PREVALENCE AND ANTIMICROBIAL SUSCEPTIBILITY OF BACTERIA ISOLATED FROM RAW MILK CONTANING BY ANTIBIOTICS AND CHEMICAL PRESERVATIVES IN KHARTOUM STATE, SUDAN By Ahmed Eltyeb Ahmed Saeed B.Sc. (Honours) Animal production University of Gezira, 2003 A thesis submitted in fulfillment for requirements of the Degree of Master of Science in Animal Production Supervisor Dr. Ibtisam Elyas Mohamed El Zubeir Co-Supervisor Dr. Osman Ali Osman El Owni Department of Dairy production Faculty of Animal Production University of Khartoum August 2006
125
Embed
PREVALENCE AND ANTIMICROBIAL …PREVALENCE AND ANTIMICROBIAL SUSCEPTIBILITY OF BACTERIA ISOLATED FROM RAW MILK CONTANING BY ANTIBIOTICS AND CHEMICAL PRESERVATIVES IN KHARTOUM STATE,
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
PREVALENCE AND ANTIMICROBIAL SUSCEPTIBILITY
OF BACTERIA ISOLATED FROM RAW MILK CONTANING BY ANTIBIOTICS AND CHEMICAL
PRESERVATIVES IN KHARTOUM STATE, SUDAN
By Ahmed Eltyeb Ahmed Saeed
B.Sc. (Honours) Animal production University of Gezira, 2003
A thesis submitted in fulfillment for requirements
of the Degree of Master of Science in Animal Production
Supervisor Dr. Ibtisam Elyas Mohamed El Zubeir
Co-Supervisor
Dr. Osman Ali Osman El Owni
Department of Dairy production Faculty of Animal Production
University of Khartoum
August 2006
i
Dedication
To my dear family
Mother and father
My brothers and sisters
To my dear friends and colleagues
With love and respect
Ahmed
ii
ACKNOWLEDGEMENT
My special praises and thanks to Allah the almighty, most gracious and
most merciful who gave me the health, strength and patience to finish this
thesis.
I would like to express my deep thanks and gratitude to my supervisor
Dr. Ibtisam Elyas Mohamed El Zubeir for her advice, continuous support and
constructive criticism throughout the period of study.
I’m very much indebted to my Co-Supervisor: Dr. Osman Ali Osman El
Owni for his continues help, encouragement and support during the study.
I wish to express my deep thanks and gratitude to all the technical
staff members of the department of Dairy Production, Faculty of Animal
Production for their advices and help. It is important here also to thank
Miss. Lymia, the secretary of Dairy Production Department for her help.
Deepest thanks are also extended to the owners, of the farms and sale
points from whom, we collected milk samples. Thank is also extended to Mr.
Abdel basit Elamin for his assistance during statistical analysis of the data.
My since thank and appreciation go also to my father, mother, sisters and
brothers for their continuous help, encouragement and support during the
study.
It is impossible to name all those good friends who encouraged me, I
can only say thanks to them all.
iii
LIST OF CONTENTS
Pagesi DEDICATION ii ACKNOWLEDGEMENT iii LIST OF CONTENT vi LIST OF TABLES vii LIST OF FIGURES viii ABSTRACT x ARABIC ABSTRACT1 CHAPTER ONE:INTRODUCTION3 CHAPTER TWO: LITERURE REVIEW3 Milk 2.14 Importance of milk 2.25 Milk composition2.35 Water2.3.16 Fat2.3.26 Lactose2.3.37 Protein2.3.47 Minor milk components2.3.58 Microorganisms in milk2.48 Total bacterial count2.4.110 Method of milk preservation 2.510 Heat treatment of milk2.5.111 Cooling of milk2.5.212 Chemical preservatives 2.6 13 Hydrogen peroxide2.6.115 Effect of different H2O2 concentration on total bacterial count 2.6.1.116 Aldehydes 2.6.217 Carbon dioxide2.6.319 Antibiotic residues2.719 Background 2.7.119 Detection of antibiotics in milk2.7.221 Antibiotic residues in milk and human health 2.7.321 Hypersensitivity to antibiotic residues in milk 2.7.422 Factors promoting emergence of resistant to antibiotics2.7.523 Transfer (development) of resistant2.7.624 Reasons for antibiotic contamination of bulk milk tank2.7.725 Prevalence of antibiotic residues in bulk tank milk2.7.825 Relationship between antibiotic and somatic cell count (SCC) 2.7.926 Antibiotic sensitivity2.7.1026 Principles of the test 2.7.10.127 Microbial drug resistance2.7.10.231 Shelf life 2.834 CHAPTER THREE: MATERIAL AND METHODS
iv
34 Source and numberof milk samples 3.1 35 Collection of milk samples 3.2 35 Analysis of the samples 3.3 35 Detection of chemical residues 3.3.1 35 Formaldehyde 3.3.1.1 35 Hydrogen peroxide 3.3.1.2 35 Boric acid 3.3.1.3 36 Carbonate and alkalinity of ash 3.3.1.436 Physicochemical properties of milk 3.3.236 Titratable acidity 3.3.2.137 pH 3.3.2.237 Temperature 3.3.2.338 Antibiotic residues 3.4.138 Total antibiotic and sulphonamide 3.4.240 Microbiological examination 3.540 Preparation of the media 3.5.140 Types of culture media used for bacteriological examination of milk 3.5.240 Solid media 3.5.2.140 Standard plate count agar (Merck, 74 o bs) 3.5.2.1.140 Nutrient agar (S. d. Fine. Chem. ltd 74056) 3.5.2.1.241 Urea media (Hi media M112) 3.5.2.1.341 Mueller and Hinton agar 3.5.2.1.441 Semi solid media 3.5.2.241 Hugh and Leifson (OF) medium 3.5.2.2.141 Motility medium 3.5.2.2.242 Liquid media 3.5.2.342 Nutrient broth (Hi Media M 002) 3.5.2.3.142 Peptone water (Oxoid L 37) 3.5.2.3.242 MR – VP medium 3.5.2.3.342 Koser citrate medium (Hi Media-M 069) 3.5.2.3.4 43 Solutions and regents 3.5.343 Methyl red solution 3.5.3.143 Alpha naphthol 3.5.3.243 Hydrogen peroxide (BDH, UK) 3.5.3.3 43 Kovac’s reagents 3.5.3.444 Liquid paraffin 3.5.3.544 Bromocresol purple 3.5.3.644 Ringer solution 3.5.3.744 Sterilization 3.644 Sterilization of equipment 3.6.144 Preparation of sample for bacteriological examination 3.745 Examination of culture 3.7.145 Primary isolation 3.7.1.145 Subculturing of the primary isolates 3.7.1.245 From solid media to solid media 3.7.1.2.1
v
