Islamic University-Gaza ‫الجاهعت االسالهيت – غزة‬

Deanship of Graduate Studies ‫عوادة الدراساث العليا‬

Faculty of Science ‫كليت العلوم‬
Biological Sciences Master ‫بزناهج هاجستيز العلوم الحياتيت‬
Program / Microbiology ‫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
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
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
 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 2014. 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);
> 1.5-2 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
 ‫الملخص‬

‫الكشف عن هتبقياث الوضاداث الحيويت في لحوم الدجاج الالحن في قطاع غزة‬

‫اكزسجذ يزجم‪ٛ‬بد االدٔ‪ٚ‬خ انج‪ٛ‬طش‪ٚ‬خ ف‪ ٙ‬االغز‪ٚ‬خ انكض‪ٛ‬ش يٍ االْزًبو ف‪ ٙ‬انسُٕاد األخ‪ٛ‬شح ثسجت رضا‪ٚ‬ذ انًخبٔف‬
‫انًزؼهمخ ثساليخ األغز‪ٚ‬خ ػُذ انًسزٓهك‪ ،‬إر ‪ٚ‬ؤد٘ ٔعٕد يزجم‪ٛ‬بد يعبداد انغشاص‪ٛ‬ى ف‪ ٙ‬انًُزغبد انغزائ‪ٛ‬خ آصبسا‬
‫سهج‪ٛ‬خ ػهٗ صؾخ انًسزٓهك ؽ‪ٛ‬ش لذ رض‪ٚ‬ذ انفشصخ نظٕٓس انسشغبَبد ٔانطفشاد انغ‪ُٛٛ‬خ ٔص‪ٚ‬بدح انؾسبس‪ٛ‬خ‬
‫ٔكزنك ظٕٓس سالالد ثكز‪ٛ‬ش‪ٚ‬خ يمبٔيخ نهًعبداد انغشصٕي‪ٛ‬خ‪ْ ،‬زِ االسجبة رغؼم يٍ انعشٔسح ٔعٕد سلبثخ‬
‫ٔرُظ‪ٛ‬ى السزخذاو االدٔ‪ٚ‬خ انج‪ٛ‬طش‪ٚ‬خ ف‪ ٙ‬ػالط انؾ‪ٕٛ‬اَبد انًُزغخ نغزاء االَسبٌ‪.‬‬

‫ْذفذ ْزِ انذساسخ نزم‪ٛٛ‬ى ٔكشف ٔعٕد يزجم‪ٛ‬بد ثؼط انًعبداد انؾ‪ٕٚٛ‬خ ف‪ ٙ‬نؾٕو انذٔاعٍ انًشثبح ف‪ ٙ‬لطبع‬
‫غضح‪ ،‬ؽ‪ٛ‬ش شًهذ انذساسخ عً‪ٛ‬غ يؾبفظبد لطبع غضح ٔاسزًش عًغ انؼ‪ُٛ‬بد يٍ شٓش ‪ُٚ‬ب‪ٚ‬ش ؽزٗ َٓب‪ٚ‬خ شٓش‬
‫‪ َٕٕٛٚ‬نؼبو ‪ .4102‬رى عًغ صالصًبئخ ٔخًسخ ٔسز‪ ٍٛ‬ػ‪ُٛ‬خ يٍ ػعهخ صذس انذعبط يٍ يسبنخ انذٔاعٍ انًٕصػخ‬
‫ف‪ ٙ‬يُطمخ انذساسخ ٔرى انزؾش٘ ػٍ ٔعٕد اسثؼخ يغًٕػبد يٍ انًعبداد انؾ‪ٕٚٛ‬خ ْٔ‪ ٙ‬يغًٕػخ‬
‫انززشاس‪ٛ‬كه‪ُٛ‬بد‪ ،‬االي‪ُٕٛ‬عه‪ٛ‬كٕس‪ٛ‬ذاد‪ ،‬انًبكشٔن‪ٛ‬ذاد ٔيغًٕػخ انجُسه‪ُٛ‬بد ٔرنك ثبسزخذاو غش‪ٚ‬مخ انًمب‪ٚ‬سخ‬
‫انؾ‪ٕٚٛ‬خ انًٕصٗ ثٓب يٍ لجم ٔصاسح انضساػخ االيش‪ٚ‬ك‪ٛ‬خ‪ .‬رى رمس‪ٛ‬ى رثبئؼ انذعبط إنٗ صالس فئبد ٔفمب ألٔصآَب‪،‬‬
‫فئخ (أ) يٍ ‪0‬ؽزٗ‪ 0.1‬كغى‪ ،‬انفئخ (ة) اكضش يٍ ‪ 0.1‬ؽزٗ ‪ 4‬كغى ٔفئخ (ط) اكضش يٍ ‪ 4‬كغى‪.‬‬
‫اظٓشد انُزبئظ اؽزٕاء صًبَ‪ٛ‬خ ٔصًبَ‪ ٍٛ‬ػ‪ُٛ‬خ ػهٗ ٔاؽذ أ أكضش يٍ يغًٕػبد انًعبداد انؾ‪ٕٚٛ‬خ انًزؾشٖ ػُٓب‬
‫ثًب ‪ٚ‬شكم يب َسجزّ ‪ ،%42‬كًب رج‪ ٍٛ‬اٌ ‪ %1..2‬يٍ انًزجم‪ٛ‬بد كبَذ يٍ ظًٍ انًغًٕػخ (أ) ٔ ‪ %.4.3‬يٍ‬
‫ظًٍ انًغًٕػخ (ة) ٔكبَذ الم انًغًٕػبد إؽزٕاءاّ نهًزجم‪ٛ‬بد ْ‪ ٙ‬انًغًٕػخ (ط) انز‪ ٙ‬رضٌ رثبئؾٓب اكضش يٍ‬
‫اصُ‪ ٍٛ‬ك‪ٛ‬هٕعشاو‪ .‬كًب اظٓش رؾه‪ٛ‬م انؼ‪ُٛ‬بد انًؾزٕ‪ٚ‬خ ػهٗ انًزجم‪ٛ‬بد اٌ يغًٕػخ انززشاس‪ٛ‬كه‪ُٛ‬بد كبَذ اكضشْب‬
‫رٕاعذا ثُسجخ ‪ %2..0‬يزجٕػخ ثًغًٕػخ االي‪ُٕٛ‬عه‪ٛ‬كٕس‪ٛ‬ذاد ثُسجخ ‪ٔ %4...‬رهزٓب يغًٕػخ انجُس‪ٛ‬ه‪ُٛ‬بد ‪%40‬‬
‫ٔكبَذ الهٓى يغًٕػخ انًبكشٔن‪ٛ‬ذاد ‪ٕٚٔ %4.2‬صٗ ثبسزخذاو اؽذ انطشق انزأك‪ٛ‬ذ‪ٚ‬خ يضم االسزششاة انغبص٘‬
‫نزؾذ‪ٚ‬ذ كً‪ٛ‬خ انًزجم‪ٛ‬بد ٔيؼشفخ يطبثمزٓب نهؾذ االدَٗ انًسٕػ ثّ نزٕاعذْب ف‪ ٙ‬نؾٕو انذٔاعٍ‬
‫ٔخهصذ انذساسخ اٌ انُزبئظ اكذد ٔعٕد يزجم‪ٛ‬بد انًعبداد انؾ‪ٕٚٛ‬خ ف‪ ٙ‬نؾٕو انذٔاعٍ ف‪ ٙ‬لطبع غضح ثشكم‬
‫كج‪ٛ‬ش ْٔزا يٍ انًؾزًم اٌ ‪ٚ‬شكم خطشا ػهٗ صؾخ انًسزٓهك‪ٔ .ٍٛ‬ػه‪ٚ ّٛ‬غت اٌ رزخز انًؼب‪ٛٚ‬ش انالصيخ نهزأكذ يٍ‬
‫االسزخذاو انغ‪ٛ‬ذ نألدٔ‪ٚ‬خ ٔيشاػبح يشالجخ فزشح سؾت انذٔاء يٍ اعسبو انذٔاعٍ لجم رسٕ‪ٚ‬مٓب ٔ رؾس‪ ٍٛ‬انس‪ٛ‬طشح‬
‫ػهٗ االسزخذاو انؾ‪ٕٛ‬اَ‪ ٙ‬نهًعبداد انؾ‪ٕٚٛ‬خ‪ .‬كًب ‪ٕٚ‬صٗ ثزطج‪ٛ‬ك َظبو رمص‪ٔ ٙ‬فؾص نهؾٕو انذٔاعٍ ٔنهًُزغبد‬
‫انغزائ‪ٛ‬خ األخشٖ انؾ‪ٕٛ‬اَ‪ٛ‬خ انًُشأ نهزأكذ يٍ يطبثمزٓب نهًٕاصفبد انؼبنً‪ٛ‬خ انخبصخ ثؾذٔد انًزجم‪ٛ‬بد انذٔائ‪ٛ‬خ‬
‫انٕاعت ػذو رغبٔصْب‪.‬‬

‫الكلواث الوفتاحيت‪ :‬يزجم‪ٛ‬بد انًعبداد انؾ‪ٕٚٛ‬خ ‪ ،‬انؾذ االػهٗ نهًزجم‪ٛ‬بد‪ ،‬انذعبط انالؽى‪ ،‬لطبع غضح‬

‫‪IV‬‬
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 1
1.2 Objectives 2
1.2.1 General objective 2
1.2.2 Specific objective 2
1.3 Significance 2
Chapter II: Literature Review
2.1 Poultry production 3
2.2 Antimicrobials 4
2.2.1 Definition of antimicrobials 4
2.2.2 Classification of antimicrobials 4
2.2.3 Common antimicrobials used in poultry 5
2.2.3.1 Tetracyclines 5
2.2.3.2 β–lactams 6
2.2.3.3 Macrolides 7
2.2.3.4 Aminoglycosides 7
2.2.4 Antimicrobials usage in veterinary medicine 7
2.2.5 Antimicrobials resistance 8
2.2.6 Emergence of resistant bacteria in chicken 8

