2. DETECTION OF AFLATOXINS FROM ANTIFUNGAL RESISTANT STRAINS IN ANIMAL TISSUES AND THEIR PRODUCTS
تحديد سموم الافلاتوكسين من السلالات المقاومة للمضادات الفطرية من أنسجة ومنتجات الحيوانات By
Sameh Farouk Ali

3. Under Supervision of Dr. Mohamed Nabil Hassan
Professor of Microbiology,
Faculty of Veterinary Medicine,
Zagazig University
Dr. Atef Abdel Aziz Hassan
Chief Researcher and Head of Mycology and Mycotoxins Dep.,
Animal Health Research Institute, Doki, Giza

4. INTRODUCTION

5. The term mycotoxin is a combination of greek word “mykes” means fungus and latin word ”oxicum” meaning poison. The word “mycotoxin” is used for the toxic chemical secondary fungal products synthesized within and produced by filamentous fungi that readily colonize crops in field or after harvest and transport or during storage and consumption.

6. The disease was traced to moldy ration and in remarkably short time the responsible mould A. flavus and its toxic metabolites aflatoxins were isolated. Not all strains of A. flavus are aflatoxin producers, several strains are non toxogenic because aflatoxin synthesis may become unstable in these fungi.

7. Aflatoxins have attracted considerable attention after the discovery of a direct causes effect relationship between dietary levels of some alfatoxins particularly B1 and liver cancer (hepatocarcinoma).

8. It well established that aflaxtoxin B1 is among the most natural liver carcinogenic agents known. Studies in Kenya, Uganda, Sweziland, Thailand and China demonstrated strong significant positive correlation between estimated aflatoxin intake or aflatoxin level in food samples and the incidence of liver cancer.

9. Animal are variably susceptible to aflatoxins, depending on such factors as age, species, bread, sex, nutrition and certain stress. Cattle, poultry are the domestic species of greatest economic concern. The evidence of aflatoxicosis in general are unthriftiness and only some reduction in weight gain, feed efficiency, immunity and production.

10. Mycotoxin contamination in animal products may occur as tissue residue in organs and muscle tissue of beef and in meat of chicken follow administration of mycotoxins. Mycotoxin metabolic products contaminated feeds are also present in dairy products when animal fed aflatoxosin B1, the hydroxilated metabolic product M1 in milk, which approximately have the same level of toxicity as B1.

11. Milk is the animal product most vulnerable to aflatoxin contamination particularly since consumption of mycotoxins contaminated meat and lactation are concurrent event i.e. there is no withdrawal period.

12. Indeed, the content of aflatoxin M1 in milk is a good indicator of the level of aflatoxin B1 in the feed. Cheeses as milk product can not escape mycotoxin contamination especially aflatoxins by the virtue of its affinity to bind to milk protein casein

13. On the other hand, direct growth of toxigenic fungi on a rich nutrient source as meat and milk favour the production of mycotoxins on and within such commodities.
Natural spices are desirable in many feed give flavor and good taste, unfortunately representing a health hazard when contaminated with mycotoxigenic fungi.

14. Aflatoxins considered the most important and prevalent mycotoxins produced by A. flavus and A. parasiticus. Four naturally occurring aflatoxins and their metabolites are AFB1, AFB2, AFG1, AFG2, AFM1 and AFM2.
It is worth to mention that heat treatment as cooking and roasting unable to destroy aflatoxins in food.

15. Where the feed hygienic system require solution different from that which has been effective in the developed countries. The reason why detecting contamination in sample affordable ways is of critical importance.

16. Aims of the study
This study aimed for detection of aflatoxins in antifungal resistant strains in animal tissues and their products, to achieve this goal the following point were investigated:
(1) Isolation and identification of fungi that contaminate cattle meat and milk and their products.
(2) Antifungal sensitivity of A. flavus and A. parasiticus isolates to some commercial antifungal agents.

17. (3) Screening of the resistant strains of A. flavus and A
(3) Screening of the resistant strains of A. flavus and A. parasiticus for aflatoxin production.by fluorescence screening on coconut agar media (CAM).
(4) Qualitative assay of aflatoxins in collected samples by thin layer chromatography (TLC).
(5) Quantitative estimation of aflatoxins in collected samples by a Immunoaffinity-Based Method by Fluorometry.

