Prof Dr. G. Devegowda, Dr. V. Sridharand Dr. PL Sherasia
Part – I Prevalence
1.0 Executive summary
Aflatoxins are the most widespread and the most studied group of all the mycotoxins, and they are prevalent in warm and humid climatic conditions; as exists in India and in many Asian countries. Aflatoxins are primarily produced by fungi of the genus Aspergillus (Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nomius), and are found in dairy feeds and human food products. Major forms of Aflatoxins found in feeds include Aflatoxins B1, B2, G1 and G2; with Aflatoxin B1 being the most common and toxic. Aflatoxin M1 is found in milk and milk products.
Strict control of Aflatoxin B1 level in feeds for lactating dairy cattle and buffaloes is required in order to minimize the level of Aflatoxin M1 in milk and milk products. The United States Food and Drug Administration (USFDA, 2018) and Food Safety and Standards Authority of India (FSSAI, FSS Regulation 2011) set a maximum permissible level for Aflatoxin M1 in milk at 0.5 µg/ kg (ppb; parts per billion) of milk, which means that one ton of milk should not contain more than 500 micrograms of Aflatoxin M1.
Aflatoxin B1 is readily transmitted from feed to milk; approximately 1.0 to 6.0% of Aflatoxin B1 present in feed is transferred to milk as Aflatoxin M1, depending on factors such as the genetics of animals, seasonal variation, the milking process and the environmental conditions. The occurrence of Aflatoxins in commercially available milk, and milk products is of concern, as milk is a key source of nutrients for humans
2.0 Introduction
There are more than 10,000 known species offungi. Fortunately, most of them are beneficial to man in the production of bread, cheese, antibiotics etc. There are about 50 fungi species harmful to livestock, poultry, and man known to produce toxins, which are collectively called mycotoxins. Mycotoxins are metabolites produced by fungi during metabolism of nutrients present in feeds and feed ingredients.
Fungi produce mycotoxins in the field (“field toxins”: fusarium toxins) or in storage (“storage toxins”: Aflatoxins, ochratoxins), or both. Mycotoxins are mainly produced by the fungi genera of Aspergillus, Fusarium, Penicillia, and Claviceps. Their formation may occur when the fungi grow on crops in the field, at harvest, in storage or during the processing of feed when conditions are favorable.
No region of the world escapes these silent killers, and their negative impact on animal productivity and human health is enormous. According to the United Nation’s Food and Agriculture Organization (FAO), approximately 25% of World’s grain supply is contaminated with mycotoxins. Recently, a 38 member scientific task-force from the Council for Agricultural Science and Technology (CAST, 2003) in the USA released a
200 page report entitled “Mycotoxins: Risks in Plant, Animal, and Human systems”. According to the report, in the United States alone the economic costs resulting from mycotoxins exceed $ 1.5 billion annually.
3.0 Mycotoxins: An overview
The threat of mycotoxins has been described as early as the Second World War when the soldiers from the Russian army suffered severe dermal necrosis, hemorrhages and destruction of bone marrow after eating mouldy grains (Fusarium contaminated, Figure 1). However, it was not until 1960, when the entire turkey population of Britain was decimated in a fatal liver disease called ‘Turkey X Disease’, that the scientific community recognized the negative effects associated with mycotoxins. British agriculture officials later traced the source of the outbreak to Aflatoxin in a shipment of peanut (groundnut) meal that originated from Brazil.
Figure 1: Maize cobs contaminated with Fusarium fungus
The occurrence of mycotoxins in nature is considered a global problem. However, in certain geographical areas of the world, some mycotoxins are produced more readily than others. In colder, more temperate regions such as Canada, the Northern US and most of the European countries, Aflatoxins are not considered to be a major problem except in imported feedstuffs grown in warmer southern climates. Economically, in these regions the most important mycotoxins are deoxynivalenol (DON, Vomitoxin), Zearalenone (ZEA), T-2 toxin produced by Fusarium fungi (Figure 1).
In Europe, the differences in climatic conditions among the northern, middle and southern parts favor the development of different fungal species. In the maize growing areas of southern and middle Europe (Sweden, Austria and Hungary) mainly fusariotoxins (DON, ZEA, T-2 toxin) cause illness and poor performance of dairy animals.
