UNIVERSITI PUTRA MALAYSIA

DEVELOPMENT OF A RAPID MOLECULAR-BASED DIAGNOSTIC KIT FOR DIAGNOSIS OF LEPTOSPIROSIS

NARUMON SOMKUNA

FPV 2004 9

DEVELOPMENT OF A RAPID MOLECULAR-BASED DIAGNOSTIC KIT FOR DIAGNOSIS OF LEPTOSPIROSIS

By

NARUMON SOMKUNA

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Doctor of Philosophy

September 2004

DEDICATION

To my parents, parents in law, husband, uncle, sisters, nieces and nephew

ii

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Doctor of Philosophy

DEVELOPMENT OF A RAPID MOLECULAR-BASED DIAGNOSTIC KIT FOR DIAGNOSIS OF LEPTOSPIROSIS

By

NARUMON SOMKUNA

September 2004

Chairman: Professor Abdul Rani Bahaman, Ph.D.

Faculty: Veterinary Medicine

Leptospirosis is a worldwide zoonosis, affecting farm animals, wildlife as well as

humans particularly in tropical areas. It is a re-emerging infectious disease caused by

pathogenic leptospires. Diagnosis of leptospirosis usually depends upon demonstration

of serum antibodies by serological tests. These tests are relatively not specific,

sensitive and often time-consuming. Early, quick and precise diagnosis is essential so

that early and specific treatment of patients can be provided and also allows for suitable

control of infection to be implemented. The primary objective of this study to use a

polymerase chain reaction (PCR) assay as a major technique to detect and differentiate

pathogenic leptospires. In addition, another aim of this study was to develop a

diagnostic kit based on PCR assay coupled with DNA hybridization assay. The assay

used a nonradioactive leptospiral DNA probe hybridized to target leptospiral DNA in a

microtitre plate for the detection of leptospiral DNA in clinical specimens. Twenty-one

iii

reference leptospiral serovars representing four species of Leptospira (L. interrogans,

L. weilii, L. inadai and L. borgpetersenii) and clinical samples were examined.

Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP)

and Low-Stringency-PCR (LS-PCR) were applied to detect and differentiate the

leptospires. The results from PCR-RFLP showed that restriction enzymes: DdeI, NdeII,

and MspI could digest the amplified leptospiral DNA with genus specific primers

G1/G2 and allowed differentiation of the leptospiral serovars and serogroups into their

species level. It is seen that LS-PCR could produce serovar specific DNA

fingerprintings. Nonradioactive digoxigenin (DIG)-labelled leptospiral DNA probes

and dot blot hybridization (DBH) assay were successfully developed for the detection

of leptospiral DNA in cultures and clinical samples. Results showed that these probes

had a high sensitivity and specificity. The lowest limit of the DNA concentration in

PCR-DBH assay was 10-6 µg. A new and rapid diagnostic kit was thus designed. This

PCR-MHA detection method has advantages over the conventional PCR assay and

DBH assay when handling a large number of samples. The procedure has fewer steps

and it is fast, safe and easy to handle. Results showed it has high specificity, sensitivity

and reproducibility. Furthermore, the assay could be used to detect leptospiral DNA

using reagents and standard laboratory ELISA equipment. Moreover, the entire method

could be completed within a day. The technique could be directly applied to clinical

samples such as urine and serum for rapid diagnosis of leptospirosis. The technique

used in this study will be useful for detection and identification of leptospirosis in

iv

patients. Moreover, it could also be used on farm animals for screening and detection

of uninfected animals.

v

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Doktor Falsafah

PERKEMBANGAN KIT DIAGNOSIS BERASAS MOLEKUL CEPAT UNTUK DIAGNOSIS LEPTOSPIROSIS

Oleh

NARUMON SOMKUNA

September 2004

Pengerusi: Profesor Abdul Rani Bahaman, Ph.D.

Fakulti: Perubatan Veterinar

Leptospirosis ialah zoonosis sejagat yang melibatkan haiwan ternakan, hidupan liar dan

juga manusia erutama sekali dalam kawasan tropika. Ia merupakan penyakit muncul

semula yang disebabkan oleh leptospira patogenik. Diagnosis untuk leptospirosis

lazimnya bergantung kepada penentuan antibodi serum melalui ujian serologi. Ujian

tersebut secara bandingan bukan khusus atau peka, dan kerap kali memakan masa.

