January 2015

Contact details: OlmixZA du Haut du Bois56580 - BréhanFranceTel: +33 297 388 103Email: contact@olmix.comWebsite: www.olmix.com

This mycotoxin supplement is brought to you by

Effective mycotoxin control

Effective mycotoxin control

Editorial

Olmix, founded in 1995, is an expert in clay treatment since its origin, producing several products based on

clays and algae, the other major area of expertise of our company. Clay minerals are used in animal nutrition due to their adsorption properties, contributing to the health of animals as they reduce the bioavailability of heavy metals and mycotoxins. In 2000, Olmix started commercialising its first toxin binder, MT.X, based on two basic clays. However, this prod-uct has a limited performance due to its basic composition, only effective to some mycotoxins such as aflatoxins and ochratoxins. Starting to know the mycotoxin world better, Olmix realised that a wide spectrum toxin binder was needed to solve the problems that Fusarium toxins were causing all around the world. In 2002 Olmix began collaborat-ing with some French research centres such as CNRS, CEVA and Mulhouse University, within the MONALISA frame. The objective was to functionalise a montmorillonite and make it more effective. Following this research in 2006, Olmix launched its premier toxin binder, MT.X+, including Olmix’s patented technology. This innovation provides a very wide spectrum of adsorption, including the larger and more difficult fusariotoxins. The launch of this product has been a success, as Olmix is now commercialis-ing this product in more than 65 countries all over the world, with growing sales every year. In 2012 a new innovation was launched, MMi S, the only microgranulated toxin binder in the market, allowing a better homogeni-zation of the mixture in on-farm rations. The success of the Olmix toxin binders in the market relies on several aspects. First the efficacy of the products, providing extremely good field results a and helping farmers all over the world to reduce the problems derived from the presence of mycotoxins in the raw materials. Secondly, a wide commercial network, based on local distributors that are close to the customers, knowing exactly what their needs are and how to help them and staying in constant contact with the livestock producers. Finally, the technical background of the commercial network and technical support is extremely important. They are always striving to provide new tools, adapt-ed to their specific needs. Our aim is to share our knowledge and to make the lives of our customers easier by solving some of their day-to-day prob-lems. The Olmix Team is committed to their customers, providing some of the latest insights in the next two articles. We hope you find this informa-tion useful. Enjoy reading! Maria Angeles Rodríguez, Technical Service Manager, Olmix

Success of Olmix relies on several aspects

Colophon

I N T E R N A T I O N A L M A G A Z I N E O N A N I M A L N U T R I T I O N , P R O C E S S I N G A N D F E E D M A N A G E M E N T

‘Effective mycotoxin control’ is a supplement of AllAboutFeed, published by Reed Business Information, the Netherlands and Olmix, France. Copyright © 2015.

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Contributing authors: Maria Angeles Rodríguez, Julia Laurain and Eric Marengue

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2 ALLABOUTFEED Volume 23, No. 1, 2015 www.AllAboutFeed.net

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Trichothecenes:Beyond deoxynivalenolTrichothecenes are mycotoxins produced by various Fusarium

species, which can co-occur worldwide in different raw materials.

Feed analyses done in over 2,500 raw material samples will help in

understanding this type of mycotoxin better. These are the results.

By Julia Laurain (technical service, Olmix), Eric Marengue (chromatography manager,

Labocea) and Maria Angeles Rodríguez (technical service manager, Olmix)

Many analyses of the occurrence of myco-toxins in feed matrices have already demon-strated that deoxyniva-

lenol (DON), a type-B trichothecene, is one of the most prevalent feed-associated mycotoxins. It affects many species and can be present in different raw materials (Figure 1). In the past, two large-scale col-laborative studies on mycotoxin contami-nations have been conducted worldwide in 2001 (JECFA) and in Europe in 2003 (SCOOP). The SCOOP study was run on more than 40,000 feed samples, showing that DON was present in 57% of all sam-ples, and provides data on few other tri-chothecenes but not for all feed groups. In 2013, Tangini et al. reviewed all data availa-ble on mycotoxin contamination in forag-es, with very few data on trichothecenes other than DON. Trichothecenes exert their effects through three primary mecha-nisms in animals. First, trichothecenes have been shown to be potent inhibitors of protein synthesis leading to apoptosis. The second impact of trichothecenes is a reduc-tion in the amount of nutrients available for use by the animal due to lower absorp-tion with or without lower feed intake (Pinton et al, 2012). Last but not least, trichothecenes increase immune-depres-sion as described by Surai and Dvorska in 2005. This article summarises the results of LC MS/MS analyses run by Labocea, a French public lab (Accreditation