2.3 Drug residues 10

2.3.1 Drug residues definition 10
2.3.2 Effects of veterinary drug residues 11

V
 2.3.3 Maximum residue limit 11
2.3.4 Withdrawal period 12
2.4 Prohibition of some antimicrobials 13
2.5 Cooking effect on antimicrobial residues 14
2.6 Detection of drug residues 16
2.6.1 Screening methods 16
2.6.1.1 Classification of screening methods by detection principle 16
2.6.2 Confirmation methods 18
2.6.3 Microbiological assay 18
2.6.4 Microbiological assay methods 19
2.6.5 Examples of microbiological assay methods 19
2.6.5.1 Four plate Test (FPT) 19
2.6.5.2 The Calf Antibiotic and Sulfonamide Test (CAST) 20
2.6.5.3 Screening Test for Antibiotic Residues (STAR) 20
2.6.5.4 Premi's test 20
2.6.5.5 CHARM test 21
2.7 Residue control programs 21
2.8 Previous studies 22
Chapter III: Materials and Methods
3.1 Materials 24
3.1.1 Equipment 24
3.1.2 Microorganisms, media and reagents 24
3.1.3 Glassware and disposables 25
3.2 Study area 25
3.2.1 Study type and piloting 25
3.3 Antibiotic residues detection 25
3.3.1 Principle of the test 25
3.3.2 Microorganisms and media 26
3.3.3 Samples size and sample collection 26
3.3.4 Buffer preparation 26
3.3.5 Sample preparation and storage 27
3.3.6 Preparation of bacterial suspensions 28

VI
 3.3.7 Petri plates preparation 28
3.3.8 Assay procedures 29
3.3.9 Interpretation of results 30
3.3.9.1 Identification of tetracyclines residues 31
3.3.9.2 Identification of β-Lactams residues 31
3.3.9.3 Identification of Macrolides residues 31
3.3.9.4 Identification of Aminoglycosides residues 31
3.4 Questionnaire 32
3.5 Data analysis 32
Chapter IV: Results
4.1 Distribution of samples in the study area 33
4.2 Detection of antibiotic residues 33
4.3 Detection of antibiotics according to samples weight 34
4.4 Frequency of positive results of samples according to region 35
4.5 Determination of antibiotic groups 35
4.6 Antibiotic groups according to regions 36
4.7 Detection of multiple antibiotics residues. 36
4.8 Questionnaire results 37
4.8.1 Drug usage pattern in broiler breeding 37
4.6.2 Antimicrobial usage during broiler breeding. 38
4.6.3 Antimicrobial used parenterally 39
Chapter V: Discussion
5.1 Detection of antibiotic residues 43
5.1.1 Tetracylines detection 45
5.1.2 Aminoglycoside detection 46
5.1.3 β-lactams detection 47
5.1.4 Macrolides detection 47
5.2 Frequency of residues in the study area 47
5.3 Antibiotic residues and carcasses weight 48
5.4 Questionnaire analysis 49
Chapter VI: Conclusions and Recommendations
6.1 Conclusions 51

VII
6.2 Recommendations 52
References 54
Annex 1 63
Annex 2 65

VIII
List of tables
Table (2.1): Poultry production expressed as numbers from 1970-1995 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
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
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
 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
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
 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 1970-1995 in West Bank
and Gaza strip [8]
Year Layers Broilers
1970 70,000 3,400,000
1973 118,000 4,330,000
1975 120,000 3,550,000
1978 179,000 2,490,000
1981 170,000 3,500,000
1984 89,000 4,400,000
1987 217,000 16,450,000
1990 418,000 16,900,000
1993 620,000 18,800,000
1995 1,812,000 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
Table (2.2): Poultry production in Palestinian territories [9]
Region Broilers Layers Broilers Turkey
mothers
2009/2010
Gaza strip 7,556,507 311,280 22,790 34,670
West Bank 23,554,904 1,233,736 376,633 486,460
Total 31,111,411 1,545,016 399,423 521,130
2010/2011
Gaza strip 16,373,467 297,678 2,200 -
West Bank 20,174,056 1,328,779 534,988 318,420
Total 36,547,523 1,626,457 537,188 318,420
2012/2013
Gaza strip 8.218.180 351.199 - 8.093
West Bank 23.297.203 1.425.579 994.620 538.320
Total 31,515,383 1.776.778 994.620 546.413

2.2 Antimicrobials
2.2.1 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].

2.2.2 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
 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

2.2.3 Common antimicrobials used in poultry

2.2.3.1 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
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).

2.2.3.2 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
2.2.3.3 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].

2.2.3.4 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].

2.2.4 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
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].

2.2.5 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].

2.2.6 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 antimicrobials-

resistant 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
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
(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

2.3.1 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
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].

2.3.2 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.

2.3.3 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 1986. Codex has defined 590 MRLs for some fifty nine
11
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]

Poultry muscle Poultry liver
Antimicrobial
(µg/kg) (µg/kg)
Chlortetracycline/ Oxytetracycline/ 200 600
Tetracycline
Neomycin 500 500
Spectinomycin 500 2000
Streptenomycin/ dihydrostreptomycin 600 600
Procaine benzylpenicillin 50 50
Flumequine 500 500
Danofloxacin 200 400
Tylosin 100 100
Erythromycin 100 100
Spiramycin 200 600
Colistin 150 150
Lincomycin 200 500

2.3.4 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
Table (2.4): Withdrawal periods of antimicrobials used in poultry production [43]

Drug Administration Animal Withdwral period

route (Days)
Chicken 1
Tylosin tartarate D.W
Turkey 5
Erythromycin D.W Poultry 1
Chicken 35
Gentamicin sulphate SC.
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
Chicken 2
Amoxicillin D.W
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 1994. 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
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
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
2.6 Detection of drug residues
2.6.1 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].

2.6.1.1 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
Table (2.5): Advantages and disadvantages of some screening methods [54]
Test Advantages
 Easy to use
 Availability for a good number of
specific compounds.
 Availability for families of
compounds (e.g. sulfanomides,
estilbenes).
 Large number of samples (42) per
kit for a single analyte.
ELISA  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.
Disadvantages
 Increased cost
 Limited storage (few
months) under refrigeration.
 The need for waste disposal.
 Interferences giving some
false positives.
 Only one kit per residue
searched.

 Easy to use.  High operative costs chips

 Results available in short time. and equipment cost.
 Multiples residues analyzed in one  Analysis restricted to
Biochip shot (as many as in an array). available chips
array  Full automation: higher
biosensors productivity.
 High through-put technique: up to
120 samples per hour and array.

 Reduced time (few hours) to obtain  Expertise required.

results.  Needs sample preparation
 Sensitive (Extraction, filtration,
HPLC  Automation leading to higher addition of internal
productivity. standards, etc.).
 Specificity depending on a detector  Expensive.

 Can be used for large surveillance  Difficult to standardize

programmers. preparation procedures.
 Basic laboratory equipment.  Some test could not insure
 Broad spectrum. MRLs compliance.
Microbial  Easy to use.  Sample preparation required
methods  Economical. 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
On the other side residue determination by chemical methods such as
chromatography (by all its types) depends on the chemical properties [55].

2.6.2 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].

2.6.3 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
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].

2.6.5 Examples of microbiological assay methods

2.6.5.1 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
2.6.5.2 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 16–20 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].

2.6.5.3 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 11778 for tetracyclines, the plate E. coli ATCC 11303 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 10149
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].

2.6.5.4 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
detection limit), the spores will be unable to germinate and therefore no color change
will be observed [65].

2.6.5.5 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
14
origin including honey. The test employs C-labeled or 3H-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
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
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
 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.

3.1.1 Equipment
Table (3.1): Equipment used in the study
Items Manufacturer
Balance, analytical.
Adam, USA
Balance, 0.1 to 200 gram capacity
Water bath
Incubator. N-Biotek, Korea
Safety cabinet
Refrigerator. P selecta, Spain
Freezer. Bio-Equip
Autoclave Cristofoli, Italy
Spectrophotometer CharmTeck
pH meter Azzota, USA
Vortex mixer Digisystem , Taiwan
Digital camera Sony, China
Hot plate and Magnetic stirrer Dragon lab, China

3.1.2 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 Manufacturer
Bacillus cereus spores ATCC 11778
KWIK-STIK™,
Kocuria rhizophila cells ATCC 9341a
Microbiologics, USA
Staphylococcus epidermidis cells ATCC 12228
Antimicrobials assay media No 4, 8 and 11
Nutrient agar media Himedia, India
Sensitivity antibiotic disks; Te (30), P (10), E (15) and N (5).
Penicillinase BD, USA
K2HPO4
Liofilchem, Italy
KH2PO4
Te; Tetracycline P; Penicillin, E; Erythromycin and N; Neomycin.

24
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 Manufacturer
Micropipettes and suitable tips. Dragon lab, China
Sterile scalpels Medipharm, China
Sterile bags. Whirlepak, USA
Stainless steel cylinders Himedia, USA
Erlenmeyer flasks 100,250 and 500 Rasotherm, Germany
ml.
Plastic Petri dishes, 90 x 15 mm. Miniplast
Media bottles, 500 ml. Kimax, USA
Eppendorf tubes 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

3.2.1 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.

3.3.1 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
3.3.2 Microorganisms and media
Bacillus cereus spores ATCC 11778.
Kocuria rhizophila cells ATCC 9341a.
Staphylococcus epidermidis cells ATCC 12228.
Antibiotic assay media No 4, 8 and 11.