3. 1. 1 Deoxynivalenol (DON, Vomitoxin) Deoxynivalenol is produced by several Fusarium species of mould primarily Fusarium graminearum and may co-occur with other mycotoxins in contaminated commodities. DON in cattle and buffaloes has been associated with reduced feed intake and lower milk production when fed with feed containing more than 5 parts per million (ppm).
3.1.2 Zearalenone Zearalenone is produced primarily by Fusarium graminearum and Fusarium roseum. Zearalenone is responsible for reproductive disorders because of its estrogenic effect. In dairy animals, the clinical manifestations such as udder enlargement, decreased milk yield, vaginal discharge, continuous estrus, infertility and abortions are observed when the level of zearalenone in the feed is more than 0.5 ppm.
3. 1.3 Fumonisins
Fumonisins are primarily produced by Fusarium moniliforme and Fusarium proliferatum, the most toxic being fumonisin B1. Fumonisin mycotoxins are found in a wide range of commodities from millets to grains to banana fruits. They are a major concern to food and feed producers since they affect human and animal health. The toxin causes liver damage and decreased milk production in dairy cattle at levels greater than 50 ppm in the ration. Usually, level of fumonisins exist between 5-20 ppm in feed.
4.0 Global scenario of Aflatoxins
Aflatoxins; the most widespread of all the mycotoxins, are common in warm and humid climatic conditions like those existing in India, Latin American, Asian and African countries, southern regions of US, and certain parts of Australia. Extensive surveys conducted in India, Pakistan, Egypt and South Africa suggested that Aflatoxins are often encountered in substantial levels in feeds and feed ingredients (Figure 2).
In Latin American countries including Brazil, Peru, Mexico, Columbia, Venezuela and Argentina, reports exist on the presence of Aflatoxins. Due to the increase in global trading of feedstuffs, mycotoxins are no longer solely found in certain geographical regions but are now more widely distributed than before (Devegowda et al., 1998).
Aflatoxins B1, B2, G1, and G2 refer to toxins which fluoresce blue (B1, B2) or green (G1, G2) under ultraviolet light. Among all Aflatoxins, B1 is the more prevalent and toxic.
Figure 2: Maize naturally contaminated with Aspergillus fungus
5.0 Limits of Aflatoxins in feed and milk in other countries
Strictly speaking, there is no safe level. With reference to mycotoxins, the risk directly depends on the level of the major mycotoxins and also on the presence and levels of other mycotoxins in feeds. A mycotoxin level considered safe in one farm may not be safe in another farm because of differences in management and disease prevalence. Additionally, factors such as interaction of mycotoxins with pathogens, genetic variability, environmental conditions, and nutritional status etc. influence the severity of the manifestation of mycotoxicosis.
In order to reduce the toxic and economic impact of mycotoxins, several countries regulate the levels of some mycotoxins in foods and feeds. Worldwide, food and feed legislation safeguards the health of consumers and the economic interests of animal producers and traders. Virtually, all countries with fully developed market economies have regulations with the exception of some African countries.
Table 1: Limits for Aflatoxin B1 in dairy feed
| Country | Aflatoxin B1 (µg/kg or ppb) |
| United States (FDA) | 20 |
| European Union (for milking animals) | 5 |
| European Union (for calves) | 10 |
| India (BIS) | 20 |
Table 2: Limits for other mycotoxins in dairy feed
| Mycotoxins (EU Guidelines) | Limits (mg/kg or ppm) |
| Deoxynivalenol: Adult animals : Calves | 5 2 |
| Zearalenone: Adults and calves | 0.5 |
| Fumonisins B1 and B2: Adult animals : Calves | 50 20 |
Table 3: Limits for Aflatoxin M1 in milk
| Country | (µg/kg or ppb) |
| United States (USFDA) | 0.5 |
| European Union | 0.05 |
| European Union, for baby foods/ infants | 0.025 |
| Australia (FSANZ) | 0.05 |
| Australia, for infants | 0.02 |
| India (FSSAI) | 0.5 |
6.0 Overview of Aflatoxins in feed and feed raw materials in India
Aflatoxins are primarily produced by fungi of the genus Aspergillus (Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nomius; Figure 3) and are found in dairy feed and human food products. Major forms of Aflatoxins found in feeds include Aflatoxins B1, B2, G1 and G2; with Aflatoxin B1 being the most common and toxic.
