UNIVERSITI PUTRA MALAYSIA
MAGED AHMED MUTHANNA AL-GARADI
FPV 2011 15
DETECTION OF INFECTION AND DETERMINATION OF BIOMARKERS FOR BRUCELLA MELITENSIS INFECTION IN GOATS
© COPYRIG
HT UPM
i
DETECTION OF INFECTION AND DETERMINATION OF BIOMARKERS
FOR BRUCELLA MELITENSIS INFECTION IN GOATS
By
MAGED AHMED MUTHANNA AL-GARADI
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,
in Fulfillment of the Requirement for the Degree of Doctor of Philosophy
May 2011
© COPYRIG
HT UPM
ii
Abstract of thesis presented to the Senate of Universiti Putra Malaysia in
fulfillment of the requirement for the degree of Doctor of Philosophy
DETECTION OF INFECTION AND DETERMINATION OF
BIOMARKERS FOR BRUCELLA MELITENSIS INFECTION IN
GOATS
By
MAGED AHMED MUTHANNA Al-GARADI
2011
Chairman: Associate Professor Dr. Siti Khairani Bejo, PhD
Faculty: Veterinary Medicine
Brucellosis is a chronic disease caused by Brucella spp. Brucella melitensis is
one of the species of bacteria that infects animals and humans leading to
undulant fever. The interaction between B. melitensis and immune system of
the host could guide us towards the discovery of new biomarker to detect the
infection as early as possible particularly understanding the unique pathway
of B. melitensis and the intracellular features.
In this study, 288 samples of whole blood and serum were collected from a
goat farm in Kedah, Malaysia, which was suspected to have been affected by
brucellosis. Serological and molecular detections of brucellosis were
performed, using Rose Bengal Plate Test (RBPT), Complement Fixation Test
(CFT), conventional PCR and real-time PCR (RT-PCR). Each test was
evaluated with respect to its sensitivity and specificity ensuring that the
recommendations made are able to be use for the national brucellosis
eradication program in Malaysia. Isolation and identification of B. melitensis
© COPYRIG
HT UPM
iii
were also done in addition to conventional PCR and real-time PCR to detect
B. melitensis from samples collected from vaginal swabs. CDs markers and
its combination in different population of peripheral blood mononuclear cells
(PMNC) were measured in detail, in different B. melitensis stages of the
infection and in experimentally infected mice and goats by using specific
monoclonal antibody. Histopathological picture was also been described in
current study.
The sensitivity of RBPT was 89.04% whilst CFT was 97.02%. The specificity
of each of RBPT and CFT were 99.06% and 96.38% respectively. Four (4) B.
melitensis isolates were isolated from 300 vaginal samples and all isolate
belonged to B. melitensis biotype 1. The real-time PCR was the easier and
safer method for the confirmation of brucellosis in goat’s population. The
CDs biomarkers namely; CD14, CD4, CD25 markers were identified as good
markers for the different stages of B. melitensis infection. A combination of a
serological test namely RBPT and molecular technique, in particular real-time
PCR based on the IS711 region of a hypothetical protein, showed promising
results. This combination can be used to reduce the number of false positive
results, which can cause severe economical loss during the implementation of
proposed eradication programs.
Keywords: Brucella melitensis, biomarkers, Real-time PCR, CDs.
© COPYRIG
HT UPM
iv
Abstrak thesis yang dikemukakan kepada Universiti Putra Malaysia sebagai
memenuhi keperluan ijazah Doktor Falsafah
DETEKSI INFEKSI DAN PENENTUAN BIOPENANDA JANGKITAN
BRUCELLA MELITENSIS DI DALAM KAMBING
Oleh
MAGED AHMED MUTHANNA Al-GARADI
Mei 2011
Pengerusi: Profesor Madya Dr. Siti Khairani Bejo, PhD
Fakulti: Perubatan Veterinar
Brucellosis adalah penyakit kronik yang berpunca daripada Brucella spp.
Brucella melitensis adalah sejenis spesies bacteria yang menjangkiti haiwan
dan manusia lalu menyebabkan demam beralun. Interaksi antara B. melitensis
dan sistem imun perumah boleh memberi panduan kepada kita untuk
menemui biopenanda baru untuk mengesan jangkitan seawal yang mungkin.
