EZENDUKA, EKENE VIVIENNE

Transcription

1 EZENDUKA, EKENE VIVIENNE OCCURRENCE OF ANTIMICROBIAL RESIDUES IN BROILERS IN ENUGU METROPOLIS AND THE EFFECT OF TEMPERATURE ON THE CONCENTRATION OF OXYTETRACYCLINE RESIDUE FACULTY OF VETERINARY MEDICINE DEPARTMENT OF VET. PUBLIC HEALTH & PREVENTIVE MEDICINE Fred Attah Digitally Signed by: Content manager s Name DN : CN = Webmaster s name O= University of Nigeria, Nsukka OU = Innovation Centre 1

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 DEPARTMENT OF VETERINARY PUBLIC HEALTH AND PREVENTIVE MEDICINE, UNIVERSITY OF NIGERIA, NSUKKA NOVEMBER,

3 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,

4 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

5 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 Date (supervisor) Prof. J. A. Nwanta Date (supervisor) Prof. A. A. Anaga Date (Head of Department) Prof. C. U. Nwosu Date (Dean) ii

6 Prof. Junaidu Kabir (External Examiner) Date 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

7 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

8 TABLE OF CONTENTS Title Declaration Certification Dedication Acknowledgement Table of Contents List of Tables List of Figures Appendices Abstract Page i ii iii iv v xii xiii xv xvii CHAPTER ONE: INTRODUCTION 1.1 Background of Study Statement of the Problem Research Questions Study Aim and Objectives Significance of the Study 9 CHAPTER TWO: LITERATURE REVIEW 2.1 Antimicrobial Agents Definition Antibiotics 11 v

9 2.2 Antimicrobial use in Animals Classification of Antimicrobial Agents for Veterinary Use Aminoglycosides Beta-lactam Antimicrobial Agents Chloramphenicol Quinolones (Fluoroquinolones) Lincosamides Macrolids Nitrofurantoin Tetracyclines Sulphonamides Antimicrobials Banned For Use in Food Animal Production Drug Residues and Related Terms Residues Drug residues Drug Approval and Safety Evaluation of Antimicrobial Residues No Effect or Maximum no Adverse Effect Acceptable Daily Intake (ADI) Tolerance level or Maximum Residue Limit (MRL) Withdrawal Time Withdrawal Time for Veterinary Drugs Antimicrobial use in Poultry Antimicrobials as Growth Promoters Antimicrobials as Coccidiostats in Poultry Production Therapeutic Antimicrobial Use in Poultry 27 vi

10 2.12 Causes of Violative Level of Antimicrobial Residues in Animal Tissues Public Health Hazards and Harmful Effects of Antimicrobial Residues Drug Allergy and Hypersensitive Reaction Effects on Human Gut Microbiota Development of Resistance to Antimicrobial Agents Effect on Bone Marrow/Bone Marrow Depression Carcinogenic Effect Industrial Effect Other Harmful Effects Prevention and Control of Antimicrobial Residue Occurrence in Foods of Animal Origin Disease prevention Proper Diagnosis and Antimicrobial Susceptibility Testing Appropriate Use of Antimicrobials and Route of Administration Appropriate Dosage Regimen Monitoring and Surveillance of Antimicrobial Residue Antimicrobial Residues Detection and Identification Test Matrix Methods of Detection of Antimicrobial Residues in Foods of Animal Origin Microbiological Method Immunochemical Methods Chromatographic Methods Effect of heat on Residue 47 CHAPTER THREE: GENERAL MATERIALS AND METHODS vii

11 3.1 Study Area Study Design Survey Studies Experimental Studies Sample Source, Population and Sampling Technique Survey Studies Experimental Study Specimen Preparation Data Presentation and Analysis 56 CHAPTER FOUR: ANTIMICROBIAL RESIDUES SURVEY IN COMMERCIAL BROILERS USING THREE PLATE TEST 4.1 Introduction Method Isolation of Bacillus subtilis Identification Molecular Characterization of Isolates Antimicrobial Sensitivity Test The Three Plate Test Results Isolation, Identification and Sensitivity of Bacillus subtilis Sensitivity Test Prevalence of Antimicrobial Residues in Broilers Occurrence of Antimicrobial Residues in Broiler Meat and Organs Organ Distribution of Antimicrobial Residues According to ph Discussion 78 viii

12 4.5 Conclusions 79 CHAPTER FIVE: ANTIMICROBIAL RESIDUES SURVEY IN COMMERCIAL BROILERS USING PREMI TEST 5.1 Introduction Materials and method Antibiotics Residues Detection Results Antibiotic Residues Detection in Commercial Broilers Antibiotics Residues Detection in Sampled Organs Detection of Antimicrobial Residues by TPT and Premi Test Discussion Conclusions 90 CHAPTER SIX: DETECTION AND QUANTITATION OF TETRACYCLINE RESIDUES IN COMMERCIAL BROILERS 6.1 Introduction Materials and methods Organ Sample Collection And Preparation Tetracycline Residues Detection Results Calculation of OTC Concentration from Optical Density (OD) Organ Distribution of Tetracycline Residues Comparison of Mean Values of the Organs with their MRL s Discussion 106 ix

13 6.5 Conclusion 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 Materials and Methods Experimental Study Design Experimental Drug Administration Testing for OTC Residue in Experimental Birds Results TPT Detection of Oxytetracycline Residues in Group A Birds TPT Detection of Oxytetracycline Residues in Orally Treated Birds ELISA Detection of Oxytetracycline Residues in Muscle Samples ELISA Detection of Oxytetracycline Residues in Liver Samples Comparing the Detection Ability of TPT and Premi Test in OTC Residues Detection in Muscles of Birds in Group A Statistical Comparison of the Validity of TPT and Premi Test in OTC Residue Detection In Muscles of Group A Birds Comparing the Detection Ability of TPT and Premi Test in OTC Residue Detection in Muscles of Birds in Group B Statistical Comparison of the Validity of TPT and Premi Test in OTC Residue Detection in Muscles of Birds in Group B Comparing the Detection Ability of TPT and Premi Test in OTC Residue Detection in Liver of Birds in Group A Statistical comparison of the validity of TPT and Premi Test in OTC residue detection in Liver of birds in group A 128 x