18. MATERIAL AND METHODS

19. MATERIALS
Samples
A total number of 600 samples were collected from meat and milk of cattle and their products comprising (50) frozen meat, (50) minced meat, (50) liver, (50) kidney, (50) luncheon, (50) sausage, (50) hawawchi, (50) raw milk, (50) yoghurt, (50) hard cheese, (50) fresh kareish cheese and (50) samples of fresh damietta cheese

20. Culture media Sabouraud’s dextrose agar (SDA)
Used for primary isolation and identification of fungi
Czapeck -dox agar
Used for brief identification of Aspergillus species
Yeast extract Sucrose medium
Used to enhance aflatoxin production
Coconut Agar Media (CAM)
Used for the detection of aflatoxin
Potato dextroseagar media (PDA)
Used for cultivation of Aspergillus species

21. Reagents Stains Phosphate buffer
Used for prepration of spore suspension
Peptone water
Used for dilution of samples
Stains
Lactophenol cotton blue stain
Used for the morphological studies of moulds

22. Mycotoxins standard solution for TLC
Aflatoxins standard B1, B2, G1, G2and M1
Thin Layer Chromatographic (T.L.C.)
Fluorometer
Antifungal agents
Sorbic, propionic and benzoic acids

23. METHODS
Isolation and identification of fungi from the genus Aspergillus
Preparation of sample homogenate
Dilution
Plate pouring
Incubation

24. Identification of moulds
Macroscopic
Microscopic
Direct exam.
Slide culture technique
Determination of the resistant isolates of A. flavus and A. parasiticus to the selected antifungals
Preparation of spore suspension of isolates

25. Determination the potency of antifungal drugs by detection of fungus growth
Screening of resistant isolated strains of A. flavus and A. parasiticus for aflatoxins production
Fluorescence screening on coco nut agar media (CAM)
Extraction of aflatoxins by glass beads extraction method from meat, milk and their products samples
Preparation of aflatoxin B1,B2, G1 and G2 standards solutions

26. Estimation of aflatoxins
Qualitative estimation of aflatoxins by thin layer chromatography
Preparation of silica gel suspension
Coating of glass plates
Spotting and resolution

27. Quantitative estimation of aflatoxins by a Immunoaffinity-Based Method by Fluorometry
Calculation
Sample extraction
Dilution
Purification
Elution and reading

28. ●Aflatoxin, ■ , ▲ impurities
Fig. (1): Schematic illustration of the steps of fluorometeric immuno affinity method

29. Immunoaffinity Abs that is immobilized on solid support (agaros gel) in ph buffer all in plastic bridge. The sample extract is applied to an immuno affinity column (IAC) containing specific Abs to aflatoxin. The aflatoxin bind to Ab and water is passed through the column to remove any immpurities. Then passing solvent as methanol through the column, captured aflatoxin is removed from Ab and thus eluted from the column. The aflatoxin fluorescence in the methanol can be measured, substance to enhance fluorescence can be added

30. RESULTS

31. Total mould count (TMC) of the examined samples:
Mycological examination of meat and meat products
Total mould count (TMC) of the examined samples:
It was noticed that the highest total mould count (TMC) was obtained from the sausage samples, followed by hawawchi samples, luncheon and minced meat; whereas the frozen meat samples yielded the lowest count of mould.

32. Type of examined sample Total moulds count (TMC)
Table (1): Total mould count in meat and meat product samples
Type of examined sample
Total moulds count (TMC)
Min
Max
Mean
± SE
Frozen meat
1X 102
2.60 X 103
2 X 102
1.20 X 102
Minced meat
1.50 X 104
1.60 X 103
6.00X 102
Liver
2X 102
4.60 X 103
4.5 X 103
9.50 X 103
Kidney
3.00 X 103
2.20 X 103
2.97 X 103
Luncheon
4.50 X 104
3.50 X 103
3.30 X 103
Sausage
5.40 X 105
4.20 X 104
1.25 X 104
Hawawchi
3.50 X 102
3.60 X 105
5.20 X 104
4.21x 104

33. Fig. (2) Total mould count in meat and meat product samples

34. Prevalence of moulds in meat and meat product samples:
It is evident that the most commonly isolated mould species in the examined frozen meat were Aspergillus spp. (70%), followed by Penicillium spp. (50%), Alternaria spp. (4%), Mucor spp. Cladosporium spp. Rhizopus spp., Scopulariopsis spp., and Curvularia spp. (2% for each) were rarely isolated.

35. Table (2): Prevalence of moulds in meat and meat product samples
Type of sample
Identified
mould spp.
Frozen meat
Minced meat
Liver
Kidney
Luncheon
Hawawchi
Sausage
No.
(50) %
Aspergillus spp.
35\50 70
50/50
100
37/50 74 25 50 39 78 40 80
Penicillium spp. 10 20 8 16 19 38 17 34
Alternaria spp. 2 4 1 15 30
Cladosporium spp. 3 6 7 14
Curvularia spp.
2.0
Fusarium spp.
4.0
Mucor spp. 5 12
Rhizopus spp.
Scopulariopsis spp.

