Detection of Antibiotic Residues in Broiler Chickens in Gaza Strip

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2 Islamic University-Gaza Deanship of Graduate Studies Faculty of Science Biological Sciences Master Program / Microbiology الجاهعت االسالهيت غزة عوادة الدراساث العليا كليت العلوم بزناهج هاجستيز العلوم الحياتيت األحياء الدقيقت Detection of Antibiotic Residues in Broiler Chickens in Gaza Strip By Mohammed A. Albayoumi Supervisor Prof. Dr. Abdelraouf A. Elmanama Ph. D Microbiology A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Biological Sciences/ Microbiology May 2015

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5 Dedication This research is dedicated to my parents, my wife and my children The research is also dedicated to Ministry of Agriculture, Palestine. Finally, I dedicate this work to everyone who assisted in the success and achievement of this work. I

6 Acknowledgment I would like to express my appreciation to my supervisor, Professor Abdelraouf A. Elmanama, for his support, patience and encouragement throughout my research. His technical and editorial advices were essential to the completion of this dissertation and he has taught me innumerable lessons and insights on the workings of academic research in general. I am extremely grateful to Palestinian Health Research Council for providing a research grant, which helped in the accomplishment of this thesis. My thanks also go to the members of Biology Sciences and Environment & Earth Sciences Departments, Faculty of Science, Islamic University-Gaza, for their support during conducting the research. I would like to thank my wife for her understanding and patience during the past few years. Her support and encouragement were in the end what made this dissertation possible. My parents receive my deepest gratitude and love for their enthusiasm during my postgraduate studies that provided the foundation for this thesis. I would like to thank Dr. Hussein Abo Alqomsan for his help in achieving the questionnaires. Last, but not least, special thanks to Dr. Ramy Alnkhal, Director of Veterinary Lab/ Gaza strip. Special thanks to Zuhair Dardona and Mohammed Jouda for their great assistance in the laboratory work. II

7 ABSTRACT Detection of Antibiotic Residues in Broiler Chickens in Gaza Strip Residues of veterinary drugs in food have received much attention in recent years because of growing food safety concerns. There are serious effects of antibiotics residues in meat for human consumption (e.g., increase antimicrobial resistance, carcinogenicity, mutagenicity, and hypersensitivity). The presence of antibiotics residues and their associated harmful health effects on humans make the control of veterinary drug residues an important measure in ensuring consumer protection. The objective of this study is to evaluate the presence of some antimicrobial residues in broilers slaughtered in Gaza strip. The study covered the five governorates of Gaza strip and lasted from January to June Three hundred sixty five chicken breast samples were collected from poultry slaughterhouses distributed over the study area. All samples were tested for the presence of β-lactams, aminoglycosides, macrolides and tetracyclines (as groups) using a bioassay method recommended by United States Department of Agriculture (USDA). Chicken carcasses were divided into three categories according to their weights; category (A); 1.5 kg, category (B); > kg and category (C)>2 kg. Of the 365 tested samples, 88 samples were positive for one or more of antibiotic residues (24.1%), more than half of them (53.41%) were from category (A), followed by (32.95%) for category (B) and the least category contains residues were group (C) (13.63%). The most detected antibiotic residues were tetracyclines 41(43.15%) followed by aminoglycosides 26(27.36%) then 20 (21%) and 8(8.42%) for β-lactams and macrolides respectively. A confirmatory method like gas chromatography (GC) is recommended to be used to determine residues compliance with the maximum residue limits. In conclusion, results confirmed the presence of antibiotic residues in poultry meat samples collected from Gaza strip. This may pose potential hazard to public health. Thus, it is recommended that rules should be taken to ensure observing proper withdrawal periods before marketing and drug control in veterinary use. In addition, a monitoring policy should be implemented to ensure the conformity of poultry meat sold in Gaza strip with international standards. Key words: Antibiotic residues, maximum residue limit, poultry, bioassay, Gaza strip III

8 الملخص الكشف عن هتبقياث الوضاداث الحيويت في لحوم الدجاج الالحن في قطاع غزة اكزسجذ يزجم بد االد خ انج طش خ ف االغز خ انكض ش ي اال ز بو ف انس اد األخ شح ثسجت رضا ذ ان خب ف ان زؼهمخ ثساليخ األغز خ ػ ذ ان سز هك إر ؤد ع د يزجم بد يعبداد انغشاص ى ف ان زغبد انغزائ خ آصبسا سهج خ ػه صؾخ ان سز هك ؽ ش لذ رض ذ انفشصخ نظ س انسشغب بد انطفشاد انغ خ ص بدح انؾسبس خ كزنك ظ س سالالد ثكز ش خ يمب يخ نه عبداد انغشص ي خ ز االسجبة رغؼم ي انعش سح ع د سلبثخ ر ظ ى السزخذاو االد خ انج طش خ ف ػالط انؾ ا بد ان زغخ نغزاء اال سب. ذفذ ز انذساسخ نزم ى كشف ع د يزجم بد ثؼط ان عبداد انؾ خ ف نؾ و انذ اع ان شثبح ف لطبع غضح ؽ ش ش هذ انذساسخ ع غ يؾبفظبد لطبع غضح اسز ش ع غ انؼ بد ي ش ش ب ش ؽز ب خ ش ش نؼبو رى ع غ صالص بئخ خ سخ سز ػ خ ي ػعهخ صذس انذعبط ي يسبنخ انذ اع ان صػخ ف ي طمخ انذساسخ رى انزؾش ػ ع د اسثؼخ يغ ػبد ي ان عبداد انؾ خ يغ ػخ انززشاس كه بد االي عه ك س ذاد ان بكش ن ذاد يغ ػخ انج سه بد رنك ثبسزخذاو غش مخ ان مب سخ انؾ خ ان ص ث ب ي لجم صاسح انضساػخ االيش ك خ. رى رمس ى رثبئؼ انذعبط إن صالس فئبد فمب أل صا ب فئخ )أ( ي 0 ؽز 0.1 كغى انفئخ )ة( اكضش ي 0.1 ؽز 4 كغى فئخ )ط( اكضش ي 4 كغى. اظ شد ان زبئظ اؽز اء ص ب خ ص ب ػ خ ػه اؽذ ا أكضش ي يغ ػبد ان عبداد انؾ خ ان زؾش ػ ب ث ب شكم يب سجز %42 ك ب رج ا 2..%1 ي ان زجم بد كب ذ ي ظ ان غ ػخ )أ( 4.3.% ي ظ ان غ ػخ )ة( كب ذ الم ان غ ػبد إؽز اءا نه زجم بد ان غ ػخ )ط( انز رض رثبئؾ ب اكضش ي اص ك ه عشاو. ك ب اظ ش رؾه م انؼ بد ان ؾز خ ػه ان زجم بد ا يغ ػخ انززشاس كه بد كب ذ اكضش ب ر اعذا ث سجخ 0..%2 يزج ػخ ث غ ػخ االي عه ك س ذاد ث سجخ...%4 رهز ب يغ ػخ انج س ه بد %40 كب ذ اله ى يغ ػخ ان بكش ن ذاد %4.2 ص ثبسزخذاو اؽذ انطشق انزأك ذ خ يضم االسزششاة انغبص نزؾذ ذ ك خ ان زجم بد يؼشفخ يطبثمز ب نهؾذ االد ان س ػ ث نز اعذ ب ف نؾ و انذ اع خهصذ انذساسخ ا ان زبئظ اكذد ع د يزجم بد ان عبداد انؾ خ ف نؾ و انذ اع ف لطبع غضح ثشكم كج ش زا ي ان ؾز م ا شكم خطشا ػه صؾخ ان سز هك. ػه غت ا رزخز ان ؼب ش انالصيخ نهزأكذ ي االسزخذاو انغ ذ نألد خ يشاػبح يشالجخ فزشح سؾت انذ اء ي اعسبو انذ اع لجم رس م ب رؾس انس طشح ػه االسزخذاو انؾ ا نه عبداد انؾ خ. ك ب ص ثزطج ك ظبو رمص فؾص نهؾ و انذ اع نه زغبد انغزائ خ األخش انؾ ا خ ان شأ نهزأكذ ي يطبثمز ب نه اصفبد انؼبن خ انخبصخ ثؾذ د ان زجم بد انذ ائ خ ان اعت ػذو رغب ص ب. الكلواث الوفتاحيت: يزجم بد ان عبداد انؾ خ انؾذ االػه نه زجم بد انذعبط انالؽى لطبع غضح IV

