1. health and fitness
2. disease
3. allergies

Effect of Thermal Treatment on Penicillin Activity and
Detection of Antibiotic Residues in Raw Cow Milk Vendered
in Khartoum State
By
Tasneem Abdelmoneim M. Ali Bakhit
B.Sc. (Agric., Honours) – 2003
Faculty of Agriculture,
University of Khartoum
A dissertation Submitted In Partial Fulfillment of the Requirements
for the Degree of Master of Science in Food Science and
Technology
Supervisor
Prof. Hamid Ahmed Dirar
Department of Botany and Agric. Biotechnology
Faculty of Agriculture
University of Khartoum
January – 2006
DEDICATION
To whom who stood with me
through the ups and downs
My dear husband
With love and respect
gtáÇxxÅ
Acknowledgement
First all my thanks go to Allah who helps me in all my life.
Then I have to thank my supervisor, Prof. Hamid Ahmed Dirar
for his advice and encouragement through all the steps of my
study.
Thanks are also extended to my family, friends and
colleagues for their unlimited help and encouragement.
Much gratitude and profuse thanks to DAL Food Group
specially the Blue Nile Dairy Plant (CAPO), for their help.
ABSTRACT
The objective of this study is to investigate the stability of
penicillin added to milk under different thermal treatments, and to detect
antibiotic residues in raw cow milk sold in Khartoum State. Four different
concentrations of penicillin in milk were prepared. They were subjected
to three different thermal treatments, sterilization, pasteurization and
boiling. The antimicrobial activity was tested using the cup plate
diffusion method. The result showed that the pasteurization and boiling
treatments had no effect on the penicillin activity while the sterilization
treatment decreased the penicillin activity.
A total of 97 samples of milk were collected randomly from
Khartoum state through its three provinces Khartoum, Omdorman and
Khartoum North. The samples were tested to detect the antibiotic residues
using the antibiotic test kits. The test revealed that the percentage of
positive samples were 26.6, 35 and 30.5% in Khartoum, Omdorman and
Khartoum North province respectively, and the total percentage of
positive sample in Khartoum state were 30.9%.
‫ﺧﻼﺻﺔ اﻷﻃﺮوﺣﺔ‬
‫اﻟﻬ ﺪف ﻣ ﻦ ه ﺬﻩ اﻟﺪراﺳ ﺔ إﺧﺘﺒ ﺎر ﺛﺒﺎﺗﻴ ﺔ اﻟﺒﻨ ﺴﻠﻴﻦ اﻟﻤ ﻀﺎف ﻟﻠ ﺒﻦ ﺗﺤ ﺖ ﻣﻌ ﺎﻣﻼت ﺣﺮارﻳ ﺔ‬
‫ﻣﺨﺘﻠﻔﺔ واﻟﻜﺸﻒ ﻋﻦ ﻣﺘﺒﻘﻴﺎت اﻟﻤﻀﺎدات اﻟﺤﻴﻮﻳﺔ ﻓﻲ اﻟﻠﺒﻦ اﻟﺨﺎم اﻟﻤﺒﺎع ﻓﻲ وﻻﻳﺔ اﻟﺨﺮﻃﻮم‪.‬‬
‫ﺣ ﻀﺮت أرﺑﻌ ﺔ ﺗﺮاآﻴ ﺰ ﻣﺨﺘﻠﻔ ﺔ ﻣ ﻦ اﻟﺒﻨ ﺴﻠﻴﻦ ﻓ ﻲ اﻟﻠ ﺒﻦ ﺗ ﻢ إﺧ ﻀﺎﻋﻬﺎ ﻟﺜﻼﺛ ﺔ ﻣﻌ ﺎﻣﻼت‬
‫ُ‬
‫ﺣﺮارﻳﺔ ﻣﺨﺘﻠﻔﺔ " ﺗﻌﻘﻴﻢ ‪ ،‬ﺑﺴﺘﺮة وﻏﻠﻴﺎن"‪ .‬ﺗﻢ إﺧﺘﺒﺎر اﻟﻨﺸﺎط اﻟﻤﻴﻜﺮوﺑ ﻲ ﺑﺈﺳ ﺘﺨﺪام ﻃﺮﻳﻘ ﺔ اﻹﻧﺘ ﺸﺎر‬
‫ﻓﻲ اﻟﻄﺒﻖ )‪.(Cup-plate diffusion method‬‬
‫أﻇﻬﺮت اﻟﻨﺘﺎﺋﺞ أﻧﻪ ﻟﻴﺲ هﻨﺎﻟﻚ ﺗﺄﺛﻴﺮ ﻟﻠﺒﺴﺘﺮة او اﻟﻐﻠﻴﺎن ﻋﻠﻰ ﻧﺸﺎط اﻟﺒﻨﺴﻠﻴﻦ ﺑﻴﻨﻤﺎ ﻗﻠﻞ‬
‫ﻣﻌﺎﻣﻠﺔ اﻟﺘﻌﻘﻴﻢ ﻣﻦ ﻓﻌﺎﻟﻴﺔ اﻟﺒﻨﺴﻠﻴﻦ‪.‬‬
‫ﻣﺠﻤﻮع ‪ 97‬ﻋﻴﻨﺔ ﻣﻦ اﻟﻠﺒﻦ ﺟﻤﻌﺖ ﻋﺸﻮاﺋﻴًﺎ ﻣﻦ وﻻﻳﺔ اﻟﺨﺮﻃﻮم ﻣﻦ ﺧﻼل ﻣﺤﺎﻓﻈﺎﺗﻬﺎ‬
‫اﻟﺜﻼﺛﺔ اﻟﺨﺮﻃﻮم ‪ ،‬أم درﻣﺎن واﻟﺨﺮﻃﻮم ﺑﺤﺮي‪ .‬ﺗﻢ إﺧﺘﺒﺎر اﻟﻌﻴﻨﺎت ﻟﻠﻜﺸﻒ ﻋﻦ ﻣﺘﺒﻘﻴﺎت اﻟﻤﻀﺎدات‬
‫اﻟﺤﻴﻮﻳﺔ ﺑﺈﺳﺘﺨﺪام ﻣﻜﻮﻧﺎت اﻹﺧﺘﺒﺎر اﻟﺠﺎهﺰة )‪.(antibiotic test kits‬‬
‫أوﺿﺤﺖ اﻟﻨﺘﺎﺋﺞ أن اﻟﻨﺴﺒﺔ اﻟﻤﺌﻮﻳﺔ ﻟﻠﻌﻴﻨﺎت اﻟﺘﻲ ﺗﺤﺘﻮي ﻋﻠﻰ ﻣﺘﺒﻘﻴﺎت اﻟﻤﻀﺎدات اﻟﺤﻴﻮﻳﺔ‬
‫هﻲ ‪ %35 ،%26.6‬و ‪ %30.5‬ﻓﻲ اﻟﺨﺮﻃﻮم ‪ ،‬أم درﻣﺎن واﻟﺨﺮﻃﻮم ﺑﺤﺮي ﻋﻠﻰ اﻟﺘﻮاﻟﻲ‪،‬‬
‫واﻟﻨﺴﺒﺔ اﻟﻤﺌﻮﻳﺔ اﻟﻜﻠﻴﺔ ﻟﻠﻌﻴﻨﺎت اﻟﺘﻲ ﺗﺤﺘﻮي ﻋﻠﻰ ﻣﺘﺒﻘﻴﺎت اﻟﻤﻀﺎدات ﺣﻴﻮﻳﺔ ﻓﻲ وﻻﻳﺔ اﻟﺨﺮﻃﻮم‬
‫آﺎﻧﺖ ‪.%30.9‬‬
LIST OF CONTENTS
Page
Dedication……………………………………………………………………………………………...
i
Acknowledgement …………………………………………….………………………………...
ii
Abstract ………………………………………………………..……………………………………...
iii
Arabic Abstract …………………………………………………………………………………...
iv
List of Contents ………………………………..………………………………………………...
v
List of Tables……………………………………..………………………………………………...
viii
List of Figures ………………………………..…………………………………………………...
ix
List of Plates ………..…………………………..…………………………………………………...
x
CHAPTER ONE: INTRODUCTION……………………………………………...
1
CHAPTER TWO: LITERATURE REVIEW…………………….