45 From solid media to liquid media 3.7.1.2.245 From liquid to solid media 3.7.1.2.346 Incubation of cultures 3.7.346 Examination of cultures 3.7.4 46 Purification of isolates 3.847 Identification of isolated bacteria 3.947 Primary tests 3.9.147 Gram’s stain 3.9.1.148 Catalase test .9.1.2348 Oxidase test 3.9.1.3 48 Oxidation fermentation test (OF) 3.9.1.449 Glucose test 3.9.1.549 Motility test 3.9.1.649 The ability to grow in air 3.9.2.650 Anaerobic growth 3.9.2.750 Secondary biochemical tests 3.9.250 Methyl red (MR) test 3.9.2.150 Vogues Prokauer (VP) test 3.9.2.251 Indole test 3.9.2.351 Urease test 3.9.2.451 Citrate utilization 3.92.5 51 Coagulase test 3.9.2.852 Sugar fermentation test 3.9.2.952 Sensitivity test 3.1054 Statistical analysis 3.1155 CHAPTER FOUR: RESULTS 55 Microbial analyses of milk samples 4.155 Total bacterial counts 4.1.158 Determination of the physicochemical properties of milk samples 4.258 Titratable acidity 4.2.163 pH values 4.2.265 Temperature 4.2.358 Shelf life of raw milk samples collected from farms and sale points in
Khartoum State 4.3
71 Correlation between total bacterial counts (cfu/ml) and some physicochemical properties of milk samples collected in Khartoum State
4.4
71 Incidences and frequencies of preservative in milk samples collected in Khartoum State
4.5
76 Bacteriology of isolated organisms 4.683 Microbial drug resistance 4.788 CHAPTER FIVE: DISCUSSION
100 CONCLUTION AND RECOMMENDATION103 REFERENCES
vi
LIST OF TABLES
Page Title Tables
53 Antimicrobial agents used for sensitivity tests 3.1
54 Determination of susceptibility of antimicrobial agents 3.2
56 Total bacterial counts of raw milk samples collected from different farms and sales points in Khartoum State during summer and winter
4.1
59 Effect of different sources, seasons and locations on TBC of raw milk samples in Khartoum State using one-way ANOVA
4.2
60 Comparison of properties milk samples collected from different locations in Khartoum State using Duncan's multiple range tests
4.3
62 Acidity and pH of milk samples collected from different farms and sale points in Khartoum State during summer and winter
4.4
64 Physiochemical properties of milk samples collected from different sources and locations in Khartoum State during summer and winter using one way ANOVA
4.5
67 Temperature ( O C ) of milk samples collected from different farms and sale points in Khartoum State during summer and winter
4.6
70 Shelf life (days) of milk samples collected from different farms and sale points in Khartoum State during summer and winter
4.7
72 Correlation between some quality measurements of milk samples collected in Khartoum State
4.8
74 Incidence and frequencies of chemical preservation in milk samples collect from different locations in Khartoum State
4.9
80 Frequency of isolated bacteria from raw milk samples contaminated with antibiotic residue in farms and sale points in Khartoum State
4.10
81 Biochemical reactions of the isolated Gram-positive bacteria in milk samples from Khartoum State
4.11
82 Biochemical reactions of the isolated Gram-negative bacteria in milk samples from Khartoum State
4.12
85 Antimicrobial susceptibility tests for the isolated potential pathogenic bacteria from raw milk samples contaminated with antibiotics residue
4.13
vii
LIST OF FIGURE
Page Title FIGURE
39 Detection of antibiotic using disk assay method 3.1
39 Structure of total antibiotic and sulphanomide tube kits 3.2
77 Positive result for antibiotic residue 4.1
78 Negative result for antibiotic residue 4.2
79 Positive and negative antibiotic residues using total antibiotic and sulphonamide kits
4.3
86 Staphylococcus aureus exhibits varying sensitivity to antibiotics 4.4
87 Escherichia coli exhibits varying resistance to antibiotics 4.5
viii
ABSTRACT
This study was carried out on milk samples collected from farms and
sales points in Khartoum State. Two hundred and forty milk samples were
randomly collected. One-hundred and twenty milk samples were collected
during summer and 120 milk samples were collected during winter from the
same sources.
The milk samples were examined for microbial quality, chemical
preservative, drug residues and physical characteristics. Total bacterial
counts, isolation and identification of microorganisms from antibiotic
contaminated milk and resistance of isolated bacteria to antibiotics were
estimated. The chemical preservative residues examination included the
detection of formaldehyde, hydrogen peroxide, boric acid, carbonate,
alkalinity of ash and detection of antibiotics residue. The physical properties
of milk studied were temperature, acidity and pH.
The present study revealed high average of total bacterial counts (5.15
× 1011 ± 5.57 × 1011 cfu/ml) in milk samples. During summer season the total
bacterial counts of milk (1.02 × 1012 ± 3.4 × 1011 cfu/ml) was significantly
Raw milk samples collected from different sale points showed
means total bacterial counts of 5.18×1011± 5.55×1011 cfu/ml, the
means total bacterial counts was 4.32×1011 ± 4.88 ×1011 cfu/ml, for
raw milk samples collected from Khartoum, during summer and
winter the means total bacterial counts were 8.51×1011 ± 3.47×1011
cfu/ml and 1.32×1010± 1.55×107cfu/ml, respectively (Table 4.1)
samples collected from Khartoum North, revealed means total
bacterial counts of 5.03×1011± 5.41×1011cfu/ml, 9.92×1011± 3.13
×1011cfu/ml and 1.35×1010± 6.11×107cfu/ml for the total ,summer and
winter, respectively (Table 4.1). Milk collected from Omdurman
revealed mean count of 6.18×1011± 6.27×1011cfu/ml as shown in
Table 4.1. During summer and winter the mean counts were found as
1.22×1012± 1.92×1011 cfu/ml and 1.37×1010± 1.14×107 cfu/ml,
respectively (Table 4.1).
