production and characterization of monoclonal antibody...

Ref. code: 25595412030115XYLRef. code: 25595412030115XYL

PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODY AGAINST CAMPYLOBACTER SPECIES

BY

MRS. NARISSARA MUNGKORNKAEW

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF MEDICAL TECHNOLOGY GRADUATE PROGRAM

FACULTY OF ALLIED HEALTH SCIENCES THAMMASAT UNIVERSITY

ACADEMIC YEAR 2016 COPYRIGHT OF THAMMASAT UNIVERSITY


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Thesis Title PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODY AGAINST CAMPYLOBACTER SPECIES Author Mrs. Narissara Mungkornkaew Degree Master of Medical Technology Department/Faculty/University Faculty of Allied Health Sciences Thammasat University Master Advisor Associate Professor Worada Samosornsuk, D.V.Sc. Master Co-Advisors Professor Wanpen Chaicumpa, D.V.M. (Hons.), Ph.D. Assistant Professor Anek Pootong, Ph.D. Academic Year 2016

ABSTRACT

Campylobacter species are an important cause of gastroenteritis in human. Immunological tools are needed for accurate and rapid identification of Campylobacter. We produced a monoclonal antibody (MAb) against Campylobacter by the fusion of P3X myeloma cells and spleen cells from BALB/c mice immunized with whole cell lysate of C. jejuni. The MAb, designated MAb2D10 and of the immunoglobulin G1 isotype, was produced at high titer. The specificity of MAb2D10 against the Campylobacter species was determined using indirect ELISA and dot blot ELISA. MAb2D10 reacted with C. jejuni, C. coli, C. lari, C. upsaliensis and C. helveticus but did not react with C. fetus, C. hyointestinalis and non-Campylobacter species. Western blotting showed that MAb2D10 bound to proteins migrating at molecular masses ranging from 19–72 kDa. MAb2D10 may be useful for the development of simple and rapid diagnostic tools for detection of human Campylobacter infection.

Keyword: Campylobacter; Monoclonal antibody; Gastroenteritis


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ACKNOWLEDGEMENTS

I would like to express my sincere appreciation and gratitude to my advisor, Associate Professor Dr. Worada Samosornsuk, for her supervision, encouragement and for allowing me to grow as a research scientist. Your advice on both research as well as on my career have been invaluable. I am deeply grateful to my Co-advisor Professor Dr. Wanpen Chaicumpa and Assistant Professor Dr. Anek Pootong who gave useful suggestion and helpful discussion. I am also deeply grateful to my committee members, Associate Professor Dr. Nitat Sookrung, Assistant Professor Dr. Pongsri Tongtawe, and Assistant Professor Dr. Sekson Samosornsuk for their constructive comments and suggestions in this study. I express special thanks to Assistant Professor Dr. Srinuan Somroop for teaching and helping me every step. Without her guidance and constant feedback this thesis would not have been achievable. I wish to thank laboratory members of Medical Technology Department, Faculty of Allied Health Sciences, Ms. Nattharee Thanawan, Mr. Sompoch Prachan, Ms. Benja Narapong, Ms. Mattika Phunhkrachuy for assisting in laboratory work. I would especially like to thank microbiology laboratory members of Thammasat University Hospital. All of you have been there to support me, work hard for me when I studied. Last but not least, I would like to express my deep gratitude to my family, especially my father who inspired me to intend the educations. I would also like to say a heartfelt to my family for always believing in me and encouraging me to follow my dreams.

Mrs. Narissara Mungkornkaew


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TABLE OF CONTENTS

Page ABSTRACT (1) ACKNOWLEDGEMENTS (2) LIST OF TABLE (8) LIST OF FIGURE (9) LIST OF ABBREVIATIONS (10) CHAPTER 1 INTRODUCTION 1

1.1 General introduction 1 1.2 Objective 3

CHAPTER 2 REVIEW OF LITERATURE 4 2.1 Campylobacter 4 2.1.1 History 4 2.1.2 Taxonomy 5 2.1.3 Morphology 8 2.1.4 Biochemical properties 10 2.2 Culturing of Campylobacter 11 2.3 Pathogenesis of Campylobacter 12 2.3.1 Motility 15 2.3.1.1 Flagella 15 2.3.1.2 Chemotaxis 15 2.3.2 Adhesion 18 2.3.3 Invasion 20 2.3.4 Toxin production 21 2.3.5 Carbohydrate structures 22 2.3.6 Iron uptake system 22 2.3.7 Multidrug and bile resistance 22


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2.3.8 Antimicrobial resistance 22 2.3.9 Stress response and survival 22 2.4 Epidemiology of Campylobacter 24 2.5 Antibiotic resistance and sensitivity 26 2.6 Identification and detection of Campylobacter 28 2.6.1 Isolation of Campylobacter 28 2.6.1.1 Selective media for isolation 28 2.6.1.2 Passive filtration 29 2.6.1.3 Incubation 29 2.6.2 Confirmation 29 2.6.2.1 Identification on solid medium 29 2.6.2.2 Microscopic examination of morphology 30 and motility 2.6.2.3 Detection of oxidase 30 2.6.2.4 Microaerobic growth at 25°C 30 2.6.2.5 Aerobic growth at 42°C 30 2.6.2.6 Latex agglutination tests 30 2.6.3 Identification of Campylobacter to the 30 species level 2.6.3.1 Detection of hippurate hydrolysis 30 2.6.3.2 Detection of indoxyl acetate hydrolysis 31 2.6.4 Molecular detection of Campylobacter 32 2.6.4.1 Detection and speciation of 32 thermophilic Campylobacter species by molecular techniques 2.6.4.2 Subtyping of Campylobacter spp. 32 2.6.4.3 Flagellin typing (fla typing) 33 2.6.4.4 Pulsed Field Gel Electrophoresis (PFGE) 33 2.6.4.5 Ribotyping 34 2.6.4.6 Random Amplyfied polymorphic DNA (RAPD) 34 2.6.4.7 Amplified Fragment Length 35 Polymorphism (AFLP) 2.6.5 Immunological methods for detection of 35 Campylobacter spp. 2.6.5.1 Identification of Campylobacter spp. 37 using immunoassays


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(1) Clinical stool samples 37 (2) Food samples 38

2.6.5.2 Increasing concentration of campylobacters 39 prior to immunoassay identification

(1) Enrichment of the samples 39 (2) Use of filter membranes to separate 39

contaminating bacteria 2.7 Antibody 40 2.8 Monoclonal antibody 44 2.8.1 History 44 2.8.2 Monoclonal antibody production 44 2.8.2.1 Immunization schedule 45 2.8.2.2 Myeloma cell line culture 47 2.8.2.3 Fusion 48 2.8.2.4 Growth and selection of 48 monoclonal antibodies 2.8.2.5 Long-term maintenance and 49 cryopreservation of MAbs 2.8.3 Monoclonal antibody characterization 49 2.8.3.1 Physicochemical characterization 49 2.8.3.2 Immunological properties 50 2.8.3.3 Biological activity 50 2.8.3.4 Purity, impurity and contaminants 50 2.8.3.5 Quality 51 2.8.4 Monoclonal antibodies application 51 2.8.4.1 Diagnostic application 51 (1) MAbs in biochemical analysis 51 (2) MAbs in diagnostic imaging 52 2.8.4.2 Therapeutic application 52 (1) MAbs as direct therapeutic agents 52 (2) MAbs as targeting agents in therapy 52 2.8.4.3 Protein purification 53 2.8.4.4 Miscellaneous applications 53 (1) Catalytic MAbs (ABZYMES) 53 (2) Autoantibody fingerprinting 54 2.9 Monoclonal antibodies against Campylobacter spp. 54


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CHAPTER 3 RESEARCH METHODOLOGY 56 3.1 Experimental animals and bacterial strains 56 3.1.1 Experimental animals 56 3.1.2 Bacterial strains 56 3.2 Antigens preparation 66 3.2.1 Bacteria culture and storage 66 3.2.2 Whole cell lysate preparation by 66 ultrasonication technique 3.2.2.1 Preparation of the cell suspension 66 3.2.2.2 Ultrasonic lysis 66 3.2.3 Dry weight measurement of whole cell lysate 67 3.2.3.1 Aluminum dishes preparation 67 3.2.3.2 Measurement 67 3.2.3.3 Calculation 67 3.3 Immunization 68 3.4 Hybridoma production 70 3.4.1 Splenectomy 70 3.4.2 Preparation of myeloma cells 70 3.4.3 Fusion 70 3.4.3.1 Feeder preparation 71 3.4.3.2 Hybridoma screening 72 3.5 Monoclonal antibody production 72 3.5.1 Selection of positive hybridomas 72 3.5.2 Limiting dilution 72 3.6 Storage and revival of hybridomas 73 3.7 Monoclonal antibody characterization 75 3.7.1 Isotyping immunoglobulin subclasses 75

3.7.2 Sodium Dodecyl Sulfate Polyacrylamide Gel 76 Electrophoresis (SDS-PAGE) 3.7.3 Western blot 78 3.7.4 Indirect ELISA 80 3.7.5 Dot blot ELISA 81


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CHAPTER 4 RESULTS AND DISCUSSION 83 4.1 Monoclonal antibody production 83 4.2 Monoclonal antibody characterization 85 4.2.1 Isotyping immunoglobulin subclasses 85 4.2.2 SDS-PAGE and western blot profile 85 4.2.3 Cross reactivity of MAb 89 4.3 Discussion 90 CHAPTER 5 CONCLUSIONS AND RECOMMENDATIONS 93 REFERENCE 94 APPENDIX 108 BIOGRAPHY 118


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LIST OF TABLES

Tables Page 2.1 A list of species, subspecies, and biovars within the 6 Campylobacter genus. 2.2 Campylobacter motility and chemotaxis factors. 17 2.3 Campylobacter adhesion factors. 18 2.4 Campylobacter invasion factors. 20 2.5 Other virulence factors in Campylobacter. 21 2.6 Multidrug and bile resistance and stress response 23 virulence factors in Campylobacter. 2.7 Confirmatory tests for thermophilic Campylobacter. 31 2.8 Basic phenotypic characteristics of selected thermophilic 31 Campylobacter species. 2.9 Characteristics of human immunoglobulin isotypes. 41 2.10 Monoclonal antibodies against Campylobacter spp. 55 3.1 Bacteria used in this study. 57 4.1 Indirect ELISA reactivity of hybridoma supernatant 83 against C. jejuni43. 4.2 Indirect ELISA reactivity of 2D10 reclone supernatant 84 against C. jejuni43 and E. coli. 4.3 Cross reaction of MAb 2D10 with whole cell homogenate 89 of bacteria by indirect ELISA and dot-blot ELISA.


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LIST OF FIGURES

Figures Page 2.1 Morphology of Campylobacter: A small gram-negative rod 9 with comma, S, or gull wings shapes. 2.2 Colonial morphology of Campylobacter. 9 2.3 Most important routes for human infection by C. jejuni. 13 2.4 Schematic representation of interaction of C. jejuni with 14 epithelial host cells. 2.5 Flagellar assembly showing the main components and 16 associated proteins. 2.6 Examples of C. jejuni adhesins and their host receptors. 19 2.7 Diagram of a prototypic immunoglobulin (Ig) G monomer. 42 2.8 Production of monoclonal antibody by 46 hybridoma technology. 3.1 BALB/c mouse. 56 3.2 Whole cell homogenate preparation by ultrasonication technique. 67 3.3 Dry weight measurement of whole cell homogenate. 68 3.4 Immunization schedules of BALB/c mice with 69 whole cell homogenate antigen. 3.5 Cell fusion procedure. 71 3.6 Limiting dilution of hybridoma cells. 73 3.7 Storage of hybridomas. 74 3.8 Immunoglobulin isotyping by rapid ELISA mouse mAb isotyping kit. 75 3.9 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. 77 3.10 Protein transfer to nitrocellulose membrane. 78 3.11 Western blot procedure. 79 3.12 Indirect ELISA procedure. 80 3.13 Dot blot ELISA procedure. 81 3.14 Monoclonal antibody production and characterization 82 In this study. 4.1 SDS-PAGE profile of whole cell homogenate of C. jejuni43. 86 4.2 SDS-PAGE and Western blot analysis of 87 whole cell homogenate of Campylobacter. 4.3 Western blot analysis of C. jejuni and C. coli 88 reacted with MAb2D10.


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LIST OF ABBREVIATIONS

Symbols/Abbreviations Terms α Alpha δ Delta γ Gamma ε Epsilon κ Kappa λ Lambda µ Mu % Percent µg Microgram(s) µl Microliter(s) µm Micrometer(s)

C Degrees Celsius / per $ U.S. dollar(s) AP Alkaline phosphatate bp Base pairs BCIP/NBT 5-bromo-4-chloro-3-indolyl-phosphate/ Nitro blue tetrazolium BSA Bovine serum albumin CDT Cytolethal distending toxin CFU Colony forming unit(s) cm2 Square centimeter DNA Deoxyribonucleic acid ECDC European Centre for Disease Prevention and Control EFSA European Food Safety Authority ETEC Enterotoxigenic Escherichia coli EU European Union FQ Fluoroquinolone


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Symbols/Abbreviations Terms g Gram HRP Horseradish peroxidase iu International unit(s) kb Kilobase kDa Kilodaltons l liter(s) LOS Lipooligosaccharide LPS Lipopolysaccharide M Molar(s) MAb(s) Monoclonal antibody(-ies) mg Milligram(s) ml Milliliter(s) MLST Multilocus sequence typing mm Millimeter(s) mM Millimolar(s) MS Members nm Nanometer(s) PBS Phosphate buffer saline PCR Polymerase chain reaction pg Picogram(s) PK Proteinase-K PNGase F Peptide-N-glycosidase F rpm Revolutions per minute RT Room temperature SDS Sodium dodecyl sulfate TEMED Tetramethylethylenediamine UDW Ultra deep water V Voltage v/v volume by volume w/v weight by volume


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Chapter 1

Introduction

Campylobacter spp. have emerged during the last three decades as significant clinical pathogens, particularly of human public health concern, as they concern a majority of bacterial enteritis. The bacteria are responsible for an estimate of 5-14% of the occurrence of human diarrhea worldwide, which translates into 400-500 million cases annually (1,2). In the United States, an estimated number of persons infected each year with Campylobacter is 1.70-2.45 million (1% of the population), 13,000 hospitalizations, and over 100 deaths (3). In the European Union (EU), more than 200,000 confirmed cases of human campylobacteriosis were reported by the 24 member states (MS), with an EU notification rate of 45.2 cases per 100,000 inhabitants (4). According to European Food Safety Authority (EFSA) 2010, clinical cases of campylobacteriosis is under reported in EU (27 Members): “There may be not less than 2 million and possibly as high as 20 million cases of clinical campylobacteriosis per year in the EU 27 MS” (5). In New Zealand, the incidence of campylobacteriosis was 161.5/100,000 population and the maximum rate was 353.8/100,000 population (6). In developing countries, Campylobacter isolation rates were ranging from 5-20%. Some developing countries according to WHO regions studied diarrhea have provided estimates of 40,000 to 60,000 /100,000 in children <5 years old (7). In Thailand, Campylobacter was identified in 9-15% of children <5 years old with diarrheal disease and was the most common bacterial dysentery (28%) in children up to 12 years of age whereas Salmonella, Shigella and enterotoxigenic Escherichia coli (ETEC) were isolated from 18%, 9% and 6% respectively (8). Pig, cattle, sheep, poultry, wildlife, and humans are all capable of harboring Campylobacter in their intestines and therefore shedding this pathogen in their feces. Transmission of Campylobacter to humans can occur through consumption of fecally contaminated water or food, contact with animals and person-to person contact (9). In Thailand, Campylobacter was isolated from 12% of various food samples including pork, chicken and vegetables and 50.6% from intestinal parts of chicken (10,11). Antimicrobial resistance in human Campylobacter isolates has become increasingly common in developing countries (12). In Thailand quinolone are widely used for bacterial infection in humans and animals. Resistance to the quinolone


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group in humans isolates in Thailand has been reported, and the prevalence of quinolone resistant Campylobacter spp. was found to increase from 0% in 1987 to 84% in 1995 (13). Campylobacteriosis has already been recognized as a public health problem in Thailand. Detection of Campylobacter infections is critical for both treatment and epidemiological surveillance. The conventional methods for detecting Campylobacter spp. involve enrichment and selective culture for initial isolation, followed by biochemical tests, which are uninteresting and may give ambiguous results. Numerous molecular techniques, such as multiplex PCR (14,15) realtime PCR (16), PCR-enzyme linked immunosorbent assay (17), PCR hybridization (18), DNA hybridization (19), and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (20) are able to identify Campylobacter spp., but they may be too expensive, time consuming and complicate for routine use. Simple, rapid, and economical methods, such as an immunoassay, are need for identification of Campylobacter on routine basis use in the microbiology laboratory. Monoclonal antibodies (MAbs) are a single type of antibody that are identical and directed against a specific epitope as they can recognize and bind specifically and strongly with respective antigens. MAbs are considered useful tool for development of rapid and simple methods for identifying bacteria without requiring specialized equipment and skills. In this study, the MAb to Campylobacter was produced. The MAb may be useful for developing simple and rapid diagnostic test for the identification of human Campylobacter infection.


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Objective

The objective of this study was to produce and characterize mouse monoclonal antibody against Campylobacter spp.


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Chapter 2

Review of Literature

Infectious diarrhea is a major public health concern worldwide. Gastroenteritis refers to syndromes of diarrhea or vomiting due to non-inflammatory infection in the upper small bowel or inflammatory infection in the colon. It may be caused by bacteria, viruses, or parasites. Bacteria are responsible for 20-40% of diarrheal episodes, contributing to high rates of childhood mortality in developing regions, and substantial morbidity and economic losses in developed regions. Many of the most common and life-threatening bacteria causing diarrheal diseases include Salmonella spp., Shigella spp., diarrheagenic Escherichia coli, Vibrio cholerae, Staphylococcus aureus, Bacillus spp., Clostridium spp. and Campylobacter spp. Campylobacter spp. are foodborne pathogens that cause diarrheal diseases and gastroenteritis, accounting for 400 million cases in adults and children worldwide each year (21). In the last 10 years, the prevalence of campylobacteriosis has increased, representing an extensive public health issue with high hospitalization rates. More invasive manifestations of the disease may occur in elderly or very young individuals and are responsible for important economic losses. 2.1 Campylobacter 2.1.1 History Campylobacter was first described in 1880 by Theodor Escherich (1). In 1886, Escherich described spiral bacteria in the colons of children who had died of what he called ‘cholera infantum’. In 1909, two veterinarians, McFadyean and Stockman described the association of a microorganism with epizootic abortion in ewes. Few years later, it was shown that the same Vibrio can be found in infectious abortions in sheep and pregnant cows (22,23), and the same microaerophillic spirillum bacterium was isolated by Smith in 1919 from aborted calf tissues (24). Due to its comma-shaped morphology, Smith and Taylor proposed the name “Vibrio fetus” and the disease was called vibrionic abortion. In 1927, Smith and Orcutt named a group of bacteria, isolated from the feces of cattle with diarrhea, as Vibrio jejuni. Seventeen years later, in 1944, Doyle isolated a different vibrio from feces of pigs with diarrhea and classified them as Vibrio coli (25,26). The three identified vibrio microorganisms, V. jejuni, V. coli and V. fetus, were named in


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association with specific disease in animals (27). In 1957, King was grouping the Vibrio bacteria and the clinical entities of bovine vibrionic abortions started to be better understood. In the same year, the first taxonomic differentiation of Vibrio started when he demonstrated that catalase positive microaerophilic Vibrios could be differentiated by their ability to grow at different temperatures (27). In 1963, Sebald and Véron classified these microaerophilic bacteria as a new genus called Campylobacter (28). Ten years later, the first comprehensive taxonomy of Campylobacter was published by Véron and Chatelain and they recognized the microorganisms Vibrio jejuni and Vibrio coli in their classification system of the genus, later these names were accepted by the International Committee of Systematic Bacteriology (29). However, Campylobacter were not generally recognized as fecal pathogens in human until the late 1970s. In 1974, in France the organism was isolated from a woman who had suffer from septic abortion, but an event that took place in Illinois in May 1983 is now regarded as the first well documented incident of the human Campylobacter infections.