14 Comparing the Detection Ability of TPT and Premi Test in OTC Residue Detection in Liver of birds in Group B Statistical Comparison of the Validity of TPT and Premi Test in OTC Residue Detection in Liver of Birds in Group B Discussion Conclusion 139 CHAPTER EIGHT: EFFECT OF HEATING PROCESSES (COOKING METHODS) AND FREEZING ON OTC RESIDUES IN CHICKEN MEAT AND ORGANS 8.1 Introduction Materials and Methods Sample Preparation Effect of Heat Treatment Effect of Freezing on OTC Residue Results Effect of Cooking Methods on OTC Residue in Muscle Using TPT Effect of Cooking Methods on OTC Residue in Liver using the TPT Effect of Cooking Methods on OTC Concentration in Muscle Tissues Effect of Cooking Methods on OTC Concentration in Liver Tissues Effect of Freezing on OTC Concentration in Both Tissues Discussion Conclusion 172 CHAPTER NINE: GENERAL CONCLUSIONS AND RECOMMENDATIONS 9.1 Prevalence Study with Three Plate Test (TPT) and Premi Test Organ (matrix) Distribution of Antimicrobial Residues Comparative Study on the Sensitivity of TPT and Premi 174 Test in OTC Detection in Broilers xi

15 9.3 Effect of Temperature on OTC Residues in Broiler Muscle and Liver 174 REFERENCES 176 APPENDICES 205 LIST OF TABLES Table Title Page 4.1 Positive Samples According to ph Tetracycline Residues Detection According to Concentration OTC Residue Detected by the Three Methods in Muscle of Birds in group A (Injected) OTC Residue as Detected by the Three Methods in Muscle of Birds in Group B (Orally administered) OTC Residue Detected by the Three Methods in Liver of Birds in Group A OTC Residue Detected by the Three Methods in the Liver of Group B Birds Proportion of the Effect of Cooking Methods on OTC in Muscle using TPT Proportion of the Effect of Cooking Methods on OTC in Liver using TPT Effect of Cooking Methods on OTC Concentration in Muscle tissue Effect of Cooking Methods on OTC Concentration in Liver Tissue Effect of Freezing Time on OTC Concentration in Muscle and Liver Tissues 168 xii

16 LIST OF FIGURES Figure Title Page 3.1 Geographical positioning of the three major poultry markets in Enugu metropolis with their coordinates Schematic Presentation of Sampling Population and Technique Schematic Presentation of Experimental Design Microscopic Morphology of Bacillus Organism API 50 Plates Amplification of 595 bp 16S rrna of Bacillus subtilis Antimicrobial Susceptibility Plates Prevalence of Antimicrobial Residues in Broilers Organ Distribution of Antimicrobial Residues Prevalence of Antimicrobial Residues in Commercial Broilers Organ Distribution of Antimicrobial Residues Detection of Antimicrobial Residues with TPT and Premi Test Tetracycline Calibration Curve: Optical Density Versus Concentration of Standard a Tetracycline Residues Concentration in the Muscle b Tetracycline Residues Concentration in the Liver c Tetracycline Residues Concentration in the Kidney d Tetracycline Residues Concentration in the Gizzard Daily mean concentration of OTC residue detected in muscle tissues of birds in both groups A and B Daily OTC residue detection and quantification with ELISA in liver tissues of birds in both groups A and B Validity of TPT and Premi Test in OTC Detection in Muscle of Injected Birds 119 xiii

17 7.4 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 Birds given in Drinking water Correlation between the Inhibition Zone of TPT and the Tetracycline Residue Concentration in Muscle of Group B Birds Validity of TPT in OTC Detection in Liver of Injected Birds Correlation between the Inhibition Zone of TPT and the OTC Residue Concentration in Liver of Group A Birds Validity of TPT in OTC Detection in Liver of Birds Given in Drinking Water Correlation between the Inhibition Zone of TPT and the Tetracycline Residue Concentration in Liver of Group B Birds General Correlation between the Inhibition Zone of TPT and Tetracycline Residue Concentration Effect of Microwaving on OTC Concentration in Muscle using TPT at ph6.0 and ph Effect of Roasting on OTC Conc. in Muscle using TPT at ph 6.0 and ph Effect of Boiling on OTC Conc. in Muscle using TPT at ph 6.0 and ph Effect of Boiling on OTC Conc. in Liver using TPT at ph 6.0 and ph Effect of Microwaving on OTC Conc. in Liver using TPT at ph 6.0 and ph Effect of Roasting on OTC Conc. in Liver using TPT at ph 6.0 and ph Effect of Cooking Methods on OTC Concentration in Muscle using ELISA Effect of Cooking Methods on OTC Concentration in Liver Tissue Effect of Freezing Time on OTC Residues on Muscle and Liver 170 xiv

18 APPENDICES Appendix Title Page I. Mechanism of action of Aminoglycosides 205 II. Mechanism of action of Beta-lactam antimicrobial 206 III. Mechanism of action of Chloramphenicol 208 IV. Mechanism of action of quinolones 209 V. Mechanism of action of Lincosamides 210 VI. Mechanism of action of Macrolids 211 VII. Mechanism of action of Nitrofurans 212 VIII. Mechanism of action of Tetracyclines 213 IX. Mechanism of action of Sulphonamides 214 X. Antimicrobials banned for use in animals intended for food production 215 XI. XII. 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 XVI. Antimicrobial residues detection with TPT in 100 sampled broiler birds 224 XVII. XVIII. 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 XIX. Elisa Work Sheet 227 XX. Calculation of Tetracycline Concentration from Standard Curve 228 xv

19 XXI. XXII. XXIII. 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 XXIV. Work In Progress, Supervision by Supervisors 232 XXV. Tetracycline ELISA Kit 233 xvi

20 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

21 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 < 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

22 (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

23 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

24 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

25 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

26 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

27 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

28 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

29 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

30 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

31 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

32 CHAPTER TWO LITERATURE REVIEW 2.1 Antimicrobial agents 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

33 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 d) Erythromycin from Streptomyces erythreas in e) Chlortetracycline from Actinomyces aureofaciens in (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

