specific when compared to IFAT . Recently , a commercial ELISA
for the detection of antibodies to Toxoplasma has also been validated in
swine (Gamble
et al., 2005 ) . Subsequently ,it is found that
the
validated
ELISA is the most sensitive test for the analysis of sera . An IgG-avidity ELISA
which
may
help
in
the
identification
of
active
or
recent
T . gondii
Chapter One
Introduction and Literatures review
36
infections , has been described (Hill et al., 2006) . The IgM-ELISA detects recently
acquired acute congenital toxoplasmosis (Ghoneim et al., 2010). In these tests , the
patients antibodies are attached to plastic wells , and Toxoplasma antigen labeled
with an enzyme , usually horseradish peroxidase , is then added to the wells
containing
the
patients antibodies . The wells are then rinsed to remove
antigen that has not been bound by the patients antibodies . A substrate for the
peroxidase is then added , and the bound Toxoplasma antigen can be detected by
the enzymatic activity (Koneman et al ., 1992 ).
1-2-10-2-8:-VITEK Immunodiagnostic Assay System (miniVIDAS)
The miniVIDAS T. gondii IgG Assay (TXG) , was performed by using the VIDAS
instrument according to the direction of the manufacturer. Solid -phase receptacles
(SPRs) , pipette tip-like disposable devices , serve both as the solid phase as
well as the pipettor for the assay . The interior surface of each SPR is coated with
inactivated tachyzoites of the RH strain of T. gondii from peritoneal extracts of
infected mice . All of the assay steps are performed automatically
by the
miniVIDAS TXG module in less than 60 min. Anti-T. gondii IgG antibodies that
may be present in the test serum bind to the T. gondii antigens that coat the
SPRs ,while unbound sample components are washed away . The detector
antibody conjugate (goat anti-human IgG conjugated to alkaline phosphate ) is then
cycled in and out of the SPRs to attach to any serum IgG bound to the antigen on
the SPR wall . The final well , is an optically clear cuvette containing a fluorescent
substrate , 4-methylumbelliferyl
phosphate . The intensity of fluorescence is
measured by the miniVIDAS optical scanner . Results are subsequently analyzed
automatically by the computer interface (Sandin et al.,1991).
1-2-10-2-9:Enzyme Immunoassay (EIA)
A wide range of EIA-based assays are available for the detection of Toxoplasmaspecific immunoglobulins, in particular, IgG, IgM, and IgA classes, although assays
Chapter One
Introduction and Literatures review
37
detecting IgE are also used successfully by some laboratories. In some
immunoglobulin class-specific assays, the patient's antibody is captured onto the
well of a microtitre plate, usually by a class-specific monoclonal antibody (e.g.
antihuman IgG, IgA, or IgM) that has been previously attached chemically to the
well. Toxoplasma antigen that has been labeled with a suitable enzyme (e.g. with
horseradish peroxidase, alkaline phosphatase, biotin, etc.) is added to the well and is
bound by any Toxoplama-specfic antibody in the patient's serum. After washing to
remove unbound antigen, bound antigen is detected colorimetrically by addition of
the appropriate enzyme substrate or biotin-detection system. Measurement of the
amount of colored product formed permits quantification of the amount of
Toxoplasma-specific antibodies in the patient's serum(Joynson and Wreghitt,2001).
1-2-10-2-10:Immunosorbent agglutination assay (ISAGA)
This is a commercial assay (bio-Merieux) for the detection of Toxoplasmaspecific IgM (ISAGA-M) and IgA (ISAGA-A) that is typically significantly more
sensitive than standard EIA methods. This increased sensitivity can be a major
advantage in clinical scenarios where low levels of antibodies might be expected, e.g.
HIV infection, immunosuppression, in neonates, etc. (Joynson and Wreghitt,2001).
1-2-10-3: Others
1-2-10-3-1:-Skin Test
This test is also known as Delayed Hypersensitivity Test (DHT). It was first
described by Frenkel in (1948) , and it is one of the useful
methods used in
population surveys ( Frenkel ,1991). The reaction is measured 48 hours after
antigen administration . A positive skin test means that the patient has been in
contact with T. gondii , but the negative result does not always indicate the
absence of antibodies or infection (Fleger et al .,1996).
Chapter One
Introduction and Literatures review
38
1-2-10-3-2:-Polymerase Chain Reaction (PCR)
A milestone in the field of molecular biology was set in the 1980 with the
development of the Polymerase Chain Reaction (PCR) ( Saiki and Scharf , 1985 ;
Jalal and Nord , 2004 ). Several polymerase
chain
reaction ( PCR) assays ,
including real-time PCR , targeted to different genes of Toxoplasma have been
described ( Hurtado et al., 2001) . They can be highly specific , but the small
size of the sample required for the tests may limit their sensitivity , since the
distribution of the tissue cysts is random , and the density of the parasite in
affected tissues can be low ( Hill et al., 2006). Today the PCR is frequently
employed to detect T. gondii DNA in clinical samples , and is performed by direct
detection of the parasite DNA , while the results do not depend on the
immunological status of the patient ( Peterson and Edvinsson , 2006). Since its
introduction , PCR has been very much refined , and at present low amounts T.
gondii DNA can be detected ( Edvinsson and Jalal , 2004 ) . The sensitivity and
specificity of the PCR depend on multiple factors , such as the DNA extraction
protocol , the characteristics of the DNA sequence that is amplified , and the
optimization of the reaction conditions , but the main problem is the lack of a
standardized protocol ( Martino and
Bretagne, 2005 ) .Three different PCR
principles for the detection of T. gondii DNA have been described , which are
conventional PCR, PCR oligochromatography and real-time PCR (Edvinsson and
Lappalainen , 2006).
1-2-10-3-3:- Diagnosis by Computerized Tomography (CT) and Magnetic
Resonance Imaging (MRI)
It is the most methods which is used for the diagnosis of any infections
like encephalitis , which caused by T . gondii or other causative agent. The MRI
is more sensitive than Computerized Tomography (CT) scan , so that it is used for
diagnosis of infection by T. gondii in susceptible patient ( Warrant , 1993 ) .
CHAPTER TWO
MATERIALS AND METHODS
39
Chapter Two
Materials, and Methods
2-Materials and Methods:
2-1: Subjects :
This study, concerned schizophrenic patients performed in Iraq admitting :Al-Rashad
Teaching Hospitals during the period the first of December 2012
until the end of
February, 2013. In this study, 300 blood samples were collected, 200 of them from
schizophrenic patients( 100 men and 100 women) and 100 of them from ordinary healthy
people (50 male and 50 female) use as a control for comparison . Blood serum was
detected for specific T.gondii antibodies.
Questionnaire sheet regarding information about the patients was filled for each person
(appendix 18) .
2-1-1: Blood Sample collection:A volume of 5 ml of basilic vein blood were collected from each male and female . The
blood sample was placed in a plain tube and left standing for 20 minutes at room
temperature to clot . Then the tube was centrifuged at 3000 rpm for 10 minutes to
collect the serum . The obtained serum was divided into 3 portions for different
serological tests to avoid repeated freezing and
thawing of the samples which is not
recommended because this may affect the quality of the results . All sera were stored at
-20Cº until being
analyzed for Toxoplasma antibodies . Detection of parasite antibody
was achieved by using LAT , MLAT and (ELISA - IgG and ELISA- IgM ). IL-12 was also
detected to perform its effect during toxoplasmosis infection.
40
Chapter Two
Materials, and Methods
2-2: Materials:
2-2-1: Instruments:
Instruments used in this study are present in table (2-1)
Table (2-1) : Instruments used in this study.
Instruments
Origin
Centrifuge
Germany
Deep freeze
Turkey
ELISA reader
Germany
ELISA washer
Germany
Incubator
Turkey
Refrigerator
Europe
Sensitive balance
Water bath
Japan
Germany
41
Chapter Two
Materials, and Methods
2-2-2: Equipments:
Equipments used in this study are shown in table (2-2) .
Table (2-2): Equipments used in this study
Equipment
Origin
Beakers
England
Centrifuge glass tube 10 ml
Germany
Cool box
India
Disposable plastic tubes
Jordan
Disposable syringes 5 ml
Turkey
Eppendorf- tubes
Germany
Filter paper
Germany
Gloves
U.S.A
Graduated glass cylinder 500 ml
England
Pipette (1,5,10 )ml
Germany
Pipette tips
England
Rack
England
Serological micro pipette
Germany
Tourniquet
Syria
42
Chapter Two
Materials, and Methods
2-2-3: Chemicals, Solutions and Kits :
Chemicals , solution and kits used in this study are shown in table(2-3)
Table (2-3) :- Chemicals and solutions used in this study.
Chemical and solution
Company
Origin
Phosphate buffer saline
Roche
Germany
2-Mercaptoethanol
Roche
Germany
Human Toxo IgM ELISA
BioCheck
Europe
Human Toxo IgG ELISA
BioCheck
Europe
Linear
Spain
CusaBio
China
Humatex Toxo latex
Human Interleukin 12
2-3:- Methods
2-3-1:-Latex Agglutination Test (LAT)
The latex agglutination test kit ( Toxo-Latex ) was used. A direct agglutination test
performed on a card for serodiagnosis of toxoplasmosis .
2-3-1-1:-The principle of the method
The Toxo latex reagent is a suspension of polystyrene coated with soluble T. gondii
antigen . By mixing sera containing anti-Toxoplasma antibodies in sufficient concentration
with Toxo latex reagent, a distinct agglutination will occur . Latex particles allow a visual
observation of the antigen -antibody reaction . If the reaction occurs , latex suspension
changes and a clear agglutination becomes evident , due to the presence of Toxoplasma
antibodies .
43
Chapter Two
Materials, and Methods
2-3-1-2:- Techniques

Reagent and specimens were brought to room temperature before use.

