Present status, prevalence and seasonal variations of aflaxtoxin in cattle feed, Bihar, India

Background/ Objectives: Aflatoxin B1, a potent carcinogen is produced by Aspergillus flavus Link ex Fries. Natural contamination of aflatoxin in Bihar is fairly high due to socio-economic backwardness and also outdated agricultural and storages practices. Methods/ Statistical analysis: In the present investigation cattle feeds collected in different seasons (Winter, Summer and Monsoon) from three different localities (Bhagalpur, Banka and Naugachia) were screened for aflatoxin B1. Analysis of Variance has been done with the help of Microsoft Excel, 2013 (USA). CD has been calculated at 5%. Findings: The amount of aflatoxin B1 was significantly high (P<0.05%) in household cattle feeds followed by commercial cattle feeds, where the mean value of AFB1 was 5.02 ± 2.46μg/kg and 4.19 ± 1.05μg/kg, respectively. Freshly harvested cattle feeds (maize, paddy husk, paddy straw, maize straw and green grasses) were comparatively safe. Seasons had marked influence on aflatoxin B1 contamination on various types of cattle feeds. Maximum levels of aflatoxin B1 was detected during wet seasons in household cattle feeds (Mean value = 7.79 ± 2.07μg/kg). Maize grains because of high nutritional value and production, it is one of the major ingredients of cattle feeds. In the locality of Naugachia, flood and frequent rains delay the natural drying of maize kernels during harvesting in monsoons resulting thereby, high mould infestation and aflatoxin synthesis. Novelty/ Applications: In order to check the risk of aflatoxinM1 toxin entrance in food chain and subsequent human/ cattle health and economic losses, frequent evaluation of AFB1 in cattle feeds be monitored.


Introduction
Mycotoxins are secondary metabolites of fungi which contaminate food and feed and can cause toxic effects in cattle and human beings (1)(2)(3)(4)(5) . FAO and WHO have estimated that 25% of the world's crops (maize, cereals, rice, nuts, cattle feeds etc.) are contaminated by moulds and mycotoxins (6)(7)(8) . Aflatoxin synthesized by Aspergillus flavus, A. parasiticus and A. nominus (9) is most potent human carcinogen, classified as Class I carcinogen (IARC 2002). It is also mutagenic, teratogenic and immunosuppressive. A. flavus infestation and aflatoxin synthesis on food and feed commodities are a global problem especially in tropical and subtropical countries like India, where the climatic conditions are quite congenial for its growth. Food and feed safety and security are the major concern in the current scenario of population growth. Aflatoxin contamination in cereal grains and other agricultural commodities being utilized as ingredients of animal feed can occur at different stages throughout the food chain. The presence of aflatoxin producing fungi could be influenced by different factors such as plant genotype, availability of inoculum, insect activities, cultural practices, climatic and weather conditions during planting, growing, harvesting, processing, transport and storage periods (10)(11)(12)(13)(14) . However, in finished animal feed any contaminated ingredient could cause contamination of the entire feed lot. When aflatoxin contaminated feedstuffs are consumed by cattle, it may cause liver toxicity, mutation, alteration in metabolism, decreased disease resistance and reduced milk production (15,16) . Aflatoxin B 1 is biotransformed in cattle liver and is transferred into the milk in the form of aflatoxin M 1 (also called milk toxin). Several reports have confirmed that the levels of aflatoxin M 1 in milk is directly correlated to the dietary intake of aflatoxin B 1 in cattle feed (17,18) . Milk being the complete food, is mostly prescribed to infants, children and old aged people (19,20) . However, those people in turn become more susceptible to the adverse effects of milk toxin (AFM 1 ) as they have comparatively low immunity. Due to the genotoxic nature of aflatoxin M 1 , it has been categorised as class 2 B human carcinogens (IARC, 2002). Considering the significant health risk, stringent regulations have been imposed by most of the countries of the world. European commission (EU) has set a maximum limit of 5µg/kg and 20 µg/kg for aflatoxin B 1 and total aflatoxins (aflatoxin B 1 , B 2 , G 1 and G 2 ), respectively.
Cattle rearing are one of the major sources of income in rural as well as semi-urban areas of Bihar. Various agricultural produce such as maize, wheat, paddy (grains, husk or straw) and green grasses are the main dietary food of cattle. Natural contamination of mycotoxins on agricultural produce of Bihar is fairly high because of the socio-economic backwardness and faulty, unhygienic, outdated agricultural and storage practices, prevalence of high temperature and fairly high humidity. In addition to those conditions, flood in the river of Ganga, Kosi and their tributaries is the regular feature of this terrain. This situation leads to excessive moistening of the grains resulting in substantially intense mould growth and mycotoxin elaboration (21,22) . Previous studies have revealed that in Bihar no edible commodities are absolutely safe from the attack of toxigenic fungal strains and/ or aflatoxin contamination (12,(23)(24)(25) . However, there is no comprehensive research on the occurrence of aflatoxin in different types of cattle feeds of this locality.
The present study was undertaken to observe the incidence of aflatoxin associated with various types of cattle feeds of Bhagalpur and surrounding areas of Bihar, India. In the selected region, variation in temperature and humidity is notable with the change of seasons. Hence, the effect of seasons on the management of feedstock was necessary to be monitored. In the selected localities (Bhagalpur and Banka districts) of Bihar, farmers utilize cattle feeds in three different ways viz. purchase from market (commercial feed), feedstock stacked in farmyard (household storage) and freshly harvested feeds (fresh fodder). Survey and surveillance of the different types of cattle feeds collected during different seasons of the year were monitored for the prevalence of aflatoxin B 1 and the associated health risk.

