Nutritionist > Poultry
What are mycotoxins?
The word mycotoxin stems from the Greek word "mykes", meaning mould, and "toxicum" meaning poison. Human cases of ergotism or St. Anthony's Fire have been described in Europe since the Middle Ages and are now known to be caused by alkaloids produced in rye by the mould Claviceps purpurea. In 1960, an outbreak of Turkey X disease in England and the subsequent discovery of the aflatoxins stimulated great interest in the field of mycotoxin research (Bullerman, 1979). Since then many more mycotoxins, such as trichothecenes, zearalenone, ochratoxins and fumonisins have been discovered.
Mycotoxins are toxic secondary metabolites produced by fungi growing on crops in the field, during handling and in storage. They enter the animal production system via feed (concentrate, silage or forage) or via bedding. Mycotoxins negatively affect animal performance, animal health and product quality. Thus mycotoxin control is crucial for production economics, animal welfare, product quality and food safety reasons.
Mycotoxins are chemically different representing a variety of chemical families and range in molecular weight from about 200 to 500 kD. There are hundreds of known mycotoxins, but few have been extensively researched and fewer still have good methods of available analysis. Mycotoxins vary greatly in their severity.
Mycotoxins exert their effects through four primary mechanisms:
- Intake reduction or feed refusal
- Alteration in nutrient content of feed in terms of nutrient absorption and metabolism
- Effects on the endocrine and exocrine systems
- Suppression of the immune system
In order to effectively identify mycotoxicosis, experience with mycotoxin-affected animals is important. This experience, combined with adequate feed and tissue analyses, provide the basis for the most accurate diagnosis of mycotoxicosis.
Moulds grow by producing long filaments called hyphae, which are important for the survival and dispersal of fungi. The hyphal network is responsible for cementing kernels together, which in stored grain or feed can result in clumps of grain that cannot be separated. Grain-mould fungi also produce spores (conidia) capable of aerial dispersal in the field as well as within a grain storage bin. It is usually masses of these spores that give the mould a characteristic colour. Spores can lay dormant for months or years until the proper conditions for fungal development are available.
Fungal species are often divided into two groups:
- Field fungi
- Storage fungi
Storage fungi (also called storage moulds) are those that invade grains or seeds during storage. They need less moisture than field fungi (13-18%) and usually do not present any serious problem before harvest. Storage fungi include species of Aspergillus and Penicillium.
While the field/storage terminology is generally used to indicate the differences in temperature and moisture required by various fungi, in fact the proper conditions for growth of a specific organism can occur in either the field or storage bin. Ideal conditions for fungal growth depend on the species, but normally moulds require high temperature and moisture.
Mycotoxins are produced as secondary metabolites. Under field conditions, stress and subsequently reduced vigour often predispose plants to infestation and colonisation by toxigenic fungi. In stored grain, toxigenic fungal infection and mycotoxin production result from a complex interaction between moisture, temperature, substrate, oxygen (O2) and carbon dioxide (CO2) concentration, fungal abundance and insect presence. Insects can influence a differentiation of fungal species, that is a specific insect determines the presence of a specific fungal species.
Mycotoxins can be found in forage. On one side plants can modify the concentration of mycotoxins due to different enzymatic systems. On the other side, it seems that they can move substances from the site of production to the hull and the stem and then to the leaves.
In general, most fungi need at least 1-2% oxygen and usually grow at temperatures between 20 and 30°C. It is important to note that if the grain is at high temperature at harvest, it can maintain that high temperature for several days or weeks after harvest unless the storage facility has cooling capabilities. Normally, in storage conditions fungi grow at 13-18% moisture. However, in case of grains with high levels of oil (e.g. peanuts) fungal growth occurs at moisture contents as low as 7%.
Overview of mycotoxins
Under the appropriate conditions, fungi proliferate, grow colonies and mycotoxin levels become high. As conditions for fungal growth vary greatly between field and storage, different fungal populations may be present, resulting in cocktails of mycotoxins being produced. This must be taken into consideration when conducting an appropriate risk assessment and implementing preventative measures. Although several hundred mycotoxins are known, the mycotoxins of most concern, based on their toxicity and occurrence, are aflatoxin, ochratoxin A, trichothecenes (DON, T-2 toxin, DAS, etc), zearalenone, fumonisin, and moniliformin (Table 1).