2.1.2 Taxonomy The family Campylobacteraceae consists of two genera, Campylobacter and Arcobacter. At present, the genus Campylobacter contains 26 species two provisional species and eight subspecies (30). A full list of Campylobacter species is presented in Table 2.1. Campylobacter jejuni subsp. jejuni, C. jejuni subsp. doylei, Campylobacter coli, Campylobacter lari, Campylobacter upsaliensis and Campylobacter helveticus form a genetically close group of species which are the most commonly isolated from human and animal diarrheas. Campylobacter hyoilei, isolated from lesions of porcine proliferative enteritis, was later identifed by a wide range of phenotypic and genotypic methods as a strain of C. coli (31). The hydrogen-requiring species Campylobacter concisus, Campylobacter showae, Campylobacter curvus, Campylobacter rectus, Campylobacter gracilis, Campylobacter sputorum and Campylobacter hominis appear to be closely related phylogenetically. Most occur in the human oral cavity, although Campylobacter hominis has been found only in the human lower intestine and C. sputorum is also found in the enteric and reproductive tracts of various production animals. Campylobacter mucosalis is, by DNA±DNA hybridization studies, most similar to C. sputorum (32), with which it shares a highly similar phenotype and a common source (pig intestine). Campylobacter fetus subsp. fetus, C. fetus subsp. venerealis, Campylobacter hyointestinalis subsp. hyointestinalis and C. hyointestinalis subsp. lawsonii are also similar by phenotype and genotype. Campylobacter fetus is mainly


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found in bovine reproductive disorders, whereas C. hyointestinalis subsp. hyointestinalis is mainly enteric in origin and C. hyointestinalis subsp. lawsonii occurs in the pig stomach. C. hyointestinalis shows remarkable diversity at the 16S rRNA genetic level (33), a finding with implications for Campylobacter lanienae, from abattoir workers (34). Table 2.1 A list of species, subspecies, and biovars within the Campylobacter genus(30). No. Campylobacter species 1 Campylobacter avium 2 Campylobacter canadenis 3 Campylobacter coli (including the proposed infrasubspecific designation

‘hyoilei’ 4 Campylobacter concisus 5 Campylobacter cuniculorum 6 Campylobacter curvus 7 Campylobacter fetus subsp. fetus 8 Campylobacter fetus subsp. venerealis 9 Campylobacter fetus subsp. venerealis biovar intermidius 10 Campylobacter gracilis 11 Campylobacter homonis 12 Campylobacter helviticus 13 Campylobacter hyointestinalis subsp. hyointestinalis 14 Campylobacter hyointestinalis subsp. lawsonii 15 Campylobacter insulaenigrae 16 Campylobacter jejuni subsp. doylei 17 Campylobacter jejuni subsp. jejuni 18 Campylobacter lanienae 19 Campylobacter lari subsp. concheus 20 Campylobacter lari subsp. lari 21 Campylobacter mucosalis 22 Campylobacter peloridis 23 Campylobacter rectus 24 Campylobacter showae


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Table 2.1 A list of species, subspecies, and biovars within the Campylobacter genus(30) (Cont.). No. Campylobacter species 25 Campylobacter sputorum biovar faecalis 26 Campylobacter sputorum biovar paraureolyticus 27 Campylobacter sputorum biovar sputorum 28 Campylobacter subantracticus 29 Campylobacter troglodytis 30 Campylobacter upsaliensis 31 Campylobacter ureolyticus 32 Campylobacter volucris 33 Campylobacter sp. Dolphin DP Provisional 34 Campylobacter sp. Prairie Dog Provisional

REFERENCES

30. Man SM. The clinical importance of emerging of Campylobacter species. Nat Rev Gastroenterol Hepatol. 2011;8:669-85.


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2.1.3 Morphology The name Campylobacter is derived from the Greek word

“Kampylos,” which means curved. Campylobacter spp. is non-spore forming and gram negative bacteria. They can be spiral, curved or occasionally straight rods, with size ranging from 0.2 to 0.8 µm wide and 0.5 to 5 µm long. Campylobacter may appear as spiral, S-, comma-shaped forms and may also be found in short or occasionally long chains (Figure 2.1). As Campylobacter cells begin to age, they become coccoid in shape (35). The cells are highly motile by means of single or occasionally multiple flagella at one or both ends (36). Extremely rapid, darting motility of comma-shaped cells can be seen with a phase contrast microscope. According to On et al. (37), the number of flagella should not be considered as an important taxonomic criterion for the Campylobacter genus because of high variation in the flagella arrangement of certain species or strains. Both mono- and bi- flagellated cells of the same general shape and size within the same culture of strain have been observed. It has been speculated that genetic changes as a result of spontaneous mutation and other mechanism such as natural or plasmid–borne transformation may cause considerable phylogenetic diversity which is observed within the genus. These microaerophilic organisms grow best in an atmosphere containing 5 to 10% oxygen and an optimum temperature for their growth ranges from 30 to 42C. Colony morphology should not be used as an important distinguishing factor because several factors including bacterial strain, basal medium, level of moisture on the surface of the agar, incubation temperature and incubation time may affect colony morphology of this organism. Colonical morphology is quite variable, from a thick translucent white growth to spreading film-like transparent growth (Figure 2.2) which can be visible on the plating media within 24 to 48 hours of incubation (38). It is difficult to isolate this organism from fecal specimens without using selective techniques because campylobacters tend to multiply slower than other enteric bacteria (38).


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Figure 2.1 Morphology of Campylobacter: A small gram-negative rod with comma, S, or gull wings shapes.

Figure 2.2 Colonial morphology of Campylobacter.


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2.1.4 Biochemical properties Campylobacter do not ferment carbohydrates and usually obtain

energy from amino acids or tricarboxylic acid cycle intermediates. Typical biochemical reactions include the reduction of fumerate to succinate, negative methyl red, acetoin, and indole production. Most species reduce nitrate and are oxidase positive but only C. jejuni is hippurate positive. C. jejuni is quite sensitive to drying and storage at room temperature, but at refrigeration temperatures and appropriate humidity, large number of bacteria may survive. Campylobacter can be distinguished from Arcobacter due to its key features which include; Arcobacter grows at 15C but not at 42C, its optimal temperature for aerobic growth is 30C, and its G+C content of the DNA ranges from 27 to 30 mol% (36), while in Campylobacter it ranges from 28 to 46 mol%.

Genus Campylobacter is divided initially into two groups based on the presence or absence of catalase. The catalase-negative species are generally considered to be saphrophytes. C. fecalis and C. sputorum are catalase-negative species. C. fecalis is shown by DNA hybridization to be a subspecies of C. sputorum. DNA hybridization studies demonstrated they belonged to a new species "C. upsaliensis" (39,40). All "C. upsaliensis" strains were hippurate negative, and six of seven were susceptible to cephalothin. This new species could be a potential human pathogen associated with gastroenteritis and bacteremia. The catalase-positive species include C. jejuni, C. coli, and C. fetus subspecies fetus and venerealis, C. laridis and C. hyointestinalis. C. hyointestinalis has been isolated from pigs with proliferative ileitis, but its role in human disease has not been established. C. fetus subspecies fetus is only rarely isolated from stools, but is more commonly reported to be a blood and tissue pathogen. C. jejuni and C. coli are the most common, human intestinal pathogens, although other catalase-positive species are occasionally responsible for cases of human gastroenteritis (41). Generally, the human intestinal pathogens (C. jejuni, C. coli) can be distinguished by their thermophilic growth properties (i.e. growth at 42C). C. jejuni and C. coli can be defferentiated by the hippurate hydrolysis, hydrogen sulfide production in triple sugar iron agar slants (TSI), and the growth in minimal medium. C. jejuni are hippurate-positive except a few strains, and the test is 80-90% reliable, while C. coli are hippurate-negative. None of the C. jejuni isolates, but all C. coli isolates produced hydrogen sulfide on TSI slants.


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2.2 Culturing of Campylobacter There is lack of consensus on the issue of the standard culturing medium for the growth of Campylobacter in the laboratory. Special requirements for growth temperature, gaseous environment and nutrient-rich basal medium are major obstacles to develop an optimum medium for this fastidious organism. An ideal Campylobacter medium should provide excellent recovery and substantial selectivity against background flora with an easy and quick differentiation of bacterial species. It should be cost effective, easy to prepare and it must have good shelf-life with a possibility to minimize the risk of contamination of the medium, especially, when adding supplements following heat-steam sterilization.

A wide variety of media have been used and modified for isolation of Campylobacter by different researchers and laboratories. Skirrow formulation, Butzler's Agar, Campylobacter Blaser Agar, Preston, Semisolid Blood-free Selective Medium (SSM), Campylobacter Thioglycollate medium, Campy-Brucella Agar Plate (Campy-BAP), Campylobacter Cefoperazone Desoxycholate Agar (CCDA), Abeyta-Hunt-Bark Agar (Campy-FDA), Brucella Broth with 0.16% Agar, Semi-Solid Campylobacter Medium, Skirrow formulation, Butzler's Agar, Preston, Semisolid Blood-free Selective Medium (SSM), and Campy-Cefex (42-48). The above mentioned media differ in several aspects, for examples, the amount of basal medium, concentration of antibiotics, presence or absence of growth enhancers such as horse or sheep blood and presence of special components like charcoal. All these nutrient-rich media support growth of a large number of fastidious organisms without much selectivity towards Campylobacter spp. In one study, five different selective media Skirrow's, Butzler's, Blaser's, Campy-BAP and Preston's were compared for the isolation of Campylobacter (49). It was reported that Preston medium preceded by enrichment on modified Preston Enrichment Broth was the most effective selective medium for Campylobacter while Butzler’s agar was the least effective.

Thermophilic Campylobacter species are able to grow between 37 and 42C, but incapable of growth below 30C (absence of cold shock protein genes which play a role in low-temperature adaptation), with an optimum temperature of 41.5C. However, a study revealed that C. jejuni survived for more than 4 hours at 27C and 60–62 % relative humidity on some common clean or soiled food contact surfaces. These characteristics reduce the ability of Campylobacter to multiply (i) outside of an animal host and (ii) in food during their processing and storage. Freezing–thawing also reduces the population of Campylobacter spp. In previous


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studies, the researcher (50,51) revealed that aged C. jejuni cells survived the longest at 4C. Campylobacter spp. do not survive below a pH of 4.9 and above pH 9.0 and grow optimally at pH 6.5–7.5. These non-spore-forming and fastidious bacteria are essentially microaerophilic, growing best in an atmosphere with low oxygen tension (5% O2, 10% CO2, and 85% N2) (52).

2.3 Pathogenesis of Campylobacter Campylobacter are found ubiquitously in the environment, wild birds, and mammals. C. jejuni can colonize the intestine of chickens and-less frequently-turkeys and ducks. In poultry, microaerophilic conditions and temperatures around 41C allow continuous replication of C. jejuni. For human infection, consumption of contaminated chicken meat products and cross-contamination from this source as well as contact with cattle including consumption of beef and milk seems to be responsible for more than 90% of all sporadic human cases. To a much lesser extent, other sources for human infection include sheep, contact with wild birds, contaminated water, and pet animals (Figure 2.3) (53). Contaminated surface water has recently suggested water activities as a potential source for human infections. This and consumption of raw milk seem to contribute significantly to outbreak of campylobacteriosis. Although direct infection from man to man might occur; it is of no larger epidemiological relevance (1). Campylobacter pathogenesis in humans is not completely understood. The symptoms caused by the pathogen indicate the existence of inflammation of the gastrointestinal tract. Human volunteer studies using clinical isolates confirmed that C. jejuni causes dysenteric symptoms with high numbers of leukocytes in the feces (54). Pathology on tissues of patients that have died from C. jejuni enteritis showed hemorrhagic inflammation and congestion of the jejunum and first half of the ileum. Biopsies from the colon indicated diffuse neutrophilic infiltration of the lamina propria with superficial crypt abscesses and histo-pathological features similar to Salmonella- and Shigella induced colitis (55). Additionally, C. jejuni has been demonstrated to be located inside colonic mucosal cells (56). This indicates that Campylobacter is able to invade human epithelial cells in vivo (Figure 2.4) (57). Less is known about C. coli infection in humans as it is less frequently observed, but as the symptoms are similar and most C. jejuni virulence factors can be found in C. coli, it is highly likely to invade the epithelial cells in a similar manner as C. jejuni. In a small number of cases, Campylobacter infection is followed by Guillain-Barre


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Syndrome (GBS). This post- infectious autoimmune-mediated neuropathy is most often associated with C. jejuni infection (58) and is thought to be caused by molecular mimicry of the LPS of the pathogen and host gangliosides (59). Clinical symptoms of GBS include weakness, sensory loss and even respiratory distress in the more severe cases. GBS is lethal in approximately 10% of the cases. Figure 2.3 Most important routes for human infection by C. jejuni(53).

REFERENCE

53. Wilson DJ, Gabriel E, Leather AJH, Cheesbrough J, Gee S, Bolton E, et al. Tracing the sources of campylobacteriosis. PLoS Genet. 2008;4:e1000203.


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Figure 2.4 Schematic representation of interaction of C. jejuni with host epithelial cells. C. jejuni can translocate the epithelial cells layer either by a paracellular route i.e., after disruption of tight junctions the bacterium travels in between cells to the basolateral side or by a transcellular route, i.e., after adhesion, the bacterium travels in a vacuole through epithelial cell or M-cell and by exocytosis ends up on the basolateral cell side. Subsequent basolateral invasion might occur(57).

REFERENCE

57. van Alphen L. Virulence strategies of Campylobacter jejuni (dissertation). (Utrecht): Utrecht University; 2007.


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The primary colonization site in poultry is the ceca, where the Campylobacter population may reach 106-108 CFU/g (60). In human, infection occurs predominantly in the small intestine. When colonizing the intestines, enteric campylobacters are predicted to express several putative virulence factors. 2.3.1 Motility The motility system in Campylobacter requires flagella and a chemosensory system that drives flagella movement based on the environmental conditions. 2.3.1.1 Flagella Motility is essential for survival under the different chemotactic conditions encountered in the gastrointestinal tract and for colonization of the small intestine. Campylobacter spp. show unusual motility, especially in viscous substances. This has been attributed to the presence of one or two polar flagella and the helical cell shape. The former provides propulsive torque and/or rotary cell movement, while the helical shape facilitates corkscrew rotation (61). The flagellum is composed of a hook basal body and the extracellular filament structural components. The hookebasal body includes; (1) a base embedded in the cytoplasm and inner membrane of the cell; (2) the periplasmic rod and associated ring structures and (3) the surface localized hook. The hookebasal body complex is composed of several different proteins (Figure 2.5). The Campylobacter motility factors are summarized in Table 2.2 (62). 2.3.1.2 Chemotaxis Chemotaxis is a mechanism by which motile bacteria sense and move towards more favourable conditions and many pathogenic bacteria depend on this process to invade their hosts. It is generally accepted that C. jejuni colonises the avian gut as a commensal, specifically the mucus filled crypts of the ceca and uses chemotaxis to locate these primary colonisation sites (63). During colonisation, the primary chemoattractants are the mucins and glycoproteins, that are the primary constituent of mucus (64). This process is very efficient at delivering a colonizing population in the cecum within 24 hours after ingestion (65,66). Campylobacter chemotaxis factors are summarized in Table 2.2 (62).


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Figure 2.5 Flagellar assembly showing the main components and associated proteins(62).

REFERENCE

62. Bolton DJ. Campylobacter virulence and survival factors. Food Microbiol. 2015;48:99-108.


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Table 2.2 Campylobacter motility and chemotactic factors(62).

Motility virulence factors Chemotactics virulence factors FlaA, the major flagellin protein Chemotaxis proteins; Che A, B, R, V, W, & Z FlaB, the major flagellin protein Methyl-accepting chemotaxis proteins

(MCPs) also called transducer-like proteins FliF, hookebasal body protein CheY, response regulator controlling

flagellar rotation FliM & FliY, flagellar motor proteins Campylobacter energy taxis system

proteins CetA (Tlp9) and CetB (Aer2) FlgI, P-ring in the peptidoglycan AI-2 biosynthesis enzyme FlgH, L ring in the outer membrane AfcB, MCP protein required for persistence

in the cecum FlgE & FliK, minor hook components s28 promoter regulates flaA gene expression

s54 promoter regulates flaB gene expression

Proteins involved in flagellin O-linked glycosylation

REFERENCE



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2.3.2 Adhesion Adherence of Campylobacter to host gastrointestinal epithelial cells is a prerequisite for colonisation which is mediated by several adhesins on the bacterial surface (67). The adhesion factors of Campylobacter are shown in Table 2.3 (62). Several adhesins in C. jejuni have been shown to mediate attachment to host cells. Of these, three, JlpA, CadF and FlpA, have known host cell receptors, whereas for a further nine C. jejuni proteins implicated in mediating adhesion to host cell surfaces; the host receptors remain unknown (Figure 2.6) (68). Table 2.3 Campylobacter adhesion factors(62). CadF, outer membrane protein CapA, Campylobacter adhesion protein A Phospholipase A Lipoprotein Peb1, periplasmic binding protein Peb3, transport protein Peb4, chaperone playing an important role in exporting proteins to the outer memberane FlpA, fibronectin-like protein A Type IV secretion system possibly involved in adhesion JlpA, 42-kDa lipoprotein involved in adhesion to Hep-2 cells

REFERENCE



19

Figure 2.6 Examples of C. jejuni adhesins and their host receptors. The jejuni lipoprotein A (JlpA) of C. jejuni has been shown to bind to the cell surface heat shock protein (Hsp) 90α of HEp-2 cells. Fibronectin-like protein A (FlpA) and Campylobacter adhesion to fibronectin (CadF) of C. jejuni bind to host fibronectin. The flagella of C. jejuni and other Campylobacter species, including C. concisus and C. showae, mediate attachment to host cell surface via an unknown receptor. An array of putative adhesins also have unknown receptors(68).

REFERENCE 68. Rubinchik S, Seddon A, Karlyshev AV. Molecular mechanisms and biological role of Campylobacter jejuni attachment to host cells. Eur J Microbiol Immunol. 2012;2:32-40.


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2.3.3 Invasion It is believed that flagella have a second function, in addition to motility, which is to serve as an export apparatus (type III Secretion System; T3SS) in the secretion of non-flagellar proteins during host invasion (69). The Campylobacter invasion factors are shown in Table 2.4 (62). Table 2.4 Campylobacter invasion factors(62). FlhA, FlhB, FliO, FliP, FliQ & FliR, components of the flagellar T3SS FlaC protein secreted into the host cells and essential for colonization and invasion CiaB, 73-kDa protein involved in adhesion CiaC, protein required for full invasion of INT-407 cells CiaI, reported role in intracellular survival IamA, invasion associated protein HtrA, chaperone involved in the proper folding of adhesins VirK, may have a role in protection against antimicrobial proteins FspA, protein with a role in apoptosis

REFERENCE



21

2.3.4 Toxin production Campylobacter produce several different cytotoxins, of which only the cytolethal distending toxin (CDT) has been studied in detail (70). Many Gram negative bacteria have the ability to produce CDT, a tripartite toxin composed of three subunits encoded by the cdtA, cdtB and cdtC genes (Table 2.5). All three gene products are required for the toxin to be functionally active (71). Lipo-oligosaccharides (LOS) are responsible for triggering the immune response that underpins the Guillain-Barre and MillereFisher Syndrome neuropathies following C. jejuni infection. Table 2.5 Other virulence factors in Campylobacter(62). Toxin Cytolethal distending toxin (CDT) subunits 1,3 galactosyltransferases involved in lipopolysaccharide production Capsule Capsular polysaccharide transport gene M Capsule biosynthesis gene N-linked glycosylation system N-linked glycosylation Iron uptake system Outer membrane ferric enterobactin (FeEnt) receptors CeuE, lipoprotein involved in iron acquisition Cj0178 putative transferring bound iron utilization outer membrane recpetor Ferric uptake regulator Outer membrane receptor for hemin and hemoglobin

REFERENCE



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2.3.5 Carbohydrate structures Carbohydrate structures such as lipooligosaccharides (LOS), a capsule and O- and N-linked glycans on the surface of the Campylobacter cell that facilitate colonization are shown Table 2.5. The LOS molecule, composed of a core oligosaccharide and lipid A, serves several functions including immune evasion, host cell adhesion and invasion. Sialylation of the LOS increases invasive potential and reduces immunogenicity (72,73). There are different classes of sialylated LOS. Class C is associated with high invasive potential and is common to both human and poultry strains belonging to multilocus sequence typing (MLST) clonal complex CC-21. The surface of C. jejuni cells are surrounded by a polysaccharide capsule that facilitates survival, adherence and evasion of the host immune system (74,75). 2.3.6 Iron uptake system Iron uptake system virulence factors are shown in Table 2.5 (62). 2.3.7 Multidrug and bile resistance The Campylobacter multidrug resistance, bile resistance virulence factor are summarised in Table 2.6 (62). Resistance to bile salts, heavy metals and a broad range of other antimicrobial agents is often mediated by the Campylobacter multidrug efflux pump (CME). 2.3.8 Antimicrobial resistance Campylobacter may encounter specific antibiotics during commensal carriage in broilers (or other food animals) or during infection in humans. There is strong evidence linking the indiscriminate usage of antibiotics in animal production to the emergence and spread of antibiotic resistance in Campylobacter spp. Most humans suffering campylobacteriosis recover without therapeutic intervention other than fluid and electrolyte replacement. More severe cases are usually treated with macrolide, tetracycline or (fluoro)quinolone antibiotics. However, the efficacy of such treatments is currently compromised by the increasing resistance to these antibiotics in C. jejuni and C. coli (76,77). 2.3.9 Stress response and survival As Campylobacter are primarily foodborne, the stresses encountered along the food chain are important in survival, transmission and infection in humans. The Campylobacter stress response virulence factors are shown in Table 2.6 (62).