Dengan cara mamahami laluan unik B. melitensis dan ciri-ciri intraselularnya
membantu untuk menemui penanda baru bagi jangkitan B. melitensis.
Asai reaksi rantai polimerase (PCR) telah terbukti sebagai pilihan yang baik
untuk diagnosis bruselosis. Dalam kajian ini, 288 sampel darah penuh dan
serum diambil dari ladang kambing di negeri Kedah, Malaysia, yang disyaki
menghidap bruselosis. Pengesanan serologi dan molekul terhadap bruselosis
telah dijalankan dengan menggunakan Ujian Plat Rose Bengal (RBPT),
Ujian Pengikatan Pelengkap (CFT), PCR konvensional dan PCR masa nyata
© COPYRIG
HT UPM
v
(RT-PCR). Setiap ujian telah dinilai berhubung dengan kepekaan dan
kekhususan untuk menentukan ujian mana yang patut digunakan dalam
program pembasmian bruselosis di Malaysia. Pengasingan B. melitensis
adalah tahap utama bagi pengenalpastian dan pengesahan bruselosis haiwan.
Walau bagaimanapun, di Malaysia jangkitan Brucella spp. di dalam kambing
semakin meningkat sejak kebelakangan ini dan tiada terdapat bukti bagi
diagnosis biotip Brucella spp. yang menyebabkan penyakit dalam populasi
kambing kecuali pengesanan kaedah serologi. Pengasingan dan
pengenalpastian B. melitensis telah dijalankan melalui kaedah bakteriologi
disamping PCR konvensional dan PCR masa nyata untuk pengesanan
Brucella melitensis daripada sampel-sampel yang diambil daripada swab
vagina di ladang yang disyaki. Penanda CD dan gabungannya dalam populasi
yang berlainan bagi sel mononuklear darah periferi (PMNC) telah diukur
dengan terperinci pada peringkat jangkitan B. melitensis yang berbeza
didalam tikus dan kambing yang dijangkitkan secara kajian dan dengan
menggunakan antiboldi monoklonal yang khusus. Gambar histopatologi turut
dihuraikan dalam kajian ini.
Sebagai rumusan, gabungan ujian serologi dan teknik molekul, terutamanya
PCR masa nyata berdasarkan kawasan IS711 daripada protein hipotesis
adalah menggalakkan. Gabungan ini boleh digunakan untuk mengurangkan
bilangan keputusan positif palsu, yang boleh menyebabkan kerugian ekonomi
yang besar semasa perlaksanaan cadangan program pembasmian. Kepekaan
RBPT adalah 89.04% sementara CFT adalah 97.02%. Kekhususan setiap
RBPT dan CFT masing-masing adalah 99.06% dan 96.38%. Empat (4) isolat
© COPYRIG
HT UPM
vi
Brucella melitensis telah diasingkan daripada 300 sampel vagina dan
kesemua isolat tergolong dalam biotip 1 B. melitensis. PCR masa nyata
adalah kaedah yang mudah dan selamat bagi pengesahan bruselosis di dalam
populasi kambing. Biopenanda CD seperti; penanda CD14, CD4, CD25 telah
dikenal pasti sebagai penanda yang baik untuk peringkat jangkitan Brucella
melitensis yang berbeza. Gabungan kaedah serologi dan molekul adalah perlu
untuk mencapai cadangan pembasmian menyeluruh bruselosis di Malaysia.
Kata kunci: Brucella melitensis, biopenanda, PCR masa nyata, CD.
© COPYRIG
HT UPM
vii
ACKNOWLEDGEMENTS
I am deeply indebted to my supervisors Assoc. Prof. Dr. Siti Khairani Bejo,
Prof. Abdul Rahman Omar and Assoc. Prof. Dr. Zunita Zakaria for their
encouragements, patience fairly guidance and understanding during the
course of this study. Appreciation is also extended to Prof. Abdul Rani
Bahman for his help and encouragement.