34 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

35 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

36 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 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

37 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) : 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) 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

38 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) 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) Lincosamides (Appendix V) 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) : 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

39 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) 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

40 2.5 Drug Residues and Related Terms 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 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

41 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

42 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

43 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

44 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

45 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

46 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 This 24

47 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) 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 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

48 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 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) 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

49 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 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 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

50 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

51 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) 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 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

52 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; Raison- Peyron 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

53 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) 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 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

54 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) 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

55 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) 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

56 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) 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

57 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

58 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) 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

59 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 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

60 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) 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

61 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. Easy to use and handle (Pikemaat et al., 2007; Javadi et al., 2009) II. 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. Reduced time to obtain the result (Fagbamaila et al., 2012) VI. Good sensitivity and specificity (Pikemaat et al., 2007) VII. Detection capability (CCβ) with an error Probability (β) < 5% (Mylliniemi, 2004) 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

62 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 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 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

63 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

64 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 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

65 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

66 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

67 (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) 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

68 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 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

69 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), β- lactams 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) 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

70 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 %, 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, % by roasting except for sulfadiazine and % 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

71 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 802,295km 2 and a population of about 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

72 Figure 3.1: Geographical positions of the three major poultry markets in Enugu Metropolis 50

73 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: 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 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 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

74 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

75 Enugu metropolis (poultry markets) Artisan Gariki Ogbete v 5w 4b 30 2v 5w 3b 30 2v 5w 3b ] v- visit; w- week; b- birds Figure 3.2: Schematicc presentation of sampling population and technique 53

76 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

77 50 4 Residue test 46 Group A(23) OTC (Inj.) Group B(23) OTC (water) 2b/daily Residue test 2b/daily Residue test Figure 3.3: Schematic presentation of experimental design 55

78 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 ( 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 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

79 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

80 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

81 4.2 Method The three plate test was used for the antimicrobial residues detection with Bacillus subtilis as the test organism 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 90 o C 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 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 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

82 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 API 50 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 Molecular Characterization of isolates The characterization of the isolates follows three basic processes: Extraction of the DNA from the sample PCR amplification of the target DNA, Detection of target DNA by gel-electrophoresis. i. 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

83 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 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 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 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

84 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 H 2 O) and mixed with PCR H 2 O (Forward + Reverse + PCR H 2 O). 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

85 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 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

86 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 hours and examined for inhibition around each discw. The diameter of the zone of complete inhibition was measured by millimetre scale The Three Plate Test Reagents and procedure Test sample: broiler meat as sampled in section 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

87 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 hrs, clear zone of inhibition with annular diameter 2mm indicates positive for antimicrobial residues (Mylliniemi et al., 2001). 4.3 RESULTS 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

88 Gram positive short rods in chains Single short rods Figure 4.1: Microscopic morphology of Bacillus organism 66

89 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

90 A. Before inoculation (all sugars were red) B: After inoculation (Yellow colour shows fermentation) Figure 4.2: API 50 Plates 68

91 Molecular characterization Bacillus subtilis was identified at 595bp region of 16s rrna of the bacteria (Figure 4.3). 595 bp Lanes 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

92 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

93 A. Gram positive discs B. Gram negative discs Figure 4.4: Antimicrobial susceptibility plates 71

94 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

95 36% % Positive % Negative 64% Figure 4.5: Prevalence of antimicrobial residues in broilers 73

96 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 = < ) 74

97 Chi-square Chi-square, df P value P value summary One- or two-sided Statistically significant? (alpha<0.05) 64.50, 3 < *** NA Yes Positive Negative Frequency Kidney Liver Gizzard Muscle Organs Figure 4.6: Organ distribution of antimicrobial residues 75

98 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

99 Table 4.1: Positive samples according to ph Matrix No ph positive Multiple 6.0/ / /8.0 All three Muscle Liver kidney Gizzard Total

100 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

101 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

102 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

103 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 Antibiotics residues detection The antibiotics residues were detected on the same 100 broilers and 400 organs as sampled in section 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

104 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. The ampoules were incubated for 3-4 hours until the negative control turned yellow 5.3 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

105 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

106 Figure 5.1: Prevalence of Antimicrobial residues in commercial broilers 84

107 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 = < ) 85

108 80 Chi-square Chi-square, df P value P value summary 41.30, 6 < *** 60 Frequency Positive Negative Douptful 0 Kidney Liver Gizzard Muscle Organs Figure 5.2: Organ distribution of antimicrobial residues 86

109 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

110 80 P value P value summary One- or two-sided Statistically significant? (alpha<0.05) ns Two-sided No Prevalence Positive Negative 0 TPT Premi Test Test type Figure 5.3: Detection of antimicrobial residues with TPT and Premi Test 88

111 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

112 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

113 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

114 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 Organ sample collection and preparation The sample collection and preparation was as described in chapter 3: sections 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 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

115 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

116 6.5 Results 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

117 Slope Y-intercept when X= ± ± Optical density Conc (ng/kg) Figure 6.1 Tetracycline calibration curve: optical density versus concentration of standard 95

118 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

119 Table 6.1: Tetracycline residues detection according to concentration Organ Frequency [Concentration (ng/kg)] Proportion < 0.1(undetected) > 0.5 detected (%) Muscle (96) Gizzard (82) Kidney (88) Liver (96) Total (90.5) 97

120 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 = < ). 98

121 1.5 Wilcoxon Signed Rank Test Theoretical median Actual median P value (two tailed) Exact or estimate? Significant (alpha=0.05)? < Gaussian Approximation Yes 1.0 Conc Muscle Tissue MRL Figure 6.2a: Tetracycline residues concentration in the muscle 99

122 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 was significantly (P < 0.05) higher than its MRL at (P = < ) 100

123 1.5 Wilcoxon Signed Rank Test Theoretical median Actual median P value (two tailed) Exact or estimate? Significant (alpha=0.05)? < Gaussian Approximation Yes Conc (ng/kg) Liver Tissue MRL Figure 6.2b Tetracycline residues concentration in the liver 101

