UNIVERSITI PUTRA MALAYSIA - psasir.upm.edu.mypsasir.upm.edu.my/41846/1/FPV 2011 15R.pdfBrucellosis adalah penyakit kronik yang berpunca daripada Brucella spp. Brucella melitensis adalah

UNIVERSITI PUTRA MALAYSIA

MAGED AHMED MUTHANNA AL-GARADI

FPV 2011 15

DETECTION OF INFECTION AND DETERMINATION OF BIOMARKERS FOR BRUCELLA MELITENSIS INFECTION IN GOATS

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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

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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

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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.

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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

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(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

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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.

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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).

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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



(Internal Examiner)

Abdul Aziz Saharee, PhD

Professor



(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,


Date: 23 August 2011

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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



(Chairman)

Abdul Rahman Omar, PhD

Professor



(Member)

Zunita Zakaria, PhD

Associate Professor



(Member)

________________________________

HASANAH MOHD. GHAZALI, PhD

Professor and Dean

School of Graduate Studies


Date:

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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

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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

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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


blood and serum by RT-PCR

46

3.3 Results 46

3.3.1 Detection of Brucella melitensis

infection by serological methods

46


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


4.2.1 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

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5 BIOMARKERS OF BRUCELLA MELITENSIS

INFECTION BASED ON CELL MEDIATED

IMMUNITY IN EXPERIMENTALLY INFECTED

MICE

5.1 Introduction 78


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


5.3.3 Real-time PCR 87


level in deferent stages of Brucella

melitensis infection

89



6 BIOMARKERS OF BRUCELLA MELITENSIS

INFECTION BASED ON CELL MEDIATED

IMMUNITY IN EXPERIMENTALLY INFECTED

GOATS

6.1 Introduction 103


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



different samples by Real-time PCR

113


level in different stages of Brucella

melitensis infection in goats

115



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7 GENERAL DISCUSSION AND CONCLUSION 130

REFERENCES 136

APPENDICES 152

BIODATA OF STUDENT 158

LIST OF PUBLICATIONS

159

Documents

UNIVERSITI PUTRA MALAYSIA - psasir.upm.edu.mypsasir.upm.edu.my/41846/1/FPV 2011 15R.pdfBrucellosis adalah penyakit kronik yang berpunca daripada Brucella spp. Brucella melitensis adalah