Mycotoxins : Control Measures & Their Management for Mycotoxins in Animal Feeds

Mycotoxins : Control Measures & Their Management for Mycotoxins in Animal Feeds

The so called “Mycotoxins” is not a new subject to Indian poultry industry. Perhaps, it is not an overstatement to say that Indian poultry feed millers, integrators and farmers understand mycotoxin challenges much better than their counterparts from the other countries. This is due to the extensive applied research carried on mycotoxins in poultry as well as high occurrence of aflatoxicosesin the field.
Mycotoxins are secondary metabolites of molds and their presence in the raw materials and feeds depends on many factors. Today scientists have discovered more than 500 mycotoxins but all of them may not be of economic significance. Some of these factors include moisture, temperature, relative humidity, insect damage etc. during plant growth as well as during storage of raw materials and feeds in feed mills.

Mycotoxin is hidden enemy for poultry farming. Mycotoxin become worldwide problem due to high incidence and level of occurrence in animal feed. Increase in feed cost has just aggravated this problem. Mycotoxin have high potential risk to public health as well. According to the FAO approximately 25% of the world’s agricultural products is contaminated with mycotoxins, and this contamination maybe due to saprophytic fungi before harvest of these crops while they are still in the field, during the process of harvest, and even after harvest during the storage of these.

The most common poultry feed ingredients contaminated by mycotoxins include – Maize, Wheat and wheat by-products and Soybean meal. Storage condition of grains and environment factors are major factors for development of mycotoxicosis. Aflatoxins (AF), zearalenone (ZEN), ochratoxin A (OTA), fumonisin (FUM), trichothecenes such as deoxynivalenol (DON), and T-2 toxin are some of the mycotoxins that can significantly impact the health and productivity of poultry. In general, contaminated feeds usually contain more than one mycotoxin.

Extreme weather conditions, heavy rain and drought lead to plant stress making them more susceptible to fungal infections. Mycotoxins are small and stable metabolites produced by fungi which can contaminate a wide variety of crops. The contamination of food and feed by mycotoxins is a global safety issue due to their adverse effects on human and animal health.

Mycotoxins vary in their chemical structures, which results in vast differences regarding their chemical, physical, and biochemical properties. While considering the great variety of mycotoxin structures there is no single method, which can be used to deactivate mycotoxins in feed. Therefore, different strategies have to be combined in order to specifically target individual mycotoxins without impacting the quality of feed. The best-known method for mycotoxin deactivation is “binding” with the use of binding agents, which are referred to as mycotoxin binders, adsorbents.

Mycotoxins are secondary fungal metabolites of worldwide concern due to their potential toxicity to animals and human alike. Fungal growth occurs in an aerobic condition in numerous raw feed ingredients. There are almost two hundred species of fungi that are capable of producing mycotoxins. Mainly, three genera of fungi, viz., Penicillium, Aspergillus, and Fusarium are associated with production of potent mycotoxins. While more than 500 known mycotoxins are formed by these fungi, only a few of them are pathogenic. Season and harvest time play a critical role in fungus growth and mycotoxin formation. It has been estimated by FAO that mycotoxins contaminate almost 25 percent of crops worldwide every year resulting in the wastage of roughly one billion metric tons of feed and feed products. Mycotoxins are given special attention because of their adverse health effects, food spoilage, and economic consequences shown in international food and food products. Feed quality, organoleptic characteristics, and nutritional quality are affected by the presence of microscopic fungi.

TYPES OF MYCOTOXINS:

The word mycotoxin has been derived from the Greek word mykes or mukos which means “fungus” and the Latin toxicum meaning a “poison”. It is a toxic secondary metabolite produced by some fungi, commonly known as molds. Most fungi are aerobic (use oxygen) and are found almost everywhere in extremely small quantities due to the minute size of their spores.

Several different types of mycotoxins are recognized: aflatoxins that are produced by Aspergillus flavus; T2 fusariotoxins produced by Fusarium spp. (mouth lesions and thin eggshells); Ochratoxins produced by Aspergillus ochraceus (interferes with functions of kidney, proventriculus, and gizzard) etc.

