Monograph On

Monograph

On Mycotoxigenic Fungi and Mycotoxins in Foods and Feeds with

Synopsis of studies done by the authors on mycotoxigenic fungi and mycotoxins in foods and feeds

Prepared by

Mohamed K. Refai1 and Atef A.Hassan2

1 Profesor of Microbiology, Faculty of Veterinary Medicine, Cairo University, Egypt, e-mail [email protected], 2 Profesor of Mycology and Mycotoxins, Animal Health Research Institute, Dokki, Egypt, e- mail, [email protected]

2013

Preface This monograph is designed as a laboratory guide for the food microbiologists to assist in the isolation and identification of common mycotoxigenic foodborne fungi and reporting their respective mycotoxins.We emphasise the fungi which cause food spoilage and mycotoxicosis but also devote space to the fungi commonly encountered in foods and feeds at harvest, and in the food factory. As far as possible, we have kept the monograph simple. The identification keys have been designed for use by microbiologists with little or no prior knowledge of mycology. For identification to genus level, they are based primarily on the cultural and physiological characteristics of fungi grown under a standard set of conditions. The microscopic features of the various fungi become more important when identifying isolates at the species level. Nearly all of the suspected mycotoxigenic species have been illustrated with colony photographs, together with photomicrographs or line drawings. The tables of mycotoxins produced by these fungi were sited briefly as rapid and simple illustration for graduate students.The knowledge about the target organs, systems and clinical symptoms due to toxicosis of each mycotoxin are also available in this monograph. Among the colleagues who helped us to prepare this monograph, we wish to particularly thank Dr. Manal A.Hassan, Chief Researcher of mycology , Animal Health Research Institute, Cairo, Egypt, for her assistant in collection of the papers about the methods of control of mycotoxicosis. We are indebted to Dr. Rasha H. Sayed El-Ahl, Reasercher of mycology, Animal Health Research Institute, Cairo, Egypt, who assisted in literature searches and some cultural and photographic work We wish to express our appreciation to all staff members of Mycology Department, who generously provided facilities, cultures and advice on some of the genera studied. Mohamed K. Refai and Atef A. Hassan November, 2013, Egypt

My great respect to the great Disability challenger Prof. Dr. Atef Abdelaziz Hassan

When he came to my office 1988 he gave me a paper on which he wrote that he wanted to register for M.V. SC. In the field of mycotoxins and hoped to accept him under my supervision, in spite of his impairment in hearing and talking. I accepted on the spot . He began working and our communication was continued by writing. When he finished his MS thesis (1990), he asked me to let one of his friends to make his presentation, but I insisted that he has to do it only by pointing to the figures and tables (Fig.1). The examiners were satisfied (Fig. 2). It was a wonderful day . He was happy and thankful to all (Fig. 2). The same was done in the discussion of his Ph. D. (Fig 3), also on mycotoxins (1994). He continued to do research assending the academic ladder till he became a chief researcher and head of the Department of Mycology and Mycotoxins (2006-2009) and shared with me as co-supervisor on several theses and examiner of postgraduate students (Fig. 4) in the field of mycotoxins. Now he is the main researcher in this field in Veterinary Medicine in Egypt and he still works by himself in the laboratory (Fig. 5).

I am happy that Prof. Atef Abdelaziz is co-author of this monograph

M. Refai

Contents Introduction 5 Classification of mycotoxins 7 Mycotoxins and mycotoxigenic fungi 8 Aspegillus toxins 8 Keys for identification of Aspergillus groups 14 Common toxigenic aspergilli 16 Penicillium toxins 27 Description of the most common toxigenic Penicillium 29 Fusarium toxins 35 Description of some Fusarium species 37 Toxins of black fungi 40 Trichoderma toxins 45 Mycotoxicoses 46 Aspergillotoxicosis 46 Aflatoxicosis 46 Ochratoxicosis 49 Penicillotoxicosis 50 Fusariotoxicosis 51 Facial Eczema (Pithomycotoxicosis) 54 Stachybotryotoxicosis 55 Management of mycotoxins contamination in feed 56 Detection of aflatoxins 57 Prevention of mycotoxin formation58 Detoxification of mycotoxins in food and feedstuffs 60 Physical methods 60 Biological detoxification of mycotoxin 62 Control of mycotoxins during food processing 62 Limits for mycotoxins in foods and feeds 68 Future studies and recommendation 72 Synopsis on studies done by authors on mycotoxigenic fungi and mycotoxins in foods and feeds 73 References 159 Annexes 1171 Annex 1: Media , reagent and stains used for isolation , identification 171 Annex 2: Methods for Isolation, Enumeration and Identification of mycotoxigenic moulds 175 Annex 3 : Determination of mycotoxins in feed and food samples 178 Annex 4: Identification of fungal isolates by polymerase chain reaction (PCR):182 Annex 5: Rapid detection of mycotoxigenic fungi:187 4

1. Introduction

Mycotoxins are secondary metabolites produced by filamentous fungi that have deleterious effects on human and animal consumers. They are structurally diverse, deriving from a number of biosynthetic pathways and their effect upon consumers is equally diverse ranging from acutely toxic to immunosuppressive or carcinogenic. The production of a particular mycotoxin is restricted to a limited number of fungal species and, in some instances, may be limited to particular strains within a species. Although over 400 mycotoxins have been described, relatively few are of major concern with respect to human and animal health, as they are responsible for the production of the great majority of the mycotoxins. The majority of these fungi infect plants and can be regarded as phytopathogens,

Mycotoxin contamination is an economic problem for live stock and feed industries. The presence of mycotoxins in feedstuffs reduces the feed quality in terms of both energy and protein value.High moisture content (>12%) and grain damage favour mould growth. Mycotoxins may get concentrated from 30-500 times in broken grain as compared to whole grain.

Risk from mycotoxin not only depends on the degree of contamination, but also on duration of exposure. There are no safe levels for mycotoxins as the damage depends on too many factors including health and nutritional status of the animal or bird, stress and other disease conditions and presence of other mycotoxins. Testing the feed for one particular toxin may not give a clear picture as there could be number of other mycotoxins. In practical feeding situations, it is rare to find a single mycotoxin. Mycotoxins generally have synergistic effects. So the damage caused by the combination is more devastating. Multiple mycotoxins at low to moderate levels may produce symptoms often distinctly different from those associated with individual mycotoxins. 5

Immunosuppression is one of the serious outcome of Mycotoxin contamination, often unnoticed, making animal or bird susceptible to infection and complex disease problems. Many mycotoxins are harmful to humans and animals when inhaled, ingested or brought into contact with human skin. Mycotoxins can cause a variety of short term as well as long-term health effects, ranging from immediate toxic response to potential long-term carcinogenic and teratogenic effects. Symptoms due to exposure to mycotoxins include dermatitis, cold and flu symptoms, sore throat, headache, fatigue, diarrhea, and impaired or altered immune function, which may lead to opportunistic infection. Mycotoxins are a known agent in biological warfare as a moderate illness compared to the other biologicals.

2. Classification of mycotoxins

2.1. Classification according to their mode of action:  Cytotoxic: Patulin and penicillic acid.  Emetic: Vomitoxin and T-2 toxin  Immunosuppressive: Aflatoxin B1  Carcinogenic: Aflatoxins, fusarenon, patulin, rugulosin.  Mutagenic: Aflatoxins, sterigmatocystin, trichothecenes.  Oestrogenic: Zearalenon.  Teratogenic: Aflatoxins, citrinin, ochratoxin A. 2.2. Classification according to the target organs:  Hepatotoxins (liver tissues): Aflatoxins, ochratoxins, rubratoxins, sporidesmin, sterigmatocystin, luteoskyrin, rugulosin, islanditoxin.  Nephrotoxins (kidney tissues): Citrinin, ochratoxin A, oxalic acid.  Neurotoxins (nervous system tissues) : Citreoviridin, patulin, cyclopiazonic acid, fumitremorgen, territrems, verruculogen.  Enterotoxins (alimentary organs and canal), dermotoxins(skin): Trichothecenes, verrucarin. 2.3. Classification according to the target systems:  Vascular system :(Increased vascular fragility, hemorrhage into body tissues, or from lung, e. g., aflatoxin, satratoxin, roridins).  Digestive system: (Diarrhea, vomiting, intestinal hemorrhage, liver effects, necrosis, fibrosis: aflatoxin; caustic effects on mucous membranes: T-2 toxin; anorexia: vomitoxin.

 Respiratory system: Respiratory distress, bleeding from lungs e. g., trichothecenes.  Nervous system, Tremors, incoordination, depression, headache, e. g.,tremorgens, trichothecenes.  Cutaneous system: Rash, burning sensation sloughing of skin, photosensitization, e. g., trichothecenes.  Urinary system: Nephrotoxicity, e. g. ochratoxin, citrinin.  Reproductive system; Infertility, changes in reproductive cycles, e. g. T-2 toxin, zearalenone.  Immune system: Changes or suppression: many mycotoxins.

3. Mycotoxins and mycotoxigenic fungi

3.1. Aspegillus toxins:

More than 40 Aspergillus species are known to produce more than 60 toxins:

Toxins Aspergillus species producer 1. 3-Nitropropionic Acid A. flavus, A. oryzae, A. wentii 2. 9-deacetoxyfumigaclavine C A. fumigatus (sartorya fumigata) 3. AcT1 A. Candidus 4. Aflatoxin B1 : A. arachidicola, A. bombycis, A. flavus, A. minisclerotigenes, A nomius,. A.ochraceoroseus, A. parasiticus, A. pseudotamarii, A. rambellii, Emericella venezuelensis 5. Aflatoxin B2: A. arachidicola, A. flavus, A. minisclerotigenes, A. nomius, A. parasiticus 8

6. Aflatoxin B2a: A. flavus

7. Aflatoxin B3: Aspergillus sp. (species not defined) 8. Aflatoxin G1 A. arachidicola, A. flavus, A.minisclerotigenes, A. nomius, A. Parasiticus A. arachidicola, A. flavus, A. minisclerotigenes, A. nomius, 9. Aflatoxin G2: A. parasiticus

10. Aflatoxin M1 A. flavus, A. parasiticus 11. Aflatrem: A. flavus, A. minisclerotigenes

12. Aspertoxin: A. flavus 13. Austdiol: A. ustus 14. Austin: A. niger, A. ustus 15. Austocystin A A. ustus 16. Brevianamide A: A. ustus 17. Citreoviridin A. terreus 18. Citrinin: A. candidus, A. carneus, A. flavipes, A. niveus, A. terreus 19. Cyclopiazonic Acid : A. flavus, A. lentulus, A. minisclerotigenes, A. oryzae, A. pseudotamarii, A. versicolor 20. Cytochalasin E A. clavatus 21. Dechloronidulin A. fumigatus, Emericella unguis 22. Echinuline A. amstelodami, A. amstelodami, A. chevalieri, A. herbariorum ser. minor, Eurotium chevalieri 23. Emestrin: Emericella striata 24. Emodin/Archin/Emodol/Frandulic A.Acid aureus,: A. terreus, A. wentii

25. Festuclavine: A. fumigatus 26. Fumagillin: A. fumigatus 26. Fumigaclavine A: A. fumigatus, A. tamarii 9

27. Fumigaclavine C: A. fumigatus 28. Fumitremorgin A: A. caespitosus, A. fumigatus, Neosartorya fischeri 29. Fumitremorgin C A. fumigatus, Neosartorya aureola, Neosartorya fischeri 30. Fumonisin B2: A. niger 31. Fumonisin B4: A. niger 32. Gliotoxin A. flavus, A. fumigatus, A. niger, A.terreus, Eurotium chevalieri, Neosartorya pseudofischeri 33. Helvolic Acid: A. fumigatiaffinis, A. fumigatus, A. novofumigatus 34. Kojic Acid A. arachidicola, A. candidus, A. minisclerotigenes, A. oryzae 35. Malformin C A. niger 36. Malformins: A. niger 37. Maltoryzine A. oryzae var. microsporus 38. Methyl-sulochrin A. fumigatus 39. Neoechinuline A. amstelodami 40. Ochratoxin A: A. albertensis, A. alliaceus, A. auricomus, A. carbonarius, A. citricus, A. fonsecaeus, A. lanosus, A. melleus. A. niger, A. ochraceopetaliformis, A. ochraceus, A. ostianus, A. petrakii, A. sclerotiorum, A. sulphureus 41. Ochratoxin B A. acanthosporus, A. albertensis, A. alliaceus, A. auricomus, A. carbonarius, A. ochraceopetaliformis, A. ochraceus, A. sclerotiorum, A. sulphureus, A. wentii 42. Ochratoxin C: A. ochraceus 43. Patulin: A. clavatus, A. longivesica, A. terreus 10

44. Penicillic Acid: A. melleus, A. ochraceopetaliformis, A. ochraceus, A. ostianus 45. Phthioic Acid: A. fumigatus, A. niger, A. terreus 46. Pyripropene A A. fumigatus, A. lentulus 47. Restrictocin: A. fumigatus, A. restrictus 48. Secalonic Acid D: A. aculeatus, A. ochraceus, A. uvarum 49. Sterigmatocystin: A. discophorus, A. flavus, A. multicolor, A. nidulans, A. olivicola, A. parasiticus, A. rambellii, A. ustus, A. versicolor, Emericella nidulans, Emericella venezuelensis 50. Terpeptin A: Aspergillus sp. (species not defined) 51. Terpeptin B: Aspergillus sp. (species not defined) 52. Terreic Acid: A. terreus

53. Terrein: A. fischeri, A. lentulus, A. novofumigatus, A. olivicola, A. terreus, Emericella variecolor, Emericella venezuelensis 54. Terretonin: A. terreus

55. Territrem A A. terreus 56. Tryptoquivaline A A. clavatus, A. fumigatus, Neosartorya fischeri 57. Verruculogen: A. fumigatus, Neosartorya fischeri 58. Versicolorin A A. flavus, A. versicolor 59. Viomellein A. melleus, A. ochraceus 60. Viriditoxin A. brevipes, A. fumigatus, A. viridinutans 61. Xanthocillin: A. candidus, A. chevalieri, Eurotium chevalieri 11

62. Xanthomegnin A. melleus, A. ochraceus, A. sulphureus

It is clear from the above mentioned data that the same toxin can be produced by different Aspergillus species

• The most important toxins are:

a. Aflatoxins: Aflatoxins are produced mainly by Aspergillus flavus and Aspergillus parasiticus, but several other Aspergillus species can produce aflatoxins. Aflatoxin B has been reported from Aspergillus flavus, A. flavus var. parvisclerotigenus, A. parasiticus, A. toxicarius, A. nomius, A. pseudotamarii, A. zhaoqingensis, A. bombycis and from the ascomycete genus Petromyces (Aspergillus section flavi), Emericella astellata and E. venezuelensis from the ascomycete genus Emericella (Aspergillus section Nidulantes). All aflatoxin producing members of section Flavi produce kojic acid and most species, except A. bombycis and A. pseudotamarii, produce aspergillic acid. Aflatoxins have been found as natural contaminants in many types of food including peanut, corn, rice, Soya beans, wheat, sorghum and barely and in animal feeds.The most important factor in the growth and production of aflatoxins by A. flavus is the relative humidity surrounding a natural substrate. A. flavus grows and produces aflatoxins when the relative humidity is over 70% and the moisture content of the substrate is over 15%. The optimum temperature for growth of A. flavus is 36-38oC, while the optimum temperature for toxin production is around 25oC. There are at least 10 different types of aflatoxins, which well studied, namely B1, B2, G1, G2, M1, M2, GM1, B2a, G2a and aflatoxicol. Aflatoxins are detected and identified by thin layer chromatography,HPLC, ELISA mass spectroscopy and fluoroumetric methods . Under UV. light the spots of aflatoxin B1 or B2 fluoresce blue and that of aflatoxins G1 or G2 fluoresce green

12 and hence the name B or G simply indicating the colour of detected spots fluoresce.

b. Sterigmatocystin: Sterigmatocystin is the major metabolite of Aspergillus versicolor, Aspergillus nidulans and Bipolaris species. It shares in the biogenetic pathway with aflatoxins. It is also of hepatotoxic and hepatocarcinogenic potentials.

c. Ochratoxins: Ochratoxins are a group of related compounds produced mainly by Aspergillus ochraceus and Penicillium viridicatum. The predominant member of the group is ochratoxin A which is a potent nephrotoxin and hepatotoxin. Ochratoxin A is produced also by A. albertensis, A. alliaceus, A. auricomus, A. carbonarius, A. citricus, A. fonsecaeus, A. lanosus, A. melleus, A. niger, A. ochraceopetaliformis, A. ochraceus, A. ostianus, A. petrakii, A. sclerotiorum, A. sulphureus. It may produce ochratoxicosis in humans. This is also known as Balkan nephropathy. The toxin is produced at optimum growth conditions at 25 degrees C and high moisture conditions. Ochratoxin B is produced by A. acanthosporus, A. albertensis, A. alliaceus, A. auricomus, A. carbonarius, A. ochraceopetaliformis, A. wentii, A. ochraceus, A. sclerotiorum and A.sulphureus,

d. Cyclopiazonic acid Cyclopiazonic acid is acutely toxic to rats. Intraperitoneal injection produces hyperesthesia and convulsions followed by death in about 30 min. The LD50 in males is 2.3 mg/kg. Oral administration results in death in 1–5 days with an LD50 of 36 mg/kg in males and 63 mg/kg in females. The lesions produced include degenerative changes and necrosis in the liver, spleen, exocrine and endocrine pancreas, kidney, salivary glands, myocardium, and skeletal muscle. An unusual lesion, seen in the bile ducts and other ducts, was enlargement of the nucleus with 13 margination of the chromatin and subsequently enlargement of the cells lining the ducts.

E. Gliotoxin Gliotoxin is ciliotoxic and cause damage to the respiratory epithelium from humans cultured in vitro. It is produced by A. flavus, A. fumigatus, A. niger, A. terreus, Eurotium chevalieri, Neosartorya pseudofischeri

4.Keys for identification of Aspergillus groups:

4.1. Key based on colour of Aspergillus groups

Colonies black or dark brown

Heads with metulae and phialides conidia rough, spheroidal, 4-5 µm diam A. niger conidia large, 7-10 µm diam, spiny or tuberculate A. carbonarius Heads with phialides only Vesicles large, 22-55 (up to 100) µm diam; conidia ellipsoidal A. aculeatus Vesicles smaller, 15-30 (up to 45) µm diam; conidia spheroidal A. japonicus Vesicles globose to elliptical, 20-30 µm diam; conidia globose to subglobose A. uvarum

Colonies blue-green

Very rapid growth at 37°C (>60 mm diam) A. fumigatus Colonies leaf green, yellow-green or yellow

Metulae usually present (majority of heads); conidia finely to moderately roughened A. flavus Conidia ellipsoidal, subglobose, pale green, smooth walled to echinulate sclerotia 150-300 µm in diam A. minisclerotigenus Heads uniseriate or biseriate, vesicles globose to subglobose, conidia globose to subglobose, echinulate, greenish A. arachidicola Heads usually with phialides only; conidia definitely rough walled A. parasiticus

Colonies brown or olive

Colonies yellow-green at first, becoming greyish-yellow or olive in age A. oryzae Colonies olive-brown, conidia 5-8 µm, walls very rough A. tamarii Colonies white A. candidus

Colonies in other colours Colonies growing slowly (less than 25 mm) on all media; small, metulate A. versicolor Conidia blue-green, ellipsoidal (3-4.5 µm); vesicles up to 250 µm long , club- shaped A. clavatus d A. clavatus Colonies ochre-coloured, Conidia light brown (ochre) or golden,, sclerotia A. ochraceus Sclerotia becoming greyish black A. alliaceus

4.2. Key based on micromorphology of common Aspergillus groups 4.3. •Sterigmata strictly uniseriate, Conidiophore smooth

Heads clavate A. clavatus Heads columner, no cleistothecia A. fumigatus Heads columner, white cleistothecia A. fischeri

•Sterigmata uni- or biseriate or both, Conidiophore smooth

Heads globose, Colonies black A. niger Colonies white to cream A. candidus Heads radiate, Colonies yellow-brown A. wentii •Sterigmata uni- or biseriate or both, Conidiophore rough

Heads radiate, Colonies yellow-green A. flavu Heads globose, Colonies yellow or ochraceus A. ochraceus Colonies olive-brown A. tamarii •Sterigmata strictly biseriate, Conidiophore smooth

Colonies definitely green, Heads columner A. nidulans Heads radiate A. versicolor

Colonies in other colours, Heads radiate, dull brown A. ustus Heads columner,brown A. flavipes Heads compact columner, white A. terreus

• The most common toxigenic aspergilli are: i. Aspergillus flavus: A. flavus colonies are commonly powdery masses of yellow- green spores on the upper surface and reddish-gold on the lower surface (underneath). Growth is rapid and colonies appear downy or powdery in texture. Hyphal growth usually occurs by thread-like branching and produces mycelium. Hyphae are septate and hyaline. Once established, the mycelium secretes degradative enzymes or proteins which can break down complex nutrients. The conidiophores of A. flavus are rough and colorless. Phialides are both uniseriate (arranged in one row) and biseriate. A. flavus produces aflatoxins B1, B2, and the animal metabolite M1. M1 has been detected in the milk of humans and animals that presumably consumed contaminated grain. In addition, aflatoxins have been reported in tissue biopsy/autopsy and urine from humans exposed in to Aspergillus spp Aflatoxin B1 is carcinogenic and adducts to the guanine basis. In addition, it readily adducts to proteins, particularly serum album. Antibodies to aflatoxin-albumin adducts have been demonstrated in humans. Hepatocellular carcinoma has been associated with Aflatoxin B1 and individuals with Hepatitis B are more susceptible to the carcinogenic effects of this mycotoxin. Finally, A. flavus has been shown to produce gliotoxin and has been detected in the sera of cancer patients with aspergillosis.

Aspergillus flavus

ii. Aspergillus parasiticus

Aspergillus parasiticus is known to produce aflatoxins. It is closely related but separable (based on DNA sequencing and Amplified fragment length polymorphismfingerprinting) to Aspergillus flavus. A. parasiticus produces aflatoxins B1, B2, G1 and G2, unlike A. flavus which produces only aflatoxin B1.

Aspergillus parasiticus iii. Aspergillus nomius Aspergillus nomius is described and represents a new aflatoxigenic species phenotypically similar to A. flavus. Separation from A. flavus is based on the presence of indeterminate sclerotia and a lower growth temperature. Recently, the comparisons of DNA relatedness reported that a relative differences between DNA paterns of A. nomius from that of A. flavus and A. tamarii.

Aspergillus nomius iv. Aspergillus pseudotamarii

A recent report of an aflatoxin producing isolate of Aspergillus tamarii prompted a taxonomic re-examination of aflatoxigenic and non-aflatoxigenic isolates identified as A. tamarii

18 as well as the closely related A. caelatus. Representatives of each species, including atypical isolates, were compared morphologically, for mycotoxin production, and for divergence in ITS, 28S, b-tubulin and calmodulin gene sequences. Because of genetic, morphological, and mycotoxin differences, the aflatoxin producing isolates of A. tamarii are given species rank as Aspergillus pseudotamarii sp. nov.

v. Aspergillus pseudocaelatus and A. pseudonomius

Two new species, A. pseudocaelatus sp. nov. and A. pseudonomius sp. nov. have been discovered, and can be distinguished from other species in this section based on sequence data and extrolite profiles. Aspergillus pseudocaelatus is closely related to the non-aflatoxin producing A. caelatus, and produces aflatoxins B & G, cyclopiazonic acid and kojic acid, while A. pseudonomius is related to A. nomius, and produces aflatoxin B(1) (but not G-type aflatoxins), chrysogine and kojic acid. In order to prove the aflatoxin.