58
Raw milk samples collected from different locations and
seasons showed significant variations for (P< 0.001) total bacterial
counts (TBC) as shown in Table 4.2 and Table 4.3. Moreover the
interaction between seasons and locations showed significant
variations (P< 0.001) for TBC as shown in Table 4.2. The interaction
between seasons and sources showed significant variations (P<0.05)
for TBC as shown in Table 4.2. Similarly the raw milk samples
collected from different sources showed significant variations
(P< 0.01) for TBC as shown in Table 4.2. Also the interaction
between locations and sources showed significant variations (P< 0.01)
for TBC as shown in Table 4.2.
4.2 Determination of the physicochemical properties of milk samples
4.2.1 Titratable acidity
The raw milk samples collected from different locations and
sources (Table 4.4) showed that the mean values for acidity was 0.20±
0.31%, during summer the mean acidity was 0.20 ± 0.22%, the
minimum and maximum were 0.20% and 0.23% respectively (Table
4.4). During winter, the acidity was 0.19 ± 0.22%, the minimum was
0.13% and the maximum was 0.25% (Table 4.4). The raw milk
samples collected from different farms showed that the total means
value for acidity was 0.19 ± 0.25%, the mean value for acidity were
59
Table 4.2 Effect of different sources, seasons and locations
on TBC of raw milk samples in Khartoum State using one-way
ANOVA
* : Significant at (P< 0.05). ** : Significant at (P< 0.01). ***: Significant at (P< 0.001).
Measurements Mean square Sig.
Seasons 6.0569×1023 0.001***
Locations 9.49833×1023 0.001***
Sources 1.2020×1021 0.026*
Seasons × locations 9.5183×1023 0.001***
Seasons × sources 8.4840×1023 0.052**
Locations × sources 2.0605×1023 0.001***
60
Table 4.3: Comparison of properties milk samples collected from different locations in Khartoum State using Duncan's multiple range tests
Locations TBC
(cfu/ml) Acidity pH Temperature Shelf
life
Khartoum 3.92×1011a 0.20a 6.74b 25.78 a 4.87a
Khartoum North 5.96×1011b 0.22a 6.74b 26.70b 5.26a b
Omdurman 5.58×1011b 0.25b 6.517a 28.60c 5.60b
In this and the following tables, means within each column bearing the same superscripts are not significantly different (p<0.05) TBC= Total bacterial count
61
0.18± 0.24% for milk samples collected from Khartoum (Table 4.4).
During summer it was 0.19 ± 0.20% and during winter it was 0.17±
0.27%, (Table 4. 4). In Khartoum North the mean value of acidity of
milk was 0.19± 0.20%, during summer it was 0.20± 0.21% and during
winter was 0.19±0.16% (Table 4.4). Similarly, the raw milk samples
collected from Omdurman revealed mean value for acidity of 0.21±
0.23%, during summer it was 0.21± 0.29% and during winter it was
0.21± 0.11% (Table 4.4). The raw milk samples collected from
different sale points (Table 4.4) showed that the total mean value for
acidity was 0.20± 0.19%. The mean value for acidity was 0.20±
0.17% for milk sample collected from Khartoum (Table 4.4). During
summer it was 0.21± 0.77% and during winter it was 0.19± 0.46%
(Table 4. 4). In Khartoum North the mean value of acidity was 0.20±
1.19%, during summer it was 0.21± 0.17% and during winter it was
0.19±0.18% (Table 4.4). Similarly the raw milk samples collected
from Omdurman showed mean value for acidity of 0.21± 0.19%
during summer it was 0.22± 0.10% and during winter it was 0.21±
0.23% (Table 4.4).
The milk samples collected from different locations showed
significant variations (P<0.001) for acidity as shown in Table 4.5.
62
Table 4.4: Acidity and pH of milk samples collected from different farms and sale points in Khartoum State during summer and winter
*Over all mean (summer and winter) for all samples
acidity PHWinter Summer Winter Summer
source location
Mean±std Min. Max. Mean± td Min. Max. Total Mean±std Mean±std Min. Max. Mean±std Min. Max.
Similarly significant variations were found between milk samples
collected during different seasons (P< 0.01). Moreover significant
variations (P<0.001) for acidity were found in the interaction between
seasons, locations and sources as shown in Table 4.5.
4.2.2 pH values
The raw milk samples collected from different locations and
sources showed that the total mean pH value were 6.6± 0.44, during
summer it was 6.7± 0.23, the minimum and maximum values were
5.84 and 6.99, respectively (Table 4.4). During winter the pH was
6.6± 0.58, the minimum was 5.91 and the maximum was 6.97 (Table
4.4). The raw milk samples collected from different farms showed that
the total mean values for pH was 6.6± 0.59, The mean value of pH
were 6.7± 0.11 for milk samples collected from Khartoum, during
summer it was 6.8± 0.13 and during winter it was 6.7± 0.14 (Table
4.4). In Khartoum North the mean value of pH for the raw milk
sample was 6.8±0.10, during summer it was 6.8± 0.10 and during
winter was 6.7± 0.01 (Table 4.4). Similarly the raw milk samples
collected from Omdurman showed mean value of pH of 6.4± 0.98,
during summer and winter the pH was found as 6.4± 0.35 and 6.3±
1.36, respectively (Table 4. 4).
