research article

Egypt. J. Exp. Biol. (Bot.), 15(2): 145 – 154 (2016) DOI: 10.5455/eg yjebb.20160710115119

© Th e Eg yptian Society of Exp erimen tal Biolog y

RESEARCH ART ICLE D a li a A . M . A bd o u R a sh a M . O t hm an N a s r . E . El - Bo r d en y N e vi n A . Ib r a h im M o h am e d A. Ab o u zei d

M ON IT OR ING PR OC ESSIN G MY C OT OXIN S

IM POR T ED GR AIN - B AS ED IN G R ED IEN T S U SED IN F EED F OR T OXI G EN I C M OU LD S AN D N AT U R AL L Y OC CU R R ING

ABSTRACT: Samples of imported feeds and feed ingredients were collected every three months during the study time from feed factories in two governorates, Egypt. Samples were cultured and the recovered isolates of Fusarium and Aspergillus were mycologically identified using the universal keys. Species of Fusarium were the prevalent with relative density of 52.9%. The majority of samples analyzed (67.7%) had moderate fungal counts (around 8.9 × 10 3 CFU g -1) whereas 26.2% contained counts over the feed hygienic quality limits of 1×10 4 CFU g −1. Based on frequency in tested feed samples as well as relative density, F. proliferatum was the predominant Fusarium species (23.8%) whereas 36.9% yielded isolates of A. flavus. Applying high performance liquid chromatography technique, Applying chromatographic techniques, T-2 toxin (0.0493 mmol/L) was detected in the culture filtrate of a single isolate of F. sporotrichioides whereas two different isolates of F. verticillioides and an isolate of F. proliferatum produced fumonisin B1 in amounts of 0.094 mmol/L, 0.1530 mmol/L, and 0.212 mmol/L. Two A. flavus isolates were capable of producing aflatoxin B1 (0.0580 mmol/L and 0.0446 mmol/L) and aflatoxin B2 (0.0202 mmol/L and 0.0236 mmol/L). Aflatoxins G1 (0.0744 mmol/L) and G2 (0.0433 mmol/L) were produced in the culture filtrate of a single isolate of A. parasiticus. Only three feed samples were naturally contaminated with traces of mycotoxins, a sample of Argentinean corn which contained T-2 toxin in addition to two locally produced samples of broiler finisher feed which contained traces of fumonisin B1 and dairy cattle CFM which contained aflatoxin B2 and a trace amount of aflatoxin B1.

D a li a A . M . A bd o u * R a sh a M . O t hm an * N a s r . E . El - Bo r d en y * * N e vi n A . Ib r a h im * * Dep artment of Microbiolog y, F acult y of Science, Ain S hams Univ ersit y, 11566 Abbassia, C airo, Egypt, 11566 Abbassia, Cairo, Eg ypt

** Departm ent of Anim al Prod ucti on, F ac ult y of Agricult ure, Ai n Shams U niv ersit y, 11241 Hadaek S houbra, Cair o, Egypt .

ARTI CLE CODE: 15.02.6

I NTRODUCTI ON : In Egypt, local poultry production was on average 874 thousand tons (Hassan, 2013) whereas total beef production is anticipated to reach 300 thousand metric tons (Egypt: Livestock and Products Annual, 2014). Most feed ingredients used in the processing of live stocks and poultry industries are imported from Latin America, Eastern Europe, Canada and USA (El-Sayed, 2012). Such imported ingredients are prone to fungal contamination either when transported or stored in Egyptian factories. Fungal contamination in animal feeds of cereal grains, oil-seed meals and forages represents the major animal health risk KEY WORDS: throughout the world (D’Mello, 2004). Three major important genera of mycotoxin -producing feed stuf fs, t oxigenic moulds, grain's fungi, Aspergillus, Penicillium and Fusarium contami nation l evel, F us arium and appear at different stages of grain production. Aspergillus m yc otoxi ns. Fusarium species invade grains during growth while species of Aspergillus and Penicillium CORRESP ONDEN CE: generally develop during grain storage (Miller, M o h am e d A. Ab o u zei d 1995). Mold growth can affect grain's nutritional Department of Micr obiolog y, F ac ult y of quality in several ways and may adversely affect Science, Ain S hams Univ ersit y, 11566 health of animals, some species are able to Abbassia, C airo, Egypt, 11566 Abbassia, produce highly toxic compounds called Cairo, Eg ypt. mycotoxins (Marguardt, 1996). However, the E-mail : [email protected] presence of fungal growth doesn't necessarily ISSN: 1687-7497 On Line ISSN: 2090 - 0503 http://my.ejmanger.com/ejeb/