Table 1: Occurrence of different key mycotoxins
|Mycotoxin||Fungi Produced||Commodities affected|
|Corn, cotton seed, peanuts, soy|
|Ochratoxin A||Aspergillus ochraceus
|Wheat, barley, oats, corn, others|
|Trichothecenes (DON, T-2, DAS, etc)||Fusarium graminearum
|Corn, wheat, barley|
|Zearalenone||Fusarium graminearum||Corn, wheat, barley, grass|
|PR toxin, patulin||Penicillium roqueforti||Silage, Grass|
Adapted from Bhatnagar et al., 2004
Factors affecting mycotoxin formation in the field
As temperature and moisture levels are key factors for fungal growth and subsequent mycotoxin production, the climate plays a key role in the occurrence of mycotoxins. Crop surveys show large variations in contamination levels from one year to another due to varying climatic conditions. However, in addition to climatic conditions, agronomic practices also have a pronounced effect on mycotoxin formation as they affect the presence of fugal spores in the field as well as fungal growth. Three key agronomic factors have been shown to affect mycotoxin presence and concentration significantly:
- Crop presence and rotation: Monocultures or planting of closely related crops one after the other will enhance the risk of mycotoxin formation, as spores will transfer to the subsequent crop and thus allow fungal growth to establish quickly and strongly.
- Soil cultivation: Ploughing harvest residues will reduce spore contamination of the subsequent crop and thus reduce fungal infestation and mycotoxin formation. No-till systems will enhance the risk.
- Crop and crop variety: Crop varieties that are more resistant to fungal foliar diseases reduce fungal infection and thus mycotoxin formation of the crop.
Mycotoxin formation during storage
Factors mostly influencing fungal growth on plants and hence mycotoxin production are free water, temperature and the time of colonisation during storage.
Fusarium mycotoxins (zearalenone, trichothecenes, fumonisin, etc) are produced mainly during the field (cultivation) phase. Aspergillus and Penicillium mycotoxins (aflatoxin, ochratoxin, etc) are produced, for the most part, during storage.
Contrary to what happens during cultivation aflatoxin synthesis during storage can take place in tropical and sub-tropical conditions.
Principal factors influencing mycotoxin production are:
- Intrinsic factors, connected to the fungal stock
- The power of generating toxins which can vary inside each stock from 1 to 103-104
- The fungal species which determine the category of mycotoxin produced
- The initial contamination level which influences the amount of mycotoxins produced (the more moulds, the greater the potential for more mycotoxins)
- Extrinsic factors, i.e. environmental conditions. These factors determine fungal growth and therefore mycotoxin production
- Chemical, physico-chemical and physical factors such as humidity, free water, temperature, the substrate, the gas composition (atmosphere) and the mechanical damages to the caryopsis
- Biological factors, such as insects, either as vectors of fungal spores or as vectors responsible for mechanical damage to the caryopsis, favouring the entry of moulds; the microbiological flora, and the competition between mycological strains; the plant stress (drought); the resistance of the layer, either as genetic strength or caryopsis integrity.
The ideal growth conditions are summarised below:
Environmental humidity or free water
The most interesting benchmark is free water. Fungal colonisation of feedstuffs is more frequent when the bacterial load at free water levels is lower than 0.85. This not because fungi cannot grow at higher levels, but rather because bacteria are strongly competitive and become the predominant microflora at values between 0.85 and 1.00, and in particular at levels over 0.90-0.93.With a level of free water between 0.85 and 0.93 only some bacteria can rapidly increase in number (lactic ones and cocci in particular) so the invasion of moulds and yeasts predominates.
According to the behavioural differences related to water availability, fungi species can be classified as follows:
- Hydric (i.e.Epicoccum nigrum, Trichothecium roseum, Mucor circinelloides): The spore can only germinate at free water levels above 0.90 (the optimal growth takes place at 1.00).
- Medium level (i.e. Alternaria tenuissima, Cladosporium cladosporioides, Penicillium cyclopium): The spores germinate at free water levels between 0.80 and 0.90. The best and optimal growth takes place at 0.95-1.00.
- Xerofile (i.e. Aspergillus repens, Aspergillus restrictus, Aspergillus versicolor): The spores germinate at free water levels less than 0.80. The best and optimum growth takes place at 0.95.
