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RAPID DETECTION OF AFLATOXIGENIC PRODUCING ATRAINS OF
ASPERGILLUS FLAVUS FROM POULTRY FEES BY UV LIGHT AND
AMMONIA
Raed Najeeb Alkhersan, Mohammed H.Khudor, Basil A. Abbas
Microbiology Department, College of Veterinary Medicine, Uni. Of Basrah, Iraq.
(Received 14 January 2016 ; Accepted 9 March 2016)
Keywords; (PDA) , Poultry feed, Aspergillus flavus.
ABSTRACT
A total 180 samples of pellet poultry feed were collected from poultry feed stores and local
markets .They were cultured on potato dextrose agar(PDA) and malt extract agar (MEA) for
isolation and identification of Aspergillus flavus.They were Sub-cultured on sabouraud dextrose
agar(SDA) .Then the isolates were cultured on coconut agar medium(CAM) . The rapid detection
of 50 selected isolates of contaminated poultry feed samples with A.flavus were achieved by blue
–green fluorescence under UV light and by ammonia vapor on CAM medium to determine the
aflatoxigenic isolates of A.flavus. The detection by UV light revealed that 26 (52%) of isolates
were aflatoxigenic (positive) by produce blue-green fluorescence under UV light at 356nm , and
also 26 (52%) of isolates were aflatoxigenic (positive) by turned the colony reverse to pink color
by exposure to ammonia vapor.
INTRODUCTION
Aspergillus flavus is widely distributed in nature and is largely found at cereal and grains.
Before harvest or during storage, A. flavus grows on agricultural crops (1). Its growth is affected
by the environmental condition such as temperature and relative humidity (2).
Aflatoxins are difuranocumarin derivatives.They are very slightly soluble in water (10–30 μg/mL),
in non-polar solvents they are insoluble , and soluble in moderately polar organic solvents (e.g.
chloroform and methanol) and extremely soluble in dimethyl sulfoxide (3) .Under the influence of
ultraviolet light they are unstable and in the presence of oxygen, to extremes of pH (< 3, > 10)
and to oxidizing agents (4).
Aflatoxins are produced only by a closely related group of Aspergilli: A. flavus, A.parasiticus and
A.nomius strains (5).Other species such as A.bombycis, A.pseudotamari and A.ochraceoroseus
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are also aflatoxin-producing species, but they are found less frequently (6).Aflatoxins cause a
problem concerning many commodities also aflatoxin B1(AFB1) act as carcinogenicity,
mutagenicity and acute toxicology and determined it to be a human carcinogen . Aflatoxins are
common occurrence in feedstuffs, feeds. Aflatoxicoses , the disease caused by exposure to
aflatoxin have made severe economic losses in the poultry industry, affecting ducklings, broilers,
layers, quail and turkeys to cause clinical signs include anorexia, decreased weight gain,
decreased egg production, hemorrhage, embryotoxicity, and increased susceptibility to
environmental and microbial stressors (7).
Blue fluorescence a method used for developing qualitative cultural methods for detecting
aflatoxigenic Aspergillus species grown on suitable media. This techniques use either solid media,
such as coconut agar medium (CAM) and potato dextrose agar (PDA) or liquid medium, like
aflatoxin producing-ability medium( APA) , and a medium with steep liquor (8,9), and achieved
by cut a small plugs from Aspergillus colonies on medium to culture on the other media . The
aflatoxins producer Aspergillus were detected under long-wave UV light (365nm) . This rapid
identification to determine aflatoxigenic isolates from non-aflatoxigenic by appear blue to blue –
green fluorescent to aflatoxigenic , and nonaflatoxigenic is non-produce fluorescent (10).
Ammonium hydroxide vapor-induced color change a rapid and sensitive method , also for
detection of aflatoxigenic and nontoxigenic strains of Aspergillus (11). In this method a single
colony was grown in the centre of Petri dish. The reverse of colony of aflatoxigenic Aspergillus
strains turned to pink color when their medium were exposed to ammonia vapor by dropped of
ammonia hydroxide on it but nonaflatoxigenic is no color produce (12).This study aims to detect
the aflatoxigenic Aspergillus flavus by UV light test and by ammonia vapor test which were
isolated from poultry feed.