Gently the latex toxo reagent was shaked ,to disperse the latex particles.

The reagent was checked against the positive and negative controls.

A quantity of 50µ of the sample serum was placed into circle of the slide .

A quantity of 50µ of the toxo latex reagent was added the next to the serum .

Both drops were mixed spreading them over the full surface of the circle, the slide was
rotated for 5 minutes .
The presence or absence of visible agglutination was noticed within this period of time .A
homogenous mixture indicates negative reaction which mean the absence of Toxoplasma
antibodies,while a clear
agglutination (positive reaction ) indicates the presence of
Toxoplasma antibodies .
2-3-2:-Toxoplasmosis latex agglutination test with 2-Mercaptoethanol (2-ME)
(MLAT).
This test was used to increase specificity and sensitivity of the agglutination test by
using
2-Mercaptoethanol
component,
the
chemical
composition
is
2-Hydroxy
Ethylmercaptan ,B-Mercaptoethanol , which acts to reduce the disulphate which bound
five units formation to immunoglobulinm (IgM) and remain the IgG . In this method the
infection can be diagnosed as acute or chronic infection.
A volume of 50µl positive serum previously detected using LAT was mixed with 50µl
of 2-ME then incubated at 37ºC for 1 hour then 50µl of the mixture was transferred
into the circle of the slide, then 50µl of the toxo latex reagent was added to it , mixed
well and spread over the full surface of the circle. The slide was rotated for 5 minutes
and the presence or absence of visible agglutination in this period of time was recorded .
A homogenous mixture means (positive reaction) which indicates the presence of
Toxoplasma IgM antibodies, while a clear agglutination (negative reaction ) means the
absence of Toxoplasma antibodies IgM .
Chapter Two
44
Materials, and Methods
2-3-3:Enzyme linked Immunosorbent Assay (ELISA-IgG) :The bioChek Toxoplasma IgG ( BC-1085) kit determines quantitatively IgG antibodies against
Toxoplasma (Fig. 2-1)(Turune et al., 1983).
2-3-3-1: The principle of the method
Purified T. gondii antigen is coated on the surface of microwell. Diluted serum sample
was added to the well, and the T. gondii IgG specific antibody, if present, binds to the
antigen. All unbound materials are washed away. HRP-conjugate is added, which binds to
the antibody-antigen complex. Excess HRP-conjugate is washed off and a solution of TMB
Reagent was added . The enzyme conjugate catalytic reaction was stopped at a specific
time. The intensity of the color generated is proportional to the amount of IgG and IgMspecific antibody in the sample.
2-3-3-2:Enzyme Linked Immunosorbent Assay Reagent

Microtiter plate :Toxoplasma antigen-Coated wells (12x8 wells).

Enzyme conjugate reagent(red color): 1 vial (12 ml).

Sample diluents (green color): 1 bottle (22 ml).

Negative calibrator : 01 IU/ml. natural cap (100 µL /vial).

Cut-off calibrator:32 IU/ml. yellow cap (100 µL /vial).

Positive calibrator : 100 IU/ml. red cap (100 µL /vial).

Positive calibrator : 300 IU/ml. red cap (100 µL /vial).

Negative control: range state on lable. Blue cap(100 µL /vial).

Positive control: range state on lable. Purple cap(100 µL /vial).

Wash buffer Concentrate(20x): 1 bottle (50 ml).

TMB reagent (one-step): 1 vial (11 ml).

Stop solution : 1 N HCL : natural cap. 1vial(11 ml).
Chapter Two
45
Materials, and Methods
2-3-3-3: Method:The manufacturer instructions of the procedure was as follows:
1- The desired number of coated well was placed into the holder.
2- Test samples, negative control, positive control and calibrator were prepared in 1: 40
dilution by adding 5 µL of the sample to 200 µL of diluent, mixed well.
3- A volume of 100 µL of each diluted sera, calibrators and controls were dispensed into the
appropriate wells. For the reagent blank, dispense 100 µL Sample diluents in 1A well
positive. The holder was tapped to remove air bubbles from the liquid and mixed well.
4- The wells were then incubated at 37°C for 30 minutes.
5- At the end of incubation period, the liquid was removed from all wells. Rinsed and flickd
the microtiter wells 4 times with diluted wash Buffer (1x) and then one time with distilled
water.
6- A volume of 100 µL of enzyme conjugate was dispensed into each well, mixed gently
for10 sec. and incubated at 37٠C for 30min. in humid chamber.
7- Enzyme conjugate was removed from all wells. Rinsed and flicked the microtiter wells 4
times with diluted Wash Buffer (1x) and then one time with distilled water.
8- A volume of 100 µL of TMB Reagent was dispensed into each well. Mixed gently for 10
seconds.
9- The wells were then incubated at 37 °C for 15 minutes.
10- A volume of 100 µL of stop solution (1N HCL) was added to stop reaction and mixed
gently for 30 seconds. It is important to determine that all the blue color changes to yellow
color completely.
11- The O.D was read at 450 nm within 15 minutes by ELISA reader.
2-3-3-4: Calculation and interpretation of results:

The mean of duplicate cut-off (32 IU/ml) calibrator value Xc. was calculated

The mean of duplicate positive control (Xp), negative control (Xn) and patient sample (Xs)
was calculated.

The Toxoplasma IgG Index of each determination obtained by dividing the mean values of
each sample by calibrator value Xc.
46
Chapter Two
Materials, and Methods

Negative : Toxo G index less than 0.90 indicated absence of prior exposure to Toxoplasma
(< 32 IU/ml).

Equivocal : Toxo G index between 0.91-0.99 is equivocal, sample should be retested.

