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VETERINARY PUBLIC HEALTH AND PREVENTIVE MEDICINE,
PREVALENCE OF AND AWARENESS OF THE RISKS ASSOCIATED WITH ANTIMICROBIAL DRUG RESIDUES IN EDIBLE TISSUES OF CATTLE AND PIGS IN ENUGU STATE, NIGERIA
NJOGA, EMMANUEL OKECHUKWU PG/M.Sc/09/51840
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PREVALENCE OF AND AWARENESS OF THE RISKS
ASSOCIATED WITH ANTIMICROBIAL DRUG RESIDUES IN
EDIBLE TISSUES OF CATTLE AND PIGS IN ENUGU STATE,
NIGERIA
BY
NJOGA, EMMANUEL OKECHUKWU
PG/M.Sc/09/51840
A RESEACH DISSERTATION SUBMITTED TO THE DEPARTMENT OF
VETERINARY PUBLIC HEALTH AND PREVENTIVE MEDICINE, FACULTY OF
VETERINARY MEDICINE, UNIVERSITY OF NIGERIA, NSUKKA IN PARTIAL
FULFILMENT FOR THE AWARD OF MASTER OF SCIENCE IN VETERINARY
PUBLIC HEALTH AND PREVENTIVE MEDICINE OF THE UNIVERSITY OF
NIGERIA
JULY, 2012
3
CERTIFICATION
NJOGA, Emmanuel Okechukwu, a postgraduate student in the Department of Veterinary
Public Health and Preventive Medicine, with registration number, PG/M.Sc/09/51840, has
satisfactorily completed the requirements for course and research work for the award of degree
of Master of Science in Veterinary Public Health and Preventive Medicine. This work embodied
in this dissertation is original and has not been submitted in part or full for any other degree of
this or any other University.
_______________________ _______________
Dr. J.A. Nwanta Date (Supervisor) _______________________ _______________ Prof. K.F Chah Date (Supervisor) ________________________ ______________
Dr. J.A. Nwanta Date (Head of Department)
_______________________ ________________ Prof. M.A. Dipeolu Date (External examiner) _________________________ __________________
Prof. C. N. Uchendu Date (Dean, FVM)
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DEDICATION
This work is dedicated to my parents, Mr and Mrs S.O. Njoga, for giving me formal education
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ACKNOWLEDGEMENTS
I wish to acknowledge my supervisors: Dr. J.A. Nwanta and Prof. K.F. Chah, for their tireless
effort and invaluable guidance in the course of this research work despite their busy schedules. I
sincerely appreciate their strict and meticulous approach to this work which had greatly
improved its quality.
I am particularly grateful to Dr. J.A. Nwanta for providing some reading materials on my
research topic. He ordered a book from the United Kingdom and bore the costs just to ensure that
I have access to relevant study materials.
I am grateful to DSM food specialists of the Netherlands and their business associate,
Biopharm®, Germany for subsidizing the cost of Premi® Test kit used in this research work and
for timely delivery of the product which facilitated the early completion of this work.
I also wish to acknowledge the entire staff of the Department of Veterinary Public Health and
Preventive Medicine for their support and encouragement during the course of this work.
My fellow graduate students in the Department and the entire Faculty of Veterinary Medicine
have been wonderful in providing ideas and moral support in the course of this M.Sc
programme.
May the Good Lord bless you all and many others all the days of your lives.
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TABLE OF CONTENTS
Title page ------------------------------------------------------------------------------------------------------------------i
Certification--------------------------------------------------------------------------------------------------------------ii
Dedication--------------------------------------------------------------------------------------------------------------- iii
Acknowledgements---------------------------------------------------------------------------------------------------- iv
Table of contents------------------------------------------------------------------------------------------------------- vi
List of tables------------------------------------------------------------------------------------------------------------ vii
List of figures and plates----------------------------------------------------------------------------------------------vii
Appendices ------------------------------------------------------------------------------------------------------------ viii
Abstract ----------------------------------------------------------------------------------------------------------------- ix
CHAPTER ONE: Introduction ------------------------------------------------------------------------------------ 1
1.1 Background of the study------------------------------------------------------------------------------------------- 1
1.2 Statement of problem ----------------------------------------------------------------------------------------------4
1.3 Research questions--------------------------------------------------------------------------------------------------6
1.4 Objectives of the study---------------------------------------------------------------------------------------------6
1.5 Research hypotheses ----------------------------------------------------------------------------------------------- 7
1.6 Scope of the study --------------------------------------------------------------------------------------------------8
CHAPTER TWO: Literature review------------------------------------------------------------------------------ 9
2.1 Definition of terms -------------------------------------------------------------------------------------------------9
2.1.1 Antimicrobial agents ---------------------------------------------------------------------------------------------9
2.1.2 Antimicrobial residues -------------------------------------------------------------------------------------------9
2.1.3 Unintentional residues ------------------------------------------------------------------------------------------10
2.1.4 Tolerance levels or maximum residue limits (MRLs) -----------------------------------------------------11
2.1.5 Acceptable daily intake (ADI) --------------------------------------------------------------------------------13
2.1.6 Violative or illegal residues -----------------------------------------------------------------------------------14
2.1.7 Extra label use of drug------------------------------------------------------------------------------------------14
2.1.8 Limits of detection (LOD) -------------------------------------------------------------------------------------15
2.1.9 Withdrawal period ----------------------------------------------------------------------------------------------16
2.2 Antimicrobials -----------------------------------------------------------------------------------------------------18
2.2.1 History of antimicrobials ---------------------------------------------------------------------------------------18
2.2.2 Use of antimicrobials in food animals ----------------------------------------------------------------------- 19
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2.2.3 Tetracyclines -----------------------------------------------------------------------------------------------------21
2.2.4 Betalactams ------------------------------------------------------------------------------------------------------22
2.2.5 Aminoglycosides ------------------------------------------------------------------------------------------------23
2.2.6 Quinolones -------------------------------------------------------------------------------------------------------23
2.2.7 Amphenicols -----------------------------------------------------------------------------------------------------24
2.2.8 Sulphonamides --------------------------------------------------------------------------------------------------25
2.2.9 Regulation of agricultural use of antimicrobials in Nigeria -----------------------------------------------25
2.3 Antimicrobial residues monitoring and surveillance in Nigeria --------------------------------------------26
2.4 Antimicrobial resistance ------------------------------------------------------------------------------------------28
2.5 Effects of cooking or cooling on antimicrobial residues in meat -------------------------------------------30
2.6 Antimicrobial residue detection in animal products ----------------------------------------------------------31
2.6.1 Microbiological methods --------------------------------------------------------------------------------------32
2.6.1.2 Limitations of microbiological methods -------------------------------------------------------------------32
2.6.2 Immunochemical methods -------------------------------------------------------------------------------------33
2.6.3 Physico-chemical methods ------------------------------------------------------------------------------------33
2.7 The choice of Premi® Test kit ----------------------------------------------------------------------------------33
2.7.1 The principle behind Premi® Test ----------------------------------------------------------------------------35
2.8 Prevalence of antimicrobial residues in animal products in Nigeria ---------------------------------------35
2.9 Public health hazards of antimicrobial residues in animal products ----------------------------------------36
2.9.1 Antimicrobial drug resistance ---------------------------------------------------------------------------------36
2.9.2 Allergic reactions -----------------------------------------------------------------------------------------------37
2.9.3 Carcinogenic effect- --------------------------------------------------------------------------------------------38
2.9.4 Mutagenic effect ------------------------------------------------------------------------------------------------38
2.9.5 Teratogenic effect -----------------------------------------------------------------------------------------------39
2.9.6 Bone marrow depression ---------------------------------------------------------------------------------------39
CHAPTER THREE: Materials and methods ------------------------------------------------------------------40
3.1 The study area -----------------------------------------------------------------------------------------------------40
3.2 The study design --------------------------------------------------------------------------------------------------41
3.2.1 Questionnaire survey and interview --------------------------------------------------------------------------41
3.2. Experimental study------------------------------------------------------------------------------------------------42
3.2.3 Cross sectional study -------------------------------------------------------------------------------------------43
3.2.3.1 Sample population --------------------------------------------------------------------------------------------43
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3.2.3.2 Sample size determination -----------------------------------------------------------------------------------43
3.2.3.3 Sampling area selection---------------------------------------------------------------------------------------44
3.2.3.4 Sampling method for sample collection--------------------------------------------------------------------44
3.2.3.5 Processing and screening of meat samples for antimicrobial residues---------------------------------45
3.2.3.6 The principle behind Premi® Test--------------------------------------------------------------------------46
3.2.3.7 Interpretation of Premi® Test results-----------------------------------------------------------------------46
3.3 Data presentation---------------------------------------------------------------------------------------------------46
3.4 Data analysis--------------------------------------------------------------------------------------------------------46
CHAPTER FOUR: Results-----------------------------------------------------------------------------------------48
4.1 Result of the questionnaire survey-------------------------------------------------------------------------------48
4.1.1 Socio-economic characteristics of livestock farmers-------------------------------------------------------48
4.1.2 Status of antimicrobial drug use in food animals------------------------------------------------------------49
4.1.3 Awareness of health risks associated with the consumption of AMRs in animal tissues--------------49
4.1.4 Knowledge of withdrawal periods among the respondents------------------------------------------------50
4.1.5 Observance of withdrawal period-----------------------------------------------------------------------------50
4.2 Result of the experimental study --------------------------------------------------------------------------------50
4.3 Result of the cross sectional study-------------------------------------------------------------------------------51
4.3.1 Prevalence of antimicrobial residues in cattle and pigs-----------------------------------------------------51
4.3.2 Organ distribution of antimicrobial residues in cattle ------------------------------------------------------51
4.3.3 Organ distribution of AMRs in pigs---------------------------------------------------------------------------51
CHAPTER FIVE: Discussion, Conclusion and Recommendations ----------------------------------------62
5.5 Discussion ---------------------------------------------------------------------------------------------------------62
5.1.1 Questionnaire survey ------------------------------------------------------------------------------------------ 62
5.1.2 Cross sectional study -------------------------------------------------------------------------------------------65
5.2 Conclusion ---------------------------------------------------------------------------------------------------------68
5.3 Recommendations -------------------------------------------------------------------------------------------------68
References --------------------------------------------------------------------------------------------------------------69
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LISTS OF TABLES
Table 1: Maximum residue limits for foods of animal origin in selected antimicrobials -------------------12
Table 2: Limits of detection (LOD) of Premi® Test kit for selected antimicrobials ------------------------15
Table 3: Withdrawal periods of various antimicrobial agents as indicated by the manufacturers----------17
Table 4: Some antimicrobial growth promoters used in cattle production ----------------------------------21
Table 5: Methods of residue testing in animal tissues ----------------------------------------------------------- 31
Table 6: Distribution of animals sampled in each of the three slaughter houses surveyed------------------ 44
Table 7: Socio-economic characteristics of livestock farmers and veterinary practitioners in Enugu State -
---------------------------------------------------------------------------------------------------------------------------52
Table 8: Antimicrobial drug use among livestock farmers and veterinary practitioners in Enugu State--53
Table 9: Distribution of responses on awareness of health risks associated with consumption of animal
products containing AMRS among livestock farmers and veterinary practitioners in Enugu State-------- 54
Table 10: Distribution of responses on knowledge of withdrawal period among livestock farmers in
Enugu State ----------------------------------------------------------------------------------------------------------- 55
Table 11: Distribution of responses on observance of withdrawal period among livestock farmers and
veterinary practitioners in Enugu State --------------------------------------------------------------------------- 56
Table 12: Prevalence of antimicrobial residues (AMRs) in cattle and pigs slaughtered for human
consumption in three major slaughter houses (SH) surveyed in Enugu State -------------------------------- 57
Table 13: Organ distribution of antimicrobial residues (AMRs) in cattle slaughtered for human
consumption in three major slaughter houses (SH) surveyed in Enugu State ------------------------------ 58
Table 14: Organ distribution of antimicrobial residues (AMRs) in pigs slaughtered for human
consumption in three major slaughter houses (SH) surveyed in Enugu State -------------------------------- 59
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LIST OF FIGURES AND PLATES
Figure 1: Theoretical representation of withdrawal period -----------------------------------------------------18
Figure 2: Premi® Test kit (as supplied by Biopharm®, Netherlands) used in the study ------------------- 34
Figure 3: Incubation of Premi® test Ampoules treated with meat fluid from the test samples ------------42
Plate 1: Positive and negative cases from the validation of Premi® Test kit used in the study---60
Plate 2: Positive and negative cases from the antimicrobial residues (AMRs) screening test---------------61
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LIST OF APPENDICES
Appendix 1: Questionnaire ---------------------------------------------------------------------------------------- 82
Appendix 2: Measurement of association between the prevalence of AMRs in cattle and pigs slaughtered
for human consumption in Enugu State using Chi-square analysis----------------------------------------------84
Appendix 3: Measurement of association between the prevalence of AMRs in the organs (kidney, liver,
muscle) of pigs slaughtered for human consumption in Enugu State using Chi-square analysis ---------- 85
Appendix 4: Measurement of association between the prevalence of AMRs in the organs (kidney, liver,
muscle) of cattle slaughtered for human consumption in Enugu State using Chi-square analysis ---------86
Appendix 5: Measurement of association between educational levels and observance of withdrawal
period among livestock farmers in Enugu State using Chi-square analysis----------------------------------- 87
Appendix 6: Measurement of association between educational levels and awareness of public health risks
associated with consumption of AMRs in animal products among the respondents using Chi-square
analysis-----------------------------------------------------------------------------------------------------------------88
Appendix 7: Measurement of association between working experience and observance of withdrawal
period among livestock farmers in Enugu State using Chi-square analysis -----------------------------------89
Appendix 8: Measurement of association between working experience and observance of withdrawal
period among veterinary practitioners in Enugu State using Chi-square analysis ----------------------------90
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ABSTRACT
This study was aimed at determining the prevalence of and awareness of health risks associated
with antimicrobial residues (AMRs) in edible tissues of cattle and pigs slaughtered for human
consumption in Enugu State. In the first phase, the awareness of public health problems
associated with consumption of animal tissues containing AMRs and compliance to specified
withdrawal period (WP) by livestock farmers and veterinary practitioners were determined. This
was carried out using a total of 200 copies of structured questionnaire designed to obtain
information on the status of antimicrobial drug use, observance of WP and awareness of public
health problems associated with consumption of animal products containing AMRs. A minimum
of sixty (60) copies of the questionnaire were distributed to willing respondents in each of the
three senatorial zones of the state. One hundred and eighty-two (182) copies of completed
questionnaire were recovered and the responses collated and analyzed. The second phase of the
study which was the determination of the prevalence of AMRs in edible tissues (kidney, liver
and muscle) of cattle and pigs in the study area was carried out using Premi® test kit. A total of
285 tissue samples (180 from cattle and 105 from pigs) were randomly collected from animals
slaughtered in Nsukka, Akwata and 9th Mile slaughter houses. Chi-square (χ2) test of
independence was used to determine the strength of association between educational level of the
respondents and observance of withdrawal period, and awareness of public health risks
associated with consumption of animal products containing AMRs. Job experience and
observance of withdrawal period were also compared among the respondents using the same test.