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mean that mycotoxins are present. As mentioned by Lawlor and Lynch (2005), mycotoxins are estimated to affect as much as 25% of the world’s crops each year. Mycotoxin production is unavoidable, unpredictable and contamination by such toxic compounds leads to greater losses in production, even when mold is not readily apparent (Adams et al., 1993). They have negative impacts on animal performance, growth and reproductive rates, immunological defense, as well as being carcinogenic, mutagenic and teratogenic (Ratcliff, 2002). Many of such natural toxic compounds become concentrated in meat, egg and even milk of animal and consequently may pose a threat to human health, this explains the major concern of food and feed industries in preventing them from entering the food chain (Akande et al., 2006). The most commonly encountered mycotoxins in feedstuffs are aflatoxins, fumonisins, zearalenones, ochratoxin, and T2toxin (Surai et al., 2008), this wide range of mycotoxins contaminating animal/poultry feed in addition to the variation in their chemi cal compositions make protection against their hazards a difficult task. Also, there are many problems complicating safety in feed manufacturing process: 1) the mixing of various batches of different raw materials together thus producing a totally new matrix with a new risk profile; 2) mycotoxins can contaminate practically all feed ingredients, for example, Fusarium species have been found in wheat, maize, barley, oats and rye whereas aflatoxins contaminate oilseeds and other feed ingredients; 3) most of mycotoxins are stable compounds that do not degrade during storage, milling or hightemperature feed manufacturing processes (Surai et al., 2008). The present study was designed in the interest of safety and quality assurance, with the aim of providing useful information on the mycological and toxicological risks associated with consumption of some imported agricultural raw materials locally used in animal feed processing. Objectives were: (i) developing a sampling plan to include all imported feed ingredients used in Egyptian feed factories (ii) isolating, enumerating, identifying toxigenic Fusarium and Aspergillus species in collected feed samples (iii) analyzing the most significant mycotoxins contaminating imported and processed feed.

Egypt. J. Exp. Biol. (Bot.), 15(2): 145 – 154 (2016)

Brazil, US A). T he s ec ond group c omprised of 24 samples (52. 2%) of imported s oybean and corn whic h ar e loc all y us ed in processi ng of soybean meal and c orn glut en. Moist ure content w as det ermined immediat ely af ter collecti on; s amples wer e divided i nto tw o parts: one w as c ultur ed immediatel y for myc ological examination and t he ot her w as stored at - 20 o C f or m yc ot oxin anal yses . M ycol ogi cal anal yses: Quantitative enumeration of fungal propagules was performed on solid media using the surface spread method. 10 g of each sample were homogenized in 90 mL 0.1% peptone water solution for 30 min. Serial dilutio ns (10- 2 10- 4) were prepared and 0.1 mL aliquots were inoculated in triplicates onto the following media: dichloran rose bengal chloramphenicol agar (DRBC) (Pitt and Hocking, 1997) for total culturable mycobiota and dichloran chloramphenicol peptone agar (DCPA) (Andrews and Pitt, 1986) for Fusarium species. DRBC and DCPA plates were incubated at 25 º C for 7–10 days and for 7 days, respectively. Only plates containing 10–100 CFU were used for counting and the results were expressed as CFU per gram of sample; feed hygienic quality limit is 1 × 10 4 CFU g -1 (GMP, 2008). Selected colonies were sub-cultured on plates with fresh agar media for purification then transferred on slants of potato dextrose agar (PDA) and Czapek yeast extract agar (CYA). Fusarium species were identified on the basis of the macroscopic and microscopic characteristics using the universal taxonomic keys of Leslie and Summerell (2006), whereas Aspergillus species using Klich (2002). Mycotoxin analysi s: Reagents and standards: All reagents w ere of anal ytical grade. Solv ents used f or c hrom atograp hy w ere of HPLC grade (ELG Trad e P harmac eutic als and Chemic als C o, Egypt). Myc otoxi n s tandards: aflatoxi n B1, B 2, G1, G 2, Fum onisi n B 1, T -2 toxin, and m onilif ormin w ere p urchased from Sigma Aldrich, St Louis e, U SA . S epar ation was carried out b y using HP LC (S himadz u, K yoto, J apan) consisti ng of an LC -10 AD V P pump and a S urveyor FL spec trophot ometer detec tor. S upelc o, U SA . T he immune - affi nit y colum ns f or was h up of the isol at ed toxi ns were purc hased from Vicam L. P., US A and Ver atox, N eog en, US A. Toxigenicity of the recovered Fusarium isolates:

M ATERI AL AND M ETHODS: For solid culture, corn and rice grains were Sam pl e col l ecti on pl an: prepared according to the method described by A t ot al of one hundred and thirt y Idris et al. (2003). 100 g of corn/rice were placed samples i ncluding proc ess ed f eed s amples in 500 mL Erlenmeyer flasks with 45 mL of (84) and feed ingredients (46) were c ollec ted distilled H 2 O. Flasks were stored overnight at from diff erent f eed fac tori es i n El -Fayoum and room temperature, steamed for 20 min, and then El-Qal ubea gov ernorates , Egypt . Samples of sterilized for 20 min at 121ºC before inoculating feed ingredient s wer e divided into 2 groups each with 5 squares (1 cm 2) of agar containing acc ording to t heir origin: t he first group actively growing mycelia and incubated at 2 5°C consisted of 22 samples ( 47. 8%) of imported for 4 weeks. Duplicates were made for each feed ingredients (Ukrai ne, Croatia, Argentine, ISSN: 1687-7497 On Line ISSN: 2090 - 0503 http://my.ejmanger.com/ejeb/

