By Dr Hannes Viljoen Animal Nutrition & Health (ANH)
Mycotoxins are a relatively large, diverse group of naturally occurring, fungal toxins, many of which cause toxic diseases in humans and animals. They are unavoidable contaminants in foods and feeds and are a major problem all over the world.The word mycotoxin means a toxin produced by a fungus or mould, which can infect grains such as maize and wheat in the field, during harvesting, during handling, and in storage. Although many mycotoxins have been identified, the most significant in naturally-contaminated foods and feeds are aflatoxins, ochratoxins, zearalenone, T-2 toxin, vomitoxin and fumonisins.
Plants can be affected by more than one fungus, and each fungus can produce more than one mycotoxin so the chances are good that most feedstuffs will contain one or more mycotoxins. These toxins may interact with one another causing problems greater than their individual effects, and this is of great concern in livestock health and productivity. Because of the many different production, handling, transport and storage conditions for feedstuffs used in the balanced feed industry, toxin profiles can differ substantially between area, locality, season and even countries. Consequently, the trade in food and feed commodities has resulted in a worldwide distribution of contaminated materials with different toxin profiles.
From the large number of identified mycotoxins, only a few are believed to affect swine performance. Risk to the pig from mycotoxin-contaminated feed depends on the age and health of the pig and level of toxin in the feed. The most severe effect is death, but low levels of mycotoxin can hurt pig performance and general well-being. When pigs eat feed containing a harmful mycotoxin, a number of organs or systems may be affected, such as the central nervous system, liver, kidney, immune system, or reproductive process.
Current status of mycotoxin awareness in South Africa
In the formal animal feed industry, The Animal Feed Manufacturers Association (AFMA) addressed the problem of mycotoxin contamination in order to give guidance as far as mycotoxin management was concerned, the purpose being the responsible production of safe animal feeds for safe food in South Africa. AFMA published a “Code of practice for the control of mycotoxins in the production of animal feed for livestock, June 2003”. The code provides an overview on mycotoxins, guidelines for establishing good practices for the control of mycotoxins in the feed industry, and interim guidelines on maximum acceptable levels of mycotoxins in animal feeds until local and/or internationally accepted regulations are set.
More efforts are currently under way by a National Mycotoxin Group supported and funded by the Maize Trust with participation by interested industries. Five focus areas were identified in the field of Mycotoxins research:
1. Guidelines for mycotoxins in food and feed;
2. A MycoMap to be established for mycotoxins;
3. A prediction model to be established for mycotoxins;
4. Identification of the risk areas where mycotoxins occurs; and
5. Identifying new areas of research in mycotoxins.
Due to the fact that the Maize Trust indicated its willingness to fund projects related to the above focus areas it was confirmed that as an initial phase, all the above research should focus on maize and maize related products. Other trusts may well follow suit.
What are the most common mycotoxins, and which fungi are most responsible for their production?
The majority of the known mycotoxin-producing fungal species fall into three recognized genera: Aspergillus, Penicillium, and Fusarium (Table 1).
The Southern African Grain Laboratory (SAGL) annually publishes analytical results of the South African grain crop (maize and wheat) (www.sagl.co.za). As grains (maize) are among the biggest contributors to mycotoxins in animal feeds the information is handy in order to guide the different feed and animal production industries on the status of the South African crop (and imports) on levels and tendencies.
The most important mycotoxins, based on toxicity and prevalence, may be carcinogenic (aflatoxin B1, ochratoxin A, fumonisin B1), oestrogenic (zearalenone), neurotoxic (fumonisin B1), nephrotoxic (ochratoxins, citrinin, oosporeine), dermonecrotic (trichothecenes) or immunosuppressive (aflatoxin B1, ochratoxin A, T-2 Toxin). Most are relatively stable and are not destroyed by processing. Although we only analyse or screen for a few mycotoxins, the existence of one mycotoxin in a feedstuff or animal feed can indicate the presence of others, which may be synergistic in nature.
It is apparent from Table 2 that the contribution of maize to Aflatoxin and Fumonisin is low and below the maximum acceptable levels for pigs. It should be noted that a high value of 9 ppb for Aflatoxin was determined during the 2006/2007 season. This value is less than half of the maximum acceptable level, but much higher than the average. This emphasises that although the history may show a low prevalence for a particular mycotoxin, regular screening for particular toxins is imperative.
The only Trichothecene mycotoxin tested for by SAGL, was DON. The analysis showed higher than the maximum acceptable levels for all classes of pigs in all seasons (marginal for 2007/2008 season), with exceptionally high values for the 2005/2006 season. These extreme values must certainly be of concern to pig producers, because with normal maize inclusion levels, the maximum acceptable mycotoxin levels will be reached or exceeded. Obviously, the other ingredients in the feed will help to dilute these high concentrations, but may also contribute other mycotoxins to the feed. Although T-2-toxin analysis is not reported by the SAGL, DON can play a role as a “marker” toxin to show general infestation.