64
Table 4.5: Physiochemical properties of milk samples collected from different sources and locations in Khartoum State during summer and winter using one way ANOVA
Variable Mean square Sig. Seasons Acidity 1.926 0.018** pH 0.165 0.345NS Temperature 24.337 0.001*** Shelf life 8.004 0.001*** Location Acidity 5.126 0.001*** pH 1.479 0.001*** Temperature 1.479 0.001*** Shelf life 6.704 0.017** Sources Acidity 4.845 0.001*** pH 2.091 0.737NS Temperature 5.104 0.440NS Shelf life 4.167 0.968NS Seasons× locations Acidity 5.798 0.001*** pH 4.193 0.797NS Temperature 10.212 0.303NS Shelf life 0.554 0.803NS Seasons × sources Acidity 2.185 0.012** pH 0.260 0.237NS Temperature 5.704 0.414NS Shelf life 2.204 0.351NS Locations× sources Acidity 7.250 0.123NS pH 0.464 0.083* Temperature 91.554 0.001*** Shelf life 5.779 0.103NS
NS: Non-significant (P>0.05). **: Significant at P<0.01.
*: Significant at P<0.05. ***: Significant at P<0.001
65
The raw milk sample collected from different sale points
showed that the total mean value for pH was 6.6± 0.19 for milk
samples collected from Khartoum State (Table 4.4). The mean value
of pH of milk samples collected from Khartoum was 6.7± 0.16, during
summer it was 6.7± 0.60 and during winter it was 6.6± 0.19 (Table 4.
4). In Khartoum North the mean value of pH for the raw milk sample
was 6.7±0.27, during summer it was 6.6± 0.36 and during winter was
6.7± 0.47 (Table 4.4). The mean value of pH for raw milk samples
collected from Omdurman was found to be 6.6± 0.25; during summer
and winter it showed values of 6.5± 0.27 and 6.6± 0.24, respectively
(Table 4.4).
The raw milk samples collected from different locations showed
significant variations (P<0.001) for pH values as shown in Table 4.5.
However non significant differences were reported in the pH values
for the milk samples collected from Khartoum and Khartoum North
(Table 4.5).
4.2.3 Temperature
The raw milk samples collected from different locations and
sources showed that the average mean value for the temperature of
milk was 27± 3.79° C. During summer it was 28± 2.44° C, the
minimum and maximum were 21° C and 33° C, respectively (Table
66
4.6). During winter it was 25± 3.86° C, the minimum was 10° C and
the maximum was 32° C (Table 4.6). The raw milk samples collected
from different farms showed that the total average mean value of
temperature was 26± 4.04° C, the average mean value of temperature
was 26± 2.84° C for milk samples collected from Khartoum (Table
4.6). During summer it was 28± 1.16° C and during winter it was 24±
2.35° C (Table 4.6). In Khartoum North the average mean value of
temperature of milk was found to be 26± 5.25° C (Table 4.6). During
summer it was 29± 1.94° C and during winter was 23 ±6.26° C (Table
4.6). The average mean values for temperature of milk samples
collected from Omdurman was 27± 3.74° C, during summer and
winter it showed that the average values for the temperature were 28±
2.75° C and 25± 4.14° C, respectively (Table 4.6).
The raw milk sample collected from different sale points
showed that the total average mean value of the temperature was 24±
3.76° C. The averages mean values for the temperature of milk was
24± 2.87° C for milk sample collected from Khartoum (Table 4.6).
During summer it was 26± 2.47° C and during winter it was 23± 2.60°
C (Table 4.6). In Khartoum North the average mean value for the
temperature of milk was 26 ±2.58° C, during summer it was 28± 1.21°
C and during winter it was 24± 2.05° C (Table 4.6). The average mean
67
Table 4.6: Temperature (O C) of milk samples collected from different farms and sale points in Khartoum State during summer and winter
(50%), tetracycline (50%), cephalexin (33.3%) and clindamicin
(33.3%). Kelbesilla aergenes showed resistance towards penicillin,
tetracycillin, colxacillin, erythromycin, clindamicin, pipercillin and
84
ampicillin, showed resistance at 50%, 50%, 50%, 50%, 50%, 50%
and 50% respectively. Pseudomonades earginosa showed 100%
100%, 100%, 50%, 50%, 50%, 50%, 50% and 50% resistance
towards gentamicin, chloramphenciol, ampicillin, Penicillin,
cephalexin, tetracycillin, colxacillin, clindamicin, and pipercillin
(Table 4.11).
In the present study the isolated Proteus mirabilis, showed
100% resistance to penicillin, cloxacillin and ampicillin.
85
Table 4.13: Antimicrobial susceptibility tests for the isolated potential pathogenic bacteria from raw milk samples contaminated with antibiotics residue.
Bacteria Staphylococus
aurous (%)
Streptococcus pyogenes
(%)
Corynobacterium ovis
(%)
Escherichia coli (%)
Citrobacter Kasseri
(%)
Kelbesilla aergenes
(%)
Pseudomonas earginosa (%)
Proteus mirabilis
(%)
Antibiotic S I R S I R S I R S I R S I R S I R S I R S I R
earginosa, Proteus mirabilis) showed wide range of multiple resistances to
the tested antimicrobial agents. The highest resistance was to pencillin
(63.33%), collxacillin (63.33%) clindamycin (56.66%), and ampicillin
(56.66%) showed the highest resistance, while cloramphenicol (3.33%)
showed the highest antimicrobial activity against the test organisms
followed by pipercillin (13.33%) and gentamicin (20.00%).
Recommendations
1/ Farmers should improve their milking practices to reduce mastitis
infection. Moreover vaccination programmes for epidemic diseases
should be applied in order to minimize the need for antibiotic
treatment.
2/ Education programmes on the uses of antibiotic and withdrawal
period should be implemented for farms owners and lobours.
3/ The official authorities should control the chemical residues in milk
and milk hygiene though the milk chains by constructing of quality
control measures for milk and milk products.
4/ Future work on evaluation of milk should be focused on the
following:
102
a/ Milk should be cooled immediately after milking, during
transportation and storage to eliminate growth and
multiplication of microorganisms.
b/ Introduction of proper collection centers and milk
pasteurization factories to ensure the distribution of safe
products to the consumers.
c/ Payment for milk according to quality should be introduced in
Khartoum State to encourage the production good quality
milk.
103
REFERENCES
Abd-El-Hady, S. M. (1995). Hydrogen peroxide as a milk preservative. Proceedings of the Egyptian Conference for Dairy Science and Technology, Cairo, 144-154.
Abd-Elfrag, Mona, M. (2004). Physiochemical and micro-biological respects of raw milk sold at Khartoum State. M.Sc. Thesis, University of Khartoum, Sudan.
