UNIVERSITI PUTRA MALAYSIA DETECTION OF INFECTION AND DETERMINATION OF BIOMARKERS FOR BRUCELLA MELITENSIS INFECTION IN GOATS

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1 UNIVERSITI PUTRA MALAYSIA DETECTION OF INFECTION AND DETERMINATION OF BIOMARKERS FOR BRUCELLA MELITENSIS INFECTION IN GOATS MAGED AHMED MUTHANNA AL-GARADI FPV

2 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,, in Fulfillment of the Requirement for the Degree of Doctor of Philosophy May 2011 i

3 Abstract of thesis presented to the Senate of 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 Chairman: Faculty: By MAGED AHMED MUTHANNA Al-GARADI 2011 Associate Professor Dr. Siti Khairani Bejo, PhD 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 ii

4 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. iii

5 Abstrak thesis yang dikemukakan kepada sebagai memenuhi keperluan ijazah Doktor Falsafah DETEKSI INFEKSI DAN PENENTUAN BIOPENANDA JANGKITAN BRUCELLA MELITENSIS DI DALAM KAMBING Pengerusi: Fakulti: Oleh MAGED AHMED MUTHANNA Al-GARADI Mei 2011 Profesor Madya Dr. Siti Khairani Bejo, PhD 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 iv

6 (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 v

7 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. vi

8 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 ). vii

9 I certify that Examination Committee has met on 31 st May 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 [P.U.(A) 106] 15 March 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 (Chairman) Mohd Zamri Saad, PhD Professor Faculty of Veterinary Medicine (Internal Examiner) Abdul Aziz Saharee, PhD Professor Faculty of Veterinary Medicine (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 viii

10 This thesis was submitted to the Senate of 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 (Chairman) Abdul Rahman Omar, PhD Professor Faculty of Veterinary Medicine (Member) Zunita Zakaria, PhD Associate Professor Faculty of Veterinary Medicine (Member) HASANAH MOHD. GHAZALI, PhD Professor and Dean School of Graduate Studies Date: ix

11 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 or at any other institution. MAGED AL-GARADI Date: 31 May 2011 x

12 ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS TABLE OF CONTENTS CHAPTER 1 INTRODUCTION 1 Page ii iv vii viii x xv xvii xxii 2 LITERATURE REVIEW 2.1 Goat Brucellosis Goats Brucellosis in Malaysia Pathogenesis of Brucellosis Reproductive tract localization Placentitis and abortion Fetal stress due to Brucella spp. infection Invitro Brucella infection of 17 chorioallantoic membrane 2.4 Brucellosis and immunity Zoonotic aspects of Brucella melitensis 21 infection 2.6 Diagnosis of Brucella melitensis in goats Direct microscopic examination Isolation and identification Serological test Cell mediated immunity based test Molecular diagnostic methods Biomarker for detection of brucellosis in goats Strategies for the control and eradication of brucellosis in goats Uniform brucellosis eradication 35 program Program components 36 3 DETECTION OF BRUCELLA MELITENSIS IN CLINICAL SAMPLES COLLECTED FROM GOATS 3.1 Introduction Materials and Methods Animal and Study Herds Serum and whole blood samples Detection of Brucella melitensis infection by serological methods 40 xi

13 3.2.4 DNA extraction from blood and 41 serum samples Detection of Brucella melitensis in blood and serum by conventional PCR Melting curve analysis and 45 evaluation of RT-PCR Detection of Brucella melitensis in blood and serum by RT-PCR Results Detection of Brucella melitensis 46 infection by serological methods Detection of Brucella melitensis in blood by conventional PCR Melting curve analysis and 48 evaluation of RT-PCR Detection of B. melitensis in blood by RT-PCR Discussion and Conclusion 55 4 ISOLATION AND IDENTIFICATION OF BRUCELLA MELITENSIS IN GOATS 4.1 Introduction Materials and Methods Vaginal samples Isolation of B. melitensis from 63 vaginal samples Identification of Brucella melitensis isolates Characterization of Brucella 67 melitensis isolates 4.3 Results Bacterial isolation and identification 68 of B. melitensis from vaginal samples using biochemical test Identification of B. melitensis 69 isolates from vaginal samples using PCR Detection of Brucella melitensis by 69 RT-PCR Sequencing of IS711 region of B. melitensis isolates Discussion and conclusion 75 xii

14 5 BIOMARKERS OF BRUCELLA MELITENSIS INFECTION BASED ON CELL MEDIATED IMMUNITY IN EXPERIMENTALLY INFECTED MICE 5.1 Introduction Materials and Methods Preparation of inoculums Experimental infection Sampling Bacteriological examination Real time-pcr (RT-PCR) Determination of CDs markers 83 level in different stages of Brucella melitensis infection Histopathology Statistical analysis Results Experimental infection Bacteriological examination Real-time PCR Determination of CDs markers 89 level in deferent stages of Brucella melitensis infection Histopathology Discussion and conclusion 97 6 BIOMARKERS OF BRUCELLA MELITENSIS INFECTION BASED ON CELL MEDIATED IMMUNITY IN EXPERIMENTALLY INFECTED GOATS 6.1 Introduction Materials and Methods Determination of CDs markers in 106 acute stage of Brucella melitensis infection in goats Determination of CDs markers in 109 chronic stage of Brucella melitensis infection in goats Statistical analysis Results Bacteriological examination Detection of Brucella melitensis in 113 different samples by Real-time PCR Determination of CDs markers 115 level in different stages of Brucella melitensis infection in goats Histopathology Discussion and conclusion 124 xiii

15 7 GENERAL DISCUSSION AND CONCLUSION 130 REFERENCES 136 APPENDICES 152 BIODATA OF STUDENT 158 LIST OF PUBLICATIONS 159 xiv