I am grateful to the staff of Faculty of Veterinary Medicine, UPM. I am also
thankful to the Dean of Graduate School, Prof. Hasanah Mohod Ghazali, and
her staff members for their valuable help during the study. I’m also grateful to
Dr. Maha Abdullah and Miss Norsharina, Miss Pooi Leong for their asistance
during all my study time. Deep honor are also given to technicians Mr. Hajar,
Miss. Hepzibah Kuppusamy, Miss. Lateifah and also technicians Azri and
Siti Khadijah. I would also like to thank my best friends Shahaza Othman,
Zanior, Seven, Nabila and Fairuz.
Finally, I am greatly thankful to my father, mother, wife and brothers
especially Waleed AL- Garadi for their support during my study period here.
This project was funded by the Government of Malaysia through the RUGS
Fund (Grant no. 91120).
© COPYRIG
HT UPM
viii
I certify that Examination Committee has met on 31stMay 2011 to conduct the
final examination of MAGED AHMED MUTHANNA Al-GARADI on his
PhD thesis entitled “DETECTION OF INFECTION AND
DETERMINATION OF BIOMARKERS FOR BRUCELLA MELITENSIS
INFECTION IN GOATS" in accordance with the Universities and University
Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia
[P.U.(A) 106] 15 March 1998. The Committee recommends that the
candidate be awarded the Doctor of Philosophy.
Members of Examination Committee are as follows:
Abdul Rani Bahman, PhD
Professor,
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(Chairman)
Mohd Zamri Saad, PhD
Professor
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(Internal Examiner)
Abdul Aziz Saharee, PhD
Professor
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(Internal Examiner)
Ian Robertson, PhD
Professor,
School of Veterinary and Biomedical Sciences University Murdoch,
Murdoch, Australia
(External Examiner)
________________________________
NORITAH OMAR, PhD
Associate Professor and Deputy Dean
School of Graduate Studies,
Universiti Putra Malaysia
Date: 23 August 2011
© COPYRIG
HT UPM
ix
This thesis was submitted to the Senate of Universiti Putra Malaysia has been
accepted as fulfilment of the requirement for the degree of Doctor of
Philosophy. The members of the Supervisory Committee were as follows:
Siti Khairani Bejo, PhD
Associate Professor
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(Chairman)
Abdul Rahman Omar, PhD
Professor
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(Member)
Zunita Zakaria, PhD
Associate Professor
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(Member)
________________________________
HASANAH MOHD. GHAZALI, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
© COPYRIG
HT UPM
x
DECLARATION
I declare that the thesis is my original work except for quotations and citation
which have been duly acknowledged. I also declare that it has not been
previously, and is not concurrently, submitted for any other degree at
Universiti Putra Malaysia or at any other institution.