COFRAC 1-0632) between January 2013 and November 2014. The Labocea data-base contains 2,580 raw materials and feed samples with more than 40 myco-toxins analysed per sample. Feed sam-ples sent to Labocea are not always randomly selected, some of the samples are selected because they represent a high risk. The high number of tri-chothecenes analysed (10) per sample by Labocea, allows better understand-ing of the profile of contamination in trichothecenes per feed matrix. This article focuses only on corn (high-moisture, grain and silage), wheat, bar-ley, grass (silage, haylage and hay) and soy (total of 1,111 samples). This article also reviews available data on tri-chothecenes toxicity.

Deoxynivalenol (DON)DON is the most important trichothecene worldwide. Fusarium graminearum and Fusarium culmorum are the main DON producers; their optimum temperature of growth is 25-27°C and 22-25°C respec-tively (Doohan et al., 2003). In a review, Petska (2007) concludes that all animal species are susceptible to DON according to the rank order of swine > mice > rats > poultry > ruminants. As shown in the SCOOP study, corn is the most affected material by DON. In our study we show that high-moisture corn and silage are more often contaminated with DON than corn grain (dry) (Table 1). The same trend is observed for other trichothecenes. In 2011, Eckard et al. measured the specificity in colonisation of different Fusarium spe-cies. It was concluded that Fusarium graminearum develops more on rachis, stalk and husks than on kernel; this can explain why corn silage is more often con-taminated with DON than corn grain. Fusarium moulds are known as field moulds as they require a high level of water to develop contrary to Aspergillum and

DON

DON DON DON DON DON DON1052.5210.5

Type

MYCOTOXIN

TOXICITY

++

++

++

+

+

+(DON Equivalent)

SENSITIVITY

FEEDPRODUCTS

AT RISK

3ADONType B-trichothecenes Type A-trichothecenes

15ADON/NIV MAS DAST-2/HT-2

Figure 1 - Assessment of the risk associated with trichothecenes according to the molecule, the specie and the raw materials.

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whereas fusarenone X is 24 times less toxic than NIV (Eriksen et al., 2004).

• In short: NIV is produced by other fungi than

DON and remains more toxic.

DON, a limited indicator of type B-TrichothecenesAs described above, DON is the most prevalent feed-associated type B-trichothecene. Fusarium fungi are the natural producers of type B-trichothecenes and always produce several toxins at once, leading to polycontamination with DON as the major toxin. In order to identify the potential correlation between DON and type B-trichothecenes, we used all DON positive samples of Labocea to measure correlation (Table 2). Unsurprisingly, the highest correlations measured are between DON and its derivatives (acetyl forms and DOM-1). Moreover, as NIV is mainly pro-duced by different fungi strains, it is poorly correlated to DON. Because of the high toxicity of NIV and its prevalence, we rec-ommend to control its level in feed as DON is not a good indicator of NIV contamination.

• DON derivatives presence can be estimated for

some raw materials based on DON presence, while

NIV cannot as it is produced by other fungi.

T-2 and HT-2 toxinsT-2 and HT-2 toxins are mainly produced by Fusarium sporotrichioides and Fusarium langsethiae. These moulds grow between -2°C and +35°C with maximum produc-tion at temperatures below 15°C and with high water activity (Thrane et al., 2004). According to JECFA (2001), it is not possi-ble to draw any conclusions on a link between climate and levels of T-2 and HT-2 toxins in cereals. Nevertheless, as