1. Aflatoxin:

It is produced by Aspergillus flavus, A. parasiticus, and some Penicillium spp. It is generally produced in a variety of mold-contaminated food like peanuts, rice etc. There are two major types of aflatoxins: B1 and G1, which show blue and green fluorescence, respectively, when exposed to UV light. Other types of aflatoxin include B2, G2, M1, M2, and P1 which are derivatives of B1 and G1. Among them, B1 is the most toxic. After ingestion, aflatoxin binds to chromosomal DNA, and causes point and frame-shift mutations. By inducing mutation, they cause cancer of the liver.

2. Trichothecenes

Trichothecenes are produced by a variety of fungi from the order Hypocreales, including Fusarium, Myrothecium, Stachybotrys and Trichoderma species. Trichothecenes are common field toxins found in grains and silages. Examples include deoxynivalenol (DON or vomitoxin), nivalenol, T-2/HT-2 toxins, and diacetoxyscirpenol (DAS). Susceptibility to trichothecenes varies between mycotoxin types, mycotoxin concentration, animal species/breed, and management systems. Swine are often considered one of the more sensitive species to DON, while T-2/HT-2 toxins are toxic to most species.

3. Ochratoxin:

There are at least seven types of structurally related ochratoxin, of which type A is the most common and the most toxic. It is produced by many Aspergillus and Penicillium species like – A. ochraceus, A. alliaceous, A. mellis, P. viridicatum and P. cycloplum. Like other mycotoxins, it is heat-stable and is not damaged during processing. When ochratoxin is ingested, it induces mitosis and causes cancer of the kidney.

4. Zearalenone:

Zearalenone is produced by fungi of the genus Fusarium. It  often occurs in combination with DON in naturally contaminated cereals or forages. This mycotoxin mimics the activity of hormones (as an estrogen analog), causing most of the reproduction-related symptoms seen in pregnant animals.

MANAGEMENT OF MYCOTOXINS IN FEED:

Management practices like withdrawal of mycotoxin-contaminated feed/feed ingredients or change of feed at the farm could provide partial protection to poultry from the toxicity as well as from mycotoxin residues. In other words, if there is a foul smell from the feed with lump formation, then it’s use must be avoided. Bulk storage of grains must be well-ventilated, and the materials should be well-protected from rain. Aflatoxins and other mycotoxins are not produced at temperatures below 5 to 8°C. The moisture level of the grain should be kept below 13% to prevent mold formation. Periodic cleaning of all handling equipment with 5-10% bleach solution will help control mold growth as well as to some extent destroy the aflatoxins present. The level of feed holding time should be kept as short as possible as the level and incidence of mycotoxin contamination increases after the feed is manufactured.

Routine analysis (mycotoxin screening):

Screening feed ingredients and feedstuff for mycotoxin contamination before formulation of poultry ration is important. There are two methods of toxin analysis; the biological method- qualitative and identification method, and the chemical method also called as quantitative method. The quantitative method includes chromatographic and non-chromatographic. Following are a few methods of mycotoxin detection through the chromatographic methods: Microcolumns, Thin layer chromatography (TLC), High-pressure liquid chromatography (HPLC), Column chromatography, and Gas Layer chromatography (GLC). Thin Layer Chromatography is commonly used, which involves the separation of a mixture of compounds between two phases, stationary and mobile, with an estimation of mycotoxin fluorescence in reference to the standard mycotoxin concentration. Non-chromatographic methods of toxin analysis includes Fluorotoximeter, mass spectrometry, and Radioimmunoassay.

Mycotoxin decontamination methods:

There are three methods of mycotoxin decontamination.

