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EZENDUKA, EKENE VIVIENNE
PG/Ph.D/09/52261
OCCURRENCE OF ANTIMICROBIAL RESIDUES IN
BROILERS IN ENUGU METROPOLIS AND THE EFFECT
OF TEMPERATURE ON THE CONCENTRATION OF
OXYTETRACYCLINE RESIDUE
DEPARTMENT OF VETERINARY PUBLIC HEALTH AND
PREVENTIVE MEDICINE
FACULTY OF VETERINARY MEDIC
CINE
Nwamarah Uche
Digitally Signed by:: Content manager’s Name
DN : CN = Weabmaster’s name
O= University of Nigeria,
a, Nsukka
OU = Innovation Centre
1
OCCURRENCE OF ANTIMICROBIAL RESIDUES IN BROILERS IN ENUGU
METROPOLIS AND THE EFFECT OF TEMPERATURE ON THE
CONCENTRATION OF OXYTETRACYCLINE RESIDUE
BY
EZENDUKA, EKENE VIVIENNE
PG/Ph.D/09/52261
DEPARTMENT OF VETERINARY PUBLIC HEALTH AND
PREVENTIVE MEDICINE, UNIVERSITY OF NIGERIA,
NSUKKA
NOVEMBER, 2014
2
OCCURRENCE OF ANTIMICROBIAL RESIDUES IN BROILERS
IN ENUGU METROPOLIS AND THE EFFECT OF
TEMPERATURE ON THE CONCENTRATION OF
OXYTETRACYCLINE RESIDUE
By
EZENDUKA, EKENE VIVIENNE
(PG/Ph.D/09/52261)
A THESIS SUBMITTED TO THE DEPARTMENT OF PUBLIC
HEALTH AND PREVENTIVE MEDICINE, FACULTY OF
VETERINARY MEDICINE IN PARTIAL FULLFILLMENT OF
THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF
PHILOSOPHY IN VETERINARY PUBLIC HEALTH AND
PREVENTIVE MEDICINE, UNIVERSITY OF NIGERIA, NSUKKA
NOVEMBER, 2014
3
DECLARATION
I, Dr. Ezenduka Ekene Vivienne hereby declare that this work was carried out
by me and has not been submitted somewhere else.
--------------------------------------------
-----------------------------
Dr. Ezenduka, Ekene Vivienne
Date
i
CERTIFICATION
We certify that Dr. Ezenduka, Ekene Vivienne carried out this research work in
the Department of Veterinary Public Health and Preventive Medicine at the
University of Nigeria, Nsukka.
The work presented herein is original and has not been previously reported
anywhere else
----------------------------------Prof S.I Oboegbulem
(supervisor)
------------------Date
----------------------------------Prof. J. A. Nwanta
(supervisor)
------------------Date
----------------------------------Prof. A. A. Anaga
(Head of Department)
------------------Date
----------------------------------Prof. C. U. Nwosu
(Dean)
------------------Date
----------------------------------Prof. Junaidu Kabir
(External Examiner)
------------------Date
ii
DEDICATION
This work is dedicated to God Almighty, “Jehovah over Do”, who does much more than we
ask for.
To my family: my husband, Charles, my unmovable rock, who always provided a shoulder to
lean on when the huddle became tough in the course of this work.
My children: Oge; Zubby; Chidiogo and Chikaosolu, who I denied enough motherly care and
attention just to make this work a reality. Thank you my angels, God will continue to bless
you.
iii
ACKNOWLEDGEMENT
My heartfelt gratitude goes to my supervisors: Prof. S. I. Oboegbulem, for his profound
supervision filled with precision and thoroughness. I just want to say a warm thank you for
being not just a supervisor but also a father; Prof. J.A. Nwanta for your insistence on carrying
out the work even when I got frustrated for lack of sensitive facilities to advance the work.
You single handedly fished out a very good laboratory where the majority of this work was
done. Thank you, for believing in my ability to deliver.
I am indebted to Dr. Emmanuel Nna and the rest of the staff and management of Safety
Molecular Pathology Laboratory, Enugu for the support and help they rendered to me during
the course of the laboratory work. Thanks guys, I’ll never forget the manner we all worked as
a family, you all made my stay a memorable one.
I’ll not fail to appreciate the efforts of Prof. DenChris Onah, who is not just a senior
colleague but also a friend indeed, for all his encouragement and assistance.
iv
TABLE OF CONTENTS
Title
Page
Declaration
i
Certification
ii
Dedication
iii
Acknowledgement
iv
Table of Contents
v
List of Tables
xii
List of Figures
xiii
Appendices
xv
Abstract
xvii
CHAPTER ONE: INTRODUCTION
1.1
Background of Study
1
1.2
Statement of the Problem
4
1.3
Research Questions
7
1.4
Study Aim and Objectives
8
1.5
Significance of the Study
9
CHAPTER TWO: LITERATURE REVIEW
2.1
Antimicrobial Agents
10
2.1.1
Definition
10
2.1.2
Antibiotics
11
2.2
Antimicrobial use in Animals
11
2.3
Classification of Antimicrobial Agents for Veterinary Use
14
2.3.1
Aminoglycosides
15
v
2.3.2
Beta-lactam Antimicrobial Agents
15
2.3.3
Chloramphenicol
16
2.3.4
Quinolones (Fluoroquinolones)
16
2.3.5
Lincosamides
16
2.3.6
Macrolids
16
2.3.7
Nitrofurantoin
16
2.3.8
Tetracyclines
17
2.3.9
Sulphonamides
17
2.4
Antimicrobials Banned For Use in Food Animal Production
17
2.5
Drug Residues and Related Terms
18
2.5.1
Residues
18
2.5.2
Drug residues
18
2.6
Drug Approval and Safety Evaluation of Antimicrobial Residues
19
2.7
No Effect or Maximum no Adverse Effect
20
2.8
Acceptable Daily Intake (ADI)
21
2.9
Tolerance level or Maximum Residue Limit (MRL)
22
2.10
Withdrawal Time
25
2.10.1 Withdrawal Time for Veterinary Drugs
25
2.11
Antimicrobial use in Poultry
26
2.11.1 Antimicrobials as Growth Promoters
26
2.11.2 Antimicrobials as Coccidiostats in Poultry Production
37
2.11.3 Therapeutic Antimicrobial Use in Poultry
27
2.12
Causes of Violative Level of Antimicrobial Residues in Animal Tissues
27
2.13
Public Health Hazards and Harmful Effects of Antimicrobial Residues
29
2.13.1 Drug Allergy and Hypersensitive Reaction
vi
29
2.13.2 Effects on Human Gut Microbiota
31
2.13.3 Development of Resistance to Antimicrobial Agents
32
2.13.4 Effect on Bone Marrow/Bone Marrow Depression
33
2.13.5 Carcinogenic Effect
34
2.13.6 Industrial Effect
34
2.13.7 Other Harmful Effects
35
2.14
Prevention and Control of Antimicrobial Residue
Occurrence in Foods of Animal Origin
35
2.14.1 Disease prevention
36
2.14.2 Proper Diagnosis and Antimicrobial Susceptibility Testing
36
2.14.3 Appropriate Use of Antimicrobials and Route of Administration
37
2.14.4 Appropriate Dosage Regimen
37
2.14.5 Monitoring and Surveillance of Antimicrobial Residue
38
2.15
38
Antimicrobial Residues Detection and Identification
39
2.15.1 Test Matrix
2.16
Methods of Detection of Antimicrobial Residues in
Foods of Animal Origin
40
2.16.2 Microbiological Method
40
2.16.3 Immunochemical Methods
45
2.16.4 Chromatographic Methods
46
2.17. Effect of heat on Residue
47
CHAPTER THREE: GENERAL MATERIALS AND METHODS
3.1
Study Area
49
3.2
Study Design
51
3.2.1
Survey Studies
51
3.2.2
Experimental Studies
51
vii
3.3
Sample Source, Population and Sampling Technique
51
3.3.1
Survey Studies
51
3.3.2
Experimental Study
54
3.4
Specimen Preparation
56
3.5
Data Presentation and Analysis
56
CHAPTER FOUR: ANTIMICROBIAL RESIDUES SURVEY IN COMMERCIAL
BROILERS USING THREE PLATE TEST
4.1
Introduction
57
4.2
Method
59
4.2.1
Isolation of Bacillus subtilis
59
4.2.2
Identification
59
4.2.3
Molecular Characterization of Isolates
60
4.2.4
Antimicrobial Sensitivity Test
63
4.2.5
The Three Plate Test
64
4.3
Results
65
4.3.1
Isolation, Identification and Sensitivity of Bacillus subtilis
65
4.3.2
Sensitivity Test
70
4.3.3
Prevalence of Antimicrobial Residues in Broilers
72
4.3.4
Occurrence of Antimicrobial Residues in Broiler Meat and Organs
74
4.3.5
Organ Distribution of Antimicrobial Residues According to pH
76
4.4
Discussion
78
4.5
Conclusions
79
viii
CHAPTER FIVE: ANTIMICROBIAL RESIDUES SURVEY IN COMMERCIAL
BROILERS USING PREMI® TEST
5.1
Introduction
80
5.2
Materials and method
81
5.2.1
Antibiotics Residues Detection
81
5.3
Results
82
5.3.1
Antibiotic Residues Detection in Commercial Broilers
83
5.3.2
Antibiotics Residues Detection in Sampled Organs
85
5.3.3
Detection of Antimicrobial Residues by TPT and Premi® Test
87
5.4
Discussion
89
5.5
Conclusions
90
CHAPTER SIX: DETECTION AND QUANTITATION OF TETRACYCLINE
RESIDUES IN COMMERCIAL BROILERS
6.1
Introduction
91
6.2
Materials and methods
92
6.2.1
Organ Sample Collection And Preparation
92
6.2.2
Tetracycline Residues Detection
92
6.3
Results
94
6.3.1
Calculation of OTC Concentration from Optical Density (OD)
94
6.3.2
Organ Distribution of Tetracycline Residues
96
6.3.3
Comparison of Mean Values of the Organs with their MRL’s
98
6.4
Discussion
106
6.5
Conclusion
107
ix
CHAPTER SEVEN: COMPARATIVE VALIDITY AND RELIABILITY OF TPT AND
PREMI® TEST IN DETECTION OF OXYTETRACYCLINE RESIDUE USING
ELISA AS GOLD STANDARD.
7.1
Introduction
108
7.2
Materials and Methods
110
7.2.1
Experimental Study Design
110
7.2.2
Experimental Drug Administration
110
7.2.3
Testing for OTC Residue in Experimental Birds
111
7.3
Results
111
7.3.1
TPT Detection of Oxytetracycline Residues in Group A Birds
111
7.3.2
TPT Detection of Oxytetracycline Residues in Orally Treated Birds
112
7.3.3
ELISA Detection of Oxytetracycline Residues in Muscle Samples
112
7.3.4
ELISA Detection of Oxytetracycline Residues in Liver Samples
114
7.3.5
Comparing the Detection Ability of TPT and Premi® Test in
OTC Residues Detection in Muscles of Birds in Group A
116
Statistical Comparison of the Validity of TPT and Premi® Test
in OTC Residue Detection In Muscles of Group A Birds
118
Comparing the Detection Ability of TPT and Premi® Test in
OTC Residue Detection in Muscles of Birds in Group B
121
Statistical Comparison of the Validity of TPT and Premi® Test in
OTC Residue Detection in Muscles of Birds in Group B
123
Comparing the Detection Ability of TPT and Premi® Test in
OTC Residue Detection in Liver of Birds in Group A
126
7.3.6
7.3.7
7.3.8
7.3.9
7.3.10 Statistical comparison of the validity of TPT and Premi® Test in
OTC residue detection in Liver of birds in group A
128
7.3.11 Comparing the Detection Ability of TPT and Premi® Test in
OTC Residue Detection in Liver of birds in Group B
131
7.3.12 Statistical Comparison of the Validity of TPT and Premi® Test in
OTC Residue Detection in Liver of Birds in Group B
133
x
7.4
Discussion
137
7.5
Conclusion
139
CHAPTER EIGHT: EFFECT OF HEATING PROCESSES (COOKING METHODS)
AND FREEZING ON OTC RESIDUES IN CHICKEN MEAT AND ORGANS
8.1
Introduction
141
8.2
Materials and Methods
142
8.2.1
Sample Preparation
142
8.2.2
Effect of Heat Treatment
142
8.2.3
Effect of Freezing on OTC Residue
143
8.3
Results
143
8.3.1
Effect of Cooking Methods on OTC Residue in Muscle Using TPT
143
8.3.2
Effect of Cooking Methods on OTC Residue in Liver using the TPT
151
8.3.3
Effect of Cooking Methods on OTC Concentration in Muscle Tissues
159
8.3.4
Effect of Cooking Methods on OTC Concentration in Liver Tissues
163
8.3.5
Effect of Freezing on OTC Concentration in Both Tissues
167
8.4
Discussion
171
8.5
Conclusion
172
CHAPTER NINE: GENERAL CONCLUSIONS AND RECOMMENDATIONS
9.1
Prevalence Study with Three Plate Test (TPT) and Premi® Test
173
9.1.1
Organ (matrix) Distribution of Antimicrobial Residues
174
9.2
Comparative Study on the Sensitivity of TPT and Premi®
Test in OTC Detection in Broilers
174
9.3
Effect of Temperature on OTC Residues in Broiler Muscle and Liver
174
REFERENCES
176
APPENDICES
205
xi
LIST OF TABLES
Table
Title
Page
4.1
Positive Samples According to pH
77
6.1
Tetracycline Residues Detection According to Concentration
97
7.1
OTC Residue Detected by the Three Methods in Muscle
of Birds in group A (Injected)
117
OTC Residue as Detected by the Three Methods in Muscle
of Birds in Group B (Orally administered)
122
OTC Residue Detected by the Three Methods in Liver of
Birds in Group A
127
7.4
OTC Residue Detected by the Three Methods in the Liver of Group B Birds
132
8.1
Proportion of the Effect of Cooking Methods on OTC in
Muscle using TPT
144
Proportion of the Effect of Cooking Methods on OTC in
Liver using TPT
152
8.3
Effect of Cooking Methods on OTC Concentration in Muscle tissue
160
8.4
Effect of Cooking Methods on OTC Concentration in Liver Tissue
164
8.5
Effect of Freezing Time on OTC Concentration in Muscle and Liver Tissues
168
7.2
7.3
8.2
LIST OF FIGURES
Figure
Title
Page
xii
3.1
Geographical positioning of the three major poultry
markets in Enugu metropolis with their coordinates
50
3.2
Schematic Presentation of Sampling Population and Technique
53
3.3
Schematic Presentation of Experimental Design
55
4.1
Microscopic Morphology of Bacillus Organism
66
4.2
API®50 Plates
68
4.3
Amplification of 595 bp 16S rRNA of Bacillus subtilis
69
4.4
Antimicrobial Susceptibility Plates
71
4.5
Prevalence of Antimicrobial Residues in Broilers
73
4.6
Organ Distribution of Antimicrobial Residues
75
5.1
Prevalence of Antimicrobial Residues in Commercial Broilers
84
5.2
Organ Distribution of Antimicrobial Residues
86
5.3
Detection of Antimicrobial Residues with TPT and Premi® Test
88
6.1
Tetracycline Calibration Curve: Optical Density Versus
Concentration of Standard
95
6.2a
Tetracycline Residues Concentration in the Muscle
99
6.2b
Tetracycline Residues Concentration in the Liver
101
6.2c
Tetracycline Residues Concentration in the Kidney
103
6.2d
Tetracycline Residues Concentration in the Gizzard
105
7.1
Daily mean concentration of OTC residue detected in
muscle tissues of birds in both groups A and B
113
Daily OTC residue detection and quantification with
ELISA in liver tissues of birds in both groups A and B
115
Validity of TPT and Premi® Test in OTC Detection
in Muscle of Injected Birds
119
7.2
7.3
7.4
7.5
Correlation between the Inhibition Zone of TPT and the
Tetracycline Residue Concentration in Muscle of Group A Birds
Validity of TPT and Premi® Test in OTC Detection in Muscle of
xiii
120
Birds given in Drinking water
124
Correlation between the Inhibition Zone of TPT and the
Tetracycline Residue Concentration in Muscle of Group B Birds
125
7.7
Validity of TPT in OTC Detection in Liver of Injected Birds
129
7.8
Correlation between the Inhibition Zone of TPT and the
OTC Residue Concentration in Liver of Group A Birds
130
Validity of TPT in OTC Detection in Liver of Birds Given
in Drinking Water
134
Correlation between the Inhibition Zone of TPT and the
Tetracycline Residue Concentration in Liver of Group B Birds
135
7.6
7.9
7.10
7.11
General Correlation between the Inhibition Zone of TPT and Tetracycline Residue
Concentration
136
8.1
Effect of Microwaving on OTC Concentration in Muscle
using TPT at pH6.0 and pH7.2
146
Effect of Roasting on OTC Conc. in Muscle using TPT at
pH 6.0 and pH 7.2
148
Effect of Boiling on OTC Conc. in Muscle using TPT at
pH 6.0 and pH 7.2
150
Effect of Boiling on OTC Conc. in Liver using TPT at
pH 6.0 and pH 7.2
154
Effect of Microwaving on OTC Conc. in Liver using TPT
at pH 6.0 and pH 7.2
156
Effect of Roasting on OTC Conc. in Liver using TPT at
pH 6.0 and pH 7.2
158
Effect of Cooking Methods on OTC Concentration in
Muscle using ELISA
162
8.8
Effect of Cooking Methods on OTC Concentration in Liver Tissue
166
8.9
Effect of Freezing Time on OTC Residues on Muscle and Liver
170
8.2
8.3
8.4
8.5
8.6
8.7
APPENDICES
xiv
Appendix
I.
Title
Page
Mechanism of action of Aminoglycosides
205
Mechanism of action of Beta-lactam antimicrobial
206
III.
Mechanism of action of Chloramphenicol
208
IV.
Mechanism of action of quinolones
209
Mechanism of action of Lincosamides
210
VI.
Mechanism of action of Macrolids
211
VII.
Mechanism of action of Nitrofurans
212
Mechanism of action of Tetracyclines
213
Mechanism of action of Sulphonamides
214
X.
Antimicrobials banned for use in animals intended for food production
215
XI.
The Codex alimentarius Maximum Residue Limits for antimicrobials
on chicken According to the FAO/WHO Food Standards
216
Some Veterinary Antimicrobials and Generics with their Varying
Withdrawal Periods and Limitations for use in Poultry
218
XIII.
Premi® Test Indicative Data on Detection Level of Meat Types and Egg
220
XIV.
Api®50 CH Record Sheet for Identification of Bacillus subtilis
222
XV.
Three Plate Test Clear Zone of Inhibition Indicative of Positive
Samples for Antimicrobial Residue
223
Antimicrobial residues detection with TPT in 100
sampled broiler birds
224
Premi® Test Color Indication for Positive and Negative Result for
Antimicrobial Residues
225
Antimicrobial residues detection with Premi® Test in
100 sampled broiler birds
226
Elisa Work – Sheet
227
Calculation of Tetracycline Concentration from Standard Curve
228
II.
V.
VIII.
IX.
XII.
XVI.
XVII.
XVIII.
XIX.
XX.
xv
XXI.
XXII.
XXIII.
XXIV.
XXV.
Daily Detection of OTC Residue with TPT and Premi® Test in
Organs of Group A Birds Injected Intramuscularly
229
Daily Detection of OTC Residue with TPT and Premi® Test in
Organs of Group B Birds Given in Drinking Water
230
Effect of Freezing Time on OTC Concentration of Samples
Positive for OTC Residue
231
Work In Progress, Supervision by Supervisors
232
Tetracycline ELISA Kit
233
xvi
ABSTRACT
The high turn-over rate and quest for white meat have given more impetus to poultry
production. The need to increase production and meet the demand for poultry meat has
necessitated the use of veterinary drugs, especially antimicrobials, for therapeutic,
prophylactic and growth promotion purposes in poultry farming. These drugs tend to
accumulate in tissues and organs as residues. The presence of drug residues in tissues above
maximum residue limit (MRL) becomes violative if withdrawal periods are not observed.
The consumption of violative levels of antimicrobial residues could result in the development
of antibiotic-resistant strains of microorganisms, allergic reaction in sensitised individuals,
distortion of activities of the intestinal flora, carcinogenesis and mutagenesis. There is
evidence of excessive prescription, overuse and abuse of antimicrobial drugs in veterinary
practice in Nigeria, and legislation regarding drug use in veterinary practice is hardly
enforced. It has become necessary to monitor the presence and level of antimicrobial residues
in poultry in Nigeria using reliable screening tests. This is imperative since there is presently
no established surveillance programme for detecting drug residues. Oxytetracycline (OTC) is
the most widely used antimicrobial in poultry production. Although thorough cooking is part
of our food culture, studies have shown that some drugs are heat stable, hence there is need to
investigate the effects of heat and freezing on the stability of this drug in tissues. The study
to: (i) assessed the occurrence of antimicrobial residues in broilers retailed in Enugu
metropolis, (ii) evaluated the effects of cooking methods and freezing on OTC residue, (iii)
compared the sensitivity of detection of OTC residues in tissues using Three Plate Test (TPT)
and Premi® test. The study involved both a cross sectional survey of broilers retailed in
Enugu metropolis and two experiments. The survey was an assay of antimicrobial residues in
broiler meat and organs using qualitative screening methods (TPT and Premi® Test) and a
quantification of tetracycline (TC) residues using specific enzyme-linked immunosorbent
xvii
assay (ELISA) technique. The three major markets (Artisan, Gariki and Ogbete) in which
broilers are retailed in Enugu metropolis, were used for the study. A total of 100 broilers were
proportionately selected according to the sale capacity of each of the markets as follows:
Artisan (40), Gariki (30) and Ogbete (30). The birds and retailers were selected using
systematic random sampling technique. Muscle, liver, kidney and gizzard were collected
from each of the 100 broilers for the survey. For the experiments, TPT, Premi® Test and
ELISA were used to detect OTC residue in tissues of OTC treated birds and to evaluate the
effects of various cooking methods (boiling, microwave grilling and roasting) and freezing
time on the level of OTC residues. Fifty 5-week old broilers were raised for 3 weeks for the
experiment. Four birds were sacrificed and their organs screened for residues. In the absence
of residues, the remaining 46 birds were assigned into 2 equal groups (A and B). Group A
birds were injected with long acting OTC at the dose of 20mg/kg body weight and group B
birds were given OTC in drinking water at the dose of 4g/l for 5 days. Their organs were
screened for OTC residue 24 hours after treatment. For each organ, meat juice was extracted
by maceration and centrifugation. The API50 and polymerase chain reaction (PCR) were
used to identify and characterize the Bacillus subtilis used for the TPT. Premi® test and
ELISA were done following the manufacturer’s instructions. Graphpad Prism 5 statistical
package was used to analyse the data generated. Chi-square was used to determine
associations between occurrence of residues and organ types. One way analysis of variance
was used to analyse other data as appropriate. Dunn’s multiple comparism was used for post
hoc analysis. Three plate test inhibition zones were correlated to OTC concentration.
Significance was accepted at p < 0.05. For the survey, TPT detected antimicrobial residues in
64% of broilers and the organ distribution were as follows: kidney (60%), liver (54%),
gizzard (30%) and muscle (11%). Premi® test detected residues in 60% of broilers and
distribution in specific organs were: kidney (49%), liver (25%) gizzard (22%) and muscle
xviii
(14%). The ELISA detected tetracycline residues in 90% of broilers and detection in specific
organs were: liver (96%), muscle (96%), kidney (88%) and gizzard (82%). The residue level
was above MRL in 84% of liver, 100% of gizzard and 86% of muscle, whereas residues in
100% of positive kidney samples were below the MRL. For the experimental study,
microwaving, boiling and roasting significantly (p < 0.05) reduced the inhibition zones
produced by raw liver in TPT. There was a significant (p < 0.05) decrease in OTC residue
concentration in roasted and boiled liver samples tested with ELISA. The three cooking
methods had no significant (p > 0.05) effect on residue levels in muscle samples. Freezing
had no significant (p > 0.05) effect on residue levels in muscle and liver samples. Three plate
test had a higher sensitivity in detecting OTC residue than Premi® test in muscle and liver of
birds treated by injection and those administered OTC in drinking water. There was a positive
and significant correlation (r = 0.94; p < 0.05) between the TPT inhibition zones and OTC
concentration.
xix
CHAPTER ONE
INTRODUCTION
1.1
Background of Study
There is a worldwide increase in the consumption of poultry products (meat and eggs). The
consumption of chicken meat has trippled over the last quarter of a century (Jordan and
Partisons, 1996). Poultry products constitute a major source of animal protein in Nigeria and
a source of healthy meat worldwide because of its white meat constituent. Poultry production
is an important source of livelihood for rural and urban dwellers in Nigeria, as it provides
employment and income. It is the most commonly kept livestock accounting for up to 70% of
livestock production (Amar-Klemesu and Maxwell, 2000). The FAO report of 1988 as cited
by Nwanta et al. (2012) stated that Nigeria recorded the lowest animal protein intake with an
average of 6g per head per day and estimated in 2012, that in an average Nigerian meal,
animal protein contributes only 3% as against 12% recommended by WHO for healthy living.
To meet up with the high animal protein demand, there is wide application of veterinary
drugs in commercial poultry designed to increase the production of poultry meat and eggs.
Veterinary drugs are used primarily to prevent and control infectious and non-infectious
poultry diseases and assist in combating stress occasioned by vaccination, debeaking and
other management practices (Dafwang et al., 1987; Kabir et al., 2003). As additives to feed
and drinking water, the drugs are used for improved performance in growth especially in
poultry broiler production and to promote growth and increase egg production in layer farms
(Choi & Ryu, 1997; Furusawa, 1999) etc. The remnants of these drugs may remain in tissues
of the animals for some time and are termed residues.
1
The residues of these antimicrobial drugs are excreted in body fluids of food animals and
tend to accumulate in tissues/organs and eggs (Droumev, 1983: Geersema et al., 1987).
Although the use of these drugs in livestock production benefit producers and consumers
alike, their indiscriminate use may result in the presence of residues of these drugs in meat
and other animal food products (milk and eggs) at a violative level that may be harmful to
man.
The presence of drug residues in foods of animal origin is one of the most important issues in
food safety because of its public health implications. They pose potential allergic reactions in
sensitized individuals and alteration of the human intestinal flora which ordinarily act in
competition inhibition of colonization of pathogenic bacteria (Vollard & Classener, 1994:
Nisha, 2008). A link has been noted between the use of antibiotics in food animals and the
development of bacterial resistance to these drugs (Stark, 2000: Reig & Toldra, 2008) and
perhaps, of greater public health relevance. Other noted pathological effects produced by
antimicrobial residues in food include autoimmunity, carcinogenicity, mutagenicity, bone
marrow toxicity (Pavlov et al., 2008: Nisha, 2008) etc. These documented effects and others
have led to the ban of some of these drugs for use in food animals by regulatory agencies.
Chloramphenicol was banned because of its involvement in bone marrow toxicity while
furazolidone and nitrofurans were also restricted for use in food animals because of both
carcinogenic and mutagenic effects they may cause.
In the developed countries, consumer awareness of the established and potential public health
implications of antimicrobial residues in food animal products and the desire of producers to
avoid litigation had led to the development of several biological and chemical tests to monitor
the presence, type and level of antimicrobial residues in animal tissues and products
(Pennycott, 1987; Oboegbulem and Fidelis, 1996). There are several programmes,
organizations and agencies concerned with food safety and drug residues in foods of animal
2
origin. The Residue Avoidance Programme (RAP) was initiated in 1981 by the Extension
Service and Food Safety and Inspection Service (FSIS) of The United States Department of
Agriculture (USDA) with the goal to prevent residue through educational programme
directed at the livestock industry and people serving the industry. The Ministry of
Agriculture, Fisheries and Food Standards Agency (FSA) also has a mapped out strategy for
the testing of chemical and antimicrobial residues in foods of animal origin. In the major food
exporting countries of the world such as the USA and Canada, the European Union has well
developed abattoir – based programmes for the surveillance and monitoring of antibacterial
residue in meat (Kindred and Hubbert, 1993).
Antimicrobial residues in animals are conventionally detected by microbiological tests which
include Bacillus stearothermophillus Disc assay(BsDA), the European Four Plate Test (FPT),
the German Three Plate Test (TPT), the Premi® test, and a number of other commercial kits.
These tests which are essentially qualitative are based on bacterial inhibition and are used
primarily as screening tools for presence of antimicrobial residues in meat, milk and eggs.