Positive : Toxo G index of 1.00 or greater, or WHO IU/ml value greater than 32 IU/ml is
seropositive.
Fig. (2-1): ELISA-IgG kit used in this study.
2-3-4: Enzyme linked Immunosorbent Assay (ELISA-IgM):
The bioChek Toxoplasma IgM ( BC-1085) kit determines quantitatively IgM antibodies
against Toxoplasma, In this test the same procedure and the same calculation and
interpretation of results were used as in (ELISA-IgG). (Section 2-3-3)(Fig. 2-2)(Turune et
al., 1983)
47
Chapter Two
Materials, and Methods
Fig. (2-2): ELISA-IgM kit used in this study
2-3-5:Determination of Human Interleukin 12 ELISA Kit :
This immunoassay kit (CSB-E04598h) allows for the In vitro quantitative determination
of human IL-12\P40 concentration in serum (Fig. 2-3).
2-3-5-1: Reagent :
Reagents used are demonstrated in table ( 2-4).
Table (2-4) : Reagent used :
Reagent
Quantity
Assay plate
1
Standard
2
Sample Diluent
1 x 20 ml
Biotin-antibody Diluent
1 x 10 ml
HRP-avidin Diluent
1 x 10 ml
Biotin-antibody
1 x 120 µL
HRP-avidin
1 x 120 µL
48
Chapter Two
Materials, and Methods
Wash Buffer
1 x 20 ml
(25 x concentrate)
TMB Substrate
1 x 10 ml
Stop Solution
1 x 10 ml
2-3-5-2:Assay procedure:
Reagents and samples were brought all to room temperature before use. It is
recommended that all samples, standards, and controls be assayed in duplicate. All the
reagents should be added directly to the liquid level in the well. The pipette should avoid
contacting the inner wall of the well.
 An aliquot (100μl) of Standard, Blank, or Sample was dispersed per well cover with the
adhesive strip incubate for 2 hours at 37°C.
 The liquid was removed from each well, without washing.
 A volume of 100μl of Biotin-antibody working solution was added to each well. Incubated
for 1 hour at 37°C. Biotin-antibody working solution was add may appear cloudy. Warmed
up to room temperature and mixed gently until solution appears uniformed.
 Each well was aspirated and washed, the process was repeated three times for a total of
three washes. Washing was determined by filling each well with wash buffer (200μl) and
let it stand for 2 minutes, then the liquid was removed by flicking the plate over a sink. The
remaining drops were removed by patting the plate on a paper towel. Complete removal of
liquid at each step was essential to good performance.
 A volume of 100μl of HRP-avidin working solution was added to each well. The microtiter
plate was coverd with a new adhesive strip incubated for 1 hour at 37°C.
 The aspiration was repeated and washed five times as mentioned earlier .
 A volume of 90μl of TMB Substrate was added to each well incubated for 10-30 minutes
at 37°C.
 Aliquot of 50μl of Stop Solution was added to each well where the first four wells contained the
highest concentration of standards develop obvious blue color. If color change does not
appear uniformed, tap gently the plate to ensure thorough mixing.
49
Chapter Two
Materials, and Methods
 The optical density of each well was determined within 30 minutes, using a microplate
reader set to 450 nm.
2-3-5-3:Calculation of results:
Duplicate reading for each standard, control, and sample were averaged and subtracted
from the averaged zero standard optical density.
A standard curve was created by
reducing the data using computer software capable of generating a four parameter logistic
(4-pl) curve-fit.
Fig. (2-3): Human Interleukin 12 ELISA (IL-12) kit used in this study
2-3-6: Statistical Analysis
The Statistical Analysis System- SAS (2010) was used to effect of difference factors in study
parameters. Chi-square test was used to significant compare between percentage of this study.
CHAPTER THREE
RESULTS AND DISCUSSION
Chapter Three
Results and Discussion
50
3: Results and Discussion
3-1:Demographic presentation of the studied groups:
The results presented in this study were based on analysis of data determined
from detection of specific antibodies for T.gondii
in the serum of individuals
collected from a total of 300 cases : 200 schizophrenic patients (100 males and 100
females ) with different age groups (10 - > 60 )years , and 100 heathy individuals
used as controls ( 50 males and 50 females ) with matched age groups. Also based
on information collected from the investigated subjects (Appendix
18 ) and
characterize them demographically in terms of age , gender, educational status,
location , presence of pets or breeding animals , consuming milk ,different types of
fresh meat or canned meat and using sterile or tap water .
3-2:Serological diagnosis of T. gondii antibodies in sera of schizophrenic
patients and healthy individuals using LAT\MLAT tests
The results observed from detecting sera of schizophrenic patients and healthy
individuals for the presence of T.gondii antibodies by LAT test , showed 143(71.5%)
seropositive for the patients and 45(45%) for the healthy individuals (Figure 3-1 )
and appendex (1).
Chapter Three
Results and Discussion
% of anti-T.gondii Abs
80
51
+Ve
-Ve
71.5
70
55
60
45
50
40
28.5
30
20
10
0
Schizophrenic patient
Control
Fig. (3-1) :- The percentage distribution of T.gondii antibodies
in the sera of Schizophrenic patients and healthy individuals
(control) as measured by LAT (Abs) test.
The statistical analysis showed
significant (p<0.05) differences between
LAT
detected antibodies between Schizophrenic patient and healthy individuals . Serum
positive individuals detected using LAT (188 serum positive ) were further checked
using MLAT to determine the presence of IgM and IgG which indicates acute or
chronic infections with toxoplasmosis (Figure 3-2) appendix (2). The results
revealed 134 (93.7%) seropositivity for IgG in schizophrenic patients and 45(100%)
in healthy individuals, which revealed 9(6.3%) seropositivity for IgM in patients
compared to zero % in healthy individuals with no significant differences (p<0.01)
but with significant differences between IgG and IgM in the same group.
Chapter Three
Results and Discussion
52
+Ve…
-Ve…
100
% of anti- T.gondii Abs
100
93.7
90
80
70
60
50
40
30
20
6.3
0
10
0
Schizophrenic patient
Control
Fig. ( 3-2): The percentage distribution of T.gondii antibodies in
sera of Schizophrenic patients and healthy individuals (control)
as measured by MLAT(Abs) test.
In this study, the use of LAT(Abs) testing was a preliminary examination to determine
antibodies of the parasite T.gondii in the patients serum whereas the use of MLAT was
used for screening to indicate the type of antibodies in the serum whether being of type IgG
or IgM Antibodies . Others (Choi et al., 1983 ; Shin et al., 2009; AL- Mayahi ,2011; ALObeady, 2011 ; AL-Saadii ,2013) used the same methods determining the percentage of the
patients antibodies in the serum with some difference.These differences might be
explained by differences in assay systems, the characteristics of the surveyed populations,
and the location of the sample populations. According to our data and a previous work
(Ajioka and Soldati ,2007), the increase of infection rate in Latex test is due to decrease of
it's specificity, also the false positive samples were increased in this test ( Oshima , 1982).
When comparing the seropositivities of Latex with ELISA, the difference in seropositivity
according to assay method may have resulted partly from different antigenic determinants
( epitopes ) being recognized in each method, and partly from different limits of sensitivity
of each test (Evans et al., 2002).
Chapter Three
Results and Discussion
3-3: Serological diagnosis
53
of anti-T. gondii antibodies in sera of schizophrenic
patients and healthy individuals by using ELISA IgG\IgM tests :The results of ELISA - IgG\IgM Abs tests for 143 serum samples of schizophrenic
patients and 45 from healthy individuals positive in LAT is shown in figures (3-3) and (34), (appendix 3 and 4 ). The number of serum positive test for IgG ELISA was 114(79.7%)
in schizophrenic patients and 33(73.3%) in healthy individuals and the number of positive
test in IgM ELISA was 6(4.19%) in schizophrenic patients and Zero in healthy individuals.
The statistical analysis revealed that there were no significance differences in seropositive
individuals but found significant differences in seronegative subjects .
% of anti-T.gondii Abs
+Ve (IgG)
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
-Ve (IgG)
79.7
73.3
26.7
20.3
Schizophrenic patients
Control
Fig.(3-3): The percentage distribution of anti-T.gondii antibodies in
sera of Schizophrenic patients and healthy individuals (control) as
measured by ELISA-IgG (Abs) test.
Chapter Three
Results and Discussion
54
100
% of anti-T.gondii Abs
95.81
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
+Ve (IgM)
-Ve (IgM)
4.19
0
Schizophrenic patient
Control
Fig .( 3-4):The percentage distribution of anti-T.gondii antibodies in
sera of Schizophrenic patients and healthy individuals (control) as
measured by ELISA-IgM (Abs.) test.
In this study, prevalence of anti-T. gondii Abs in schizophrenic patients and healthy
individuals was 79.7% , 73.3% (IgG) and 4.19%, 0% (IgM) respectively and the difference
was not statistically significant. This study results
conferms the result of Saraei-
Sahnesaraei et al. (2009) and Daryani et al.(2010) studies in Iran, showed seropositive for
IgG specific antibodies to T. gondii and the differences were not statistically significant.
Prevalence of T. gondii in both studies carried out in Iran showed the same results in both
patients and control group. Some researchers such as Cook and Derrick (1961) in Australia;
Garrido and Redondo (1968) in Spain; Qiuying et al. (1999) in China; Boronow et al. (2002)
in the United States, Torrey and Yolken (2003) in Ireland ;Emelia et al. (2012) in Malaysia
reported that the differences between the two groups were not statistically significant. In
contrast, other researchers showed that the differences between the two groups were
statistically significant (Gu et al., 2001 in China; Yolken et al., 2001 in Germany; Leweke et
al., 2004 in Germany; Alvarado-Esquivel et al., 2006 in a northern Mexican city; Cetinkaya
et al., 2007 in Turkey; Mahmoud and