3
2.1 Antibiotics…………………..……………………………………..……………………………
3
2.1.1 Definitions and general characteristics………………………………………
3
2.1.2. Groups of antibiotics……………………………..……………………………………
3
2.2 Uses of antibiotics……………………………………..……………………………………
4
2.2.1 Therapeutic uses……………………………………..……………………………………
4
2.2.2 Growth promotion……………………...…………..……………………………………
6
2.2.3 Food preservation………………………….………..……………………………………
6
2.3. Residues of veterinary drugs…………………..……………………………………
7
2.3.2 Safe residue levels…………………..…………………..………………………………
9
2.3.2.1 Acceptable daily intake (ADI) …………………..……………………………
9
2.3.2.2 Maximum residue limits (MRLs) …………………..………………………
10
2.4 Health hazards of antibiotic residues…………………..………………………
10
2.4.1 Allergic reactions…………………..………………….…………………………………
10
2.4.2 Microbial drug resistance……………………....……………………………………
10
2.4.3 Changing the flora…………………..……………………...……………………………
12
2.4.4 Aplastic anemia…………………..……………………………..…………………………
13
2.5 Residues detection methods…………….………..……………………………………
14
2.6. Penicillin…………………..…………………………………….………………………………
15
2.6.1 Identity…………………..…………………………………..…………………………………
15
2.6.2. Chemical name………………………….…………..……………………………………
15
2.6.3. Synonyms………………………………………….…..……………………………………
16
2.6.4. Structural formula…………………..……………..……………………………………
16
2.6.5. Molecular formula………………………….……..……………………………………
16
2.6.6. Molecular weight…………………..……………………………………………………
16
2.6.7. Manufacture of penicillin…………….………..……………………………………
16
2.7 Factors affecting penicillin stability…………………..…………………………
17
2.8. Penicillin residues…………………………………….……………………………………
17
2.9. The effect of thermal treatment on the antibiotic residue in food
18
2.10. Antibiotic residues surveillance studies…………………..…………………
20
2.11 Veterinary drug residues monitoring programmes……………………
21
CHAPTER THREE: MATERIALS AND METHODS……..…………
23
3.1 Materials…………………..………………………………………………..……………………
23
3.1.1 Penicillin…………………..………………………………..…………………………………
23
3.1.2 Test organisms……………………………………..……………………………………
23
3.1.3 Culture media…………………..…………………………..………………………………
23
3.1.3.1 Diagnostic sensitivity test agar (D.S. T. Agar) ……..………………..
23
3.1.3.2 Nutrient broth…………………..………………………………………..………………
23
3.1.4 Antibiotic free-milk…………………..…………………………………………………
24
3.1.5 Survey milk samples…………………..……………..…………………………………
24
3.1.6 Antibiotics test kits………………………………...……………………………………
24
3.2 Methods…………………..………………………………………………………………………
24
3.2.1 Experimental methods………….………………..……………………………………
24
3.2.1.1 Preparation of different milk concentration of penicillin………
24
3.2.1.2 Thermal treatment…………………..…………….……………..……………………
24
3.2.2 Antibiotic sensitivity test…………………..……...…………………………………
25
3.2.2.1 Microorganism staining method……….…………..…………………………
25
3.2.2.1.1 Preparation of smears…………………..……..…………………………………
25
3.2.2.1.2 Grams staining method…………………....……………………………………
26
3.2.2.2 The cup-plate agar diffusion technique…………………..………………
26
3.2.2.3 Antibiotic test kit technique…………………..…………………………………
27
CHAPTER FOUR: RESULTS AND DISCUSSIONS…………….……
31
4.1 Effect of thermal treatments in penicillin activity…………………..……
31
4.2 Antibiotic residues in raw cow milk sold in Khartoum state…….…
37
CHAPTER FIVE: CONCLUSIONS AND RECOMMENDATIONS
43
5.1. Conclusions…………………..…………………………………..……………………………
43
5.2. Recommendations………………………………….....……………………………………
43
REFERENCES…………………………………………………………………………………...
44
LIST OF TABLES
Table Title
3.1
Sensitivity of kit……………………...………………………………………
4.1
Antimicrobial activity of milk containing penicillin with
concentration 1.0 I.U. /ml………………………………………………..
4.2
33
34
Antimicrobial activity of milk containing penicillin with
concentration 4.0 I.U. /ml…………………………………………………
4.4
28
Antimicrobial activity of milk containing penicillin with
concentration 2.0 I.U. /ml…………………………………………………
4.3
No.
35
Antimicrobial activity of milk containing penicillin with
concentration 8.0 I.U. /ml………………………………………………
36
4.5
Khartoum province samples……………………………………………
39
4.6
Omdorman province samples……………………………………………
40
4.7
Khartoum North province samples……………………………………
41
4.8
Percentage of positive samples in Khartoum State………………
42
LIST OF PLATES
Fig.
Title
No.
3.1
Dry block heater……………………………………………………………..
29
3.2
Colours card…………………………………………..………………………
30
3.3
Positive and negative samples…………………………………………
30
4.1
Antimicrobial activity of milk containing penicillin with
concentration 1.0 I.U. / ml…………………….………………………
4.2
Antimicrobial activity of milk containing penicillin with
concentration 2.0 I.U. / ml……………….……………………………..
4.3
34
Antimicrobial activity of milk containing penicillin with
concentration 4.0 I.U. / ml……………………………………………..
4.4
33
35
Antimicrobial activity of milk containing penicillin with
concentration 8.0 I.U. / ml……………………………………………
36
CHAPTER ONE
INTRODUCTION
Increasing awareness of public health and food safety issues in
recent years has lead to a greater interest in milk quality. The growing
globalization of the world's markets is making it necessary to meet the
most stringent requirements in order to sustain trade.
From time to time the quality of milk has been lowered by addition
of water and abstraction of fat. It may be necessary to consider the
presence of additives, e.g. preservatives, colours, thickeners and
contamination, e.g. detergents, antibodies and dirt (Pearson, 1976).
Unfortunately, many of the least developed countries suffer from a
lack of effective regulatory control of uses of veterinary drugs. The
presence of antimicrobial residues in milk could cause serious health
problems for consumers in the form of antibiotic resistance or allergies
(EMEA, 1999) as well as for dairy industry, in the form of delays in
bacteriological processes used to manufacture dairy products (Mäyrä –
Makinen, 1995).
Residues may occur due to bad veterinary practices or by direct
addition of some antibiotics to food as preservative substances to prolong
the shelf life of milk, for example, which is not always a permissible
practice.
There is evidence that penicillin is added to fresh milk to delay its
spoilage by microorganisms, so, more attention should be given to detect
and investigate its residues in milk.
Our objective in this study was to investigate the stability of
penicillin added to milk under different thermal treatments and to detect
antibiotic residues in raw cow milk sold in Khartoum State.
CHAPTER TWO
LITERATURE REVIEW
2.1 Antibiotics:
2.1.1 Definitions and general characteristics:
Antibiotics are chemical substances produced by certain microorganisms that inhibit or kill other microorganisms (Madigan et al.,
2000). Brander and Pugh (1977) defined antibiotics as a group of organic
chemicals, which in minute quantities have a detrimental effect on other
micro-organisms.
Antibiotics should be non-toxic to the host and without undesirable
side effects. An example for an ideal selectively toxic agent is penicillin
(Gringauz ,1978). Selective toxicity means being harmful to the parasite
without injuring the host (Baker et al., 1980).
An antibiotic should not eliminate the normal flora of the host, it
should be non-allergic to the host, and should be able to reach the part of
human body where the infection is occurring. It should also be chemically
stable (Todar, 1996).
Antibiotics can be either cidal (killing organism) or static
(inhibiting growth). It should have a wide spectrum of activity with
ability to destroy or inhibit many different species of pathogens (Brander
and Pugh, 1977).
2.1.2. Groups of antibiotics:
Madigan et al, (2000) mentioned that antibiotics can be grouped
based on the chemical structure. In bacteria the important targets of
antibiotics action are the cell wall, the cytoplasmic membrane, the
biosynthetic processes of protein synthesis and nucleic acid synthesis.
Brander and Pugh (1977) illustrated that antimicrobial agents can
be divided into four groups as they affect the synthesis of:
• Nucleic acid.