Confirmation and quantification of specific antimicrobial residues are performed by using
more sensitive chromatographic and/or immunochemical methods such as validated High
Performance Liquid Chromatography (HPLC) (Popelka et al, 2005) and Enzyme Linked
Immunosorbent assay (ELISA).
Most available information on drug residues in foods of animal origin is mostly related to the
concentration of these drugs or their metabolites in raw samples. Since most of these foods
are cooked before consumption, information on the effect of heat is required to give a more
accurate estimate on the concentration of these residues the consumers may be exposed to.
For years, many researchers have been interested in determining whether antibiotic residues
can be destroyed or concentration reduced by different cooking procedures, pasteurization, or
canning processes (Rose et al., 1995; Isidori et al., 2005; Hassani et al., 2008; Hseih et al,
3
2011). Traditionally, heat stabilities of antibiotics have been studied based on either the
evaluation of the decrease in antimicrobial activity or by specific chromatographic analysis of
change in concentration after heat treatments. Relatively few studies have been carried out
using both microbiological and chemical analyses in evaluating the heat stability of
veterinary drug residues (Franje et al., 2010). Moreover, whether or not the heating of these
compounds and any structural changes generated result in altered genotoxicity that could
contribute to the mutagenicity of bacteria remains unclear.
Tetracyclines rank among the antimicrobial substances most frequently used in the animal
food production (Schmidt & Rodrick, 2003). Tetracyclines display a wide spectrum of
antimicrobial action: apart from a stronger action on the gram-positive bacteria and a weaker
one on the gram-negative ones, they exercise action also on mycoplasmas, chlamydiae,
rickettsias, spirochetes, actinomycetes, and some protozoa (Sundin, 2003). The sum of
tetracycline action is bacteriostatic.
Adverse effects on human health after the therapeutic use of tetracyclines are well known.
Tetracyclines should not be used by children up to the age of 6–8 years or by pregnant
women because of the risk of developing secondary tooth discoloration. Other chronic effects
include nephrotoxicity, hepatotoxicity, skin hyperpigmentation in the sun exposed areas,
hypersensitivity reactions. Tetracyclines have also been reported to cause hypouricemia,
hypokalemia, proximal and distal renal tubular acidosis (Goldfrank et al., 2002).
1.2 Statement of the Problem
In Nigeria, as in most African countries there is excessive prescription, overuse and abuse of
antimicrobial drugs in veterinary practice and human medicine. This problem is compounded
by largely unrestricted availability of antimicrobial drugs and the practice of self-medication
by both poultry farmers and human patients. Unauthorized and unprofessional exposure of
4
poultry to veterinary drugs without adherence to recommended dose and/or withdrawal time
ensures the accumulation of violative residues in meat and eggs.
In Enugu State, poultry products are about the cheapest source of animal protein, unlike what
is obtainable in the North where there is high availability of beef and fresh milk. In the state,
the fear of consuming antimicrobial contaminated milk is not much of a problem because
already processed liquid or powdered milk is being used. Instead the bother lies on the
poultry meat readily available to us. Poultry production is a common agricultural practice in
Enugu State. There is so much reliance on veterinary drugs in the management of poultry
farming, being a major source of protein and does not require much space to manage;
individuals go into rearing birds to meet up with the growing human population and demand
for white meat. This much reliance on drugs in poultry industry in Nigeria creates conditions
that allow for development of drug resistant strains of organisms thereby encouraging the use
of multiple antibiotics in the treatment of one disease condition. Potentially, this practice
could give rise to accumulation of these drugs in poultry meat and eggs. In an earlier survey
in the study area, violative levels of antimicrobial residues were detected in table eggs
sampled from the commercial poultry farms and retail outlets. (Ezenduka et al., 2011).
In 1971, the British Government implemented The Swann committee recommendations on
the use of antimicrobiasl in animal production and thus banned the use of antimicrobials
meant for therapeutics for growth promotion following legistlation for use of growth
promoters. Although there is legislation regarding drug use in veterinary practice in Nigeria,
it is hardly if ever enforced. A good example is the ban of furazolidone and nitrofurans for
use in food animals by WHO and even NAFDAC because of both carcinogenic and
mutagenic effects, the drug is very much in use in this country, particularly in poultry
production.
5
Few studies have been done in Nigeria regarding residues of veterinary drugs, some have
been reported in slaughtered chicken and commercial eggs in the East (Oboegbulam and
Fidelis, 1996; Ezenduka et al., 2011) and in the North by Kabir et al. (2003), Fagbamila et al.
(2012) and Mbodi et al. (2014). The occurrence of residue of oxytetracycline, a commonly
used antibiotic in Nigeria, was demonstrated in meat and eggs of chicken fed recommended
levels of oxytetracycline in the west by Dipeolu & Alonge (2001) and Dipeolu & Dada
(2005).
In the developed countries, several programmes have been mapped out with respect to food
safety and drug residues in foods of animal origin. The Residue Avoidance Programme
(RAP) was initiated in 1981 by the Extension Service and Food Safety and Inspection Service
(FSIS) of The United States Department of Agriculture (USDA) with the goal to prevent
residue through educational programme directed at the livestock industry and people serving
the industry. The Ministry of Agriculture, Fisheries and Food Standards Agency (FSA) also
has a mapped out strategy for the testing of chemical and antimicrobial residues in foods of
animal origin. In the major food exporting countries of the world such as the USA and
Canada, the European Union has well developed abattoir-based programmes for the
surveillance and monitoring of antibacterial residue in meat (Kindred and Hubbert, 1993).
However, in Nigeria there is no national programme in place for monitoring drug residues in
food animals in farms and abattoirs.
Microbiological screening methods are preferably used for monitoring the presence of
antimicrobial residues in large numbers of slaughter animals. Principally, two types of
microbiological (plate and tube) tests are available. The plate tests were earlier and routinely
used to detect different antimicrobials (Heitzman, 1994; Okerman et al., 2001; Jabbar, 2004).
The tube tests are more exoti because of the fast growing properties of the organism at
elevated temperature, it is possible to obtain an analysis result within a few hours with the
6
test (Pikkemat et al., 2009). The use of spores instead of vegetative cells allows prolonged
storage and enables commercial distribution.
Since tetracyclines are the most widely used antimicrobials in poultry production, it became
necessary to gain more insight on the ability of the more convenient tube (Premi®) to detect
Oxytetracycline residue in poultry tissues.
Heat treatment of food is a primary and important method for rendering edible animal
products safe for human consumption. Literature abounds with the value of heat treatment in
inactivating pathogenic microorganisms. Similarly, freezing is commonly done to inhibit or
retard microbial growth in foods while retaining their natural constituents. There is limited
information on various temperature based treatments, persistence, levels and activities of
residues in edible foods and products.
There is urgent need for extensive surveillance of veterinary drug residue in poultry meat and
egg in Southeastern Nigeria, where poultry production is a primary form of agriculture. In
many developed countries, the public outcry against much use of antimicrobial drugs in food
animals is based on the increased awareness of the public health hazards of residues of these
drugs. Because there is no national programme for routine monitoring of veterinary drug
residues in Nigeria and because public awareness of the adverse effects of the drug residue is
low, this study became necessary to address these problems in the study area.
1.3
Research Questions
1. Do commercial poultry (broiler) meat and organs sold at Enugu metropolis contain
antimicrobial residues, and at what proportion?
2. What is the prevalence of tetracycline residues and do the concentrations in different
organs differ from their maximum residue limit?
7
3. Are there differences in the concentration of antimicrobial residues in chicken muscle,
gizzard, liver and kidney.
4. What are the effects of different cooking methods (boiling, microwaving and roasting)
on the concentration of oxytetracycline residue in broiler muscle and Liver?
5. Does freezing affect the concentration of oxytetracycline residues?
6. What is the validity and reliability of the two antimicrobial residue screening tests
(FPT and Premi test) in OTC residue detection?
1.4 Study Aim and Objectives
Based on the questions raised above, these studies are designed to achieve the main aim of
determining the occurrence of antimicrobial residues in broiler meat (muscle, gizzard, liver
and kidney) in Enugu State, and the effect of freezing and cooking methods on the
concentration of oxytetracycline residue. The following are the specific objectives:
1. To screen for the presence and determine the prevalence of antimicrobial residues in
broiler meat and organs (Muscle, gizzard, liver and kidney) using the German Three
Plate Test and the Premi® test kit.
2. To establish the relative distribution of antimicrobial residues in the muscle, gizzard,
liver aznd kidney.
3. To determine the prevalence and relative concentration of tetracycline residues in
broiler meat and organs (muscle, gizzard, liver and kidney) using Enzyme Linked
Immunosorbent Assay (ELISA) immunoassay quantitative method.
4. To compare the validity and reliability of the microbiological methods (TPT and
Premi® Test) in OTC residue detection using ELISA as gold standard.
5. To determine the effect of cooking methods (boiling, microwaving and roasting) on
oxytetracycline residues concentration in broiler meat and organs
8
6. To determine the effect of freezing time on oxytetracycline residues concentration in
broiler meat and organs
1.5
Significance of the Study.
•
The findings will add to baseline information on existing data on the prevalence, types
and levels of veterinary drug residues in the poultry industry.
•
This
study
will
provide
further
evidence,
requested
or
public
health
education/awareness amongst stake holders as a first and necessary step towards
reducing excessive application of ntimicrobials and other veterinary drugs in animal
production.
•
The information from the study will serve as a tool for veterinarians, NAFDAC and
the government in decision making and policy formation geared towards designing
appropriate intervention to checkmate the occurrence of antimicrobial residues by
routine screening and monitoring of food of animal origin.
•
The result will also educate stakeholders on the best method to process poultry meat
to reduce exposure to violative level of antimicrobial residues.
9
CHAPTER TWO
LITERATURE REVIEW
2.1
Antimicrobial agents
2.1.1 Definition
Antimicrobial agents or simply put antimicrobials are drugs that act against microorganisms
(bacteria, fungi, rickettsia and viruses) either by inhibiting their growth and multiplication,
through various mechanisms, or by completely destroying them (Brander et al., 1993).
Antimicrobials that are sufficiently non-toxic to the host are used as chemotherapeutic agents
in the treatment of infectious diseases of humans, animals and plants. Such chemical agents
have been present in the environment for a long time, and have played a role in the battle
between man and microbes. In the last century, the discovery and introduction of
antimicrobial agents: sulphonamides in the 1930s and penicillin in the 40s, revolutionized the
treatment of infectious diseases by a dramatic reduction in mortality and morbidity and
contributing significantly to improvements in the health of the general population (Serrano,
2005).
Antimicrobial agents can be naturally produced by microorganisms which could be:
•
Mould and fungal metabolites (Penicillin spp, Streptomyces spp) otherwise called
antibiotics, from bacteria (e.g Bacillus spp.).
•
Semisynthetic variants of natural products (e.g, formation of amoxicillin from
benzylpenicillin
•
Synthetic organic chemical compounds (e.g sulphonamides) (Brander et al., 1993;
Merck, 1998)
10
2.1.2
Antibiotics
According to the original definition by Nobel laureate S.A. Waksaman, the term antibiotic
only refers to those substances which are produced by synthesis of one living organism
(growth of microorganism) and are toxic for another. However the term is often used as a
synonym for any antimicrobial agent by both professionals and laypersons alike (Guardabassi
and Kruse, 2008). Examples of natural compound (antibiotics) of microbial origin include:
a) Penicillins; the first antibiotic to be discovered in 1929 by Sir Alexander Flemming
from the mould, Peniciilium spp.
b) Cephalosporin from Cephalosporium spp. In the 1960’s.
c) Streptomycin from Actinomycesspp (since renamed Streptomyces) in 1944.
d) Erythromycin from Streptomyces erythreas in 1952.
e) Chlortetracycline from Actinomyces aureofaciens in 1948. (Brander et al., 1993)
2.2
Antimicrobial Use in Animals
Antimicrobial use in animals originated over 50 years ago when chlortetracycline
fermentation waste was found to enhance animal growth and health (Schwarz and Dancla,
2001). Since then major changes have taken place in both companion animal medicine and
food animal production. The increase in human population has led to the intensification of
food animal production with changes in size, structure and management, thereby having high
animal densities and stressful conditions. The use of antimicrobials has become widespread
in both animal production and veterinary medicine. Today, it is estimated that half of all
antimicrobials produced worldwide are used in animals (Guardabassi and Kruse, 2008).
Information and the quantitative figures on the consumption of antimicrobial agents for
veterinary use in most countries are very rare and estimates are available for only a few
countries. Antimicrobial consumption in animals in the USA showed an increase from 110
11
tonnes in 1951 to 5580 tonnes in 1978 (Black, 1984). Antimicrobials are used for the
following purposes in animals:
Therapeutic (treatment): antimicrobials are used and administered individually to animals
for the purposes of treatment. They are used in the treatment of specific diseases caused by
microorganisms. Their use in specific conditions is justified because the role of microbial
agents is mainly to kill the rapidly dividing invading cells.
Prophylactic (prevention): antimicrobials are administered in sub-therapeutic doses to
prevent possible infections. Administration of antimicrobials to clinically healthy animals
belonging to the same flock or pen as animals with clinical signs is a form of prophylaxis
called metaphylaxis (Antimicrobial resistance learning site, 2014). Metaphylaxis is typically
used during disease outbreaks in a flock especially in poultry and aquaculture but also used in
cattle and swine production (Guardabassi and Kruse, 2004).
Growth promotion: antibiotics are used nowadays for improved performance in growth
especially in poultry and as fatteners in other species (Nisha, 2008). The feeding of
antibiotics is associated with decreases in animal gut mass, increased intestinal absorption of
nutrients due to thinning of the gut mucous membrane and energy sparing. This results in a
reduction in the nutrient cost for maintenance, so that a larger portion of consumed nutrients
can be used for growth and production, thereby improving the efficiency of nutrient use.
Antibiotics act by eliminating the subclinical population of pathogenic microorganisms
(Chambers and Deck, 2009). Eradicating this metabolic drain allows more efficient use of
nutrients for food production. Antibiotics alter the non-pathogenic intestinal flora, producing
beneficial effects on digestive processes and more efficient utilization of nutrients in feeds
(Serrano, 2005). It has been estimated that around 6 percent of the energy in a pig’s diet
could be lost due to microbial fermentation occurring in the stomach and small intestine.
12
Intestinal bacteria inactivate pancreatic enzymes and metabolize dietary protein with the
production of ammonia and biogenic amines. Antibiotics inhibit these activities and increase
the digestibility of dietary protein (Nisha, 2004; Serrano, 2005). Experimental results
obtained with some antibiotics commonly used as growth promoters (chlortetracycline,
penicillin and sulfamethazine) have shown that treated pigs have higher serum levels of an
insulin-like growth factor. In this way, the effect may extend beyond digestion in the intestine
and stimulate metabolic processes (Committee on Drug Use in Food Animals, 1999; Doyle,
2001).
The most common antimicrobial drugs used presently and in the past as growth promoters
include macrolids (tylosin and spiramycin), polypeptides (bacitracin), glycolipids
(bambermycin), streptogramins (virginiamycin), glycopeptides (avorparcin), quinoxalines
(carbadox and olaquindox) and ionophores (monensin and salinomycin) (Buttery et al.,
1986). Since growth promoters also contribute to prevention of certain diseases, and can be
administered for this purpose, there is no clear distinction between growth promotion and
prophylactic use of antimicrobials. Some countries allow antimicrobials used therapeutically
to be also used as growth promoters in subtherapeutic doses. Penicillin, tylosin tetracycline
and erythromycin are allowed for both therapeutic and growth promotion in the USA
(Guardabassi and Kruse, 2008). In Europe, antimicrobials for therapeutics were not allowed
for growth promotion following legistlation for use of growth promoters as per recommended
by the Swann report, (1969).
The swan report is the report of the joint committee on the use of antimicrobials in animal
husbandry and veterinary medicine to consider the implications for animal husbandry and
for human and animal health and to make recommendations for the use of antimicrobials.
The concern about possible influence of antimicrobial use in animals upon human health led
to the appointment of the committee. Swan report recommended that antimicrobial agents be
13
excluded from animal feed if they were used as therapeutic agents in human and animal
medicine or if they were associated with the development of cross resistance to drugs that
were used in human. The Swann committee recommendations were implemented by The
British Government in 1971.
Following the Swan report, antimicrobials were officially classified into two groups: agents
approved for use as growth promoters in animal feeds (bacitracin, virginiamycins and
barnbermycins) and agents for therapeutic purposes whose use was restricted to specific
prescription by a medical or veterinary practitioner. Hence therapeutic antimicrobials were
removed from sub-therapeutic use (Guardabassi & Kruse, 2008).
2.3 Classification of Antimicrobial Agents for Veterinary Use
Antimicrobials used in animals are generally as, or closely related to, antimicrobials used in
humans. Tetracyclines constitute the antimicrobial class quantitatively most used in animals,
followed by macrolids, lincosamides, penicillins, aminoglycosides, flouroquinolones,
cephalosporins and phenicols (Schwarz and Chaslus-Dancla, 2001). The types of
antimicrobial agents used in humans and animals vary between countries. Chlortetracycline, a
member of the tetracycline group is used by 87% of the farms sampled in Trinidad and
Tobago as a feed additive; the medicated feed is given as prophylaxis or for therapeutic
purposes, the dosage depending on what purpose it is used for (Adesuyin et al., 2004).
Tetracycline (chlortetracycline) was the most common residue detected in chicken in a work
done in Barbados in other words, the most commonly used drug (Hall et al., 2004). In
Nigeria, Kabir et al. (2003) and Ezenduka et al. (2011) also indicated oxytetracycline as the
most widely used antimicrobial in poultry. In Denmark, penicillins account for 70% of all
dosages given to humans whereas the most commonly used agents in swine production are
macrolids (70%) and tetracyclines (21%) (Anonymous, 2005). In Norway in 2004, of the
total antimicrobial usage in terrestrial animals 24% were pure penicillins preparations and
14
only 3% tetracycline was used (NORMV-VET, 2005 cited by Guardabassi and Kruse, 2008).
In many countries, drugs licenced for human use are administered to animals and veterinary
products are used in animal species that are not indicated as appropriate on the label (extralabel use).
Mechanisms of antimicrobials fall into four categories: inhibition of cell wall synthesis,
damage to cell membrane function, inhibition of nucleic acid synthesis or function, and
inhibition of protein synthesis (Bywater, 1993; Chambers and Deck, 2009). The aim of
antimicrobial therapy is to rapidly produce and then to maintain an effective concentration of
drug at the site of infection for sufficient time to allow host specific and nonspecific defences
to eradicate the pathogen (Prescott, 2000a; Prescott and Walker, 2000).
The classification of antimicrobials below is based on that of the United States’
Pharmacopoeia (USP, 1999, 2000a–m).
2.3.1: Aminoglycosides
This class inhibits bacterial protein synthesis by binding to protein receptors on the
organism’s 30S ribosomal subunit. This process interrupts several steps including initial
formation of the protein synthesis complex, accurate reading of the mRNA code and
formation of the ribosomal- mRNA complex. Anaerobic bacteria cannot take up these agents
intracellularly, so they are usually not inhibited by aminoglycosides (Appendix I).
2.3.2 Beta-lactam antimicrobial agents
These agents are those that contain the four-membered, nitrogen-containing, beta-lactam ring
at the core of their structure. This drug class comprises the largest group of antimicrobial
agents and dozens of derivatives are available for clinical use. Their bactericidal activity and
lack of toxicity to humans gained them popularity among antimicrobials; also their molecular
structure can be manipulated to achieve greater activity for wider therapeutic applications
(Forbes et al., 2007) (Appendix II)
15
2.3.3 Chloramphenicol
Chloramphenicol inhibits the addition of new amino acids to the peptide chain by binding to
50S ribosomal subunit. It’s highly effective against a wide variety of gram-positive and gramnegative bacteria. However, because of the serious toxicity associated with it, FDA
regulations ban chloramphenicol from use in animals intended for food production. The
Canadian Health Protection Branch, the European Union, Japan and Nigeria apply the same
measure (Appendix III).
2.3.4 Quinolones and Fluoroquinolones
These are derivatives of nalidixic acid, an older antimicrobial agent. They bind to and
interfere with DNA gyrase enzymes involved in the regulation of bacterial DNA
supercoiling, a process that is essential for DNA replication and transcription. Quinolones are
bactericidal agents with a broad spectrum activity against both gram-positive and gramnegative bacteria. (Appendix IV)
2.3.5 Lincosamides (Appendix V)
2.3.6 Macrolides
This class of antimicrobials inhibit protein synthesis by binding to receptors on the bacterial
50S ribosomal subunit and subsequent disruption of the growing peptide chain. The
macrolids are generally not effective against most genera of gram-negative bacteria, primarily
due to uptake difficulties associated with gram-negative outer membranes (Appendix VI).
2.3.7: Nitrofurantoin
The mechanism of action of nitrofurantoin is not completely known. This agent may have
several targets involved in bacterial protein and enzyme synthesis and the drug also may
directly damage DNA (Appendix VII).
16
2.3.8 Tetracyclines
Tetracyclines inhibit protein synthesis by binding to the 30S ribosomal subunit so that
incoming tRNA-amino acid complexes cannot bind to the ribosome, thereby putting peptide
chain elongation to a halt. They are broad spectrum in action (both gram-positive and gramnegative) and several intracellular bacteria pathogens such as chlamydia, rickettsia, and
rickettsia-like organisms (Appendix VIII).
2.3.9 Sulphonamides
The folic acid pathway is used by bacteria to produce precursors important for DNA
synthesis. Sulphonamides target and bind to one of the enzymes dihydropteroate synthase and
disrupt the folic acid pathway (Appendix IX).
2.4 Antimicrobials Banned For Use in Food Animal Production
Some antimicrobial agents were banned for use in animals intended for food by WHO, FAO,
NAFDAC and other agencies. List of the antimicrobials banned for use in different countries
are stated in Appendix X.
The public health risk associated with the use of growth promoters in animal production
attracted the attention of international scientists and the documentations made showed that,
some countries including the EU have banned or are in the process of phasing out the use of
growth promoters. This policy is in accordance with the recommendation proposed by WHO
in 2000 (WHO, 2000) and endorsed by FAO and OIE in 2003 (WOAH, 2004). The
investigations carried out in Denmark on the effect on total antimicrobial consumption that
resulted from the ban of growth promoters in 1995 showed that the total consumption of
antimicrobial was reduced by approximately half in the period between 1994(206 tons) to
2004 (101 tons) (Anonymous, 2004 as cited by Guardabassi and Kruse, 2008).
17
2.5 Drug Residues and Related Terms
2.5.1 Residues
Residues on a broader note may be defined as undesirable substances present in meat. These
substances could be chemical or biological in nature and present in small amounts in foods
and foods of animal origin. These chemicals or biological agents can be introduced into food
as a result of incorrect storage of food stuffs, by various technological practices or as a result
of medications given to livestock or of processing methods (Biswas et al., 2009). The
residues that can be found in meat and food animal products include: pesticide, drug and
mycotoxin residues.
2.5.2 Drug residues
Veterinary drugs are generally used in farm animals for therapeutic (treatment of animal
diseases), prophylactic (preventive) purposes or as feed additives for growth promotion and
feed efficiency. They include a large number of compounds which can be administered in the
feed or drinking water or administered individually depending on the purpose of medication.
The use of these substances in food animals may accumulate to form residues (Droumev,
1983; Reig and Toldra, 2007). These residues may be deposited or stored within the cells,
tissues and organs of an animal, body fluids and products such as egg (Booth, 1988). Residue
may also occur when drugs are intentionally or unintentionally added to food products. All
residues, including parent drug metabolites or decomposition products or any composition of
these three are of potential toxicologic importance (Jackson, 1980). Residual quantities of a
drug and its derivatives are expressed in parts by weight or volume such as;
Milligrammes per kilogramme (mg/kg) or milligramme per liter (mg/l) i.e. parts per
million (ppm).
18
Microgramme per kilogramme (µg/kg) or microgramme per liter (µg/l) i.e. parts per
billion (ppb).
Nanogramme per kilogramme (ηg/kg) or nanogramme per liter (ηg/l) i.e. parts per
trillion (ppt).
Constantly improving analytical methods make it possible to detect minute quantities of drug
residues in animal tissues ranging from a fraction of ppm to a few ppb or ppt (Dey et al.,
2003)
2.6
Drug Approval and Safety Evaluation of Antimicrobial Residues
The use of antibiotics in developed countries is strictly regulated by national and international
bodies on food regulations. In the US, the Food and Drug Administration (FDA) is the
regulatory agency responsible for approval of antibiotic use in animals. Along with the sister
agency, USDA, both are involved in active surveillance and compliance programme to ensure
prudent use of antimicrobials and the safety of the food supply once the antimicrobial is
approved for use (Donoghue, 2003). In the EU this work is performed by the Committee for
Veterinary Medicinal Products (CVMP) of the European Agency for the Evaluation of
Medicinal Products (EMEA) (Myllyniemi, 2004). The FDA is saddled with the responsibility
of ensuring safety and efficacy of drugs prior to approval. To assess the safety of ingested
antimicrobial residues national and international committees evaluate data based on extensive
chemical, pharmacology, toxicology studies and other, e.g. antimicrobial, properties of the
drugs derived from studies of experimental animals and observations in humans (Woodward,
1998; Silley, 2007). The type of toxicological studies required to approve the use of an
antimicrobial in an animal species will depend on the existing knowledge on the drug. For a
new untested drug, extensive safety testing will be required, however for an already existing
drug which has a history of safe use in a food animal will require less extensive toxicology
19
testing for use in another species. Thus the drug approval process may vary for different
antimicrobials.
Acute (immediate reaction like allergy) and chronic (cancer) toxicology testing has to be
performed on animals for a particular drug. These tests will determine no observable effect
level (NOEL): a dose that does not create any health problem. This level is the basis for
calculating an acceptable daily intake (ADI). After an ADI has been determined, maximum
residue limits (MRLs) are determined for various food commodities so that overall residue
intake remains below the set ADI in a standard food basket. Finally, to ensure that drug
residues have declined to a safe concentration in various tissues, a specified period of drug
withdrawal is set for a veterinary medicinal product.
In the European Union (EU) maximum residue limits (MRLs) must be established for all
pharmacologically active substances for the concerned animal species and relevant tissues or
products. These MRLs are the basis for the determination of limits of detection (LODs) of
various analytical methods used in residue surveillance.
2.7 No Effect or Maximum no Adverse Effect
Before a tolerance level or concentration for a drug or chemical residue can be established, it
is necessary to obtain the no-effect [sometimes referred to as no adverse effect (NOAEL) or
no observed effect level (NOEL)] concentration of compound in the most sensitive
mammalian species. This is necessary so that a comfortable margin of safety can be
established between the level of exposure that does not affect experimental animals (i.e. the
established no effect concentration) and the quantity of exposure allowable for the human
(Booth, 1988).
To the toxicologist, no effect and maximum no adverse effect generally refer to a
concentration of a drug or chemical that produces no harmful effect. The terms are also used
to denote no change or effect upon physiological activity, no change in organ weight or body
20
weight as well as in rate of growth, and no change or effect upon cellular structure or
enzymatic activity of cells. In most cases, a drug must show no effect when fed for two years
to the most sensitive species before it can be permitted in the human diet (Booth, 1988).
2.8 Acceptable Daily Intake (ADI)
The Acceptable Daily Intake (ADI) is the daily concentration of a drug or chemical residue
that during the entire lifetime of a person appears to be without appreciable risk; the practical
certainty that a life time exposure to the residue will not result in a deleterious or injurious
effect, to health on the basis of all the facts known at the time (Myllyniemi et al., 2001). The
ADI approach was originally developed to take account of effects based on classical
toxicology, and it was applied to the results of standard toxicity studies. These studies were
used to derive a NOEL. The ADI was calculated by dividing this by a suitable safety factor,
usually 100, which assumes that humans are 10 times more sensitive than animals and that
within the human population there is a 10-fold range of sensitivity (IPSC, 1987; Woodward,
1998). For a drug or chemical residue, the ADI is established to provide a guide for the
maximum quantity that can be taken daily in food without appreciable risk to the consumer
(EC, 2001). ADI values are always subject to revision whenever new information becomes
available and are expressed in mg of drug or chemical per Kilogram of food (mg/kg) (Booths,
1988).
Since the toxicological studies alone are said to be inadequate in evaluating adverse effects of
antimicrobials, it is therefore necessary to determine the microbiological end points of the
agents (Perrin-Guyomard et al., 2001). The microbiological properties of residues are
included in their safety evaluation at national and international level (WHO, 1991; US FDA,
1996; CVMP, 2001a and 2001b).
