Volume 1 Number 4 (December 2009) 18-22

Aflatoxin production by Aspergillus flavus isolates from green– tiger

shrimps (Penaeus semisulcatus)
Yousefi S 1, Dadgar S 2, Safara M 3, Zaini F 3*
1
Agriculture Research and Education Organization, Tehran, Iran; 2Department of Aquaculture,Iranian
Fisheries Research Organization Tehran, Iran; 3Department of Medical Mycology and Parasitology School
of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
Received: October 2009, Accepted: December 2009.

ABSTRACT

Backgrounds and Objectives: to obtain information about aflatoxigenicity of isolated Aspergillus flavus strains from
shrimps.
Material and Methods: Forty – three isolates of Aspergillus flavus from cultured green tiger shrimps of Persian Gulf were
examined for their ability to produce aflatoxins. Initially two media; Aflatoxin Producing Ability medium and Coconut Agar
medium were used to detect fluorescence under UV light, later the presence of aflatoxin in culture extract was confirmed and
quantified by high pressure liquid chromatography.
Results: Only 2 (4.6%) isolates fluoresced on Aflatoxin Producing Ability medium and Coconut Agar medium under UV
light. In sum, 9 (20.93%) isolates (including the 2 above mentioned isolates) were confirmed to be aflatoxigenic by High
Performance Liquid Chromatography. Eight (18.7%) of isolates produced aflatoxin B1 ranging from 0.32 to 12.18 ppb, while
1(2.3%) of isolates produced 18.88 ppb and 0.36 ppb of aflatoxin B1 and aflatoxin B2 respectively. Aspergillus oryzae did not
produce any detectable aflatoxins. Although highest level of aflatoxin B1 (18.88 ppb) was detected in an isolate from a hepato-
pancreatic sample, no histopathological change was observed in that tissue.
Conclusion: Some Aspergillus flavus strains which were isolated from shrimps showed aflatoxin producing ability without
any histopathological changes in tissues of contaminated shrimps.

Keyword: Green Tiger shrimp, aflatoxin, HPLC, Iran.

INTRODUCTION climate provides optimal conditions for the growth of

moulds (4). Because moulds are present in soil and
The aflatoxins are a group of mycotoxins produced
plant debris and are spread by wind currents,
by certain Aspergillus species, in particular A. parasiticus,
insects, and rain, they are frequently found in /on foods
A. flavus, A. nomius, and A. pseudotamarii (1). Asper-
together with their associated mycotoxins (1). On the
gillus flavus is an important Aspergillus species with
other hand, the culture of various species of shrimp
aflatoxin (AF) producing capability. Aflatoxins (AFs)
has an economical importance in many parts of the
B1, B2, G1, G2 (AFB1, AFB2 , AFG1 and AFG2) are a
world as well as in Iran. One of the problems facing the
group of toxic, mutagenic, carcinogenic and terato-
culture of shrimp farming is widespread disease which
genic polypeptide secondary metabolites with health
lead to heavy losses to industry. Additionally, disease
hazards to humans and animals and can adversely
caused by other factors, such as culture environments
affect agricultural productivity (2,3). AFB1 was evalu-
and feed, also influence the success of shrimp culture
ated as a class 1 human carcinogen (1). The incidence
(5). One such factor with a global food safety concern
of AF in food and feed is relatively high in tropical
is AF, a contaminant produced by Aspergillus during the
and subtropical regions where the warm and humid
processing and storage of feed. The green tiger shrimp
* Corresponding author: Farideh Zaini Ph.D (Penaeus semisulcatus) is the endemic species of Persian
Address: Department of Medical Mycology and Parasitology
Gulf and is cultured in Helleh shrimp farm complex,
School of Public Health, Tehran University of Medical Sciences,
located in the humid tropical environment of Bushehr in
Tehran, Iran.
Tel: +98-21-88951583 south of Iran. Therefore, aflatoxin producing potency of
Fax: +98-21-66462267 the A. flavus isolates from above mentioned shrimps and
E-mail: fzaini@Tums.ac.ir water of their culture ponds were examined.