Diagnosis awal, cepat dan tepat adalah perlu supaya rawatan awal dan khusus terhadap

pesakit boleh dilakukan dan juga untuk melaksanakan kawalan yang sesuai terhadap

jangkitan ini. Justeru itu, matlamat kajian ini ialah untuk mengguna asai tindak balas

rangkaian polimerase (PCR) sebagai suatu teknik utama dalam pengesanan dan

pembezaan leptospir patogenik. Sebagai tambahan, matlamat lain ialah untuk

mengembangkan kit diagnosis berasaskan asai PCR yang terganding dengan asai

vi

penghibridan DNA. Asai ini menggunakan kuar DNA leptospira bukan radioaktif yang

terhibrid kepada DNA leptospira sasar dalam plat mikrotiter untuk pengesanan DNA

leptospira dalam spesimen klinik. Dua puluh satu serovar leptospira yang mewakili

empat spesies Leptospira (L. interrogans, L. weilii, L. inadai dan L. borgpetersenii) dan

sample klinik telah diperiksa. Tindak Balas Rangkaian Polimerase-Polimorfisme

Panjang Serpihan Pengehadan (PCR-RFLP) dan PCR Keketatan Rendah (LS-PCR)

telah diguna untuk mengesan dan membeza leptospira. Hasil daripada PCR-RFLP

menunjukkan yang enzim pengehadan: DdeI, NdeII, dan MspI boleh mencerna DNA

leptospira teramplifikasi dengan primer khusus genus G1/G2 dan membolehkan

pembezaan serovar leptospira dan serokumpulan dilakukan sehingga pada aras spesies.

Apa yang dapat dilihat ialah, LS-PCR boleh menghasilkan sidik jari DNA khusus

serovar. Kuar DNA leptospira terlabel digoksigenin (DIG) dan asai penghibridan sap

titik (DBH) telah dapat dikembangkan dengan jayanya untuk pengesanan DNA

leptospira dalam kultur dan sample klinik. Hasil kajian menunjukkan yang kuar-kuar ini

adalah tinggi kepekaan dan kekhususannya. Had paling rendah untuk kepekatan DNA

dalam asai PCR-DBH ialah 10-6 µg. Satu kit diagnosis baru dan cepat telah direka

bentuk. Kaedah pengesanan ini adalah lebih baik daripada asai PCR konvensional dan

asai DBH apabila mengendali bilangan sample yang besar. Prosedur ini tidak banyak

langkah-langkahnya dan adalah cepat, selamat dan mudah dikendalikan. Hasil kajian

juga menunjukkan ia tinggi kekhususan, kepekaan, dan kebolehulangannya. Lagipun,

assai ini boleh diguna untuk mengesan DNA leptospira dengan mengguna reagen dan

peralatan ELISA makmal standard. Tambahan pula kaedah ini dapat dilengkapkan

vii

dalam tempoh sehari sahaja. Teknik ini boleh diguna secara langsung pada sampel

klinik seperti urin dan serum untuk diagnosis cepat leptospirosis. Teknik yang diguna

dalam kajian ini adalah berguna dalam pengesanan dan pengenalpastian leptospirosis

dalam pesakit. Teknik ini juga boleh diguna terhadap haiwan ternakan untuk

penyaringan dan pengesanan haiwan bukan terjangkit.

viii

ACKNOWLEDGEMENTS

I would like to give my special acknowledgement to my dedicated Supervisor

Committee comprising of the chairman, Professor Dr. Abdul Rani Bahaman, Professor

Dr. Mohd. Azmi Mohd. Lila, and Associate Professor Dr. Abdul Rahim Mutalib for

their helpful advice, guidance and constant encouragement given throughout the

planning and execution of this study. I am grateful to the staffs of Faculty of Veterinary

Medicine, UPM. I also acknowledge my deep appreciation to the following people who

have been of great help: Dr. Zuridah Hassan, Dr. Siti Khairani Bejo, Dr. Tan Do Yew,

Mr. Cheng Kim Sing, Ms Sushil Kuar, Ms Yap May Ling and friends. My special

thanks Ms Apinya Vanichpun, Ms Lee Yock Ann, Mr. Ng Kok Hong and his family,

Mr. Yeong Ming Tat, Ms Tongted Phumoona, and all thai students in UPM, who have

been a constant source of support and encouragement throughout my study. Finally, I

wish to express my deepest appreciation and thank to my parents, parents in law, uncle

Nakhonrat, all sisters: Jittrawadee, Thittaya, Sureerat, Katcharine, Wipawan and

Wichuda, brother Wichit, nieces and nephew. Special thanks my husband, Mr. Eakkasit

Somkuna, for his patient, supportive, and encouragement towards the pursuance of PhD.

study at Universiti Putra Malaysia, UPM.