Furthermore, the strength of association in the occurrence of AMRs among the species (cattle
and pigs) and organs in each of the species were also determined. All the respondents (100%)
had used at least one antimicrobial drug in food animals between January and April, 2011 but
only 25.99% of the respondents administered these drugs based on veterinary prescription. The
antimicrobial drugs used in food animals and the percentage of respondents that utilized them
were oxytetracycline (23.44%), penicillin (21.16%), streptomycin (16.81%) tylosin (11.62%),
sulphadimidines (10.17%), enrofloxacin (8.92%) and chloramphenicol (7.88%). Only 33.12% of
the respondents administered the drugs for chemotherapeutic purposes while the rest (66.88%)
used the drugs for non-chemotherapeutic purposes (prophylaxis or growth promotion).
Irrespective of educational levels, majority (84.68%) of the livestock farmers were not aware of
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the public health risks associated with consumption of animal products containing AMRs. All the
veterinary practitioners surveyed were aware of the health problems associated with the
consumption of AMRs in edible tissues. Majority of the livestock farmers (88.37%) and some
(30.19%) of the veterinary practitioners do not observe WP. There were significant association
(p<0.05) between educational levels and observance of WP, and awareness of public health
problems associated with the consumption of animal tissues containing AMRs. However, no
significant association (p>0.05) existed between job experience and observance of WP in both
the livestock farmers and veterinary practitioners. The prevalence of AMRs was significantly
higher (p<0.05) in cattle (30.00%) than in pigs (22.86%). There were no significant association
(p>0.05) in the organ (kidney, liver and muscle) distributions of AMRs in both cattle and pigs.
The general public in the study area are at risk of the health hazards associated with consumption
of animal tissues containing AMRs. Withdrawal period should be observed and strict
enforcement of veterinary drug laws in Nigeria is recommended to safeguard human health.
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CHAPTER ONE
INTRODUCTION
1.1 Background of the study
Antimicrobials are agents that act against microorganisms either by inhibiting their
growth and multiplication or by complete destruction through various mechanisms of actions
(Brander et al., 1993; Guardabassi and Kruse, 2008). Antimicrobials are used in farm animals for
3 main purposes: therapy, prophylaxis and growth enhancement (Gutafson and Bowen, 1997).
These agents are used extensively in veterinary practice in Nigeria to prevent or control
infectious diseases, to minimize post surgical infections and as growth enhancer by incorporation
in feed at sub-therapeutic doses (Kabir et al., 2002). Gains made in food production capacity in
many parts of the world, in the past decades, would not have been possible if not for the ability
of antimicrobials to contain the threats of diseases to animals (WHO, 1997).
Antimicrobials seem to be very important not just for sustainable livestock production but
in the control of some zoonoses. Guardabassi and Kruse (2008), are of the opinion that one of
the major benefit to public health in the proper use of antimicrobials in animals, is the ability of
these drugs to combat pathogens in animal transmissible to humans by direct contact,
consumption of contaminated animal products or proliferation of these zoonotic pathogens into
the environment. Despite the gains in food animal production capacity and zoonoses control due
to antimicrobial drug use in animals, the administration of these drugs in food animals is not
without possible health risks especially when regulations guiding veterinary drug use are not
complied with (Vandenberge et al., 2011). This probably explains why the use of antimicrobials
15
in food producing animals has become a very important public health issue in rescent time. In
fact, the occurrence of antimicrobial residues (AMRs) in foods of animal origin is one of the
most important indexes for their safety (Pavlov et al., 2008) as these residues tend to accumulate
in animal tissues and predispose consumers of such tissues to diverse health problems
(Donoghue, 2003). These health problems include development of antimicrobial resistance,
allergic reactions, bone marrow depletion, teeth coloration, disruption of normal intestinal flora
and organ toxicity (Donoghue, 2003; Doyle, 2006; Vandenberge et al., 2011).
For every case of antimicrobial use in food animals, heavy responsibility is placed on
veterinary practitioners and livestock farmers to safeguard human health by observing
withdrawal period prior to slaughter or sale of such animal for human consumption. Withdrawal
period is the minimum period of time in which animals that received treatment are required to be
held before slaughter, for AMRs in the tissues to deplete to safe levels (Vranic et al., 2003). To
prevent the harmful effects of AMRs on the consumers, Food and Agricultural Organization
(FAO), World Health Organization (WHO) and the European Union (EU) have established the
maximum residue limits (MRLs) under Council Regulation (EEC) No. 2377/90 for different
animal species and tissues (EEC, 1990; European commission, 2001). Antimicrobial residues
above the approved limits have the potential to cause harm in humans, animals and even the
environment (WHO, 1997; Sundlof and Fernandez, 2000) although very little amount of some
AMRs (such as penicillin), far below the MRLs can elicit allergic reaction in sensitized
individual or may even encourage the development of resistant bacteria (Donoghue, 2003).
Humans as non target population of veterinary drugs receive various amounts of these
drugs as residues through consumption of animal products containing antimicrobial residues.
This can cause alterations in the intestinal micro flora balance thereby predisposing man to
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disease conditions and development of antimicrobial resistant pathogens (Chah and
Oboegbulem, 2007; Nonga et al., 2009). Antibiotic-resistant strains of meat-borne bacteria
pathogens such as Salmonella spp, Eschericha coli and Campylobacter spp are zoonotic, causing
human diseases that are difficult to treat (Aarestup, 2005). Even when some of these antibiotic
resistant organisms are not zoonotic, they may pass their resistant gene to other pathogenic
bacteria (Lester et al., 2006). Antimicrobial residues from animal manure may also constitute a
great environmental problem as this may enhance the proliferation of pathogenic microbes in the
environment (Lee et al., 2001).
Antimicrobial drug resistance appears to be the most important hazard associated with the
consumption of food animal tissues containing AMRs (Dipeolu, 2010). This may become a
greater public health problem especially in developing countries where the people concerned
may not have access to substitute drug due to unavailability of these drugs or financial
constraints (Dipeolu, 2010). Resistant bacteria can cause diseases that are very difficult to treat
and may also transfer resistant genes to human micro flora and pathogens (Lester et al., 2006;
Olatoye and Ehinmowo, 2010).
Antimicrobial residues above the approved limits in meat and other animal products do
not only pose health risks to the consumers but are also of economic concern because
palatability, aroma and quality of meat could be affected by the presence of drug residues (Aliu,
2004). Drug and pesticide residue concerns are among the reasons adduced for denial of Africa’s
livestock products access into the European and American livestock markets (Aliu, 2004). This
may have negative impact on the economic growth of the continent, especially in countries that
derive most of their foreign exchange from livestock products.
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1.2 Statement of problem
Food animals treated with antimicrobials are required to be held for a specified minimum
period of time called withdrawal period within which drug residues are expected to deplete to
safe levels in animal tissues intended for human consumption (KuKanchi et al., 2005; Olatoye
and Ehinmowo, 2010). In Nigeria, just like most other developing countries of the world,
withdrawal periods are usually not observed and there is no programme for
monitoring/surveillance of veterinary drug residues such that, a number of food animals
slaughtered for human consumption may contain an undetermined magnitude of residue
(Oboegbulem and Fidelis, 1996; Kabir et al., 2004).
Olatoye and Ehinmowo, (2010) recorded that a greater proportion of cattle in Nigeria are
reared by nomadic herdsmen and other animal handlers who administer veterinary drugs without
prescription. When such people use antimicrobial agents, incorrect dosage is most likely and
withdrawal time may not be observed. Antimicrobials can readily be purchased over the counter
without prescription in Nigeria and this encourages indiscriminate use of these drugs, especially
in food animals (Dina and Arowolo, 1991 as cited by Olatoye and Ehinmowo, 2010).
Apart from these, extra-label use of veterinary drugs (whereby these drugs are used in a
manner that is not in accordance with the drug labeling) is very common in Nigeria (Dipeolu,
2010). As the country’s agriculture becomes more and more intensive, larger quantities of these
drugs will undoubtedly be used. This may expose consumers of food animals to some health
problems associated with the consumption of AMRs in animal products, such as antimicrobial
drug resistance, allergic reaction, mutagenicity, teratogenicity and carcinogenicity (Riviere and
18
Sundlof, 2001; Ibrahim et al., 2010; Ezenduka et al.,, 2011). These problems may be
compounded when livestock farmers or veterinary practitioners are neither aware of the possible
adverse effects of accumulation of these residues in animal tissues nor the health risks associated
with its consumption (Kabir et al., 1999).
Although violative levels of AMRs have been reported in edible cattle tissues in Nigeria
(Oboegulem and Fidelis, 1996; Dipeolu and Alonge, 2002; Kabir et al., 2002; Olatoye and
Ehinmowo, 2010; Ibrahim et al., 2010), there are however no published report on the prevalence
of AMRs in pigs in Southeast, Nigeria. In addition, most of these works were carried out using
the European four plate test, which is less sensitive (Stead et. al., 2004) and unlike the Premi®
test kits, takes a minimum of two days to produce result. This makes it difficult to use for AMRs
screening during ante-mortem inspection. Also, these studies did not consider the awareness
status of livestock farmers and veterinary practitioners on the health risks associated with
consumption of AMRs in meat. Furthermore, the extent of farmers’ compliance with specified
withdrawal period may not have been in the scope of these works.
In developed countries where the consumer awareness of potential health risks of AMRs
exist, livestock producers and marketers of livestock products are conscious of litigations that
may arise due to violation (Oboegulem and Fidelis, 1996; Kabir et al., 2002). Continuous and
regular monitoring of drug residue levels in animal products are therefore carried out to avoid
such litigations. There is no such programme for routine monitoring or surveillance of AMRs in
food animals in Nigeria (Oboegulem and Fidelis, 1996; Dipeolu and Alonge, 2002; Kabir et al.,
2002), despite large scale prescription and use of various antimicrobial including Nitrofuran and
19
Chloramphenicol prohibited for use in food animals (WHO, 1995; NAFDAC, 1996; Ezenduka,
et al., 2011).
The public health problems associated with the consumption of AMRs in food animals,
such as antimicrobial drug resistance could reach an alarming level should the trend of extra-
label use of antimicrobials in food animals is left unchecked.
1.3 Research questions
The following research questions guided the study.
1. What percentage of livestock farmers and veterinary practitioners are aware of the health
risks associated with the consumption of meat containing AMRs in the study area?
2. What percentage of livestock farmers and veterinary practitioners observe withdrawal
period before sale or animal slaughter for human consumption in Enugu State?
3. What are the organ (kidney, liver and muscle) distribution of AMRs in cattle and pigs in
the study area?
1.4 Objectives of the study
The main purpose of this study was to determine the prevalence of and awareness of health risks
associated with AMRs in edible tissues of cattle and pigs slaughtered for human consumption in
Enugu State.
The specific objectives of the study include:
1. To determine the awareness status of livestock farmers and veterinary practitioners on the
health hazards associated with AMRs in meat
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2. To assess compliance in withdrawal period among livestock farmers and veterinary
practitioners in the study area.
3. To determine the prevalence and organ distribution of AMRs in cattle and pigs
slaughtered for human consumption in Enugu state
4. To make recommendations, based on findings of this work, in order to safeguard public
health
1.5 Research Hypotheses
The following null hypotheses (H0) further guided the study and were tested at 5% level of
significance.
1. There is no significant association in the occurrence of AMRs in animal species
slaughtered for human consumption in Enugu State.
2. There is no significant association in the organ (kidney, liver and muscle) distribution of
AMRs in cattle and pigs slaughtered for human consumption in Enugu State.
3. There is no significant association between educational levels and awareness of public
health risks associated with consumption of animal products containing AMRs among
livestock farmers in the study area.
4. There is no significant association between educational levels and observance of
withdrawal period among the respondents in Enugu State.
5. There is no significant association between job experience and observance of withdrawal
period among the respondents in Enugu State.
21
1.6 Scope of the study
Although each of the 3 senatorial zones of Enugu State (Enugu north, Enugu east and
Enugu west) was visited, only the major slaughter house in each zone was purposively selected
for meat sample collection. The species of animal screened for AMRs were limited to cattle and
pigs only. This is because beef is widely consumed in Enugu State while pork is cheap and
enjoys wide acceptance unlike in some parts of country where there is religious restriction to the
rearing of pig and consumption of pork. In each of the species, only the kidney, liver and muscle
tissues were screened for AMRs.
22
CHAPTER TWO
LITERATURE REVIEW
2.1 Definition of terms
2.1.1 Antimicrobial agents
Antimicrobial agents or antimicrobials are chemical compounds that kill or inhibit the
growth and multiplication of microorganisms. They may be naturally produced by
microorganisms such as fungi (e.g. penicillin) and bacteria (e.g. tetracycline and erythromycin),
or can be synthetically (e.g. sulfonamides and fluoroquinolones) or semi-synthetically (e.g.
amoxicillin, clarithromycin and doxycycline) produced (Guardabassi and Kruse, 2008).
Antimicrobial agents having either bacteriocidal or bacteriostatic activity against bacteria are
referred to as antibacterials, although some of them (e.g. sulfonamides and tetracyclines) have
activities against protozoa (Guardabassi and Kruse, 2008). An antibiotic is a term used to
describe those substances (e.g. penicillin) produced by a microorganism which is toxic to another
unrelated microorganism. Some antimicrobials affect both bacterial and animal cells due to lack
of selective toxicity, and are therefore used only on inanimate objects (disinfectants) or on
external surfaces of the body (antiseptics).
2.1.2 Antimicrobial residues
Residues of veterinary medicinal products are pharmacologically active substances (whether
active principles, excipients or degradation products) and their metabolites which remain in
foodstuffs obtained from animals to which the veterinary medicinal product in question has been
administered (European commission, 2001). Riviere and Sundlof, (2001) defined antimicrobial
23
residue as either the parent compound or its metabolites that may deposit, accumulate or
otherwise be stored within the cells, tissues, organs or other edible tissues of animals after using
it to prevent, control or treat animal disease or to enhance performance. Irrespective of the route
or purpose of administration, antimicrobials can accumulate as residues, for some times in
animal tissues, before they are completely metabolized or excreted from the body. The
occurrence of these residues in meat or other animal tissues is most probable, when animals are
slaughtered for human consumption while on medication or shortly after medication before the
withdrawal period elapses (Tollefson and Miller, 2000). The consumption of such animal tissues
may result in many health problems (Lee et al., 2001; Ezenduka, 2011).
