AEROBIC BACTERIA ASSOCIATED WITH FRESH

AND PROCESSED OF CAMEL’S MEAT

By

Hanadi Ahmed Sayed Hussien B.V. Sc. University of Khartoum

A thesis submitted as the Requirements of Master Degree of Public and Environmental Health (MPEH) in Food Hygiene and Safety

Supervisor: Dr. Tawfig ElTigani Mohamed

Department of Food Hygiene and Safety, Faculty of Public and Environmental Health, University of Khartoum

2005 ﺒﺴﻡ ﺍﻟﻠﺔ ﺍﻟﺭﺤﻤﻥ ﺍﻟﺭﺤﻴﻡ

ﻗﺎل ﺘﻌﺎﻟﻰ: ♦ ﺃَﻓﹶﻠﹶﺎ ﻴ ﻨ ﻅﹸ ﺭ ﻭ ﻥ ﺇِﻟﹶﻰ ﺍ ﻟﹾ ﺈِ ﺒِ لِ ﻜﹶ ﻴ ﻑﹶ ﺨﹸ ﻠِ ﻘﹶ ﺕﹾ ♥

ﺼﺩﻕ ﺍﻟﻠﺔ ﺍﻟﻌﻅﻴﻡ (ﺴﻭﺭﺓ ﺍﻟﻐﺎﺸﻴﺔ ﺍﻻﻴﺔ17 ) ﻗﺎل ﺘﻌﺎﻟﻰ: ﻭﻟﹶﻜﹸـﻡ ﻓِﻴﻬـﺎ ♦5♥" ﻭ ﺍ ﻷَ ﻨﹾ ﻌ ﺎ ﻡ ﺨﹶﻠﹶﻘﹶﻬﺎ ﻟﹶ ﻜﹸ ﻡ ﻓِﻴﻬﺎ ﺩِ ﻑﹾ ﺀ ﻭ ﻤ ﻨﹶ ﺎ ﻓِ ﻊ ﻭﻤِﻨﹾﻬﺎ ﺘﹶ ﺄْ ﻜﹸ ﻠﹸ ﻭ ﻥ ﻭ ﺘﹶ ﺤ ﻤِ لُ ﺃَﺜﹾﻘﹶﺎﻟﹶﻜﹸﻡ ﺇِﻟﹶﻰ ﺒ ﻠﹶ ﺩٍ ﻟﱠ ﻡ ﺘﹶ ﻜﹸ ﻭ ﻨﹸ ﻭ ﺍﹾ ♦6♥ ﺠ ﻤ ﺎ لٌ ﺤِ ﻴ ﻥ ﺘﹸ ﺭِ ﻴ ﺤ ﻭ ﻥ ﻭ ﺤِ ﻴ ﻥ ﺘﹶ ﺴ ﺭ ﺤ ﻭ ﻥ ♦7♥ ﺒ ﺎ ﻟِ ﻐِ ﻴ ﻪِ ﺇِ ﻻﱠ ﺒِ ﺸِ ﻕﱢ ﺍ ﻷَ ﻨ ﻔﹸ ﺱِ ﺇِ ﻥ ﺭ ﺒ ﻜﹸ ﻡ ﻟﹶ ﺭ ﺅُ ﻭ ﻑﹲ ﺭ ﺤِ ﻴ ﻡ ﺼﺩﻕ ﺍﷲ ﺍﻟﻌﻅﻴﻡ (ﺴﻭﺭﺓ ﺍﻟﻨﺤل ﺍﻵﻴﺎﺕ 5 ، 6 ،7)

This work was carried out at the Department of Food

Hygiene and Safety, Faculty of Public and Environmental

Health, University of Khartoum, under the supervision of

Dr.Tawfig Eltigani " Department of Preventive Medicine and Veterinary Public Health, Faculty of Veterinary Science,

University of Khartoum.

To my family and to all my friends with my deeply love

First, thank to Allah for giving me the health and strength to complete this work.

My deepest gratitude to my supervisor Dr. Tawfig

ElTigani Mohamed, for his guidance, encouragement and help which made this work possible.

Also thanks are extended to any person helped me.

In conclusion, I would like to pay tribute to my colleagues for their help and Tahani Hassan for typing this thesis.

CONTENTS

Subject Page

Preface ------ii

Dedication ------iii

Acknowledgement------iv

Contents------v

List of Tables------xii

List of figures ------xiv

Abstract (English) ------xv

Abstract (Arabic) ------xvii

Introduction ------1

Chapter one: Literature Review

1. The camel------4

1.1 The distribution of dromedary------4

1.2 Population, Geographical location & distribution in

Sudan------4

1.3 Camel pasture ------8 1.4 Camel breeds ------9

1.5 Importance of the camel ------10

1.5.1 Meat & milk production ------11

1.5.1.1 Meat production ------11

1.5.1.2 Milk production------13

1.6 Diseases of camels ------16

1.6.1 Bacterial diseases of camels include ------17

1.6.1.1 Brucellosis ------17

1.6.1.2 Salmonellosis------18

1.6.1.3 Clostridial infection and

Enterotexaemia ------18

1.6.1.4 Pneumonia in camels ------18

1.6.1.5 Anthrax------21

1.6.1.6 Tuberculosis ------21

1.6.1.7paratuberculosis------22

1.6.1.8 Contagious skin necrosis------22

1.6.1.9 Leptospirosis------22

1.6.1.10 Mastitis------22 1.7 Contamination of meat ------23

1.8 Growth of microorganisms in meat------25

1.9 Food poisoning------26

1.10 Microbiological studies of camel's meat ------26

1.11 Contamination of processed meat ------28

1.12 Hazard Analysis Critical Control Point (HACCP)

concept in meat production ------29

1.13 effect of temperature on meat------32

1.14 Antibiotics sensitivity tests------33

Chapter two: Material and methods

1. Samples for bacteriological examination------36

1.1 Sources of samples------36

1.2 Collection of samples------36

1.3 Distribution of samples------37

2. Processing of camel's meat (sausage)------39

2.1 The steps of sampling------39 2.2 Processing of sausage------39

3. Sterilization------39

3.1 Sterilization by heat------40

3.1.1 Sterilization by moist heat ------40

3.1.2 Sterilization by hat air oven ------40

3.1.3 Sterilization by flaming ------40

4. Disinfections ------39

5. Preparation of media ------40

5.1 Solid media------40

5.1.1 Blood Agar base ------40

5.1.2 MacConkey agar ------41

5.1.3 Nutrient agar------42

5.1.4 Urea Agar base (Oxoid)------42

5.1.5 Salmonella shigella agar (Difco)------43

5.1.6 Dnase agar ------43

5.1.7 Eosin methylene blue (EMB)(oxoid)------43

5.1.8 Brillent Green agar------43

5.1.9 Aesculin agar medium------44 5.1.10 Diagnostic Sensitivity Test (DST) ------44

5.1.11 Edwards media------45

5.1.12 Manitol salt agar------45

5.2 Semi solid media ------45

5.2.1 Nutrient gelatin ------45

5.2.2 Hugh and leifson's (O/F ) medium------45

5.2.3 Motility medium. ------46

5.3 Liquid media------47

5.3.1 Peptone water------47

5.3.2 Carbohydrate fermentation medium------47

5.3.3 Koser liquid citrate ------47

5.3.4 MR-VP test medium------48

5.3.5 Nutrient broth ------38

5.3.6 Selenite broth ------48

5.1.7 Nitrate broth ------49

6. Reagents ------49

6.1 Hydrogen peroxide------49 6.2 Tetramethyl-P.phenylene diamine dihydro chloride ------49

6.3 Kovac's reagent------50

6.4 Voges Proskauer test reagent (KOH)------50

6.5 Methyl red solution ------50

6.6 Nitrate reagent ------50

7. indicators ------51

7.1 Andraders indicator ------51

7.2 Bromothymal blue ------51

7.3 Lead acetate papers ------51

8. Preparation of material for bacteriological examination ---- 51

8.1 Preparation of the samples ------51

9. Cultural methods------52

9.1 primary isolation ------52

9.1.1 Solid media ------52

9.1.2 Broth media------52

9.2 Sub culturing of primary isolates ------52

9.2.1 From solid to solid media------52 9.2.2 From broth to solid ------52

10. Incubation of cultures ------52

11. Examination of culture ------53

11.1 Motility------53

11.2 Purification of cultures------53

12. Staining------53

12.1 Gram's stain------53

12.2 Reagents ------54

12.2.1 Amonium oxalate- Crystal violet------54

12.2.2 Carbol fuchasins------54

12.2.3 Lugal' iodine ------54

12.3 Staining technique ------55

13. Identification of bacteria ------55

14 Biochemical methods for identification of isolated bacteria------56

14.1 Potassium hydroxide test ------56

14.2 Gelatin hydrolysis ------56

14.3 Indole production ------56 14.4 Urease activity ------57

14.5 Aesculin hydrolysis------57

14.6 Oxidation and fermentation ------57

14.7 Nitrate reduction ------57

14.8 Coagulate test ------58

14.9 Oxidase test------58

14.10 Catalase test------58

14.11 Dnase test------59

14.12 Acid from carbohydrate ------59

14.13 Voges Proskauer (VP) ------59

14.14 Citrate utilization ------60

14.15 Hydrogen sulphide (H2S production)------60

14.16 Methyl Red------60

14.17 Antibiotic sensitivity tests------60

Chapter three: Results

A. Bacterial isolation------62

I- From samples collected from Elbugaa slaughter house ------62 II- From samples collected from ElKadaro slaughter house------70

B. Bacterial isolation of camels' meat processing------77

C. Antibiotics sensitivity tests ------86

Chapter Four: Discussion ------91

Conclusion------105

Recommendation ------105

References ------108

LIST OF TABLES

Tables Page

Table 1. Estimated numbers of camel in Eastern Africa. 6

Table 2. Numbers of domestic ruminants and camels in the world (1989) in million head. 6

Table 3. The number of livestock in the Sudan. 7

Table 4 . Livestock distribution by state. 7

Table 5 . Meat Production & consumption in the Sudan1. 13

Table 6 . Milk production and consumption in the Sudan. 15

Table 7. Samples collected and their localities. 38

Table 8 . Bacterial groups isolates from samples collected from Elbugaa slaughter house. 64

Table 9 . Distribution of isolated bacteria genera among different types of samples 65

Table 10 shows. Distribution of isolates bacteria genera among different types of samples. 67

Table 11. Distribution of the gram-negative organisms among different samples collected from Elbugaa. 69 Table 12 . bacterial groups isolates from samples collected from ElKadaro slaughter house. 72

Table (13 a,) Distribution of isolated bacteria genera among different type of samples. 73

Table (13,b) Distribution of isolated bacteria genera among different type of samples. 74

Table 14 . distribution of the Gram-positive organisms among different samples collected from Elkadaro. 75

Table 15. Distribution of the Gram-negative organisms among different samples collected from ElKadaro. 76

Table 16 . Bacterial groups isolated from different stages of processing of camel's meat. 79

Table 17. Percentage of different types of isolates in the total No. of samples. 80

Table (18.a) .Distribution of isolated bacteria genera among different types of samples gram-positive. 81

Table (18.,b) .Distribution of isolated bacteria genera among different types of samples gram-positive. 82 Table 19. Distribution of the gram-positive among different samples 83

Table 20. Distribution of the Gram-negative isolates organism among different samples. 84

Table 21 . The effect of refrigeration at 4 °C for 5 days on isolates. 85

Table 22 . Antibiotics sensitivity tests to some Gram- positive bacteria. 87

Table 23. Antibiotics sensitivity test to some Gram-negative bacteria. 88

LIST OF FIGURES

NO Page

1. Diagram showing sources of contamination with bacteria and critical control points (CCP) in red meat production 31

2. ß "clear" haemonlysis of staphylococcus aureus on

Blood Agar medium after 24 hours incubation at 37°C 89

3. Alpha heamolysis of streptococcus pneumoniae on

Blood Agar medium after 24 hours incubation at 37 °C 89

4. Antibiotic sensitivity test to corynebacterium pseudotuberculasis cultured on nutrient agar medium after 24 hours incubation at 37 °C 90

This study was carried out to determine the aerobic bacteria associated with camel's meat. The samples collected from different stages of camel slaughtered at Elbugaa slaughter house (Omdurman) for local consumption and

Elkadaro slaughter house (Kh. North ) for export. Also samples were collected from workers , knives and equipments which were used to cut Carcass (saw and cutter machine).

A total of 157 samples collected from both areas comprised of 20 thigh muscles, 20 back muscles, 20 shoulder muscles, 20 swabs from outer skin, 20 swabs from internal cavity, 20 from surfaces of carcasses, 13 swabs from workers' hands, 6 swabs from saws, 15 swabs from knives and one swab from carcass cutter machine in Elkadaro slaughter house. All these samples were cultured for the isolation of aerobic bacteria.

Gram positive bacteria (373 isolates) and Gram- negative bacteria (181 isolates) were isolated from different samples collected from both areas.

Also other samples were collected from different sites for sausage processing and from workers, spices and machines at Regional training Center for Meat Inspection, hygiene and grading.

All these samples were examined to determine aerobic bacteria. The isolates were comprised of Gram-positive and

Gram-negative isolates. Gram-positive isolated were

Staphyllococcus, Corynebacterium, Streptococcus,

Micrococcus, Bacillus, and Gram-negative isolated were

E.coli, Pseudomonas proteus , Citrobacter, Edwardsiella tarda, Acinetobacter, and Klebsiella. The spices, machines and workers during processing were found to be sources of contamination of the meat during processing.

Also the end product of the processed meat was stored at

4°C for 5 days and reexamined . and then found that micrococcus , Streptococcus and Pseudomonas were increased and Corynebacterium, E.coli were decreased after storage but Staphylococcus and Bacillus were still constant

Edwardseilla tarda was disappeared after cold storage.

Different types of antibiotics were used for sensitivity tests of bacteria isolated from fresh meat as well as from processed meat. All Gram-positive and Gram-negative were found to be sensitivity to Gentamycin and Ampicillin. Gram- positive bacteria were found to be highly sensitive to

Tetracycline.