2011 in Iran ) .
Hasan 2009 ;Jassam 2010 in Iraq; Alipour et al.
Chapter Three
Results and Discussion
55
IgM antibodies in this study showed no significant differences between the groups. This
is due to the fact that IgM is an indicator of recent infection and becomes negative within
4–12 weeks, hence presumably is not associated with the increased risk of schizophrenia
(Hamidinejat et al., 2010). Generally IgM antibodies are detected within the first 2 weeks of
infection and reduce to negligible levels within 6 months after exposure. However, in
toxoplasmosis, IgM titres can remain elevated up to a year or even more. Thus, the mere
presence of IgM antibodies is not diagnostic of an acute toxoplasmosis infection. However,
a negative IgM antibody test rules out recently acquired infection unless the serum is
tested too early after exposure so that antibodies have not as yet developed.
A single positive IgG antibody test indicates chronic infection, which might have been
acquired before conception, thus posing no risk to the foetus (Subasinghe et al., 2011) .
Because antibody titers to Toxoplasma IgG may remain elevated for significant periods of
time, an increase in IgG antibody may reflect an active primary infection, reactivation of
infection, or a persistent immune response to a dormant infection(Remington et al., 2001).
Increased IgG titers to Toxoplasma have been associated with both severe and subtle
neuropsychiatric abnormalities (Sever et al., 1988). Different results in various studies may
be due to many reasons which include geographical conditions, using only serological tests
with no DNA detection, selection of control group, source of infection (oocyst or tissue
cyst), differences in genetic susceptibility, timing of the infection, different strains of
Toxoplasma and consumption of antischizophrenia drugs, in some studies there was an
association between T. gondii infection and schizophrenia. It may be because of certain
circumstances how the infection occurs or because of secondary manifestation (Daryani et
al.,2010).
In Spain, schizophrenia patients showed a high rate of seropositivity to T. gondii because
they worked in the hospital garden that had been faecally contaminated by the hospital’s
cats (Garrido and Redondo, 1968). On the other hand, institutionalized schizophrenia
patients may be fed undercooked meat, thereby increasing their exposure to T. gondii
(Daryani et al.,2010). Alternatively, in some instances increased T. gondii antibodies in
schizophrenia patients are secondary to immune system
abnormalities, such as in
individuals infected with HIV, a pathogen which is a primary agent of schizophrenia,
Chapter Three
Results and Discussion
56
causing reactivation of T. gondii tissue cysts in different organs and generation of antibody
to T. gondii which is a secondary manifestation (Torrey et al., 2006).
Horacek et al. (2012) found that the gray matter density in the brain of schizophrenia
patients who latently infected with T. gondii was significantly reduced than Toxoplasma
negative schizophrenia patients in the caudate, median cingulate, thalamus and occipital
cortex and in the left cerebellar hemispheres. Torrey et al. (2007), in their meta-analysis of
11 studies, showed that individuals with schizophrenia have an increased prevalence of
antibodies to T. gondii and suggested that this, as well as genetic and environmental
factors, could be associated with a large number of cases of schizophrenia. One study
showed that mothers having antibodies to T. gondii late in pregnancy, even though the
infection was not necessarily recent, had an increased risk of giving birth to offsprings who
later were diagnosed with a schizophrenia spectrum disorder (Mortensen et al., 2007).
In this study
no significant association revealed between the precence of T. gondii
antibodies and schizophrenia, some possible reasons including the fact that more than 9٥%
of patients in the present study received anti-schizophrenia treatment. Leweke et al.
(2004) a study on three groups including schizophrenia patients receiving antischizophrenia treatment, those who had never received any drug and those of the control
group, they showed that the antibody levels for the treated group were intermediate
between the levels of the never-treated group and those of the control group, it suggests
that anti-schizophrenia medication may have decreased the antibody levels. It is noted that
in terms of the possible effect of medications some of the therapeutic agents commonly
employed for the treatment of schizophrenia and bipolar disorder have the ability to
inhibit the replication of T. gondii tachyzoites in cell culture (Jones-Brando et al., 2003).
The difference in seropositivity according to assay method (LAT,ELISA) may have resulted
partly from different antigenic epitopes being recognized by each method, and partly from
different limits of sensitivity for each test (Rigsby et al., 2004).
Chapter Three
Results and Discussion
57
3-4:Frequencies of anti-T.gondii antibodies in sera of Schizophrenic patients and
healthy individuals(control) according to educational level as measured by ELISAIgG (Abs.) test:
The study results showed the percentage differences in seropositive IgG antibodies
according to educational levels as follows: illiterate (88.57 %),primary school (77.55
%),secondary school (79.41 %), institute and college (66.67 %)
, with significant
differences between studied groups at (p<0.01).Figure (3 - 5), (appendex 5).
90
88.57
77.55
80
-Ve
66.67
70
% of anti-T.gondii Abs
+Ve
79.41
60
50
33.33
40
30
20
22.45
20.59
11.43
10
0
Illiterate
Primary school
Secondary school Vocation(Institute
,College)
Fig. (3-5): Frequencies of anti-T.gondii antibodies in sera of
Schizophrenic patients and healthy individuals(control) according
to educational level as measured by ELISA-IgG test.
The results were in agreement with several studies (AL-Qurashi, 2004 ; Ertug et al., 2005
and AL-Obeady, 2012). Mohamed et al. (2012) from Sudan showed that women who
were uneducated were more expected to infection than women who learn at any level with
significant differences . Although, 90% of women participated in his study were educated
but uneducated women still are more suspected to infection by Toxoplasma gondii. This
might be due to the fact that uneducated women were unaware of the ideal health
conditions to be followed in eating and drinking or may be not caring about that. But there
Chapter Three
Results and Discussion
58
are disagreement with several studies that recorded no significant differences between the
education status and infection by toxoplasmosis ( Karem, 2007; AL-Mayahi, 2011) .This
may be due to the method of transmission and how to avoid the infection and because they
studied aborted women in Sulaimania , Kut cities . AL-Shikhly, (2010) showed that the
number of infected educated females with toxoplasmosis was higher than uneducated
women , who concluded that the lower levels of education with an increased risk for
toxoplasmosis, moreover, lower levels of education are associated with socioeconomic
status and may be related to employment in jobs with greater soil exposure. These
differences may be due to variation in the specimens used by each study and their variable
conditions and time of study, or the use of different laboratory methods in addition to
sample size, time of sampling and to geographical location which plays an important role in
difference of seroprevalence rate of toxoplasmosis (AL-Mayahi, 2011).
3-5:Frequencies of anti-T.gondii antibodies in sera of Schizophrenic patients and
healthy individuals(control) according to occupation as measured by ELISA-IgG
(Abs.) test:
The result showed that
the percentage of T. gondii infection
was 54 (75%)
seropositive employed individuals out of 72 individual tested , while it showed the
percentage between unemployed individuals was 93 (80.17%) seropositive out of 116
individual tested .( Figure 3-6) , ( appendix 6) . The present study showed
that the
number of unemployed individuals infected with T. gondii was higher than employed
individuals, but with
no
significant
differences between both
groups, but found
significant differences between seropositive and seronegative in the same group at
(p<0.01).
Chapter Three
Results and Discussion
59
90
% of anti-T.gondii Abs
80
+Ve
-Ve
80.17
75
70
60
50
40
25
30
19.83
20
10
0
Employed
Unemployed
Fig. (3-6): Frequencies of anti-T.gondii antibodies in sera of
Schizophrenic patients and healthy individuals according to occupation
as measured by ELISA-IgG (Abs.) test.
The results were in agreement with
several previous studies ( AL-Qurashi , 2004
;Karem , 2007 ; AL-Mayahi, 2011) , and were in disagreement with others (AL-Obeady,
2012 ; AL-Saadii ,2013). In Iran Mohraz et al., (2011) revealed that the seroprevalence of
toxoplasmsis were clerks (20%) merchant (58.6%) students (33.3%) unemployed (50%)
other jobs (48.91%). Zhou et al.,(2009) in China showed that some occupations required
people to have contact with animals and meats and these frequently posses higher risk of
infection with the parasite, such as dairy workers (45.0%) slaughter house workers
(25.6%) veterinarians (12.5%), meat processing workers (13.7%) meat sellers and cooks
(29.7%). Another investigators demonstrated that out of the 200 fruit and vegetable
workers, 15(7.5%) of them, and 31(7.8%) of the 400 controls were positive for antiToxoplasma IgG antibodies while, anti-Toxoplasma IgM, antibodies were found in 2(1%) of
the dealing with fruit and in 11(2.8%) of the control subjects (Alvarado-Esquivel et al.,
2011). In Mexico Alvarado-Esquivel et al. (2011) revealed (7%) of the 124 butchers and 22
Chapter Three
Results and Discussion
60
(9%) of the 248 controls were positive for anti-T. gondii IgG antibodies with no statistically
significant differences in the IgG seroprevalences . Alvarado-Esquivel et al. (2008) showed
an association between T. gondii infection and consumption of unwashed fruits was found
in workers occupationally exposed to water, sewage, and soil .Many