This project was supported by the Intensification of Research in Priority Area (IRPA)

Program, Ministry of Science, Technology and Environment, Project No. 54169.

ix

I certify that an Examination Committee met on date of viva to conduct the final examination of Narumon Somkuna on his Doctor of Philosophy thesis entitled “Development of a Rapid Molecular-Based Diagnostic Kit for Diagnosis of Leptospirosis” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows: Dato Sheikh Omar Abd. Rahman, Ph.D. Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Chairman) Abdul Rahman Omar, Ph.D. Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) Siti Khairani Bejo, Ph.D. Lecturer Faculty of Veterinary Medicine Universiti Putra Malaysia (Member)

Victor Lim Kok Eow @ Azman Lim, Ph.D. Professor International Medical University Kuala Lumpur, Malaysia. (Independent Examiner) ___________________________________

GULAM RUSUL RAHMAT ALI, Ph.D. Professor/Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date:

x

This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirements for the degree of Doctor of Philosophy. The members of the Supervisory Committee are as follows: Abdul Rani Bahaman, Ph.D. Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Chairman) Mohd. Azmi Mohd. Lila, Ph.D. Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member)

Abdul Rahim Mutalib, Ph.D. Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) ___________________________________

AINI IDERIS, Ph.D. Professor/Dean School of Graduate Studies Universiti Putra Malaysia Date:

xi

DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations which have duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

___________________

NARUMON SOMKUNA

Date:

xii

TABLE OF CONTENTS

Page DEDICATION ii ABSTRACT iii ABSTRAK vi ACKNOWLEGEMENTS ix APPROVAL x DECLARATION xii LIST OF TABLES xviii LIST OF FIGURES xix LIST OF ABBREVIATIONS xxii CHAPTER I INTRODUCTION 1 II LITERATURE REVIEW 8 Leptospirosis 8 Biology of Leptospires 9 Taxonomy and Classification 11 Molecular Biology of Leptospires 12 Pathogenesis of Leptospirosis in Humans and Animals 14 Epidemiology of Leptospirosis 15 Clinical Leptospirosis in Humans and Animals 16 Leptospirosis in Malaysia 17 Treatment, Control and Prevention of Leptospirosis 19 Diagnosis of Leptospirosis 22 Clinical Laboratory Techniques in the Diagnosis of Leptospirosis 23 Dark Field Microscopy (DFM) 23 Microscopic Agglutination Test (MAT) 24 Enzyme-linked Immunosorbent Assay (ELISA) 25 Indirect Haemagglutination Assay (IHA) 26 Immunofluorescence 27 Histological Staining Techniques 27 Culturing Methods 28 Animal Inoculation 29 Monoclonal Antibodies for Diagnosis of Leptospirosis 30 Simple Assays for the Rapid Screening Test for

Leptospirosis 30 Molecular Techniques for the Detection and Identification of Leptospires 32 Molecular Typing 33

xiii

Ribotyping 33 Bacterial Restriction Endonuclease DNA Analysis (BRENDA) 34 Pulse Field gel Electrophoresis (PFGE) 36 Nucleic Acid Probes and DNA Hybridization Techniques 36 Polymerase Chain Reaction assay (PCR) 40

Other PCR-Based Techniques 43 PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) 43 Nested Polymerase Chain Reaction Assay (N-PCR) 44 Arbitrarily Primed-Polymerase Chain Reaction Assay (AP-PCR) 45 Low-Stringency Polymerase Chain Reaction Assay (LS-PCR) 46 Random Amplified Polymorphic DNA Analysis (RAPDs) 46 Detection of PCR Products 47 Detection of PCR Products by Gel Electrophoresis And Fluorescence Staining 47 Detection of PCR Products by PCR-Dot Blot Hybridization Assay (PCR-DBH) 48 Detection of PCR Products by Microplate Hybridization Assay (MHA) 49 Nucleic Acid Sequencing Techniques 50