The EU requires classical toxicology tests which include single dose toxicity, repeated dose
toxicity, tolerance in the target species, reproductive toxicity, mutagenicity and
21
carcinogenicity. Studies on other effects include immunotoxicity, microbiological properties
of residues, observations in humans, and neurotoxicity (EC, 2001). Various in vivo and in
vitro methods have been developed for testing the effects of the drug residues on human
intestinal microbiota in the determination of a microbiological ADI. In vitro studies, such as
those to determine the MIC, are simple and inexpensive procedures, but are not always
representative of the relevant bacteria, and may not take into account factors such as pH,
anaerobiosis and the barrier effect (Boisseau, 1993; Cerniglia and Kotarski, 1999), thus
failure to accept in vitro MIC data for establishing a microbiological no-effect level by the
US FDA.
2.9 Tolerance level or Maximum Residue Limit (MRL)
Tolerance is the ability of an organism to show less response to a specific dose of a chemical
or drug than was demonstrated on a previous exposure (Merck, 1998). Tolerance level is
therefore the maximum allowable level or concentration of a drug or chemical in or on feed
or food at a specified time of slaughter and harvesting, processing, storage and marketing up
to the time of consumption by animal or human. Tolerance levels are expressed in parts by
weight of the drug or chemical residue per million (mg/kg or mg/l) or billion parts by weight
(µg/kg or µg/l) of the food and are never greater than the permissible level for the feed or
food in question (Booth, 1988). Tolerances are established based on extensive toxicological
studies of potential hazards of consumption to humans (Donoghue 2003; Myllyniemi, 2004;
Nisha, 2008). In determining the Tolerance level/MRLs, a safety factor of 100 is usually used
for antibiotics already in use with a known safety record. Otherwise, a safety factor of 1,000
is used.
•
Tolerance = NOEL × safety factor (100 or 1000 times less than NOEL dose) × ADI
× 60kg adult weight (Donoghue, 2003)
22
In calculating a MRL, the ADI, the residue depletion patterns of a compound in the edible
tissues of a particular food-producing animal and the theoretical food intakes are taken into
account. Possible persistence of residues in organs or at the injection site is also considered
(Fitzpatrick et al., 1995; EC, 2001). Most countries have established tolerance or safe levels
(T/SL) of drugs below which it is considered that the drug may be safely used without
harming the consumer. The Codex Alimentarius Commission is the accepted international
agency responsible for food safety issues and has established Tolerance/Safety Level and
MRL listings for many antibiotics (MacNeil, 1998) as shown in appendix XI. A country may
set its own levels or may accept those set by Codex Alimentarius. In the EU, maximum
residues limits based on microbiological effects have been set for e.g. cephalexin, CTC,
doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, kanamycin, lincomycin,
marbofloxacin, nafcillin, novobiocin, OTC, pirlimycin, sarafloxacin, spiramycin and
tetracycline (EMEA, 2004). An ADI not based on microbiological effects has been set only
for a few antimicrobials, e.g. for danofloxacin, difloxacin, dihydrostreptomycin (DHS),
neomycin, streptomycin and tiamulin. MRLs for penicillins are based on immunological
effects and on the sensitivity of bacteria used in the dairy industry. No maximum residue
limit can be established for some substances (e.g.chloramphenicol) because residues of these
substances, at whatever limit, in foodstuffs of animal origin constitute a hazard to the health
of the consumer (WHO, 1991). So, their use is prohibited for production animals(GESAMP,
1997: SANCO 2001a).
The codex Alimentarius Commission is a body under the auspices of FAO and WHO that
develops food standards, guidelines and related texts such as codes of practice under the
joint FAO/WHO food standards programme. The main purposes of Codex are to protect the
health of consumers and to ensure fair trade practices in the international food trade
(Guardabassi and kruse, 2005).
23
In defining allowable concentrations of drug residues, three major types of tolerance are
considered:
i.
Finite tolerance: A finite tolerance is defined as a measurable amount of drug or chemical
(non-carcinogen) residue that is permitted in food. In establishment of finite tolerance, the
acceptable daily intake (ADI) for the human is generally determined by applying a safety
factor of 1:100. Toxicity tests that are required to establish a finite tolerance include:
a.
Lifetime studies (i.e., two year chronic toxicity) in the rat and mouse.
b.
A 6 month or longer study in a non-rodent mammalian species (usually the
dog)
c.
A 3 generation reproductive study with teratogenic phase; and other special
toxicity study that may be indicated, depending upon the particular drug or
chemical to be tested (Booth, 1988).
ii.
Negligible tolerance: This is toxicologically insignificant amount of residue resulting in a
daily intake that is a small fraction of the maximum ADI (Jackson, 1980; Booth, 1988).
Negligible tolerances are generally derived from the lowest limit detecting a residue with
the most sensitive analytical procedure that can be used. Duration of this study is
generally based on 10% of the test animal’s lifetime: for a rat with a 30 month lifespan,
this would be 3 months or 90 days (Maugh, 1982; Booth, 1988). The maximum
concentration of total residue considered to be negligible is 0.1ppm in meat and 10ppb in
milk or egg, the lower value becomes the established negligible tolerance for that product.
iii.
Zero tolerance: This is determined on the basis that no residue is permitted in food or feed
because of extreme toxicity or most often because the compound is carcinogenic (Mercer,
1975). For example, diethylstilbestrol (DES) before now (in the 70’s) was used as a
growth promoter in food producing animals because the method used in detection of
carcinogen then was the Mouse Uterine Bioassay procedure developed in 1958. This
24
method was incapable of detecting DES below 2ppb; lower concentrations could not be
detected and were referred to as zero. But with the evolution of more sensitive analytical
method, DES has been banned since Nov 1, 1979 (Booth, 1988).
2.10
Withdrawal Time
Various antibiotics take different time periods to be excreted from the body. This time period
is known as withdrawal period for the particular antibiotic and has to be observed before
slaughtering animals, before taking eggs from birds and before milking lactating animals
(Jackson, 1980). The withdrawal time is synonymous with depletion or clearance period, preslaughter withdrawal period or holding time. It is the time required for the residue of
toxicologic concern to deplete or reduce to safe concentration as defined by the tolerance or
the time which passes between the last dose given to the animal and the time when the
concentration of residues in the tissues: muscle, liver, kidney, skin/fat or products milk, eggs,
honey is lower than or equal to the MRL (Cholas, 1976; Jackson, 1980; Nouws, 1981). It is
also referred to as the interval from the time an animal is removed from medication until the
permitted time of slaughter. This interval is required to minimize or prevent violative levels
of drug residues in edible tissues for human consumption rather than safeguarding the health
of the animal (Booth, 1988). Withdrawal time intervals vary with each drug preparation and
among the different animal species. Depending on the drug product, dosage form, and route
of administration, the withdrawal time may vary from a few hours to several days or weeks.
2.10.1 Withdrawal Time for Veterinary Drugs
Withdrawal of medication is necessary so that violative or illegal levels of drug residue above
tolerance level are avoided in meat, milk, and eggs marketed for human consumption thereby
safeguarding humans from unnecessary exposure to antimicrobials (Nisha, 2008). Whenever
drug preparations are administered to food producing animals, the veterinarian may alert
owners to the necessity of withholding animals from market or slaughter during or following
25
the treatment period. From a public health welfare standpoint, veterinarians have the
awesome responsibility of ensuring the proper use of drug they prescribe or use in food
animals (Booth, 1988).
Withdrawal time vary with each drug preparation and among the different animal species for
the same drug, ranging from a few hour to several days or weeks or even months.
2.11. Antimicrobial use in Poultry
Antimicrobial agents used in poultry are in the form of growth promoters, coccidiostats and
antimicrobial for therapeutic and prophylactic purposes (Lohren et al., 2007).
2.11.1 Antimicrobials as growth promoters
Antimicrobials were first used for growth promotion purposes as early as the late 1940s when
it was discovered that chickens grew faster when fed tetracycline fermentation by-products
(Gustafson and Bowen, 1997). Subsequently, other antimicrobials were approved for growth
promotion and performance enhancement over the years. Initially, before the publication of
the Swann Report in 1969, antimicrobials like tetracycline, bacitracin and tylosin were used
in poultry at low concentrations as feed additives for growth promotion and higher doses
restricted for veterinary use. Based on the recommendations of the Swann Report that
therapeutic antimicrobials should not be used as growth promoters, most countries in Western
Europe and North America adapted their legislation over time and certain antimicrobials were
banned for use as growth promoters but can only be used as feed additives or as therapeutic
agents under the prescription of a veterinarian (Casewell et al., 2003). In 2006, all remaining
growth promoters were banned from use in animal feeds in the EU, but the USA and other
Third world countries have not introduced similar restrictions (Lohren et al., 2007).
26
2.11.2 Antimicrobials as coccidiostats in poultry production
Intensive production of commercial poultry over the past 60 years has been largely due to the
introduction of coccidiostats in the feed. These products interfere with the various stage(s) of
intestinal development of coccidia (Mortier et al., 2005). In the early days, sulphonamides
were primarily used as coccidiostats, and they are still registered for veterinary prescription
for treatment of coccidiosis. In 1980s, a new group of coccidiostats was added: the polyether
ionophores, since a vast majority of coccidiostats were regulated by feed legislation as feed
additives. Ionophores are used almost exclusively as coccidiostats because they have limited
antibacterial activity (AVMA, 2005). Their significance has increased in EU since
antimicrobial growth promoters were banned and are not perceived as antimicrobials by most
public health authorities as they are not used in human medicine.
2.11.3 Therapeutic antimicrobial use in poultry
The use of antimicrobials for therapeutic purposes in most developed countries is regulated
by specific veterinary or pharmaceutical legislation where their use is restricted to veterinary
prescription. Misuse and overuse of antimicrobials occurs more easily in countries where the
farmer has easy access to antimicrobials not requiring veterinary prescription (Dipeolu &
Dada, 2004; Kabir et al., 2003, Ezenduka et al., 2011). In these cases, antimicrobials tend to
be used on a trial and error basis to find which will give a favourable response.
2.12 Causes of violative level of antimicrobial residues in animal tissues
The occurrence of violative level of residue in food animal tissues is multifactorial.
According to Taylor (1965), most of the violative residues found in food animals are due to
misuse of drugs or chemical preparations. These include:
27
•
Non-observance of withdrawal periods: The most likely cause of violative drug
residues is the failure to observe withdrawal times (Paige and Kent, 1987; Van
Dresser and Wilke, 1989; Guest and Paige, 1991; Paige, 1994).
•
Improper use of a licensed product or through the illegal use of an unlicensed
substance (Higgins et al., 1999)
•
Using drugs to mask clinical signs at the time of slaughter to avoid condemnation of
animals at ante-mortem inspection.
•
Unintentional residues may also occur in calves fed milk and/or colostrum from cows
receiving antimicrobials (Guest and Paige, 1991). Faecal recycling, where the drug
excreted in faeces of treated animals contaminates the feed of untreated animals, can
be the cause of residues of certain antimicrobial groups (Bevill, 1984; McCaughey et
al., 1990). Housing of unmediated pigs in boxes where pigs had previously been
treated orally with sulfamethazine resulted in residues in urine, kidney and diaphragm
(Mc Caughey et al., 1990; Elliott et al., 1994; Kietzmann et al., 1995).
•
Contamination of animal feeding stuffs with a variety of compounds also occurs. The
significance of this contamination depends on the pharmacodynamics of the
compound and the species affected (McEvoy, 1994). Indeed, contamination of
feeding stuffs seems to be an important source of unintended application of
antimicrobials. In a survey carried out in Northern Ireland antimicrobials were
detected in 44% of feeds declared by the manufacturers to be free of medication
(Lynas et al., 1998). Residual quantities of medicated meal may be retained at various
points along the production line, contaminating subsequent batches of meal as they
are processed (Kennedy et al., 2000). Data from a sulfamethazine residue program
suggested that 25% of violations were due to inadequate cleaning of feed mixers
(Guest and Paige, 1991).
28
•
Feeding unapproved or unauthorized medicated feed to animals.
•
Extra-label dosages, wrong route of administration of drugs and use of drugs which
have not been approved for the species in question may lead to violative residues
(Papich et al., 1993; Kaneene and Miller, 1997; Higgins et al., 1999).
2.13
Public health hazards and harmful effects of antimicrobial residues
According to Crosby (1991), if man consumes animal products containing traces of
antibiotics, harmful effect might arise in exactly the same way as if the equivalent dose has
been administered directly. It has been established that antimicrobial residues may result in
side effects, such as direct toxicity, allergic reaction, development of antimicrobial resistance
amongst bacterial pathogens (Dupont and Steel, 1987). The public health concern of these
side effects according to Booth (1988) is the potential carcinogenicity, mutagenicity and
teratogenic effects of these drug residues on human.
2.13.1 Drug Allergy and Hypersensitive Reaction
Drug hypersensitivity is defined as an immune-mediated response to a drug agent in a
sensitized patient, and drug allergy is restricted to a reaction mediated by IgE (Riedl and
Casillas, 2003). For drugs to elicit immunogenic reaction, they must act as haptens, which
must combine with carrier proteins to be immunogenic and elicit antibody formation due to
their small molecular weight (Dewdney et al., 1991).
Allergic reactions to drugs and chemicals may range from life threatening anaphylactic
reaction, serum sickness to milder cutaneous reactions (rashes and itches) and delayed
hypersensitivity (Booth, 1988). Drug-induced allergic reactions may occur acutely (within 60
min of challenge), subacutely (1-24 h), or as latent responses (1 day to several weeks). The
acute and some subacute disorders are often due to Type I (IgE)-mediated reactions and,
more rarely, due to IgG antibodies (Type II). Immune complex disorders (Type III) are much
rarer in this context. Type IV (cell mediated) responses develop more slowly. The principal
29
types of disorder are: Type I: anaphylactic shock, asthma and angioneurotic oedema; type II:
haemolytic anaemia and agranulocytosis; type III: serum sickness and allergic vasculitis, and
type IV: allergic dermatitis (Dayan, 1993; Riedl and Casillas, 2003).
Exposure of human beings to antibiotic residue in animal products may produce allergic or
anaphylactic reactions in susceptible and sensitized individuals (with sulphonamides and
penicillin being of particular importance) (Hubber, 1986; Stark 2000 & Hall et al., 2004). In
relation to primary sensitisation, it is unlikely that residues could contribute to the overall
immune response in view of the very low concentrations that are likely to be encountered.
The duration of exposure is also short (Dewdney et al., 1991; Sundlof et al., 2000).
Notwithstanding their non-toxic nature, ß-lactams appear to be responsible for most of the
reported human allergic reactions to antimicrobials (WHO, 1991; Sundlof, 1994; Fein et al.,
1995). About 10% of human population is considered to be sensitive to penicillin (Irvin &
Roger, 1994). Aminoglycosides, sulphonamides and tetracyclines may also cause allergic
reactions (Paige et al., 1997). Certain macrolides may, in exceptional cases, be responsible
for liver injuries, caused by a specific allergic response to macrolide metabolite-modified
hepatic cells (Dewdney et al., 1991). However, only a few cases of hypersensitivity have
been reported as a result of exposure to residues in meat. Anaphylactic reactions to penicillin
in pork and beef have been described (Tscheuschner, 1972; Kanny et al., 1994; RaisonPeyron et al., 2001). Severe anaphylactic shock was reported in a consumer who ate chicken
treated with penicillin (Teh & Rigg, 1992). Consumption of eggs containing high
concentration of sulphonamides has been shown to cause skin allergy to subjects sensitive to
sulphonamides (WHO, 1988). A case of anaphylaxis was reported by Bevill (1984) as
possibly caused by streptomycin residues. Among the antibiotics commonly applied as feed
additives or in chemotherapy , penicillin and streptomycin and to a lesser extent novobiocin
30
and oleandomycin appear from clinical use to be more inclined to produce hypersensivity or
allerginicity than others in contemporary use (WHO, 1971).
Failure to associate minor reactions, e.g. urticaria, with exposure to allergenic residues may
be one reason for the lack of reported cases, although it may also be due to a genuine dearth
of reactions (Woodward, 1998).
2.13.2 Effects on Human Gut Microbiota
The microbiota in the human gastrointestinal tract is a complex, yet relatively stable,
ecological community, containing more than 400 bacterial species (Carman et al., 1993). The
concentration of anaerobic microbiota is 1011-1012 CFU/g of faeces, and the concentration of
aerobic microbiota much lower, less than 0.1% of the normal microbiota (Vollard and
Clasener, 1994).
The gut flora acts by Barrier effect which can also be referred to as bacterial antagonism,
antagonistic effect, colonization resistance or competitive exclusion (Tancrède, 1989). Barrier
effect means natural defence by normal microbiota/flora (strict anaerobes of dominant
indigenous flora) against colonisation and translocation by exogenous potentially pathogenic
microbes or against the overgrowth of indigenous opportunists (Van der Waaij et al., 1971;
Barza et al., 1987; Corpet, 1993; Vollard and Clasener, 1994) and has an important role for
food digestion. Administration of antimicrobial agents may cause disturbances in the normal
function of the normal flora (Nord and Edlund, 1990). To what extent disturbances in the
ecological balance between host and microorganisms occur depends on the spectrum of the
antimicrobial agent, the dose, pharmacokinetic and pharmacodynamic properties, and in-vivo
inactivation of the agent (Sullivan et al., 2001). Four microbiological endpoints have been
identified that could be of public health concern:
•
Modification of the metabolic activity of microbiota,
•
Changes in bacterial populations,
31
•
Selection of resistant bacteria, and
•
Perturbation of the barrier effect (Boisseau, 1993; Gorbach, 1993; Sundlof et al.,
2000; Perrin- Guyomard et al., 2001).
Tetracyclines may, in relatively low doses, have some impact on the faecal anaerobic
microbiota of humans (WHO, 1991; Waltner-Toews and McEwen, 1994). A close
relationship between tetracycline, streptomycin, gentamicin and chloramphenicol residues
and the resistance of bacteria isolated from the samples was found, suggesting that the
presence of low levels of antimicrobials might exert a positive pressure towards the selection
and expression of resistance in bacteria colonizing animal tissues (Vázquez-Moreno et al.,
1990). Therefore, the consumption of trace levels of antimicrobial residues in foods from
animal origin may have consequences on the indigenous human intestinal microflora (Reig
and Toldra, 2008).
2.13.3 Development of Resistance to Antimicrobial Agents
Emerging antimicrobial resistance, due to use of antimicrobials, is a public health concern in
human and animal medicine worldwide. According to the Centre for Disease Control and
Prevention (CDC), resistant strains of three micro-organisms causing human illness:
Salmonella sp., Campylobacter sp. and Escherichia coli are linked to the use of antibiotics in
animals (Serrano, 2005). The public health implication of the use of antimicrobials in animals
could be direct or indirect. It is direct when humans are exposed to low doses of
antimicrobials and consequent development of resistant strains of microorganisms (Nisha,
2007). Ladefoged (1996) stated that consumption of meat containing residues of antibiotics
over a protracted period of time may lead to emergence of resistant gut flora and pathogens in
human beings such as E. coli and Salmonella sp. Indirect exposure could be from
consumption of foods contaminated with resistant microorganism originating from the use of
antimicrobials in animals. Humans also get indirectly exposed to resistant bacteria
32
disseminated by animals in the environment. Studies have shown that the feeding of
antimicrobials to chicken and pigs resulted in an increase in a population of resistant E. coli
(Corpet, 1987). Although in developed and high income countries, withdrawal periods and
residue controls are conducted in slaughterhouses to prevent harmful drug residuals in food
that humans consume (McEwen and Ferdorka-Cray, 2002), these waiting periods do not
apply to carcasses and other wastes disposed of in dumps exploited by scavengers. Therefore,
scavengers may consume drug residues in livestock carrion. Indirect effects on health may
include the acquisition of antibiotic-resistant bacteria (Langelier, 1993; Thomas, 1999;
McEwen and Ferdorka-Cray, 2003; Oaks et al., 2004; Blanco et al., 2007) and the alteration
of normal protective flora through the acquisition of transient flora that may include
pathogenic bacteria (Winsor et al., 1981; Blanco et al., 2006; Blanco et al., 2007).
Furthermore, antibiotic residues ingested by avian scavengers may select for antibiotic
resistance, the emergence, dissemination and persistence of which represents a major health
problem in human and veterinary medicine worldwide (McEwen and Ferdorka-Cray, 2002).
2.13.4 Effect on Bone Marrow/Bone Marrow Depression
Hazards of chloramphenicol observed in association with clinical use in humans include
dose-related, reversible suppression of the bone marrow, gray baby syndrome, which is a
circulatory collapse in children less than 30 days on high doses, and irreversible,
idiosyncratic, non-dose related aplastic anaemia (WHO, 1988; Waltner-Toews and McEwen,
1994). Aplastic anaemia can occur in susceptible individuals exposed to concentrations of
chloramphenicol that might remain as residues in edible tissues of chloramphenicol-treated
animals (Settepani, 1984). Because of the toxicity of chloramphenicol (aplastic anemia), its
use in food animal production particularly lactating cows and laying birds has been banned
for use in many countries upon the recommendation of WHO (Settepani, 1984; WHO, 1988,
Hall et al., 2004).
33
2.13.5 Carcinogenic Effect
Carcinogenic effect refers to an effect produced by a substance having cancer producing
activity (WHO, 1971). Since all chemical carcinogens pose a hazard, human exposure must
be reduced to a practical minimum (Booth, 1988). It is generally recognized that there is no
relationship between the toxicity and carcinogenicity of chemical compounds. The potential
hazard of carcinogenic residues is related to their interaction or covalent binding with various
intracellular components such as proteins, DNA, ribonucleic acid (Booth, 1988).
Use of animal drugs with carcinogenic potential is of concern because of possibility that
residues in meat, milk and egg would add to the body burden. There is no question that
authorities involved in animal and human health endeavors desire a food, water and air
supply as free as possible from chemical contaminants. As desirable as this may be, it is
impossible to achieve, just as absolute safety itself is impossible to guarantee (Wildavsky,
1979; Campbell, 1980). Some drugs and/or their metabolites possess carcinogenic potential
e.g. sulphamethazine residues containing meat preserved with sodium nitrate may develop a
triazine complex that has a considerable carcinogenic potential (Maqbool, 1988; Ladefoged,
1996). Residues of sulphamethazine, tetracycline and furazolidone have been implicated in
carcinogenicity in humans (Nisha, 2004).
2.13.6 Industrial Effect
The presence of antimicrobials in food may influence starter cultures in food industries
(Cunha, 2001; Kirbis, 2006; Javadi et al., 2009). In milk and yoghurt production for instance,
presence of antimicrobial in milk may inhibit bacterial fermentation process and cause
problems for producers and subsequent losses in the food industry and loss of consumer
confidence (Sierra et al., 2009; Karamibonari and Movassagh, 2011).
34
2.13.7 Other Harmful Effects
There have been increasing concerns that drugs as well as environmental chemicals may pose
potential hazards to the human population by production of gene mutations or chromosome
aberrations (WHO. 1971). Nitrofurans were banned for use in food animals in many countries
because of its mutagenic effect (WHO, 1997; Nisha, 2004; Ali, 1999; Cooper et al., 2005).
Aminoglycosides can produce damage in urinary, vestibular and auditory functions (Clark,
1977; Shaikh and Allen, 1985). Toxic and allergic reactions in humans and animals caused by
tetracyclines have been observed at therapeutic doses (Berends et al., 2001) though prolonged
ingestion of tetracycline present in the broiler meat has detrimental effects on teeth and bones
in growing children (Aamer et al., 2000). Tetracycline has also been found to cause Fanconi
Syndrome and excessive flatulence (George et al.,1963)
2.14 Prevention and Control of Antimicrobial Residue Occurrence in Foods of Animal
Origin
Various international organisations such as the WHO, OIE, FAO and the EU Commission
have emphasized the importance of prudent and rational use of antimicrobials use in animals
intended for food in order to reduce to the barest minimum the impact of animal
antimicrobial usage on public and animal health (Guardabassi and Kruse, 2008). Identified
basic principles for prudent and rational veterinary use of antimicrobials are as follows:
•
Disease prevention
•
Proper and accurate diagnosis and antimicrobial susceptibility testing
•
Appropriate use of antimicrobials and route of administration (Nisha, 2008)
•
Appropriate dosage regimen
•
Monitoring and surveillance of antimicrobial residues (Nisha, 2008)
35
2.14.1 Disease prevention
Antimicrobials are used in animal production for therapeutic (disease treatment) and
prophylactic (disease prevention) purposes. To reduce the use of these agents and minimize
dependence on them is to prevent disease occurrence. Successful disease control will depend
on good animal husbandry, management, nutrition, vaccination and strict biosecurity
measures, especially in poultry.
A good example of reduction in antimicrobial use sequel to adequate measures of disease
prevention is provided in Norway. The annual usage of antimicrobials in terrestrial animals in
this country gradually decreased by 40% from 1995 to 2001 and has remained at a constant
level since then. The reduction was brought about by the campaign of professional
organisations within animal husbandry focused on preventive veterinary medicine and
prudent use of antimicrobials. The disease prevention strategy in aquaculture, particularly
vaccination, yielded a massive increase in total production of farmed fish and decline in
antimicrobial use by 98% from 1987 to 2004 (NORM/NORM-VET, 2005).
2.15.2 Proper Diagnosis and Antimicrobial Susceptibility Testing
Antimicrobials should be administered for use based on prescription by a veterinarian. The
prescription should be on the basis of accurate laboratory diagnosis and antimicrobial
susceptibility testing. Antimicrobial susceptibility testing should be done according to
internationally recognised standards. Standardized methods currently available include those
of the Clinical Laboratory Standards Institute (CLSI) in the USA, the British Society for
Antimicrobial Chemotherapy (BSAC), the Swedish Reference Group for Antibiotics
(SRGA), the Deutsches Institut fur Normung (DIN) etc (Guardabassi and Kruise, 2008).
36
2.15.3 Appropriate Use of Antimicrobials and Route of Administration
Clinical efficacy requires not only that the pathogen is susceptible to the selected drug, but
also that the drug is able to penetrate and be active at the site of infection. Host related factors
like low immunity, pregnancy, age and allergies should be considered in order to avoid
undesirable effects on the health of the animal. It is also very important to consider species
and route of administration, local treatment should be preferred to systemic treatment when
the infection is localised and accessible by topical products. When systemic treatment is
necessary in animal production, intramuscular and intravenous injections are preferable to
oral administration to avoid disturbance of the normal gut flora.
In all circumstances, veterinary practitioners should only prescribe antimicrobial formulations
that are approved for the species and the indication concerned. Off-label use of antimicrobials
should be exceptional and always under the professional responsibility of the veterinarian.
2.15.4 Appropriate Dosage Regimen
Appropriate dosage regimen including dose level, dose interval and duration of treatment is
important to ensure rational antimicrobial use and guard against residue. Antimicrobials
should be administered according to the recommended dosage regimen to minimize therapy
failures and compliance to the regulated withdrawal times is very essential to avoid
occurrence of residue of these drugs. Non-compliance in antimicrobial treatment regimes in
animal production usually occurs when the farmers or animal handlers tend to administer
drugs themselves without the instructions of a veterinarian. Therefore, veterinarians have the
important role of informing/educating farmers and livestock production owners or managers
about the importance of complying with the prescribed dosage regimens.
37
2.15.5 Monitoring and surveillance of antimicrobial residue
In developed and high income countries, government and stake holders are involved in
routine monitoring of farms for observance of withdrawal periods and slaughter houses and
food industries for drug residues. In the USA, the National Residue Programme conducts two
types of residue testing programmes. Under the monitoring program, a statistically based
selection of random samples from normal animal population is collected. The surveillance
programme focuses on obtaining samples from animals suspected to contain violative drug
residues in their tissues (Sundlof et al., 2000; Dey et al., 2003). The USDA-FSIS and the
FDA are responsible for monitoring meat and poultry products for animal drug residues. The
USDA-FSIS conducts the National Residue program (NRP) to prevent animals containing
violative amounts of drug residues into the market through extensive on-site sampling
technique. The samples are also sent to FSIS laboratories for further testing. The FDA centre
for Veterinary Medicine (CVM) is responsible for approving new animal drugs, setting
tolerances and enforcement actions depends on result of FSIS findings (Bruns et al., 1991:
Biswas et al., 2009).
In Europe, Under Council Directive 96/23/EC every EU Member State must monitor a set
proportion of the total annual production of different food commodities of animal origin for
residues. The number of samples depends on the animals slaughtered or on the animal
products produced during the year. Veterinary drugs are monitored for MRL compliance. The
directive establishes the groups of substances to be controlled for each food commodity.