Adesiyun, A. A.; Webb, L. A.; Romain, H. and Kaminjolo, J. S. (1997). Prevalence and characteristic of stran of Eschericia coli isolation from milk and fesses of cow on dairy farms in Trinidad. Journal of Food Protection, 60: 1174-1181.
Al Dabbagh, W.; Al Raiab, W. J. and Almallah M. K. (1984). Some observations on the activity of lactic cultures in milk due to H2O2. Indian Journal of Comparative Microbiology Immunology and Infectious Diseases, 5:(2); 60-62.
Aleksieva, V. and Krusher, B. (1981). Quality of raw cow's milk. Vet. Med Nouki, 18 (3): 65-71.
Ali, A. M. (1988). The pasteurization efficiency of milk, M.Sc, Thesis, University of Khartoum, Sudan.
Ali, F. M.; Elaish, N. and Mohamed, T. A. (1993). Incidences of nasal carriers of Staphylococcus aureus in and out side hospital environmental and antibiotic sensitivity of isolated Staphylococcus strain. Egypt Public Health Assoi., 68 (1-2): 33-42.
Allison, J. R. D. (1985). Antibiotic residues in milk. Br. Vet. J., 141: 121-124.
Al -Tarazi, Y.; Al –Zamil, A.; Shaltout, F. and Abdel-Samei, H. (2005). Sanitary status of raw cow milk marked in northern Jordan. Assiut Veterinary Medical Journal, 49 (96): 180 – 194.
AOAC (1990). Official methods of analysis, 15th edition. Association of Official Analytical Chemist, Washington, D. C. USA.
Australian Academy of Science. (2004). chemical preservation. Cited in: http://www.yahoo.com/search?publications/abstracts/september2004.htm.
104
Ayres, H. M.; Furr, G. R., Russell. A. D. (low resistance1999). Effect of permeabilizers on antibiotic sensitivity of Pseudomonas aeruginosa. Lett. Appl. Microbiol., 28 (1):13-16.
Baker, F. J. and Breach, M. R. (1980). Antibiotic susceptibility testing. Medical microbiology techniques. Buffer Worth and Co. Publishers Ltd , pp. 343-350.
Baner, A W.; Kirby, W. M. and Sheris, J. C. (1966). Antibiotic susceptibility testing by a standard disk method. Am. J. Clin. Path, 45: 493-496.
Barakat, E. A. (1995). Evaluation of milk hygiene in Khartoum State. M. Sc. University of Khartoum, Sudan.
Barrow, G. I. and Feltham, R. K. (1993). Cowan and Steel manual for the identification for medical bacteria, 3rd ed., Cambridge.
Boonk, W. and Van schaik, W. G. (1982). The role of penicillin in the pathogenesis of chronic urtearia. Br. J. Dermatology, 106: 183-190.
Booth, J. A. and Harding F. (1986). Testing for antibiotic residues in milk. Vet. Rec., 119: 565-569.
Booth, J. M. (1998). Antibiotic residues in milk. Impract, 4: 100-109.
Buckwoold, F. J. and Ronald, A. R. (1979). Antimicrobial misuse effects and suggestions for control. J. Antimicrobial. Chemother., 5: 129-136.
Caraviello, D. (2004). Financial losses and management practices associated with BTSCC. J. Dairy Sci, 87 (Suppl.): 375.
Chamberlain, Anne, (1990). An introduction to animal husbandry in the tropics. Fourth Edition, pp 752-754. Longman, Singapore.
Chu, H. D. and Leeder, J. G. (1975). Immobilized catalase reaction for use in hydrogen peroxide sterilization of dairy products. J. Food Sci., 40: 641-643.
Clarence, E.H; Combm, W.B and Macym, H. (1982). Milk and milk products. University of Minnesota. Fourth edition.
Cogan, T. M. (1972). Susceptibility of cheese and yoghurt starter bacteria to antibiotics. Appl. Microbiol., 23: 960-965.
105
Committee on Chemistry and Public Affairs (1980). Chemistry and the food system, Washington: American Chemical Society, page 8.
De Weck, A. L. (1983). Penicillin and cephalosporins. In: Allergic reactions to drugs. Ed. A. L. de Weck and H. Bungaard. pp 423-482. Springer– Verlag.
Dewdney, J. M. and R. G. Edward. S. (1984). Penicillin hypersentivity, is milk a significant hazards, a review. J. Royal Soc. Med., 77: 866-877.
Dewdney, J. M.; Maes, L.; Raynand, J. P.; Blance, F.; Scheid J. P.; Jackson, T.; Lens, S. and Verschueren, C. (1991). Risk assessment of antibiotic residues of B-lactams and macrolides in food products with regard to their immuno– allergic potential. Food Chem. Toxicol., 29: 477-483.
Dirar, A. A. (1975). Study on the hydrogen peroxide preservation of raw milk. Sudan J. Food Sci. Technol., 7: 66– 71.
Douglas, J. R. (2000). Bulk tank cultures are the dairy man’s best friends. University of Wisconsin Milking Research and Instruction Lab.
Eckles, H. C.; Cow, B. W. and Macy, H. (1982). Milk and milk products of cowpea (Vignauculata). J. Sci. Food Agric., 54, 1-7.
El Barbary, A. S. A.; Al hakim, M. K. and Issa, H. H. (1983). Studies on milk composition of Karadi caws. Indian Vet. J., 60: 291-296.
Elghoul, S. M. (1995). Preservation of raw milk by hydrogen peroxide, M.Sc. Thesis, University of Khartoum, Sudan.
Elmagli, A. A. and El Zubeir, E. M. (2006). Study on the hygienic quality of pasteurized milk in Khartoum State (Sudan), Research Journal of Animal and Veterinary Sciences, 1 (1): 12-17.
El Zubeir, Ibtisam, E. M.; Kutzer, P. and Elowni, O. A. O. (2006). Frequencies and antibiotic susceptibility patterns of bacteria causing mastitis among cows and their environment in Khartoum State. Research Journal of Microbiology, 1 (2): 101-109.