9 Table of contents Title Page Dedication I Acknowledgement II English abstract III Arabic abstract IV Table of contents V List of tables IX List of figures X List of abbreviations XI Chapter I: Introduction 1.1 Overview Objectives General objective Specific objective Significance 2 Chapter II: Literature Review 2.1 Poultry production Antimicrobials Definition of antimicrobials Classification of antimicrobials Common antimicrobials used in poultry Tetracyclines β lactams Macrolides Aminoglycosides Antimicrobials usage in veterinary medicine Antimicrobials resistance Emergence of resistant bacteria in chicken Drug residues Drug residues definition Effects of veterinary drug residues 11 V

10 2.3.3 Maximum residue limit Withdrawal period Prohibition of some antimicrobials Cooking effect on antimicrobial residues Detection of drug residues Screening methods Classification of screening methods by detection principle Confirmation methods Microbiological assay Microbiological assay methods Examples of microbiological assay methods Four plate Test (FPT) The Calf Antibiotic and Sulfonamide Test (CAST) Screening Test for Antibiotic Residues (STAR) Premi's test CHARM test Residue control programs Previous studies 22 Chapter III: Materials and Methods 3.1 Materials Equipment Microorganisms, media and reagents Glassware and disposables Study area Study type and piloting Antibiotic residues detection Principle of the test Microorganisms and media Samples size and sample collection Buffer preparation Sample preparation and storage Preparation of bacterial suspensions 28 VI

11 3.3.7 Petri plates preparation Assay procedures Interpretation of results Identification of tetracyclines residues Identification of β-lactams residues Identification of Macrolides residues Identification of Aminoglycosides residues Questionnaire Data analysis 32 Chapter IV: Results 4.1 Distribution of samples in the study area Detection of antibiotic residues Detection of antibiotics according to samples weight Frequency of positive results of samples according to region Determination of antibiotic groups Antibiotic groups according to regions Detection of multiple antibiotics residues Questionnaire results Drug usage pattern in broiler breeding Antimicrobial usage during broiler breeding Antimicrobial used parenterally 39 Chapter V: Discussion 5.1 Detection of antibiotic residues Tetracylines detection Aminoglycoside detection β-lactams detection Macrolides detection Frequency of residues in the study area Antibiotic residues and carcasses weight Questionnaire analysis 49 Chapter VI: Conclusions and Recommendations 6.1 Conclusions 51 VII

12 6.2 Recommendations 52 References 54 Annex 1 63 Annex 2 65 VIII

13 List of tables Table (2.1): Poultry production expressed as numbers from in West 3 Bank and Gaza strip. Table (2.2): Poultry production in Palestinian territories. 4 Table (2.3): MRLs for some antimicrobials. 12 Table (2.4): Withdrawal periods of antimicrobials used in poultry production. 13 Table (2.5): Advantages and disadvantages of some screening methods. 17 Table (3.1): Equipment used in the study. 24 Table (3.2): Microorganisms, media and reagents used in the study. 24 Table (3.3): Glassware and disposables used in experimental work. 25 Table (3.4): Preparation of Phosphate Buffers with varying ph values. 27 Table (3.5): Bacterial suspension concentrations in plates. 29 Table (3.6): Sample extracts and their specific plates. 30 Table (3.7): Interpretation of results of five-plate bioassay. 31 Table (4.1): Distribution of samples according to chicken weight in the study 33 area. Table (4.2): Distribution of positive results according to regions. 35 Table (4.3): Distribution of positive results by region and weight categories. 35 Table (4.4): Distribution of detected antibiotic groups according sample 36 weight categories. Table (4.5): Distribution of detected antibiotic groups according to regions. 36 Table (4.6): Multiple detection of antibiotic groups. 36 Table (4.7): Breeders' responses to the questionnaire. 37 Table (4.8): Breeders' behavior in dealing with antibiotics in farms. 38 IX

14 List of figures Figure (2.1): Tetracyclines: Three members of tetracycline family. 6 Figure (2.2): Typical structure of a macrolide member (Erythromycin A). 7 Figure (2.3): Muscle samples on a plate of (FPT). 19 Figure (3.1): Distribution of samples according to study area. 26 Figure (3.2): KWIK-STIK device. 28 Figure (3.3): Stainless steel bioassay cylinders. 29 Figure (3.4): Five bioassay cylinders on an inoculated agar plate surface. 30 Figure (4.1): A positive sample showing 20 mm inhibition zone on plate 5 33 (that detects aminoglycosides residues). Figure (4.2): Overall positive samples of antibiotic residues. 34 Figure (4.3): Positive samples distribution according to carcass weight. 34 Figure (4.4): Antimicrobials used in broiler chickens therapy. 39 Figure (4.5): Antimicrobials used parenterally in broiler chickens therapy. 40 X

15 List of abbreviations ADI Allowed Daily Intake AMR Antimicrobial Residues ATCC American Type Culture Collection BSDA Bacillus stearothermophilus disc assay CAC Codex Alimentarius Commission CAST Calf Antimicrobial and Sulfonamide Test CFU Colony Forming Unit DAD Diode Array Detection DNA Deoxyribonucleic Acid ELISA Enzyme Linked Immunosorbent Assay EU European Union FDA Food and Drug Administration FPT Four Plate Test FSIS Food Safety and Inspection Service GC Gas Chromatography HPLC High Performance Liquid Chromatography LC-MS Liquid Chromatography Mass Spectrometry MRL Maximum Residue Limit ph Power of Hydrogen RNA Ribonucleic Acid SPSS Statistical Package for the Social Science STAF Swab Test on Animal Food STAR Screening Test for Antimicrobial Residues TLC Thin Layer Chromatography USDA United States Department of Agriculture UV Ultra Violet WHO World Health Organization WP Withdrawal Period ZI Zone of Inhibition XI

16 Chapter I Introduction 1. Overview Antimicrobials are generally used in farm animals for therapeutic and prophylactic purposes. They include a large number of different types of compounds, which can be administered either in feed, in drinking water or by injection. Some practices involve in the use of cocktails (mixtures of small amounts of several substances). Residues of these substances or their metabolites in meat and other foods of animal origin may cause adverse effects to consumers. The presence of residues and associated harmful health effects on humans make the control of veterinary drug residues an important measure in ensuring consumers protection [1]. In the recent years, residues of veterinary drugs in food have received much attention because of increasing concerns of food safety by consumers [2]. There are potential hazards of ingesting antimicrobial residues (AMR) in food for human consumption which include; carcinogenicity, mutagenicity, bone marrow toxicity (Chloramphenicol) and allergy (Penicillin) [3]. Also AMR in food disrupt the intestinal microbiota and increase the development of resistant bacteria in the general population. Drug resistance has gained its importance due to its ability of transmission to other enteric pathogens which have posed a serious public health problem [4]. In addition soil microbiota which receives AMR via birds manure may affect the human health as a source of developing resistant microorganisms [5]. Maximum Residue Limit (MRL) means the maximum concentration of residue resulting from the use of a veterinary medicinal product (expressed in mg/kg or µg/kg on a fresh weight basis) which may be accepted by the community to be legally permitted or recognized as acceptable in or on a food [6]. Inappropriate use of veterinary drugs can possibly leave residues in edible tissues or food products, which may have a potential risk to consumers because of allergic reactions of individuals to antimicrobials and/or their metabolites [7]. 1

17 Antimicrobial residues are detected by chemical, biological and immunological methods. Detection methods can be classified by their degree of quantification into qualitative, semi-quantitative and quantitative methods. In this study, the bioassay method was used as a screening method for identification of AMR in poultry meat. Chicken meat, rather than other commodities, such as milk or beef, was chosen for a variety of reasons; one of them is that poultry meat is largely consumed in Gaza strip by consumers of all ages. To the best of our knowledge, this is the first study which attempt to tackle this issue in Gaza strips. 1.2 Objectives General objective: The main objective of this study is to determine the presence of four antibiotic group residues in broilers slaughtered in Gaza strip. Specific objectives 1. To determine the types of antimicrobials used in poultry industry in Gaza strip. 2. To screen chicken samples for the presence of four antibiotic groups residues. 3. To compare the incidence of antibiotic residues according to carcasses weight. 1.3 Significance Given the potential hazards presented by the presence of antimicrobial residues in poultry on human health, and that there is an intensive, un-regulated and uncontrolled use of antimicrobials in the poultry industry in Gaza strip, it is of utmost importance to investigate this issue and to generate data that would serve as a baseline data for researchers as well as for decision makers. This study is the first in Gaza strip and is expected to indirectly increase awareness of adverse effects of antimicrobial residues in poultry, and could help in the reduction of antimicrobial residues effects. 2