Aspergillus pseudocaelatus 19

vi. Aspergillus versicolor:

A.versicolor produces gliotoxin, cyclopiazonic acid, sterigmatocystin, and methoxy sterigmatocystin. The Sterigmatocystins are related to the aflatoxins and are considered carcinogens.

A. versicolor

vii. Aspergillus fumigatus:

A. fumigatus readily produces gliotoxin which has been found on building materials from water affected buildings as well as in dust from similarly affected buildings. Other mycotoxins produced by A. fumigatus are: fumagillin, helvolic acid, fumatremorgin.

A. fumigatus viii. Aspergillus niger:

A. niger produces gliotoxin, which has been identified in the sera of humans and mice with aspergillosis. It produces also ochratoxin A,, fumonisin B2, B4, malformins and pethioic acid.

Aspergillus niger

ix. Aspergillus carbonarius

Ochratoxin A (OTA) production has been repeatedly reported in Aspergillus section Nigri; OTA has been occasionally detected in pistachio nuts in high concentrations. Fungi responsible for the presence of Ochratoxin A (OTA) in grapes have been identified as 21 belonging section Nigri, among which Aspergillus carbonarius is the main producer.

A. carbonarius

x. Aspegillus ochraceus

Aspergillus ochraceus is the major species responsible for OA production. Other toxins which can be produced by this fungus include penicillic acid, xanthomegnin and viomellein.

Aspergillus ochraceus

xi.Aspergillus sydowii

Aspergillus sydowii is a saprophytic fungus found in soil that can contaminate food and is occasionally pathogenic to humans. It is the predominant fungus found on wheat Qu, the most widely used source of raw microorganisms and crude enzymes for Chinese rice wine brewing. It has been found to be present in sea water in the Caribbean region and has been shown to be the cause of aspergillosis in sea fans. Aspergillus sydowii has been implicated in the pathogenesis of several human diseases, including aspergillosis, onychomycosis, and keratomycosis. Several indole alkaloids have recently (2012) been isolated from laboratory-grown cultures of the fungus. The compounds [4-(2- methoxyphenyl)-1-piperazinyl][(1-methyl-1H-indol-3-yl)]-methanone, cyclotryprostatin B, fumiquinazoline D, fumitremorgin B, fumiquinazoline C, fumiquinazoline B, fumiquinazoline A, fumiquinazoline F, fumiquinazoline G are produced by this fungus. Other bioactive compounds known to be unique to this fungus include aspergillusenes A and B, (+)-(7S)-7-O- methylsydonic acid, and hydrogenated xanthone derivatives aspergillusones A and B.

Aspergillus sydowii

Xii. Aspergillus wentii Aspergillus wentii develops conidiogenous cells (phialides) that sometimes arise directly from the vesicle (i.e. uniseriate) as in Aspergillus fumigatus, but colonies are brown. The fungus produces emodin, emolol, frandulic acid, ochratoxin B and citrinin.

Aspergillus wentii

xiii. Aspergillus ustus

Aspergillus ustus produces austin, austdiol, sterigmatocystin and brevianamide.

Aspergillus ustus 24

xiv. Aspergillus terreus

Aspergillus terreus is a cosmopolitan fungus which is primarily isolated from compost, plant material, and from soil. It is more common in tropical or sub – tropical areas and is an occasional causative agent of pulmonary aspergillosis among immunocompromised patients. A few cases of cerebral infection have been reported. Aspergillus terreus is isolated occasionally from outer ear canal colonizations. It produces citreoviridin, citrinin, patulin, pethioic acid and gliotoxin.

Aspergillus terreus

xv. Aspergillus flavipes

Two novel fungal metabolites, N-benzoyl-L-phenylalaninol and asperphenamate were isolated from the culture filtrate and mycelium of Aspergillus flavipes . N-benzoyl-L-phenylalaninol was identified by direct comparison with an authentic sample. Three new cytochalasans, namely, aspochalasins I, J and K and four known cytochalasans, aspochalasins C D and E and TMC-169 were isolated from extract of the fungus. All compounds exhibited weak

25 to moderate cytotoxicity against NCI-H460, MCF-7, and SF-268 cancer cell lines,

l Aspergillus flavipes

xvi. Aspergillus candidus Aspergillus candidus is found in warm soils, grain, and secondary decay of vegetation. It produces AcT1, citrinin and petulin which may be associated with disease in humans and other animals.

Aspergillus candidus

5.Penicillium toxins

1. Anacine: P. aurantiogriseum, P. nordicum 2. Asteltoxin:P. cavernicola, P. concentricum, P. confertum, P. formosanum and P. tricolor. 3. Auranthine: P. aurantiogriseum 4. Aurantiamine: P. aurantiogriseum, P. freii 5. Botryodiploidin: P. brevicompactum and P. paneum 6. Brevianamide A: P. brevicompactum, P. viridicatum 7. Chaetoglobosin A, B: P. discolor, P. expansum 8. Chrysogine: P. chrysogenum, P. nalgiovense 9. Citrinin: P. citrinum , P. expansum, P. radicicola and P. verrucosum. 10. Citromycetin: P. glabrum 11. Communesins: P. expansum 12. Compactins: P. solitum 13. Cyclochlorotine: P. islandicum 14. Cyclopaldic acid: P. commune 15. Cyclopenin: P. aurantiocandidum, P. crustosum, P. discolor, P. echinulatum, P. freii, P. polonicum, P. solitum 16. Cyclopenol: P. aurantiocandidum, P. crustosum, P. discolor, P. echinulatum, P. freii, P. polonicum, P. solitum 17. Cyclopiazonic acid: P. camemberti, P. commune, P. dipodomyicola, P. griseofulvum and P. palitans 18. Emodin: P. islandicum 19. Erythroskyrin: P. islandicum 20. Fumitremorgin A* & B: P. brasilianum, P. palitans 21. Islanditoxin: P. islandicum 22. Isofumigaclavine A & B: P. roqueforti 23. Italicic acid: P. italicum 24. Luteoskyrin: P. islandicum 25. Marcfortines: P. paneum 26. Meleagrin: P. chrysogenum 27. Mycophenolic acid: Penicillium stoloniferum, Penicillium roqueforti, Penicillium brevicompactum, Penicillium pinophilum , P. bialowiezense, P. carneum, P. rugulosum 28. Ochratoxin A: P. nordicum, P. verrucosum 29. Oxaline: P. atramentoseum, P. oxalicum 30. Patulin: P. carneum, clavigerum, P. concentricum, P. coprobium , P. dipodomyicola , P. expansum, P. glandicola, P. gladioli, P. griseofulvum, P. marinum, P. paneum,, P. sclerotigenum and P. vulpinum. 31. penitrem A: P. carneum, P. clavigerum, P. crustosum, P. flavigenum, P. glandicola, P. melanoconidium and P. tulipae. 27

32. Penicillic acid: P. aurantiogriseum, P. aurantiocandidum, P. brasilianum, P. carneum, P. cyclopium, P. freii, P. melanoconidium, P. neoechinulatum, P. polonicum, P. radicicola, P. tulipae and P. viridicatum 33. Penitrem A: P. crustosum, P. melanoconidium 34. PR-toxin : P. chrysogenum and P. roqueforti. 35. Puberulic acid: P. aurantiocandidum 36. Raistrick phenols: P. brevicompactum 37. Roquefortine C: P. carneum, P. chrysogenum, P. crustosum, P. expansum, P. griseofulvum, P. hirsutum, P. hordei, P. melanoconidium, P. paneum, P. roqueforti 38. Rubratoxin: P. crateriforme 39. Rugulosin: P. islandicum, P. rugulosum, P. variabile 40. Rugulovasine A & B: P. crateriforme, P. atramentoseum, P. commune 41. Secalonic acid A: P. chrysogenum and P. confertum 42. Secalonic acid D & F: P. oxalicum, 43. Spiculisporic acid: P. crateriforme 44. Tanzawaic acid A: P. citrinum 45. Terrestric acid: P. crustosum, P. hirsutum, P. hordei 46. Territrems: P. cavernicola and P. echinulatum. 47. Tryptoquivalins: P. digitatum 48. Verrucins: P. verrucosum 49. Verrucologen: P. brasilianum 50. Verrucolone: P. italicum, P. nordicum, P. olsonii, P. verrucosum 51. Verrucosidin: P. aurantiogriseum, P. melanoconidium, P. polonicum 52. Viomellein: P. clavigerum, P. cyclopium, P. freii, P. melanoconidium, P. tricolor and P. viridicatum 53. Vioxanthin: P. clavigerum, P. cyclopium, P. freii, P. melanoconidium, P. tricolor and P. viridicatum 54. Viridic acid: P. nordicum and P. viridicatum 55. Viridicatumtoxin: P. brasilianum, P. aethiopicum. 56. Xanthomegnin: P. clavigerum, P. cyclopium, P. freii, P. melanoconidium, P. tricolor and P. viridicatum.

• Description of the most common toxigenic Penicillium spieces:

The genus Penicillium consists of about 350 species, most of them are saprophytes. The genus Penicillium is divided into two subgenera, Aspergilloides and Penicillium, and 25 sections. Very few species are incriminated as human or animal pathogens, several species are known to produce toxins and many species are used in the industry of cheese, organic acids, enzymes, antibiotics… etc. Penicilli are among the most ubiquitous of all fungi. They constitute the normal and an abundant part of the mycoflora of all soils and they play an active role in decomposition and deterioration processes.The colony of Penicillium on Sabouraud dextrose agar may be plane, deeply furrowed or wrinkled, may be velvety, lanose, funiculose, with colours such as green, yellow, blue, white, brown or red and mostly present various shades and degrees of these colours.

a. Colony characters of some Penicillium species

Penicillium chrysogenum Penicillium citrinum Penicillium digitatum

Penicillium expansum Penicillium funiculosum Penicillium glabrum

Penicillium italicum Penicillium purpurogenum Penicillium roqueforti

Penicillium verrucosum Penicillium viridicatum Penicillium brevicompactum

The basic structures of Penicillium species consist of vegetative mycelium, septate, submerged or partially submerged and partially aerial. Conidiophores arise from submerged or aerial hyphae, septate, smooth or rough, terminating in a boom-like whorl of branches, the penicillus, which carries chains of conidia.

The penicillus consists of a single whorl of spore-bearing organs, called phialides, which arise from one or several branches of the conidiphore. The branches carrying the phialides are called metulae and those supporting the metulae are called rami. The branching system may be symmetrical or asymmetrical.

• The conidiophore branching types: a. Monoverticillate conidiophores: consist of a single whorl of phialides. b. Divaricate conidiophores consist of numerous subterminal branches, where conidiophore parts are divergent c.Biverticillate conidiophores have a whorl of three or more metulae between the end of the stipe and the phialides; the metulae may be of unequal or equal length, vary in their degree of divergence. d.Terverticillate conidiophores have another level of branching between the stipe and the metulae, often just a continuation of the stipe axis and one side branch, sometimes a true whorl of three or more branches.

31 e. Quaterverticillate conidiophores are produced by only a few species, and have one extra level of branching beyond the terverticillate pattern. f. Terverticillate and quaterverticillate conidiophores tend to be conspicuously asymmetrical

Conidiophore branching patterns observed in Penicillium. A. Conidiophores with solitary phialides. B. Monoverticillate. C. Divaricate. D, E. Biverticillate. F. Terverticillate. G. Quaterverticillate,

b. Micromorphology of some Penicillium species

P. brevicompactum P. chrysogenum P. citrinum P. digitatum

P. expansum P. funiculosum P. glabrum P. italicum

P. purpurogenum P. roqueforti P. verrucosum P. viridicatum

6. Fusarium toxins

Fusarium species have been important for many years as plant pathogens causing diseases such as crown rot, head blight and scab on cereal grains, vascular wilts, root rots etc. In the last years, Fusarium species have been studied extensively because the mycotoxins they produce can be of threat to man and animal health. More recently, Fusarium species have become important as pathogens of human patients with compromised immune system, where they can cause invasive diseases. Fusarium toxins make up a diverse group of relatively potent compounds allegedly responsible for both human and animal diseases. Fusarium species grow profusely at 24-27oC and once grains are sufficiently invaded by the fungus, most rapid production of toxins takes place especially at lower temperature near 12oC. They are also toxic to plants.

•The most important Fusarium toxins are: a. T2-toxin is produced by Fusarium tricinctum. It causes irritation of the skin and inhibits protein synthesis. T-2 toxin occurs in a fairly low proportion of feed samples (3% to 5%). b. Doxynivalenol (DON) or Vomitoxin is produced by Fusarium graminearum and is one of the more commonly detected mycotoxins. Incidence may be as high as 50% to 80% of feeds. c. Zearalenon is produced by Fusarium scirpi and Fusarium tricinctum. Zearalenone and zearalenol are produced almost exclusively by Fusarium species that contribute to the ear and stalk rot that occurs in the ears of corn and on the heads of cereal grains (scab) standing in the field or in stored ear corn. Such Fusarium species require a minimum of 22 to 25 percent moisture to grow in cereal grains. Generally, shelled corn stored at these moistures is 35 likely to be colonized by a mixture of other fungi, yeasts, and bacteria with which F. species competes poorly.. zearalenone is more prevalent in wet and cool seasons. Zearalenone has been found in 10% to 20% of feeds in some surveys. d. Fusarenon is produced by Fusarium nivale and inhibits protein synthesis. e. Butenolide is produced by Fusarium tricinctum and Fusarium nivale and causes tail necrosis in cattle. f. Poaefusain and sporofusain are produced by Fusarium poae and Fusarium sporotrichioides. They cause alimentary toxic aleuka (ATA) in man. In animals they cause inflammation of the skin and defective haemopiosis. g. Trichothecines are produced by various species of Fusarium, Cephalosporium, Myrothecium, Trichothecium, Trichoderma and Stachybotrys. They have quite similar biological activities. All of them are capable of inducing dermal reaction consisting of severe local irritation, inflammation and desquamation. Most of them are cytotoxic and phytotoxic h. Fumonisin is a toxic metabolite of the Fusarium moniliforme and Fusarium proliferatum which are soil borne fungi. These fungi grow rapidly from single cell spores into the thread-like masses that produce fumonisin. Fumonisin is found primarily in corn and corn- based products. Fumonisin may be found in several chemical forms suc , B1, B2 and B3 are the types of most concern. Fumonisin is generally produced in warm to hot climates. i.Fusarochromanone is produced mainly by Fusarium wquiseri’. Fusarochromanone is probably the cause of tibial dyschondroplasia (TDP) in poultry.

• Description of some Fusarium species

Colonies are cottony or woolly and entire with delicate lavender, purple or rose-red colours, as seen in the following:

F. oxysporum F. subglutinans Fusarium dimerum

F. verticilloides F. solani F. sporotrichiodes

F. anthophilum F. proliferatum F. nygamai

Fusarium species may produce three types of spores called macroconidia, microconidia and chlamydospores. Some species produce all three types of spores, while other species do not. The macroconidia are produced in a specialized structure called a 37 sporodochium in which the spore mass is supported by a superficial cusionlike mass of short monophialides bearing the macroconidia. Macroconidia may also be produced on monophialides and polyphialides in the aerial mycelium. The morphology of the macroconidia is the key characteristic for characterization, not only of the species but also of the genus Fusarium. Microconidia are produced in the aerial mycelium but not in sporodochia.. Microconidia are of various shapes and sizes, they may be single or in chains. The presence or absence of microconidia is a primary character in Fusarium. The chlamydospores are thick-walled spores, filled with lipid-like material. The chlamydospores may be born singly, in pairs, in clumps or in chains., and the outer wall may be smooth or rough. The presence or absence of chlamydospores is a primary characteristic.

F. andiyazi. F. acuminatum F. anthophilum

F. avenaceum F. chlamydosporum F. fujikuroi

F.globosum F. graminearum F. napiforme 38

F. oxysporum F.poae F. proliferatum

F. scirpi F. solani F. sporotrichioides

F. thapsinum F. tricinctum F. verticillioides Accepted species Common Synonyms ______Fusarium acuminatum F. scirpi var. acuminatum, F. gibbosum var.acuminatum Fusarium andiyazi Fusarium moniliforme, Fusarium verticillioides Fusarium anthophilum Fusarium moniliforme var. anthophilum Fusarium chlamydosporum F. sporotrichioides var. chlamydosporum, F.fusarioides Fusarium fujikuroi Fusarium proliferatum Fusarium scirpi Fusarium equiseti var. bullatum, F. chenopodium Fusarium sporotrichioides F. tricinctum, F. sporotrichiella var. sporotrichioides Fusarium tricinctum F. sporotrichioides, F. sporotrichioides var. tricinctum Fusarium verticillioides Fusarium moniliforme Fusarium thapsinum Fusarium moniliforme

7.Toxins of black fungi

a. Alternaria toxins

The most important secondary metabolites with mammalian toxicity produced by Alternaria species are the dibenzo--pyrones altenuene (AE), alternariol (AOH), alternariol monomethyl ether (AME) and a derivative of tetramic acid, tenuazonic acid. The comparisons between growth and mycotoxin production show that absolute aw limit for germination is about 0.86 aw, and for growth and mycotoxin production about 0.88–0.89 for all types of Alternaria mycotoxins. Optimum temperature production was at about 25 ºC. The tenuazonic acid (TA) production by A. alternata and A. tenuissima species shows minimum water availability conditions for production were found to be about 0.93–0.90 aw and temperature of 20 ºC. Alternariol (AOH) and alternariol monomethyl ether (AME) are the main benzopyrone mycotoxins produced by Alternaria alternata. Other species of Alternaria , Stagonospora nodorum and Phomopsis strains have also been found to produce AOH and AME. The toxicological database on AOH and AME is limited. Although their acute toxicity in animals is low, they are mutagenic in vitro and there is also some evidence for carcinogenic properties in unconventional assays.

The three important mycotoxins of A. alternata are alternariol, alternariol-mono-methyl-ether and altertoxin I

Alternaria is a wide-spread dematiaceous fungus commonly isolated from plants, soil, food, and indoor air environment. The production of melanin-like pigment is one of its major characteristics.The genus Alternaria currently contains around 50 species. Among these, Alternaria alternata is the most common one 40 isolated from human infections. Alternaria chartarum, Alternaria dianthicola, Alternaria geophilia, Alternaria infectoria, Alternaria stemphyloides, and Alternaria teunissima are among the other Alternaria spp. isolated from infections

Microscopically, Alternaria spp. have septate, brown hyphae. Conidiophores are also septate and brown in colour, occasionally producing a zigzag appearance. They bear simple or branched large conidia, which have both transverse and longitudinal septations (muriform conidia). They are dark in colour, elongated and found in chains. The conidia (7-10 x 23-34 µm) may be observed singly or in acropetal chains and may produce germ tubes. They are ovoid to obclavate, darkly pigmented, muriform, smooth or roughened. The end of the conidium nearest the conidiophore is round while it tapers towards the apex.

Alternaria alternate

b. Macrocylic trichothecenes

S. chartarum produces a variety of macrocylic trichothecenes and related trichoverroids: roridin E and L-2; satratoxins F, G, and H; isosatratoxins F, G, and H; verrucarins B and J; and the trichoverroids, trichoverrols A and B and trichoverrins A and B. The satratoxins are generally produced in greater amounts than the 41 other trichothecenes, but all compounds are produced in low quantities. They apparently occur in all parts of the fungus (53). The difficulty in obtaining, identifying, and purifying these toxins has slowed extensive studies on their biological activity

Macrocyclic trichothecenes are highly toxic compounds with a potent ability to inhibit protein synthesis. Numerous studies have demonstrated the toxicity of toxins from S. chartarum on animals and animal and human cells. Satratoxin G was the most cytotoxic of eight trichothecenes tested on mammalian cells, even more toxic than the well known T-2 toxin associated with alimentary toxic aleukia. Other researchers have also reported the high toxicity of satratoxins compared to other trichothecenes. The LD50 in mice for satratoxins is ~1 mg/kg (32).

In addition, S. chartarum us produces nine phenylspirodrimanes (spirolactones and spirolactams) and cyclosporin, which are potent immunosuppressive agents. It was suggested that the combination of trichothecenes and these immunosuppressive agents may be responsible for the observed high toxicity of this fungus. New biologically active compounds are still being discovered in cultures of S. chartarum, e.g. atranones A- G, dolabellane diterpenes and stachylysin, a hemolysin (compounds that lyse erythrocytes), and a hydroxamate siderophore. They suggest these compounds could be pathogenicity factors involved in pulmonary hemorrhage in infants exposed to S. chartarum.

M. verrucaria produced large amounts of macrocyclic trichothecenes when cultured on solid rice medium, including epiroridin E (16.8 mg/g crude extract), epiisororidin E (1 mg/g), roridin E (8.7 mg/g), roridin H (31.3 mg/g), trichoverrin A (0.6 mg/g), trichoverrin B (0.1 mg/g), verrucarin A (37.4 mg/g), and verrucarin J (2.2 mg/g). Most of these toxins were also isolated from M. verrucaria spores and mycelia grown on potato dextrose

agar medium, including epiroridin E (32.3 mg/g), epiisororidin E (28.6 mg/g), roridin E (0 mg/g), roridin H (60 mg/g), trichoverrin A (1.3 mg/g), trichoverrin B (1.8 mg/g), verrucarin A (13.8 mg/g), and verrucarin J (131 mg/g). Two new trichothecenes, 14'-hydroxymytoxin B (1) and 16- hydroxyroridin E were isolated from a fermentation extract of Myrothecium roridum

Stachybotrys chartarum, also called S. atra, S alternans or Stilbospora chartarum, is a black mould that produces its conidia in slime heads. It is sometimes found in soil and grain, but the mould is most often detected in cellulose-rich building materials from damp or water-damaged buildings. S. chartarum requires high moisture content in order to grow and is associated with wet gypsum material and wallpaper.

Stachybotrys chartarum

Myrothecium roridum M. verrucaria Microscopic structure

c.Trichoderma toxins

The common house mould, Trichoderma longibrachiatum, produces small toxic peptides containing amino acids not found in common proteins, like alpha-aminoisobutyric acid, called trilongins (up to 10% w/w). Their toxicity is due to absorption into cells and production of nano-channels that obstruct vital ion channels that ferry potassium and sodium ions across the cell membrane.

Trichoderma

Cultures are typically fast growing at 25-30°C, but some species of Trichoderma will grow at 45°C but not all. Colonies are transparent at first on media such as cornmeal dextrose agar or white on richer media such as potato dextrose agar . Mycelium are not typically obvious on CMD, conidia typically form within one week in compact or loose tufts in shades of green or yellow or less frequently white. A yellow

8. Mycotoxicoses

Mycotoxicoses are diseases caused by toxins produced by many fungi, mainly those belonging to the genera Aspergillus, Penicillium and Fusarium which are common contaminants of food and feed and affect man and animals.

•The mycotoxicoses may be divided into three general forms : (1) Acute primary mycotoxicoses are those which develop when high to moderate amounts of mycotoxins are consumed. Specific symptoms and signs of the toxic effect of the mycotoxins can be seen. (2) Chronic primary mycotoxicoses result from moderate to low levels of mycotoxin intake. Often non-specific effects such as reduced weight gain and reproductive efficiency occur. (3) Secondary mycotoxic diseases result from lesser levels of mycotoxin intake, which do not cause overt mycotoxicoses but which predispose to infectious diseases through impairment of immunogenesis and native mechanisms of resistance.

i. Aspergillotoxicosis

a. Aflatoxicosis:

Aflatoxins are hepatotoxic and hepatocarcinogenic to man, animals and birds. Ducklings are extremely susceptible, G. pigs are very susceptible, while chickens are less susceptible. Some lesions have been reported also in calves, sheep, dogs and cats.