124 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 was significantly (p<0.05) lower than its MRL at 1.20 as shown in Fig 6.2c (P = < ). 102

125 1.5 Wilcoxon Signed Rank Test Theoretical median Actual median P value (two tailed) Exact or estimate? Significant (alpha=0.05)? < Gaussian Approximation Yes Conc (ng/kg) Kidney MRL Figure 6.2c Tetracycline residues concentration in the kidney 103

126 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 was significantly (p < 0.05) higher than its MRL at (P = < ) as shown in Fig 6.2d. 104

127 Wilcoxon Signed Rank Test Theoretical median Actual median P value (two tailed) Exact or estimate? Significant (alpha=0.05)? < Gaussian Approximation Yes Conc (ng/kg) Gizzard MRL Figure 6.2d: Tetracycline residues concentration in the gizzard 105

128 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

129 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

130 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

131 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), an organization that mainly certifies 109

132 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 Experimental Study design: as stated in section 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

133 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 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 2 nd day (Appendix xxii). 111

134 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 1 st 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) 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 1 st until the 7 th day in the injected group and from 1 st until the 6 th 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 ng/kg in decreasing order and Group B, had lower OTC concentrations ranging from ng/kg. 112

135 Conc. (ng/kg) Gp A (Injection) Gp B (Oral) Days Figure 7.1: Daily mean concentration of OTC residue detected in muscle tissues of birds in both groups A and B 113

136 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 6 th day in group a birds and from the 1 st day in group B birds (Figure 7.2). In Group A, the concentration gradually increased from the 1 st day (0.578ng/kg) peaking at day 5(0.900ng/kg) and gradually decreased to 0.318ng/kg on the 10 th day. Group B showed a steady daily decrease ranging from

137 Conc. (ng/kg) Days Gp A (Injection) Gp B (Oral) Figure 7.2: Daily OTC residue detection and quantification with ELISA in liver tissues of birds in both groups A and B 115

138 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 4 th day, while the TPT detected for the first five days (Table 7.1). 116

139 Table 7.1: OTC residue detected by the three methods in muscle of Injected birds Days/Test method TPT(IZ) 7.0(+) 6.7(+) 3.9(+) 3.25(+) 3.0(+) NI(-) NI(-) NI(-) NI(-) NI(-) Premi Test (-) ELISA (ng/kg) (+) (+) (+) (+) (+) (+) (+) (-) (-) (-) IZ: inhibition zone, NI: no inhibition; NI: no inhibition 117

140 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

141 Result Sensitivity and specificity Sensitivity 95% confidence interval Specificity 95% confidence interval Positive Predictive Value 95% confidence interval Negative Predictive Value 95% confidence interval to to to to ELISA Positive ELISA Negative Result Sensitivity and specificity Sensitivity 95% confidence interval Specificity 95% confidence interval Positive Predictive Value 95% confidence interval Negative Predictive Value 95% confidence interval to to to to ELISA Positive ELISA Negative 1 0 TPT Positive TPT Negative 0 Premi test positive Premi test negative Test method Test method Fig 7.3a: Validity of TPT in OTC detection in muscle of injected birds Fig 7.3b: Validity of Premi Test in OTC detection in muscle of injected birds 119

142 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. Inhibition zone(mm) Slope Y-intercept when X=0.0 Number of XY Pairs Spearman r ± ± 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

143 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

144 Table 7.2: OTC residue as detected by the three methods in muscle of birds orally administered Days/Test method TPT(IZ) 4.5(+) 4.0(+) 2.5(+) 1.5(-) NI(-) NI(-) NI(-) NI(-) NI(-) NI(-) Premi Test. +(-) ELISA (ng/kg) (+) (+) (+) (+) (+) (-) (-) (-) (-) (-) IZ: Inhibition zone, NI: no inhibition; NI: no inhibition +(-): Douptful, partially positive 122

145 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

146 6 Sensitivity and specificity Sensitivity 95% confidence interval Specificity 95% confidence interval Positive Predictive Value 95% confidence interval Negative Predictive Value 95% confidence interval to to to to ELISA Positive ELISA Negative 6 Sensitivity and specificity Sensitivity 95% confidence interval Specificity 95% confidence interval Positive Predictive Value 95% confidence interval Negative Predictive Value 95% confidence interval to to to to ELISA Positive ELISA Negative 4 4 Result 2 Result 2 0 TPT Positive TPT Negative 0 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

147 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) ± ± Inhibition zone(mm) 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

148 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 1 st until the 8 th day. 126

149 Table 7.3: OTC residue detected by the three methods in Liver of birds in group A (injected) Days/T est method TP(IZ) NI (-) 3.6(+) 5.0(+) 5.75(+) 8.0(+) 7.5(+) 4.75(+) 2.0(+) NI(-) NI(-) Premi - - +(-) Test ELISA (-) (+) (+) (+) (+) (+) (+) (+) (+) (-) IZ: inhibition zone; NI: no inhibition; + (-): Douptful (partially positive) 127

150 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

151 8 6 Sensitivity and specificity Sensitivity 95% confidence interval Specificity 95% confidence interval Positive Predictive Value 95% confidence interval Negative Predictive Value 95% confidence interval to to to to ELISA Positive ELISA Negative Sensitivity and specificity Sensitivity 95% confidence interval Specificity 95% confidence interval Positive Predictive Value 95% confidence interval Negative Predictive Value 95% confidence interval to to to to ELISA Positive ELISA Negative Result 4 Result TPT Positive TPT Negative 0 Premi Test Positive Premi Test Negative Test method Test Method Fig. 7.7a: Validity of TPT in OTC detection in liver of injected birds Fig. 7.7b: Validity of Premi Test in OTC detection in liver of injected birds 129

152 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) ± ± Inhibition zone(mm) 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

153 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 1 st until the 6 th day. 131

154 Table 7.4: OTC residue detected by the three methods in the liver of group B (orally administered) birds Days/Test method TPT(ZI) 5.75(+) 5.0(+) 4.5(+) 4.0(+) 3.75(+) 1.8(-) 1.0(-) NI(-) NI(-) NI(-) Premi Test +(-) ELISA (+) (+) (+) (+) (+) (+) (-) (-) (-) (-) IZ: inhibition zone, NI: no inhibition; +(-): Douptful (partially positive) 132