36. Fig. (3) Prevalence of mould species in meat and meat product samples

37. Frequency of identified Aspergillus species isolated from examined meat and meat products:
It is clear that the examined minced meat and liver samples had the highest total isolated Aspergillus spp. The most common isolates of Aspergilli in frozen meat were A. flavus (30%), A.niger and A. candidus (24% for each), while A.versicolor was recovered from (8%) and A.fumigatus from (4%). Whereas, A. ochraceus and A. parasiticus were present in few of samples (2% for each).

38. Table (3): Frequency of identified Aspergillus spp
Table (3): Frequency of identified Aspergillus spp. isolated from the examined meat and meat products
Type of sample
Aspergillus spp.
Frozen meat
Minced
meat
Liver
Kidney
Luncheon
Hawawchi
Sausage
No.
(50) %
A. candidus 12 24 10 20 11 22 9 18 7 14 3 6 13 26
A. flavus 15 30 17 34 16 32
A. parasiticus 1 2 4 5
A. fumigatus 8
A. niger 28
A. ochraceus
A. terreus
A. versicolor

39. Fig. (4) Frequency of identified Aspergillus spp
Fig. (4) Frequency of identified Aspergillus spp. isolated from the examined meat and meat products

40. Total mould count of the examined in milk and milk product samples:
Mycological examination of milk and milk products
Total mould count of the examined in milk and milk product samples:
It was noticed that the highest total mould count was obtained from the Fresh Damietta cheese samples, followed by Fresh Kareish cheese samples, hard cheese, yoghurt whereas the Raw milk samples yielded the lowest count of mould.

41. Type of examined sample
Table (4): Total mould count in milk and milk products
Type of examined sample
Total moulds count /g
Min
Max
Mean
± SE
Raw milk
1X 102
2.60 X 102
6.10 X 10
1.20
Yoghurt
0.3 X 102
1X 103
1.60 X 102
1.000
Hard cheese
2.50 X 103
0.50 X 103
2.30
Fresh Kareish cheese
8X 102
1X 104
3.00 X 103
1.25
Fresh Damietta cheese
5 X 102
3.20 X 103
5.20 X 103
2.10

42. Fig. (5): Total mould count of the examined in milk and milk product samples

43. Prevalence of moulds in milk and milk products:
Genera were isolated from examined raw milk represents by genus Aspergillus. (30%), followed by Penicillium spp. (24%), Alternaria spp. (4%), Mucor spp. Cladosporium spp., Scopulariopsis spp. (2% for each).

44. Table (5): Prevalence of moulds in milk and milk product samples
Type of sample
Identified
mould spp.
Raw milk
Yoghurt
Hard cheese
Fresh Kareish cheese
Fresh Damietta cheese
No.
(50) %
Aspergillus spp. 15 30 18 36 19 38 72 35 70
Penicillium spp. 12 24 10 20 17 34 40
Alternaria spp. 2 4 1 5
Cladosporium spp. 3 6
Botryotrichum spp.
Curvularia spp.
Mucor spp. 8 16
Scopulariopsis spp.

45. Fig. (6): Prevalence of moulds in milk and milk products

46. Frequency of identified Aspergillus species isolated from examined milk and milk product samples:
It is clear that the examined milk and milk product samples were predominated, respectively Aspergillus spp.. The most common isolated spp. of Aspergillus from raw milk was A. flavus (20%) A. niger (16%), A. candidus (8%), A. fumigatus (4%) and lastly by both. A. ochraceus and A. parasiticus were (2% for each).

47. Table (6): Frequency of identified Aspergillus species isolated from the examined milk and milk product samples
Type of sample
Aspergillus spp.
Raw milk
Yoghurt
Hard cheese
Fresh Kareish cheese
Fresh Damietta cheese
No.
(50) %
A. flavus 10 20 9 18 5 11 22 36
A. parasiticus 1 2
A. fumigatus 4 3 6
A. niger 8 16 12 24 15 30 17 34
A. ochraceus
A. candidus

48. Fig. (7): Frequency of identified Aspergillus spp
Fig. (7): Frequency of identified Aspergillus spp. isolated from the examined milk and milk product samples

49. Photo (1): A. flavus on SDA agar showing yellow green surface colour and granular consistency
Photo (2): Microscopy of A. flavus showing radiated head, biseriate phialide and rounded vesicle

50. Photo (3): A. fumigatus on SDA with blue- green to gray surface colour and velvety texture
Photo (4): Microscopy of A. fumigatus with uniseriated columnar head flask shape vesicle and short smooth conidiphore