The ideal temperature for mould development is between 15 and 30°C, with optimal values of 20-25°C. Some species such as Cladosporium herbarium have an apparent growth at -6°C. Others, such as some species of Penicillium can develop in frozen fish at -20°C. Within the literature some spores (Rhizopus nigricans, Mucor mucedo, Aspergillus niger, Aspergillus glaucus) are reported to persist after the immersion for 77 hours in liquid hydrogen at -253°C and for 492 hours in liquid air at -190°C.
The colony does not develop at high temperatures, except for Aspergillus fumigatus, which can visibly contaminate the first respiratory tract at body temperature. At 35-40°C Monilia sitophila, a typical contaminant of bread, can still survive. Other species, for example Bortytis cinerea, can persist at refrigeration temperatures and can still replicate at 5°C.
pH and Oxygen
The development of fungal colonies takes place at pH values between 4 and 8. However some moulds can grow at lower or higher values, modifying the acidity of the medium, during the development of the colony.
Moulds are generally aerobic organisms that develop above the surface of the medium. Some species can however develop in deep mediums, such as Stachybotrys, or in liquid mediums, with a low oxygen rate, assuming a jelly appearance, or even in a modified atmosphere, with CO2 and N2.
The environmental conditions and the moisture content influence the production of mycotoxins. Toxin production is improved by levels of free water of 0.90. Aspergillus flavus can start to produce aflatoxin at 0.83. A. ochraceus needs at least 0.97 to produce ochratoxins.
The humidity of the sub stratus is the principal factor to be considered in the prevention of contamination and its control has become fundamental, for example, in feed production.
Aspergillus flavus easily produces aflatoxins at approximately 25°C. Under 10°C the toxin production has never been demonstrated. Fusarium tricinctum can produce T2 toxin at temperatures between 1 and 4°C, to a maximum of 15°C. Aspergillus ochraceus produces ochratoxin from 20 to 30°C, but never below 12°C. The same mycotoxin is produced by Penicillium viridicatum between 4 and 31°C
Thus it is difficult to define temperature values to control mycotoxin production, except aflatoxin, which is never produced under 10°C, even in deeply mouldy conditions.
Oxygen concentration and the acidity of substratum are not relevant for mycotoxin production.
An important factor to be taken into consideration is the type of substrate. Vegetal substrates improve mycotoxin production, more than the animal ones and those of animal origin.
In particular the presence of starch seems to help mycotoxin generation. Moreover the presence of zinc, only referred to the aflatoxin production. Cereals, oleaginous seeds and dried fruit are the foods most frequently contaminated by aflatoxins. The more frequent products contaminated are maize, peanuts and cotton seeds.Fruit and juices are the principal carriers of patulin, and cereals of ochratoxin.
Economic losses associated with mycotoxicosis include:
- Poor growth
- Reduced egg production
- Reduced feed conversion
- Increased mortality
- Poor egg shell quality
- Reduced fertility
- Leg problems
- Carcass condemnation
- Increased susceptibility to disease
In poultry production, feed is the key vector bringing mycotoxins into the production system and control strategies should mainly focus on optimising feed quality. However, cases have been reported where significant concentrations of mycotoxins have been introduced into the production systems through litter. Straw may already be contaminated with significant concentrations of mycotoxins at the time of harvest, although any type of litter can be contaminated during unfavourable storage conditions.
In order for you to effectively recognise mycotoxicosis, flocks have to be carefully inspected for symptoms. Symptoms are often very general and can greatly vary between mycotoxins, making proper diagnoses a difficult undertaking. Careful recognition of symptoms and post-mortem analyses combined with adequate feed analyses, provide the most accurate mean of a mycotoxicosis diagnosis.
Aflatoxins are of concern in warm and humid climatic conditions. Although aflatoxins are not considered to be a major problem in cold or more temperate regions, caution must be exercised in colder climates when using feedstuffs imported from warm and humid countries.