155 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

156 Sensitivity and specificity Sensitivity 95% confidence interval Specificity 95% confidence interval Positive Predictive Value 95% confidence interval Negative Predictive Value 95% confidence interval to to to to Sensitivity and specificity Sensitivity 95% confidence interval Specificity 95% confidence interval Positive Predictive Value 95% confidence interval Negative Predictive Value 95% confidence interval to to to to ELISA Positive ELISA Negative 6 ELISA Positive ELISA Negative Result 4 2 Result TPT Positive TPT Negative 0 Premi Test Positive Premi Test Negative Test Method 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

157 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) ± ± Inhibition zone(mm) 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

158 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) to < *** Gaussian Approximation Yes 10 8 TPT Inhibition zone(mm) ELISA Conc (ng/kg) Figure 7.11: General Correlation between the inhibition zone of TPT and tetracycline residue concentration 136

159 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 2 nd 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 5 th and 6 th 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

160 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

161 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

162 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

163 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

164 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 were used for this study 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 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

165 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 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 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

166 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 (%) Mean ph 7.2 Inhibition zones (mm) Difference from Raw Difference (%) Raw Microwaved Roasted Boiled

167 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

168 10 Mann Whitney test P value Exact or approximate P value? P value summary Are medians signif. different? (P < 0.05) Gaussian Approximation ns No 8 Inhibition zone Raw Micro 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) Gaussian Approximation ns No 6 Inhibition zone Raw Microw. Cooking method (ph 7.2) Figure 8.1: Effect of microwaving on OTC concentration in muscle using TPT at ph6.0 and ph

169 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

170 10 Mann Whitney test P value Exact or approximate P value? P value summary Are medians signif. different? (P < 0.05) Gaussian Approximation ns No 8 Inhibition zone Raw Roasted Cooking method (ph 6.0) 8 Mann Whitney test P value Exact or approximate P value? P value summary Are medians signif. different? (P < 0.05) Gaussian Approximation ns No 6 Inhibition zone Raw Roasted Cooking method (ph 7.2) Figure 8.2: Effect of roasting on OTC conc. in muscle using TPT at ph6.0 and ph

171 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

172 Mann Whitney test P value Exact or approximate P value? P value summary Are medians signif. different? (P < 0.05) Gaussian Approximation * Yes 10 8 Inhibition zone Raw Boiled 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) Gaussian Approximation * Yes 8 6 Inhibition zone Raw Boiled Cooking method (ph 7.2) Figure 8.3: Effect of boiling on OTC conc. in muscle using TPT at ph6.0 and ph

173 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

174 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 Boiled Microwaved Roasted

175 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

176 Mann Whitney test P value Exact or approximate P value? P value summary Are medians signif. different? (P < 0.05) Gaussian Approximation ns No 5 4 Inhibition zones Raw Boiled 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) Gaussian Approximation * Yes 6 Inhibition zones Raw Boiled Cooking method (ph 7.2) Figure 8.4: Effect of boiling on OTC conc. in liver using TPT at ph6.0 and ph7. 154

177 Fig 8.5: The difference between the median (mean) values of raw and microwaved liver at both ph 6 (p = ; Mann Whitney U = 1.5) and ph 7.2 (p = 0.003; Mann Whitney U = 5.5) was statistically significant (p < 0.05) 155

178 5 Mann Whitney test P value Exact or approximate P value? P value summary Are medians signif. different? (P < 0.05) Gaussian Approximation ** Yes 4 Inhibition zone Raw Cooking method Microw. ph 6.0 Mann Whitney test P value Exact or approximate P value? P value summary Are medians signif. different? (P < 0.05) Gaussian Approximation *** Yes 6 Inhibition zones Raw Microw Cooking method ph 7.2 Figure 8.5: Effect of microwaving on OTC conc. in Liver using TPT at ph6.0 and ph

179 Fig 8.6: The difference between the median (mean) values of raw and roasted liver at both ph 6 (p = ; Mann Whitney U = 0.0) and ph 7.2 (p < ; Mann Whitney U = 1.5) is statistically significant (p < 0.05) 157

180 Mann Whitney test P value Exact or approximate P value? P value summary Are medians signif. different? (P < 0.05) Gaussian Approximation *** Yes 4 Inhibition zones Raw Cooking method Roasted ph Mann Whitney test P value Exact or approximate P value? P value summary Are medians signif. different? (P < 0.05) < Gaussian Approximation *** Yes Inhibition zones Raw Cooking method Roasted ph 7.2 Figure 8.6: Effect of roasting on OTC conc. in Liver using TPT at ph6.0 and ph

181 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 ng/kg (0.3%) by roasting. The mean concentration of OTC was reduced from to ng/kg (3.5%) by boiling. 159

182 Table 8.3: Effect of cooking methods on OTC concentration in muscle tissue OTC concentration (ng/kg) Cooking Mean values Mean difference Percentage methods from raw value Difference (%) Raw Microwaved Boiled Roasted

183 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

184 Mann Whitney test P value Exact or approximate P value? P value summary Are medians signif. different? (P < 0.05) Gaussian Approximation ns No Conc (ng/kg) RAW MICROW. 1.1 Mann Whitney test P value Exact or approximate P value? P value summary Are medians signif. different? (P < 0.05) Cooking method (Microwaving) Gaussian Approximation ns No 1.0 Conc (ng/kg) RAW BOILED Cooking method (Boiling) Mann Whitney test P value Exact or approximate P value? P value summary Are medians signif. different? (P < 0.05) Gaussian Approximation ns No Conc (ng/kg) RAW ROASTED Cooking method (Roasting) Figure 8.7: Effect of cooking methods on OTC concentration in muscle using ELISA 162

185 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 ng/kg (1.85%) by microwaving, to ng/kg (2.83%) by boiling and to ng/kg (3.17%) by roasting (Table 8.4) 163

186 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 MICROWAVED BOILED ROASTED