• Protein
• The cell formation of the cell wall
• Cell membrane.
2.2 Uses of antibiotics:
2.2.1 Therapeutic uses:
Antibiotics are effective against living bacteria, some ricketsiae,
some viruses, some fungi and a few helminthes (Brander and Pugh,
1977). The use of antibiotic therapy to treat and prevent udder infections
in cows is a key component of mastitis control in many countries
(Hillerton, 1999).
A broad spectrum antibiotic, like chloramphenicol, is one which is
effective against a wide variety of organisms, gram-positive and gramnegative bacteria. A narrow spectrum antibiotic is one in which the
antibacterial effect is restricted to a small number of organisms; a good
example is penicillin which is active mainly against gram-positive
organisms (Brander and Pugh, 1977).
Antibiotics may also be given to prevent outbreaks of diseases in
particular circumstances when animals are known to be more susceptible
to an infection (Select Committee on Science and Technology, 1998).
2.2.2 Growth promotion:
A major non-medical use of antibiotics is addition to animal feeds
which stimulates animal growth, shortening the period required to get
animal to the market (Madigan et al., 2000).
This effect was discovered by accident when chickens were fed
vitamin B which was produced by fermenting bacteria. The birds grew
faster than usual. It was then realized that the bacteria also produced the
antibiotic chlortetracycline (Bonner, 1997).
Growth promoters are used at low concentrations. Their use
increases the average daily growth and food conversion ratios by 3 – 11%
depending on species. Their mode of action is said to be by suppressing
commensal bacteria, which would divert nutrient from animal and by
maintaining more effective and absorptive gut lining (Select Committee
on Science and Technology, 1998).
2.2.3 Food preservation:
Antibiotics have a great success in controlling pathogenic
microorganisms in living animals and they are natural which will lead to
extensive investigations in their potential use in food preservation
(ICMSF, 1980).
Two antibiotics are approved for food uses in many countries
(nisin and neomycin) and three others (tetracycline, subilin and tylosin)
have been studied and found effective for various food applications.
Some risks may be anticipated from the use of any food additive, but the
risk should not outweigh the benefits (Jay, 1986).
There are many considerations noted on the uses of antibiotics as
food preservatives by Ingram et al., and several of the key ones are
summarized below as summarized by Jay (1986).
●
The antibiotic should kill not inhibit the flora and ideally
decompose into innocuous products, or be destroyed by cooking
for products that required cooking.
●
The antibiotic should not be inactivated by food components or
products of microbial metabolism.
●
The antibiotic should not readily stimulate the appearance of
resistant strains.
●
The antibiotic should not be used in foods if used therapeutically or
as an animal feed additive.
2.3 Residues of veterinary drugs:
Residues of veterinary drugs include the parent compounds and/ or
their metabolites in any edible portion of the animal product and include
residues of associated impurities of the veterinary drug concerned
(FAO/WHO, 1993).
Food residues is a matter (material or substances) remaining in
meat, milk, eggs, formed, fish or honey after any treatment or preparation
as food species origin (Prescott and Baggot, 1988).When Park (1997)
defined the food additives, the definition included animal feed adjuncts
which may result in residues in human food and components and may
find their way incidentally through farming practice.
Due to the widespread use of antibiotic treatment of mastitis in
dairy cows, much effort and concern has been directed towards the proper
management and monitoring of antibiotics used in such treatment in order
to prevent contamination of raw milk (Popelka et al., 2002). About 15%
of milk samples tested in Britain has antibiotic residues. The reason given
by farmers for these failures as summarized by Prescott and Baggot
(1988) were:
• Poor records of treatment.
• Not withholding milk for the time recommended.
• Early calving, this leads to a short the dry period.
• Accidental transfer of contaminated milk.
• Prolonged secretion of antibiotics after intra-mammary treatment.
• Contamination of jars by treated milk.
• Lack of warning toward the withholding time.
2.3.1 Withdrawal time:
The definition adopted by the Codex Alimentarius Commission for
the withdrawal time and withholding time is the period of time between
the last administration of a drug and the collection of edible tissue or
products from a treated animal that ensures the contents of residues in
food comply with the maximum residue limit for this veterinary drug
(FAO/WHO, 1993).
Withdrawal period is the time when animal must be held free of the
drug before it can be marketed so as to allow the drug to be eliminated
from tissues. In the case of milk, the term withholding period is
commonly used. This term states the period that milk cannot be sent for
human consumption following the treatment of the animal with a drug so
as to allow any residues in milk to be eliminated before it is placed on the
market (Blood and Radostits, 1987).
2.3.2 Safe residue levels:
The safe levels of residues established by (FAO/WHO) Codex
Alimentarius programme were carried on the basis of toxicology studies.
In addition to conventional toxicological effects, immune system and
pharmacological effects should be taken into account. Also it includes
specific effects of residue of veterinary antibiotics on the human gut flora
(Boisseau, 1993).
Acceptable daily intake (ADI) and maximum residues limits
(MRLs) can be used to establish milk and meat withholding times for
animals treated with antibiotics (European Scientific Conference, 1999).
2.3.2.1 Acceptable daily intake (ADI):
Acceptable daily intake (ADI) as estimated by JECFA is the
amount of a veterinary drug, expressed on a body weight basis that can be
ingested daily over a life time without appreciable health risk
(FAO/WHO, 1993).
2.3.2.2 Maximum residue limits (MRLs):
Maximum residue limits is the maximum concentration of residue
resulting from the use of a veterinary drug (expressed in mg/kg or µg/kg
on a fresh weight basis), that is recommended by the Codex Alimentarius
Commission to be legally permitted or recognized as acceptable in food
(FAO/WHO, 1993).
2.4 Health hazards of antibiotic residues:
2.4.1 Allergic reactions:
Allergy is the condition in which tissue shows an increased
capacity to react to some foreign substances (Bigger, 1962).
Because of the possible health hazard to consumer, the presence of
antibiotic residues in milk is highly undesirable. Of these penicillin
residues are the most common and are of particular concern because they
may cause allergic reactions in individuals sensitized to penicillin
(Deweck, 1971).
Henderson (1971) reported that certain infants were allergic to
penicillin in amounts that were occasionally found in milk. Allergic
reactions can occur in those consumers who may be allergic to these
substances at as low concentrations as 1 ppb (Jones, 1999).
2.4.2 Microbial drug resistance:
Foods of animal origins are considered an important factor for
transfer of antibiotic resistance from animal to man (Rechcigl, 1983).
Most resistance genes are acquired through a process of genetic exchange
from the antibiotic producers, in order to protect themselves from the
antibiotics they produce; under right circumstances, resistance genes can
be transferred to other organisms (Madigan et al., 2000).
Franklin and Snow (1974) reported that the agricultural uses of
antibiotics are responsible for some cases of bacterial resistance. Subtherapeutic doses of broad spectrum antibiotics that have been fed to
animals for prophylactic reasons were a source of bacterial-resistance
(WHO, 1997).
Hillers and Knuston (1992) believe that resistant strains of specific
organisms that cause illness are linked to the use of antibiotics in animals.
The treatment of animals with penicillin is stated to result in strains of
penicillin-resistant staphylococci, cause of bovine mastitis, becoming
common and if transmitted to man such disease might not be responsive
to treatment with antibiotics (Herschedoerter, 1968).
Administration of antimicrobial drugs to food-producing animals
can promote emergence of resistance in bacteria that may not be
pathogenic to animals, such as Salmonella, Campylobacter and
Escherichia coli (WHO, 1982). These bacteria are common and exist in
the intestinal flora of various food-producing animals without causing
disease. However, all three bacteria can cause severe food illness in
humans (Todar, 1996). Difficulties may occur in treatment of infected
humans, particularly slaughterhouse workers, food handling workers and
farmers feeding antibiotics to animals (WHO, 1997).
2.4.3 Changing the flora:
Antibiotic residues may alter the intestinal flora and affect vitamin
synthesis (Graham et al., 1968). Brooks et al., (1998) explained that
antimicrobial drugs affect not only the infecting microorganisms but also
susceptible members of the normal microbial flora of the body. An
imbalance is thus created that in itself may lead to disease. For example
in hospitalized patients who receive antimicrobials, the normal microbial
flora is suppressed. This creates a partial void that is filled by the
organisms most prevalent in the environment, particularly drug-resistant
gram-negative aerobic bacteria, e.g., pseudomonads, staphylococci, fungi,
etc. Such super-infecting organisms subsequently may produce serious
drug-resistant infections.