51. Photo (5): A. ochraceus on SDA with buff or golden yellow surface colour
Photo (6): Microscopy of A. ochraceus showing rounded, biseriated head and large septated conidiophores

52. Photo (7): Fusarium species on SDA with cottony texture, white surface colour and rose pigments on the center
Photo (8): Microscopy of Fusarium species with characteristic slender, multicelled macroconidia and microconidia

53. Photo (9): Microscopy of Rhizopus species showing long, branched sporangio-phores and terminate with rhizoids
Photo (10): P. citrinum on SDA showing deep bluish green surface colour and velvety texture

54. ITS based PCR for differentiation Aspergillus species from the other member of genera
The molecular diagnosis for the critical mycotoxigenic species (A. flavus and A. parasiticus) is considered, with utilizing unique internal transcribed spacer (ITS-1) regions for genus and species-specific detection for identifying Aspergillus species producing aflatoxin based on oligonucleotides.

55. Eleven isolates of identified Aspergillus species that isolated from frozen meat, liver, kidney, hawawchi, sausage, cheese, fresh kariesh cheese, yoghurt and raw milk were selected for genotypic identification and amplified product was obtained at 195 bp.

56. Photo (11): Representative electrophoretic band patterns of PCR products for ITS region of Aspergillus species, lane M: molecular size marker, lanes 1- 11: Aspergillus species at 195 bp

57. Screening of aflatoxin production from resistant strains of A
Screening of aflatoxin production from resistant strains of A. flavus and A. parasiticus on Coco nut agar media (CAM)
For determination of aflatoxin production based on presence or absence of fluorescence on the reverse side of the colonies was determined by exposing the petri dishes to u.v (365 nm) and expressed as positive (blue fluorescence) or negative.

58. Photo (12): Aflatoxin producing A
Photo (12): Aflatoxin producing A. flavus on showing blue fluorescence on reverse side of the plate after exposure to u.v. rays
Photo (13): Negative control CAM plate

59. Potency of Benzoic acid:
Detection of potency of antifungal drugs against A. flavus and A. parasiticus isolated from meat and its products
Potency of Benzoic acid:
When the concentration of benzoic acid increased gradually from µg/ml, the growth of mould was inhibited. Considering frozen meat, when the antifungal benzoic acid increased in concentration from µg/ml, the growth of Aspergilli were particularly inhibited.

60. Where growth of 6/15 isolates of A
Where growth of 6/15 isolates of A. flavus was inhibited by benzoic acid at concentration of 3.00 µg/ml. But 9/15 isolates were resistant to this antifungal. On the other hand A. parasiticus isolates were resistant.

61. Potency of Sorbic acid:
The growth of the majority of A. flavus and A. parasiticus isolates were inhibited by propionic acid at concentration of 3 µg/ml. reminder were resistant to this antifungal.
The growth of the majority of A. flavus and A. parasiticus isolates were inhibited by propionic acid at concentration of 3 µg/ml. reminder were resistant to this antifungal

62. Table (7): Potency of benzoic acid against fungi isolated from meat and its products
Source of isolation
Tested strains numbers
Number of sensitive and resistant strains at different concentrations of tested antifungals
0.5
0.75
1.0
2.0
3.0 S R
Frozen meat
Aspergillus flavus (15) 14 1 - 15 3 12 5 10 6 9
Aspergillus parasiticus (1)
Minced meat
Aspergillus flavus (17) 17 7
Aspergillus parasiticus (2) 2
Liver
Aspergillus flavus (16) 16 11
Aspergillus parasiticus (3)
Kidney
Aspergillus flavus (11)
Aspergillus parasiticus (5) 4
Luncheon
Sausage
Hawawchi
Aspergillus flavus (12)

63. Table (8): Potency of sorbic acid against fungi isolated from meat and its products
Source of isolation
Tested strains numbers
Number of sensitive and resistant strains at different concentrations of tested antifungals
0.5
0.75
1.0
2.0
3.0 S R
Frozen meat
Aspergillus flavus (15) 8 7 9 6 15 -
Aspergillus parasiticus (1) 1
Minced meat
Aspergillus flavus (17) 11 12 5 17
Aspergillus parasiticus (2) 2
Liver
Aspergillus flavus (16) 10 16
Aspergillus parasiticus (3) 3
Kidney
Aspergillus flavus (11) 4
Aspergillus parasiticus (5)
Luncheon
Sausage 14
Hawawchi
Aspergillus flavus (12)