Antimicrobial residues in animal tissues may be quantified in parts by weight as follows:
Parts per million (ppm) i.e mg/kg or mg/l,
Parts per billion (ppb) i.e µg/kg or µg/l or
Parts per trillion (ppt) i.e ηg/kg or ηg/l.
2.1.3 Unintentional residues
Unintentional antimicrobial residues are ones that accumulate in animal tissues due to
feed or water contamination or from other means other than the actual administration of the
antimicrobial in the animal (Nisha, 2008; Booth, 1988). Residues of drugs or chemicals that
occur as environmental contaminants but cannot be differentiated from residues resulting from
actual use of the drugs or chemicals in animals are also called unintentional residues (Nisha,
2008).
24
2.1.4 Tolerance levels or maximum residue limits (MRLs)
Tolerance level is the maximum allowable concentration of antimicrobial residue in
tissues of food animals at the time of slaughter, processing, storage or marketing up to the time
of consumption. Tolerance level is the same as the maximum residue limits (MRLs) which was
established by Council regulation EEC/2377/90 (EEC, 1990). MRLs are the maximum levels of
residues of veterinary drug that may be present in foodstuffs of animal origin without presenting
any harm to the consumer (European commission, 2001). The European Union definition of
MRL is the same as that adopted by Codex Alimentarius committee for residues of veterinary
drug in foods but most developing countries are yet to develop their own MRLs (European
commission, 2001).
Although efforts have been made to harmonize maximum residues limits worldwide
under the aegis of World Trade Organization (WTO) and the Codex Alimentarius, MRLs still
vary from one geographical location to another. In fact, MRLs in a particular animal product
may differ from one country to another depending on the local food safety regulatory agencies.
The MRL for any substance is determined from safety data submitted by manufacturers or
supplied to residue regulatory bodies, the most important of such data being the Acceptable daily
intake (ADI). The MRLs for foods of animal origin in selected antimicrobial agents are
presented in Table 1.
25
Table 1: Maximum residue limits (MRLs) for foods of animal origin in selected antimicrobials
Antimicrobials Feedstuffs of animal origin MRLs (mg/kg or mg/l) Amoxicillin Cow milk 0.01 Edible offal 0.01 Eggs 0.01 Poultry meat 0.01 Poultry edible offal 0.01 Amprolium Egg 4 Poultry edible offal 1 Poultry meat 0.5 Ampicillin Cow milk 0.01 Bacitracin Poultry meat 0.5 Egg 0.5 Cow milk 0.5 Benzyl penicillin G Mammalian edible offal 0.06 Mammalian meat 0.06 Milk 0.0015 Chlortetracycline Cattle and pig kidney 0.6 Cattle and pig liver 0.3 Beef, Pork and Poultry meat 0.1 Egg 0.2 Cloxacillin Cow milk 0.01 Erythromycin Mammalian meats and edible offal 0.3 Milk and poultry meat 0.4 Poultry edible offal 0.3 Florfenicol Cattle kidney and pork 0.5 Cattle and pig liver 3 Pig kidney, fat and skin 1 Lincomycin Mammalian meats, cow milk and egg 0.02 Poultry meat 0.1 Neomycin Cattle and pig kidney 10 Poultry meat, egg, cattle and pig liver 0.5 Milk 1.5
26
Oxytetracycline Poultry meat, mammalian meat and milk 0.1 Cattle and pig kidney 0.6 Honey, cattle and pig liver 0.3 Fish muscle 0.2 Procaine penicillin G Mammalian meats and edible offal 0.1 Cow milk 0.0025 Spiramycin Pig and poultry edible offal 1 Pork and poultry meat 0.1 Streptomycin and dihydrostreptomycin
Cow milk 0.2
Mammalian meats and edible offal 0.3 Sulphonamides Edible tissues of all species 0.01 Tetracycline Cow milk 0.1 Meats 0.1 Toltrazuril Pork, cattle edible offal 1 Poultry meat 2 Beef 0.25 Egg 0.05 Tylosin Beef and cattle edible offal 0.1 Poultry meat, Pork, pig edible offal or eggs 0.2 Cow milk 0.05 Source: Australian pesticides and veterinary drug authority, 2011)
2.1.5 Acceptable daily intake (ADI)
The acceptable daily intake is the amount of a drug or its residue that may be consumed
daily by an individual without causing any obvious health risk (Woodward, 1991; Donoghue,
2006). The ADI is based on No Observable Effect Level (NOEL), divided by a safety factor,
often 100 (European commission, 2001). NOEL is the maximum level of exposure at which
chemical substances such as drug residues have no observable adverse effect on the consumer
27
and with practical certainty that a life-long exposure to the residue, at that level will not result in
any adverse health effect (European commission, 2001; Raison-Peyron et al., 2001). The ADI
values are subjected to revision from time to time based on the availability of new information
and are expressed as milligram of the drug or its residue per kilogram of food (mg/kg).
2.1.6 Violative or illegal residues
This is the occurrence of residues, its metabolites or other substances in animal tissues, in
excess of the approved safe levels following intentional or unintentional administration of such
drug in animals (Tyler and Cullor, 1989; Brown, 1992).
2.1.7 Extra-label use of antimicrobial drug
The use of antimicrobial drug outside the label specifications or in a way, manner or
purpose not recommended by the manufacturer is referred to as extra-label use. The labels of
antimicrobial drugs should contain all necessary information regarding the use of the drug such
as species in which it may be used, conditions for which it may be administered, dosage and
route of administration as well as withdrawal period (Schwartz and Chaslus-Dancla, 2001).
Antimicrobial residues could occur in food animals when the drugs are administered outside the
labeled dose or recommendations (Van Dresser and Wilcke, 1989). Extra-label use of drugs in
veterinary practice is only allowed at the permission or supervision of a veterinary doctor but this
is not so in Nigeria where farmers and non-veterinarians administer veterinary drugs without
prescription (Dipeolu, 2002; 2010)
28
2.1.8 Limits of detection (LOD)
The limit of detection of a test kit refers to the minimum amount of residues or analytes
needed to be present in the test samples in order to obtain a positive result. The LOD of most
available test kits are usually set at or below the MRLs. When the amount of the analyte such as
antimicrobial residue present in the test sample falls below the LOD of the test kit, the kit
recognizes or interprets the result as a negative case but vice versa if the concentration of the
analyte equals the LOD or falls above it. The Limits of detection of Premi® Test kit for selected
antimicrobials are presented in Table 2.
Table 2: Limits of detection (LOD) of Premi® Test for selected antimicrobials
Antimicrobials LOD of Premi® Test (µg/kg) MRL in pork (µg /kg) MRL in beef (µg /kg)
Penicillin G 25 50 50
Amoxicillin 25 50 50-100
Ampicillin 25 50 50
Cloxacillin 50 100 100
Chlortetracycline 50 100 100
Oxytetracycline 50 100 100
Tetracycline 50 100 100
Doxycycline 50 100 100
Adapted from Nisha, (2008)
29
2.1.9 Withdrawal period
Withdrawal period also known as depletion time, holding time, clearance period, pre-
slaughter time, etc is the time required for residues levels of toxicological importance in food
animal tissues to deplete to safe levels (Vranic, et al., 2003; Cannavan, 2004). It is the minimum
period of time between the last medication and the time of slaughter, which must elapse in order
for residues in animal tissues (muscle, liver, kidney, and skin/fat) or other products (milk, eggs,
honey) to equal or fall below the MRLs for the tissues (Reyes-Herrera, et al., 2005). Until the
withdrawal period elapses, medicated food animal should not be slaughtered for human
consumption.
The withdrawal period is set out in the data sheet for the medicine and in the instructions
for use which are part of the product packaging. Farmers and veterinary practitioners are
required by law to observe withdrawal period or ensure that it is observed by their client.
Withdrawal periods exist so that MRLs are not exceeded and to ensure consumers’ safety. When
extra-label use of antimicrobials is allowed by veterinarians, it is expected that withdrawal period
should be adjusted accordingly, most times extended, to minimize the chances of accumulation
of the residues in animal tissues (Vranic, et al., 2003). Food safety is the sole reason why both
MRLs and withdrawal periods are established (Kaferstein, 2003). The withdrawal periods of
selected antimicrobials used in cattle and pigs as indicated by the manufacturers are presented in
Table 3. Theoretical representation of tolerance level and withdrawal period is shown in figure
1.
30
Table 3: Withdrawal periods of various antimicrobial agents as indicated by the manufacturers
Antimicrobial agents Producer Constituent Withdrawal Period (days)
Meat Milk
Oxytetra-200 LA® Pantex Oxytetracycline 21 7
Tylovet® 20% Cossyvet Tylosin 5 3
Gentreat 10% Kepro Gentamycin 7 3
Oxytetracyclin® Tennyson Oxytetracycline 15 -
Tylonor 20% Jubaili Agrotec Tylosin 7 3
Oxytrox® LA Tennyson Oxytetracycline 21 6
Streptopen® Pantex Procaine penicillin-G,
Dihydrostreptomycin
9 4
Alrocin-10® Mudra pharma Enrofloxacin - -
Conflox inj® Animal care Enrofloxacin 14 3
SULPHAvet 33.3% Cossyvet Sulphamethazine 14 3
SULMIDIN® Eagle chemicals Sulphadiazine 15
Norflox-150® Interchemie Norfloxacin 8 4
Penstrep-400® Interchemie Procaine penicillin-G,
Dihydrostreptomycin
21 3
Limoxin-200 LA® Interchemie Oxytetracycline 28 7
Biocillin-150 LA® Interchemie Amoxicillin 21 3
Biogenta vet® Interchemie Amoxicillin,
Gentamycin
30 2
Genta-100® Interchemie Gentamycin 30 3
Macrolan-200® Interchemie Tylosin 10 3
Sulfa-333® Interchemie Sulphadimidine 10 4
Procaben-LA® Interchemie Procain penicillin-G, 14 3
Source: Field survey, 2011(Unpublished report)
31
Figure 1: Theoretical representation of withdrawal period
Adapted from: Donoghue, (2003)
2.2 Antimicrobials
2.2.1 History of antimicrobials
Antimicrobials are substances capable of inhibiting the growth or multiplication of
micro-organisms, with the largest group being those that are effective against bacteria (Prescott,
1997). It was the discovery of penicillin, a fungal metabolite, by Fleming in 1929 and its later
development by Ernst Chain and Howard Florey during World War II that lead to the
antimicrobial revolution and development of many other classes of antimicrobial (Phillips et al.,
2004; Guardabassi and Kruse, 2008). Today, antimicrobials play a major role in modern
livestock production and their use has been on the rise in many countries due to poor
management practices and endermicity of diseases (Witte, 1998; Sarmah et al., 2006).
32
2.2.2 Use of antimicrobials in food animals
The use of antimicrobials in food animals began over 50 years ago when
chlortetracycline fermentation waste was found to enhance animal health and performance
(Guardabassi and Kruse, 2008; Phillips et al., 2004). Antimicrobial use in animal is generally the
same as or closely related to its use in humans (Lee et al., 2001; Olatoye and Ehinmowo, 2010).
Because of the fact that many of the antimicrobials used in animals are also used in humans, the
use of these drugs in food animals is part of the global problem of antimicrobial resistance
pathogens such as methcillin-resistant bacteria (Kim, et al., 2005; Doyle, 2006; Olatoye and
Ehinmowo, 2010).
Antimicrobial agents can be administered to animal individually for treatment purposes
(therapy) or to prevent (prophylaxis) disease. The drug can be administered to clinically healthy
animals belonging to the same flock or group of animals with clinical signs of illness (a form of
prophylaxis called metaphylaxis mostly carried out in acquaculture, poultry and swine
production), or for growth enhancement (Guardabassi and Kruse, 2008). Antimicrobials have
been in use as feed additives in low doses in farm animals for decades to improve growth rate or
feed efficiency in poultry, pigs and feedlot cattle (Nisha, 2008). The use of antimicrobials for
growth enhancement increases feed efficiency by 17% in beef cattle, 15% in swine and poultry
and 10% in lambs (Gutafson and Bowen, 1997; Nisha, 2008). Although the mechanism by
which antimicrobials exert their growth promotion property is not properly understood, Nisha
(2008) suggested that they may produce improved performance in livestock production in
various ways which includes:
Thinning of the mucous membrane of the gut to facilitate nutrient absorption,
Altering gut motility to enhance assimilation,
33
Enhancement of conditions beneficial to gut microbes by destroying pathogenic
bacteria,
Partitioning proteins to muscles accretions via suppression of monokines,
Decrease waste of nutrients and toxin formation.
In most of all these cases, only young growing animals are responsive enough to the growth
enhancing property of antimicrobial drugs. Report have shown that the claimed benefits derived
from the use of antimicrobials as growth promoters may no longer be realized in modern animal
production system (Barua, et al., 2006) and this practice tends to thrive only in farms where
management practices and hygienic conditions are poor (Gutafson and Bowen, 1997).
Although the use of antimicrobials as growth promoters has been prohibited in the
European countries, it is still being widely used as feed additives in sub-therapeutic doses in
many African countries for growth enhancement in food animals and to compensate for
mismanagement practices that often predispose animals to diseases (Byarugaba, 2004; Barua et
al., 2006). Banning the use of antimicrobial agents in livestock production or veterinary practice
may cause great welfare problems in animals or may even reverse the gains being made in the
production of foods of animal origin. Witte (1998) is of the view that the use of antimicrobials
for growth enhancement in livestock production can be gradually phased out by adopting better
farm management practices.
Several guidelines are available in developed countries for judicious use of antimicrobials
in food animals but very little is being done in most developing countries to minimize irrational
use of drugs in food animals (Byarugaba, 2004). Teteracycline is the antimicrobial class
quantitatively most used in animals followed by ß-lactams (Schwartz, and Chaslus-Dancla, 2001;
34
Donoghue, 2003). Some antimicrobials growth promoters being used in food animals are
presented in Table 4.