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Table 2.6 Multidrug and bile resistance and stress response virulence factors in Campylobacter(62). Multidrug and bile resistance CME efflux pumps consisting of a periplasmic protein (CmeA), inner membrane efflux transporter (CmeB) and an outer membrane protein (CmeC) CmeR, CME efflux pump transcriptional repressor Stress response Stringent control Kat A, Catalase (converts hydrogen peroxide to water and oxygen) AhpC, alkyl hydroperoxide reductase Tpx, thiol peroxidase Cytochrome c peroxidases SOD proteins, antioxidant proteins Cj545c, an NADPH quinine reductase Other proteins that protect against reactive oxygen species Heat shock protein

REFERENCE



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2.4 Epidemiology of Campylobacter

In developed countries, Campylobacter is one of the most frequently reported causes of acute bacterial gastroenteritis. Significant variations in incidence rates have been observed between different countries. Several factors, including differences in infection rates in food animals, food production system, or different patterns of food consumption can be responsible for these variations. These differences in incidence rates can also occur because of differences in diagnosis, reporting systems, or case definitions used in each country’s surveillance systems

(78). In the United States, it is estimated that 2.5 million cases of campylobacteriosis occur annually. Although accounting for only 5% of estimated food-related deaths, campylobacters are responsible for approximately 17% of hospitalizations resulting from foodborne infections (79). Annual economic cost of Camyplobacter-associated illnesses is estimated up to $8 billion in the United States alone (80). In a recent study in the United Kingdom, it was estimated that the total number of cases of C. jejuni was 450,000. This figure agrees closely with other community-based studies in both, the United Kingdom and United States that estimated a population-based incidence of approximately 1% (81,82).

A report from the U.S. Centers for Disease Control and Prevention estimated that each year Campylobacter infection causes 124 deaths in the United States (80). In 2009, the number of reported infections and incidence per 100,000 population by Campylobacter was 6,033 and 13.02, respectively. However, many more cases remain undiagnosed or unreported. Estimates are that Campylobacter causes more than two million illnesses (or 1% of the population), 13,000 hospitalizations, and over 100 deaths each year. In an epidemiological survey conducted in the European Union, it was reported that fifteen out of 18 EU countries reported 134,971 Campylobacter infections in 1999 alone (83). During the period from 1995 to 1999, 11 countries reported 154 outbreaks and the highest number of the reported outbreaks was in 1997. The reports of outbreaks varied greatly by country. However, these numbers reflect a rough estimate of the true situation. Forty-eight percent of the outbreaks were linked with food, which served as a vehicle for transmission. Consumption of unpasteurized milk was responsible for 15% and another 15% were water-borne. During the year 2005, the number of reported cases of Campylobacter in Germany was highest than ever before, surpassing the number of 60,000, which made Campylobacter a number one bacterial pathogen responsible for food poisoning in Germany. European Food Safety Authority (EFSA) and European


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Centre for Disease Prevention and Control (ECDC) reported in the last 5 years, campylobacteriosis has been the most commonly reported zoonosis in the EU followed by salmonellosis and yersiniosis (84,85). According to EFSA (86), clinical cases of campylobacteriosis is under-reported in the EU (27 MS): “There may be not less than 2 million and possibly as high as 20 million cases of clinical campylobacteriosis per year in the EU 27 MS.” From 2010 until 2015, the UK Government increased the priority of “Innovation strategy for Campylobacter.” In the UK, Campylobacter is actually considered the most common cause of food poisoning, responsible for 321,000 estimated cases in England and Wales in 2008, with more than 15,000 hospitalizations and 76 deaths.

In developing countries, Campylobacter is the most commonly isolated bacterial pathogen from <2-year-old children with diarrhea. The disease does not appear to be important in adults. In contrast, infection occurs in adults and children in developed countries. Poor hygiene and sanitation and the close proximity to animals in developing countries all contribute to easy and frequent acquisition of any enteric pathogen, including Campylobacter. Generally, developing countries do not have national surveillance programs for campylobacteriosis; therefore, incidence values in terms of number of cases for a population do not exist. Most estimates of incidence in developing countries are from laboratory-based surveillance of pathogens responsible for diarrhea. Campylobacter isolation rates in developing countries range from 5 to 20% (86). Despite the lack of incidence data from national surveys, case-control community-based studies have provided estimates of 40,000 to 60,000/100,000 for children <5 years of age (87,88). Campylobacter is isolated relatively frequently with another enteric pathogen in patients with diarrhea in developing countries. In some cases half or more patients with Campylobacter enteritis also had other enteric pathogens (89,90). Organisms reported include Escherichia coli, Salmonella, Shigella, Giardia lamblia, and Rotavirus. C. jejuni and C. coli are the two main species isolated in developing countries. The isolation rate of C. jejuni exceeds that of C. coli, similar to observations in most developed countries (82,91).

In Thailand, the incidence of Campylobacter enteritis is as high as 40,000 per 100,000 for children below 5 years of age (92). Campylobacter species were isolated from 93 (15%) of 632 Thai children with diarrhea; 10%, 2%, and 3% of Campylobacter isolates were C. jejuni, C. coli, and atypical Campylobacter, respectively. Therefore, C. jejuni is the predominant enteric Campylobacter species (92). C. jejuni occurs more frequently than do infections caused by Salmonella


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species, Shigella species, or Escherichia coli O157:H7 and most of C. jejuni infections are acquired by the consumption and handling of poultry (93).

2.5 Antibiotic resistance and sensitivity

Most Campylobacter infections cause acute, self-limiting diarrheal disease; however, severe, prolonged, or relapsing campylobacteriosis does occur, especially in the very young, the elderly, and people with underlying diseases. When antimicrobial therapy is indicated, macrolides (primarily erythromycin, or alternatively one of the newer macrolides, such as clarithromycin or azithromycin) remain the frontline agents for treating culture-confirmed Campylobacter cases (94). Fluoroquinolones (e.g., ciprofloxacin) are also commonly used because they are the drugs of choice for empirical treatment of undiagnosed diarrheal illness, such as travelers' diarrhea (95,96). A meta-analysis indicated that early treatment with macrolides or fluoroquinolones shortened the duration of Campylobacter intestinal symptoms by 1.32 days (97). Tetracycline, doxycycline, and chloramphenicol are alternative drugs that can be used for treatment (98). Serious systemic infections should be treated with an aminoglycoside such as gentamicin or a carbapenem such as imipenem (98,99). Third-generation cephalosporins are used widely as alternatives to fluoroquinolones for empirical treatment of community- acquired bacterial diarrhea, but they have not been proven effective for treating bacteremia due to Campylobacter species other than Campylobacter fetus (100). Antimicrobial susceptibility testing continues to play a critical role in guiding therapy and epidemiological monitoring of resistance. The emergence of antimicrobial resistance in Campylobacter, particularly to fluoroquinolones, has underscored the importance of in vitro antimicrobial susceptibility testing.

Campylobacter jejuni and C. coli are susceptible to nalidixic acid, ciprofloxacin, norfloxacin, and ofloxacin (101). Furazolidone is another drug that has been shown to be effective against Campylobacter species. All C. jejuni and C. coli isolates are intrinsically resistant to a number of antibiotics, including bacitracin, novobiocin, rifampin, streptogramin B, trimethoprim, vancomycin, and usually cephalothin. According to Taylor and Courvalin (101), the Campylobacter genus, has apparently been able to acquire resistance determinants from both gram–positive and gram–negative organisms, although the former seem to be the more common source. Campylobacter spp. and Enterococcus spp. occupy a common niche (the human and animal gastrointestinal tracts) and DNA exchange between these two


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species within this environment is very likely to occur. Campylobacter spp. might acquire resistance against tetracycline, minocycline, kanamycin, chloramphenicol, streptomycin, spectinomycin, erythromycin, ampicillin and nalidixic acid. Erythromycin resistance is accompanied by cross–resistance to spiramycin, tylosin, and clindamycin (101). A rapid increase in the proportion of Campylobacter strains resistant to antimicrobial agents, particularly to fluoroquinolone (FQ), has been reported in many countries worldwide. Prior to 1992, FQ resistant Campylobacter was rarely observed in the USA and Canada, but several recent reports have indicated that approximately 19–47% of Campylobacter strains isolated from humans were resistant to ciprofloxacin (102,103). A steady increase in FQ resistance among Campylobacter isolates has also been observed in many European countries and 17–99% of Campylobacter strains isolated from humans and animals in this region were resistant to FQs, with the highest resistance levels reported in Spain (104). FQ-resistant Campylobacter has also become prevalent in Africa and Asia. In both continents, FQ resistance among clinical Campylobacter isolates was not detected before 1991. However, since 1993 the frequency of FQ-resistant Campylobacter strains has increased remarkably and the FQ-resistance rates have reached more than 80% in Thailand and Hong Kong (105,106). Although FQ resistance in Campylobacter isolates was also observed in Australia and New Zealand, the rate of FQ resistant Campylobacter isolates in this region is significantly lower than that in other regions (107,108). A trend for increased macrolide resistance in Campylobacter has been observed in some countries. Generally, the prevalence of erythromycin resistance among Campylobacter strains (including both C. jejuni and Campylobacter coli) isolated from humans, broilers and cattle in the USA and Canada has been reported at 10% or lowers (109). In contrast, more than 40% of C. coli, isolated from turkeys and swine in the USA, were resistant to this antimicrobial agent. Likewise, macrolide resistance among Campylobacter isolates from humans and C. jejuni isolates from chickens and cattle has been low and stable in most European countries, especially in Scandinavia, but a high prevalence of macrolide resistance, ranging from 15–80%, was observed in C. coli, isolated from chickens and swine (110,111). Interestingly, high erythromycin resistance levels were observed among human clinical Campylobacter isolates from Africa, but low resistance levels to this antibiotic were noticed in C. jejuni and C. coli isolated from food-producing animals (112). In Asia, less than 5% of C. jejuni isolated from humans, broilers, swine and cattle were resistant to


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macrolides, while 14–62% of C. coli isolated from humans, broilers and swine were resistant to this class of antimicrobial agents (113,114). Similar to findings from other continents, macrolide resistance was mainly observed among C. coli isolates from swine in Australia (115). 2.6 Identification and detection of Campylobacter

2.6.1 Isolation of Campylobacter

For the isolation of Campylobacter from fecal/cecal or intestinal samples, no pretreatment is needed; samples can be plated on to selective medium or the filtration method on non-selective agar can be used. In the case of caecal samples, ceca are aseptically opened by cutting the end with a sterile scissors and squeezing out the material to be processed. Enrichment is recommended to enhance the culture sensitivity of potentially environmentally stressed organisms or in the case of low levels of organisms in feces, for example from cattle, sheep or pigs. However, enrichment from the latter samples is not carried out routinely and in research setting only. 2.6.1.1 Selective media for isolation Many media can be used in the recovery of Campylobacter spp. Modified charcoal, cefoperazone, desoxycholate agar (mCCDA), is the recommended medium, although alternative media may be used. A detailed description on Campylobacter detection by culture and the variety of existing media is given by Corry et al. (116). The selective media can be divided into two main groups: blood-containing media and charcoal-containing media. Blood components and charcoal serve to remove toxic oxygen derivatives. Most media are commercially available. The selectivity of the media is determined by the antibiotics used. Cefalosporins (generally cefoperazone) are used, sometimes in combination with other antibiotics (e.g. vancomycin, trimethoprim). Cycloheximide (actidione) and more recently amphotericin B are used to inhibit yeasts and molds. The main difference between the media is the degree of inhibition of contaminating flora. All the selective agents allow the growth of both C. jejuni and C. coli. There is no medium available that allows growth of C. jejuni and inhibits C. coli or vice versa. To some extent, other Campylobacter species (e.g., C. lari, C. upsaliensis, C. helveticus, C. fetus and C. hyointestinalis) will grow on most media, especially at the less selective temperature of 37C.


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2.6.1.2 Passive filtration Passive filtration, a method developed by Steele and McDermott (117) obviates the need for selective media; thus it is very useful for the isolation of antimicrobial-sensitive Campylobacter species. As the method does not use expensive selective media, it may be used in laboratories with fewer resources. For passive filtration, feces are mixed with PBS (approximately 1/10 dilution) to produce a suspension. Approximately 100 µl of this suspension are then carefully layered on to a 0.45 or 0.65 µm filter, which has been previously placed on top of a non-selective blood agar plate. Care must be taken not to allow the inoculum to spill over the edge of the filter. The bacteria are allowed to migrate through the filter for 30–45 minutes at 37C or room temperature. The filter is then removed, the fluid that has passed through the filter is spread with a sterile glass or plastic spreader, and the plate is incubated microaerobically at 42C. 2.6.1.3 Incubation Microaerobic atmospheres of 5–10% oxygen, 5–10% carbon dioxide are required for optimal growth. Appropriate atmospheric conditions may be produced by a variety of methods. In some laboratories, (repeated) gas jar evacuations followed by atmosphere replacement with bottled gasses are used. Gas generator kits are available from commercial sources. Variable atmosphere incubators are more suitable if large numbers of cultures are undertaken. Media may be incubated at 37C or 42C, but it is common practice to incubate at 42C to minimize growth of contaminants and to select for optimal growth of Campylobacter. Campylobacter jejuni and C. coli usually show growth on solid media within 24–48 hours at 42C. As the additional number of positive samples obtained by prolonged incubation is very low, 48 hours of incubation is recommended for routine diagnosis. 2.6.2 Confirmation

A pure culture is required for confirmatory tests, but a preliminary confirmation can be obtained by direct microscopic examination of suspect colony material (Table 2.7) (118).

2.6.2.1 Identification on solid medium

On Skirrow or other blood-containing agars, characteristic Campylobacter colonies are slightly pink, round, convex, smooth and shiny, with a regular edge. On charcoal-based media such as mCCDA, the characteristic colonies are greyish, flat and moistened, with a tendency to spread, and may have a metal sheen.


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2.6.2.2 Microscopic examination of morphology and motility The material from a suspect colony is suspended in saline and evaluated, preferably by a phase-contrast microscope, for characteristic, spiral or curved slender rods with a corkscrew-like motility. Older cultures show less motile coccoid forms. 2.6.2.3 Detection of oxidase A portion of suspect colony was placed on to a filter paper moistened with oxidase reagent. The appearance of a violet or deep blue colour within 10 seconds is a positive reaction. If a commercially available oxidase test kit is used, the manufacturer’s instructions must be followed. 2.6.2.4 Microaerobic growth at 25C The pure culture is inoculated on to a non-selective blood agar plate and incubate at 25C in a microaerobic atmosphere for 48 hours. 2.6.2.5 Aerobic growth at 42C The pure culture is inoculated on to a non-selective blood agar plate and incubate at 41.5C in an aerobic atmosphere for 48 hours. 2.6.2.6 Latex agglutination tests

The pure cultures of C. jejuni/C. coli (often also including C. lari) was confirm by commercial kit. 2.6.3 Identification of Campylobacter to the species level Among the Campylobacter spp. growing at 42C, the most frequently encountered species from samples of animal origin are C. jejuni and C. coli. However, low frequencies of other species have been described. Generally, C. jejuni can be differentiated from other Campylobacter species on the basis of the hydrolysis of hippurate as this is the only hippurate-positive species isolated from veterinary or food samples. The presence of hippurate-negative C. jejuni strains has been reported (118). Table 2.8 (118) gives some basic classical phenotypic characteristics of the most important thermophilic Campylobacter species. Sensitivity to nalidixic acid used to be one of the most commonly tested characteristics, but nowadays may give difficulties in interpretation, both due to an increase in nalidixic acid-resistant strains of C. jejuni and C. coli and to the isolation of nalidixic acid-sensitive genogroups of C. lari. 2.6.3.1 Detection of hippurate hydrolysis A loopful of growth from a suspect colony was suspended in 400 µl of a 1% sodium hippurate solution (care should be taken not to


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incorporate agar) and incubated at 37C for 2 hours. Then 200 µl 3.5% ninhydrin solution were added to the side of the tube to form an overlay. The preparation was re-incubated at 37C for 10 minutes, and was read the reaction. Positive reaction: dark purple/blue. Negative reaction: clear or grey. If commercially available hippurate hydrolysis test disks are used, follow the manufacturer’s instructions. 2.6.3.2 Detection of indoxyl acetate hydrolysis A suspect colony was placed on an indoxyl acetate disk and was added a drop of sterile distilled water. If indoxyl acetate is hydrolysed a colour change to dark blue occurs within 5–10 minutes. No colour change indicates hydrolysis has not taken place. If commercially available indoxyl acetate hydrolysis test disks are used, follow the manufacturer’s instructions. Table 2.7 Confirmatory tests for thermophilic Campylobacter (118).

Confirmatory test Result for thermophilic Campylobacter Morphology Small curved bacilli

Motility Characteristic (highly motile and cork-screw like) Oxidase +

Aerobic growth at 41.5C -

Microaerobic growth at 25C - Table 2.8 Basic phenotypic characteristics of selected thermophilic Campylobacter species(118).

Characteristics C. jejuni C. coli C. lari Hydrolysis of hippurate + - -

Hydrolysis of indoxyl acetate + + -

REFERENCE

118. Steinhauserova I, Ceskova J, Fojtikova K, Obrovska I. Identification of thermophilic Campylobacter spp. by phenotypic and molecular methods. J Appl Microbiol. 2001;90:470–75.


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2.6.4 Molecular detection of Campylobacter Most molecular methods based on the polymerase chain reaction (PRC) are fast, simple, and reliable. However an evaluation of the sensitivity and specificity of the developed detection and specification of the methods is needed. It has been recognized for a long time that campylobacters are extremely diverse in phenotype, and, as became clear more recently, also in genotype. A number of genotyping techniques have been developed for genetic subtyping of campylobacters. 2.6.4.1 Detection and speciation of thermophilic Campylobacter species by molecular techniques Detection of microorganisms by the polymerase chain reaction has long been predicted as the modern-time alternative for bacteriological culture. However, just as it required over 15 years to identify proper culture methods for Campylobacter (and these are probably still not optimal) it will require time to find the best PCR detection procedure. PCR is the amplification (multiplication of the amount of DNA) of a specific piece of DNA, making use of specific primers and a DNA polymerase that is extremely thermostable (normally Taq polymerase). The technique is very powerful and can be hightly sensitive, however, depending on the type of sample, different problems have to be solved. In food products, the low numbers of organisms require pre-enrichment and the presence of potential PCR inhibitors require robust PCR Protocols (119). Purification procedures to remove PCR inhibitors are effective but add extrawork. In feces, numbers of campylobacters may vary and the presence of high numbers of other microorganisms demands a high specificity. Some PCR detection methods developed for Campylobacter spp. detect directly at the species level. Other methods detect C. jejuni and C. coli without differentiation, and in some C. lari is included as well. Most methods are based on amplification of (fragments of) flagellin genes or ribosomal genes. In some described methods the target gene for species-specific amplification has been selected by hybridization experiments and has not been further characterized. 2.6.4.2 Subtyping of Campylobacter spp. The diversity of biochemical and phenotypic properties within Campylobacter species have been recognized for a long time. In the past, phenotypic differences between isolates were used to develop subtyping schemes. For Salmonella and other Enterobacteriacae serotyping had proved valuable, and therefore in the 80’s serotyping was developed for Campylobacter (120,121). The serotyping scheme based on heat-stable (HS) antigens (120) is still in use in few


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laboratories and now encompasses over 60 serotypes. Since most phenotypic characteristics are somehow represented by differences in the genome, genotypic differentiation is a straightforward alternative. The advantages are obvious: genotypes are generally stable and independent on culture conditions or expression of antigens; most genetic methods have higher typeability than phenotypic methods; and computer-aided technology allows excellent compatibility. Genotypic methods are diverse and can be devided in methods that depend on a single locus (or several loci) within a genome, and those methods that depend on the complete genome. Some methods rely on the absence or presence of recognition sites for restriction enzymes, other methods are based on PCR amplification. Unfortunately, some - in principal identical - methods are known under different names and different methods share identical or confusing names. 2.6.4.3 Flagellin typing (fla typing) This method is based on restriction fragment length polymorphism (RFLP) of PCR products derived from the flagellin genes (fla) of C. jejuni. Briefly, fla specific PCR primers are used to obtain a PCR fragment which is digested with restriction enzymes. The obtained banding pattern after agarose gel electrophoresis is determined by the choice of primers and the restriction enzyme used. Fragments are generated in the range of 0.1-1 kilo base pairs (kb). Since C. jejuni contains two flagellin genes (that are next to each other on the genome), the PCR can detect either one fla gene or two, depending on which primers are used. The primers are designed to bind to strongly conserved sequences (but the sequence inbetween the primers is highly variable) and the primers were found to work for C. coli and C. upsaliensis as well. The different fla typing schemes mainly differ in the choice of primers and enzymes. Typeability can be improved when purified DNA is used instead of cell lysates, but this increases the amount of work. The discriminatory power can be in creased by using more than one restriction enzyme. 2.6.4.4 Pulsed-Field Gel Electrophoresis (PFGE) Also known as genomic fingerprinting or macrorestriction profiles. A method based on the presence or absence of recognition sites for restriction enzymes that cut infrequently in the genome. PFGE is dependent on complete chromosomal DNA, which is isolated in a protective gelling agent to avoid shearing. After digestion the DNA is analyzed on agarose gels using specialized equipment that generates a pulsing electrical field. In this way large (20-200 kb long) fragments can be separated. The obtained banding pattern depends on the choice of