64. Table (9): Potency of propionic acid against fungi isolated from meat and its products
Source of isolation
Tested strains numbers
Number of sensitive and resistant strains at different concentrations of tested antifungals
0.5
0.75
1.0
2.0
3.0 S R
Frozen meat
Aspergillus flavus (15) 8 7 9 6 15 -
Aspergillus parasiticus (1) 1
Minced meat
Aspergillus flavus (17) 10 11 12 5 17
Aspergillus parasiticus (2) 2
Liver
Aspergillus flavus (16) 4 16
Aspergillus parasiticus (3) 3
Kidney
Aspergillus flavus (11)
Aspergillus parasiticus (5)
Luncheon
Sausage
Hawawchi
Aspergillus flavus (12)

65. Detection of Potency of antifungal drugs against A. flavus and A
Detection of Potency of antifungal drugs against A. flavus and A. parasiticus isolated from milk and its product
Potency of Sorbic acid
When the concentration of Sorbic acid increased gradually from µg/ml, the growth of mould was inhibited, where Sorbic acid at concentration of 3 µg/ml inhibit the growth of most A. flavus isolates while the others were resistant.

66. Potency of Sorbic acid
On the other hand, all A. parasiticus isolated from different milk and its products were sensitive to the used sorbic acid effect.
Potency of Benzoic acid
When the concentration of benzoic acid increased gradually from µg/ml, the growth of mould was inhibited. MIC of benzoic acid was 3 µg/ml as the growth of most A. flavus isolates were inhibited by this concentration.

67. Potency of Benzoic acid
All isolated A. parasiticus from different milk and its products were sensitive to the used benzoic acid antifungal effect.
Potency of Propionic acid
Most of isolated A. flavus and A. parasiticus isolates were sensitive to treatment with different concentrations of propionic acid.

68. Growth of isolates isolated from raw milk and hard cheese were inhibited by 3µg/ml of propionic acid. In yoghurt, fresh Kareish and fresh Damietta cheese samples MIC of propionic acid was 2 µg/ml. All isolates of A. parasiticus were sensitive to the used propionic acid antifungal effect.

69. Tested strains numbers Aspergillus parasiticus (1)
Table (10): Potency of sorbic acid against fungi isolated from milk and its product
Source of isolation
Tested strains numbers
Number of sensitive and resistant strains at different concentrations of tested antifungals
0.5
0.75
1.0
2.0
3.0 S R
Frozen meat
Aspergillus flavus (10) - 10 4 6
Aspergillus parasiticus (1) 1
Youghurt
Aspergillus flavus (9) 5 3 8 9
Hard cheese
Aspergillus flavus (5) 2
Fresh karesh
Aspergillus flavus (11) 11
Fresh damietta
Aspergillus flavus (18) 14 13 17 18

70. Tested strains numbers Aspergillus parasiticus (1)
Table (11): Potency of benzoic acid against fungi isolated from milk and its product
Source of isolation
Tested strains numbers
Number of sensitive and resistant strains at different concentrations of tested antifungals
0.5
0.75
1.0
2.0
3.0 S R
Frozen meat
Aspergillus flavus (10) 4 6 10 -
Aspergillus parasiticus (1) 1
Youghurt
Aspergillus flavus (9) 5 9
Hard cheese
Aspergillus flavus (5) 2 3
Fresh karesh
Aspergillus flavus (11) 11
Fresh damietta
Aspergillus flavus (18) 13 7 18

71. Tested strains numbers Aspergillus parasiticus (1)
Table (12): Potency of propionic acid against fungi isolated from milk and its product
Source of isolation
Tested strains numbers
Number of sensitive and resistant strains at different concentrations of tested antifungals
0.5
0.75
1.0
2.0
3.0 S R
Frozen meat
Aspergillus flavus (10) 2 8 3 7 4 6 10 -
Aspergillus parasiticus (1) 1
Youghurt
Aspergillus flavus (9) 5 9
Hard cheese
Aspergillus flavus (5)
Fresh karesh
Aspergillus flavus (11) 11
Fresh damietta
Aspergillus flavus (18) 18

72. Prevalence of resistant strains of A. flavus and A
Prevalence of resistant strains of A. flavus and A. parasiticus for all tested antifungals that isolated from meat and meat products samples
The high incidence of the resistant strain of A. flavus to the antifungal agents (that were isolated from sausage (64.7%), minced meat (58.8%), hawawchi (58.3%) liver (56.2%) and kidney (54.5%) the resistant strains of A.flavus from Frozen meat and Luncheon samples were of low incidence (53.3%).