Abdou et al., Monitoring imported grain-based ingredients used in feed processing for toxigenic moulds and naturally occurring mycotoxins

isolate. Cultures of each isolate were dried in a forced-air draft oven at 55ºC for 48 hours, crushed with a mortar and pestle, packed in plastic bags, labeled and stored at 4ºC until use. Extraction was carri ed out as follows: 25 g sample were dried and ground, extracted with 100 mL methanol/water (55:45, v/v, 1% NaCl) and filtered through a Whatman grade no. 1 filter paper. The filtrate (50 mL) was defatted twice with n-hexane and extracted three times with methylene chloride. Extracts were combined, dried (Na 2 SO4), evaporated under reduced pressure and dissolved in 1.0 mL methanol for further analysis. For liquid culture, 500 mL Erlenmeyer flasks containing 200 mL of potato dextrose broth medium were used. Each flask was inoculated with 3 squares (1cm 2) of actively growing mycelia selected from the margin of cultures. Flasks were incubated in static conditions at 25ºC for 4 weeks in the dark, then filtered and kept lyophilized until extraction. Lyophilized culture filtrate corresponding to 100 mL was dissolved in 100 mL ultra-pure water, and extracted (4 × 100 mL) with ethyl acetate under acidic conditions (pH 2) using HCOOH 1M. Organic extracts were combined, dried and then evaporated under reduced pressure ( Idris et al., 2003). Toxigenicity of the recovered Aspergillus isolates: Erlenmeyer flasks each containing 50 mL of the YES broth medium described by Scott et al. (1970) and Ezzat and Sarhan (1991) were inoculated with 3 squares (1 cm 2) cut from the margin of actively growing colonies of the tested isolates grown on PDA medium. Three flasks were prepared for each isolate and all flasks were incubated at 25 oC under static conditions for 10 days. Aflatoxins were extracted by adding equal amount of chloroform t o the obtained culture filtrate. The lower chloroform layer was collected and organic extracts were dried at 40 oC using a rotary evaporator and the dry weights of each extract were recorded. The samples and standards were spotted on commercially precoated silicagel 60 TLC glass plate, 20 x 20 cm, 0.25 mm thickness, (E. Merck, Germany) . The plate was first eluted with anhydrous ethyl ether,

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dried up in a fume hood for 5 min., and developed with chloroform: acetone (9 :1, v /v) (Gimeno and Martins, 1983) at s ame direction. The TLC plate was visually examined under ultraviolet light at 366 nm (Chromato-VueÒ C70G, Ultra-violet Products, USA). The R f values for develop ed spots in comparison to authentic samples of Aflatoxin G2, G1, B2, and B1 (Sigma Aldrich, St Louise, USA). The sample showed similar R f values with those of the standards: B1: 0.7; B2: 0.64; G1: 0.61; G2: 0.52. Extraction and analyses of naturally occurring mycotoxins from raw feed samples: Extraction and cl ean-up of feed sam pl es for HPLC analyses: For Fusarium mycotoxins, Extraction and purification were performed as described by Hietaniemi and Kumpulainen (1991). After extracting and filtering the sample, the filtrate was defatted twice with n -hexane and evaporated to dryness. The residue was washed through a Florisil column with methanol, mycotoxins were eluted with chloroform methanol (90: 10, vol/vol) and evaporated to give a residue which was dissolv ed in 1 ml methanol-water (60: 40, vol/vol) and filtered through Sep-Pak C18 cartridges. Elution from the cartridge with 15 ml methanol-water (60: 40, vol/vol) followed with evaporation to dryness yielded a residue which was reserved in methanol for chromatographic analysis. For Aspergillus mycotoxins, based on the method described by Stroka et al. (2000). 50 mL of n-hexane was added to the extract before filtration, 25 mL of the filtrate was diluted with 150 mL of deionized water and was cleaned up on a Afla test IAC column preconditioned with 10 mL of phosphate buffered saline (3 mL/min) and washed with 25 mL water. Aflatoxin was eluted from the column using HPLC grade methanol followed by addition of HPLC grade water. HPLC Analyses of Fusarium mycotoxins: Chromatographic columns and running conditions used in HPLC analysis of organic extracts of various Fusarium solid and liquid cultures are recorded in table 1.