ﺃﺠﺭﻴﺕ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ﻟﺘﺤﺩﻴﺩ ﺍﻟﺒﻜﺘﺭﻴﺎ ﺍﻟﻬﻭﺍﺌﻴﺔ ﺍﻟﻤﺭﺘﺒﻁﺔ ﺒﻠﺤﻭﻡ ﺍﻹﺒل ﻤﻥ ﺨﻼل ﺍﻟﻤﺭﺍﺤل ﺍﻟﻤﺨﺘﻠﻔﺔ ﻟﺫﺒﺢ ﺍﻹﺒل ﺒﻜل ﻤﻥ ﻤﺴﻠﺦ ﺍﻟﺒﻘﻌﺔ ﺒﺄﻤـﺩﺭﻤﺎﻥ ﻭﻤـﺴﻠﺦ ﺍﻟﻜﺩﺭﻭ ﻟﻠﺼﺎﺩﺭﺍﺕ ﺒﺎﻟﺨﺭﻁﻭﻡ ﺒﺤﺭﻱ . ﻭﻜﺫﻟﻙ ﻤﻥ ﺍﻴﺩﻱ ﺍﻟﻌـﺎﻤﻠﻴﻥ ﻭﺍﻟـﺴﻜﺎﻜﻴﻥ ﻭﺍﻟﻤﻌﺩﺍﺕ ﺍﻟﻤﺴﺘﻌﻤﻠﻪ ﻟﻘﻁﻊ ﺍﻟﺫﺒﺎﺌﺢ " ﺍﻟﺴﺎﻁﻭﺭ ﻭﺍﻟﻤﻨﺸﺎﺭ" . ﻭﻗﺩ ﺘﻡ ﺠﻤﻊ 157 ﻋﻴﻨﺔ ﻤﻥ ﺍﻟﻤﺴﻠﺨﻴﻥ ﻭﻗﺩ ﺍﺸﺘﻤﻠﺕ ﻋﻠﻲ 20 ﻋﻴﻨﻪ ﻤـﻥ ﻋﻀﻠﺔ ﺍﻟﻔﺨﺫ ، 20 ﻤﻥ ﻋﻀﻠﺔ ﺍﻟﻅﻬﺭ ، 20 ﻤﻥ ﻋﻀﻠﺔ ﺍﻟﻜﺘﻑ ﻜﻤﺎ ﺘﻡ ﺍﺨﺫ 20 ﻤﺴﺤﺔ ﻤﻥ ﺍﻟﺠﻠﺩ ﺍﻟﺨﺎﺭﺠﻲ ﻗﺒل ﺍﻟﺫﺒﺢ ﻭ 20 ﻤﺴﺤﺔ ﻤﻥ ﺍﻟﺴﻁﺢ ﺍﻟﺨﺎﺭﺠﻲ ﻟﻠﺫﺒﻴﺤﻪ ﻭ20 ﻤﺴﺤﺔ ﻤﻥ ﺍﻟﺘﺠﻭﻴﻑ ﺍﻟﺩﺍﺨﻠﻲ ﻭ 15 ﻤﺴﺤ ﺔ ﻤﻥ ﺍﻴﺩﻱ ﺍﻟﻌـﺎﻤﻠﻴﻥ ﻭ 15 ﻤـﻥ ﺍﻟﺴﻜﺎﻜﻴﻥ ﺍﻟﻤﺴﺘﻌﻤﻠﺔ ﻭ 6 ﻤﺴﺤﺎﺕ ﻤﻥ ﺍﻟﺴﺎﻁﻭﺭ ﺍﻟﻤﺴﺘﻌﻤل ﻓـﻲ ﻤـﺴﻠﺦ ﺍﻟﺒﻘﻌـﻪ ﻭﻋﻴﻨﻪ ﻭﺍﺤﺩﺓ ﻤﻥ ﺍﻟﻤﻨﺸﺎﺭ ﺍﻟﻤﺴﺘﻌﻤل ﺒﻤﺴﻠﺦ ﺍﻟﻜﺩﺭﻭ ﻭﻜل ﻫـﺫﻩ ﺍﻟﻌﻴﻨـﺎﺕ ﺘـﻡ ﺯﺭﻋﻬﺎ ﻟﻌﺯل ﺍﻟﺒﻜﺘﺭﻴﺎ ﺍﻟﻬﻭﺍﺌﻴﺔ. ﻭﻗﺩ ﺒﻠﻎ ﻋﺩﺩ ﺍﻟﻌﺘﺭﺍﺕ ﺍﻟﻤﻭﺠﺒﺔ 373 ﻋﺘﺭﻩ ﻭﻋﺩﺩ ﺍﻟﻌﺘﺭﺍﺕ ﺍﻟﺴﺎﻟﺒﺔ 181 ﻋﺘﺭﺓ ﺴﺎﻟﺒﻪ ﺘﻡ ﻋﺯﻟﻬﺎ ﻤﻥ ﺍﻟﻌﻴﻨﺎﺕ ﺍﻟﻤﺨﺘﻠﻔﺔ ﺍﻟﺘﻲ ﺘﻡ ﺠﻤﻌﻬﺎ ﻤﻥ ﺍﻟﻤﻨﻁﻘﺘﻴﻥ. ﻜﻤﺎ ﺘﻡ ﺠﻤﻊ ﻋﻴﻨﺎﺕ ﻤﻥ ﺍﻟﻤﺭﺍﺤل ﺍﻟﻤﺨﺘﻠﻔﺔ ﻟﺘﺼﻨﻴﻊ ﺍﻟﺴﺠﻙ ﺍﻟﻤﺼﻨﻊ ﻤـﻥ ﻟﺤﻭﻡ ﺍﻻﺒل ﻭﻜﺫﻟﻙ ﻤﻥ ﺍﻴﺩﻱ ﺍﻟﻌﺎﻤﻠﻴﻥ ﻭﺍﻟﺒﻬﺎﺭﺍﺕ ﺍﻟﻤﻀﺎﻓﺔ ﻭﺍﻟﻤﺎﻜﻴﻨﺎﺕ ﺍﻟﻤﺴﺘﻌﻤﻠﺔ ﺒﻤﻌﻬﺩ ﺘﺼﻨﻴﻑ ﺍﻟﻠﺤﻭﻡ ﺒﺎﻟﻜﺩﺭﻭ . ﻜل ﻫﺫﻩ ﺍﻟﻌﻴﻨﺎﺕ ﺘﻡ ﻓﺤﺼﻬﺎ ﻟﺘﺤﺩﻴـﺩ ﺍﻟﺒﻜﺘﺭﻴـﺎ ﺍﻟﻬﻭﺍﺌﻴﺔ. ﻭﻗﺩ ﺍﺸﺘﻤﻠﺕ ﺍﻟﺒﻜﺘﺭﻴﺎ ﺍﻟﻤﻌﺯﻭﻟﺔ ﻋﻠﻲ ﺒﻜﺘﺭﻴﺎ ﻫﻭﺍﺌﻴﺔ ﻤﻭﺠﺒﺔ ﻟﺼﺒﻐﺔ ﺠﺭﺍﻡ ﻭﺍﺨﺭﻱ ﺴﺎﻟﺒﺔ ﻭﺍﺸﺘﻤﻠﺕ ﺍﻟﻤﻭﺠﺒﺔ ﻋﻠﻲ Staphylococcus , Bacillus, Micrococcus, Streptococcus, , Corynebaterium ﻭﺍﺸﺘﻤﻠﺕ ﺍﻟﺴﺎﻟﺒﻪ ﻋﻠـﻲ , Pseudomonas ,Ecoli Edwardsiella Tarda, Citrobacter, Kilebsiella, Acinetobacter

ﻟﻘﺩ ﻭﺠﺩﺕ ﺍﻟﺒﻬﺎﺭﺍﺕ ﺍﻟﻤﻀﺎﻓﺔ ﻭﺍﻴﺩﻱ ﺍﻟﻌﺎﻤﻠﻴﻥ ﻭﺍﻟﻤﺎﻜﻴﻨﺎﺕ ﺍﻟﻤـﺴﺘﻌﻤﻠﺔ ﺍﺜﻨﺎﺀ ﺍﻟﺘﺼﻨﻴﻊ ﻤﺼﺩﺭ ﻟﺘﻠﻭﺙ ﺍﻟﻠﺤﻭﻡ ﺍﺜﻨﺎﺀ ﺘﺼﻨﻴﻌﻬﺎ . ﻜﻤﺎ ﺘﻡ ﺤﻔﻅ ﺍﻟﻨﺎﺘﺞ ﺍﻟﻨﻬﺎﺌﻲ ﻟﻠﺤﻭﻡ ﺍﻟﻤﺼﻨﻌﺔ ﻓﻲ ﺩ ﺭﺠﺔ 4°ﻡ ﻟﻤﺩﺓ 5 ﺃﻴﺎﻡ ﻭﺘﻡ ﺇﻋﺎﺩﺓ ﻓﺤـﺼﻬﺎ ﻭﺘـﻡ ﻋـﺯل ﺍﻟﺒﻜﺘﺭﻴــﺎ ﻤــﺭﺓ ﺃﺨــﺭﻱ ﻭﻭﺠــﺩ ﺃﻥ ﺍﻟـــ , Streptococuus ﻭ ﺍﻟـــ Pseudomonas ﻗــﺩ ﺯﺍﺩ ﻤﻌــﺩﻟﻬﺎ ﺒﻌــﺩ ﺍﻟﺘﺒﺭﻴــﺩ ﺒﻨــﺴﺒﺔ ﻀــﺌﻴﻠﺔ ﻭﺍﻟـCorynebacerium, E.cloi ﻗﺩ ﻨﻘﺹ ﻤﻌﺩﻟﻬﺎ ﺒﻌـﺩ ﺍﻟﺘﺨـﺯﻴﻥ ﺍﻟﻤﺒـﺭﺩ ﻭﺍﻟـــStaphyllococcus ﻭﺍﻟـــBacillus ﻅﻠــﺕ ﻜﻤــﺎ ﻫــﻲ ﺍﻤــﺎ ﺍﻟـEdwardsiella tarda ﻗﺩ ﺍﺨﺘﻔﺕ ﺘﻤﺎﻤﺎ ﺒﻌﺩ ﺍﻟﺘﺨﺯﻴﻥ ﺍﻟﻤﺒﺭﺩ. ﻜﻤﺎ ﺍﺠﺭﻱ ﺍﺨﺘﺒﺎﺭ ﺍﻟﺤﺴﺎﺴﻴﺔ ﻟﺒﻌﺽ ﺍﻨﻭﺍﻉ ﺍﻟﻤﻀﺎﺩﺍﺕ ﺍﻟﺤﻴﻭﻴـﺔ ﻋﻠـﻲ ﺍﻟﻌﻴﻨﺎﺕ ﺍﻟﻤﻌﺯﻭﻟﺔ ﻤﻥ ﺍﻟﻠﺤﻭﻡ ﺍﻟﻁﺎﺯﺠﺔ ﺍﻟﻤﺴﺘﻌﻤﻠﺔ ﻓﻲ ﺍﻟﺘﺼﻨﻴﻊ ﻭﻭﺠﺩ ﺍﻥ ﺠﻤﻴﻊ ﺍﻟﻤﻴﻜﻭﺭﺒﺎﺕ ﺍﻟﻤﻭﺠﺒﺔ ﺍﻟﺴﺎﻟﺒﺔ ﺍﻟﺘﻲ ﺘﻡ ﺍﺨﺘﺒﺎﺭﻫﺎ ﺤﺴﺎﺴﺔ ﻟﻜل ﻤـﻥ ﺍﻟﺠﻨﺘﺎﻤﻴـﺴﻴﻥ ﻭﺍﻻﻤﺒﺴﻠﻴﻥ ﻭﻭﺠﺩ ﺍﻥ ﺍﻟﺒﻜﺘﺭﻴﺎ ﺍﻟﻤﻭﺠﺒﺔ ﻟﺼﺒﻐﺔ ﺠـﺭﺍﻡ ﺫﺍﺕ ﺤـﺴﺎﺴﻴﺔ ﻋﺎﻟﻴـﺔ ﻟﻠﺘﺭﺍﺴﻴﻜﻠﻴﻥ . INTRODUCTION

The camel population in the world is estimated to be 19 million.

Sudan and Somalia contain about 70% of Africans camels and 55% of that of the world. Sudan ranks second after Somalia in numbers. (AbdelMajed,

1999).

Camels are playing an important role in the gross national product

(GNP) of the Sudan, through exportation to the Arab countries as a source of meat (Egypt) and for camel racing (the gulf countries) (Bashir, 1992).

Babiker and Tibin in 1989 explained that the ability of the camel to survive in semiarid condition makes it an important source of food in drought areas of the world where famine is endemic to over 20 million people each year.

Skar and AbdelMajed, (1998) concluded in their study that, camels are more important than the other livestock species, and there fore it is necessary to give special attention to this animal which can produce meat and milk under harsh desert conditions for a large section of the society.

Camel production is known to be facing many problems and camel habitats are stricken by severe and prolonged droughts which force pastoralists and their camels to migrate to new areas where they face other problems. However camels' numbers are increasing in many areas due to the camel adaptation and economic traits which attract the attention of new investors and concerned organizations (Wardeh, 1992). Recently camel production has attracted the attention of regional and international organization. The Arab Center for the studies of Arid zones and Dry land (ACSAD) has paid much attention to developing camel production as one of the major elements of food security in desert and dry ecosystems. So it has been conducting studies in the field of camel production since the early 1980 and has established the camel Applied research and Development Net work (CARDN) with a financial support from International Fund for Agricultural Development (IFAD) and a technical assistance from French government (ACSAD, 2003).

Bacterial Flora responsible for meat spoilage and illness due to consumption of camel meat need more investigation.

Jay, 1986 explained that, for national public health safety, there should be a follow up off food product from its first step of production till the final product for hygiene, and that what is called Hazard Analysis

Critical Control points (HACCP). Introduction of Hazard Analysis Critical

Control points (HACCP) concept can be helpful to maintain high standards of slaughter and dressing hygiene based on an assessment of the risks to human and animals health (.FAO , 2004).

In Sudan, the bacteriological studies in camel meat are very few, so that they need additional research.

This study will follow up camel meat to determine the kind of bacteria at the different stages of its preparation (HACCP). This study (research) followed up the following objectives:

• To investigate and identify aerobic bacteria associated with camel

meat in order to add more knowledge in this area of food hygiene.

• Camel meat prepared for consumption requires more information

about its processing and storage.

• To perform in vitro antibiotic sensitivity tests to some of the

isolated bacteria at the different stages of processing and storage.

CHAPTER ONE

LITERATURE REVIEW

1. The camel

The camelidae (order Artiodactyla, Suborder Tyloboda (pad footed) are divided into two genera.

The genus camelus (true or old- world Camels). And the genus

Lama (new- world camels). The true camels include the one humped or

Arabian camel (camelus dromedarius), commonly called the dromedary, and the two-humped or Bactraim camel (Camelus bactrianus). (Schwartz and Dioli, 1992).

1.1 Distribution of the dromedary

The dromedary camel has wide geographical distribution in the arid areas of Africa, particularly in the arid lowlands of Eastern Africa, i.e.

Somalia, Sudan, Ethiopia, Kenya and Djibouti. Approximately 11.5 million animals in this region, represent over 80% of the African and tow thirds of the world's population of camels ( table 1&2) (Schwartz,1992).

1.2 Population, Geographical location &distribution in Sudan

The camel population in the Sudan estimated to be 3 million; this makes it the second largest camel population in Africa. This represents

23.8% of camels of Africa and 17% of the world camel population (FAO,

1989). The camel is considered as an animal of the tropics but much of its present day normal range is extra-tropical. Camels are mainly distributed in the arid and semi arid parts of the Sudan which is limited to about 14 °N in the west (Darfur& Kordofan States) and about 16°N in the East (ElAmin,

1979).

Babiker, (1984) explained that the camels in the Sudan can survive in a geographical zone of 1.5 million km2, but the area inhabited by camels extend between latitudes 10 and 20 North and is bounded by the Ethiopia mountains and Red sea hills in the East and by the ingassana mountains and Bahr ElArab in the South. This large area is differentiated into two regions, Nile, the Butana in the East which is enclosed by the Atbra River,

Nile and Blue Nile River & the west comprising Darfur & Kordofan states.

Table 3. Shows the number of livestock in the Sudan. Table 4

Shows the livestock distribution by state.

Table 1: Estimated numbers of camel in Eastern Africa

COUNTRY NUMBER %

Djibouti 60,000

Ethiopia 1,030,000

Kenya 780,000

Somalia 6,350,000

Sudan 3,100,000

Source: FAO production year book 1989

Table 2: Numbers of domestic ruminants and camels in the world

(1989) in million head

SPECIES WORLD AFRICA EASTERN

(WHOLE) AFRICA

Cattle 1,264 181 72

Sheep 1,173 200 65

Goats 520 167 63

Camels 18.9 14.2 11.5

Source: FAO production year-book (1989)

Table 3: the number of livestock in the Sudan

CATTLE 31.669.000

Sheep 37.202.000

Goat 35.215.000

Camels 03.915.000

Total 108.001.000

Source: Ministry of Animal Recourses, report 1998 General ADM. Of

Quarantines and meat production& trade

Table 4: livestock distribution by state

State Cattle% Sheep% Goat% Camel%

Kordufan 15.6 20.2 21.0 36.2

Eastern 4.4 11.2 12.2 28.1

Darfur 23.8 16.2 20.5 15.2

Central 19.2 27.8 21.2 11.0

Northern 0.4 07.0 3.7 7.2

Khartoum 0.4 01.8 3.8 0.5

Southern 36.2 15.8 17.6 1.8

Total 100 100 100 100

Source: Ministry of Animal Resources, report 1998 General

ADM, of Quarantines and meats, Sudan livestock and meat production & trade. 1.3 Camel pasture

The pasture utilized by camels include semi desert and desert communities such as thorn bushes and desert shrubs. Camels feed mainly on shrubs, bushes and desert trees which represent a very nutritious and reliable forage sources (Osama, 1988).

In the Sudan, rain fall in the camels areas occurs seasonally and it varies between 100 mm and 800 mm annum in southern parts and from traces to over 110mm /annum in the North. (Babiker, 1984).

Camel owing tribes are of nomadic nature. The various tribes have different traditional camel migration routes but, the pattern is generally similar between tribes. In winter they migrate to Gizzu grazing land at the

North West of Sudan. The plant species of Gizzu belong to various families, they all have a high percentage of water content coupled with low temperature make the camels independent of free water for a long period .

During summer (March & June) they return to their homes where they spend the rainy season. (Wilson,1984).

Musa and Abbas, (1989) explained that about 41.000 people of northern Butana desert in Sudan depend on camel and sheep for survival, the vegetative cover was scarce and consisted mainly of Acacia species and

Aristida species. Droughts forced camel herders to migrate towards the agricultural areas for settlement. Small herders (20-30 camels) are setting along Nile utilizing crop residues. Large herders (50-100 camels) are moving towards New Halfa and

Rahad schemes. While medium herder (30-50camels) are continuing the transhuman traditional grazing system in the area.

1.4 Camel breeds

Camels or the families of camel, the camelidae include the one-

humped camel (Camelus Dromedarius) and the two-humped camel

(Camelus Bactrianus) are the two breeds in habitat of the arid regions of

Africa and Asia.

