Mycopathologia (2012) 174:341–349 DOI 10.1007/s11046-012-9545-8
The Effect of Substrate, Season, and Agroecological Zone on Mycoflora and Aflatoxin Contamination of Poultry Feed from Khyber Pakhtunkhwa, Pakistan Sahib Alam • Hamid Ullah Shah • Habibullah Khan • Naresh Magan
Received: 22 January 2011 / Accepted: 8 April 2012 / Published online: 29 April 2012 Ó Springer Science+Business Media B.V. 2012
Abstract To study the effects of and interactions among feed types, seasons, and agroecological zones on the total fungal viable count and aflatoxins B1 (AFB1), B2 (AFB2), G1 (AFG1), and G2 (AFG2) production in poultry feed, an experiment was conducted using three-factorial design. A total of 216 samples of poultry feed ingredients, viz. maize, wheat, rice, cotton seed meal (CSM), and finished products, that is, starter and finisher broilers’ rations, were collected from Peshawar, Swat, and D. I. Khan districts of Khyber Pakhtunkhwa, Pakistan, during the winter, spring, summer, and autumn seasons of the year 2007/2008. Analysis of variance showed that there was a complex interaction among all these factors and that this influenced the total fungal viable count and relative concentrations of the aflatoxins produced. Minimum total culturable fungi (6.43 9 103 CFUs/g) were counted in CSM from D. I. Khan region in winter season while maximum (26.68 9 103 CFUs/g) in starter ration from Peshawar region in summer. Maximum concentrations of AFB1 (191.65 ng/g), AFB2 (86.85 ng/g), and AFG2 (89.90 ng/g) were S. Alam (&) H. U. Shah H. Khan Department of Agricultural Chemistry, Khyber Pakhtunkhwa Agricultural University Peshawar, Peshawar, Pakistan e-mail:
[email protected] N. Magan Institute of BioScience and Technology, Cranfield University, Cranfield, UK
examined during the summer season whereas the concentration of AFG1 was maximum (167.82 ng/g) in autumn in finisher ration from Peshawar region. Minimum aflatoxins were produced in the winter season across all the three agroecological zones. Keywords Poultry feed Aflatoxin Mycoflora Aspergillus flavus Toxigenic fungi
Introduction Poultry feed is an essential part of the poultry industry. Its composition includes mixtures of home grown cereals, cotton seed meal, pulses, and other additives. In Pakistan, poultry feed is produced by commercial feed mills as well as by home mixture. The feed mills owners purchase a huge bulk of these ingredients in the production season and store them for feed production throughout the year. The ingredients are usually directly purchased from fields having high moisture contents. Warm climate, long post-harvest period, and moist environmental conditions are known to favor molds growth that produces toxins [20, 34]. Aflatoxins (AFs) are a family of highly toxic secondary metabolites produced by several species of Aspergillus which frequently contaminate agricultural products all over the world [8]. Optimum condition for aflatoxins production by Aspergillus species is 33 °C and 0.99aw, while that for growth is 35 °C and 0.95aw [2, 34]. There are as many as 20 fractions of AFs but
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the important ones are aflatoxin B1, B2, G1, and G2 [21]. They are designated so because they fluorescence either blue or green in ultraviolet light [41]. The toxicity of AFs in broiler chickens has been investigated for their carcinogenic, mutagenic, teratogenic, immunosuppressive, and growth inhibitory effects [28, 31, 39, 40]. When AFs contaminated feeds are offered to birds, it causes aflatoxicosis in poultry production characterized by listlessness, anorexia with lowered growth rate, poor feed utilization, decreased weight gain, decreased egg weight and production, increased susceptibility to environmental and microbial stresses, and increased mortality [25]. Birds grown with toxins containing feed is not only loss for farmers but it also affects the consumers’ health through its residues. Begum et al. [10] reported a significantly higher level of aflatoxins in liver as compared to kidney and meat. A positive correlation of AFs ingestion and liver cancer in human beings has been reported by International Agency for Research on Cancer (IARC) in Asia and Africa [19]. Therefore, toxins in animal feed are also important from public health point of view. The purpose of this study was to examine the fungal flora and natural occurrence of AFs in poultry feeds over different seasons of the year.