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restriction enzyme and the electrophoretic conditions, both of which are in need of standardization. PFGE is one of the most commonly used methods and is often presented as a ‘gold standard’ for genotyping, although there is no clear advantage of this method over others, and the method is rather laborious. Some strains are not typeable because of DNAse production, which can be overcome by adaptation of the method. Discriminatory power can be increased by the use of more than one restriction enzyme. A small percentage of strains have DNA that is undigestable by commonly used enzymes, presumably by restriction/modification systems. Such strains are sometimes typeable using alternative enzymes. 2.6.4.5 Ribotyping This method is based on the presence or absence of restriction sites in or around the three ribosomal loci, which are visualized by Southern blot hybridization. Briefly chromosomal DNA is isolated, digested, and separated on agarose gels. From these gels a Southern blot is obtained (a ‘blueprint’ of the separated DNA bands on a nitrocellulose filter) which is hybridized with a labeled DNA fragment specific for the ribosomal RNA (rRNA) genes. The rRNA specific labeled fragment (the ‘probe’) is usually produced by PCR. The obtained banding pattern depends on the choice of restriction enzymes and the choice of the labeled fragment, which can be obtained from the genes encoding 16S rRNA, 23S rRNA, or both. The method is rather laborious and the discriminatory power is relatively low. The reason for this is not completely understood. In comparison to other species (e.g. Salmonella), where ribotyping proved to have excellent discriminatory power, C. jejuni contains less ribosomal gene loci (3 as compared to 5 for Salmonella). The fragments detected by ribotyping are 0.5 - 5 kb and the resolution of the gels is poorer than gels used for fla typing. Ribotyping has not been used as much as fla typing or PFGE. An automatic device (commercially available under the name ‘riboprinter’) allows high throughput with little handling, at high costs for equipment and materials. 2.6.4.6 Random Amplyfied Polymorphic DNA (RAPD) This method also called Arbitrarily Primed PCR fingerprinting (AP-PCR), is based on PCR but does not amplify specific loci. Instead, arbitrary developed primers are used to amplify randomly distributed fragments. The amplification conditions are chosen at low stringency so that fragments can be amplified even when the primers do not perfectly fit. The obtained PCR products are separated by agarose gel electrophoresis and the obtained patterns (consisting of bands with varying intensity) depend on the presence, orientation, and location of


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primer sites. The major problem with RAPD is the lack of reproducability. The low stringency required for the PCR makes the method very sensitive to experimental conditions (purity and concentration of the DNA, inhibitors, PCR apparatus, etc.). The original method used one primer but variants have been described for Campylobacter using two primers, one of which may be specific for enterobacterial repetitive sequences (REP primers). A classical REP-PCR amplifies fragments between repetitive sequences to which REP primers bind with high specificity. Since such repetitive sequences are absent in Campylobacter, a classical REP-PCR cannot be used and the primers are used at low stringency instead, which resembles RAPD. The lack of reproducability greatly limits compatability of the method, and therefore RAPD is mainly in use in individual laboratories, where good results are reported. The method is fast and simple. 2.6.4.7 Amplified Fragment Length Polymorphism (AFLP) This genotyping method should not be confused with methods that determine the size of bands of PCR products (PCR RFLP), which is known under the same name. In AFLP a combination of PCR amplification and restriction enzyme recognition is used in a relatively complex way. Chromosomal DNA is isolated and digested with two restriction enzymes that cut relatively frequently. After ligation of linkers, a subset of these fragments are amplified by PCR in an ingenious way in which the restriction sites serve as the primer-specific sequences, with the addition of one or more specific nucleotides. The difference with PFGE is that the obtained fragments are much smaller (50-500 bp) and that they are analyzed on acrylamide gels at very high resolution. The difference with RAPD is that the PCR reaction is carried out under stringent conditions which results in high reproducability. The method is relatively new but compatibility proved to be high. Automated gel reading and data processing by computer has greatly aided to objective interpretation of the results, and the high number of generated bands gives a certain leeway in band variation due to artefacts that are averaged out. AFLP has excellent typeability and discrimination power, and may well become the ‘gold standard’ of the future. However specialized equipment for acrylamide electrophorese and automated gel-reading is needed and the method is not fast, although the throughput is reasonable. 2.6.5 Immunological methods for detection of Campylobacter spp. Current methods of identification of Campylobacters in stool and food samples rely on their growth on selective agar plates with and without prior broth enrichment. These methods are labor intensive and time consuming, taking


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four or more days for completion and require robust bacterial growth, which may limit the detection of stressed bacteria that do not grow well but are still infective. More rapid and accurate methods are necessary to identify Campylobacters in stool samples in order to treat campylobacteriosis early, and to detect outbreaks as campylobacteriosis is a notifiable disease in the USA. Moreover, rapid detection of Campylobacters in environmental samples is important for trace-back investigations to mitigate outbreaks and in food samples to catch contamination during commercial food processing. During the 1960s and early 1970s, the search for alternative techniques to replace radioactive label reporters to identify biological molecules led to the development of the enzyme-linked immunosorbent assay (122). In the mid-1970s the, the development of the hybridoma technique for monoclonal antibody synthesis (123) helped expand the new field of immunodiagnosis and initiate development of immunoassays for the identification of bacterial pathogens. Since then, a variety of immunoassays have been developed for the detection of different foodborne pathogens in food and stool samples, including a few immunoassays for Campylobacter spp. detection. The simplest is latex agglutination, in which antibody-coated colored latex beads or colloidal gold particles are used for rapid confirmation of culture results or serotyping of culture isolates. The EIA, the most popular immunoassay used for pathogen detection in foods and stool, is typically designed as a “sandwich” assay, in which an antibody bound to a solid matrix is used to capture the bacterium and a second antibody conjugated to an enzyme then binds to the bacterium. Multi-well microplates are a commonly used solid support but other formats include dipsticks, paddles, membranes, or other solid matrices. The lateral flow immunochromatographic method is a modified EIA, packaged in a simple device (dipstick or within a plastic casing) and used for rapid pathogen detection. Typically, “sandwich” assays are used for large analyses such as bacteria. Samples migrate from the sample pad through a conjugate pad where the target analyze binds to the antibody conjugated to colored particles. The sample is drawn across the membrane to the capture zone where the target/conjugate complex binds to immobilized antibodies producing a visible line on the membrane. To ensure a working test, the sample migrates further until it reaches the control zone, where excess conjugate is bound to produce a second visible line on the membrane. Two clear lines on the membrane are a positive result. A single line in the control zone is a negative result. Lateral flow immunoassays have many advantages including their simplicity, production of a result within 15 minutes,


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stability with a long shelf life even in some cases without refrigeration and their low cost, but they may have a higher threshold of detection compared to EIA. 2.6.5.1 Identification of Campylobacter spp. using immunoassays The presence of campylobacters in food and stool samples is based on the growth of the bacteria on a selective agar, incubated at 42C under a reduced oxygen atmosphere (~5% O2). The limitations of the culture method, especially the need for up to four days to identify campylobacters (124), have dictated the development of culture-independent methods, including immunoassays, for the detection of Campylobacter spp. in clinical stool and food samples. Although a variety of immunoassays have been developed for testing clinical and food samples for Campylobacter spp., these assays require approval by regulatory bodies, necessitating comparison of immunoassays to culture-based methods, considered the reference methods. The validation of immunoassays for detection of Campylobacter spp. includes testing for inclusivity, to assure that different strains of C. jejuni and C. coli are detected, and exclusivity, to assure that closely related, non-target bacteria do not cross-react with the assay (125).

(1) Clinical stool samples Several commercial immunological assays are available for identification of C. jejuni and C. coli in stool samples, including some designed to confirm culture results and others that are culture-independent. Assay formats include latex agglutination, EIAs, and lateral flow formats. Immunoassays based on latex agglutination, developed in the late 1980s, are of limited use because they can only confirm culture results, and may detect closely-related organisms (126). Several EIAs are commercially available for use directly with clinical stool samples, and in some studies have performed as good or better than the standard culture techniques for detecting C. jejuni and C. coli, and possibly C. upsaliensis, and are comparable to nucleic acid tests (127) but not in all cases. Of the four EIAs commercially available in the USA for direct detection of Campylobacter in stool specimens, two are microplate-based and two are incorporated in lateral flow devices. These methods are reasonably rapid, from 20 minutes for the lateral flow assays to 2-4 hours for the microplate assays, and identify specific Campylobacter antigens common to C. jejuni and C. coli. Currently, none of the described assays can be recommended for standalone identification of campylobacters in stool samples, in part because of the limited and conflicting findings regarding the performance of these immunoassays. The performance of these EIAs is variable and suggests a high


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probability of non-acceptable levels of false negatives in certain situations. The Centers for Disease Control and Prevention (CDC) has recently released preliminary data concluding that EIAs should not be used as standalone tests for direct detection of Campylobacter in stool samples. Therefore, a positive EIA test alone is not sufficient to consider a case "confirmed” and laboratories must confirm positive EIA results by culture methods (128). (2) Food samples

Current identification methods for Campylobacter in foods rely on bacterial growth on one of a variety of selective agar plates which contain antimicrobials to allow for the growth of the target organism, a method adapted from that used for stool samples. Besides increasing bacterial concentration, enrichment is important because campylobacters are randomly distributed and can be present in clumps or aggregates in foods, limiting direct detection. Also, enrichment ensures that stressed or injured bacteria have a chance to recover prior to plating on selective agars. Culture-based methods are the accepted methods outlined in the Food and Drug Administration (FDA)‘s Bacteriological Analytical Manual (BAM), the Microbiology Laboratory Guidebook (MLG) of the Food Safety and Inspection Services of the U.S. Department of Agriculture (FSIS USDA) and the International Organization for Standardization (ISO). These culture-based methods for food screening suffer from the same constraints as those for stool samples: low specificity resulting in false positives, and long lag-time for results. However, a new performance standard aimed at limiting the prevalence of Campylobacter-contaminated poultry meat products in the USA requires the screening of processed carcasses for the presence of this pathogen. Thus, there is a critical need to develop and validate rapid methods for Campylobacter identification, including those that are antibody-based. Although antibody-based methods would speed assay time, only a few latex agglutination assays are included in reference manuals and only for confirmation of positive Campylobacter isolates. Based upon the studies, commercial immunoassays show promise as one of the tools to speed food sample screening. However, more studies of these assays with naturally contaminated samples from a variety of foods is necessary. The effect of bacterial stress that occurs during food processing and storage on assay performance should also be studied. Additionally, the antigen targets of the antibodies used in these assays need to be identified. A concerted effort to improve the performance of these assays should be undertaken now that a new standard for food screening poultry meat for Campylobacter spp. has been established in the USA.


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2.6.5.2 Increasing concentration of campylobacters prior to immunoassay identification Because EIA assays have a sensitivity of ≥ 104 CFU/ml or CFU/g, there is a need to increase the number of the target organisms in food samples prior to assay. The enrichment of the sample is the most common method to increase the number of bacterial cells. No enrichment protocol is 100% selective for the organism of concern; therefore other methods are used to separate or concentrate the target bacteria from the rest of the contaminants. This section will review enrichment broths, filtration, and immunomagnetic separation as techniques to increase the number of Campylobacter spp. in the food samples prior to identification by EIA.

(1) Enrichment of the samples The enrichment of food samples is imperative for the

isolation of Campylobacter spp. from poultry or raw milk products. The most commonly used enrichment broth is Bolton broth, which has a basal component made up of meat peptone, lactalbumin hydrolysate, yeast extract, alphaketoglutaric acid, ssodium chloride, sodium pyruvate, metabisulphite and carbonate. The use of buffered peptone water performs similarly to Bolton broth for the isolation Campylobacter spp., suggesting that the basal medium does not need to be rich in nutrients to support the growth and multiplication of Campylobacter spp.(129). An important requirement is the incubation of food samples for at least 48 hours in enrichment broth before identification of positive cultures by immunoassay because 24 hours incubation results in a high number of false negative samples (130). The antimicrobials added to the basal broth are cefoperazone, trimethoprim and vancomycin, at concentrations of 20 mg/l each, and cycloheximide, at a concentration of 50 mg/l. Originally, Bolton broth was supplemented with 5% lysed horse blood, but research has shown that the addition of blood is not necessary for isolation of Campylobacter spp. from retail broiler meat. Most importantly, blood in the enrichment broth is not necessary if an EIA-based method is employed to detect positive samples (130).

(2) Use of filter membranes to separate contaminating bacteria Filter membranes were used for the first isolation of C. jejuni from human stools (131). In this report, the use of 0.65 µm filters was aimed at retaining most of the contaminating bacteria in fecal suspensions while allowing Campylobacter organisms to pass through the filter for isolation on agar plates. This


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differential filtration is different from most of the other filtration protocols in which the target organisms are concentrated by filter retention for subsequent identification. Although filters may not help isolate Campylobacter spp. from samples with low number of cells such as water samples, filters help isolate Campylobacter spp. from highly contaminated samples. A filtration method coupled with a sandwich EIA that uses polymyxin B sulfate (PMB) instead of an antibody to capture C. jejuni and C. coli was developed by one of the authors. Filter concentration coupled with PMB-capture ELISA was able to detect 102 CFU/ml of Campylobacter spp. 2.7 Antibody

The immune system in vertebrates is continuously evolving to protect itself from different intruding pathogens. The immune responses rotate around some innate mechanisms, including adaptive processes such as producing antibody (Ab) molecules that can bind to all molecular structures of the microbial pathogen (bacteria, viruses, fungi, nematodes, and other parasites) and can keep pace with the diversified mutations in an organism. Antibodies are antigen-reactive proteins, designated immunoglobulins, present in the plasma and in extracellular fluids. They bind their nominal antigens with exquisite specificity and potentially neutralize their harmful effects. Antibodies were the first elements of the immune system to be identified. They are present in an immune serum, called antiserum, and obtained after exposure of the vertebrate host to a given antigen, called an immunogen. In humans, five classes of immunoglobulins, also called isotypes (IgG, IgM, IgA, IgD and IgE), differ in their physicochemical and serological properties, and in the amino acid sequence of their constant regions (Table 2.9) (132). In their monomeric form, all classes exhibit the same basic structure: two heavy (H) and two light (L) chains. While IgG, IgD and IgE consist of a single monomeric unit, the IgM and IgA classes are composed of monomers that associate to form pentamers and dimers, respectively. Basically, the immunoglobulin molecule is a Y-shaped tetrameric protein characteristically composed of two H and two L polypeptide chains held together by covalent (disulfide) and noncovalent bonds (Figure 2.7) (132). Treatment of immunoglobulins with enzymes and chemical reagents capable of cleaving peptide bonds breaks them up into fragments. Three types of treatment are particularly important in understanding antibody structure. First, agents that cleave disulfide bonds generate two H-chain polypeptides of approximately 50 kDa and two L chains


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of 25 kDa. Second, papain splits the basic molecule into three fragments of approximately 45 kDa. Two of them retain the antibody ability to recognize the antigen and are referred to as Fab fragments (for ‘fragment antigen binding’). Each fragment possesses only one combining site and can bind, but cannot precipitate, the antigen. The third fragment produced by papain digestion can be crystallized from a solution and is therefore referred to as Fc fragment (for ‘fragment crystalline’). Finally, treatment with pepsin gives rise to several small fragments and a large fragment, the F(ab)’2 fragment, of molecular weight double that of one Fab fragment, and capable of binding and precipitating the antigen. The Fab is the antigen binding constituent and the Fc fragment is the anchoring site for proteins of the complement system and for receptors of various effector cells. Table 2.9 Characteristics of human immunoglobulin isotypes(132).

IgG1 IgG2 IgG3 IgG4 IgM IgA1 IgA2 IgD IgE Heavy chain γ1 γ2 γ3 γ4 µ α1 α2 δ ε No. of constant domains 3 3 3 3 4 3 3 3 4 Light chain Κ / λ Κ / λ Κ / λ Κ / λ Κ / λ Κ / λ Κ / λ Κ/ λ Κ/ λ

Molecular weight (kDa) 150 150 165 150 970 160 160 175 190 Serum concentration (mg mL–1)

9 3 1 0.5 1.5 3 0.5 0.04 0.0003

Half-life (days) 23 23 7 23 5 6 6 3 2.5 Placental transfer +++ + +++ +++ - - - - - Binding to macrophages and other phagocytic cells

+ - - + - - - - +

Binding to basophils and mast cells

- - - - - - - - +

Complement activation (classical pathway)

++ + +++ - +++ - - - -

Complement activation (alternate pathway)

- - + - - + - - -

REFERENCE

132. Zouali M. Antibodies. ENCYCLOPEDIA OF LIFE SCIENCES internet. 2001. cited 2016 Jan 18. Available from http:// gdou.edu.cn/nxy/dwmyx/resources\pdf.


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Figure 2.7. Diagram of a prototypic immunoglobulin (Ig) G monomer. Each rectangle represents an immunoglobulin domain. The complementarity determining regions (CDRs) are highlighted. The green bars depict the hinge region. The Fc fragment (for ‘fragment crystalline’) is produced by papain digestion of IgG and can be crystallized from a solution. It is the anchoring site for proteins of the complement system and for receptors of various effector cells. The Fab fragment (for ‘fragment antigen binding’) is produced by papain digestion of IgG. It possesses one combining site and can bind, but cannot precipitate, the antigen. The F(ab)’2 fragment is produced by pepsin digestion of IgG. Its molecular weight is double that of one Fab fragment, and it is capable of binding and precipitating the antigen(132).

REFERENCE

132. Zouali M. Antibodies. ENCYCLOPEDIA OF LIFE SCIENCES internet. 2001. cited 2016 Jan 18. Available from http:// gdou.edu.cn/nxy/dwmyx/resources\pdf.


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Each millilitre of normal serum contains approximately 1016 immunoglobulin molecules. Even antibodies specific for the same epitope are heterogeneous. An antiserum raised against a given immunogen contains many different antibodies which bind to the antigen in slightly different fashions, and some of them may cross-react with related antigens and even with antigens exhibiting no obvious structural similarity with the immunogen. The antibodies are extraordinarily and the main component in the immunoassay which is a technology for identifying and quantifying organic and inorganic compounds that relies on a special reaction between an antibody and a target compound. Their highly specific binding to a wide range of molecules, and their ability to identify specific antigenic determinants in these molecules make them extremely valuable. The technology has been used widely for field analysis in the environmental field including bacterial detection because the antibodies can be highly specific to the target compound and because immunoassay is relatively quick and simple to use. They are two types of antibodies used in immunoassay, namely monoclonal antibodies and polyclonal antibodies. Polyclonal antibodies are a mixture of different immunoglobulin types binding to multiple sites on the antigen used for immunization, but the monoclonal antibodies consist of a single specific site on the antigen used for immunization. Early researcher used polyclonal antibodies for studying the antibody-antigen reaction. However, many immunochemist have investigated the interaction between antibodies and antigens using monoclonal antibodies since 1975. For analysis of trace levels of environment contaminants using monoclonal antibodies, the antibodies must be characterized for their specificity and affinity for antigen targets. The polyclonal antibodies are less than ideal for the study the antibody-antigen relationship due to their heterogeneity.