Among poultry, ducks are the most susceptible to aflatoxin, followed by turkeys, broilers, laying hens and quail. In all species, aflatoxins are hepathotoxic with fatty changes, causing hepathocyte degeneration, necrosis, and altered liver function. Suppression of hepatic protein synthesis is the main factor resulting in growth suppression and reduced egg production. Aflatoxin is also known to interfere with vitamin D metabolism, contributing to reduced bone strength and leg weakness. By reducing bile salt production, aflatoxin negatively affects lipid and pigment absorption. Additionally the metabolism of other minerals including iron, phosphorus and copper are also affected by aflatoxin. Aflatoxin increases the fragility of capillaries, reducing prothrombin levels thereby drastically increasing the incidence of bruising in carcasses and carcass downgrading. Due to the transfer of aflatoxin into edible products and its carcinogenic effects, most countries have set upper legal limits for aflatoxin in feed. See the regulations page on this web site for more information
Clinical signs of aflatoxin toxicity include:
- Decreased weight gain / anorexia
- Decreased egg production
- Reduce feed conversion efficiency
- Increased mortality
- Immune suppression and increased disease susceptibility
- Reduced fertility and hatchability
- Embryo toxicity
- Specific visceral haemorrhage
- Increased susceptibility to environmental and microbial stressors
- Leg weakness and reduced bone strength
- 'Pale bird syndrome'
- Fatty liver
- Liver necrosis
- Bile duct hyperplasia
- Increased incidence of bruising and downgrading
Ochratoxins are important storage toxins. They are produced by different fungi and are prevalent in temperate as well as in tropical regions.
Ochratoxin A is the most important of the ochratoxins. The primary effect of ochratoxin A in all poultry species is nephrotoxicity. In poultry the proximal tubules are mainly affected and the kidney is pale and grossly enlarged. As with aflatoxin, fatty liver can also occur due to ochratoxin exposure. In acute cases mortalities can occur due to acute renal failure. In young chicks, ochratoxin A is approximately three times more toxic than aflatoxin.
Ochratoxin has been implicated in significant field outbreaks of mycotoxicosis in poultry.
Clinical signs of ochratoxin toxicity include:
- Reduced feed intake
- Reduced growth rate and egg production
- Reduced feed conversion efficiency
- Mortality due to acute renal failure
- Poor egg shell quality and higher incidence of eggs with blood spots
- Reduced embryo viability and decreased hatchability
- Reduced feathering
- Polyurea with large volumes of wet faeces
- Pale and grossly enlarged kidney
- Fatty liver
- Urate deposition in joints and abdominal cavity (at high exposure levels)
- Depletion of lymphocytes and with it strong suppression of cellular immunity, thus enhanced susceptibility to viral infections.
Trichothecenes (T-2 toxin, diaceptoxyscripenol (DAS), deoxynivalenol (DON), HT-2 toxin, etc)
Trichothecenes are typical field mycotoxins and are produced on crops entering the feed via contaminated ingredients. Trichothecenes are proven tissue irritants with the major observation associated with their ingestion being oral lesions, dermatitis and intestinal irritation.
The major physiological response to trichothecenes mycotoxins is loss of appetite, thus earning them the name, feed refusal toxin.
Of the different trichothecenes, poultry are most sensitive to T-2 toxin and DAS. Trichothecenes are strong immune suppressive mycotoxins affecting cellular immune response by direct effects on bone marrow, spleen, lymphoid tissues, thymus and intestinal mucosa, where actively dividing cells are damaged.
Clinical signs of trichothecenes toxicity include:
- Oral lesions: circumscribed proliferate yellow caseous plaques occurring at the margin of the beak, mucosa of the hard palate and the angle between the mouth and the tongue.
- Reduced feed intake
- Reduced weight gain and egg production
- Poor shell quality
- Reduced female fertility and hatchability of fertile eggs
- Immune suppression, reduced vaccination response
- Tibia dyschondroplasia
- Gizzard erosion
- Necrosis of proventricular mucosa
- Regression of ovaries
- Increased liver weight
Zearalenone often occurs with DON in naturally-contaminated cereals. Zearalenone is responsible for reproductive disorders due to its oestrogenic effect at high concentrations. However, in general ZEA has limited toxicity to birds.
At high concentrations the following symptoms have been observed:
- Vent enlargement
- Enhanced secondary sex characteristics
Broilers and turkeys seem relatively resistant to acute toxic effects of fumonisins. Fumonisin mycotoxicosis leads to a very specific increase in sphiganine:sphingosine ratio. However, as sphinganine and sphingosine analyses are quite complex this ratio is seldom used as a biomarker in field situations.