1. a) Physical methods:

• Antimycotic agents like sorbic acid and sorbate, propionic acid and propionate, benzoic acid, benzoates, and parabens, and acetic acid and its derivatives prevent mold growth and interfere with mycotoxins production.
• Herbs and spices like cloves, cinnamon oil, mustard, garlic, and oregano.
• Low oxygen concentration (<1%) and/or increased concentrations of other gases (90% of Carbon dioxide) depress mold growth and mycotoxin formation (Bullerman et al., 1984).
• Density segregation of contaminated grain and oilseed involving sorting and delineating good versus contaminated kernels by floatation.
• Irradiation involving gamma or electronic irradiation is highly effective for destroying fungal spores. Simple exposure of contaminated grains to sunlight substantially reduces mycotoxin levels.
• Mechanical separation of contaminated grains.
• Processing of food: wet milling, malting, autoclaving, brewing, dry and oil roasting.
• Solvent extraction

1. b) Chemical methods:

• Ammoniation: Treatment with aqueous ammonia or ammonium hydroxide, with or without heat and pressure is currently the best and most effective way to destroy mycotoxins. This method not only detoxifies mycotoxins but also prevents mold growth. It has been shown that 5% NH₃detoxify orchatoxins in the feed at 70 °C in 96 hours.
• Ozonization: several studies indicate that ozone degrades aflatoxins in corns and cottonseed meals and degrades toxins, , deoxynivalenol and moniliformin.
• Structural degradation: numerous chemicals including acids, bases, aldehydes, bisulfites, oxidizing agents, and various gases have been tested for the ability to degrade or detoxify AFB1 and other mycotoxins.

1. c) Biological methods/ amelioration:

• Mycotoxin-binding agents: like activated carbons, bentonites, clay, hydrated sodium, calcium alumina silicate, and zeolite have currently been used to counteract mycotoxicosis in poultry. The commercially available synergistic blends of mycotoxin binders are found to be beneficial against the commonly occurring mycotoxins in poultry species. These sorbents are nutritionally inert and reduce the bioavailability of various mycotoxins by absorption on their surface in the intestinal tract.
• Microbiological binding agents: Mannan oligosaccharide (MOS) extracted from the cell wall of Saccharomyces cerevisiaehas shown broad-spectrum efficacy against most mycotoxins. Certain strains of Bifidobacterium, lactic acid bacteria, and propionic bacteria have cell wall structures that can bind mycotoxins and limit mycotoxin bioavailability in the body. Bifidobacterium animalis and Lactobacillus acidophilus were found to be effective against OTA and patulin, respectively.

 Rule of Thumb for Mycotoxin Analyses
Given that molds are ubiquitous, it is impossible to find any grains or feeds without one or the other mycotoxins. Since there are more than 500 mycotoxins that are known today, it is practically impossible to analyze all of them. It is well accepted globally that the analyses of feeds and raw materials for aflatoxins, ochratoxins, fumonisins, T-2 toxin, DON (vomitoxin) and zearalenone will provide a fair understanding of potential toxicity in animals. Since mycotoxin analysis can be expensive, the following rule of thumb should be applied while considering mycotoxin analyses;

1. When feed millers need to make a decision about accepting or rejecting incoming raw materials, individual raw materials especially grains should be analyzed for mycotoxins.
2. When bird performance and health is questioned in the field, poultry feed should be analyzed first for mycotoxins. If the feed contains more than accepted levels of mycotoxin/s, then raw materials should be analyzed to ascertain the root cause of the problem.

Types of Molds and Mycotoxins
The molds and mycotoxins that are produced in the field during crop growth

are respectively called as “field molds” and “field mycotoxins”.Aspergillus, Penicillium and Fusarium are the most studied field molds (Figure 1 and 2). Aspergillus generally grows in tropical countries such as India while Fusarium molds are capable of growing both in tropical, semi tropical and temperate regions of the world. Penicillium molds are more common in semi tropical regions such as Eastern Europe.
Aspergillus and Penicillium molds are well known to be grown during storage of raw materials and feeds (Figure 3). Storage molds and themycotoxins these producecreatea significant problem in tropical regions such as India.

From Table 1, it can be clearly understood that India being a tropical country (some parts are semi-tropical in winter season) the type of mycotoxins those can be expected in raw materials and feeds are aflatoxins, ochratoxins, cyclopiazonic acid, T-2 toxin, HT-2 toxin, DAS, fumonisins and moniliformin. As mentioned earlier, today Indian poultry industry has the practice of analyzing raw materials and feeds for aflatoxins, ochratoxins, T-2 toxin, DON, zearalenone and fumonisins. This practice may not be relevant to Indian poultry (Table 2) and that raises few questions.