Mycopathologia (2012) 174:341–349
December 2008. To facilitate handling and for further storage, each sample was reduced to 1 kg and sent to Cranfield University, UK, for mycofloral and aflatoxins analysis. To determine the effect of each factor on total fungal viable count and aflatoxins production and the interaction among the factors, a three-factorial completely randomized design was used. All analyses were carried out in triplicate. Mycoflora Determination The total fungal viable counts of each sample were determined by using malt extract agar (MEA) media [13, 32, 35]. One gram each of well-ground sample was taken in 9 ml of sterilized distilled water containing 0.01 % Tween 80 in glass Universal bottles. The contents were agitated for 2 min with a Worley mix. A serial dilution series (10-1–10-4) was made using 1 in 9 ml. 100 ll of the suspension from the appropriate dilutions was transferred to Petri plates containing MEA and was spread plated using sterilized bent Pasteur pipettes. All the Petri plates were incubated at 25 °C for 7–10 days. The numbers of colonies of different fungi were counted and the total fungal viable count per gram of sample calculated. Extraction and Analysis of Aflatoxin
Materials and Methods Experimental Design The factors and levels of each factor used in this study included seasons [winter (December to February), spring (March to May), summer (June to August), and autumn (September to November)], agroecological zones (Peshawar, Swat, and D. I. Khan districts of Khyber Pakhtunkhwa province of Pakistan), and poultry feeds (maize, wheat, rice, cotton seed meal, starter and finisher broiler’s rations). To determine the effect of each factor on total fungal viable count and aflatoxins production and the interaction among the factors, a three-factorial completely randomized design was used. Poultry feeds samples 2–3 kg each of ingredients, that is, maize, wheat, rice, cotton seed meal, and finished products viz. starter and fisher rations, were randomly collected from local markets of selected locations in triplicate. Sampling was carried out during the four seasons of the year. A total of 216 samples were collected from December 2007 to
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The extraction of AFs was carried out with a modified procedure of Stroka et al. [38]. Each of the wellground sample (50 g) was extracted with 250 ml of acetonitrile–water (60:40, v/v) using high-speed blending for 2 min. The extract was filtered through Whatman No. 4 filter paper, and an aliquot of 5 ml was diluted with 120 ml of 0.01 M PBS buffer (pH 7.4). The immunoaffinity column (IAC AflaTestÒ, Vicam, USA) for aflatoxins analysis was conditioned with 20 ml of PBS buffer by gentle syringe pressure at a flow rate of 5 ml/min. Then, the mixture of the filtrate diluted extract (120 ml) was passed through the IAC column, followed by washing with 10-ml deionized water. Aflatoxins were then slowly eluted from IAC with 2-ml methanol into a glass vial. The eluate was evaporated to dryness with a gentle stream of N2 at 52 °C, re-dissolved with 100 ll of trifluoroacetic acid (TFA) for 3 min, re-evaporated to dryness with N2 at 52 °C, and reconstituted in 500 ll of the mobile phase for HPLC analysis. TFA was added to AFB1 and B2 working standards in the same conditions as the
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343
extract samples to derivatize them. An HPLC system (Waters 600E System Controller) with a fluorescence detector (Waters 470) was used for aflatoxins quantification. A reversed phase LC-18 column (Novapak) was used with a mobile phase consisting of a mixture of acetonitrile–water–acetic acid (18:82:1, v/v/v) at a flow rate of 1 ml/min and fluorescence detection: Ex 365nm, Em 440nm [3, 9]. Statistical Analyses The data were analyzed by using the analysis of variance (ANOVA) using three factors completely randomized (CR) design [37]. All the analyses were carried out by statistical software package M-State-C Ver.2.