18 Chapter II Literature Review 2.1 Poultry production The animal production sector, especially poultry production is one of the most important sectors of Palestinian agriculture. Its importance comes from the increasing investments in the livestock sector. During the last two decades the number of both layers and broilers has increased dramatically as illustrated in (Table 2.1) [8]. Table (2.1): Poultry production expressed as numbers from in West Bank and Gaza strip [8] Year Layers Broilers ,000 3,400, ,000 4,330, ,000 3,550, ,000 2,490, ,000 3,500, ,000 4,400, ,000 16,450, ,000 16,900, ,000 18,800, ,812,000 31,790,000 The Palestinian Central Bureau of Statistics in cooperation with Ministry of Agriculture conducts an annual agricultural statistics survey. The following (Table 2.2) demonstrates poultry production within three productive years [9]. It is clear that poultry production is becoming more and more prominent sector and it needs to be improved and developed. Intensive farming, which is a natural response to increasing demand, may put pressure on farmers and veterinarians to use more and more antimicrobials in terms of types and quantities. 3

19 Table (2.2): Poultry production in Palestinian territories [9] Region Broilers Layers 2009/2010 Broilers mothers Turkey Gaza strip West Bank Total Gaza strip West Bank Total Gaza strip West Bank Total 7,556,507 23,554,904 31,111,411 16,373,467 20,174,056 36,547, ,515, ,280 1,233,736 1,545, / ,678 1,328,779 1,626, / , , ,423 2, , , , , , , , Antimicrobials Definition of antimicrobials Antimicrobial agents are chemical compounds that kill or inhibit the growth of microorganisms but cause little or no damage to the host. They are naturally produced by microorganisms such as fungi (e.g. penicillin) and bacteria (e.g. tetracycline) or can be semi-synthetically produced (e.g. amoxicillin) or totally synthetically produced (e.g. sulfonamides) [10] Classification of antimicrobials According to Wang, 2012 [11] antimicrobials can be classified by many ways; 1. According to the spectrum of activity: Broad spectrum antimicrobials. Narrow spectrum antimicrobials. 2. According to the mode of action: Inhibiting cell wall synthesis. Inhibiting protein synthesis. Inhibiting nucleic acid synthesis. Inhibiting the synthesis of essential metabolites. Injuring the plasma membrane. 4

20 3. According to their effects on microorganisms Bactericidal antimicrobials. Bacteriostatic antimicrobials. 4. According to the chemical structure: β-lactams Nitroimidazoles Aminoglycosides Phenicols Lincosamides Ionophores Tetracyclines Polypeptides Quinolones Quinoxalines Macrolides Phosphoglycolipids Nitrofurans Sulfonamides Common antimicrobials used in poultry Tetracyclines The tetracyclines were discovered in the 1940s, they are a family of antimicrobials that inhibit protein synthesis by preventing the attachment of aminoacyl-trna to the ribosomal acceptor (A) site [12]. Tetracyclines consist of a common four-ring structure to which a variety of side chains are attached (Figure 2.1) [13]. Chlortetracycline and oxytetracycline were the first members of the tetracycline group to be described. Subsequently, a number of important semisynthetic tetracyclines were developed, e.g. doxycycline and minocycline [14]. Tetracyclines are the most commonly prescribed antimicrobials; they have played an important role in veterinary medicine. Because of their broad spectrum activity and low cost, tetracyclines (TCs) including tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC) and doxycycline (DC) are widely used in animals for both prevention, treatment and as feed additives to promote growth [15]. The widespread utilization of TCs leads to an increasing resistance factor, so accurate monitoring by public health agencies is required [16]. Three different tetracycline resistance mechanisms have been described; active efflux of the antimicrobial, ribosomal protection and enzymatic inactivation of the drug. All these mechanisms are based on the acquisition of one or several tetracycline resistance 5

21 determinants, which are widely distributed among bacterial genera. Additionally, mutations in the rrna, multidrug transporter systems or permeability barriers may be involved in the resistance to several antimicrobials including tetracyclines [14]. Figure (2.1): Tetracyclines: Three members of tetracycline family [14]. Tetracycline lacks both of the groups that are shaded. Chlortetracycline differs from tetracycline in having a chlorine atom (blue); doxycycline consists of tetracycline with an extra hydroxyl (purple) β-lactams The β-lactam group is one of the most important families of antimicrobials used in veterinary medicine and has been widely used for decades in animal husbandry. This group consists of penicillins and cephalosporins. The most common members of the penicillins used in veterinary practice are benzyl penicillin, amoxicillin, ampicillin and penicillin G. The extensive use of penicillins may cause the presence of their residues in food products of animal origin and may have side effects to consumers. Moreover, penicillin residues in food products may be responsible of allergic reactions in humans and promote the occurrence of antimicrobials resistant bacteria [17]. The cephalosporins are chemically related to the penicillins and both share the β-lactam ring structure. A number of cephalosporins, including cefalexin, cefuroxime, ceftiofur, cefquinome and cefotaxime are used in veterinary medicine in food animals [18]. Due to increased emergence of cephalosporin resistant bacteria (specially E. coli and Salmonella) [19, 20] the FDA prohibited the usage of cephalosporins in food producing animals including poultry [21]. 6

22 Macrolides Macrolides constitute a very important class of antibacterial compounds widely used in veterinary medicine to treat respiratory diseases. These antimicrobials are molecules with a central lactone ring bearing 12 or 16 atoms to which several amino and/or neutral sugars are bound (Figure 2.2) [22]. The antibacterial action of macrolides is through the inhibition of protein synthesis by binding to the 50S ribosomal subunit of prokaryote organisms. Resistance to macrolides is usually plasmid-mediated, but modification of ribosomes may occur through chromosomal mutation, resistance can occur either by decreasing entry into bacteria, synthesis of bacterial enzymes that hydrolyze the drug or modification of the target (ribosome) [23]. Figure (2.2): Typical structure of a macrolide member (Erythromycin A) [23] Aminoglycosides Aminoglycosides are a large class of antimicrobials that are characterized by two or more amino sugars linked by glycosidic bonds to an aminocyclitol component, Aminoglycosides are broad-spectrum antibiotics and act primarily by impairing bacterial protein synthesis through binding to prokaryotic ribosomes [24]. In veterinary medicine and animal husbandry, aminoglycosides are widely used in the treatment of bacterial infections, and have been added to feeds for prophylaxis and for growth promotion. Those most commonly used are gentamicin, neomycin, streptomycin and dihydrostreptomycin [25] Antimicrobials usage in veterinary medicine Antimicrobials are used largely for three purposes in animals: therapeutic use to treat sick animals, prophylactic use to prevent infection in animals and as growth promoters to improve feed utilization and production. In general, therapeutic 7

23 treatment involves treatment of individual animals over a short period with doses of antimicrobials exceeding the minimal inhibitory concentration of the known or suspected pathogen [26]. Sometimes, with intensively farmed animals, therapeutic treatment is delivered through feed or drinking water. Prophylactic treatment involves moderate to high doses of antimicrobials, often given in feed or water for a defined period to a group of animals. Antimicrobials used as growth promoters tend to be given in feed at sub-therapeutic levels over extended periods to entire herds and flocks [27] Antimicrobials resistance Due to the excessive and inappropriate use of antimicrobials, there has been a gradual emergence of populations of antimicrobials resistant bacteria, which pose a global public health problem. A resistant microbe is one which is not killed by an antimicrobial agent after a standard course of treatment [28]. Antimicrobials used to combat infection forces bacteria to either adapt or die irrespective of the dosage or time span. The surviving bacteria carry the drug resistance gene, which can then be transferred either within the species/genus or to other unrelated species. Clinical resistance is a complex phenomenon and its manifestation is dependent on the type of bacterium, the site of infection, distribution of antimicrobials in the body, concentration of the antimicrobials at the site of infection and the immune status of the patient [29] Emergence of resistant bacteria in chicken In animals, antimicrobials resistant enteropathogens (e.g., Salmonella, Campylobacter, Yersinia, and some strains of Escherichia coli) are of special concern to human health because these bacteria are most likely to be transferred through the food chain to humans, or resistance genes in commensal bacteria may be transferred to the zoonotic enteropathogens [30]. The most important antimicrobials-resistant strains are the multiply antimicrobialsresistant Salmonella, macrolide or fluoroquinolone-resistant Campylobacter, and multiply antimicrobials-resistant E. coli. In all cases, the hypothesis is that the food chain is the main mean of transmission [31]. Direct physical contact, shared 8