important
roles
in determining
seroprevalence ratio of
factors
toxoplasmosis such
play
as ,
wearing the gloves when cutting meat and gardening or touching soil or sand because
of possible presence of cat feces , washing cutting border , dishes , counters
hands with
hot soapy
water
after
being
and
in contact with raw meat , poultry or
seafood or with unwashed fruits or vegetables . Frequent consumption and type of
meat (pig , sheep , and goat) were identified as the principle risk factor in several
studies of T. gondii infection in human (Baril et al. , 1999).
3-6: Frequencies of anti-T.gondii antibodies in sera of Schizophrenic patients
according to drinking water as measured by ELISA-IgG( Abs.) test:
The result noticed that the rate of toxoplasmosis infection between individuals who
used tap water for drinking was higher [102(91.89%)] from those who used the sterile
water [12(37.5%)], with a significant difference between them at ( p<0.01), (Figure 3-7)
and (appendix 7).
Chapter Three
Results and Discussion
61
91.89
100
% of anti-T.gondii Abs
+Ve
-Ve
90
80
62.5
70
60
50
37.5
40
30
20
8.11
10
0
Minerals water
Tap water
Fig. (3-7): Frequencies of anti-T.gondii antibodies in sera
of Schizophrenic patients and control according to
drinking water type as measured by ELISA-IgG (Abs.)
test.
The results appeared were in agreement with
AL-Obeady ( 2012) and AL-Shikhly (
2012) but disagree with AL-Mayahi(2011). This finding may be due to low levels of
education between men and women concerning the risk of T. gondii infection and method
of transmission from environment to the women and may be due to that most of the study
groups were uneducated or with primary education levels, in other hand most of the
families depends on a water reservoir kept upside the roofs as a source of drinking water
that may be not well covered which make it in exposure to direct contact with air or
contamination with cat or chiken feces (AL-Obeady, 2012) . Oocysts of T.gondii can remain
viable for long periods of time in water and can resist freezing and moderately high water
temperatures (Lindsay and Dubey , 2009). They are not killed by chemical and physical
treatments currently applied in water treatment , including chlorination and ozone
treatment (Dumetre et al ., 2008). Robert-Gangneux and Darde (2012) observed a positive
correlation
between the consumption of unboiled well water and the presence of
Chapter Three
Results and Discussion
62
Toxoplasma antibodies, especially in farms with poor-hygiene conditions surrounding
shallow wells.
3-7:Frequencies of T.gondii antibodies in sera of Schizophrenic patients and healthy
individuals according to presence of cats and pets as measured by ELISA-IgG test:
The results showed that the presence of T.gondii antibodies in the individuals lived with
the presence of the cats 107 (88.43%) have higher seropositivity than those who do not
have cats 40 (59.7%)
. Breeding different animals ( chicken, sheep and rabbit) in the
study revealed elevated number of individuals who were seropositive for T. gondii
[110(91.67%)] compared to individuals who did not[37(54.41% )].With significant
difference between them (P<0.05) Figures (3-8),(3-9) and (appendix 8, 9).
+Ve
-Ve
88.43
%of anti-T.gondii Abs
90
80
70
59.7
60
50
40.3
40
30
20
11.59
10
0
Presence of cats
No Presence of cats
Fig. (3-8): Frequencies of anti-T.gondii antibodies in sera
of Schizophrenic patients according to the presence of
cats as measured by ELISA-IgG test.
Chapter Three
Results and Discussion
63
+Ve
-Ve
% of anti-T.gondii Abs
100
91.67
90
80
70
54.41
60
45.59
50
40
30
20
8.33
10
0
Chicken, sheep ,rabbit
No breeding animals
Fig. (3-9): Frequencies of anti-T.gondii antibodies in sera of
Schizophrenic patients and healthy individuals according to
presence of pets animals as measured by ELISA-IgG test.
The results observed were in agreement with several studies ( Frenkel and Ruiz ,1980
; AL-Najjar , 2005 ; Karem , 2007 ; AL-Mayahi, 2011; AL-Obeady ,2012 ) which showed
the
risk of T . gondii infection in
women
increased
by increasing
contact with
animals . While the results were in disagreement with previous other studies (AbdelHameed , 1991 ; AL-Wattari , 2005 ; AL-Shikhly, 2010 ) that recorded no significant
difference between infected women depending on contact with animals. AL-Shikhly
(2012) found no significant differences between students contact with animals in different
universities , that revealed high rate of infection between women not in contact with
animals , the owners and
transmission of
breeders of animals did not have the active role in
infection , but the infection may happened by exposure to the
million of oocysts by the cat feces that contaminated the environment (Lopez et al. ,
2010) . Stanford et al.(1990) showed that keeping cats as pets in the general population
revealed high prevalence of T. gondii antibodies, an increase in Toxoplasma antibodies in
patients with schizophrenia may be less apparent. In contrast Remington et al.(2001) in
Chapter Three
Results and Discussion
64
China showed that keeping cats is uncommon and prevalence of T.gondii antibodies was
low and higher prevalence of antibodies in patients with schizophrenia may be apparent .
This difference can be attributed that most of samples were collected from women who
had direct contact with animals through having pets , Or indirect by ingestion of
vegetable or fruits contaminated with oocyst by cat feces that may be encountered
in gardens (Qublan et al ., 2002 ; Jones and Dubey, 2010) .
Individuals with occupations requiring contact with soil in environments frequented by
cats are significantly more likely to contract toxoplasmosis (Jones et al., 2001). However,
the more significant risk factor is in contact with cats and cat litter. Cats infection may be
asymptomatic, increasing the likelihood of accidental infection (Elmore et al., 2010 ; Lilly
and Wortham, 2013 ).
3-8: Frequencies of anti- T.gondii antibodies in sera of Schizophrenic patients and
healthy individuals according to consuming milk as measured by ELISA-IgG test:
The observed
results noticed that the rate of toxoplasmosis infection between
individuals who consume milk were [133(81.60%)] and[14(56%)] with non consuming
milk with a significant difference between them at (p>0.01) (Figure 3-10) and ( appendix
10).
Chapter Three
Results and Discussion
90
65
+Ve
-Ve
81.6
% of anti-T.gondii Abs
80
70
56
60
44
50
40
30
18.4
20
10
0
Consuming milk
non consuming
milk
Fig. (3-10): Frequencies of anti-T.gondii antibodies in sera of
Schizophrenic patients and healthy individuals according to
consuming milk as measured by ELISA-IgG test.
The results showed significant differences between milk consumer and non milk consumer
patients with schizophrenia and healthy individuals , this may be due to the contamenated
tools used for drinking milk or the milk itself by T. gondii .
In Norway, consumed undercooked goat meat and unpasteurized goat milk are potential
sources of T. gondii infection (Stormoen et al., 2012). Milk may serve as a potential source
for
human toxoplasmosis (Asgari et al., 2011). The association between infection and
unpasteurised milk or milk products was unexpected, T. gondii tachyzoites have been
isolated from goats' milk and cows' colostrum(Dubey, 1988). But are destroyed within
minutes by gastric juices (Remington et al., 1995).
Chapter Three
Results and Discussion
66
3-9:Frequencies of T.gondii antibodies in sera of Schizophrenic patients and healthy
individuals according to type of meat (fresh or canned) consumed as measured by
ELISA-IgG test:
The results demonstrated that the rate of toxoplasmosis infection between individuals who
consume different types of meat showed seropositivity percentage of (86.24%),
(70.42%),(37.50%), for chicken, sheep and beef consuming meat respectively . On the
other hand the results found different percentages of seropositivity between individuals
who consumed canned meat 125( 82.24%) and 22(61.11%) who do not consume canned
meat , with a significant difference between them ( p<0.01), (Figure 3-11 , Figure 3-12 )and
( appendix 11, 12).
90
+Ve
-Ve
86.24
% of anti-T.gondii Abs
80
70.42
70
62.5
60
50
37.5
40
29.57
30
20
13.76
10
0
Chicken
Sheep
Beef
Fig.(3-11): Frequencies of anti-T.gondii antibodies in sera of
Schizophrenic patients and healthy individuals according to type
of meat consumed as measured by ELISA-IgG test.
Chapter Three
Results and Discussion
67
+Ve
-Ve
90
82.24
% of anti-T.gondii Abs
80
70
61.11
60
50
38.89
40
30
17.76
20
10
0
Consuming canned meat
Non consuming canned meat
Fig. (3-12):Frequencies of anti-T.gondii antibodies in sera of
Schizophrenic patients and healthy individuals according to
consumed canned meat as measured by ELISA-IgG test.
From the results mentioned , it was observed there is a relationship between the presence
of T.gondii antibodies and type of meat consumed by the individuals. Asgari et al.(2011)
presented the total prevalence of Toxoplasma infection as 33.3 % among 22 goats (22.7 %)
and 56 sheep (37.5 %) by PCR. Ghorbani et al.(1983) have reported the Serological
prevalence of toxoplasmosis in sheep in Kuzestan (southwest of Iran), Mazandaran and
Gilan (North of Iran) which showed 12.6 %, 32.5-35.8 % and 29-31 % respectively . It has
been assumed that infected sheep, will remain persistently infected for their whole life
(Uggla and Buxton , 1990). Sharif et al.(2007) found 30 %, 35 % sero-positivity in goats and
sheep respectively by IFA test from Mazandaran Province . These altered results indicate
that animals were exposed to different environmental contamination with T. gondii
oocytes. Rahdar et al.(2012) found 4 % of cattle were positive for T. gondii and the infection
was more frequent in sheep compared with cattle. Dubey and Thulliez (1993) have
reported that Toxoplasma infection in cattle does not usually cause clinical symptoms
because of high natural resistance to the parasite . These results indicate that meat products probably have low importance as a source of infection for human toxoplasmosis in the