shown on Figure 2, it is admitted that there is a strong trend of higher T-2/HT-2 levels in feed samples from Northern countries. T-2 toxin, HT-2 toxin, deoxynivalenol and nivalenol appear to cause similar effects at the biochemical and cellular levels. The acute toxicity of T-2 and HT-2 tox-ins are within the same range in the gut and T-2 toxin is rapidly metabolised into HT-2 toxin. Based on HT-2, further metabolisation leads to a variety of prod-ucts such as T-2 tetraol (inactive form). Acute toxicity of T-2 toxin is higher than DON (Cavret et al., 2006, Galtier, 2006 and Gunnar Sundstol, 2004): T-2 toxin is between 5 and 10 times more toxic than DON, poultry being more sensitive. In the Labocea database, a high number of samples originates from France and this explains the low percentage of type-A trichothecenes positive samples. As shown in SCOOP study, corn is the most affected feed material. Barley may tend to be more affected than wheat, but this needs to be confirmed.

• In short: T-2 and HT-2 toxins must be carefully

controlled in northern countries as they are highly

toxic for animals especially poultry.

DAS and its derivativesDiacetoxyscirpenol (DAS, also called anguidin) is produced by Fusarium graminearum (Jimenez, 2000) but also Fusarium sporotrichioides (Thrane et al., 2004). DAS is metabolised in animals (by intestinal flora) into 15-monoacetoxy-scirpenol (MAS). Very few data are availa-ble on DAS and MAS toxicity and inci-dence. In a study on rats, Chi (1978) dem-onstrated that DAS is at least twice as toxic as T-2. According to Ademoyero (1991), MAS is 5 times less toxic than DAS on mouth lesions in broilers. Our incidence

data (Table 1) are similar to the one of Schollenberger (2006) as he also observed a higher incidence of MAS than DAS on 125 grains and by-products samples. Type A- and type B-trichothecenes are produced by different fungi strains such that there is no significant correlation between DON level and type A-trichothecenes level. Not all tri-chothecenes are well documented; more data are needed on metabolism, toxicity per specie and incidence. In addition, it is known that depending on climate and region, the profile of mycotoxins may vary significantly. Most of the quality control of trichothecenes in feed materials are based on DON test and decisions on the fate and use of those feed materials are based on a single contamination. Even if DON is often used as an indicator of polycontamination, it often occurs with other trichothecenes that can be more toxic even if they are pre-sent in a lower level. Type A-trichothecenes are also an important threat for animal performances, particularly in poultry, and they deserve special attention in Northern countries.

• In short: Type A-trichothecenes are also composed

of DAS and MAS but with a lower incidence than

T-2 and HT-2 whereas they are more toxic.

ConclusionMycotoxin analyses are often expensive and time consuming. It is good to have some indicators of polycontamination such as DON for some trichothecenes B and T-2/HT-2 for some trichothecenes A. However, in order to understand better the risk of the feed materials that are used in animal feeds and moreover the conse-quences that they may have on animal health and performance, a more extensive analysis is needed. AAF

100%

80%

60%

40%

20%

0%

ESTONIARUSSIA

UKRAINE

HUNGARYSPAIN

POLANDFRANCE

TURKEY

CZECH REPUBLIC

SLOVAKIA

% of

pos

itive

sam

ples

T-2 Toxin DON

Figure 2 - T-2 toxin and DON levels in feed samples in ten European countries.

Pennicillium. Ensiling is a forage preserva-tion method based on spontaneous lactic acid fermentation under anaerobic condi-tions that lowers the pH to a level at which Clostridia and most moulds growth are inhibited (Weinberg and Ashbell, 2003). This process of storage permits preserva-tion of materials with high level of mois-ture. On silages, visible moulds usually develop in the surface layers of the silage, where oxygen is present (Driehuis et al., 2008). But under poor storage conditions, mould can develop in the core of the silo, particularly if silage is exposed to air (eg: drier silages do not pack well; Muck, 1988). Last but not least, it is known that cleaning systems permit removal of broken kernels and thus significantly reduce the DON level, by 10-20% (Bennett et al., 1976). As corn silage and high moisture corn are poorly cleaned before storage, they present a higher risk of containing significant levels of DON than corn grain. It is often consid-ered that soya cannot be affected by DON as Fusarium poorly develops on soya plants. Nevertheless, in our study, 26.5% of the 34 soya samples analysed were positive in DON (Table 1). • In short: DON is the most frequent trichothecene

worldwide, mainly affecting corn. Corn silage

represents a higher risk than corn grain.