The other toxin of importance is Ochratoxin. Average values for the 2006/2007 and 2007/2008 seasons (0.5 ppb and 0.23 ppb) are higher than the maximum acceptable level of 0.2 ppb. The grain crop for the last four seasons had lower average Zearalenone levels than the minimum acceptable levels. Maximum analyses values were however higher than these maximum acceptable levels for the 2004/2005 and 2005/2006 seasons and warrant further attention.
Damaged feedstuffs and foreign material are readily available food sources for mould growth, which then encourages mycotoxin infection. Many mycotoxins are concentrated in the outer covering of seeds and therefore increase the chances of mycotoxin related problems when such materials are used in animal diets, for example, during the wheat milling process. DON occurs in highest concentrations in the bran fraction, and lowest in flour.
The SAGL analysis of wheat for the 2007/2008 season showed an average DON content of 1.36 ppm with the highest value of 2.70 ppm. The average ochratoxin content was 0.33 ppb (µg/kg) with the highest value being 2.8 ppb. The high toxin concentration in wheaten bran is of considerable concern.
Mycotoxicoses in pigs
Mycotoxicoses in pigs are well described in the literature. The condition can be complicated, as a number of different moulds can produce an array of mycotoxins, all affecting the wellbeing of pigs. Because of the large number of structurally unrelated mycotoxins that are produced by the various fungi it is difficult to pinpoint which toxin(s) is responsible for a particular outbreak, even if a mycotoxicoses is strongly suspected.
Furthermore, there are a multiplicity of factors such as breed, sex, environment, nutritional status, and other toxic entities that can cause the infection. The economic impact of lowered productivity, decreased weight gain and decreased feed efficiency, increased disease incidence because of immune system suppression, subtle damage to vital body organs and interference with reproduction, is many times greater than that of immediate morbidity and lethality.
A short description of the role and main attributes of different mycotoxins are given below, but it must be stressed again that a single toxin is rarely responsible for the toxic effects.
Aflatoxins are potent liver toxins and most animal species exposed to these mycotoxins show signs of liver disease (degeneration, necrosis, and altered liver function) ranging from acute to chronic. These toxins may be lethal when consumed in large doses. Generally, young animals are more susceptible than older ones to the toxic effects of aflatoxins. The effects of aflatoxins in pigs vary, depending on age, diet, concentration, and length of exposure. Aflatoxin at 20 to 200 ppb suppresses the immune system and makes pigs more susceptible to bacterial, viral, or parasitic diseases.
Long-term consumption of contaminated feed may cause cancer, liver damage, jaundice, and internal bleeding. Profits are reduced because of loss in feed efficiency, slower growth, and increased medical costs. The EU and the AFMA guidelines (AFMA 2003) specify an upper limit of 20 ppb aflatoxin B1 in pig feed.
Due to the transfer of aflatoxin into edible products and its carcinogenic effects, most countries have set upper legal limits for aflatoxin in feed. High Aflatoxins levels are not common in the South African grain crop (Table 2).
Trichothecenes (Don, T-2 toxin and DAS)
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. Deoxynivalenol (DON) is the most common of the trichothecenes group causing animal disease and effects range from feed refusal and vomiting to immunosuppression and loss of productivity.
Monogastric animals, particularly swine, exhibit the greatest sensitivity to DON, while poultry, followed by ruminants, appear to have higher tolerance. A concentration of 1.3 ppm DON in diet causes feed intake by growing pigs to be significantly decreased, followed by complete feed refusal at 12 ppm and vomiting at 20 ppm. Extensive lesions are not typically documented in field cases, because pigs regulate toxin ingestion by adjusting their feed intake. It has been estimated that feed intake is reduced by 7.5% for every 1 ppm DON found in the diet.
At lower dietary DON concentrations, reduction in food intake is transitory, lasting only a few days before animals begin to compensate for initial losses. With increased DON levels in feed, animals may not return fully to control intake but the extent of feed refusal diminishes with time. Although T-2 and DAS data are scarce, as part of the Trichothecenes group it stays important as pigs are sensitive to T-2 toxin and very sensitive to DAS.
There is a tendency for feed intake and gain to be reduced when T-2 toxin is included in the diet at 0,4; 0,8; 1,6 and 3.2 ppm. Although negative effects were observed at the lower levels, the reduction in performance may occur at approximately 3 ppm. The AFMA guideline for T-2 toxin is however 0.2 ppm for all pigs. The T-2 toxin consumption over time by breeding sows has caused drastically decreased conception rates and weak piglets. High levels of T-2 toxin can also cause dermatitis of the skin on the snout, dorsal part of the nose, behind the ears, around the prepuce as well as on the mucous membrane of the oral cavity and the tongue.