Erkmen, O. (2000). Effect of carbon dioxide pressure on Listeria monocytogenes in physiological saline and foods. Food Microbiology, 17(6): 589-596
106
Erskine, R. J.; Cullor, J.; Schaellibaum, M., Yancey, R. and Zecconi, A. (2004). Bovine mastitis pathogens and trends in resistance to antibacterial drugs. Pp 400-414 Proc. 43th Ann. Meeting Natl. Mastitis Count. Madison, WI.
Espie, W. E. (1996). Carbon dioxide longer life milk. Dairy Industries International, 63 (3): 32-33.
Fal, N. L. (1997). Antibiotic sensitivity of microflora isolated in ENT diseases. Antibiotic, 24 (3): 189-193.
FAO (1957). Report on the meeting of experts on the use of H2O2 and other preservatives in milk. FAO 157/18655, September 1957.
FAO (1984). Manual of food quality control, Food inspection. FAO, pp. 59-106.
FAO (1990). The technology of traditional milk products in developing countries. Animal Production and Health Paper, 85: 25-64.
FDA (1997). Grade A Pasteurized Milk Ordinance. Revision PHS FDA publication No. 229. Cited in http://www. National dairy concil.org/medcent, newer – khowledge/nk65.html.
Fernandez, E.; Gonzalez, D, E.; Liano, D. and Garcia, E, F. (1996). Preservation of the microbiological and biochemical quality of raw milk by carbon dioxide addition. J. of. Food Protection, 59 (5): 502-508.
Filer, L. J. and Reynold, W. A. (1997). Lessons in comparative physiology: Lactose intolerance. Nutr. Today, 32: 79. Cited in http://www. National dairy council.org/medcent/newer– knowedge/nk64thml.
Foly, J.; Buchley, J. and Murphy, M. F. (1974). Commercial testing and product control in the dairy industry. University College Cork.
Fudle Elseed, M. F. (2005). A study on bacterial contamination of raw milk in small dairy producing units in Omdurman, Khartoum State, M.Sc. Thesis, University of Khartoum, Sudan.
Giovannini, A. (1998). Important of milk hygiene to public health. Report of MZCP Workshop on the management of milkborne zoonoses surveillance and control in the MZCP countries. Cephlonia Islands, Greece.
107
Godefay, B. and Molla, B. (2000). Bacteriological quality of raw cow’s milk from four dairy farms and milk collecting centre in and around Addis Ababa. Berl. Munch. Tieraztl Wochenschr, 113 (7-8): 276-278.
Goldbeawrg, J.; John, P.; Joseph,W.; Drescheler, A. P.; Murelough, A. P. and Howard, B. D. (1990). An update survey of bulk tank milk quality in Vermont. J. Food Protection, 54: 549 – 553.
Goma, M. S. (1998). Detection of formaldehyde in milk, effect of heat treatment and addition of Sacchromyces cerevisiae. Proceedings of the 7th Egyptian Conference for Dairy Science and Technology, Cairo, 16: 55-67.
Gran, H. M. (2002). Aspects of production hygiene in the small scale food industry in sub Saharan Africa with focus on the dairy industry in Zimbabwe.
Gruetzmacher, T. J. and Bradlley, R. L. R. (1999). Identification and control of processing variables that affect the quality and safety of fluid milk. J. Food Protection, 62 (6): 625-630.
Haj Mahmoud, Assagad, H. (2002). Milk quality and aerobic bacteria in raw fluid milk in El Obeid (North Kordofan State). M. Sc. Thesis, University of Khartoum, Sudan.
Harding, F. (1999). Milk quality, first edition, Chapman and Hall Food Science Book, pp. 102-104.
Harold, M. C. (2004). On food and cooking, Chapter 1: Milk and dairy products. USA, Inc used by Simon and Schuster, pp 55-67.
Harrigan, W. F. and McCance, Margaret, E. (1976). Laboratory methods in food and dairy microbiology. Pp. 25-29. Academic Press. London.
Harvey, W. M. E. and Hill, H. (1967). Milk production and control, 4th ed. London. H. K. Lewis and Co. Ltd: page 539-431.
Hayes, M. C.; Ralyea, R. D.; Murphy, S. C.; Carey, N. R., Scarlett, J. M. and Boor, K. J. (2001). Identification and characterization of elevated microbial count in bulk tank raw milk. J. Dairy Sci., 84 (1): 292-298.
Heeschen, W.; Suhrem, G. and Reichmuth, J. (1987). Evaluation of the bacteriological quality or raw milk. Cited in DSA, 45: 4564.
108
Hosseain, M. S. (1990). Studies on the preservation of milk with hydrogen peroxide. Bangladesh J. Anim. Sci., 18 (1-2): 75-80.
Ibrahim, E. A. (1970). Basic sanitary principle applicable to milk production in the Sudan. Sudan, J. Vet. Sci. Anim. Husb., (10): 64-68.
IDF (1994). Pasteurization and heat treatment processes. In: Recommendation for the hygienic manufacture of milk based products. International Dairy Federation, Brucell, Belgium.
Idris, O. F.; Mustafa, A.A. and Wahbi, A. A. (1975). Physiochemical and bacterial composition of raw milk supply to the three towns. Sudan. J. Vet. Sci. and Anim. Husb., (16): 82 – 93.
Igoe, R. S. and Hui, Y. H. (1996). Dictionary of food ingredients, 3rd ed. Chapman of Hall, New York.
Janetschke, P. (1992). Method of preserving foods. DSA, 54 (8): 195.
Jensen, R. G. (1995). Handbook of milk composition. Academic Press, New York, pp. 465-470.
John, H. K. (2001). Antibiotic residues. University of California Davis Veterinary Medicine Extension.
Jones, G. M. (1999). Preventing drug residues in milk and cull dairy cows. Milk quality and milking management, Virginia Tech, 403-404.
Kailasapathy, K. and Wijayakan, S. (1987). Study of the microflora at various stages of processing of fluid milk in Srilanka. D.S.A. 50: 2549.
Kalman, M.; Szollosi, E.; Czermann B.; Zimanyi, M. and Szekeres, S. (2000). Milk borne campylobacter infection in Hungry. J. Food Protection, 63: 1426-1429.
Karmakar, B. (1977). Effect of chemical preservatives on different constituents of cow milk during storage under refrigerated conditions. Cheriron, 26 (5-6): 89-93.