Clinical signs of fumonisin toxicity include:
- Spiking mortality (paralysis, extended legs and neck, wobbly gait, gasping)
- Reduced growth rate
- Increased organ weights
- Hepatocellular hyperplasia
- Poor vaccination response
- Increased liver sphinganine : sphingosine ration (biomarker)
Co-contamination and further mycotoxins
Contaminated feeds or ingredients typically contain more than one known and probably several unknown mycotoxins. The toxic responses and clinical signs observed in poultry when more than one mycotoxin is present in feed are complex and diverse.
Co-contamination of mycotoxins appears to exert greater negative effects on health and productivity than do single mycotoxins. For this reason, symptoms typical of mycotoxicosis are often seen in poultry despite analyses of the feed indicating only very low or zero concentrations of individual mycotoxins. Toxicity may be due to interactions between different mycotoxins that exaggerate the toxicity symptoms.
With mycotoxins the risk directly depends on the level of the major mycotoxins in the feed, the co-occurrence and level of other mycotoxins as well as the avian species, their age and health status. Therefore strictly speaking it is not possible to define safe levels of mycotoxins. This complex situation makes it critical to take the necessary precautions.
Sampling and testing for mycotoxins
If clinical signs of mycotoxicosis are observed, it is important to properly collect a grain or feed sample and send it to a laboratory to determine the presence and level of the suspected mycotoxin(s). Sampling accounts for 80 - 90% of the error associated with measuring mycotoxins in grain or feed. Analytical tests for mould spore counts are of little or no value.
Random samples (10 to 30) should be collected from several locations within a batch of grain or feed and combined thoroughly to provide a composite sample for submission. Using a grain probe at several evenly distributed locations will provide the most representative sample.
Samples can also be collected periodically from grain being augured which can also be an effective form of sampling. Paper bags should be used to transport sample(s), since plastic bags retain moisture, and therefore can promote additional fungal growth.
Contact the laboratory for specific sampling requirements prior to submission.
It is important to remember that based on the uncertainties associates with any mycotoxin test procedure, it is difficult to determine the true concentration of a bulk lot.
Mycotoxins are difficult to measure for a number of reasons:
- Many different mycotoxins can be present simultaneously, making analysis difficult and expensive. Under commercial conditions analyses is normally limited to a couple of indicator mycotoxins.
- Sampling of bulk feeds is difficult. Mycotoxins are present in 'hot' spots and are not evenly distributed throughout the feed. Therefore strict sampling procedures should be followed with many samples taken from a particular batch to get a realistic reading.
- Latest research has identified complexes of mycotoxins and their metabolites for which there is no accurate analysis method.
As the risk if mycotoxicosis is very difficult to predict or evaluate, prevention strategies should be initiated when assessing even a low risk situation. Prevention strategies must primarily aim at minimising mycotoxin formation in the field and during storage.
A significant reduction in mycotoxin formation can be achieved by good agronomic practices, for example:
- Selection of crop varieties that are more resistant to fungal foliar diseases
- Ploughing up harvest residues
- Avoiding no-till soil management practices
- Proper crop rotation
- Avoiding monoculture
- Moisture content below 12%
- Relative humidity below 60%
- Storage temperature below 20 °C
- Clear grain, avoid broken kernels
- Control insects and rodents
- Avoid stress (frost, heat, pH changes)
Mycotoxin adsorbents and binders
As we know mycotoxins are usually found in combinations in complete animal feeds. A broad substrate binding capacity will ensure at least some fraction of all the mycotoxins will be rendered non-bioavailable and the bioavailable mycotoxins will be below the threshold of biological activity. Broad substrate binding capacity of a binding agent will also minimise the potential for toxicological synergy between mycotoxins.
Speciality feed additives, known as mycotoxin adsorbents or binding agents are the most common approach to prevent and treat mycotoxicosis in animals. It is believed that the agents bind to the mycotoxin preventing them from being absorbed. The mycotoxins and the binding agent are excreted in the manure.
The effective level of dietary inclusion for mycotoxin adsorbents will depend on the mycotoxin binding capacity of the adsorbent and the degree of contamination of the feed in question. A high binding capacity will minimise the level of inclusion and minimise the reduction in nutrient density caused by the feeding of the adsorbent. High levels of inclusion of adsorbents can also alter the physical properties of the feed which might impair feed processing such as pellet formation, in addition to altering the actual diet specification.