Ochratoxin is particular serious for the poultry and swine industries because monogastric animals lack the ability to degrade ochratoxin rapidly, as compared to ruminants. Ochratoxin A, a nephrotoxic mycotoxin produced by several Aspergillus and Penicillium species, primarily affects the kidneys in animals exposed to naturally occurring levels. Changes in the renal function of pigs include impairment of proximal tubular function, altered urine excretion, and increased excretion of urine glucose. Extra renal effects may have occurred in animals exposed to levels of Ochratoxin A in feed greater than 5 to 10 ppm.
Ochratoxin is hazardous to swine at low levels, typically 0.2 ppm. If ingested over a long enough period of time this toxin can contaminate most of the edible tissues, and can produce enough kidney damage to result in carcass condemnation. As ochratoxin residues in animal products are transmissible to consumers, some national governments have taken stringent measures to allay consumer fears regarding their pork products.
Ochratoxin tested positive in thirteen maize samples for the 2006/2007 season in the SAGL analysis report. Positive samples tested between 0.97 ppb and as high as 6.5 ppb. These values are way higher than the 0.2 guideline given by AFMA (2003) for piglets.
Tests involving intubation with pure fumonisins, or feeding highly contaminated feed, showed pulmonary oedema, accompanied by liver damage and pancreatic lesions. Some cases of pulmonary oedema were observed in weaned pigs at contamination concentrations, between 10 and 40 ppm, but with longer feeding periods of 28 days. Fumonisins tested positive in most of the SAGL samples (Table 2). All these levels are lower than the maximum 10 ppm guidelines for all classes of pigs by AFMA, (2003), although a sample analyzed as high as 6.5 ppm.
Reproductive effects (Zearalenone and Ergot alkaloids)
Two of the most prominent mycotoxins that can cause reproductive effects are Zearalenone and Ergot alkaloids. Zearalenone often occurs with DON in naturally-contaminated cereals.
The major effects of zearalenone are estrogenic and primarily involve the urogenital system. Swine are the most commonly affected animals. Hyperestrogenism in female swine may be manifested as swelling of the vulva and enlargement of the mammary glands, especially in prepubescent gilts. Dietary concentrations of 1.0 ppm zearalenone or more may produce these symptoms.
Zearalenone has been associated with feminization in young male swine, including testicular atrophy, swollen prepuce, and mammary gland enlargement; decreased libido may be a variable consequence. In severe cases, this syndrome may progress to rectal and vaginal prolapse. Other effects related to higher concentrations include anoestrus, nymphomania, and pseudo pregnancy. The South African grain crop for the last three seasons had lower average Zearalenone levels than the guideline minimum acceptable levels by AFMA (2002).
Maximum analysis values were however higher than these maximum acceptable levels for the 2004/2005 and 2005/2006 seasons and warrant attention in order to be aware of any toxicity signs.
Abortion may be a variable and controversial sequel to ergot ingestion, depending on species affected and alkaloid content of sclerotia. Ergot-contaminated diets of pregnant swine are associated with decreased piglet birth weights and increased stillborn rates; gestation time may be shorter or longer than normal. Ergot also inhibits prolactin secretion in pregnant swine, resulting in diminished udder development and agalactia at farrowing.
Individual versus multiple mycotoxin contamination
The scientific literature is replete with information on the effects of individual mycotoxins in various livestock species. Concern arises from the fact that concentrations of individual mycotoxins associated with poor livestock performance and/or disease syndromes in commercial operations usually are lower than those reported to cause toxic effects in controlled laboratory studies Additionally, in some of these reports the feed contained more than one mycotoxin. Many fungal species also are capable of simultaneously producing several mycotoxins. Therefore, an individual grain may be naturally contaminated with more than one mycotoxin, or the incorporation of numerous grain sources, which are each contaminated with a different mycotoxin(s), into a single feed may result in a diet that contains a number of different mycotoxins.
Preventing Mycotoxin formation
The prevention of mould growth, mycotoxin production and the detoxification/binding of mycotoxins is a multistage process on which different role-players in the chain have an effect. The pig producer does not always have control over many of the stages such as pre-harvest control, (cultivar selection), harvesting and initial handling and storage of grains in order to prevent mould growth and mycotoxin production.
On farm control
Storage conditions are important. The critical point for controlling fungal growth in storage is grain /feed moisture levels. Grain that is dry when placed in storage and kept dry (less than 14% moisture) is unlikely to support growth of fungi that produce mycotoxins. Storage bins need to be kept clean and in good repair. Be aware of condensation, which sets the stage for mould growth and toxin production. Clean feed and grain storage bins frequently to prevent feedstuffs from bridging and forming hot spots.