Commission Decision 97/747/EC provides further rules for certain animal products: milk,
eggs, honey, rabbit and game meat (Myllyniemi, 2004).
2.15 Antimicrobial Residues Detection and Identification.
Antimicrobial residues detection in foods of animal origin and the protocol for control is
usually based on a two steps procedure: first, screening for presence of different antimicrobial
38
groups and second, confirmating by identification of the specific antimicrobial in the sample.
A screening method should be able to detect residues beyond the MRL of a drug and must
also reduce, to the barest minimum, the number of false positives. The main requirements for
a screening method are as follows:
I.
II.
Easy to use and handle (Pikemaat et al., 2007; Javadi et al., 2009)
Low set-up and running costs (Javadi et al., 2009)
III.
High throughput (Okerman et al., 1998; Pikemaat et al., 2007)
IV.
Possibility of automation
V.
VI.
VII.
Reduced time to obtain the result (Fagbamaila et al., 2012)
Good sensitivity and specificity (Pikemaat et al., 2007)
Detection capability (CCβ) with an error Probability (β) < 5% (Mylliniemi, 2004)
2.15.1 Test Matrix
Pre- and post-slaughter matrices can both be used for the detection of antimicrobial residues
in animals though the majority of antimicrobial screening procedures use post-slaughter
matrices. Post-slaughter matrices used are kidney, liver and muscles. In chicken gizzard can
also be used. The pre-slaughter matrices such as serum, urine and eggs (detection of residues
of drugs in birds) have been used. Microbial inhibition tests are generally conducted directly
on the sample itself without any preliminary isolation steps (Boison and MacNeil, 1995). The
sample can be applied directly to the medium, on filter paper discs impregnated with liquid
sample (Mitchell et al., 1998; Javadi et al., 2011) or liquid samples put in wells dug in the
media (Nonga et al., 2009).
The pharmacokinetic data on the antimicrobials are needed in order to select a suitable test
matrix. Some matrices like egg and kidney contain natural inhibitors, which may be
proteinous in nature and so may give a false positive result in microbiological assay. To avert
this, pre-treatment such as protein denaturization is employed (Boison and MacNeil, 1995;
39
Isoherranen and Soback, 1999). If organ tissue, renal pelvis fluid, urine etc. is used as the test
matrix, the test result is expected to predict residue concentrations in muscle tissue. The
reason for the use of other test matrices than muscle tissue is that higher residue
concentrations are usually found in those tissues.
2.16
Methods of Detection of Antimicrobial Residues in Foods of Animal Origin
Three main methods are known for the detection of antimicrobial residues in food animals
and products: Microbiological, immuno-chemical and chromatographic methods.
2.16.1 Micrologbioical method
This is also known as bacteriological assay (bioassay) and it is based on the principle of
inhibition, where growths of microorganisms (test bacteria) are inhibited due to the presence
of inhibitors (antimicrobial residues) in the test sample. It is usually, the first-hand method for
the analysis of a sample to establish the presence or absence of residues (Aerts et al., 1995)
and the method used for monitoring the presence of veterinary drug residues in foods of
animal origin (Hussein 2004). This method is a low-cost and high-sample throughput (ability
to analyse a large number of samples simultaneously) method, optimised to prevent falsenegative results and to have an acceptable number of false-positive results. In order to
prevent false-negative results, it should be positive for all samples that contain residues at
MRL levels; preferably at 50% of the MRL (Heitzman, 1994; Korsrud et al., 1998).
Microbiological method is suitable for large scale screening because of its convenience and
ability to detect a wide spectrum of antibiotics (Aerts, et al., 1995; Haasnoot et al., 1999).
This method needs relatively short time for sample preparation as no purification procedures
are required, easy to use and can be efficiently adopted by laboratory staff and do not require
expensive equipment.
Most microbiological methods used in detecting antimicrobial residue are based on growth
inhibition through agar diffusion. Some methods are based on growth inhibition in liquid
40
media, but only a few tests based on alternative microbiological methods, have been
described (Myllyniemi, 2004).
A. Test organisms
The limit of detection of a microbiological test for a given antimicrobial depends primarily on
the sensitivity of the test bacterium. Sporulating bacteria from the Genus, Bacillus are
normally used specifically:
•
Bacillus subtilis: this organism is sensitive to a wide range of antimicrobials and the
spore samples are commercially available. It has been widely used for many studies (Huber et
al., 1969; Fabiansson and Rutegård, 1979; Thamdrup Rosdal et al., 1979; Johnston et al.,
1981; USDA, 1983; Nouws et al., 1988; Koenen-Dierick et al., 1995) and still very much in
use till date.
•
Bacillus megaterium: The test organism for the Calf Antibiotic and Sulfa Test
(CAST) as well as the Fast Antimicrobial Test (FAST)(USDA, 1984).
•
Bacillus cereus strains have been used to screen for tetracycline residues (McCracken
et al., 1976; Bugyei et al., 1994; Okerman et al., 2001 and 2004).
•
Bacillus stearothermophilus has also been widely used (Kundrat, 1968; Kabay, 1971;
Bielecka et al., 1981; Braham et al., 2001; Ayse et al., 2010). It is the test organism used in
the manufacture of commercial test kits like Premi® Test (DSM Food Specialities, Delft,
Netherlands), Charm Farm Test (CFT) (Charm Sciences Inc., Malden, MA, USA), and
Brilliant Black Reduction Test Kit (BR Test)(Enterotox Laboratories, Germany; Lloyd and
van der Merwe, 1987). The limitation of B. stearothermophilus is its sensitivity to the
inhibitory activity of lysozyme (van Schothorst and Peelen- Knol, 1970), which may give rise
to false positive results. This makes the bacterium less suitable for residue detection in kidney
tissue.
41
•
Non-sporulating bacteria are also used as test organisms. Micrococcus luteus (Huber
et al., 1969; van Schothorst and van Leusden, 1972; McCracken et al., 1976; Nouws, 1979;
Bogaerts and Wolf, 1980; Okerman et al., 2001) is especially sensitive to β-lactams and
macrolides (Okerman et al., 1998a). M. luteus 9341, 9341a and 15957 are currently classified
as Kocuria rhizophila. Escherichia coli strains (Ellerbroek, 1991; Choi et al., 1999; Okerman
et al., 2001) are used to detect fluoroquinolone residues. A bioluminescent bacterium
Photobacterium phosporeum has been used for the detection of chloramphenicol and oxolinic
acid (Tsai and Kondo, 2001).
The size of inoculum has a direct bearing on the zone of inhibition and needs to be
meticulously standardized (Cooper, 1972; Barry, 1976). A low to moderate inoculum
improves sensitivity (Renard et al., 1992); however, too low inoculum tends to produce
granular and poorly defined zone edges (Davis and Stout, 1971).
B. Some Tests Based on Microbiological Principle of inhibition
These tests have the same basic principle of inhibition. They can be prepared in plates (plate
tests) or in tubes (tube tests)
I.
Plate tests
A plate test consists of a layer of inoculated nutrient agar, with samples applied on top of the
layer, or in wells in the agar. Bacterial growth will turn the agar into an opaque layer, which
yields a clear growth-inhibited area around the sample if it contains antimicrobial substances.
In Europe this has been the main test format since screening of slaughter animals for the
presence of antibiotics started (Pikemaat, 2009). The following are some examples:
a. Swab Test on Premises (STOP)
This is a rapid method of analysis instituted by the United States Department of Agriculture,
Food Safety and Inspection Services (USDA-FSIS) in 1977 as a tool for monitoring and
detecting antibiotics in carcasses at places of slaughter (Dey et al., 2003). STOP test makes
42
use of kidney as the test matrix and Bacillus subtilis as the test organism. If the stop test is
positive, then presence of antimicrobial is suspected in the carcass; samples of the kidney,
muscle and liver are collected from the carcass and sent to FSIS lab for further confirmatory
analysis (White et al., 1998a). STOP, was originally used only on dairy cattle with particular
attention to culled cows that have mastitis, clinical signs of other diseases or visible injection
sites, but later it was used in all food animals and poultry, except ‘’bob’’ veal calves ( calves
less than 3 weeks of age or weighs less than 150 pound) (Anonymous, 1984).
b. Calf Antibiotic and Sulfa Test (CAST)
CAST was developed and introduced in slaughter establishments in 1985 for the detection of
antibiotic and sulfonamide residues in bob veal calf carcasses. An increase of violative levels
of sulfonamide residue in bob veal carcasses with a sharp rise in 1981and continued to
increase until 1984 when the FDA set a tolerance level of sulfamethazine and sulfatriazole in
tissue at 0.1 ppm, led to the development of CAST. CAST has the same procedure with
STOP except that the test organism is Bacillus megatarium, and the incubation temperature is
41°C (White et al.,, 1998b).
c. Fast Antimicrobial Test (FAST)
FAST was developed in 1994 because neither STOP nor CAST was sensitive enough to
detect sulfonamides at a violative level. The procedure is similar to that of CAST except that
FAST uses a different medium. FSIS initiated the use of FAST to replace CAST because
results for FAST can be visualized after 6 hours and the inhibition zones remain unchanged
till 18 hours of incubation. In addition to being faster FAST is more sensitive to sulfonamides
and can detect greater variety of antimicrobials than STOP (Dey et al., 2003).
d. The Four Plate Test (FPT)
FPT is a reference microbiological method for the screening of antibiotic residues and it still
used in European countries (Heitzman, 1994). It can detect the presence of a number of
43
antibiotic classes at the same time; these include beta-lactams, aminoglycosides, macrolides,
tetracycline and sulphonamides. Although the method is widely known as the “four-plate
method”, many variations are used and most laboratories apply a specific approach with a
different number and types of bacterial strains and therefore a different number of test plates
(Myllyiemi et al., 1999; 2001; Ferrini et al., 2006;). Methods using between one and eighteen
plates have been described by many studies in different literatures. There are also differences
in incubation periods, pH values of media and the quantity of media on which the bacteria are
cultured, and, most importantly, differences in detection levels (Hofz, 1994; Okerman et al.,
1998; Okerman et al., 2001; Okerman et al., 2004).
II.
Tube Tests
From a practical perspective, tube tests form an attractive alternative to multiplate methods.
Almost without exception these tests use B. stearothermophilus var. calidolactis as the
indicator organism. The only equipment needed is a device (e.g., garlic press) to obtain tissue
fluid and an incubator or water bath at the appropriate temperature. Assay results are
available within 4 h, and the use of spores instead of vegetative cells allows prolonged shelf
life, which makes commercial distribution feasible. Initially, commercially available B.
stearothermophilus tube tests were developed for the analysis of milk, but for several years
tests intended for other animal matrices have also become commercially available; e.g.,
Premi®Test (DSM), Delvotest® (DSM), Explorer (Zeu-Inmunotech), and Kidney Inhibition
Swab (KIS™) test (Charm Sciences). The only tests for which a substantial amount of
literature is available is Premi®Test and the Delvotest®
1. Premi® test
Premi® test is a new broad spectrum screening test recently developed by DSM, Netherlands
for the detection of antimicrobials in food animal products (Rebroeck, 2000). The test is a
microbiological test still based on the principle of inhibition of growth of microorganisms
44
(Bacillus stearothermophilus), a thermophillic bacterium sensitive to many antimicrobials
(Stead et al., 2004). Premi® test comes in ampoules, containing Bacillus stearothermophilus
var calidolactics spores imbedded in agar containing bromocresol purple colour indicator. It
combines the principle of agar diffusion test with colour change of the indicator. The test
changes colour from purple to yellow when there is active metabolism of the imbedded
organism. The test remains purple if there is inhibition of the growth of the imbedded
organism due to presence of an antibiotic at or above the level of detection (LOD). Premi®
test has been used in the detection of antimicrobial residues in meat (Rebroeck, 2000;
Popelka et al., 2005) and in eggs (Ezenduka et al., 2011). Premi® test indicative data
(Appendix XIII)
2. Delvotest®
Delvotest or Delvo – P test is also based on the principal of inhibition. The test organism and
the test procedure is the same as Premi® Test but with a shorter incubation period of 2hrs, 30
minutes. The test is basically for detection of antimicrobial residues in milk. The
manufacturers claim that the test is suited for all kinds of bulk milk (Hassig, 2003). Delvo®
SP, a newer test developed by the Delvo® P manufacturing company has block method using
the 96-well plate which supersedes the Delvo® P. the Delvo® SP is a broad spectrum
screening test for the detection of antibiotic residues and sulphonamides in milk and is
presently the standard test method required by the DAFRD for the detection of antibiotics and
sulphonamides in milk. It is the current test method carried out in Ireland by dairy companies
(Food safety, Ireland, 2002).
2.16.5 Immunochemical Methods
Immunochemical methods are based on the principle of immunologic reaction where
antibodies detect and bind specifically to different substances (antigens). The antigenantibody interactions are bound by weak molecular binding forces like Coulomb and Van der
45
Waals forces as well as hydrogen bonding and hydrophobic binding (Märtlbauer et al., 1994).
There are different techniques for antigen quantification but the usual technique used for drug
residue detection is the enzyme-linked immunosorbent assay (ELISA). The technique is
performed by bringing the antibodies into contact with the analyte and adding an amount of
radio-, enzyme-, or fluorescent-labeled analyte. The use of radio-labeled analyte is based on
measurement of the radioactivity of the immunological complex and is known as
Radioimmunoassay (RIA) (Samaraajewa et al., 1991). The use of fluorescent or
chemiluminescent compounds makes use of a flourimeter and luminescence detector for
enhanced detectability (Roda et al., 2003). The use of enzyme-labeled analyte to detect
immunological response could be double antibody/sandwich ELISA or competition with the
non-labeled analyte for the available binding sites (Blake and Gould, 1984; Boison and Mac
Neil, 1995), otherwise known as competitive ELISA. When an enzyme substrate is used,
enzyme activity proportional to the number of enzymes catalyzing the reaction is measured
(Boison and Mac Neil, 1995). There are different types of ELISA kits commercially available
for a large number of substances. These kits allow for the analysis of large number of
samples per kit, do not require sophisticated instrumentation, short time of analysis and are
quite specific and sensitive to detect specific residues or group of related compounds. ELISA
kits have been used in so many studies for the detection of tylosin and tetracycline (De
Wasch et al., 2001; Lee et al., 2001; Kumar et al., 2004; karamiburani, and Movassagh 2011)
sulphonamides (Wang et al., 2006; Mahgoup et al., 2006), chloramphenicol (Guadin et al.,
2003) residues in meat and other animal food products.
2.16.3 Chromatographic methods
These are chemical methods which allow both quantitative and qualitative detection of multiresidues of antimicrobials. They usually proceed with a preliminary extraction in order to
isolate the drugs of interest from the biological matrix. The extraction is basically done to
46
remove macromolecules and other matrix constituents that may interact and adversely affect
the chromatographic system or interfere with the detection, and enrichment of the analytes
(Aerts et al., 1995). Commonly used procedures for the detection of veterinary drug residues
include HPLC, gas chromatography (GC), thin layer chromatography (TLC) and mass
spectrometry (MS) (Aerts et al., 1995; McCracken et al., 2000). The low solubility of some
antimicrobials in organic solvents has made it difficult to develop procedures to extract and
concentrate their residues from biological matrices. Other antimicrobials are either
insufficiently volatile or are too thermally unstable (or both) to permit their analysis using GC
or GC-MS. Liquid chromatography (LC) has emerged as the method of choice for
determination of antimicrobials which are rather polar, non-volatile, and sometimes heat
sensitive (Shaikh and Moats, 1993; Kennedy et al., 1998). High Performance Liquid
Chromatography (HPLC) is a separation technique with high selectivity and sensitivity. With
HPLC, typical detections of multi-residues in meat samples are relatively simple and rapid
requiring a preliminary clean-up through solid-phase extraction. HPLC has been used in the
detection of sulphonamides (Pecorelli et al., 2004), tetracyclines (Samanidou et al., 2005), βlactams and Macrolids (Nagata et al., 2004) and quinolones (Kirbi et al., 2005) in animal
food products. The steps involved in HPLC analysis include: extraction of the drug with a
specific solvent, separation of the drug on the solid phase by HPLC, detection of the effluent
from the solid phase by spectrometry and quantitation of the amount of antimicrobial present
by peak height or peak area analysis. UV absorbance is the simplest and most widely used
detection method for LC analysis (Moats, 1997).
2.17. Effect of Heat on Residue
Rose et al. (1999) demonstrated that residues of a range of veterinary drugs have varying
degrees of stability during cooking and therefore, the cooking influences the level of risk
posed by such residues. Some researchers indicated that except for some tetracyclines, most
47
therapeutic antibiotics are relatively heat stable and resist both pasteurization and cooking
process (Aamer et al., 2000; Booth and McDonald, 1986). Heat stability of antimicrobial
residues in meat and meat products have been studied by several researchers with varying
levels of stability. Substantial net reduction of OTC concentration from 34-94% was
observed on investigation of the effect of cooking methods (cooking, frying, microwaving,
roasting, grilling and brazing) on meat incurred OTC residues by Rose et al. (1996). In their
study, cooking temperature had the largest impact on the loss. The effect of pasteurizing
procedure in milk spiked with OTC, TC and CTC by Locksuwan (2002) showed varying
significant (p ≤ 0.05) reduction of 97.36-86.77%, 22.97-54.75 for OTC and TC respectively.
No significant (p>0.5) reduction of CTC was found.
Lan et al. (2001) found that microwaving and roasting of tilapia meat had up to 90 and 85 %
of sulfamethazine reduction, respectively. Furusawa and Hanabusa (2002) carried out the
heat treatments on sulfadiazine, sulfamethoxazole, sulfamonomethoxine and sulfaquinoxaline
in chicken thigh muscle; the sulfonamides had reduction of 45-61% by boiling, 38-40 % by
roasting except for sulfadiazine and 35-41 % by microwaving. The study by Javadi et al.
(2001) also showed reduction in concentration of sulfadiazine + trimethoprim residue after
different cooking processes. Although heat treatments tend to reduce the concentration of
some antibiotics, full elimination of these drugs from food animals is not guaranteed
(Locksuwan, 2002; Javadi, 2011). Since most foods of animal origin are always cooked
before consumption, findings about the effect of heat on antimicrobial residues are needed to
accurately determine consumer exposure to these drugs.
48
CHAPTER THREE
GENERAL MATERIALS AND METHODS
3.1
Study Area
The study was carried out in Enugu metropolis of Enugu State. Enugu State is located in the
Southeast geopolitical zone of Nigeria between latitudes 5° 56΄North and 7°55´ North and
longitudes 6° 53΄ East and 7° 55´ East. It covers a total land area of about 802,295km2 and a
population of about 3.257.298 with a population density of 248 persons per square kilometer
(National Population Commission [NPC], 2006).
The State is bounded in the North-east by Benue state, in the North-west by Kogi State, in the
west by Anambra State, in the South by Imo and Abia States and East by Ebonyi State. The
State is made up of 3 senatorial districts (Enugu East, Enugu North and Enugu West) and 17
Local Government Areas. Ecologically, tropical forest and savannah predominate the area.
The residents are predominantly Igbo speaking.
The indigenes are involved in agriculture producing mainly oil palm, rice, groundnut, cassava
and livestock. Poultry farming is the livestock farming mostly practiced in this state; most
households have backyard poultry or just local birds on free range around the compound.
Consumption of poultry meat and eggs is part of the food habit/culture of the population.
Enugu, being the State capital has the largest human population in Enugu State. Most Federal
and State parastatals are located in the State capital and the staffs reside within Enugu
metropolis. The metropolis consisting Enugu North, Enugu South and Enugu East Local
Government Areas, also has a number of schools (primary, seconadary and tertiary) in which
both staff and students reside.
49
Figure 3.1: Geographical positions of the three major poultry markets in Enugu Metropolis
50
3.2 Study Design
The research involved cross sectional survey and experimental studies using both qualitative
and quantitative approach. The studies done are as follows:
3.2.1 Survey Studies
o Study i: Survey of antimicrobial residues in the meat and organs of market broilers
using the Three Plate Test (TPT).
o Study ii: Survey of antimicrobial residues in the meat and organs of market broilers
using Premi® Test.
o Study iii: Survey and quantification of tetracycline residues in the meat and organs of
market broilers using specific ELISA immunoassay.
3.2.2 Experimental Studies
o Study iv: Determination of comparative sensitivity of two microbiological methods
(TPT and Premi® Test) in the detection of residues in meat and organs of
oxytetracycline (OTC) treated birds.
o Study v: Determination of the effect of cooking methods on the concentration of OTC
residue in the treated birds using both microbiological (TPT and Premi® Test) and
immunoassay (ELISA) methods.
o Study vi: Determination of the effect of freezing on the concentration of OTC residue
in treated birds using microbiological (TPT and Premi® Test) and immunoassay
(ELISA) Methods
3.3. Sample Source, Population and Sampling Technique.
3.3.1. Survey Studies
Enugu Metropolis has three major poultry markets serving the densely populated metropolis,
namely: Artisan market, Ogbete main market and Gariki market with Artisan market having
the highest bird capacity (figure 3.1). One market was visited twice a week and on each visit,
51
a 1 in 4 systematic random sampling technique was used to select two out of 8 to 10 broiler
retailers usually present. 2 birds were selected using simple random sampling technique from
each selected retailer. In Artisan market, 4 birds were purchased at each visit, i.e 8 birds per
week for 5 weeks. In Main market and Gariki, 3 birds were purchased at each visit i.e 6 birds
per week for 5 weeks. A total of 100 birds were purchased; 40 from Artisan market which has
the highest retail bird capacity and 30 each from Main market and Gariki. Four organs were
tested from each bird making a total of 400 organ specimens (Figure 3.2).
52
Enugu metropolis
(poultry markets)
100
Artisan
Gariki
Ogbete
40
30
30
2v×5w×4b
2v×5w×3b
2v×5w××3b
160
120
120
40×4
30×4
30×4
4
]
400
v- visit; w- week; b- birds
Figure 3.2: Schematic presentation of sampling population and technique
53
3.3.2 Experimental study
Fifty 5-week old broilers were bought from a major poultry farmer who had not used
antimicrobial on the birds. The birds were fed ad-libitum with antimicrobial free feed and
water for three weeks. After the three weeks waiting period, 4 were slaughtered; the breast
muscles, liver, kidney and gizzard were collected. The organs were tested for the presence of
antimicrobial residues using TPT, Premi® test and ELISA. All the tested organs were
negative. The remaining birds were then randomly assigned into two equal groups, Groups A
and B for drug adminsteration (Figure 3.3).
54
50
4
Residue test
46
Group A(23)
Group B(23)
OTC (Inj.)
OTC (water)
2b/daily
2b/daily
Residue test
Residue test
Figure 3.3: Schematic presentation of experimental design
55
3.4 Specimen preparation
Five grams of each organ was macerated using sterile pestle and mortar, emulsified with 5mls
of distilled water (Nonga et al., 2009) and centrifuged at 5000rpm for 10 minutes. The
supernatant was decanted into dark coloured eppendorf tubes and stored for analysis.
3.5 Data analysis and presentation
Data generated from the study were presented in tables and charts and analysed using
descriptive statistics and in GraphPad Prism Statistical software version 5.02
(www.graphpad.com). Gaussian distribution of data sets was tested for, using D’agostino
Omnibus Normality test before choosing the most appropriate statistical tests. Significance
was accepted at p ≤ 0.05. Specifically,
•
Chi square was used to determine association between type of organ and the
occurrence of antimicrobial residues
•
Wilcoxon Signed Rank Test was used to analyse the difference in the median
concentrations of different organs and their MRLs
•
Mann Whitney test (t test for non parametric values) was used to determine the
difference in the mean concentration of the raw values and each of the different
cooking methods
•
Kruskal Wallis One way ANOVA was used to determine the difference in the means
of different freezing time and Dunn’s Multiple comparison used for post hoc analysis
•
Sensitivity, Specificity and Predictive value was used to determine the validity of the
screening tests.
56
CHAPTER FOUR
SURVEY OF ANTIMICROBIAL RESIDUES IN COMMERCIAL BROILERS USING
THREE PLATE TEST
4.1 Introduction
Veterinary drugs, specifically antimicrobials, are widely used in poultry production for
treatment of infections, managing stress and are given as feed addictives for prophylaxis and
growth promotion (Black, 1984). These drugs tend to accumulate in tissues to form residues
and become violative if the withdrawal periods are not observed. Antimicrobial residues in
food are potential allergens and may result in severe reactions in sensitized individuals (Stark,
2000; Nisha, 2008). Furthermore, they can influence the microbial composition and
metabolic activity of the intestinal microflora (Vollard & Classener, 1994: Nisha 2008).
Probably of greatest importance is the fact that a link has been established between the use of
antibiotics in food animals and the development of bacterial resistance to these drugs (Stark,
2000; Reig and Toldra, 2008). Other recorded pathological effects produced by antimicrobial
residues in food include autoimmunity, carcinogenicity, mutagenicity, bone marrow toxicity
(Pavlov et al., 2008; Nisha, 2008).
To underscore the importance of drug residues and reduce the adverse effects, tolerance
limits or maximum residue limits (MRLs) for antimicrobial residues have been set in most
developed countries. However, that which was set by Codex Alimentarius commission has
been adopted by the WHO and is generally being used by countries that do not have any
(MacNeil,1998). Several programmes have been mapped out with respect to food safety and
drug residues in foods of animal origin. For instance, the Residue Avoidance Programme
(RAP) was initiated in 1981 by the Extension Service and Food Safety and Inspection Service
(FSIS) of The United States Department of Agriculture (USDA) with the goal to monitor the
food supply in order to ensure that antibiotic residue concentrations do not exceed MRLs.
57
The informations generated by RAP are disseminated to farmers and people working in the
livestock industry through an educational programme, thus preventing and reducing the
occurrence of drug residues. Similarly, the European Union has well developed abattoir –
based programmes for the surveillance and monitoring of antibacterial residues in meat
(Mylliniemi, 2004). In contrast, Nigeria has no national programme for monitoring drug
residues in food animals in farms and abattoirs and no strict regulation on the use of
antimicrobials in livestock production (Oboegbulem and Fidelis, 1996).
Several methods are available for determination of antimicrobial residues in different
matrices, but many of these methods are relatively expensive and time consuming.
Antimicrobial residues in animals are conventionally detected by microbiological tests, some
of which include Bacillus stearothermophillus Disc assay(BsDA), the European four Plate
Test (FPT), the German Three Plate Test(TPT), the Premi® test, and a number of other
commercial kits. These tests which are essentially qualitative are based on bacterial inhibition
and are used primarily as screening tools for presence of antimicrobial residues in meat, milk
and eggs. The TPT, like the FPT, determines the presence of antimicrobials in a sample and
also identifies the specific antimicrobial group/class (Haasnoot et al., 1999; Javadi et al.,
2009). The test is prepared with Bacillus subtilis as the test organism at pH 6, 7.2 and 8 of the
method hence the name Three Plate Test. The pH 6 plates are to detect, in particular, betalactams and tetracyclines, pH 7.2 plates are to detect sulphonamides while aminoglycosides
are particularly detected by pH 8 (Chang et al., 2000). The only variation of TPT from the
EU FPT is the fourth plate which contains Micrococcus luteus at pH 8 detecting in particular
beta-lactams and macrolides. The objective of the study therefore, is to detect the presence
and determine the prevalence of antimicrobial residues in poultry meat and organs using the
German Three Plate Test (TPT).
58
4.2
Method
The three plate test was used for the antimicrobial residues detection with Bacillus subtilis as
the test organism.
4.2.1 Isolation of Bacillus subtilis
Five grams (5g) of soil sample from cassava waste dump site was weighed into 45 ml of
Ringer’s solution to form a stock mix and heated at 90oC for 1 hr to encourage the formation
of spores of Bacillus if present as well as eliminating other unwanted microorganisms in the
sample. One tenth of the mix was inoculated unto already prepared 0.4% dextrose nutrient
agar and incubated at 37°C for 24hrs.
4.2.2 Identification
i.
Morphological and microscopic identification
White wide colonies with serrated ends were purified by subculturing on fresh 0.4% nutrient
agar and incubated at 37ºC for 18-24 hours. Smears of the freshly prepared pure colonies
were made and gram stained following standard gram staining procedure. The slides were
viewed under light microscope in oil immersion.
ii.
Biochemical identification
Colonies of the isolated suspected Bacillus organisms were subjected to API50 CH
biochemical tests (Biomeriuex, UK) for further identification of the organism.
Principle:
API50 CH is a standardized commercial system involving 50 biochemical tests for the study
of carbohydrate metabolism, substrate utilization and enzyme production of microorganisms.