Table 1. HPLC instrumentation and running conditions used in analyses of organic extraxts* from solid and liquid culture filtrates of some isolated Fusarium species. MYCOTOXIN HPLC COLUMN

SOLVENT SYSTEM

FLOW RATE WAVE LENGTH RETENTION TIME TOTAL TIME RUN

FUMONISIN reversed-phase C18 (4.6X250 mm) Catalog No. 1612124 Buffer (Sodium phosphate, pH 3.5): Methanol (MeOH): Acetonitrile (ACN) (85: 0:15 0.0 min) (85: 0:15 5.0 min) (55: 30:15 5.1-20 min) (40:6 0: 0 20.1-23 min) (40:6 0: 0 23.1-40 min) (20: 0:80 40.1-60min) 1 mL/ minute 330 nm – 465 nm 7.85-8.60 min 30 min

*Three injections each of 20 µL for each sample ISSN: 1687-7497 On Line ISSN: 2090 - 0503

T2 TOXIN reversed-phase Hypersil C18-BDS column (250×4.60) mm, 5 μm particle size

Methanol: Water (60:40, v/v)

1.5 mL/ minute 381 nm 26.13-26.30 min 55 min

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HP LC Anal yses of afl atoxi ns: Stock standard solutions of AFB 1 , AFB 2 , AFG 1 , and AFG 2 (~10 µg/mL) were prepared by dissolving the solid standard in benzene: acetonitrile (98:2, v/v) and the exact concentration were measured by spectrophotometer (Shimadzu UV -1601PC, Shimadzu Scientific Instruments, Japan) . Chromatographic run was proceeded through Supelcosil GOLD Lc -18 (15 cm × 4.7 mm) column and solvent s ystem wat er: acetonitrile: methanol (63: 22: 15 v/v/v ) at a flow rate of 900 µL/min. Det ection was at λ ex: 365 nm λem: 455 nm. Order of elution (R t ) is: aflatoxin G2: 3.64 min; aflatoxin G1: = 3.27 min; aflatoxin B2: = 4.18 min; aflatoxin B1= 4.69 min. Stati sti cal an al yses: IBM SPSS statistical software package (V. 20.0, IBM Corp., USA, 2011) was used for data analyses. Data were expressed as both number and percentage for categorized variables. Crosstabulation with Chi-square was used to study the association between variables or comparison between independent groups as regards the categorized data. The probability of error ( P value) at 0.05 or less was considered significant, while at 0.01 and 0.001 highly significant .

RESULTS: Mycological analyses: One hundred twenty-seven (97.7%) out of 130 tested samples were mould-contaminated. Total fungal counts observed on DRBC medium ranged from < 102 to 7.4×104 CFU g-1 with an average count of 8.9 × 103 CFU g-1. The Majority of samples (67.7%) investigated had fungal counts of moderate values above the detection limit (1 × 102 CFU g-1). 26.2% of samples contained fungal counts exceeding the feed hygienic quality limits of 1 × 104 CFU g−1. The relationship between the different types of feed samples examined during the study and their level of fungal contamination was highly statistically significant (p < 001). All corn samples showed fungal contamination levels over the maximum recommended level. Yellow corn contained the highest counts (ranging from 1.2 × 104 to 7.4 × 104 CFU g−1) with an average 3.58 × 10 3 CFU g−1. Corn gluten had the lowest mould counts ranging from 0 to 6.8 × 103 CFU g−1 with an average 3.18 × 10 3 CFU g−1. Mycological examination of the 130 samples tested indicated the presence of 10 different species of the genus Fusarium and two species of the genus Aspergillus. Total fungal counts in the investigated feed samples are illustrated in figure 1.

35000

Average count CFUg-1

30000

2.9 × 104

25000 20000 1.5 × 104

1.5× 104

15000

9.1×103

10000 5000

7.6 ×103 3.1 × 10

3.9 × 103 3

4 ×1

5.1×103

03

3.2×103

4.1×103

Maximum recommended level (104 )

0

Type of feed material Fig. 1. Fungal counts* (CFUg -1) in the investigated feed samples. *: Results are given as mean value ± standard deviation; Detection limit: 1× 102 CFU g−1; Maximum recommended level: 1 × 104 CFU g−1.

Frequ ency of o ccurr ence of th e recover ed Fusari um and Asp ergi llu s speci es: Is olates of F us arium and As pergillus species w ere rec ov ered from 70 s amples (53. 8%) out of 130 t ot al f eed s amples (46 samples out of 84 proc ess ed f eed s amples and 24 samples out of 46 f eed ingredients). Tot al c ount of Fusarium and As per gillus isolat es r ec over ed from t hes e feed s amples were 90 from proc ess ed f eeds and 48 from ISSN: 1687-7497

ingredients . F usarium and As per gillus speci es were the predominant, a highl y s tatis ticall y significant diff erenc e b etween the i ncidence of both speci es and t ypes of feed as w ell as the origin of import ed feed ingredients and incidenc e of differ ent F us arium and Aspergillus speci es r ec over ed from t hes e ingredients (p < 0. 01) and (p < 0.05), respectiv el y. N umber of is olat es, isol ation frequenc y and relative densit y of t he identified Fusari um and As per gillus is olates are

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calcul at ed in imported f eed i ngredients in relation t o t he countr y of origin (Table 2) and

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in loc ally proc ess ed f eed c omposed imported ingredients (T able 3).

of

Table 2. Isolation frequency and relative density of Fusarium and Aspergillus species in feed ingredient samples according to the country of origin Locally extracted ingredientsa

Argentineb

Brazilb

Ukraineb

USAb

Croatiab

Fusarium species No. of isolates

Frc (%)