and seasons. The locations, alone, had no significant effect (P [ 0.01). However, all two- and three-factor interactions were highly significant (P B 0.05). It was examined (Fig. 1) that in Peshawar region minimum numbers (6.52 9 103 CFUs/g dry weight) of fungal colonies were found in maize sample in autumn while maximum numbers of fungal colonies (26.68 9 103 CFUs/g) were observed in starter ration in the summer season. Total number of culturable fungi in Swat region varied from 6.80 9 103 CFUs/g in CSM in winter to 16.90 9 103 CFUs/g dry weight in rice samples collected in the autumn. In D. I. Khan region, minimum numbers of culturable fungi (6.43 9 103 CFUs/g) were observed in CSM samples in the winter while maximum numbers (25.58 9 103 CFUs/ g) were counted in maize samples in the autumn. Interactive Effects of Substrate, Agroecological Zone, and Season on Aflatoxins Production
Results Interactive Effects of Substrate, Agroecological Zone, and Season on Total Fungal Population The ANOVA for total fungal viable counts (Table 1) revealed that the most significant single factors (P B 0.01) affecting the mold incidence in commercial poultry feeds and their ingredients were feed types
Table 1 ANOVA for total fungal count (TFC) in commercial poultry feeds and their ingredients Source
dfa
Mean square
Fb
P value
Feeds
5
0.149
19.844
Locations
2
0.022
2.936
Feeds 9 locations Seasons Feeds 9 seasons
0.000 0.0563(NS)c
10
0.037
4.999
0.000
3
0.895
119.446
0.000
15
0.077
10.265
0.000
Locations 9 seasons
6
0.058
7.781
0.000
Feeds 9 locations 9 seasons
30
0.024
3.177
0.000
Error
144
0.007
Total
215
a
df degree of freedom
b
F variance ratio
c
NS not significant (P [ 0.05)
The ANOVA for AFB1 production (Table 2) showed that all the single-factor and two- and three-factor interaction were highly significant at P B 0.01. Among the single factors tested, location had the greatest effect (F = 2222.16). The amount of B1 produced was determined by the complex interaction of feed type, agroecological zones, and seasons, and their effects on B1 production are shown in Fig. 2. Minimum AFB1 contents were examined in rice (13.71 ng/g), wheat (13.37 ng/g), and rice (6.96 ng/g) samples collected in the winter season from Peshawar, Swat, and D. I. Khan district, respectively, while AFB1 contents were maximum in finisher ration from Peshawar and Swat regions (191.65 and 147.34 ng/g, respectively) and CSM samples (58.31 ng/g) from D. I. Khan region in the summer season. The ANOVA for AFB2 production (Table 3) indicated that all the single-factor and two- and three-factor interactions were highly significant at P B 0.01. Location had the greatest effect (F = 4246.17) on B2 production among all the single factors tested. The highest concentration of B2 (86.85 ng/g) was observed in finisher ration samples collected from Peshawar region in the summer region whereas minimum concentration of B2 (3.63 ng/g) was examined in wheat samples from district Swat in the winter season (Fig. 3). Similar trend was observed for aflatoxin G1 production (Fig. 4). The ANOVA (Table 4) showed
123
344
Mycopathologia (2012) 174:341–349
*