The dromedary is found in the warmer areas of North & East

Africa, India, Afghanistan and the southern region of USSR.

The bacterian camel is found in the arid colder regions of Asia,

adjoining the areas of the dromedary camel (Wilson, 1984).

In the Sudan, the largest population of camels is classified mainly

into riding and packing camels according to the function ( Gillispie, 1962

and Elamin,1979).

The riding camels refer to the Bashri and Anafi types. The Bashari camel is mainly owned by the Baja tribe in eastern Sudan where they have two types of riding camels, a southern type has good conformation than the northern one.

Bashari camels have certain characteristic features which makes it more popular than Anafi: the well covered shoulders, the chest is wide with shorter back, the well developed loin, broad thick thighs and muscular fore arms.

The Anafi type is owned mainly by the Rashida in the Butana area

&to some extent by Shukria, Lahawian and Batahin. This type of camel is higher than Basharian camel with a long narrow head (Wilson, 1984).

The pack camels of the Sudan usually referred to as:

A. Arab camel which are heavier with well developed hump and short

legs.

B. The Rashaidi camel, a much rare breed, being reared only by the

Rashida tribe north of Kassala town. It is sturdy, relatively short

legged beast, not quite so heavy as the Arab (Bennett, John and

Bewison, 1948).

1.5 Importance of the camel

The camel plays significant socio-economic role as milk, meat and transport animal in arid and semi-arid areas, especially during drought and famine. It survives and produces when other livestock classes cease to produce & die (Harbi, 1993).

Camels are used in Sudan for other purposes including utilization of their wool and hides, traction for agricultural purposes, traditional oil milling& pulling water from surface wells. Camels are also exported to other countries, namely Egypt, Libya& the Arabian Gulf (Absin, 1982).

The relative importance of the camel is significant compared to the total animal units in many countries. They make up about 42% in

Somalia, 39% in Qatar, 30% in the United Arab Emirates, 30% in

Mauritania, 24% in Sudan, 20% in Oman, 15% in Saudi Arabia, 12% in

Chad, 10% in the Niger and 6% Kenya (Wardeh, 1992).

1.5.1 Meat & milk production

1.5.1.1 Meat production

The camel is a good producer of meat and in Tunisia it is the main product of camels and represent about 2.5% of the total red meat production in the country.

The average annual meat production has increased from 2150 tons in 1987 to nearly 3000 tons in 2001, (ACSAD, 2003).

In Sudan Harbi, (1993) indicated that camel meat had certain dietary & producing characteristics which makes it an ideal diet.

- It is low in fat and cholesterol content - It has a superior water holding capacity which is ideal for

meat processing.

- It is high in total protein compared with beef.

Babiker and Yousif (1990) explained that camel meat chemically had significantly lower sarcoplasmic proteins & non-protein nitrogen than beef.

The concentration of Mycofibrillar protein was similar in the lorngism dorsi of the two species. The L.dorsi of the camel carcass had more moisture & significantly (P<0,001 ) less fat than that of the beef carcass. Processing properties such as water holding capacity and cooking loss were superior in camel than beef. Similarly sheer force was lower in camel meat that in beef. The red colour of camel meat was lighter (Paler) than that of beef. (Babiker and Tibin, 1989).

Table (5). Shows meat production and consumption in the Sudan.

Table 5: Meat production & consumption in the Sudan

Item 1991-1992 production Percapita 2001-2002 Percapita

1000 tons consumption production consumption

Beef& veal 261 67 808 144

Mutton 124 04 1441 41

Goat 69 2.5 510 14.4

Camel 27 1.0 05 0.1

Source: paper presented to the 2nd Arab Veterinary Medical conference 1993.

"The camel an important milk& animal" by Harbi.

1.5.2 Milk production

Milk is the most important food for the herders. The milking of she camel occurs twice day, morning &evening. The average milk production of camels is 5-6 litres a day. The amount depends on the type of the camel, its lactation period, the season and the water situation.

In the Sudan Harbi, (1993) explained that, the camel milk is unique diet in the desert and semi-desert areas due to the following:

-It is not affected by hot and dry desert climate

- It is high in water content 90% - She-camel is a high producer of milk ( 200kg/lactation of 10-12

months).

She-camel in the Sudan can produce as much as four Baggara cows.

Sabiel, (1999) indicated that, pack camels yielded more milk than riding camels.

In all groups milk was significantly higher in autumn and winter compared to hot dry summer. The fat, protein and ash contents were highest in summer. Lactose & PH levels were highest in autumn. Moisture content showed no seasonal differences.

Hassan, (1994) explained that average milk production of she-camel is estimated at 1500-3000 hg/annum. The total milk in Sudan was estimated as 1.399.000 tons of which 625000 tons were available for human consumption.(table No. 6. Shows milk production and consumption in the Sudan ).

Table 6: Milk production and consumption in the Sudan

Class Production Consumption Production Consumption

(tons) (tons) (tons) (kg)

1992-93 2000-2001 2000-2001 2000-2001

Cow 3306 119 9290 262

Goat 841 30 5871 165

Sheep 206 7 2815 79

Camel 384 132* 446 153

Source: paper presented to the 2nd Arab Veterinary Medical

Conference 1993.

"The camel an important milk & meat animal" by Harbi

* The camel milk consumption is mainly confined to camel nomads, half of the production is consumed by them while the other half is consumed by the calf.

In Somalia, beside economic importance of camel as a milk, meat and transport animal, camels have social and cultural importance for

Somalis. They are traditionally given as bride wealth by the groom and his relatives to the bride's family, a custom known as yarad. (Hussien, 1987).

1.6 Diseases of camels:

Camels are subjected to infection by parasites, bacteria and viruses.

The most common parasitic diseases are trypanosomasis (Jufar), mange

(Jarab), Haemonchosis & hydatosis. Viruses represent minor hazards and are rarely diagnosed in the camel. (Hussein, 1988).

Agab (1993) reported that out of 10518 camels in Butana area examined throughout the years of study (1991-1992), 3934 were affected with different disease conditions giving diseases incidence rate of 37.4%.

Also Agab, (1998) explained that, the most common diseases caused mortality among camels herds in the Butana area. Calf diarrhea was the main cause of death among camel calves during summer season (39.1%) followed by helminth infections (31%) then tick paralysis (24.1%). During autumn, helminthes infection was the main killing diseases (41.1%) followed by calf diarrhoea (20.6%), tick paralysis (17.8%) and camel contagious ecthyma (16.8%). Calf diarrhoea, was the sole cause of death during the winter (80%).

1.6.1 Bacterial diseases of camels include

1.6.1.1 Brucellosis

Was reported in all camel breeds. In some Africa countries, the prevalence of brucellosis in camel was detected serologically

In Egypt, reported by Fahmy (1990) a reactor rate of 10.24% among camels, using the slide agglutination test.

In Sudan, the incidence of the disease was investigated by Mustafa

& Awad Elkarim (1971)in Kassala and Butana area and their result showed a prevalence rate between 1.75% in Kassala and 5.75% in Butana area among camels.

Abu Damir and Tageldin, (1989) reported that, six camels were experimentally infected with tow strains of Brucella abortus, four with S-

19 and two with a field bovine strain. On post mortem examination no gross lesions were observed although histopathological sections showed focal granuloma in the liver and generalized lymphadenitis. The organism was recovered mainly from the lymph nodes of the head and genital tract.

Bornstein, and Musa (1988) investigated the prevalence of antibodies to Brucella abortus in serum from camels in Sudan and their results showed a prevalence rate of 5.9% to Brucella abortus.

1.6.1.2 Salmonellosis

Salmonella "food poisoning" through camel meat has been reported from different Arab countries like Egypt, Sudan and the United Arab

Emirates. (ACSAD, 2003).

In Egypt, Fahmy,(1990) explained that Salmonellosis was characterized by rise in temperature and increased pulse rate accompanied by swelling of the L.N and muscular contractions of the head and neck region. There was diarrhea and congestion of mucous membranes of the gastro-intestinal tract.

Also Hamada, (1963) isolated two strains of Salmonella typhimurium from spleen, livers, Iliac and mesenteric lymph nodes of camels slaughtered at Cairo abattoir.

In Sudan, Gurasson, (1918) reported that an enteric type of infection and another outbreak of enteritis in camel during the dry season. Since this report no outbreak of camels Salmonellosis were recorded. In united Arab

Emirates, Omer, (1990) reported that, 187 Salmonella isolates were isolated from 4006 camels samples, representing 28 different serovars.

Seventeen different Salmonella servers were detected in both humans and camels, 44% originating from humans and 63% originating from camels.

This indicated that camel meat play an important role as a reservoir for

Salmonellosis in man in the Emirates.

1.6.1.3 Clostridial infections and Enterotoxaemia

symptoms of clostridial infections in camels were fever, lameness and respiratory distress followed by death after 3-4 days of infection.

Enterotoxaemia in camels referred to cl. Perfrirgens.(Fahmy, 1990)

In Sudan, Haroun, Galal and Suliman,(1990) reported that a total of

132 Clostridium perf. strains were isolated from camel livers, spleens, small intestines and abomasums. All strains were catalase negative.

1.6.1.4 Pneumonia in camels

Bashir, (1992) reported that "Pneumonia like other camel diseases has not been well exposed in the literature. However, some work was published in clarification of the situation of the diseases in camels.

In Egypt, Fahmy, (1990) studied pneumonia in camels and isolated the following microorganisms: Coryn. pyogens 20%, Haemolytic streptococci (21%), Diphtheroids (16%),Streptococcus viridans (20%)

Salmonella spp (2%) coliform bacilli (16%) Alkaligens Faecalis (22) and

Bacillus pyogenes.

Also Fahmy, (1990) stating that incidence of pneumonia in camels was low in comparison to infection of cattle, sheep and goats under the same environmental conditions.

In Sudan, Shigidi, (1973) studied microflora of the respiratory tract of 64 apparently healthy camels. Cultures were made from swabs, lungs& bronchial lymph nodes. The most common organisms were found: Bacillus spp. coagulase positive Staphylococci, Diphtheroids, Aspergillus and

Streptomyces. Gross examination of lungs showed no abnormalities and it was concluded that the respiratory tract of camels can harbour some microorganisms without apparent pathological changes.

Bacteriological examination of infested nasopharyngeal mucosa in six cases yielded Pasteurella haemolytica, Klebsiella ozaenac,

Corynebacterium, Bacillus and Proteus spp. Musa et al (1989).

Abu El Gasim, (1992) collected 514 samples from three areas with high camel population (Kordufan,Butana and Tamboul). The samples including (146) nasal swabs, (148) tracheal swabs and (170) pneumonic lungs data analysis demonstrated that the incidence of pneumonia was at its highest in autumn and lowest in summer. Older camels were the most to be affected. The organisms isolate were Staph aureus,

Corynebacterium pyogenes, Bacillus spp, Klebsiella pneumoniae, Staph albus and mixed Flora.

Moallin and Zessin, (1990) studied diseases of the dromedary at

Belotweyne abattoir in central Somalia and from 79 camels slaughtered, 25 positive pneumonia. Staphylococcus spp. were isolated from pneumonic lungs with Pseudomonas. aeruginosa and Citrobacter freundii. 1.6.1.5 Anthrax

Anthrax is the most serious bacterial disease that affects camels. It is caused by Bacillus anthracis. The disease is usually contracted by ingestion of contaminated feed or water and transmitted by mean of biting flies (Tabanus sp) (Higyens, 1983).

In Ethiopia, anthrax in camels was reported by Richard (1979), and in India by Rawat (1990).

In Sudan, Musa et al (1993) reported that an out break of Anthrax in February, 1988, near Kulbus town and Chad border affected 6 dromedaries & 10 people. The ten people had participated in the slaughtering, cooking or eating of an infected dromedary; 5 died despite antibiotics treatment. Since this report no out break of anthrax was recorded in Sudan.

1.6.1.6 Tuberculosis

Tuberculosis in camels has received little attention in comparison with other domesticated animals (Bashir, 1992).

In Somalia and Ethiopia the disease was reported by Pellegrini

(1945) & Richard, (1979). They found caeous nodules in lungs, liver, spleen and lymph nodes of affected camels.

1.6.1.7 Paratuberculosis

Para tuberculosis (Johne's disease) in camel is scarcely reported.

In Sudan, there was no reports present about John's disease.

1.6.1.8 Contagious skin necrosis

This disease is wide spread in Sudan, Egypt, Ethiopia, India and

Somalia.

In Russia, semushkin, (1968) called it " contagious skin abscesses" and described that , Staphylococcus cameli. as causative agent of this disease.

Sana, (1996), isolated 388 bacterial organisms from 131 infected camels. The prevalence of the disease was found to be 5.7%, the highest prevalence was observed during winter (8.3%). Young camels (less than 5 years) are more susceptible to the disease (74.8%).

The isolates from lesions. of skin necrosis were almost pure cultures of Streptococci & Staphylococcus spp (Domenech, 1977).

1.6.1.9 Leptospirosis

This disease was reported in Tunisia and Afghanistan but in Sudan, a serological survey for Leptospirosis from 55 camels was carried out and the disease was not detected in camels (Shigidi, 1974).

1.6.1.10 Mastitis

Mastitis in she camels is reported in India, USSR, Somalia, Egypt&

Sudan. In Sudan, Obied, (1983) investigated 764 she-camels for the prevalence of mastitis, identification of its bacterial causes and determination of leucocytes content of camel's milk. He isolated

Streptococcus, Staphylococcus, Micrococcus, Aerobacter spp and coli- form.

A bdurahman, (1995), examined 391 quarter milk samples from 101 she camels to study the occurrence and causes of mastitis in tradition managed camels in Eastern Sudan. Streptococcus agulactiae, other

Streptococcus spp, Staphylococcus aureus, coagulase Staphylococcus, and

E.coli were isolated from milk.

Maycoplasmal, bacterial and fungal mastitis in she-camel was reported by Soheir 2004.

1.7 Contamination of meat

The healthy inner flesh of meat contain no microorganisms although they have microorganisms in lymph nodes, bone marrow and even flesh. Staphyllococcus, Streptococci, Clostridia and Salmonella were isolated from lymph nodes of different slaughtered animals. (Frazier and

Westhoff, 1978).

The important contamination, however comes from external sources during bleeding, dressing and cutting. The main sources of microorganisms are the exterior of the animals (hides, hoof & hair and even its intestinal tract). (Frazier and Westhoff, 1978). The hide and hair of the animal are important sources of contamination of the surface of the carcass during skinning. The knife used to slaughter animals may introduce organisms to the still-circulating blood stream. Contamination is not only from the hide during skinning but also from knives and from workers and their clothes. During evisceration, contamination may be come from the animal intestine, the air, the water for washing carcass, the various knives, saws, etc used, and the hands clothing of the workers, further contamination during cutting and trimming comes from knives saws, conveyors tables, air, water and workers. (Jay,

1970 and Stringer; Bilskie and Numan, 1969).

Also many researchers have found that the source of bacteria contamination of meat are hooves, hides, soil adhering to the hides, intestinal content, air, water supply, knives, floor & personnel. (Haines,

1933).

Empey & Scott, (1939) investigated bacterial contamination of hides, air and water supply in slaughter-houses and found that hides contained 3.3 ×106 organism/cm2 of hide surface.

Jay (1970) reported that contamination of carcasses come from different sources including environment & equipments with which meat comes in contact during slaughtering and processing but hid remain an important source of contamination of carcasses. Salih and Ibrahim, (1977) investigated bacterial contamination in

Omdurman central abattoir and found that hair, skin, water supply and air contained bacterial number in the range of 104-109/inch for skin, 107-

109/ml for water and 104 CFU/m3 for air.

1.8 Growth of microorganisms in meat

Meat is an ideal cultural medium for many organisms because it is high in moisture, plentifully supplied with minerals and accessory growth factors, usually has some fermentable carbohydrates (glycogen) and is at a favorable pH for most microorganisms. (Frazier and Westhoff, 1978).

In Sudan earlier microbiological studies of fresh and refrigerated bovine meat were presented by Salih, (1971).

Hussien, (1987) isolated from fresh meat samples Staphylococcus epidermidis, Micrococcus spp, E.coli, Proteus spp, Aermonas spp,

Pseudonas spp, No Salmonella or coagulase positive Staphylococcus were isolated.

Intisar, (1998) isolated Gram-positive Micrococci at the preparation level of the meat and Gram-negative enterobacteria at the marketing level of the meat, and she reported that the initial load of aerobic bacteria was

106 colony forming unit (CFU) per gram at the preparation level and 109

(CFU) per gram at the marketing level. Fatima, (1982) isolated Salmonella spp, Clostridium prefringens,

Staphylococcus aureus and E.coli from processed meat examined during that study.