In women taking antibiotics by mouth, the normal vaginal flora
may be suppressed, permitting marked overgrowth of Candida. This
leads to unpleasant local inflammation (vaginitis) and itching that is
difficult to control.
In the presence of urinary tract obstruction, the tendency to bladder
infection is great. When such urinary tract infection due to a sensitive
microorganism (e.g. Escherichia coli) is treated with an appropriate drug,
the organism may be eradicated. However, very often re-infection due to
another drug-resistant gram-negative bacillus occurs after the drugsensitive microorganisms are eliminated. A similar process accounts for
respiratory tract super infections in patients given antimicrobials for
chronic bronchitis.
In persons receiving antimicrobial drugs by mouth for several days,
parts of the normal intestinal flora may be suppressed. Drug-resistant
organisms may establish themselves in the bowel in great numbers and
may precipitate serious enterocolitis (Clostridium difficile, staphylococci,
etc.).
2.4.4 Aplastic anemia:
Meyle and Herxheimer (1972) deduced that few of the antibiotics
used in animal have few toxic effects and may threat human health like
chloramphenicol which is the reason of cytological and hematological
changes in bone marrow and blood and they indicated that the bone
marrow toxicity are of two types a dose-related reversible depression of
the formation of erythrocytes, thrombocytes and granulocytes or a rare
but very serious and in most cases irreversible pancytopenia (aplastic
anemia).
Chloramophenicol which is consumed by humans from eating meat
eggs or from drinking milk is the reason of the hematological and
cytological changes in the bone marrow (Allen, 1985).
The basic problem is failure of the stem cells to a varying degree,
producing hypoplasia of the marrow elements. Causes of aplastic anemia
are: drugs (cytotoxic drugs, idiosyncratic antibiotics–chloramphenicol
and sulphonamides), chemicals (insecticides as organophosphates and
carbamates, benzene flume solvent), abuse radiation, viral hepatits,
pregnancy and paroxysmal nocturnal haemoglobinuria (Mackie et al.,
2000).
2.5 Residues detection methods:
Many methods were used to detect antimicrobial agents. Factors to
be considered to choose the most suitable method of residue detection are
the type of antibiotic used, expected time limitations, sensitivity and costs
(Senyk et al., 1990). The antibiotic residue detection assay systems that
are currently available use different methods and test organisms (Van
Eenennaam et al., 1993).
The measurement of residues of veterinary drugs is carried out
using either bioassay or physico-chemical methods. Microbiological
methods measure the ability of the drug to inhibit the growth of selected
bacteria (FAO/WHO, 1993). The presence of antimicrobial substance is
indicated by zones of inhibition (Myllniemi et al., 1999). The
microbiological method involves a standard culture of a test organism in
agar growth media that is inoculated with a milk sample and incubated
for periods of up to several hours. If the milk contains sufficient
concentrations of inhibitory substances, the growth of the organism will
be reduced or eliminated (Harvey and Hill, 1967).
Delvotest SP is a multiple microbial inhibitor test usable to detect
antimicrobial agents such as beta-lactam and sulpha compounds, (Suhren,
1998). In the last few years, a new microbiological assay (the Tet-Lux
test) has been developed for the detection of tetracycline residue in raw
milk. It uses Escherichia coli bacteria carrying a sensor plasmid, in which
a tetracycline-specific control unit regulates the expression of bacterial
luciferase genes. The presence of tetracycline residues in the sample
causes an increase in the light emission of the test bacteria. This assay is
able
to
detect
4-35
ug/ml
of
tetracycline,
oxytetracycline,
chlortetracycline,
doxycycline,
demeclocycline,
methacycline
and
minocycline (Kurittu, 2000).
The biochemical assay methods are based on using antibiotics as
substrates for the enzymes, like using penicillin as substrates for the
enzyme
penicillinase.
Physico-chemical
methods
make
use
of
chromatographic techniques of which HPLC has superceded TLC as the
more suitable method. HPLC is very specific, precise and has lower
limits of sensitivity of 50g/kg in tissues and 10g/l in milk (FAO/WHO,
1993). Antibiotic residues in milk were also identified by high voltage
electrophoresis in 1% agarose gel and bio-autographic strain for the
detection, pH of the media was 8. The condensation of samples by freezedrying increased the sensitivity of the method (Krcmar and Ruzickova,
1996).
2.6 Penicillin identity:
This identity of Benzyl penicillin was mentioned by the
(FAO/WHO 1993).
2.6.1 Chemical name: 3, 3,-Dimethyl-7-oxo-6[(phenylacetyl) amino]–4–
thia –azabicyclo [3.2.0] heptane-2-carboxlic acid.
2.6.2 Synonyms: Free benzylpenicillin; PenicillinG; Penicillin II.
2.6.3. Structural formula:
H
H
CH3
S
CH2CONH
CH3
O
N
COO–
The basic structure of penicillins consists of thiazolidine ring
connected to a B-lactam ring to which is attached a side chain. The
penicillin nucleus itself is the chief structural requirement for biological
activity, whereas the side chain varies in different penicillins and
determines many of the antibacterial and pharmacological properties of
the different penicillins.
2.6.4 Molecular formula: C16N18N2O2S (Normally used as the
Sodium salt of the carboxylic acid).
2.6.5 Molecular weight: 334.38.
2.7 Manufacture of penicillin:
Penicillin, originally made on a small scale from surface broth
cultures of the mould, is now manufactured on a vast scale by deep vat
culture methods using various medium and various strains and species of
penicillin moulds. Purification processes are such that, instead of the
originally manufactured yellow, amorphous impure penicillin, the
product marketed is a white crystalline substance with a high degree of
purity (Brander and Pugh, 1977).
2.8 Factors affecting penicillin stability:
Penicillin is the least stable of the commonly used antibiotics.
(Brander and Pugh, 1977) reported the following factors that affected
penicillin stability:
●
Moisture: Penicillin is hygroscopic, although the potassium salt is
less so than the sodium or calcium salt and deterioration by
hydrolysis is rapid.
●
pH: Acids and alkalis cause rapid deterioration in the penicillin
solutions. Penicillin is most stable within a range of pH (6 to 6.5).
●
Temperature: Deterioration rate increases with temperature.
●
Oxidizing: All oxidizing agents rapidly destroy penicillin.
●
Enzymes: The enzyme penicillinase produced by some organisms
rapidly destroys the penicillin.
●
Miscellaneous: Penicillin is markedly affected by a number of heavy
metals, alcoholic groups and thiol containing
compounds.
2.9 Penicillin residues:
The presence of antibiotic residues in milk is highly undesirable.
Of these, penicillin residues are the most common and are of particular
concern because they may cause allergic reactions in individuals
sensitized to penicillin or may bring about sensitization of those
previously not allergic to the antibiotic (Deweck, 1971).
Table 2.1 The penicillin residues estimated by JECFA (FAO/WHO,
1993).
Definition of
Substance
residues on which
Commodity
MRL
ADI
MRL was set
Benzylpencillin
Benzylpencillin
Liver, kidney and
muscle (cattle and pigs)
Milk (cattle)
50 µg/kg
4µg/kg
30µg/kg/
person/day
30µg/kg/
person/day
2.10 The effect of thermal treatment on the antibiotic residue in food:
Rose et al., (1997) studied the stability of benzyl penicillin to
heating and cooking. Stability of this compound in water at 100ºC and
65ºC, 5% ethanol, 5% sodium bicarbonate, pH 5.5 buffer at 100ºC and in
hot cooking oil (at 140º C and 180ºC) was established. Benzyl penicillin
was stable at 65 ºC but not stable at higher temperature with half-life
times varying between 15 and 60 minutes in the solution investigated.
This drug was not stable to cooking, losses being proportional to were
released during the cooking process, sometimes in to the cooking
medium.
Small volumes of Oxytetracycline, benzyl penicillin and tylosin
were added to known volumes of pure milk. The same volumes of
antibiotic were added to distilled water as a control. All containers of
milk and distilled water were heated to 80ºC and boiled for 10 minutes to
evaluate the role of heating on antibiotic activity. The observations
indicated the absence of any effect of heating or boiling on tylosin and a
very slight influence of boiling on oxytetracycline and penicillin in milk.