RDd No. of (%) isolates

F. andiyazi

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

F. fujikuroi

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

F. globosum

1

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

F. napiforme

-

-

-

1

33.3

25

-

-

-

1

7.7 11.1

-

-

-

1

100

50

F. nygamai

-

-

-

-

-

-

-

-

-

1

7.7 11.1

-

-

-

-

-

-

F. oxysporum

1

4.3 11.1

-

-

-

F. proliferatum

2

8.7 22.2

1

33.3

25

1

F. solani

1

4.3 11.1

-

-

-

-

-

-

-

-

F.sporotrichioides

-

1

33.3

25

-

-

-

-

-

F. verticillioides

4

1

33.3

25

-

-

-

2

4.3 11.1

-

-

17.4 44.4

Frc (%)

RDd No. of Frc (%) isolates (%)

RDd No. of (%) isolates

100 100

Frc (%)



1

7.7 11.1

-

-

-

-

-

-

4

30.8 44.4

1

20

25

-

-

-

-

-

-

-

1

100

50

-

-

-

-

-

-

-

3

60

75

-

-

-

1

100

50

1

100

50

15.4 22.2

Total isolates

9

4

1

9

4

2

Total samples

23

3

1

13

5

1

A. flavus

6

A. parasiticus

-

26.1 100 -

-

1

33.3 33.3

-

2

66.7 66.7

1

Total isolates

6

3

Total samples

23

3

-

-

100 100

3

23.1

60

2

2

15.4

40

-

1 1

ª

RDd (%)

b

40 100 -

-

5

2

2

13

5

1

c

: Seeds from USA and processed into meal in Egypt; : country of origin; : Frequency = (ns/N) × 100, ns: the number of samples where a s genus/species occurred, N: the total number of collected samples; d: Relative density = (ni/Ni) × 100, ni: the number of isolates of a genus/species, Ni: the total number of fungal isolates Table 3. Isolation frequency of Fusarium and Aspergillus species recovered from different processed feed materials

Fusarium species

Poultry layer feed

Broiler finisher Broiler starter Rabbit growing feed feed mixture

Rabbit does mixture

No. of isolates

Fra (%)

No. of isolates

Fra (%)

No. of isolates

Fra (%)

No. of isolates

Fra (%)

No. of isolates

Fra (%)

F. andiyazi

-

-

-

-

-

-

-

-

-

-

F. fujikuroi

-

-

-

-

-

-

-

-

-

-

F. globosum

2

1.7

-

-

1

8.3

-

-

-

-

F. napiforme

4

33.3

1

8.3

-

-

-

-

-

-

F. nygamai

-

-

-

-

-

-

-

-

-

-

F. oxysporum

-

-

-

-

-

-

-

-

-

-

F. proliferatum

3

25.0

6

50.0

4

33.3

2

1.7

2

1.7

F. solani

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

8.3

2

1.7

3

25

1

8.3

1

F. sporotrichioides F. verticillioides

1

Total isolates

10

Total samples

9

12

A. flavus

7

A. parasiticus

3

8

12 58.3

6

25.0

2

3

12 50.0

7

16.7

1

3

8.3

-

No. of isolates

Fra (%)

-

-

1

8.3

1

8.3

-

-

-

-

-

-

1

8.3

-

-

-

-

1

8.3

1

8.3

-

-

1

8.3

4

33.3

1

8.3

-

-

-

-

-

-

-

8.3

-

-

-

5

12 25.0

2

-

3

Beef cattle CFM

Fra (%)

No. of isolates

3

12 58.3

Dairy cattle CFM

12 17.6

6

25.0

1

6 12

50.0

5

8.3

1

41.7 8.3

Total isolates

10

8

8

3

5

7

6

Total samples

12

12

12

12

12

12

12

a

: Frequency = (ns/N) × 100, ns: the number of samples where a genus/species occurred, N: the total number of collected samples

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150

M ycotoxi n producti on Aspergillu s i sol at es:


by

Fu ariu m

and

In comparison to authentic samples of different mycotoxins, chromatographic analyses of aliquots of organic extracts from solid cultures and liquid culture filtrates, eleven Fusarium isolates b elonging to five species namely F. proliferatum, F. verticillioides, and F. sporotrichioides F. oxysporum, and F. nygamai were found to be producers of 8 different types of mycotoxins. Isolates of F. proliferatum were producers of fumonisins, moniliformin whereas F. verticillioides and F. sporotrichioides were producers of fumonisin and T-2 toxin, respectively. Only three isolates of A. flavus out of the 12 tested were able to produce aflatoxins B1 and B2 and one single isolate out of the 4 tested of A. parasiticus was found to be a producer of aflatoxins G1 and G2. Anal yses of m ycotoxi n's di stri buti on i n raw feed sam pl es: A c onc entration of 0. 0493 mmol/ L w as recorded f or T-2 toxi n (Fig. 2) by an is olat e of

F. s por otrichi oides r ecov ered from a sample of corn imported from Argentina whereas a higher amount (0.212 mmol/L) was recorded for FB1 detected in the culture of F. proliferatum isolate (Fig. 3) recovered from a sample of locally processed broiler finisher feed. Compared with authentics of aflatoxins (Fig. 4), only two isolates contaminating two different samples of locally produced broiler starter feed and dairy cattle CFM produced aflatoxin B1 (0.0580 mmol/L and 0.0446 mmol/L) and aflatoxin B2 (0.0202 mmol/L and 0.0236 mmol/L), a single isolate from soya bean meal imported from America and prepared in Egypt was aflatoxin B2 (0.0088 mmol/L) producer and of A. parasiticus from a locally processed sample of layer poultry feed produced aflatoxin G1 (0.0744 mmol/L) and G2 (0.0433 mmol/L). Distribution of detected mycotoxins and toxigenic isolates of Fusarium and Aspergillus among the investigated feed samples is fully illustrated in tables 4 and 5.