Table 4: Some antimicrobial growth promoters used in cattle production
Antimicrobial agents
Classes of animal
Level (mg/head/day)
Purpose
Bacitracin Beef cattle, 35-70 Growth promotion, improved feed efficiency
Chlortetracycline Calves Growth promotion, improved feed efficiency
Beef cattle 70 Growth promotion, improved feed efficiency
Oxytetracycline Calves 25-75 Increased weight gain and Growth promotion, improved feed efficiency
Beef cattle 75 Increased weight gain and Growth promotion, improved feed efficiency
Monensin Beef cattle 5-30g/ tone of feed
Growth promotion, improved feed efficiency
Adapted from Dipeolu, (2010)
2.2.3 Tetracyclines
Tetracyclines are broad spectrum antimicrobials produced naturally by Streptomyces of
the genus Actinobacteria (Alekshun and Levy, 2007). Tetracyclines work by binding to the 30s
ribosomal subunit thereby inhibiting protein synthesis in bacteria cell walls. Kabir, et al., (2004)
identified tetracyclines as the quantitatively most used antimicrobials in Nigeria while Stead et
al., (2004) listed chlortetracycline, oxytetracycline and doxycycline as the members of
tetracyclines family mostly used in food animals and veterinary practice world-wide.
However, the use of this class of antimicrobial in food animals may result in
accumulation of its residues or metabolites in animal derived food products, especially if the
withdrawal period is not observed. These residues may pose health hazards to consumers,
35
depending on the type of food and the amount of residue present. The maximum residue limits
(MRLs) for oxytetracycline as recommended by the joint FAO/WHO expert committee on food
additives (1999) are 0.2, 0.6 and 1.2 μg g-1 for muscle, liver and kidney tissues, respectively.
Tetracycline residues that exceed the MRLs may be of public health concern. Other human
health problem that could arise from the consumption of tetracycline residues in meat and other
animal products include gastrointestinal disturbances (Baker and Leyland, 1983), teratogenicity
and allergic reactions (Woodward 1991; Akbar-Shahid et al., 2007) and development of resistant
pathogens in human and animals (Mishra et al., 2011). Tetracyclines residues in meat are also
associated with teeth discoloration in neonates and peripheral blood changes upon prolong
exposure (Waltner-Toews and McEwen, 1994; Walton et al., 1994).
2.2.4 Betalactams
This class of antimicrobial has bacteriocidal activity and act by the inhibition of cell wall
synthesis in both Gram negative or Gram positive bacteria. Residues of this class of drug in meat
or other animal products can cause severe allergic reaction in susceptible individuals as well as
development of antimicrobial resistance (Raison-Peyron, et. al., 2001). About 10-15 percent of
human population is considered to be sensitive to betalactam antimicrobials especially penicillin
(Cochrane et al., 1995) and may suffers allergic reactions like skin rashes, hives, asthma and
anaphylactic shock upon exposure to the drug or its residue. There is no evidence that exposure
to penicillin residue in food animals can cause sensitization to the drug or its residue but as little
as 0.6µg or 101 IU of penicillin residue in animal products can cause allergic reactions in
sensitized individuals (Waltner-Toews and McEwen, 1994; Raison-Peyron, et. al., 2001).
However, the degree of the reaction varies with individuals and types of food consumed due to
36
different rates of absorption of the residues in these individuals and types of food consumed
(JECFA, 1990, Doyle, 2006). The overall prevalence of allergy to penicillin residues in foods of
animal origin in different populations has been estimated at 3-10% (Doyle, 2006).
2.2.5 Aminoglycosides
Aminoglycosides act bacteriocidally by inhibiting protein synthesis at the 30s RNA of
susceptible Gram-negative bacteria. Gentamycin and streptomycin are the quantitatively most
used members of this class of antimicrobial in both humans and animals (Nisha, 2008).
Aminoglycoside residues in animal tissues are associated with organ toxicity especially
nephrotoxicity (JECFA, 1997). Kim and Park, (1998) reported severe damage in the cranial
nerves of neonates which later resulted in congenital deafness following the treatment of
pregnant mothers, suffering from tuberculosis with 1g of streptomycin, twice weekly during the
first trimester. Aminoglycoside residues can also cause tinnitus, a side effect of this class of drug
usually described as “ringing in the ear”. It may also facilitate the development of antimicrobial
resistance bacteria in animal, transferable to man, especially when administered at sub-
therapeutic doses (JECFA, 1997).
2.2.6 Quinolones
Quinolones are an important group of synthetic antimicrobials with bacreriocidal action
due to their selective inhibition of bacterial DNA synthesis. The quinolones are relatively new
antimicrobial agents and resistance to these drugs remains largely low (Al-Ghamdi and Al-
Mustapha, 2000). However, there is presently a worrisome world-wide trend of increased
resistance to these agents among bacteria responsible for both hospital and community-acquired
infections including methicillin-resistant Staphylococcus aureus, Klebsiella pneumoniae,
37
Pseudomonas aeruginosa, Escherichia coli, Salmonella spp., Campylobacter spp. and Neisseria
gonorrhoeae (Kim, et. al., 2005) such that the use of this class of antimicrobial agents in food-
producing animals has become a very important public health issue (Belloc, et al., 2005). The
use of Enrofloxacin, a second generation quinolone, in animal is prohibited in USA and UK as it
readily induces resistance in zoonotic Campylobacter spp (Al-Ghamdi and Al-Mustapha, 2000;
Strolker & Brinkman, 2005).
2.2.7 Amphenicols
Chloramphenicol is an effective antimicrobial drug widely used in veterinary and human
medicines as it acts against many pathogens by inhibiting protein synthesis at the 50s rRNA.
However, resent reports of aplastic anaemia in humans arising from its use had led to the ban of
its use in food animals in most parts of the world (WHO, 1995; 1999). Aplastic anaemia is an
irreversible type of bone marrow depression in susceptible individuals, which is usually fatal.
Inhibition of protein synthesis in the mitochondria of bone-marrow cells has been suggested as
the mechanism by which chloramphenicol induces bone-marrow depression in man and animal
(Nagata and Saeki, 1992; Rappeport and Bunn, 1994; Young, 2002). Florfenicol, which is
structurally similar to chloramphenicol but with no history of aplastic anaemia is used as
substitute for chloramphenicol. Apart from aplastic anaemia, other public health hazards
attributable to residues of amphenicol class of drugs in animal products are antimicrobial
resistance, decreased haemoglobin concentrations, blood dyscrasia and reticulocytopenia
(FAO/WHO, 1999; Plumb, 2002).
38
2.2.8 Sulphonamides
Sulphonamides are bacteriostatic antimicrobial agents that exert their effect through the
inhibition of DNA synthesis in microbial cells by blocking folic acid production in the cell. It is
effective against coccidia oocysts and Salmonellae species in animals especially when combined
with trimethoprim (Brander et al., 1993). This class of antimicrobial drugs, are widely used for
therapeutic and prophylactic purposes in both human (Kim and Park, 1998) and animals
(Schwarz and Chaslus-Dancla, 2001), and sometimes as additives in animal feed because
prolonged ingestion of sulfonamides may have a growth-promoting effect (Long et al., 1990).
The main public health risk of sulphonamide residue in foods of animal origin is the
development of antimicrobial resistance (Paige, et al., 1999; Kozarova, et al., 2004). In addition,
some sulphonamides residues are carcinogenic (Nue, 1992) and its use in food animals has
become a cause of considerable debate in food safety. It has been reported that 10-15% of human
patients treated with sulphonamides or those who ingested the residue in animal products
received unwanted effects from the drug (JECTA, 1990; Kozarova, et al., 2004) such that
sulphonamide residues in foods of animal origin has become a major food safety concern.
2.2.9 Regulation of agricultural use of antimicrobials in Nigeria
Government at all levels in Africa recognize the importance of food safety especially
foods of animal origin, and have laws and regulations such as meat inspection laws but
enforcement of such laws and regulations is usually poorly done (FAO/WHO, 2005) probably
due to lack of funding and motivation. Veterinary drugs are sold in the open market in Nigeria
and in a bid to reduce the cost of veterinary services, farmers buy and administer veterinary drug
without a prescription (Fagbamila et al., 2010). In Nigeria just like most developing countries of
39
the world, antimicrobials are used in animals indiscriminately for the prevention and treatment of
bacterial infection (Kabir et al., 2004; Dipeolu, 2010).
Financial resources are usually inadequate for law enforcement agencies to carry out
their works effectively and support facilities such as laboratories are usually ill-equipped in both
personnel and research equipment (FAO/WHO, 2005). This is further compounded by the fact
that in terms of resource allocation, public health issues of livestock production is not prioritized
in most African countries (FAO/WHO, 2005).
2.3 Antimicrobial residues monitoring and surveillance in Nigeria
In order to address the public health concerns of AMRs in food animal, it is imperative to
have knowledge of the occurrence of AMRs in human food chain. This knowledge needs to be
updated regularly so that appropriate responses can be adopted. Activities involved in such a
system are referred to as “Monitoring” and “Surveillance”. Monitoring is the performance and
analysis of routine measurements, aimed at detecting epidemiological changes in the
environment or health status of a population. Wong, et al., (2004) defined surveillance as the
ongoing systematic collection, collation, analysis and interpretation of data, for dissemination of
the information generated to all stake holders so that appropriate measures may be taken to
safeguard public health.
Information on the effects of antimicrobial residues is scanty in Nigeria although
NAFDAC in 1996 cautioned on the mutagenic potentials of Nitrofuran drugs used in the
treatment of salmonellosis in poultry (Fagbamila, et al., 2010). An official monitoring of drug
residues is lacking in Nigeria and consumers response towards the health risks posed by
antimicrobial residues in animal products is passive (Kabir, et al., 1999). The presence of AMRs
40
in foods of animal origin should be continually monitored to reduce the health hazards posed to
consumers by such residues as well as the negative impacts of AMRs to the environment
(Popelka, et al., 2005).
Australia had a monitoring programme on drug residues in animal products since the
1960s through the national residue survey (NRS) (Nicholls, et al., 1994) while China had
regulated the use of antimicrobials in animals since 1989 (Jin, 1997). Agricultural use of
antimicrobials in the USA and Canada are also regulated. Feed antimicrobials including
performance enhancers, coccidiostats, and therapeutic antimicrobials are licensed for specific
purposes in poultry, pigs, calves or feedlot cattle (Prescott, 1997). In the UK and other European
countries, veterinary medicinal products and performance enhancers are subjected to assessment
for safety, emergence of antimicrobial resistance, cross resistance to therapeutic antimicrobials
and transferable resistance via an agency called the Veterinary medicine directorate (Okerman et
al., 1998; Al-Ghamdi and Al-Mustapha, 2000).
There is no national surveillance or monitoring programme on drug residues in animal
products in Nigeria (Oboegbulem and Fidelis, 1996; Dipeolu, 2002; Fagbamila et al., 2010)
despite the large scale misuse and abuse of antimicrobials (Oboegbulem and Fidelis, 1996) just
as legislation regarding veterinary drug use and residue control is lacking (Kabir et al., 2004).
Basic facilities for detection of antimicrobial residues in foods of animal origin are also
unavailable at the level of abattoirs, meat processing plants and meat markets in Nigeria (Kabir
et al., 2004).
41
2.4 Antimicrobial resistance
The development of antimicrobial resistance bacteria is a global public health problem.
Scientific evidence suggests an increase in the trend of development of bacteria resistance and its
negative impact on both human and animal health (Guardabassi and Kruse, 2008). Peter et al.,
(2008) defined bacterial resistance as the development of bacterial defenses against therapeutic
agents. The most common of these bacterial defenses are enzymatic drug inactivation,
modification or replacement of drug target, active drug efflux and reduce drug uptake (Peter et
al., (2008)
Bacterial resistance can be intrinsic (constitutive or natural) or acquired. Intrinsic
resistance is due to a structural or functional trait inherently associated with a bacterial species,
genus or even a larger group. Acquired resistance is due to genetic changes in the bacterial
genome consequent upon random mutation in housekeeping gene or horizontal acquisition of
foreign genes (Nayak et al., 2004; Lars et al., 2008). Schwarz and Chaslus-Dancla, (2001)
reported that bacteria can acquire antimicrobial resistance genes by uptake of free DNA
(transformation), via bacteriophage (transduction) or by cell-to-cell transfer (conjugation).
Conjugation is by far the most important mechanism of transfer of bacterial resistant gene due to
its broad-host range and the frequent location of resistant genes on conjugate elements such as
plasmids and transposons (Peter et al., 2008) but resistance can also result from mutation and
gene transfer (Kim and Park, 1998). Regardless of the method by which resistance is acquired,
the use of antimicrobial agents may create optimal conditions for the emergence and
dissemination of resistant bacteria.
While there is still no consensus on the degree to which usage of antimicrobials in
animals contributes to the development and dissemination of antimicrobial resistance in human
42
bacteria (Amaechi, and Ezeronye, 2006), epidemiological and molecular studies point to a direct
relationship between antimicrobial use and the emergence of resistant bacterial strains in animals
(Lee et al., 2001), and their spread to humans, especially via the food chain (FAO/WHO, 1999).
WHO in 1997, warned that the use of growth promoters in food animals encourages the
development of antibiotic resistant organisms. She recommended that antimicrobial use in
agriculture as growth promoter should be discontinued especially if the same or similar
antimicrobial is being used in human therapy or known to select for cross-resistance to
antimicrobials used in humans.
Multi-drug-resistant Salmonella typhimurium definitive phage type (DT) 104 is
responsible for numerous infections in humans in the United Kingdom, Europe, the United States
of America and Canada (Amaechi, and Ezeronye, 2006). The infections in the United Kingdom
were regarded as zoonotic in origin as DT 104 is the most common Salmonella strain in cattle,
sheep and pigs and second most common in poultry in the United Kingdom (Bolton, et al., 1999;
Walker et al., 2000). Resistance problem in one country can spread to another country since
microbes do not respect geographical boundaries.
Resistance to one antimicrobial agent can be selected for by another agent by either
cross-selection or co-selection. Cross selection refers to the ability of a single resistant bacteria
gene to exhibit resistance to two or more antimicrobial agent (cross resistance), usually
belonging to the same antimicrobial class (Bolton, et al., 1999). Co-selection is due to co-
existence of distinct genes or mutations in the same bacterial strain, each conferring resistance to
a different class of drug (Bolton, et al., 1999. Multidrug resistance bacteria isolates in animals
have been reported in Enugu State (Chah et al., 2003; Okoli et al., 2005; Chah and Oboegbulem,
43
2007; Akwuobu et al., 2010) and this was attributed to indiscriminate use of antimicrobials in
livestock production and non-compliance to withdrawal period (Abiade-Paul, et al., 2006).