3.3.3 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;
1-1.5 kg, category B; > 1.5-2 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

The Middle
33%

Khanyounis and Rafah

Figure (3.1): Distribution of samples according to study area.

3.3.4 Buffer preparation

Four types of potassium phosphate buffers were prepared using Dipotassium
hydrogen phosphate (K2HPO4) and Potassium dihydrogen phosphate (KH2PO4) [78]
as shown in (Table 3.4).

26
Table (3.4): Preparation of Phosphate Buffers with varying pH values [78]

Buffer strength K2HPO4(gm) KH2PO4(gm)

(A) 0.1M Phosphate buffer solution pH 4.5 ------------ 13.61

(B) 0.1M Phosphate buffer solution pH 6.0 2.8 11.2

(C) 0.1M Phosphate buffer solution pH 8.0 16.73 0.523

(D) 0.2M Phosphate buffer solution pH 8.0 33.46 1.046

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.

3.3.5 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
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].

3.3.7 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
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

3.3.8 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
 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
1 4.5 0.1M Tetracycline Te (30)
2 6 0.1M Penicillin P (10)
3* 6 0.1M Penicillin P (10)
4 8 0.2M Erythromycin E (15)
5 8 0.1M 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].

3.3.9 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
3.3.9.1 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 5.

3.3.9.2 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.

3.3.9.3 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.

3.3.9.4 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]

Aminoglycosides
Tetracyclines

Macrolides
β-lactams

Plate No and Microorganism Agar

H L H&L H L H L
1- B. Cereus ATCC 11778 8 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 12228 11 R R R S S S S
* Without penicillinase, H: high concentration, L: low concentration.

31
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
 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 49 48 37 134 36.7
Middle 39 50 32 121 33.1
Khanyounis and 41 39 30 110 30.1
Rafah
Total number 129 137 99 365 100

% 37.5 35.3 27.1 100

4.2 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
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%

53%
33%

Figure (4.3): Positive samples distribution according to carcasses weight.

34
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 134 30 (22.38%)
Middle 121 34 (28.1%)
Khanyounis and Rafah 110 24 (21.8%)
Total 365 88 (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
1≥1.5 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
 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%) 95

4.6 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 29 ---
Category C 11 1
Total 81 7

36
4.8 Questionnaire results
Ninety breeders responded to our questionnaire

4.8.1 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. 61 67.7 29 32.3
I have good knowledge of the nature of all used drugs. 71 78.9 19 21.1
I have good knowledge of the instructions of the used drugs. 51 56.6 39 43.4
I have good knowledge of the WP of used drugs. 68 75.5 22 24.5
I accelerate healing by using a double dose of drugs. 49 54.4 41 45.6
I use more than one antimicrobial in a single treatment. 70 77.8 20 22.2
I'm aware of the negative effect of drug residues on human health. 76 84.4 14 15.6
I use steroids during breeding. 18 20 72 80
I do sell chickens during a treatment period. 41 45.5 49 54.5

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
Table: (4.8): Breeders' behaviors in dealing with antibiotics in farms
Subject Options Response of Percentage
90 breeders
Consulting veterinarians Always 33 36.6%

Sometimes 52 57.8%
Never 5 5.6%
Method of antibiotic Feed 0 0%
administration. Water 90 100%
Parenteral 56 62%
Stop giving drugs before One day 8 9%
marketing. Two days 11 12.2%
More 71 79%
Waiting of marketing after A week 30 53.6%
parenteral treatment. Ten days 13 23.2%
Two weeks 13 23.2%
Reason of using Prevention 48 53.3%
antimicrobials Treatment 50 55.5%
Enhancement 0 0%

4.6.2 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
 Florfenicol 10

Lincosamides 26.6

Beta-lactams 26.6

Sulphonamides 34.4

Macrolides 53.3

Polymyxins 56.6

Flouroquinolones 66.6

Tetracyclines 70

Aminoglycosides 72.2

0 10 20 30 40 50 60 70 80 90 100
Percentage
Figure (4.4): Antimicrobials used in broiler chickens therapy.

4.6.3 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
 60%

50%
50% 46.40%

40% 37.50%

30.40%
30%
25%

20%

10%
5.30%
3.60%
1.80%
0%

Figure (4.5): Antimicrobials used parenterally in broiler chickens therapy.

40
 Chapter V
Discussion

Antimicrobial residues in food of animal origin have received much attention in

developed countries to ensure food safety, many countries have monitoring programs
to avoid AMR in food of animal origin [81]. Currently, Palestine has no regulations
regarding the use of antimicrobials or the maximum allowable antimicrobial
concentrations in food. Additionally, there are no systems to monitor the presence of
AMR in animal products in Palestine. Therefore, screening of food products from
animal origin intended for human consumption for the presence of AMR is essential
to ensure food safety.

In this study, AMR in poultry meat slaughtered in Gaza strip was evaluated. Poultry
meat was chosen because it is the most meat consumed by Gaza strip inhabitants.
The average chicken meat consumed each month in Gaza strip amounts to 2,000 to
2,800 tons [82]. No previous studies done concerning antimicrobial residue in meat
and the only published study conducted in Palestine reported the detection of AMR
in food was carried out in the West Bank to detect β-Lactams and Tetracyclines in
raw milk [83].

Concentration of drug residue may vary between different tissues and most
monitoring programs use the term "muscle tissues" without specifying which type of
muscles that should be used. Enrofloxacin residues were proved to be higher in
breast muscles than in thigh muscles [84], also there isn't difference in levels of
enrofloxacin residues between breast sections, therefore samples can be collected
from any breast section to evaluate fluoroquinolone residue concentrations [85]. This
situation may be compatible with other AMR.

Anyhow, breast muscles were selected since they do not contain fats and because of
their easiness of handling. Furthermore, the widely usage of breast muscle in
preparation of different local meat dishes e.g. Shawarma, Thai chicken and Shish
Taouk made pectoral muscle tissue a good choice to be screened.

41
The most commonly sold antimicrobial classes in the major livestock especially in
poultry production in 15 countries from Europe, Asia and Australia were penicillins,
tetracyclines, macrolides and aminoglycosides, especially since each of these classes
has been in use for more than 50 years [86]. In Saudi Arabia, enrofloxacin,
oxytetracycline, ampicillin, neomycin and sulphamethoxazole were the most
veterinary drugs used in poultry production [87]. These antimicrobials are
administered to broilers by injections (intramuscularly or subcutaneously) and orally
in food or water [88]. Although many antimicrobial groups are used in Gaza strip,
tetracyclines, β-lactams, aminoglycosides and macrolides were chosen to be screened
because they are extensively used in poultry medication, have long WPs and can be
used in various production periods.

Screening techniques are the first step in determination the presence of

antimicrobials in food of animal origins, these techniques may use biological
methods, biochemical methods and physicochemical methods [89]. Microbiological
inhibition tests are cheap and permit to analyze a large number of samples in a short
time. Microbiological screening relies on a common property of all antibacterials;
they inhibit growth of microorganisms [90].

The National Residue Program conducted by Food Safety and Inspection Service
(FSIS) of the United States Department of Agriculture (USDA) uses a 7-plate
method to detect AMR in meat and poultry tissues [79].

In this study, the 7-plate method was used with a slight modifications; five plates
were used instead of seven, two plates were excluded, one of them for streptomycin
detection and the other for erythromycin confirmation. Streptomycin is not
commonly used in Gaza strip to treat poultry, while, erythromycin belongs to
macrolides and can be detected by the specific plate which detect macrolides group
without necessity for confirmation whether a residues is erythromycin or tylosin.

Since poultry producers distribute their products in farm origin area and neighboring
places and to increase collection feasibility, the study area was divided into three
regions, North Gaza and Gaza governorates, the middle governorate and Khanyounis
and Rafah governorates.
42
5.1 Detection of antibiotic residues
Antibiotic residues were detected in 88/365 (24.1%) samples. This study used a
screening method to detect the presence of antibiotic residues; therefore it could not
be possible to proof their exceedance of the maximum residue limits. A confirmatory
method like HPLC or GC is required to determine the type and the level of AMR in a
sample [91]. These methods were not used in this research due to budget limitations.

Similar results were recorded in Egypt in a study on one hundred randomly collected
fresh and frozen broiler fillet samples (50 of each) and evaluated the AMR level
qualitatively by microbiological inhibition assay using B. subtilis (ATCC 6633). It
was found that the sum of positive samples for AMR in both fresh and frozen fillet
representing 21% of total examined samples [76].

In another study also in Egypt using FPT high percentages of AMR were detected in
broiler meat 90%, 88% and 72% in fresh, local frozen and imported frozen meat
respectively. These high results may be due to the fact that the researcher
investigated the presence of two additional groups of antimicrobials, sulfonamides
and quinolones in addition to the four groups investigated in the current study [92].
In the aforementioned study, 72% frozen imported broiler meat showed presence of
AMR, this may be as a result of importing meat from countries that do not have
monitoring programs, also the small sample size (25) may interferes with results.

A high percentage of AMR was recorded (70.8% of samples were positive for AMR)
using Premi®Test [93]. This test is based on the growth inhibition of B.
stearothermophilus and is capable of detecting β-lactams, cephalosporines,
macrolides, tetracyclines, sulphonamides, aminoglycosides, quinolones,
amphenicoles and polypeptides, this property may explains the high reported results
[65]. In a similar study conducted in Sudan, a microbiological bioassay using three
plates method revealed that 27% of 221 samples contained AMR; in muscles 28
(28%) samples had AMR, this result is similar to what obtained from the current
study [94].
In Saudi Arabia, 69.7% of 33 broilers farms in the eastern province were proved to
contain AMR detected by a microbiological assay using four different reference
strains of bacteria. Interestingly one or more compounds of tetracyclines were

43
detected in all farms [49]. The high percentage in that area may be attributed to the
fact that tetracyclines are added to water as a feed additive to control mycoplasma
infection.