Figure 3: Growth of Aspergillus flavus on rice
Extensive surveys conducted in India suggest that Aflatoxins are often encountered in substantial levels in feeds and feed ingredients. Aflatoxins are rarely found in forages. To study the incidence of Aflatoxin B1 in different raw materials, a survey was conducted by the Division of Animal Sciences, University
of Agricultural Sciences, Bengaluru. The results of the survey showed that out of 246 samples analyzed by Thin Layer Chromatography (TLC), 206 samples were found to be positive for the presence of either Aflatoxin.
The cereal and cereal byproducts analyzed were maize, de- oiled rice bran, rice polish and wheat bran. The samples were found 88% positive for Aflatoxins. The oilseed meals analyzed were soybean meal, full-fat soy, sunflower meal, groundnut cake/ extract/ expeller, rapeseed meal and til cake. The oilseed meals contain higher incidence of Aflatoxin (96%) contamination as compared to cereals and cereal byproducts.
Aflatoxin B1 content was analysed in some samples of cattle feed (n=254) suspected to be contaminated with Aflatoxins at NDDB, Anand (NDDB, 2018). The results found that the average level of Aflatoxin B1 was 68 ppb in these samples.
7.0 Metabolism of Aflatoxin in ruminants and conversion of Aflatoxin B1 to Aflatoxin M1
In milk, Aflatoxin appears as Aflatoxin M1, one of its metabolites. Aflatoxin B1 is metabolized by enzymes found primarily in the liver (Cytochrome P450) to Aflatoxin M1. Strict control of Aflatoxin B1 level in feeds for dairy animals is required in order to minimize the level of Aflatoxin M1 in milk and milk products.
In the US (FDA) and in India (FSSAI), it is required by the law that Aflatoxin M1 in milk should be less than 0.5 ppb; which means that one ton of milk should not contain more than 500 micrograms of Aflatoxin M1. In European Union, the regulations are much more stringent, and maximum levels are set
at 0.05 ppb (EC, 2002). Aflatoxin is readily transmitted from feed to milk; approximately 1.0 to 6.0% of Aflatoxin B1 present in feed is transferred to milk as Aflatoxin M1 (Figure 4). After Aflatoxin M1 is formed, it is excreted in milk and urine of the dairy animals.
Aflatoxin B1 Aflatoxin M1
Figure 4: Conversion of Aflatoxin B1 to M1
Diaz et al. (2004) have indicated that Aflatoxins appear in milk within 12 hours following oral administration of Aflatoxin B1. The peak concentration in milk occurs after 24 hrs. Clearance is also very rapid, since Aflatoxin disappears from the milk four days after cessation of oral administration (Diaz et al., 2004). These results confirm the rapid absorption and metabolism of Aflatoxins in ruminants.
The USFDA and BIS (India) stipulate a maximum level of 20 ppb of Aflatoxin in dairy feed, which means that one ton of feed should not contain more than 20 milligrams of Aflatoxin B1. The European Union regulations are much more stringent and the maximum level set at 5.0 ppb. Like Aflatoxin B1, Aflatoxin M1 is toxic, although toxicity of Aflatoxin M1 is somewhat lower than that of Aflatoxin B1.
8.0 Impact of feeding Aflatoxin-contaminated feed in dairy animals
Milk producers may not be able to see visual symptoms of aflatoxicosis in the animals at low level Aflatoxin in the feed. However, high concentrations of Aflatoxins and/ or prolonged duration may cause visual symptoms in dairy animals. Feed refusal, reduced growth rate and decreased feed conversion efficiency are the predominant signs of chronic Aflatoxin poisoning. In addition, listlessness, weight loss, rough hair coat and mild diarrhea may occur. The disease may also impair reproductive efficiency, including abnormal estrous cycles (too short and too long) and abortions. Other symptoms include impaired immune system response, and increased susceptibility to diseases.