MATERIALS AND METHODS
Collection of samples
A total 180 samples of pellet poultry feed were collected from poultry feed stores and local
markets at Basrah governorate during one year from Sep. 2014 to Apr. 2015 . Samples were stored
for 2-3 days in sterile plastic containers at room temperature (22-25ºC) in laboratory . After
stored, they were prepared for fungi isolation and identification.
Isolation and identification of A.flavus
Suspension of 20 g of the poultry feed samples with 180 ml of saline solution (0.85% Sodium
Chloride) with 0.05% Tween 80 on a horizontal shaker for 30 minutes. Then 0.1 ml of suspension
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was inoculated on PDA and MEA(13,14). The pure culture was incubated at 25 °C ± 2 and after 7
days (15). After incubation , the macroscopic and microscopic distinct colonies by using
lactophenol cotton blue were done. The morphological characteristics of A.flavus isolates were
described microscopically according to Domsch and Gams (16) and Klich (17) .Sub-culture on
SDA was done , and also on CAM according to Davis et al (18). Then the cultures were
incubated at 25◦C ± 2 for 7 days (19).
Coconut based medium test
It was done by fluorescence on coconut agar medium(CAM). A preliminary screen for
aflatoxin producer Aspergillus was performed on the basis of emission of blue to blue – green
fluorescence after UV light excitation at 365 nm after growing the isolates on coconut agar
medium , because this agar is inductive of aflatoxin production (20). Producer isolates can be
identified by fluorescence in the reverse side of the culture CAM in glass Petri dishes(18,
21).Five millimeter diameter sterile cork borer was used to make a hole in the center of CAM
medium in petri dish .The isolate was inoculated of a mass of conidia by cork borer to the hole at
the central point Petri dish of CAM , then they were incubated at 28 °C for 7 days.
nonaflatoxigenic Aspergillus niger was used as control (20,22).
Ammonia vapor test
The fungal isolates were inoculated on CAM as single colonies by cork borer (5mm) diameter
in the center of plate and incubated in the dark at 28 °C. for 7 days. The dish was inverted and 1 or
2 drops of concentrated ammonium hydroxide solution are placed on the inside of the lid of Petri
dish. Then the Petri dish inverted over the lid containing the ammonium hydroxide. A control as
was mentioned in previous test was prepared.
RESULTS
Fifty A.flavus isolates were considered out of 180 samples. The detection of aflatoxigenic and
nonaflatoxigenic Aspergillus flavus by using UV light and ammonia vapor revealed that 26
(52%) of isolates were aflatoxigenic (positive) and 24(48%) of isolates were nonaflatoxigenic
(negative) for both methods (table1,2) . The detection by UV light 365nm recognized
aflatoxigenic by produce blue-green fluorescent colonies in the center of glass Petri dish of CAM
in the reverse , from nonaflatoxigenic which were nonproducing fluorescent colonies , similar to
the control isolates of nonaflatoxigenic A.niger ,( figure 1).
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Table (2) : The results of aflatoxigenic A.flavus isolates recovered from poultry feed samples
which was obtained by UV light detection , ammonia vapor detection on CAM.
No. of
isolate
Coconut
based
medium test
Ammonia
vapor test
No. of
isolate
Coconut based
medium test
Ammonia vapor
test
1 + + 26 + +
2 + + 27 - -
3 + + 28 + +
4 + + 29 + +
5 - - 30 + +
6 - - 31 - -
7 - - 32 + +
8 - - 33 + +
9 + + 34 + +
10 + + 35 + +
11 + + 36 + +
12 - - 37 - -
13 - - 38 - -
14 + + 39 + +
15 + + 40 - -
16 + + 41 - -
17 + + 42 + +
18 + + 43 - -
19 - - 44 - -
20 - - 45 - -
21 + + 46 - -
22 - - 47 - -
23 + + 48 - -
24 - - 49 - -
25 + + 50 - -
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Figure(1): Showed the detection of aflatoxigenic A.flavus by CAM under UV light at 365nm
.(a) control of nonaflatoxigenic isolate of A.niger , (b) nonaflatoxigenic A.flavus (negative)
isolate, and (c) aflatoxigenic A.flavus (positive) isolate , showing a blue-green fluorescent
ring around the colony .