III DETECTION AND DIFFERENTIATION OF LEPTOSPIRAL SEROGROUPS AND SEROVARS BY POLYMERASE CHAIN REACTION-RESTRICTION FRAGMENT LENGTH POLYMORPHISM (PCR-RFLP) AND LOW-STRINGENCY- POLYMERASE CHAIN REACTION (LS-PCR) 53 Introduction 53 Materials and Methods 56 Leptospiral strains 56 Clinical samples 56 Preparation and purification of leptospiral DNA 57 Determination of DNA concentration and purity of extracted

Leptospiral DNA 58 Specific polymerase chain reaction (PCR) assay 58 Detection of leptospiral 16S rRNA gene polymorphism by PCR-restriction fragment length polymorphism (16S rRNA gene PCR-RFLP) 59 Low-stringency polymerase chain reaction (LS-PCR) 60 Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis 61 Results 64

xiv

Detection and differentiation of leptospiral serogroups and Serovars by PCR-restriction fragment length polymorphism (PCR-RFLP) 64 Differentiation of leptospiral serovars by low-stringency

polymerase chain reaction (LS-PCR) 71 Specific PCR amplification 71 Low-stringency polymerase chain reaction (LS-PCR) 71

Discussion 75 IV DEVELOPMENT OF NONRADIOACTIVE LEPTOSPIRAL

DNA AND DOT BLOT HYBRIDIZATION (DBH) ASSAY FOR THE DETECTION OF LEPTOSPIRAL DNA

Introduction 81 Materials and Methods 86 Polymerase chain reaction (PCR) amplification 86 Labelling DNA with DIG-dUTP 86 Determination of the sensitivity of DIG-labelled DNA probes 87 Test of the sensitivity of the detection of genomic DNAs and amplified DNAs by agarose gel electrophoresis and dot blot hybridization assay 88 Test of specificity and efficacy of DIG-labelled leptospiral DNA probes by dot blot hybridization assay 88 Preparation of samples, dot blot hybridization and Immunological assay 89 Preparation of biological/clinical samples 90 Urine samples 90 Blood samples 90 Enzyme-linked immunosorbent assay (ELISA) 91 Dot blot hybridization assay 92 Nucleotide sequence analysis 93 Results 94 Sensitivity of DIG-labelled leptospiral DNA probes 94 Comparison of the sensitivity of the detection of DNA samples by agarose gel electrophoresis with dot blot hybridization assay 95 PCR amplification assay 94 Specificity of the PCR amplification assay 96 Test of the sensitivity and specificity of the DIG-labelled genomic DNA probe by DBH assay 96 Enzyme-linked immunosorbent assay (ELISA) 96 The efficacy of DIG-labelled DNA probes in the detection of Leptospiral DNA in clinical samples 97 Discussion 104

xv

V DEVELOPMENT OF POLYMERASE CHAIN REACTION- MICROPLATE HYBRIDIZATION ASSAY (PCR-MHA) AS A DIAGNOSTIC KIT FOR THE DETECTION OF

LEPTOSPIRAL DNA 110 Introduction 110 Materials and Methods 114 Preparation of samples and DNA extraction 114 Primers and specific polymerase chain reaction amplification 114 Optimization and standardization of microplate hybridization assay 114 The cut-off value 116 Sensitivity and specificity of the polymerase chain reaction and microplate hybridization assay 116 Detection of PCR products by agarose gel electrophoresis 117 Detection of PCR products by developed microplate hybridization assay 117 Reproducibility of PCR-MHA detection method 118 Extraction and purification of leptospiral DNA from clinical 118 Samples

A. Boiling method Urine samples 118 Serum samples 119 B. Extraction of leptospiral by Wizard genomic DNA

Purification kit 119 Serological tests for the clinical samples 119 DNA concentration and purify of extracted leptospiral DNA and amplified leptospiral DNA 120 Detection of leptospiral DNA in clinical samples by PCR-MHA 120 Analysis of sensitivity, specificity, positive predictive values (PPV), negative predictive values (NPV), and efficiency of the detection method 120 Results Optimization of PCR-MHA assay 122 Determination of sensitivity of PCR-MHA assay 123 Determination of specificity of PCR-MHA assay 123 Reproducibility of PCR-MHA assay 124 The cut-off value 124 Detection of leptospiral DNA from clinical samples by Agarose gel electrophoresis 126 Detection of leptospires in clinical samples by serological tests 126 Sensitivity, specificity, positive predictive values (PPV), negative predictive values (NPV), and efficiency of the detection method 126

xvi

Comparison of the results of detection by gel electrophoresis, PCR-DBH assay, and PCR-MHA detection method 127 Discussion 138