24 environments, and exposure through vectors and fomites are all routes for bacterial transmission between animal species. Poultry is considered a leading source for foodborne infections caused by Campylobacter and Salmonella. Food surveillance most commonly isolates Salmonella from fresh meat, commonly from poultry and less frequently from eggs, beef, fishery products, vegetables and milk [32]. Elmanama and Abdelatif, (2012) conducted a study to investigate the antimicrobial resistance for enteric pathogens isolated from acute gastroenteritis patients in Gaza strip. The study showed that diarrhea was more frequent among peoples living in houses rearing poultry and pigeons. They isolated Salmonella, Campylobacter coli/jejuni, Aeromonas hydrophilia, Shigella boydii and Yersinia enterocolytica. All isolates were resistant for more than one antimicrobials especially Campylobacter coli/jejuni [33]. Many researchers worldwide studied the prevalence and antimicrobial resistance for bacteria isolated from chicken meat. In 2010, a study was carried out to investigate the prevalence and antimicrobial resistance profiles of Salmonella, Campylobacter and Yersinia spp. from retail chicken in Tehran, Iran. They revealed that a high proportion of chicken in markets were contaminated with Campylobacter and Salmonella. From 190 chicken samples, Campylobacter, Salmonella and Yersinia were isolated from 94(49.5%), 86(45%) and 41(21.5%) of samples, respectively. Concerning antimicrobial resistance of isolated microbes, nalidixic acid resistance in Campylobacter and Salmonella isolates was greater than in Yersinia isolates. Resistance of Campylobacter to nalidixic acid (quinolone) was largely associated with ciprofloxacin (fluoroquinolone) resistance and resistance to nalidixic acid and tetracycline was found in Salmonella. All Salmonella isolates were sensitive to ciprofloxacin. Tetracycline was the second most frequently observed type of antimicrobial resistance among the different genera tested [34]. Other researchers from Iran determined the prevalence and antimicrobial resistance of Campylobacter spp. that were isolated during different stages of broiler processing. Samples were collected from four sites along the processing line including de-feathering stage, evisceration stage, twenty minutes after the chilling period started and 24 h after the chilling period completed. 186 of 336 carcasses 9

25 (55.4%) were positive for Campylobacter spp. ten antimicrobial agents were used to asses isolates sensitivity. Of the 198 Campylobacter isolates tested, 178 (92.9%) were resistant to one or more antimicrobial agents. Resistance to tetracycline was the most common finding (78.3%), followed by resistance to ciprofloxacin (62.1%), nalidixic acid (58.6%), and enrofloxacin (44.4%) [35]. A Korean study investigated the prevalence and antimicrobial resistance of Salmonella isolated from chicken meat produced by different integrated broiler operations. 210 samples from seven brands of conventional chicken meat were collected. There were differences in the number of bacteria isolated from different brands, but in general, 47 (22.4%) samples were positive for Salmonella. S. enteritidis was the dominant (57.4%) of the Salmonella-positive chickens. Twenty antimicrobial agents were used to determine antimicrobial resistance of isolates. Isolates were resistant to cephalothin 41(87%), nalidixic acid 41(85%), and streptomycin 33 (70%). All isolates of Salmonella were susceptible to amikacin, ciprofloxacin, imipenem, enrofloxacin, and trimethoprim [36]. A recent survey to estimate the prevalence of antimicrobial resistance in Salmonella spp., E. coli, Enterococcus spp. and S. aureus in meat in Saudi Arabia was published. A total number of 288 unprocessed meat samples of four different types (beef, camel, lamb and poultry) were analyzed. They were divided into domestic chilled (144) and imported frozen (144). All types of meat analyzed contained the four types of bacteria with E. coli being the most prevalent overall at 72.2%, Enterococcus prevalence was 26.2%, S. aureus prevalence was 24.6% and Salmonella prevalence was 10.7%. These bacteria were resistant to a number of antimicrobials and some were multidrug resistant. They concluded that bacterial contamination of meat is a multi-country problem and consideration should be made to improve methods of decontaminating food animals and work surfaces during meat processing [37]. 2.3 Drug residues Drug residues definition The term "residues" is used to describe all active principles and their metabolites, which persist in meats or other food products from animals that have been treated 10

26 with the drug in question. The term metabolite has not been defined, it is generally accepted that it applies to any by-product of biotransformation of the initial active principle [38] Effects of veterinary drug residues A number of possible adverse health effects of veterinary drug residues have been suggested. These may include but not limited to the following [39]: 1. Allergic or toxic reactions to residues. 2. Chronic toxic effects occurring with prolonged exposure to low levels of antimicrobials. 3. Development of antimicrobial-resistant bacteria in treated animals. These bacteria might then cause difficult-to-treat human infections. 4. Disruption of normal human microbiota in the intestine. The bacteria that usually live in the intestine act as a barrier to prevent incoming pathogenic bacteria from getting established and causing disease. Antimicrobials might reduce total numbers of these bacteria or selectively kill some important species Maximum residue limit Maximum residue limit means the maximum concentration of residue resulting from the use of a veterinary medicinal product, which may be legally permitted or recognized as acceptable in or on a food, allocated to individual food commodities. It is based on the type and amount of residue considered to be without any toxicological hazard for human health as expressed by the Allowed Daily Intake (ADI), or on the basis of a temporary ADI that utilizes an additional safety factor [40]. The Codex Alimentarius Commission (CAC) is a commission jointly sponsored by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO). It is a collection of international food standards, guidelines, and codes of practice that protect the health of consumers and ensure fair practices in food trade. The Codex Alimentarius covers food safety matters (residues, hygiene, additives, contaminants, etc.) and quality matters (product descriptions, quality classes, labeling, and certification). Codex established the Codex Committee on Residues of Veterinary Drugs in Food in Codex has defined 590 MRLs for some fifty nine 11

27 veterinary drugs. Most countries use Codex MRLs as a basis for establishing their national regulations for veterinary drug use, but still other organizations make their own MRLs to be used in their countries. (Table 2.3) shows the MRLs of some antimicrobial residues as stated by Codex Alimentarius Commission [41]. Table (2.3): MRLs of some antimicrobials [41] Antimicrobial Poultry muscle Poultry liver (µg/kg) (µg/kg) Chlortetracycline/ Oxytetracycline/ Tetracycline Neomycin Spectinomycin Streptenomycin/ dihydrostreptomycin Procaine benzylpenicillin Flumequine Danofloxacin Tylosin Erythromycin Spiramycin Colistin Lincomycin Withdrawal period (WP) The withdrawal period is defined as the interval between the time of the last administration of a drug and the time when the animal can be safely slaughtered for food, milk or eggs can be safely consumed. The withdrawal period provides a high degree of assurance to both producers and consumers that concentration of residues in foods derived from treated animals will not exceed the MRLs [42]. Each antimicrobial has a WP which depends on drug type, drug concentration, route of administration, animal kind and the animal product [23] as demonstrated in (Table 2.4) [43]. All antimicrobials are labeled with the appropriate WP, whether it is hours, days or weeks. 12

28 Table (2.4): Withdrawal periods of antimicrobials used in poultry production [43] Drug Administration route Animal Withdwral period (Days) Tylosin tartarate D.W Chicken 1 Turkey 5 Erythromycin D.W Poultry 1 Gentamicin sulphate SC. Chicken 35 Turkey 63 Neomycine sulphate D.W Poultry 0 Streptomycin D.W Chicken 4 Lincomycin D.W Chicken 0 Oxytetracycline HCl D.W Poultry 7-14 Chloretetracyclines D.W Poultry 1 Enrofloxacin D.W Poultry 8 Amoxicillin D.W Chicken 2 Turkey 5 Sulfaquinoxaline D.W Poultry 10 D.W. drinking water, SC subcutaneously 2.4 Prohibition of some antimicrobials The extensive use of antimicrobials as feed additives for long time may contribute to the development of resistant bacteria to drugs that are used to overcome infections. These microbes pose a potential risk for humans if they are transferred to people. Many European countries banned using antimicrobials as food additives. Sweden prohibited in 1986 the use of additives belonging to the groups of antimicrobials in feeding stuffs. Avoparcin was banned in Denmark (1995) and Germany (1996), spiramycin was prohibited in Finland (1998) because this product was used in human medicine, and virginiamycin was prohibited in Denmark (1998). Also zinc bacitracin was banned because its use in human medicine as treatment skin infections [44]. Chloramphenicol, a broad-spectrum antimicrobial, was previously widely used in veterinary and human medicine. Reports of aplastic anemia in humans arising from its use led to its ban in the USA and European Union (EU) in Thiamphenicol and florfenicol were permitted as substitutes [22]. Nitrofurans, particularly furazolidone, furaltadone, nitrofurantoin and nitrofurazone for livestock production was completely prohibited in the EU in 1995 due to concerns about the 13