2.5 Effects of cooking or cooling on antimicrobial residues in meat
Research has shown that freezing and cooking reduce the levels of antimicrobial residues
in foods of animal origin but conventional heat treatment such as cooking, do not eliminate most
AMRs in meat (Dipeolu and Ayo-Adisa, 2006). Van Egmond et al., (2000) reported that the
mean biological activity of enrofloxacin in pork tissues only reduced to 68% after heat treatment
at 134˚C for 20 minutes. Al-Mustafa and Al-Ghamdi, (2000) in another study involving
Norfloxacin reported that 40.5% and 72.1% of muscle and liver tissues of cattle respectively
retained AMRs above MRL after cooking at 100˚C for 20 minutes. Mishra, (2011) reported that
pasteurization of milk at 65˚C for 30 minutes produced no significant reduction in cloxacillin
residues in milk. Although cooking degraded tetracycline residues (chlortetracycline and
oxytetracycline) in meat containing as much as 10 ppm (FAO/WHO, 2005), Javadi et al., (2011)
are of the opinion that cooking process cannot eliminate AMRs present in meat because the
temperature and time duration required are not attainable during normal cooking process. Most
of the residues are excreted from tissue to cooking fluid during cooking process (Javadi, et al.,
2011).
While heat treatment, such as cooking, may be useful in reducing the amount of some
AMRs in meats, effort should be geared towards total elimination of drug residues in foods of
animal origin. This is because minute quantities of some residues such as penicillin can produce
immunological effects (e.g allergic reactions) in sensitized individuals (Mishra, 2011). Health
problems associated with consumption of AMRs in animal tissues may be reduced by proper
44
cooking of meat and discarding the cooking water as AMRs or its metabolites tend to
concentrate in the water (Javadi, et al., 2011).
2.6 Antimicrobial residue detection in animal tissues
The availability of a simple and reliable screening tool for the detection of antimicrobials
in food animal tissues is essential to food safety and public health (Lohajova et al., 2006).
Methods for surveillance testing of antimicrobial residues may be classified into screening
methods and confirmatory methods. Screening methods are used to detect the presence of
analytes of interest such as AMRs in large number of samples but most screening methods lack
specificity (Popelka et al., 2005). Confirmatory methods such as high performance liquid
chromatography (HPLC) are used to identify and quantify specific AMRs in samples positive to
screening methods (Stead et al., 2004). Methods of analysis of antimicrobial residue present in
animal tissues may be microbiological, immunochemical, or physico-chemical methods. Details
on some of these methods are as listed in Table 5 below.
Table 5: Methods of residue testing in animal tissues
Microbiological method Immunochemical method Physico-chemical methods
Premi® Test kits (PTK) Enzyme linked immuno-zobent assay (ELIZA) test kit
High Performance Liquid Chromatograpgy (HPLC)
Four Plate Test (FPT) Radioimmunoassey High Performance Thin-Layer Chromatograpgy (HPTLC)
Live animal swab test (LAST) Multi-array tests Mass spectrometer
Bacillus sterothermophilus vas calidolactic disk (BsDA),
Biosensors Gas chromatography
Test On Premises (STOP test)
Delvotest – P Source: Stead et al., (2004)
45
2.6.1 Microbiological methods
Microbiological method also known as bioassay or microbial inhibition test is based on
the inhibition of the growth of microorganisms due to the presence of inhibitors (AMRs) in test
samples (meat, milk, egg, etc). Microbial inhibition tests are multi-residue screening tests for
detection of AMRs in meat and other animal products due to their high sensitivity but they may
lack specificity (Popelka et al., 2005). Live animal swab test (LAST) is a type of microbial
inhibition method used for detection of potential AMRs in meat before an animal is slaughtered
(Seymour et al., 1988) so that withdrawal period in positive cases may be enforced before
slaughter. Microbiological inhibition tests are widely used for AMR screening in food animals
and it is unlikely that these tests will be replaced by other screening techniques in near future
(Gaudin, et al., 2009) because, they are relatively cheap, broad-spectrum and permits screening
of large number of samples in a short period of time (Gaudin, et al., 2008)
2.6.1.2 Limitations of biological methods
Biological methods are mostly used as screening tool. Some of these methods have been
shown to give false positive result up to the tune of about 16% in milk when subjected to
confirmatory test such as the HPLC (Sischo and Burns, 1993). Such false positive results
associated with microbial inhibition tests are due to high concentration of somatic cells in milk
samples from mastitic udder (Sischo and Burns, 1993). Jones and Seymour, (1988) observed that
in meat and other animal tissues that are not from inflamed tissue, false positive results may be
due to the inhibition of the test organism by inhibitors other than AMRs. However, Popelka, et
al., (2005) in their own study attributed the false positive results of microbiological inhibition
methods to the type of test organism used and demonstrated that tests utilizing Bacillus
46
sterothermophilus such as Premi® test had highest true positive results than ones utilizing other
microbial test organisms.
2.6.2 Immunochemical methods
Some of the immunochemical methods of AMRs testing are listed in table 5. They are
rapid, selective, sensitive and of considerable utility in some areas of AMRs analysis. Using
bioreceptors from biological organisms or their receptors, scientists have developed new means
of immunochemical analysis with high specificity such as the biosensors (Vo-Dinh & Cullum,
2000). These methods can identify specific antimicrobial residue or recognize structurally similar
metabolites through the antibody-antigen/enzyme interaction (Vo-Dinh & Cullum, 2000).
Advanced immunodiagnostic assay such as immunoflourescence and radioimmunoassay can
detect less than 10-9 mg/dl of antimicrobial residue of interest in edible tissues but
immunochemical methods are generally very expensive (Vo-Dinh & Cullum, 2000).
2.6.3 Physico-chemical methods
The physico-chemical methods such as the HPLC are regarded as confirmatory tests for
AMRs detection and quantification in edible tissues. The HPLC analysis involves extraction of
analytes, sample clean-up and sample analysis and have become the most widely accepted
confirmatory technique for AMRs in meat and other animal products (Petz et al., 2002; Biswas
et al., 2007). However, HPLC system is very expensive. The personnel and facilities required to
carry out this test are not readily available in most developing countries.
2.7 The choice of Premi® Test kit
The procedures for conducting antimicrobial screening tests need to be reliable, simple,
inexpensive and requires very simple equipment. Premi® Test kit (fig 2) has been used as a
47
method of first choice for qualitative screening of meat samples for AMRs (Popelka, et al.,
2005). Premi® test kit is a simple, sensitive and cost-effective screening tool capable of
detecting a wide range of AMRs from a variety of tissues in different species within a short
period of about 3 hours (Stead et al., 2004; lohajova et al., 2006, Stead et al., 2007). In a study
carried out by Guadin, et al., (2008) to compare various microbial tests used for the detection of
AMRs in meat, it was found that Premi® Test results correlated with the confirmatory results of
HPLC than other microbiological inhibition tests. The kit is qualitative but semi-quantitative in
nature, being able to detect AMRs at the European MRL (Stead et al., 2004; Guadin, et al.,
2008).
Figure 2: Premi® Test kit (as supplied by Biopharm®, Netherlands)
48
2.7.1 The principle behind Premi® Test
Premi® test is a commercially available agar diffusion screening test based on inhibition
of the growth of, Bacillus stearothermophilus, a thermophilic bacterium that is very sensitive to
many antimicrobial compounds (Stead et al., 2004; 2007). A standardized number of spores of
the test organism are imbedded in the agar medium with selected nutrients contained in an
ampoule. When Premi® test ampoule is heated at 64°C, the spores will germinate. The
germinated spores will multiply with the production of acid if inhibitory substances such as
AMRs are absent. This will be manifested by a change in colour of the bromocresol purple
indicator from purple to yellow coloration. When antimicrobial compounds are present in
sufficient amount (above limit of detection) no growth will obtain and the colour of the lower 2/3
of the ampoule remains purple.
2.8 Prevalence of antimicrobial residues in animal tissues in Nigeria
Unlike in developed countries, where the amount of antimicrobial drugs used in a year
can readily be determined by measuring sales and prescriptions (McCaig & Hughes, 1995), it is
much more difficult to measure the amount of drugs used in developing countries where farmers
are often unaware of the drug prescribed, purchased, or administrated by a veterinarian. The task
is even made more difficult by the wide range of available products and mixtures, adulteration,
and uncontrolled sale of drugs (Bojalil & Calva, 1994). Medication records may also be
unavailable.
A number of studies have been conducted to determine the prevalence of AMRs in
foodstuffs of animal origin in Nigeria. Oboegbulem and Fidelis (1996), Kabir et al., (2002),
Dipeolu and Alonge (2002), Ibrahim et al., (2010), Olatoye and Ehinmowo (2010) reported
49
antimicrobial prevalence of 8.3%, 7.4%, 16.7%, 44% and 34.4% respectively in cattle in various
states of Nigeria. There is paucity of information concerning antimicrobial residues in pigs in
Nigeria probably due to some religious restrictions that forbids rearing and handling of pigs but
Dipeolu and Alonge (2002) recorded a prevalence of 6.67% for streptomycin residues in pigs
slaughtered for human consumption in some states of Southwest, Nigeria. Ezenduka et al.,
(2011) and Fagbamila et al., (2010) recorded prevalence of 36% and 3.6% in table eggs sold for
human consumption respectively.
2.9 Public health problems of antimicrobial residues in animal products
It has been documented that consumption of Mexican beef liver containing residues of
illegal growth promoter, clenbuterol, produced symptoms of trembling, headache and general
malaise in at least 225 consumers in Jalisco in 2005 (Martínez, 2005). Antimicrobials in food
animals resulted in unwanted effects, such as allergic reaction and the development of
antimicrobial resistance bacteria pathogens following consumption of food animal tissues with
history of recent treatment (Dupont, & Steel, 1987). Other public health concerns of these
residues include carcinogenicity, mutagenecity and teratogenic effects in human.
2.9.1 Antimicrobial drug resistance
Antimicrobial resistance is the ability of certain bacteria which are normally destroyed by
a particular antimicrobial, to survive exposure to that antimicrobial. This means that the bacteria
no longer respond to antibacterial treatment. Investigations have identified increasing resistance
in several types of bacteria which can be transmitted from animals to humans through food chain
(Chah et al., 2003; Okesola and Oni, 2009). This resistance was attributed to indiscriminate use
50
of antimicrobial and lack of compliance to the regulations guiding the use of antimicrobials in
food animals (Okesola and Oni, 2009).
Many of the antibiotics used to treat bacterial infections in humans also have veterinary
applications; being used to treat infections in sick and injured animals or in lower concentrations
for prophylaxis and growth promotion purposes (Lee et al., 2001). The use of low doses of these
drugs for prophylaxis or growth enhancement encourages the development of antibiotic resistant
strains of bacteria (Simonsen et al, 1998). There is a serious public health concern that resistant
organisms shed by animals may transfer resistance-genes to enteric organisms of humans which
could complicate the treatment of human diseases. Smith et al., (2007) reported higher incidence
of antimicrobial resistance in individuals who consume antibiotic-treated animal tissues than in
the unexposed populations.
2.9.2 Allergic reactions
Exposure of humans to AMRs (especially suphonamides and penicillins) in animal
products may produce allergic reactions in susceptible and sensitized individuals (Wang, et al,
2006). Allergic reactions to drugs and chemicals may include anaphylaxis, serum sickness,
cutaneous reaction (rashes and itches) and delayed hypersensitivity (Booth, 1998). Consumption
of meat or other animal products containing high concentrations of residues of penicillins and
sulphonamides have been shown to cause skin allergy to subjects sensitive to the drugs (Popelka
et al., 2001).
In a survey of all antimicrobial violative cases in milk in the United States, Paige et al.,
(1999) reported that the most frequent detected antimicrobial residues were penicillin (20%),
streptomycin (10%), and sulfamethazine (9%). Cases of allergic reactions (e.g., skin rashes) in
51
individuals previously sensitized to penicillin-G residues in milk and meat have been
documented, with strong evidence linking a widespread agricultural use of antibiotics to an
increase in antibiotic resistance among animal and human pathogens (Kindred et al. 1993).
2.9.3 Carcinogenic effect
Certain AMRs such as sulphonamides may be carcinogenic (Booth, 1988). Since all
chemical carcinogens can lead to uncontrollable cell division in the body, human exposure must
be reduced to the barest minimum (Booth, 1988). 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). It is generally recognized that there is no
relationship between the toxicity and carcinogenicity of chemical compounds, ordinarily the
toxic dose of a carcinogen is higher than the carcinogenic dose (Kindred et al. 1993). Since
cancer induction is humans may take several decades, a drug or chemical agent may be
extensively used for sometime before evidence of carcinogenicity appear (WHO, 1995). This
long latency period complicates evaluation or study of drug residues that have known
carcinogenic activity. Use of animal drug with carcinogenic potential is of great public health
concern because of the possibility that residues of such drug in animal products may add to the
human burden. Consumers desire that foods of animal origin be free from contaminants such as
AMRs. As desirable as this may be, it is impossible to achieve, just as absolute safety itself is
impossible without genuine commitments on the part of all stake holders (Campbell, 1980).
2.9.4 Mutagenic effect
The term mutagen is used to describe chemical agents that damage the genetic components of a
cell or organism. Genetic material of all living organisms, with the exception of some viruses is
52
the DNA. There has been increasing concerns that drug residues may pose a potential hazard to
the human population by production of non-dose-dependent gene mutations or chromosome
aberrations (WHO. 1997). Residues of antimicrobials which excert their action by interference
with DNA synthesis may cause gene mutation in individuals who consume animal products
containing such residues.
2.9.5 Teratogenic effect
Teratogens refer to drug residues or chemical agents that may produce a toxic effect on
the embryo or fetus during the gestation period. As a consequence, a congenital malformation
that affects the structural and functional integrity of the organism is produced. The case of
thalidomide episode involving several children in Europe is direct testimony to the hazards that
may be produced when such an agent is administered (intentionally or unintentionally as in the
case of AMRs) during pregnancy. Approximately 5 years after the introduction of thalidomide
into clinical use, this drug was identified as the aetiologic agent of Phocomelia or ‘seal limbs’
(Booth, 1988) which led to series of litigations, extensive compensation, withdrawal and ban of
the drug. The teratogenic effect of methallibure, an anterior pituitary activator or estrus cycling
control chemical for swine used in the U.K, has been reported (Booth, 1988).
2.9.6 Bone marrow depression
Another potential health risk of residues of antimicrobials in food animals is the
development of aplastic anemia resulting from bone marrow depression associated with
chloramphenicol residues (Settepani, 1984; Gantverg, 2003). Prohibition of the use of
chloramphenicol in all food producing animal, was due to its unwanted effects on blood forming
tissues (Settepani, 1984; Rappeport and Bunn, 1994; WHO, 1997).