In another study, thin layer chromatography (TLC) was used to detect

oxytetracycline, sulfadiazine, neomycin, and gentamycin in stored poultry product,
44% of muscles (breast and thigh) contained oxytetracycline and sulfadiazine but no
neomycin and gentamycin were detected [74].

Microbial inhibition test with B. cereus ATCC 11778 and M. luteus ATCC 9341 was
used to investigate the presence of antimicrobials in slaughtered chickens in Kaduna
State, Nigeria. About 33% of broilers had AMR [95]. However, researchers of this
study used chicken feces instead of muscles or other organs and this may not express
or correlate real antimicrobial accumulations in tissues since some antimicrobials are
poorly absorbed from gut such as aminoglycosides.

In another study carried out also in Nigeria, 70 commercial broiler birds were
screened by Premi®Test, four organs were screened from each carcass (muscles,
kidneys, livers and gizzards), overall results revealed that 42(60%) were positive.
Regarding muscle samples, 14.3% were positive and 10% doubtful [77]. Comparable
research carried out on 500 broiler carcasses in Pakistan using Swab Test on Animal
Food (STAF) method revealed that residues were detected in 32.0% of the breast
meat and found to be 30% of the thigh samples from the same carcass [96]. These
results are not much different from the recorded in the current research.

A Bulgarian study utilised FPT to determine the presence of AMR in chicken tissues
(breast muscles, liver and kidneys). From 115 meat samples (pectoral muscles), only
2 samples (1.7%) were identified as AMR-positive while 17 (8.8%) livers and 33
(21.%) kidneys were positive [73]. In Belgium, a study used a combination of three
plates, seeded with strains of M. luteus, B. cereus or E. coli to detect residues of β-
lactam antimicrobials, tetracyclines and fluoroquinolones respectively in poultry
meat. Eighteen out of the 228 (7.9%) broilers contained antimicrobials [70].

44
Investigations in countries where official regulations have been established
demonstrate that regulations have been successful for controlling this issue. For
example in the United Kingdom the surveillance for veterinary residues in food in
2007 revealed that violations found for all products were less than 0.5%. In broiler
muscles, one sample out of 1158 was had a sulfadiazine residues over MRLs [97].

In USA, the Food Safety and Inspection Service (FSIS) is the public health agency in
the USDA responsible for ensuring that the nation's commercial supply of meat,
poultry, and egg products is safe, healthy, and perfectly packaged. FSIS analyzes
samples of meat and poultry for specific chemical compounds. In 2012, 1474
samples from broilers and turkey were screened for chemical compounds that include
veterinary drugs, none of these samples was found to have any of the investigated
chemical compounds [98].

These findings necessitate the establishment of local continuous monitoring

programs to ensure food safety.

5.1.1. Tetracylines detection

In this study out of 95 positive samples, tetracyclines group constituted 41(43.15%).
This high result may be as a result of tetracyclines properties; since they are broad
spectrum antimicrobial and can defend Rickettsia, Spirochetes, Chlamydia, and
Mycoplasma infections [43]. In addition, they are produced by many drugs
manufacturers and available in all veterinary clinics. These results are consistent with
the results of the study questionnaire which revealed that 70% of chicken breeders
had used tetracyclines in drinking water for chicken treatment. It has been reported
that tetracyclines are the most predominantly prescribed antimicrobials in Africa, and
of all antimicrobial-associated residues they represent 41% of cases [99].

Similar results were found in Egypt, tetracyclines group was recorded in 52% of
fresh and frozen local broiler meat [92]. In another study, 6 samples from 21(31.5%)
containing AMR confirmed to be for oxytetracycline residues, all of them where
exceeding MRLs [76]. A survey comprised of twenty three poultry farms and nine
veterinary clinics about the antimicrobial agents available for poultry use in the
Eastern Province of Saudi Arabia revealed that enrofloxacin was found to be the

45
most commonly used agent followed by tetracyclines, especially oxytetracycline and
doxycycline [87].

Tetracyclines were recorded as the most detected group in broilers in Saudia Arabia.
One or more compound of tetracyclines were detected in all positive farms screened
for AMR (69.7% of 33 broilers farms) in the eastern province [49].

A lower result than detected in this study was recorded in Kuwait. Using Charm II
method to investigate the presence of tetracycline residues in 263 chicken parts, 12
(5%) samples contained tetracyclines. All of detected samples were confirmed by
LC/MS/MS and found to be above MRLs [100]. This recorded low results may be as
a result of using Charm II method whereas the detection limit of the mostly used
members of tetracycline group )oxytetracycline and chlortetracycline( is equal to the
established MRLs (100ug/kg) [101].

In a study carried out in Pakistan, tetracyclines were detected in 29% of broilers

tissue samples, upon confirmation by HPLC six out of ninety nine were above MRLs
[102]. Using HPLC technique, one hundred broiler breast muscles were screened for
the incidence of tetracyclines residues in Iran. The study showed 60% of samples
were positive for tetracycline residues. The concentration of tetracycline in ten
samples were significantly higher than MRLs (100μg/kg) [103].

5.1.2 Aminoglycoside detection

The most commonly used aminoglycosides in Gaza strip are gentamicin, neomycin
and kanamycin. Aminoglycosides are bactericidal against Gram negative aerobes but
gentamicin is an extended spectrum aminoglycoside with activity against
Pseudomonas, Proteus, Staphylococcus, and Corynebacterium spp. Aminoglycosides
are not absorbed from the gastrointestinal tract, they are administered orally for
gastrointestinal infection or parenterally (intramuscular or subcutaneous) for
systemic infections [43]. In the present study out of 95 positive results,
aminoglycosides group was defined in 26 samples (27.36%).

46
5.1.3 β-lactams detection
β-lactams residues were detected in 20/95 (21%) of positive samples. In Gaza strip,
the most commonly used β-lactams are cephalosporins, amoxicillin and ampicillin.
The questionnaire revealed that 26.5% of breeders used β-lactams to treat chickens;
approximately 90% belong to the cephalosporins and were used parenterally.
Aminopenicillins have a short WP (1-2 days) in broiler chicken. Little information is
available about WP of cephalosporins when used parenterally. Ceftiofur, a third
generation cephalosporin, is used parenterally in one day old chickens to prevent E.
coli infection [104]. Treated chickens must not be slaughtered before five days from
the last dose of repeated administration of ceftiofur to withdraw the drug residues
from all tissues of treated chickens [105].

5.1.4 Macrolides detection

Macrolide group was detected in 8/95 (8.42%). According to the questionnaire
results the main macrolides used in Gaza strip are tylosin and erythromycin. These
two drugs are used primarily by oral route. Although 53% of breeders used
macrolide both orally and parenterally, results showed that only 8/95 (8.4%)
samples contained macrolides residues. Withdrawal period for tylosin and
erythromycin is one day when used orally, and it was determined to be two days for
erythromycin when used by injection [106]. As a result of the short WP and about
79% of breeders said that they retail chickens after more than two days, macrolides
group was the least detected one.
The Brazilian National Residues and Contaminants Control Plan (PNCRC) using
FAST method reported the occurrence of residues of antimicrobials with
confirmation for macrolides and aminoglycosides. 212 (21.9%) were positive for
FAST test, upon confirmation by liquid chromatography mass spectrometry 45%
were macrolides and 25% for aminoglycosides, but none of these residues exceeded
the established MRLs [107].

5.2. Frequency of residues in the study area

The highest percentage of antibiotic residue detection was recorded in The Middle
region 34(28.1%), percentages of samples contained residues in the other regions

47
were approximately similar to each other 22.38% and 21.8% for (Gaza and North
Gaza) and (Rafah and Khanyounis) respectively.
This decrease may as a result of seasonal variation since samples from the middle
region were collected from January to March (cold weather). Respiratory diseases as
Chronic Respiratory Disease (CRD) and Infectious Coryza are more prevalent in
winter [33], using antimicrobials may be increased in this period, the rest of samples
where collected from April to June which is a warmer weather than the previous
period. About 20% of chicken breeders answered in the study questionnaire that
diseases are more prevalent in winter season.

In a study, researchers screened chicken meat and offals in summer and winter
seasons and revealed that 2.8%, 11.2% and 27.5% of muscles, livers and kidneys
respectively contained AMR in winter, and AMR were detected in summer in 0%,
6% and 14.6% of muscles, livers and kidneys respectively, the researchers concluded
that season variation affects the presence of AMR [73].

5.3. Antibiotic residues and carcass weight

In this study broilers weight were used as a reflection of age since in standard
conditions of closed broiler houses, broilers reach one and half kilos in about five
weeks, and within the seventh week broilers may reach to two kilograms live body
weight [108].
Samples were divided into three categories depending on broilers live weight.
category (A) (≤ 1.5 kg), category (B) (>1.5- 2 kg) and Category (C >2 kg). The most
AR were found in category (A), 47 samples out of 88 positive samples (53.4%) were
from broiler carcasses weigh less or equivalent to 1.5 kilograms, category (B) had 29
(28.4%) positive samples and the least detected samples 12 (12.6%) were found in
category (C) from chickens weigh two kilograms and above.
Chickens breeding needs highly specific environmental conditions e.g. temperature,
ventilation and humidity, although theses conditions are important all over the period
of breeding they are more crucial in the early stages of breeding, any fault in the
management of a farm adversely effect chickens health specially to the respiratory
system, therfore antimicrobials are used frequently, this condition may explains the
high AR detection in category (A) samples.