SDM for feeds =±0.155; locations =±0.160 and seasons =±0.121
4.4 4.3 4.2 4.1 4.0 3.9 3.8
PE SW DK PE SW DK PE SW DK PE SW DK Maize PE SW DK PE Wheat SW DK Rice CSM Starter Finisher
Autumn Summer Spring Winter
Season
Log10 CFUs/g dry weight
4.5
Fig. 1 Seasonal total fungal viable counts (Log10 CFUs/g dry weight) in sample of commercial poultry feeds and their ingredients collected from PE Peshawar, SW Swat, and DK D. I. Khan districts of Khyber Pakhtunkhwa
Table 2 ANOVA for AFB1 production (ng/g) in commercial poultry feeds and their ingredients
dfa
Source
Feeds
5
Locations
F variance ratio
Fig. 2 Seasonal occurrence of aflatoxin B1 (ng/g) in commercial poultry feeds and their ingredients collected from PE Peshawar, SW Swat, and DK D. I. Khan districts of Khyber Pakhtunkhwa
37791.576
14786.6086
0.0000
2
56808.110
22227.1566
0.0000
17966.615 2493.308
7029.7492 975.5499
0.0000 0.0000
Feeds 9 seasons
15
441.590
172.7796
0.0000
6
447.065
174.9219
0.0000
79.3269
0.0000
30
202.743
Error
144
2.556
Total
215
200
*
SDM for feeds =±29.521; locations =±10.374 and seasons =±49.275
180 160 140
AFB1(ng/g)
df degree of freedom
P value
10 3
Feeds 9 locations 9 seasons
b
Fb
Feeds 9 locations Seasons Locations 9 seasons
a
Mean square
120 100 80 60
20 0 PE SW DK PE SW DK PE SW DK PE SW DK Maize PE SW DK PE Wheat SW DK Rice CSM Starter Finisher
123
Autumn Summer Spring Winter
Season
40
Mycopathologia (2012) 174:341–349 Table 3 ANOVA for AFB2 production (ng/g) in commercial poultry feeds and their ingredients
345 dfa
Source
4022.872
2278.7619
2
7496.090
4246.1720
0.0000
10
3387.370
1918.7812
0.0000
Locations 9 seasons Feeds 9 locations 9 seasons
Fig. 3 Seasonal occurrence of aflatoxin B2 (ng/g) in commercial poultry feeds and their ingredients collected from PE Peshawar, SW Swat, and DK D. I. Khan districts of Khyber Pakhtunkhwa
Error Total
90
3
301.965
171.0484
0.0000
15
136.357
77.2394
0.0000
6
170.283
96.4571
0.0000
30
86.572
49.0390
0.0000
144 215
1.765
*
SDM for feeds =±11.761; locations =±4.469 and seasons =±18.938
80 70
AFB2 (ng/g)
F variance ratio
0.0000
5
Feeds 9 seasons
b
P value
Locations Seasons
df degree of freedom
Fb
Feeds Feeds 9 locations
a
Mean square
60 50 40 30
0 PE SW DK PE SW DK PE SW DK PE SW DK Maize PE SW DK PE Wheat SW DK Rice CSM Starter Finisher
200
Autumn Summer Spring Winter
*
SDM for feeds =±24.199; locations =±7.588 and seasons =±46.147
180 160
AFG1 (ng/g)
Fig. 4 Seasonal occurrence of aflatoxin G1 (ng/g) in commercial poultry feeds and their ingredients collected from PE Peshawar, SW Swat, and DK D. I. Khan districts of Khyber Pakhtunkhwa
Autumn Summer Spring Winter
Season
10
Season
20
140 120 100 80 60 40 20 0 PE SW DK PE SW DK PE SW DK PE SW DK Maize PE SW DK PE Wheat SW DK Rice CSM Starter Finisher
123
346 Table 4 ANOVA for AFG1 production (ng/g) in commercial poultry feeds and their ingredients
Mycopathologia (2012) 174:341–349
Source
37140.154
19082.5582
0.0000
2
43014.012
22100.5381
0.0000
10
14555.100
7478.3897
0.0000
3
1151.443
591.6098
0.0000
15
540.616
277.7679
0.0000
Locations 9 seasons Feeds 9 locations 9 seasons
F variance ratio
Error Total