API50 CH strip consists of 50 microtubes used to study fermentation of substrates belonging
to the carbohydrate family and its derivatives. It is used in conjunction with API50CHB/E
medium for the identification of Bacillus. The fermentation tubes are inoculated with
59
API50CHB/E medium which rehydrates the substrate. During incubation, fermentation is
revealed by a colour change in the tube caused by the anaerobic production of acid, detected
by the pH indicator present in the medium. The first tube, which does not contain any active
ingredient, is used as a negative control.
Procedure:
•
The strips were prepared by setting out the incubation box and lid and filling the
honey combed wells with about 10mls of distilled water to create a humid
atmosphere.
•
The 5 small strips each containing 10 numbered tubes were gently placed on the
incubation box
•
A suspension of the organism was made from a 24 hour culture of B subtilis in 10mls
of API50 CHB/E medium equivalent to 0.5% McFarland standard,
•
The suspension was homogenized by vortexing, and 200µl of the homogenate
inoculated into each of the 50 tubes
•
The tray was then covered and incubated at 24°C for 24 hours.
4.2.3 Molecular Characterization of isolates
The characterization of the isolates follows three basic processes:
i.
•
Extraction of the DNA from the sample
•
PCR amplification of the target DNA,
•
Detection of target DNA by gel-electrophoresis.
DNA extraction
Two fresh colonies of the Bacillus organism isolates were put into T/E elution buffer and
heated at 70°C for 20 minutes to enhance the separation of the DNA of the organism. Nextec
Bacterial DNA extraction kit (Nextec UK) was used to extract the DNA of the isolates
according to the following procedure:
60
Separation
•
200µl of sample was dispensed into 200µl of ATL buffer in a 2ml Eppendorf tube
containing 20µl of proteinase K, vortexed and incubated in the heating block at 56°C
for 10 minutes.
•
20µl of Pt-L2p was added and vortexed for about 15 secs, incubated on ice for 10
minutes and then centrifuged at room temperature for 5 mins at 13000 rpm.
•
The clear supernatant was carefully transfered into a fresh 2ml tube and the pellet
containing impurities was discarded.
DNA precipitation
•
60µl of absolute ethanol (molecular grade) at room temperature was added to the
supernatant and mixed gently by inversion 10 times. The mixture was allowed to
stand at room temperature for 10 minutes for full precipitation of the DNA
•
The mixture was centrifuged at room temperature for 3 minutes at 13000 rpm and the
supernatant carefully removed without altering the DNA pellet
DNA wash
•
250µl of 70% ethanol was added and allowed to stand at room temperature for 1
minute
•
The tube was centrifuged at 13000 rpm for 5 minutes and the ethanol completely
removed without altering the DNA pellet.
•
The pellet was dried in the heating block at 56°C for 3 minutes.
Re-dissolving the DNA
•
60µl of AE buffer was added to dissolve the DNA pellet and incubated at room
temperature for 5 minutes and vortexed gently for 5 seconds (solution may still
contain insoluble fragments like membrane or protein)
•
The mixture was centrifuge at 8000 rpm for 2 minutes to separate insoluble materials.
61
•
The supernatant (DNA) was removed into a 1.5µl tube and stored at 4°C for PCR
amplification.
ii.
PCR amplification
Amplification of unique targeted DNA sequence was done using specific primers and
conventional Polymerase Chain Reaction (PCR).
Materials
o The extracted sample DNA
o DNA nucleotide primers (Forward and Reverse)
BSu bsf: AAG TCG AGC GGA CAG ATGG (19)
BSu bsr: CCA GTT TCC AAT GAC CCT CCCC (22)
o Tag polymerase
o PCR-cycler machine for amplification of target DNA,
Procedure
•
The lypholysed primers are dissolved with Tris EDTA buffer, vortexed and stepped
down from 100µm to 10µm
•
The Primer mix was prepared by making a 1:10 dilution of the primers (mixing 0.1ml
to 0.9ml Distilled H2O) and mixed with PCR H2O (Forward + Reverse + PCR H2O).
•
The PCR mix was prepared by mixing the Primer mix + Master mix(Tag polymerase)
+ extracted genomic DNA
•
The PCR mix was loaded into the machine which was set with initial cycle
temperature of 95°C to run for10 minutes. The denaturing cycle temperature was set
at 95°C for 30 seconds. The annealing temperature was set at 65°C for 30 seconds.
The initial extension temperature was set at 72°C for 1 minute.
62
iii.
Agarose-Gel electrophoresis.
Principle
This is used to read and interprete the PCR product to detect the presence of the isolate.
Agarose allows for the separation of DNA molecules based on size and conformation. The
negatively charged DNA migrates to positive charge when subjected to electricity.
Procedure
o 2% Agarose was prepared by mixing 2g of the powder to 100mls 1× TBE buffer
o The mixture was heated for about 3 minutes for even emulsification and 10µl of
Ethidium bromide, added.
o The prepared gel was poured into the electrophoresis apparatus and allowed for 30
minutes to gel, producing 8 wells
o The 1×TBE buffer was then poured into the electrophoresis tank and the gel placed on
the buffer to float
o 2µl of dye was mixed with 10µl of the sample product
o The bp ladder(marker) was added to the first well and the dyed samples were added
into the remaining wells
o The machine was switched on and allowed for 30 minutes.
o The presence of the organism was indicated with a band and read following the ladder
marker.
4.2.4
Antimicrobial sensitivity test
Susceptibility of the isolated and identified Bacillus subtilis was tested with commercially
produced antibiotic discs using Kirby and Bauer disc diffusion methods of determining
susceptibility as per recommendation of Clinical Laboratory Standard Institute (CLSI, 2011).
The organism was tested against the following antimicrobials: Ampicillin (30µg),
ciprofloxacin (5µg), tetracycline (30µg), gentamicin (10µg), erythromycin (10µg),
63
Ceftriaxone (30µg), cefixin (5µg) levofloxacin (5µg), norfloxacin (10µg), ofloxacin (5µg),
clindamycin (10µg), nitrofurantoin (100µg), pefloxacin (5µg), augmentin (30µg),
clarithromycin (30µg), and chloramphenicol (10µg). This method allows for rapid
determination of invitro efficacy of a drug by measuring the diameter of the zone of
inhibition that results from diffusion of the agent into the medium surrounding the disc.
Procedure
•
Fresh 24 hour culture of the isolate was prepared in nutrient agar
•
2 Mueller-Hinton agar plates were prepared according to manufacturer’s instructions,
for determination of sensitivity of B subtillis on both gram positive and gram negative
discs
•
One colony was used to make a suspension in 1ml of sterile distilled water in a bijoux
bottle to correspond to 0.5 McFarland standards for each plate.
•
The 1ml suspension was poured onto the Mueller-Hinton agar plates, rotated on the
working table to allow even spread and the excess decanted. Inoculated plates were
allowed to dry for approximately 3-5 minutes and then the antibiotic discs applied
aseptically to the surface of the inoculated agar with the help of a sterile forceps.
•
The plates were then incubated at 37ºC for 18-24 hours and examined for inhibition
around each discw. The diameter of the zone of complete inhibition was measured by
millimetre scale.
4.2.5 The Three Plate Test
Reagents and procedure
•
Test sample: broiler meat as sampled in section 3.3.1
•
Organ specimens: extracts of muscle, gizzard, liver and kidney, as prepared in section
3.4
•
Media: Nutrient agar of pH 6, 7.2, and 8
64
•
Test organism: Bacillus subtilis
•
pH adjusting reagents: NaOH (Base) & dilute HCL (acid)
•
Universal bottles, Petri dishes, pH meter, hole borer
Three batches of nutrient agar broth was prepared and adjusted to pH 6, 7.2 and 8, the media
were poured onto sterile petri dishes and allowed to solidify. Each plate was seeded with the
isolated Bacillus subtilis seeded. 5 holes were bored on each agar plate. 80µl of the organ
extracts were inoculated in the 4 holes, each hole representing an organ, the remaining hole
was inoculated with 80µl of distilled water as negative control. The plates were incubated at
30ºC for 18-24 hrs, clear zone of inhibition with annular diameter ≥ 2mm indicates positive
for antimicrobial residues (Mylliniemi et al., 2001).
4.3 RESULTS
4.3.1 Isolation, identification and sensitivity of Bacillus subtilis
Cultural and microscopic characteristics
Organisms showing large flat colonies with ground-glass appearance on nutrient agar that are
gram positive short rods (Figure 4.1) with centrally located refractile spores were isolated.
65
Gram positive short rods
in chains
Single short rods
Figure 4.1: Microscopic morphology of Bacillus organism
66
Biochemical test
Figure 4.2 A shows the plates before inoculation of the organism. The organism fermented
glycerol, D-glucose, D- fructose, D-Manose, D-sorbitol, inositol, and 15 other carbohydrates
after inoculation and incubation as shown in Figure 4.2 B. API WEB read the organism with
API CHB 4.0 software to be Bacillus subtillis at 99.9% accuracy (Appendix xv).
67
A. Before inoculation (all sugars were red)
B: After inoculation (Yellow colour shows fermentation)
Figure 4.2: API®50 Plates
68
Molecular characterization
Bacillus subtilis was identified at 595bp region of 16s rRNA of the bacteria (Figure 4.3).
595 bp
Lanes
1
2
3
4
5
Figure 4.3: Amplification of 595 bp 16S rRNA of Bacillus subtilis.
Lane 1 and 5 are 100 bp DNA Ladder.
Lanes 2 and 3 are positive samples.
Lane 4: Negative sample
69
4.3.2. Sensitivity test
Figure 4.4 shows that the isolate, Bacillus subtilis is sensitive to all the tested gram positive
antimicrobial discs except cefixime. The isolate is also sensitive to all the gram negative discs
except augmentin.
70
A. Gram positive discs
B. Gram negative discs
Figure 4.4: Antimicrobial susceptibility plates
71
4.3.3. Prevalence Antimicrobial residues in broilers
A bird with at least one positive organ is regarded as positive for antimicrobial residue.
Antimicrobial residues occurence in one organ alone in descending order of frequency:
kidney (10), liver (5), gizzard (1) and muscle (1); occurrence in two organs alone is as
follows: liver and kidney (17), liver and gizzard (2), kidney and gizzard (1); occurrence in
three organs is as follows: liver, kidney and gizzard (20), liver, gizzard and muscle (5),
kidney, gizzard and muscle (1). Antimicrobial residues occurred twice (2) in the four organs
(Appendix xvii). Out of the 100 birds sampled for antimicrobial residues, sixty four (64%)
were positive while the remaining thirty six (36%) were negative as shown in figure 4.5.
72
36%
% Positive
% Negative
64%
Figure 4.5: Prevalence of antimicrobial residues in broilers
73
4.3.4 Occurrence of antimicrobial residues in broiler meat and organs
A total of 155 (38.8%) out of 400 organ samples, tested positive for antimicrobial residues.
The proportion of organs positive for antimicrobial residues in descending order of frequency
is as follows: kidney (60%), liver (54%), gizzard (30%) and muscle (11%). A strong
association as shown in Figure 4.6 was found between the type of organ and occurrence of
antimicrobial residues at p < 0.05 (χ 2 = 64.5; p = < 0.00018)
74
Chi-square
Chi-square, df
P value
P value summary
One- or two-sided
Statistically significant? (alpha<0.05)
64.50, 3
< 0.0001
***
NA
Yes
100
Positive
Negative
Frequency
80
60
40
20
0
Kidney
Liver
Gizzard
Muscle
Organs
Figure 4.6: Organ distribution of antimicrobial residues
75
4.3.5 Organ distribution of antimicrobial residues according to pH
Out of the one hundred and fifty five (155) positive organ samples, twenty five (25) were
detected at pH 6.0 (best detects tetracycline and β-lactams), twenty six (26) at pH 7.2 (best
detects sulphonamides) and thirty five (35) at pH 8.0 (best detects aminoglycosides).
Residues were detected in sixty nine (69) samples with multiple pH: ten (10) by both pH 6.0
and 7.2; eight (8) by pH 6.0 and 8.0; ten (10) by pH 7.2 and 8.0 and 41 detected by the three
pH levels (table 4.1).
76
Table 4.1: Positive samples according to pH
Matrix
No
positive
pH
6.0
7.2
8.0
Multiple
6.0/7.2 6.0/8.0 7.2/8.0
All three
Muscle
11
2
0
5
1
1
0
2
Liver
54
6
11
15
2
3
3
14
kidney
60
11
10
9
6
3
3
18
Gizzard
30
6
5
6
1
1
4
7
Total
155
25
26
35
10
8
10
41
77
4.4 Discussion
Up to 64% of the investigated commercial broilers in this study had detectable levels of
antimicrobial residues at the point of purchase using the TPT method. This does not agree
with the earlier work done in Northern Nigeria by Kabir et al. (2004) who recorded 15.7%
prevalence in sampled broilers. The disparity may be due to the authors’use of only faeces as
the sample matrix in their study. The present study also shows low prevalence of 11% using
only muscle matrix. Another possible factor may be the level of antimicrobial use by the
farmers in the area of study. High presence of different antimicrobials in chicken has been
established as well in the Western Nigeria (Dipeolu and Alonge, 2002; Dipeolu and Dada,
2005). In other developing countries, high prevalence of antimicrobial residues in broilers has
also been recorded: 39.4% and 70% in Pakistan (Muhammad et al., 2007 and Jabbar, 2004),
respectively; 52% in Iraq (Shareef et al., 2009) and 70% in Tanzania by Nonga et al. (2009).
The high prevalence generally, indicates excessive prescription, overuse and non-observance
of withdrawal period in veterinary practice in developing countries. This problem inturn is
due to unrestricted availability of antimicrobial drugs and the practice of self-medication by
both poultry farmers. This unauthorized and unprofessional exposure of poultry to veterinary
drugs without adherence to recommended dose promotes accumulation of violative residues
in tissues.
Among the organs tested, the kidney and the liver had very high prevalence of residues at
60% and 54% respectively. This finding is similar to studies of Myllyniemi et al. (1999) and
Aerts et al. (1995), who also reported high prevalence in these organs. The two organs are
used as the primary matrix in many countries because the liver is the organ of drug
detoxification and the kidney, the major excretory organ of most drugs. However, there may
be effect of tissue matrix containing bacteriocidal tissue components like the kidney which is
78
said to have natural inhibitors to antimicrobials (Mylliniemi, 2004), with evidence of false
positive results.
Detection of antimicrobials at different pH in the same organ implies that different classes of
antimicrobials are being administered to a bird at the same time. Shareef et al. (2009),
confirmed in their study, that all the broiler farms surveyed, use different drugs at the same
time to either treat or prevent diseases in their farms. The use of multiple classes of
antimicrobials may be associated with the fact that farmers attempt to curb the problem of
concurrent-infections in their farms, as also reported by Riviere et al. (2001) and Ibrahim et
al. (2010). Bacillus subtilis at pH 6.0 is said to best detect oxytetracycline (OTC) which is the
most widely used drug in poultry production in Nigeria (Kabir et al., 2003; Ezenduka et al.,
2011) but the detection of tetracyclines (25) alone was lowest in this study as compared with
sulphonamides (26) detected at pH 7.2 and aminoglycosides (35) detected at pH 8.0. A
contributory factor may be that most positive pH 6.0 samples may have fallen into the
multiple pH categories.
4.5 Conclusion
There is a widespread indiscriminate use of antimicrobials as well as non-implementation of
recommended withdrawal periods in poultry production in Enugu State, Nigeria. This study
has also shown that using only muscle as a sample matrix may underestimate the occurrence
of antimicrobial resiudes. There is also evidence of use of more than one class of
antimicrobials in the poultry farms.
79
CHAPTER FIVE
SURVEY OF ANTIMICROBIAL RESIDUES IN COMMERCIAL BROILERS USING
THE PREMI® TEST
5.1 Introduction
Poultry industry is one of the fastest means of ameliorating the animal protein deficiency in
Nigeria. The high turn-over rate and the quest for white meat has given more credence to
poultry among livestock farming. The need to meet up with the demand for poultry meat has
necessitated the use of veterinary drugs especially antimicrobials which are particularly used
in poultry farming for therapeutic purposes and added in feed and water in sub-therapeutic
doses for prophylaxis and growth promotion (Dipeolu and Alonge, 2002). Presence of drugs
or antibiotics residues in food above the maximum level is recognized worldwide by various
public health authorities as being illegal (Kempe and Verachtert, 2000) and their consumption
could result in public health hazards. Jones (1999), Cunha (2001) and Kirbis (2006) have
suggested economic losses in the food industry especially in interfering with starter culture in
yoghurt and cheese production consequent upon the presence of antibiotics residues in milk.
Therefore, detection of these residues in foods of animal origin intended for human
consumption is essential for the safety of consumers.
Drug residue detection could be quite an expensive, time consuming and laborious venture.
Microbiological methods are quite suitable for the detection of antimicrobial residues
especially as they are less expensive than immunochemical and chromatographic methods,
and are able to screen a large number of samples at minimal cost (Pikkemaat et al., 2007).
Many microbiological tests have been developed for detection of antimicrobial residues.
Premi® Test, like the Three Plate Test is a microbial screening test for the detection of
antimicrobial residues in foods of animal origin. The test is based on the inhibition of growth
80
of Bacillus stearothermophilus, a thermophilic bacterium sensitive to many antibiotics and
sulpha compounds (Stead et al, 2004). The Premi® Test allows for the screening of meat
(beef, pork, poultry) for the residues of β-lactam, cephalosporins, macrolids, tetracycline,
sulphonamides, aminoglycosides, quinolones, amphenocols and polypeptides. The kit
recently received French Association for Normalization (AFNOR) certification. The AFNOR
validation mark certifies the analytical effectiveness of commercial methods for a defined
field of application, which should be comparable to the effectiveness of a reference method
(Pikkemaat, 2009; Fagbamaila et al., 2012). It has a detection limit in line with the EU
Maximum Residue Limits (MRL’s) and needs little time for incubation. The objective of the
study therefore is to detect the presence and determine the prevalence of antimicrobial
residues in poultry meat and organs using the The Premi® Test Kit.
5.2
Materials and method
5.2.1 Antibiotics residues detection
The antibiotics residues were detected on the same 100 broilers and 400 organs as sampled in
section 3.3.1 and extracted in section 3.4, using Premi® Test kit.
The kit comprises the following:
o Polystyrene boxes in quantities of 25 ampoules of agar containing Bacillus
stearothermophilus spores and Bromocrescol purple colour indicator.
o A heating block incubator that incubates at optimum temperature of 64ºC .
o A pair of scissors
o A thermometer
o Plastic cover foils
o Sterilized pipette tips
o Tuberculin needle with syringe
81
Principle
The ampoules contain nutrient agar imbedded with standardized number of spores of Bacillus
stearothermophilus var calidolactis organism and a colour indicator. Premi® Test combines
the principle of an agar diffusion test with colour change of the indicator. If there is an active
metabolism of the included microorganism, the test will change from purple to yellow colour.
If growth of the microorganism is inhibited (due to presence of an antibiotic at or above the
Limit of Detection (LOD)) the test will remain purple. In Premi® Test interpretation, the
negative (yellow colouration) is due to the growth of the spores at 64°C that initiates an
acidification process which causes the turning of a pH indicator from purple to yellow. The
presence of antibiotic residues on the other hand will cause delay or inhibition of the spores,
depending on the concentration of the residues, In the presence of residues therefore, the
spores will not multiply and the pH indicator will remain purple.
Procedure
•
The incubator containing ten wells for the ampoules was pre-heated for 20 minutes
while using the thermometer provided to ensure optimium temperature of 64 ± 1°C
•
100µl of each sample was inoculated into an ampoule and labeled
•
Two controls (negative and positive) were used for each test series. The negative
control was ampoule containing 100µl of distilled water (which is the meat extraction
diluent) while the positive control, meat extract spiked with tetracycline.
•
5.3
The ampoules were incubated for 3-4 hours until the negative control turned yellow
RESULTS
Those ampoules that remained purple after incubation were recorded as positive for
antimicrobial residues, those that turned yellow were negative and those that were bluish
yellow were recorded as douptful for antibiotic residues (Appendix xviii).
82
5.3.1 Antibiotic residues detection in commercial broilers
A positive bird is a bird with at least one organ positive. Antimicrobial residues occurence in
one organ alone in descending order of frequency: kidney (20), liver (2), gizzard and muscle
(0); occurrence in two organs alone is as follows: gizzard and muscle (9), kidney and gizzard
(7) and none detected from the rest of two organ combinations; occurrence in three organs is
as follows: liver, kidney and gizzard (6), liver, kidney and muscle (6). Antimicrobial residues
occurred ten times (10) in the four organ combination (Appendix xix). Out of the 100 broilers
sampled, 60% were positive while 40% were negative for antibiotic residues as shown in
figure 5.1.
83
Figure 5.1: Prevalence of Antimicrobiall residues in commercial broilers
84
5.3.2 Antibiotics residues detection in sampled organs
A total of 107 (26.8%) out of 400 organ samples, tested positive for antimicrobial residues
and 21(5.4%) were douptful. The proportion of organs positive and doubtful for antimicrobial
residues in descending order of frequency is as follows: kidney (49%), liver (23%; 2%),
gizzard (22%; 9%) and muscle (14%; 10%) (fig 5.1), A strong association (p < 0.05) as
shown in Figure 5.2 was found between the type of organ and occurrence of antimicrobial
residues at 5% probability (χ 2 = 41.3; P = < 0.0001)
85
Chi-square
Chi-square, df
P value
P value summary
41.30, 6
< 0.0001
***
80
Frequency
60
Positive
Negative
40
Douptful
20
0
Kidney
Liver
Gizzard
Muscle
Organs
Figure 5.2: Organ distribution of antimicrobial residues
86
5.3.3 Detection of antimicrobial residues by TPT and Premi® Test
TPT detected 64(chapter 4) and Premi® Test detected 60 out of the 100 sampled birds
positive for antimicrobial residues with 64% and 60% prevalence respectively. Figure 5.3
shows that there is no difference (p > 0.05) in the proportion of positive and negative samples
between the two microbiological tests in general screening for antimicrobial residues (p =
0.66).
87
80
P value
P value summary
One- or two-sided
Statistically significant? (alpha<0.05)
0.6622
ns
Two-sided
No
Prevalence
60
Positive
Negative
40
20
0
TPT
Premi Test
Test type
Figure 5.3: Detection of antimicrobial residues with TPT and Premi®Test
88
5.4. Discussion
The 60% prevalence of antibiotics residues in commercial broilers in this study is a clear
indication that consumers are being exposed to violative levels of drug residues as detected
by Premi® Test. Although the test is not a quantitative method, it detects most antibiotics at
or above their MRLs (Pikkemat, 2009) as set by the World Health Organisation (WHO).
However, the test organism, Bacillus stearothermophilus is relatively not sensitive to
quinolone antibiotics, therefore, to cover the whole antibiotic spectrum, additional testing
method will be required (Pikkemat, 2009). Occurrence of antimicrobial residues in tissues is
dependent on the organ as the study clearly states that a strong association exists between
occurrence of antimicrobial residues and the type of organ. The kidney has the highest
proportion of antibiotic residues and apart from it being the major excretory organ of most
drugs, the premi test organism, Bacillus stearothermophilus is sensitive to the inhibitory
activity of the lysozymes present in the kidney (Kirbis, 2007). There was no statistical
difference (P > 0.05) in the proportion of antimicrobial residues detected by both tests (TPT
and Premi® Test). The tests have their detection deficiencies, TPT does not have a fourth
plate of Micrococcus luteus at pH 8.0 and therefore cannot detect macrolids as well as it
detects other antibiotic groups and Premi® Test cannot detect quinolones.
Generally, microbiological methods are basically screening methods for detecting the
presence of antimicrobial residues in food and plate tests are able to differentiate only
between antibiotic groups, they are the earliest methods used for the detection of antibiotic
residues and are still widely used. Microbiological based tests are very cost-effective and in
contrast to, for example, immunological or receptor-based tests, have the potential to cover
the entire antibiotic spectrum within one test.
89
5.5
Conclusion
The Premi® Test detection of antimicrobial residues in commercial poultry in this study
indicates that the poultry consumers in the study area may be at risk of taking violative level
of antimicrobial residues since the test detects residues at or above the WHO recommended
MRLs. Careful attention should therefore be given to indiscriminate use of antimicrobials in
animal production, to avoid problem of reduction in their potency and effectiveness since
they are the vital drugs used in treating human infections.
90
CHAPTER SIX
DETECTION AND QUANTITATION OF TETRACYCLINE RESIDUES IN
COMMERCIAL BROILERS
6.1. Introduction
The tetracyclines (TCs) are a group of antibiotics commonly used as veterinary drugs and as
growth promoters in food producing animals, they rank among the antimicrobial substances
most frequently used in the animal food production (Schmidt & Rodrick, 2003). A lot of
antibiotics were used in veterinary medicine in the EU countries and TCs were among the
most frequently used (Kools, et al., 2008; Moulin et al., 2008). Moreover, in the USA, the
largest quantity of antibiotics were sold in 2007 and 2009 for use in food animals and the
commonest group, the TCs (Salah, 2013). Chlortetracycline, a member of the group is used
by 87% of the farms sampled in Trinidad and Tobago as a feed additive given as prophylaxis
or for therapeutic purposes (Adesuyin et al., 2004). Chlortetracycline was also the most
common residue detected in chicken in a work done in Barbados in other words, the most
commonly used drug (Hall et al., 2004). In Nigeria, the tetracycline group of drugs is also the
most widely used antimicrobials in poultry farming in Kaduna State (Kabir et al., 2003),
Ezenduka et al. (2011) reported the use of Oxytetracycline by 100% of poultry farms in
Enugu State. The common use of OTC as prophylaxis in poultry production is expected,
since it is also an anticoccidial as well as an antibiotic (Harold et al., 1998). Because of their
wide application, there is a concern about their residues in foods of animal origin which may
result from excessive and improper use, or a shortened withdrawal time. One of the most
worrisome problems associated with TCs residues is idiosyncratic reactions especially in
hypersensitive consumers (Loksuman, 2002). Tetracycline residues in meat potentially may
stain teeth of young children (Mesgari Abasi et al., 2009). Development of resistant strains of
bacteria following the ingestion of subtherapeutic doses of antimicrobials and distortion of
91
the metabolic activity of the microflora are some of the hazardous effects associated with
tetracycline residues (Wilson et al., 2003; Dayan, 1993; Mateu and Martin, 2001).To prevent
any harmful health effects on consumers, Food and Agriculture Organization, World Health
Organization and European Union (EU) have established the maximum residual limits
(MRL) for veterinary drugs. The maximum residual limit set by the codex alimentarius for
FAO/WHO legislation for tetracycline (TTC) in chicken is as follows: muscle (0.2 ng/kg),
kidney (1.2 ng/kg), liver (0.6 ng/kg), and egg (0.4 ng/kg). There are basically two methods
that can be used to quantify the concentration of antimicrobial in foods: chromatographic and
immunochemical methods. Both methods are quantitative and specific. They are used to
confirm screened samples that are positive, and also determine the concentrations of the
incriminated antimicrobials (Plumb, 2008). Immunochemical methods like Enzyme Linked
Immunosorbent Assay (ELISA) can be used both qualitatively and quantitatively. Compared
to HPLC, it offers the advantage of having low cost, simpler to perform and tests more
samples at a time (Amir, 2011). Based on these advantages and owing to lack of information
on tetracycline levels in poultry in Nigeria, ELISA was used in this study with the objectives
of determining the prevalence and level of TC residues in commercial broiler meat and
organs.
6.4
Materials and methods
6.4.1 Organ sample collection and preparation
The sample collection and preparation was as described in chapter 3: sections 3.3.1 and 3.4.
Out of the 400 organs from 100 broilers, whose organ extracts were used in the other survey
studies, 200 of the same organs from 50 birds were used for this study.
6.2.2 Tetracycline residues detection
The tetracyclines were detected with tetracycline ELISA kit (ABRAXIS USA). In
preparation, the microtitre plates and the reagents were adjusted to room temperature before
92
use. The lypholysed conjugate was reconstituted first with 1ml of conjugate diluent, vortexed
and diluted with the same conjugate diluents at 1:10 ratio. The standards and control were
reconstituted with 1ml of deionized water. The wash buffer (5X) concentrate was diluted at a
ratio of 1:5. For each plate, a working scheme (Appendix XX) was prepared, the standards
and samples were run in duplicates.
Procedure
•
Twelve strips, each containing 8 wells were fixed on the plate
•
50 µl of assay buffer solution was first added to the individual wells with a multichannel pipette
•
100 µl of each of the six provided standard solutions (0.1, 0.2, 0.3, 0.4, 0.6, and 0.8)
in ng/kg was added in duplicate wells according to the working scheme. 100 µl of
each sample was added in duplicate wells following the standards according to the
working scheme.