• Aflatoxicosis in poultry primarily affects the liver, but can involve immunologic, digestive, and hematopoietic functions. It affects weight gain, feed intake, feed conversion efficiency, 46 pigmentation, processing yield, egg production, male and female fertility, and hatchability. Some effects are directly attributable to toxins, while others are indirect, such as reduced feed intake. Susceptibility to aflatoxins varies, but in general, ducklings, turkeys, and pheasants are susceptible, while chickens, Japanese quail, and guinea fowl are relatively resistant. Clinical signs vary from general unthriftiness to high morbidity and mortality. At necropsy the lesions are found mainly in the liver, which can be reddened due to necrosis and congestion or yellow due to lipid accumulation. Hemorrhages may also occur. In chronic aflatoxicosis, the liver becomes yellow to gray and atrophied, but tumor formation is rare with the natural disease, probably because the animals do not live long enough for this to occur.

• Aflatoxicosis in mammals includes inappetence, lethargy, ataxia, rough hair coat, and pale, enlarged fatty livers. Symptoms of chronic aflatoxin exposure include reduced feed efficiency and milk production, jaundice, and decreased appetite. Aflatoxin lowers resistance to diseases and interferes with vaccine-induced immunity. Although no level of aflatoxin is considered safe, the degree of toxicity is related to level of toxin, duration of feeding, and the amount of other stresses affecting the animal. Levels above 300 ppb to 700 ppb are considered toxic to cattle. There is some suggestion that dairy cattle may be affected at levels as low as 100 ppb, especially if fed for an extended period and if other stressors are present.

• Aflatoxicosis in fish includes poor growth, pale gills, reduced RBCs, anemia, impaired blood clotting, damage to liver, decreased immune responsiveness and increased mortality. In rainbow trout, prolonged feeding of a low concentration of aflatoxin B1 (AFB1) causes liver tumors. Rainbow trout is one of the most sensitive animals to AFB1, so LD50 for 50 g rainbow trout is 500 – 47

1000 ppb. However, warm water fish, such as catfish, are less sensitive to AFB1 (it has IP-LD50 of 11.5 mg/Kg body weight). Feeding catfish on at least 10 ppm AFB1 – contaminated feed for 10 weeks had adverse effects on the fish. Growth rate, PCV%, Hb concentration and erythrocyte count were lower than those from the other treatments (0, 100, 500, 2000 ppb). At the highest level, AFB1 caused necrosis and basophilia of hepatocytes, enlargement of blood sinusoids in the head kidney, accumulation of iron pigments in the intestinal mucosa epithelium, and necrosis of gastric glands.

• Aflatoxicosis in man is characterized by immunosuppressive, mutagenic, teratogenic and carcinogenic effects. The main target organ for toxicity and carcinogenicity is the liver. Several outbreaks of aflatoxicosis have occurred in tropical countries, mostly among adults in rural populations with a poor level of nutrition for whom maize is the staple food. The clinical picture presented by cases indicated acute toxic liver injury, which was confirmed by morphological changes in liver autopsy specimens that were indicative of toxic hepatitis. Mortality rates in the acute phase were 10-60 %. At the end of one year, surviving patients had no jaundice, and most of them had recovered clinically. Aflatoxins have been detected in the blood of pregnant women, in neonatal umbilical cord blood, and in breast milk in African countries, with significant seasonal variations. Levels of aflatoxins detected in some umbilical cord bloods at birth are among the highest levels ever recorded in human tissue and fluids. Aflatoxins have been suggested as an etiological factor in encephalopathy and fatty degeneration of viscera, similar to Reye syndrome, which is common in countries with a hot and humid climate. The clinical picture includes enlarged, pale, fatty liver and kidneys and severe cerebral oedema. Aflatoxins have been found in blood during the acute phase of the disease, and in the liver of affected children.

48 b. Ochratoxicosis:

• Ochratoxins cause nephrosis in swine and dogs, bovine abortion and death of horses, probably due to renal failure. They cause also severe liver lesions in ducklings and they cause in man what is called endemic Balkan nephropathy. All kinds of laboratory animals tested have been sensitive to injury by ingested ochratoxins. Regular consumption of a ration containing several hundred ppb of ochratoxin results in poor feed conversion, reduced growth rate, and general unthriftiness, accompanied by reduced immunity to infection by bacteria and viruses. Other prominent features of ochratoxin poisoning are increased water consumption and increased urine production because of kidney damage. The increased urine production in pigs results in the floor of the pig pen being constantly wet and needing to be cleaned daily.

• In cattle, OTA is rapidly degraded in the rumen and thus thought to be of little consequence unless consumed by young pre- ruminant calves, With high-grain diets, less of the dietary ochratoxin may be degraded in the rumen and thus be more toxic in those situations. Moldy alfalfa hay containing A. ochraceus was implicated as producing OTA associated with abortions in cattle. OTA in mouldy forage has also been implicated in cattle deaths.

• In man, acute renal failure in one person, possibly caused by inhalation of ochratoxin A in a granary which had been closed for 2 years, was reported in Italy. The symptoms developed after 24 hours of transitory epigastric tension, respiratory distress, and retrosternal burning. Acute tubular necrosis was found on biopsy, but the blood was not analyzed for ochratoxin A. The presence of the mycotoxin in wheat from the granary was proved qualitatively by thin-layer chromatography.

Owing to the similarity of morphological and functional kidney lesions in ochratoxin A-induced porcine nephropathy and endemic nephropathy, this mycotoxin has been proposed as the causative agent of endemic nephropathy, although the evidence for this is not substantial. This fatal renal disease occurs among rural populations in Croatia, Bosnia and Herzegovina, Yugoslavia, Bulgaria, and Romania, where it has been estimated that about 20,000 people are either suffering from or are suspected to have the disease. ii. Penicillotoxicosis:

Some mycotoxicoses, caused by Penicillium toxins, have disappeared owing to more rigorous hygiene measures. For example, citreoviridin-related malignant acute cardiac beriberi ("yellow rice disease" or shoshin-kakke disease in Japanese) has not been reported for several decades, following the exclusion of mouldy rice from the markets. Citreoviridin is a metabolic product of Penicillium citreonigrum, which grows readily on rice during storage after harvest , especially in the colder regions of Japan.

The Penicillium toxins are either hepatotoxic causing liver necrosis as in case of rubratoxins,luteoskyrin, and rugulosin, or neurotoxic as in case of patulin, cyclopiazonic acid,tremorgenic toxin and citreoviridin or nephrotoxic as in citrinin.

•Citrinin is produced by Penicillium and Aspergillus species and is a natural contaminant of corn, rice, and other cereal grains, is nephrotoxic and causes a diuresis that results in watery fecal droppings and reductions in weight gain. At necropsy, lesions are generally mild and involve the kidney. In cattle, the toxicity of citrinin causes necrosis of tubular epithelial cells in the kidney, and in some cases, hepatotoxicity

• Cyclopiazonic acid causes, in poultry, impaired feed conversion, decreased weight gain, and mortality. Lesions develop in the proventriculus, gizzard, liver, and spleen. The proventriculus is dilated and the mucosa is thickened and sometimes ulcerated. Cyclopiazonic acid fed for 10 weeks at a concentration of 100 µg/kg of diet had significantly growth-suppressing effect on catfish and a concentration of 10 mg/kg caused accumulations of proteinaceous granules in renal tubular epithelium and necrosis of gastric glands

iii. Fusariotoxicosis:

a.T-2 toxin has been associated with reduced feed consumption, loss in yield, gastroenteritis, intestinal haemorrhages, and death. T-2 is known to suppress immunity and interfere with protein synthesis. T-2 is toxic to the intestine, lymphoid tissues, liver, kidney, spleen, and bone marrow. T-2 is a severe gastrointestinal tract irritant, which can cause haemorrhage and necrosis of the intestinal tract. Diarrhoea is usually present but may not be hemorrhagic. T-2 may not occur alone and thus, naturally- contaminated feeds may contain other similar toxins. Cattle data are not sufficient to establish a tolerable level of T-2; but, a practical recommendation is to avoid more than 100 ppb of T-2 toxin in the diet.

b.DON is almost the primary mycotoxin associated with swine health problems including feed refusals, diarrhoea, vomiting, reproductive failure, and deaths. DON has been associated in cattle with reduced feed intake and milk production. DON may serve as a marker which indicates exposure of feed to a situation conducive to mould growth and mycotoxin formation. A dietary level of 300 ppb to 500 ppb DON may cause problems when fed to cattle.

c.Zearalenone induces estrogenic response in ruminants. Large doses are associated with abortions in cattle. Other cattle responses may include vaginitis, vaginal secretions, poor reproductive performance, and mammary gland enlargement of virgin heifers. Zearalenone may be associated with poor feed intake, a loss of milk production, poor conception, and increased reproductive tract infections. Establishment of a tolerable level of zearalenone for cattle is difficult based on a meager amount of data. As with DON, zearalenone may serve as a marker for toxic feed. Zearalenone above 200 ppb to 300 ppb in the diet may be of concern. Broiler chicks and laying hens, unlike swine and dairy cows, are affected very little by dietary zearalenone even when fed massive doses. Pure zearalenone fed to broiler chicks and finishing broilers at rates from 10 to 800 ppm produced no effect on weight gain, feed consumption, and feed-to-gain ratio.

d.Fumonisin ingestion affects different animals in different ways. Humans, horses, cattle, swine, poultry, mice, rats and rabbits have been found to be affected by fumonisin. Horses are extremely sensitive to fumonisin. Very low concentrations can cause leukoencephalomalacia (ELEM) or liquefication of the brain. It is also known as "crazy horse disease" and "the blind staggers." Affected horses display symptoms such as blindness, head butting and pressing, constant circling and ataxia, followed by death. In swine, fumonisin attacks the cardiopulmonary system, causing pulmonary edema and liver and pancreatic lesions. Fumonisins have been linked to cancer in humans.

In India a single outbreak of acute food-borne disease possibly caused by fumonisin has been reported. In the 27 villages involved, the individuals affected were from the poorest social strata, who had consumed maize and sorghum harvested and left in the fields during unseasonable rains. The main features of the disease were transient abdominal pain, borborygmus and diarrhea,

52 which began half an hour to one hour following consumption of unleavened bread prepared from moldy sorghum or moldy maize. Patients recovered fully when the exposure ceased and there were no fatalities. Fumonisin was found in much higher concentrations in the maize and sorghum from the affected households than from controls.

e.Fusarochromanone, when added to the diet of broiler chicks at 75 ppm, 100 percent of the chicks showed symptoms of TDP, it also kills chick embryos in fertilized eggs. TDP is a common and economically important bone deformation in growing broiler chickens and turkeys. The lesion appears in a cone of cartilage extending distally from the proximal tibiotarsalphysis.,

f.Trichothecenes are mycotoxins produced mostly by members of the Fusarium genus, although other genera (e.g. Trichoderma, Trichothecium, Myrothecium and Stachybotrys) are also known to produce these compounds. To date, 148 trichothecenes have been isolated, but only a few have been found to contaminate food and feed. The most frequent contaminants are deoxynivalenol (DON), also known as vomitoxin, nivalenol (NIV), diacetoxyscirpenol (DAS), while T-2 toxin is rarer. Fusariotoxicosis in poultry caused by the trichothecenes results in feed refusal, caustic injury of the oral mucosa and areas of the skin in contact with the mold, acute digestive disease, and injury to the bone marrow and immune system. Lesions include necrosis and ulceration of the oral mucosa, reddening of the GI mucosa, mottling of the liver, atrophy of the spleen and other lymphoid organs, and visceral hemorrhages. In laying hens, egg production decreases, accompanied by depression, recumbency, feed refusal, and cyanosis of the comb and wattles. Ducks and geese develop necrosis and pseudomembranous inflammation of the esophagus, proventriculus, and gizzard.

Common manifestations of trichothecene toxicity are depression of immune responses and nausea, sometimes vomiting. The first recognized trichothecene mycotoxicosis was alimentary toxic aleukia in the USSR in 1932; the mortality rate was 60%. In regions where the disease occurred, 54% of grain samples cultured showed the presence of Fusarium sporotrichoides, while in those regions where the disease was absent this fungus was found in only 2-8% of samples. The severity of mycotoxicosis was related to the duration of consumption of toxic grain. Such severe trichothecene mycotoxicoses, the consequence of continuous ingestion of toxins, have not been recorded since this outbreak.

In several cases, trichothecene mycotoxicosis was caused by a single ingestion of bread containing toxic flour or rice. In experimental animals, trichothecenes are 40 times more toxic when inhaled than when given orally. Trichothecenes were found in air samples collected during the drying and milling process on farms, in the ventilation systems of private houses and office buildings, and on the walls of houses with high humidity. There are some reports showing trichothecene involvement in the development of "sick building syndrome". The symptoms of airborne toxicosis disappeared when the buildings and ventilation systems were thoroughly cleaned.

iv. Facial Eczema (Pithomycotoxicosis)

Sporidesmins is a group of related mycotoxins produced by the pasture fungus Pithomyces chartarum (formerly Sporidesmium bakeri); causes liver damage and facial eczema in cattle and sheep. In this mycotoxic disease of grazing livestock, the toxic liver injury commonly results in photodynamic dermatitis. In sheep, the 54 face is the only site of the body that is readily exposed to ultraviolet light, hence the common name. The disease is most common in New Zealand but also occurs in Australia, France, South Africa, several South American countries, and probably North America. Sheep, cattle, and farmed deer of all ages can contract the disease, but it is most severe in young animals . v. Stachybotryotoxicosis

In the late 1930s, stachybotryotoxicosis was reported in humans working on farms in Russia. People who were affected are those who handled hay or feed grain infested with S. chartarum. Some of the individuals who were infected had burned the straw or even slept on straw-filled mattresses that had rampant growth of Stachybotrys chartarum. The infested straw can be described as black in colour from growth of the fungus. Common symptoms in humans that have stachybotryotoxicosis are rashes, especially in areas subject to perspiration, dermatitis, pain and inflammation of the mucous membranes of the mouth and throat, conjunctivitis, a burning sensation of the eyes and nasal passages, tightness of the chest, cough, bloody rhinitis, fever, headache, and fatigue. The workers who were infected developed symptoms within two to three days of exposure to the fungus. Some members of the Russian teams investigating this disease rubbed the fungus onto their skin to determine its direct toxicity. The fungus induced local and systemic symptoms similar to those observed in naturally occurring cases.

9. Management of mycotoxins contamination in feed

1. It is proposed that, where a mild contamination is suspected, a practical response might be to increase nutrient density in the formulation to compensate for the lower intake expected. Increase the vitamins, minerals and amino acids by between 5-20%, it said, depending on the severity of the feed refusal.

2. Diluting mouldy material with clean grains is often mentioned as a potential strategy, but risks are attached to it. One is that the diluting grain harbours other mycotoxins which combine synergistically with aflatoxins to have a more damaging impact on the animals. The other is that the initial contamination will be allowed to spread and so deteriorate a previously clean ingredient.

3. Decontaminating grain is rarely an option for the farm-mixer. Heat treatment can destroy unstable agents such as ergot alkaloids, yet has little effect on aflatoxins or on a range of other mycotoxins including zearalenone, vomitoxin and ochratoxin A. Other processes sometimes applied in feedmills and grain stores expose the grains to ammonia or radiation, neither of which has an easy application at farm level.

a. Simple rules that may be applied when dealing with moulds include. i. to pass the grain through a screen that extracts fine particles in which any mycotoxins are likely to be most concentrated.

ii. to be particularly cautious of grain by-products because toxin concentrations tend to be highest in the hulls or outer layers. iii. to consider using one of the rapid mycotoxin detection kits now on the market, together with an action plan that establishes both the maximum concentrations which can be tolerated and the use permitted for the ingredient according to its mould burden.

10. Detection of aflatoxins

a. Sampling

1. The sample must be adequate. Proper sampling is essential because one aflatoxin-contaminated kernel in 1,000 kernels of grain may be a source of significant contamination. 2. The sample must then be finely ground so that it will pass through a 15- to 20-mesh screen and be thoroughly blended to obtain a subsample appropriate for analysis. The sample should be representative. 3. A representative sample may require random sampling of plants in all areas of a production field, whereas, in freshly mixed grain (after harvest or following handling), a representative sample may be easily acquired by a few subsamples. 4. Samples stored for analysis should be placed in a paper bag or cardboard box and kept under cool, dry conditions that will not permit fungal growth or the possible continued production of mycotoxins. 5. Care must be taken to keep samples in the same condition as at the time of sampling. For example, moist grain samples stored in plastic bags under warm, humid conditions may have significant aflatoxin contamination occur during sample storage. 6. When probing a ship hold, grain bin, vehicle, or hopper car, numerous random probes may be required and site-selective

probing should be done if signs of moisture leakage, insect activity, or hot spots are identified.

b. Methods of analysis

The methods of aflatoxin analysis fall into three categories :

I. Visual inspection of the grain, which may locate lots presumed to be contaminated with aflatoxin (blacklight test); II. Rapid screening procedures to determine the presence or absence of aflatoxin (the fluorometric iodine rapid screening and minicolumn tests III. Laboratory procedures quantifying the actual amounts of toxin present (thin-layer chromatography, gas-liquid chromatography, high-pressure liquid chromatography, fluorometric iodine, or ELISA tests). Various commercial, state, and federal laboratories perform aflatoxin analyses on a fee basis

7. Prevention and control of mycotoxin formation in feed:

Prevention of mycotoxin formation ideally should begin from the point of harvesting, then storage and transportation. However, many times, it goes beyond control. Harvest at maturity and as soon as the moisture content allows minimum grain damage. The harvesting equipment should be adjusted for minimum seed or kernel damage and maximum cleaning. All grain should be dried to at least 15-percent moisture as rapidly as possible, not to exceed a 24- to 48-hour period after harvest. Grains should be stored under dry storage conditions in water, insect and rodent-tight structures:

1. Elimination of oxygen is a very important mycotoxin preventative management practice. 2. Some additives may be beneficial in reducing mycotoxins because they are effective in reducing mould growth. Ammonia, propionic acid, and microbial or enzymatic silage additives are reported to be at least partially effective in inhibiting mould growth. 3. Silo size should be matched to herd size to ensure daily removal of silage at a rate faster than deterioration. 4. Feed bunks should be cleaned regularly. 5. Care should be taken to ensure that high moisture grains are stored at proper moisture content and in a well-maintained structure. 6. Grains or other dry feed, such as hay, should be stored at a moisture content (<14%) below which moulds do not readily grow. 7. Aeration of grain bins is important to reduce moisture migration and to keep the feedstuffs in good condition. 8. Mouldy feed should be avoided. If unacceptably high levels of mycotoxins occur, dilution or removal of the contaminated feed is preferable. However, it is often a problem to completely replace some feeds in the ration, particularly the forage ingredients. Use of mould inhibitors and toxin binders provides practical solution. However, make sure, the selected product is effective at the field condition. 9. Mould inhibitors are commonly acids in liquid form as propionic, sorbic, formic, acetic acid or their salts such as probionate or sorbate salts . 10. Adsorbent materials, such as hydrated sodium calcium aluminosilicate and bentonite clays, are added to mycotoxin- contaminated diets of rats, poultry, swine, and cattle. Adsorbent materials bind some mycotoxins, reducing their availability to the animal. Researches showed that the addition of adsorbents to diets containing aflatoxin(AFB1), significantly reduced aflatoxin residues in milk (AFM1).

8. Detoxification of mycotoxins in food and feedstuffs: a. Physical methods:

1. Cleaning methods:

It consists of scouring, aspiration, sieving, and specific gravity separation. Dust, husks, hair and loose superficial particles are blown away by scouring and aspiration. Extensive aspiration can considerably reduce the quantity of mould spores and mycelia on the kernel surface. Sieving and specific gravity separations remove small kernels, kernel debris, weed seeds and other impurities which differ in size and gravity in comparison to normal kernels.

2. Heat:

• Aflatoxins are relatively resistant to thermal inactivation and are destroyed only at temperatures of around 250 ºC. Therefore, processes which include heating should reach that temperature range of toxin destruction. For example, roasting of green coffee beans at 200 ºC for 12 min caused a reduction of 79 % in aflatoxin content, whereas after 15 min, 94 % of the toxin had disappeared • Ochratoxin A is highly stable to heat treatment and it is not destroyed even at 200 ºC. However, heating in the presence of NaOH resulted in decomposition and detoxification of the toxin (by using HeLa cells). • Most of the Fusarium mycotoxins are relatively resistant to heat. Zearalenone was not destroyed in corn, even after 44 h, at 150 ºC and fumonisin B1 required a high temperature (150–200 ºC) to achieve 87–100 % destruction in corn cultures. Canned, whole-kernel corn showed a significant decrease in fumonisins at an average rate of 15 %. Canned, cream-style corn and baked corn bread showed

60 significant decreases in fumonisin levels at average rates of 9 and 48 %, respectively. DON was not usually completely destroyed during baking – if any reduction in the amount of DON occurred, it was not more than 50.

3. Microwaves

• A reduction of at least 95 % in aflatoxin content in peanuts occurred following a 16 min treatment at a microwave power level of 1.6 kW or a 5 min treatment at a power level of 3.2 Kw. • Microwave treatment was only partially successful in lowering deoxynivalenol levels but it was most effective at the highest temperatures •There is no additional evidence of the destruction of other mycotoxins by microwaves.

4.Irradiation

Despite the advantages associated with the use of irradiation, and apart from the difficulties involved (special equipment and safety issues), the dosages required to degrade most mycotoxins are well above those permitted for use in food preservation (up to 10 kGy). Therefore, at the present time, GI cannot be regarded as a practical means for mycotoxin elimination. b. Biological detoxification of mycotoxins:

• Biological detoxification’ of mycotoxins occur by two major processes:. i. Sorption:

• Live micro-organisms can absorb either by attaching the mycotoxin to their cell wall components or by active internalization and accumulation. • Dead microorganisms absorbed mycotoxins, and this phenomenon can be exploited in the creation of biofilters for fluid decontamination or probiotics to bind and remove the mycotoxin from the intestine.

ii. Enzymatic degradation:

• Mycotoxin degradation by enzymes has also been reported. • It can be performed by either extra- or intra-cellular enzymes. The degradation can be complete, the final product being CO2 and water. Alternatively, enzymatic modification can alter, reduce or completely eradicate toxicity.

9. Control of mycotoxins during food processing i. Beverages processing:

• Patulin is produced by Penicillium expansum in apples and some other fruits; it can thus occur in apple juice which may be produced directly through crushing to a pulp. The processing of apples usually involves removal of decayed apples, initial washing, crushing/pulping, clarification using rotary vacuum and pasteurization. Also, washing and handling were critical steps in reducing patulin in apples since up to 54 % could be removed by high-pressure water spraying.

• Reduction of patulin was shown to be due to binding to solid substrates. Pressing followed by centrifugation proved the most effective, removing 89 % of themycotoxin. Also. the complex clarification procedure could reduce patulin levels by about 40. Complete destruction of patulin by the alcoholic fermentation using

Saccharomyces cerevisiae for 48 hours or by treatment with 0.125 % sulphur dioxide. . • Ochratoxin A often occurs in green coffee beans which during processing. is cleaned, roasted, typically for 20 minutes at about 200 ºC, ground, and then extracted with hot water, concentrated and spray-dried. The ochratoxin A in coffee. level was only slightly reduced after roasting, with most of the mycotoxin then eluting into the brew. Roasting time varied from 2.5 to 10 minutes and the roast colour varied from light medium to dark. The reduction was about 69 % over the combined results. ii. Milk and dairy products:

Aflatoxin M1 concentrations falls in the cheese during the ripening process. Aflatoxin M1 concentrations falls between 13 and 22 % when cows’ milk is fermented to produce yoghurt and by 16 and 34 % after storage of yoghurts of PH4.6 and 4.0 respectively. iii. Dried fruit:

The transfer of aflatoxins from contaminated figs to fig molasses and a 65 % reduction of aflatoxin B1 was occurred during transfer , but smaller losses for aflatoxins B2, G1 and G2. iv. Beans:

Up to 84 % loss of ochratoxin A was detected in processing beans (Phaseolus vulgaris L.), although smaller losses of about 53 % were reported previously after bleaching, salting and heat treatment. Greater losses were observed when beans were soaked in water for 12 hours before cooking under pressure at 115 ºC for 45 minutes.