The results indicated a human hazard of antibiotic contaminated milk
even after boiling (Abdulrahman, 2001).
The stability of sixteen antibiotics during the destruction process of
animal and offal was investigated. The antibiotics were added to a
mixture of pork meat, pork kidney, and pork liver. Subsequently, these
were pasteurized at 80ºC (15 min), sterilized at 134°C (3bar, 20 min) and
dried at 100°C (4 hours). During the different stages of this process,
samples were taken and analyzed for antimicrobial activity by bioassay.
The remaining activity after the full destruction process was for
lincomycin 80%, flumequine 69%, enrofloxacin 68%, neomycin 46%,
tylosin, 44%, sulfamethazine 38% and spiramycin 15%. Penicillin,
ampicillin,
cloxacillin,
oxytertracyline,
doxycycline,
colistin,
dihydrostrptomycin and sulfamethoxazole were fully degraded (less than
10% remaining activity) after the sterilization step (134°C). It is
concluded that the high temperature destruction process does not
guarantee a full break- down of residues of veterinary drugs in
condemned animal. (Van Egmond et al., 2000).
The B-lactam compounds are the most important clinical
antibiotics. This group includes penicillins and the cephalosporin is a
narrow-spectrum antibiotic which, in its mainly various forms is officially
recognized in all the pharmacopoeias of the world.( Brander and Pugh
,1977).
2.11 Antibiotic residues surveillance studies:
Surveillance of antibiotic residues objectives were, testing for
compliance with nationally and/or internationally set safety standards,
checking on with effectiveness of licensing and other control procedures
and estimating the exposure of consumers to veterinary drug residues in
diet. These objectives were not comprehensive but, the approach taken in
surveillance depends largely upon the objectives.
In Sudan a total of 220 samples of milk were collected from
Khartoum State. Some of the samples were taken from cows which had
received antibiotic treatment and milked before the completion of the
withdrawal period. The remainders of the samples were collected from
bulk milk, either from farm cans or from groceries. All the samples were
examined to detect the antibiotic residues. The examination revealed that
all the samples collected randomly from farms and groceries were free of
antibiotic residues while 76.6% of samples collected from treated cows
were positive for antibiotic residues (Abdulrahman, 2001). Tajelsir
(2001) found that the percentage of the positive samples was 10.7%.
Chewulukei (1987) found that 15% of milk samples in Nairobi area
contained antimicrobial inhibitors. Another study in Nairobi indicated
that all samples were free of any inhibitors (Ombui, 1994), while in
Washington D.C. the percentage of positive samples were 0.3% in 1994
(Smuker, 1996).
Eltayeb (1999), studying the status of antibiotic residues in meat,
reported that 11 out of 74 (14.9%) of tissue samples from bovine
carcasses and 29 out of 78 (17.95%) of tissue samples from ovine
carcasses contained antibiotic residues.
2.12 Veterinary drug residues monitoring programmes:
Antibiotic residues in food of animal origin, particularly milk are a
food safety concern and require practical analytical methods for
detecting, quantifying and identifying residues that may be present at
levels above established safe residues limit (WHO, 1997).
Governments need regulatory control programmes to ensure their
citizens of a safe and wholesome food supply. Specifications of a residue
control programmes are determined by the importance of the various
health risks that could be incurred by consumers of products derived from
animal food products (FAO/WHO, 1994).
Strategies for the detection of veterinary drug residues must be
characterized by the following as mentioned by Teagase (1997):
●
Direction to determine the residues of concern from a toxicological
point of view.
●
Direction to ensure the safety of the food supply with the least
amount of interference with food production and processing using
methods and analytical systems which are rapid, easy to do,
sensitive, reliable, cost-effective and precise with development in
extraction purification and in determination technologies.
Teagase (1997) also explained that, regulations on veterinary
drugs were conditioned according to the substances that should be
detected. If the substance is forbidden, this will direct the analyst to
improve the development methods with lowest possible limits of
detection (chasing zero), while in the case of permitted substances
the analyst’s concern might be better directed towards more rapid
and specificity methods with adequate limits of detection to police
the maximum residue levels (MRLs).
CHAPTER THREE
MATERIALS AND METHODS
3.1 Materials:
3.1.1 Penicillin:
Penicillin G sodium was obtained from Harbin Pharmaceutical
Factory (China), each vial contained sodium penicillin G equivalent to
1,000,000 I.U of penicillin G. The serial dilution technique was used to
prepare stock solution with concentration of 50 IU/ml from the original
penicillin G sodium concentration.
3.1.2 Test organisms:
Standard strain of Staphylococcus aureus (gram positive cocci),
American Type Collection Culture (A.T.C.C. 25923) was obtained from
the National Health Laboratories, Khartoum.
3.1.3 Culture media:
3.1.3.1 Diagnostic sensitivity test agar (D.S.T. Agar):
D.S.T. agar was obtained as a dry powder from Oxiod Ltd,
England, and was prepared according to manufacturer’s instructions by
suspending 40 gm in 1000 ml distilled water and boiling till it was
completely dissolved, then the medium was sterilized.
3.1.3.2 Nutrient broth:
Nutrient broth was obtained as a dry powder from Bio Mark
(India). It was prepared according to manufacturer’s instructions by
suspending 13 gm in 1000 ml distilled water, and boiling to dissolve the
medium completely, then the medium was sterilized.
3.1.4 Antibiotic free-milk:
Two packages of pasteurized cow milk free of antibiotics produced
by the Blue Nile Dairy Plant (CAPO) was purchased from local market,
each package contained 1000 ml of milk.
3.1.5 Survey milk samples:
Ninety seven samples of raw milk were collected randomly from
different distribution points at Khartoum State. These samples were
collected in sterile containers and kept in cooled boxes till taken for
testing.
3.1.6 Antibiotics test kits:
Kits used in this test were offered by the Blue Nile Dairy Plant
(CAPO) which were obtained from CHR. HANSEN (Italy).
3.2 Methods:
3.2.1 Experimental methods:
3.2.1.1 Preparation of different milk samples containing different
concentrations of penicillin:
Four different concentrations 1.0, 2.0, 4.0, 8.0 I.U./ml were
prepared by adding, 1.0, 2.0, 4.0, 8.0 ml from the stock solution to, 49.0,
48.0, 46.0, 42.0 ml of antibiotics free milk, respectively.
3.2.1.2 Thermal treatment:
From each concentration 200 ml were prepared and divided into
four flasks each one containing 50 ml of milk with a certain
concentration.
Three flasks from each concentration were subjected to three
different thermal treatments namely sterilization, pasteurization and
boiling.
Sterilization was carried out by autoclaving at 121oC for 15 min.
under a pressure of 15 Ib/ squared inch (Vieira, 1996), while
pasteurization was done by heating flasks in a thermostatically controlled
water bath to 62.8 and was held at this temperature for 30 min. and then
rapidly chilled to 4oC in an ice bath (Vieira, 1996), and boiling was done
by heating the tested flask on an electric heater till boiling. The fourth
flask of each concentration, the control, was not subjected to any thermal
treatment.
3.2.2 Antibiotic sensitivity test:
The different penicillin concentrations in milk which were
subjected to the thermal treatments were examined to evaluate their
antimicrobial activity, by using the Cup-plate Agar Diffusion Technique,
while the survey collected samples were examined by using the
antibiotics test kits.
3.2.2.1 Microorganism staining method:
3.2.2.1.1 Preparation of smears:
For preparation of smear from solid culture of the microorganism,
a drop of sterile normal saline was placed with a sterile loop on the center
surface of a clean slide. A small portion of a colony from an agar culture
was picked up with a sterile loop, mixed with a saline drop and spread on
a slide. The smear formed was allowed to air-dry and was fixed by gentle
flaming (Harrigan, 1998).
3.2.2.1.2 Gram staining method:
This is a differential double-staining method. It was employed for
the diagnostic differentiation of gram-positive and gram-negative
organisms and for the confirmation of the purity of the test organism
stock cultures.