73. Percent of resistant strains of A.flavus and A. parasiticus
Table (13): Percent of resistant strains of A. flavus and A. parasiticus for all tested antifungals isolated from meat and meat products samples
Source of isolation
Percent of resistant strains of A.flavus and A. parasiticus
A. flavus
A. parasiticus
All tested strains R
% of R
Frozen meat 15 8
53.3% 1
100%
Minced meat 17 10
58.8% 2
50%
Liver 16 9
56.2% 3
33%
Kidney 11 6
54.5% 5
40%
Luncheon
Sausage
64.7%
Hawawchi 12 7
58.3%

74. Fig. (8): Prevalence of resistant strains of A. flavus and A
Fig. (8): Prevalence of resistant strains of A. flavus and A. parasiticus for all tested antifungals isolated from meat and meat products samples

75. Prevalence of resistant strains of A. flavus and A
Prevalence of resistant strains of A. flavus and A. parasiticus for all tested antifungals isolated from raw milk and milk products samples
The majority of resistant strain of A. flavus to antifungal agents used were recovered from yoghurt (44.4%), hard cheese (40%), fresh Kareish cheese (27.3%), fresh Damietta cheese (22.2%) and that isolated from raw milk (20%), No resistant strains of A. parasiticus were detected, all the isolates were sensitive to used antifungal agents Sorbic, Benzoic and Propionic Acids.

76. Percent of resistant strains of A.flavus and A. parasiticus
Table (14): Percent of resistant strains of A. flavus and A. parasiticus for all tested antifungals isolated from raw milk and milk products samples
Source of isolation
Percent of resistant strains of A.flavus and A. parasiticus
A. flavus
A. parasiticus
All tested strains R
% of R
Raw milk 10 2
20% 1
Yoghurt 9 4
44.4%
Hard cheese 5
40%
Fresh Kareish cheese 11 3
27.3%
Fresh Damietta cheese 18
22.2%
Total 53 15
28.3%

77. Fig. (9): Prevalence of resistant strains of A. flavus and A
Fig. (9): Prevalence of resistant strains of A. flavus and A. parasiticus for all tested antifungals isolated from raw milk and milk products samples

78. Aflatoxins detection from resistant strains of A. flavus and A
Aflatoxins detection from resistant strains of A. flavus and A. parasiticus isolated from meat and meat product samples
The resistant isolates of A. flavus (87.5%) and A. parasiticus (100%) that recovered from frozen meat produced significant levels of Aflatoxins. The levels of produced Aflatoxins by A. flavus ranged from ( ppb) for AFB1, and ( ppb) for AFB2, while, the production of AFs by A. parasiticus that isolated from frozen meat ranged from (43.0 ppb) for AFB1, (32.0 ppb) for AFB2, ( ppb) for AFG1 and ( ppb) for AFG2.

79. Quantitative detection of aflatoxins (µg/1000 ml) (ppb)
Table (15): Aflatoxins detection from resistant strains of A.flavus and A. parasiticus isolated from meat and meat product samples
Source of isolation
Strains
Total No. of isolates
Aflatoxin +ve
samples
Quantitative detection of aflatoxins (µg/1000 ml) (ppb) No % B1 B2 G1 G2
Frozen meat
A. flavus 8 7
87.5%
A. parasiticus 1
100%
43.0 32
0-43
0-26
Total 9
88.8
Minced meat 10
80%
0-31
0-22
0-33
0-16
Liver
77.7%
0-37
0-36
100.0% 55 22
0-77
0-35
Kidney 6 5
83.3%
0-15
0-20
100.00% 12 18 16
85.7%
Luncheon
62.5%
12-24
0-27
Sausage 11
81.8%
9.5-25
0-23
0-9
Hawawchi
71.4%
0-41

80. Fig. (10): Prevalence and levels of aflatoxins produced by A
Fig. (10): Prevalence and levels of aflatoxins produced by A. flavus and A. parasiticus isolated from examined meat and meat product samples (µg/1000 ml) (ppb)

81. Aflatoxins detection from resistant strains of A. flavus and A
Aflatoxins detection from resistant strains of A. flavus and A. parasiticus isolated from milk and milk product samples
The isolated A. flavus (50%) from raw milk produced significant levels AF ranged from (0-56 ppb) for AFB1 and (0-15 ppb) for AFB2. Whereas, the result of AFs production by the isolated A. flavus (100%) from yoghurt produce the levels ranged from ( ppb) for AFB1, and ( ppb) for AFB2. AFG1 and AFG2 were not detected at all.