53
CHAPTER THREE
MATERIALS AND METHODS
3.1 The study area
This study was carried out in Enugu State which is situated in the Southeast geo-political
zone of Nigeria. It lies between latitude 60 25′ North and longitude 70 27′ East (Oformata, 1975).
The state is bounded in the north by Benue State, in the west by Anambra State, in the south by
Imo State and in the east by Ebonyi State. Enugu State is made-up of 17 local government areas
with a population of 3,253,298 as at 2006 (NPC, 2006).
The state is characterized by two seasons- the wet and dry season. The wet season occurs
between April and October with a break in August while the dry season lasts from late
November to April with cold harmattan wind between December and February. Enugu State has
semi-tropical rain forest vegetation which changes gradually northwards from rain forest to
savannah (Oformata, 1975).. Apart from the chains of low hill, the rest of the state is separated
by numerous streams and rivers.
Igbo language is the indigenous language in the study area but English language is widely
used due to the multi-ethnicity nature of the inhabitants. Slaughter houses and meat markets in
the state are patronized daily by the general public for purchase of meat and other animal
products.
This study was carried out in three senatorial zones of the state namely, Enugu north,
Enugu east and Enugu west. Three major slaughter houses, Nsukka, Akwata and 9th Mile
slaughter houses, located within each of the senatorial zones of the state were visited for sample
54
collection during the study. Enugu State has a lot of tertiary institutions including the University
of Nigeria Nsukka, the citadel of learning, where this research work was carried out. Livestock
farming, civil service and crop farming are the major occupation of the inhabitants of the state.
3.2 The study design
This research work was carried out in three stages:
Questionnaire survey and interview
Experimental study
Cross sectional study
3.2.1 Questionnaire survey and interview
This was carried out using a total of 200 copies of structured questionnaire designed to
obtain information on pattern of antimicrobial drug use, observance of withdrawal period,
awareness of public health risks associated with the accumulation of antimicrobial drug residues
in animal tissues, etc. Prior to the administration of the questionnaire, the respondents were
assured of the confidentiality of their responses since the survey is solely for research purposes.
A minimum of sixty (60) copies of the questionnaire were distributed to willing
respondents (livestock farmers and veterinary practitioners) in each of the three zones of the
state. The content of the questionnaire was communicated in Igbo language, in the form of
interview, to willing respondents who were unable to read or write. Aspects of the questionnaire
that raises questions from the respondents were also explained. Thereafter, the copies of the
questionnaire were retrieved and the responses collated and analyzed.
55
3.2.2 Experimental study
This was carried out to confirm the potency or validity of Premi® test kit (figure 2),
purchased directly from Biopharm®, Germany used in this study. Three hens (about 1 year old)
were purchased from Nsukka market in Enugu State and housed in a poultry cage for 24 hours.
Thereafter, each of the birds were given intramuscular (IM) injection of one of the following
drugs: Long acting oxytetracycline (Limoxin-200 LA®), combination of procaine penicillin-G
and Dihydrostreptomycin (Penstrep®) and sulphadimidine (Sulphavet®) at the recommended
therapeutic dose for 3 consecutive days.
The birds were then sacrificed 8 hours after the drug administration on the third day and
vital organs such as kidney, liver and muscle tissues were harvested and screened for AMRs
using Premi® test kit according to the manufactures instructions (figure 3). This was carried out
in the laboratory of Veterinary Public Health and Preventive Medicine, Faculty of Veterinary
Medicine, University of Nigeria, Nsukka.
Figure 3: Incubation of Premi® test Ampoules treated with meat fluid from the test samples
56
1.2.3 Cross sectional study
This study was carried out to determine the prevalence of AMRs in edible tissues
(kidney, liver and muscle) of cattle and pigs in Enugu State. Premi® test kit was used for the
screening. During the screening, all meat samples that retained the purple coloration of the Premi
test ampoule or had very slight colour change after for 3 hours were regarded to be positive,
contain detectable amounts of AMRs while those that changed from purple to yellow (Figure 4
and 5) were considered to be negative, containing no or undetectable amount of AMRs.
3.2.3.1 Sample population
Cattle and pigs slaughtered in the study area for human consumption were the sample
populations. Beef is widely consumed in Enugu State due to its affordability unlike chicken that
is expensive and not usually retailed. Pork is relatively cheap and readily available as there is no
cultural or religious restriction to its consumption in Enugu State.
3.2.3.2 Sample size determination
A total of 95 animals which was made-up of sixty (60) cattle and thirty-five (35) pigs
were used for this study (table 6). Three samples each consisting of kidney, liver and muscle
tissues were collected per animal. The minimum sample size of 212 which is made-up of 116
and 96 for cattle and pigs respectively was calculated using the formula: N = Z2PQ÷D2 as
described by Margaret, (2004). The calculations were based on the prevalence of AMRs in pigs
and cattle in Nigeria as recorded by Dipeolu and Alonge, (2002) and Oboegbulem and Fidelis,
(1996) respectively.
N=the desired sample size (where the population is ≥ 10,000)
Z=standard normal deviate, usually set at 1.96
57
P= the proportion in the target population estimated to have a particular characteristic.
Q=1−P
D= degree of accuracy desired, usually set at 0.05 level of significance.
3.2.3.3 Sampling area selection
Nsukka, Akwata and 9th Mile slaughter houses were purposefully selected for this study
being the largest slaughter houses in Enugu north, Enugu east and Enugu west senatorial zones
of the state respectively. In Akwata and 9th Mile slaughter houses, slaughtering of pigs was
carried out in a nearby extension called “artisan” and “new market” respectively. Each of the
slaughter houses were visited once every week for eight consecutive weeks (between April and
June, 2011) for sample collection.
3.2.3.4 Sampling method for sample collection
Systematic random sampling method was used in this study such that sample specimen
(kidney, liver and muscle tissues) were collected from every 5th animal (cattle and pigs)
slaughtered. The number of animals sampled in each of the slaughter houses visited is shown in
the Table 6 below and was based on the individual slaughter capacities of the slaughter houses
visited.
Table 6: Number of animals sampled in each of the three slaughter houses visited in Enugu State
Animal species Nsukka SH Akwata SH 9th Mile SH Total
Cattle 25 20 15 60
Pigs 15 10 10 35
Total 40 30 25 95 SH = Slaughter house. Source: Field survey, 2011
58
About 50g each of the liver, kidney and muscle tissues from each selected animal were
collected, packaged separately, labeled and transported in an air-tight container with ice-packs to
the Veterinary Public Health Laboratory, Faculty of Veterinary Medicine, University of Nigeria,
Nsukka for AMRs screening using Premi® Test kit as described by the manufacturer.
3.2.3.5 Processing and screening of meat samples for antimicrobial residues
Premi® Test kit is made up of:
1. Polystyrene boxes in quantities of 25 ampoules of agar containing Bacillus
stearothermophilus spores and bromo-cresol purple indicator
2. A heating block incubator that incubates at fixed temperature of 64 ± 1°C.
3. A pair of scissors
4. A meat press
5. A thermometer
6. Plastic foils
7. Sterilized pipette tips
8. Tuberculin syringe.
The steps involved in the screening were:
1. The required number of Premi® Test ampoules was cut open using a pair of scissors.
2. About 2ml of meat juice from the meat sample were extracted using meat press.
3. About 1ml of the meat juice was slowly pipette unto the agar in the ampoule.
4. The mixture was allowed to stand at room temperature for about 20 minutes for pre-
diffusion to occur.
59
5. The meat juice was then carefully flushed out of the ampoules by gentle washing with de-
mineralized water.
6. Thereafter, the opened parts of the ampoules were covered with foil to limit evaporation
and then incubate at 64˚C for 3 hours.
7. After the incubation, the ampoules were removed and the colour of the lower 2/3 of the
agar observed and interpreted
3.2.3.6 Interpretation of Premi® Test results
During the experimental and screening procedures, all meat samples that retained the
purple coloration of the ampoule or had very slight colour change after about 3 hours of
incubation were regarded to be positive, contain detectable amounts of AMRs while those that
changed from purple to yellow were considered to be negative, containing no or undetectable
amount of AMRs (Plates 1 and 2).
3.3 Data presentation
Data generated in this work were presented using descriptive tools such as tables,
histogram and percentages. The distribution of antimicrobial residues in organs of cattle and pigs
was presented as the percentage of positive samples in relation to the total number of samples.
3.4 Data analysis
Chi-square (χ2) test of independence was used to determine the strength of association
between educational levels of the respondents and observance of withdrawal period and
awareness of public health risks associated with the consumption of AMRs in animal products.
The association between years of working experience and observance of withdrawal period
60
among the respondents was also tested using the same Chi-square statistics. The species
distribution of AMRs in cattle and pigs and the organ distribution of the residues in each of the
species were also compared. All the statistical analyses were done using SPSS package version
17.0 for windows (SPSS Inc, Chicago, Illinois) at 5% probability level.
61
CHAPTER FOUR
RESULTS
4.1 Result of the questionnaire survey
Out of the 200 copies of structured questionnaire distributed to willing respondent in
Enugu State, 182 copies, representing 91% were retrieved; the responses were then collated and
analyzed.
4.1.1 Socio-economic characteristics of the respondents
The socio-economic characteristics of the respondents are presented in Table 7. One
hundred and twenty-nine of the respondents (70.88%) were livestock farmers while fifty-three
(29.12%) were veterinary practitioners. About 29% of the livestock farmers had no formal
education, 34.11% had primary education, 30.23% had post primary education while only 6.98%
had tertiary education. Most of the livestock farmers (52.83%) were into pig farming only. About
8% were cattle farmers only while the remaining respondents (39.15%) combined cattle or pig
farming with other livestock farming such as poultry, goat, sheep, rabbit production, etc.
On working experience, 19.38% of the farmers had less than 1 year farming experience,
17.83% had been in the business for 1-5 years, 35.66% had 6-10 years experience while 27.13%
had over 10 years livestock farming experience. On the other hand, 26.42% of the veterinary
practitioners had less than one year job experience, 37.74% had 1-5 years experience while
33.96% and 1.88% had been in veterinary practice for 6-10 years and above 10 years
respectively.
62
4.1.2 Status of antimicrobial drug use in food animals
All the respondents (livestock farmers and veterinary practitioners) had administered at
least one antimicrobial drug in food animals between January and April, 2011(Table 8). The
antimicrobial drugs used in food animals and the percentage of respondents that utilized these
drugs are oxytetracycline (23.44%), penicillin (21.16%), streptomycin (16.81%) gentamycin
(11.62%), sulphadimidine (10.17%), enrofloxacin (8.92%) and chloramphenicol (7.88%). Many
(48.38%) of the respondents administered the antimicrobial drugs for prophylaxis while 33.12%
and 18.51% of them used the drug for therapy and performance enhancement respectively (Table
8).
On prescription status, 10.17% received their prescriptions from animal handlers, 57.63%
were either involved in self prescription or received their prescriptions from other farmers,
6.21% did not disclose their prescription status while only 25.99% of the farmers administered
the drugs based on veterinary doctor’s prescription (Table 8). On administration status, only
22.97% of the farmers admitted that veterinarians administered the drugs for them (Table 8).
About 16% of the respondents did not disclose who administered the drug for them, 10.04%
utilized the services of animal handlers while 50.72% administered the drugs themselves.
4.1.3 Awareness of health risks associated with consumption of AMRs in animal tissues
The percentage distribution of respondents according to their awareness of health risks
associated with consumption of AMRs in animal tissues is shown in Table 9. About 15% of the
livestock farmers were aware of the health risk(s) associated with consumption of animal tissues
while majority (84.68%) of the farmers were ignorant of the health risks. All the 53 (100%)
63
veterinary practitioners were aware of the possible health problems that may arise following the
consumption of AMRs in animal tissues.
4.1.4 Knowledge of withdrawal period among the respondents
The survey revealed that although 61 (47.29%) of the livestock farmers were aware of
withdrawal period, only 35 of them, equivalent to 57.38% of the “Yes” respondents had
knowledge of withdrawal period (Table 10). All the veterinary practitioners surveyed had
knowledge of withdrawal period (Table 10).
4.1.5 Observance of withdrawal period
Majority of the livestock farmers (88.37%) do not observe withdrawal period while only
11.63% do observe it (Table 11). The survey showed that 69.81% of the veterinary practitioners
observe withdrawal period, while 30.19% do not observe it (Table 11).
4.2 Result of the experimental study
All the organs (kidney, liver and muscle tissues) harvested from the birds injected with
antimicrobials and screened with Premi® Test ampoules retained the purple coloration of the
ampoules even after incubation at 64˚C for 3 hours (Plate 1). The negative control ampoules
containing sterile water changed from purple to yellow colour within the 3 hours of incubation at
64˚C (Plate 1).
64
4.3 Result of the cross sectional study
4.3.1 Prevalence of antimicrobial residues in cattle and pigs
The prevalence of AMRs in cattle and pigs slaughtered for human consumption in the
study area is shown in Table 12. Edible tissues from 30.00% of cattle and 22.85% of pigs
examined contained detectable amounts of AMRs. Nsukka slaughter house recorded the highest
prevalence of AMRs in cattle (15%) while Akwata and 9th mile slaughter houses recorded
prevalence of 8.33% and 6.67% respectively (Table 12). In pigs, the prevalence of AMRs was
lowest (5.71%) in Nsukka slaughter house but equal prevalence of 8.57% was recorded for both
Akwata and 9th Mile slaughter houses.
4.3.2 Organ distribution of antimicrobial residues in cattle
Liver tissues accounted for the total prevalence of 30% as against 26.67% and 16.66%
recorded for kidney and muscle tissues respectively (Table 13). In Nsukka slaughter house, the
prevalence of 13.33%, 15.00% and 10.00% were recorded for kidney, liver and muscle tissues
respectively. Akwata slaughter house recorded prevalence of 6.67%, 8.33% and 3.33% for
kidney, liver and muscle tissues respectively. In 9th mile slaughter house, equal prevalence of
6.67% was recorded for liver and kidney tissues while that of muscle tissue was 3.33%.
4.3.3 Organ distribution of AMRs in pigs
The percentage of AMRs in organs of pigs is presented in Table 14. The prevalence of
AMRs in liver, kidney and muscle tissues of pig slaughtered for human consumption in Enugu
State were 22.85%, 14.28% and 11.43% respectively. In Nsukka slaughter house, equal
prevalence of 5.71% was recorded for all the 3 tissue (kidney, liver and muscle). In Akwata
slaughter house, 5.71% prevalence was recorded for kidney and muscle tissues while liver
tissues yielded 8.57% prevalence. The prevalence of 2.86%, 8.57% and 0.00% were recorded for
kidney, liver and muscle tissues respectively in 9th mile slaughter house.