The fixed smear was covered with a few drops of 1% aqueous
crystal violet stain and allowed to act for two minutes. Lugol iodine
solution was used to tip off the stain and for stain fixation. Fresh iodine
solution was allowed to act for one minute then tap water was used for
gentle washing of iodine and excess stain. Decolourization was
performed with absolute alcohol by tilting the slide from side to side until
colour ceased to come out of the preparation, for a few seconds. The
decolourized slide was washed off with tap water and few drops of 2%
aqueous solution of safranine (counter-stain) was added to the prepared
slide and allowed to act for 10 seconds and counter-stain allowed to dry
in the air. The gram-stained film was then examined under oil immersion.
Gram-positive bacteria were stained with dark violet colour while gramnegative bacteria should be stained with a pink colour (Harrigan, 1998).
3.2.2.2 The cup-plate agar diffusion technique:
The cup-plate agar diffusion technique was used to evaluate the
antimicrobial activity of penicillin. About five ml of nutrient broth were
inoculated with the test organism Staphylococcus aureus, and incubated
at 37oC overnight, and then added to solidified DST media in Petri dishes
and left to stand for 30 min. then the excess broth was taken carefully by
using a microtitre pippete. Cups were cut out by using a flamed and
cooled 7 mm cork-borer in the inoculated agar plate. On each plate four
cups were done. Two hundred µl of milk were added to each cup by a
microtitre pippete (Brander and Pugh, 1977). The plates were then
incubated in the upright position. The diameters of the resultant growth
inhibition zones were then viewed against a suitable background and
measured with a suitable ruler.
3.2.2.3 Antibiotic test kit technique:
It is a qualitative ready-to-use test for the detection of inhibitory
substances in milk. It contains spores of Bacillus stearothermophilus var.
calidolactis and is sensitive to different limits of detection for different
antimicrobials (Table 3.1). The method was carried out according to the
manufacturer's instructions by adding 100 µl of milk by a special pipette
to the test tube. The tube was incubated at 64 ± 1oC in a water bath or dry
block heater for 3 hours (Fig. 3.1) (www.CHR-Hancen). The yellow
colour represents negative result while purple and red represent positive
result (Fig. 3.2) (refer to the colours card) (Fig. 3.3).
Table 3.1 Sensitivity of antibiotic test kit.
Antimicrobial
Na penicillin G
Limit of
detection (ppb)
2.5
Antimicrobial
Limit of
detection (ppb)
Sulphadiazine
50
Ampicillin
4
Chlorotetracycline
100
Amoxicillin
4
Oxteteacyciciline
150
Cloxacillin
25
Tetracycline
100
Dicloxacillin
20
Erythromycin
200
Oxacillin
15
Spiramycin
Cephapirin
10
Tylosin
100
Ceftiofur
50
Tylmicosine
100
Sulphamethazine
125
Gentamycin
250
Sulphadimethoxin
50
Neomycin
600
Sulphathiazole
50
Dihydrostreptomycin
2,000
Trimethoprim
200
Streptomycin SO4
2,000
1,500
Plate 3.1. Dry block heater
Plate 3.2 Colours card
1
2
Plate 3.3 Positive and negative samples
1. Kit contains sample with antibiotic
residue.
2. Kit contains sample without antibiotics.
CHAPTER FOUR
RESULTS AND DISCUSSION
4.1 Effect of thermal treatments on penicillin activity:
Figure 4.1 shows the results of the antimicrobial activity of the
milk containing penicillin with concentration 1.0 I.U./ ml. Zones diameter
of Staphylococcus aureus reflect that there were no differences between
pasteurization, boiling and the control (Table 4.1), and the statistical
analysis confirmed that the slight decrease in the inhibition zones
diameter has no significant difference. However, the sterilization
treatment showed a clear effect on the inhibition zone’s diameter, and the
presence of the inhibition zone around the sterilized sample indicated that
the penicillin was still active even after the sterilization treatment.
These results appeared again on the other three concentrations with
a clear increase of the zones diameters according to the increase of the
penicillin concentration. Tables 4.2, 4.3 and 4.4and Figures 4.2, 4.3 and
4.4 show the results of the antimicrobial activity of the milk containing
penicillin with concentrations 2.0, 4.0 and 8.0 I.U./ ml., respectively.
The results obtained are similar to those obtained by Abdulrahman
(2001) who observed that there were no substantial effect of heating or
boiling on antibiotics activity and he also indicated that the penicillin
mixed with distilled water was affected by heat and denatured, but if it
was mixed with milk it may conjugate with milk protein and will not be
denatured by heating.
Plate. 4.1 Antimicrobial activity of milk containing 1.0 I.U. / ml
penicillin.
A: Concentration (1.0. I.U. /ml)
1: Sterilized sample.
2: Boiled sample.
3: Pasteurized sample.
0: Control sample.
Table 4.1 Antimicrobial activity of milk containing 1.0 I.U. /ml
penicillin.
Control
Zone diameter
mm
20.6
Treated sample
Sterilized
Boiled
Pasteurized
15.0
19.78
20.0
Plate 4.2 Antimicrobial activity of milk containing 2.0 I.U. / ml
penicillin.
B: Concentration (2.0 I.U./ml).
1: Sterilized sample.
2: Boiled sample.
3: Pasteurized sample.
0: Control sample.
Table 4.2 Antimicrobial activity of milk containing 2.0 I.U. / ml
penicillin.
Control
Zone diameter
mm
23.0
Treated sample
Sterilized
Boiled
Pasteurized
15.0
22.0
22.89
Plate 4.3 Antimicrobial activity of milk containing 4.0 I.U. / ml
penicillin.
C: Concentration (4.0 I.U./ml).
1: Sterilized sample.
2: Boiled sample.
3: Pasteurized sample.
0: Control sample.
Table 4.3 Antimicrobial activity of milk containing 4.0 I.U. / ml
penicillin.
Control
Treated sample
Sterilized
Boiled
Pasteurized
24.0
28.67
27.0
Zone diameter
mm
29.0
.
Plate 4.4 Antimicrobial activity of milk containing 8.0 I.U. / ml
penicillin.
D: Concentration (8.0 I.U./ ml.).
1: Sterilized sample.
.
2: Boiled sample.
.
3: Pasteurized sample.
0: Control sample.
Table 4.4 Antimicrobial activity of milk containing 8.0 I.U. / ml
penicillin.
Control
Zone diameter
mm
30.0
Treated sample
Sterilized
Boiled
Pasteurized
26.0
29.6
29.8
In another study of stability of antibiotics in foods there were similar
results reported by Van Egmond et al., (2000) who studied the stability of
antibiotics in meat during a simulated high temperature destruction
process, they concluded that the high temperature destruction process
does not guarantee a full breakdown of veterinary drugs residues in meat.
The results obtained in this study are similar to those reported by
Rose et al., (1997) who studied the stability of benzyl penicillin to heat
and cooking in water (at 100oC and 65oC), 5% ethanol, 5% sodium
bicarbonate, pH 5.5 buffer at 100oC and hot cooking oil at 140oC and
180oC. Benzyl penicillin was stable at 65oC but not stable at higher
temperatures with half life times varying between 15 and 60 minutes in
solutions investigated.
4.2 Antibiotic residues in raw cow milk sold in Khartoum State:
The results of this study indicate that there are antibiotic residues in
about one third of the collected samples.
Table 4.5 shows milk samples which were collected from
Khartoum Province. Of 30 collected samples, 8 samples contained
antibiotic residue.
Thirty one milk samples were collected from Omdurman Province,
11 of which contained antibiotic residues (Table 4.6).
Table 4.7 shows the samples collected from Khartoum North
Province, which were 36 samples, 10 of which contained antibiotics
residue.
Table 4.8 shows the total samples and the percentage of positive
samples in each province which were 26.6, 35.4 and 30.9% in Khartoum,
Omdurman and Khartoum North, respectively. Also the table shows that
out of the 97 samples collected from Khartoum State there were 30
samples containing antibiotic residue which represent 30.9% of all
samples.