18
 AFLATOXIN PRODUCTION BY ASPERGILLUS FLAVUS
MATERIALS AND METHODS
Fungal isolates. A total of 43 A. flavus strains that
were isolated as mycoflora from cultured green tiger
shrimps and water of their ponds were included in
the present study. These strains were identified based
on morphological characteristics during our previous
study (6) and their aflatoxin producing ability was
examined in the current study. Two additional toxigenic
A. parasiticus (NRRL 2999) with capability to
produce both aflatoxin B (B1 & B2) and G (G1 & G2)
and also non–toxigenic A. oryzae (IMI 126842) were
also included as positive and negative quality controls.
These isolates were stored in distilled water and kept
at room temperature until required for use.
Cultivation and observation of fluorescence.
Stock cultures were maintained on plates containing
potato dextrose agar (PDA) (E.Merck, Germany).
Inoculations were done by conidial transfer to center of
plates containing Aflatoxin Producing Ability (APA)
medium supplemented with 0.3% β-cyclodextrin and
Coconut agar medium (CAM) as described by Hara
et al., Davis et al. and Gupa et al. respectively (7-9).
The APA and CAM plates were then incubated at 28 ˚C
in the dark for 2-5 days and 7 to 10 days respectively. The
reverse side of colonies were periodically observed
under long – wave (365 nm) UV light for blue
fluorescence.
AF Production and Analysis. For assessment
of the AFs, Koehler et al. method (10), with some
modification, was used. All isolates were cultured in
250 ml Erlenmeyer flasks containing 50 ml yeast
extract–sucrose (YES) broth medium (2% yeast extract,
20% sucrose) (E.Merck, Germany). The cultures were
incubated at 30˚C for 8 days in the dark. Afterwards,
the culture was filtered through Whatman no.1 filter
paper. AFs were extracted from culture filtrates by
adding 50 ml of chloroform to each flask and shaking
for 15 min in gyro rotary shaker. The chloroformic
extracts were then concentrated by rotary evaporator
(Eyela N-1000 Japan) near to dryness.
The residue of each sample was resolved in 10 ml
of 40% methanol (HPLC grade) and diluted in 60 ml
PBS (pH 7.8). Regarding the AOAC official methods
999.07 (11), aflatest immunoaffinity columns (IACs)
(Vicam, Water Town, MA) were used for clean-up
of samples. For this purpose, 10 ml PBS was passed
through the IAC at a flow rate of 2-3 ml / min. Whole
diluted extract was then applied to the column, at a
steady flow rate of 3 ml / min. Thereafter, after washing
the column with 15 ml of double-distilled water, air
19
was drawn through the column until dry. AF was
eluted from the column into a 3 ml flask in a two step
procedure. Initially, 0.5 ml methanol was applied on
the column and passed by gravity. After 1 min, the
second portion of 0.75 ml methanol was applied and
collected. Finally, eluate was diluted with water (final
volume 3 ml) and analyzed by High – Pressure Liquid
Chromatography (HPLC).
Analysis of AFs using HPLC. Reverse- phase
HPLC was mainly applied to quantitate AFs along
with fluorescence detector followed by post column
derivatization (PCD) involving bromination using a
water HPLC system (pump 1525; fluorescence detector
2475, analytical column Nova – pack – (18 250 × 406
mm : 4 µm)) (12). We used Kobra cell and added
bromide to the mobile phase to achieve PCD. 100 µl
of diluted AF eluate was then injected into HPLC.
Mobile phase was water–methanol- acetonitrile
mixture with the 600: 300: 200 (V/V/V) ratio in addition
to 350 µl of nitric acid 4mol /l and 120 mg potassium
bromide with a 1 ml / min flow rate. The fluorescence
detector was operated at wavelengths of 365 nm and
435 nm as excitation and emission respectively. AF
levels were calculated by measuring the areas under
the chromatogram and standard curves with C-R3A
chromatopac (Shimadzu).
Histopatholoigical Examination. Tissue sections
were obtained from the other half of the specimens of
hepato-pancreas, gills and external shells of shrimps
which were previously collected and kept in Davidson, s
fixative (13). At this stage, only tissue specimens were
included in the study, from which the aflatoxigenic
A. flavus strains were isolated and their aflatoxi-
genicities established and quantitated by HPLC. Tissue
sections were stained with haematoxylin and eosin
and examined microscopically for presence of
histopathological alterations.
RESULTS
Of 43 isolates of A. flavus, 9 (20.93%) produced
some AFs on YES medium with mean concentration
of 4.38 ± 6.95 ppb when analyzed by HPLC. Whereas
only 2 (4.6 %) isolates were determined to be
aflatoxigenic by presence of blue fluorescence on
the reverse side of colonies after exposure to UV light
on APA and CAM. AF production of these two isolates
was also confirmed by HPLC. The results revealed
that only the A. flavus isolates which produced AFs
at concentrations of 19.25 and 12.18 ppb fluoresced
on APA and CAM media. From these AF producer
strains, 8 (18.7%) isolates produced only AFB1 and
20 ZAINI ET AL . IRAN. J. MICROBIOL. 1 (4) : 18-22