10. Biological detoxification of mycotoxins:

Biodegradation may enable the removal of mycotoxins under mild conditions, without using harmful chemicals and without significant impairment of the nutritive value or palatability of the detoxified food or feed. The entire organism can be used as a starter culture, as in the fermentation of beer, wine and cider, or in lactic acid fermentation of vegetables, milk and meat. The purified enzyme can be used in soluble or immobilized (biofilter) forms. The gene encoding the enzymatic activity can be transferred and overexpressed in a heterologous system; interesting candidates for this application include yeasts, probiotics and plants. Biological detoxification’ of mycotoxins occur by two major processes:. a. Sorption:

Live micro-organisms can absorb either by attaching the mycotoxin to their cell wall components or by active internalization and accumulation. Dead microorganisms absorb mycotoxins, and this phenomenon can be exploited in the creation of biofilters for fluid decontamination or probiotics to bind and remove the mycotoxin from the intestine.

b. Enzymatic degradation:

Mycotoxin degradation by enzymes has also been reported: aflatoxin degradation by maize was suggested , but the reduction in toxin level may have been due to conjugation or absorption and there is no clear evidence of aflatoxin-degrading enzymes in plants. It can be performed by either extra- or intra-cellular enzymes. The degradation can be complete, the final product being CO2 and

64 water. Alternatively, enzymatic modification can alter, reduce or completely eradicate toxicity.

Mycotoxins can be detoxified by the plant during microbial fermentation (alcoholic or lactic) or by probiotic or other symbiotic micro-organisms; the producer might degrade its own toxin; and soil or water micro-organisms (mixed or pure culture) can be very effective in degradation.

Organisms that have been demonstrated to detoxify aflatoxin include the bacterium Corynebacterium rubrum, the yeast Candida lipolytica, and several fungal species, Aspergillus niger, Trichodermaviride and Mucor ambiguous . In addition, several Rhizopus species, A. niger and Neurospora species were found to reduce aflatoxin levels. A number of fungal species have been shown to prevent aflatoxin biosynthesis in culture, as well as to degrade the toxin. Among these, a Phoma species was the most efficient, destroying about 99 % of the aflatoxin .

Under certain conditions, some of the mycotoxin-producing moulds are able to degrade their own toxin. Aflatoxin was degraded by A. parasiticus and A. flavus.The degradation involved lactoperoxidase , peroxidases and P450 monooxygenases.

A non- toxigenic strain of A. flavus has been found to degrade the aflatoxin produced by a toxigenic strain . Cell-free enzymatic preparations that exhibit mycotoxin-degrading activity can be used as food or feed additives, biological filters or food- processing additives.

• Enzymatic transformation by the producing fungi was observed when a Fusarium moniliforme strain reduced fumonisin B1 concentrations in liquid culture . Also, the black yeasts Exophiala spinifera and Rhinoclodiella atrovirensa hydrolyzed ester bonds of fumonisin B1 and the hydrolyzed

65 fumonisin exhibited cytotoxicity to rat hepatocytes and human colonic cells . •The degradation of mycotoxins by micro-organisms in silage and other moist feed is an attractive method for the decontamination of crops.

• Bacteria, fungi and yeasts have been investigated for their ability to degrade mycotoxins.

• About 1000 micro-organisms was screened for their ability to detoxify aflatoxin and found that Flavobacterium aurantiacum was effective. Apart from F.aurantiacum, a number of bacterial and especially fungal species have been found to detoxify aflatoxin and Rhizopus sp. has been claimed to be particularly suitable for large- scale detoxification of aflatoxin-contaminated feeds by solid-state fermentation .

• Ochratoxin A is rapidly degraded by micro-organisms in the rumen. Acinetobacter calcoaceticus to be able to degrade ochratoxin in an ethanol-containing medium.

• Different strains of Lactobacillus, Bacillus and Saccharomyces have also been shown to degrade ochratoxin in vitro to varying degrees.

Some strains were able to degrade up to 94 %. They were also tested for degradation of trichothecenes, but with less success. Additional yeast strains have also beentested, and some were able to partly degrade ochratoxin, nivalenol, deoxynivalenol, zearalenone and fumonisins .

• Reduction of zearalenone has been shown in ruminal fluid and for many mixed and pure cultures of bacteria, yeast and fungi, but the

66 transformation cannot be considered as a detoxification as the zearalenols still show estrogenic activity.

•The degradation product will, in some cases, probably regain its estrogenic potential when fed to animals.

• Soil bacteria have been investigated for the degradation of trichothecenes but only a single active culture and bacterium could be isolated.

• Microbial degradation of trichothecenes to their non-toxic de- epoxides has been found in ruminal fluid and intestinal contents from pigs, hens and rats

• From ruminal fluid , an anaerobic bacterium belonging to the genus Eubacterium, is able to degrade trichothecenes to de-epoxy metabolites . This bacterium has been cultured, produced and stabilized in order to be used as a feed additive.

• Yeasts in grass silage have been found to degrade patulin in silage inoculated with Paecilomyces sp. to induce patulin production. Both bacteria and yeasts from maize silage have also been shown to be able to degrade fumonisins .

• The degradation of mycotoxins during alcohol fermentation for the production of ethanol has been investigated only in a few studies, but many papers have appeared on the fate of mycotoxins during the production of beer and wine.

• Enzyme preparations from Saccaromyces telluris, which are claimed to contain activities for hydrolyzing the lactone bond of zearalenone and to destroy the epoxide group in trichothecenes, are included in Mycofix Plus (Biomin,Herzogenburg, Austria).

• The intestinal microflora of chicken with a deoxynivalenol de- epoxidation capacity was used to detoxify deoxynivalenol contaminated maize which was fed to pigs. Feed intake, weight gain and feed efficiency were significantly improved.

11. Limits for mycotoxins in foods and feeds:

Maximum limits for mycotoxins in foods in various European countries and USA(2002)

Mycotoxin Country Maximum limit Foods (_g/kg or _g/l) Aflatoxin B1 Finland 2 All Germany 2 All

The 5 All

Belgium 5 All

Portugal 25 Peanuts

5 Children’s food

20 Others

Austria 1 All 2 Cereals, nuts Switzerland 1 All

2 Maize, cereals

Spain 5 All

Luxembourg 5 All

Ireland 5 All

Denmark 5 All

Greece 5 All

Aflatoxin M1 Sweden 0.050 Liquid milk products

Austria 0.050 Milk

Germany 0.050 Milk

Netherlands 0.050 Milk

0.020 Butter

0.200 Cheese

Russia 0.5

Switzerland 0.020 Baby food

0.050 Milk and milk products 0.250 Cheese

Belgium 0.050 Milk USA 0.50 Milk

Czech Rep. 0.1 Children’s milk 0.5 Adult’s milk

France 0.03 Children’s milk 0.05 Adult’s milk

Bulgaria 0.5

Deoxynivalenol USA 1000 (monitoring) Wheat

Russia 1000 Cereals

Austria 750 Wheat

Ochratoxin A Romania 5 All

Czech Rep. 1 Children’s 20 food

Denmark 5 Cereals

25 Pigs

Austria 5 Cereals

Switzerland 2 Cereals

Greece 20 All

France 5 All (proposal 70

The 0 Cereals Netherlands Fumonisin Switzerland 1000 Maize B1+B2 Zearalenone Romania 30 Cereals, vegetable oils Austria 60 Cereals France 200 Cereals, vegetable oils Russia 1000 Cereals, vegetable oils

T2-toxin Russia 100 All

12. Future studies and recommendation

• Most research effort has concentrated on the means for prevention of mycotoxigenic moulds growth and mycotoxin formation, and this must remain the best defence for protecting the consumer.

• Achieving the correct limit minimizes any unnecessary restriction on the use of valuable food commodities without compromising human health. Thus introduction of new approach for controlling the growth and mycotoxin production become critical demands , particularly the mycotoxins that pose the greatest potential risk for humans. In some instances it may then be possible to introduce modifications to commercial processes that result in a significant reduction of mycotoxin content in feed and food product.

• Recently, nanotechnology was introduced by the authors successfully in therapeutic and prophylactic control of mycotoxigenic moulds, mycotoxin and other fungal pathogens. The further evaluation of metal nanoparticles in degradation and detoxification will be monitored.

Synopsis of studies done by authors on mycotoxigenic fungi and mycotoxins in foods and feeds Contents 1. Fungal contamination of meat 2. Fungal contamination of meat products 3. Fungal contamination of meat environment 4. Fungal contamination of feeds and feed ingredients 5. Mould infection of fresh and smoked fish 6. Mould infection in milk products 7.Toxigenicity of moulds isolated from foods and feeds a. Studies on the toxins of A. niger isolated from mouldy smoked fish b. Production of aflatoxins by A. flavus on natural and synthytic media c. Aflatoxin production by A.flavus isolated from feeds d. Ochratoxin production by A.ochraceus isolated from feeds e. Production of toxins produced by Fusarium species isolated from feeds

8. Detection of mycotoxins in foods and feeds a. Detection of aflatoxins in meat and meat products b. Detection of mycotoxins in milk and milk products c. Detection of aflatoxin B1 and ochratoxins in feed samples d. Detection of aflatoxins by biological technique (Bioassay)

9. Detection of mycotoxins in diseased birds, animals and fish

10. Detection of mycotoxins in diseased birds, animals and fish 11. Mycotoxins effects on immune and endocrine systems 12. Miscellaneous studies on mycotoxins 13. Control of fungal growth and mycotoxins

1. Fungal contamination of meat

In the year 1969, an extensive study of mould contamination of meat was carried out. 3617 swabs were taken from the surfaces of fresh meat in slaughter houses, butcher’s shops, cold stores and transport means. As shown Table 1, moulds were equally distributed on the surfaces of different types of meat. The 1748 isolates recovered were identified into 12 mould genera, where the genera Aspergillus, Penicillium and Rhizopus were the most common. Aspergillus niger was by far the most prevalent, constituting 38.9% of the total isolates, followed by Penicillium species with 26.7%. However, A. fumigatus and A. flavus.5

Table 1: Number and types of moulds isolated from meat

The fungal flora of fresh and chilled meat were studied in modern abattoirs in the year 1993. The culturing of 200 swabs from the surfaces of the meat revealed the isolation of 299 fungal isolates (Table 2). Aspergillus (24.75%), dematiaceous fungi (22.41%) and Penicillium species (15.0%) were the most predominant on the surface of fresh meat.. A. niger and Cladosporium species were the most common. More fungi were

74 isolated from region of the shoulder than from that of the thigh. It is to be noted that Penicillium species were the most frequent mould isolates recovered from chilled meat.22

Table 2: Fungi isolated from fresh and chilled carcasses (100 swabs each)

2. Fungal contamination of meat products

Attention was directed particularly to basterma, as early as 1968, because it can be eaten directly without being subjected to any heat treatment. A. niger, A. candidus, A. flavipes as well as Penicillium, Hormodendrum, Stemphylium, Trichothecium, Monilia,Pullularia and Chaetomium species were isolated from samples of basterma collected from shops. The experimental infection of fungal free basterma with the isolated moulds, revealed that \only Penicillium species showed visible growth which started at the inoculated sites, then spread all over the piece of basterma (Fig. 1).2 75

Fig. 1: Heavy growth of Penicillium species on basterma

The problem of fungal contamination was thoroughly studied by the authors in 2003, where the product was examined before and after processing, also the additives were tested individually. As shown in Table 3, the contamination was higher in samples collected during summer, particularly in the coat, and all additives showed high fungal counts.30

Table 3: Total mould countnof basterma and its components

It is to be noted that , the meat used for preparation of basterma was free from Aspergillus and Penicillium species, but these fungi showed high incidence in the additives, particularly coriander, fenugreek, capsicum and pepper. This explains the source of contamination of the meat in the final product (Table 4).

Table 4: Incidence of mould genera in basterma and its components

The authors carried out several studies on mould contamination of meat products. In one of the studies they found that the predominant fungi isolated from 40 samples of sausage were Aspergillus and Penicillium, but Fusarium, Cladosporium, Mucor, Rhizopus spp. were also found. The TMC was 3.8 x 102 / g. A. niger and A. flavus constituted the most fungal contamination (65.0% and 32.5%) (Table 5 and 6).45

Table 5: Mycoflora of sausage samples

Table 6: Members of Aspergillus species isolated from sausage samples

The examination of 81 samples of frozen meat, chickens meat and meat products (minced meat, sausage, luncheon and kofta) revealed that the highest mould count was recovered from minced meat (5 x 102 / g). while chicken meat and frozen meat samples showed the lowest mould count (3.3 x 101, 4.1 x 101 / g, , respectively ). Aspergillus and Penicillium species were detected in 79 all examined samples while Fusarium, Rhizopus and Mucor species were detected in some examined samples. A. niger, A. flavus and A. fumigatus were the most predominant species isolated from the examined samples (Tables 7 and 8).49

Table 7: Total mould counts of meat and meat product samples

Table 8: Aspergillus species isolated from meat and meat product samples

In another study, 100 samples of spiced minced meat (hawawshy), raw and cooked meat (shawerma) were investigated for mycological contamination. The mean colony count of A. niger was ( 8.3 x 10 and 2.7 x 10), A. flavus was (9.0 x 10 and 3 x 10) and Penicillium species was ( 6.4 x 10 and 1 x 10) for both raw and cooked hawawshy, respectively. A. flavus and A. niger showed the highest colony count in raw shawerma (4.5 x 10 and 3.6 x 10 colony / g), while in cooked shawerma, Penicillium showed the highest colony count (4.1 x 10 colony / g) (Tables 9 and 10).53

Table 9: Total colony counts of fungi in raw and cooked hawawshy

Table 10: Total colony counts of fungi in raw and cooked shawerma

In a recent study (2008), 200 samples of meat and meat products including imported frozen beef cuts (50) and processed meat products (30 each of basterma, hamburger, luncheon and sausage were tested for mould contamination. As shown in Table 11, the commonly isolated moulds were Aspergillus spp (75%), followed by Penicillium species (35.0%) and Mucor (12.0%). Other moulds such as Alternaria spp, Cladosporium spp, Curvularia spp, Fusarium spp. Geotrichum species, Paecilomyces species, Rhizopus spp., Scopulariopsis spp and Trichoderma spp. were rare.

Table 11: Frequency of identified moulds isolated from meat and meat products

From Table 12, it is evident that the examined frozen and sausage samples had the highest total Aspergillus species (80%), with A. niger on the top (24%), followed by A. flavus (20%), while A. fumigatus, A. terreus, A. versicolor were isolated at a rate of 8% each, followed by A, candidus, A. ochraceus and A. parasiticus (4%). In sausage samples, also A. niger was the most common, followed by A. ochraceus, A. candidus, A. fumigatus, A. parasiticus and A. terreus.34

Table 12: Frequency of identified Aspergillus spp. isolated from meat and meat products

3. Fungal contamination of meat environment

In an extensive study of mould contamination of meat environment 2005 samples were collected from the air, water, walls, floors, utensils, workers’clothes in slaughter houses, butcher’s shops, cold stores and transport means . It is clear from Table 7 that moulds were found in a high percentage in the surroundings, especially in the air, on walls and floors; these were equal in distribution in slaughter houses and cold stores, but slightly less in shops and more frequent in transport means. On the contrary, water played no role in the contamination. Aspergillus niger and Penicillium species were the most common, but A. fumigatus and A. flavus were in low numbers (0.03 and 0.009%).5

Table 13: Number and types of moulds isolated from the environment of slaughter houses, butcher’sshops, cold stores and transport means

As demonstrated in Table 14, the samples taken from the meat surroundings in modern abattoirs revealed that the air was the main source of contamination in both freshly slaughtered and chilled carcases. The air yielded 2136 isolates. Penicillium and Cladosporium species were the most common moulds isolated from the air of chilling halls. Other genera were Paecilomyces, Scopulariopsis, Trichoderma, Mucor, Rhizopus, Fusarium, Acremonium and Geotrichum. 22

Table 14: Frequency distribution of fungi from air of a modern abattoir

The .Aspergillus species were identified as A. niger, A. flavus, A. fumigatus, A. ochraceus, A. tamari, A. terreus and A. parasiticus. Of the demtiaceous fungi isolated from the air, Cladosporium spp. were the most common, followed by Alternaria, Curvularia, Helminthosporium, Nigrospora, Stemphylium, Epicoccum spp. and Stachybotrys atra. A total of 1535 fungal isolates were recovered from the floors, walls and utensils of slaughter, chilling, deboning and processing halls of the modern abattoirs. Aspergillus species, dematiaceous moulds and penicillium species were the most common isolates.. Of particular interest was the frequent recovery of Cladosporium from the samples (Table 15).22

Table 15: Frequency distribution of moulds isolated from meat and its surroundings

4. Fungal contamination of feeds and feed ingredients

Attention was directed to the mould contamination of poultry feeds as early as 1968, where fifty six samples of poultry feeds (fish, blood, meat and bone meal) were collected from different farm-stores in Egypt. Thirty-seven strains of molds were isolated which could be grouped in the genera Rhizopus, Aspergillus, Penicillium, Mucor, Scopulariopsis and Paecilomyces. The different species are tabulated in Table 16. It was clear that most fungi were frequently isolated from fish meal, whereas in other animal byproducts they were rarely met with. The predominant fungus was Rhizopus nigricans. Penicillium species were not met with in blood meal. Aspergillus fumigatus and A. flavus which were reported to 87 cause infection in poultry in Egypt were isolated only once from fish and blood meal, respectively.3

Table 16: Types of fungi isolated from different poultry feeds

The examination of grains and feed samples in 1985 and1988 revealed that in grains the genus Aspergillus was the most predominant one followed by Fusarium, Penicillium, Rhizopus and Mucor. While in feed samples the most frequent fungi were Aspergillus, Rhizopus, Penicillium, Mucor and Fusarium.9,12

The occurrence of ochratoxigenic mould contamination of feed and feedstuffs in Egypt revealed a rather high incidence of Aspergillus ochraceus and Penicillium spp. in samples tested. Straw samples were the most frequently contaminated with A. ochraceus (92%), whereas wheat bran were the most samples contaminated with Penicillium species (90%).26

The examination of eighty food and feed samples (20 of each of lentil, broad bean, yellow corn and poultry feed containing Soya bean meal, wheat-bran, crushed corn and vitamins) collected from different localities of Cairo markets showed that the mean total count of moulds ranged from 1.8 – 7.3 x 103 / gram. The most predominant fungi isolated were A. flavus, A. fumigatus, A. candidus, A. ochraceus, A. niger, A. ustus, A. glaucus, Fusarium species, Penicillium species, Mucor species, Rhizopus species, Alternaria and Cladosporium (Table 17). 50

Table 17: Prevalence of fungi of poultry feeds and grains

Two hundreds and fifty samples of feeds (125 of each of ingredients of plant origin “yellow corn, white corn, Soya bean, soya bean meal, wheat and beans” and compound manufactured feed and animal protein concentrates (meat-bone meal, fish meal, poultry offal and mixed feed) were collected from various poultry farms of Giza and Cairo Governorates. Fungal isolation from ingredients of plants origin revealed lower rate of mould contamination in comparison to compound manufactured feed and animal feed protein concentrates. For instance, Aspergillus species was isolated from 48 – 68% and 80 – 100% of feed ingredients and animal concentrates, respectively.61 90

Imported animal feed were also evaluated for fungi. Nine genera and eight species of moulds were isolated. The genus Mucor and Penicillium (66% and 65%) were predominantly isolated, followed by Aspergillus (40%), Fusarium (20%) and Cladosporium (14%). A. flavus was frequently isolated (28%) but F. graminarium was obtained only from 10% of samples. Species of Rhizopus, Scopulariopsis and Alternaria were yielded from 4, 2 and 2% of samples, respectively (Table 18).57

Table 18: Prevalence of moulds in imported animal feeds

Poultry feeds received a great attention by the authors. The prevalence of fungi isolated from single poultry feeds was studied by examining 200 feedstuffs (20 of each of yellow corn, wheat, Soya bean, hay, tibn and layer’s concentrates. The isolated fungi from single feeds represented 12 genera. Members of the genera Aspergillus, Penicilium and Cladosporium were isolated from all types of feeds. Aspergillus species were recovered from all samples (100%), while Penicillium species were recovered from (30-90%) of the samples.33

Table (19) demonstrates that Aspergillus isolates (225) were the most common, followed by Penicillium (68) and Fusarium isolates (24). Aspergillus niger was the most common (86 isolates) and was found in all types of feeds with the highest rate of isolation (100%) from hay and tiben, followed by Soya bean (90%), yellow corn and wheat (70%). The second most common Aspergillus species was A. flavus (64 isolates), which was recovered from all hay samples (100%) , followed by Soya bean and tiben samples (80%), then yellow corn (60%). A. ochraceus (13 isolates), and A. parasiticus (4 isolates) were rare. The (68) Penicillium isolates were identified into (8) species with P. thomii at the top (30 isolates), followed by P.chrysogenum (11 isolates), P. digitatum (7 isolates), P. viridicatum and P. funiculosum (6 isolates each), while P. verrucosum was isolated only once from a sample of Soya bean. Twenty one isolates of Fusarium species were identified as F.solani (8 isolates), F. tabacium (5 isolates), F. violacium and F. oxisporium (each 4 isolates) and F. tricinctum (3 isolates), Fusarium species were not found in Soya bean.33

Table 19: Prevalence of members of Aspergillus, Penicillium and Fusarium species in single poultry feeds

The results of the study on prevalence of fungi in compound feeds are shown in Table 20. It is clear that fungi isolated from compound feeds were identified in 9 genera. The least number of genera was recorded in bone and meat meals (5 genera) and the highest was in processed animal feeds (8 genera). Aspergillus and Penicillium species were recovered from all types of feeds, where Aspergillus species were found in 94 samples. Aspergillus species were the most common in poultry ration, followed by broiler's concentrates, layer's concentrates bone, meat meals and 93 processed animal feeds. The 203 isolates could be identified into 8 species, with A. flavus at the top of the list, followed by A. niger, A. terreus, A. candidus, A. ochraceus, A. fumigatus, A. parasiticus and A. glaucus. Penicillium isolates recovered from 68 samples could be identified into 8 species, where P. digitatum was the most common, followed by P. chrysogenum, P. thomii, P. viridicatum, P. restrictum, P. citreoviride and P. purpurogenum. Fusarium isolates were isolated mainly from poultry ration. They were identified as F. solani, F. oxysporium, F. tricinctum and F. moniliform.33

Table 20: Prevalence of members of Aspergillus, Penicillium and Fusarium species in compound poultry feeds

Fusarium species The Fusarium was always in the back mind of the author since the mid sixties, when several donkeys died in the Sharkia Govornorate at that time with nervous manifestations as the main symptoms. The pathologists diagnosed the cases as encephalomalacia and the virologists failed to isolate a virus. The author had the chance to visit the places, where the donkeys died and noticed that donkeys were eating the corn stumps, which were covered with pink fungal growth, which was identified as Fusarium species. Regrettably, the author had no facilities at that time to continue studying the problem and resorted to education of the farmers to prevent their animals from consuming such mouldy stumbs through lectures and announcement in the daily news paper Al-Ahram (Fig. 2). The farmers responded positively, so that in a short time the problem was solved.