VI GENERAL DISCUSSION AND CONCLUSION 144 REFERENCES 152 APPENDICES A 176 B 178 C 181 D 183 E 184 BIODATA OF THE AUTHOR 185 PUBLICATIONS 186

xvii

LIST OF TABLES

Table Page

3.1

Reference leptospiral serovars used in this study

63

3.2 Leptospira spp. groups according to the PCR-RFLP patterns of the 285 bp DNA fragments

66

4.1

Other bacterial strains used in this study

94

5.1

Comparative evaluation of MAT at cutoff value ≥ 320 dilutions, gel electrophoresis, PCR-DBH assay, and PCR-MHA in the detection of 64 human urine samples

135

5.2 Comparative evaluation of DFM, gel electrophoresis, PCR-DBH assay, and PCR-MHA in the detection of 64 human urine samples

136

5.3 Comparison of the detection of leptospiral DNA by gel electrophoresis, PCR-BDH assay and PCR-microplate hybridization assay

137

xviii

LIST OF FIGURES

Figure Page

3.1 Ethidium bromide stained 2% agarose gel showing the specific PCR products amplified with primers G1/G2

67

3.2

Ethidium bromide stained 2% agarose gel of restriction endonuclease enzyme: DdeI

67

3.3

(a) and (b) Ethidium bromide stained 2% agarose gel electrophoresis of HhaI digested PCR products shows the PCR-RFLP profiles

68

3.4

Ethidium bromide stained 2% agarose gel electrophoresis of MspI digested PCR products shows the PCR-RFLP profile

69

3.5 (a)

Ethidium bromide stained 2% agarose gel electrophoresis of NdeII digested product shows the PCR-RFLP profiles

69

3.5 (b)

Ethidium bromide stained 2% agarose gel electrophoresis of NdeII digested product shows the PCR-RFLP profiles

70

3.6

Ethidium bromide stained 2% agarose gel electrophoresis of NdeII digested PCR products shows the PCR-RFLP profiles of reference strains and unknown samples

70

3.7

Ethidium bromide stained 2% agarose gel showing the specific PCR products amplified with primers G1/G2

72

3.8

Ethidium bromide stained 10% polyacrylamide gel showing the LS-PCR products derived from primers G1/G2

73

3.9

Ethidium bromide stained 10% polyacrylamide gel showing the LS-PCR products derived from primers G1/G2

74

xix

3.10 Ethidium bromide stained 10% polyacrylamide gel showing the LS-PCR products derived from primers G1/G2

74

4.1 Determination of the sensitivity of the DIG-labelled leptospiral DNA probes

98

4.2

(a) and (b) Sensitivity of the detection of leptospiral genomic DNA in 2% agarose gel electrophoresis compared with dot blot hybridization assay

99

4.3

(a) and (b) Sensitivity of polymerase chain reaction detection and dot blot hybridization assay

100

4.4

Ethidium bromide stained 2% agarose gel showing the specific PCR products amplified with primers G1/G2

101

4.5

Sensitivity and specificity of DIG-labelled genomic DNA probe in the detection of leptospiral DNA by dot blot hybridization assay

101

4.6

Specificity of the DIG-labelled leptospiral PCR-DNA probe in the detection of leptospires by dot blot hybridization assay

102

4.7

Ethidium bromide stained 2% agarose gel showing the specific PCR products from clinical samples amplified with primers G1/G2

102

4.8

Sensitivity of the DIG-labelled leptospiral PCR-DNA probe in the detection of leptospiral DNA by dot blot hybridization assay

103

5.1

The method of PCR-MHA detection

121

5.2

Determination of the optimum anti-DIG-AP in PCR-MHA

128

5.3

The cut-off value of PCR-MHA detection method

129

5.4 (a)