65
Table 7: Socio-economic characteristics of livestock farmers (n=129) and veterinary
practice (n=53) in Enugu State
S/no. Items required Number (%) of respondents
1 Job description
Livestock farmers 129 (70.88) Veterinary practitioners 53 (29.12) 2
Educational levels of livestock farmers
No formal education 37 (28.68) Primary education 44 (34.11) Post primary education 39 (30.23) Tertiary education 9 (6.98) 3 Species of animal reared Cattle only 17 (8.02) Pigs only 112 (52.83) Others 83 (39.15) 4 Farming experience (livestock farmers only) Less than 1 year 25 (19.38) 1-5 years 23 (17.83) 6-10 years 46 (35.66) Above 10 years
35 (27.13)
5 Experience in veterinary practice (Vets only) Less than 1 year 14 (26.42) 1-5 years 20 (37.74) 6-10 years 18 (33.96) Above 10 years 1 (1.88)
66
Table 8: Antimicrobial drug use among livestock farmers and veterinary practitioners in Enugu
State
S/no. Items required Number (%) of respondents
1 Have administered an antimicrobial drug in pigs or cattle
between January and April, 2011
Yes 182 (100) No 0 (0.00)
2 Antimicrobial drug (s) administered Oxytetracycline 113 (23.44) Penicillin 102 (21.16) Sulphadimidine 49 (10.17) Chloramphenicol 38 (7.88) Gentamycin 56 (11.62) Streptomycin 81 (16.81) Enrofloxacin 43 (8.92)
3 Reason(s) for the drug administration Therapy 102 (33.12) Prophylaxis 149( 48.38) Performance enhancement 57 (18.51)
4 Prescription of the drug(s) used by livestock farmers Farmers/Self prescription 102 (57.63) Animal handlers 18(10.17) Veterinary doctors 46 (25.99) No response 11 (6.21)
5 Administration of the drug(s) used by livestock farmers Farmers 106 (50.72) Animal handlers 21 (10.04) Veterinary doctors 48 (22.97) No response 34 (16.27)
67
Table 9: Distribution of responses on awareness of health risks associated with consumption of animal products containing AMRs among livestock farmers (n=129) and veterinary practitioners
(53) in Enugu State
Category of respondents Awareness
Yes (%) No (%) No response
Livestock farmers 19 (15.32) 105 (84.68) 5
Veterinary practitioners 53 (100) 0 (0.00) 0
Total (%) 72 (40.68) 105 (59.32) 5
68
Table 10: Distribution of responses on knowledge of withdrawal period (WP) among livestock farmers (n=129) in Enugu State
S/no Items required Number (%) of respondents
1 Awareness of WP
Yes 61 (47.29)
No 68 (52.71)
2 Knowledge of WP among the Yes respondents
Time to stop given drug because of overdose 17 (27.87)
Time to stop given antibiotics before vaccination 05 (8.20)
Time to stop given drugs before slaughter 35 (57.38)
No response 04 (6.56)
69
Table 11: Distribution of responses on observance of withdrawal period among livestock farmers (n=129) and veterinary practitioners (n=53) in Enugu State
Category of respondents Observance of withdrawal period
Yes (%) No (%)
Livestock farmers 15 (11.63) 114 (88.37)
Veterinary practitioners 37 (69.81) 16 (30.19)
Total 52 (28.57) 130 (71.43)
70
Table 12: Prevalence of antimicrobial residues (AMRs) in cattle (n=60) and Pigs (n=35) slaughtered for human consumption in three major slaughter houses (SH) in Enugu State
Animal Species No. (%) positive for AMRs
Nsukka SH Akwata SH 9th Mile SH Total
Cattle 9 (15.00) 5 (8.33) 4 (6.67) 18 (30.00)
Pigs 2 (5.71) 3 (8.57) 3 (8.57) 8 (22.85)
Total 11 (20.71) 8 (16.90) 7 (15.24) 26 (52.86)
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Table 13: Organ distribution of antimicrobial residues (AMRs) in cattle (n=60) slaughtered for human consumption in three major slaughter houses (SH) in Enugu State
Organs No. (%) positive for AMRs
Nsukka SH Akwata SH 9th Mile SH Total
Kidney 8 (13.33) 4 (6.67) 4 (6.67) 16 (26.67)
Liver 9 (15.00) 5 (8.33) 4 (6.67) 18 (30.00)
Muscle 6 (10.00) 2 (3.33) 2 (3.33) 10 (16.66)
Total 23 (38.33) 11 (18.33) 7 (16.67) 44 (73.33)
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Table 14: Organ distribution of antimicrobial residues (AMRs) in pigs (n=35) slaughtered for human consumption in three major slaughter houses (SH) in Enugu State
Organs No. (%) positive for AMRs
Nsukka SH Akwata SH 9th Mile SH Total
Kidney 2 (5.71) 2 (5.71) 1 (2.86) 5 (14.28)
Liver 2 (5.71) 3 (8.57) 3 (8.57) 8 (22.85)
Muscle 2 (5.71) 2 (5.71) 0 (0.00) 4 (11.43)
Total 6 (17.13) 7 (20.00) 4 (11.43) 17 (48.56)
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Positive cases Negative cases
Plate 1: Positive and negative cases of AMRs during the validation of Premi® Test kit used in this study
74
Positive cases Negative cases
Plate 2: Positive and negative cases of AMRs during the screening test
75
CHAPTER FIVE
DISCUSSION, CONCLUSION AND RECOMMENDATIONS
5.1 Discussion
5.1.1 Questionnaire survey
The questionnaire survey revealed that most of respondents were pig farmers. This could
probably be due to the absence of religious or aesthetic restriction to the rearing of pig or
consumption of pork in Southeast Nigeria (Nwanta et al., 2011; Marire, 1997). Adesehinwa et
al., (2010), was of the opinion that the massive participation in pig farming in some parts of
Nigeria was due to the high fecundity of pigs in terms of litter size and shorter gestation period in
comparison to cattle. Furthermore, the marketability of pork in Enugu State may have been a
major boost to pig farmers as pork is one of the major delicacies in most restaurants in the study
area (Nwanta et al., 2011).
The poor participation in cattle farming in Enugu State could be due to a misconception
among the “Igbo” tribe that cattle rearing is an occupation of the Fulani man, alien to Igbo
culture and tradition. Apart from this, the unpopularity of cattle farming in Southeast Nigeria
could be attributed to heavy tse-tse fly infestation of the area (Samudi et al, 2010) which is a
major burden to livestock farmers as trypanosomosis, transmitted by the tse-tse fly, is a major
disease problem in cattle (Samudi et al, 2010) usually reared under extensive-transhuman
husbandary systerm.
Lack of formal education among the farmers may have been responsible for the poor
awareness of the health risks associated with the consumption of AMRs in animal tissues. It
may have also been responsible for non-compliance to drug label instructions and observance of
76
withdrawal period recorded in this study, since such farmers may not be able to read and
understand instructions on drug labels.
The high level of indiscriminate use of antimicrobials in food animals recorded in this
study suggests that emphases may have shifted from basic disease prevention management
practices such as biosecurity, vaccination, good nutrition and farm hygiene to drug
administration, in order to make-up for such mismanagement practices in farms. This finding
agrees with the view of Dipeolu, (2010) who reported that there is massive abuse and misuse of
veterinary drugs in Nigeria due to mismanagement practices in the farms. This was further
supported by the findings of Olatoye and Ehinmowo, (2010); Ezenduka et al, (2011) and Ibrahim
et al., (2010) who reported high rate of drug abuse and misuse among livestock farmers in
Southwest, Southeast and Northern Nigeria respectively.
The high level of indiscriminate use of antimicrobials in food animals revealed in this
study may facilitate the accumulation of AMRs in food animals. Since direct relationship
between antimicrobial use in animals and deposition of residues in animal tissues has been
reported (Tollefson and Miller, 2000; KuKanich et al., 2005; Smith et al., 2005), consumers of
animal products in the study area are at risk of the health problems associated with the
consumption of AMRs in food animals.
Drug administration without veterinary prescription as recorded in this study might be to
avert the cost of veterinary services. Olatoye and Ehinmowo, (2010) reported that livestock
farmers, especially the Fulani herdsmen, administer chemotherapeutic agents without veterinary
prescription to save cost. The use of chloramphenicol class of antimicrobial among the
respondents, despite the ban of its use in food animals in Nigeria by the National Agency for
Food and Drug Administration and Control (NAFDAC) in 1996, suggests that there is no
77
compliance to drug laws among Nigerians especially livestock farmers. Residue of
chloramphenicol in animal products may cause aplastic anaemia and other haematological
problems in people who consume the products (Young, 2002).
The poor awareness (15.32%) of public health risks associated with the consumption of
AMRs in meat products recorded among livestock farmers in this study contradicts the findings
of Resurreccion and Gavez (1999) in which 77% of respondents in the United States of America
identified AMRs in food stuffs of animal origin to be of serious public health concern. This poor
awareness among the farmers may be due to poor educational status of the farmers. This may
exacerbate the public health problems associated with consumption of AMRs in animal products
as consumers may not adopt measures to reduce exposure to these residues in foods of animal
origin.
The non-compliance to withdrawal period by the respondents, especially, livestock
farmers could also be due to poor awareness and illiteracy. Aliu, (2004) and Fagbamila et al.,
(2010) suggested that the nonchalant attitude of farmers in observing withdrawal period could be
due to lack of enforcement of veterinary drug laws in Nigeria that would have compelled them to
comply with label instructions in order to safeguard public health. Van Dresser and Wilke,
(1989) observed that failure to observe withdrawal periods is a major cause of accumulation of
drug residue in food animal tissues to the detriment of unsuspecting consumers.
Apart from the health problems, the accumulation of AMRs or their metabolites in animal
products hinders international trade (Aliu, 2004). There has not been any deliberate effort put in
place in Nigeria (by government or non-governmental organizations) to sensitize farmers on the
public health problems that may result from non-observance of withdrawal period or
indiscriminate use of antimicrobials in food animals (Kabir et al., 1999; Fagbamila et al., 2010).
78
This problem is further compounded by the fact that in most African countries, resource
allocation to public health issues such as drug residue control has never been prioritized (FAO/
WHO ( 2005).
5.1.2 Cross sectional study
The 30% prevalence of AMRs recorded in cattle in this study corroborates with the
findings of Olatoye and Ehinmowo, (2010) who recorded 34.4% prevalence in cattle slaughtered
for human consumption in Akure municipal abattoir. The result is higher than the findings of
other researchers in Nigeria such as Oboegbulem and Fidelis (1996) and Kabir et al., (2002)
who recorded prevalence of 8.3% and 7.4% respectively in cattle. The disparity in the results
could be due to increase in the trend of indiscriminate use of antimicrobials (Aliu, 2004;
Dipeolu, 2010; Ezenduka et al., 2011). It could also be due to the higher sensitivity of Premi®
Test used in this study in contrast to the European four plate test utilized in the other studies
(Popelka et al., 2004; Stead, et al., 2004).
This 30% prevalence of AMRs in cattle is however lower than 44% prevalence recorded
by Ibrahim et al., (2010) in slaughter cattle in Sokoto. Most cattle farmers in Nigeria do so under
the extensive-transhumane husbandry system. Since the animals are not usually given
concentrate feed supplement, the AMRs observed is this study are likely to have resulted from
the use of the antimicrobials for the purposes of disease prevention and treatment. Since these
farmers are not trained to administer veterinary drugs, it is very likely that the drugs were not
administered according to label instruction thereby resulting in the high prevalence of AMRs in
cattle. The 30% prevalence is however lower in comparison to the reports of Muriuki et al.,
(2001) and Sasanya et al., (2008) from slaughter cattle in Kenya and Uganda respectively.
79
The 22.86% prevalence of AMRs recorded in pigs in this study is higher than the findings
of Dipeolu and Alonge (2002) and Van de Water and Haagsma, (1991) in which they recorded
6.67% and 2.7% prevalence respectively in pigs. The reasons given for the high prevalence of
AMRs in cattle is also applicable to pigs. Furthermore, unintentional administration of
antimicrobials is most likely in pigs, via feeding of medicated concentrates, as pigs are mostly
raised in intensive management system.
Relationship between antimicrobial drug use in food animals and accumulation of AMRs
in animal tissues, and development of antimicrobial resistance has been reported (Smith et al.,
2005). It is common knowledge that these drugs are easily purchased and administered without
veterinary prescription by livestock farmers and this makes these animals prone to the
accumulation of AMRs since withdrawal period may not be observed (Paul et al., 1982). This is
reflected in the higher prevalence of AMRs recorded in cattle and pigs in this study. Since beef
and pork are widely consumed in Enugu State, the consumers are at risk of antimicrobial
resistance, allergic reactions, etc associated with consumption of animal tissues containing
AMRs.
The liver tissues yielded the high prevalence of AMRs among the organs screened for
AMRs in both cattle and pigs. This agrees with the results of Dipeolu and Alonge (2001) who
recorded prevalence of 16.63%, 15.0% and 13.34% from liver, kidney and muscle samples
respectively. The high prevalence of AMRs recorded in liver in both cattle and pigs in this study
agrees with the findings of Rao et al., (2001). Drug biotransformation occurs in the liver and this
may have facilitates the accumulation of these residues in the liver.
It was not surprising to note that the prevalence of the residues in the kidney ranked next
to that of liver in both species in view of the high percentage of respondents using beta-lactam
80
(21.16%) and aminoglycosides (16.81%) which are antimicrobials known to be excreted via the
urinary system. The high concentration of these residues in liver and kidney, mostly cherished in
homes and restaurants, is of a great public health concerns. This is because similar antimicrobials
are still being used for the treatment of microbial diseases in humans. This may aggravate the
problem of antimicrobial resistance; prolong duration of medication and increased cost of
treatment in humans who consume these organs.
From the statistical analysis, education seems to be a very important factor in the
epidemiology of AMRs. Uneducated livestock farmers may not be able to read and comply with
drug label instructions aimed at safeguarding public health. Given that there were only few
livestock farmers who had tertiary education, consumers of food animals in Enugu State are at
risk of the health problems associated with the consumption of AMRs in animal tissues since
withdrawal period and other precautionary measures aimed at reducing the accumulation of
AMRs in animal tissues may not be obeyed.