Therefore, antibodies are useful research tools in diagnosis and therapy, as they can recognize and bind specifically and strongly with respective antigens. Polyclonal antibodies mixtures contain different antibodies developed in the blood of immunized animals from different cell types. As most antigens bear multiple epitopes, they can stimulate the proliferation and differentiation of a variety of B-cell clones. Thus a heterogeneous pool of serum antibodies can be produced with specificity for particular epitope(s) of the antigen. In contrast, monoclonal antibodies (MAbs) are a mixture of homogenous antibody molecules with affinity towards a specific antigen, often generated using a hybridoma by fusing a B-cell with a single lineage of cells containing a definite antibody gene. Finally, a population of identical cells (or clones) is produced that secrete the same antibody. Due to their specificity


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and high reproducibility using culture techniques, MAbs offer advantage over polyclonal antibodies. MAbs are increasingly used in applications such as research and diagnosis, therapeutic tools in cancer and immunological disorders, and pharmacy, thus generating a great demand in industry. The essential characteristics that confer the clinical applicability of MAbs include their specificity of binding and homogeneity, as well as their ability to be produced in unlimited quantities (133). Another unique advantage of hybridoma production is that a mixture of antigens can be employed to generate specific antibodies. This also enables one to screen an antibody of choice from a mixture of antibody population with a purified antigen; thus, a single cell clone can be isolated (133,134). For these reasons, the objective of this review was to dissect the diverse facets of applicability of MAbs in disease monitoring and diagnosis. 2.8 Monoclonal antibody

2.8.1 History The production of MAbs by hybridoma technology was discovered in 1975 by Georges Kohler of West Germany and Cesar Milstein of Argentina, who jointly with Niels Kaj Jerne of Denmark were awarded the Nobel Prize for Physiology and Medicine in 1984. In 1976, Kohler and Milstein developed a technique to fuse splenocyte cells (separated from the spleen of an immunized mouse) with tumorous myeloma cells. The hybrid cells were clones of antibody producing cells against a desired antigen and propagate rapidly to produce very large amounts of antibody. The hybridoma is capable of rapid propagation and high antibody secreting rates such as in myeloma cells, which can maintain the antibody genes of mouse spleen cells. In 1988, Greg Winter used the first humanized MAbs to avoid reactions/responses observed in patients injected with murine derived MAbs (133-135). 2.8.2 Monoclonal antibodies production The main objective is to produce a homogenous population of MAbs against a pre-fixed immunogen (Figure 2.8) (133). The basic strategy includes (i) purification and characterization of the desired antigen in adequate quantity, (ii) immunization of mice with the purified antigen, (iii) culture of myeloma cells which are unable to synthesize hypoxanthine-guanine-phosphoribosyl transferase (HGPRT) enzyme necessary for the salvage pathway of nucleic acids, (iv) removal of spleen cells from mice and its fusion with the myeloma cells, (v) following fusion, the


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hybridomas were grown in hypoxanthine aminopterin thymidine (HAT) medium. The fused cells are not affected in the absence of HGPRT unless their de novo synthesis pathway is also disrupted. In the presence of aminopterin, the cells are unable to use the de novo pathway and thus these cells become auxotrophic for nucleic acids as a supplement to HAT medium. In this medium, only fused cells will grow. Unfused myeloma cell does not have ability to grow in this HAT medium because they lack HGPRT, and thus cannot produce DNA. Unfused spleen cells cannot grow because of their short life spans. Only fused hybrid cells or hybridomas can grow in HAT medium. Hybrid cells have the capacity to grow in the HAT medium since spleen cell partners produce HGPRT. (vi) The hybrid cell clones are generated from single host cells (vii) the antibodies secreted by the different clones are then tested for their ability to bind to the antigen using an enzyme-linked immunosorbent assay (ELISA). (viii) The clone is then selected for future use (133,134). 2.8.2.1 Immunization schedule Depending on the purity and nature of the purified antigen, an immunization protocol is determined. For immunization, the desired protein should be available in adequate quantity (a few milligrams). However, in case of a complex multi-molecular antigen, it is quite stringent to purify it in adequate quantity. Thus, depending on its screening and selection abilities, MAbs can purify a target antigen from an antigen mixture. Mice must be immunized with antigen 6–10 weeks before fusion to allow them to develop a robust immune response before generating hybridomas. The injection schedule and the actual timing may vary depending on the antigen used for the immunization as well as other factors. It is desirable to immunize mice with a pure antigen, as this simplifies the screening of hybridomas. However, complex antigenic mixtures can be used (133-135).


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Figure 2.8 Production of monoclonal antibody by hybridoma technology. The hybridoma technology outline involves the isolation of spleen cells from immunized mice, their fusion with immortal myeloma cells and the production and further propagation of monoclonal antibodies from the hybrid cells (133).

REFERENCE

133. Tyagi S, Sharma PK, Kumar N, Visht S. Hybridoma technique in pharmaceutical science. Int J Pharm Tech Res. 2011;3(1):459–63.


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Collection of pre-immune serum is required prior to immunization to use as a baseline control for antibody screening. The mouse is bled by cutting approximately 1–2 mm off the tip of the tail thereby collecting 100–200 µl of blood in a capillary tube from where the serum is collected and can be cryo-preserved. A typical immunization schedule includes intra-peritonial injections of 2–4 adult mice (eg, BALB/c mice) with 20–100 µg of purified antigen in a total volume of 200 µl (ie, 200 µl of a 1:1 emulsion of antigen in saline: adjuvant). A stable emulsion is critical for generating a strong immune response. The injection is repeated 14–30 days later and booster doses are administered for 2–3 times until a good titer of antibody is obtained. Next, 10–14 days after the last injection, 100–200 µl of blood is collected from the tip of the tail or from the eye and serum is separated. The antibody levels in serum can be detected by applying different immune-techniques such as ELISA, immunofluorescence, flow cytometry, and immuno blotting, and the antibody titer of the post-immune serum is compared with the pre-immune serum from the same animal. The mouse with the highest antibody titers was selected for fusion. Between one and four days prior to fusion, the selected mouse is boosted intravenously via the tail vein. Following this step, spleen cells are prepared for fusion (Figure 2.8) (133-135). 2.8.2.2 Myeloma cell line culture In myeloma culture, hybridomas should grow continuously and selectively by suppressing the growth of the parent myeloma. It is desirable to obtain a parental myeloma cell that has been proven to yield stable hybridomas. The selected myeloma cell lines should have lost their capability to produce nucleotides using the salvage pathway. Myeloma cells are cultured in presence of 8-azaguanine so that they are unable to synthesize the HGPRT enzyme necessary for the salvage pathway of nucleic acids. Parental myeloma cells are cultured for at least one week prior to fusion to ensure that the cells are well-adapted to HGPRT negative conditions. Cells are seeded at a density of approximately 5 × 104 cells/ml and passaged every 2 days; those growing in the early-mid log phase prior to fusion are selected for fusion. After fusion, by using the drug aminopterin, de novo synthesis pathways can be blocked and thus the myeloma cells (where salvage pathway was previously blocked) cannot produce RNA or DNA and die. On the other hand, hybridomas have a functional salvage pathway (derived from the spleen cells of mouse) and can grow when they are cultured in medium containing the substrates


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for the pathway, ie, thymidine and hypoxanthine. This selective culture medium is HAT medium containing hypoxanthine, aminopterin, and thymidine respectively. 2.8.2.3 Fusion The parental myeloma cells used to make the hybridoma must match the strain of mouse being immunized (eg, for BALB/c mice the myeloma cells must be of BALB/c origin) and must not secrete any of their own immunoglobulin chains. The parental myeloma cells should be mycoplasma-free, fuse well, and allow the formation of stable hybridomas that continually secrete specific MAbs. SP2/0 and X63Ag8.653 are widely used parental myeloma cells that meet all of these criteria. There are various agents that induce the somatic cell to fuse. There are some physical agents, such as electro-fusion and chemical agents, including polyethylene glycol (PEG) and calcium ions, among others. Large numbers of cells can be fused in the presence of PEG within a short time. During electrofusion, a continuous electric potential is maintained in the fusion medium. Current is applied in short pulses at high voltage with short duration or in low voltage with long duration. The factors that are controlled during electro-fusion were specific resistance, osmotic strength, field strength, and ionic composition of the fusion medium. The cells should be given proteolytic pretreatment. An immunized mouse, 48–72 hours after tail vein injection, is euthanized and the spleen can be removed and disaggregated into a single cell suspension under sterile conditions. At the same time, the myeloma cells are harvested and added to fusion medium and mixed with spleen cells together with PEG solution to yield single hybridoma colonies. The fused cell mixture is plated in culture plates containing a feeder layer prepared from control un-immunized mice. 2.8.2.4 Growth and selection of MAbs Within 7–14 days after fusion, the growth of hybridomas occurs gradually together with the addition of interleukin 6 (IL-6), the hybridoma growth factor. Unfused myeloma cells, after 7 days of fusion, die in presence of aminopterin. Aminopterin accumulates gradually in the culture medium, while hypoxanthine and thymidine are continuously depleted, so it is essential to replace the HAT medium with hypoxanthine and thymidine (HT) medium. The qualitative and quantitative levels of the secreted antibody in the supernatant are screened by flow cytometry or ELISA. Selected cultures are cloned and re-cloned in order to achieve a pure clone population. Pure clones are grown in a larger culture flask to obtain a large amount of antibody and cells are used for future research and evaluation.


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2.8.2.5 Long-term maintenance and cryo preservation of MAbs The selected hybridomas are cryo-preserved in liquid nitrogen in ampules which can be thawed for use in future studies. The cryo-preserved hybridomas are thawed and re-cultured only when fresh antibody is needed. For long-term maintenance, it is always desirable to evaluate the quality of the produced antibody regularly because there may be some deterioration. 2.8.3 Monoclonal antibodies characterization The monoclonal antibody should be characterised thoroughly. This characterisation should include the determination of physicochemical and immunochemical properties, biological activity, purity, impurities and quantity of the monoclonal antibody. 2.8.3.1 Physicochemical characterization A physicochemical characterisation program will generally include a determination of the class, subclass, light chain composition (kappa and/or lambda chain) and primary structure of the monoclonal antibody. The amino acid sequence should be deduced from DNA sequencing and confirmed experimentally by appropriate methods (e.g. peptide mapping, amino acid sequencing, mass spectrometry analysis). The variability of N- and C- terminal amino-acid sequences should be analysed (e.g. C-terminal lysine(s)). Free sulphydryl groups and disulfide bridges should be determined. Disulfide bridge integrity and mismatch should be analysed. The carbohydrate content (neutral sugars, amino sugars and sialic acids) should be determined. In addition, the structure of the carbohydrate chains, the oligosaccharide pattern (antennary profile), the glycosylation site(s) and occupancy should be analysed. Typically, monoclonal antibodies have one N-glycosylation site on each heavy chain located in the Fc region. The light chain is usually not glycosylated. However, additional glycosylation site(s) in the heavy chains may occur, and thus their presence or absence should be confirmed. Glycan structures should be characterised, and particular attention should be paid to their degree of mannosylation, galactosylation, fucosylation and sialylation. The distribution of the main glycan structures present (often G0, G1 and G2) should be determined. Higher-order structure of the monoclonal antibody should be characterised by appropriate physicochemical methodologies. 2.8.3.2 Immunological properties The immunological properties of the antibody should be fully characterised. Binding assays of the antibody to purified antigens and defined regions of antigens should be performed, where feasible, to determine affinity,


50

avidity and immunoreactivity (including crossreactivity with other structurally homologous proteins). The complementary determining regions (CDR) should be identified, unless otherwise justified. The epitope and molecule bearing the relevant epitope should be defined. This should include a biochemical identification of these structures (e.g. protein, oligosaccharide, glycoprotein, glycolipid), and relevant characterisation studies (amino acid sequence, carbohydrate structure) to the extent possible. The ability for complement binding and activation, and/or other effector functions should be evaluated, even if the intended biological activity does not require such functions. 2.8.3.3 Biological activity The biological activity (i.e. the specific ability or capacity of a product to achieve a defined biological effect) should be assessed by in vitro and/or in vivo assays as appropriate. The mechanism of action and the importance (or consequences) of the product effector functions with regards to the safety and efficacy of the product should be discussed. For antibodies where effector function may play a role in the mechanism of action, and/or have an impact on the product safety and efficacy, a detailed analysis of ADCC, cytotoxic properties (e.g. apoptosis), ability for complement binding and activation and other effector functions, including Fc gamma receptor binding activity, and neonatal Fc receptor (FcRn) binding activity should be provided, as appropriate. 2.8.3.4 Purity, impurity and contaminants Monoclonal antibodies commonly display several sources of heterogeneity (e.g. C-terminal lysine processing, N-terminal pyroglutamate, deamidation, oxidation, isomerisation, fragmentation, disulfide bond mismatch, N-linked oligosaccharide, glycation), which lead to a complex purity/impurity profile comprising several molecular entities or variants. This purity/impurity profile should be assessed by a combination of orthogonal methods, and individual and/or collective acceptance criteria should be considered for relevant product-related variants. These methods generally include the determination of physicochemical properties such as molecular weight or size, isoform pattern, extinction coefficient, electrophoretic profiles, chromatographic data and spectroscopic profiles. In addition, suitable methods should be proposed to qualitatively and quantitatively analyse heterogeneity related to charged variants. Multimers and aggregates should also be appropriately characterised using a combination of methods. The formation of aggregates, sub-visible and visible particulates in the drug product is important and should be investigated and closely monitored on batch release and during stability


51

studies. In addition to the pharmacopoeial test for particulate matter, other orthogonal analytical methods may be necessary to determine levels and the nature of particulates. Potential process-related impurities (e.g. HCP, host cell DNA, cell culture residues, downstream processing residues) should be identified, and evaluated qualitatively and/or quantitatively, as appropriate. Contaminants, which include all adventitiously introduced materials not intended to be part of the manufacturing process (e.g. microbial species, endotoxins) should be strictly avoided and/or suitably controlled. Where non-endotoxin pro-inflammatory contaminants, such as peptidoglycan, are suspected, the use of additional testing, such as the monocyte activation test, should be considered. 2.8.3.5 Quantity Quantity should be determined using an appropriate physicochemical and/or immunochemical assay. It should be demonstrated that the quantity values obtained are directly related to those derived using the biological assay. When this correlation exists, it may be appropriate to use measurement of quantity rather than the measurement of biological activity in the product labelling and manufacturing processes, such as filling. 2.8.4 Monoclonal antibodies application MAbs have proved to be extremely valuable for basic immunological and molecular research because of their high specificity. They are used in human therapy, commercial protein purification, suppressing immune response, diagnosis of diseases, cancer therapy, diagnosis of allergy, hormone test, purification of complex mixtures, structure of cell membrane, identification of specialized cells, preparation of vaccines, and increasing the effectiveness of medical substances. The four types of applications are diagnostic applications, therapeutic applications, protein purification, and miscellaneous applications. 2.8.4.1 Diagnostic applications Monoclonal antibodies have revolutionized the laboratory diagnosis of various diseases. For this purpose, MAbs may be employed as diagnostic reagents for biochemical analysis or as tools for diagnostic imaging of diseases.

(1) MAbs in biochemical analysis Diagnostic tests based on the use of MAbs as reagents

are routinely used in radioimmunoassay (RIA) and enzyme-linked immunosorbent assays (ELISA) in the laboratory. These assays measure the circulating concentrations of hormones (insulin, human chorionic gonadotropin, growth hormone, progesterone, thyroxine, triiodothyronine, thyroid stimulating hormone, gastrin, renin), and several


52

other tissue and cell products (blood group antigens, blood clotting factors, interferon’s, interleukins, histocompatibility antigens, tumor markers). In recent years, a number of diagnostic kits using MAbs have become commercially available. For instance, it is now possible to do the early diagnosis of the following conditions/diseases.

(2) MAbs in diagnostic imaging Radiolabeled-MAbs are used in the diagnostic imaging of diseases, and this technique is referred to as immunoscintigraphy. The radioisotopes commonly used for labeling MAb are iodine-131 and technetium-99. The MAb tagged with radioisotope are injected intravenously into the patients. These MAbs localize at specific sites (say a tumor) which can be detected by imaging the radioactivity. In recent years, single photon emission computed tomography (SPECT) cameras are used to give a more sensitive three dimensional appearance of the spots localized by radiolabeled- MAbs. Immunoscintigraphy is a better diagnostic tool than the other imaging techniques such as CT scan, ultrasound scan and magnetic resonance. For instance, immunoscintigraphy can differentiate between cancerous and non-cancerous growth, since radiolabeled-MAbs are tumor specific. This is not possible with other imaging techniques. Monoclonal antibodies are successfully used in the diagnostic imaging of cardiovascular diseases, cancers and sites of bacterial infections. 2.8.4.2 Therapeutic applications Monoclonal antibodies have a wide range of therapeutic applications. MAbs are used in the treatment of cancer, transplantation of bone marrow and organs, autoimmune diseases, cardiovascular diseases and infectious diseases. The therapeutic applications of MAbs are broadly grouped into 2 types.

(1) MAbs as direct therapeutic agents Monoclonal antibodies can be directly used for enhancing the immune function of the host. Direct use of MAbs causes minimal toxicity to the target tissues or the host.

(2) MAbs as targeting agents in therapy Toxins, drugs, radioisotopes etc., can be attached or conjugated to the tissue-specific monoclonal antibodies and carried to target tissues for efficient action. This allows higher concentration of drugs to reach the desired site with minimal toxicity. In this way, MAbs are used for the appropriate delivery of drugs or isotopes.


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2.8.4.3 Protein purification Monoclonal antibodies can be produced for any protein. And the so produced MAb can be conveniently used for the purification of the protein against which it was raised. MAbs columns can be prepared by coupling them to cyanogen bromide activated Sepharose (chromatographic matrix). The immobilized MAbs in this manner are very useful for the purification of proteins by immunoaffinity method. 2.8.4.4 Miscellaneous applications (1) Catalytic MAbs (ABZYMES) Catalysis is the domain of enzymes. The most important common character between enzymes and antibodies is that both are proteins. Further, the binding of an antibody to its antigen is comparable to the binding of an enzyme to its substrate. In both instances, the binding is specific with high affinity and involves weak and non-covalent interactions (electrostatic, hydrogen and van der Waals forces). The striking difference is that the enzyme alters the substrate (to a product) while the antigen bound to antibody remains unaltered. Certain similarities between enzyme-substrate interaction and antibody-antigen interaction have tempted researchers to explore the possibility of using antibodies in catalysis. The antibody enzymes, appropriately regarded as abzymes, are the catalytic antibodies. There is a difference in the antibody recognition of an antigen and enzyme recognition of a substrate. While the antibodies recognize in ground state, the enzymes recognize in a transition state (associated with a conformational change of protein). In fact, it is in the transition condition the catalysis occurs. If a molecule resembling the transition state and conformation (between substrate and product) could be used as a hapten the antibodies so produced should bring about catalysis. This is what precisely is done to create abzymes. Researchers have produced a hapten-carrier complex which resembles the transition state of an ester undergoing hydrolysis. This hapten conjugate is used to generate anti-hapten monoclonal antibodies. These MAbs could bring about hydrolysis of esters with great degree of specificity (to the transition state to which MAbs were raised). Besides ester hydrolysis, there are several other types of reactions wherein antibodies can be used. These include hydrolysis of amides and carbonates, cyclization reactions, elimination reactions and bio-molecular chemical reactions. Certain enzymes require cofactors for their catalytic function. MAbs incorporating metal ions have been developed to carry out catalysis. Lerner and his associates carried out pioneering work in the development of abzymes. They could create a large number of immunoglobulin-


54

gene libraries for the production of antibodies that could be screened for their catalytic function. Abzymes represent a major biotechnological advancement that will have a wide range of applications (cutting of peptides and DNAs, dissolution of blood clots, killing of viruses etc.). (2) Autoantibody fingerprinting The occurrence of autoantibodies and their involvement in certain diseases is well known (e.g. rheumatic arthritis). A new category of individual specific (IS) autoantibodies have been discovered in recent years. These IS-autoantibodies are produced after birth and reach maximum in number by 2 years, and then remain constant for the later part of life. Monoclonal antibodies produced against IS-autoantibodies can be used for their detection, and identification of individuals. This technique referred to as autoantibody fingerprinting, is particularly useful for the detection of criminals, rapists etc. The autoantibodies collected from samples such as blood, saliva, semen and tears can be used. 2.9 Monoclonal antibodies against Campylobacter spp. Polyclonal antibodies have traditionally been used for detection and serotyping of bacteria isolates. These polyclonal reagents contain antibodies to many bacteria components, some of which cross-react with other bacteria, and may result in false positive reactions. Problems associated with low specificity and batch-to-batch variations have also been reported with the use of polyclonal antibodies for detection bacteria. Polyclonal antibodies are more sensitive than monoclonal antibodies in detecting environmental contaminants. For example, the detection level of paraoxon with polyclonal antibodies was 28 pg/ml (136) while the detection level with monoclonal antibodies was 10 ug/ml (137). Nowadays, monoclonal antibodies against bacteria have been produced and shown to be useful for detection and characterization of Salmonella spp. (138), V. cholera (139) and other bacteria. However, there have been relatively few MAbs published which are specific to Campylobacter and are shown in Table 2.10.


55

Table 2.10 Monoclonal antibodies against Campylobacter spp.

Bacteria Antigen Characterization Reference C. jejuni Purified antigen ELISA, Immunoblot 140 C. jejuni Veronal extract ELISA, Immunoprecipitation 141 C. jejuni rMOMP* Dot blot, Western blot 142 C. jejuni Whole cell component ELISA, Western blot 143 C. jejuni Sonicated cells ELISA, Western blot 144 C. jejuni Recombinant hippurate hydrolase ELISA, Western blot 145 C. coli Whole cell bacteria ELISA, Western blot 146 C. fetus Lipopolysaccharide ELISA, Immunoblot 147 C. fetus Whole cell lysates ELISA, Western blot 148 * Recombinant Major Outer Membrane Protein


56

Chapter 3 Research methodology

3.1 Experimental animals and bacterial strains

3.1.1 Experimental animals Female BALB/c mice 6-8 weeks old (Figure 3.1) from National

Laboratory Animal Center, Mahidol University. 3.1.2 Bacterial strains

One hundred and thirteen Campylobacter spp. and 148 non- Campylobacter spp. used in this study are listed in Table 3.1. The bacterial strains were provided by Osaka Prefecture University, Thammasat University Hospital and The Faculty of Allied Health Sciences, Thammasat University. All bacterial strains were maintained at the Faculty of Allied Health Sciences, Thammasat University. Figure 3.1 BALB/c mouse


57

Table 3.1 Bacteria species used in this study.