48
5.4 Questionnaire analysis
Ninety questionnaires were filled in by breeders themselves and with the assitance of
a veterinarian for questions concerning drugs names or their trade names. About 68%
of breeders use antimicrobials without any sickness signs on chickens and 54.5%
used double dose to accelerate healing. These two practices by breeders may cause
two significant adverse effects to chickens health, firstly they may affect the stability
of microbiota in chickens intestinal tract which play an important role in the benefit
of feed and reducing enteric diseases [109, 110], secondly emergence of resistace
microorganism may be increased as a result of these practices[111, 112].
Most breeders 68/90 (75.5%) claimed that they have knowledge about WPs and
51/90 (56.6%) do fully understand the instructions of the used drugs. Howeve, it
seems that they did not follow these instructions since 41/90 (45.5%) of them retail
chickens during treatment period and 49/90 (54.5%) use double doses to accelerate
healing which are completely incompatible with drugs usage instructions.

On another hand, 71/90 (78.9%) of breeders indicated that they retail broilers after
more than two days from the termination of drugs administration, 11/90 (12.2%) and
8/90 (8.9%) of breeders said that they retail broilers after two and one day from the
termination of drugs administration respectively.
Withdrawal period varies according to drugs type, drug preparation, route of
administration and animal species. For broiler chicken drugs, it can be from zero day
up to five weeks [43], thus it cannot be judged whether breeders follow the
instructions or not except by identifying each drug and the rout of administration.
In the same context, lincospectin (lincomycin and spectinomycin) and gentamicin
have a long WP; 10 and 35 days respectively when used by injection. The
questionnaire showed that 50% of breeders used lincospectin and about 46% used
gentamicin parenterally. About 13/56 (23.2%), 13/56 (23.2%) and 30/56 (53.6%) of
breeders indicated that the longest period of waiting for marketing after treatment
parentally was 14, 10 and 7 days or less respectively.

From this point, it is certain that chickens injected with gentamicin are sold
containing high levels of gentamicin residues and this situation may be expected for

49
lincospectin residues as 35% of breeder wait a week or less after treatment parentally
to retail broiler chickens. Lincospectin was not investigated in this study.

The ninety participants of the questionnaire with assistance of veterinarians listed the
antimicrobials used in chicken therapy either orally or parenterally, data revealed that
aminoglycosides 72% followed by tetracyclines 70% were the most commonly used
groups, while florfenicol was 10%. These data supports the results of the bioassay
detection of these groups. Although aminoglycosides were recorded to be the most
used group, tetracyclines were the most detected group from meat samples followed
by aminoglycosides. These result might be appeared because that the WP of
tetracyclines extends from one to two weeks, also because all aminoglycosides that
were used orally were not absorbed through intestine but the test detects only
parenterally used aminoglycoside.

Macrolides and β-lactams have short WPs (1-2) days [43] when used orally, no
information available about their WPs when used parenterally. Despite the fact that
breeders reported higher usage of macrolides than β-lactams, β-lacatms were
detected by the bioassay method more frequently than macrolides. This result may
correlate to WPs for these two groups when used parenterally.

A few proportion of breeders (18/90) 20% said that they used hormonal steroids to
enhance growth. In the EU, the use of hormones in poultry production is forbidden
[113]. In a study carried out in Egypt, the researches revealed that the anabolic
steroids residues level including testosterone and progesterone were within the
permissible limit which refers to no illegal use of hormones as growth promoting
agents in broiler production [76].
Also a study conducted in Costa Rica to assess serum concentrations of some steroid
hormones and comparing them between commercial and control group of broiler,
results revealed that no significant differences were observed in mean serum
concentrations of steroid hormones [114].
It could be missunderstanding by breedes between vitamins and steroids, since they
think that they used vitamins to activate chicken hormones. Hormone residues were
not included in this research. However, this should be investigated further, to
determine the extent of this issue.
50
 Chapter VI
Conclusions and Recommendations
6.1 Conclusions
1. The study revealed that 24.1% of local broiler chickens retailed in Gaza strip
contained one or more antibiotic agents belonging to one of the four
investigated antibiotic groups (Tetracyclines, Aminoglycosides, β-Lactams
and Macrolides).
2. Out of the 88 positive samples, seven samples (7.95%) contained multiple
antibiotic residues.
3. Antibiotic residues detected in higher frequency in lighter chickens than
heavier ones. It seems that broiler weighing more than two kilograms is the
safest category.
4. Tetracyclines group is the most commonly detected antibiotic group 41/95
(43.15%) followed by aminoglycoside 26/95 (27.36%) then β-lactams 20/95
(21%) and the least detected group was macrolides 8/95 (8.42%).
5. A large number of breeders (67.6%) use antimicrobials without signs of
sickness.
6. About (56.6%) of breeders claimed that they had a good knowledge of the
instructions of the used drugs.
7. A large number of breeders (77.8%) used more than one antimicrobial in a
single treatment.
8. Nearly half of breeders (45.5%) sell chickens during a treatment period.
9. Veterinary drugs are misused in poultry production. Breeders do not
appropriately follow indications and instructions.
10. Withdrawal periods are not observed when broiler chickens are marketed.

51
6.2 Recommendations
In light of the results that are revealed by the bioassay detection of AMR and the
information collected by questionnaire tool, the followings are recommended:
1. It is recommended to investigate the presence of antimicrobial residues which
belong to the other used antimicrobial groups in poultry treatment.
2. Since screening methods detect the presence of AMR, it is recommended to
quantify their amounts by confirmatory methods to determine if these AMR
are in accordance to the legal limits established by Codex.
3. In an action to reduce using antimicrobials, it is recommended to import and
utilize all available vaccines used in poultry breeding which are not found in
Gaza strip such as coccidiosis vaccines.
4. It is recommended to ensure importing hatching eggs free of vertical
transmitted disease such as mycoplasma, this action also decrease the need of
antimicrobials.
5. The effective prevention of infectious diseases and applying strict hygiene
standards and rearing skills may reduce the need of antimicrobials.
6. Reduce unnecessary antibiotic use in poultry production, avoid antibiotic use
for the treatment of viral disease in animals, and reduce prophylactic
antibiotic use.
7. Strict observation of antibiotic withdrawal periods should be made; the
avoidance of antimicrobials lacking clearly documented pharmacokinetic and
pharmacodynamic properties must be considered.
8. Off-label use of antibiotics by breeders and some veterinarians should be
stopped such as using oral preparations drugs parenterally.
9. Avoid using antimicrobials in the veterinary field without a veterinarian’s
prescription.
10. National monitoring of antimicrobial residues in foods and establishing of the
maximum residue limits of these residues is highly recommended.
11. It is recommended to carry out subsequent studies on other AMR in a variety
of animal products intended for human consumption.
12. It is strongly recommended to investigate hormonal steroids usage as growth
promoters in poultry, this issue is highly important and should be investigated
either by researches or by the Ministry of Agriculture.

52
13. Launching of awareness campaigns about human health hazards associated
with antibiotic residues in food of animal origin by concerned authorities
(Ministry of Agriculture, Ministry of Health, consumer protection
organizations, breeders).

53
References:

1. Reig, M. and F. Toldrá, Veterinary drug residues in meat: Concerns and rapid
methods for detection. Meat Science, 2008. 78(1): p. 60-67.

2. Jafari, M., et al., Determination of veterinary drug residues in chicken meat

using corona discharge ion mobility spectrometry. Analytica Chimica Acta,
2007. 581(1): p. 147-153.

3. Nisha, A.R., Antibiotic Residues - A Global Health Hazard. Veterinary World,

2008. 1(12): p. 375-377.

4. Singh, S., et al., Antibiotic Residues: A Global Challenge. Pharma Science

Monitor, 2014. 5(3): p. 184-197.

5. Tajick, M.A. and B. Shohreh, Detection of antibiotics residue in chicken meat

using TLC. International Journal of Poultry Science, 2006. 5(7): p. 611-612.

6. McGlinchey, T.A., et al., A review of analytical methods for the determination

of aminoglycoside and macrolide residues in food matrices. Analytica Chimica
Acta, 2008. 624(1): p. 1-15.

7. Yu, H., et al., Development of an HPLC–UV method for the simultaneous

determination of tetracyclines in muscle and liver of porcine, chicken and
bovine with accelerated solvent extraction. Food Chemistry, 2011. 124(3): p.
1131-1138.

8. Shanty, H., J.A. Omar, and R.A. Othman. Feed industry in Palestine. in 3rd All
Africa Conference on Animal Agriculture and 11th Conference of the Egyptian
Society of Animal Production. 6-9 November 2000. Alexandria, Egypt: p.373-
378.

9. Palestinian Central Bureau of Statistics, Livestock Survey, 2013, Main Results.

2014: Ramallah, Palestine.

10. Guardabassi, L., L.B. Jensen, and H. Kruse, Guide to antimicrobial use in
animals. 1st ed. 2008, Oxford, UK: Blackwell.

11. Wang, J., J.D. MacNeil, and J.F. Kay, Chemical analysis of antibiotic residues
in food. 2012, Hoboken, N.J.: Wiley & Sons.

12. Chopra, I. and M. Roberts, Tetracycline antibiotics: mode of action,

applications, molecular biology, and epidemiology of bacterial resistance.
Microbiology and Molecular Biology Reviews, 2001. 65(2): p. 232-260.

13. Prescott, L.M., J.P. Harley, and D.A. Klein, Microbiology. 5th ed. 2002, Boston,
UK: McGraw-Hill.

14. Michalova, E., P. Novotna, and J. Schlegelova, Tetracyclines in veterinary

medicine and bacterial resistance to them. A review. Veterinarni Medicina
(Czech) 2004. 49(3): p. 79-100.

54
15. Mesgariabbasi, M., et al., Simultaneous Determination of Tetracyclines
Residues in Bovine Milk Samples by Solid Phase Extraction and HPLC-FL
Method. Advanced Pharmaceutical Bulletin, 2011. 1(1): p. 34-39.