•
50 µl of enzyme conjugate solution was added to each of the wells.
•
The plate (wells) was covered with a paraffin tape and the content mixed by circular
motion on the bench top for 30 seconds and then incubated at room temperature for
60 minutes
•
After incubation, the strips were washed four times with 250 µl of the wash buffer
and the plates patted on stacked paper towels to dry.
•
150 µl of substrate solution was then added to the wells, covered, mixed for 30
seconds on the bench top and left to incubate at room temperature for 30 minutes. The
strips were covered with black plastic to protect from direct sunlight.
•
100 µl of stop solution was added to the wells in the same sequence as the substrate
solution
•
The absorbence was read within 15 minutes of adding the stop solution at 450 nm.
93
6.5
Results
6.3.1 Calculation of Tetracycline concentration from Optical Density (OD)
The standard curve for Tetracycline is given in figure 6.1. The concentrations of the standards
(0.1, 0.2, 0.3, 0.4, 0.6, and 0.8) in ng/kg were plotted against their ODs. The caliberation
curve was used to extrapolate the concentration of the samples where the absorbtion is
inversely proportional to the drug concentration of the samples with the formula: Y= MX + C
(Appendix xxi)
94
Slope
Y-intercept when X=0.0
-1.506 ± 0.07277
1.529 ± 0.03387
Optical density
1.5
1.0
0.5
0.0
0.0
0.2
0.4
0.6
0.8
1.0
Conc (ng/kg)
Figure 6.1 Tetracycline calibration curve: optical density versus concentration of standard
95
6.3.2 Organ distribution of tetracycline residues
Out of 200 organs sampled, 19 were undetected (no residue), 22 were within the detection
limit (control) of the tetracycline kit while 181(90.5%) were above the detection limit.
Approximately, 96% of muscle, 96% of liver, 88% of kidney and 82% of gizzard were
positive for tetracycline residue.
96
Table 6.1: Tetracycline residues detection according to concentration
Organ
Frequency [Concentration (ng/kg)]
Proportion
detected (%)
< 0.1(undetected)
0.1 – 0.5
> 0.5
Muscle
2
9
39
48 (96)
Gizzard
9
6
35
41 (82)
Kidney
6
2
42
44 (88)
Liver
2
5
43
48 (96)
Total
19
22
159
181 (90.5)
97
6.3.3 Comparison of mean values of the organs with their MRL’s
Figures 6.2 (a-d) are showing the mean concentration of TCs in different organs compared to
their MRL. Wilcoxon Signed Rank Test was used to analyse the difference in the median
values of the organs. The median values of each organ are designated the actual values while
the MRLs are termed the theoretical values.
a. The residue concentrations of 43(89.5%) out of 48 detected muscle samples were
above the MRL. The median value of TC residues in muscle at 0.742ng/kg is higher
than its MRL at 0.2ng/kg and the difference is statistically significant (p<0.05) as
shown in Fig 6.2a (P = < 0.0001).
98
1.5
Wilcoxon Signed Rank Test
Theoretical median
0.2000
Actual median
0.7420
P value (two tailed)
< 0.0001
Exact or estimate?
Gaussian Approximation
Significant (alpha=0.05)?
Yes
Conc.
1.0
0.5
M
us
cl
e
M
R
L
0.0
Tissue
Figure 6.2a: Tetracycline residues concentration in the muscle
99
b. The residue concentrations of 42(87.5%) out of 48 detected liver samples were above
the MRL as shown in Fig 6.2b. The median concentration of the detected TC residues
in liver at 0.873 was significantly (P < 0.05) higher than its MRL at 0.60. (P = <
0.0001)
100
Wilcoxon Signed Rank Test
Theoretical median
0.6000
Actual median
0.8735
P value (two tailed)
< 0.0001
Exact or estimate?
Gaussian Approximation
Significant (alpha=0.05)?
Yes
1.0
0.5
M
R
L
0.0
Li
ve
r
Conc (ng/kg).
1.5
Tissue
Figure 6.2b Tetracycline residues concentration in the liver
101
c. The concentrations of all (100%) the 44 detected kidney samples were below the
MRL. The median concentration of the detected TC residues in kidney at 0.901 was
significantly (p<0.05) lower than its MRL at 1.20 as shown in Fig 6.2c (P = <
0.0001).
102
Wilcoxon Signed Rank Test
Theoretical median
1.200
Actual median
0.9010
P value (two tailed)
< 0.0001
Exact or estimate?
Gaussian Approximation
Significant (alpha=0.05)?
Yes
1.0
0.5
M
R
L
0.0
K
id
ne
y
Conc (ng/kg).
1.5
Figure 6.2c Tetracycline residues concentration in the kidney
103
d. The TC concentrations of 39(97.5%) of the 40 detected gizzard samples were above
the MRL of muscle tissue. The median value of the detected TC residues in gizzard at
0.7555 was significantly (p < 0.05) higher than its MRL at 0.20. (P = < 0.0001) as
shown in Fig 6.2d.
104
Wilcoxon Signed Rank Test
Theoretical median
0.2000
Actual median
0.7555
P value (two tailed)
< 0.0001
Exact or estimate?
Gaussian Approximation
Significant (alpha=0.05)?
Yes
1.0
0.6
0.4
0.2
0.0
M
R
L
G
iz
za
rd
Conc (ng/kg).
0.8
Figure 6.2d: Tetracycline residues concentration in the gizzard
105
6.4 Discussion
The tetracycline ELISA kit detects the presence of drugs in the tetracycline class of drugs
including, oxytetracycline, chlortetracycline, tetracycline, doxycycline etc in foods in varying
degrees, hence the residues detected in this study could be any of the tetracyclines. The 90%
prevalence of tetracycline residues in chicken organs in this study is very suggestive of
constant use of tetracycline group of drugs either added as feed additives for prophylaxis or
used therapeutically for treatment. This conforms to the findings of Kabir et al. (2004) and
some other studies in Nigeria, that tetracyclines are the most widely used antibiotic in poultry
production in the country, owing to its wide application in treating many disease conditions,
they are also inexpensive and readily available. A similar study by Farahmand et al., (2006)
in Iran also reported a high prevalence (100%) of TC residues in chicken tissues and also
indicated that TC’s are the most widely used antibiotics in poultry production.
All the organ types with the exception of the kidney records tetracycline concentrations
statistically (p < 0.05) higher than the WHO recommended MRL. This implies that
consumers are exposed to violative levels of tetracyclines and therefore, exposed to the health
hazards associated with consumption of drugs residues in human food chain. Although the
urinary system is the main excretory route of tetracyclines and almost all the TC residues
detected in kidney samples were above the control limit of 0.5ng/kg, however, the median
concentration was statistically (p < 0.05) lower than the WHO recommended MRL of
1.2ng/kg. This may be associated with the fact that OTC is excreted by both bile and kidney.
Bile excrets 40% while kidney excrets 60% (Brander et al., 1993), hence, the residue
concentration in the kidney may not be as high as 1.2ng/kg. In particular, orally administered
tetracyclines must have been biotransformed and metabolized in the liver (first pass effect)
and the concentration reduced before excretion.
106
6.5
Conclusion
Commercial broilers sold in Enugu metropolis contain violative levels of tetracycline
residues and consumers in the area may be exposed to deleterious levels of these residues.
Despite their low toxicity, residues of TCs in meat should be avoided because they may pose
potential health threats to consumers. The importance of antibiotics in livestock production
cannot be overemphasized as they represent a key component in the strategy used in disease
control in humans and animals. Therefore, their prudent use in livestock production is of
utmost importance to avoid the occurrence of violative level of antimicrobial residues in
human food chain and subsequent development of resistant strains of microorganisms.
107
CHAPTER SEVEN
COMPARATIVE VALIDITY AND RELIABILITY OF TPT AND PREMI®
TEST IN DETECTION OF OXYTETRACYCLINE RESIDUE USING ELISA
AS GOLD STANDARD.
7.1 Introduction
The use of antibiotics in livestock production has generated a lot of concern in the developed
world because their application in food producing animals may lead to the presence of
residues in edible products. The major concern regarding the use of antibiotics in food
animals is the development of resistance due to the transfer of antibiotic resistance genes to
human pathogens. A recent report by the Centre for Disease and Control (CDC) in September
2013 gave a confirmation on a connection between the use of antibiotics in livestock and a
growing number of microorganisms that are resistant to most antibiotics such as methicillin
resistant Staphylococcus aureus (MRSA)(Hubbard, 2013). The CDC also stated that this
growing bacterial resistance results in the death of at least 23,000 people and causes
morbidity in about 2 million people per year (Hubbard, 2013).
Microbiological methods were the earliest used for the detection of antibiotic residues
(Pikkemaat, 2009) and are still widely used. The Food Safety and Inspection Service (FSIS),
United States Department of Agriculture (USDA), for the National Residue Programme
developed Swab Test on Premises (STOP), Fast Antimicrobial Screen Test (FAST) and Calf
Antibiotic Sulfonamide Test (CAST). The Swab test on Premises makes use of Bacillus
subtilis and was developed as early as 1977 to screen large numbers of meat samples and
modified to CAST in 1980, for use in slaughter establishment. FAST was later developed in
1994 due to its higher sensitivity and shorter analytical time (Dey et al., 2003). Both CAST
and FAST use Bacillus megatarium as the test organism. The European Union (EU), on the
other hand has developed several tests for antimicrobial residues detection based on the
108
microbiological principle of inhibition using two main test formats: the plate and the tube
tests. The plate test has been the test format in Europe since the inception of screening for
antimicrobial residues in slaughter animals (Bogaerts et al., 1980 cited by Pikkemaat, 2009).
In the Netherlands in 1973, the Sarcina lutea (Micrococcus luteus) kidney test of van
Schothorst became the first official method used for screening, followed by The German
Three Plate Test (TPT), that was adopted by other countries (Nouws et al, 1979, cited by
Pikkemaat, 2009). The TPT comprises three plates of agar medium inoculated with B. subtilis
BGA spores at pH 6, 7.2, and 8. The pH 6 plates are to detect, in particular, beta-lactams and
oxytetracyclines, pH 7.2 plates are to detect sulphonamides while aminoglycosides are
particularly detected by pH 8 (Chang et al., 2000). In order to incorporate detection of other
antibacterials, the EU four-plate was proposed by the Scientific Veterinary commission of the
European Commission with the addition of Kocuria rhizophila (formerly known as
Micrococcus luteus (Tang and Gillevet, 2003) ATCC 9341 plate at pH 8 for the detection of
beta-lactams and macrolids and supplemented pH 7.2 medium with trimethoprim for
increased sensitivity to sulphonamides (Pikkemaat, 2009). Tube tests were later developed
because of their shorter incubation periods, and the feasibility of commercial distribution of
spores instead of vegetative cells of Bacillus stearothermophillus test organism which has
prolonged shelf life (Pikkemaat, 2009). Among the known tube tests are Premi®Test by
DSM, Explorer by Zeu-Inmunotech, and Kidney Inhibition Swab (KIS™) test by Charm
Sciences. Among these, Premi®Test is the only test for which a substantial amount of
literature is available indicating its frequent use. B. stearothermophilus is said to be very
sensitive to the β-lactam group of antibiotics, Popelka et al. (2005), in their study showed that
Premi® Test exhibits excellent sensitivity for penicillin, amoxicillin, ampicillin, oxacillin,
and cloxacillin. Premi®Test has currently received validation certificate from the French
Association
for
Normalization (AFNOR),
109
an
organization
that
mainly
certifies
microbiological detection methods in food and water. The test was validated for satisfactory
detection of amoxicillin, ceftiofur, and tylosin at their respective MRLs.
The focus antibiotic in the present study is oxytetracycline which in so many studies in
different parts of the world is known to be the most widely used antimicrobial in poultry
production (Hall et al., 2002, Schmidt & Rodrick 2003, Kabir et al., 2003, Adesiyun et al.,
2004, Ezenduka et al., 2011). The test organisms mostly used for detection of tetracycline
residues in plate methods are Bacillus subtilis (Javadi et al., 2009; Javadi, 2011) and Bacillus
cereus (Kirbis, 2007), although Okerman (2009), reported a remarkably small difference in
the sensitivity of tetracyclines between the two organisms. Tsai and Kondo (2001) also
showed that better sensitivity to tetracyclines may be noticed with Bacillus subtillis grown on
minimum medium.
Both plate test (TPT) and Premi Test are qualitative microbiological methods. It has been
established that they can identify the presence of antimicrobial residues in meat samples but
will not quantify the concentration of the residue and the incriminating antimicrobial. ELISA
on the other hand is specific to a particular antimicrobial and can perform both qualitative
and quantitative tests. This study will therefore determine the validity of the FPT and
Premi®Test (microbiological methods) in detection of OTC in experimentally administered
broiler birds
7.2 Materials and methods
The samples were tested for residues with the TPT, Premi® Test and ELISA immunoassay
7.2.1
Experimental Study design: as stated in section 3.3.2
7.2.2
Experimental Drug administration
Birds in group A were injected intramuscularly at the breast muscle with long acting
Oxytetracycline (Coophavet, France) at the label therapeutic dose of 20mg/kg body weight.
110
Birds in group B were given OTC (Penta-oxytetra 200: 200mg OTC HCL/gm, Pantex,
Holland) in drinking water at the dose of 4g/L of drinking water for 5 days.
7.2.3 Testing for OTC residue in experimental birds
After 24 hours of injecting the birds in group A, 2 birds were slaughtered, the liver and
muscle harvested and juice from each organ was extracted as stated in section 3.4. Each
organ extract was tested for OTC residues, first after 24 hours and subsequently, 2 birds were
sacrificed daily and tested for residue until no residue was detected in the organs. After 5
days of drug administration for birds in group B, 2 birds were sacrificed daily and the organ
extracts tested for residues until no residue was detected.
7.3 RESULTS
7.3.1: TPT detection of Oxytetracycline residues in Group A birds.
OTC residue was detected at pHs 6.0 and 7.2 but none was detected at pH 8.0. The drug
residue was detected in muscle tissue from day1 to day 5 with gradual decrease in the
inhibition zones. Detection in the liver tissue started from day 2 with a gradual increase that
peaked at day 5 and started decreasing untill day 8. Detection in the kidney started from day 2
with a gradual increase that peaked at day 7 and started decreasing until day 9. Detection in
the gizzard started from day 2, gradually increased and peaked at day 5 and gradually
decreased until day 9 when OTC was last detetcted. Premi Test on the other hand detected
OTC in muscle from day 1 to 3 and a partial detection on day 4. Detection in liver and kidney
was on days 1 and 2 and a partial detection on day 3. Detection in gizzard was only on day
1and a partial detection on the 2nd day (Appendix xxii).
111
7.3.2: TPT detection of Oxytetracycline residues in orally treated (Group B) birds
TPT detected the presence of OTC residue in all the organs from the 1st day. Detection
stopped in muscle on day 3, liver: day 6, kidney and gizzard: day 7. There was a gradual
daily decrease in inhibition zones in all the organs (Appendix xxiii).
7.3.3 ELISA detection of Oxytetracycline residues in muscle samples of birds in both
groups (A and B)
As shown in Figure 7.1, ELISA detected OTC residue above the MRL (0.2ng/kg) in muscles
from the 1st until the 7th day in the injected group and from 1st until the 6th day, in birds given
in drinking water. There was a daily reduction in OTC concentration in both groups. Group A
had higher OTC concentrations ranging from 1.004 - 0.133ng/kg in decreasing order and
Group B, had lower OTC concentrations ranging from 0.739 - 0.088ng/kg.
112
1.2
Gp A (Injection)
Conc. (ng/kg)
1
Gp B (Oral)
0.8
0.6
0.4
0.2
0
1
2
3
4
5
6
7
8
9
10
Days
Figure 7.1: Daily mean concentration of OTC residue detected in muscle tissues of birds in
both groups A and B
113
7.3.4 ELISA daily detection of Oxytetracycline residues in liver samples of birds in
both groups (AandB)
ELISA detected OTC residue above the MRL (0.600ng/kg) in liver from the 2nd until the 6th
day in group a birds and from the 1st day in group B birds (Figure 7.2). In Group A, the
concentration gradually increased from the 1st day (0.578ng/kg) peaking at day 5(0.900ng/kg)
and gradually decreased to 0.318ng/kg on the 10th day. Group B showed a steady daily
decrease ranging from 0.813 - 0.254.
114
Conc. (ng/kg)
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Gp A (Injection)
Gp B (Oral)
1
2
3
4
5
6
7
8
9
10
Days
Figure 7.2: Daily OTC residue detection and quantification with ELISA in liver tissues of
birds in both groups A and B
115
7.3.5 Detection of OTC residues in muscles of injected birds by the three Test methods
For the TPT, inhibition zones ≥ 2mm are regarded as positive, while concentrations of OTC
above the MRL (0.2ng/kg) of muscle (as evaluated by ELISA) are regarded as positive for
OTC. ELISA detected the presence of OTC in muscle for the ten days, Premi® Test detected
fully the first three days and partially on the 4th day, while the TPT detected for the first five
days (Table 7.1).
116
Table 7.1: OTC residue detected by the three methods in muscle of Injected birds
Days/Test
1
2
3
4
5
6
7
8
9
10
method
TPT(IZ)
Premi®Test
7.0(+) 6.7(+) 3.9(+) 3.25(+) 3.0(+)
+
+
+
+(-)
-
ELISA
1.004
0.813
0.682
0.595
0.506
(ng/kg)
(+)
(+)
(+)
(+)
(+)
IZ: inhibition zone, NI: no inhibition; NI: no inhibition
117
NI(-)
NI(-)
NI(-)
NI(-)
NI(-)
-
-
-
-
-
0.397 0.212 0.194 0.182 0.133
(+)
(+)
(-)
(-)
(-)
7.3.6. Statistical comparison of the validity of TPT and Premi® Test in OTC residue
detection in muscles of injected birds
Figure 7.3 is showing the sensitivity and specificity of both microbiological tests in OTC
detection from muscle of injected birds using ELISA as gold standard. In fig.7.3a, 5 samples
were both TPT and ELISA positive; none was TPT positive and ELISA negative (no false
positive). Three samples were both TPT and ELISA negative and 2 were TPT negative and
ELISA positive (2 false negatives); TPT therefore has a sensitivity of 71.4%. In fig.7.3b, 4
samples were both Premi® Test and ELISA positive and no Premi® Test positive and ELISA
negative (no false positive). Three samples were both Premi® Test negative and ELISA
positive (3 False negatives) and 3 were both Premi® Test and ELISA negative. The Premi®
test therefore, has a sensitivity of 57%. Both tests have 100% specificity.
118
6
Sensitivity and specificity
Sensitivity
95% confidence interval
Specificity
95% confidence interval
Positive Predictive Value
95% confidence interval
Negative Predictive Value
95% confidence interval
0.7143
0.2904 to 0.9633
1.000
0.2924 to 1.000
1.000
0.4782 to 1.000
0.6000
0.1466 to 0.9473
5
Sensitivity and specificity
Sensitivity
95% confidence interval
Specificity
95% confidence interval
Positive Predictive Value
95% confidence interval
Negative Predictive Value
95% confidence interval
ELISA Positive
ELISA Positive
4
ELISA Negative
ELISA Negative
Result
4
Result
0.5714
0.1841 to 0.9010
1.000
0.2924 to 1.000
1.000
0.3976 to 1.000
0.5000
0.1181 to 0.8819
3
2
2
1
0
TPT Positive
0
TPT Negative
Premi test positive
Premi test negative
Test method
Test method
Fig 7.3a: Validity of TPT in OTC detection in
muscle of injected birds
119
Fig 7.3b: Validity of Premi®Test in OTC
detection in muscle of injected birds
Figure 7.4 shows that there is a strong correlation (r = 1) between the inhibition zones of
TPT and ELISA concentrations in OTC detection in muscle of injected birds.
Slope
Y-intercept when X=0.0
Number of XY Pairs
Spearman r
9.297 ± 1.956
-1.923 ± 1.449
5
1.000
Inhibition zone(mm)
8
6
4
2
0
0.0
0.5
1.0
1.5
Conc (ng/kg)
Figure 7.4: Correlation between the inhibition zone of TPT and the tetracycline residue
concentration in muscle of group A birds
120
7.3.7 Detection of OTC residues in muscles of group B (given OTC in drinking water)
birds by the three Test methods
In Table 7.2, ELISA detected the presence of OTC in muscle for the ten days, Premi® Test
partially detected only on the first day, while the TPT detected for the first four days.
121
Table 7.2: OTC residue as detected by the three methods in muscle of birds orally
administered
Days/Test
1
2
3
4
5
6
7
8
9
10
NI(-)
NI(-)
NI(-)
NI(-)
NI(-)
NI(-)
-
-
-
-
-
method
TPT(IZ)
4.5(+) 4.0(+) 2.5(+) 1.5(-)
Premi®Test. +(-)
-
-
-
-
ELISA
0.739
0.744
0.643
0.397
0.238 0.139 0.126 0.102 0.094 0.088
(ng/kg)
(+)
(+)
(+)
(+)
(+)
IZ: Inhibition zone, NI: no inhibition; NI: no inhibition
+(-): Douptful, partially positive
122
(-)
(-)
(-)
(-)
(-)
7.3.8. Statistical comparison of the validity of TPT and Premi® Test in OTC residue
detection in muscles of birds in group B (drug adminidistered in drinking water)
Figure 7.5 is showing the sensitivity and specificity of both microbiological tests in OTC
detection from muscle of injected birds using ELISA as gold standard. In fig.7.5a, 3 samples
were both TPT and ELISA positive; none was TPT positive and ELISA negative. 2 were TPT
negative and ELISA positive and 5 samples were both TPT and ELISA negative; TPT
therefore has a sensitivity of 60% to OTC and 100% specificity in muscle tissue of birds
administered orally. In fig.7.5b, 1 sample was both Premi® Test and ELISA positive and
none (0) was Premi® Test positive and ELISA negative. 4 samples were Premi® Test
negative and ELISA positive and 5 were both TPT and ELISA positive; The Premi® test
therefore, has a sensitivity of 20% with a100% specificity.
123
Sensitivity and specificity
Sensitivity
95% confidence interval
Specificity
95% confidence interval
Positive Predictive Value
95% confidence interval
Negative Predictive Value
95% confidence interval
Sensitivity and specificity
Sensitivity
95% confidence interval
Specificity
95% confidence interval
Positive Predictive Value
95% confidence interval
Negative Predictive Value
95% confidence interval
0.6000
0.1466 to 0.9473
1.000
0.4782 to 1.000
1.000
0.2924 to 1.000
0.7143
0.2904 to 0.9633
ELISA Positive
6
ELISA Positive
6
ELISA Negative
ELISA Negative
4
Result
Result
4
2
2
0
0.2000
0.005051 to 0.7164
1.000
0.4782 to 1.000
1.000
0.02500 to 1.000
0.5556
0.2120 to 0.8630
TPT Positive
0
TPT Negative
Premi Test Positive
Premi Test Negative
Test Method
Test Method
Fig. 7.5a Validity of TPT in OTC detection in
Muscle of birds given in Drinking water
of injected birds
Fig. 7.5b Validity of Premi test in OTC detection in
Muscle of birds given in Drinking water
of injected birds
124
Figure 7.6 shows that there is a strong correlation (r = 0.87) between the inhibition zones of
TPT and ELISA concentrations in OTC detection in muscle of group B birds.
Slope
Y-intercept when X=0.0
Spearman r
P value (two-tailed)
8.519 ± 2.400
-2.696 ± 1.410
0.8721
0.0833
Inhibition zone(mm)
6
4
2
0
0.2
-2
0.4
0.6
0.8
Conc (ng/kg)
Figure 7.6: Correlation between the inhibition zone of TPT and the tetracycline residue
concentration in muscle of group B birds
125
7.3.9 Detection of OTC residues in Liver of birds in group A (Injected with OTC) by the
Three test methods
For the TPT, inhibition zones ≥ 2mm are regarded as positive, while concentrations of OTC
above the MRL (0.6ng/kg) of liver (as evaluated by ELISA) are regarded as positive for
OTC. In Table 7.3, ELISA detected the presence of OTC on liver for the ten days, Premi®
Test detected for three days starting with a partial detection on the third day, while the TPT
detected from the 1st until the 8th day.
126
Table 7.3: OTC residue detected by the three methods in Liver of birds in group A
(injected)
Days/T 1
2
3
4
5
6
7
8
9
est
method
NI(-)
TP(IZ) NI (-) 3.6(+) 5.0(+) 5.75(+) 8.0(+) 7.5(+) 4.75(+) 2.0(+)
Premi
®Test
ELISA
-
-
0.578
(-)
0.701
(+)
+(-)
+
+
-
0.841
(+)
0.860
(+)
0.900
(+)
0.790
(+)
IZ: inhibition zone; NI: no inhibition; + (-): Douptful (partially positive)
127
0.706
(+)
0.640
(+)
10
NI(-)
-
-
0.781
0.318
(+)
(-)
7.3.10. Statistical evaluation of the reliability of the two screening test methods (TPT
and Premi® Test) in OTC residue detection in Liver of birds in group A
Figure 7.7 is showing the sensitivity and specificity of both microbiological tests in OTC
detection from Liver of OTC injected birds using ELISA as gold standard. In fig.7.7a, 7
samples were both TPT and ELISA positive; none was TPT positive and ELISA negative. 1
was TPT negative and ELISA positive and 2 samples were both TPT and ELISA negative;
TPT therefore has a sensitivity of and specificity of 87.5% and 100% respectively to OTC in
liver tissue of birds injected. In fig.7.7b, 3 samples were both Premi® Test and ELISA
positive and none (0) was TPT positive and ELISA negative. 5 samples were Premi® Test
negative and ELISA positive and 2 were both Premi® Test and ELISA negative; The Premi®
test therefore, has a sensitivity of 37.5% with a 100% specificity.
128
Sensitivity and specificity
Sensitivity
95% confidence interval
Specificity
95% confidence interval
Positive Predictive Value
95% confidence interval
Negative Predictive Value
95% confidence interval
Sensitivity and specificity
Sensitivity
95% confidence interval
Specificity
95% confidence interval
Positive Predictive Value
95% confidence interval
Negative Predictive Value
95% confidence interval
0.8750
0.4735 to 0.9968
1.000
0.1581 to 1.000
1.000
0.5904 to 1.000
0.6667
0.09430 to 0.9916
8
ELISA Negative
ELISA Negative
4
4
Result
Result
6
ELISA Positive
6
ELISA Positive
0.3750
0.08523 to 0.7551
1.000
0.1581 to 1.000
1.000
0.2924 to 1.000
0.2857
0.03669 to 0.7096
2
2
0
TPT Positive
0
TPT Negative
Test method
Premi Test Positive
Premi Test Negative
Test Method
Fig. 7.7a: Validity of TPT in OTC detection in
liver of injected birds
129
Fig. 7.7b: Validity of Premi® Test in OTC
detection in liver of injected birds
Figure 7.8 shows that there is a strong correlation (r = 0.92) between the inhibition zones of
TPT and ELISA concentrations in OTC detection in liver of injected birds.
.
Slope
Y-intercept when X=0.0
Spearman r
P value (two-tailed)
21.10 ± 4.316
-11.29 ± 3.278
0.9286
0.0022
Inhibition zone(mm)
10
8
6
4
2
0
0.5
0.6
0.7
0.8
0.9
1.0
Conc (ng/kg)
Figure 7.8: Correlation between the inhibition zone of TPT and the tetracycline residue
concentration in liver of group A birds
130
7.3.11 Detection OTC residues in Liver of birds in group B (administered with OTC in
drinking water)by the three test methods
In Table 7.4, ELISA detected the presence of OTC in the liver for the ten days, Premi® Test
partially detected on the first day, while the TPT detected from the 1st until the 6th day.
131
Table 7.4: OTC residue detected by the three methods in the liver of group B (orally
administered) birds
Days/Test
1
2
3
4
5
6
7
8
9
10
method
TPT(ZI)
5.75(+) 5.0(+) 4.5(+) 4.0(+) 3.75(+) 1.8(-)
1.0(-) NI(-)
NI(-)
NI(-)
Premi®Test
+(-)
-
-
-
-
-
-
-
-
ELISA
0.813
0.801
0.789
0.747
0.644
0.604
0.545 0.496 0.396 0.256
(+)
(+)
(+)
(+)
(+)
(+)
(-)
IZ: inhibition zone, NI: no inhibition; +(-): Douptful (partially positive)
132
-
(-)
(-)
(-)
7.3.12. Statistical comparison of the validuty of TPT and Premi® Test in OTC residue
detection in Liver of birds in group A
Figure 7.9 is showing the sensitivity and specificity of both microbiological tests in OTC
detection from Liver of injected birds using ELISA as gold standard. In fig.7.9a, 5 samples
were both TPT and ELISA positive; none was TPT positive and ELISA negative. One was
TPT negative and ELISA positive and 4 samples were both TPT and ELISA negative; TPT
therefore has a sensitivity and specificity of 83.3% and 100% respectively to OTC in liver
tissue of birds injected. In fig.7.9b, 1 sample was both Premi® Test and ELISA positive and
none (0) was Premi® test positive and ELISA negative. 5 samples were Premi® Test
negative and ELISA positive and 4 were both Premi® Test and ELISA negative; The Premi®
test therefore, has a sensitivity of 16.6% with a 100% specificity.