No Code Organism Source Origin 1 OPU A2-24h-40-1 Campylobacter jejuni meat OPU* 2 OPU A3-24h-41-2 C. jejuni meat OPU 3 OPU A4-24h-42 C. jejuni meat OPU 4 OPU A5-24h-43 C. jejuni meat OPU 5 OPU A6-24h-44 C. jejuni meat OPU 6 OPU A7-24h-45 C. jejuni meat OPU 7 OPU A8-24h-46 C. jejuni meat OPU 8 OPU A10-24h-48 C. jejuni meat OPU 9 OPU A39-48h-78 C. jejuni meat OPU 10 OPU B3-48h-21 C. jejuni meat OPU 11 OPU B4-48h-22 C. jejuni meat OPU 12 OPU B5-48h-23 C. jejuni meat OPU 13 OPU B6-48h-24 C. jejuni meat OPU 14 OPU B7-48h-25 C. jejuni meat OPU 15 OPU B8-48h-26 C. jejuni meat OPU 16 OPU B9-48h-27 C. jejuni meat OPU 17 OPU C5-5 C. jejuni meat OPU 18 OPU D3-MJ08-101 C. jejuni meat OPU 19 OPU D4-MJ08-103 C. jejuni meat OPU 20 OPU D5-MJ08-105 C. jejuni meat OPU 21 OPU D7-MJ08-108 C. jejuni meat OPU 22 OPU D8-MJ08-110 C. jejuni meat OPU 23 OPU D9-MJ08-111 C. jejuni meat OPU 24 OPU D10-MJ08-112 C. jejuni meat OPU 25 OPU D11-MJ08-114 C. jejuni meat OPU 26 OPU D12-MJ08-116 C. jejuni meat OPU 27 OPU D14-MJ08-140 C. jejuni meat OPU 28 OPU E1-P4724 C. jejuni meat OPU 29 OPU E2-P4727 C. jejuni meat OPU 30 OPU E3-P4728 C. jejuni rectal swab OPU


58

Table 3.1 Bacteria species used in this study (Cont.)

No Code Organism Source Origin 31 OPU E4-P4729 C. jejuni rectal swab OPU 32 OPU E5-P4792 C. jejuni rectal swab OPU 33 OPU E6-P4794 C. jejuni rectal swab OPU 34 OPU E7-P4814 C. jejuni rectal swab OPU 35 OPU E8-P4819 C. jejuni rectal swab OPU 36 OPU E9-P4825 C. jejuni rectal swab OPU 37 OPU E10-P4835 C. jejuni rectal swab OPU 38 OPU E19-P4545 C. jejuni rectal swab OPU 39 OPU E20-P4150 C. jejuni rectal swab OPU 40 C1 C. jejuni gamecock AHS TU** 41 C2 C. jejuni gamecock AHS TU 42 C3 C. jejuni gamecock AHS TU 43 C9 C. jejuni gamecock AHS TU 44 C10 C. jejuni gamecock AHS TU 45 C13 C. jejuni gamecock AHS TU 46 C14 C. jejuni gamecock AHS TU 47 C15 C. jejuni gamecock AHS TU 48 C17 C. jejuni bantam AHS TU 49 C20 C. jejuni bantam AHS TU 50 C23 C. jejuni gamecock AHS TU 51 C33 C. jejuni chicken AHS TU 52 C34 C. jejuni chicken AHS TU 53 C41 C. jejuni patient AHS TU 54 C43 C. jejuni patient AHS TU 55 C44 C. jejuni patient AHS TU 56 C46 C. jejuni patient AHS TU 57 C47 C. jejuni patient AHS TU 58 C48 C. jejuni patient AHS TU 59 C52 C. jejuni patient AHS TU 60 C54 C. jejuni patient AHS TU


59


No Code Organism Source Origin 61 C55 C. jejuni patient AHS TU 62 C57 C. jejuni patient AHS TU 63 C58 C. jejuni patient AHS TU 64 C60 C. jejuni patient AHS TU 65 C62 C. jejuni patient AHS TU 66 C63 C. jejuni patient AHS TU 67 C67 C. jejuni patient AHS TU 68 C79 C. jejuni patient AHS TU 69 C80 C. jejuni patient AHS TU 70 C93 C. jejuni chicken AHS TU 71 C94 C. jejuni patient AHS TU 72 C101 C. jejuni patient AHS TU 73 C106 C. jejuni patient AHS TU 74 C109 C. jejuni patient AHS TU 75 C. jejuni ATCC 33560 C. jejuni ATCC 33560 standard AHS TU 76 OPU A1-24h-39 C. coli meat OPU 77 OPU A9-24h-47 C. coli meat OPU 78 OPU A16-48h-27 C. coli meat OPU 79 OPU A22-48h-33 C. coli meat OPU 80 OPU A24-48h-35 C. coli meat OPU 81 OPU B1-24h-7 C. coli meat OPU 82 OPU D1-MJ06-4 C. coli meat OPU 83 OPU D2-MJ06-24 C. coli meat OPU 84 OPU D6-MJ08-107 C. coli meat OPU 85 OPU D13-MJ08-118 C. coli meat OPU 86 OPU D15-MJ08-152 C. coli meat OPU 87 OPU D16-MJ08-154 C. coli meat OPU 88 OPU D17-MJ09-f1 C. coli meat OPU 89 OPU D18-MJ09-f28 C. coli meat OPU 90 OPU D19-NNF07-1 C. coli meat OPU


60


No Code Organism Source Origin 91 OPU E11-P3291 C. coli rectal swab OPU 92 OPU E12-P3344 C. coli rectal swab OPU 93 OPU E13-P3458 C. coli rectal swab OPU 94 OPU E14-P3682 C. coli rectal swab OPU 95 OPU E15-P3810 C. coli rectal swab OPU 96 OPU E16-P3828 C. coli rectal swab OPU 97 OPU E17-P3961 C. coli rectal swab OPU 98 OPU E18-P4428 C. coli rectal swab OPU 99 C11 C. coli gamecock AHS TU 100 C32 C. coli chicken AHS TU 101 C45 C. coli patient AHS TU 102 C. coli ATCC 33559 C. coli ATCC 33559 standard AHS TU 103 OPU C1-NNF08-7 Campylobacter fetus meat OPU 104 OPU C6 C. fetus cow stool OPU 105 OPU B055 C. fetus cow stool OPU 106 CF TUH1 C. fetus patient TUH*** 107 CF PRA C. fetus patient AHS TU 108 C. fetus ATCC 27374 C. fetus ATCC 27374 standard AHS TU 109 C. lari ATCC 35221 C. lari ATCC 35221 standard OPU 110 C. upsaliensis C. upsaliensis standard OPU 111 OPU C2-32 C. hyointestinalis meat OPU 112 OPU C3-32 C. hyointestinalis meat OPU 113 OPU A11-24h-52 Arcobacter butzleri meat OPU 114 OPU A12-48h-12 A. butzleri meat OPU 115 OPU A13-48h-24 A. butzleri meat OPU 116 OPU A14-48h-25-1 A. butzleri meat OPU 117 OPU A15-48h-26 A. butzleri meat OPU 118 OPU A17-48h-28-1 A. butzleri meat OPU 119 OPU A18-48h-28-2 A. butzleri meat OPU 120 OPU A19-48h-30 A. butzleri meat OPU


61


No Code Organism Source Origin 121 OPU A20-48h-31 A. butzleri meat OPU 122 OPU A21-48h-32 A. butzleri meat OPU 123 OPU A23-48h-34 A. butzleri meat OPU 124 OPU A25-48h-36-1 A. butzleri meat OPU 125 OPU A26-48h-36-2 A. butzleri meat OPU 126 OPU A27-48h-37-1 A. butzleri meat OPU 127 OPU A28-48h-38 A. butzleri meat OPU 128 OPU A29-48h-39 A. butzleri meat OPU 129 OPU A30-48h-40 A. butzleri meat OPU 130 OPU A31-48h-41 A. butzleri meat OPU 131 OPU A32-48h-42 A. butzleri meat OPU 132 OPU A33-48h-43 A. butzleri meat OPU 133 OPU A34-48h-44 A. butzleri meat OPU 134 OPU A35-48h-45 A. butzleri meat OPU 135 OPU A36-48h-46 A. butzleri meat OPU 136 OPU A37-48h-47 A. butzleri meat OPU 137 OPU A38-48h-63 A. butzleri meat OPU 138 OPU A40-WT-5 A. butzleri water OPU 139 OPU A41-WT-6 A. butzleri water OPU 140 OPU A42-WT-8 A. butzleri water OPU 141 OPU A43-WT-10 A. butzleri water OPU 142 OPU A44-WT-12 A. butzleri water OPU 143 OPU B10-48h-28 A. butzleri meat OPU 144 OPU B11-48h-29 A. butzleri meat OPU 145 OPU B12-48h-30 A. butzleri meat OPU 146 OPU B13-48h-31 A. butzleri meat OPU 147 OPU B14-48h-32 A. butzleri meat OPU 148 OPU B15-48h-33 A. butzleri meat OPU 149 OPU B16-48h-34 A. butzleri meat OPU 150 OPU B17-48h-35 A. butzleri meat OPU


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No Code Organism Source Origin 151 OPU B18-48h-36 A. butzleri meat OPU 152 Sal1 Salmonella Tennessee patient AHS TU 153 Sal2 S. Derby patient AHS TU 154 Sal3 S. Stanley patient AHS TU 155 Sal4 S. Weltevreden patient AHS TU 156 Sal5 S. Muenchen patient AHS TU 157 Sal6 S. Agona patient AHS TU 158 Sal7 S. Brunei patient AHS TU 159 Sal8 S. Virchow patient AHS TU 160 Sal9 S. Krefeld patient AHS TU 161 Sal10 S. Poona patient AHS TU 162 Sal11 S. Welikade patient AHS TU 163 Sal12 S. Radar patient AHS TU 164 Sal13 S. Newport patient AHS TU 165 Sal14 S. Anatum patient AHS TU 166 Sal15 S. Infantis patient AHS TU 167 Sal16 S. Saint-paul patient AHS TU 168 Sal17 S. Bareilly patient AHS TU 169 Sal18 S. East bourne patient AHS TU 170 Sal19 S. Mbandaka patient AHS TU 171 Sal20 S. Livingstone patient AHS TU 172 Sal21 S. Kedougou patient AHS TU 173 Sal22 S. Lexington patient AHS TU 174 Sal23 S. Orion patient AHS TU 175 Sal24 S. Anatum patient AHS TU 176 Sal25 S. London patient AHS TU 177 Sal26 S. Blockley patient AHS TU 178 Sal27 S. Hvittingfoss patient AHS TU 179 Sal28 S. Heidelberg patient AHS TU 180 Sal29 S. Javana patient AHS TU


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No Code Organism Source Origin 181 Sal30 S. Typhimurium patient AHS TU 182 Sal31 S. Typhimurium patient AHS TU 183 Sal32 S. Paratyphi b patient AHS TU 184 Sal33 S. Emek patient AHS TU 185 Sal34 S. Choleraesuis patient AHS TU 186 Sal35 S. Enteritidis patient AHS TU 187 Sal36 S. Enteritidis patient AHS TU 188 Sal37 S. Enteritidis patient AHS TU 189 Sal38 S. Enteritidis patient AHS TU 190 S1 S. Enteritidis patient TUH 191 S2 S. Enteritidis patient TUH 192 S3 S. Enteritidis patient TUH 193 S4 S. Enteritidis patient TUH 194 S5 S. Enteritidis patient TUH 195 S6 S. Enteritidis patient TUH 196 S7 S. Enteritidis patient TUH 197 S8 S. Enteritidis patient TUH 198 S9 S. Enteritidis patient TUH 199 S10 S. Enteritidis patient TUH 200 S11 S. Enteritidis patient TUH 201 S12 Bacillus cereus patient TUH 202 S13 Aeromonas spp. patient TUH 203 S14 Plesiomonas spp. patient TUH 204 S15 Vibrio cholera patient TUH 205 S16 V. parahaemolyticus patient TUH 206 S17 Staphylococcus aureus patient TUH 207 S18 Escheria coli patient TUH 208 S19 E. coli patient TUH 209 S20 E. coli patient TUH 210 S21 E. coli patient TUH


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No Code Organism Source Origin 211 S22 E. coli patient TUH 212 S23 E. coli patient TUH 213 S24 E. coli patient TUH 214 S25 E. coli patient TUH 215 S26 E. coli patient TUH 216 S27 E. coli patient TUH 217 S28 E. coli patient TUH 218 S29 E. coli patient TUH 219 S30 E. coli patient TUH 220 S31 E. coli patient TUH 221 S32 E. coli patient TUH 222 S33 E. coli patient TUH 223 S34 E. coli patient TUH 224 S35 E. coli patient TUH 225 S36 E. coli patient TUH 226 S37 E. coli patient TUH 227 S38 Klebsiella pneumoniae patient TUH 228 S39 K. pneumoniae patient TUH 229 S40 K. pneumoniae patient TUH 230 S41 K. pneumoniae patient TUH 231 S42 K. pneumoniae patient TUH 232 S43 K. pneumoniae patient TUH 233 S44 K. pneumoniae patient TUH 234 S45 Proteus mirabilis patient TUH 235 S46 Enterobacter spp. patient TUH 236 S47 Citrobacter spp. patient TUH 237 S48 Citrobacter spp. patient TUH 238 S49 Citrobacter spp. patient TUH 239 S50 Citrobacter spp. patient TUH 240 OPU P1-GTC2020 Providencia alcalifaciens Gifu. Uni.v. OPU


65


No Code Organism Source Origin 241 OPU P2-Thai74-1 P. alcalifaciens patient OPU 242 OPU P3-NNF6 P. alcalifaciens beef OPU 243 OPU P4-TM3 P. alcalifaciens chicken OPU 244 OPU P5-AS-1 P. alcalifaciens patient OPU 245 OPU P6-GTC1501 P. heimbachae Gifu. Uni.v. OPU 246 OPU P7- GTC1263 P. rettgeri Gifu. Uni.v. OPU 247 OPU P8-P2234 P. rettgeri patient OPU 248 OPU P9-Thai51-3 P. rettgeri patient OPU 249 OPU P10-TM28 P. rettgeri beef OPU 250 OPU P11-GTC1054 P. rustigianii Gifu. Uni.v. OPU 251 OPU P12-con3 P. rustigianii human OPU 252 OPU P13-GTC1444 P. stuartii Gifu. Uni.v. OPU 253 OPU P14-TM9 P. stuartii beef OPU 254 S51 Acinetobacter baumannii patient TUH 255 S52 Enterococcus spp. patient TUH 256 S53 Shigella sonnei patient TUH 257 S54 Enterotoxigenic E. coli patient ASH TU 258 S55 Enteropathogenic E. coli patient ASH TU 259 S56 Enteroaggregrative E. coli patient ASH TU 260 S57 Enterohemorrhagic E. coli patient ASH TU 261 C. helveticus C. helveticus No data OPU

*OPU = Osaka Prefecture University. **AHS TU = Allied Health Science Faculty, Thammasat University. *** TUH = Thammasat University Hospital.


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3.2 Antigen preparation

3.2.1 Bacteria culture and storage C. jejuni, C. coli, C. lari, C. upsaliensis and C. helveticus isolates

were cultured on sheep blood agar (SBA; Blood agar base no. 2 [Oxoid Ltd., Basingstoke, United Kingdom] supplemented with 7% sterile sheep blood) under microaerophilic conditions (5% O2, 5% H2, 10% CO2, and 80% N2) at 42C for 48 hours. C. fetus and C. hyointestinalis were cultured on SBA at 37C for 48 hours under a microaerobic condition. Of non-Campylobacter spp., A. butzleri were culture at 37C for 48 hours under a microaerobic condition, other bacterial strains were culture on SBA at 37C, overnight. Bacterial cultures were harvested and suspended in 1 ml of phosphate-buffered saline (PBS; pH 7.0). Bacterial suspension was centrifuged at 10,000 rpm for 10 minutes. The supernatant was discarded then the pellet was dispensed in distill water and stored at -20C (Figure3.2).

3.2.2 Whole cell homogenate preparation by ultrasonication technique

3.2.2.1 Preparation of the cell suspension Cell pellet was suspended in distill water and mixed the

suspension gently. The suspension container was placed in an ice bath that completely covered the walls of the tube. A small amount of water was added into the ice bath.

3.2.2.2 Ultrasonic lysis The sonicator was setted time for 20 minutes and pulse

on/off for 20/25 seconds per cycle. The probe was cleaned with ethanol and placed it inside the tube. The bacterial suspension was sonicated until sample got clear (Figure 3.2).


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Figure 3.2 Whole cell homogenate preparation by ultrasonication technique.

3.2.3 Dry weight measurement of whole cell homogenate (Figure 3.3) 3.2.3.1 Aluminium dishes preparation Aluminium dishes were dried at 37C for overnight. Three

Dishes were used for one sample. The three empty aluminium dishes were dried on hotplate for 10 minutes and sequentially placed on balance. The empty aluminium dishes were weight (W1) were recorded (Figure 3.3 A). 3.2.3.2 Measurement

Whole cell homogenate was added into the aluminium dishes for 100 µl and dried on hotplate for 20 seconds. The sample aluminium dishes were removed from hotplate and placed on balance. Then the sample aluminum dishes were weighted (W2) and recorded (Figure 3.3 B). 3.2.3.3 Calculation

Whole cell homogenate dry weight was calculated by formula (mg/ml): Dry weight = (W2-W1) X 1000 W1 = weight of empty dish in milligrams 100 W2 = weight of sample and dish in milligrams


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Figure 3.3 Dry weight measurement of whole cell homogenate. A: aluminum dishes preparation and B: whole cell homogenate measurement. 3.3 Immunization Female BALB/c mice (6-8 weeks old) were immunized intraperitoneally with 0.2 ml of a mixture of 50 µg of C. jejuni 43 whole cell homogenate 0.15 ml emulsified in Imject Alum Adjuvant (Thermo Scientific, USA) 0.1 ml and normal saline 0.05 ml. At 14, and 28 days after the first immunization, the mice were boosted intraperitoneally with 100 µg of the immunogen that was similarly prepared as above. Mice were bled from the submucosal plexus to check their immune status. The antibody titers against C. jejuni43 were determined by indirect ELISA. Two-fold serial dilutions of the serum was prepared i.e. 1:200 to 1:409,600 and 100 µl of each dilution was added to 96-well plates precoated with the C. jejuni43 whole cell homogenate which was plated at 1 µg in 100 µl of coating buffer per well (10 µg/ml in 0.05 M carbonate-bicarbonate buffer, pH 9.6) and allowed to bind the plate overnight at 37C. After blocking the wells with 200 µl of bovine serum albumin (1% BSA in 0.01 M PBS, pH 7.4) at 37C, 1 hour, and 100 µl of serial dilution of serum


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from the immunized mice was added to each well at room temperature for 1 hour. Following three washes with PBS-T (0.05% Tween20 in 0.01 M PBS, pH 7.4) 100 µl of a 1:3,000 goat anti-mouse immunoglobulin (Ig)-horseradish peroxidase (Sigma, USA) dilution in PBS (0.2% BSA, 0.2% gelatin) was added and incubated at room temperature, 1 hour. The unbound conjugate was removed by washing with PBS-T, and then 70 µl of substrate (ABTS peroxidase substrate) was added and incubated in darkness at room temperature, 30 minutes. The reaction was stopped by adding 100 µl of 1% SDS, and the absorbance was measured at 405 nm. It was considered appropriate to perform a fusion if the titer of immune serum was more than 12,800. Three days before the fusion, the selected mouse was given intravenous booster with 50 µg of C. jejuni43 whole cell homogenate in normal saline solution without adjuvant (Figure 3.4). Figure 3.4 Immunization schedules of BALB/c mice with whole cell homogenate antigen.