Chapter Three
Results and Discussion
68
studied region. Warnekulasuriya et al.,(1998) detected one positive sample out of 67 cured
meat samples, including dried and semi-dried sausages and hams using PCR in UK. In
Fallah et al., (2011) study, samples (sausages, hamburgers) have been selected from the
factories in Tabriz province. Despite the dramatic differences in pollution levels in different
areas of Iran due to different weather conditions, especially favorable temperature for the
maintenance of parasite oocytes, it seems that high pollution in his study is justifiable.
Besides, beef supplies are used more in the preparation of these products and based on
studies in Iran and the world, contamination of cattle is much lower than that sheep.
Among food animals, pigs are considered to be the major source of T. gondii for humans,
this probably is one reason for different results, in different regions, could be using meat
products that contain pork meat (Dubey , 1994). These findings may be related to various
ways of infection prevalence in meat-producing animals, or different eating habits; it has also been indicated that T. gondii is killed by salting, curing, freezing, or heating procedures
that are used in meat processing, these products are not a likely source of human exposure
to the infection. It is difficult to find T. gondii tissue cysts in large animal species for several
reasons, including sampling bias and preferred parasite sites. Dubey et al. (1996) has
estimated that less than 1 tissue cyst/50 g of tissue is likely to be found in T. gondii-infected
pigs . The seropositivily rate of T.gondii is very high in countries such as France and
Ethiopia , where undercooked or raw meat is regularly consumed , yet schizophrenia had
not been found to be unusually prevalent in these countries , possible explanations include
the fact that transmission by eating tissue cysts in undercooked meat is a more benign
mode of infection (Ledgerwood et al., 2003).
The results in this study showed , a high rate of infection with T. gondii for persons who
consume chicken meat, Chickens are considered one of the most important hosts in the
epidemiology of T. gondii infection because they are an efficient source of infection for cats
that excrete the environmentally resistant oocysts and because humans may become
infected with this parasite after eating undercooked infected chicken meat (Dubey , 2010 ).
Chickens play an important role in the epidemiology of T. gondii in the rural environment,
perhaps more than rodents, because the chickens are clinically resistant to T. gondii and
live longer than rodents. Cats fed naturally infected chicken tissues can shed millions of
Chapter Three
Results and Discussion
69
oocysts (Dubey et al., 2002 ). Chickens fed T. gondii oocysts were killed 1–2 weeks later and
their tissues were fed to cats. Cats fed tissues of chickens that had been infected 10 days
earlier shed oocysts (Ruiz and Frenkel, 1980). Chickens can harbour mouse-virulent T.
gondii without showing any clinical signs (Dubey et al., 2002). In many instances, especially
in developing countries, these chickens are killed at home or in unsupervised slaughter
facilities and the viscera are left for scavengers or are improperly disposed off, T. gondii
infection can be transmitted if care is not taken to wash hands thoroughly after cutting
meat and during cooking of meat (Dubey ,2010 ). In this study it was found that the
potential risk of the disease transmission by consumption of contaminated meat should
still be considered as a public health problem. Based on the obtained results, it is suggested
that not only schizophrenic patient and immunocompromised patients should be
addressed but also the whole population should be informed on how to prevent infection.
3-10: Frequencies of anti- T.gondii antibodies in sera of Schizophrenic patients and
healthy individuals according to age and gender group as measured by ELISA-IgG
test
The results by ELISA-IgG seropositive for T. gondii were 1(50%) ,7 (38.89%), 28(80%),
76(85.39%), 25(78.13%), 10(83.33%) for (10-20 ), (21-30), (31-40), ( 41-50), (51-60),
(>60) years age groups in Schizophrenic patients and healthy individuals , which indicated
increase in anti-T. gondii seropositivity by age progression with significant difference
(P<0.01) between IgG levels in different age groups. The results showed seropositive
percentages for both men and women Schizophrenic patients
were 53(76.8%) and
61(82.43%) respectively, and healthy individuals(control) the seropositivity in men and
women were 9(56.2%) and 24(82.76%) respectively, with significant differences between
men in both groups but with no significant differences between women in both groups
(p>0.01). Figure (3-13),(3-14) and( appendix 13, 14).
Chapter Three
Results and Discussion
90
+Ve
-Ve
% of anti-T.gondii Abs
80
70
60
70
85.39
83.33
80
78.13
61.11
50 50
50
38.89
40
30
21.87
20
16.67
14.61
20
10
0
10-20.
21-30.
31-40.
41-50.
51-60.
<60.
Fig. (3-13): Frequencies of anti-T.gondii antibodies in sera of
Schizophrenic patients and healthy individuals according to age
group as measured by ELISA-IgG test.
80
Schizophrenic
patients
Control
82.76
82.43
90
76.81
% of ant-T.gondii Abs
70
60
56.25
43.75
50
40
23.19
30
17.57
17.24
20
10
0
+ve
-ve
Men
+ve
-ve
Women
Fig. (3-14) :Frequency of anti-T. gondii antibodies in sera o Schizophrenic
patients and healthy individual according to gender as measured by ELISA
–IgG test.
Chapter Three
Results and Discussion
This study was disagree with
71
Emelia et al., (2012) which observed that the highest
positivity rate in schizophrenic patients for anti-T. gondii IgG antibody was seen in those
aged >40 years old by a percentage of 37(25.7%) using ELISA BioRAD kit , and with ALObeady (2012) who found high percentages of seropsitive IgG in women detected by LAT
test were13 (13 %), 9(9%) ,6(6%), 4(4%) for age groups 20-24 , 25-29 , 30-34 and 35- 39
years respectively, with significant differences between groups .And also with AL-Saadii
(2013) who showed the highest positive percentage of 30 (24.79%) in ELISA IgG test at
age group of both (18-25) and (26-33) years whereas the lowest one was 7(5.78%) as
noticed at the age group of (50-57) years . While, a previous study by Jasim (1979)
indicated that the incidence of toxoplasmosis increases with age not exceeded 50 years
whereas Williams et al., (2005) pointed that the incidence increases with age but to a peak
of 34 years. The seroprevalence of T. gondii infection increased significantly with age
(Alvarado-Esquivel et al., 2011 ). In contrast this study agreed with Jassam (2010) who
found that the lowest positive rates for Anti-Toxoplasma IgG Ab determined by ELISA test,
were recorded in age groups of schizophrenia patients it was (20-29) years with the rates
of (28.6%), while the age groups of (30-39) , (40-49), (≥ 50) years with the rates of
(48.6%), (44%), (58.1%) respectively, with no significant differences . The variation in the
results may be because samples of her study were taken from men only . Other study
results showed no significant difference with age factor but the highest infection rate
occurred in age group (31-35) years (Fernands,2010).
In relation with gender , this study disagrees with Khademvatan et al., (2013) who found
no significant difference in prevalence of IgG positivity between men and women this
differences may be due to the samples taken from students of Medical Science University
in Iran. while, Lindová et al.(2006) reported higher seroprevalance in male compared to
female students .Also, a higher prevalence in males than in females, were found by Xiao et
al.(2010).
Chapter Three
Results and Discussion
72
Some studies have reported higher rates of schizophrenia among the relatives of female
patients with schizophrenia than among the relatives of male patients. (Kendler and Walsh,
1995 ) . Jablensky and Eaton ( 1995) found no support for such differences. Finding of an
excess number of male with schizophrenia is consistent with the results of other registerbased studies. The effects of the risk factors included in were identical for male and female
(Mortensen et al., 1999). Ochoa et al.(2012) found differences in age of onset are the most
replicated finding in studies into gender differences in schizophrenia male usually develop
the illness at age 18–25 years while in female, the mean age of onset is 25–35 years.
Furthermore, the onset distribution curves for males and females are not isomorphic.
Female seem to have two peaks in the age of onset of disease: the first after menarche and
the second once they are over 40 years. However, Castle et al.(1998) found that earlyonset age distribution is similar between male and female . The major prevalence of female
once they are over 40 years could be explained by the reduction of estrogens after
menopause according to the estrogenic hypothesis of schizophrenia (Riecher- Rossler et
al., 1994). However, a number of studies found no gender difference in the age of onset
(Naqvi et al., 2005; Ochoa et al.,2012).
These differences between previous results and current result may be due to the
differences in the specificity and sensitivity of detecting methods used for diagnosis and
response of each host to the strain of the parasite, the variation in parasite strains may play
an important role in the stimulation of host immune response against the parasite (Suzuki
and John, 1994). And also the differences may be due to different samples , gender,
geographical location , season of sample collection ,and also because of the fact that most of
the age groups in this study are of ages of >30 years.
3-11:Frequencies of anti- T.gondii antibodies in sera of Schizophrenic patients and
healthy individuals according to type of locality as measured by ELISA-IgG test
The distribution percentage of anti-T. gondii antibodies in Schizophrenic patients
and healthy individuals who live in city (urban area ) and country side (rural) was 106
(80.92%) and 8(66.67)
seropositive . The study noticed the rate of infection by