Fig. 2

The authors had the chance to consider Fusarium in the studies done later on feeds, which are mentioned above. The evaluation of 160 samples of feed (80 of each of yellow corn and mixed feed) at different seasons of the year for fungal contamination indicated that the Fusarium species were isolated 96 only during winter in tested samples (5, 15%) of yellow corn and mixed feeds respectively.47

Moreover, two studies were done on Fusarium. The first study was concerned with the incidence of Fusarium in equine feeds. In this study 100 equine feeds ( 48 barley, 16 pelleted feeds,14 Soya bean, 12 yellow corn and 10 hay samples) were collected from different farms and clubs and subjected to mycological examination for isolation and identification of Fusarium species. All samples examined were contaminated with moulds. The highest total mould count/g was obtained from the hay samples, followed by barley, Soya bean, yellow corn and pelleted feeds samples. TMC/g in hay samples ranged from 4.20 x 103 to 5.30 x 105with a mean count of 1.415 x 105 , while in barley the TMC/g ranged from 1.00x10 to 1.28x105 with mean count of7.42x103. However, in soya the count ranged from9.00x10 to 7.50x104 with mean count of 2.805x104 , TMC/g of yellow corn samples ranged from 1.40x103 to 5.40x104 with mean count of 2.25x104, in pelleted feeds the count ranged from 2.00x102 to 2.40x104 with a mean count of 4.48x103. Fusarium species were recovered from 23 samples (23%).35

The highest total Fusarium count/g was obtained from the Soya bean samples, however, the Fusarium colony counts constituted the highest percentage of total fungal count in barley, which reached to 71.43% in one sample, 50.0% in one samples, 10-18% in 6 samples and 5.55% in one sample. In Soya bean, the highest contribution of Fusarium in total fungal count was 33.33% in one sample and 18.75% in another sample, while the rest of the samples the contribution varied from 1.52-3.57%. The lowest Fusarium count was observed in corn and hay where it varied from 0.88-11.90% and 2% respectively of the total fungal count only. The incidence of Fusarium was the highest in samples of yellow corn 58.33%, followed by soya, in which the incidence of Fusarium was 42.85%; in barley it was 18.75% and in hay it was 10%. Samples of

97 pelleted feeds were all negative for Fusarium. 44 isolates of Fusarium were recovered from equine feeds. The isolates were identified into 9 Fusarium species. The most common Fusarium species was F. verticillioides (21 isolates), followed by F. anthophilum (9 isolates), F. proliferatum and F. solani (4each), F. dimerum (2 isolates) and one isolate of each of F. nygamai, F. oxysporum, F. poae and F. sporotrichoides. Only one isolate was recovered from hay, which was identified as F. verticillioides.35

The results of the second study were published in the year 2001, where 14 Fusarium species were isolated feedstuffs and identified (Table21)29.

Table 21: Prevalence of Fusarium species in feedstuffs

5. Mould infection of fresh and smoked fish

During 1966, 6420 kg imported smoked herring , packed in 642 wooden boxes were found to be mouldy on inspection. There was a musty odour and black and white fungal spots covering the surface of the herring (Fig. 3), the wrapping papers and sides of the boxes. The uppermost layers were heavily contaminated with the fungi. The proportion of the white spots to the black ones was 1:8. Direct microscopic examination of smears made from the spots revealed the presence of Aspergillus and Penicillium species. The microculture of the black spots presented black and globose heads of A. niger group. The Penicillium isolate was identified as P. funiculosum.1

Fig.3 Smoked herring with fungal spots

These findings initiated the interest to make a study on the local fish.Two studies were carried out, the first one was done on freshwater fish Oreochromis species and Clarias gariepinsi and the second study was carried out on fresh Tilapia nilotica. Mycological examination revealed the isolation of 2081 isolates from 150 diseased and 210 apparently healthy fish samples, of which 1334 were isolated from Oreochromis species and 747 isolates from 100

Clarias gariepinsi. Identification of fungi revealed that the percentage of moulds was slightly higher in Oreochromis species (80.5%) in comparison to that in Clarias gariepinsi (78.2). The isolated moulds belonged to the genera Saprolegnia, Aspergillus, Fusarium, Mucor, Penicillium, Rhizopus, Scopulariopsis, Paecilomyces and Curvularia.37

A total of one hundred fish samples including; 40 of fresh fish (Tilapia nilotica), 30 each of (smoked fish and salted fish) was randomly collected from different shops and retail markets at different sanitation levels at Giza Governorate. Also, one hundred and fifty samples of fish feeds, worker hands and water surrounding the collected fish (50 of each) were collected. All collected samples were subjected for detection of fungal contamination. The results showed that 7 genera of mould were recovered from different types of fish. The most commonly isolated mould species in the examined Tilapia nilotica were Alternaria spp. (90%), followed by Penicillium spp., Cladosporium spp. and Candida spp. (70.0% for each).Other moulds were recovered in a variable frequency. However, in salted fish samples, Candida spp., Rhodotorula spp. and Aspergillus spp. were the most common isolates (93.3%, 80% and 83.3 %).Of genus Aspergillus; A. flavus was recovered from (66.6%) of salted fish. On the other hand, in smoked fish samples, members of Aspergillus spp. were also the most common isolates (100%), A. flavus was recovered from (70%), A. niger (36.6%), followed by Candida spp.(`73.3%), Rhodotorula spp.(66.6%), Penicillium spp. (60%), P. citrinum and P. expansum (33.3% and 26.6%), respectively. Six genera of fungal spp. and one genus of yeast were recovered from fish feeds; worker hands and utilized water with a nearly similar to the incidence of contamination in fish particularly genus Aspergillus spp., where A. flavus was predominantly recovered from fish feed. (Table 22).77

101

Table 22: incidence of fungal species fresh and smoked fish Tilapia nilotica Salted fish Smoked fish Identified moulds (40) (30) (30) spp. No of +ve % No of +ve % No of +ve % samples samples samples Aspergillus spp. 20 50 25 83.3 30 100 A. flavus 20 50 20 66.6 21 70 A. niger 16 40 22 73.3 11 36.6 A. terreus 0 0 4 13.3 - 0 Penicillium spp. 28 70 12 40.0 18 60 P. citrinum 8 20 10 33.3 10 33.3 P. expansum 6 15 10 33.3 8 26.6 Alternaria spp. 36 90 0 0.00 5 16.6 Cladosporium spp. 28 70 6 20.0 14 46.6 Rhizopus spp. 8 20 2 6.6 8 26.6 Fusarium spp. 2 5 4 13.3 3 13.3 Mucor spp. 8 20 4 13.3 6 20.0 Candida spp. 28 70 28 93.3 22 73.3 Rhodotorula spp. 28 70 24 80 20 66.6

6. Mould infection in milk products

Thirty samples of hard cheese and twenty eight samples of skimmed milk soft cheese collected randomly were examined mycologically. The most prevalent fungi were Aspergillus and Penicillium species species. A. niger was the most common, particulry in hard cheese, but also A. flavus and A. parasitucus were isolated (Tables 23 and 24)46

102

Table 23: Incidence of moulds in hard cheese

______

103

Table 24: Incidence of moulds in skimmed milk soft cheese

104

The results obtained when milk powder and soft cheese from it were examined, revealed that A. flavus was the most common in milk and cheese (Table 25).54

Table 25: Prevalence of fungi in milk powder and soft cheese

The screening of milk products samples for contamination with fungi and detection of aflatoxins, indicated that the most prevalent fungi belonged to members of genus Aspergillus spp. which were recovered from fresh Kareish cheese, fresh Damietta cheese and Yoghurt samples at rates of (70 %,65% and 60%) respectively . The species of A. flavus were recovered from (30% of Yoghurt, 25% of fresh Damietta cheese and 15% of fresh Kareish cheese, followed

105 by A. niger which was recovered at the rates of (25%, 15%, and 30% ), respectively (Table 26).80

7. Toxigenicity of moulds isolated from foods and feeds a. Studies on the toxins of A. niger isolated from mouldy smoked fish

The presence of toxins in the mycelial extract and cultural filtrate of A. niger was confirmed chemically by the formation of a white ring at the junction of the ether and the mixture of extract or filtrate with 2 N Sodium hydroxide solution. Moreover, both filtrate and extract showed strong fluorescence when exposed to U.V.

106 light. The application of dermal test in rabbits caused mild acute dermatitis. Histologically, there was necrosis and neutrophilic infiltration (Fig. 4). The 4 dogs fed with A. niger contaminated bread showed icterus of mucous membranes, enlargement of the liver and haemorrhages in the gastro-intestinal tract. The uterus was inflamed in the 2 females. Histopathologically, the liver showed cloudy swelling (Fig. 5) and fatty metamorphosis (Fig. 6). The brain showed perivascular cuffs (Fig. 7). Fig. 8 shows the mortality dose response curve. It was found that 0.65 ml of the extract/.100 g body weigt was the smallest dose that caused death of 10% of the rats in 24 hours, when injected i.p. All rats died when 1.8-2.4 ml/100 g b.w. were injected. \the slope of linear regression of the dose mortality curve proved to be very highly significant.6

Fig. 4 : Skin of a rabbit showing necrotic Fig. 5: Cloudy swelling in liver changes and neutrophilic infiltration of dogs

Fig.6: Fatty metamorphosis in liver of dogs Fig. 7: Perivascular cuffs in brain of dogs

107

Fig. 8: Mortality % in response to lethal dose

b. Production of aflatoxins by A. flavus on natural and synthytic media In this study spore suspension of A. flavus was added to natural substrate (rice and corn) and to yeast extract sucrose (YES) broth incubated at 25oC for 8 days. The result of aflatoxin production is seen in Table 13. A. flavus was capable of producing 4 types of aflatoxins. The highest concentration of yielded aflatoxins was of G1, while the lowest one was obtained for aflatoxin B2.

108

Aflatoxin B1 and G1 were better produced on rice, while aflatoxins B2 21 and G2 were better produced on corn and YES broth (Table 27).

Table 27: Quantity of aflatoxins produced on natural and yeast extract sucrose ______

______

An extensive study for the production of aflatoxins by A. flavus and A. parasiticus isolated from meat and meat products was carried out on 43 isolates of A. flavus and 8 isolates of A. parasiticus recovered from frozen meat and meat products. It was found that 22 isolates of A. flavus and 6 isolates of A. parasiticus produced aflatoxins. (Table 28). Twenty out of the 22 A. flavus isolates (90.9%) produced aflatoxins B1, B2, G1, G2, and only 2 isolates produced both B1 and B2. On the other hand, aflatoxin B1 was produced by one isolate of A. parasiticus, B1, B2 by another isolate, 2 04 3 toxins by the remaining 4 isolates.34

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Table 28: Results of aflatoxin production by A. flavus and A. parasiticus isolated from meat and meat products

c. Aflatoxin production by A.flavus isolated from feeds : The results of aflatoxin B1 production by A. flavus isolated from different feed samples revealed that the higher incidence of toxigenic A. flavus was recorded in layer’s concentrates (50%), followed by broiler’s concentrates (40%) and from hay (40%), whereas, the A. flavus isolated from poultry ration and Soya bean samples represented low incidence (Table 29). The relations between the prevalence of A. flavus in feeds, colony count and their toxigenicity were irregular. In some cases as in hay samples and layer’s concentrate samples the results showed higher prevalence of A. flavus (100%and 60%), colony count (1.9 x 101±0.05 and 75 x 101±0.039 ), high incidence of toxigenic isolates (40% and 50%) and maximum level of aflatoxin produced (2.3 ppm and 4.1 ppm) respectively. On the other hand, the isolated A. flavus from poultry ration showed maximum rate of incidence (100%) and higher colony count (1.9 x 101± 0.049) but the toxigenicity of isolates was (10%) and the produced level of aflatoxin was comparatively low (0.625).33

110

Table 29: Aflatoxin production by A. flavus isolated from feeds

d. Ochratoxin production by A.ochraceus isolated from feeds : The result of ochratoxin A production by A. ochraceus isolated from different feed samples revealed that the higher incidence of toxigenic A. ochraceus was recorded in poultry ration (100%) followed by processed animal feed (50%), from broiler’s concentrate (50%) and from yellow corn samples (50%). Whereas the incidence of toxigenic A.ochraceus isolated from hay and layer’s concentrate samples represented low incidence (25% for both) (Table 30). The relations between the prevalence of A. ochraceus in feeds, colony count and their toxigenicity were also irregular, where in some cases as in poultry ration, the results showed low prevalence of A. ochraceus (15%), colony count (0.5 x 101±0.028), high incidence of toxigenic isolates (100%) and low level of ochratoxin produced (0.5 ppm). On the other hand, the isolated A.

111 ochraceus from layer’s concentrates showed a low incidence (20%), colony count (0.75 x 101±0.029) and low toxigenicity of isolates (25%) but had a maximum level of ochratoxin produced (1.2 ppm).33

Table 30: Ochratoxin production by A. ochraceus isolated from feeds

e. Production of toxins produced by Fusarium species isolated from feeds

In this study a total of 27 isolates were tested for T2 production. As seen in Table 31, 13 isolates produced the toxin (48%). The isolates recovered from white and yellow corn showed the highest rate of toxin-producers (57-62.5%). The highest amount of toxin was 4.0 ppm and the lowest was 0.5 ppm, with a mean of 2.9 ppm. On the other hand, all isolates produced zearalenone in amounts that ranged between 1.2-5.6 ppm (Table 32), while

112 fumonisine was produced only by 7 out of the tested 18 F. moniliforme (Table 33).29

Table 31: Quantitative determination of T2 toxin produced by Fusarium species isolated from feedstuffs

113

Table 32: Quantitative determination of zearalenone produced by Fusarium species isolated from feedstuffs

114

Table 33: Quantitative determination of fumonisine produced by Fusarium species isolated from feedstuffs

Fumonisin B1 mycotoxin was detected in 33% of tested samples (30 samples of commercially mixed poultry feed) with the mean of 25 ppm, whereas T2 (one of trichothecenes group) and zearalenone toxins were obtained at relatively low levels (0.2 for T2 and 0.5 ppm for zeralenone).55

T2 toxin (member of trichothecenes) was detected in 30% of fifty samples of imported animal feed with the mean amount of 60 ppb and aflatoxin in 20% with the mean amount of 3.4 ppb, whereas zeralenone and fumonisin B1 toxins were found in 6 and 2% with mean levels of 22 and 70 ppb, respectively.57

In the study done on equine feeds, all the 21 tested Fusarium isolates, namely F. anthophilum, F. proliferatum and F. verticillioides were mycotoxigenic and produced fumonisin B1 in vitro in amounts varyied from 0.42 ppm. to 21 ppm. The statistical analysis of these results revealed that fumonisin produced by F.verticillioides ranged from 0.45 ppm to 21 ppm with a mean value of 10.72±14.53, while that produced by F.anthophilum ranged from

115

2.4 ppm to 12 ppm with a mean value of 7.2±6.78 , and that produced by F.proliferatum ranged from 0.42 ppm to 3.1 ppm with a mean value of 1.76±1.89 (Table 34).35

All the 21 Fusarium isolates were subjected to PCR analysis. The use of a primer specific for the conserved ITS DNA region of Fusarium genus and all tested isolates were positive to this region (Fig. 9). All the tested isolates exhibited the fum1 gene of fumonisin production (Fig. 10). Depending on the data obtained from the random amplified polymerase DNA analysis it was possible to descriminate between the three Fusarium species.

Fig. 9

116

Fig. 10

117

Table 34: Results of fluorometeric assay for fumonisine producing Fusarium spp.

118

8. Detection of mycotoxins in foods and feeds a. Detection of aflatoxins in meat and meat products Several studies were done by the authors to determine the incidence of aflatoxins in meat and meat products. Different aflatoxins could be detected at different rates in sausage samples, namely, 280 ppb AFB1 in 6 samples, 160 ppb AFB2 in one sample, 84 ppb AFG1 in 2 samples and 586 ppb AFG2 in 3 of the samples. The detected aflatoxin was associated with higher pH (5.2-6.5) and rich moisture content (36%-60%).46 Aflatoxin were detected in minced meat, sausage and luncheon. The highest amount was found in luncheon (200 ppb for B1 and 320 ppb for B2, 49 G1 and G2). In other studies, aflatoxin B1 was detected in 6.6% and 13.3% of raw and cooked hawawshy (spiced minced meat) with the mean value of 40 and 32 ppb, respectively. 53

In an extensive studies on the prevalence and levels of aflatoxins in meat products, it was found that aflatoxins were detected in 40% of hamburger samples, 33.3% in basterma samples and in 26.66% of luncheon samples. The samples of sausage had the highest incidence of aflatoxins (46.66%) . The detected levels of aflatoxins in meat and meat products (Table 35), showed that the sausage samples had the highest levels of total AFs (ranged from 12.88 ppb to 29.95 ppb), particularly ( 23.95 ppb) in case of AFG1, (18.22 ppb) in AFB2, in AFG2 and (12.19 ppb) in AFB1. However, in hamburger samples, the total AFs ranged from 4.80 ppb to 14.89 ppb with a highest levels detected of 12.39 ppb in AFB1 , 10.95 ppb in AFB2 , 7.20 ppb in AFG1 and 5.17 ppb in AFG2 .Also, in basterma samples, the total AFs ranged from 13.15 ppb to 18.55 ppb with a highest individual AFs of 13.55 ppb in AFB1 , 12.45 ppb in AFB2 and 8.70 ppb in each of AFG1and AFG2. In luncheon samples, the total AFs ranged from 6.80 ppb to 14.50 ppb with a highest individual AFs of 7.25 ppb in each of AFB1 and AFG1, 6.30 ppb in AFB2 and 5.10 ppb in AFG2.Whereas, in minced meat samples, the total AFs varied from 2.97 ppb to 8.52 ppb with a

119 highest individual AFs of 3.29 ppb in AFB1 2.65 ppb in AFB2 , 2.13 in AFG1 and 0.99 ppb in AFG2. In frozen meat samples, the total AFs revealed 5 ppb with AFs of 2.50 ppb in each of AFB1 and 34 AFG1.

Table 35: Levels of aflatoxins residues (ug/Kg) in meat and meat products

120

The results of a study on frozen meat, hamburger and sausage samples revealed that the rate of contamination with aflatoxins was comparatively higher (75.0%) in sausage than in hamburger (55.0%) and frozen meat (25.0%). The highest levels of aflatoxins was also detected in sausage samples. Aflatoxins B1, B2, G1and G2 were detected.69

In another work, it was evident that sausage samples were highly contaminate with aflatoxins, particularly AFB, which was detected in 7 samples, AFG1 in 6 samples, AFB2 in 5 samples and AFG2 in 4 samples. However, six hamburger samples were contaminated with aflatoxins, where AFB1 was detected in 6 samples, AFG1 in 5 samples, AFB2 in 4 samples and AFG2 in 3 samples.Two frozen meat samples were contaminated with aflatoxins (AFB1 and G1). The detected levels of aflatoxins in meat and meat products, showed that the sausage samples had the highest levels of total AFs (ranged from 12.88 ppb to 29.95 ppb). However, in hamburger samples, the total AFs ranged from 4.80 ppb to 14.89 ppb. In frozen meat samples, the total AFs revealed 5 ppb. The detection of the levels of aflatoxins in this work revealed that the highest mean values of aflatoxin residues (mg/kg) B1, B, G1 and G2 detected in the sausage samples were 6.84 ± 1.20, 6.70 ± 2.21; 17.50 ± 1.24 and 10.50 ± 1.86 respectively; followed by hamburger 7.82 ± 2.24, 6.24 ± 1.20, 4.85 ± 2.35 and 2.89 ± 2.21 and the lowest levels detected in frozen meat 0.55 ± 1.02 and 0.17 ± 0.59

The studies done on basterma for the detection of aflatoxins are depicted in Table 36. It is evident that 40% of the basterma coat samples collected in summer months were contaminated with aflatoxins and only 25% of samples were contaminated in winter. In summer season 8 samples were contaminated with total aflatoxins at levels from 9.6 to 12o ug/kg, only one meat sample had total aflatoxins at a level of 24ug/kg and 121

8 of coat and meat samples together contained total aflatoxins at levels from 2.8 to 47 ug/kg. In winter season the total aflatoxins decreased in all basterma samples.30 Table 36 Levels of aflatoxins in individual basterma samples (ug/kg) after processing

As shown in Table 37, aflatoxins residues could be detected in pepper (100%), coriander (30%), fenugreek (50%), capsicum (10%) and garlic (60%). The total aflatoxin residues was highest (285.6 ug/kg), followed by garlic (224.4 ug/kg), fenugreek (194.2 ug/kg), coriander (166.4 ug/kg) and capsicum (42.4 ug/kg).30

122

Table 37: Levels of aflatoxin residues detected in the examined spices used for prepation of basterma (ug/kg)

b. Detection of mycotoxins in milk and milk products

The detection of aflatoxin B1 residues in milk products was in a mean level of (9.24±0.2 ppb) in Yoghurt and a mean level of (8.50±0.5 ppb) in fresh Damietta cheese, while, no AFB1 was detected in fresh Kareish cheese. Whereas, AFM was detected in all kinds of milk products at mean levels of (6.03±0.5 ppb) in Yoghurt , (10.5±0.8 ppb) fresh Kareish cheese and (15.67±1.8 ppb) in fresh Damietta cheese. The samples of Yoghurt contained all 80 types of aflatoxins (B1, B2, G1, , G2 and M (Table 38). 123

Mycotoxins were detected in hard cheese. Aflatoxins M1,B1,B2,G1 and G2, ochratoxin A, citrinin and T-2 toxins were detected in hard cheese prepared from contaminated skim milk (Table 39). Soft cheese prepared from the source of milk showed the same toxins (Table 40)46 Table 39: Incidence of mycotoxins in hard cheese prepared from skim milk

124

Table 40: Incidence of mycotoxins in soft cheese prepared from skim milk

Detection of mycotoxins in milk powder and soft cheese revealed that aflatoxins M1, B1, B2, G1 and G2 were detected in 4 out of 30 samples of milk powder tested. The same toxins were detected in soft cheese prepared from this milk, but in 6 out of the 30 samples tested (Table). T-2 toxin was detected in both milk powder and cheese, but ochratoxin A was detected in only one sample of milk powder (Table 41).54

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Table 41: Comparison between mycotoxin residues in milk powder and soft cheese

c. Detection of aflatoxin B1 and ochratoxins in feed samples

Testing of 50 samples off animal feeds and feed ingredients for aflatoxin B1 showed that 99 samples (19.8%) contained aflatoxin B1 at a rate of 125 ppb, 45 samples (9%) 25-50 ppb, 32 samples (6.4%) 101-200 ppb and 19 samples (3.8%) contained aflatoxin B1 at a rate of 201-2000 ppb (Table 42)32

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Table 42: Aflatoxin B1 detection in animal feed samples

The analysis of 160 samples of feeds , 80 each of yellow corn and mixed feed for mycotoxins, revealed that aflatoxins and ochratoxins could be detected in visible amount specially in winter and summer seasons. For instance, aflatoxins in yellow corn (27 ppb in winter, 35 ppb in summer seasons) and ochratoxins (300 ppb in winter, 250 in autumn and 300 ppb in summer seasons) (Table 43).47

Table 43: Mycoyoxins in yellow corn at different seasons

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Aflatoxin B1 was detected in all kinds of tested food and feed samples (20 of each of lentil, broad bean, yellow corn and poultry feed containing Soya bean meal, wheat bran, crushed yellow corn and vitamins). The average level of aflatoxins in samples was 50 – 130 ppb.(Table 44)50

Table 44: aflatoxins in lentil, broad bean, yellow corn and poultry feed

In another study, ochratoxin A was detected in 98 of 295 samples of stored feedstuffs at levels ranging from 700 to 8000 ppb (Table 45). The highest amount of ochratoxins (8000 ppb) was detected in straw, Yellow and white corn as well sorghum szamples presented amounts of 4800 ppb. Kidney beans and barley samples were free of ochratoxins28

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Table 45: Detection of ochratoxins in different types of feedstuffs

129 d. Detection of aflatoxins by biological technique (Bioassay)

A bioassay was conducted using five standards bacterial strains (Table 46). All strains except P. denitrificans showed varied degrees of inhibition when applied with crude aflatoxins at 5-40 ug/ml. The minimum concentration of aflatoxins to inhibit P. denitrificans was 10ug/ml. Both diluted and undiluted bbacterial broth cultures showed a direct relationship between the diameter of inhibition zones and the concentration of aflatoxins. Field trials were applied to testify the validity of our data. Samples of contaminated corn were tested by this method in parallel with TLC analysis. Data indicated that the most sensitive organism inhibited by as low as 7.5 ug aflatoxins/ml was B. megaterium. Of 14 wheat and 10 corn samples naturally contaminated with aflatoxins, as proved by TLC, only 4 wheat and 2 corn samples were found to be positive (Table 47)21.