1.9 Food poisoning

A microbial food borne illness may result from ingestion of a food containing either pathogenic microorganism or toxin. When the etiological agent is a pathogen the illness is called infection, but if the etiological agent is toxin it is called intoxication or food poisoning (Ban Wart, 1981).

Also Ban wart, (1981) stated that the organisms which cause food borne diseases or poisoning are Staphylococcus aureus, Colstridium,. Perfringes,

Salomonella spp, Shigella spp, Esherichia Coli, Bacillus sereus

Campylobacter foetus. Subspecies Jejuni, Klebsiella pneumoniae and

Yersinia entrocolitica.

Giracey, (1980). Reported that many other bacteria occasionally cause outbreaks of food poisoning, including Streptococci, Proteus,

Pseudomonas, Providencia, Citrobacter , Aeromonas hydrophila, Yersinia entrocolitica, Campylobacter and Shigella sonnei.

1.10 Microbiological studies of camel meat

The literature concerning microbiological studies of camel meat is scarce because the studies in camel meat have received little attention in comparison with other domesticated animals. All studies which carried out in Sudan were aimed only to isolate aerobic bacteria from lymph nodes or from condemned camel livers.

Salih, (1971) isolated proteus spp at the rate of 100% from livers of camels slaughtered at Omdurman slaughter house.

Bashir, (1992) revealed the presence of aerobic bacteria in 98.2% of lymph nodes of camels slaughtered at Tamboul slaughter area and 100% of that at El Gadaref slaughter house. The isolates were grouped into Gram- positive rods, gram-negative rods and gram positive cocci.

The Gram-positive rods isolated at Tamboul area were Listeria murrayi, C.ovis and Bacillus spp. From El Gadaref another isolates were

E.Coli, Klebsiella aerogens and Shigella sonnie.

Abdel Rahim, (1990) examined a total of 204 Libyan camels slaughtered at Soug El Guma'a slaughter house; he found that, an infection rate of 6.9% in the prepectoral and prescapular lymph nodes.

Staphylococcus aureus was isolated from infected lymph nodes.

Adam, (1996) isolated aerobic bacteria from 89 samples of condemned camel livers. The isolates included different types of bacteria many of which are known to be implicated in meat borne infections and intoxications. There are some evidences that Salmonella is the most likely incriminated organism when compared with others.

1.11 Contamination of processed meat

Jay, (1970) reported that, sausages are usually contained more varied flora than most other processed meat due to different stages of processing employed.

Frazier and Westholf (1988) reported that, during processing contamination will result from special equipments (grinders, sausage stuffers and casing) and some ingredients in special products (fillers and spices).

Also food safety and inspection service (U.S.A, 1997) pointed out that microbiological contamination during processing steps came from handling , grinding, boning and addition of ingredients (spices, bread and ground nuts).

Fatima, (1990) found that the Aerobic plate count of the meat before processing was 1×102-1×103 CFU/g and after processing was

1.0×107-1.0×108 cfu/g .

Amani, (2000) found that, the quantitative bacterial finding revealed

2.20×1010 colony forming unit (cfu) per milliliter for fresh meat samples,

0.95×1010 cfu/ml for minced meat samples, 2-28×1010 (cfu)/ml for mixed meat samples and 3.50×1010 (cfu)/ml for end product samples.

Fatima (1982) indicated that pathogenic bacteria in processed meat which she studied included Salmonella spp. Clostridium perfringes,

Staphylococcus aureus and E.coli. Amanie, (2000) reported that, the bacterial isolates were

Micrococcus spp, Bacillus spp, Aerococcus spp and Staph. lentus as

Gram+ve. Gram-ve isolates were Proteus spp, Acinetobacter spp,

Pseudomonas spp and Escherichia coli. Also she indicated that, the spices and additives were sources of contamination to the meat during processing.

Frazier and Westhoff, (1988) reported that the inhibitory effect of spices is different with the kind of spices and the microorganism being tested.

1.12 Hazard Analysis Critical Control Point (HACCP) Concept in

Meat Production

Nagah, (2004) explained that, HACCP system, which is science based and systematic, identified specific hazard and measures for their control to ensure food safety; also she indicated that, the Hazard Analysis and Critical Control Point system is a management tool for food safety assurance focuses on prevention rather than relying mainly on end product testing.

FAO, (2004) reported that, the Hazard Analysis Critical Control

Point (HACCP) concept was introduced in the food industry to ensure that there would be effective control of the quality of processed foods.

The world health organization (1995), recommended that this concept also be applied to meat inspection and meat hygiene in particular to control salmonellosis. It can also be used to reduce bacterial contamination during slaughtering and dressing and to ensure quality control in meat inspection.

FAO, (2004) indicated that, it would be advantageous to use the

HACCP concept to identify the critical control points at which bacterial groups and other spoilage organisms may contaminate the carcasses, so that appropriate action can be taken. Salwa, (2004) reported a, high rate of bacteria of beef samples collected from Khartoum Air Port at shipping time. This is due to lack of HACCP system during handling and slaughtering inside the abattoir.

The critical control points that have been identified for bacterial groups contamination in red meats shown in Fig-1.

Fig.1 Flow Diagram showing sources of contamination with bacteria and critical control points (CCP) in Red Meat Production.

CCP2 Animals feeds* Farm animals

Transportation to Contaminated * slaughter + streams and Pastures

Lairage +

Stunning

Killing CCP2

CCP2 Skinning *

CCP 1 Evisceration *

Chilling

Cutting, Deboning processing +

* Major contamination

+ Possible contamination

CCP1 effective CCP

CCP2 Ineffective CCP

Source: FAO organization report 2004 1.13 Effect of temperature in Meat

The factors that determine microflora growing on meat are temperature, moisture, pH and inhibitors such a nitrites or concentration of gases in the environment (Grau, 1981). Refrigeration of foods without freezing is an extremely important method of food processing and is possibly the most import method of preservation. (Nekerson and Sinskey,

1974).

Ayres, (1960) found that after storing meat at 5°C for 24 hours and at 0°C for 48 hours, the microbial population was almost invariably less than it was initially.

He attributed this to the fait that, most of the contaminating bacteria are unable to survive at low temperature.

Warriss, (2000) reported that, because lower temperature reduce or prevent microbial growth, cooling of carcass as soon as possible after dressing, and keeping meat at low temperature, can considerably reduce the rate of spoilage and the growth of pathogenic bacteria.

Ingram and Daintly, (1971) reported that spoilage of fresh meat should be considered in relation to temperature because there are importance differences between spoilage under warm and under chill conditions. Under chill conditions (0°C) mesophiles will not be able to grow and the few psychrotphic clostridia grow relatively very slowly, under these conditions spoilage of meat is therefore usually caused by the development of more or less aerobic psychrotroph predominantly on the surface.

1.14 Antibiotics sensitivity test

There are three methods commonly employed routinely in diagnostic bacteriology laboratories.

a. Standard disc diffusion method:

Commercially prepared disc of absorbent paper, impregnated with

defined antibiotics are available for sensitivity testing. The general

procedure is to place discs two centimeters apart on the surface of

nutrient agar plate (Riedner, Albuquerque, Badke and Weiblen, 1987)

which were inoculated with the test organism.

b. Cylinder plate method:

This similar to the previous method except that instead of discs, a

series of small sterile glass cylinders is placed on the surface of

inoculated medium. Into these cylinders, measured volumes of known

concentration of antibiotics prepared in proper solvent and diluted two

folds , using sterile distilled water, are added. The antibiotic diffuses

causing a zone of inhibition of growth (Cowan and Steel 1993).

c. Broth dilution method:

Stock solutions of antibiotics with known concentrations are

prepared with paper solvents and twofold diluted with liquid medium

suitable for growth of the test organisms. Then aloopful of actively growing broth culture is inoculated into each dilution. Inhibition of

growth is shown by the absence of turbidity . the greatest dilution of

antibiotics showing concentration (MIC). (Cowan and Steel, 1993)

Staphylococcus species

Buxton and Fraser, (1977) indicated that, Novobiocin, penicillin, streptomycin, tetracycline, Erythromycin and Neomycin were used in the treatment of staphylococcal infections.

Isam, (2003) reported that, the isolates of Staphylococcus species were highly sensitive to tetracycline, gentamycin, chloramphenical and moderately sensitive to streptomycin and Ampiciliin.

Streptococcus species

Buxton and Fraser, (1977) reported that, penicillin, sulfonamides and chlormphenical were effective in streptococcus infection.

Klebseilla Pneumonia

Elhassan, (1999) reported that, klebsiella pneumonia is highly sensitive to Ciprofloxacin, Chloramphenicol, gentamycin and streptomycin and weakly sensitive to Ampicillin and tetracycline.

Isam, (2003) reported that, klebseilla pneumoniae was highly sensitive to gentamycin and Ampicillin and moderately sensitive to tetracycline, chloramphericol and streptomycin, and resistant to penicillin, erythromycin and cloxacillin.

Pseudomonas aeruginosa

Isam, (2003) indicated that, Pseudomonas aeruginosa is highly sensitive to penicillin and chloramphericol and moderately sensitive to

Gentamycin and resistant to ampicillin, streptomycin, erythromycin, tetracycline and cloxacillin. Queen, (2000) reported that, Ps. aeruginosa is resistatnt to many commonly used antibiotic.

Corynebacterium pseudo tuberculosis

Isam, (2003) reported that, Corynebacterium pseudotuberculosis was highly sensitive to Ampicillin, chloramphenicol and streptomycin and moderately sensitive to Gentamycin and tetracycline.

Lubna, (2003) indicated that, Corynebacterium was resistant to slufafurazole, trimethoprim and oxacillina and sensitive to streptomycin, gentamycin and tetracycline.

CHAPTER TWO

MATERIAL AND METHODS

1. Samples for bacteriological examination

1.1 Sources of samples

The samples used in this study were obtained from camels slaughtered at Elbugaa slaughter house (Omdurman) and ElKadro slaughter house (Khartoum North).

The animals used were at different ages and appeared normal at the time of the antemortern before slaughtering. A total of 120 samples collected from slaughtered animals and samples (37swabs) were collected from workers's hands (15 samples), their knives (15 samples) and saws (6 samples) which used to cut carcasses of slaughtered animals and one swab from carcass cutter machine.

Table No. (7) Shows the different varieties of samples and their locality.

1.2 Collection of samples

A. Muscles

Samples of muscles were collected from camels slaughtered at

Elbugaa (Omdurman) for local consumption and ElKadaro (Khartoum

North) slaughter house for export

Methods

With use of sterile scalpel, forceps, scissors and burner, the muscles were removed and kept in sterile screw capped bottle and labeled.

Instruments were sterilized by dipping in alcohol and flaming between samples. All samples were placed in ice in theromoflask immediately after collection.

B. Swabs

Swabs were taken from outer skin, surface of carcasses, internal cavity, surfaces of workers's hand, surfaces of their knives, saws and carcass cutter machine which were used to cut carcasses.

Methods

With use of sterile swabs. The swabs were obtained from surfaces or inserted to obtain the samples then labeled and put in ice box immediately.

1.3 Distribution of samples

90 samples (75%) were collected from 15 camels slaughtered at

Elbugaa slaughter house and 30 samples (25%) were collected from 5 camels slaughtered in Elkadaro for export, beside 24 swabs (80%) from surfaces of hands of workers and their knives and 6 swabs from surface of swas in Elbugaa. Also 5 swabs (20%) from surface of workers's hands

,their knives and one swabs from carcass cutter machine in Elkadaro slaughter house.

Table No 7: Samples collected and their localities

Locality No. of Oute Surface Internal Thigh Back Shoulder Hands animals r skin of carcass cavity muscle muscle muscle of workers Elbugaa 15 15 15 15 15 15 15 12 Slaughter house

ElKadaro 5 5 5 5 5 5 5 3 Slaughter house

Total 20 20 20 20 20 20 20 15

2. Processing of camel's meat

2.1 The steps of sampling

Samples were collected from sausage and minced meat at the different steps of processing starting from fresh meat till the final product and storage.

Also from workers, spices and machines which were used in processing.

The samples were collected from processing unit in the Regional

Training Center for Meat Inspection, Hygiene and Grading (R.T.C.M.I.HG) in

ElKadaro (Khartoum North).

Samples collected as swabs and examined with in one hour from the time of collection.

2.2 processing of sausage

Fresh meat were brought from butcher shop and cooled for 24 hours.

Then meat was minced by mincer machine, spices, dry bread were added to the meat and mixed with naked hands of workers, then meat was staffered in intestinal casing by special machine.

3. Sterilization

It means the freeing of article from all living organisms, pathogenic and non-pathogenic (Omer, 1986).

3.1 Sterilization by heat

3.1.1 Sterilization by moist heat Media in bottles, and test tubes and the discarded culture were sterilized in the autoclave at 15 Ib 121°C for 15 minutes but sugars were sterilized in the autoclave at 10 Ib at 121°C for 10 minutes.

3.1.2 Sterilization by hot air oven

Petri-dishes, graduated pipettes, flasks, test tubes and bottles were sterilized in the hot air oven at 160°C for one hour.

3.1.3 Sterilization by flaming

This was done for the metal, scissors, spatula, scalpels and forceps were sterilized by flaming after dipping in spirit.

The wire loops were heated to redness with direct flaming.

4. Disinfections

This is freeing of an article from some living organisms. (Omer, 1986).

Laboratory benches cleaned and disinfected by ethyl alcohol solution (70%).

This step was done by cotton before, during and after each work in the laboratory. Forceps, scissors, scalpels and pipettes were put in disinfectant after use, hands were also washed with soap and disinfected.

5. Preparation of media

5.1 Solid media

5.1.1 Blood agar base (oxaid ) (Cowan and steel,1993)

Lab-lemcopowder 10g

Peptone 10g

Sodium chloride 5g

Agar No. 3 13g Distilled water 1000g

PH 7.4 (approx.)

40 grams of dehydrated powder were suspended in one litre of distilled water, brought to boil to dissolve the medium completely, mixed and sterilized by autoclaving at 121 °C for 15 minutes. Then cooled to 50-55 and 100 ml of fresh defibrinated sheep blood added, mixed gently and distributed into sterile

Petri dishes, 20 ml in each dish.

5.1.2 MacConkey's agar (oxoid)

Peptone 20 grams

Lactose 10g

Bile salts 5g

Sodium chloride 5g

Neutral red 0.075g

Distilled water 1000ml

PH 7.4 (approx)

52 grams of dehydrated medium were suspended in one litre of distilled water, brought to boil to dissolve the ingredients, then sterilized by autoclaving at 121°Cfor 15 minutes and poured into sterile Petri-dishes.

5.1.3 Nutrient agar

Lab-Lemco powder 1g

Yeast extract 2g

Peptone 5g Sodium chloride 5g

Agar No.3 15g

Distilled water 1000ml

PH 7.4 (approx)

13 grams of dehydrated medium were dissolved in alitre of distilled water, sterilized by autoclaving at 121°C for 15 minutes, cooled to 50-55°C and then distributed into sterile Petri-dishes, 20 ml in each.

5.1.4 Urea agar base (oxoid)

This medium was used to test ability of organism to split urea. The medium consisted of urea agar base (oxoid) to which 5 ml. of 40% urea solution was added. The based urea medium consisted of peptone, Dextrose, sodium chloride, disodium phosphate, potassium dihydrogen phosphate, phenol red and agar No.3. The basal medium was prepared according to the producer by dissolving 2.4 grams in 95 ml distilled water. The medium was sterilized and cooled to 42-45°C then 5 ml sterile urea solution was added and mixed thoroughly.

5.1.5 Salmonella-shigella agar (Difco)

The medium obtained in dehydrated form. It contained Bacto-beef – extract-proteose peptone-bacto lactose-Bile salts- Sodium citrate-Sodium thiosulphate- Ferric citrate- Brilliant green- neutral red and agar. The medium was prepared by adding 60 gm to one litre of distilled water and heated to dissolve according to the directions of the producer.

5.1.6 D Nase agar

The medium was prepared by dissolving 39g. per litre and sterilized by autoclaving, 15 Ib at 121°Cfor 15 minutes. The medium was cooled to 45°C and distributed in the sterile Petri-dishes 20 ml in each dish.

5.1.7 Eosin Methlene Blue (EMB) (oxiod)

An amount of 36 grams of dehydrated medium were suspended in one litre of distilled water dissolved by heating. Sterilized by autoclaving to 15 Ib for 15 minutes at 121°C. cooled to 45°C. then poured in sterile Petri-dishes

(20ml each).

5.1.8 Brillent Green Agar

Sucrose 10,0000

Lactose 10,0000

Phenol red 0,0800

Brilliant green 0,0125

Agar 15,0000

PH 7,0(±0,2)

Suspend 53g of powder in IL of distilled water and heat to boiling with continuous stirring. Dispense into containers and sterilize at 121°C for 15 min.