King, J. O. (1980). Factors affecting the butter fat content of cow’s milk. Vet. Rec., (113): 132-134.
Kirk, J. H. (2001). Antibiotic residues, Tulare: University of California Davis, Page 1.
109
Koenen-Dierick, K.; Okerman, L.; De Zutter, L.; Degroodt, J.M.; Van Hoof, J. and Srebrnik, S. (1995). A one-plate microbiological screening test for antibiotic residues testing in kidney tissue and meat an alternative to EEC four-plate method. Food Additives and Contaminants, 12: 77- 82.
Kolosova, A. Y.; Samsonova, J. V.; Blintosov, A. and Egorov, A. M. (1989). Enzyme linked immunosorbent assay of antibiotics and principle of its application in medicine and for food control. Moscow: University of Vorobigovygory.
Komorowski, E. S. and Early, R. (1992). Liquid milk and cream. In: R. Early, (ed.).The Technology of Dairy Products, VCH Publishers Inc. New York, USA, pp. 1-23.
Kon, S. K. (1972). Milk and milk products in human nutrition. FAO nutritional studies, No. 27, FAO, Rome.
Kordylas, M. (1991). Processing and preservation of tropical and subtropical foods. Educational Low Priced Book. Hong Kong pp. 309.
Kvoger, M.; Kurman, J. A. and Rasic, J. L. (1989). Fermented milk based and soy milk based yoghurt. J. Food Sci., 55 (2): 502-536.
Lim, Y. S.; Nagan, C. C. and Tay, L. (1992). Enteropathogenic E. coli as occause of diarrhea among children in Singapore, J. Trop. Med. Hyg., 95 (15): 339-342.
Lubis, D. (1983). Effect of concentration and time of addition of hydrogen peroxide on the keeping quality of milk at room temperature. D S A, 45 (7): 5171.
Ma, Y.; Ryan, C.; Barbano, D. M.; Galton, D. M.; Ruden, M. A. and Boor, K. J. (2000). Effects of somatic cell count on quality and shelf-life of pasteurized fluid milk. J. Dairy Sci., 83: 264-274.
Madigan, M. T.; Martiko, J. M. and Parker, J. M. (2000). Antibiotic sensitivity. J. Biology of Microorganisms, 44: 3249-3256.
Makovec, J. A. and Ruegg P. L. (2003). Antimicrobial resistance of bacteria isolated from dairy cow milk samples submitted for bacteria culture: 8.905 samples (1994-2001). Am. Vet. Med. Assoc., 222: 1582-1589.
110
Manie, T.; Brozel, V. S.; Veith, W. J. and Gouws, P. A. (1999). Antimicrobial resistance of bacterial flora associated with bovine products in South Africa. J. Food Protection, 62: 615-618.
Marshall, R. T. (1992). Standard methods for the examination of dairy products, 16th ed., Public Health Assoc., Inc., Washington DC, USA.
Mitchell, J. M.; Griffiths, M. W.; McEwen, S. A., Me Nab, W. B. and Yee, A. J. (1998). Antimicrobial drug residues in milk and meat: cause, prevalence, regulations, test and performance. J. of Food Protection, 61 (6): 741-756.
Mitchell, M.; Bodkin, B. and Martin J. (1995). Detection of beta lactams antibiotic in bulk tank milk. J. Food Protection, 58: 577-578.
Moats. W. A. (1999). The effect of processing on veterinary residues in foods. In impact of processing on food safety, edited by Jackson et al., Plenum Pub. New York, 1999, pp. 233-241.
Mocquot, G. and Ducluzeau, R. (1968). In “low temperature biology of food stuffs” Hawthorn, J. and Rolfe, E. J. (eds.), pp. 235-250.
Mohamed, H. H. (1988). Quality of raw milk offered for sale in Khartoum. M.Sc. Thesis, University of Khartoum, Sudan.
Mohamed, Ibtisam, E.; Mohamed, E.G. and El Owni, O.A.O. (1993). Study on incidence and etiological agent of mastitis of Friesian cattle in Sudan. Proceedings of the Second Scientific Conference, Assuit, Egypt.
Mohamed, Maha, S. D. (2000). Effect of heat intensity and duration on the chemical composition and microbial quality of milk. M. Sc. Thesis, University of Khartoum, Sudan.
Mohamed, Nahid, N. I. (2004). Evaluation of the quality of milk sold in Khartoum State. M. Sc. Thesis, University of Khartoum, Sudan.
Murphy, S. C. and Boor, K. J. (2000). Trouble shooting sources and causes of high bacteria counts in raw milk. Dairy Food and Environmental Sanitation, 20 (8): 606-611.
Mustafa, Soad, E. (1994). The economics of milk production in Khartoum State. M.Sc. Thesis, University of Khartoum, Sudan.
111
Myllyniemi, A. L.; Rintula, R.; Niemi, A. and Backman, C. (1999). Microbiological identification of antimicrobial drugs in kidney and muscle samples of bovine cattle and pigs. J. of Food Additives and Contaminants, 16 (8): 339-351.
New-lander, J. A. and Atherton, H. V. (1984). The chemistry and testing of dairy product, 3rd ed. Olsen Publishing Co. Milwaukee, Wisconsin. USA.
Norman, H. D.; Miler, R.H. H.; Wright, J. R. and Wigganas, C. R. (2000). Herd and state means for somatic cell count from dairy herd improvement. J. Dairy Sci., 83: 2782-2788.
Odoi, A. (2003). Effectiveness and affordability of hydrogen peroxide in milk preservation under tropical conditions. Milchwisenschaft, 58: 1-2, 65-67.
Olanoet, A.; Reguena, T.; Juarez, M. and Fuente, M. A. (1998). Salt balance and rennt clotting properties of cows, ewes, and goots milks preserved with carbon dioxide. J. of Food Protection, 61 (1): 66-72.
Olukoya, D. K.; Daini, D. A. and Niemogha, M. T. (1993). Preliminary epidemiological studies on tetracycline resistant plasmids isolated from enteric bacteria in Nigeria. Trop. Gerg. Med., 43 (3): 117-120.