that all the factors alone and interactively significantly (P B 0.01) affected G1 production in commercial poultry feeds and their ingredients. It is evident from Fig. 4 that average G1 contents in maize, wheat, rice, CSM, and starter and finisher rations samples collected from Peshawar region were examined to be 16.00, 10.85, 19.03, 17.62, 135.95, and 151.62 ng/g, respectively, over the four seasons of the year. Maximum G1 content (167.82 ng/g) was examined in finisher ration samples collected during autumn while minimum concentration (8.55 ng/g) in wheat samples collected during winter season. In Swat region, average G1 contents were found to be 8.68, 6.18, 9.53, 6.45, 31.46, and 90.47 ng/g in maize, wheat, rice, CSM, and starter and finisher rations samples over all seasons. The highest amount of G1 (143.33 ng/g) was quantified in finisher ration during summer whereas minimum in CSM samples during winter which was 4.27 ng/g. In D. I. Khan district, both the commercial poultry feeds and their ingredients viz. maize, wheat, cotton seed meal, and starter and finisher rations were found contaminated with G1 in all the four seasons of the year. The average G1 contents were found to be 12.03,
Table 5 ANOVA for AFG2 production (ng/g) in commercial poultry feeds and their ingredients
Source
144 215
1.946
6.85, 6.29, 18.07, 12.70, and 8.83 ng/g in maize, wheat, rice, cotton seed meal, and starter and finisher rations, respectively. Minimum G1 content (4.41 ng/g) was examined in wheat in autumn and the same amount in rice samples in winter whereas maximum concentration (24.54 ng/g) was recorded in CSM samples during autumn season. The ANOVA (Table 5) indicated that all the singlefactor and the two- and three-way interaction significantly (P B 0.01) affected AFG2 production in commercial poultry feeds and their ingredients. Among all the single factors tested, feed type had the highest effect (F = 6524.58) on G2 production. The combined effect of feed type, location, and season on the production of G2 is shown in Fig. 5. In Peshawar and Swat regions, maximum G2 contents were examined in finisher ration, that is, 89.90 ng/g and 49.72 ng/g, respectively. In D. I. Khan region, G2 concentration was maximum in maize which was noted to be 4.66 ng/g. The toxin production was greatly favored by hot summer season across all the agroecological locations. On the contrary, minimum G2 contents in Peshawar and Swat regions were observed in maize samples
dfa
Mean square
Fb
P value
5425.530
6524.5825
0.0000
12141.245
14600.7025
0.0000
10
2864.533
3444.8029
0.0000
3
611.871
735.8179
0.0000
Feeds 9 locations 9 seasons
123
0.0000 0.0000
2
Feeds 9 seasons
F variance ratio
264.1801 149.7986
5
Locations 9 seasons
df degree of freedom
514.171 291.551
Locations Feeds 9 locations
b
6 30
Feeds
Seasons
a
P value
5
Feeds 9 seasons
b
Fb
Locations Seasons
df degree of freedom
Mean square
Feeds Feeds 9 locations
a
dfa
Error Total
15
272.167
327.2995
0.0000
6
241.973
290.9899
0.0000
30
123.240
148.2049
0.0000
144 215
0.832
Mycopathologia (2012) 174:341–349
(5.14 and 1.95 ng/g, respectively) in autumn, while in D. I. Khan region, CSM contained minimum concentration (0.02 ng/g) of AFG2.