16. Oka, H., Y. Ito, and H. Matsumoto, Chromatographic analysis of tetracycline

antibiotics in foods. Journal of Chromatography A, 2000. 882(1–2): p. 109-133.

17. Kowalski, P. and L. Konieczna, Determination of penicillin antibiotics in

poultry muscle by capillary electrophoresis. Bulletin-Veterinary Institute In
Pulawy, 2007. 51(4): p. 595-598.

18. Woodward, K.N., Veterinary pharmacovigilance : adverse reactions to

veterinary medicinal products. 1st ed. 2009, Oxford, UK: Wiley-Blackwell.

19. Forward, K.R., et al., Recovery of cephalosporin-resistant Escherichia coli and

Salmonella from pork, beef and chicken marketed in Nova Scotia. The
Canadian Journal of Infectious Diseases & Medical Microbiology, 2004. 15(4):
p. 226-230.

20. Dhanji, H., et al., Cephalosporin resistance mechanisms in Escherichia coli

isolated from raw chicken imported into the UK. Journal of Antimicrobial
Chemotherapy, 2010. 65(12): p. 2534-2537.

21. Schmidt, C.W., FDA Proposes to Ban Cephalosporins from Livestock Feed.
Environmental Health Perspectives, 2012. 120(3): p. a106-a106.

22. Stolker, A. and U.T. Brinkman, Analytical strategies for residue analysis of
veterinary drugs and growth-promoting agents in food-producing animals—a
review. Journal of Chromatography A, 2005. 1067(1): p. 15-53.

23. Riviere, J.E. and M.G. Papich, Veterinary pharmacology and therapeutics. 9th
ed. 2009, Ames, Iowa, USA: Wiley-Blackwell.

24. Mingeot-Leclercq, M.-P., Y. Glupczynski, and P.M. Tulkens, Aminoglycosides:

Activity and Resistance. Antimicrobial Agents and Chemotherapy, 1999. 43(4):
p. 727-737.

25. Stead, D.A., Current methodologies for the analysis of aminoglycosides.

Journal of Chromatography B: Biomedical Sciences and Applications, 2000.
747(1–2): p. 69-93.

26. Barton, M.D., Antibiotic use in animal feed and its impact on human health.
Nutrition Research Reviews, 2000. 13(02): p. 279-299.

27. Wallace, R.J. and A. Chesson, Biotechnology in animal feeds and animal
feeding. 1st ed. 1995, Weinheim ; Cambridge: VCH.

28. Levy, S.B. and B. Marshall, Antibacterial resistance worldwide: causes,

challenges and responses. Nature Medicine, 2004. 10: p. S122-S129.

55
29. Ahmed, M., Antibiotic Resistance: An Emerging Global Headache, in
Antibiotic Resistant Bacteria - A Continuous Challenge in the New Millennium,
M. Pana, Editor. 2012, InTech. p. 3-14.

30. McEwen, S.A. and P.J. Fedorka-Cray, Antimicrobial use and resistance in
animals. Clinical infectious diseases : an official publication of the Infectious
Diseases Society of America, 2002. 34 Suppl 3: p. S93-S106.

31. Phillips, I., et al., Does the use of antibiotics in food animals pose a risk to
human health? A critical review of published data. Journal of Antimicrobial
Chemotherapy, 2004. 53(1): p. 28-52.

32. Rosengren, L., S. Gow, and J. Weese, Antimicrobial use and resistance in pigs
and chickens: A review of the science, policy, and control practices from farm
to slaughter - executive summary. Canadian Journal of Infectious Diseases &
Medical Microbiology, 2010. 21(3): p. 123 - 127.

33. Elmanama, A.A. and N. Abdelateef, Antimicrobial Resistance of Enteric

Pathogens Isolated from Acute Gastroenteritis Patients in Gaza strip,
Palestine. The International Arabic Journal of Antimicrobial Agents, 2012. 2(4):
p. 1-4.

34. Dallal, M.M.S., et al., Prevalence and antimicrobial resistance profiles of

Salmonella serotypes, Campylobacter and Yersinia spp. isolated from retail
chicken and beef, Tehran, Iran. Food Control, 2010. 21(4): p. 388-392.

35. Rahimi, E., et al., Prevalence and antimicrobial resistance of Campylobacter

species isolated from chicken carcasses during processing in Iran. Poultry
Science, 2010. 89(5): p. 1015-1020.

36. Kim, M.-S., et al., Prevalence and antimicrobial resistance of Salmonella

species isolated from chicken meats produced by different integrated broiler
operations in Korea. Poultry Science, 2012. 91(9): p. 2370-2375.

37. Greeson, K., et al., Frequency of antibiotic resistant Salmonella, Escherichia

coli, Enterococcus, and Staphylococcus aureus in meat in Saudi Arabia.
African Journal of Microbiology Research, 2013. 7(4): p. 309-316.

38. Burgat-sacaze, V., P. Delatour, and A. Rico, Bound residues of veterinary

drugs: bioavailability and toxicological implications. Annales de Recherches
Vétérinaires, 1981. 12(3): p. 277-289.

39. Doyle, M., Veterinary drug residues in processed meats—potential health risk,
a review of the scientific literature. Food Research Institute (FRI
Briefings),University of Wisconsin–Madison, March 2006. Last accessed
[15/3/2014], https://fri.wisc.edu/files/Briefs_File/FRIBrief_VetDrgRes.pdf.

40. Myllyniemi, A.-L.,Development of microbiological methods for the detection

and identification of antimicrobial residues in meat, Faculty of Veterinary
Medicine, University of Helsinki, 2004, Master Thesis, Unpublished data.

56
41. Codex Alimentarius Commission, Maximum residue limits (MRLS) and risk
management recommendations (RMRs) for residues of veterinary drugs in
foods. Updated as at the 37th Session of the Codex Alimentarius Commission,
2014. CAC/MRL 2.

42. Almanama, M.A.,Prevalence of Mycoplasma gallisepticum in the Ten licensed

Hatcheries in Gaza strip, Palestine, Biological Sciences, Islamic University
Gaza, 2011, Master Thesis, Unpublished data.

43. Hsu, W.H., Handbook of veterinary pharmacology. 1st ed. 2008, Ames, Iowa,
USA: Wiley-Blackwell.

44. Castanon, J., History of the use of antibiotic as growth promoters in European
poultry feeds. Poultry Science, 2007. 86(11): p. 2466-2471.

45. Vass, M., K. Hruska, and M. Franek, Nitrofuran antibiotics: a review on the
application, prohibition and residual analysis. Veterinarni Medicina, 2008.
53(9): p. 469-500.

46. Davis, J.L., et al., Update on drugs prohibited from extralabel use in food
animals. Journal of the American Veterinary Medical Association, 2009. 235(5):
p. 528-34.

47. O'brien, J., N. Campbell, and T. Conaghan, Effect of cooking and cold storage
on biologically active antibiotic residues in meat. Journal of Hygiene, 1981.
87(03): p. 511-523.

48. Abou-Raya, S.H., et al., Effect of Ordinary Cooking Procedures on

Tetracycline Residues in Chicken Meat. Journal of Food & Drug Analysis,
2013. 21(1): p. 80-86.

49. Al-Ghamdi, M., et al., Residues of tetracycline compounds in poultry products

in the eastern province of Saudi Arabia. Public Health, 2000. 114(4): p. 300-
304.

50. Lolo, M., et al., Effect of cooking on enrofloxacin residues in chicken tissue.
Food Additives & Contaminants, 2006. 23(10): p. 988-993.

51. Javadi, A., H. Mirzaie, and S.A. Khatibi, Effect of roasting, boiling and
microwaving cooking methods on Enrofloxacin residues in edible tissues of
broiler. African Journal of Pharmacy and Pharmacology 2011. 5(2): p. 214-218.

52. Aerts, M.M.L., A.C. Hogenboom, and U.A.T. Brinkman, Analytical strategies
for the screening of veterinary drugs and their residues in edible products.
Journal of Chromatography B: Biomedical Sciences and Applications, 1995.
667(1): p. 1-40.

53. Community Reference Laboratories (CRLs), Guidelines for the validation of

screening methods for residues of veterinary medicines (initial validation and
transfer). 2014. Last accessed [10/12/2010],
http://ec.europa.eu/food/food/chemicalsafety/residues/Guideline_Validation_Scr
eening_en.pdf.

57
54. Sirdar, M.M.,Antibiotc residues in comercial layer hens in Khartoum state,
Sudan 2007-2008., Veterinary Science, University of Pretoria, 2010, Master
Thesis, Unpublished data.

55. Mitchell, J., et al., Antimicrobial drug residues in milk and meat: causes,
concerns, prevalence, regulations, tests, and test performance. Journal of Food
Protection®, 1998. 61(6): p. 742-756.

56. Tothill, I.E., Rapid and on-line instrumentation for food quality assurance. 1st
ed. 2003, Boca Raton, Cambridge, England: CRC Press ; Woodhead.

57. Watson, D.H., Pesticide, veterinary and other residues in food. 1st ed. 2004,
Boca Raton, Cambridge, England: CRC Press ; Woodhead.

58. Pikkemaat, M.G., Microbial screening methods for detection of antibiotic

residues in slaughter animals. Analytical and Bioanalytical Chemistry, 2009.
395(4): p. 893-905.

59. Bovee, T.F. and M.G. Pikkemaat, Bioactivity-based screening of antibiotics and
hormones. Journal of Chromatography A, 2009. 1216(46): p. 8035-8050.

60. Okerman, L., K. De Wasch, and J. Van Hoof, Detection of antibiotics in muscle
tissue with microbiological inhibition tests: effects of the matrix. Analyst, 1998.
123(11): p. 2361-2365.