The significant association noted in the occurrence of AMRs in cattle tissue is of great
public health concern as beef is widely accepted and consumed in the study area. Since beef is
widely consumed in Enugu State, the general public who consume cattle products such as beef
and cow milk is at risk of the health problems associated with consumption of AMRs in animal
tissues.
81
5.2 Conclusion
• There is poor awareness of the health risks associated with consumption of AMRs among
livestock farmers in the study area
• Majority of the respondents do not observance WP prior to animal slaughter or sale for
human consumption
• AMRs occur in edible tissues of cattle and pigs slaughtered for human consumption in
Enugu state
• Beef and pork consumers in Enugu State are at risk of the health problems associated
with consumption of AMRs in animal tissues such as antimicrobial drug resistance,
allergic reactions, etc
5.2 Recommendations
1. Emphasis should be placed on good farm management practices such as biosecurity,
proper vaccination, good nutrition and farm hygiene to reduce the need for drug
administration in food animals
2. Nationwide awareness campaign should be carried out on the health risks associated with
the accumulation of AMRs in food animals
3. There is the need for legislative control over the sale and administration of Veterinary
drugs, especially antimicrobials, with functional machinery put in place for strict
implementation and compliance
4. The NAFDAC Act should be reviewed to incorporate all professionals under both animal
and human health as is done in the developed world for effective regulation of veterinary
products.
5. Ante-mortem screening of food animals for AMRs should be incorporated in routine
meat inspection in slaughter houses and abattoirs.
82
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95
Appendix 1: Questionnaire University of Nigeria Nsukka
Faculty of Veterinary Medicine Department of Veterinary Public Health & Preventive Medicine
SECTION A: SOCIO-ECONOMIC CHARACTERISTICS OF RESPONDENTS
Please, answer the questions bellow by ticking in the appropriate box. The confidentiality of your responses is guaranteed as this survey is solely for research purposes.
1. Occupation / Job description Veterinary practitioner Livestock farmer
2. Please, indicate your highest educational qualification No formal education Primary education Post primary education Tertiary education (specify e.g DVM, B.Sc)
3. Indicate your year(s) of experience in veterinary practice or livestock
farming. Less than one year 1-5 years 6-10 years above ten years
5. Which type (species) of animal rearing / practice are you involved in?
Cattle Pigs Others (Specify)
SECTION B: STATUS OF ANTIMICROBIAL DRUG USE AMONG THE RESPONDENTS
6 Have you administered antimicrobial drug(s) in food animals in the last 4 months? Yes No
7 If yes, what was the purpose of using the drug(s)?
Disease prevention Disease treatment Growth enhancement Others (specify)
8 Who prescribed or recommended the drug? Farm worker Fellow farmer Self (livestock farmer) Animal handler Veterinary doctor
9 Which drug(s) did you administer? Tetracyclines eg L.A Penicillin Enrofloxacin Sulphadimidine Tylosin Gentamycin Others (specify)
96
10 Who administered the drug(s)? Farm workers Self (livestock farmer) Animal health superintendent Veterinary doctor
SECTION C - OBSERVANCE OF WITHDRAWAL PERIOD AND AWARENESS OF HEALTH RISKS ASSOCIATED WITH CONSUMPTION OF AMRs IN ANIMAL PRODUCTS
11 Have you heard about withdrawal period (WP)? Yes No
12 If yes, what do you think WP is all about? Time to sell off all the animals in the farm Time to stop giving drugs to animals to avoid over dose Time to stop giving antibiotics before vaccination Minimum period of time in which an animal that received medication must be allowed prior to sale or slaughtering for human consumption
13 Do you observe withdrawal period? Yes No
14 If yes, please state the average withdrawal period (in pig or cattle) for the drug(s) you use in the last 4 month as indicated in 9 above. Tetracyclines Penicillin Sulphadimidines Tylosin Gentamycin Enrofloxacin Others (please specify)
15 Can Antimicrobial residues (AMRs) accumulate in animal products such as meat following antimicrobials treatment in food animals? Yes No
16 If yes, is there any health risks associated with the consumption of such animal products? Yes No
17 If yes, which of these is a possible health risk associated with the consumption AMRs in animal products? Decrease / cessation of egg lay in laying birds Stunted growth / weight loss in animals Development of antimicrobial drug resistance organisms Others (specify) Thank you for your response!
97
Appendix 2: Measurement of association between the occurrence of AMRs in cattle and pigs
slaughtered for human consumption in Enugu State
Null hypothesis (H0): There is no significant association in the occurrence of AMRs in cattle
and pigs slaughtered for human consumption in Enugu State.
Alternative hypothesis (Ha): There is significant association in the occurrence of AMRs in
cattle and pigs slaughtered for human consumption in Enugu State.
Pigs Cattle Total
Observed frequency (F0) 8 18 26
Expected frequency (Fe) = ∑(cattle +ve + pigs +ve) ÷ 2
13 13 26
At 5% alpha level (α = 0.05) and degree of freedom (df) = R (No. of rows) -1 x C (No. of
columns) - 1 = (2-1) x (2-1) = 1;
χ2table = 3.841
χ2cal = ∑ (F0 -Fe)2 ÷ Fe
= (8-13)2/13 + (18-13)2/13 = 3.846
Since χ2
cal (3.846) > χ2table (3.841), we reject the H0 and accept the Ha which states that there is
significant association in the occurrence of AMRs in cattle and pigs slaughtered for human
consumption in Enugu State.
98
Appendix 3: Measurement of association between the occurrence of AMRs in the organs
(kidney, liver, muscle) of pigs slaughtered for human consumption in Enugu State
Null hypothesis (H0): There is no significant association in the occurrence of AMRs in the
organs (kidney, liver, muscle) of pigs slaughtered for human consumption in Enugu State.
Alternative hypothesis (Ha): There is significant association in the occurrence of AMRs in the
organs (kidney, liver, muscle) of pigs slaughtered for human consumption in Enugu State.
Kidney Liver Muscle Total
Observed frequency (F0) 5 8 4 17
Expected frequency (Fe) = ∑(kidney +ve + liver +ve + muscles +ve) ÷ 3
5.67 5.67 5.67 17
At 5% alpha level (α = 0.05) and degree of freedom (df) = R (No. rows) -1 x C (No. of columns)
-1 = (2-1) x (3-1) = 2
χ2table = 5.99
χ2cal = ∑ (F0 -Fe)2 ÷ Fe
= (5-5.67)2/5.67 + (8-5.67)2/5.67 + (4-5.67) 2/5.67 = 1.529
Since χ2
cal (1.529) < χ2table (5.99), we accept the H0 which states that there is no significant
association in the occurrence of AMRs in the organs (kidney, liver, muscle) of pigs slaughtered
for human consumption in Enugu State.
99
Appendix 4: Measurement of association between the prevalence of AMRs in the organs
(kidney, liver, muscle) of cattle slaughtered for human consumption in Enugu State
Null hypothesis (H0): There is no significant association in the occurrence of AMRs in the
organs (kidney, liver, muscle) of cattle slaughtered for human consumption in Enugu State.
Alternative hypothesis (Ha): There is significant association in the occurrence of AMRs in the
organs (kidney, liver, muscle) of cattle slaughtered for human consumption in Enugu State.
Kidney Liver Muscle Total
Observed frequency (F0) 16 18 10 44
Expected frequency (Fe) = ∑(kidney +ve + liver +ve + muscles +ve) ÷ 3
14.67 14.67 14.67 44
At 5% alpha level (α = 0.05) and degree of freedom (df) = R (No. of rows)- 1 x C (No. of
columns) -1 = (2-1) x (3-1) = 2
χ2table = 5.99
χ2cal = ∑ (F0 -Fe)2 ÷ Fe
= (16-14.67)2/14.67 + (18-14.67)2/14.67 + (10-14.67) 2/14.67 = 2.363
Since χ2
cal (2.363) < χ2table (5.99), we accept the H0 which states that there is no significant
association in the occurrence of AMRs in the organs (kidney, liver, muscle) of cattle slaughtered
for human consumption in Enugu State.
100
Appendix 5: Measurement of association between educational levels and observance of withdrawal period among livestock farmers in Enugu State
Null hypothesis (H0): There is no significant association between educational levels and
observance of withdrawal period among livestock farmers in Enugu State
Alternative hypothesis (Ha): There is significant association between educational levels and
observance of withdrawal period among livestock farmers in Enugu State
Educational levels of respondents Observance of withdrawal period No formal
education Primary education
Post primary education
Tertiary education
Total
YES Observed frequency (F0) 0 2 10 3 15 Expected frequency (Fe) 4.3 5.1 4.5 1.0 14.9
NO Observed frequency (F0) 37 42 29 6 114 Expected frequency (Fe) 32.7 38.9 34.5 8.0 114.1
Total Observed frequency (F0) 37 44 39 9 129 Expected frequency (Fe) 37 44 39 9 129
At 5% alpha level (α = 0.05) and degree of freedom (df) = R (No. of rows) – 1 x C (No. of
columns) -1 = (2-1) x (4-1) = 3;
χ2table = 7.81
χ2cal = ∑ (F0 -Fe)2 ÷ Fe = (0-4.3)2/4.3 + (37-32.7)2/32.7 + (2-5.1) 2/5.1 + (42-38.9)2/38.9 + (10-
4.5)2/4.5 + (29-34.5) 2/34.5 + (3-1)2/1 + (6-8)2/8 =
4.3+0.565+1.884+0.247+6.722+0.877+4+0.5 =
19.096.
Since χ2cal (19.096) > χ2
table (7.81), we reject the H0 and accept the Ha, which states that there is
significant association between educational levels and observance of withdrawal period among
livestock farmers in Enugu State
101
Appendix 6: Measurement of association between educational levels and awareness of public health risks associated with consumption of AMRs in animal products among livestock farmers
in Enugu State Null hypothesis (H0): There is no significant association between educational levels and
awareness of public health risks associated with consumption of AMRs in animal products
among livestock farmers in Enugu State
Alternative hypothesis (Ha): There is significant association between educational levels and
awareness of public health risks associated with consumption of AMRs in animal products
among livestock farmers in Enugu State
Educational levels of respondents Awareness of public health risks Associated with the consumption of AMRs in animal products
No formal education
Primary education
Post primary education
Tertiary education
Total
YES Observed frequency (F0) 1 6 8 4 19 Expected frequency (Fe) 5.4 6.5 5.7 1.3 18.9
NO Observed frequency (F0) 36 38 31 5 110 Expected frequency (Fe) 31.6 37.5 33.3 7.7 110.1
Total Observed frequency (F0) 37 44 39 9 129 Expected frequency (Fe) 37 44 39 9 129
At 5% alpha level (α = 0.05) and degree of freedom (df) = R (No. of rows) – 1 x C (No. of
columns) -1 = 2-1 x 4-1 = 3
χ2table = 7.81
χ2cal = ∑ (F0 -Fe)2 ÷ Fe = (1-5.4)2/5.4 + (36-31.6)2/31.6 + (6-6.5) 2/6.5 + (38-37.5)2/37.5 + (8-
5.7)2/5.7 + (31-33.3) 2/33.3 + (4-1.3)2/1.3 + (5-5.7)2/5.7 =
3.585+0.613+0.007+0.928+0.159+5.608+0.947 =11.88 Since χ2
cal (11.88) > χ2table (7.81), we reject the H0 and accept the Ha which states that there is
significant association between educational levels and awareness of public health risks
associated with consumption of AMRs in animal products among livestock farmers in Enugu
State
102
Appendix 7: Measurement of association between working experience and observance of
withdrawal period among livestock farmers in Enugu State
Null hypothesis (H0): There is no significant association between working experience and
observance of withdrawal period among livestock farmers in Enugu State
Alternative hypothesis (Ha): There is significant association between working experience and
observance of withdrawal period among livestock farmers in Enugu State
Working experience Observance of withdrawal period Less than
1 year 1-5 years 6-10 years Above 10
years Total
YES responses
Observed frequency (F0) 3 5 4 3 15 Expected frequency (Fe) 2.9 2.7 5.3 4.1 15
NO responses
Observed frequency (F0) 22 18 42 32 114 Expected frequency (Fe) 22.1 20.3 40.7 30.9 114
Total Observed frequency (F0) 25 23 46 35 129 Expected frequency (Fe) 25 23 46 35 129
At 5% alpha level (α = 0.05) and degree of freedom (df) = R (No. of rows) – 1 x C (No. of
columns) -1 = 2-1 x 4-1 = 3
χ2table = 7.81
χ2cal = ∑ (F0 -Fe)2 ÷ Fe = (3-2.9)2/2.9 + (22-22.1)2/22.1 + (5-2.7) 2/2.7 + (18.20.3)2/20.3 + (4-
5.3)2/5.3 + (42-40.7) 2/40.7 + (3-4.1)2/4.1 + (32-30.9)2/30.9 =
0.0034+0.0005+1.959+0.261+0.319+0.042+0.295+0.039 = 2.92
Since χ2
cal (2.92) < χ2table (7.81), we accept the H0 which states that there is no significant
association between working experience and observance of withdrawal period among livestock
in Enugu State
103
Appendix 8: Measurement of association between working experience and observance of withdrawal period among veterinary practitioners in Enugu State
Null hypothesis (H0): There is no significant association between working experience and
observance of withdrawal period among in Enugu State veterinary practitioners
Alternative hypothesis (Ha): There is significant association between working experience and
observance of withdrawal period among veterinary practitioners in Enugu State
Working experience Observance of withdrawal period Less than
1 year 1-5 years 6-10 years Above 10
years Total
YES Observed frequency (F0) 8 16 13 0 37 Expected frequency (Fe) 9.8 14.0 12.6 0.7 37.1
NO Observed frequency (F0) 6 4 5 1 16 Expected frequency (Fe) 4.2 6.0 5.4 0.3 15.9
Total Observed frequency (F0) 14 20 18 1 53 Expected frequency (Fe) 14 20 18 1 53
At 5% alpha level (α = 0.05) and degree of freedom (df) = R (No. of rows) – 1 x C (No. of
columns) -1 = 2-1 x 4-1 = 3
χ2table = 7.81
χ2cal = ∑ (F0 -Fe)2 ÷ Fe = (8-9.8)2/9.8 + (6-4.2)2/4.2 + (16-147) 2/14 + (4-6)2/6 + (13-12.6)2/12.6 +
(5-5.4) 2/5.4 + (0-0.7)2/0.7 + (1-0.3)2/0.3 =
0.331+0.771+0.286+0.667+0.013+0.029+0.7+1.633 = 4.43
Since χ2
cal (4.43) < χ2table (7.81), we accept the H0 which states that there is no significant
association between working experience and observance of withdrawal period among veterinary
practitioners in Enugu State