Mycotoxin binding is achieved through both:
- Relatively weak bonding involving van der Waals interactions and hydrogen bonding
- (Chemisorption) is a stronger interaction which involves ionic or covalent bonding.
There are two types of mycotoxin adsorbent/binder:
- Inorganic binders
- Organic adsorbents
Inorganic mycotoxin binders are silica based polymers. Examples could include:
- bleaching clays from the refining of canola oil
- hydrated sodium calcium aluminosilicates (HSCAS)
- diatomaceous earth
- numerous clays
- Phyllosilicates are characterised by alternating layers of tetrahedral silicon and octahedral aluminium coordinated with montmorillonite oxygen atoms
- Isomorphous substitution leads to a net negative charge which must be satisfied by the presence of inorganic cations (Na, Ca, Mg, K)
- Applications: Adsorbents for heavy metals, suspension-stabilising agents in coatings, bonding agents for foundry sands and washes, binder in pelletisation processes, desiccants in feed products.
- Tectoalumosilicates of alkali and alkaline earth cations that have an infinite three-dimensional cage-like structure
- Isomorphous substitution leads to a net negative charge which is satisfied by the presence of inorganic cations (Na, Ca, Mg, K)
- Applications: Adsorbents for ammonia, heavy metals, radioactive cesium and mycotoxins.
The amount of organic acids in clays is often very small. Does the small amount of organic acid(s) really work in inhibiting moulds? The answer is NO; and it can actually do more harm than good: The small amount of acids quite often has no effect. Worst of all, if the acids do work, due to such small amounts, they are not enough to kill the mould. Instead, the acids change the pH of the environment and bring pH stress to the moulds. The pH stress can actually stimulates the moulds to produce MORE mycotoxins. (REMEMBER, mycotoxins are the secondary metabolites from moulds produced due to stress from environmental factors, such as pH)
Organic mycotoxin adsorbents are carbon based polymers. Examples could include:
- fibrous plant sources such as:
- oat hulls
- wheat bran
- alfalfa fibre
- extracts of yeast cell wall
The efficacy of glucomannan-containing yeast products as mycotoxin adsorbents in feeds has been investigated globally with several studies with all animals [Click here to see in vivo research]. Research conducted in France at the National Institute for Agricultural Research (INRA) identified four Saccharomyes cerevisiae yeast strains that differed greatly in their glucan/mannan ratio. It was found that large differences existed in adsorptive capacity between the yeast strains with the amount of mycotoxin adsorbed strongly related to the beta-D-glucan content. This research confirms earlier work carried out in Alltech which led to the selection of a yeast strain high in insoluble beta-D-glucan content for the design and production of glucomannan-containing yeast product. (A. Yiannikouris et al., 2004) Advanced molecular techniques were used to elucidate the spatial conformation and molecular sites of interaction between zearalenone and glucomannan-containing yeast product. Molecular modelling was used to locate the interaction sites. Both hydrogen bonds and van der Waal's stacking interactions were identified as key interactions between mycotoxins and glucomannan-containing yeast product (Figure A)
(A. Yiannikouris et al., 2004; Biomacromolecules, 5:2176-2185)
Mycotoxin adsorbents offer an attractive short-term solution to the challenge of mycotoxin-contaminated animal feeds. The only complete solution to the mycotoxin challenge will be the long-term goal of eliminating mycotoxins from the food and feed chains through improved quality control based on better analytical techniques coupled with genetic advances in plant resistance to fungal infestation.
If you are considering adding a mycotoxin adsorbent to your feed you need to look for the following:
- Proven efficacy in vivo as well as in vitro
- Low effective inclusion rate
- Stable over a wide pH range (This is necessary so that the mycotoxin stays attached to the adsorbent throughout the gut and is excreted.)
- High affinity to adsorb low concentrations of mycotoxins
- High capacity to adsorb high concentrations of mycotoxins
- Ability to act rapidly before the mycotoxin can be absorbed into the blood stream.
Comment posted by: Y. Man, Malaysia
On a global basis, the most significant feed-borne mycotoxins affecting poultry production are aflatoxin and the many Fusarium mycotoxins. Determining the threat posed by feeding aflatoxin-contaminated feedstuffs is further simplified by the analytical techniques that are available. They are rapid, reproducible and sensitive.