61. Crosby, N.T., Determination of veterinary residues in food. 1st ed. 1991, New
York: Ellis Horwood.

62. Heitzman, R.J., Veterinary drug residues : residues in food producing animals
and their products : reference materials and methods : final report. 2nd ed.
1994, Oxford: Blackwell Scientific.

63. Dey, B.P., et al., Calf antibiotic and sulfonamide test (CAST) for screening
antibiotic and sulfonamide residues in calf carcasses. Journal of AOAC
International, 2005. 88(2): p. 440-6.

64. Gaudin, V., et al., Validation of a Five Plate Test, the STAR protocol, for the
screening of antibiotic residues in muscle from different animal species
according to European Decision 2002/657/EC. Food Additives and
Contaminants, 2010. 27(7): p. 935-52.

65. Gaudin, V., et al., AFNOR validation of Premi® Test, a microbiological-based

screening tube-test for the detection of antimicrobial residues in animal muscle
tissue. Food Additives and Contaminants, 2008. 25(12): p. 1451-1464.

66. Navrátilová, P., Screening methods used for the detection of veterinary drug
residues in raw cow milk – a review. Czech Journal of Food Science, 2008.
26(6): p. 393-401.

58
67. Botsoglou, N.A. and D.J. Fletouris, Drug residues in foods : pharmacology,
food safety, and analysis. Food Science and Technology. 2001, New York:
Marcel Dekker.

68. Cunningham, F.M., J. Elliott, and P. Lees, Comparative and veterinary

pharmacology. 1st ed. Handbook of experimental pharmacology. Vol. 199.
2010, Heidelberg ; New York: Springer.

69. Paige, J.C., M.H. Chaudry, and F.M. Pell, Federal surveillance of veterinary
drugs and chemical residues (with recent data). The Veterinary clinics of North
America. Food animal practice, 1999. 15(1): p. 45-61.

70. Okerman, L., et al., Inhibition tests for detection and presumptive identification
of tetracyclines, beta-lactam antibiotics and quinolones in poultry meat. Food
Additives & Contaminants, 2001. 18(5): p. 385-393.

71. Tin, T.M., Detection of Antibiotic Residues and Isolation of Antibiotic

Resistant Escherichia Coli from Chicken Meat and Chickens in Malaysia,
Veterinary Medicine, Universiti Putra Malaysia, 2003, Ph.D Thesis, Unpublished
data.

72. Shahid, M.A., et al., Evaluation of a Microbiological Growth Inhibition Assay

as a Screening Test for the Presence of Antibiotic Residues in Poultry Meat.
American Journal of Food Technology, 2007. 2(5): p. 457-461.

73. Pavlov, A., et al., Residues of antimicrobial drugs in chicken meat and offals.
Trakia Journal of Sciences, 2008. 6(1): p. 23-25.

74. Shareef, A.M., Z.T. Jamel, and K.M. Yonis, Detection of antibiotic residues in
stored poultry products. Iraqi Journal of Veterinary Sciences, 2009. 23(1): p. 45-
48.

75. Silfrany, R., et al., Detection of quinolones in poultry meat obtained from retail
centers in Santiago Province, the Dominican Republic. Journal of Food
Protection®, 2013. 76(2): p. 352-354.

76. Hussein, M.A. and S. Khalil, Screening of some antibiotics and anabolic
steroids residues in broiler fillet marketed in El-Sharkia governorate. Life
Science Journal, 2013. 10(1): p. 2111-2118.

77. Ezenduka, E.V., O.S. Ike, and N.J. Anaelom, Rapid detection of antimicrobial
residues in poultry: A consequence of non-prudent use of antimicrobials.
Health, 2014. 6(2): p. 149-152.

78. USDA Food Safety and Inspection Service, Media and Reagents, in
Microbiology Laboratory Guidebook. 2013.

79. USDA Food Safety and Inspection Service, Bioassay for the Detection,
Identification and Quantitation of Antimicrobial Residues in Meat and Poultry
Tissue, in Microbiology Laboratory Guidebook. 2011.

59
80. Microbiologics. KWIK-STIK™ Microorganisms, instructions for use. Last
accessed [12/5/2015], http://microbiologics.com/Products/KWIK-STIK.

81. Ellis, R.L., Development of veterinary drug residue controls by the Codex
Alimentarius Commission: a review. Food Additives and Contaminants, 2008.
25(12): p. 1432-1438.

82. Saint, E., Broiler Chicken Market System,Gaza Strip, Occupied Palestinian
Territory. 2013, Emergency Market Mapping & Analysis (EMMA).

83. Al Zuheir, I.M., Detection of β-Lactams and Tetracyclines Antimicrobial

Residues in Raw Dairy Milk for Human Consumption in Palestine. Walailak
Journal of Science and Technology (WJST), 2012. 9(3): p. 277-279.

84. Reyes-Herrera, I., et al., Concentrations of antibiotic residues vary between

different edible muscle tissues in poultry. Journal of Food Protection, 2005.
68(10): p. 2217-2219.

85. Reyes-Herrera, I. and D.J. Donoghue, Antibiotic Residues Distribute Uniformly

in Broiler Chicken Breast Muscle Tissue. Journal of Food Protection, 2008.
71(1): p. 223-225.

86. Pagel, S. and P. Gautier, Use of antimicrobial agents in livestock. Revue

Scientifique et Technique-OIE, 2012. 31(1): p. 145-188.

87. Al-Mustafa, Z.H. and M.S. Al-Ghamdi, Use of antibiotics in the poultry
industry in Saudi Arabia: implications for public health. Annals of Saudi
Medicine, 2002. 22(1/2): p. 4-7.

88. Kirbiš, A., Microbiological screening method for detection of aminoglycosides,

β-lactames, macrolides, tetracyclines and quinolones in meat samples.
Slovenian Veterinary Research, 2007. 44(1/2): p. 11-18.

89. Cháfer-Pericás, C., Á. Maquieira, and R. Puchades, Fast screening methods to

detect antibiotic residues in food samples. TrAC Trends in Analytical
Chemistry, 2010. 29(9): p. 1038-1049.

90. De Brabander, H., et al., Residue analysis: Future trends from a historical
perspective. Journal of Chromatography A, 2009. 1216(46): p. 7964-7976.

91. Balizs, G. and A. Hewitt, Determination of veterinary drug residues by liquid

chromatography and tandem mass spectrometry. Analytica Chimica Acta,
2003. 492(1): p. 105-131.

92. Karmi, M., Detection and Presumptive Identification of Antibiotic Residues in

Poultry Meat by Using FPT. Global Journal of Pharmacology, 2014. 8(2): p.
160-165.

93. Abdalbagi, A.Y.A.,Detection of antibiotic residue in Poultry Meat by Thin

Layer Chromatography and Microbiological method in Khartoum state,
Medical Laboratory Science, Sudan University of Science and Technology,
2011, Master Thesis, Unpublished data.

60
94. Hind, A.E., M.S. Adil, and A.E. Samah, Screening of Antibiotic Residues in
Poultry Liver, Kidney and Muscle in Khartoum State, Sudan. Journal of
Applied and Industrial Sciences, 2014. 2(3): p. 116-122.

95. Kabir, J., et al., Veterinary drug use in poultry farms and determination of
antimicrobial drug residues in commercial eggs and slaughtered chicken in
Kaduna State, Nigeria. Food Control, 2004. 15(2): p. 99-105.

96. Asad, F.,Antibiotic Residue in Poultry Products, Chemistry and Biochemistry,

University of Agriculture, Faisalabad, 2012, Ph.D Thesis, Unpublished data.

97. Veterinary Residues Committee, Annual Report on Surveillance for Veterinary

Residues in Food in the UK 2007. VRC, Surrey 2008.

98. USDA Food Safety and Inspection Service, United States National Residue
Program for Meat, Poultry, and Egg Products 2012 Residue Sample Results.
The Red Book, ed. N. Abdelmajid. 2014.

99. Darwish, W.S., et al., Antibiotic residues in food: the African scenario.
Japanese Journal of Veterinary Research, 2013. 61(Supplement): p. S13-S22.

100. Al-Mazeedi, H.M., et al., Screening for tetracycline residues in food products
of animal origin in the State of Kuwait using Charm II radio-immunoassay
and LC/MS/MS methods. Food Additives and Contaminants, 2010. 27(3): p.
291-301.

101. Charm Sciences Inc, Charm® II Antibiotic Analysis for Tissue (Brochure).
Last accessed [12/4/2015], http://www.charm.com/resource/file/142.

102. Shahid, M.A., et al., Status of Oxytetracycline Residues in Chicken Meat in

Rawalpindi/Islamabad Area of Pakistan. Asian Journal of Poultry Science,
2007. 1(1): p. 8-15.

103. Ehsani, A., et al., Measurement of Tetracycline residue in consumed Broiler

meats in Ahvaz City by HPLC. Journal of Veterinary Medicine & Laboratory
2010. 2: p. 119-129.

104. Hornish, R.E. and S. Katarski, Cephalosporins in veterinary medicine-ceftiofur

use in food animals. Current Topics in Medicinal Chemistry, 2002. 2(7): p. 717-
731.

105. El-Sayed, M.G., et al., Pharmacokinetics and Tissue Residues of Ceftiofur in

Normal and Escherichia Coli Infected Chickens. Journal of Physiology and
Pharmacology Advances, 2015. 5(3): p. 574-582.

106. Goudah, A., K. Abo El Sooud, and A.M. Abd El-Aty, Pharmacokinetics and
tissue residue profiles of erythromycin in broiler chickens after different routes
of administration. Deutsche Tierarztliche Wochenschrift, 2004. 111(4): p. 162-
165.