29 carcinogenicity of the drug residues and their potential harmful effects on human health [45]. Due to emergence of fluoroquinolone-resistant bacteria especially Campylobacter and Salmonella, the Food and Drug Administration (FDA) in 1977 banned the use of fluoroquinolones in treating poultry but the use of sarafloxacin and enrofloxacin in poultry was permitted, but an increase in fluoroquinolone-resistant Campylobacter spp. in poultry was linked to increased incidence of infection with resistant Campylobacter spp. in humans. Finally, FDA in 2005 prohibited the usage of enrofloxacin in poultry and sarafloxacin were withdrawn by the producer, thus usage of any members of fluoroquinolones in poultry species is illegal by FDA [46]. 2.5 Cooking effect on antimicrobial residues To determine the effect of cooking process on AMR, a study investigated the effect of cooking and cold storage on ampicillin, chloramphenicol, oxytetracycline, streptomycin and sulphadimidine residues in meat, the study showed that active AMR might be detected in animal tissue after roasting, grilling and prolonged cold storage. They concluded that it would be unwise to rely on cooking or cold storage to minimise or destroy such residues. The only way to ensure no residues would appear to be the strict observance of the WP for each drug administered to domestic animals [47]. In another study, researchers investigated the effects of various ordinary cooking procedures (boiling, roasting and microwaving) on tetracyclines (TC) residues in chicken meat. The obtained data revealed that the reduction of TC residues in cooked samples was related to cooking procedures, cooking time and TC agents. The losses of TC residues increased with prolonged cooking time. Doxycycline was the most heat stable of TCs, less than 50% of the initial residues concentration was decreased in boiling and microwaving for 40 and 80 minutes respectively [48]. In contrary, a different study concluded that oxytetracycline was the most heat labile. The time required to destroy more than 90% of the initial level of oxytetracycline (OTC) in breast meat was 15, 40 and 60 minutes for microwaving, boiling and 14

30 roasting, respectively, OTC residues in breast meat were not detected after microwaving for 20 minutes. Generally, sufficient cooking temperature and time can have a significant effect on the losses of TC residues and provide an additional margin of safety for consumers [49]. To determine the effect of different cooking processes (microwaving, roasting, boiling, grilling and frying) on enrofloxacin residues in chicken muscle, investigators used liquid chromatography mass spectrometry (LC-MS) method to evaluate stability of enrofloxacin in natural incurred chicken samples after cooking. They conducted the study on different parts of chicken (breast muscles, thigh muscles and liver). The study showed that enrofloxacin remained stable in boiling water for three hours. On the other hand, the amount of residue increased in the case of roasting and grilling. Also they noticed that when there was a reduction in residues percentage, the lost amount of analyte was found in water or exudates. These results rendered the investigators to inferred that cooking procedures did not affect the levels of quinolones [50]. In another study also evaluated the effects of different cooking processes on enrofloxacin residues in chicken muscle, liver and gizzard tissue from broiler chickens, results showed that enrofloxacin residues were reduced after different cooking processes. In cooked meat and gizzard, the most reduced levels of the residue were due to the boiling method. A high residue levels remained stable after microwave cooking/ heating. They concluded that cooking processes cannot destroy the total amounts of this drug but it can only decrease their amounts and most of the residues in boiling process are excreted from tissue to cooking fluid during the boiling process. Thus, exposure to residues can be reduced by discarding any juice that come from the edible tissues as they are cooked. Among the various agents affecting antimicrobial residues after the cooking process, it was found that cooking time and temperature can play major roles [51]. 15

31 2.6 Detection of drug residues Screening methods A screening method is defined as the first procedure that is applied to sample analyses. The purpose is to assure the presence or absence of veterinary drugs residues. This procedure should be as simple as possible. Still, it may be rather complex, due to, e.g. the properties of the drugs of interest or the desired limit of detection, and in certain cases, will provide (semi) quantitative next to the qualitative data [52] Classification of screening methods by detection principle [53] 1. Biological methods: detect cellular responses to analytes (e.g. inhibition of bacterial growth). These methods are not selective and can cover several chemical classes of active analytes (e.g. hormones, antimicrobials). They do not allow the identification of individual analytes. 2. Biochemical methods: detect molecular interactions (e.g. antigens, proteins) between analytes and antibodies or receptor proteins (e.g. ELISA), chemical labeling of either the analyte or antibody/receptor allows the interaction to be monitored and measured. These methods are either selective for a family of analytes having related molecular structures or are sometimes analyte specific. 3. Physicochemical methods: distinguish the chemical structure and molecular characteristics of analytes by separation of molecules (e.g. TLC, GC, HPLC) and the detection of signals related to molecular characteristics (e.g. UV, DAD,..etc). They are able to distinguish between similar molecular structures and allow the simultaneous analysis of several analytes. Table 2.5 demonstrates advantages and disadvantages of different screening methods of residues analysis [54]. 16

32 Table (2.5): Advantages and disadvantages of some screening methods [54] Test Advantages Disadvantages Easy to use Increased cost Availability for a good number of Limited storage (few specific compounds. months) under refrigeration. Availability for families of The need for waste disposal. compounds (e.g. sulfanomides, Interferences giving some estilbenes). false positives. Large number of samples (42) per Only one kit per residue ELISA kit for a single analyte. searched. Reduced time to obtain the results (2-2.5 h for most kits). High sensitivity and specificity. Possibility to use within the food processing facility. Biochip array biosensors Easy to use. Results available in short time. Multiples residues analyzed in one shot (as many as in an array). Full automation: higher productivity. High through-put technique: up to 120 samples per hour and array. High operative costs chips and equipment cost. Analysis restricted to available chips HPLC Reduced time (few hours) to obtain results. Sensitive Automation leading to higher productivity. Specificity depending on a detector Expertise required. Needs sample preparation (Extraction, filtration, addition of internal standards, etc.). Expensive. Microbial methods Can be used for large surveillance programmers. Basic laboratory equipment. Broad spectrum. Easy to use. Economical. Difficult to standardize preparation procedures. Some test could not insure MRLs compliance. Sample preparation required to remove false positives due to protein bacterial inhibitors. Low sensitivity. Determination of antimicrobial residues in food products such as meat, milk, and eggs by microbiological methods depends on the effect on a specific microorganism, the spectrum and the mode of action of the antimicrobials which will be determined. 17

33 On the other side residue determination by chemical methods such as chromatography (by all its types) depends on the chemical properties [55] Confirmation methods Various confirmation methods have been described for the detection of veterinary drugs in various matrices. Most techniques comprise a chromatographic separation and a detection method. Liquid chromatography (LC) is often combined with ultraviolet detection (UV), fluorescence detection and mass spectrometry, Gas chromatography (GC) can be combined with electron capture detection, infrared detection and mass spectrometry [56]. Confirmation methods can be both qualitative and quantitative. Quantitative methods are necessary to detect veterinary products that are permitted in some matrices in a maximum concentration; these methods need to confirm if the concentration of an analyte is below or above this limit. The quantification limit should be approximately 0.5 times the MRL. Qualitative methods are used for forbidden substances and violative use of veterinary medicinal products [57] Microbiological assay Microbiological assay screening methods for AMR exploit the primary property of these compounds, their selective toxicity towards specific bacteria. Growth inhibition assays for the detection of antimicrobials mainly concern two types of formats: The tube test and the (multi) plate assay. Briefly, the first type comprises a growth medium inoculated with bacterial spores and a ph or redox indicator. In the absence of AMR, the test bacterium will start to grow, acidify the medium and cause a color change [58]. A plate assay comprises a layer of inoculated growth medium. Samples can be applied on top of, or in a well in the agar layer. After over-night incubation, the presence of an antimicrobial residue becomes visible as an inhibition zone around the sample. The size of the inhibition zone depends on the type of residue and its concentration, while the sensitivity of the test is affected by many factors, such as indicator organism, ph, type of growth medium, and thickness of the agar layer [59]. 18

34 2.6.4 Microbiological assay methods Microbiological assays can be classified depending on their mode of detection; growth inhibition and luminescence. If food samples do not contain AMR, or the concentrations are below the load of detection, the organisms grow producing acid compounds that change the indicator color, permitting visual or photometric detection. Nevertheless, if an antimicrobial is present in the sample no color change is observed [58]. Most of the microbiological inhibition tests with agar diffusion are based on inhibition-diameter measurement using a caliper. In these tests, samples are applied to plates of agar media inoculated with specific bacteria. Diffusion of an antibacterial substance is shown by the formation of inhibition zones [60] Examples of microbiological assay methods Four plate Test (FPT) This test was developed as a mean of import control within the European Commission primarily to monitor residues of antimicrobials in fresh meat from Third World countries for use at national borders [61]. The test is comprised of four plates of agar medium inoculated with Bacillus subtilis (BGA) spores (at ph 6.0, 7.2 and 8.0) and Kocuria rhizophila ATCC 9341 (at ph 8.0). Meat samples are cut into small cylinders and applied to the plates (Figure 2.3). Trimethoprim is incorporated into the ph 7.2-medium to enhance the test's sensitivity toward sulfonamide residues. After incubation, diffusion of an antibacterial substance is shown by the formation of inhibition zones on any seeded plate [62]. Figure (2.3): Muscle samples on a plate of FPT [58]. 19