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3.4 Hybridoma production

3.4.1 Splenectomy A BALB/c immunized mouse was sacrificed by cervical dislocation and placed onto its right side in a sterile tray. The mouse was liberally washed with 70% ethanol. An incision was made posterior to the left side of the ribcage. The skin was separated and scissors and forceps were used to remove a large skin flap over the area of the spleen. The facia and musculature were opened and the spleen was removed and washed in a dish containing 10 ml RPMI 1640 (Gibco, USA) medium containing 100 iu of penicillin and 100 µg streptomycin/ml (Pen Strep, Gibco, USA). The spleen was then placed into RPMI 1640 medium with another dish and was poked full of holes with a 26 gauge needle, five ml of RPMI 1640 media were gently forced through the spleen causing ejection of the spleen cells. The spleen cells are confirmed to be removed when the spleen color becomes relatively clear and then sieved through a sterile metal net to obtain a fine suspension. The cells and medium were placed into a 50 ml tube and centrifuged at 800 rpm for 5 minutes. The supernatant was removed. The cell was tapped loose and was counted in a cell counting chamber in preparation for fusion with P3X myeloma cells. 3.4.2 Preparation of Myeloma cells

P3X63Ag8.653 cells are permanently stored in liquid nitrogen. When needed, the P3X cells were removed from the liquid nitrogen tank and warmed rapidly to 37C for two minutes. The cells were resuspended into 8 ml of complete medium. These cells were allowed to grow for approximately 7 days in preparation for fusion with the B cells from the splenectomy. 3.4.3 Fusion

Complete medium was used to mix 5.1 X 108 spleen cells and 1.2

X 107myeloma cells. The cells were centrifuged at 1,200 rpm for 5 minutes and the

supernatant was discarded. The pellet was broken by gently tapping the bottom of the tube. A 1 ml volume of polyethylene glycol was routinely prepared on the day of the fusion to limit the formation of toxic peroxides. It was prepared by dissolving 1.5 g polyethylene glycol (PEG: average molecular weight 1,500), (Sigma, USA) with 3 ml PBS by heating in a microwave for 1 minute. It was added drop wise to the cell mixture with gentle shaking during 1 minute. After further 1 minute incubation, 1 ml of complete medium was added for 1 minute, followed by another 1 ml of medium for 1 minute. Finally, 8 ml of complete medium were added and the cells were


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centrifuged at 600 rpm for 5 minutes. The supernatant was removed and the cells were diluted to 10 ml with RPMI 1640 containing 20% fetal bovine serum (FBS), (Hyclone, Thermo Scientific, UK), 2 mM glutamine (Gibco, USA), 100 units/ml penicillin-streptomycin, HAT medium (100 µM Hypoxanthine, 4 X 10-7M Aminopterin, 1.6 X 10-5 M Thymidine) and 10% feeder. The cells were gently resuspended, added 100 µl to microtitre plate wells and incubated in a tissue culture incubator at 37C with 80% humidity and 5% CO2 (Figure 3.5). The hybridoma cells were monitored for everyday. HAT medium was replaced in every 3 days. At day 20, complete medium was replaced. The first observation of acidic pH adjustment in the media color and the cells were tested for antibody after at least 25% confluency was reached. Figure 3.5 Cell fusion procedures

3.4.3.1 Feeder preparation BALB/c mouse were sacrificed by cervical dislocation and

washed with 70% ethanol. The skin was separated and the spleen was removed. The RPMI 1640 medium was injected into the spleen and was then drawn back into the syringe, this process was repeated more three times. The macrophages suspended in the RPMI 1640 medium were then collected by centrifugation at 700 rpm for 5-8


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minutes. The supernatant was removed and the pellet was resuspended into 100 ml of complete media. The macrophages were used as feeder cells for the B-cell hybridomas. 3.4.3.2 Hybridoma screening by indirect ELISA

Supernatant from each well exhibiting hybridoma growth was tested by indirect ELISA for the presence of anti-C. jejuni 43 antibody using C. jejuni 43 whole cell homogenate. For indirect ELISA, C. jejuni 43 was plated at 1 µg in 100 µl of coating buffer per well and incubated overnight at 37C. After blocking the wells with 200 µl of bovine serum albumin at 37C for 1 hour. The plate was then emptied and unadsorbed antigen was removed by inverting the plate and tapping on paper towel. One hundred microlitre of hybridoma culture supernatants was added to each well. Following one hour incubation at room temperature, the primary antibody was dumped out and the plate was washed again three times with PBS-T followed by tapping on paper towel between each wash. Then, 100 µl of a 1:3,000 goat anti-mouse immunoglobulin (Ig)-horseradish peroxidase (HRP) dilution in PBS was added and incubated at room temperature for 1 hour. The secondary antibody was dumped and the plate was washed five times in PBS-T. A substrate was used to develop a deep blue color when reacted with HRP. The plate was allowed to develop for 30 minutes in the dark after which 1% SDS was added to stop the reaction. The reaction was read at a wavelength of 405 nm in an ELIA reader. The supernatant which produced an absorbance of at least 0.2 were initially considered positive. 3.5 Monoclonal antibody production

3.5.1 Selection of positive hybridomas Positive hybridoma was selected which based on the appearance of a clone with a strong reaction in an Indirect ELISA (3.4.3.2).

3.5.2 Limiting dilution Five tubes were prepared. A complete medium with feeder was

added into each tube for 1.5 ml. Hybridoma cells suspension were added to tube No. 1 for 15 µl and was transfer for 300 µl to tube No. 2 and was diluted in serial dilution until tube No. 5. The suspension of each tube was transfer to 96 wells tissue culture plate with volume 100 µl/well. (Figure 3.6) The plates were incubated for 5 days without changing the medium. Six days after cloning 50% of the complete medium with feeder was replaced. The first cloning plates were examined visually


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for the presence of only one clone per well. Up to 6 single clones ELISA positive wells were transferred to individual wells of a 24-well plate containing complete medium with feeder. After 2 to 3 days each clone was rescreened to monitor the level of antibody production and determine the hybridomas were selected for a second round of cloning. Hybridomas were cloned at least 3 times by limiting dilution.

Figure 3.6 Limiting dilutions of hybridoma cells 3.6 Storage and revival of hybridomas Long term storage of useful clones was achieved by employing a freezing solution containing 10% (v/v) dimethyl sulphoxide (DMSO) (Sigma, USA) in complete media. When the cells were 80% confluent in 25 cm2 flasks, the side of a 25 cm2 flask was vigorously tapped until most of the adherent cells had been released from the plastic. This was also facilitated by swirling the media in the flask in a circular notion. Ten millilitres of complete media was removed and transferred into a sterile 10 ml centrifuge tube. The cells were then pelleted by centrifugation at 800 rpm for 10 minutes and the supernatant was collected for diagnostic use. The pelleted cells


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were resuspended in 1 ml of pre-cooled freezing down solution (≤4C) by flicking. The cells were subsequently transferred into pre-cooled ampoules with appropriate labels. The ampoules were transferred to a sealed polystyrene box within a -80C freezer in order to freeze down the cells as slowly as possible, thus preventing the formation of ice crystals. Finally, the ampoules were transferred to canes in liquid nitrogen for long term storage (Figure 3.7). As necessary, a frozen hybridoma was revived by taking the required ampoule out of a cane, and soaking it in ethanol for 30 seconds to sterilize the outside of the ampoule. The ampoule was rapidly thawed in a 37C water bath. The lid was taken off in the biohazard hood and the cells were transferred to a flask and grown up overnight in a tissue culture incubator at 37C. Figure 3.7 Storage of hybridomas


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3.7 Monoclonal antibody characterization

3.7.1 Isotyping immunoglobulin subclasses (Thermo Fisher Scientific) A plate strips and TMB substrate were equilibrated to room

temperature. MAb was diluted with tris buffer saline (TBS) in dilution 1:50. A diluted MAb was added to each well of the 8-well strip in a volume 50 µl, then 50 µl of the goat anti-mouse IgG+IgA+IgM HRP conjugate was added to each well and mixed by gently tapping the palte. The plate was covered and incubated for 1 hour at room temperature and then was washed 3 times of wash buffer. TMB substrate was added for 75 µl to each well, a blue positive response were visible after 1 minute. After 5-15 minutes, stop solution was added for 75 µl. The stop solution was changed the color from blue to yellow. The plate was measured with a spectrophotometer at 450 nm, an absorbance were reading ≥0.2 is a positive response (Figure 3.8).

Figure 3.8 Immunoglobulin isotyping by rapid ELISA mouse mAb isotyping kit.


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3.7.2 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis The approximate molecular weight of C. jejuni 43 whole cell

homogenate was determined using sodium dodecylsulfate gel electrophoresis (SDS-PAGE) under reducing conditions using a 30% acrylamide resolving gel. Molecular weight markers (3.5-245 kDa) were used as standards to estimate the molecular weight of the C. jejuni 43 whole cell homogenate (BLUelf Prestained Protein Ladder, GeneDirex).

The gel casting apparatus was thoroughly cleaned. The buffer, water and acrylamide were mixed together in the appropriate proportions to make a 12 % (v/v) saperating gel. An ammonium persulphate solution 10% (w/v) was prepared and added to the gel solution which was vigorously degassed at room temperature for 10 minutes. The chemical catalyst, 10 µl of TEMED, was added to the gel. The resultant gel solution was swirled and poured into the gel casting mold until the gel solution was up to 3 cm of the top of the glass plates. An overlay solution of water was gently applied onto the gel surface. The gel was then left to set for 45 minutes. An upper, stacking gel was prepared in the same manner as the separating gel, using a 4 % (v/v) acrylamide solution. After removal of the overlay solution, the upper gel was poured onto the lower gel and a well-forming comb gently inserted without trapping air bubbles. The surface of the upper gel was covered with an overlay solution of water and then left at room temperature to set for 45 minutes. When the gel was set the comb was gently removed and the overlay solution poured off.

The casting mold was dismantled and the gel inserted into the running chamber. Tris-glycine buffer was poured into both the inner chamber and the outer chamber. The samples were diluted to 1:4 in sample buffer and then heated at 100C for 5 minutes and centrifuged at 10,000 rpm for 5 minutes before loading. Samples were also treated with proteinase K (PK), and peptide-N-glycosidase F (PNGase F). Briefly, 20 µg of proteinase K (Sigma-Aldrich, USA, 2.5 mg.mL-1 in lysis buffer) solubilized in 10 µl of lysing buffer was added to each boiled whole cell lysate and incubated at 37C for 60 minutes. Lysing buffer without PK (10µl) was added to paired controls before incubation at 37C for 60 minutes. For PNGase F (BioLabs inc., USA), whole cell lysate was denatured at 100C combined with buffer (2 µl GlycoBuffer 2 (10X), 2 µl 10% NP-40 and 6 µl H2O), then 1 µl of PNGase F was added and incubated at 37C for 60 minutes. Using an auto pipette, 5 µl of samples were injected into the wells. The middle lanes were used for the samples and the outer lane was used for pre-stained SDS-PAGE standards. The gel was run at constant voltage of 200V for the first half hour, or until the sampler front entered the lower


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gel, at which point the voltage was lowered to 45V. The gel was run at this voltage for 3 hours (Figure 3.9). Coomassie blue staining was done by soaking the gel in fixing solution at room temperature for 2 hours and then staining overnight. The gel was destained by constant stirring of the destaining solution at room temperature until the background became clear. The gel was photographed and stored in a zip lock plastic bag with a small amount of water. Silver staining was done by soaking the gel in fixing solution for 30 minutes then the fixing solution was removed. The sensitizing solution was added and leaved shaking for 30 minutes. The gel was washed with distill three times for 5 minutes each time. The silver solution was added and leaved shaking for 20 minutes the gel was washed with distill water two times for one minute each time. The developing solution was added and leaved shaking for 5 minutes, the gel was transferred to stop solution when the bands have reached desired intensity and leaved shaking for 10 minutes. The gel was washed with distill water three times for 5 minutes. The gel was photographed and stored in a zip lock plastic bag with a small amount of water. Figure 3.9 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis.


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3.7.3 Western blot The polyacrylamide gel was removed from the plates and

equilibrated in 300-400 ml of the transfer buffer for 30 minutes. When setting up the transfer blotting cartridges it was essential that all the air bubbles were removed between the polyacrylamide gel and the nitrocellulose membrane. Electrophoretic transfer was carried out 90 minutes at room temperature with 100V constant voltage (Figure 3.10). The sheet of nitrocellulose was removed from the polyacrylamide gel and any unreacted binding sites on the membrane blocked by incubating the membrane for 60 minutes in 5% skimmed milk. An aliquot of 10 ml MAb supernatant was added to the nitrocellulose sheet for 1 hour at room temperature. The nitrocellulose was then washed with 4 times of PBS-T. Goat anti-mouse IgG alkaline phosphatase was used at a dilution of 1:3000 in diluents. The nitrocellulose sheet was incubated with the secondary antibody solution for 1 hour at room temperature. The nitrocellulose was again washed with 5 times of PBS-T. The nitrocellulose sheet was then incubated with BCIP/NBT substrate solution (Amresco, USA). The reaction was stopped by pouring off the substrate solution and washing the nitrocellulose sheet with distilled water (Figure 3.11).

Figure 3.10 Protein transfer to nitrocellulose membrane.


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Figure 3.11 Western blot procedure.


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3.7.4 Indirect ELISA All bacteria strains (Table 3.1) were plated at 1 µg in 100 µl of

coating Buffer (10 µg/ml in 0.05 M carbonate-bicarbonate buffer, pH 9.6) per well and allowed to bind the plate overnight at 37C. After blocking the wells with 200 µl of bovine serum albumin (1% BSA in 0.01 M PBS, pH 7.4) at 37C, 1 hour, 100 µl of MAb2D10 was added to each well at room temperature for 1 hour. Following three washes with PBS-T (0.05% Tween20 in 0.01 M PBS, pH 7.4) 100 µl of a 1:3,000 goat anti-mouse immunoglobulin (Ig)-horseradish peroxidase (Sigma, USA) dilution in PBS (0.2% BSA, 0.2% gelatin) was added and incubated at room temperature, 1 hour. The unbound conjugate was removed by washing with PBS-T, and then 70 µl of substrate (ABTS peroxidase substrate) was added and incubated in darkness at room temperature, 30 minutes. The reaction was stopped by adding 100 µl of 1% SDS, and the absorbance was measured at 405 nm. An absorbance at least 0.5 were initially considered positive (Figure 3.12). Figure 3.12 Indirect ELISA procedure.


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3.7.5 Dot blot ELISA Whole cell homogenate of bacteria in Table 3.1 (1µg/dot) was applied on nitrocellulose membrane in volume 3 µl. After dotting onto the membrane, the Ag was left to dry for several minutes. Subsequently, the membrane was blocked using 5% Bovine serum albumin (BSA) in PBS-T (PBS with 0.05% Tween 20) for 1 hour at room temperature (RT) under gentle orbital shaking. The blocking reagent was then poured off and then they were incubated with 10 ml of MAb at RT under gentle orbital shaking for 1 hour. The membranes were washed with PBS-T. The secondary antibody (Goat anti-mouse IgG alkaline phosphatase) diluted 1:3,000 with diluent was incubated with the membrane for 1 hour at room temperature under gentle orbital shaking. After incubation, the membranes were washed with PBS-T for 3 times and the reagent was replaced by Tris pH 9.6 for 1 time. The BCIP substrate solution was added, after short incubation, development of a purple-pink color of insoluble substrate product on the dot was observed by naked eye (Figure 3.13).

Figure 3.13 Dot blot ELISA procedure.


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Figure 3.14 Monoclonal antibody production and characterization in this study.


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Chapter 4 Results and Discussion

4.1 Monoclonal antibody production Female BALB/c mice were immunized with whole cell homogenate of C. jejuni 43 (Cj43). The antibody titers were assessed by indirect ELISA after the third immunization. The mouse with the highest titer (1:102,400) was boosted and used as

a splenocyte donor in the hybridoma production. A total of 5.1 x 107

spleen cells

were obtained from this mouse and were fused with about 1.2 x 108 myeloma cells.

Four hybridoma clones were selected and were tested for antibodies against the whole cell homogenate of C. jejuni 43 by indirect ELISA (Table 4.1). From the screening results, only one hybridoma clone, name 2D10, were found to retain the antibody producing capability after sub-culturing and did not react with whole cell homogenate of E. coli by indirect ELISA (Table 4.2). The hybridoma was recloned by limiting dilution method. The clone designed 2D10B10E9D7B9D9F3 produced high antibody titer (A405 = 1.924) and the monoclonal antibody (MAb) was named MAb2D10. Another hybridoma clones were storage into liquid nitrogen tank. Table 4.1 Indirect ELISA reactivity of hybridoma supernatant against C. jejuni43.

Hybridoma clone Absorbance at 405 nm. 6F3 0.238

2D10 1.410 2F5 0.274 4F4 0.169


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Table 4.2 Indirect ELISA reactivity of 2D10 reclone supernatant against C. jejuni43 and E. coli.

2D10 clone Absorbance at 405 nm. C. jejuni 43 E. coli

A6 2.041 0.02 A10 1.931 0.014 B2 0.795 0.014 B4 0.305 0.013 B5 1.867 0.017 B10 1.910 0.022 B12 1.869 0.009 C5 0.031 0.004 C7 0.040 0.005 C9 0.078 0.009 C11 1.98 0.008 D4 2.109 0.012 D6 0.05 0.012 D9 0.065 0.009 D12 1.789 0.005 F10 1.919 0.003 F11 1.848 0.001


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4.2 Monoclonal antibody characterization 4.2.1 Isotyping immunoglobulin subclasses MAb2D10 was isotyped by using the mouse typer subisotyping kit (Thermo Scientific). The isotypes was IgG1 and carried kappa type light chain. 4.2.2 SDS-PAGE and western blot profile SDS-PAGE and western blotting were used to analyze the antigen recognized by MAb2D10. MAb2D10 was used to probe nitrocellulose membranes containing cellular proteins of Campylobacter that had been separated in SDS-PAGE. Treatment of whole cell lysate of C. jejuni 43 with proteinase K and peptide-N-glycosidase F showed that the antigens were digested by proteinase K but, no effect was observed with peptide-N-glycosidase F enzyme (Figure 4.1). Western blotting revealed that MAb2D10 bound to proteins migrating at molecular masses ranging from 19-72 kDa. Three major protein bands were observed with molecular masses 25, 28 and 31 kDa. Using whole cell lysate of C. coli, C. lari and C. upsaliensis, the MAb reacted with different protein profile at molecular masses ranging from 20-72, 25-70 and 19-62 kDa, respectively. The 25, 28 and 31 kDa were observed as major protein band of C. lari, C. upsaliensis and C. coli, respectively (Figure 4.2). Moreover, MAb 2D10 recognized protein profile common of four C. jejuni and three C. coli strains tested (Figure 4.3).


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Figure 4.1 SDS-PAGE profile of whole cell homogenate C. jejuni43. Lane1: molecular weight standard; lane2: whole cell homogenate C. jejuni43; lane3: whole cell homogenate C. jejuni43 treated with proteinase K; lane4: whole cell homogenate C. jejuni43 (proteinase K negative control); lane5: whole cell homogenate C. jejuni43 treated with PNGase F; lane5: whole cell homogenate C. jejuni43 (PNGase F negative control). A: coomassie stain, B: silver stain.


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Figure 4.2 SDS-PAGE and Western blot analysis of whole cell homogenate of Campylobacter. A coomassie brilliant blue-stained gels are shown on the left (lanes2-7) with the corresponding blots, probed with MAb2D10, on the right (lanes8-13). Lane 1: molecular weight standard; lanes 2,8: C. jejuni; lanes 3,9: C. coli; lanes 4,10: C. lari; lanes 5,11: C. upsaliensis; lanes 6,12: C. fetus and lanes 7,13: C. hyointestinalis.


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Figure 4.3 Western blot analyses of C. jejuni (lanes1-4) and C. coli (lanes5-7) reacted with MAb2D10, showed common protein profile in different isolates. Lane 1 C. jejuni 43; lane 2 C. jejuni ATCC 33560; lane 3 C. jejuni 70; lane 4 C. jejuni H604; lane 5 C. coli 45; lane 6 C. coli cc1; lane 7 C. coli ATCC 33559.


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4.2.3 Cross reactivity of MAb The specificity of MAb2D10 was assayed by indirect ELISA and dot blot ELISA with bacteria whole cell lysate listed in table 3.1. MAb2D10 reacted with all C. jejuni, C. coli, C. lari and C. upsaliensis but did not react with C. fetus, C. hyointestinalis and non Campylobacter species (Table 4.3). Table 4.3 Cross reaction of MAb 2D10 with lysate of bacteria by indirect ELISA and dot-blot ELISA

Bacteria No. of positive isolates/ total isolates (%) Indirect ELISA Dot blot ELISA

C. jejuni 68/68 (100) 75/75 (100) C. coli 27/27 (100) 27/27 (100) C. lari 1/1 (100) 1/1 (100) C. upsaliensis 1/1 (100) 1/1 (100) C. helveticus 1/1 (100) 1/1 (100) C. fetus 0/6 (0) 0/6 (0) C. hyointestinalis 0/2 (0) 0/2 (0) A. butzleri 0/39 (0) 0/39 (0) Salmonella spp. 0/39 (0) 0/49 (0) S. sonnei 0/1 (0) 0/1 (0) Bacillus cereus 0/1 (0) 0/1 (0) Aeromonas spp. 0/1 (0) 0/1 (0) Plesiomonas spp. 0/1 (0) 0/1 (0) V. cholerae 0/1 (0) 0/1 (0) V. parahaemolyticus 0/1 (0) 0/1 (0) S. aureus 0/1 (0) 0/1 (0) E. coli 0/1 (0) 0/24 (0) K. pneumonia 0/1 (0) 0/7 (0) P. mirabilis 0/1 (0) 0/4 (0) Enterobacter spp. 0/1 (0) 0/3 (0) Citrobacter spp. Not done 0/4 (0) P. alcafaciens 0/14 (0) 0/14 (0) A. baumannii 0/1 (0) 0/1 (0) Enterococcus spp. 0/1 (0) 0/1 (0)


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4.3 Discussion The purpose of the present study was to generate Monoclonal antibody (MAb) specific to Campylobacter species as only few researchers have ever developed MAbs for detection of these bacteria. Brook et al.(143), produced nine MAbs from whole cell lysate components of C. jejuni. These MAbs, reacted with one or more proteins in the molecular masses 10, 23, 29, 30, 31, 33, 62 and 92 kDa, and were specific to C. jejuni and C. coli. In the study of Chaiyaroj et al.(141), a MAb was produced from veronal extract of C. jejuni which reacted with 30, 45 and 55 kDa proteins and was able to detect C. jejuni and C. coli. Lu et al.(140) reported four MAbs from whole cell lysate components of C. jejuni. Two MAbs reacted with 62 kDa while the two other reacted with 92 and 31 kDa, respectively. These four MAbs were specific to C. jejuni, C. coli and C. lari. However, all MAbs were found to cross-react with non-Campylobacter strains. Heo et al.(144) produced MAb from sonicated antigen of C. jejuni. The MAb, recognized antigens of 62 and 43 kDa, and was specific to C. jejuni and C. coli. Qian et al.(142) produced MAb directed against the major outer membrane protein (MOMP) of C. jejuni. The MAb bound to a single protein band approximately 43 kDa and reacted specifically for C. jejuni. Huang et al.(149) reported MAb reacted with surface components of at approximately 81, 61, 54, 51, 45 and 34 kDa. The MAb was specific to thermotolerant Campylobacter spp. In this study, whole cell lysate of C. jejuni were used as antigen, MAb 2D10 was produced. The MAb, recognized protein at the molecular masses ranging from 19-72 kDa and, was specific to C. jejuni, C. coli, C. lari, C. upsaliensis and C. helveticus. Most of MAbs were produced for detection and identification of Campylobacter spp. were designed only for C. jejuni and C. coli (141,142,144,145) but rare MAbs specific for C. jejuni, C. coli, C. lari, C. upsaliensis and C. helveticus . MAb2D10 had ability to detect these five species and did not show any cross reaction to non-Campylobacter bacterial strains. According to the study of Huang et al.,(149) a MAb was produced and characterized. The MAb was specific to thermotolerant Campylobacter spp. (C. jejuni, C. coli, C. lari, C. upsaliensis and C. helveticus).