Fig. 2. HPLC pattern of the organic extract of F. sporotrichioides showing a characteristic peak of T-2 toxin at Rt of 26.26 min.

Fig. 3. HPLC pattern of the organic extract of F. verticillioides showing a characteristic peak of fumonisin B1 at Rt of 8.64 min.

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Fig. 4. HPLC pattern of the reference samples of aflatoxins showing peaks of aflatoxins G2, G1, B2 and B1 at Rt of 3.27 min., 3.64 min. 4.18 min, and 4.69 min., respectively. Table 4. Distribution of mycotoxins and toxigenic Fusarium isolates according to type and origin of feed samples Isolate code

Type of mycotoxin (s) produced

Detection limit (mmol/L)

Source sample

Origin of samples

F. proliferatum

PRO.S16

Fumonisin B1

0.212

Broiler Finisher Feed

F. sporotrichioides

SPR.S34

T-2 toxin

0.0493

VER.S28

Fumonisin B1

VER.S31

Fumonisin B1

Species

F. verticillioides

Mycotoxin (s) in raw feed samples Occurrence

Detection limit

Locally processed

+ ve

0.107

Corn

Argentine

+ ve

0.0255

0.0941

Broiler Finisher Feed

Locally processed

ND

ND

0.1530

Soya Bean Meal

USA (Locally extracted)

ND

ND

Table 5. Distribution of aflatoxins and toxigenic isolates of A. flavus and A. parasiticus according to type and origin of feed samples Species

Isolate code

Type of mycotoxin Detection limit Source sample (s) produced (mmol/L) Afla B1 (traces)

0.0446

Afla B2 (traces)

0.0236

Afla B1

0.0580

Afla B2

0.0202

Afla B2

0.0088

Afla G1

0.0744

Afla G2

0.0433

Flav.S02

A. flavus Flav.S26

Flav.S50

A. parasiticus

Par.S57

DI SCUSSI ON: Ubiquitous nat ure of mold cont aminati ng feedstuf fs w orldwide r es ults m ainl y from the raw materials used i n their produc tion and make t heir total eliminati on from f eed impossible (Accensi et al., 2004). Pr ocessed feed contai ns cor n, s oybean and oil s eed meals as m ajor ingredients w hich are exc ellent s ubstrates f or num erous fungi. W hen long -term f avor able c onditions f or fungal growth are provided, m yc ot oxins may be produced whic h c annot be complet el y ISSN: 1687-7497

Mycotoxin (s) in raw feed samples

Origin of samples

Broiler starter feed

Locally processed

Dairy cattle CFM

Locally processed

Soya bean meal

America (locally extracted)

Layer poultry feed

Locally processed

Occurrence

Detection limit (mmol/L)

ND

ND

ND

ND

+ ve (traces)

0.0406

+ ve

0.0115

ND

ND

ND

ND

ND

ND

remov ed from t he f eed (Bankol e and Kpod o, 2005). Ingredients us ed during proc essing may hav e various m yc ot oxins present i n a mixed feed. Suc h f ungal t oxins c an b e st ored in meat, milk and egg and fi nall y tr ans ferred to hum an bei ngs (Brag ulat et al., 1995). In this s tud y, m old and differ ent t yp es of myc ot oxins cont aminating import ed grains locall y us ed in proc ess ed animal f ee ds in Egypt w ere inv estigated. Likewis e, m yc otoxi n production b y s ome of the identifi ed fungal species w as examined.