Ormerod, A. D.; Reid, T. M. and. Main, R. A. (1987). Penicillin in milk and its importance in urticaria. Clinical Allergy, 17: 229-234.
Park, K. (1997). Park textbook of preventive and social medicine, 15th ed. India: M/S manarsidas.
Payne, A. J. W. (1990). Introduction to animal husbandry in the tropic and sub tropical, 24th edition Longman, Singapore, p. 758.
Rechcigl, M. (1983). Handbook of natural occurring food toxicants. Florida: CRC Press, Inc.
Robinson, R. K. (1981). Milk and milk proceeding In: Dairy Microbiology. Vol. (1): Elsevier Applied Sconce London and New York.
Rodrigues, A. C.; Caraviell, O. Z. and Ruegg, P. L. (2004). Financial losses and management practices associated with BTSCC. J. Dairy Sci. (Suppl), 87: 375.
112
Roundy, Z. D. (1958). Treatment of milk for cheese with H2O2. J. Dairy Sci., 41: 1460-1476.
Ruas, P.; Reys, C. G.; Olano, A. and Villamiel, M. (2000). Influence of refrigeration and carbon dioxide addition to raw milk on microbial levels, free monsacharides and myoinositol content of raw and pasteurized milk. European Food Research and Technology, 212 (1): 44-47.
Rushing, J. E. (1994). Preventing antibiotic residues in milk. Food Safety, 95 (2): 1-95.
Saha, B. K.; Ali, M. X.; Chakraorty, M.; Islams, Z. and Hira, A. K. (2003). Study on the preservation of raw milk with hydrogen peroxide for rural dairy farmer. Pakistan. J. of Nut., 2 (1): 39- 42.
Sargean, J. M.; Schukken, Y. H. and Leslie, K. E. (1998). Ontario milk somatic cell count reduction progress and outlook. J. Dairy Sci., 81: 1545-1554.
Sartwell, P. E. (1977). Preventive medicine and public health. 10th edition, pp 1007-1021.
Seymour, E. H.; Jones, G. M. and Megilliard, M. L. (1988). Persistence of residues in milk following antibiotic treatment of cattle. J. Dairy Sci., 69: 292- 296.
Shitandi, A. and Sternesjo, O. (2004). Factors contributing to the occurrence of antimicrobial drug residues in Kenyan milk. J. Food Protection, 67: 399-402.
Sid Ahmed, A. M. E. (2006). Assessment of microbial load and antibiotic residues in milk supply in Khartoum State. M. Sc. Thesis, University of Khartoum, Sudan.
Singh, K. B. and Boxi, K. K. (1982). Studies on the actiology in vitro sensitivity and treatment of subclinical mastitis in milch animals. Indian Veterinary Journal, 59: 191-198.
Snedaecor, G. W. and Cochran, N. G. (1980). Statistical methods, 7th Ames. Lowa. The Lowa State, University Press. US A.
Sudanese Laws Commission (1975). Laws of the Sudan. P. 546.
Tajelsir, S.M. (2001) Antibiotic residues in raw cow milk at Khartoum State. M. Sc. Thesis, University of Khartoum, Sudan.
113
Talley, M. R. (1999). The national milk safety program and drug residues in milk. Food Anim. Practice, 15: 63-73.
Tanwani, S. K. and Yodava, R. (1983). Diseases of man communicable through milk. Livestock Advisor, 8: 39-42.
Teuber, M. and Perreten, V. (2000). Role of milk and meat products as vehicles for antibiotic resistant bacteria. Acta Vet. Scand. (Suppl.), 93: 75-87.
Tikofsky, L. L.; Barlow, J. W.; Santisteban, C. and Schukken, Y. H. (2003). A comparison of antimicrobial susceptibility patterns for Staphylococcus aureus in organics and conventional dairy herds. Micro Drug Resistance Mech. Epi. and Disease, 9 (Suppl.): 539-545.
Titini, R. S.; ElHabaz, A. and Griffiths, M. (1991). Rapid detection of psychotrophic bacteria in manufacturing grade of raw milk. J. Food Protection, 54: 861-867.
Tortra, G. J.; Funk, B. R. and Case, C. L. (1997). Microbiology. An introduction, 6th ed. Benjomin/ Cummings Publishing company. Colifornia, USA.
Vaara, G.S. (1995). Outer membrane permeability barrier to a zithromycin, clarithromycine, and roxithromycin in Gram– negative enteric bacteria. Antimicrob. Agent’s Chemother, 37 (2): 354-356.
Van schaik, G. M. (2002). Trends in somatic cell counts, bacterial counts and antibiotic residues violations in New York State during 1999-2000, J. Dairy Sci., 85: 782-798.
Verheijen, R.; Stouten, P.; Cazemier, G. and Hassnoot, W. (1998). Development of one step strip test for the detection of sulphadimidine residues. Analyst, 123 (12): 2437-2441.
Vilgalys, R. (2001). Method to determine antibiotic sensitivity. J. Microbiology, 103 (18): 25-28.
Vollhardt, K P.C and Schore, N. E. (1998). Chemical preservative for additive. Thorson publisher limited. third edition.
Walstra, P. R. (2002). Dairy chemistry and physics, John Wiley Sons, New York.
114
Webb, B. B.; Johnson, A. H. and Alford, J. A. (1983). Fundamentals of dairy chemistry. The AVI publishing company, West Port, Conneneticut, USA.
Webb, B. H.; Johnson, R. H. and Alford, J. A. (1980). Fundamentals of dairy chemistry. Second edition, third printing, the Avi Publishing Company, West port, Connect cute U. S. A
Wicher, K.; Reisman, R. E. and Arbesman, C. E. (1969). Allergic reaction to penicillin present in milk. Am. Vet Med. Assoc., 208: 143-145.
Wilson, G. S. (1935). The bacteriological grading of milk. Medical research council special report series, No. 206. London: H.M.S.O.
Yagoub, S. O.; Awadlla, N. E. and El Zubeir, Ibtisam, E. M. (2005). Incidence of some potential pathogens in raw milk in Khartoum North (Sudan) and their susceptibility to antimicrobial agents. Journal of Animal and Veterinary Advances, 4 (3): 341-344.