Sensitivity of PCR-MHA for the detection of amplified leptospiral DNA

130

5.4 (b)

Sensitivity of polymerase chain reaction detection

130

xx

5.5 Specificity of PCR-MHA detection 131

5.6 Efficacy of PCR-MHA detection when tested on the serum samples from cattle

132

5.7

Efficacy of PCR-MHA detection when tested on the human urine samples

133

5.8

PCR-microplate hybridization assay in the detection of human urine samples

134

xxi

LIST OF ABBREVIATIONS

A

Adenine or adenosine; one- letter code for alanine

Ab antibody Ag Antigen A260 Absorbance at 260 nm ABTS 2,2-Azino-bis (3-Ethylbenzthaizoline-6-Sulfonic Acid)

Diammonium Salt AFLPs Amplified fragment length polymorphisms AP Alkaline phosphatase bp Base pair BRENDA Bacterial restriction endonuclease deoxyribonucleic acid

analysis BSA Bovine serum albumin BCIP 5-bromo-4chloro-3-indolyl phosphate C Cytosine or cytidine; one-letter code for cysteine cDNA Complementary deoxyribonucleic acid cpm Counts per minute cm centimeter ˚C Degree Celsius dATP Deoxyadenosine triphosphate dCTP deoxycytosine triphosphate dGTP Deoxyguanosine triphosphate dNTP Deoxynucleotide triphosphate DNA Deoxyribonucleic acid DBH Dot blot hybridization DIG Digoxigenin EDTA Ethylenediaminetetraacetic acid ELISA Enzyme-linked immunosorbent assay EtBr Ethidium bromide EMJH Ellinghausen, McCullough, Johnson and Harris G guanine HCl Hydrochloric acid IgG Immunoglobulin G IgM Immunoglobulin M JS medium Johnson-Seiter medium kbp Kilobase pair LS-PCR Low-stringency polymerase chain reaction LSPs Low-stringency products M Molar MAT Microscopic Agglutination Test mM Milimolar ml Mililiter

xxii

xxiii

mmol/l Milimol per liter Mg2+ Magnesium ion MgCl Magnesium chloride MW Molecular weight mg/l Milligram per liter mol/l Mol per liter MHA Microplate hybridization NBT nitroblue tetrazolium salt ng nanogram OD Optical Density PAGE Polyacrylamide gel electrophoresis PBS Phosphate-buffer saline PCR Polymerase Chain Reaction pg picogram pH Puissance hydrogen (Hydrogen-ion concentration) RAPD Random amplified polymorphic DNA RFLP Restriction fragment length polymorphism REA Restriction endonuclease analysis RE Restriction enzyme RNA Ribonucleic acid RNase Ribonuclease rpm Round per minute SDS Sodium dodecyl sulfate TBS Tris buffer saline Tris-HCl Tris (hydroxymethyl) aminomethane hydrochloride TEMED N′ , N, N, N- tetra-methylethylenediamine UV ultraviolet v/v Volume per volume w/v Weight per volume µ micron µg microgram µl microlitre µ micron µl microliter SDW Sterile distilled water 5-FU 5- fluorouracil

CHAPTER I

INTRODUCTION

Leptospirosis is a worldwide zoonosis that affects animals, wildlife and humans. It is an

infectious disease caused by pathogenic spirochaetes of the genus Leptospira. It is a

disease of great concern for both humans and veterinary medicine because the causative

organisms can persist in wildlife and domestic animals such as cattle, swines, and dogs.

Moreover, pathogenic leptospires can change host specificity and virulence in response

to selective pressure from the environment (Christopher et al., 2000). Rats are the most

important source of human infection. An infected animal can remain asymptomatic and

continue to shed pathogenic leptospires in the urine for a long time (Plank and Dean,

2000). Infected animals with leptospires are also occupational hazards to farmers,

butchers, sewer workers, field agricultural workers, fishermen, pet shop owners,

slaughterhouse workers, housewives, soldiers and veterinarians (Heath and Johnson,

1994).

Leptospirosis causes abortion, stillbirth, infertility, decreased milk production, and

sometimes death in livestock (Thiermann, 1984). Infection in man is not common

except in tropical regions and occurs mostly through occupational or accidental contact

with infected animals or through the ingestion of water contaminated with their urine

(Heath and Johnson, 1994; Farr, 1995).