35 The Calf Antibiotic and Sulfonamide Test (CAST) CAST is a microbial inhibition screening test for the detection of antibiotics and sulfonamides in veal calf carcasses. The test uses Bacillus megaterium ATCC 9885 as the indicator organism and Mueller Hinton agar as the growth medium. A sterile cotton swab is inserted into a kidney of a freshly slaughtered calf and the swab is allowed to soak in the kidney fluid for 30 min. Then the swab is removed from the kidney and placed on a plate seeded with specific concentration of B. megaterium. The plate is incubated at 45 C. After h incubation, swabs are removed from the plate and the zone of inhibition (ZI) around each swab is measured vertically and horizontally and recorded [63] Screening Test for Antibiotic Residues (STAR) The STAR protocol is intended for the qualitative detection of residues of substances with antimicrobial activity in milk and muscle of pig, cattle, sheep, and poultry by using bacterial strains sensitive to antimicrobials. This method is based on five different plates (Five-Plate Test) to detect specific families of antimicrobials, the plate B. cereus ATCC for tetracyclines, the plate E. coli ATCC for quinolones, the plate B. subtilis B.G.A for aminoglycosides, the plate K. rhizophila ATCC 9341 for macrolides and the plate Bacillus stearothermophilus ATCC for sulfonamides and β-lactams. Slices of muscle samples of 2 mm in thickness and 8 mm in diameter are placed onto the plates. Then plates are incubated. If there is AMR, a zone of inhibition (ZI) around the meat sample will appear [64] Premi's test The Premi Test is a commercial growth inhibitor test used for the detection of AMR in fresh meat, kidneys, fish and eggs in less than four hours. Premi test is an ampoule, containing a specific agar medium, imbedded spores of B. stearothermophilus var. calidolactis and a color indicator. The meat juice is placed in the ampule and after 20 min of pre-diffusion at room temperature; the meat juice is removed by washing step. Finally, the ampoule is incubated for approximately 3 h at 64 C. If no inhibitory substances are present, the germinated spores will multiply with the production of acid. This will be visible by a color change from purple to yellow. When anti-microbial compounds are present in sufficient amount (above the 20

36 detection limit), the spores will be unable to germinate and therefore no color change will be observed [65] CHARM test The CHARM test, a commercial test, is based on the irreversible binding reaction between functional groups of antibacterials and receptor sites on or within the cell of added microorganisms. For example, β-lactams bind to D-alanine carboxypeptidase on the cell wall, whereas other binding sites are found on ribosomes [66]. The Charm I test was developed exclusively for β-lactams in milk, further CHARM II test was developed to test a variety of antimicrobials in both milk and other food of animal origin including honey. The test employs 14 C-labeled or 3 H-labeled antibacterials to compete for the binding sites. This competition for receptor sites prevents the radiolabeled antibacterial from binding. Thus the more radiolabeled compound bound the less analyte in the sample [67]. 2.7 Residue Control Programs Residue control programs are designed in accordance with country regulations. These programs generally control both domestic and imported products. Veterinary drugs for inclusion in these programs are selected on the basis of their risk profiles. Only the domestic residue sampling program includes steps for addressing the occurrence of violative residues in food-producing animals, on-farm. The import residue sampling program is primarily a verification program to determine that the domestic residue sampling program of an exporting country is operating effectively [68]. Control programs have two principal components: monitoring and surveillance. Residue monitoring program randomly collect sample tissues from animals at slaughters then tissue samples are screened for residues of veterinary drugs, pesticides and environmental contaminants, and the residues are assessed for compliance with the applicable MRL or environmental standard. Surveillance programs collect sample tissues from animals suspected of violative residues depending on clinical signs or herd history. If monitoring reveals a potential residue problem, the action taken will vary in accordance with country rules [69]. 21

37 2.9 Previous studies A Belgium study used a combination of three plates, seeded with strains of Micrococcus luteus, B. cereus and E. coli to detect residues of β-lactams, tetracyclines and fluoroquinolones in poultry meat. Confirmation and quantification of positive samples were performed using a validated HPLC method with fluorescence detection. 18 out of the 228(7.9%) broilers contained inhibiting substances. Seventeen samples inhibited B. cereus. Doxycycline was detected in the 16 samples that were investigated with HPLC with fluorescence detection. One sample inhibited M. luteus and was confirmed to be amoxicillin. No fluoroquinolones were detected [70]. In a study conducted to investigate AMR in chicken, three microbial screening tests were used; fast antimicrobial screening test (FAST), B. stearothermophilus disc assay (BSDA) and a commercial test kit (TAT). Four hundreds chicken meat samples were screened; the prevalence of AMR in chicken meat was from 11.1% to 21.7%. Test performances were evaluated on sensitivity, specificity, positive predictive value and negative predictive value, the researcher concluded that BSDA is the screening test of choice, in addition to simplicity, short incubation period as well as the low cost [71]. In a study done in Pakistan using B. subtilis as a test organism, screening of AMR in a total of 100 broiler tissue samples (33 livers, 33 kidney and 33 muscles) reveald that 13(39.4%) livers, 9(27.3%) kidneys and 7(20.6%) muscles contained antimicrobial residus [72]. A Bulgarian study carried out to investigate the presence of antimicrobial drugs residues in chicken (breast muscles, liver and kidneys). Samples from meat (breast muscles), liver and kidneys were taken as follows:115, 192, 155 for meat, liver and kidneys respectively, samples were screened using FPT method, 2 samples (1.7%) from meat were identified as antimicrobials-residue-positive while 17(8.8%) from liver and 33(21.%) from kidney [73]. 22

38 Shareef and colleagues used thin layer chromatography (TLC) to screen the presence of oxytetracycline, sulfadiazine, neomycin, and gentamycin in stored poultry products in Mosul, Iraq. 25 samples from each (livers, thigh muscle, and breast muscle) were screened. Total of 75 samples of stored poultry products were tested. 39 (52%) of the samples were positive. In more details, 56% of samples were positive for each liver and breast muscle while 44% of samples were positive in thigh muscle. In that study neither gentamicin nor neomycin were detected. On the other hand, oxytetracycline and sulfadiazine were detected in equal number of positive results, 18 for each type [74]. A study done in the Dominican Republic, Santiago province to determine whether retail broiler meat contained quinolone residues, a total of 135 chicken breast samples were screened using colorimetric assay based on the inhibition of an E. coli strain which is sensitive to quinolones. 9(6.6%) of samples were containing quinolones above MRL [75]. An Egyptian study carried out to assess the safety of broiler fillet through residues monitoring of antimicrobials especially (oxytetracycline & enrofloxacin). In that study, two methods were used for the determination of AMR in broiler fillet, a screening method by microbiological inhibition assay using B. subtilis (ATCC-6633) as indicator organism and a confirmation method using HPLC analysis. From one hundred random broiler fillet samples (50 fresh and 50 frozen), the screening test found that 21% of total examined samples contained AMR. HPLC method for confirmation and quantification proved that six samples were containing oxytetracycline and three samples were containing enrofloxacin, all samples except one had violative values of AMR comparing to MRLs determined by European Union Commission [76]. A study was done in Nigeria to determine the prevalence of AMR in commercial broiler chickens using Premi Test Kit. From 70 sampled commercial birds from three major poultry markets in the study area, 42 (60%) of birds contained antimicrobial residues. It detected also residues in 90 out of the 280 different organ matrices made up of 70 samples of each organ, kidney was the highest at 48.6%, gizzard (30.1%), liver (25.8%), and muscle (24.3%) [77]. 23

39 Chapter III Materials and Methods This chapter describes the materials and methods used to achieve the objectives of the study. This is a cross-sectional analytical study that aimed at detecting four antibiotic groups residues in broiler chickens sold in Gaza strip. 3.1 Materials Equipment listed in (Table 3.1) were used in the biological sciences and Environmental and earth sciences departments of the Islamic University-Gaza Equipment Table (3.1): Equipment used in the study Items Balance, analytical. Balance, 0.1 to 200 gram capacity Water bath Incubator. Safety cabinet Refrigerator. Freezer. Autoclave Spectrophotometer ph meter Vortex mixer Digital camera Hot plate and Magnetic stirrer Manufacturer Adam, USA N-Biotek, Korea P selecta, Spain Bio-Equip Cristofoli, Italy CharmTeck Azzota, USA Digisystem, Taiwan Sony, China Dragon lab, China Microorganisms, media and reagents Microorganisms used in this study are ATCC strains. Reagents are of analytical grade. Media were purchased from HiMedia, India and were prepared according to manufacturer's recommendation (Table 3.2). Table (3.2): Microorganisms, media and reagents used in the study Reagent Bacillus cereus spores ATCC Kocuria rhizophila cells ATCC 9341a Staphylococcus epidermidis cells ATCC Antimicrobials assay media No 4, 8 and 11 Nutrient agar media Sensitivity antibiotic disks; Te (30), P (10), E (15) and N (5). Penicillinase K 2 HPO 4 KH 2 PO 4 Te; Tetracycline P; Penicillin, E; Erythromycin and N; Neomycin. 24 Manufacturer KWIK-STIK, Microbiologics, USA Himedia, India BD, USA Liofilchem, Italy