82. Quantitative detection of aflatoxins (µg/1000 ml) (ppb)
Table (16): Aflatoxins detection from resistant strains of A.flavus and A. parasiticus isolated from milk and milk product samples
Source of isolation
Strains
Total No. of isolates
Aflatoxin +ve samples
Quantitative detection of aflatoxins (µg/1000 ml) (ppb) No % B1 B2 G1 G2
Raw milk
A. flavus 2 1
50%
0-56
0-15
0-0
A. parasiticus
Total
Yoghurt 4
100%
2-15.5
Hard cheese
0-25
0-10
Fresh Kareish cheese 3
66.6%
0-27
0-12
66.6
Fresh Damietta cheese
75%
0-22 75

83. Fig. (11): Prevalence and levels of aflatoxins produced by A
Fig. (11): Prevalence and levels of aflatoxins produced by A. flavus and A. parasiticus isolated from examined milk and milk product samples (µg/1000 ml) (ppb)

84. Statistical analysis of aflatoxin residues (ppb) detected in examined meat and meat product samples
The highest mean values of aflatoxin residues (µg/kg), B1, B2, G1 and G2 were detected in the kidney samples (12.36 ± 1.89, 9.84 ± 1.63, 5.38 ± 1.36 and 6.84 ± 1.39, respectively), followed by the liver samples (13.81 ± 1.96, 3.26 ± 0.92, 2.51 ± 0.63 and 1.36 ± 0.38), luncheon samples (3.71 ± 1.35, 3.59± 1.12, 5.24 ± 1.12 and 6.77 ±1.49), hawawchi samples (11.03 ± 2.43, 2.25± 0.52, 2.54 ± 0.99 and 2.56 ± 0.27),

85. minced meat samples (3. 62 ± 0. 88, 3. 40 ± 0. 82, 4. 24 ± 0. 85, 2
minced meat samples (3.62 ± 0.88, 3.40 ± 0.82, 4.24 ± 0.85, 2.83 ± 0.60) frozen meat samples which had the lowest level of AFs (4.80 ± 0.89, 5.3 ± 2.1, 1.71 ± 0.60 and 0.0) respectively.

86. Table (17): Statistical analysis of aflatoxin residues (ppb) detected in examined meat and meat product samples
Types of aflatoxins
Type of examined sample B1 B2 G1 G2
Min
Max
Mean
±SE
Frozen meat
1.2
9.25
4.80
0.89 00 22
5.30
2.1
6.0
1.71
0.60
Minced meat
0.0
7.5
3.62
0.88
7.7
3.40
0.82
8.1
4.24
0.85
5.5
2.83
Liver 20
13.81
1.96
6.25
3.26
0.62 7
2.51
0.63
3.50
1.36
0.38
Kidney
3.75 24
12.36
1.89
17.9
9.84
1.63
1.5 18
5.38
15.5
6.84
1.39
Luncheon 11
3.71
1.35
11.2
3.59
1.12
10.5
5.24 15
6.77
1.49
Sausage 19
6.05
2.11
8.5
2.20
0.84
5.34
1.50
6.75
2.36
0.71
Hawawchi 21
11.03
2.43 5
2.25
0.523
2.54
0.99
2.56
0.76

87. Fig. (12): Statistical analytical results of various aflatoxin residues (ppb) detected examined meat and meat product samples

88. Statistical analytical results of total (B1-B2-G1-G2) aflatoxin residues (ppb) detected in positive examined meat and meat product samples
The highest mean values of total aflatoxin residues (µg/kg) were detected in kidney samples (31.68 ± 3.13), followed by liver (19.98± 2.85), luncheon samples (18.9 ± 3.00), l hawawchi samples (18.56 ± 2.99), minced meat (14.78 ± 3.06) and the lower levels observed in frozen meat samples (11.4 ± 1.25).

89. Table (18): Statistical analysis of aflatoxin residues (ppb) detected in examined meat and meat product samples
Types of aflatoxins
 Type of examined sample
Aflatoxin (ppb)
Min
Max
Mean
±SE
Frozen meat
7.05 20
11.4
1.25
Minced meat 30
14.78
3.06
Liver
19.98
2.85
Kidney 14 44
31.68
3.13
Luncheon
34.5
18.9
3.00
Sausage
30.3
15.92
3.96
Hawawchi 27
18.56
2.99

90. Fig. (13): Statistical analytical results of total aflatoxin residues (ppb) detected in positive examined meat and meat products samples

91. Statistical analysis of aflatoxin residues (ppb) detected in examined milk and milk product samples
The residues of AFs in milk and milk product. Where the highest mean values of aflatoxin residues (µg/kg), M1, B1, B2, G1 and G2 were detected in the hard cheese samples (5.4 ± 1.2, 8.3 ± 1.9, 1.4 ± 1.4, 0.0 and 0.0, respectively), followed by the fresh damietta cheese samples (4.6 ± 1.6, 6.4 ± 0.66, 1.0 ± and 0.0),

92. yoghurt samples (1. 3 ± 1. 0, 2. 3± 1. 4, 0. 8 ± 0. 55 0. 0 and 0
yoghurt samples (1.3 ± 1.0, 2.3± 1.4, 0.8 ± and 0.0), raw milk samples (4.39 ± 1.55, 0.0, 0.0, 0.0 and 0.0), fresh kareish cheese samples (2.9 ± 1.09, 0.0, 0.0, 0.0 and 0.0), respectively.