61
107. Nonaka, C.K., et al., Occurrence of antimicrobial residues in Brazilian food
animals in 2008 and 2009. Food Additives and Contaminants. Part A, 2012.
29(4): p. 526-534.

108. Haitook, T., Study on chicken meat production for small-scale farmers in
Northeast Thailand. Journal of Agriculture and Rural Development in The
Tropics and Subtropics, 2006. supplement(87): p. 178.

109. Patterson, J.A. and K.M. Burkholder, Application of Prebiotics and Probiotics
in Poultry Production. Poultry Science, 2003. 82: p. 627-631.

110. Lan, Y., et al., The role of the commensal gut microbial community in broiler
chickens. World's Poultry Science Journal, 2005. 61(01): p. 95-104.

111. Ahmed Geidam, Y., et al., High prevalence of multi-drug resistant bacteria in
selected poultry farms in Selangor, Malaysia. Asian Journal of Animal and
Veterinary Advances, 2012. 7(9): p. 891-897.

112. Amaechi, N., Correlation between the Use of Antimicrobials and the
Occurrence of Antimicrobial Resistant Bacteria in Poultry and Pig Farms.
Global Journal of Biology, Agriculture and Health Sciences, 2014. 3(3): p. 50-
54.

113. Mulder, R.W.A.W. and H. Hupkes, European Legislation in Relation to Food

Safety in Production of Poultry Meat and Eggs. The Journal of Applied Poultry
Research, 2007. 16(1): p. 92-98.

114. Holst-Schumacher, I., et al., Serum sexual steroid hormones and lipids in
commercial broilers (Gallus domesticus) in Costa Rica. The Journal of Applied
Poultry Research, 2010. 19(3): p. 279-287.

62
 ‫‪Annexes‬‬

‫‪Annex 1‬‬
‫االستبانت بالغت العزبيت‬

‫هذه االستبانت تتناول هوضوع بقايا الوضاداث الحيويت في لحوم الدواجن والوقدم كجزء هن هتطلباث الحصول‬
‫على درجت الواجستيز في العلوم الحياتيت ‪ /‬الجاهعت االسالهيت‪-‬غزة‬
‫انًُطمخ ‪...........................‬‬ ‫انًذ‪ُٚ‬خ‪..........................‬‬

‫ػًش انط‪ٕٛ‬س‪....................‬‬ ‫ػذد انط‪ٕٛ‬س ‪.............‬‬ ‫انًسبؽخ انكه‪ٛ‬خ ‪.................‬‬

‫‪ ‬ال‬ ‫‪َ‬ؼى‬ ‫‪ْ -0‬م رسزخذو انًعبداد انؾ‪ٕٚٛ‬خ ؽزٗ نٕ نى ‪ٚ‬ظٓش أػشاض يشظ‪ٛ‬خ ػهٗ انط‪ٕٛ‬س؟‬
‫‪ ‬ال‬ ‫‪َ‬ؼى‬ ‫‪ْ -4‬م نذ‪ٚ‬ك ػهى ثطج‪ٛ‬ؼخ كم األدٔ‪ٚ‬خ انًسزخذيخ نهؼالط اصُبء انزشث‪ٛ‬خ؟‬
‫‪ ‬ال‬ ‫‪َ‬ؼى‬ ‫‪ْ -.‬م رؼزمذ أٌ انذٔاء ف‪ ٙ‬عسى انذعبط نّ رأص‪ٛ‬ش سهج‪ ٙ‬ػهٗ صؾخ اإلَسبٌ؟‬
‫‪ ‬ال‬ ‫‪َ‬ؼى‬ ‫‪ْ -2‬م رؼشف أ٘ رؼه‪ًٛ‬بد أٔ إسشبداد غ‪ٛ‬ش انغشػخ السزخذاو انذٔاء؟‬
‫‪ ‬ال‬ ‫‪َ‬ؼى‬ ‫‪ْ -1‬م نذ‪ٚ‬ك يؼشفخ ثفزشح انسًبػ نألدٔ‪ٚ‬خ انًسزخذيخ؟‬
‫‪ ‬ال‬ ‫‪َ‬ؼى‬ ‫‪ْ -6‬م رؼزمذ اٌ اسزخذاو عشػخ يعبػفخ يٍ األدٔ‪ٚ‬خ رؼغم يٍ شفبء انذعبط انًش‪ٚ‬ط؟‬
‫‪ ‬ال‬ ‫‪َ‬ؼى‬ ‫‪ْ -.‬م رسزخذو أكضش يٍ يعبد ؽ‪ ٕ٘ٛ‬ف‪ ٙ‬فزشح ػالط ٔاؽذح؟‬
‫‪ ‬ال‬ ‫‪َ‬ؼى‬ ‫‪ْ -4‬م رسزخذو يُشطبد ْشيَٕ‪ٛ‬خ أصُبء فزشح انزشث‪ٛ‬خ؟‬
‫‪ ‬ال‬ ‫‪َ‬ؼى‬ ‫‪ْ -3‬م رٕعذ فزشح ركضش ثٓب إصبثخ انمط‪ٛ‬غ ثبأليشاض؟‬
‫إرا كبَذ اإلعبثخ ثُؼى ؽذد رهك انفزشح انضيُ‪ٛ‬خ ‪..................................‬‬ ‫‪‬‬

‫‪ ‬أؽ‪ٛ‬بَب‬ ‫‪ ‬ال‬ ‫‪َ ‬ؼى‬ ‫‪ْ -01‬م رمٕو ثج‪ٛ‬غ انذعبط أصُبء فزشح انؼالط؟‬
‫‪ ‬يُشػ‬ ‫‪ٔ ‬لب‪ٚ‬خ‬ ‫‪ ‬ػالط‬ ‫‪ -00‬يب ْٕ سجت اسزخذاو انًعبداد انؾ‪ٕٚٛ‬خ؟‬
‫‪ ‬يكبٌ اخش‬ ‫‪ ‬انج‪ٛ‬ذ‬ ‫‪ ‬انًضسػخ‬ ‫‪ -04‬أ‪ ٍٚ‬رمٕو ثزخض‪ ٍٚ‬انذٔاء‬
‫‪ ‬انؼهف‬ ‫‪ ‬انؾمٍ‬ ‫‪ ‬يبء انششة‬ ‫‪ -0.‬يب ْ‪ ٙ‬غش‪ٚ‬مخ إػطبء انذٔاء‬
‫‪َٓ ‬بئ‪ٛ‬ب‬ ‫‪ ‬اؽ‪ٛ‬بَب‬ ‫‪ ‬دائًب‬ ‫‪ْ -02‬م رسزؼ‪ ٍٛ‬ثبنطج‪ٛ‬ت انج‪ٛ‬طش٘ نٕصف انذٔاء؟‬
‫‪ ‬اكضش‬ ‫‪ٕٚ ‬ي‪ٍٛ‬‬ ‫‪ٕٚ ‬و‬ ‫‪ -01‬يزٗ رزٕلف ػٍ إػطبء انذٔاء لجم انزسٕ‪ٚ‬ك؟‬
‫‪ -06‬يب ْ‪ ٙ‬أغٕل يذح رُزظشْب نهزسٕ‪ٚ‬ك ثؼذ انؼالط ثبنؾمٍ؟‬
‫‪.....................................‬‬
‫‪ -0.‬يبْ‪ ٙ‬االدٔ‪ٚ‬خ انز‪ ٙ‬رسزخذيٓب ؽمُب؟‬
‫‪.............................................-4 ..............................................-0‬‬
‫‪.............................................-2 .............................................-.‬‬
‫‪63‬‬
 ‫‪ -04‬يب ْ‪ ٙ‬األدٔ‪ٚ‬خ انز‪ ٙ‬اسزخذيزٓب يُز ثذا‪ٚ‬خ رشث‪ٛ‬خ انمط‪ٛ‬غ؟‬

‫‪-6‬‬ ‫‪-0‬‬
‫‪-.‬‬ ‫‪-4‬‬
‫‪-4‬‬ ‫‪-.‬‬
‫‪-3‬‬ ‫‪-2‬‬
‫‪-01‬‬ ‫‪-1‬‬

‫‪64‬‬
 Annex 2

An English version questionnaire

This questionnaire is about antimicrobial residues in broiler chickens in Gaza strip
submitted as part of the requirements for the degree of master of biological sciences
Islamic University-Gaza.

1. Do you use antimicrobials on birds, despite the absence of infection signs?

□ No □ Yes
2. Do you know the nature of all drugs used for treatment during breeding?
□ No □ Yes
3. Do you believe that drugs in chicken body have a negative effect on human
health? □ No □ Yes
4. Do you know any instructions apart from dosage instructions for the used drugs?
□ No □ Yes
5. Do you know the withdrawal period of used drugs? □ No □ Yes
6. Do you think using a double dose of drugs accelerate the healing of sick chickens?
□ No □ Yes
7. Do you use more than one antimicrobial in a single treatment?
□ No □ Yes
8. Is there a period of increasing the incidence of diseases □ No □ Yes
• If yes, determine the period. …………………………………………
9. Do you retail chickens during treatment periods? □ No □ Yes
10. What is the reason for using antimicrobials?
□Treatment □Prevention □ Enhancement
11. Where do you store drugs? □Farm □House □Elsewhere
12. How do you administer medication?
□Drinking water □ Injection □ Feed
13. Do you consult a veterinarian to describe medications?

65
 □ Always □ Sometimes □ Never
14. Would you cease giving drugs before marketing?
□ Day □ Two □ More
15. What is the longest period of waiting for marketing after treatment parentally?
.....................................
16. What are the medicines you use parentally?
1. …………….
2. …………….
3. …………….
4. …………….
17. What medications are used since the beginning of the breeding herd?
1. 6.
2. 7.
3. 8.
4. 9.
5. 10.

66