Table 1. Limit of detection of aflatoxins by various cultural methods and HPLC in Aspergillus flavus isolates from Green-
tiger shrimps (Penaeus semisulcatus) .

Isolate Fungal Fluorescence

Concentration of AFs (ppb) on YES medium
number isolates ( UV light )

PDA APA CAM AFs AFB1 AFB2 AFG1 AFG2

16 A. flavus - + + 19.25 18.88 0.36 ND ND

5 A. flavus - + + 12.18 12.18 ND ND ND

24 A. flavus - - - 2.08 2.08 ND ND ND

33 A. flavus - - - 1.88 1.88 ND ND ND

14 A. flavus - - - 1.45 1.45 ND ND ND

9 A. flavus - - - 1.08 1.08 ND ND ND

43 A. flavus - - - 0.68 0.68 ND ND ND

29 A. flavus - - - 0.57 0.57 ND ND ND

21 A. flavus - - - 0.32 0.32 ND ND ND

2999 A. parasiticus
± + + 28.98 27.79 0.55 0.43 0.21
(+control) NRRL

126842 A. oryzae
- - - ND ND ND ND ND
(-control) IMI

AF: Aflatoxin, +: Positive fluorescence, -: Negative fluorescence, PDA: Potato dextrose agar, APA: Aflatoxin–producing
ability, CAM: Coconut agar medium, YES: Yeast extract sucrose, ND: Not detected, ppb: Part per billion, HPLC: High Perfor-
mance Liquid Chromatography, IMI=International Mycological Institute, NRRL: Northen Regional Research Laboratory.