Deoxynivalenol derivativesThe major derivatives of DON are either formed by fungi (the acetylated derivatives: 3-acetyl deoxynivalenol (3ADON), 15-acetyl deoxynivalenol (15ADON), or bacteria (the de-epoxide derivatives of DON: DOM-1). The Fusarium fungi pro-duce more or less derivatives depending on their chemotype: the 3ADON chemo-type produces DON and primarily

3ADON while a 15ADON chemotype generates DON and primarily 15ADON. Scientists identified highly divergent pop-ulations of Fusarium producing more or less derivatives, which explains the varia-bility of contamination profile in type B-trichothecenes (Puri et al., 2010). In our study 15ADON is the most frequent derivative as shown by SCOOP, 2004. The 15ADON LD50 on mice was measured as 2.3 folds more toxic than DON (Forsell et al., 1987). In the pig model, Pinton et al., 2012, also concluded that 15ADON is more toxic for epithelial cells than DON. As shown for DON, in our study, high-moisture corn and silage are more often contaminated in 15ADON than corn grain (dry) (Table 1), but contrary to DON, barley is more contaminated in 15ADON than wheat. This is in accord-ance with Rishi et al. (2008) findings showing that all barley isolates had the 15ADON chemotype whereas it was only present in 62% of wheat isolates. The hydroxyl on carbon 3 plays a major role in DON toxicity, leading to the fact that 3 acetyl-DON is less toxic than DON. Following different studies (Danicke et al.,2008; Pinton et al., 2012; Kimura et al., 1998) it is considered that 3ADON is twice less toxic than DON. In all feed matrices, 3ADON is the least frequent derivative (SCOOP 2004; Table 1). Unlike 15ADON, 3ADON is more frequent in corn grain and barley than in high-mois-ture corn, silage and wheat (Table 1). The de-epoxidation of DON is performed by microbial strains under anaerobic condi-tions with fastidious nutritional demands and under specific conditions (pH). This de-epoxidation leads to the production of DOM-1 which is a detoxified metabolite of DON. This de-epoxy reaction mainly

occurs in the animal digestive system, the rumen being the most efficient organ, thanks to the presence of protozoa (Kiessling et al., 1984). A recent finding indicates that some of the anaerobic bac-terial strains de-epoxifying DON may be facultative aerobes (Karlovsky et al., 2011). Some of the feed samples analysed by Labocea, mainly corn-feed and cereals, contain low levels of DOM-1 that could be produced by this facultative aerobes bacteria or under unexpected aerobic conditions due to poor storage conditions (Table 1). On the contrary, grass-feed and soya do not contain any DOM-1.

• In short: DON derivatives can be detoxified into

forms that are safer for the animals but also into

forms that are more toxic for animal health and

performances.

Nivalenol (NIV) and its derivativesNivalenol is a mycotoxin produced main-ly by fungi Fusarium cerealis and Fusarium poae, and to a lesser extent Fusarium cul-morum and Fusarium graminearum (Eriksen, 2003). In contrast to DON, NIV occurs more frequently in years with dry and warm growing seasons (Pettersson, 1995). Nivalenol is more common in Europe, Australia and Asia than in America, where the occurrence of NIV is limited. Both mean levels and incidence of positive samples of NIV are lower than for DON (SCOOP, Eriksen and Alexander, 1998, Table 1). NIV is mainly present in corn-feed and to a lesser extent in wheat and barley and even less frequently in grass-feed (Table 1). The C-4 acetylated derivative of NIV, also called fusarenone X (FusX), is very rare in feed material (Table 1). According to different studies (NTP 2009), NIV is twice as toxic as DON

Type of Feed Number of

samples

Type A-Trichothecenes Type B-Trichothecenes

DAS MAS T-2 Toxin HT-2 Toxin T-2 tetraol DON DOM-1 15ADON 3ADON NIV Fus X

Limit of quantification (ppm) 0.010 0.010 0.010 0.010 0.020 0.010 0.010 0.010 0.010 0.010 0.010