_____________________
MAGED AL-GARADI Date: 31 May 2011
© COPYRIG
HT UPM
xi
TABLE OF CONTENTS
Page
ABSTRACT ii
ABSTRAK iv
ACKNOWLEDGEMENTS vii
APPROVAL viii
DECLARATION x
LIST OF TABLES xv
LIST OF FIGURES xvii
LIST OF ABBREVIATIONS xxii
CHAPTER
1 INTRODUCTION 1
2 LITERATURE REVIEW
2.1 Goat Brucellosis 6
2.2 Goats Brucellosis in Malaysia 10
2.3 Pathogenesis of Brucellosis 11
2.3.1 Reproductive tract localization 14
2.3.2 Placentitis and abortion 14
2.3.3 Fetal stress due to Brucella spp.
infection
16
2.3.4 Invitro Brucella infection of
chorioallantoic membrane
17
2.4 Brucellosis and immunity 17
2.5 Zoonotic aspects of Brucella melitensis
infection
21
2.6 Diagnosis of Brucella melitensis in goats 24
2.6.1 Direct microscopic examination 25
2.6.2 Isolation and identification 26
2.6.3 Serological test 26
2.6.4 Cell mediated immunity based test 28
2.6.5 Molecular diagnostic methods 28
2.7 Biomarker for detection of brucellosis in goats 30
2.8 Strategies for the control and eradication of
brucellosis in goats
32
2.8.1 Uniform brucellosis eradication
program
35
2.8.2 Program components 36
3 DETECTION OF BRUCELLA MELITENSIS IN
CLINICAL SAMPLES COLLECTED FROM GOATS
3.1 Introduction 38
3.2 Materials and Methods 40
3.2.1 Animal and Study Herds 40
3.2.2 Serum and whole blood samples 40
3.2.3 Detection of Brucella melitensis
infection by serological methods
40
© COPYRIG
HT UPM
xii
3.2.4 DNA extraction from blood and
serum samples
41
3.2.5 Detection of Brucella melitensis in
blood and serum by conventional
PCR
43
3.2.6 Melting curve analysis and
evaluation of RT-PCR
45
3.2.7 Detection of Brucella melitensis in
blood and serum by RT-PCR
46
3.3 Results 46
3.3.1 Detection of Brucella melitensis
infection by serological methods
46
3.3.2 Detection of Brucella melitensis in
blood by conventional PCR
47
3.3.3 Melting curve analysis and
evaluation of RT-PCR
48
3.3.4 Detection of B. melitensis in blood
by RT-PCR
49
3.4 Discussion and Conclusion 55
4 ISOLATION AND IDENTIFICATION OF
BRUCELLA MELITENSIS IN GOATS
4.1 Introduction 61
4.2 Materials and Methods 63
4.2.1 Vaginal samples 63
4.2.2 Isolation of B. melitensis from
vaginal samples
63
4.2.3 Identification of Brucella melitensis
isolates
63
4.2.4 Characterization of Brucella
melitensis isolates
67
4.3 Results 68
4.3.1 Bacterial isolation and identification
of B. melitensis from vaginal
samples using biochemical test.
68
4.3.2 Identification of B. melitensis
isolates from vaginal samples using
PCR
69
4.3.3 Detection of Brucella melitensis by
RT-PCR
69
4.3.4 Sequencing of IS711 region of B.
melitensis isolates
71
4.4 Discussion and conclusion 75
© COPYRIG
HT UPM
xiii
5 BIOMARKERS OF BRUCELLA MELITENSIS
INFECTION BASED ON CELL MEDIATED
IMMUNITY IN EXPERIMENTALLY INFECTED
MICE
5.1 Introduction 78
5.2 Materials and Methods 81
5.2.1 Preparation of inoculums 81
5.2.2 Experimental infection 81
5.2.3 Sampling 82
5.2.4 Bacteriological examination 83
5.2.5 Real time-PCR (RT-PCR) 83
5.2.6 Determination of CDs markers
level in different stages of Brucella
melitensis infection
83
5.2.7 Histopathology 85
5.2.8 Statistical analysis 85
5.3 Results 85
5.3.1 Experimental infection 85
5.3.2 Bacteriological examination 86
5.3.3 Real-time PCR 87
5.3.4 Determination of CDs markers
level in deferent stages of Brucella
melitensis infection
89
5.3.5 Histopathology 95
5.4 Discussion and conclusion 97
6 BIOMARKERS OF BRUCELLA MELITENSIS
INFECTION BASED ON CELL MEDIATED
IMMUNITY IN EXPERIMENTALLY INFECTED
GOATS
6.1 Introduction 103
6.2 Materials and Methods 106
6.2.1 Determination of CDs markers in
acute stage of Brucella melitensis
infection in goats
106
6.2.2 Determination of CDs markers in
chronic stage of Brucella melitensis
infection in goats
109
6.2.3 Statistical analysis 111
6.3 Results 112
6.3.1 Bacteriological examination 112
6.3.2 Detection of Brucella melitensis in
different samples by Real-time PCR
113
6.3.3 Determination of CDs markers
level in different stages of Brucella
melitensis infection in goats
115
6.3.4 Histopathology 121
6.4 Discussion and conclusion 124
© COPYRIG
HT UPM
xiv
7 GENERAL DISCUSSION AND CONCLUSION 130
REFERENCES 136
APPENDICES 152
BIODATA OF STUDENT 158
LIST OF PUBLICATIONS
159