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Table 46: Mean value of inhibition zones induced by crude aflatoxins

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Table 47: Mean values of inhibition zones (mm) obtained from 6 TLC positive samples of contaminated wheat and corn

9. Detection of mycotoxins in diseased birds, animals and fish

Fusariotoxicosis in sheep attracted the attention of the authors. One hundred cases of diseased sheep at desert districts in th governorates of (Giza; 6 . October and El-Wadi-El-Gadid), were investigated. Sixty percent of these sheep sera had mean levels of T-2, zearalenone and fumonisins (2.5±0.2, 4.3±0.5 and 25.0±2.0) respectively. The used feeds and underground water in breeding of sheep were examined mycologically which revealed that all examined samples gave a variable rates of contamination. The most predominant isolates belonged to members of genus Aspergillus with a range of (5-100%), followed by Fusarium spp. with a range of (40-90%), Penicillium spp. with a range of (10-55%) and Mucor spp. with a range of (10-50). The Fusarium toxins were detected in same feed samples, the largest amount estimated in crushed yellow corn (60%) namely FB1, T2 and zearalenone with the mean levels of (48.4±1.0; 3.0±0.1 and 0.84±0.03) respectively. The significant high levels of FB1 in the present feed samples and serum of diseased sheep gave a large possibility that FB1 was responsible for this disease outbreak in sheep (Tables48) 73

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Table 48: Determination of Fusarium toxins in serum of diseased sheep Prevalence of Mean levels of Fusarium toxins Fusarium toxins (ppm) Animals No. No. % Fumonisins T-2 Zearalen tested +ve one

Sheep 100 60 60 25.0±2.0 2.5±0.2 4.3±0.5

Mycotoxicosis in cows was investigated during 4 years (2000-2003). Out of 50 cases of diseased cows, aflatoxins were recovered from milk and sera (64% and 42%, respectively) with mean levels of (0.6± 0.01 ppb) and 4.7± 0.1 ppb), respectively. Ochratoxins were detected at lower levels. Aflatoxins were detected in muscles, livers and kidneys (Tables 49 and 50)63

Table 49: Detection of aflatoxins and ochratoxins in serum and milk of disesded cows

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Table 50: Residues of aflatoxins and ochratoxins in organs of dead cows

A study on mycosis and mycotoxicosis in cattle was conducted on forty diseased and apparently healthy cases of cattle (20 se-rum samples of each) at Cairo Governorate. Sera of diseased cattle contained significant levels of aflatoxins and zearalenon. Meanwhile, 60% of cattle had the mean levels of aflatoxins (15.20±0.01 ppb) and zearalenon in 80% cattle with the mean level of (62±0.10). The used feed samples in breeding of these animals had the amounts of AFB1; OA; ZEAR and T2 detected in (60%; 40%; 32%; and 36%) of feed samples, with the mean levels of (55.00±1.50; 45.00±0.30; 31.00±0.20 and 26.00±0.02 ppb), respectively (Table 51).83

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In another study, samples of sera of cattle and sheep (one hundred samples of each) were collected from farms at Minufiya, El-Behira and Assiute governorates in which animals (cattle and sheep) suffered from loss of weight gain, low productivity and disturbance in fertility.The detection of mycotoxins in sera of diseased cattle and sheep showed that the most prevalent mycotoxins in cattle sera was aflatoxin B1 which was detected in 40% of cases with the mean level of (5.4 ± 0.1), followed by ochratoxin A in 33% of cases with the mean level of (8.2 ± 0.1), T2 in 17% with the mean level of (26 ± 0.2) and zearalenon in (10%) with mean level of (19 ± 0.2). Also , the pattern of incidence of mycotoxins in sheep sera was nearly similar to those in cattle with the exception that the FB1 was not detected at all in sheep (Table 52 and 53).

Table 52:Prevalence of mycotoxins in serum of diseased cattle

Table 53: Prevalence of mycotoxins in serum of diseased sheep

The aflatoxins, ochratoxins and zearalenone were given to male albino rats in the doses of 0.5, 1.0 and 2.5 ppm in feeds(respectively), for up to 6 months of age to investigate their 135 effects on the growth rates and hormones regulating fertility (FSH, LH, Testosterone, T3 and T4). The results indicated the obvious adverse effects of mycotoxins on the secretion of these hormones and productivity of animals. The environmental pollutions particularly feed contamination was suggested to be the main source of the problem. Hence, regulatory measures must be undertaken to prevent such contaminants to reach the feed of animals69.

The presence of high contamination of turkey feeds directed the attention of the authors that mycotoxins might be the cause of sudden death in turkey farms at El-Wadi El-Gadid and Giza Governorates. The Post mortems examination of forty representative cases, showed enlargement and hemorrhages of liver, spleen, lung, kidney and muscles. Mycotoxins were detected in sera of diseased live birds, where 40% of turkey sera had a mean levels of 4.74±0.01 ppb aflatoxins, 20% had 1.200±0.03 ppb of ochratoxins and 50% of examined diseased turkey sera had a mean levels of 52.0±0.2 ppb T-2 toxins (Table 54 ). These results give a large probability that the feed, water, litters are the sources of these toxicosis.76

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Table 54: Determination of mycotxoins in serum of representative cases of diseased turkeys. Aflatoxins Ochratoxin A T-2 +v +v +v Mean Mean Mean Turkey e e e % levels % levels % levels cas cas cas ppb ppb ppb es es es Appare 5 1 0.57± 4 8 0.100± 8 1 5.7±0 ntly 0 0.2 0.01 6 .03 healthy (50) Disease d 4 4.74± 2 1.200± 5 52.0± 20 10 25 cases(5 0 0.1 0 0.03 0 0.2 0) Ppb: Part per billion

Detection of mycotoxins in fish was undertaken on one hundred fish samples including; 40 of fresh fish (Tilapia nilotica), 30 each of (smoked fish and salted fish), which were randomly collected from different shops and retail markets at different sanitation levels at Giza Governorate. aflatoxin B1 was detected in higher levels in muscle tissues of salted fish (25±0.1 ppb) than fresh fish (18 ±0 ppb). Ochratoxin A was yielded at a mean level of (22±0.5 ppb). Moulds of A. flavus isolated from different types of fish and fish feed were able to produce aflatoxins. On the other hand, smoked fish was highly contaminated with aflatoxins producing strains, followed by the isolated strains from salted fish and Tilapia nilotica (53.3, 45 and 40%) respectively. Aflatoxins were detected in fish feeds and different types of fish in significant higher levels. Forty percent of fish feeds and salted fish

137 were contaminated with aflatoxin at mean levels of (105.2±1.3 and 44.1±0.4 ppb) respectively (Table 55).77

Table 55: levels of aflatoxins in fish and fish feed samples. Types Prevalence of Levels of aflatoxins(ppb) examined aflatoxins samples No. of % Max. Min. Mean± SE +ve samples Fish feeds 20 40 150 52.5 105.2±1.3 (50) Tilapia 10 33.3 70.5 22.0 49.8±0.021 nilotica (30) Salted 12 40.0 50.0 18.5 44.10±0.4 fish(30) Smoked 8 26.6 96.0 32.0 65.1±0.05 fish(30)

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10. Mycotoxins effects on immune and endocrine systems i. The effect of mycotoxins (aflatoxin B1 and ochratoxin A) on the immune system of chickens was experimentally studied. The first work was concerned with the impairment of CMI and phagocytic function in 300 chickens immunized with the living attenuated Cu fowl cholera vaccine and toxicated with aflatoxin B1 and/or ochratoxin A. Different parameters have been considered to evaluate the influence of these toxins on CMI in fowl cholera vaccinated and non-vaccinated chickens against fowl cholera. Both mycotoxins (singly or combined) could significantly decrease the total leucocytic count till 50 days of age. The blastogenic response of peripheral blood lymphocytes of toxicated birds to T-cell mitogens (PHA and CON.A) showed that in combined form they could suppress the T-cell blastogenesis at 30 days till the end of the experiment. When given singly, these mycotoxins exerted their suppressive effect earlier. B-cell blastogenesis by pockweed mitogen was also significantly depressed at 20, 30 and 40 days. The percentage and activity of phagocytosis in chicken mononuclear cells were depressed till 30 days of age without any significant effect at 40 and 50 days in all toxicated groups. 19 ii. The second work was concerned with the capability of aflatoxin B1 and/or ochratoxin A either single or combined to alter the chicken humoral immune response to Cu fowl cholera vaccine, beside evaluating the resistance of toxicated vaccinated chickens to challenge infection with the virulent P. multocida. The toxins greatly depressed the immune response to the vaccine at 30 and 40 days of age. At 50 days, the effect of ochratoxin A was non-significant. Both toxins, singly or combined, had the ability to suppress the resistance of birds to challenge infevtion with the virulent P. multocia.20 iii. The immune suppressive effect of aflatoxins in chickens was studied. Broiler chicks fed a diet contaminated with aflatoxins

139 at doses 0,1,1.5 and 2 ppm frpm one day to 34 days of age revealed histopathological changes in the form of lymphocytic necrosis in bursa of fabricous, thymus gland, liver and spleen. When these chicks were vaccinated aginst Newcastle disease virus, slight or no decrease in the geometrical mean titres of haemagglutination inhibition antibodies was observed.On the other hand, chicks vaccinated against Gumboro disease virus showed a sharp decrease in the development of humoral immunity.48 iv. The influence of aflatoxins and zearalenone on the immune response of cattle naturally infected with brucellosis and G. pigs vaccinated with S19 was studied. The results indicated that the toxins decreased the humoral immune response in naturally infected cattle and may play a role in enhancing abortion. The same findings were obtained in experimentally vaccinated G. pigs fed on ration contaminated with mycotoxins.60, 68 v. Effect of aflatoxin B1, zearalenone and ochratoxin A on some hormones related to fertility in male rats was studied. The mycotoxins were given to male albino rats in the doses of 0.5, 1.0 and 2.5 ppm in feeds(respectively), for up to 6 months of age to investigate their effects on the growth rates and hormones regulating fertility (FSH, LH, Testosterone, T3 and T4). The results indicated the obvious adverse effects of mycotoxins on the secretion of these hormones and productivity of animals. The environmental pollutions particularly feed contamination was suggested to be the main source of the problem. Hence, regulatory measures must be undertaken to prevent such contaminants to reach the feed of animals.70

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11. Miscellaneous studies on mycotoxins

i. The effect of ochratoxin A on different tissue culture cell types was studied. The study revealed that primary chicken embryo fibroblast cells and baby hamster kidney cell lines were more sensitive to ochratoxin A than the primary lamb testis cella and the African green monkey cell lines. Microscopic examination of the exposed confluent cell sheet revealed cytotoxicity in the form of cell rounding, aggregation and detachment of the cells from the surface.23

ii. The influence of media and temperature on the production of ochratoxin A was studied. In YES broth, all tested A. ochraceous strains produced ochratoxin A when incubated at 28oC. A. ochraceous NRRL 3174 produced the highest level (30 ug/100 ml), while each of A. ochraceous 6330 and a strain, isolated locally from corn produced lower amount (18 ug/100 ml). On the other hand, M4, 824, 112 strains, in addition to isolates recovered locally from rice, poultry feeds, hay and tibn produced the lowest level ( 11.5-16.0 ug/100 ml). All tested strains failed to produce ochratoxin A in YES incubated at 5oC. There was no correlation between the weight of mycelial mats and the amount of toxin produced.25 iii. Biochemical changes in blood and urine of beef buffaloes fed on A. flavus and Penicillium species contaminated concentrate mixture was studied. 28 out of the 38 male buffalo calves showed nervous symptoms. Biochemical changes were in the form of increased GOT, serum urea nitrogen, slight albuminurea, urobilin and urine acetone. There was a decrease in the serum calcium, magnesium and phosphorus as well as in the serum iron.8 iv. A study was carried out to investigate the ability of dietary selenium to control the toxicosis by fumonisin B1 in broiler chickens. The chickes were fed on fumonisin contaminated feeds (300 ppm) with and without selenium. The biochemical alterations due to fumonisin toxicosis consisted of increased level of aspartate

141 aminotransferase, alanine aminotransferase, alkaline phosphatase, calcium and phosphorus. These alterations were significantly reduced when adequate level of selenium (015 ppm) was added in poultry feeds.55 v. Experimental feeding of aflatoxin B1 and ochratoxin A in quails at a rate of 1 and 2 ppm in the diet, respectively, revealed significant alterations in tested serum biochemical parameters concerning hepatorenal functions.58 vi. Clinicopathological studies on caprine aflatoxicosis revealed the detection of aflatoxins in serum samples of 40 out of 80 goats examined. Examination of 600 samples of feed utilized by these animals yielded aflatoxins and toxigenic A. flavus in 52.2% of the samples. Experimental induction of aflatoxicosis in goats gave similar symptoms which were observed in field animals.. Biochemical and haematological examinations of blood of experimentally intoxicated animals revealed significant alterations in most of the examined values including liver and kidney function tests, total protein, albumin and globulin. Histopathological examination of internal organs showed obvious changes in all tissues, particularly in the liver and kidney.64 vii. The hepatoprotective effect of dimethyl 4,4- dimethoxy 5,6,5,6- dimethylene dioxy-biphenyl- dicarboxylate (D.D.B.) on aflatoxin B1 induced liver injury in rats. The effect of dimethyl 4, 4- dimethoxy 5, 6, 5, 6- dimethylene dioxybiphenyl 2, 2- dicarboxylate (D.D.B.) in degradation of AF was evaluated . The administration of DDB to rats fed on diet contaminated with aflatoxin B1 effectively improved haematological alterations and prevent serum biochemical changes, ameliorated, the toxic effect of aflatoxin B1 and had hepatoprotective effect on AFB1 induced liver toxicity.71 viii. The effect of a specific combination of Mannan- oligosaccharides (MOS) and β-glucans extracted form the cell wall

142 of a specific strain of Saccharomyces cerevisiae (AGRIMOS®) was investigated on ochratoxicosis and immune dysfunction caused by ochratoxin in broiler chickens. Three hundred and sixty, one day-old chickens were randomly allocated in a 2x2 factorial design for 5 weeks: supplementation of 2kg/ton of MOS (presence or absence) and feed contamination (presence or absence) with 50 μg/kg of ochratoxin A (OTA) for the first 3 weeks of life was done. Obtained results revealed that OTA did affect bird’s growth one week after the contamination, although the final weight gain after 5 weeks was not different from the control. The use of AGRIMOS® stimulated the overall daily gain compared to the OTA group. Feed intake and feed conversion were not affected by the dietary treatments. Cumulative mortality was similar between treatments and performance indexes significantly improved with AGRIMOS® for the OTA challenged regimes. AGRIMOS® supplementation reduced macroscopic and microscopic lesion scores associated with ochratoxicosis. Also, it corrected the depression in phagocytosis induced by ochratoxin intoxication and it had strong immunomodulation as it stimulated the immune response to vaccination. It could be concluded that administration of a specific combination of Mannanoligosaccharides and β-glucans extracted form yeast cell wall (AGRIMOS®) to chickens had a potent immunomodulatory effect, evoked immune response and enhanced vaccination effectiveness. It helps not only in controlling chicken ochratoxicosis but also can play a positive role in treating chicken immune dysfunction.74

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12. Control of fungal growth and mycotoxins i. Decontamination and detoxification of feeds by γ-radiation were the focus of several studies done by the authors. In one of these studies it was reported that the doses of 1.0 or 2.0 KGY gamma radiation reduced the level of mould growth of Aspergillus flavus and aflatoxin production, whereas, the dose of 4.0 kgy eliminated all viable fungi. Aflatoxin B1 production decreased with increased levels of irradiation and was negligible at 4.0 Kgy.52 ii. In another study it was revealed that the total viable population of Aspergillus parasiticus and aflatoxin B1 production decreased significantly by increasing gamma irradiation doses and no growth 42 or aflatoxin B1 production occurred at 4.0 Kgy. iii. The application of irradiation on mouldy basterma decreased the viable count of moulds by increasing the radiation dose. The effective dose was 3 kGy, which decreased the counts by about 2-4 log cycles. Complete destruction of moulds in basterma meat and spices was achieved at a dose level of 5kGy (Tables 56 and 57). Irradaion of 10 basterma samples at a dose level of 3 kGy destructed aflatoxin B1 in all except one sample each of pepper, fenugreek and basterma paste, which were contaminated with aflatoxin B1 at concentarion of 8.6, 8.6 and 5.6 ug/kg, respectively. All basterma samples and the spices were free from aflatoxins at an irradiation dose level of 5kGy.30

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Table 56: Effect of γ-irradiation on the total mould count of basterma samples and its components

Table 57: Levels of aflatoxin B1 in γ-irradiated basterma and its components for 2 weeks at room temperature

iv. The influence of solar simulator, gamma irradiation and laser rays on the growth and aflatoxin production of Aspergillus flavus and Aspergillus parasiticus was studied. the rays of solar simulator and 145 light emitting diodes (led ) in the presence of phloxine b as photosensitizer caused complete inhibition of mycelium growth and aflatoxin B1 production at a dose level of 2.0 mg% phloxine b in case of solar simulator. On the other hand, the application of led resulted in complete inhibition of mycelium growth and afb1 production at a dose level of 1 and 2 mg% phloxine b.72 v. The biodegradation of aflatoxin B1 by bacteria and fungi was studied. Lactobacillus casi, L. bulgaricus and L. halvaticus were used for testing the ability of these bacteria to bind and subsequently remove aflatoxin B1 from liquid YES medium. It was found that acidic and heat treatment of bacterial pellets significantly enhanced their ability to bind aflatoxin B1, but the heat treatment was not as effective as acid treatment. Screening the ability of either intact, fragmented mycelium orculture cell-free system of non- aflatoxin producing A. flavus and A. parasiticus indicated that fragmentation increased the ability of the tested strains to degrade aflatoxin B1. Culture cell-free system showed the highest percent of aflatoxin B1 degradation. A. flavus showed higher percent of degradation than A. parasiticus (Fig 11 and 12).32

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Fig. 11: Percentage of degraded aflatoxin B1 by fragmented mycelium of A. flavus and A. parasiticus

Fig. 12: Percentage of degraded aflatoxin B1 by non-heated cell free system of A. flavus and A. parasiticus 147 vi. The antifungal effects of stationary or the exponential culture filtrate obtained from the strain of Streptomyes sp. were evaluated against the pathogenic fungi. The results indicated that the stationary culture filtrate possessed higher antifungal potential than the exponential culture filtrate, where, the filtrate of the stationary phase of Streptomyes sp. yielded significantly wider range of antifungal activity zones ranged from 7±0.69 to 11±1.41 mm diameter compared with antifungal activity zone of the culture filtrate of the exponential phase which ranged from 5±0.64 to 8±1.58 mm diameter in comparison with benzoic acid as control which ranged from 3±0.55to 8±0.83 mm diameter (P < 0.05) (Table 58).75

Table 58: Evaluation of the antifungal effect of bioactive compounds of Streptomyces compared with benzoic acid

vii. Detoxification of ochratoxin A in different contaminated feeds by some physical and chemical treatments was studied. The rate of detoxification of feed samples by sun rays was of low value, the destruction of ochratoxin A was 3-8% only after 9 weeks exposure. Treating feed samples with dry heat at 200oC for 3 hours caused destruction of 4% in yellow corn, 20% in Soya beans and cotton seed cake. Moist heat at 150 oC for 2 hours destructed 31% 148 of the toxin in in yellow corn, 41% in Soya beans and 52% in broiler’s concentrates and the toxin was completely eliminated by autoclaving. Ammonium, calcium and sodium hydroxides, formaldehyde, acetic and propionic acids were successfully used to inactivate the fungi in the contaminated feeds (Table 59).24

Table 59: Influence of some physical and chemical treatments on the detoxification of ochratoxin A in different feeds

viii. The effects of antimicrobial agents on the growth of toxigenic A. ochraceous A NRRL 3174 was investigated on Sabouraud agar. The growth was completely inhibited by 0.005% sodium EDTA, 0.05% each of gentian violet (100%), copper sulphate, mono- and heptohydrate and benzoic acid, and 1% each of potassium sorbate, sorbic and propionic acids. In poultry feeds, the growth of Aspergillus ochraceus was completely inhibited by 4% each of gentian violet (100%), copper sulphate, mono- and heptohydrate. Bio-Add,Mycocurb at 0.5% andAflagen at 2% inhibited the growth on SDA. In poultry feeds, Mycocurb (3%), Bio- Add (4%) and Aflagen (5%) completely inhibited the growth of Aspergillus ochraceus (Tables 60 and 61).27

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Table 60: Effects of antimicrobial agents on the growth of toxigenic A. ochraceous A on synthetic medium

150

Table 61: Effects of antimicrobial agents on the growth of toxigenic A. ochraceous A on natural medium (poultry feeds)

ix. This study was undertaken to investigate the effect of some minor elements and extract of Lupinus termis seeds on aflatoxin production by aflatoxigenic moulds isolated from frozen and canned fishes. The majority of the used minor elements reduced the growth rate and aflatoxin production by A. flavus isolated from fish samples. The inhibitory effect increased by increasing the concentation of Zn, Mn, Ba and Sel., while low concentration of Fe and Cu (5 mg/L) had a slight stimulatory effect on the growth and toxin production. Increasing the concentration of 151 both elements to 20 mg/ml inhibited the growth and toxin production (Table 62) . The antifungal capacity of aqueous extract of salinated Lupinus temis seeds was evident when used at a concentration of 3%. It reduced the growth and toxin production significantly (Table 63).65

Table 62: Influence of some elements on growth of A. flavus and production of aflatoxins

Table 63: Influence of Lupinus termis seed extract on growth of A. flavus and production of aflatoxins

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x.The testing of antifungal activity of buckthorn (Rhamnus cathartica) plant extract revealed that the chrysophanol was at the top of all other extracts against A. parasiticus . Penicillium species was most sensitive to chrysophanol glycosides and 2-aldehydo-4- hydroxy-methyl-chrysophenol (Table 64).66

Table 64: Antifungal activity of anthraquinone compounds from Rhamnus cathartica leaves ______

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xi. The effect of heat on stability of aflatoxin B1 in frozen meat was studied. From Table 65 it is evident that boiling caused reduction in aflatoxin B1 concentration by 11.8-16.8%., while frying reduced the toxin by 16-22.8% and roasting by 7.8-13.6%. The combination of boiling and frying reduced the toxin by 25-42.4%.34

Table 65: Effect of heat treatment methods on stability of aflatoxins B1 in the examined samples Type of Aflatoxin B (µg) Reduction No. of 1 heat Before After samples (µg/Kg) % treatment treatment treatment 1 20 17.63 2.37 11.85 2 20 17.19 2.81 14.05 Boiling 3 20 16.64 3.36 16.80 * 20 17.15 2.85 14.25 1 20 16.69 3.31 16.55 2 20 16.47 3.53 17.65 Frying 3 20 15.43 4.57 22.85 * 20 16.20 3.80 19.02 1 20 18.43 1.57 7.85 2 20 17.59 2.41 12.05 Roasting 3 20 17.27 2.73 13.65 * 20 17.76 2.24 11.18 1 20 14.97 5.03 25.15 Boiling 2 20 13.57 6.43 32.15 and frying 3 20 11.52 8.48 42.40 * 20 13.35 6.65 33.25

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xii. Effect of potassium sorbate as antifungal chemical preservatives on total mould contamination was studied. The addition of potassium sorbate eliminated the moulds from the examined samples of frozen meat, when it was added at a concentration of 1% (Table 66).34

Table 66: Effect of potassium sorbate as antifungal chemical preservatives on total mould contamination. No. of TMC/g After treatment with Pot. Sorbate at samples before conc. treatment 0.125% 0.250% 0.50% 1.0% 1 105 5.00 X 2.50 X 1.25 X 6.25 X 104 103 102 10 2 105 3.13 X 1.56 X 1.22 X 0 104 103 102 3 105 3.91 X 1.95 X 9.80 X 0 104 103 102 The initial total mould spores were adjusted as 105/ ml treatment. xiii. Efficacy of use of forskolin plant extract in control of toxic effects of aflatoxicosis in food was studied. The evaluation of the efficacy of use of forskolin plant in control of the dangerous changes caused by aflatoxicosis was undertaken. Forskolin has been isolated from the roots of Coleus Forskohlii, a plant rich in alkaloids which are considered to have a high probability of influence on the biological systems. Forskolin has a unique property of activating almost all hormone sensitive adenylate cyclase enzymes in a biological system. For experimental evaluation the effect of forskolin against aflatoxicosis, forty rats

155 were divided into 4 equal groups, where, rats of the first group were given normal feed (free from mycotoxins and without any treatment) and kept as a negative control. While, rats of the other groups were given single dose of AFB1 intra-peritoneal at the rate of 1.5 ppm. Then on the second day, rats of the third and fourth were dosed orally by 50 and 100 mg of forskolin for 2 weeks), while those of the second group were left without any treatment and kept as positive control.