These results are different from that reported by Abdulrahman
(2001) who concluded that there was no antibiotic residue in any of the
110 samples he collected randomly from groceries. The percentage of
positive samples obtained is regarded as high when compared with the
percentage of positive samples studied by Tajelsir (2001) which were
10.7%. Other results are high when compared with similar studies in
other countries, where Chewulukei (1987) found that 15% of samples in
Nairobi, Kenya, contained antimicrobial inhibitors. Another study in
Nairobi indicated that all samples were free of any inhibitors (Ombui,
1994), while in Washington,D.C. the percentage of positive samples were
0.03% in 1994 (Smuker, 1996). These low detected levels in these
countries may relate to the high level of awareness among milk
producers. By contrast the bad agricultural practices and wrong uses of
veterinary drugs may be behind the high levels of antibiotic residues.
Moreover, the low level of awareness towards the veterinary drugs
withdrawal period after animal treatment may lead to these high levels of
antibiotic residues. This idea is confirmed by looking at previous studies
showing the situation of veterinary drugs residues in other animal
products in Sudan. The status of antibiotic residues in meat reported by
Eltayeb (1999) revealed that 11 out of 74 (14.9%) of tissue samples from
bovine carcasses and 24 out 78 (17.95%) of tissue samples from ovine
carcasses were positive for antibiotic residues.
The season of collection may also affect these results to some
extent due to the high rate of infections with inflammation during the
rainy season. In addition to that, methods followed in production and
distribution in Sudan are so poor, the dealers tend to use any means to
prolong the shelflife of milk even if it is not permissible especially in the
absence of monitoring programmes.
Table 4.5: The presence (+) or absence (-) of antibiotic residue in
milk samples collected from Khartoum Province.
Sample
Area of
No.
collection
Original source of sample
Antibiotics test
result
1
Alsog Elarabi
Hillt Kuku
-
2
Almogran
Hillt Kuku
-
3
Burri
Edbabiker
-
4
Burri-Edraisa
Alamab Bahrabid
-
5
Emtedad Naser
Hillt Kuku
-
6
Almanshia
Hillt Kuku
-
7
Alriad
Alnoba
+
8
Altaif
Hillt Kuku
-
9
Algireif
Hillt Kuku
-
10
Almamora
Aljazira, Almasid
-
11
Arkawit
Aljazira
-
12
Alamarat street 54
Hillt Kuku
-
13
Aldaim
Hillt Kuku
-
14
Alsahafa sharig
Alhaj Yousef
+
15
Alazhari
Alsalama
-
16
Khartoum 2
Suba
-
17
Alseka hadeed
Ed Babiker
-
18
Khartoum 3
Hillt Kuku
-
19
Al Sagana
Taiba Elhasanab
+
20
Abuhamama
Al Sagana
+
21
Alhilla Aljadeeda
Aljazira
+
22
Al Remaila
Hillt Kuku
-
23
Alamab bahrabid
Hillt Kuku
(+, -)
24
Alshagra
Alamab Bahrabid
+
25
Aluzozab
Abukasawi
-
26
Alushara
Taibat Alhasanab
+
27
Alkalakla Elgoba
Jebal Awlia
-
28
Al Kalakla Sungat
Taibat Alhasanab
-
29
Jabra
Taibat Alhasanab
-
30
Alsahafa Zalt
Alamab Bahrabid
-
Table 4.6: The presence (+) or absence (-) of antibiotic residue in
milk samples collected from Omdurman Province.
Sample
Area of
No.
collection
Original source of sample
Antibiotics test
result
1
Almolazmeen
Hillelt Kuku
-
2
Bit Elmal
Aldroshab
-
3
Aburoof
Aldroshab
-
4
Wadarw
Alfaky Hashim
-
5
Wad Nobawy
West of Alharat
-
6
Althawra 6
Omdurman
-
7
Wad El Bakhit
Alfaky Hashim
-
8
Althawra 21
Hillt Kuku
-
9
Althwara 34
Aljaily
-
10
Althawra 10
West of Elharat
-
11
Ashingity-Alroomy
Alfaky Hashm
(+, -)
12
Ashingity 11
Unknown
-
13
Alshingity 4
Althawra 39
-
14
Hay Elarab
Alzakiab
-
15
Alhashmab
Unknown
-
16
Almawrada
Hillt Kuku
(+, -)
17
Banat
Hillt Kuku
-
18
Almohandeseen
Hillt Kuku
-
19
Alfitaihab
Jabal Toria
(+, -)
20
Abu Seid
Jabal Toria
-
21
Ombada Elsabil
Hillt Kuku
-
22
Ombada 8
Hillt Kuku
+
23
Sog Libia
JabalToria
+
24
Ombada 12
Unknown
+
25
Ombada 11
Unknown
-
26
Ombada 10
Almarkhiat
(+, -)
27
Ombada Eljemiab
Almarkhiat
+
28
Ombada Madani
Unknown
-
30
Al-Arda (North)
Unknown
+
31
Al-Arda (South)
Unknown
+
Table 4.7: The presence (+) or absence (-) of antibiotic residue in milk
samples collected from Khartoum North Province.
Sample
Area of
No.
collection
Original source of sample
Antibiotics test
result
1
Shambatl Al Hela
Al Faki Hashim
-
2
Alsamrab
Alhalfaia
+
3
Alhalfaia
Alhalfaia
+
4
Aldroshab South
Alhalfaia
+
5
Aldroshab North
Alhalfaia
-
6
Al Kadarw
Aldroshab
+
7
Shambat North
Alhalfaia
-
8
Alsoug Almarkazy
Alhalfaia
(+, -)
9
Shambat South
Aljaily
-
10
Alsafia North
Alhalfaia
-
11
Elsafia
Shambat
-
12
Alshabia North
Alhalfaia
-
13
Alshabia South
Alfaki Hashim
-
14
Aldanagla North
Hillt Kuku
-
15
Aldanagla South
Hillt Kuku
-
16
Hillt Kuku
Algadisia
-
17
Almazad
Unknown
-
18
Hillt kuku
Edbiada
-
19
Kafori
Ebabiker
-
20
Alsog Almarkazi
Alhalfia
+
21
Alsog Almarkazi
Alfaki Hasim
-
22
Hillt Kuku
Edbabiker
+
23
Alsog Almarkazi
Alzakiab
-
24
Alhagyousif Mygoma Unknown
-
25
Hillt Kuku
Edelshamla
+
26
Alhagousif St. 1
Unknown
(+, -)
27
Alsababy
Alsababy
-
28
Hillt Khogaly
Hillt Khogaly
-
29
Hillt Hamad
Hillt Khogaly
-
30
Elamlak West
Edbabiker
-
31
Elamlak East
Shambat
-
32
Almogtarbin
Dardoog
-
33
Alkhatmia
Aldoroshab
-
34
Alhgyousif Alshigla
Alshegla
-
35
Hillt Kuku
Ramallah
+
36
Hillt Kuku
Hillt Kuku
+
Table 4.8: The Percentage of milk samples testing positive for
antibiotic residue in Khartoum state.
Total of
Area of collection
collected
samples
Positive
Negative
samples
samples
Percentage of
positive
samples (%)
Khartoum Province
30
8
22
26.6
Omdurman Province
31
11
20
35
Khartoum North Province
36
11
25
30.5
Total
97
30
67
30.9
CHAPTER FIVE
CONCLUSIONS AND RECOMMENDATIONS
CONCLUSIONS:
The results obtained in this study indicate that the pasteurization
and boiling treatment had no marked effect on the penicillin activity,
while the sterilization treatment lowered the penicillin activity.
The data shown in the survey study indicate that there were
antibiotic residues in about one third of the collected samples with a
percentage of 30.9%.
RECOMMENDATIONS:
Because of these high levels of residues in milk and the high
stability of antibiotic residues to heat treatment we recommend the
following:
●
Increase the awareness of the milk producers and distributors
towards the hazard of the improper uses of antibiotics.
●
Increase the awareness of the consumers towards their right to
consume safe and healthy foods.
●
Government should establish efficient inspection and detection
programmes.
●
Introduce the methods of the detection of antibiotics in milk
production sector.
●
Induce the adoption of quality programmes by the processors to
produce healthy and safe milk and other dairy products.
REFERENCES
Abdulrahman, M. A. (2001). Detection of Antibiotics in Milk and the
Effect of Heating on the Antibacterial Activity. M.S. c. Thesis,
Faculty of Veterinary Science, University of Khartoum. Sudan.
Allen, E. H. (1985). Chromatographic Methods for Chloramphenicol
Residues in Milk, Eggs and Tissue from Producing Animals.