Sampling
A total of 1130 cattle feed samples were collected randomly (following RND methods) from different localities of Bhagalpur, Banka, and Naugachia during three different seasons i.e. Winters (November -January), Summers (April -June) and Monsoons (July -September) of the year 2017-18. Source of collections were categorized as commercial cattle feed (from shops), household stored feed (cattle feed stored in mud houses or covered with hay stacks or stacked open in farmyard) and fresh feed (seasonally available fresh grasses, freshly harvested crop residues/ grains or crop straw from field, however, collected within 24 -48 hours). Samples of commercial feed included grains of maize, crushed maize, wheat bran, wheat grains and mustard oil cake. Household stock of cattle feed comprised of maize grains, maize straw, paddy husk, paddy straw and wheat bran. Fresh fodder, however, included freshly harvested wheat grain, maize/ paddy straw and husk, crushed maize, maize grain and green grasses. Approximately 250 gms of each samples were collected in sterilized (already dried) polythene bags and were subsequently taken to the laboratory for further analysis within 1-2 days of collections. After monitoring the moisture contents, samples were dried in the laboratory at 60ºC for removal of moisture. Further, those samples were stored in deep freezer to restrict the fungal growth. Each dried sample was ground and mixed to obtain 60 gms sub-sample for aflatoxin analysis.
For the isolation of Aspergillus flavus, the samples were subjected to blotter test and on sterilized PDA medium and also through dilution plating technique, depending on the nature of the substrates. https://www.indjst.org/

Aflatoxin extraction
Samples were dried (at 60ºC) and were separately ground to obtain 50gms of sub samples. Extraction of aflatoxin was made (26) . The residue was finally extracted using chloroform which was further dried on water bath. The extract was then stored in screw tight glass container for thin layer chromatography.

Qualitative and Quantitative analysis
The determination of aflatoxin was done through TLC technique. Standard aflatoxin solution (obtained from Sigma Company, St. Louis, USA) diluted in benzene-acetonitrile (9:1) at the concentration of 0.5µg aflatoxin B 1 was used as reference spot on HPTLC. The extracts as well as standard aflatoxin B 1 were spotted on HPTC and the chromatographs were developed. The HPTLC plates were further developed by using solvent mixtures of toulene, isoamyl alcohol and methanol in ratio of 90:32:2 (27) . Triflouroacetic acid was used for the chemical confirmation of aflatoxin. The quantity of aflatoxin B 1 was determined through CAMAG TLC scanner (Camag, Muttenz, Switzerland), using a D 2 lamp and K-400 filter along with a Sklar integrator.

Statistical analysis
The mean ± standard deviation values were calculated using Microsoft Office Excel (ver. 2013, Microsoft, USA). The data was obtained from the factorial analysis of variance (ANOVA), performed in order to estimate variation among feed types and seasons. The significance level was determined at P<0.05.