one strain produced both AFB1 and AFB2. In no case
did aflatoxin G1 or G2 contribute to the total toxin
produced. Production patterns of AFs by aflatoxigenic
A. flavus isolates are presented in Table 1.
The results indicated that AFB1 was the most
commonly detected AF from A. flavus isolates
ranging from 0.32 to 19.25 ppb (mean ± StD = 4.36
± 6.58). Although in the present study highest level of
AFB 1 (18.88 ppb) was detected in an isolate
from hepato-pancreatic sample, no hisotopathologi-
cal changes were observed in that tissue. Moreover,
none of the other shrimp specimens from which
toxigenic A. flavus strains (with AFB1concentrations
of 0.32-12.18 ppb) were isolated, showed any histo-
pathological changes.
DISCUSSION
The AFs are extremely potent mutagens and
suspected human carcinogens. They can adversely
affect animal health and agricultural productivity. In
aquatic animals, AF can cause abnormalities such as
poor growth, physiological disorders and histological
changes that decrease production. Problems can be
caused by many factors such as low quality of feed
ingredient and inappropriate methods of feed storage,
which lead to contamination of food and feed with
toxigenic fungi. Additionally, feed contamination
indirectly causes a risk to human health through the
ingestion of AF residue in animal products. Due to the
importance of this group of mycotoxins to human and
animal health, screening of food and feed for presence
of the AFs particularly AFB1 , the most potent member
of the group is routinely considered by many countries
(14). Production of AFs is mainly reported to occur
by some strains belonging to the three major species;
A. flavus, A. parasiticus and A. nomius (3) and less
frequenty by some other Aspergillus species including
A. bombycis, A. pseudotamarii and A. ochraceus as
well as two Emericella species (1). Aflatoxigenic
A. flavus strains have been reported from various
crops, agricultural commodities, and soils (15, 16)
and also from clinical specimens, but little is known
about shrimp isolates (17, 18). In the present study,
AF producing ability of A. flavus isolates from green
tiger shrimps was investigated.
HPLC analysis indicated that 18.7% of all
af latoxigenic A. flavus isolates were able to produce
only AFB1. However, only 1 (2.3%) was capable
 AFLATOXIN PRODUCTION BY ASPERGILLUS FLAVUS
of producing both AFB1 and AFB2 .Vaamonde et al.
reported that A. flavus strains from various crops may
be different in their AF producing ability (15). They
showed that 29% of the total A. flavus isolates were
able to produce aflatoxins. In the same manner, Wei
and Jong (20) and Koehler et al. (10) reported 34.91
and 54% of A. flavus strains as aflatoxin producer
respectively.
On the other hand it has been established that
cultured shrimps generally suffer from toxic effects
of dietary AFs (4, 21–25) but no information on the
contamination of shrimp’s tissue with toxigenic
A. flavus strains was found. Although in the current
study HPLC analysis revealed that 9 (20.93%) of A.
flavus isolates are aflatoxin producers, no abnormali-
ties were detected in gills, shells and hepato-pan-
creatic tissue samples of shrimps examined. These
findings suggest that the possibility of aflatoxicosis
occurring in shrimp via toxigenic A. flavus strains is
limited. However, the definite role of colonization
with such fungi in shrimp must be investigated to
achieve a better conclusion in the future.
Histopathological studies indicated that no
alterations have been observed in the hepato-pan-
creatic tissue of shrimps. These results are in agree-
ment with findings of Bintvihok et al. (4), who dem-
onstrated that higher degrees of hepato-pancreatic
damage occurred by 20 and 10 ppb of the AFB1 in
diet. In contrast to Bintvihok et al. (4), Boomyarat-
palin et al. (24) showed that AFB1 at 0.17 and 37 ppb
caused no histological changes in hepato-pancreatic
tissues, while alterations of hepato-pancreatic tissue
occurred at concentrations of 74, 126 and 220 ppb.
Ostrowski – Missner et al. (22) reported abnormal he-
pato-pancreatic and antennal gland tissues by AFB1, at
50 ppb. Wiseman et al. (25) as well as Ostrowski (22)
showed histologic change in shrimps fed 50 to 300 mg of
AFB1. Histopathological changes were also observed
by Boutista et al. (20) in tissue of shrimps given diets
with 26.5 µg / kg AFB1.
Although the APA and CAM were reported as
special aflatoxin detecting media (7- 9), the present
study showed that these two media are only capable
of detecting AF concentrations above the 12 ppb.
In conclusion, although aflatoxin–producing A.flavus
strains have been reported to contaminate shrimps, no
pathological changes were observed due to those fungi.
ACKNOELEDGEMENTS
This research was supported by Tehran University of
21
Medical Sciences. Kind cooperation of shrimp culture
of Helleh complex at Bushehr province is appreciated.
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