The serum NO level significantly increased in AF B1 treated rats. Also, a significant decreased in catalase activity , GSH levels and increased TBARS levels were shown after Af treatment. However, supplementation of forskolin extract for toxicated rats with AFB1 increased CAT activities and TBARS and eliminates the possibility of oxidative stress due to the administration of AF B1to rats. Receiving of forskolin to aflatoxicated rats decreased the destructive effect of AFB1 in the tissues examined specially liver, where, cytoplasmic regeneration of hepatocytes was detected and had a significant improvement in all lesions appeared in most organs which represented by minimizing of histopathological and biochemical alteration. Hence, the supplementation of forskolin in food and feed is valuable in reduction of the severity of the toxicity and the histopathological and biochemical alteration produced by aflatoxin B1 (Tables 67 and 68).78

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Table 67: Chemoprevention of AFB1-induced hepatocarcinogenesis by Forskolin(n=10 for each group). ______

Table 68: Detection of aflatoxins residues in the internal organs of rats after administration of aflatoxin alone or in combination with forskolin extract.

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Table69: Influence of different concentrations of ZnO-NPs on growth and mycotoxins production by toxigenic moulds

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Fig. 13: Scanning Electron Microscopy(SEM) images of toxigenic Asperagillus spp.:(1)(R) Control normal conidial cells. (2)(M) Cells treated by 8 μg/ml ZnO-NPs. (3)(L) Cells treated by 10 μg/ml of ZnO-NPs.

13. References : 1. Refai, M., El Mossalami, E. and Loot, A. : Mould infection of smoked herrings. Mykosen 11, 83-86 (1968) 2. Refai, M. and Sadek, I. : Studies on mould contamination of different foods in Egypt. Mykosen 11, 625-630 (1968) 3. Refai, M. and El-Bahay, G. (1968): Incidence of moulds in poultry feeds in Egypt. Mykosen., 11:459-462 4. El-Bahay, G., Elmossalami, E. and Refai, M. : The use of some disinfectants as fungicides. Mykosen 11, 807-810 (1968) 5. Refai, M. and Loot, A. : Studies on mould contamination of meat in slaughter houses, butcher’s shops and cold stores. Mykosen 12, 621-624 (1969) 6. Kamel, S., Refai, M. and Loot, A. : Studies on the toxins of A. niger isolated from meat. Castellania 4, 159-161 (1976) 7. Saif, A. and Refai, M. : The use of thiabendazole to control moulds in poultry farms. Castellania 5, 183-187 (1977) 8. El-Sherif, M., Moutwaly, M., Moustafa, M. and Refai, M. : Biochemical changes in blood and urine of beef buffaloes fed mouldy concentrate mixtures. Ernst-Rodenwaldt-Archiv. 2, 41- 43(1975)

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vaccinated with fowl cholera vaccine. I. Impairment of cell- mediated immunity and phagocytic function J. Egypt. Vet. Med. Ass. 53, 227-233 (1993) 20. Refai, M., El-Sanousi, S., Gergis, S., Abdel-Hamid, S. and Hashad, M. : Experimental afla- and ochratoxicosis in chickens vaccinated with fowl cholera vaccine. II. Influence of humoral response and resistance to challenge with virulent multocida organism. J. Egypt. Vet. Med. Ass. 53, 235-241 (1993) 21. Refai, M., Hatem, M. Sharaby, E. and Saad, M. : Detection and estimation of aflatoxins using both chemical and biological techniques. Mycotoxin Res. 9, 47-52 (1993) 22. Refai, M., Mansour, N., El-Naggar, A. and Abdel-Aziz, A. Fungal flora in modern Egyptian abattoirs. Fleischwirtsch. 73, 172-174 (1993) 23. Refai, M., Hammad, H., Hassan, A. and Moustafa, E.: Effect of ochratoxin A on different culture types. J. Egypt. Vet. Med.Ass. 54, 113-122 (1994) 24. Refai, M., Abdel-Aziz, A., Aziz, N., Hammad, H. and Hassan, A. : Influence of some physical and chemical treatment on the detoxification of ochratoxin A in different feedstuffs. J. Egypt. Vet. Med. Ass. 55, 795-806 (1995) 25. Hammad, H., Refai, M., Abdel-Aziz, A., Hassan, A. and Aziz, N. : Influence of media and temperature on production of ochratoxin A. J. Egypt. Vet. Med. Ass. 55, 807-817 (1995) 26. El-Far, F., Hassan, A., Kotb, H., Hegazi, E., Hamouda, A. and Refai, M. : Occurrence of ochratoxins and ochratoxigenic moulds in different feeds and feedstuffs in Egypt. J. Egypt. Vet. Med. Ass. 55, 855-856 (1995) 27. Refai, M., Kotb, H., El-Far, F., Hassan, A., Hegazi, E. and Hamouda, A. : Effect of antimicrobial agents on the growth of toxigenic Aspergillus ochraceus in poultry feedstuffs. J. Egypt. Vet. Med. Ass. 55, 867-879 (1995) 28. Refai, M.K.; Aziz, N.H.; Ferial, El Far and Hassan, A.A. (1996):"Detection of ochratoxin production by Aspergillus

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ochraceus in feed stuff and its control by gamma irradiation."App. Radiat. Isot., 47 (7): 617- 621. 29. El-Hamaky, A.A.; Hassan, A.A. and Refai, M.K. (2001):Incidence of moulds in feedstuffs with particular references to Fusarium species and their toxins. J. Egypt. Vet. Med. Ass., 61 (6B) : 261-271. 30. Refai, M. Niazi, Z.M., Aziz, N.H. and Khafaga, N.E.M. Incidence of aflatoxin B1 in the Egyptian cured meat basterma and control by γ-irradiation. Nahrung/Food 47 (6), 377-382 (2003). 31. Refai, M, S.A Attia, R.M. Salem and E.M. Al-Dahshan (2004): Studies on the pathogenicity and enzymatic activities of Aspergillus fumigatus, A. flavus and A. niger isoated from chickens and their environment. Egypt. G. Comp. Path.&Clin.Path. 17 (2) 193-205. 32. Rania M. Azab, W.M. Tawakkol, A. M. Hamad, M.K. Abou-Elmagd, H. M. El-Agrab and M.K. Refai (2005): Detection and estimation of aflatoxin B1 in feeds and its biodegradation by bacteria and fungi. Egypt. J. Natural Toxins 2, 39-56 33. Rasha ,H.Sayed El Ahl ; Hassan , A.A. *; El Barawy, A.M ; R.T.Salem ; W.M.Tawakkol ; H.A.Abdel- Lateif and. Refai , M.K. ; (2006): Prevalence of fungi and toxigenicity of A.flavus and A.ochraceus isolates recovered from feed and their control . Egyp. J.Agric.Reas. , 84 (4),1303-1318(2006). 34. Heidy, Abou El- Yazeid; Atef, A. Hassan; Nahed , W.Tawakkol ; Howayda El-Shafei and M. Refai (2008): Contamination of meat and meat products with Asperagillus species and aflatoxins production and their control. Bull. Fac.Pharm. Cairo Univ., Vol. 46, No. 12 (2008) (Special Issue) 35. Heidy Abo Al-Yazeed, Atef Hassan, Mahmoud EL- Hariri, Mai Hamed and Mohamed Refai (2010): Studies on Fusarium species and fumonisins-producing isolates in horse’s feeds in Egypt.Mycotoxin Conference. NRC. Oct. 2010. 162

36. Heidy Abo El Yazeed; Atef Hassan; Reda E.A. Moghaieb, Mai Hamed and Mohamed Refai (2011). Molecular Detection of Fumonisin-producing Fusarium Species in Animal Feeds Using Polymerase Chain Reaction (PCR). Journal of Applied Sciences Research,7(4): 420-427, 2011 37. Refai, M., Mohamed, Amany Kenawy and Shimaa, Ali (2008): The assessment of mycotic settlement of freshwater fishes in Egypt. 7. International Conference on Recirculating Aquaculture, July 25- 27, 2008, pp. 499-507, Blacksburg, V A 24051, USA. 38. Magan, N and M. Olsen (2004): Mycotoxins in food, Detection and control. CRC. Press Boca Raton Boston New York Washington, DC. 39. Food and Agriculture Organization (2001), Safety Evaluation of Certain Mycotoxins in Food. Prepared by the 56th meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). The FAO Food and Nutrition Paper 74, Rome, Food and Agriculture Organization of the United Nations. 40. World Health Organization (2002), Evaluation of Certain Mycotoxins in Food. Fifty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives, WHO Technical Report Series 906, Geneva, World Health Organization. 41. Anon (2001): Manual of the application of the HACCP system in mycotoxin prevention and control, FAO Food and Nutrition Paper No. 73, Rome, FAO. 42. Aziz N H, Attia E S A and Farag S A (1997), Effect of gamma-irradiation on the natural occurrence of Fusarium mycotoxins in wheat, flour and bread, Nahrung-Food, 41(1), 34–7. 43. European Commission (2002): Commission Directive 2002/27/EC of March 2002 amending Directive

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98/53/EC laying down the sampling methods and the methods of analysis for the official control of the levels for certain contaminants in foodstuffs. Official Journal of the European Communities, L75, 44–5. 44. Salem, A.L.; Hammad, H.A.; Abd El-Haleem, M.M.; Ragheb, R.R.; Aida Abdel-Aziz; Ahlam, A. Farghaly and A.A. Hassan (1995):Some serological studies on rabbits immunized with Fusarium species. Egypt. J. Immunology, 2 (1): 101-106. 45. Hassan , A. A. and Ragheb, R.R. (1996): Identification of some fungi and mycotoxins in Sausage.Vet. Med. J. Giza, 44 (2): 2 15-220. 4th Scientific Congress proceeding, Fac. Of Vet. Med., Cairo University. 46. Abouzeid, A.M.; A. A. Hassan and Ragheb, R.R. (1996):Mycological studies on hard (Roume) and skim soft cheese (kariesh) with quantitative evaluation of the existing mycotoxins. (1996). Vet. Med. J. Giza, 44(2): 113-121. 4th Scientific Congress proceeding, Fac. of Vet. Med., Cairo University 47. Hassan , A. A. and R.M.A. Omran (1996): Seasonal variation in mycoflora and mycotoxins in feeds and pathological changes due to ochratoxins. J. Egypt. Vet. Med. Ass., 56 (1): 73-96. (1996). 48. Hassan , A. A.; M. Hussain; M.H. El-Azzawy and A.E. Saad (1997): Immunosuppression effect of aflatoxins in chickens. 23” Arab Vet. Med. Congress, J. Egypt. Vet. Med. Ass., 57 (1): 917-931. 49. Hassan A. A ; Waffia, H. Abdallah and Safaa Abd El- Aziz (1997): Mycological examination of frozen meat, chickens and meat products. Zagazig Vet. J., 25 (4): 33- 39. 50. Hassan, A. A and R. El-Sharnouby (1997): Antimicrobial effect of some chemicals and plant extracts on Egyptian food. J. Egypt. Vet. Med. Ass., 57 (4): 1331-1349.

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51. Hassan , . A. A. (1998):Mycosis in turkeys. 5th Scientific Congress proceeding, Fac. of Vet. Med., Cairo University, Vet. Med. J. Giza, 46 (4B): 857-865. 52. Hassan, A. A and N.H. Aziz (1998): Influence of moisture content and storage temperature on the production of aflatoxin by Aspergillus flavus EA-8 1 in maize after exposure to gamma radiation. J. of Food Safety, 18(3): 159-17 1. (USA). 53. Wafia, H. Abdallah and Hassan, A.A. (2000):Sanitary status of some ready to eat meat meals in Ciaro and Giza Governorates. J Egypt. Vet. Med. Ass., 60 (7): 95-104 54. Hassan, A. A. and Hammad, A.M. (2001):Fungi and mycotoxins in milk powder and its product (soft cheese). J. Egypt. Vet. Med. Ass., 61 (2): 303-309, 25th Arab Vet. Med. Congress, Cairo, Egypt. 55. Hassan, A.A ; K.M. Koratum and Amal I.Y. El-Khawaga (2002):Effect of selenium in broiler chicken fed a diet containing F. moniliforme culture material supplied known level of Fumonisin B1. Egypt. J. Comp. Path. & Clinic. Path., 15 (1): 98-110, (13th Sci. Conference). 56. Hassan, A.A. and Nariman A. Rahmy (2002): Mycotic pneumonia in buffaloes and cattle.Egypt. J. Comp. Path. &Clinic. Path., 15 (1): 111 – 122 57. Mogda, K., Mansour, A.A., Hassan and M.A., Rashid (2002): The fungi recorded in imported feed samples with reference to control of T-2 toxicosis by antioxidant substances in chicks. Vet. Med. J., Giza. 50 (4): 485-499, (7th Sci. Congress, Fac. of Vet. Med., Cairo University). 58. Hassan, A.A.; Gab-Allah, H.M. and Ahmed, E.E.K. (2002): Biochemical alterations associated with toxicity of fungi isolated from quails meat. Suez Canal Vet. Med. J., V (1): 103-120 (2nd Sci. Conference, Fac. of Vet. Med., Suez Canal University). 59. Hassan, A.A. and Mogeda, K. Mansour (2003): New trials of use of molasses and garlic extracts for compating

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mycotoxicosis. Kafr El-Sheikh Vet. Med. J., 1 (1): 653- 680 (1st Sci. Congress, Fca. Vet. Med., Tanta University, Kafr El-Sheikh branch). 60. Hassan, A.A.; A.M. Montassser and K.M. Koratum (2003):Influence of aflatoxin and zearalenone on biochemical assay and immune response on cattle naturally infected with brucellosis and experimentally vaccinated Guinea pigs with S19. Egypt. J. Agric. Res., 81 (1): , Special Issue: 2nd Scientific Congress for Provincial Laboratories, Animal Health Research Institute, 7-10 September 2003. 61. Hassan , A.A. (2003):Detection of some mycotoxins and mycotoxins producing fungi in both macro- and microenvironment of diseased animals. 7th Sci. Cong. Egyptian Society for Cattle Diseases, pp. 112 – 119, 7-9 Dec., 2003, Assiut , Egypt. 62. Hassan , A.A. and Abdel- Dayem, R.H. (2004): Prevalence of fungi and mycotoxins in fresh and salted fish. J. Egypt. Vet. Med. Assoc., 64 (1): 59-68. 63. Hassan, A.A.; Ragheb, R.R. and Rahmy, Nariman, A. (2004):Pathological changes in cows spontaneously fed on some mycotoxins. Egypt. J. Comp. Path. & Clinic. Path., 17 (1): 282- 293 64. Manal, A.H.;H., Kamel and Hassan, A.A (2004): Clinicopathological studies on caprine aflatoxicosis. Vet. Med. J.Giza Vol. 52 , No.4 : 535-550 . 65. Hassan ,A.A ; El Barawy,A.M and Manal Hassan.(2007):Screening of meat and dairy byproducts for Candida albicans and effect of some antifungal on growth and germ tube formation of its isolstes in Giza Governorate. Egypt. J. Comp. Path. & Clinic. Path., 20 (1):333-342. 66. Hassan ,A.A.;Hammad,A.M; El Barawy,A.M.**and Manal,A.H*(2007) Incidence of aflatoxigenic fungi in frozen and canned fishes and trials to inhibit aflatoxin

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production by use of some minor elements and lupinus termis seeds. Egypt. J. Appl. Sciences, Vol. 22 No. (10 B)Oct. 2007(351-360) 67. Hassan, A. A.; El Shorbagy, M.M. and El-Barawy, A.M. and Manal , A. Hassan.(2008a): Study the availability of using buckthorn(Rhamnus cathartica) plant extract in laboratory control of some bacterial and fungal diseases. The 5 th Scientific Congress, Minufiya Vet. J.Vol.5 (1): 27-39. 68. Hassan, A.A.; Ramadan M. Khoudair and EL Sayed E. Youniss ( 2009): The Effect of Some Mycotoxins on Immunity of Cattle Vaccinated against Brucellosis and Guinea Pigs Experimentally Vaccinated With S19 Vaccine Egypt. J. Appl. Sciences ,Vol. 24 No. (2 A) 2009 (1-13) 69. Hassan, A.A.; Nahed , M. El-Mokhtar ; Manal, A. Hassan; Noha, M. El-Shinawy and R.H., Abdel Dayem(2009): Hygienic significance of fungal contamination and aflatoxins production in frozen meat, sausage and hamburger in Cairo Governorate. J. Egypt. Vet. Med. Assoc., 69 (1): 123-133. 70. Hassan, A.A.; M.A. Rashid and Kh. M. Koratum (2010) Effect of aflatoxin B1, Zearalenone and Ochratoxin A on some hormones related to fertility in male rats. Int. J. Life Sciences, Vol. 7 No. (3) (64-72). 71. Hassan, A.A.; Wael M. Tawakkol ; Elbrawy A.M. (2010): The hepatoprotective effect of dimethyl 4,4- dimethoxy 5,6,5,6- dimethylene dioxy-biphenyl - dicarbxylate (D.D.B.) against liver injury induced by aflatoxin B1 in rates . Int. J. Life Sciences, Vol. 7 No. (3) (148-153). 72. Hassan, A.A.; Howayda, M. El Shafei; Rania, M. Azab. (2010): Influence of solar simulator, gamma irradiation and laser rayes on the growth and aflatoxin production of

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14. Annexes

Annex 1: Media , reagent and stains used for isolation , identification of toxigenic moulds and mycotoxin production

1. Sabouraud dextrose agar (SDA) medium with chloramphenicol: Dextrose 40.0 g Peptone 10.0 g Chloramphenicol 250.0 mg Agar agar 20.0 g Distilled water up to 1000.0 ml Preparation: The ingredients are suspended in 1000 ml distilled water and mixed thoroughly to give a uniform suspension. The mixture is boiled with frequent agitation. After heating the medium and before autoclaving 250 mg of chloramphenicol are dissolved in 5 ml ethanol and added to the medium. The pH of the medium is adjusted to 6.5 at room temperature and the medium is sterilized by autoclaving at 121°C for 15 minutes.

2. Sabouraud dextrose (SD) broth: (37) Dextrose 40.0 g Peptone 10.0 g Distilled water up to 1000.0 ml Preparation: The ingredients are suspended in 1000 ml distilled water and mixed thoroughly to give a uniform suspension. The mixture is boiled with frequent agitation. The pH of the medium is adjusted to 5.7 at room

171 temperature and the medium is sterilized by autoclaving at 121°C for 10 minutes.

3-Aspergillus flavus and parasiticus agar (AFPA) Peptone, bacteriological 10 g Yeast extract 20 g Ferric ammonium citrate 0.5 g Chloramphenicol 100 mg Agar 15 g Dichloran 2 mg (0.2% in ethanol, 1.0 ml) Distilled water up to 1000.0 ml After addition of all ingredients, sterilise by autoclaving at 1218C for 15 min. The final pH of this medium is 6.0–6.5.

4- Czapek yeast extract agar (CYA) K2HPO4 1 g Czapek concentrate 10 ml Trace metal solution 1 ml Yeast extract, powdered 5 g Sucrose 30 g Agar 15 g Distilled water up to 1000.0 ml Refined table grade sucrose is satisfactory for use in CYA provided it is free from sulphur dioxide. Sterilize by autoclaving at 1218C for 15 min. The final pH is 6.7.

5-Czapek yeast extract agar with 20% sucrose K2HPO4 1 g Czapek concentrate 10 ml Yeast extract 5 g Sucrose 200 g Agar 15 g Distilled water up to 1000.0 ml

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Sterilize by autoclaving at 1218C for 15 min. The final pH is 5.2.

6-Malt extract agar (MEA) Malt extract, powdered 20 g Peptone l g Glucose 20 g Agar 20 g Distilled water up to 1000.0 ml Commercial malt extract used for home brewing is satisfactory for use in MEA, as is bacteriological peptone.Sterilise by autoclaving at 121oC for 15 min. Do not sterilize for longer, as this medium will become soft on prolonged or repeated heating. The final pH is 5.6.

7- Potato dextrose agar (PDA) Potatoes 250 g Glucose 20 g Agar 15 g Distilled water up to 1000.0 ml PDA prepared from raw ingredients is more satisfactory than commercially prepared media. Wash the potatoes, which should not be of a red skinned variety, and dice or slice, unpeeled, into 500 ml of water. Steam or boil for 30–45 min. At the same time, melt the agar in 500 ml of water. Strain the potato through several layers of cheese cloth into the flask containing the melted agar. Squeeze some potato pulp through also. Add the glucose, mix thoroughly, and make up to 1 l with water if necessary. Sterilize by autoclaving at 1218C for 15 min.

8-Yeast extract sucrose liquid medium for ochratoxin production:.

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Yeast extract 20 gm Sucrose 400 gm Distilled water up to 1000.0 ml Sterilize by autoclaving at 110 °C for 10 minutes.

9- Yeast extract sucrose liquid medium for aflatoxin production

Yeast extract 20.0 gm Sucrose 150.0 gm Distilled water up to 1000.0 1000.0 ml ml After complete dissolution of the medium in worm water bath P. cresol is added at conc. of 0.019 % and then autoclaved at 110°C for 10 minutes.

10- Buffered peptone water (BPW): Peptone 10.0 gm Sod. Chloride 5.0 gm Disodium hydrogen phosphate 9.0 gm Potassium dihydrogen phosphate 1.5 gm Distilled water up to 1000.0 ml

pH was adjusted to 7.0 dispensed in portions of 225 ml into bottles of 500 ml capacity and of 9 ml in tubes, then sterilization was done for 20 minutes at 121°C.