Results and Discussion
Eight genera of moulds were isolated from cattle feed samples. Mould spectrum comprised of Aspergillus, Fusarium, Penicillium, Rhizopus, Alternaria, Curvularia, Cladosporium and Mucor spp [ Table 1]. The incidence and diversity of fungi varied with the change in the climatic conditions of various seasons. The incidence of Aspergillus flavus was maximum (87%) during the wet seasons (monsoons) followed by Summers (76%) and cold seasons (19%). The moisture contents of the samples ranged from 16 to 46% depending upon the seasons and nature of the samples.
Out of 1130 cattle feed samples (381 commercial feeds, 390 fresh feeds, 359 household stored feeds) tested, 753 (66.6 %) were contaminated with aflatoxin B 1 . Altogether 390 cattle feed samples (143 commercial feeds, 117 fresh feeds and 130 household stored feeds) were collected during the winters. Out of 383 samples of summers 132, 136 and 115 were collected from market (commercial feeds), fresh feeds and household stored feeds, respectively. During the wet seasons, 357 samples (106 commercial feeds, 137 fresh feeds and 114 household stored feeds) were collected and screened for aflatoxin contamination. The amount of aflatoxin B 1 was high (5.02 ± 2.46 µg/kg) in the household cattle feed samples [ Table 2]. This amount of toxin was significantly high (p<0.05%) compared to the level of toxin found in the samples of commercial and fresh feeds. 33% of the household cattle samples contained aflatoxin B 1 level exceeding the European Union (EU) limit. While considering different types of samples and the different seasons together, it was observed that maximum level of aflatoxin B 1 was detected in monsoons of household cattle feed samples (Mean 7.79 ± 2.07 µg/kg) [ Table 1 Figure 1] followed by commercial feed (5.17 ± 0.75 µg/kg). However, in fresh cattle feed mean value of aflatoxin level was 3.04 ± 0.68 µg/kg during this period. Low level (1.1 ± 0.34 µg/kg) of aflatoxin was found during winters in freshly harvested cattle feed. During the dry weather (Summers) aflatoxin contamination was maximum (4.34 ± 0.37 µg/kg) in commercial feed followed by household feed (4.22 ± 0.6 µg/kg). Considering the level of toxins in different cattle feeds, it was obvious that the freshly harvested feedstuffs were comparatively safe for consumption as because 83-95% of the samples contained aflatoxin below the EU limit (5µg/kg). During the present climatic changes, seasonal assessment of aflatoxin B 1 level in different types of cattle feeds can be an important indicator of aflatoxicoses risk. In earlier studies also an interaction between seasons and cattle feed types in relation to aflatoxin contamination have been observed (5,(28)(29)(30)(31) . Choudhary and Kumari (2014) in an investigation made in Bihar during 2006 -2010 found that the climatic conditions had correlated well with the incidence of aflatoxin in field crops. Per cent of viable conidia of toxigenic A. flavus in wet months (monsoons) was comparatively greater than in cold or dry months. High temperature (25 -45ºC), high humidity (>80%) and improper storage systems prevailing in this area provide congenial condition for A. flavus growth and aflatoxin synthesis. Further, seasonal variation in aflatoxin level might be due to the variation in the fungal community as the co-inhabiting mycoflora may have antagonistic or synergistic effects (32)(33)(34) .

Aflatoxin B 1 in different types of cattle feeds
The samples collected from the commercial centres had 4.19 ± 1.05 µg/kg of mean aflatoxin B 1 content, however, the range of aflatoxin B 1 was from 0.85µg/kg to 7.56µg/kg. 23.3 % of commercial feed samples were above the acceptable limit (5µg/kg) of https://www.indjst.org/ European Union. The risk of aflatoxin in commercial feeds increases as because maize is being utilized as the major ingredients of cattle feed (13,35) . Maize grains have high nutritional value and also it has high quantity of production in Bihar. In this state, maize is one of the most important staple crops, which is consumed as food and feed in various ways (22) . It is cultivated throughout the year (summer, monsoon and winter), covering an area of 0.6 million hectares with the yearly production of 2.0 million tonnes. The harvested crop is stored for future consumption as food and feedstuffs, however, the storage condition is mostly traditional, poor and unhealthy. In finished animal feed if contamination of an ingredient occur at any stage of crop development, it may cause contamination of the entire feed lot. In household stored cattle feed mean level of aflatoxin was 5.02 ± 2.46 µg/kg and the range of contamination varied from 1.2 µg/ kg to 11.2 µg/ kg. In household stores, it was observed that the feeds were mostly stacked in open places in farmyard, in which later were used before those stacked earlier. Thus earlier stacked feeds were left undisturbed throughout the year till being utilized for cattle consumption. Poor and unhealthy management of drying of feeds were observed in household storage of cattle feeds. Unseasonal rain was the recurrent feature of this locality, which resulted high moistening of the feed and thereby, providing optimal condition for mould infestation and aflatoxin contamination. Freshly harvested feeds showed the least contents of aflatoxin while considering the three sources of feeds. The mean total of the aflatoxin B 1 content in fresh feeds was 2.06 ± 0.97 µg/kg (ranging from 1.5 µg/ kg to 4.4 µg/ kg). About 12.1 % of samples contained aflatoxin B 1 above EU acceptable limit. Cattles fed with fresh fodder had comparatively reduced chance of aflatoxin poisoning. It is evident [ Figure 1] that fresh feed was comparatively safe for the consumption, however, even in wet seasons where the maximum (83%) samples were within the limit of acceptance. CD value for types of cattle feed at 5% = 2.08.
https://www.indjst.org/ At p value < 0.05, for season,the mean data for incidence of aflatoxin showed significant difference betweenwinters and monsoons for all three districts under observation. For statistical analysis of Season X Feed, CD is 2.08 (at 5%) which clearly show that during summerand monsoon the mean value of aflatoxin is significantly higher compared towinter. In case of feeds procured from household and commercial centres showedsignificantly higher level of aflatoxin B 1 compared to fresh feed.