133
Sensitivity and specificity
Sensitivity
95% confidence interval
Specificity
95% confidence interval
Positive Predictive Value
95% confidence interval
Negative Predictive Value
95% confidence interval
Sensitivity and specificity
Sensitivity
95% confidence interval
Specificity
95% confidence interval
Positive Predictive Value
95% confidence interval
Negative Predictive Value
95% confidence interval
0.8333
0.3588 to 0.9958
1.000
0.3976 to 1.000
1.000
0.4782 to 1.000
0.8000
0.2836 to 0.9949
0.1667
0.004211 to 0.6412
1.000
0.3976 to 1.000
1.000
0.02500 to 1.000
0.4444
0.1370 to 0.7880
ELISA Positive
6
ELISA Negative
6
ELISA Positive
ELISA Negative
4
Result
Result
4
2
0
2
TPT Positive
0
TPT Negative
Premi Test Positive
Test Method
Premi Test Negative
Test Method
Fig. 7.9a Validity of TPT in OTC detection in
liver of birds given in Drinking water
Fig. 7.9b Validity of Premi test in OTC detection in
liver of birds given in Drinking water
134
Figure 7.10 shows that there is a strong correlation (r = 1) between the inhibition zones of
TPT and ELISA concentrations in OTC detection in liver of birds given OTC in drinking
water.
Slope
Y-intercept when X=0.0
Spearman r
P value (two-tailed)
15.20 ± 2.231
-7.055 ± 1.591
1.000
0.0004
Inhibition zone(mm)
8
6
4
2
0
0.5
0.6
0.7
0.8
0.9
Conc (ng/kg)
Figure 7.10: Correlation between the inhibition zone of TPT and the tetracycline residue
concentration in liver of group B birds
135
Figure 7.11 shows that there is a strong correlation (r = 0.93) between the inhibition zones of
TPT and ELISA concentrations in OTC detection generally, irrespective of the organ and
route of OTC administration.
Spearman r
95% confidence interval
P value (two-tailed)
P value summary
Exact or approximate P value?
Is the correlation significant? (alpha=0.05)
0.9336
0.8497 to 0.9714
< 0.0001
***
Gaussian Approximation
Yes
10
TPT Inhibition zone(mm)
8
6
4
2
0
0.5
-2
1.0
1.5
ELISA Conc (ng/kg)
Figure 7.11: General Correlation between the inhibition zone of TPT and tetracycline
residue concentration
136
7.4
Discussion
The comparison study was set up to find out better and more efficient microbiological
screening tool between TPT and Premi test for the detection of oxytetracycline in broiler
birds. The TPT detected OTC in both groups (A and B) at pH 6.0 and 7.2 but not pH 8.0 with
B. subtilis isolate. Contrary to the report that Bacillus subtilis at pH 6.0 best detects
teracyclines (Chang et al., 2000), this study shows that the detection of OTC in chicken at pH
7.2 is as good as the detection at pH 6.0, but pH 8.0 did not detect OTC (no inhibition zone).
This indicates that the isolate (B. subtilis) may not be sensitive to OTC at an alkaline or high
pH of ≥ 8.0.
The detection of OTC in muscle of injected and orally administered birds started right from
24 hours after the drug administration, with higher concentration in injected than orally
administered birds. The higher level of OTC in the injected group is because samples for the
test were taken from the site of injection (breast muscle). The concentration gradually
reduced by the day as the oil base drug gradually dissipated and entered the system. By the
2nd day, the drug was detected in the rest of the organs with a gradual build up in
concentration. The increase in concentration peaked in the liver and kidney on the 5th and 6th
days due to accumulation for biotransformation and excretion respectively. For the orally
administered birds on the other hand, there was a gradual daily decrease in concentration in
all the organs because of the daily dose drug administration. There was no peaking in the
liver because metabolism of the drug may have started taking place from day one of drug
administration due to the first pass effect; the drugs therefore lasted longer in the system of
injected birds than those orally administered. i.e, with oral administration of drug, absorption
from the gut means that the drug passes through the portal veins to the liver before it enters
the systemic circulation. It is thereby exposed to extraction and inactivation by the drug
metabolizing enzymes in the liver therefore impairing the absorbtion of tetracyclines in the
137
intestines (Dwight and Yuan, 1999). This effect explains some of the most dramatic reduction
in bioavailability of drug given orally and may contribute to nearly all cases of difference
between oral and parenteral bioavailability of the same drugs (Brander et al, 1993).
In comparism, TPT has a higher sensitivity to OTC in both liver and muscle of injected and
orally administered birds than Premi® Test with very few false negative results. Premi® Test
showed very poor sensitivity and high false negative results. This finding is in agreement
with the work of Okerman et al. (2004), who compared several test methods, including
Premi®Test, for the detection of tetracyclines in animal tissue. The Premi®Test results of
chicken muscle spiked with 100 µg kg-1 of all four veterinary used tetracyclines, were
negative. Pikkemaat et al. (2009), also compared two multi plate tests with Premi® test in
monitoring the presence of five different antibiotics in meat and found out that Premi® Test
could not detect the presence of tetracyclines, which was the highest occurring antibiotic as
detected by the multi-plate tests. The study shows also that Premi® test was able to detect
OTC on the days the tissues had very high concentrations. This finding agrees with the result
of the test performed for AFNOR validation of the Premi® Test kit, which indicated that
OTC was only adequately detected at twice the MRL of an organ (Pikkemaat, 2009).
Although TPT has a good sensitivity to OTC in broiler tissues, it appears to have a better
sensitivity to liver than muscle tissues for both routes of administration. This may be
attributed to the report by Mylliniemi (2004), that muscle tissue has low innate sensitivity to
B. subtillis organism. With good sensitivity of TPT to OTC, there is a strong correlation
between TPT and OTC concentration as quantified by ELISA. There is therefore a chance of
predicting the concentration of OTC with the diameter of zone of clearance (inhibition zone)
in TPT, as the zone of clearance increases with increase in OTC concentration and decreases
with decrease in OTC concentration. However, both tests had 100% specificity since all the
negative results were detected as negative by both tests.
138
An efficient screening test method for the detection of antimicrobial residues in food needs to
be of low-cost and high-throughput, and allow detection of a wide spectrum of antibiotics
(Aerts et al., 1995; Haasnoot et al., 1999; Pikkemaat, 2009). Although Premi® Test needs
relatively shorter time for preparation of samples and incubation, TPT seems to be more cost
effective and a better screening test for OTC. Since TPT was able to detect more than 50% of
OTC in the study, the occurrence of false negative result was at the barest minimum given
that it concurred with the reports of Heitzman, (1994) and Korsrud et al. (1995) who
suggested that In order to prevent false-negative results, a screening test method should be
positive for all samples that contain residues at MRL levels; preferably at 50% of the MRL.
The screening tests were carefully performed though there may be some margin of error since
the zone of inhibition of the TPT may be dependent on the texture and quantity of media
poured, as stated by Currie et al. (1998) and Koenen-Dierick and De Beer. (1998), quoted by
Mylliniemi et al. (2001), that the thickness of the agar is inversely related to the zone size,
which in turn determines the depth of the hole bored and the quantity of extract put in the
holes. The zone of inhibition may also be dependent on the the mode of administration of the
sample: putting raw samples, putting sample extract on bored holes in the medium or
absorbing the meat juice with a paper disc before placing on the agar. For the Premi®,
changes in color were sometimes difficult to interpret and the incubation time appeared to be
quite important, as continued incubation past the time at which a negative control turned
yellow could lead to false negative samples
7.5
Conclusion
Chemical and immunological methods generally were considered too specific and expensive
to be applied as an initial screening test. However, in particular for those countries that rely
upon a monitoring infrastructure including dozens of routine field laboratories, it can be
concluded that there is still a strong need for the development and implementation of
139
adequate microbial screening test methods, and more regular proficiency testing to reveal the
shortcomings in the currently applied screening methods. It should be realized that these
methods form the first line of defense in antibiotic residue monitoring, so it is essential to
have accurate screening methods in place. Since TPT is comparable to ELISA with higher
sensitivity and good predictability, it may therefore serve as a better screening test for OTC in
broiler chickens with a certain degree of reliability.
140
CHAPTER EIGHT
EFFECT OF TEMPERATURE (COOKING AND FREEZING) ON THE
CONCENTRATION OF OTC RESIDUES IN CHICKEN MEAT
8.1 Introduction
In developed countries, many researchers have been interested in evaluating whether
antibiotic residues can be destroyed by cooking procedures, pasteurization, or canning
processes (Ibrahim and Moats, 1994; Rose et al., 1995; Isidori et al., 2005; Hassani et al.,
2008; Hsie et al., 2011). Traditionally, heat stability of antibiotics has been studied based on
change in concentration using microbiological test methods. Few studies evaluating the heat
stability of veterinary drug residues have been carried out using both microbiological and
immunoassay (Franje et al., 2010).
Previous
studies
have
suggested
that
sulfamethazine
(SMZ),
oxacillin
(OXA),
chloramphenicol, aminoglycosides, quinolones, clindamycin, novobiocin, trimethoprim,
vancomycin, and azlocillin are heat-stable (Traub and Leonhard, 1995; Rose et al., 1995;
Papapanagiotou et al., 2005)) while oxytetracycline (OTC) and erythromycin were shown to
be heat-labile (Hassani et al., 2008). On the other hand, several β-lactams such as penicillin
(PCN) G, ampicillin (AMP) and amoxicillin (AMX) appear partially heat-labile (Traub and
Leonhard, 1995). Antibiotics of the same class were also reported to show different heat
stability depending on different matrices and heating treatments involved (Kitts et al., 1992;
Rose et al., 1996; Franje et al., 2010).
Since meat is always heated before consumption, few reports have been published about the
effect of heating on the stability of TCs residues in chicken. The fate of drug residues during
heat processing is however unclear. There is dearth of information on the effect of freezing
with time on the concentration of antimicrobial residues in foods of animal origin. Although
freezing is a form of preservation method of meat by impeding the growth of micro-
141
organisms, the fate of antimicrobial residues concentration when frozen with time is
unknown.
This study will thus investigate the effect of heat treatment and freezing on OTC residues in
liver and muscle tissues of broiler chicken.
8.2
Materials and methods
Tissue samples (muscle and liver) obtained from the same birds experimentally treated in
section 3.3.2 were used for this study.
8.2.1 Sample preparation
Each positive organ with high concentration of OTC residue was divided into four parts by
weight; the first part was analysed for the effect of freezing and the remaining three parts for
the effect of heat treatment (boiling, microwaving and roasting). After each treatment, 2-5g
piece (depending on the initial weight of the organ) of each organ was macerated using sterile
pestle and mortar and emulsified with equal volume of distilled water at 1:1 ratio (Nonga et
al., 2009). The emulsified samples were centrifuged at 5000rpm for 10 minutes and the
supernatant, decanted into eppendorf tubes and stored for analysis with ELISA immunoassay.
8.2.2 Effect of heat treatment
o Boiling: The weighed sample was placed into a strainer, immersed in 10 ml of water
bath preheated to 100°C and cooked for 30 minutes and allowed to cool before meat
juice extraction.
o Roasting: The weighed sample was placed on a metal baking tray and cooked to well
done in the center of electric oven at 200°C for 30 minutes and allowed to cool before
extraction.
142
o Microwaving: The sample portin was placed on a turned table in the microwave and
cooked under full power (900 W) for 3 min, removed, and allowed to cool before
extraction
o All the heat treated samples were tested for residue with TPT and ELISA
immunoassay.
8.2.3 Effect of freezing on OTC residue
o The extracted meat juice from the first portion of the organ was kept frozen at -20°C
and tested for residue first after 24hrs, and every 3 days for 10 days
8.3
RESULTS
8.3.1 Effect of Cooking Methods on OTC Residue in Muscle Using TPT
Table 8.1 shows the effect of different cooking methods on the concentration of OTC
residues using TPT at two different pH levels. The mean inhibition zones of raw muscles at
pH 6.0 was reduced from 7.00mm to 3.56mm (49.1%), to 3.25 (53.5%) and to
2.12mm(69.6%) for microwaving, boiling and roasting respectively. The mean inhibition
zones of raw muscles at pH 7.2 were reduced from 6mm to 3.94mm (34.4%), to 2.81mm
(53.25%) and to 1.94mm (67.7%), for microwaved, boiled and roasted muscle samples
respectively.
143
Table 8.1: Proportion of the effect of cooking methods on OTC in muscle using TPT
Cooking
methods
pH 6.0 Inhibition zones (mm)
Mean
Difference
from Raw
Difference
(%)
pH 7.2 Inhibition zones (mm)
Mean
Difference
from Raw
Difference
(%)
Raw
7.00
_
_
6.00
_
_
Microwaved
3.56
3.44
49.11
3.94
2.07
34.38
Roasted
3.25
3.75
53.57
2.81
3.19
53.25
Boiled
2.13
4.88
69.64
1.94
4.06
67.71
144
Figure 8.1: There were no statistical differences (P > 0.05) in the median (mean) values of
raw and microwaved muscle at both pH 6 (p = 0.1; Mann Whitney U = 16) and pH 7.2 (p =
0.18; Mann Whitney U = 19).
145
Mann Whitney test
P value
Exact or approximate P value?
P value summary
Are medians signif. different? (P < 0.05)
10
0.1015
Gaussian Approximation
ns
No
Inhibition zone.
8
6
4
2
M
ic
ro
R
aw
0
Cooking methods (pH 6.0)
8
Mann Whitney test
P value
Exact or approximate P value?
P value summary
Are medians signif. different? (P < 0.05)
0.1870
Gaussian Approximation
ns
No
Inhibition zone.
6
4
2
M
ic
ro
w
.
R
aw
0
Cooking method (pH 7.2)
Figure 8.1: Effect of microwaving on OTC concentration in muscle using TPT at pH6.0 and
pH7.2
146
Figure 8.2: There were no significant differences (p > 0.05) in the median (mean) values of
raw and roasted muscle at both pH 6 (p = 0.1; Mann Whitney U = 16.55) and pH 7.2 (p =
0.07; Mann Whitney U = 14.5).
147
10
Mann Whitney test
P value
Exact or approximate P value?
P value summary
Are medians signif. different? (P < 0.05)
0.1109
Gaussian Approximation
ns
No
Inhibition zone.
8
6
4
2
R
oa
st
ed
R
aw
0
Cooking method (pH 6.0)
Mann Whitney test
P value
Exact or approximate P value?
P value summary
Are medians signif. different? (P < 0.05)
0.0714
Gaussian Approximation
ns
No
8
Inhibition zone.
6
4
2
R
oa
st
ed
R
aw
0
Cooking method (pH 7.2)
Figure 8.2: Effect of roasting on OTC conc. in muscle using TPT at pH6.0 and pH7.2
148
Figure 8.3: The difference between the median (mean) values of raw and boiled
muscle at both pH 6 (p = 0.02; Mann Whitney U = 10) and pH 7.2 (p = 0.01; Mann
Whitney U = 8) was statistically significant (p < 0.05)
149
Mann Whitney test
P value
Exact or approximate P value?
P value summary
Are medians signif. different? (P < 0.05)
0.0211
Gaussian Approximation
*
Yes
10
Inhibition zone.
8
6
4
2
B
oi
le
d
R
aw
0
Cooking methods (pH 6.0)
Mann Whitney test
P value
Exact or approximate P value?
P value summary
Are medians signif. different? (P < 0.05)
0.0130
Gaussian Approximation
*
Yes
8
Inhibition zone.
6
4
2
B
oi
le
d
R
aw
0
Cooking method (pH 7.2)
Figure 8.3: Effect of boiling on OTC conc. in muscle using TPT at pH6.0 and pH7.2
150
8.3.2: Effect of cooking methods on OTC residue in Liver using the TPT
Table 8.2 shows the effect of different cooking methods on the concentration of OTC
residues using TPT at two different pH levels. The mean inhibition zone of raw liver at pH
6.0 was reduced from 4mm to 1.6(57.75%), to 0.81(79.75%), and to 0.44(89%) for boiling,
microwaving and roasting respectively. At pH 7.2, the mean inhibition zone of raw liver was
reduced from 4.9mm to 2.09(57.40%), to 1.0(79.63%) and to 0.55(88.89%) for boiled,
microwaved and roasted samples respectively.
151
Table 8.2: Proportion of the effect of cooking methods on OTC in liver using TPT
Cooking
pH 6.0 Inhibition zones (mm)
pH 7.2 Inhibition zones (mm)
methods
Mean values
Difference
Diff (%)
Mean values
Difference
Diff (%)
Raw
4.0
_
_
4.91
_
_
Boiled
1.69
2.31
57.75
2.09
2.82
57.40
Microwaved
0.81
3.06
79.75
1.00
3.91
79.63
Roasted
0.44
3.39
89.00
0.55
4.36
88.89
152
Figure 8.4: There were no statistical differences (P < 0.05) in the median (mean) values of
raw and boiled liver at pH 6.0 (p = 0.09; Mann Whitney U = 15.5) and at pH 7.2, the
difference in the mean values were slightly significant (p = 0.01; Mann Whitney U = 22.5),
153
Mann Whitney test
P value
Exact or approximate P value?
P value summary
Are medians signif. different? (P < 0.05)
0.0878
Gaussian Approximation
ns
No
5
Inhibition zones
4
3
2
1
B
oi
le
d
R
aw
0
Cooking method ( pH 6.0)
Mann Whitney test
P value
Exact or approximate P value?
P value summary
Are medians signif. different? (P < 0.05)
0.0135
Gaussian Approximation
*
Yes
Inhibition zones
6
4
2
B
oi
le
d
R
aw
0
Cooking method (pH 7.2)
Figure 8.4: Effect of boiling on OTC conc. in liver using TPT at pH6.0 and pH7.
154
Fig 8.5: The difference between the median (mean) values of raw and microwaved liver at
both pH 6 (p = 0.0014; Mann Whitney U = 1.5) and pH 7.2 (p = 0.003; Mann Whitney U =
5.5) was statistically significant (p < 0.05)
155
5
Mann Whitney test
P value
Exact or approximate P value?
P value summary
Are medians signif. different? (P < 0.05)
0.0014
Gaussian Approximation
**
Yes
Inhibition zone
4
3
2
1
M
ic
ro
w
.
R
aw
0
Cooking method
pH 6.0
Mann Whitney test
P value
Exact or approximate P value?
P value summary
Are medians signif. different? (P < 0.05)
0.0003
Gaussian Approximation
***
Yes
Inhibition zones
6
4
2
M
ic
ro
w
R
aw
0
Cooking method
pH 7.2
Figure 8.5: Effect of microwaving on OTC conc. in Liver using TPT at pH6.0 and pH7.2
156
Fig 8.6: The difference between the median (mean) values of raw and roasted liver at both
pH 6 (p = 0.0007; Mann Whitney U = 0.0) and pH 7.2 (p < 0.0001; Mann Whitney U = 1.5)
is statistically significant (p < 0.05)
157
Mann Whitney test
P value
Exact or approximate P value?
P value summary
Are medians signif. different? (P < 0.05)
0.0007
Gaussian Approximation
***
Yes
5
Inhibition zones
4
3
2
1
R
oa
st
ed
R
aw
0
pH 6.0
Cooking method
Mann Whitney test
P value
Exact or approximate P value?
P value summary
Are medians signif. different? (P < 0.05)
< 0.0001
Gaussian Approximation
***
Yes
Inhibition zones
6
4
2
R
oa
st
ed
R
aw
0
Cooking method pH 7.2
Figure 8.6: Effect of roasting on OTC conc. in Liver using TPT at pH6.0 and pH7.2
158
8.3.3 Effect of cooking methods on OTC concentration in muscle tissues
Table 8.3 shows that microwaving slightly increased the concentration of OTC in muscle
tissues from 0.991ng/kg to 1.003ng/kg (1.2%) and a slight increase to 0.992 ng/kg (0.3%) by
roasting. The mean concentration of OTC was reduced from 0.991 to 0.956 ng/kg (3.5%) by
boiling.
159
Table 8.3: Effect of cooking methods on OTC concentration in muscle tissue
OTC concentration (ng/kg)
Cooking
Mean values
methods
Mean difference
Percentage
from raw value
Difference (%)
Raw
0.991
_
_
Microwaved
1.003
-0.012
-1.23
Boiled
0.956
0.034
3.5
Roasted
0.992
-0.003
-0.28
160
Figure 8.7 shows that there is no significant (p > 0.05) difference between the mean
concentration of raw muscle and the cooking methods. (For microwaving, p = 0.948; Boiling,
p = 0.608; Roasting, p = 0.746)
161
Mann Whitney test
P value
Exact or approximate P value?
P value summary
Are medians signif. different? (P < 0.05)
0.9488
Gaussian Approximation
ns
No
1.10
Conc (ng/kg).
1.05
1.00
0.95
0.90
M
IC
R
O
W
.
R
A
W
0.85
Cooking method (Microwaving)
Mann Whitney test
P value
Exact or approximate P value?
P value summary
Are medians signif. different? (P < 0.05)
0.6085
Gaussian Approximation
ns
No
1.1
Conc (ng/kg).
1.0
0.9
0.8
0.7
B
O
IL
E
D
R
A
W
0.6
Mann Whitney test
P value
Exact or approximate P value?
P value summary
Are medians signif. different? (P < 0.05)
Cooking method (Boiling)
0.7466
Gaussian Approximation
ns
No
1.10
Conc (ng/kg).
1.05
1.00
0.95
0.90
R
O
A
ST
ED
R
A
W
0.85
Cooking method (Roasting)
Figure 8.7: Effect of cooking methods on OTC concentration in muscle using ELISA
162
8.3.4
Effect of cooking methods on OTC concentrations in liver tissues
Microwaving reduced the mean concentration of OTC in raw liver from 1.02 to 1.002 ng/kg
(1.85%) by microwaving, to 0.993 ng/kg (2.83%) by boiling and to 0.985 ng/kg (3.17%) by
roasting (Table 8.4)
163
Table 8.4: Effect of cooking methods on OTC concentration in liver tissue
Cooking methods
Mean
Mean difference Percentage
concentration
from raw
difference (%)
(ng/kg)
RAW
1.021
_
_
MICROWAVED
1.002
0.019
1.85
BOILED
0.993
0.028
2.83
ROASTED
0.985
0.032
3.17
164
Figure 8.8: There are significant (p < 0.05) differences in the mean concentration of OTC
between raw and boiled (p = 0.006) and between raw and roasted (p = 0.005) liver tissues.
The difference between raw and microwaved (p = 0.681) liver is not significant (p > 0.05).
165
Mann Whitney test
P value
Exact or approximate P value?
P value summary
Are medians signif. different? (P < 0.05)
0.6814
Gaussian Approximation
ns
No
Conc (ng/kg).
1.1
1.0
0.9
M
IC
R
O
W
R
A
W
0.8
Cooking method (Microw aving)
Mann Whitney test
P value
Exact or approximate P value?
P value summary
Are medians signif. different? (P < 0.05)
0.0060
Gaussian Approximation
**
Yes
1.10
Conc (ng/kg).
1.05
1.00
0.95
0.90
B
O
IL
E
D
R
A
W
0.85
Cooking method (Boiling)
Mann Whitney test
P value
Exact or approximate P value?
P value summary
Are medians signif. different? (P < 0.05)
0.0053
Gaussian Approximation
**
Yes
1.10
Conc (ng/kg).
1.05
1.00
0.95
R
O
A
S
TE
D
R
A
W
0.90
Cooking method (Roasting)
Figure 8.8: Effect of cooking methods on OTC concentration in liver tissue
166
8.3.5 Effect of freezing on OTC concentration in Muscle and Liver of broilers
Table 8.4 shows that the freezing times have no steady decreasing or increasing effects on the
tissues
167
Table 8.5: Effect of freezing time on OTC concentration in muscle and liver tissues
Period
Median OTC Concentrations
(ng/kg) in muscle
Median OTC Concentrations (ng/kg)
in liver
Values
Difference
from day 0
Values
Difference
from day 0
Day 0(prefreezing)
1st day
0.763
_
0.766
_
0.761
0.002
0.766
0.0
3rd day
0.764
-0.001
0.767
-0.001
6th day
0.764
-0.001
0.765
0.001
9th day
0.762
0.001
0.764
0.002
168
Figure 8.9: There is no significant difference (p > 0.05) in the effect of the different freezing
times on the concentration of OTC residue in both liver and muscle tissues (Kruskal-Wallis =
0.09; P = 0.999).
169
25% Percentile
Median
75% Percentile
Day 0
0.6768
0.7435
0.8283
1st day
0.6843
0.7380
0.8170
3rd day
0.6810
0.7430
0.8218
P value
Exact or approximate P value?
P value summary
Do the medians vary signif. (P < 0.05)
Number of groups
Kruskal-Wallis statistic
6th day
0.6855
0.7455
0.8210
9th day
0.6818
0.7390
0.8240
0.9990
Gaussian Approximation
ns
No
5
0.09037
1.2
0.8
0.6
y
h
9t
da
y
h
6t
da
y
da
d
3r
td
1s
ay
0
ay
0.4
D
Conc.
1.0
Time
Figure 8.9: Effect of freezing time on OTC residues on muscle and liver
170
8.4
Discussion
The effect of cooking procedures on oxytetracycline in poultry was determined by both TPT
and ELISA immunoassay. Although the TPT is said to detect antibiotics around their
maximum residue limits (MRL), it cannot quantify the concentration of the drug. ELISA on
the other hand quantifies and gives the concentration of the drug. The result of the TPT for
both pH in muscle gave a net mean inhibitory zone of 42-68% reduction with the maximum
(68%) reduction shown by boiling and the minimum shown by microwaving but the
difference between the inhibitory zones of raw and cooked samples were not statistically
significant (P<0.05). ELISA also shows that the changes in the concentrations of OTC with
the cooking methods are not statistically different. A similar work done by Javadi et
al.,(2011) on doxycycline, had a net decrease in mean inhibitary zones of 33-100% with
maximum and minimum inhibitory zone also shown by boiling and microwaving
respectively, the decrease again was not statistically significant. The highest reduction of
OTC in muscles by boiling in this study also agrees with the work of Rose et al. (1996), with
observed substantial net reductions in OTC of 35–94 % and also in pig muscle with a net
reduction of 48-60% (Nguyen et al., 2013). The reduction of OTC in liver samples on the
other hand had a higher net mean inhibitory reduction for both pH at 58-89% with roasting
having the highest (89%) reduction and boiling the least (58%). The difference in the mean
inhibitory values between raw and cooked samples were significant (P<0.05) with significant
decrease between raw and microwaved and raw and roasted. The ELISA analysis also shows
significant decrease in the concentration of OTC in the liver when given heat treatment; the
significance was between raw and roasted samples. Although Javadi et al. (2009), had total
(100%) reduction of inhibitory zones for both muscle and liver samples at pH 6.0 and 7.2, the
mean inhibitory zones of the raw samples were less than 2mm in diameter as opposed to
mean value of more than 4mm in this study.
171
The results of this study are consistent with several other studies that reported decrease in
antimicrobial residues concentrations in foods following heat treatment. Therefore, the
possibility of reducing the apparent toxic effect of these drugs to consumers is increased.
Based on the effect of cooking on the OTC surveyed samples in raw meat from market and
farms in Saudi Arabia, the mean detectable concentration of OTC in raw muscle and liver
samples were above the MRL but some decreased below the MRL after cooking for 20
minutes (Al Ghamdi et al., 2000) while some were still above the MRL after cooking.
Reduction in TC concentrations during boiling was due to migration of the TC from the meat
to cooking medium (water) while during the microwaving and roasting processes, reduction
was due to juice exuding out from the meat. The overall loss of TC residues was due to
denaturation of protein - TC compounds. From the safety and toxicological point of view,
these findings show an additional advantage of cooking as a food processing method.