Mycotoxin Survey in India           
Many surveys have been conducted over the years on mycotoxin contamination in Indian raw materials and feeds. One such survey (Biomin Mycotoxin Survey 2017, Table 2) clearly shows that the occurrence of fumonisins was the highest (88%) followed by aflatoxins (81%) and ochratoxins (76%).

LD50 Values for Mycotoxins
Mycotoxin toxicity depends on the species of animal in question, age of the animal, type and number of toxins detected, and the concentration of mycotoxins in feed. LD50 values are commonly used in laboratory animals and poultry to determine the severity of toxicity of mycotoxins to a specific species. LD50 (mg/kg body weight) means the amount of single dose of toxin that is required to kill 50% of experimental population. In poultry, such measurements are generally made using one-day-old broiler chickens. Table 3 shows LD50 values for some of the mycotoxins of importance in poultry. According to Table 3, poultry species is more sensitive to ochratoxins followed by T-2 toxin, aflatoxins and DON. Poultry is quite resistant to DON.
Taking into account the kind of mycotoxin analyzed in India today, type of mycotoxins actually detected in survey and LD50 values of mycotoxins in poultry, the following questions can raise. An honest effort has been to answer them with scientific facts.

Why are we analyzing DON and Zearalenone in Indian poultry raw materials and feeds?
Table 2 clearly shows that the concentrations of these two toxins are quite low (76 and 21ppb). Moreover, these two toxins are very less toxic to poultry. Please refer to LD50 value in Table 3. Zearalenone levels as high as 800ppm did not cause much toxicity in poultry (Leeson et al., 199%).
Does the high occurrence of fumonisins in Indian feeds warrant its regular analysis?
The answer is “No” as the concentrations detected are too low (Table 2) to cause toxicity in poultry. Weibking et al., (1993) proposed minimum effective dose of 75ppm (75,000ppb) fumonisins to cause toxicity in poultry. This is about 180 times that of the average fumonisins detected in Indian raw materials and feeds (Table 2).
Do we need to analyze T-2 toxin?
There is no doubt that poultry is sensitive to T-2 toxicity (please see Table 3). However, the concentrations are too low to cause toxicity (Table 2). Given their high toxicity in poultry, it is recommended to analyze T-2 toxin case-by-case basis. This means whenever the birds are experiencing immunity and gut health challenges, the analysis of T-2 toxin in the feedis recommended.
Analysis of Mycotoxins
Rapid analysis of mycotoxins at feed mills is very critical to maintain quality of raw materials and poultry feeds. In this regard, a reliable and relatively cheaper technology has been developed which can analyze mycotoxins in less than 15 minutes.
Conclusions
Mycotoxins continue to pose threat to Indian poultry industry by compromising feed quality and health and performance of broilers, layers and breeders. Quality control measures at feed mill such as analyzing moisture and mycotoxin levels of incoming raw materials will help to accept or reject raw materials. Analysis of feed is warranted only when poor bird performance is reported in the field. Frequent analysis of raw materials is recommended only for afltaoxins and ochratoxins and T-2 toxin analysis can be done case-by-case basis. Taking into consideration the scientific facts, the analysis of poultry raw materials and feeds for DON, zearalenone and fumonisins is not required.

The problem of mycotoxicosis is not so easy to solve and requires constant attention throughout the entire process of grain harvest, shipping, storage, feed manufacturing and formulation. The complete elimination of molds and their toxins is practically impossible; so, finding such agents that can bind the toxins selectively in the gut is the need. The possible presence of toxic residues in poultry products, which enter into the food chains, may have a potential risk of their detrimental effects on human health. So, there is an urgent need to set a rational limit for such mycotoxins for the economic growth of the poultry industry.

Compiled  & Shared by- This paper is a compilation of groupwork provided by the

Team, LITD (Livestock Institute of Training & Development)

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 Reference-On Request

Mycotoxins in Poultry feed

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