Aflatoxin detection by ammonia vapor to characterize as aflatoxigenic isolates A.flavus which
produced pink to red color colonies in inverted petri dish by applying 1or 2 drops of concentrated
ammonia hydroxide solution on the inside of the lid , but no color change occurred in
nonaflatoxigenic isolates, (figure 2).
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DISCUSSION
By using UV light technique in this study , 26 isolates (52%) from a total 50 isolates of A.flavus
were aflatoxigenic can colored with blue –green fluorescence ( positive ) on reverse of glass
Petri dish of CAM with compared to nonaflatoxigenic showed no color (negative) results and
considered as negative. The same results above by ammonia vapor detection were reported in
which the colony of aflatoxigenic A.flavus turned to pink color , while no change in color with
non aflatoxigenic isolates. This mean that the number of aflatoxigenic isolates of A.flavus were
equal by UV light at 365 nm and ammonia vapor on CAM. This result is similar with those
obtained by Yazdani et al (11) , Saito et al (12) , Zarari et al (24), Nair et al(25) and Sudini et al
(26). While there was difference with Riba et al (23) whose confirmed that the cultures of
aflatoxigenic Aspergillus were tested for 365 nm UV light fluorescence and for bright orangeyellow
colony reverse coloring, and also this study disagrees with study of Fani et al (27) , which
reported only (25.6 %) positive isolates of aflatoxigenic A.flavus by fluorescence detection on
CAM , while less isolates(12 %) were identified as aflatoxigenic using ammonium vapor
detection.
CONCLUSIONS
Aflatoxin is a major problem in developing countries where contaminated food commodities may
readily reach food stores and homes . It is important to know that the effect of aflatoxin on
animals extend beyond the symptoms. There are high percentage of aflatoxigenic A.flavus in
poultry feed product.
المنتجة للافلاتوکسین المعزولة من أعلاف Aspergillus flavus الکشف السریع عن سلالات الفطر
الدواجن بواسطة ضوء الأشعة فوق البنفسجیة وبخار الأمونیا
رائد نجیب الخرسان، محمد حسن خضر ،باسل عبد الزھرة عباس
فرع الاحیاء المجھریة، کلیة الطب البیطری ، جامعة البصرة
الخلاصة
تم جمع 180 عینة من اعلاف الدواجن المرکزة من مخازن أعلاف الدواجن و الأسواق المحلیة . تم زرعھا على الوسطین
وقد تم اعادة زرعھا على وسطین ثانویین . Aspergillus flavus لعزل وتحدید سلالات الفطر (MEA) و (PDA)
بواسطة ضوء A.flavus تم الکشف السریع لت 50 سلالة مختارة من عینات علف الدواجن المتلوثة بن .(CAM) و (SDA)
المنتجة A.flavus لتحدید سلالات الفطر CAM الأشعة فوق البنفسجیة بطول موجی 356 نانومتر وبخار الأمونیا على الوسط
aflatoxigenic 52 ٪) من العزلات کانت ) للافلاتوکسین وغیر المنتجة .اثبت الکشف بواسطة الأشعة فوق البنفسجیة أن 26
(إیجابیة) من خلال انتاج الضوء الفلورسنتی الاخضر المزرق تحت ضوء الأشعة فوق البنفسجیة فی 356 نانومتر، وأیضا لوحظ
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ایجابیة) من خلال تحول لون ظھر المستعمرة الى اللون الوردی عند ) aflatoxigenic 52 ٪) من العزلات کانت ) ان 26
التعرض لبخار الأمونیا.
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