152

According to G ood Manufacturi ng Practic es ( G MP, 2008), t otal f ungal count should not exceed 1 × 10 4 CFU g − 1 . During this s tud y, 26. 2% of c ollec ted feed s amples (34 samples) did not m eet micr obiological adequac y st andards and exc eeded t he f eed hygienic qualit y limits. Tot al f ungal loads in the anal yzed finis hed feed s amples w ere around 8. 9 × 10 3 CFU g - 1 w hich is higher t han those obt ained fr om Slov akian poultr y f eed mixt ures examined b y Labuda and Tanči nov á (2006) but w ere similar wit h findings on fungal counts in f eed s amples from Argentina (Mag noli et al., 1998) and Serbia ( Krnjaj a et al., 2011). Althoug h t he m ajorit y of t he anal yz ed samples ( 67.7%) s how ed mod erat e fungal counts , high fr equenci es of F us arium (50. 8%) and Aspergillus (47.7%) wer e notic eable. Many res earchers have prov ed t hat the majorit y of feeds ar e cont aminat ed with species from Aspergillus and/ or Fus arium gener a ( Os ho et al ., 2007; Krnjaj a et al., 2011; P ereyr a et al., 2011; R eddy and S alleh, 2011). Out of t he 130 inv es tigated s amples, Fusari um is olates w ere rec ov ered from 66 feed s amples ( 50.8%). This c oincides wit h the finding of Krnj aja et al. (2011) t hat Fus arium species w ere t he m ost c ommon f ungi isolated from poultr y f eed samples wit h frequencies of 56.09% in 2007 and 63. 40% i n 2008 at S erbia, and with an earlier r eport by Osho et al. (2007) w ho f ound t hat t he F usari um speci es were t he s ec ond frequent isol at es (42%) in poultr y f eed samples anal yzed in Nigeria. In t he pres ent st ud y, F. prolifer at um was found t o be the m ost predomi nant Fus arium species (43%) f ollow ed by F. v erticillioides (25%) of t otal Fus arium is olates and w ere recov ered fr om 31 s amples ( 23.8%) and 18 samples (13. 8%), r espectiv el y out of t he total feed samples . S uc h results s upport previous findings or surv eys on t he mos t enc ount ered Fusari um species eit her in Egypt (Abo El Yaz eed et al., 2011) or in ot her countries including Argenti na ( D alcero et al., 1998), Slov ekia (Labuda and Tanči nov á, 2006) and Malaysi a (R eddy and S alleh, 2011). F. prolifer at um and F. verticillioi des are renowned for t heir role as important maiz e ears pat hog ens t hat are comm onl y diff used in maize-produci ng c ountries w orldwide (Ghiasi an et al. , 2004; Glenn, 2007; Iglesi as et al., 2010; Chil aka et al., 2012). In the present st ud y, F. napiform e, F. oxy sporum, F. solani, F. nygamai and F. s por otrichi oides were also isol at ed from f eed samples , t his is in acc ordance wit h ot her st udies c oncer ned


with c ont aminated ani mal feed ( Labud a and Tanči nová 2006; Attit alla et al., 2010; Azizi et al., 2012). Myc ological examinati on of 130 feed samples recov ered 48 As per gillus flav us isolat es (36. 9%) and 17 As per gillus par asiticus isol at es ( 13. 1%). T hes e r esult s are almost identical t o thos e rec orded previousl y in Eg ypt by A zab et al. (2005) w ho is olat ed A. flavus and A. parasitic us fr om 36% and 13% out of 500 of anim al f eed samples and ingredients . They i nvestigat ed the level of aflatoxi n B 1 c ontami nating anim al feeds and recorded t he fr eq uenc y of t oxigenic A. flav us and A. par asiticus in t he examined f eed samples. W orldwide, sev eral s tudies on f ungi contami nating f eedst uf fs prov ed onl y a high frequenc y of A. flav us (Labuda and Tanči nová, 2006; Azar akhsh et al ., 2011; Reddy and S alleh, 2011). A . par asitic us w as onl y enc ount ered in feed mi xt ure s amples studied b y D alc ero et al. (1998) and Mag noli et al. (1998) in Argenti na but was missing in the rest of st udies on Slov aki an, Ir ani an , and Malaysi an samples . Monit oring the pres enc e of m yc ot oxins in various f eed samples is not onl y important for cons umer prot ecti on, but also for producers of the r aw produc ts prior t o transp ort. Met hods bas ed on high performanc e liquid chrom at ography (H PLC) hav e been report ed (Ali et al., 2005; Manett a et al., 2005). H PLC t echniques are simple, eas y, rapid, w ell proven and widel y acc epted even if havi ng some disadv antages (Bad ea et al., 2004). In t he pres ent st ud y, HP LC provided a rapid v aluable tool i n d et ecting myc ot oxins including T - 2 toxi n, f umonisin, as well as afl at oxins B 1, B2, G1, and G 2, relativ el y measuri ng their content in fungal cultur es and i n raw f eed. F our out of 73 Fusari um isolates ( 5. 5%) were found t o be myc ot oxigenic. Thes e isolates w er e recov ered from f our s amples (3. 1%) and bel onged t o three speci es: F. pr oliferatum , F. verticillioides and F. S por otrichi oides . Toxi ns produced b y differ ent sp ecies of Fusarium are of mos t conc ern f or animal healt h (Oliveira et al., 2006). S urveys c onc erni ng Fus arium myc ot oxins , even if s till limited, all cl earl y show that feed should be acc urat el y inves tigat ed fr om t he m ycotoxic ological poi nt of view (Eriks en and P ett erss o, 2004; Lab uda et al., 2005). ACKNOWLEDGM ENTS: The authors thank Dr Ahmad Kamal, Consultant at ASSH for precious suggestions on statistical analyses and statistical elaboration.