93. Table (19): Statistical analysis of aflatoxin residues (ppb) detected in examined milk and milk product samples

94. Fig. (14): Statistical analytical results of various aflatoxin residues (ppb) detected in positive examined milk and milk product samples

95. Statistical analytical results of total aflatoxin residues (ppb) detected in examined milk and milk product samples
The highest mean values of total aflatoxin residues (µg/kg) were detected in hard cheese samples (13.96 ± 3.29), followed by fresh damietta cheese samples (9.41 ± 2.36), yoghurt samples( 5.39 ± 1.93), raw milk (4.39 ± 1.55) and the lower levels observed in fresh kareish cheese samples (3.30 ± 2.21).

96. Table (21): Statistical analysis of aflatoxin residues (ppb) detected in examined milk and milk product samples
Types of aflatoxins
Type of examined sample
Aflatoxin (ppb)
Min
Max
Mean
± SE
Raw milk 13
4.39
1.55
Yoghurt
12.5
5.39
1.93
Hard cheese
29.2
13.96
3.29
Fresh Kareish cheese 14
3.30
2.21
Fresh Damietta cheese 19
9.41
2.36

97. Fig. (15): Statistical analytical results of total aflatoxin residues (ppb) detected examined milk and milk product samples

98. Comparison between the total aflatoxins residues detected in examined samples of meat and meat products and the permissible limits of WHO and FAO and FDA
In frozen meat, 25% of the samples showed higher levels of aflatoxins than the permissible limits recommended by (WHO) (>15ppb) and 10% of the samples contained aflatoxins more than the permissible limits of (>20ppb) (FAO&FDA).

99. Table (22): Comparison between the total aflatoxin residues detected in examined samples of meat and meat products and recommended permissible limits of WHO (15 ppb) and FAO and FDA (20 ppb)
Type of examined sample
No. of examined samples
No. of positive samples
Samples contained aflatoxins > 15 ppb (WHO)
Samples contained aflatoxins
> 20 ppb (FAO) & FDA
No. %
Frozen meat 20 11 5 25 2 10
Minced meat 15 7 35 4
Liver 8 40 50
Kidney 13 9 45
Luncheon 65
Sausage 17 75 6 30
Hawawchi 14

100. Fig. (16): Comparison between the total aflatoxins residues detected in positive examined samples of meat and meat products and recommended permissible limits of WHO (15 ppb) and FAO & FDA (20 ppb)

101. Comparison between the total aflatoxins residues detected in examined samples of milk and milk proucts and recommended permissible limits of the European Community (EC) limit (0.5 ug/kg for all AFs and AFM1.), WHO (15 ppb) and FAO and FDA (20 ppb)
The samples of milk and milk products unfit for consumption according to the European Community. Where, their AF content more than 0.5 ppb, the high rate of rejected samples detected in domietta cheese (80%), hard cheese (60%), raw milk (50%), fresh kariesh cheese (30%) and the lowest incidence detected in 10% of yoghurt samples.

102. Comparing our results to the limits of FAO and FDA (15-20 ppb), only the rejected samples decrcted in 50% of hard cheese and 30% of fresh kareish cheese samples.

103. CONCLUSION

104. It is concluded that
(1) Mycotoxin contamination is worldwide problem; it is of special importance in developing countries of Africa, Asia and south America.
(2) The contamination of meat and milk and their products with different species of fungi and its products (mycotoxins) can be occurred very widely constituting a public health hazards.

105. (3) Comparatively the higher mould counts were detected in meat and milk and their products may be attributed to the variation in the sanitary measures adopted in handling of fresh raw meat and milk and during prolonged storage, transportation, packaging.
(4) Aflatoxin production depends on not only the genetic makeup of the strains but also on the temperature,humidity,composition of growth and nutrients limitation.

106. (5) The development of a Immunoaffinity-Based Method by Fluorometry fluometric methods for the detection of amount of mycotoxins is the most important step towards safer foods and feeds.

107. Thank you