5.1.9 Aesculin agar medium

Peptone 1 grams

Ferric citrate 0.5g Agar 20g

Distilled water 1000ml

Solid ingredient were dissolved in peptone water, sterilized by autoclaving at 115°C for 10 minutes, distributed into sterile Petri-dishes.

5.1.10 Diagnostic Sensitivity Test (DST)

Proteose (oxoid L.) 10g

Dextrose 2.0g

Sodium chloride 3.0g

Disodium phosphate 2.0g

Sodium acetate 1.0g

Adenine sulphate 0.01g

Guanine hydrochloride 0.01g

Distilled water 1000ml

PH 6.8 (approx)

128 grams were added to one litre of distilled water, dissolved by heating, sterilized by autoclaving at 121°C for 15 minutes, well mixed, then distributed in 15 ml amounts into sterile Petri-dishes and cooled below 20°C.

5.1.11 Edwards medium

41 grams were added to one litre of distilled water, dissolved by heating, sterilized by autoclaving at 115 °C 20 minuets. Cool to 50°C, then distributed in 20 ml amount into sterile Petri dishes.

5.1.12 Manitol salt agar 111 grams of dehydrated medium were dissolved in a litre of distilled water, sterilized by autoclaving at 121°C for 15 minutes and then distributed into sterile Petri-dishes, 20 ml in each one.

5.2 Semi-solid media

5.2.1 Nutrient gelatin (oxoid)

The medium contained lablemco, peptone and gelatin. The medium was prepared according to the manufacture's instructions by dissolving 128g in one litre of distilled water by boiling. It was distributed in 10 ml amount in test tubes and sterilized by autoclaving at 121°C for 15 minutes.

5.2.2 Hugh and liefson's (O/F) medium

As directed by Cowan and Steel,(1993)

Ingredients 9/L

Sodium chloride 5.0

Dipotassium hydrogen Phosphate 0.3

Peptone 2.0

Agar 3.0

Distilled water 1000L

Bromthymol blue 15.00ml

0.2% aqueus solution

Dissolve the solids by steaming adjust to PH 7.1, filter and add the indictor and sterilize at 115°C and 10 pounds per square inch pressure for 15 minutes. Sterile solution of glucose was added aseptically to give final concentration of 1% Aseptically the mixture was distributed into sterile tubes of 10 ml volumes and scaled with cotton plugs.

5.2.3 Motility medium (Cowan, 1993)

Ingredients g/L

Nutrient broth 13.0 grams

Agar 1.1 grams

The fourteen point one grams were suspended in litre of distilled water and brought to boil to dissolve completely. The PH was approximately adjusted to 7.2. The medium was distributed in 3.5 ml portions into test tubes contain craigie tubes and the prepared media was sterilized by autoclaving at 115°C for

15 minutes.

Peptone water

The medium was prepared by dissolving 10 grams peptone in powder form and 5 grams sodium chloride in one litre of distilled water.

5.3.1 Carbohydrate fermentation medium

These were prepared according to the Cowan and Steel, (1993). When

Andrad's indicator was used, 900 ml of peptone water was prepared, and it's PH was adjusted to 7.1. 10 ml Andrad's indicator was added and the medium was sterilized. The colour of the medium was pink, but it faded with cooling. 10 grams of sugars were added in 90 ml of distilled water and sterilized, this amount was added aseptically to the sterile peptone water and the indicator. It was then distributed in 5 ml amount in test tubes with inverted Durham's tubes and sterilized by autoclaving at 115°Cfor 10 minutes. The carbohydrate used were salicin, Glucose, Manitol, Sucrose, Lactose, xylose, Glactose,Maltose and

Trehalose.

5.3.3 Koser's liquid citrate medium (oxoid)

This medium was used to test the ability of culture to utilize citrate and ammonium salt as the sole sources of carbon and energy for growth. The medium contained sodium ammonium phosphate, potassium dihydrogen phosphate, magnesium sulphate, sodium citrate and promothymol blue indicator. The medium was prepared by dissolving 17 g of medium in one litre of distilled water according to the description of the producer.

5.3.4 MR-VP test medium (Difco)

Peptone 5g

KMPO 5g

Distilled water 100ml

PH 7.1 (approx)

15 dehydrated powders were added to one litre of distilled water, mixed well, then distributed into bottle, and sterilized by autoclaving at 115°C for 10 minutes.

5.3.5 Nutrient broth (oxoid)

Lab. Lemco powder 1 gram Yeast extract 2g

Peptone 5g

Sodium chloride 5g

Distilled water 1000ml

PH 7.4 (approx)

13 grams of dehydrated powder were added to one titre of distilled water, well mixed and distributed in 5ml amounts into sterile test tubes and sterilized by autoclaving at 121°C for 15 minutes.

5.1.6 Selenite broth

The medium contained:

Sodium acid selenite 4.0 gm

Peptone 5.0 gm

Lactose 4.0gm

Disodium hydrogen phosphate 9.5gm

Distilled water 1 litre

Sodium hydrogen phosphate 0.5

23 grams of medium were added to one litre of distilled water and distributed in 10 ml in tubes and sterilized by boiling at 100°C for 20 minutes.

5.1.7 Nitrate broth

This medium contained

KNO3 1.0 gm

Nutrient broth 1000ml KNO3 was dissolved in nutrient broth then distributed into sterile test tubes and sterilized at 10 pounds per square inch for 20 minutes.

6. Reagents

6.1 Hydrogen peroxide

This was prepared in a concentration of 3% aqueous solution for catalase test.

6.2 Tetramethyl p. phenylenediamine dihydrochoride

This was prepared as 3% a queous solution and was used for oxidase test.

6.3 Kovac's reagent

This reagent is composed of 5g paradimethylamino-benzaldehyde, 75 ml amyl alcohol and 25 ml concentrated hydrochloric acid. It was prepared as described by Barrow and Feltham (1993) by dissolving the aldehyde in alcohol through heating in water bath. It was then stored at 4°C for later use in indole test.

6.4 Voges proskauer test reagent (KOH)

This reagent is composed of 40% potassium hydroxcide and 5% &* naphthol in absolute ethanol.

6.5 Methyl red solution

This solution was prepared as described by Barrow and Feltham, (1993) by dissolving 0.04g methyl red powder in 40 ml of ethanol and the volume was topped to 100 ml distilled water. It was used in methyl red test.

6.6 Nitrate reagent

This reagent is composed of solution A (0.33% sulpharilic acid in 5 N- acetic acid) dissolved by gentle heating and solution B (0.6%dimethyl & naphthylamine in 5 N- acetic acid ). It was used in nitrate reduction test.

7.1 Andrade's indicator This was prepared according to Barrow and Feltham, (1993) by dissolving 9g of acid Fuchsine in one litre of distilled water and 150 ml alkali solution was added. It was used in peptone water sugar media.

7.2 Bromothymol blue

This was prepared by dissolving 0.2g of bromothymol blue powder in

100ml distilled water.

7.3 Lead acetate papers

Small strips of filter paper (5-10 mm wide 50-60 mm long) were cut and impregnated in lead acetate saturated solution (10 mg lead acetate in 100 ml hot water). The impregnated strips were dried at 50-60°C and stored in screw- capped container. It was used for production of hydrogen sulphide.

8. Preparation of Material for bacteriological examination

8.1 Preparation of the samples

The samples were prepared using of scissors and forceps. The muscles were put in sterile Petri dish, and then were cut into two parts.

9. Cultural methods

9.1. Primary isolation

9.1.1. Solid media The prepared samples were cut into two parts with sterile scissors. The internal surface was streaked on to part of blood agar and MacConkey agar and then the streaking over the plate was completed with wire loop.

9.1.2 Broth media

The swabs samples dipped in 10 ml of sterile nutrient broth and incubated at 37°C for 24 hours. Then sub cultured in Blood Agar and

MacConkey agar.

9.2 Sub culturing of primary isolates

9.2.1 from solid to solid media

A part of atypical and well isolated colonies was picked with wire loop and streaked on the surface of afresh plate of the medium.

9.2.2 From broth to solid

A loop full broth culture streaking on the solid medium "sub culturing from broth to solid media".

10. Incubation of cultures

All inoculated solid media and broth media were incubated aerobically at 37°C for 18-24 hours except MR-VP and indole medium, which were incubated for 48 hours.

11. Examination of cultures

All cultures on solid media were examined with naked eye for growth, colonical morphology and any changes in the medium. The broth media used examined for turbidity, colour changes, and accumulation of gas in case of

carbohydrate media.

11.1 Motility

The isolates were studied for motility by craigie's technique (Cruick

Shank, 1975) in which the bacteria was incubated into central tube containing

semi solid agar placed test tube using straight wire. After incubation at 37°C

for 24 hours.

The tubes were examined for migration of the bacteria out the craigie

tube.

11.2 Purification of cultures

Purification of cultures was made by sub culturing of atypical and well isolated colony on nutrient agar. This process was repeated twice. The resulting growth was checked for purify by examining smears stained by gram method.

12. Staining

12.1 Gram's stain

Clean microscopic slides, normal saline, forceps, bacteriological loop

and staining set were used. (Cowan and Steel, 1981).

12.2 Reagents

12.2.1.Ammonium oxalate- crystal violet

Solution A

Crystal violet 10gm

Ethanol (95%) 100gm Mix and dissolve

Solution B

Ammonium oxalate 10% aqueous solution.

For use mix 20 ml of solution A and 80 ml of solution B.

12.2.2 Carbol Fuchsins "strong"

Basic fuchsin 10gm

Ethanol (95%) 100ml

Mix and dissolve. For use dilute 1/9 with distilled water.

12.2.3 Lugol's iodine

10dine 5.0gm

Potassium iodide 10gm

Distilled water 100ml

Dissolve the potassium iodide in 10 ml of the distilled water and adjust to volume with distilled water. For use dilute 1/5 with distilled water.

12.3 Staining technique

Clean microscopical slides, normal saline, bacteriological loop and Bunsin burner were used. Apart of an isolated colony was emulsified in drop saline in the center of slides and spread thinly, left to dry and fixed by passing 2-3 times over the flame.

The smear was stained as follows

i. Crystal violet solution was applied for two minutes ii. The smear was washed with distilled water

iii. Lugol's iodine solution was applied for one minute

iv. The smear was washed with distilled water

v. The smear was decolorized by acetone for ten seconds

vi. The smear was washed with distilled water

vii. Carbol fuchsin solution was applied for one minute

viii. The smear was washed with distilled water, dried and examined

microscopically. Gram-positive bacteria appeared violet while

Gram-negative bacteria appeared red.

13. Identification of bacteria

The purified isolated bacteria were identified according to critea out lined by Barrow and Feltham, (1993) include.

1. Reaction of gram stain

2. Shape of the bacteria colonies

3. Presence or absence of spores

4. Motility

5. The colonial characteristics on the different media and heamolysis of

blood agar.

6. Biochemical tests.

14. Biochemical methods for identification of isolated bacteria

All biochemical tests were performed according to Cowan and

Steel,(1993), (1981). They include

14.1 Potassium hydroxide test A loop full culture is taken from non-selective medium and mixed with an equal amount of 3%of potassium hydroxide (KOH) on clean microscopic slide, if bacteria are gram-negative agel viscous is formed and if they are

Gram-positive bacteria no gel formation.

14.2 Gelatin hydrolysis (Cowan and Steel,1993)

The test organism was stabbed in nutrientgelatine using sterile straight wire and incubated at 37°Cfor up to two weeks with daily examination then placed in the refrigerator for two hours.

A positive result was indicated by liquefaction of gelatin.

14.3 Indole production (Cowan and Steel, 1993)

Peptone water was inoculated with test organism and incubated at 37°C for 24 hours, then 1 ml of Kovac's reagent was rundown the side of test tube.

Pink ring within a minute, indicates appositive reaction.

14.4 Urease activity (Cowan and Steel,1993)

Slopes of Christensen's urea medium were inoculated with test organism and incubated at 37°C for seven days. Pink colour indicated positive reaction

(Urease enzyme production), while negative reaction was yellow colour.

14.5 Aesculin hydrolysis (Cowan and Steel,1993)

The Aesculin agar was inculcated with organism under test, and examined daily for up to five days. Appositive reaction was indicated by black colouration around the bacterial growth. 14.6 Oxidation and Fermention (O/F) of carbohydrates (Cowan and Steel,

1993)

Duplicated tubes of Hugh and Leifson's medium were inoculated with straight wire loop, then a layer of melted soft paraffin was added, about 3 cm above the medium surface in one tube and the other left open, incubated and examined daily for up to 14 days. If the colour is yellow in both tubes it indicates formation reaction, if the colour in the open tube is blue or green and in the sealed tube is green it indicates production of alkali, if the colour is yellow in the open tube only this indicates oxidation of glucose-No change in both tubes means negative reaction.

14.7 Nitrate reduction (Cowan and Steel,1993)

The nitrate broth was inoculated lightly and incubated for up to five days.

1 ml reagent (A) was added followed up by 1 ml reagent (B). A deep red colour indicates that nitrate has been reduced. If the tubes not showing a red colour within 5 minutes powdered zinc was added and allowed to stand. Red colour means negative reaction.

14.8 Coagulase test (Cowan and Steel, 1993)

An amount of 0.5 ml of an 18-24 hours broth culture of the test organism was added to 0.5 ml 1/10 dilution of rabbit plasma in saline in small sterile agglutination tube, then incubated at 37°C for 2-24 hours, appositive result was indicated by definite clot formation. If negative, the tube was left at room temperature over night and reexamined.

14.9 Oxidase test (Cowan and Steel,1993) Drops of 1% tetramethyl-p-phenylendiamine dihydrochleride were poured over wet strips of filter paper in Petri-dish and then sterilized in a hot air oven. Using sterile forceps, strips were laid on a clean slide. Growth on nutrient agar was picked off with a sterile glass rod and rubbed on to a small area on the filter paper. A purple colour that developed within 10 seconds was considered a positive reaction.

14.10 Catalase test (Cowan and Steel,1993)

Method 1: on a clean slide a drop of 3% aqueous solution of hydrogen peroxide was placed. A colony of the test organism in nutrient agar was picked with sterile glass rods and put on the drop of the hydrogen peroxide, evolution of gas and appearance of bubbles indicated that the organism produced enzyme.

Method 2: Hydrogen peroxide (3%) was poured over the growth on agar plate, production of gas bubbles indicated production of catalase by the organism.

14.11 DNase test

DNase agar medium was inoculated with tested bacteria, incubated at

37°C for 24 hours. The inculcated DNase agar was flooded with week HCl.

Positive reaction was indicated by clear zone around the streaking of tested bacteria.

14.12 Acid from carbohydrates (Cowan and Steel,1993) (Fermentation of

sugars). Carbohydrate media were inoculated with test organism and examined daily for seven days for acid and gas production in Durham tubes (Colour changing to pink).

14. 13 Voges-Proskauer (VP) test

This is to test the production of a acetyl methyl carbonyl . Glucose phosphate medium was inoculated with the test organism and incubated at 37C for 24 hours. O.6 ml of 3% alcoholic solution of & naphthal solution and 0.2 of

40% KOH were added to one ml of culture. A positive reaction was indicated by development of bright pink colour with in 30 minutes.

14.14 Citrate utilization (Cowan and Steel, 1993)

This test was applied to indicate the ability of organism to utilize citrate as sole source of carbon. A light suspension of organism in sterile saline was inoculated in citrate medium with wire loop and incubated at 37°C. A positive test was indicated by change of colour to blue or turbidity

14. 15 Hydrogen sulphide (H2S) production (Cowan and Steel, 1993)

Peptone water was inoculated with test culture. Filter paper impregnated with 10% lead acetate solution was placed in the neck of the tube and incubated at 37°C for 48 hours. A positive reaction was indicated by brown or black colour of the paper.

14.16 Methyl Red test (MR) Glucose phosphate medium was inoculated with the test organism and then incubated at 37°C for 48 hours. Five to six drops of methyl red solution were added and the tube was shaken. The development of red colour indicated positive reaction. This test was done for the production of prolonged activity from glucose in a buffered media.

14.17 Antibiotic sensitivity test

Sensitivity of isolates to a number of antibiotics was determined by the standard diffusion method (Buxton and Fraser, 1977). Plates of (DSTA) medium and nutrient agar were dried in incubator for 30 minutes. A plate was used for each isolates to test twelve antibiotics. Each isolates was incubated overnight at 37°C in test tubes containing 2ml nutrient broth, shaken thoroughly to obtain a homogenous suspension of the test culture. Then excess fluid of isolates was aspirated on the surface of plate with sterile swabs, the disk of antibiotic was placed on the surface of the plate and incubated over night and look for clear zone of growth inhibition around the disk.