Discussion In the present study, a total of 216 samples collected in different seasons from Peshawar, Swat, and D. I. Khan districts of Khyber Pakhtunkhwa Province of Pakistan were investigated for mycoflora and natural occurrence of aflatoxins for the first time. The presence of different fungal genera, representing both field and storage fungi and the incidence of Aspergillus, Penicillium, and Fusarium [11] in higher percentage, was particularly important, because these are known to be toxin producers [3, 4, 6, 33]. This study is in close agreement with that of [14]. Khan et al. [23] found that 59 % corn kernels were contaminated with food spoilage fungi in South West Pakistan. Our findings are also in line with [30, 42], who had earlier established aflatoxins producing genera predominate over other genera in tropical environments. Total fungal viable count significantly varied among different seasons in commercial poultry feed and its ingredients. It was noted that tropical and humid regions were more prone to fungal contamination of poultry feed raw materials and finished products. Nutrients composition also played a significant role in fungal population. These findings were in close agreement with that of [1, 7, 12, 27, 29, 35] who observed that fungi grow well on high protein and carbohydrates substrates in tropical and humid regions. It was noted in our study that aflatoxin contents varied significantly with season, agroecological zones, and substrates. On the average, poultry feeds and its ingredients from Swat and D. I. Khan regions contained aflatoxins contents with in the safe limit as recommended by FDA/WHO, while the samples from Peshawar district contained significantly higher concentration of aflatoxins. This could be explained by the fact that factors like temperature, humidity, physical condition of the grain, transportation, and storage structure all promote growth of fungi and subsequent production of mycotoxins in feed [5, 35]. The optimum temperature for aflatoxins production ranges from 25 to 35 °C [24]. Temperature values recorded in our regions about 10–45 °C, indicating favorable condition for Aspergillus growth and aflatoxins
347
production. In a survey on maize sold in rural markets in South West Pakistan, Khan et al. [23] found aflatoxin contamination ranged from 20 to [100 lg/kg. Maximum aflatoxin contamination of maize was during the rainy seasons. Feng and Tang [15] analyzed various feed for aflatoxin and recorded maximum amount (5,000 ng/g) in maize, maize bran, ground nut cakes, and feed mixture. Giray et al. [16] determined the aflatoxin B1, B2, G1, and G2 levels in wheat samples grown and consumed in some regions of Turkey. They found that total AFs were ranging from 10.4 to 643.5 ng/g. Khan [22] analyzed 331 samples of cottonseed collected from poultry farms, feed mills, and godowns/retails shops of Karachi, Pakistan, and found that 70 % of samples were positive for AFB1 with an average value of 155.7 lg/kg and the range being 3–1,629 lg/kg. In the same study, she analyzed 371 and 341 samples of broilers starter and finisher rations and found that AFB1 content varied from 2 to 384 and 1 to 353 lg/kg in them, respectively. The recommended FDA/WHO safe limit for AFs in feed is 20 ng/g [26, 36]. According to Grybauskas et al. [18], maize containing 20 ng/g AFB1 or more should not be sold commercially and in general, should not be fed to young poultry, swine, and livestock, or to lactating animals, and must not be milled for human consumption. A valid experimental design and sound statistical analysis is necessary to assess the significance of treatment combinations. Factorial designs are the most commonly used designs for the analysis of interaction between a range of different factors applied at different dose levels and are economical and save time. They have previously been used to determine the interaction among temperature, aw, substrate type, fungal colonization, insect infestation, etc., in cereal grains [17] but have not been used before to study the effect of seasons, geographical locations, and substrate type on production of aflatoxin B1, B2, G1, and G2, simultaneously. In the present investigation, the use of three-factorial design demonstrated the complex interaction between factors controlling aflatoxins production in poultry feeds and helped to explain their variable concentration in natural substrates. The findings of this study showed that although the mean aflatoxins contents in the majority of poultry feed ingredients were within the safe limit, a significant percentage of samples were above the safe limit particularly that of commercial feeds from Peshawar.
123
348
90
*
SDM for feeds =±12.344; locations =±4.868 and seasons =±20.685
80 70
AFG2 (ng/g)
Fig. 5 Seasonal occurrence of aflatoxin G2 (ng/g) in commercial poultry feeds and their ingredients collected from PE Peshawar, SW Swat, and DK D. I. Khan districts of Khyber Pakhtunkhwa
Mycopathologia (2012) 174:341–349
60 50 40 30
10 0 PE SW DK PE SW DK PE SW DK PE SW DK PE SW DK Maize Wheat PE SW DK Rice CSM Starter Finisher
So it is recommended that there is a dire need for constant monitoring of fungal and aflatoxins contamination with special reference in major feeds and crops such as corn, rice, wheat, etc., which are harvested, processed, and stored in the same agro-climatic conditions. Acknowledgments We are grateful to Higher Education Commission, Islamabad, Pakistan, for financial support and University of Cranfield, UK, for facilitating the accomplishment of the study.
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