40 3.1.3 Glassware and disposables The most frequently used glassware and disposables are listed in (Table 3.3). Table (3.3): Glassware and disposables used in experimental work Items Micropipettes and suitable tips. Sterile scalpels Sterile bags. Stainless steel cylinders Erlenmeyer flasks 100,250 and 500 ml. Plastic Petri dishes, 90 x 15 mm. Media bottles, 500 ml. Eppendorf tubes Manufacturer Dragon lab, China Medipharm, China Whirlepak, USA Himedia, USA Rasotherm, Germany Miniplast Kimax, USA Eppendorf 3.2 Study area The study covered the five governorates of Gaza strip; North Gaza, Gaza, the Middle, Khanyounis and Rafah. The study area was divided into 3 regions: 1. North Gaza and Gaza 2. The Middle area 3. Khanyounis and Rafah Study type and piloting The research is a cross sectional analytical study. A pilot study was conducted to evaluate the proposed method wherein 10 broiler chicken meat samples were collected and processed. 3.3 Antibiotic residues detection Numerous types of antimicrobials are used in veterinary medicine to treat chicken. In this research, residues of tetracyclines, β-lactams, aminoglycosides and macrolides groups were investigated Principle of the test The principle of the test is preparing plates seeded with sensitive bacteria at specific conditions that can presumptively indicate the presence of specific antimicrobial group residues depending on the presence or absence of inhibition zones on the seeded plates. 25

41 3.3.2 Microorganisms and media Bacillus cereus spores ATCC Kocuria rhizophila cells ATCC 9341a. Staphylococcus epidermidis cells ATCC Antibiotic assay media No 4, 8 and Samples size and sample collection Three hundred sixty five chicken breast samples were collected from poultry slaughterhouses distributed over study area (Figure 3.1) and packed in sterile bags and kept in a deep freezer (-22 C) until analysed. Carcasses were divided into three categories according to their weights; category A; kg, category B; > kg and category C>2 kg. From each carcass, 20 gm of breast meat were collected as a sample. Samples Distribution 30% 37% North Gaza and Gaza 33% The Middle Khanyounis and Rafah Figure (3.1): Distribution of samples according to study area Buffer preparation Four types of potassium phosphate buffers were prepared using Dipotassium hydrogen phosphate (K 2 HPO 4 ) and Potassium dihydrogen phosphate (KH 2 PO 4 ) [78] as shown in (Table 3.4). 26

42 Table (3.4): Preparation of Phosphate Buffers with varying ph values [78] Buffer strength K 2 HPO 4 (gm) KH 2 PO 4 (gm) (A) 0.1M Phosphate buffer solution ph (B) 0.1M Phosphate buffer solution ph (C) 0.1M Phosphate buffer solution ph (D) 0.2M Phosphate buffer solution ph For each type, the required weights were dissolved in about 800 ml of distilled water. The ph of the solution was adjusted if necessary by the dropwise addition of 0.1 N HCl or 0.1 N NaOH. Using a volumetric flask, solutions were diluted up to 1 liter. Buffers were autoclaved for 15 minutes at 121 C Sample preparation and storage Samples were handled so that freezing and thawing were kept to a minimum. Samples and sample extracts were kept cold at all times with allowance to remain briefly at room temperature during processing and testing. Muscles were cut into 0.5 cm pieces. Sterile bags were used; they were labelled with the sample identification and buffer ph. Four bags for each sample were used; each one has a different buffer (ph 4.5, 0.1M, ph 6, 0.1M, ph 8, 0.2M and ph 8, 0.1M) to identify tetracyclines, β-lactams, macrolides and aminoglycosides respectively. Five grams of a sample were weighed and placed into a sterile bag, then crushed thoroughly by a mortar, after that 20±0.5 ml of an appropriate buffer were added into the bag. After well mixing, grounded tissues were allowed to settle for a minimum of 45 minutes before use. Supernatant fluid was collected and transferred to Eppendorf tube and used as an extract. The sample extracts were refrigerated if they were held for more than 2 hours before use. The extracts may be stored refrigerated for 24 hours, or frozen for 14 days for additional testing [79]. 27

43 3.3.6 Preparation of bacterial suspensions KWIK-STIK is a self-contained package including a lyophilized microorganism pellet, reservoir of hydrating fluid, and inoculating swab (Figure 3.2). Bacteria were cultured according to manufacturer's instructions (Microbiologics) as follows, the ampoule at the top of the KWIK-STIK was pinched to release hydrating fluid then the fluid flowed through shaft into the bottom of the unit containing the pellet. Pellets were crushed in the fluid until pellet suspension was homogenous; the heavily saturated swab with the hydrated material was gently rolled onto one-third of Muller Hinton agar plates. Using a sterile loop, a streak was done to facilitate colony isolation. Plates were incubated at 37 C for 24 hours [80]. After incubation period, an isolated colony was picked by a sterile loop and streaked onto a nutrient agar slant then incubated at 37 C for 24 hours. This step was done for each microorganism. Within 24 hours from slants incubation, under aseptic conditions; sterile nutrient broth was added to the incubated slants, slants were shaken gently to free colonies from agar surface then suspension was returned to a nutrient broth tube. Suspensions were adjusted to equal absorbance to 0.36 at 600 nm wavelength. Figure (3.2): KWIK-STIK device [80] Petri plates preparation Plates were prepared according to Food Safety Inspection Services (FSIS) protocol [79] with few modifications ( inoculum suspension was adjusted as aforementioned and bacterial concentrations were done as shown in table 3.5). Three types of antibiotic assay media (4, 8 and 11) were prepared according to manufacturer's instructions (HiMedia) and autoclaved at 15 psi, 121 C for 15 minutes. After 28

44 completing autoclaving, media bottles were placed in a water bath at 48 C until it cooled to water bath temperature. The required quantity of prepared bacterial suspension was added into the prepared antibiotic assay media to make a final concentration of bacteria that makes a confluent growth on a Petri plate as shown in (Table 3.5). A volume of 1 ±0.1 ml of penicillinase (BD) enzyme per 100 ml of seeded medium (100,000 units per ml of agar) were added and mixed. Using a 20 ml syringe, six ml of the mixture were poured into a Petri plate to make a layer thickness of 1 mm; plates were gently swirled to ensure uniformity. Prepared plates were stored at 2 8ºC and used within 5 days after preparation [79]. Table (3.5): Bacterial suspension concentrations in plates Plate number Microorganism Suspension / 100 ml media Media 1 B. cereus 300 µl 8 2 K. rhizophila 680 µl 4 3 K. rhizophila 680 µl 4* 4 K. rhizophila 680 µl 11 5 S. epidermidis 250 µl 11 * Without penicillinase Assay procedures Standard stainless steel bioassay cylinders were used to apply the extract on agar surface; these cylinders are 10 mm high, 6 mm diameter inside and 8 mm diameter outside as shown in (Figure 3.3). Figure (3.3): Stainless steel bioassay cylinders. 29

45 Five cylinders were placed on the surface of prepared plates as shown in (Figure 3.4), 200µl of sample extract were added into the cylinders by 200 µl micropipette. Figure (3.4): Five bioassay cylinders on an inoculated agar plate surface. Each extract was added into bioassay cylinders on the five prepared petri plates as shown in (Table 3.6). In addition to sample extracts, an antibiotic sensitivity disk for each antibiotic group was placed on the specific plate for that group. Table (3.6): Sample extracts and their specific plates Plate number ph of extraction buffer Antibiotic disk M Tetracycline Te (30) M Penicillin P (10) 3* 6 0.1M Penicillin P (10) M Erythromycin E (15) M Neomycin. N (5) * Without penicillinase Plates from 1 to 4 were incubated at 29 ±1ºC for 16 to 18 h. plate 5 was incubated at 37 ±1ºC for 16 to 18 h. After incubation, the presence or absence of zones of inhibition on each plate was read and recorded [79] Interpretation of results Interpretation of results depends on four basic factors; the microorganism, media type, presence of penicillinase, and a residue level. These factors are illustrated in (Table 3.7). The following sections specify each group of residues [79]. 30