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‫رصد لمدى تعرض المكونات القائمة على الحبوب المستوردة والمستخدمة في تصنيع االعالف‬ ‫للفطريات المنتجة للسموم والسموم الفطرية‬ ،*‫ نيفين احمد إبراهيم‬،**‫ نصر السيد البردينى‬،*‫ رشا محسن عثمان‬،*‫داليا على محمود عبده‬ *‫محمد عبد المنتصر ابو زيد‬

‫ مصر‬،‫ جامعة عين شمس‬،‫ كلية العلوم‬، ‫* قسم الميكروبيولوجي‬ ‫ مصر‬،‫ جامعة عين شمس‬،‫ كلية الزراعة‬،‫** قسم تغذية الحيوان‬ ‫ كانت لها‬F._sporotrichioides ‫تم تجميع عينات من المواد الخام المستوردة أن عزلة واحدة من عزالت‬ ‫ و أن‬،)‫ لتر‬/ ‫ مليمول‬0.0493 ( T-2 ‫والمستخدمة في انتاج العالئق وأيضا من األعالف المصنعة القدرة على إفراز سم‬ ً ‫ باإلضافة‬F._verticillioides ‫ وذلك بشكل دوري كل ثالثة أشهر أثناء عزلتين مختلفتين من عزالت‬،‫المكونات‬ ‫من تلك‬ ‫ كانت قادرة‬F. proliferatum ‫فترة الدراسة من مصانع األعالف الموجودة بمحافظتين إلى عزلة واحدة من‬ ‫ لتر و‬/ ‫ مليمول‬0.0153( ‫ وتم زراعة عينات من األعالف المجمعة على على إنتاج سم الفيومونيسين‬. ‫مصريتين‬ ‫ أما بالنسبة‬.)‫ لتر‬/ ‫ مليمول‬0.212‫ لتر و‬/ ‫ مليمول‬0.094 ‫األوساط الغذائية المناسبة بهدف عزل الفطريات من تلك‬ ‫ فقد تم التحقق من قدرة عزلتين‬A._flavus ‫ كما تم تعريف العزالت لعزالت فطر‬.‫العينات وكذلك تحديد أعدادها‬ ‫المنتمية لجنسي الفيوزاريوم واألسبرجيلوس وذلك تبعاً مختلفتين من هذا النوع على انتاج كل من األفالتوكسين‬ ‫ لتر) و‬/ ‫ مليمول‬0.0446 ‫ لتر و‬/ ‫ مليمول‬0.0580( B1 ‫ل صفاتها المجهرية وللصفات الخاصة بأشكال مستعمراتها‬ ‫ مليمول‬0.0236 ‫ لتر و‬/ ‫ مليمول‬0.0202 ( B2 ‫ كانت األنواع التابعة األفالتوكسين‬.ً ‫على البيئات الغذائية المحضرة معمليا‬ G1 ‫ وقد تم التأكد من انتاج كل من األفالتوكسين‬.) ‫ لتر‬/ ‫لجنس الفيوزاريوم هي السائدة فقد تميزت بكثافة نسبية‬ 0.0433 ( G2 ‫ لتر) واألفالتوكسين‬/ ‫ مليمول‬0.0744( ‫) كانت‬67.7%( ‫ وفى معظم العينات الفطرية‬.%52.9 ‫تبلغ‬ ‫ لتر ) في مستخلص راشح المزرعة السائلة لعزلة‬/ ‫ لكل مليمول‬103 × 8.9 ‫نتائج العد الكلى معتدلة (بمتوسط قيمته‬ ‫ عينات فقط من‬3 ‫ ُوجدت‬.A._parasiticus ‫ ) من العينات فقد تجاوز العدد واحدة من‬26%( ‫ أما فى نسبة‬،)‫جم‬ ‫ وتضمنت‬.‫ األعالف محل الدراسة ملوثة بالسموم الفطرية‬104 × 1( ‫الكلى الحدود القياسية للجودة الصحية لألعالف‬ )‫ األرجنتين‬:‫ استنادا إلى تردد العزالت وكذلك الكثافة النسبية هذه العينات عينة واحدة من الذرة (المصدر‬.)‫لكل جم‬ ‫ باإلضافة إلى عينتين من األعالف‬, T-2 ‫ هو أكثر أنواع الفيوزاريوم شيوعاً ملوثة بسم‬F._proliferatum ‫فقد كان‬ ‫ من إجمالي عينات األعالف التي المصنعة محليا ً تشمل عليقة ناهي تسمين دجاج مصنعة‬%23.8 ‫حيث تم عزله من‬ ‫ وعينة من‬B1 ‫محليا ً ملوثة بآثار من سم الفيومونيسين‬ A. ‫ بينما تم الحصول على عزالت من فطر‬،‫تم اختبارها‬ ‫ تم الكشف عن السموم عليقة ماشية اللبن منتجة محليا ً والتي كانت ملوثة‬.‫ من العينات‬%36.9 ‫من‬flavus . B1 ‫ و بآثار من األفالتوكسين‬B2 ‫باألفالتوكسين‬ ‫ واثبتت النتائج‬.HPLC ‫الفطرية بواسطة تقنية الكروماتوغرافيا‬

ISSN: 1687-7497