CHAPTER THREE

RESULTS

A. Bacterial isolation

I. From samples collected from Elbugaua slaughter house

Ninety samples were collected from 15 camels slaughtered in Elbugaa slaughter house (Omdurman State) and thirty swabs collected from 12 worker's hands,12 worker's knives and 6 swabs from saws which were used to cut carcass.

12 muscles samples from the total muscles samples were negative for bacterial growth "sterile".

All these samples were taken and examined to determine aerobic bacteria. These samples yielded 399 isolates of which 142 were Gram-positive cocci. 69 were Staphylococcus spp, 39 were Streptococcus and 34 were

Micrococcus. (Table 8).

126 isolates were Gram-positive bacilli, of which 70 isolates were corynebacterium, 33 were bacillus and 23 were lactobacillus (table 9).

131 isolates were Gram-negative Bacillus which include: E.coli,

Citrobacter spp. Proteus, Pseudomonas, Edwardsiella, Klebsiella spp, Serratia spp, Acinetobacter and Salmonella (table 9).

The organisms isolated from all samples were identified as:

Staphylococcus xylosus, Staphylococcus warneri, Staphylococcus cascolyticus, staphylococcus epidermidis, Staphylococcus lentus, Staphylococcus aureus,

Staphylococcus schleferi, Corynebacterium pseudotuberculesis,

Corynebacterium xerosis, Corynebbacterium pseudodiphtheriticum

Corynebbacterium ulcerans, Streptococcus uberis, Streptococcus aglactiae,

Streptococcus disaglactiae, Streptococcus sciuri Enterococcus faecalis,

Micrococcus roscus, Micrococcus luteus Micrococcus kristinae, Micrococcus lylae, bacillus lentus, bacillus alvei, bacillus licheniformis, lactobacillus salivarius ,E.coli, Citrobacter freundii, Citrobacter koseri, Citrobacter aerogenes, Pseudomonas aerugenosa, Pseudomonas pickatti, Pseudomonas stutzeri, Edwardsiella tarda,. Proteus vulgaris, Proteus morganii, Klebsiella pneumoniae, Serratia plymthica, Serratia marcescens, Acinetobacter lowffi and

Salmonella typhi. Distribution of the above organisms among different samples are shown in tables No.,9,10, and 11.

Table (8): Bacterial groups isolates from samples collected from Elbugaa slaughter house

Isolates Gram- Gram- Gram- Total positive negative positive Rods Rods Cocci Outer skin 61% 3.23% 35.48% 38 2 22 62 9.5% 0.5% 5.5% 15.5% Surface of 41.10% 26.03% 32.88% carcass 30 19 24 73 7.5% 4.8% 6.0% 18.3% Internal 5.56% 52.78% 41.67% cavity 4 38 30 72 1.0% 9.5% (7.5%) (18.0%) Back muscle 32.5% 42.5% 25% 13 17 10 40 3.3% 4.3% 2.5% 10% Thigh 35.29% 3.5% 29.41% muscle 12 12 10 34 3% 3% 2.5% 8.5% Shoulder 28% 16% 56% muscle 7 4 14 25 1.8% 1.0% 3.5 6.3% Workers's 2.7.03% 32.43% 40.54% hands 10 12 15 37 2.5% 3% 3.8% 9.3% Workers's 24.24% 39.39% 36.36% knives 8 13 12 33 2% 3.3% 3% 8.3% Saws 1.0% 3.5% 1.3% 4 14 05 23 17.4% 60.9% 21.7% 5.8% Total 126 131 142 399 31.6% 32.8% 35.6% 100%

Table (9): Distribution of isolated bacteria genera among different types of samples- :

Types of samples Outer Surface Internal Back Thigh Shoulder skin of cavity muscle carcass Gram-positive bacteria corynebacterium 20 16 - 8 9 4 (28.6%) 22.9% 11.4% 12.91% 5.7% Staphyllococcus - 7 18 8 7 10 (10.4%) (26.1%) (11.6%) (10.4%) (14.5%)

Streptococcus 12 10 5 1 2 3 (30.8%) (25.4%) (12.8%) (2.6%) (5.1%) (7.7%) Micrococcus 10 7 7 1 1 1 (29.4%) (20.6%) (20.6%) (2.9%) (2.9%) (2.9%) Bacillus 15 9 - - - - (45.5%) (27.3%)

Lactobacillus 3 5 4 5 3 3 (13%) (21.7%) (17.4%) (21.7%) (13%) (13%)

Total 60 54 34 23 22 21 (22.4%) (20.2%) (12.7%) (8.6%) (8.2%) (7.8%)

Cont. table 9

Gram-negative bacteria E.coli 2 12 15 2 4 2 10 (3.5%) 16% (3.5%) 7% (3.5%) (17.5% Citrobacter - 2 8 2 5 1 - (9.1%) (36.4%) (9.1%) (22.7%) (4.5%) Proteus - 2 7 1 1 1 - (11.8%) (41.2%) (5.9%) (5.9%) (5.9%) Pseudomonas - 1 3 3 3 - - (6.3%) (18.8%) (18.8%) (18.8%) Edwardsiella - 1 3 1 1 - - tarda (16.7%) (50%) (16.7%) (16.7%) Acinetobacter - - 1 1 1 - 1 (16.7%) (16.7%) (16.7%) (16.7% Klebsiella - 1 1 - 2 - - (25%) (25%) (50%) Serratia - - - 2 - - (100%) Salmonella - - - - - 1 100% Total 2 19 38 12 17 4 12 (1.5%) (14.5%) (29%) (9.2%) (13%) (3.1%) (9.2%

Table 10: Shows distribution of isolated gram-positive bacteria among different samples collected from Elbugaa slaughter house.

Types of samples Outer Surface Internal Thigh Back Shoulder skin cavity Corynebcterium 8 7 - 3 1 - pseudotuberculosis (29.6%) (25.9%) (11.1%) (3.7%) Corynebacterium xerosis 6 4 - 4 3 2 (31.6%) (21.1%) (21.1%) (15.*%) (10.5%)

Corynebacterium 4 3 - 2 4 2 pseudodipheriticum (26.7%) (20.0%) (13.3%) (26.7%) (13.3%) Corynebacterium ulcerans 2 2 - - - - (22.2%) (22.2%)

Staphylococcus caseolyticus - - 3 4 2 1 (13.6%) (18.2%) (9.1%) (4.5%) Staphylococcus lentus - 2 1 2 2 4 (13.3%) (6.7%) (13.3%) (13.3%) (26.7%) Staphyllococcusepidermidis - - 5 - 3 5 (35.7%) (21.4%) (35.7%) Staphaphyllococcus lentus - 2 3 - - - (33.3%) (50%) Staphylococcus schlefri - 2 - 1 1 - (40%) (20%) (20%) Staphylococcus aureus - - 4 - - (100%) Staphylococcus warneri - 1 2 - - - (33.3%) (66.7%)

Cont. table 10

Streptococcus agalactiae 3 2 1 1 - 1 (25%) (16.7%) (8.3%) (8.3%) (8.3%) Enterococcus faecalis 5 2 3 1 1 - (41.7%) (16.7%) (25%) (8.3%) (8.3%) Streptococcus disaglactiae 2 1 1 - - 2 (28.6%) (14.3%) (14.3%) (23.6%) Streptococcus sciuri 1 4 - - - - (16.7%) (66.7%) Streptococcus uberis 1 1 - - - - (50%) (50%) Micrococcus lylae 4 1 2 - - 1 (36.4%) (9.1%) (18.2%) (9.1%) Micrococcus roseus 2 3 1 1 1 1 (20%) (30%) (10%) (10%) (10%) (10%) Micrococcus luteus 2 2 4 - - - (22.2%) (22.2%) (44.4%) Micrococcus kristinae 2 1 - - - - (50%) (25%) Bacillus lichenoformis 5 3 - - - - (41.7%) (25%) Bacillus lentus 4 4 - - - - (36.4$) (36.4%) Bacillus alvei 6 1 - - - - (60%) (10%) Lactobacillus alivarius 3 5 4 3 5 3 (13%) (21.7%) (17.4%) (13%) (21.7%) (13%) Table (11): Shows distribution of the organisms among different samples

Types No. Ou Sur Inte Thi Bac Shou Wor Wor Saw of of ter face rnal gh k lder ker;s ker;s s samples ident of of cavi mus mus mus hand knive ified ski carc ty cles cles cles s s n ass E.coli 57 2 12 15 2 4 2 10 8 2 (3. (21. (36. (3.5 (7.0 (3.5 (17.5 (17.0 (3,5 5% 1%) 3%) %) %) %) %) %) %) ) Citroba 10 - 1 3 1 2 1 - 2 - cter (10. (36. (10. (3.5 (10.0 (20.0 koseri 0%) 0%) 0%) %) %) %)

Citrobc 6 - 1 2 1 1 - - 1 - ter (16. (33. (16. (16. (16.7 freundi 7%) 3%) 7%) 7%) %) i Citroba 6 - - 3 - 2 - - - 1 cter (50. (33. (16. aerogen 0%) 3%) 7%) es Proteus 9 - - 5 - 1 1 - - 2 vulgris (55. (11. (11.1 (22. 6%) 1%) %) 2%) Proteus 8 - 2 2 1 - - - - 3 morgan (25. (25. (12. (37. ii 0%) 0%) 5%) 5%) Pseudo 9 - 1 2 2 1 - - 1 2 monas (11. (22. (22. (11. (11.1 (22. aergino 1%) 2%) 2%) 1%) %) 2%) sa Pseudo 5 - - 1 - 2 - - - 2 monas (11. (22. (22. stutzeri 1%) 2%) 2%) Pseudo 2 - - - 1 - - - 1 - monas (50. (50.0 piacetti 0%) %) Edwar 6 - 1 3 1 1 - - - - dsiella (16. (50. (16. (16. tarda 7%) 0%) 7%) 7%) Acineto 6 - - 1 1 1 - 1 - 2 bacter (16. (16. (16. (16.7 (33. lowffi 7%) 7%) 7%) %) 3%) Klebsie 4 - 1 1 - 2 - - - - lla (25. (25. (50. pneum 0%) 0%) 0%) oniae Serrati 1 - - - 1 - - - - - a (100 plymet %) hica Serrati 1 - - - 1 - - - - - a (100 marces %) cens Salmon 1 ------1 - - ella (100 Typhi %) II. Bacterial isolation from samples collected from Elkadaro slaughter house.

Thirty samples were collected from five camels slaughtered in Elkadro.

Together with seven swabs collected from three worker's hands, three from their knives and one from carcass cutter machine.

All these samples were collected and examined to determine aerobic bacteria.

These samples yielded 155 isolates of which 63 were Gram .positive

Cocci, twenty five were staphylococcus, 17 were streptococci and 21 were micrococci. (Table No 12). Forty two isolates were Gram-positive bacilli, of which 14 were bacillus and 28 were corynebacterium (Table No.13).

Fifty isolates were Gram-negative Bacilli, which include: Pseudomonas

7, Citrobacter 2, Proteus spp 6, Edwardsiella tarda 6, Acinetobacter spp 5,

Escherichia Coli 20, Serratia 3 and Klebsiella 1 (table No. 13).

The organisms isolated from all samples were identified as:

Staphylococcus epidermides, Staphylococcus lentus, Staphylococcus xylosus,

Staphylococcus warneri, Micrcoccus leteus, Micrococcus roseus,

Cornebacterium pseudotuberculosis, Corynebaeterium ulcerans , Bacillus licheniformis, Bacillus cereus, Enetrococcus faecalis, Enetrococuus pneumeniae, Streptococcus uberis, Streptococcus lentus, lactobacillus salivarius, E.Coli, Pseudomonas aeruginosa, Pseudomonas Putida, Proteus vulgaris, Edwardsiella tarda, Citrobacter freundii, Klebsiella pneumoniae,

Serratia plymethica and Acinetobacter lowffi. Distribution of the above organisms among different samples is shown in tables 14, 15.

Table (12): bacterial groups isolated from samples collected from

ElKadaro slaughter house.

Isolates Gram Gram Gram Total positive negative positive Rods Rods cocci Outer skin 19 1 8 28 (12.3%) (0.65%) (5.16%) 18.061 Surface of carcass 12 3 12 27 (7.79%) (1.44%) (1.44%) 17.42% Internal - cavity 11 9 20 (7.10%) (5.81%) 12.9% Thigh 2 11 10 23 muscles (1.29%) (7.10%) (6.45%) 14.8 Back 2 10 7 19 muscles (1.29%) (6.45%) (4.52%) 12.3 Shoulder 1 5 11 17 muscles (0.65%) (3.23%) (7.10%) 11% Worker's 3 5 3 11 hands (1.44%) (3.23%) (1.44%) 7.1% Workers's 3 4 3 10 knives (1.94%) (2.58%) (1.94%) 6.5% Carcass - - - - cutter machine Total 42 50 63 155 27% 32.3% 40.7% 100%

Table (13 a): Distribution of isolated bacterial genera among different types of samples

Types of samples Outer Surface Internal Thigh Back Shoulder Work skin of cavity muscle hand Genus carcass Gram-positivebacteria Corynebacterium 14 7 - 1 1 1 2 (9%) (4.5%) (0.7%) (0.7%) (0.7%) (1.3% staphyllococcus - 3 4 5 4 5 3 (1.9%) (2.6%) (3.2%) (2.6%) (3.2%) (1.9% Micrococcus 3 5 4 2 1 4 - (1.9%) (3.2%) (2.6%) (1.3%) (0.7%) (2.6%) Streptococcus 5 4 1 3 2 2 - (3.2%) (2.6%) (0.7%) (1.9%) (1.3%) (1.3%) Bacillus 5 5 - - - - 1 (3.2%) (3.2%) (0.7% Lactococcus - - - 1 1 - - (0.7%) (0.7%) Total 27 24 9 12 9 12 6 17.4% 15.5% (5.8%) 7.7% 5.8% 7.7% 3.9%

Table (13 b): Distribution of isolated bacteria genera among different types of samples collected from ElKadaro

Types of samples Outer Surface Internal Thigh Back Shoulder Worker' of skin of carcass cavity muscle muscle Genus Gram-negative E.coli 1 3 4 2 2 2 3 (0.7%) (1.9%) (2.6%) (1.3%) (1.3%) (1.3%) (1.9%) Pseudomonas - - - 2 2 1 2 (1.3%) (1.3%) (065%) (1.3%) Proteus - - 2 2 2 - - (1.3%) (1.3%) (1.3%) Edwardsiella - - 3 1 1 1 - (1.9%) (0.65%) (0.65%) (0.65%) Acinetobacter - - - 2 2 - - (1.3%) (1.3%) Serratia - - 1 1 - 1 - (0.65%) (0.65%) (0.65%) Citrobacter - - - 1 1 - - (0.65%) (0.65%) Klebsiella - - 1 - - - - (0.65%) Total 1 3 11 11 10 5 5 (0.65%) (1.9%) (7.1%) (7.1%) (6.5%) (3.2%) (3.2%)

Table (14): Shows distribution of G.P. bacteria isolated among different samples collected from ElKadaro slaughterhouse Organis No. Out Inte Thi Bac Shou Wor Wor Sur Cutt ms of er rnal gh k lder ker's ker's face er ident skin cavi mus mus Mus hand kniv of mac ified ty cles cles cles s es carc hine ass Staphyllo 8 - 2 1 - 2 2 - 1 - cocus (5.2 (1.3 (0.6 (1.3 (1.3 (0.6 epidermi %) %) 5%) %) %) 5%) dis Staphyllc 4 - - 1 1 1 - 1 - - occus (2.5 (0.6 (0.6 (0.65 (0.65 lentus %) 5%) 5%) %) %) Staphyloc 7 - 1 1 1 1 1 - 2 - occus (4.5 (0.6 (0.6 (0.6 (0.65 (0.65 (1.3 xylosus %) 5%) 5%) 5%) %) %) %) Staphyloc 6 - 1 2 2 1 - - - - occus (3.9 (0.6 (1.3 (1.3 (0.65 warneri %) 5%) %) %) %) Micrococ 12 2 2 2 - 2 - 1 3 - cus luteus (7.7 (1.3 (1.3 (1.3 (1.3 (0.65 (1.9 %) %) %) %) %) %) %) Micrococ 9 1 2 - 1 2 - 1 2 cus (5.8 (0.6 (1.3 (1.3 (1.3 (0.65 (1.3 roseus %) 5%) %) %) %) %) %) Coryneba 17 10 - 1 1 - 1 1 3 - cterius (11% (6.5 (0.6 (0.6 (0.65 (0.65 (1.9 pseudotu ) %) 5%) 5%) %) %) %) berclosis Coryneba 11 4 - - - 1 1 1 4 - cterium (7.1 (2.6 (0.65 (0.65 (0.65 (2.6 ulcerans %) %) %) %) %) %) Bacillus 4 2 - - - - 1 1 - - lichenifor (2.6 1.3 (0.65 (0.65 mis %) % %) %) Bacillus 6 2 ------4 - lentus (3.9 (1.3 (2.6 %) %) %) Bacillus 2 1 - - - - - 1 - cereus (1.3 (0.6 (0.6 %) 5%) 5%) Enteroco 7 2 - 2 1 - - - 2 - ccus (4.5 (1.3 (1.3 (065 (1.3 faecalis %) %) %) %) %) Streptoco 3 - 1 - 1 1 - - - - ccus (1.9 (0.6 (0.6 (0.65 pneumoni %) 5%) 5%) %) ae Streptoco 3 2 ------1 - ccus (1.9 (1.3 (0.6 uberis %) %) 5%)