Journal of A.O.A.C., 68 (5): 990 -999.
Baker, F. J., Leighton, I., Breach, M. R., and Taylor, D. (1980). Medical
Microbiologuical Techniques. London : Butterworths.
Bigger, T. W. (1962). Hand Book of Bacteriology for the Student
Practitioners of Medicine. 8th ed. London . Ballieretinal and
Cox.
Boisseau, J. (1993). Basis of the Evaluation of the Microbiological Risks
Due to Veterinary Drug Residues in Food – Vet. Microbiology
35 (3 – 4): 187 – 192.
Bonner, J. (1997). Hooked on Drugs. New Scientist.2065: 24-27.
Blood, D. C., and Radostits, O. M. (1987). Veterinary Medicine.
Saskatoon: Healthier Pearce. Pp 127 - 152.
Brander, G. C. and Pugh, D. M. (1977). Veterinary Appplied
Pharmacology and Therapeutics. Third Edition.
Balliene
Tindall : London.
Brook, G. F.; Butel, J, S. and Morse, S. A. (1998). Medical Microbiology.
21ST edition. Applleton & Lang,USA.
Chewulukei, A. k. (1987). Antibiotic Residues in Milk. M. Sc. Thesis.
University of Nairobi, Kenya.
Deweck, A. L. (1971). Allergology Proceedings. VII Congress of the
International
Assoc.
of
Allergology,
Excerpt
Medico,
Amsterdam, the Netherlands.
Eltayeb, N. (1999). Monitoring Antibiotic Residues in Beef and Mutton
in Khartoum State. M.P.E.H. thesis, Faculty of Public and
Environmental Health, University of Khartoum, Sudan.
EMEA. (1999).Antibiotic Resistance in the European Union Associated
with Therapeutic Use of Veterinary Medicines: Report and
Qualitative Risk Assessment by Committee for Veterinary
Medicine Products. European Medicine Evaluation Agency
FAO/WHO. (1993). Residue of Veterinary Drugs in Food. Food
Standards Programme, Codex Alimentarius Commission.
Volume three. Rome, Italy.
Franklin, T. J. and Snow, G. A. (1974). Biochemistry of Antimicrobial
Action. 2nd edition. Chapmans & Hall, London.
Graham, H. D., Agres, J. C. Blood, F. R., Chicester, C. D. , Poers, J. J.,
Schweigert, B. S. Mcutcheon, R. S. , Zweig, G., and Stevens,
A. D. (1968). The Safety of Food. AVI publishing company,
Inc. London.
Gringauz, A. (1978). Drugs: How They Act and Why. Saint Louis: C. V.
Mosby company.
Harrigan, W. F. (1998). Laboratory Methods in Food Microbiology. 3rd
edition. Academic Press Limited. London.
Harvey, W. C. and Hill, H. (1967). Milk Production and Control. Third
edition. H. K. Lewis and Co- Ltd. London.
Henderson, J. L. (1971). The Fluid Milk Industry. Third edition. AVI
Publishing Company Inc.London
Herschedoerter, S. M. (1968). Quality Control in the Food Industry.
Volume Two. Academic Press. New York.
Hillers, V. Kuntson, D. (1992). Antibiotics for Animals. The National
Dairy Data Base. NDB/ safe food. Washington
Hillerton, J. E., Halley ,B. I. , Neaves,P. Rose M. D. (1999). Detection of
Antimicrobial Substances in Individual Cow and Quarter Milk
Samples Using Delvotest Microbial Inhibit or tests. J. Dairy
Sci. 82: 704.
ICMSF (1980). Microbial Ecology of Foods. Factors Affecting Life and
Death of Microorganisms. International commission on microbiological
specifications for food. Volume 1. New York: Academic Press.
Jay, J.M. (1986). Modern Food Microbiology. Third edition. Van
Nostrand Reinhold Company, New York.
Jones, M. (199) On-Farm test for Drug Residues in Milk.Virginia Cooperative Extension. Page 1- 8, Publication No.401-404.
Kremar,
P.
Ruzichova.
(1996).
High
Voltage
Electrophoretic
Idendification of Residual Antibiotics in Milk. Veterinary
Medicine Praha. 44 (3):93 – 95.
Madigan, M.T.; Martinko, J. M. and Parker, J. (2000). Brock Biology of
Microorganisms. 9th edition. Prentice Hall International Inc, U.
S. A.
Makie, M. J., Ludium, C. A. and Haynes, A. P (2000). Diseases of the
Blood, in Hasleet, C.; Chilvers, E. R.; Hunter, J. A. A. (edits)
Davidsons Principles and Practice of Medicine. 18th edition.
Harcourt Publishers limited, London.
Mayra-Makinen, A.(1995). Technological Significance of Residues for
the Dairy Industry. In Residues of Antimicrobial Drugs and
Other Inhibitors in Milk. Pp 136-143. IDF Special Issue no
9505.Kiel,Germany.
Meyle, I. Herxheimer, A. (1972). A Survey of Unwanted Effect of Drug
Reported in 1968 – 1971.Amsterdam: Ezxperta medica.
Myllyniemi,
A.
L.;
Rintula,
R;
Niemi,
A.,
and
Backman,
c.(1999).Microbiological Identification of Antimicrobial Drugs
in Kidney and Muscies Samples of Bovine Cattle and Pigs .J.
of Food Additives and Contaminants.10(8):339-351.
Ombui, J. N. (1994). Antibiotic Residues in Milk Received by Dairy Cooperative Societies in Kiambu District, Kenya. East African
Medical Journal. 7: 10.
Park, K. (1997). Park Textbook of Preventive and Social Medicine. 18th
edition. India: M/S Manarsidas.
Pearson, D.(1976). The Chemical Analysis of Foods. 7th Edition.
Churchill livingstone. London.
Popelka, P.; Nagy, J.; Poelka, P.; Sokol, J.; Hajurka, J.; Cabadaj, R.;
Marcincak.S., and Bugarsky, A.(2003). Comparison of Various
Methods for Penicillin Residues Detection in Cow Milk After
Intramammary and Potential Treatment. Bull. vet. Inst.
Pulaway.47:203-209.
Prescott, J. E., Baggot, J. D. (1988). Antimicrobial Therapy in Veterinary
Medicine. Black well Scientific Publication. London.
Rechcigl, M. (1983). Handbook of Naturally Occurring Food Toxicants.
CRC press, Inc. Boca Raton, Florida.
Select Committee on Science and Technology (1998). Resistance to
Antibiotics and Other Antimicrobial Agents. Select Committee
on Science and Technology; 7th Report.
Senyk, G. F.; Davidson, J. H; Brown, J. M.; Hallstead, E. R.; Sherbon ,
Antibiotic Residues in Milk. J. Food Protection. 53: 158- 164.
Smucker, J.M. (1996). Milk Monitoring with Antimicrobial Drug
Screening Test. Center of Veterinary Medicine, Washington.
Suhren, G., and Beukers, R. (1998). Delvotest S P for Detection of
Cloxacillin and Sulfamethoxazole in Milk :IDF Interlaborotary
Study. J.AOAC.81: 978-990.
Tagelsir, S. (2001). Antibiotics Residue in Raw Cow Milk at Khartoum
State. M.P.E.H. thesis, Faculty of Public and Environmental
Health, University of Khartoum, Sudan.
Teagasc, M. (1997). Monitoring of Veterinary Drug Residues in Food.
Dublin: the Society of Food Hygiene Technology.
Todar, K. (1996). Bacterial Resistance to Antibiotics. Madison: Kennith
Todar, University of Wisconsin.
Van Eenennaam, A. L.; Cullor, J. S.; Perani, L., Gordner I. A.;Ssmith W.
L.; Dellinger J., Guterbock W. M. (1993). Evaluation of Milk
Antibiotic Residue Screening Test in Cattle with Naturally
Occurring Clinical Mastitis. J. Dairy Sci. (76):3041-3053.
Vieira, E. R. (1996). Elementary Food Science. Fourth edition. Chapman
and Hall. New York.
WHO (1992). Antibiotic Resistance in Pathogenic Bacteria. 36: 191 –
196.
WHO (1997). The Medical Impact of the Use of Antimicrobials in Food
Animals. WHO meeting 13 – 17.Berlin.