187 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

188 Mann Whitney test P value Exact or approximate P value? P value summary Are medians signif. different? (P < 0.05) Gaussian Approximation ns No Conc (ng/kg) RAW MICROW Cooking method (Microwaving) Mann Whitney test P value Exact or approximate P value? P value summary Are medians signif. different? (P < 0.05) Gaussian Approximation ** Yes 1.05 Conc (ng/kg) RAW BOILED Cooking method (Boiling) Mann Whitney test P value Exact or approximate P value? P value summary Are medians signif. different? (P < 0.05) Gaussian Approximation ** Yes 1.05 Conc (ng/kg) RAW ROASTED Cooking method (Roasting) Figure 8.8: Effect of cooking methods on OTC concentration in liver tissue 166

189 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

190 Table 8.5: Effect of freezing time on OTC concentration in muscle and liver tissues Period Median OTC Concentrations (ng/kg) in muscle Values Difference from day 0 Median OTC Concentrations (ng/kg) in liver Values Day 0(prefreezing) _ _ 1 st day Difference from day 0 3 rd day th day th day

191 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

192 25% Percentile Median 75% Percentile Day st day P value Exact or approximate P value? P value summary Do the medians vary signif. (P < 0.05) Number of groups Kruskal-Wallis statistic 3rd day th day th day Gaussian Approximation ns No Conc Day 0 1st day 3rd day 6th day 9th day Time Figure 8.9: Effect of freezing time on OTC residues on muscle and liver 170

193 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 % 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 % 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

194 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

195 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

196 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

197 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

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226 resistance: management options, March 2004, Oslo Norway. Available at Wildavasky, A. (1979). Toxicology of Drug and Chemical Residues. In: Veterinary Pharmacology and Therapeutics 6 th Edition 1988 (Booth N.H and Mcdonald, L. E;Eds). Iowa State University Press Ames, Wilson, J., Otsuki, T., Majumdsar, B Balancing food safety and risk: do drug residue limits affect international trade in beef? J. of Int. Trade Econ. Dev. 12: Winsor D. K., Bloebaum A. P, Mathewson J. J. (1981). Gram-negative, aerobic, enteric pathogens among intestinal microflora of wild turkey vultures in west central Texas. Appl Environ Microbiol 42: Woodward, K. N. (1998). The use of microbiological end-points in the safety evaluation and elaboration of maximum residue limits for veterinary drugs intended for use in food producing animals. Vet. Pharmacol. Ther. 21: World Health Organisation (2000). WHO global principles for the containment of antimicrobial resistance in animals intended for food. Report of a WHO consultation with the participation of food and Agricultural Organisation of the United Nations and the office international des Epizootics, Geneva Switzerland. Available at World Organisation for animal health (2004). World Health Organization, Food and Agricultural Organization of the United states,. Joint FAO/OIE/WHO 2nd workshop on non-human antimicrobial therapy: management options March 2004, Oslo, Norway. Available at 204

227 APPENDICES Appendix I: Mechanism of action of Aminoglycosides Antimicrobial Mechanism of action Spectrum of activity Amikacin Dihydrostreptomycin Gentamicin Kanamycin Neomycin Streptomycin Apramycin Inhibitors of protein synthesis. Proteins are synthesized on cell structures called ribosomes. Bactericidal or bacteriostatic High doses can affect animals and humans because some ribosomes are similar to those in bacteria. Used for the treatment of infections caused by aerobic Gram-negatives. Effective against some Gram-positives like Staphylococcus aureus. The use of aminoglycosides in the treatment of infection in animals has been tempered by toxicity considerations in the animal treated. Often, systemic use is limited to the treatment of serious Gram-negative 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

228 Appendix II: Antimicrobial Penicillin G Mechanism of action of Beta-lactam antimicrobial Mechanism of action Inhibitors of Spectrum of activity Active against aerobic and anaerobic Gram-positives. Penicillin G benzathine, G potassium, G procaine, G sodium Aminopenicillins Amoxicillin Ampicillin Cephalosporins First generation Cefadroxil bacterial cell wall Synthesis by binding enzymes involved in peptidoglycan production (ie, Penicillin-binding protein or PBS). Animals and humans not affected because their cells do not have walls. Susceptible aerobes include most beta-haemolytic Streptococci and beta-lactamase-negative Staphylococci. Most species of anaerobes are susceptible, including Clostridium species, but excluding beta-lactamase producing Bacteroides species. Ineffective against betalactamase producers and those bacteria that are resistant to other mechanisms, such as those having a relatively impermeable cell wall. Has therefore little activity against many Staphylococci and most Gram-ve bacteria. Active against penicillin-sensitive Gram-positive bacteria and some Gram-negative bacteria. Gram-positive spectrum includes alpha- and betahaemolytic Streptococci, some Staphylococci species and Clostridia species. Gramnegatives: Escherichia coli, many strains of Salmonella, and Pasteurella multocida. Is susceptible to destruction by betalactamases.) 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 These cephalosporins have the highest activity against Grampositive bacteria, including most Corynebacteria, Streptococci and Staphylococci, particularly Staphylococcus 206

229 Cefazolin Cephalexin Cephalothin Cephapirin Cephradine Second generation Cefaclor Cefamandole Cefmetazole Cefonicid Cefotetan Cefoxitin Cefprozil Cefuroxime Third generation Cefixime Cefoperazone(1) Cefotaxime Cefpodoxime, Ceftazidime Ceftizoxime Ceftriaxone New generation Ceftiofur aureus. Activity against Gram-negative bacteria: Escherichia coli, Klebsiella pneumoniae, Haemophillus influenzae, Pasteurella and Salmonella. Many anaerobic bacteria are susceptible to these antibacterials, with the exception of betalactamase-producing Bacteroides and Clostridium difficile. Have slightly less 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. 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 Sources: Pharmacology, 1997; Forbes et al., 2007; USP 1999, 2000a, 2000b 207

230 Appendix III Mechanism of action of Chloramphenicol Antimicrobial Mechanism of action Spectrum of activity Chloramphenicol Inhibit protein synthesis by binding to 50S ribosomal subunit Broad spectrum. Effective against: Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Proteus vulgaris, Aerobacter aerogenes, Salmonella species, Pseudomonas species, anaerobic bacteria Sources: Pharmacology, 1997; Forbes et al., 2007;US Pharmacopea, 2000e 208