8.5
Conclusion
Cooking methods generally can reduce the concentration of OTC in meat, boiling was more
effective in reducing the concentration of OTC in muscle while roasting was more effective
in reducing concentration of liver samples. Although the effect of increase in cooking time
was not done in this study, it may not be out of place to suggest that microwaving had the
greatest effect on OTC concentration reduction in tissues since it had the least cooking time.
Since some of the reductions in OTC concentrations were not statistically significant, there is
a possibility that their OTC concentrations after cooking, were still above the MRL. It is
therefore better to prevent the occurrence of violative levels of drug residues in raw meats but
where inevitable, cooking for longer time should be explored.
.
172
CHAPTER NINE
GENERAL CONCLUSIONS AND RECOMMENDATIONS
9.1
Prevalence study with Three Plate Test (TPT) and Premi® Test
This study shows that there is high prevalence of antimicrobial residues in commercial
broilers in Enugu metropolis as indicated by the TPTand Premi® tests. The organs of the
birds also contain violative levels of tetracycline residues. It is therefore presumed that
consumers in the study area may be constantly exposed to violative levels of these residues.
This situation indicates a widespread indiscriminate use of antimicrobials and suggests nonimplementation of withdrawal periods in poultry.
Recommendations
It is therefore recommended that:
a. Since the TPT and Premi test are basically qualitative, It is necessary to confirm the
presence and concentration of detected antimicrobial residues and identify the specific
antimicrobial within a class with a quantitative test method such as chromatographic
(HPLC) or immuno-enzymatic method (ELISA)(Mitchell et al., 1998; Kirbis, 2007).
b. Prudent use of antimicrobials in livestock production should be inculcated in
veterinarians and farmers: this is best advocated during vet and farmers annual
congresses/conferences
c. There should be routine monitoring of antimicrobial residues in farms and abattoirs
d. The enforcement of the legislation regarding drug use in veterinary practice and
livestock production is vital to establishing surveillance programme for detecting drug
residues in meat and other foods of animal origin. This is to prevent the introduction
of chemotherapeutic agents into the human food chain. The legistlation against the
non-prudent use of antimicrobials should therefore be enforced in this country.
173
9.1.1 Organ (matrix) Distribution of Antimicrobial Residues
The study shows that the kidney out of the four organ matrices used had the highest
prevalence of antimicrobial residues and the muscle had the lowest. The study therefore
shows that the use of only one organ as a sample matrix may be underestimating the extent of
antimicrobial residues thereby giving an incorrect prevalence.
Recommendation: The use of kidney as sample matrix is always recommended because it is
the major excretory organ of most drugs, but they usually give false positive results due to
presence of natural inhibitors of bacterial growth such as lysozyme which are often present in
kidneys (Kirbis, 2007) and so should be used along with other organ matrixes as was done in
this study.
9.2
Comparative Study on the Sensitivity of TPT and Premi® Test in OTC Detection
in Broilers
The TPT with locally sourced Bacillus subtilis is a better detection/screening test for
oxytetracycline(OTC) than the Premi® test and OTC is very well detected at both pH 6.0 and
pH 7.2 but not detected at pH 8.0. There was a strong correlation between TPT and OTC
concentration.
Recommendation: it is therefore recommended that two plate test at pHs 6.0 and 7.2 or even
one plate test at either pHs with Bacillus subtilis can be satisfactoryly used to detect
tetracyclines in broilers. Since there was a strong correlation, the concentration of OTC
residues may be predicted with the zones of inhibition of the TPT.
9.3
Effect of temperature on OTC residues in broiler muscle and liver.
Although all cooking methods are effective, microwaving and roasting had better reduction
effect than boiling on liver tissues since both methods significantly (p < 0.05) reduced the
concentration of OTC in liver, while boiling had a better reduction effect than microwaving
174
and roasting on muscle tissue with a significant (p < 0.05) reduction in muscle tissue.
Cooking methods used in the present study are similar to those widely applied to meat
during household cooking. Therefore, the observed findings may be helpful in confirming
and selecting the ideal method for cooking so as to effectively reduce TC and probably other
antibiotic residues in meat prior to consumption. Freezing had no effect on the concentration
of OTC residues in both organs.
Recommendations: It may therefore be recommended that meat be cooked with adequate
heating temperature with longer time to reduce the concentration of OTC residue.
Microwaving and roasting should be used for liver tissues since they have better reduction
effect and boiling should be used for muscle tissues.
..
175
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APPENDICES
Appendix I:
Antimicrobial
Mechanism of action of Aminoglycosides
Mechanism of action
Spectrum of activity
Amikacin
Inhibitors of protein
Used for the treatment of infections caused by
Dihydrostreptomycin
synthesis. Proteins are
aerobic Gram-negatives. Effective against
Gentamicin
synthesized on cell
some Gram-positives like Staphylococcus
Kanamycin
structures called ribosomes.
aureus. The use of aminoglycosides in the
Neomycin
Bactericidal or bacteriostatic treatment of infection in animals has been
Streptomycin
High doses can affect
tempered by toxicity considerations in the
Apramycin
animals and humans
animal treated. Often, systemic use is limited
because some ribosomes are
to the treatment of serious Gram-negative
similar to those in bacteria.
infections resistant to less toxic medications.
Acidic or purulent conditions can hamper
their effect and the presence of cations
(calcium or magnesium ions) can decrease
antibacterial effects
205
Appendix II:
Penicillin G
Mechanism of action of Beta-lactam antimicrobial
Mechanism of
Spectrum of activity
action
Inhibitors of
Active against aerobic and anaerobic Gram-positives.
Penicillin
bacterial cell wall
Susceptible aerobes include most beta-haemolytic
G benzathine,
Synthesis by
Streptococci and beta-lactamase-negative Staphylococci.
G potassium,
binding enzymes
Most species of anaerobes are susceptible, including
G procaine,
involved in
Clostridium species, but excluding beta-lactamase producing
G sodium
peptidoglycan
Bacteroides species. Ineffective against betalactamase
production (ie,
producers and those bacteria that are resistant to other
Penicillin-binding
mechanisms, such as those having a relatively impermeable
protein or PBS).
cell wall. Has therefore little activity against many
Animals and
Staphylococci and most Gram-ve bacteria.
Antimicrobial
humans not
affected because
their cells do not
have walls.
Aminopenicillins
Active against penicillin-sensitive Gram-positive bacteria and
Amoxicillin
some Gram-negative bacteria. Gram-positive spectrum
includes alpha- and betahaemolytic Streptococci, some
Staphylococci
species
and
Clostridia
species.
Gram-
negatives: Escherichia coli, many strains of Salmonella, and
Pasteurella multocida. Is susceptible to destruction by betalactamases.)
Ampicillin
Active against alpha- and beta-haemolytic streptococci,
including Streptococcus equi, non-penicillinase-producing
Staphylococcus species, and most strains of Clostridia. Also
effective against Gram-negative bacteria, such as
Escherichia coli, Salmonella and Pasteurella multocida
Cephalosporins
These cephalosporins have the highest activity against
First generation
Grampositive bacteria, including most Corynebacteria,
Cefadroxil
Streptococci and Staphylococci, particularly Staphylococcus
206
Cefazolin
aureus. Activity against Gram-negative bacteria: Escherichia
Cephalexin
coli,
Cephalothin
Pasteurella and Salmonella. Many anaerobic bacteria are
Cephapirin
susceptible to these antibacterials, with the exception of beta-
Cephradine
lactamase-producing Bacteroides and Clostridium difficile.
Second
generation
Cefaclor
Have
Cefamandole
Cefmetazole
Cefonicid
Cefotetan
Cefoxitin
Klebsiella
slightly
pneumoniae,
less
Haemophillus influenzae,
efficacy
than
first-generation
cephalosporins against Gram-positive pathogens. Are more
efficient than first-generation drugs in the treatment of
infections caused by Gram-negative organisms.
The most effective of the cephalosporins against antibioticresistant Gram-negative bacteria. Less effective than other
cephalosporins against Gram-positive bacteria.
Cefprozil
Cefuroxime
Third generation
Cefixime
Cefoperazone(1)
Cefotaxime
Cefpodoxime,
Ceftazidime
Ceftizoxime
Ceftriaxone
Broader Gram-positive activity, including good activity
against Streptococci but less activity against Pseudomonas
than third generation cephalosporins. Active against betalactamase-producing strains, as well as against anaerobes.
Broader Gram-positive activity, including good activity
against Streptococci but less activity against Pseudomonas
than thirdgeneration cephalosporins. Active against betalactamase-producing strains, as well as against anaerobes
New generation
Ceftiofur
Sources: Pharmacology, 1997; Forbes et al., 2007; USP 1999, 2000a, 2000b
207
Appendix III
Antimicrobial
Mechanism of action of Chloramphenicol
Mechanism of action
Spectrum of activity
Chloramphenicol
Inhibit protein synthesis
Broad spectrum. Effective against:
by binding to 50S
Staphylococcus aureus, Streptococcus
ribosomal subunit
pyogenes, Escherichia coli, Proteus vulgaris,
Aerobacter aerogenes, Salmonella species,
Pseudomonas species, anaerobic bacteria
Sources: Pharmacology, 1997; Forbes et al., 2007;US Pharmacopea, 2000e
208
Appendix IV
Antimicrobial
Enrofloxacin
Mechanism of action of quinolones
Mechanism of action
Spectrum of activity
Inhibit DNA synthesis by Broad spectrum. Bactericidal
binding DNA gyrases
at relatively low
concentrations,
highly bio-available
following either oral or
parenteral
administration in most
species, and achieves good
penetration of body tissues
and fluids
Sarafloxacin
Escherichia coli infections
Oxolinic acid
Against
Gram-negative
bacteria
flumequin
Against
Gram-negative
bacteria
Note: Enrofloxacin is not labelled for use in food-producing animals (United States of America and Canada).
Sarafloxacin is not labelled for use in laying hens producing eggs for human consumption. Sources: USP, 2000h, 2000m;
GESAMP, 1997.
209
Appendix V
Antimicrobial
Mechanism of action of Lincosamides
Mechanism of action
Spectrum of activity
Lincosamides:
Inhibitors of protein synthesis. Proteins
Activity against many Gram-
Clindamycin
are synthesized on cell structures called
positive bacteria and many
Lincomycin
ribosomes. Bactericidal or bacteriostatic
anaerobic bacteria, but not
Pirlimycin
High doses can affect animals and
effective against most Gram-
humans because some ribosomes are
negatives. Effective against
similar to those in bacteria.
Staphylococcus species,
Streptococcus species (except
Streptococcus feacalis
Sources: Pharmacology, 1997; Forbes et al., 2007, USP 2000j
210
Appendix VI
Antimicrobial
Mechanism of action of Macrolids
Mechanism of action
Spectrum of activity
Erythromycin:
Inhibitors of protein
Primarily against Gram-positives, such as
Base
synthesis. Proteins are
Staphylococcus and Streptococcus species,
Estolate
synthesized on cell
including many that are resistant to penicillins by
Ethylsuccinate
structures called
means of beta-lactamase production. Active against
Gluceptate
ribosomes. Bactericidal
Campylobacter and Pasteurella species. Has
Lactobionate
or bacteriostatic High
activity against some anaerobes, but Bacteroides
Phosphate
doses can affect animals
fragilis is usually resistant. Most Pseudomonas,
Stearate
and humans because
Escherichia coli, and Klebsiella strains are resistant
Thiocyanate
some ribosomes are
to erythromycin. Resistant strains of Staphylococci
similar to those in
and Streptococci have been reported. Cross
bacteria.
resistance to other macrolides can also occur.
Tilmicosin
In vitro activity against Gram-positive micro-
Phosphate
organisms and Mycoplasma. Active against certain
Gram-negatives, such as Haemophilus somnus,
Pasteurella haemolytica and P. multocida.
Gram-negatives such as Enterobacter aerogenes,
Escherichia coli, Klebsiella pneumoniae,
Pseudomonas aeruginosa, Salmonella and Serratia
species are very resistant to Tilmicosin
Tylosin Base
Spectrum of activity similar to that of erythromycin
Tylosin Phosphate
but more active than erythromycin against certain
Tylosin Tartrate
Mycoplasmas
Sources: Pharmacology, 1997; Forbes et al., 2007, US pharmacopeia, 2000c
211
AppendixVII
Antimicrobial
Mechanism of action of Nitrofurans
Mechanism of action
Spectrum of activity
Nitrofurantoin
Uncertain exact mechanism, Good activity against gram-
furazolidone
may have several bacterial positive and gram-negative
enzyme targets and directly bacteria. Specifically used in
damage the DNA
poultry for the treatment of
Salmonellosis
Source: Forbes et al., 2007.
212
Appendix VIII
Antimicrobials
Mechanism of action of Tetracyclines
Mechanism of action
Spectrum of activity
Chlortetracycline
Proteins are synthesized on
Broad spectrum with activity against Gram
Doxycycline
cell structures called
positives and Gram-negatives, including some
Oxytetracycline
ribosomes. They inhibit
anaerobics. Active also against Chlamydia,
Tetracycline
protein sunthesis by binding
Mycoplasma,
30S ribosomal subunit.
Protozoa and several Ricketsiae, including
Bactericidal or bacteriostatic
Ehrlichia and Haemobartonella. Active
High doses can affect animals against Escherichia coli, Klebsiella species,
and humans because some
Salmonella species, Staphylococcus species
ribosomes are similar to those
and Streptococcus species. However,
in bacteria.
resistance has been acquired by some
coliforms, mycoplasmas, streptococci and
staphylococci.
Source: USP 2000g. Pharmacology, 1997; Forbes et al., 2007.
213
Appendix IX
Antimicrobial
Mechanism of action of Sulphomamides
Mechanism of action
Spectrum of activity
Sulphonamides:
Inhibitors of folic acid
Antibacterial; antiprotozoal; broad
Sulfachlorpyridazine
synthesis. Folic acid is needed
spectrum; inhibiting Gram-positives
Sulfadimethoxine
to make RNA and DNA for
and Gram-negatives and some
Sulfamethazine
growth and multiplication, and
Protozoa, such as Coccidia.
Sulfanilamide
bacteria must synthesize folic
Ineffective against most obligate
Sulfaquinoxaline
acid. They are Bacteriostatic.
anaerobes and should not be used to
Sulfathiazole
Animals and humans obtain treat serious anaerobic infections.
folic acid from their diets, so Resistance of animal pathogens to
they are not affected.
sulphonamides is widespread as a
result of more than 50 years of
therapeutic use. Nevertheless, still
used in combination with other
medications.
Source:. USP, 2000i. Pharmacology, 1997; Forbes et al., 2007.
214
Appendix X
Antimicrobials banned for use in animals intended for food production.
Antibiotic
Country
Reason
Reference
Spectinomycin
USA
Its use is limited by
USP, 2000d
the ready
development of
bacterial resistance
Enrofloxacin
USA
Its use is limited by
USP, 2000h
the ready
development of
bacterial
resistance
(quinolone)
Chloramphenicol
Argentina, Canada,
Induces human
USP, 2000e;
EU, Japan, Nigeria,
aplastic anaemia
GESAMP, 1997;
USA
Rifampine
SANCO, 2001a.
Not labelled in USA
Tumorgenicity and
or Canada for use in
teratogenic effects
animals, including
on experimental
food producing
animals
USP, 2000k
animals
Nitrofurazone
Not labelled in USA
Mutagenic effect,
or Canada for use in
carcinogenic effect
NAFDAC, 1996;
WHO, 1997; Ali,
animals, including
1999; Hoogenboom
food producing
et al., 2002; Nisha,
animals. Banned for
2004; Cooper et al.,
use in Nigeria in
livestock and poultry
production
215
2005.
Appendix XI
The Codex alimentarius Maximum Residue Limits for Antimicrobials on chicken
According to the FAO/WHO Food Standards
Name of drug
Tissue
MRL (µg/kg) Year of
adoption
Avilamycin
Muscle
300
2009
Liver
200
2009
Kidney
200
2009
Fat
200
2009
Procain benzylpenicillin
Muscle
50
1999
Liver
50
1999
Kidney
50
1999
Tetracyclines (Chlor, Oxy and Muscle
200
2003
tetra)
Liver
600
2003
Kidney
1200
2003
Egg
400
2003
Collistin
Muscle
150
2008
Kidney
200
2008
Liver
150
2008
Egg
300
2008
Fat
150
2008
Danofloxacin
Muscle
200
2001
Liver
400
2001
kidney
400
2001
Fat
100
2001
Dihydrostreptomycin/streptomycin Muscle
600
2001
Liver
600
2001
Kidney
1000
2001
Fat
600
2001
Erythromycin
Muscle
100
2008
Liver
100
2008
Kidney
100
2008
Egg
50
2008
Fat
100
2008
Flumequin
Muscle
500
2005
Liver
500
2005
Kidney
3000
2005
Fat
1000
2005
Lincomycin
Muscle
200
2003
Liver
500
2003
Kidney
500
2003
Fat
100
2003
216
Monensin
Narasin
Neomycin
Sarafloxacin
Spectinomycin
Spiramycin
Tilmocosin
Tylosin
Muscle
Liver
Kidney
Fat
Muscle
Liver
Kidney
Fat
Muscle
Liver
Kidney
Egg
Fat
Muscle
Liver
Kidney
Fat
Muscle
Liver
Kidney
Egg
Fat
Muscle
Liver
Kidney
Fat
Muscle
Liver
Kidney
Fat/skin
Muscle
Liver
Kidney
Egg
Fat/skin
FAO/WHO Food Standards (Codex alimentarius)
217
10
10
10
100
15
50
15
50
500
500
1000
500
500
10
80
80
20
500
2000
5000
2000
2000
200
600
800
300
150
2400
600
250
100
100
100
300
100
2009
2009
2009
2009
2012
2012
2012
2012
1999
1999
1999
1999
1999
2001
2001
2001
2001
1999
1999
1999
1999
1999
1997
1997
1997
1997
2011
2011
2011
2011
2009
2009
2009
2009
2009
Appendix XII
Some Veterinary Antimicrobials and Generics with their Varying Withdrawal Periods
and Limitations for use in Poultry (A Compendium of Veterinary Products, 2007).
Product
name Company
Route*
Limitation for use
W/T#
(Generic)
Aureomycin® 110G
Alpharma
Feed
None
7d
Aureomycin®
220G
Alpharma
Feed
None
7d
Gallimycin®
Vétoquinol
Dw
(Drinking
water)
Do not use in birds producing 24h
food for human purposes
Feed
Do not use in laying birds
0d
Dw
None
0d
Dw
Do not use in laying birds
3d
Dw
Do not use in laying birds
3d
Dw
None
7-14d
Dw
None
7-14d
Lincomix®
premix
110 Pfizer
Lincomix®
powder
soluble Pfizer
Lincomycin
Bio
Agri
Spectinimycin 100 Mix
soluble powder
Linco-spectrin® 100 Pfizer
soluble powder
Neomix soluble
Pfizer
powder
Neomycin 325
Vétoquinol
Neo Oxymed
Medprodex
Dw
Do not feed to laying birds
7d
Oxy 250
Medprodex
Dw
Do not feed to laying birds
7d
Oxy 1000
Jaapharm
Dw
Do not use in laying birds
7d
Oxy tetra A
Dominion
Dw/Feed
Oxytetracycline
200Granular Premix
Bio Agri
Mix
Feed
Eggs taken from treated birds 7d
within 60hrs after the latest
treatment with this drug must
not be used for food
None
7d
Paracillin® SP
Intervet
Dw
Do not use in laying birds
Salinomycin
Bio Agri
Mix
Feed
Do not feed to replacement or
laying birds
218
2d
Sodium
Sulphomethazine
Solution 12.5%
Dominion
Dw
This product must not be used 12d
in laying birds
Sulfa “25”
P.V.L
Dw
This product must not be used 12d
in laying poultry
Sulfa,25% solution
BimedaMTC
Dw
Do not use in laying birds
Sulphaquinoxalin
Dominion
19.2%
liquid
concentrate
Dw
This product must not be used 12d
in laying birds
Sulphaquinoxalin-S
Dw
None
4d
Feed
None
7d
Terramycin®
Premix
Medprodex
100 Phibrio
12d
Tetracycline 250
Vétoquinol
Dw
Do not use in laying birds
5d
Tetracycline 1000
Vétoquinol
Dw
Do not use in laying birds
5d
Tetracycline HCL
Dominion
Dw
This product must not be used 5d
in laying birds
Teramed 250
Medprodex
Dw
Do not feed to laying birds
Virginiamycin 44
Premix
Bio Agri
Mix
Feed
Do not use in birds producing
eggs for food purposes
5d
The Withdrawal Times Listed Correspond To Label Doses Only.* Route of Administration of Drug # Withdrawal Period in
Meat
219
Appendix XIII
Premi® Test Indicative Data on Detection Level of Meat Types and Egg
GROUP
SUBSTANCE
DETECTION LEVEL (mg/kg)
Chicken
Pork
Beef
Egg
B-lactam
Amoxicillin
Ampicillin
B-penicillin
Penicillin
5
5
2.5
-
5
5
2.5
-
5
5
2.5
-
5
5
2.5
Cephalosporin
Cefquinome
Ceftiofor
75
100
100
200
100
100
400
Macrolides
Tylosin
Erythromycin
Lincomycin
Spiramycin
50
100
100
1000
50
100
100
1000
50
100
100
1000
50
50
-
Tetracycline
Chlortetracycline
Oxytetracycline
Doxycycline
Tetracycline
100
100
100
-
100
100
100
-
100
100
100
-
600
400
200
200
Sulphonamides
Sulphamethazine 75
Sulphadiazine
75
sulphamethaxole -
75
75
-
100
75
-
25
25
25
Amonoglycosides Gentamycine
Streptomycine
Neomycine
100
1500
300
100
1500
300
100
3000
300
100
1000
3000
Quonolones
>600
>100
>600
>100
>600
>100
-
Enrofloxacine
Flumequine
220
Chloramphenicol
Chloramphenicol
Florphenicol
Salinomycin
Naracin
Monensin
Zn-bacitracin
2,500
100
1,250
1,250
1,250
1,250
2,500
100
-
2,500
100
-
2500
-
Polypeptides
Virginiamycine
Bacitracine
500
500
500
500
500
500
-
Appendix XIV
Api®50 CH record sheet for identification of Bacillus subtilis
221
Appendix XV
Three Plate Test clear zone of inhibition indicative of positive samples for antimicrobial
residue
222
Clear zone
of inhibition
Appendix XVI
Table 4.1: antimicrobial residues detection with TPT in 100 sampled broiler birds
223
Organ(s)
No Positive
No Negative
Muscle only
1
99
Gizzard only
1
98
Liver only
5
93
Kidney only
10
83
Muscle & Gizzard
0
83
Muscle & Liver
0
83
Muscle & Kidney
0
83
Gizzard & Liver
2
81
Gizzard & Kidney
1
80
Kidney & Liver
17
63
Muscle, Gizzard, Liver
5
58
Muscle, Kidney, Liver
0
58
Muscle, Gizzard, Kidney
1
57
Kidney, Gizzard, Liver
19
38
Muscle, Gizzard, Liver, Kidney
2
36
Total
64
36
Appendix XVII
Premi test colour indication for positive and negative result for antimicrobial results
224
Douptful samples
(blue coloration)
Positive samples
(Purple coloration
coloration)
Negative samples
(yellow coloration
coloration)
Appendix XVIII
ntimicrobial residues detection with Premi® Test in 100 sampled broiler birds
Antimicrobial
225
Organ(s)
No Positive
No Negative
Muscle only
0
100
Gizzard only
0
100
Liver only
2
98
Kidney only
20
78
Muscle & Gizzard
9
69
Muscle & Liver
0
69
Muscle & Kidney
0
69
Gizzard & Liver
0
69
Gizzard & Kidney
7
62
Kidney & Liver
0
62
Muscle, Gizzard, Liver
0
62
Muscle, Kidney, Liver
6
56
Muscle, Gizzard, Kidney
0
56
Kidney, Gizzard, Liver
6
50
Muscle, Gizzard, Liver, Kidney
10
40
Total
60
40
Appendix XIX
Elisa work - sheet
226
A
2
1
Std 0 Std 4
3
4
Spl 1
B
Std 0 Std 4
Spl 1
C
Std 1 Std 5
Spl 2
D
Std 1 Std 5
Spl 2
E
Std 2 Std 6
Spl 3
F
Std 2 Std 6
Spl 3
G
Std 3 Cont.
H
Std 3
5
6
7
8
9
10
Std – standard; Spl – sample
Appendix XX
Calculation of tetracycline concentration from standard curve
227
11
12
The formula: y = mx + c.
Where: m = slope
C = intercept
Y = optical density (OD)
X = concentration
Therefore, to calculate concentration (X),
X=
Y–C
M
Since the standard curve gave: slope (M) = -1.506
Intercept (C) = 1.529
Therefore, concentration (C) = OD(Y) – 1.529
-1.506
Appendix XXI
Daily detection of OTC residue with TPT and Premi® Test in organs of group A birds
injected intramuscularly.
228
Days
1
2
3
4
5
6
7
8
9
10
Organ
TPT (pH)
Premi® Test
Muscle
Liver
Kidney
Gizzard
Muscle
Liver
Kidney
Gizzard
Muscle
Liver
Kidney
Gizzard
Muscle
Liver
Kidney
Gizzard
Muscle
Liver
Kidney
Gizzard
Muscle
Liver
Kidney
Gizzard
Muscle
Liver
Kidney
Gizzard
Muscle
Liver
Kidney
Gizzard
6.0
7.0
NI
NI
NI
6.5
3.7
3.0
2.0
3.8
5.0
5.0
3.0
3.0
6.0
5.0
4.0
3.0
8.0
5.0
5.0
NI
8.0
5.0
3.0
NI
5.0
7.0
5.0
NI
2.0
5.5
3.5
7.2
7.0
NI
NI
NI
6.0
3.5
3.0
2.0
4.0
5.0
5.0
3.5
3.5
5.5
5.0
4.0
3.0
8.0
5.0
5.0
NI
7.0
4.5
2.0
NI
4.5
7.0
4.5
NI
2,0
5.0
3.5
8.0
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
+
+
+
+
+
+
+
+(-)
+
+(-)
+(-)
+(-)
-
Muscle
Liver
Kidney
Gizzard
Muscle
Liver
Kidney
Gizzard
NI
NI
3.0
2.0
NI
NI
NI
NI
NI
NI
2.0
2.0
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
-
Appendix XXII
Daily detection of OTC residue with TPT and Premi® Test in organs of group B birds given
in drinking water
229
Days Organ
1
2
3
4
5
6
7
8
9
10
TPT (pH)
Muscle
Liver
Kidney
Gizzard
Muscle
Liver
Kidney
Gizzard
Muscle
Liver
Kidney
Gizzard
Muscle
Liver
Kidney
Gizzard
Muscle
Liver
Kidney
Gizzard
Muscle
Liver
Kidney
Gizzard
Muscle
Liver
Kidney
Gizzard
Muscle
Liver
Kidney
Gizzard
6.0
5.0
6.5
5.5
4.0
3.5
5.0
4.0
4.5
2.0
4.0
3.0
3.0
NI
4.0
2.0
2.0
NI
3.5
3.5
2.0
NI
2.0
2.5
3.2
NI
1.0
2.0
1.5
NI
NI
1.0
NI
7.2
4.0
5.0
5.5
4.5
4.0
5.0
4.0
4.0
NI
5.0
3.0
3.0
NI
4.0
3.0
2.0
NI
3.0
3.0
2.0
NI
1.5
2.5
3.0
NI
1.0
2.5
1.0
NI
NI
NI
NI
8.0
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
Muscle
Liver
Kidney
Gizzard
Muscle
Liver
Kidney
Gizzard
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
1.0
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
Premi®
Test
+(-)
+(-)
+
+
-
NI: no inhibition
Appendix XXIII
Effect of freezing time on OTC concentration of samples positive for OTC residue
230
OTC concentrations (ng/kg)
Positive samples
Day 0
Day 1
1
1.009
1.010
1.009
1.011
1.012
2
1.000
1.003
1.001
1.004
1.004
3
0.843
0.830
0.835
0.833
0.838
4
0.784
0.778
0.782
0.785
0.782
5
0.672
0.676
0.673
0.679
0.676
6
0.691
0.709
0.705
0.705
0.699
7
0.599
0.590
0.595
0.591
0.599
8
0.590
0.592
0.595
0.590
0.584
9
0.732
0.730
0.732
0.735
0.732
10
0.746
0.746
0.751
0.749
0.749
11
0.744
0.722
0.744
0.748
0.721
12
0.743
0.746
0.742
0.743
0.746
231
Day 3
Day 6
Day 9
Appendix XXIV
Work In Progress, supervision by supervisors
232
Appendix XXV
Tetracycline ELISA kit
233