Streptoco 4 1 - 1 - 1 1 - ccus (2.5 (0.6 (065 (0.65 (0.6 lentus %) 5%) %) %) 5%) Lactobaci 2 - - 1 1 - - - - - llus (1.3 (0.6 (0.6 salivarus %) 5%) 5%)

Table (15): Shows distribution of G.N. bacteria isolated among different samples collected Elkadaro slaughter house

Organisms No. of Outer Surface Internal Thigh Back Shoulder W identified of skin of cavity muscle muscle muscle h carcass E.coli 20 1 3 4 2 2 2 (12.9%) (0.65%) (1.9%) (2.6%) (1.3%) (1.3%) (1.3%) Pseudomonas 4 - - - 1 1 1 aerginosa (2.6) (065%) 0.65% 0.65% Pseudomonas 3 - - - 1 1 - Putida (1.9%) 0.65% 0.65% Proteus 6 - - 2 2 2 - vulgarisbio (3.9) (1.3%) (1.3%) (1.3%) group 3 Edwardsiella 6 - - 3 1 1 1 tarda (3.9%) (1.9%) (0.65%) (0.65%) (0.65%) Acinetobacter 05 - - - 2 2 - lowfii (3.2%) (1.3%) (1.3%) Serratia 03 - - 1 1 - 1 plymthica (1.9%) (0.65%) (0.65%) (0.65%) Citrobacter 02 - - - 1 1 - freundii (1.3%) (0.65%) (0.65%) Klebsiella 01 - - 1 - - - pneumoniae (0.65%) (0.65%) Total 50 1 3 11 11 10 5 (32.3%) (0.65%) (1.9%) (7.1%) (7.1%) (6.5%) (3.2%)

B. Bacterial isolation of camel's meat processing

Samples collected from different stages of sausage processing, three samples from mincer machine, two samples from spices, eight samples from workers and three samples from end product after storage at 4°C in refrigerator for five days.

All these samples were examined to determined aerobic bacteria.

These isolated were comprised of 84 Gram-positive and 90 were Gram negative bacteria. (Table No. 16).

Gram positive bacterial species isolated were Micrococcus lylae,

Micrococcus luteus, Micrococcus xerosis, Staphylococcus epidermidis,

Staphylococcus xylosus, Staphylococcus sciuri, Enetrococcus faecalis,

Corynebacterium Pseudotuberculosis Corynebacterium xerosis, bacillus lichiniformis, Bacillus sphericus and Bacillus pantithenticus.

Gram-negative bacterial species isolated were Proteus vulgaris, Proteus morgani, E.coli, Edwardsiella tarda, Citrobacter freundii, Acinetobacter lowffi, Pseudomonas aeruiginosa, Pseudomonas pseudoalcaligenes and

Klebsilla pneumoniae. Rates of the different isolates

Micrococcus spp were found to have high rate of isolation 26 (14.9%), then Escherichia coli 21 (12%) Pseudomonus spp 20 (11.5%), Staphylococcus

20 (11.5%) Corynebacterium spp 18 (10.4%).

Bacillus spp, Citrobacter spp, Streptococcus spp and klebsiella pneumoniae were found to have low rates of isolation. (Table No. 17).

Distribution of the above organisms among different samples are shown in tables 18, 19 and 20.

The effect of refrigeration on the isolated bacteria are shown in table

(21).

Table (16): bacterial groups isolated from different stages of processing, spices, workers and machine

Isolates Gram Gram Gram Total

Type of positive negative positive

Samples Rods Rods cocci

Fresh meat 4 15 7 26 (15.4%) (57.7%) (26.9%) (14.9%) Minced meat at first 5 15 7 27 mincing (18.5%) (55.6%) (25.9%) (15.5%) Minced meat + spices 6 17 8 31 (19.4%) (54.8%) (25.8%) (17.8%) Minced meat at 6 17 8 31 second mincing (21.4%) (60.7%) (28.6%) (17.8%) From end product 7 17 11 35 (20%) (48.6%) (31.4%) (20%) Mincer machine 1 - 2 3 (33.3%) (66.7%) (1.7%) Spices 1 2 2 5 (25%) (50%) (50%) (2.9%) Workers 3 7 6 16 (18.8%) (43.8%) (37.5%) (9.2%) Total 33 90 51 174 (19%) (51.7%) (29.3%) (100%)

Table (17): percentage of different types of isolates in the total No. of samples

Type of isolates No. of isolates in the Percentage of isolates in total No. of samples the total No. of samples

Micrococcus spp 26 14.9%

E.coli 21 12%

Staphylococcus spp 20 11.5%

Pseudomonas spp 20 11.5%

Corynebacterium spp 18 10.4%

Bacillus spp 15 8.6%

Proteus spp 15 8.6%

Edwardsiella tarda 11 6.3%

Acinetobacter spp 10 5.8%

Citrobacter spp 8 4.6%

Streptococcus spp 5 2.9%

Klebsiella spp 5 2.9%

Total 174 100%

Table (18 a): Distribution of isolated bacteria genera among different types of samples

Types of samples Fresh meat Minced Minced Minced From end M meat at meat + meat at product m Genus first spices second Gram-positive bacteria micrococcus 3 3 4 4 5 (3.6%) (3.6%) (4.8%) (4.8%) (6%) ( Staphylococcus 3 3 3 3 5 (3.6%) (3.6%) (4%) (2.7%) (6%) Corynebacterium 3 3 3 3 4 (3.6%) (3.6%) (3.6%) (3.6%) (4.8%) Bacillus 1 2 3 3 3 (1.2%) (2.4%) (2.6%) (3.6%) (4%) ( Streptococcus 1 1 1 1 1 (1.2%) (1.2%) (1.2%) (1.2%) (1.2%0 Total 11 12 14 14 18 (13.1%) (14.3%) (16.7%) (16.7%) (21.4%) (

Table (18 b): Distribution of isolated bacterial genera among different type of samples:-

Types of samples Fresh Minced Minced Minced From end Min meat meat at first meat + meat at product mac Genus spices second Gram-negative bacteria

E.coli 3 3 3 3 3 (3.3%) (3.3%) (3.3%) (3.3%) (3.3%) Pseudomonas 4 4 4 4 4 (4.4%) (4.4%) (4.4%) (4.4%) (4.4%) Proteus 3 3 3 3 3 (3.3%) (3.3%) (3.3%) (3.3%) (3.3%) Edwardsiella 2 2 2 2 2 (2.2%) (2.2%) (2.2%) (2.2%) (2.2%) Acinetobacter 2 2 2 2 2 (2.2%) (2.2%) (2.2%) (2.2%) Citrobacter - - 2 2 2 (2.2%) (2.2%) (2.2%) Klebsiella 1 1 1 1 1 (1.1%) (1.1%) (1.1%) (1.1%) (1.1%) Total 15 15 17 17 17 (16.7%) (16.7%) (18.9%) (18.9%) (18.9%)

Table (19): Shows distribution of the organisms among different samples :

ORGANISMS NO. OF FRESH MEAT AT MEAT MEAT AT END IDENTIFIED MEAT FIRST MINCED SECOND PRODUCT MINCING +SPICES MINCING Micrococcus 14 2 2 2 2 2 lylae (14.3%) (14.3%) (14.3%) (14.3%) (14.3%) Micrococcus 8 1 1 1 1 2 luteus (12.5%) (12.5%) (12.5%) (12.5%) (25%) Micrococcus 4 - - 1 1 1 xerosis (25%) (25%) (25%) Corynebacterium 10 2 2 2 2 2 psudotuberculosis (20%) (10%) (10%) (20%) (20%)

Corynebacterium 8 1 1 1 1 2 xerosis (12.5%) (12.5%) (12.5%) (12.5%) (25%) Staphylococcus 6 1 1 1 1 1 epidermidis (16.7%) (16.7%) (16.7%) (16.7%) (16.7%) Staphylococcus 10 2 2 2 2 2 xylosus (20) (20) (20) (20) (20) Staph. Sciuri 4 - - - - 2 (50%) Bacillus 7 1 1 1 1 2 lichintiformis (14.3%) (14.3%) (14.3%) (14.3%) (28.6%) Bacillus 5 - 1 1 1 1 sphericus (20%) (20%) (20%) (20%) Bacillus 3 - - 1 1 - pantothenticus (33.3%) (33.3%) Streptococcus 5 1 1 1 1 1 faecalis (20%) (20%) (20%) (20%) (20%) Total 84 11 12 14 14 18

Table (20): shows distribution of the organisms among different sample:-

ORGANISMS NO. OF FRESH MEAT AT MEAT MEAT AT END IDENTIFIED MEAT FIRST MINCED SECOND PRODUCT MINCING +SPICES MINCING E.coli 21 3 3 3 3 3 (14.3%) (14.3%) (14.3%) (14.3%) (14.3%) Pseudomonas 10 2 2 2 2 2 aeruginosa (20%) (20%) (20%) (20%) (20%) Pseudomonas 10 2 2 2 2 2 alcaligenes (20%) (33.3%) (20%) (20%) Proteus 10 2 2 2 2 2 vulgaris (20%) (25%) (12.5%) (12.5%) Proteus 5 1 1 1 1 1 morgani (20%) (20%) (20%) (20%) (20%) Edwardsiella 11 2 2 2 2 2 tarda (18.2%) (18.2%) (18.2%) (18.2%) Acinetobacter 10 2 2 2 2 2 lowffi (20%) (20%) (20%) (20%) Citrobacter 8 - - 2 2 2 freundii (25%) (25%) (25%) Klebsiella 5 1 1 1 1 1 pneumoniae (25%) (25%) (25%) (25%) Total 90 15 15 17 17 17

Table (21): shows the effect of refrigeration at 4°C for 5 days on isolates

(from end products)

Type of isolates No. of isolates No. of isolates at Rate of at End product End product after isolation after at 0 time 5 days at 4 °C storage at 4°C to 5 days Micrococcus luteus 2 3 Increased Micrococcus xerosis 1 2 Increased Corynebacterium 2 1 Decreased xerosis Corynebacterium 2 1 Decreased pseudotuberculosis Staphylococcus 1 1 Constant Epidermidis Bacillus lichinoformis 2 2 Constant Bacillus sphericeus 1 1 Constant Streptococcus faecalis 1 3 Increased E.coli 3 1 Decreased Pseudomonas 2 3 Decreased aeruginosa Proteusmorgni 2 2 Constant Edwardsiella tarda 2 Zero Disappeared Citrobacter freundii 2 1 Decreased

C- Antibiotic sensitivity tests

The results of the antibiotics sensitivity tests are shown in tables. The antibiotics were Ampicillin (20mg), Co-trimoxazole (25 mg), Cephalexin (30 mg), Tetracycline (30 mg), Cefolaxime (30 mg), Cipofloxacim (5 mg),

Pefloxacin (5 mg), Oflxacim (10 mg), Cloxacillin (1 mg), Roxcythromycin (15 mg), Linomycin (2 mg) and Gentamycin (10mg).

The results showed that, the organism tested to be sensitive or resistant depending on the measurement of the diameter of the inhibition zone.

Most of organisms tested were highly sensitive to most of the antibiotics used but resistant to three antibiotics (Cloxacillin, Roxythromycin and

Linomycin).

Pseudomonas aeruginosa was resistant to all antibiotics used except

Gentamycin. Tables 22 and 23 shows antibiotics sensitivity tests to some isolates.

Table (22) : Antibiotics Sensitivity Tests to some gram-positive isolates:-

As BA PR TE CF CP PF OF C R L G Code (20 (20 (30 (30 (30 (30 (5 (10 X F M M mg mg mg mg mg mg m mg (5 (1 (2 (10 Organis ) ) ) ) ) ) g) ) m m m mg m g) g) g) ) Staph. 2.3 2.9 1.5 2c R R R R R R R 2 Epidemid cm cm cm m is Staph. 2.5 R R 2.3 1.2 2c 2c 2.1 R R R 2.4 Xylous cm cm cm m m cm cm Enterococ 2c R 1.5 1c R R 2c 2.4 R 1c R 1.6 cus m cm m m cm m cm Faecalis Coryneba 1.3 2.5 1.2 1.4 2c 2.2 2.2 2 R R R 2.9 cterium cm cm cm cm m cm cm cm Pseudotu berculosis

R : Resistant cm: Diameter of inhibition zone

CF: cefolaxime TE: Tetracycline CX: Cloxacillin AS: Ampicillin CP: Cipofloxacim RF: Roxcythromycin BA: Co-trimoxazole PF: Pefloxacim LM: Linomycin PR: Cephalexin OF: Oflaxacim GM: Gentamycin

• Figures in brackets indicate concentration of antibiotic.

Table 23: Antibiotics sensitivity tests to some gram-negative isolates :

As BA PR TE CF CP PF OF CX RF L GM Code (20 (25 (30 (30 (30 (5 (10 (5 (15 (15 M (10 Organis mg mg mg mg mg mg mg) mg mg) mg) (2 mg) m ) ) ) ) ) ) ) mg ) E.coli 1.7 2.5 2c 2c 2.3 2c 2.1c 2.1 R R R 2cm cm cm m m cm m m cm Proteus 2c 2.7 1.4 2.4 2.4 2.6 2.1c 2.9 R 1.2c R 1.3c Vulgaris m cm cm cm cm cm m cm m m Citrobac 1.7 2.5 1.5 2.4 2.3 2.5 2.5c 2.5 R R R 2cm ter cm cm cm cm cm cm m cm freundii Pseudo R R R R R R R R R R R 2 monas cm aerugino sa Klebsiel 2.4 3 1.8 2.2 2.5 2.5 2.5c 2.7 R 1.7 R 1.3c la cm cm cm cm cm cm m cm m pneumo niae Edwards 2.2 2.7 1.8 1.9 2.4 1.5 2.5c 2c R 1.2c 0.6 1.4c iella cm cm cm cm cm cm m m m m tarda Acineto 1.5 2.5 1.2 1.2 1.4 2.6 2cm 2c R R R 1.5c bacter cm cm cm cm cm cm m m lowffi

Fig : ( 3 ) Alpha haam blood agar medium

Figure ( 4 ) : Antibiotic sensitivity test to Corynebactenium pseudotueberculosis on nutrient agar medium after 24 hours incubation at 37o C

CHAPTER FOUR

DISCUTION

Camel's meat is an important and considered as a good source of protein in some Arab countries, that , may play an appreciable role in solving food problems " Ministry of animal Resources, (2001).

Bashir, (1992) reported that camel's meat is an important source of food poisoning. This attributed to the contamination obtained from its environment and during preparation till it reaches the consumers. Bacterial flora responsible of meat spoilage and illness due to consumption of camel meat, as a source of human protein, this made the study of bacteria associated from camel's meat is important.

In Sudan, the bacteriological studies for camel meat to investigate aerobic bacteria are very few, and even little so that, they need additional research.

This work was carried out to investigate aerobic bacteria that, may constitute a potential public health hazard due to consumption of camel's meat.

All organisms gave reactions used for identification which described by

Cowan and Steel, (1993). These reactions were based on morphological, cultural and biochemical properties.

This work illustrated bacteriologically a total of 157 samples (120 from

Elbugaa slaughter house and 37 from El Kadaro slaughter house). Twelve samples from all of the collected muscles samples at Elbugaa were found to be sterile also the sample was collected from carcass cutter machine at ElKadaro was sterile that was attributed to that carcass cutter machine which was washed by soap and hot water before and after work.

The isolates from all collected samples were grouped into three groups.

Gram-negative rods, gram-positive rods and Gram-positive cocci. The same isolates represented as percent. 30.3%, 31.6% and 38.1% at Elbgaa and 32.3%,

27% and 63% in ElKadaro. (table 8 and 12).