231 Appendix IV Mechanism of action of quinolones Antimicrobial Mechanism of action Spectrum of activity Enrofloxacin Sarafloxacin Inhibit DNA synthesis by binding DNA gyrases Broad spectrum. Bactericidal 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 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,

232 Appendix V Mechanism of action of Lincosamides Antimicrobial Mechanism of action Spectrum of activity Lincosamides: Clindamycin Lincomycin Pirlimycin Inhibitors of protein synthesis. Proteins are synthesized on cell structures called ribosomes. Bactericidal or bacteriostatic High doses can affect animals and humans because some ribosomes are similar to those in bacteria. Activity against many Grampositive bacteria and many anaerobic bacteria, but not effective against most Gramnegatives. Effective against Staphylococcus species, Streptococcus species (except Streptococcus feacalis Sources: Pharmacology, 1997; Forbes et al., 2007, USP 2000j 210

233 Appendix VI Mechanism of action of Macrolids Antimicrobial Mechanism of action Spectrum of activity Erythromycin: Base Estolate Ethylsuccinate Gluceptate Lactobionate Phosphate Stearate Thiocyanate Inhibitors of protein synthesis. Proteins are synthesized on cell structures called ribosomes. Bactericidal or bacteriostatic High doses can affect animals and humans because some ribosomes are similar to those in bacteria. Primarily against Gram-positives, such as Staphylococcus and Streptococcus species, including many that are resistant to penicillins by means of beta-lactamase production. Active against Campylobacter and Pasteurella species. Has activity against some anaerobes, but Bacteroides fragilis is usually resistant. Most Pseudomonas, Escherichia coli, and Klebsiella strains are resistant to erythromycin. Resistant strains of Staphylococci and Streptococci have been reported. Cross resistance to other macrolides can also occur. Tilmicosin Phosphate In vitro activity against Gram-positive microorganisms 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 Tylosin Phosphate Tylosin Tartrate Spectrum of activity similar to that of erythromycin but more active than erythromycin against certain Mycoplasmas Sources: Pharmacology, 1997; Forbes et al., 2007, US pharmacopeia, 2000c 211

234 AppendixVII Mechanism of action of Nitrofurans Antimicrobial Mechanism of action Spectrum of activity Nitrofurantoin furazolidone Source: Forbes et al., Uncertain exact mechanism, may have several bacterial enzyme targets and directly damage the DNA Good activity against grampositive and gram-negative bacteria. Specifically used in poultry for the treatment of Salmonellosis 212

235 Appendix VIII Mechanism of action of Tetracyclines Antimicrobials Mechanism of action Spectrum of activity Chlortetracycline Doxycycline Oxytetracycline Tetracycline Proteins are synthesized on cell structures called ribosomes. They inhibit protein sunthesis by binding 30S ribosomal subunit. Bactericidal or bacteriostatic High doses can affect animals and humans because some ribosomes are similar to those in bacteria. Broad spectrum with activity against Gram positives and Gram-negatives, including some anaerobics. Active also against Chlamydia, Mycoplasma, Protozoa and several Ricketsiae, including Ehrlichia and Haemobartonella. Active against Escherichia coli, Klebsiella species, Salmonella species, Staphylococcus species and Streptococcus species. However, resistance has been acquired by some coliforms, mycoplasmas, streptococci and staphylococci. Source: USP 2000g. Pharmacology, 1997; Forbes et al.,

236 Appendix IX Mechanism of action of Sulphomamides Antimicrobial Mechanism of action Spectrum of activity Sulphonamides: Sulfachlorpyridazine Sulfadimethoxine Sulfamethazine Sulfanilamide Sulfaquinoxaline Sulfathiazole Inhibitors of folic acid synthesis. Folic acid is needed to make RNA and DNA for growth and multiplication, and bacteria must synthesize folic acid. They are Bacteriostatic. Animals and humans obtain folic acid from their diets, so they are not affected. Antibacterial; antiprotozoal; broad spectrum; inhibiting Gram-positives and Gram-negatives and some Protozoa, such as Coccidia. Ineffective against most obligate anaerobes and should not be used to treat serious anaerobic infections. Resistance of animal pathogens to 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.,

237 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 SANCO, 2001a. Rifampine Not labelled in USA Tumorgenicity and USP, 2000k or Canada for use in teratogenic effects animals, including on experimental food producing animals animals Nitrofurazone Not labelled in USA or Canada for use in animals, including food producing animals. Banned for use in Nigeria in livestock and poultry Mutagenic effect, carcinogenic effect NAFDAC, 1996; WHO, 1997; Ali, 1999; Hoogenboom et al., 2002; Nisha, 2004; Cooper et al., production 215

238 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 Procain benzylpenicillin Tetracyclines (Chlor, Oxy and tetra) Collistin Danofloxacin Muscle Liver Kidney Fat Muscle Liver Kidney Muscle Liver Kidney Egg Muscle Kidney Liver Egg Fat Muscle Liver kidney Fat Dihydrostreptomycin/streptomycin Muscle Liver Kidney Fat Erythromycin Muscle Liver Kidney Egg Fat Flumequin Muscle Liver Kidney Fat Lincomycin Muscle Liver Kidney Fat

239 217 Monensin Muscle Liver Kidney Fat Narasin Muscle Liver Kidney Fat Neomycin Muscle Liver Kidney Egg Fat Sarafloxacin Muscle Liver Kidney Fat Spectinomycin Muscle Liver Kidney Egg Fat Spiramycin Muscle Liver Kidney Fat Tilmocosin Muscle Liver Kidney Fat/skin Tylosin Muscle Liver Kidney Egg Fat/skin FAO/WHO Food Standards (Codex alimentarius)