toxoplasmosis between individuals living in city was higher than the ones live in
Chapter Three
Results and Discussion
73
country side , figure (4-15) and ( appendix 15) , with significant differences between
both groups at (p>0.01) .
90
+Ve
-Ve
80.92
80
% of ant-T.gondii Abs
66.67
70
60
50
33.33
40
30
19.08
20
10
0
City
Country side
Fig. (3-15):Frequencies of anti-T.gondii antibodies in sera of
Schizophrenic patients according to type of locality as measured
by ELISA-IgG test.
This study agrees with AL-Saadii (2013) in which had noticed significant difference and
high percentage of toxoplasmosis in males inhabited rural area which where 111(91.73%)
compared to10 (8.26%) urban residents.
The result was disagreed with several previous studies ( Karem , 2007; AL-Mayahi,2011)
that indicated no significant difference of infection by T. gondii between women lived in
urban and rural areas but the seropositive women in rural area were more than
that in the urban areas , The reason for this difference may be because their study
concerned aborted women sera in Sulaimania and Kut respectively . Other studies in Iraq
by Al-Jubori (2005) regarding the residency of the patients and its relation with
seropositive Toxoplasma Abs showed no significant difference between Toxoplasma Abs
distribution on both urban and rural areas, through which the rate were (33.98%) and
(32.08%) respectively. Sroka et al. , (2010) in Poland also showed that human living in
Chapter Three
Results and Discussion
74
farms had significantly greater percentage of anti-Toxoplasma antibodies with (59%)
compared to urban dwellers (41.0%) .The prevalence of schizophrenia is higher in urban
areas than in rural areas (Takei et al., 1995). The difference has been ascribed to selective
migration from rural to urban areas before the onset of schizophrenia, but this hypothesis
does not explain the finding of a higher risk among people born in urban areas. Other
possible explanations include increased exposure to infections during pregnancy and
childhood because of more crowded living conditions or more perinatal complications in
urban areas (Mortensen et al., 1999). The difference
between different studies in city
(urban area ) and country side (rural)) may be due to the hygiene and the difference in
samples and races, frequent ready-made foods , eat more in restaurants in the city from the
countryside, and also the fact that specimens in this study, most of them were from city
residents compared to rural areas.
3-12: Presence of IL-12 in Schizophrenic patients with toxoplasmosis infection and
healthy individuals as measured by ELISA-IL 12 kit
In this study the concentration of interleukin-12 was measured in the serum of
patients with schizophrenia and healthy individuals (mean ± SE) was (5.79 ± 0.61 pg\ml)
and (5.01 ± 0.89 pg\ml) respectively and found non-significant differences between both
groups . whereas when results separated according to gender , significant differences
were found between the two groups studied for both sexes at (p<0.05). Figure (3-16) and (
appendix 16 ,17).
Chapter Three
Results and Discussion
75
Mean Concentration of IL-12
pg\ml
5.79
5.8
5.6
5.4
5.2
5.01
5
4.8
4.6
Patients
Control
Fig. (3-16):Mean concentration of IL-12 in schizophrenic patients
and healthy individuals.
IL-12 which is secreted by the macrophages and DCs during antigen stimulation, appears to
play a major anti-Toxoplasma role during the acute phase of the infection. Indeed, it
activates the production of IFN-γ by NK cells and CD4+, CD8+ TL , the administration of IL12 combined with the recombinant T. gondii SAG1 (surface antigen 1) surface protein
directs the immune response towards predominantly type-1 profile, associated with high
IFN-γ production, this modulation of the immune response is linked to a reduction in
cerebral parasite load (Letscher-Bru et al., 1998). IL-12 is also essential during the chronic
phase of the infection, when it is responsible for maintaining a long-term immune response
(Filisetti and Candolfi, 2004). Mashayekhi et al.(2011) showed that the CD8α+ DCs were
the only cells within the innate immune system whose IL-12 production was required for
resistance to acute T. gondii infection. Miller et al.(2009) observed that IL-12 production by
DCs is rapid within a few hours of infection.Yap et al.(2000) showed that endogenous IL12 is required for the long-term maintenance of a protective type 1 T cell response against
the intracellular pathogen, T. gondii. Nevertheless, for a 2-week period after IL-12
withdrawal, pathogen-specific T cells retain the capacity to produce IFN-γ and mediate
Chapter Three
Results and Discussion
76
control of infection. The eventual loss of IFN-γ synthesis and host resistance is likely to
result from the exit of these terminally differentiated effectors from lymphoid organs into
brain tissue, where they recognize their targets and die in situ . Because T. gondii is never
cleared by the immune response, recruitment of T cells into the effector pool continues
(Ford et al., 1996). Hunter et al.(1995) highlight the importance of the protective activity,
rather than the suppressive activity, of IL-12 in toxoplasmosis. In this study, there was no
any significant differences between the seropositive patients with schizophrenia and
healthy individuals , contrary to the results obtained by Abdul-Lateef et al.(2012) who
showed high level of IL-12 in both male and female with asymptomatic toxoplasmosis in
comparison with control group . Kareem (2008) results revealed a significant difference
between female infected with T. gondii and apparently healthy woman in IL-12 level. The
reason for this contradictory results may be due to the small sample size difference and
the difference in the samples used the study patients were schizophrenic and the control
group have serum antibodies from chronic and not acute infection.
CONCLUTIONS
AND
RECOMMENDATION
Conclusion and
Recommendation
77
CONCLUSIONS:
In the light of the current study it is concluded that :
1.LATtest can be used to determine the seropositivity of the schizophrenic
patients for T.gondii antibodies .While MLAT test revealed higher
seropositivity(93.7% IgG and 6.3% IgM ) than that when detected using
ELISA (79.7% IgG and 4.19% IgM ).
2.Illiterate patients are more susceptible toT.gondiiinfections(88.57%) more
than of somewhat educated patients ( primary school 77.55% , secondary
school 79.41% , college level 66.67% ).
3.
No
differences
appeared
between
employed
and
unemployed
schizophrenic study individuals concerning susceptibility for T. gondii.
4. The use of sterile drinking water protected the study individuals from
toxoplasmosis ( 12 (37.5%) compared with those who use tap water (102
(91.89%).
5. Living with cats or other animals (chicken , sheep or rabbit )keepers
become more exposed to toxoplasmosis than those who do not live with pets.
6.Individuals who consumed contaminated milk have higher seropositivity
for T.gondiiIgG than those who do not consume milk .
7. Concerning different type of meat (chicken, sheep, beef) consumers :
chicken meat consumers are more susceptible for T.gondii infection
(86.24%), followed by sheep meat were (70.42%). Moreover canned meat
consumers also showed higher seropositivity (82.24%) than those who
prepare fresh meat .
Conclusion and
Recommendation
78
8.All age study groups were susceptible for infection withT.gondii . In
addition individuals living in the city (80.92 %) were more susceptible for
the infection than those who lived in the country side (66.6%) .
9. According to the gender , schizophrenic females shows higher
seropositivity for T.gondiiIgG that the males(76.8%) . But it must be noticed
than the healthy control women showed seropositivity similar to those of the
schizophrenic women
10. No difference appeared in the meanconcentration serum IL-12 between
(schizophrenic patients and control individuals)with toxoplasmosis.While
when patients separated according to their gender , females show higher
mean concentration of IL-12 than the males.
Conclusion and
Recommendation
79
RECOMMENDATION :
1. Special attention and hygiene care should be taken into consideration when
adopting cats or any other pets . moreover abandoned cats should be prohibited in
the hospitals and restaurants.
2.Detection ofT. gondii antibodies is very important for pregnantwomen and women
of child-bearing age. This is an effective way to find the infection, and then to provide
treatment. It is also an efficient way to stop congenital toxoplasmosis in newborns.
3.The use of molecular methods for the detection of T.gondii to better define the
relationship between the infection and the onest of the schizophrenic symptoms.
4.Study the immunological profile of schizophrenic patients .
CHAPTER FOUR
REFERENCES
Chapter four
References
80
-A Abdel-Hafez ,S . K . ; I . Shbeeb ; N . S. Ismail and F .Abdel-Rahman ( 1986 ) .
Serodiagnosis
of Toxoplasma gondii
in habitually aborting women
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APPENDICES
Appendix
(1):The percentage distribution of anti-T.gondii antibodies in 300 sample sera of
Schizophrenic patients and healthy individuals (control) as measured by LAT test:
Test - LAT
ChiSample
square
Total
+Ve
-Ve
No.
Percentage
No.
Percentage value
(%)
(%)
Schizophrenic
200
143
71.5
57
28.50
10.54 **
patient
Control
100
45
45.00
55
55.00
4.092 *
Total
300
188
--١١٢
----Chi-square
----7.946 **
--7.946 **
--value
* (P<0.05), ** (P<0.01).
(2):The percentage distribution of anti-T.gondii antibodies in sera of
Schizophrenic patients and healthy individuals (control) as measured by MLAT
test:
Test - MLAT
ChiSample
square
Total
+Ve (IgG)
-Ve (IgM)
No.
Percentage
No.
Percentage value
(%)
(%)
Schizophrenic
143
134
93.7
9
6.3
13.289
patien
**
Control
45
45
100.0
0
0.0
14.500
**
Total
188
179
--٩
----Chi-square
----1.603 NS
--1.603 NS
--value
** (P<0.01).
Appendix
(3):The percentage distribution of anti-T.gondii antibodies in sera of
Schizophrenic patients and healthy individuals (control) in as measured by ELISAIgG test:
Test - ELISA-IgG
ChiSample
square
Total
+Ve (IgG)
-Ve (IgG)
No.
Percentage
No.
Percentage value
(%)
(%)
Schizophrenic
143
114
79.7
29
20.3
12.783
patien
**
Control
45
33
73.3
12
26.7
11.471
**
Total
188
١٤٧
--٤١
----Chi-square
----1.583 NS
--1.583 NS
--value
** (P<0.01).
(4):The percentage distribution of anti-T.gondii antibodies in sera of Schizophrenic
patients and healthy individuals (control) as measured by ELISA-IgM test:
Test - ELISA-IgM
ChiSample
square
Total
+Ve (IgM)
-Ve (IgM)
No.
Percentage
No.
Percentage value
(%)
(%)
Schizophrenic
143
6
4.19
137
95.81
13.012
patien
**
Control
45
0
0.00
45
100
14.500
**
Total
188
6
--182
----Chi-square
----0.873 NS
--0.873 NS
--value
** (P<0.01).
Appendix
(5): Frequencies of anti-T.gondii antibodies in sera of Schizophrenic patients and
healthy individuals(control) according to educational level as measured by ELISAIgG test:
Test - ELISA-IgG
ChiEducation level
square
Total
+Ve
-Ve
No.
Percentage
No.
Percentage value
(%)
(%)
Illiterate
35
31
88.57
4
11.43
11.57
**
Primary school
49
38
77.55
11
22.45
10.45
**
Secondary school
68
54
79.41
14
20.59
11.39
**
Vocation(Institute
36
24
66.67
12
33.33
9.52
,Collage)
**
Total
188
147
--41
----Chi-square value
----4.83 *
--4.83 *
--* (P<0.05), ** (P<0.01).
(6): Frequencies of anti-T.gondii antibodies in sera of Schizophrenic patients and
healthy individuals according to occuption as measured by ELISA-IgG test:
Test - ELISA-IgG
ChiOccupation
square
Total
+Ve
-Ve
No.
Percentage
No.
Percentage value
(%)
(%)
Employed
72
54
75.00
18
25.00
11.69 **
Unemployed
116
93
80.17
23
19.83
11.86 **
Total
188
147
--41
----Chi-square
----1.034 NS
--1.034 NS
--value
** (P<0.01).
Appendix
(7): Frequencies of anti-T.gondii antibodies in sera of Schizophrenic patients
according to water type drinking as measured by ELISA-IgG test:
Test - ELISA-IgG
ChiWater type
square
Total
+Ve
-Ve
drinking
No.
Percentage
No.
Percentage value
(%)
(%)
Minerals
32
12
37.50
20
62.50
9.05 **
water
Tap water
111
102
91.89
9
8.11
12.48 **
Total
143
114
--29
----Chi-square
----10.43 **
--10.43 **
--value
** (P<0.01).
(8): Frequencies of anti-T.gondii antibodies in sera of Schizophrenic patients and
healthy individuals in according to presence of cats as measured by ELISA-IgG
test:
Test - ELISA-IgG
ChiPresence of
square
Total
+Ve
-Ve
cats
No.
Percentage
No.
Percentage value
(%)
(%)
Presence of
121
107
88.43
14
11.5٧
12.09 **
cats
No Presence
of cats
67
40
59.70
27
40.30
7.83 **
Total
Chi-square
value
188
---
147
---
--8.23 **
41
---
--8.23 **
-----
** (P<0.01).
Appendix
(9): Frequencies of anti-T.gondii antibodies in sera of Schizophrenic patients and
healthy individuals according to presence pets animals as measured by ELISAIgG test:
Test - ELISA-IgG
ChiPresence pets
square
Total
+Ve
-Ve
animals
No.
Percentage
No.
Percentage value
(%)
(%)
Chicken,
120
110
91.67
10
8.33
12.43 **
sheep ,rabbit
No breeding
animals
68
37
54.41
31
45.59
4.75 *
Total
Chi-square
value
188
---
147
---
--8.34 **
41
---
--8.34 **
-----
* (P<0.05),** (P<0.01).
(10): Frequencies of anti-T.gondii antibodies in sera of Schizophrenic patients and
healthy individuals according to consuming milk as measured by ELISA-IgG test:
Test - ELISA-IgG
ChiConsuming
square
Total
+Ve
-Ve
milk
No.
Percentage
No.
Percentage value
(%)
(%)
Consuming
163
133
81.60
30
18.40
11.36 **
milk
Non
25
14
56.00
11
44.00
5.19 *
consuming
milk
Total
188
147
--41
----Chi-square
----8.31 **
--8.31 **
--value
* (P<0.05), ** (P<0.01).
Appendix
(11): Frequencies of anti-T.gondii antibodies in sera of Schizophrenic patients and
healthy individuals in according to type of consuming meat as measured by
ELISA-IgG test:
Test - ELISA-IgG
ChiType of
square
Total
+Ve
-Ve
consuming
No.
Percentage
No.
Percentage value
meat
(%)
(%)
Chicken
109
94
86.24
15
13.76
12.65 **
Sheep
71
50
70.42
21
29.57
10.54 **
Beef
8
3
37.50
5
62.50
9.39 **
Total
188
147
--41
----Chi-square
----9.43 **
--9.43 **
--value
** (P<0.01).
(12): Frequencies of anti-T.gondii antibodies in sera of Schizophrenic patients and
healthy individuals in Iraq according to consuming canned meat as measured by
ELISA-IgG test:
Test - ELISA-IgG
ChiConsuming
square
Total
+Ve
-Ve
canned meat
No.
Percentage
No.
Percentage value
(%)
(%)
Consuming
152
125
82.24
27
17.76
11.58 **
canned meat
Non
36
22
61.11
14
38.89
7.91 **
consuming
canned meat
Total
188
147
--41
----Chi-square
----7.13 **
--7.13 **
--value
** (P<0.01).
Appendix
(13): Frequencies of anti-T.gondii antibodies in sera of Schizophrenic patients and
healthy individuals in Iraq according to age group as measured by ELISA-IgG test:
Test - ELISA-IgG
ChiAge group
square
Total
+Ve
-Ve
(year)
No.
Percentage
No.
Percentage value
(%)
(%)
10-20
2
1
50.00
1
50.00
0.00 NS
21-30
18
7
38.89
11
61.11
9.35 **
31-40
35
28
80.00
7
20.00
11.25 **
41-50
89
76
85.39
13
14.61
12.76 **
51-60
32
25
78.13
7
21.87
11.04 **
>60
12
10
83.33
2
16.67
12.37 **
Total
188
147
--41
----Chi-square
----9.71 **
--9.71 **
--value
** (P<0.01).
(14): Frequencies of T.gondii antibodies in sera of Schizophrenic patients and
healthy individuals(control) according to gender as measured by ELISA-IgG test:
Test - ELISA-IgG
Sample
sex
Men
Women
Total
+ve
-ve
+ve
-ve
T NO. (%) N (%)
T
N (%) NO (%)
O.
O.
.
Schizophrenic
143 69
53
76.8 16 23.1 74 61 82.43 13 17.5
patients
1
9
7
control
45
16
9
56.2 7 43.7 29 24 82.76 5 17.2
5
5
4
Total
188 85
62
--- 23 --- 103 85
--18 --Chi-square value ----8.31 -- 8.31 --- 0.35 -- 0.35
**
**
NS
NS
** (P<0.01).
Appendix
(15): Frequencies of anti-T.gondii antibodies in sera of Schizophrenic patients
according to type of locality as measured by ELISA-IgG test:
Test - ELISA-IgG
ChiType of
square
Total
+Ve
-Ve
locality
No.
Percentage
No.
Percentage value
(%)
(%)
City
131
106
80.92
25
19.08
11.36 **
Country side
12
8
66.67
4
33.33
9.75 **
Total
143
114
-29
---Chi-square
----7.12 **
--7.12 **
--value
** (P<0.01).
(16): Mean Concentration of IL-12 in Schizophrenic patients with toxoplasmosis
and healthy individuals(control)
Concentration of IL-12 (mean ± SE)
Group
Patients
5.79 ± 0.61
Control
5.01 ± 0.89
T-test
0.433 NS
NS: Non-significant.
Appendix
(17): Compare between Iraqi Schizophrenic patients with toxoplasmosis infection
and healthy individuals(control) in concentration of IL-12
Group
Mean ± SE
LSD Value
Male
Female
Patients
5.626 ± 0.93
5.976 ± 0.80
Control
3.777 ± 0.423 6.260 ± 1.62
1.734 *
LSD Value
0.893 *
---
0.722 *
* (P<0.05).
0.509 NS
Appendix
(18):Questionnaire sheet used for each individuals included in this
study .
Questionnaire
Age ………….
Locality:
City.....
Country side.....
Sex:
Female ………..
Male …….
Contact with animals: (Cat , Sheep, Chicken, Rabbit)
Cat……
others animals........
No contact……
Pets animals:( Cat , Sheep, Chicken, Rabbit)
Yes........
No......
Source of drinking water:
Minerals water………
Tap water……..
Education level:
Illiterate…..
Primary school….
Vocation(Institute, Collage)......
Secondary school.....
Appendix
Occuption :
Employed……
Unemployed……
Consuming milk:
Yes......
No.......
Type of consuming meat:
Chicken ....
Sheep.....
Beef ....
Consuming canned meat:
Yes......
No.......
‫الخالصة‬
‫االصابة بداء المقوسات الكوندية ‪ Toxoplasma gondii‬واسعة االنتشار وذات اھمية كبيرة ‪ ,‬وان مرض داء‬
‫المقوسات ال يظھر اي عالمات سريرية معينة ‪ ,‬اجريت ھذه الدراسة على ‪ 200‬شخص مصابين بمرض انفصام‬
‫الشخصية و‪100‬من االشخاص االعتيادين الذين اعتمدوا كعينات سيطرة وتم جمع عينات الدم للفترة من بداية شھر‬
‫كانون االول ‪ 2012‬لغاية نھاية شھر شباط ‪ 2013‬وكانت العينات تشمل الذكور واالناث ‪.‬تم استخدام اختبار‬
‫‪ LAT\MLAT‬وتقنية االمتزاز المناعي المرتبط باالنزيم ‪ ELISA‬لتحديد االضداد الخاصة بالطفيل نوع ‪ IgG‬و‬
‫‪ IgM‬في المصول وعالقتھا بالعديد من العوامل التي تؤثرعلى االصابة وكما تم قياس مستوى االنترلوكين‪12 -‬‬
‫لمعرفة تركيزه في مصول ‪ 40‬عينة لتحديد العالقة بين االصابة بانفصام الشخصية وداء المقوسات الكوندية كأحد‬
‫المؤشرات المناعية ‪ ,‬ولقد لخصت نتائج ھذه الدراسة كاالتي ‪:‬‬
‫‪ ‬النسبة المئوية لالمصال الموجبة لتالزن المقوسة الكوندية من المجموع الكلي في مصل الدم الوريدي لمرضى‬
‫االنفصام واالشخاص االعتيادين باستخدام اختبار ‪ LAT‬بلغت ‪ %45, %71.5‬على التوالي وكانت ھناك فروق‬
‫معنوية بين نسب االصابة ‪ .‬بينما اختبار ‪ MLAT‬كانت النسب المئوية لمرضى االنفصام ھي ‪ %93.7‬لل ‪ IgG‬و‬
‫‪ IgM %6.3‬بينما النسبة المئوية لالشخاص االعتياديين كانت ‪%100‬‬
‫‪ IgG‬وكانت ھناك فروق معنوية بين‬
‫نسب االصابة ‪.‬‬
‫‪ ‬النسبة المئوية لالمصال الموجبة لتالزن المقوسة الكوندية من المجموع الكلي في مصل الدم الوريدي لمرضى‬
‫االنفصام باستخدام اختبار ‪ ELISA‬من نوع ‪ IgG‬كانت ‪ %79.7‬بينما كانت النسبة المئوية لالشخاص االعتيادين‬
‫ھي ‪ %73.3‬ولم توجد اي فروق معنوية بين نسب االصابة ‪.‬‬
‫‪ ‬النسبة المئوية لالمصال الموجبة لتالزن المقوسة الكوندية من المجموع الكلي في مصل الدم الوريدي لمرضى‬
‫االنفصام باستخدام اختبار ‪ELISA‬من نوع ‪ IgM‬كانت ‪ %4.19‬ولم توجد اي اصابة لالشخاص االعتيادين ‪.‬‬
‫‪ ‬كانت ھناك فروق معنوية بين نسب االصابة بطفيلي المقوسات الكوندية للعينات حددت باستعمال اختبار االليزا‬
‫وكانت ھذه النسب لھا عالقة بمستوى التعليم ‪ ,‬الوظيفة ‪ ,‬نوع مياه الشرب ‪ ,‬وجود القطط ‪ ,‬تربية الحيوانات االليفة ‪,‬‬
‫شرب الحليب ‪ ,‬نوع اللحوم المتناولة ‪ ,‬العمر ‪ ,‬الجنس ‪,‬مكان السكن ‪.‬‬
‫‪ ‬ال توجد فروق معنوية لتركيز االنترلوكين ‪ 12-‬في مصول مرضى االنفصام واالشخاص االعتيادين )السيطرة (‬
‫حيث كانت متوسطات تراكيزھم )‪ . ( 5.01 ± 0.89 pg\ml),(5.79 ± 0.61 pg\ml‬لكن عند فصل الذكور‬
‫عن االناث احصائيا وجدت فروق معنوية بين متوسطات التراكيز وكانت متوسطات الذكور لمرضى االنفصام‬
‫واالشخاص االعتيادين )‪ . ( 3.777 ± 0.423) , ( 5.626 ± 0.93‬بينما متوسطات نساء المصابات باالنفصام‬
‫ونساء السيطرة كانت )‪ (6.260 ± 1.62) ,(5.976 ± 0.80‬على التوالي ‪.‬‬
‫جمھورية العراق‬
‫وزارة التعليم العالي والبحث العلمي‬
‫جامعة بغداد‪ /‬كلية العلوم‬
‫األنتشار المصلي لداء المقوسات الكوندية في عينة من مرضى‬
‫أنفصام الشخصية العراقيين‬
‫دراسة مقدمة إلى‬
‫مجلس كلية العلوم _ جامعة بغداد‬
‫كجزء من متطلبات الحصول على شھادة الماجستير‬
‫في علوم ألحياة ‪ /‬علم الحيوان‬
‫قدمت من قبل‬
‫سھيرداخل نعمة المعموري‬
‫بكلوريوس علوم حياة‪-‬جامعة بغداد‬
‫‪2009‬‬
‫ألمشرفين‬
‫أالستاذ فوزية أحمد ألشنوي‬
‫كلية العلوم _جامعة بغداد‬
‫شعبان ‪1435‬‬
‫االستاذ الدكتور أليس كريكور أغوب‬
‫كلية العلوم_ جامعة بغداد‬
‫‪ 2014‬حزيران‬