46 Aminoglycosides Macrolides β-lactams Tetracyclines Identification of tetracyclines residues The tetracyclines are identified by zones of inhibition (ZI) > 8 mm on Plate 1. When the concentration of tetracycline is low, there may be no ZI on any other plate. Higher concentrations of tetracyclines may produce zones on any or all of the plates except Plate Identification of β-lactams residues The presence of β-lactams antibiotics like penicillin in a sample is indicated by ZI > 8 mm on plate 2 and no zone on other plates Identification of Macrolides residues The presence of macrolides like erythromycin and tylosin in tissue is indicated by ZI > 8 mm on plate 4. When the concentration of erythromycin is high, inhibition zones may appear on other plates Identification of Aminoglycosides residues Neomycin and gentamicin residues present in tissue at low concentrations (<1.0 µg/g) produces ZI > 8 mm only on Plate 5. At higher concentrations, neomycin and gentamicin may also produce zones of inhibition on additional plates, but not on plate 2 and 3. Table (3.7): Interpretation of results of five-plate bioassay [79] Plate No and Microorganism Agar H L H & L H L H L 1- B. Cereus ATCC S S R S R S R 2-K. rhizophila ATCC 9341a 4 * S R S S R R R 3-K. rhizophila ATCC 9341a 4 S R R S R R R 4-K. rhizophila ATCC 9341a 11 S R R S S S R 5- S. epidermidis ATCC R R R S S S S * Without penicillinase, H: high concentration, L: low concentration. 31

47 3.4 Questionnaire A close-ended questionnaire was designed to collect data about using antimicrobials in chicken breeding. Questions included if breeders have knowledge about instructions of the used drugs and the WP of these drugs. Questions also included listing the used drugs during breeding either orally or parenterally and the reason of their usage. Questionnaires were completed by breeders themselves. See (Annex 1 and 2) for Arabic and English version of the questionnaire. 3.5 Data analysis Data obtained from the analysis of broiler chicken samples and those obtained from the questionnaire survey were entered into SPSS software. Data were summarized and crosstabulations were made. 32

48 Chapter IV Results 4.1 Distribution of samples in the study area Samples were collected from different slaughterhouses distributed in the study area and in addition, samples were collected at intervals to ensure sample representativeness. Total of 365 samples were collected from different regions and were categorized as shown in (Table 4.1). Table (4.1): Distribution of samples according to chicken weight and the study area Region Category A Category B Category C Total % 1.5 kg >1.5-2 kg >2 kg North and Gaza Middle Khanyounis and Rafah Total number % Detection of antibiotic residues A sample is considered a positive sample (containing residues) when an extract inhibits the growth of bacteria on any plate with a zone of inhibition more than 8 mm in diameter as shown in (Figure 4.1). Figure (4.1): A positive sample showing 20 mm inhibition zone on plate 5 (That detects aminoglycosides residues). 33

49 From 365 tested samples, 88 (24.1%) samples were shown to contain one or more antibiotic residues (Figure 4.2). 24% 76% Negative Positive Figure (4.2): Overall positive samples of antibiotic residues. 4.3 Detection of antibiotics according to samples weight Residues were detected in 88 samples from all categories but the most frequently detected residues was from category (A) ( 1.5 kg) 47 (53.41%), followed by 29 (32.95%) from category (B) (>1.5-2 kg) and the least was category (C) (>2 kg) which were 12 (13.63%) as shown in (Figure 4.3). Positive results within categories Category A Category B Category C 14% 33% 53% Figure (4.3): Positive samples distribution according to carcasses weight. 34

50 4.4 Frequency of positive results of samples according to region The highest percentage of antibiotic residues was detected in the middle region 34/121 (28.1%). On the other hand, Khanyounis and Rafah have the least percentage of residues that were detected in 24/110 (21.8%). Positive results distributed by regions are shown in (Table 4.2). Table (4.2): Distribution of positive results according to regions Regions Samples Positive samples North and Gaza (22.38%) Middle (28.1%) Khanyounis and Rafah (21.8%) Total (24.1%) Results showed that the most detected residues were from category (A) from all regions as illustrated in (Table 4.3). Table (4.3): Distribution of positive results by region and weight categories Region Category A Category B Category C Total kg >1.5-2 kg >2 kg North and Gaza 19 (48.7%) 6 (12.5%) 5 (13.5%) 30 (22.8%) Middle 16 (41%) 14 (28%) 4 (12.5%) 34 (28%) Khanyounis and Rafah. 12 (29.2%) 9 (23%) 3 (10%) 24 (21.8%) Total 47 (53.4%) 29 (28.4%) 12 (12.6%) 88 (100%) 4.5 Determination of antibiotic groups The most detected antibiotic residues were tetracyclines 41/95 (43.15%) followed by aminoglycosides 26/95 (27.36%) then 20/95 (21%) and 8/95 (8.42%) for β-lactams and macrolides respectively as shown in (Table 4.4). 35

51 Table (4.4): Distribution of detected antibiotic groups according sample weight categories Sample Category Tetracyclines β-lactams Macrolides Aminoglycoside Total Category A ( 1.5 kg) 22 (42.3%) 12 (23%) 3 (5.77%) 15 (28.84%) 52 Category B (>1.5-2 kg) 14 (46.66%) 6 (20%) 4 (13.33%) 6 (20%) 30 Category C ( >2 kg) 5 (38.46%) 2 (15.4%) 1 (7.7%) 5 (38.46%) 13 Total 41 (43.15%) 20 (21%) 8 (8.42%) 26 (27.36%) Antibiotic groups according to regions As mentioned earlier, tetracyclines are the most frequently detected residues among groups, they are the most detected residues in each region and macrolides are the least detected group (Table 4.5). Table (4.5): Distribution of detected antibiotic groups according to regions Region Tetracyclines β-lactams Macrolides Aminoglycosides North and Gaza. 16 (39%) 6 (30%) 2 (25%) 10 (42.1%) Middle. 13 (31.7%) 12 (60%) 3 (37.5%) 7 (36.84%) Khanyounis and Rafah. 12 (29.3%) 2 (10%) 3 (37.5%) 9 (21.05%) Total 41 (100%) 20 (100%) 8 (100%) 26 (100%) 4.7 Detection of multiple antibiotics residues Although 88 samples were containing antibiotic residues, 7 samples contained more than one residue. In category (A), six samples contained two different antibiotic residues (Table 4.6). Table (4.6): Multiple detection of antibiotic groups Sample group One More than one Category A 41 6 Category B Category C 11 1 Total

52 4.8 Questionnaire results Ninety breeders responded to our questionnaire Drug usage pattern in broiler breeding Ninety questionnaires were filled in, self-administered, by broiler breeders from different regions in the study area; the questionnaire is concerned essentially about antimicrobials usage in broiler breeding. The main breeders' responses are presented in (Table 4.7). Table (4.7): Breeder's responses to the questionnaire Subject Yes % No % I use antimicrobials without signs of sickness I have good knowledge of the nature of all used drugs I have good knowledge of the instructions of the used drugs I have good knowledge of the WP of used drugs I accelerate healing by using a double dose of drugs I use more than one antimicrobial in a single treatment I'm aware of the negative effect of drug residues on human health I use steroids during breeding I do sell chickens during a treatment period Breeders were asked about consulting veterinarians, method of antibiotic administration and time of marketing after treatment. Breeders' responses are shown in (Table 4.8). 37

53 Table: (4.8): Breeders' behaviors in dealing with antibiotics in farms Subject Options Response of Percentage 90 breeders Consulting veterinarians Always % Method of antibiotic administration. Stop giving drugs before marketing. Waiting of marketing after parenteral treatment. Reason of using antimicrobials Sometimes % Never 5 5.6% Feed 0 0% Water % Parenteral 56 62% One day 8 9% Two days % More 71 79% A week % Ten days % Two weeks % Prevention % Treatment % Enhancement 0 0% Antimicrobial usage during broiler breeding This section of the questionnaire was filled in with the assistance of veterinarians. After dividing antimicrobials to groups, nine groups were identified in usage during broiler breeding in Gaza strip. Results showed that aminoglycosides and tetracyclines were the most frequently used antimicrobials, while florfenicol was the least used antimicrobial. The used antimicrobial groups in chicken therapy in Gaza strip are illustrated in (Figure 4.4). 38

54 Florfenicol 10 Lincosamides Beta-lactams Sulphonamides 34.4 Macrolides Polymyxins Flouroquinolones Tetracyclines Aminoglycosides Percentage Figure (4.4): Antimicrobials used in broiler chickens therapy Antimicrobial used parenterally The collected data determine eight antimicrobials used parenterally, some of these antimicrobials were used also in drinking water, and others were used only parenterally as cephalosporins. Lincospectin (lincomycin and spectinomycin) is the most frequently used antimicrobial (50%), followed by gentamycin (46%) while ampicillin and colistin (1.8%) were the least used antimicrobials. Antimicrobial types and their frequency are illustrated in (Figure 4.5). 39