240 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 food for human purposes 24h Lincomix 110 premix Pfizer Feed Do not use in laying birds 0d Lincomix powder soluble Pfizer Dw None 0d Lincomycin Bio Agri Dw Do not use in laying birds 3d Spectinimycin 100 Mix soluble powder Linco-spectrin 100 Pfizer Dw Do not use in laying birds 3d soluble powder Neomix soluble Pfizer Dw None 7-14d powder Neomycin 325 Vétoquinol Dw None 7-14d 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 Eggs taken from treated birds within 60hrs after the latest treatment with this drug must not be used for food Oxytetracycline 200Granular Premix Bio Agri Mix 7d Feed None 7d Paracillin SP Intervet Dw Do not use in laying birds 2d Salinomycin Bio Agri Mix Feed Do not feed to replacement or laying birds 218

241 Sodium Sulphomethazine Solution 12.5% Dominion Dw This product must not be used in laying birds 12d Sulfa 25 P.V.L Dw This product must not be used in laying poultry 12d Sulfa,25% solution Bimeda- MTC Dw Do not use in laying birds 12d Sulphaquinoxalin 19.2% liquid concentrate Dominion Dw This product must not be used in laying birds 12d Sulphaquinoxalin-S Medprodex Dw None 4d Terramycin 100 Premix Phibrio Feed None 7d 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 in laying birds Teramed 250 Medprodex Dw Do not feed to laying birds 5d Virginiamycin 44 Premix Bio Agri Mix Feed Do not use in birds producing eggs for food purposes The Withdrawal Times Listed Correspond To Label Doses Only.* Route of Administration of Drug # Withdrawal Period in Meat 5d 219

242 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 Cephalosporin Cefquinome Ceftiofor Macrolides Tylosin Erythromycin Lincomycin Spiramycin Tetracycline Chlortetracycline Oxytetracycline Doxycycline Tetracycline Sulphonamides Sulphamethazine Sulphadiazine sulphamethaxole Amonoglycosides Gentamycine Streptomycine Neomycine Quonolones Enrofloxacine Flumequine >600 >100 >600 >100 >600 >

243 Chloramphenicol Chloramphenicol Florphenicol Salinomycin Naracin Monensin Zn-bacitracin 2, ,250 1,250 1,250 1,250 2, , Polypeptides Virginiamycine Bacitracine Appendix XIV Api 50 CH record sheet for identification of Bacillus subtilis 221

244 Appendix XV Three Plate Test clear zone of inhibition indicative of positive samples for antimicrobial residue 222

245 Clear zone of inhibition Appendix XVI Table 4.1: antimicrobial residues detection with TPT in 100 sampled broiler birds 223

246 Organ(s) No Positive No Negative Muscle only 1 99 Gizzard only 1 98 Liver only 5 93 Kidney only Muscle & Gizzard 0 83 Muscle & Liver 0 83 Muscle & Kidney 0 83 Gizzard & Liver 2 81 Gizzard & Kidney 1 80 Kidney & Liver Muscle, Gizzard, Liver 5 58 Muscle, Kidney, Liver 0 58 Muscle, Gizzard, Kidney 1 57 Kidney, Gizzard, Liver Muscle, Gizzard, Liver, Kidney 2 36 Total Appendix XVII Premi test colour indication for positive and negative result for antimicrobial results 224

247 Douptful samples (blue coloration) Positive samples (Purple coloration) Negative samples (yellow coloration) Appendix XVIII Antimicrobial residues detection with Premi Test in 100 sampled broiler birds 225

248 Organ(s) No Positive No Negative Muscle only Gizzard only Liver only 2 98 Kidney only 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 Total Appendix XIX Elisa work - sheet 226

249 A Std 0 Std 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 Std standard; Spl sample Appendix XX Calculation of tetracycline concentration from standard curve 227

250 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) = Intercept (C) = Therefore, concentration (C) = OD(Y) Appendix XXI Daily detection of OTC residue with TPT and Premi Test in organs of group A birds injected intramuscularly. 228

251 Days Organ TPT (ph) Premi Test Muscle NI + Liver NI NI NI + Kidney NI NI NI + Gizzard NI NI NI + 2 Muscle NI + Liver NI + Kidney NI + Gizzard NI +(-) 3 Muscle NI + Liver NI +(-) Kidney NI +(-) Gizzard NI - 4 Muscle NI +(-) Liver NI - Kidney NI - Gizzard NI - 5 Muscle NI - Liver NI - Kidney NI - Gizzard NI - 6 Muscle NI NI NI - Liver NI - Kidney NI - Gizzard NI - 7 Muscle NI NI NI - Liver NI - Kidney NI - Gizzard NI - 8 Muscle NI NI NI - Liver 2.0 2,0 NI - Kidney NI - Gizzard NI - 9 Muscle NI NI NI - Liver NI NI NI - Kidney NI - Gizzard NI - 10 Muscle NI NI NI - Liver NI NI NI - Kidney NI NI NI - Gizzard 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

252 Days Organ TPT (ph) Premi Test 1 Muscle NI +(-) Liver NI - Kidney NI - Gizzard NI - 2 Muscle NI - Liver NI - Kidney NI - Gizzard NI - 3 Muscle 2.0 NI NI - Liver NI - Kidney NI - Gizzard NI - 4 Muscle NI NI NI +(-) Liver NI - Kidney NI - Gizzard NI - 5 Muscle NI NI NI - Liver NI - Kidney NI + Gizzard NI - 6 Muscle NI NI NI - Liver NI - Kidney NI - Gizzard NI + 7 Muscle NI NI NI - Liver NI - Kidney NI - Gizzard NI - 8 Muscle NI NI NI - Liver NI NI NI - Kidney 1.0 NI NI - Gizzard NI NI NI - 9 Muscle NI NI NI - Liver NI NI NI - Kidney NI NI NI - Gizzard NI 1.0 NI - 10 Muscle NI NI NI - Liver NI NI NI - Kidney NI NI NI - Gizzard NI NI NI - NI: no inhibition Appendix XXIII Effect of freezing time on OTC concentration of samples positive for OTC residue 230

253 Positive samples OTC concentrations (ng/kg) Day 0 Day 1 Day 3 Day 6 Day

254 Appendix XXIV Work In Progress, supervision by supervisors 232

255 Appendix XXV Tetracycline ELISA kit 233