Fungal inhibitors such as propionic acid can be used; however, these have no effect on mycotoxins already present at the time of application. They only prevent future growth of fungi. Ground feed is an ideal source of food for fungal growth. During periods of high humidity and heat, do not store ground feedstuffs and/or pigs diets for longer than 10-14 days.
Less well understood, perhaps, is the fact that the geographic distribution of some toxins may be localized down to the farm level. Producers need to establish their own historical record of mycotoxin exposure and monitor for deviations from that pattern. The producer, then, needs to be in a position to monitor one or more ‘marker’ toxins that will allow some reasonable conclusions to be drawn as to the overall mycotoxicologic quality of the feeds and forages in use. A marker toxin derives from the fact that since many moulds produce more than one toxin, and that some toxins are far easier to assay than others, using simple means to deduce the extent of contamination is most efficient.
Troubleshooting also raises the issue of what actions to take once a problem is detected. A first approach recommended is to make maximum use of steps likely to reduce the risk of serious mould infestation and subsequent mycotoxin formation, such as listed in Table 3. It needs to be realized that application of all preventive measures does not provide for foolproof protection. “Breaks” will occur and even the most diligent producer should expect to have a problem now and then.
Furthermore, what appears to be a uniform environment is likely to be far from it. Pig diets in an on-farm storage tank might seem like a relatively constant product. However, just within the tank, temperature differentials created by rise and fall of the sun create discreet microenvironments, some of which cause moisture changes sufficient to support germination and growth of mould spores.
Sampling and Analysis
Commercial enzyme immunoassay kits (ELIZA) are available to screen commodities for DON, T-2 toxin, fumonisin B1 and zearalenone. These, TLC and HPLC measurements by Contract laboratories can perform the tests within a reasonable time. It is important to have assurance that such methods have been thoroughly validated.
The most critical part of any analytical procedure for mycotoxins is sampling the commodity in a truly representative manner (Whitaker 2000). For example, published studies of aflatoxins in peanuts have shown that only 6% of the total testing error is due to the analytical method, while 94% is due to sampling and sub sampling problems. It is strongly advised to employ well-validated protocols in getting representative samples of grain for mycotoxin testing. This would ensure that end-use decisions regarding the commodity tested are based on valid results.
Strategies to control mycotoxins in feeds
Broadly speaking, the aim of detoxification is to inactivate or remove the mycotoxin, while leaving no chemical residues from the process. Furthermore, the palatability and nutritional value of the commodity should be maintained. The market price of feed ingredients such as cereal grains requires that detoxification be cost-effective. For lowering mycotoxin contamination of feeds and foods, several strategies have been investigated, and these can be divided into biological, chemical and physical methods.
There are a number of products available for absorbing, binding and detoxifying by bio-transformation of specific mycotoxins. Most of these products are specific in detoxifying one or a few mycotoxins that renders it of utmost importance to identify the type of mycotoxin/s and apply the correct product. As an example, hydrated sodium calcium aluminosilicates (HSCAS) are effective in binding and preventing absorption of aflatoxin, but limited against zearalenone and ochratoxin A, and totally ineffective against trichothecenes such as T-2 toxin, diacetoxyscirpenol and DON.
Yeast cell wall extracts and wall components harbour different adsorption mechanisms for a wider array of mycotoxins. Biological detoxification by enzymatic degradation or biotransformation of mycotoxins is possible, and microorganisms can be used successfully to metabolize certain mycotoxins and thus remove them.
The essence is to identify the mycotoxins (group of mycotoxins) and select an appropriate in-feed product to detoxify the toxin and ensure optimum animal production. If a feed is acquired from a feed producer, ensure that they screen for the mycotoxins and take the appropriate corrective measures.
In summary, troubleshooting mycotoxicologic problems by pig producers and feed suppliers starts with a fundamental approach to quality assurance. This can be achieved by having the fundamental procedure of prevention and detection in place. Procurement of pig feeds or feed ingredients from feed producers/dealers with the necessary quality assurance programs in place is surely a sound base from which to start.
A continued responsibility at the farm level includes basic preventive measures such as storage management, turnaround time of feeds and ingredients and the development of a unit’s history of marker toxin exposures and animal performance. If the responsible, or at least major, toxin is known, then a reasonable decision can be made with respect to an appropriate feed additive. Again suppliers need to be selected who can assist in these decisions and be part of rectifying the problem.
Confirming the animal’s nutritional status and health is always warranted. Finally, success usually comes to those who recognize that no single approach will solve all mycotoxin problems. Fungal infestation, metabolism, and synthesis of toxic products are ever present, and those who recognize this and have a regular program to control it will ultimately succeed in animal productivity, and their economic returns will be higher than those who choose to wait until disaster strikes.