The bacteria isolated were Corynebaterium pseudotuberculosis,

Corynebacterium pseudodiphtheriticum, Corynebacterium ulcerans, Staphylococcus xylosus, Staphylococcus warneri, Staphylococcus caseolyticus,

Staphylococcus aureus, Staphylococcus schleferi, Streptococcus uberis,

Streptococcus disaglactiae, , Streptococcus sciuri, Streptococcus faecalis,

Micrococcus lylae, Micrococcus luteus, Micrococcus resources, Micrococcus ristinae, Bacillus lentus, Bacillusalvei, Bacillus licheniformis, Lactobacillus salivarius, Escherchia coli, Citrobacter freundii, Citrobacter koseri,

Citrobacter aerogenes, Pseudomonas aeruginosa, Pseudomonas putida,

Pseudomonas stutzeri, Edwardsiella tarda, Proteus vulgaris, Proteus morgani,

Klebsiella pneumoniae, Serratia plymthica, Serratia marcesscens,

Acinetobacter lowffi and Salmonella typhi.

The isolates were found in high rate in skin and surface of carcasses, hands of workers and knives. These findings in agreement with the findings of

Salih and Ibrahim, (1977) who, reported high rate of isolation of bacteria from hair, skin, hands of workers in Omdurman central abattoir.

Gram-negative bacteria showed a very low level of occurrence with exception of Escherichia Coli which had high incidence rate 43.5% at Elbugaa slaughter house and it had a level of 40% at ElKadaro slaughter house.

E.coli is an enterobacteria. It's presence in food indicates faecal contamination. (Omer, 1999).

E. coli is a normal inhabitant of the intestinal tract of man and animals,

(Gracey, 1980). Also he reported that, some of E.coli cause disease, one form being responsible for acute enteritis in infant and adult. Infection is acquired through the consumption of contaminated food stuffs. In our findings the appearance of high rate of E.coli in surface of carcasses, may be due to the exposure of the carcass to more handling and contact with workers naked hands and at Elbugaa the animals were slaughtered on floor which was very dirty with matter and dust. This is in agreement with

Hussien, (1987) who reported its presence in high rate in the thigh and shoulder of bovine slaughtered at Omdurman slaughter house.

Citrobacter spp. were isolated from surface of carcasses and internal cavity with high rate at Elbugaa slaughter house and with low rate from thigh and back muscl at ElKadar slaughter house.

Citrobacter koseri was isolated in high rate for citrobacter spp, it was found in surface of carcass, internal cavity, back muscle, workers's hands and was isolated only from saws. The findings were in agreement with Bashir,

(1992), who isolated C..koseri from prescapular, bronchial, mesenteric and suppramammary lymph nodes of camels slaughtered at ElGadaref slaughter house and Tambul slaughter area. Also Adam, (1989) isolated the organism from both apparently normal and abnormal bovine mesenteric, mediastinal and retropharyngeal lymph nodes, and it was isolated from pneumonic camels lungs in Somalia (Moallin& Zissin, (1990).

Gracey, (1980). Reported that Citrobacter spp. were common water and soil bacteria. The high rate to Citrobacter isolates indicated that contamination of meat may be due to contact of carcass with hands of workers or from knives or saws and from skins of animals. Proteus spp were isolated from all samples collected from Elbugaa except outer skin, worker's hands and knives , but were isolated from internal cavity, thigh and back muscle only at ElKadaro slaughter house. Proteus vulgaris and Proteus morgani isolated from Elbugaa, but Proteus vulgaris was isolated only at ElKadaro. These findings were in agreement with Salih,

(1971), who isolated Proteus spp. at rate of 100% from livers of camels slaughtered at Omdurman slaughter house. Also Shigidi, (1974) isolated the same organism from pneumanic lungs.

Pseudomonas aeuginosa was isolated from all samples collected from

Elbugaa except outer skin, shoulder and workers. At Elkadaro it was isolated from thigh, back, shoulder muscles and warker's hands.

Pseudomonas stutzeri was isolated from samples collected from Elbugaa

,but Pseudomonas putida was revealed from thigh, back and workers' hands at

Elkadaro. These findings were in agreement with the findings of Bashir, (1992) who isolated ps.aruginosa and Pseudomonas putida from mesenteric, bronchial and prescapular lymph nodes of camels slaughtered at ElGadaref and Tambul area. Also our findings in agreement with Salih (1971), who isolated pseudomonas spp at rate of 35.4% from camel's livers slaughtered at Omurman slaughter house.

Gracey, (1980) reported that, pseudomonas spp. widely distributed in soil, fresh water, sea water and decomposing matter. Bashir, (1992) reported that, pseudomonas spp as a cold living and tolerant bacterium could play considerably important role in meat spoilage. Klebsiella spp, Serratia spp, Acinetobacter spp, and Edwardsiella tarda were isolated in low rates from different samples collected at Elbugaa and

ElKadaro slughter house.

Salmonella typhi was isolated only once from worker's hands at Elbugaa and it was not isolated from any other samples. That is in agreement with standards of international commission of microbiological specification for food

(ICMSF) which recommended microbiological limit for meat equal zero for salmonella (Jay, 1986).

Gram-positive bacteria isolated in Elbugaa and ElKadaro slaughter houses comprised of 35.6% Gram-positive cocci and 31.6% gram-positive bacilli at Elbugaa and 40.7% gram positive cocci and 27% Gram-positive bacilli in ElKadaro.

Corynebacterium spp were found in high rate in both Elbugaa and

Elkadar. It was reported in high numbers in outer skin and surface of carcass.

These findings were in agreement with Jay, (1970), Haines (1933); Empey and

Scott, (1939) and Salih & Ibrahim, (1977).They reported that the hide and hair of the animal are they important sources of contamination of the carcass during skinning.

Corynebaterium spp were comprised of C.pseudotuberculosis,

C.pseudodipherticum, C.ulcerans and C.xerois from Elbugaa S.H . These findings were in agreement with Shigidi, (1973), who isolated

Corynebacterium spp from camel bronchial lymph nodes and with Bashir, (1992), who isolated it from lymph nodes of camels slaughtered at ElGadref slaughter house and Tambul area.

Bacillus spp have higher rate of isolation at Elbugaa , 33 isolates (6%) than ElKadaro 12 isolates (2.2%). It was isolated from outer skin, surface of carcass, workers' hands, knives and only one from saws.

Bacillus spp. isolates were Bacillus lentus, B.licheniformis and Bacillus alvei, they are non pathogenic spp. and have a wide distribution in nature human, animals, soil, water and food. (Omer, 1990).

Lactobacillus salivarius was isolated in all samples except workers' hands, knives and saws at Elbugaa, and from thigh, and back muscle only at

Elkadaro. This finding was not reported in the literature so that, it may be the first finding in camels. Gram-positive cocci isolated were found in high rate in both Elbugaa and Elkararo slaughter house (35.6% in Elbugaa and 40.7% in

ElKadaro slaughter house).

Staphylococcus caseolyticus was found in high rate in all collected samples except outer skin and surface of carcass at Elbugaa, and it was not found in any sample collected from Elkadaro.

Staphylococcus epidermidis and S.lentus were found in most of samples collected from both areas except outer skin. S.epidermids showed highest rate of isolation in internal cavity and thigh muscle at Elbugaa.

S.xylosus was isolated from three samples only " surface of carcass, internal cavity and workers" at Elbugaa, but at ElKadaro was isolated from all samples except outer skin and knives, that may be due to different sources for contamination. S.warneri was isolated in low rate from both areas.

S.aureus was revealed from four internal cavities of camels slaughtered at Elbugaa, and not found in ElKadaro. It was the more important bacteria isolated. This bacteria produces toxin and introtoxin, which have powerful effect on man and resulted in vomiting with in few hours, diarrhoea, sever abdominal pain and some times leads to collapse (Gracey, 1980). These findings were in agreement with some researchers who, isolated it from many pathological conditions of camels. Shigidi, (1973) isolated Co.agulase negative staphylococci from lung tissues of camels slaughtered at Tambul. Abdel

Rahim, (1990) isolated S.aureus from the prepectoral and prescapular lymph nodes of camels slaughtered at soug ElJ'ama "Omdurman".

Staphylococcus spp. also can cause pyogenic bacterial infection e.g. contagious skin necrosis. (Sana, 1996, and Domench, 1977).

Some species of staphylococcus are common cause of food poisoning than others, that depend on the type of entrotoxin produced. This condition occurs when food is left too long at room temperature. (Gracey, 1980).

Streptococci were found in high rate in outer skin at both Elbugaa and

ElKadaro samples.

Streptococcus spp. were comprised of S.agalactiae, S.faecales,

S.disagalactiae, S.sciuri. and S.uberis at Elbugaa.

Streptococcus pneumoniae, S.faecalis, S.uberis and S.lentus at Elkadaro. Also Streptococcus spp were found in diseases or pathological conditions. Shigidi, (1973) isolated it from Respiratory tract, Bashir, (1992) isolated it from lymph nodes of camels slaughtered at ElGadaref and Tambul area, Fahmy, (1990) isolated the organism from vaginal swabs and endrometritis and Sana, (1996) isolated it from contagious skin necrosis.

Our findings were in agreement with Bashir, (1992), who reported that streptococcus spp. were mostly found in the animals body awaiting the suitable moment to play a pathogenic role.

Micrococcus spp. isolates included M.lylae, M.roseus. M.luteus and micrococcus kristinae at Elbugaa slaughter house, but at Elkadaro only

M.roseus and M.luteus were isolated.

These findings were in agreement with Fahmy, (1990) who isolated the organism from camel slaughtered at Cairo and with Bashir, (1992) who isolated

M.reseus and M.luteus from camels slaughtered at ElGadaref and Tambul.

Also were agreed with Intisar, (1998) who isolated it at the preparation level of meat.

Finally we noted that during dressing of the carcass of camel it is exposed to heavy contamination from abattoir environment including implements used and the hands of the operators, also from hides of animals, stomach and gastrointestinal contents, these are attributed to that sanitation and

HACCP system which reduced contamination are not considered at abattoirs.

These notes are in agreement with Salwa, (2004) who, reported that high rate of bacteria of beef samples collected from Khartom Airport at shipping time, which is due to lack of HACCP system during handling and slaughtering inside the abattoir.

In processing of camel's meat, some isolates were also reported during processing, but not found in fresh meat samples. This indicated that it might be contaminated during preparation (Wllson, 1994).

This contamination was due to different sources like contact with hands of workers, machines equipments and addition of additives and spices. (Frazier and Westhoff, 1988).

The bacteria isolated were comprised of micrococcus spp.,

Staphylococcus spp, Streptococcus spp, Corynebacterium spp , Bacillus spp,

E.coli, Acinetobacter spp , Edwardsiella, Pseudomonas and Klebsiella. Spp.

These findings were in agreement with Intisar, (1998), Amanie, (2000) and Jay,

(1986). Table (19&20 ).

The common microorganisms which were isolated included micrococcus spp and Escherichia coli. Micrroccus has a wide distribution in nature (human, soil, water, animals and food) and exist as saprophyte. (Frazier

& Westhoff 1988and Omer, 1990).

The Pseudomonas and staphylococcus spp formed the second rate of isolates, that might be due to different sources of contamination. "naked hands of workers" (Frazier & Westhoff, 1988).

Pseudomonoas, Corynebacterium, Bacillus, Edwardsiella, Citrobacter,

Streptococcus and Klebsiella spp were reported to cause different types of spoilage of meat. (Frazeir &Westhoff, 1988). Pseudomonas and Acinetobacter spp were found to be primary causes of spoilage of fresh type of food kept at -3°C (Neckerson & Sinikey, 1974).

Some isolates were found in workers, spices and mincer machines, that may caused contamination of meat.

In our findings some isolates were found in processed meat but not found in fresh meat which was obtained from market. The fresh meat was exposed to more sources of contamination including trucks, containers, cloth, and hands of workers . (Frazier and Westhoff, 1988).

The minced meat samples did not show an increase in rate of bacterial isolates than fresh meat.

This was not in agreement with Fatima, (1990) who found a decrease in rate of bacterial isolates in the minced meat samples. She attributed that to the damage of bacterial cell on freezing and temperature shock results in the death of some theromphiles and mesophiles. (Jay, 1986).

Mixture samples and end products from meat samples showed increased in rate. This in agreement with Fatima, (1990) and Amanai (2000), who, reported that meat surface is subjected to many sources of contamination before it reaches the consumers as processed meat.

Some isolates were increased in level at end product samples after storage for five days at 4°C (refrigerator).

Micrococcus spp showed an increase in rate of isolation after refrigeration at 4°C. also Streptococcus faecalis and Pseudomonas aeruginosa were increased that was in agreement with Empey and Scott, (1939) who reported that Pseudomonas spp were responsible for spoilage of refrigerated meat.

E.coli, Corynebacterium spp and Citrobacter freundii showed a decrease in the rate of isolation after storage. But Staphylococcus epidemidis and Bacillus lichinoformis, Bacillus sphericus and Proteus morgani still has a constant level after refrigeration.

Edwardsiella tarda can not be isolated after refrigeration at 4°C for 5 days.

These findings were in accordance with Hussien, (1987), and Ingram

&Dainty, (1971) who reported that under chill conditions 0°-4°C . mesophiles will not be able to grow and the few psychrotrophic grow relatively very slowly, under this condition spoilage of meat is there fore usually caused by the development of a more or less aerobic psychrotrophs predominantly on the surface.

In our study of the antibiotics sensitivity tests that carried out for some isolates from fresh meat which was used in processing, the following antibiotics used were Ampicillin (2o mg), Co. Trimoxazole (25 mg), cephaloxin (30 mg), Tetracycline (30 mg), Cefotaxime (30), Ciprofloxacin

(5mg), Pefloxacin (10mg), Ofloxacin (5mg), Claxacillin (1mg)

Roxythromycin, (15 mg), Lincomycin (2 mg), Gentamycin (10mg).

All gram-positive and gram-negative isolates were sensitive to

Gentamycin (10 mg) and Ampicillin (20 mg) Tables (22& 23). Our findings also revealed that, all gram-positive bacteria tested were sensitive to tetracycline (30 mg), that means tetracycline may be efficient for therapy in gram positive infections, these were in agreement with Lubna,

(2003).

Staphylococcus epidermidis was sensitive to Ampicillin (20 mg),

Gentamycin. (10m) Tetracycline (30 mg), Co.Trimoxazole (25 mg) and

Cephaloxin (30 mg), but Staphylococcus sciuri was sensitive to all antibiotics except cloxacillin (1 mg) and linomycin (2 mg). staph. Xylosus was highly sensitive to tetracycline and gentamycin.

Pseudomonas aeruginosa was resistant to all antibiotics used except

Gentamycin. This finding was in agreement with Quinn, et al, (2000) who reported that, Ps. Aeruginasa is resistant to many commonly used antibiotics.

From the results it was observed that, most of the organisms tested were highly sensitive to most of the antibiotics used. This indicated that the slaughtered animals have not received antibiotics for therapy.

CONCLUSION

In this study I observed that, the rate of bacterial isolation from samples collected from skins and surface of carcass of camels slaughtered at Elbugaa slaughter house compared to that, collected from ElKadaro slaughter house was attributed to the unsatisfactory hygienic measures, to poor hygienic level of equipments and personnel and to the slaughtering of animals on the floor with poor drainage system, that lead to heavy contamination. The public health implications were not considered and precautionary measures were not institute in both local and export slaughter houses.

Also in processing or in the meat industry I observed that, the spices, workers and machines were important sources of contamination of meat during processing. Storage of processed meat at 4°C led to an increase in rate of isolated organisms and to the decrease in rate of other isolates.

Sensitivity to antibiotics revealed that, Gentamycin & Ampicillin were more effective in Gram-positive and Gram-negative bacteria infections.

RECOMMENDATIONS

1. During skinning and exposure of the carcass, the external surface of

the hide should not contact the carcass meat

2. The equipments used during slaughtering , and dressing must be

sterilized to avoid contamination or cross contamination

3. The viscera are not opened during the dressing procedures or during

evisceration

4. The opened viscera must be spread quickly from the dressing area to

avoid contamination of the floor

5. If any part of carcass is contaminated with faeces or visceral content

should be trimmed off.

6. strict hygiene measures in slaughter houses

7. Education of personnel on the importance of general and personal

hygiene " wash hands before start of work, when re-entering work area or after any other situation that will cause the hands to become

dirty and became source of contamination or cross contamination

8. It is important for the meat industry to follow the international

standards of hygiene for processed meat for human safety.

9. Rehabitation of slaughter houses and modern equipments and

technical facilities which are used in slaughtering operations must be

applied

10. Chemicals as pesticides should be used for pest control with the

required supervision in " Restricted use" and by personnel with the

required training in " General use"

11. The workers should wear the gloves during work

12. Uniforms of workers should be clean at the start of work and should

be changed when they become dirty

13. Cleaning and disinfection of knives. With safe chemicals and hot

water

14. Cleaning carcass surface by using potable running water

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