The most important Campylobacter spp. causing human infections are the thermophilic species C. jejuni, C. coli, C. lari, and C. upsaliensis (150,151). C. jejuni belong to the most frequent bacterial pathogens that cause gastrointestinal infection in human (152), while C. coli which cause similar clinical symptoms is less frequently detected than C. jejuni (153). C. lari and C. upsaleinsis were reported as a commom cause of acute bacterial gastroenteritis (154,155), both species have been recognized


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as emerging human pathogens (151). C. helveticus was isolated from faeces of diarrheic and healthy domestic cats, and more rarely from dogs. The possibility of transmission of C. helveticus from pets to man would be an important subject of investigation (173). These species are often referred to as thermotolerant campylobacters because of their optimal growth at 42C and are also associated prominently with poultry by colonizing preferentially the avian gastrointestinal tract (156). The occurrence of human campylobacter gastroenteritis has been largely attributed to the consumption of contaminated food animal products, especially poultry. Detection of thermophilic Campylobacter in food or animal products can be eliminating the infection. In this study, MAb2D10 was specific to C. jejuni, C. coli, C. lari, C upsaliensis, and C. helveticus. This may be give a tool for early detect the thermophilic Campylobacter. In previous studies, indirect ELISA and dot blot have been characterized MAbs against Campylobacter spp. (140,141,144-146) and other bacteria (139,157-159). The indirect ELISA has been shown to be a highly sensitive technique capable of detecting antibodies to a wide variety of antigens (160). Dot blot assay serve as alternative to plate ELISA and possesses distinct advantages. In this study, the specificity of MAb2D10 was characterized by indirect ELISA and dot blot. The result of both techniques showed that MAb2D10 reacted with all isolates of C. jejuni, C. coli, C. lari, C. upsaliensis, and C. helveticus. Furthermore, there was no cross-reactivity with other species of enteropathogenic bacteria. Although the specificity of the MAb by indirect ELISA and dot blot was not different but dot blot was a preferable technique. Dot blot was rapid and simple to perform, according with the previous studied, Hawks et al. (161) revealed that the dot blot assay is reported to be of equal or greater sensitivity than ELISA and can function over a wide range of antigen-antibody ratios. An additional advantage is that the results are easily interpreted without an ELISA reader. Belo et al. (162) described dot ELISA has a number of advantages over standard ELISA. The nitrocellulose membrane is capable of binding more antigens than the microtiter plates. Additionally, the reaction on nitrocellulose membrane is viewed against the membrane white background, making it much easier to identify a positive or negative reaction. The cellular components of C. jejuni that reacted with MAb2D10 was determined by western blot. The Mab reacted with protein at the molecular masses ranging from 19-72 kDa. Three major protein bands were observed with molecular masses 25, 28 and 31 kDa. In previous studied, the 28 kDa was identified as a surface protein of C. jejuni (163). The MAbs to 28 kDa were potentially suitable for rapid


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identification of bacterial cells (164). The 31 kDa was protein which may be a component of a microcapsular structure that located on the surface of the Campylobacter cell wall. The 31 kDa was identified as protein common to thermophilic Campylobacter (164-167). However, the 25 kDa protein have never been reported. The outer membrane of Campylobacter spp. includes lipopolysaccharide and specific proteins, which are exposed on the surface of the bacterium and are antigenic to human. The surface components play an important role in several virulence functions (168). C. jejuni surface components have been studied extensively and form a multiple antigen complex. Blaser et al. (169) reported the major outer membrane protein (OMP) that migrates between a molecular weight of 41-45 kDa of C. jejuni. Newell. (170) reported the 63 kDa protein that was identified as a flagella of Campylobacter spp. Pei et al. (164) reported the 28 and 31 kDa proteins were characterized as major antigenic proteins of C. jejuni and C. coli. They are similar in amino acid compositions but differ in amino-terminal sequence. According to this study, the MAb2D10 reacted with protein antigen of C. jejuni at the molecular mass 28 and 31 kDa. The protein antigens from Campylobacter spp. which MAb2D10 reacted may be surface protein. However, reactivity of MAb2D10 to the surface protein will need to be confirmed. MAb2D10 showed reactions to multiple bands as same as the study of Haung (149), the MAb (M1169) reacted with protein at approximately 81, 61, 54, 51, 45, and 34 kDa with predominant reaction to the bands at 61, 54, and 51. A mixture of eight strains of C. jejuni, C. coli, and C. lari was prepared either live or formalin-killed and used as antigens. In this study one strain of C. jejuni was socinated as whole cell homogenate antigen. Both MAb were specific to thermotolerent Campylobacter and did not show cross reaction with non Campylobacter spp.

The ability to distinguish between Campylobacter spp. is important in the identification of Campylobacter sources and transmission routes (171,172). Klena et al. described a multiplex PCR to identified and discriminated between C. coli, C. jejuni, C. lari, and C. upsaliensis (14). In this study, the western blot result showed that MAb2D10 recognized different protein profile from C. jejuni, C. coli, C. lari and C. upsaliensis. Unlike other MAbs against Campylobacter, MAb2D10 had ability to discriminate among four species by Western blot. It could be applied to identify and discriminate the thermophilic Campylobacter in food or clinical specimens.


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Chapter 5 Conclusions and Recommendations

Campylobacteriosis, a food- and water-borne zoonotic illness caused by

bacteria of the genus Campylobacter, is one of the leading cause bacterial gastroenteritis worldwide. C. jejuni is most often implicated as the causative agent of campylobacteriosis, followed by C. coli, C. upsaliensis, and C. lari. Campylobacter is increasingly an incidence and resistant to antibiotics. It has already been recognized as a public health problem. Detection of Campylobacter infection is important for both treatment and epidemiological surveillance. Laboratory diagnosis of Campylobacter requires cultural isolation the organism and identification using biochemical tests. However, cultural procedures and biochemical test are labor intensive and time consuming thus there is a need for rapid, simple and reliable diagnostic procedures for detection these organisms. Monoclonal antibodies are considered useful tools for identifying bacteria. In the present study, Monoclonal antibody against Campylobacter spp. was produced and characterized. The results of this study indicate that MAb 2D10 could be useful for detecting C. jejuni, C. coli, C. lari, C. upsaleinsis , and C. helveticus. Unlike other MAbs against Campylobacter, the MAb had ability to detect four species that recognized as the important pathogenic Campylobacter (150,151). Moreover, MAb2D10 did not show any cross reaction to non-Campylobacter bacterial strains. This MAb could potentially have other applications for developing diagnostic tests for thermotolerant Campylobacter. Further studies are requiring identifying target protein antigen of MAb and developing an immunological tool for rapid identification of Campylobacter spp. Latex agglutination is attractive technique.


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APPENDICES


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APPENDIX A CHEMICALS

Chemical Molecular weight (g/mol) Source

Absolute ethanol (C2H6O) 46.07 E.Merck, Darmstadtt, Germany. Absolute methanol (CH3OH) 32.04 E.Merck, Darmstadtt, Germany. Acetic acid (CH3COOH) 60.05 E.Merck, Darmstadtt, Germany. Acrylamide (CH2=CHCONH2) 71.08 Sigma, USA. Aminopterin (C19H20N8O5) 440.41 Sigma, USA. Ammonium persulfate ((NH4)2S2O8) 228.20 Sigma, USA. Ammonium sulfate (NH4)2SO4 132.14 Sigma, USA. Disodium phosphate (Na2HPO4) 141.96 Sigma, USA Glycerol (C3H8O3) 92.09 BDH Laboratory Supplies, England. HEPES (C8H18N2O4S) 238.30 Sigma, USA. Hydrochloric acid (HCl) 36.50 E.Merck, Darmstadtt, Germany. Hypoxanthine (C5H4N4O) 136.11 Sigma, USA. Monopotassium phosphate 136.08 Sigma, USA (KH2PO4) Potassium chloride (KCl) 74.55 Sigma, USA. Sodium acetate (CH3CooNa) 82.03 USB, Cleveland, USA. Sodium azide (NaN3) 65 E.Merck, Darmstadtt, Germany Sodium bicarbonate (Na2HCO3) 84 E.Merck, Darmstadtt, Germany Sodium carbonate (Na2CO3) 105.99 E.Merck, Darmstadtt, Germany. Sodium chloride (NaCl) 58.44 E.Merck, Darmstadtt, Germany.


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Chemical Molecular weight (g/mol) Source Sodium dodecyl sulfate (SDS) 288.38 Sigma, USA. (CH3(CH2)11OSO3Na) Sodium hydroxide (NaOH) 40.00 E.Merck, Darmstadtt, Germany. Tetramethylethylenediamine 116.21 ThermoFisher (TMED) Scientific, USA. Thymidine (C10H14N2O5) 242.23 Sigma, USA. Tris (Hydroxymethyl aminomethane) 121.14 USB, Cleveland, USA. [NH2C(CH2OH)3]


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APPENDIX B INSTRUMENTS AND REAGENTS

1. Bacteria culture

Instruments and materials - Balance (Mettle Toledo, Switzerland) - Incubators (Mettle Toledo, Switzerland) - Biosafety cabinet (Holten Lamin Air, Scientific promotion co. LTD) - Duran bottle 1,000 ml - Petri dish - Hot plate stirrer - Autoclove (TOMY, Japan)

- Magnetic bar - Inoculating loops Media - Blood agar base (Oxoid, England) - Sheep blood (CLINAG Co. LTD)

2. Whole cell lysate preparation Instruments and materials - Ultrasonicator (Vibra-Cell Ultrasonic Liquid Processor)

- Centrifuge - Microcentrifuge tube - Autopipette - Balance - Hot plate - Aluminum foil

Reagents - Ultrapure distilled water (UDW) - PBS (0.01 M, pH 7.4)

3. SDS-PAGE

Instruments and materials - Protein electrophoresis equipment (Bio RAD, Switzerland) - Power supply (Bio-RAD, USA) - Autopipette and pipette tips - Microcentrifuge tubes 1.5 ml (Trefflab, Switzerland) - Heat box

Reagents - UDW - 30% Acrylamide - 1.5 M Tris base pH 8.8 - 0.5 M Tris base, pH 6.8 - 10% SDS - 10% APS


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- TEMED - 95% glycerol - Protein marker - 1X running buffer - 10X loading buffer

4. Comassie Brilliant Blue staining

Instrument and material - Shaker (Scientific Promotion co.LTD) - Tray Reagents - Fixing solution - Comassie Brilliant Blue G-250

5. Protein transfer to nitrocellulose membrane and western blot

Instruments and materials - Protein electrophoresis equipment (Bio RAD, Switzerland) - Power supply(Bio-RAD, USA) - Autopipette and pipette tips - Filter paper - Nitrocellulose membrane - Sponge pad - Centrifuge - Gel - Ice - Shaker - Tray - Microcentrifuge tubes 1.5 ml (Trefflab, Switzerland) - Cutter - Forceps - Heat box - Plastic box - Plastic tubes - Parafilm Reagents - UDW - TEMED - 40% Acrylamide - 1% SDS - 1.5 M Tris base pH 8.8 - 0.5 M Tris base pH 6.8 - 10% SDS - 10% APS - 10X loading buffer - 1X running buffer - Protein marker - Transfer buffer - Monoclonal antibody (MAb2D10) - 5% skim milk - PBS (0.01M, pH 7.4) - PBS-T - Conjugate (Goat anti-mouse IgG-Alkaline Phosphatase) - Nitrocellolose membrane blotted with antigen - Substrate (BCIP/NBT) - Diluent


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6. Indirect ELISA

Instruments and materials - 96 well microtiter plates (Nunc-Immuno Microwell) - ELISA reader - Autopipette and pipette tips - Multichannel autopipette - Tray

Reagents - Coating buffer - Blocking solution - PBS-T - Diluent - Conjugate (Goat anti-mouse IgG-Horseradish Peroxidase) - Substrate (ABTS peroxidase substrate) - Monoclonal antibody (MAb2D10) - 1% SDS

7. Dot blot ELISA

Instruments and materials - Nitrocellulose membrane - Autopipette and pipette tips - Tray

Reagents - Blocking solution - UDW

- PBS-T - Diluent - Nitrocellolose membrane blotted with antigen - Monoclonal antibody (MAb2D10) - Tris, pH 9.6 - Conjugate (Goat anti-mouse IgG-Alkaline Phosphatase) - Substrate (5-Bromo-4-Chloro-3-Indolyl-Phosphate/NitroBlue Trteazolium)


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APPENDIX C MEDIA AND REAGENTS PREPARATION

1. Sheep blood agar

Materials - Blood agar base No. 2 (Oxoid, England) 40 g - Distilled water 1,000 ml - Sheep blood 70 ml - Duran bottle - Hot plate stirrer - Magnetic bars - Autoclave - Petridish Procedure Forty grams of blood agar base No. 2 were suspended in 1,000 ml of

distilled water. The medium was boiled to dissolve completely and was sterilized by autoclaving at 121C for 15 minutes. The medium was cooled to 45-50C then sterile sheep blood was added of volume 70 ml, mixed well gently. The medium was dispensed 15 ml into a petridish. Sheep blood agar plates were store at 2-8C.

2. SDS-PAGE reagents 2.1 Tris-HCl (1.5M, pH 8.8): stock buffer for separating gels.

Materials and reagents - Tris base 18.15 g - Conc. HCl - UDW 50 ml - pH meter - Balance - Beaker - Magnetic bar

Procedure Tris base 18.15 g were dissolved in 40 ml of UDW. The solution was adjusted the pH to 8.8 by concentrated HCl then Brought up the volume to 50 ml with UDW. 2.2 Tris-HCl (0.5M, pH 6.8): stock buffer for stacking gels.

Materials and reagents - Tris base 6.05 g - Conc. HCl - UDW 50 ml - pH meter - Balance - Beaker - Magnetic bar


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Procedure Tris base 6.05 g were dissolved in 40 ml of UDW. The solution was adjusted the pH to 6.8 by concentrated HCl then brought up the volume to 50 ml with UDW. 2.3 10% Ammonium persulfate (APS) Materials and reagents - APS 0.1 g - UDW 1,000 µl

- Beaker - Balance - Autopipette and tips Procedure Ammonium persulfate were weighted at 0.1 g and were dissolved into

800 µl of UDW, then brought up the volume to 1,000 µl. Store at 4C. 2.4 10X Tris-glycine running buffer Materials and reagents - Tris base 30.25 g - Glycine 144 g - 20% SDS 50 ml - UDW - Balance

Procedure Tris base and glycine were weighted at amount in above. Twenty percent of SDS was added in volume 50 ml, then brought up the volume to 1,000 ml with UDW. 2.5 2X sample loading buffer Materials and reagents

- 1M Tris, pH 7.3 5 ml - 20% SDS 25 ml - Glycerol 20 ml - Bromophenol blue 2 mg - UDW - Balance

Procedure All reagents at above were added and then brought up the volume to 100 ml with UDW.


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3. Coomassie brilliant blue 3.1 Coomassie brilliant blue reagent Materials and reagents - O-phosphoric acid 0.95 ml - Ammonium sulfate (NH4)2SO4 0.4 g - Coomassie blue G-250 0.005 g - Methanol 10 ml - UDW 40 ml

Procedure Coomassie blue G-250 0.005 g and ammonium sulfate 0.4 g were dissolved in 10 ml of methanol. UDW and O-phosphoric acid were added in a volume 40 ml and 0.95 ml into solution respectively. Coomassie brilliant blue reagent were kept at room temperature. 3.2 Fixing solution Materials and reagents - Absolute ethanol 25 ml - Glacial acetic acid 37.5 ml - UDW

Procedure Absolute ethanol 25 ml and glacial acetic acid 37.5 ml were pooled together and then brought up the volume to 500 ml with UDW.

4. Buffers for western blotting 4.1 10X transfer buffer Materials and reagents - Tris base 30.3 g

- Glycine 144 g - UDW

Procedure Tris base and glycine were mixed with weight above then brought up the volume to 1,000 ml with UDW. 4.2 1X transfer buffer (1,000 ml) - Cold UDW 700 ml - 10X transfer buffer 100 ml - Methanol 200 ml


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5. Indirect ELISA reagents 5.1 Coating buffer (carbonate – bicarbonate buffer, pH 9.6)

Materials and reagents - NaHCO3 2.93 g - NaCO3 1.59 g - UDW Procedure NaHCO3 2.93 g and NaCO3 1.59 g were dissolved with UDW 700 ml. The

solution was stirred for few minutes and was adjusted the pH to 9.6 with NaOH. The solution was brought up the volume to 1,000 ml with UDW and was kept at 4C. 5.2 Diluents (0.2% BSA, 0.2% gelatin) Materials and reagents - 0.01M PBS, pH 7.4 300 ml - BSA 0.6 g - Gelatin powder 0.6 g Procedure A gelatin powder 0.6 g was boiled with 0.01M PBS, pH 7.4. When the reagent was cooled at room temperature, then BSA 0.6 g. was added. The diluents were kept at -20⁰C. 5.3 Blocking solution (1% BSA in 0.01 M PBS, pH 7.4) Materials and reagents - 0.01M PBS, pH 7.4 500 ml - BSA 5 g Procedure BSA 5 g was dissolved with 0.01M PBS, pH 7.4 500 ml then the solution were kept at -20⁰C. 5.4 5% Skim milk Materials and reagents - Skim milk powder 2.5 g - UDW 50 ml Procedure Skim milk powder 2.5 g was dissolved with UDW 50 ml the solution were kept at -20⁰C.


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5.5 PBS-T (Washing solution) Materials and reagents - NaCl 8 g - Na2HPO4 1.44 g - KH2PO4 0.24 g - KCl 0.2 g - Tween20 2 ml - UDW Procedure All reagents were dissolved in 800 ml of UDW. The solution was adjusted to pH 7.2 and was brought up the volume to 1,000 ml with UDW.


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BIOGRAPHY

Name Mrs. Narissara Mungkornkaew Date of Birth February 18, 1980 Educational Attainment Academic Year 2002: Bachelor of Science

(Medical Technology), Thammasat University, Thailand

Publication 1. Pootong A, Mungkornkaew N, Chanphorng A, and Cowawintaweewat S. Evaluation of VersaTREK automated microbial detection system. J Med Tech Assoc Thailand. 2011;39.

2. Apisarnthanarak A, Khawcharoenporn T, Thongphubeth K, Yuekyen C, Damnin S, Mungkornkaew N, and Mundy LM. Postflood pseudofungemia due to Penicillium species. Clin Infect Dis. 2012;55:e9-e11.

3. Shima A, Hinenoya A, Samosornsuk W, Samosornsuk S, Mungkornkaew N, and Yamasaki S. Prevalence of Providencia strains among patients with diarrhea and in retail meats in Thailand. Jpn J Infect Dis. 2016;69:323-25.

4. Niyomdecha N, Mungkornkaew N, Samosorksuk W. Serotype and antimicrobial resistance of Salmonella enterica isolated from pork, chicken meat and lettuce, Bangkok and central Thailand. Southeast Asian J Trop Med Public Health. 2016; 47:31-9. Work Experiences

1. Medical Faculty, Thammasat University, 2002-2003. 2. Microbiology Laboratory, Thammasat University Hospital, Thammasat

University, 2003-present.


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production and characterization of monoclonal antibody...

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