Fig 1. Aflatoxin contamination in various cattle feeds
At p<0.05, the mean value of aflatoxin B 1 in household and commercial feed is significantly high compared to fresh feed.

Aflatoxin B 1 in different seasons
Season has marked influence on aflatoxin B 1 contamination on various types of cattle feeds. In all the three localities under observation and also considering the types of cattle feeds, wet period showed significantly high levels (p<0.05) of aflatoxin contamination [ Figure 2 ]. CD value for season at 5% is 2.08 At p<0.05, the mean value of aflatoxin B 1 incidence during monsoon is significantly higher compared to winter. However, the mean value of AFB 1 incidence during summer is at par with the winter considering any type of cattle feed.
During the summers and winters the mean value of aflatoxin contamination was 3.53 µg/kg and 2.55 µg/kg, respectively. However, the differences between the levels of toxin during dry and the winter seasons were non-significant [ Figure 3 ].
CD value for season at 5% is 2.08 https://www.indjst.org/  At p<0.05, the mean value of aflatoxin B 1 incidence during monsoon is significantly higher compared to winter. However, the mean value of AFB 1 incidence during summer is at par with the winter considering any type of cattle feed.

Aflatoxin B 1 contamination at different districts
Feed samples of Naugachia had the highest level of aflatoxin in all the seasons. Mean aflatoxin level was 4.2 ± 1.56 µg/kg in cattle feed of Naugachia which was followed by Bhagalpur (3.7 ± 1.45 µg/kg) and Banka (3.5 ± 1.43) [ Figure 2]. Cattle feed of Naugachia contained high levels of aflatoxin during monsoons (11.14µg/kg). Flood is the recurrent feature at Naugachia and some parts of Bhagalpur where maize (a major ingredients of cattle feed) is harvested in late August/ early September during which crops get submerged with flood water (36) . Maize crops are harvested before full maturity and thereby, kernels contained high moisture contents. Farmers have no option but they further process (peeling, shelling and drying) the crops on nearby National Highway. Drying is delayed because of frequent rains. Such situations provide ideal conditions for A. flavus infestation and aflatoxin synthesis. Once the feed ingredients (maize) get contaminated, it enters into the entire food chain.

Conclusion
Aflatoxin in cattle feed is an inevitable contaminants produced by Aspergillus group of fungi. The present study revealed that aflatoxin B 1 remained present in all types of feedstuffs obtained from the observed localities. Aspergillus flavus was most prevalent fungus especially during monsoons and summer seasons. The level of afl B 1 was comparatively high in the household feeds of farmers. Samples collected from commercial feeds also showed the alarming incidence of aflatoxin B 1 (23.3% above EU limit). However, the fresh feeds were comparatively safe for consumption. With respect to seasons, in wet seasons there was the highest incidence of aflatoxin contamination due to prevailing conducive environmental conditions for A. flavus growth and https://www.indjst.org/ aflatoxin synthesis. Maize (a major ingredient of cattle feed) is harvested during monsoons/ onset of floods, which resulted in excessive moistening of the kernels and thereby, high aflatoxins contamination.