11-Stains:- a. India Ink. (Pelikan) b. Trypan blue stain 0.4% (Difco) c. Lactophenol Cotton blue stain. Glycerol 40.0 ml 1% aqueous Cotton blue solution 2.0 ml Lactic acid 20.0 ml 174

Phenol crystals 20.0 gm Distilled water 2O Ml Preparation: The lactic acid and glycerine are added to the distilled water and mixed thoroughly. The phenol crystals are mixed and heated gently in hot water with frequent agitation until crystals are completely dissolved. 2 ml of 1% cotton blue solution are added and mixed thoroughly

Annex 2: Methods for Isolation, Enumeration and Identification of mycotoxigenic moulds

1. Isolation of fungi from tested samples

Sabouraud dextrose agar and potato dextros agar, with antibacterial antibiotics and cycloheximide are the most common media used for isolation. a. Direct Plating

Food particles are placed directly on solidified agar media. Particles should be surface disinfected before plating as chlorine solution or mercuric chloride and rinsing. Incubate plates upright, for 5 days at 25oC. Examine plate visually, counting of various genera , a stereomicroscope and experience assists in this process. b- Plate count of fungi

Of the aseptically mixed ground samples of food or feed , 25 g are put into a stomacher jar containing 225 ml of peptone water. The homogenate sample is mixed by shaking and 1ml is transferred into a tube containing 9 ml of peptone water (1:10) and is mixed carefully by vortex. Several dilutions are made to obtain

175 suitable number of colonies, which could be easily counted. After each dilution the solution is mixed thoroughly. Of each dilution 1ml is transferred into each of appropriately marked duplicate Petri- dishes.

In each Petri-dish, 15-20 ml of Sabouraud’s Dextrose Agar o tempered to 45 C is poured. The mixture is then thoroughly mixed and allowed to solidify. Inoculated plates are left to solidify at room temperature, then incubated at 25ºC for 5-7 days. During the incubation period, the plates are examined daily for the "Star- shaped" mould growth which is picked up under aseptic condition with its surrounding cultivated medium and transferred onto SDA slopes then kept at 25ºC for 5-7 days, for purification and further identification. Estimation of the total mould counts is achieved by counting the isolated colonies from each dilution of the agar separately and finally the mean of the total count is obtained.

3-Mould identification:

The incubated plates are examined visually and microscopically. The individual colonies of fungal isolates are selected depending upon their morphological characters and microscopic examination. All colonies are transferred onto a PDA media to identify them up to the species level.

Macroscopic examination includes examination of the colour, shape of colony, growth rate, texture and pigmentation on the surface and reverse sides of the Petri-dishe, over a period of 7- 10 days.

Microscopic examination is carried out through:

a) Wet preparation technique:

A piece of the colony is cut into small fragments on a clean 176

slide, then one drop of Lactophenol cotton blue is added. The slide is covered with a glass cover and examined microscopically. Lactophenol cotton blue stain is used for staining of mould for microscopic examination of septated and non-septated hyphae, structure of hyphae, any modification of the hyphae and conidia .

4 Testing the isolated strains for mycotoxins production

The suspected mycotoxigenic moulds are tested for mycotoxins production on liquid medium, yeast extract sucrose (YES) or selected synthetic medium. Or natural medium (as yellow corn)

a. Production of the toxins on the synthetic medium :

Isolated strains of A. flavus and A. parasiticus are subcultured on potato dextrose agar slants for approximately 14 days at room temperature (20-22°C) until well sporulated. Spores are harvested by adding 5 ml sterile, double distilled water and dislodging the spores aseptically with a sterile inoculating loop. The final spore concentration is adjusted to be approximately 5 x 106 spore/ml by haemocytometer.n0.05 ml spore suspension is inoculated into each flask having 25 ml of sterile yeast extract- sucrose (2% yeast extract, 15% sucrose) and supplemented with 0.019 % P. cresol. Inoculated flasks are incubated at room temperature (20-22°C) in the dark for 20 days. At the end of the inoculation period, mycelia of the cultures are carefully overlaid with 25 ml chloroform and kept 24 hours in dark. Then 25 ml chloroform are added again and cultures are shaken for ½ an hour. Chloroform layers are filtered into 500 ml round bottom flasks and cultures are extracted once more with 50 ml chloroform. Chloroform layers are combined and concentrated in a rotatory flash evaporator.

b. Production of the toxins on natural medium 177

Into a half liter conical glass flasks 200g of sterile corn kernels are added to each flask (crushed corn had been previously, autoclaved at 121°C for one hour on each of t he two consecutive days to ensure they are sterile and do not harbour ear rot fungi, 200ml of sterile distilled water are added to the flasks. The isolates are cultured on PDA and incubated at 25°C for 10 days, conidia of different isolates are suspended in sterile water and 50 ml of spore suspension inoculated on moistened corn, cultures are then incubated in the dark with shaking for 28 days at 25°C. The cultures are then dried at 45°C for 72 hours. The dried samples are finally ground using blender with ethanol cleaning in-between samples and stored at 0°C until analysis.

Annex 3 : Determination of mycotoxins in feed and food samples a. Extarction of aflatoxin B1 from feed samples:

This done according to Roberts and Patterson (1975) as follows: Each sample of feed or feed product is finely ground in an electric mill to pass sieve No. 10. Twenty five grams of the ground sample are transferred into a 500 ml Erlenmeyer flask, then covered with a thin layer of solid 6 mm diameter glass beads and extracted with a solution composed of 90 ml acetonitrile and 10 ml 4% potassium chloride solution.The mixture is shaken vigorously for 30 minutes and filtered through 9 cm Whatman No. 41 which allows rapid filtration with minimum solvent evaporation. 50 ml of filtrate is defattened by shaking with an equal volume of iso-octane (2, 2, 4- trimethyl pentone) in 250 ml separating funnel. When the two layers are clearly separated, the upper layer is discarded and the lower layer re-extracted with another 50 ml iso-octane followed by discarding the lipid extract. 12.5 ml distilled water are added to the

178 acetonitrile layer and aflatoxin B1 is extracted with 25 ml chloroform. The lower acetonitrile-chloroform layer is drained through a bed of anhydrous sodium sulphate contained in funnel lined with Whatman No. 41 paper. Extraction of aqueous layer is repeated 3 more times using 10 ml portions of chloroform. Filtrates are combined and evaporated to dryness in a rotatory evaporator. b. Estimation of aflatoxin B1: i. Estimation of aflatoxin B1 by thin layer chromatography (T.L.C.),

The silica gel suspension for coating glass plates is prepared by adding 90-100 ml distilled water to 40 grams silica gel in a conical flask and shaken vigorously for one minute.The silica gel suspension is poured into the spreader without let slit in closed position. The spreader is then placed on the starting glass plate on the tray and the lever rotated for 180 degree and immediately the five glass plates are coated with 0.25 mm thickness of silica gel suspension. The plates are left until solidified and stored in the desiccator until just before use.

Chromatographic plates, previously coated with silica gel, are activated by heating into a desiccator to cool. Starting parallel spots, 2 cm away from each side of the plate and 1.5 cm apart are made from the chloroform extract together with the standard aflatoxin B1 by means of micropipettes. The spots are left to dry in the air. The prepared TLC plates are transferred into the developing tank containing the developing solvent system [Toluene-ethylactate- 90% formic acid (60:30:10)]. When the solvent travels about 12 cm front, the plates are removed from the tank and air dried. The plates are inspected under U.V. light (256 nm and 365 nm) and the outline of each fluorescence spot is marked by a sharp pin. The Rf values, colours and intensities of the unknown spots are compared with those of standard spot.

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ii. Estimation of mycotoxin using fluorometric assay:

Setting up of the Fluorometer :

The fluorometer is calibrated and the required reagents are prepared, namely, methanol: water mixture (80:20), 1x PBS and 1x 0.1% Teeen-20/PBS buffers (washing and diluting buffers) and developer A & B mixture. It is necessary to ensure that the reagent blank (1 ml methanol+ 1ml developer A and B mixture) reads 0 ppm on a calibrated fluorometer and that 2 ml PBS in a cuvette read 0 ppm on a calibrated fluorometer.

Sample extraction:

50 g of each ground sample with 5g salt are placed in the blender jar, 100 ml of methanol: water (80:20) are added and the blender jar is covered, then the mixture is blended at high speed for 1 minute. Cover is removed from the jar and the extract is poured to be filtered and the filtrate is received in a clean container.

Extract dilution:

10 ml of the filtered extract are poured into a clean vessel and diluted with (40 ml of 0.1 %Tween-20/PBS) and mixed well. Diluted extract is filtered through 1 µm microfiber filter directly into glass syringe barrel using markings on side of barrel to measure 10ml.

Column chromatography:

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Ten ml diluted extract are passed completely through test affinity column at a rate of 1-2 drops/ second until air comes through column. 10 ml of PBS were passed through the column at a rate of 1-2 drops/ second until air comes through column for washing out the impurities.Glass cuvette is placed under test column and 1 ml HPLC grade methanol is added into glass syringe barrel. Test column is eluted at a rate of 1-2 drops/ second and all of the sample elute (1ml) is collected in a glass cuvette.1 ml of developer A&B mixture is added to the cuvette and mixed well. The cuvette is placed in the fluorometer and mycotoxins concentration is read. iii. Estimation of mycotoxins by high performance liquid chromatography (HPLC)

Estimation of mycotoxins by high performance liquid chromatography (HPLC) of extracts of commodities, foods and feeds is the most prevalent and sensitive current method for the identification and quantitation of mycotoxins. Uncanny sensitivity and precision in the detection of ppt (parts per trillion) concentrations of the fluorescent mycotoxins AFB1, AFB2, AFG1, AFG2 and sterigmatocystin, citrinin and ochratoxin A(OTA) can be achieved by careful preparation and concentration of extracts of grain/fruit samples, followed by HPLC in an apparatus equipped with a fluorescence detector.

Often, the mycotoxins extracted from field samples undergo clean-up using commercial immunoaffinity columns before their analysis by HPLC. The columns are available for all important mycotoxins, namely AFB1, AFB2, AFG1, AFG2, AFM2, ochratoxin A, T2 toxin, deoxynivalenol (vomittoxin), citrinin, fumonisins FB1, FB2, FB3, zearalenone, patulin and moniliformin. Multiplex columns are available for aflatoxins, ochratoxin A and

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zearalenone . The rationale beyond the multiplex columns and for multiplex detection methods is the frequent production of more than one mycotoxin by a single fungus, and the frequent contamination of crops or silage with several species of fungi

Annex 4: Identification of fungal isolates by polymerase chain reaction (PCR):

Fungal cultures and DNA isolation

Fungal isolates are inoculated in 500 μl potato dextrose broth in 2 ml Eppendorf tubes and incubated at room temperature for 4 days.

Mini-prep” DNA extraction protocol:

Approximately 1-2 hours prior to beginning of extraction, the extraction buffer is placed in a water bath(65°C) to warm it. Alternately, the buffer is heated to ~65°C in a microwave oven immediately prior to use. Each mycelial sample is ground to a fine powder in a mortar under liquid nitrogen. The ground mycelium is placed into a labeled 1.5-ml microcentrifuge tube to fill the tube to approximately the 500-μl level. 700 μl of hot (65°C) 2% CTAB buffer with mercaptoethanol are added to the microcentrifuge tubes containing ground mycelia. Each sample is vigorously mixed to disperse any clumps of mycelia, then the tubes are placed in a water bath( 65°C) for 30 min, the extraction tubes are gently mixed 3-4 times by inverting the tubes to ensure that the contents are well mixed.

The extraction tubes are moved from the water bath and 300-400μl of chloroform: isoamyl

182 alcohol are added (24:1, v:v) to each tube, each sample is vortexed briefly (a few seconds) to mix the aqueous and organic phases.The tubes are then placed in a microcentrifuge and centrifugated at ~11,400 x g for 5 min. to separate the organic and aqueous phases.

The tubes are carefully removed at the end of the centrifuge run to avoid disturbing the layer of cellular debris that is formed between the two liquid layers and moved to the fume hood, and with a 1000-μl pipetter, 600 μl (or as much of this layer as possible) of the aqueous (upper) phase of each sample is transferred to a fresh, sterile, microcentrifuge tube without disturbing the debris in the middle layer. The debris layer and the organic phase are discarded as appropriate(a hazardous waste) 600 μl of isopropanol (2-propanol) are added to the recovered aqueous phase of each extraction, and then each tube is inverted several times to be mixed well, the tubes are allowed to sit for ~5 min.at room temperature for the nucleic acids to precipitate. These tubes are stored overnight at -20°C.

The tubes containing the samples are cenrifugated for 5 min. at ~9200 xg to pellet the nucleic acids.The tubes are removed from the microcentrifuge. The aqueous/alcohol mixture is decanted and discarded from the pellets.The tubes are inverted onto a clean paper towel and allowed to air-dry for 4-5 min. 600 μl of 1× TE buffer are added to each crude pellet and resuspended by mixing. These tubes are stored overnight at -20°C. Once the pellets are completely resuspended, 200-300 μl of phenol: chloroform: isoamyl alcohol (25:24:1) are added to each microcentrifuge tube, and each sample is vortexed briefly (a few seconds) to mix the aqueous and organic phases.

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The tubes are placed in a microcentrifuge and centrifugated at ~11,400 x g for 5 min. to separate the organic and aqueous phases. The tubes are carefully removed from the microcentrifuge at the end of the centrifuge run to avoid disturbing the layer of denatured proteins that is formed between these two layers. With a 1000-μl pipetter, 500 μl (or as much of this volume as possible) of the aqueous (upper) phase of each sample are transferred to a fresh, sterile, microcentrifuge tube without disturbing the debris in the middle layer. 1 μl of RNAse A is added to each sample and each sample is vortexed briefly to disperse the RNAse, then incubated at 37°C for 30 min.The microcentrifuge tubes are removed from the incubator, and 500 μl of isopropanol (2-propanol) are added to each tube. Each microcentrifuge tube is inverted several times for mixing, the microcentrifuge tubes are allowed to sit for ~5 min. at room temperature for precipitation of the nucleic acids .

The tubes containing the samples are centtrifugated for 5 min. at~11,400 x g to pellet the DNA. The tubes ae removed from the microcentrifuge, and the aqueous /alcohol mixture is decanted and discarded from the pellets, the tubes are inverted onto a clean paper towel and allowed to air-dry for 4-5 min. The DNA pellets are washed with 1 ml of ice cold 70% (v/v) ethanol (the 70% ethanol kept in a -20°C freezer) by adding the ethanol, and then decanting it. The tubes are allowed to air- dry inverted on a clean paper towel for 2-3 minutes. The wash is repeated twice. After the second wash, the open tubes are left in a 65°C incubator to dry . Each pellet is resusspended in 50 μl of TE buffer and stored at 4°C.

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Estimation of purity and concentration of DNA:

The concentration and purity of extracted DNA ware determined by estimation of the optical density at wave length of 260 and 280 nm, using the spectrophotometer. The concentrtion is calculate as follows: OD. 260 nm= 50µg/ml. The purity of DNA= OD. At 260/ OD. At 280 nm. The optimum purity of DNA has a value that ranges from 1.8 to 2.

Primers for PCR amplification:

Two sets of primer are used. One set of primer is used from the conserved ITS DNA region specific to toxigenic fungal isolates. Another set of primer specific for mycotoxin production is used from ‘toxin gene’ of each fungal species (Forward ) and expected amplicon size for each toxin..

PCR mixture and conditions:

PCR is performed using thermocycler (AP,Applied Biosystems). PCR mixture (25 μl) contains 2 μl of DNA sample, 10X PCR buffer, 25 mM MgCl2, 2 mM dNTPs, 20 pmol of each forward and reverse primer and 0.5 μl (3U/μl) of Taq DNA polymerase. The PCR conditions for ITS and FUM 1 regions include 94°C for 4 min for initial denaturation, followed by 35 cycles of denaturation at 94°C for 1 min, primer annealing at 58°C for 1 min, primer extension at 72°C for 1 min. The final extension is set at 72°C for 10 min.

Screening of PCR products by agarose gel

185 electrophoresis:

Solution of molten agarose is prepared as required by adding the 1x TAE buffer to the appropriate amount of agarose powder(1.5%) in a suitable flask ,and brought to boil in the microwave oven. The agarose must be completely dissolved prior to pouring. To facilitate visualization of DNA fragments during the run, ethidium bromide solution is added to the gel to a final concentration of 0.5 μg/mL. The ends of the plastic tray are closed with masking tape and the gel is poured after it cools down to o about 55 C . After setting of the gel, the tape is removed from the casting tray and the gel comb is withdrawn carefully to avoid tearing the sample wells. The gel casting tray containing the set gel is placed in the electrophoresis tank. Sufficient 1xTAE buffer is added to cover the gel to a depth of 1mm and until the wells are just submerged. 2 μL of the gel loading buffer aere added to each 10 μl sample.

Samples (PCR products) are added to the individual wells. DNA molecular weight marker is also loaded . The tray is coverd with the safety cover and gel is run at 110 mA (7 x 10 cm tray) or 200 mA (15 x 10 cm tray) - typically 1 to 10 V/cm of gel. The power supply is turned off when the bromophenol blue has migrated a distance judged sufficient for separation of the DNA fragments. The DNA is visualized on a UV transilluminator and photographed.

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Annex 5: Rapid detection of mycotoxigenic fungi:

1. immunological and molecular assays to detect mycotoxigenic fungi:

The enzyme-linked immunosorbent assay (ELISA):

Although polyclonal antibodies (PAbs) derived from immunizing animals have largely been superseded by the use of monoclonal antibodies (MAbs). This procedure can still provide the basis for useful assays. While the cost and time required to develop MAb-based immunoassays is considerable, this is offset by the potential to produce, indefinitely, unlimited quantities of a specific MAb. A broad range of immunoassay formats have been developed, although the enzyme-linked immunosorbent assay (ELISA) is the most widely used for the detection of mycotoxigenic pathogens . Immunoassays have also been adapted for on-site field work in dip-stick or dot-blot formats providing a user friendly system for rapid pathogen detection and disease diagnosis. More recently, lateral flow devices have been produced that simplifydetection of the target molecule in a one-step procedure. In addition to detection, such immunoassays can be used for quantification of the target species.

Many antigens appear to be common to fungi of different species or genera such that polyclonal antibodies that are raised against a particular fungus may crossreact with another that is taxonomically distinct. The specificity of the antibodies may reflect that of the antigens against which they were raised. The immunodominant extracellular polysaccharide (EPS) antigens are more highly conserved than soluble macromolecules (exoantigens) obtained from surface washings of mycelium Such conservation may be exploited where fungi from more than one genus produce a particular mycotoxin. For example, the immunodominant extracellular polysaccharide (EPS) of Aspergillus and Penicillium

187 species both contain galactofuranoside residues so that antibodies raised against these targets may react to both. Indeed PAbs raised against culture filtrate of A. parasiticus cross-reacted with Aspergillus and Penicillium species but not with Fusarium species and a monoclonal antibody raised against A. flavus EPS also crossreacted with high specificity to Aspergillus and Penicillium species .

A novel approach to detecting aflatoxin producing species by polyclonal antibodies were raised to products of two genes, ver-1 and apa-2 involved in aflatoxin biosynthesis . Polyclonal antibodies to these chimeric proteins were highly specific towards A. flavus and A. parasiticus and did not cross-react with the other species tested. The authors, however, suggested that such assays may not react with mycelium in which the mycotoxin is not being synthesized and may also cross-react with related enzymes from other fungi that are not involved in mycotoxin biosynthesis.

Polyclonal antibodies raised to soluble protein fractions of F. culmorum, F. graminearum and F. poae when used in an ELISA but did not cross-react with Microdochium nivale or Tapesia species. Monoclonal antibodies have been raised against a number of Fusarium species including F. culmorum , F. avenaceum and F. graminearum and measured by ELISA .

Neucleic acid hybridization

A large number of assays have been produced to detect these fungi on the basis of nucleic acid sequences that are specific to the target organism. The authors used nucleic acid hybridization assays to detect and identify mycotoxigenic fungi as species- specific DNA probes developed towards several species as aflatoxigenic A.flavus , ochratoxin producing fungi (A. ochraceus,P.verucosum and A.niger ) and Trichothecines and Fumonosin B1 producing Fusarium .

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Nucleic acid hybridization assays involve the selection, cloning and chemical labelling (e.g. biotin, digoxigenin, 32P) of sequences specific to the target organism. These are then used as probes to detect RNA or DNA of the pathogen in extracts or tissue squashes of plant material. The assay may involve immobilization and detection of nucleic acid on a membrane or in some instances, utilize a microplate format similar to that used in immunoassays. The development and use of nucleic acid hybridization assays to detect and identify pathogenic fungi has been limited, although species-specific DNA probes have been developed towards several Fusarium species, including F. culmorum, F. graminearum and F. avenaceum

Polymerase chain reaction (PCR)-based assays for Detecting mycotoxigenic fungi:

In contrast to hybridization, the PCR has found widespread use throughout the diagnosis of diseases and a significant number of PCR-based assays have been developed for use with species associated with the production of mycotoxins. PCR involves the enzymatic amplification of a target DNA sequence by a thermo stable DNA polymerase such as Taq from yeast or mould .

PCR generally involves the denaturing of DNA strands and annealing of two oligonucleotide primers to their homologous sequences that flank the region to be amplified. The DNA polymerase extends the primers across the region and so generates a copy. The process of melting, annealing and extension is repeated up to 50 times, so producing many millions of copies (amplicons) of the target region.

The sensitivity of the PCR process lies in the amplification aspect while the specificity is determined by the choice of primers used in the reaction. Where sufficient specificity is designed into the

189 primers, individual isolates of a particular species can be detected within complex DNA mixtures. Minor differences in amplification efficiencies, such as might occur where DNA samples contain different amounts of inhibitory substances, result in large differences in amplicon yield.

Two approaches have been taken to overcome such problems:

a. Competitive-PCR:

A known quantity of ‘competitor’ template DNA is added to the sample before PCR. The ‘competitor’ and fungal target DNA have identical primer sites but differ in the length of sequence between them. During the PCR the fungal ‘target’ and ‘competitor’ template sequences compete for reagents such that the ratio of fungal ‘target’ DNA to ‘competitor’ DNA amplified increases with increasing amounts of fungal DNA. Following PCR, the two products can be size separated and the relative amount of each determined with standard image analysis software. In uninfected tissue only the ‘competitor’ DNA is amplified confirming that the PCR is functioning normally. There are relatively few competitive PCR assays for mycotoxigenic pathogens perhaps because of the difficulty of producing and maintaining competitor templates for relatively large numbers of fungal species. b. Real-time PCR:

The amount of amplicon is assayed at each cycle, provides a second means of quantifying the amount of DNA of a particular species or fungal target. In its simplest form the amplicon is quantified on the basis of fluorescence produced by an intercalating dye. Such a system is not, however, able to determine whether the

190 fluorescence is due to amplification of the correct amplicon. In order to address this, a number of fluorigenic probe-based assays have been developed to reduce interference from amplification of non- target fragments. These include TaqMan , Molecular Beacons and Scorpion primers. In all cases, the use of such assays should be accompanied by an internal control to account for differences in amplification efficiency between samples.

The authors used Real-time’ PCR and Competitive-PCR for detection of geners responsible for mycotoxin production. Species- specific DNA probes were developed towards several species as aflatoxigenic A.flavus , ochratoxin producing fungi (A. ochraceus , P.verucosum and A.niger ) as well as trichothecines and fumonosin B1 producing Fusarium.

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