All samples 1111 0.5% 25.2% 7.4% 34.6% 17.9% 85% 4% 57% 18% 58% 5%

Corn silage 337 0% 65% 4% 57% 38% 99% 3% 86% 18% 67% 0%

Corn grain 230 1% 10% 22% 33% 11% 84% 4% 73% 38% 67% 21%

High moisture grain 87 2% 33% 3% 51% 9% 100% 11% 99% 16% 74% 5%

Wheat 226 0% 1% 1% 12% 5% 92% 2% 19% 8% 52% 0%

Barley 98 0% 4% 9% 35% 17% 91% 7% 42% 21% 62% 0%

Grass silage 31 0% 6% 0% 6% 3% 19% 0% 3% 0% 16% 0%

Grass haylage 10 0% 6% 0% 6% 3% 19% 0% 3% 0% 16% 0%

Hay 58 0% 0% 5% 7% 10% 31% 0% 3% 0% 22% 2%

Soya 34 0% 0% 0% 9% 3% 26% 0% 3% 0% 6% 0%

Table 1 - Trends seen in different toxins in different types of feed. Feed analysis done by Labocea.

Eric Marengue in front of Labocea HPLC-MS machines, explaining the functioning of this state-of-the-art equipment able to detect low levels of mycotoxins in different feed or food matrices.

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whereas fusarenone X is 24 times less toxic than NIV (Eriksen et al., 2004).

• In short: NIV is produced by other fungi than

DON and remains more toxic.

DON, a limited indicator of type B-TrichothecenesAs described above, DON is the most prevalent feed-associated type B-trichothecene. Fusarium fungi are the natural producers of type B-trichothecenes and always produce several toxins at once, leading to polycontamination with DON as the major toxin. In order to identify the potential correlation between DON and type B-trichothecenes, we used all DON positive samples of Labocea to measure correlation (Table 2). Unsurprisingly, the highest correlations measured are between DON and its derivatives (acetyl forms and DOM-1). Moreover, as NIV is mainly pro-duced by different fungi strains, it is poorly correlated to DON. Because of the high toxicity of NIV and its prevalence, we rec-ommend to control its level in feed as DON is not a good indicator of NIV contamination.

• DON derivatives presence can be estimated for

some raw materials based on DON presence, while

NIV cannot as it is produced by other fungi.

T-2 and HT-2 toxinsT-2 and HT-2 toxins are mainly produced by Fusarium sporotrichioides and Fusarium langsethiae. These moulds grow between -2°C and +35°C with maximum produc-tion at temperatures below 15°C and with high water activity (Thrane et al., 2004). According to JECFA (2001), it is not possi-ble to draw any conclusions on a link between climate and levels of T-2 and HT-2 toxins in cereals. Nevertheless, as

shown on Figure 2, it is admitted that there is a strong trend of higher T-2/HT-2 levels in feed samples from Northern countries. T-2 toxin, HT-2 toxin, deoxynivalenol and nivalenol appear to cause similar effects at the biochemical and cellular levels. The acute toxicity of T-2 and HT-2 tox-ins are within the same range in the gut and T-2 toxin is rapidly metabolised into HT-2 toxin. Based on HT-2, further metabolisation leads to a variety of prod-ucts such as T-2 tetraol (inactive form). Acute toxicity of T-2 toxin is higher than DON (Cavret et al., 2006, Galtier, 2006 and Gunnar Sundstol, 2004): T-2 toxin is between 5 and 10 times more toxic than DON, poultry being more sensitive. In the Labocea database, a high number of samples originates from France and this explains the low percentage of type-A trichothecenes positive samples. As shown in SCOOP study, corn is the most affected feed material. Barley may tend to be more affected than wheat, but this needs to be confirmed.

• In short: T-2 and HT-2 toxins must be carefully

controlled in northern countries as they are highly

toxic for animals especially poultry.

DAS and its derivativesDiacetoxyscirpenol (DAS, also called anguidin) is produced by Fusarium graminearum (Jimenez, 2000) but also Fusarium sporotrichioides (Thrane et al., 2004). DAS is metabolised in animals (by intestinal flora) into 15-monoacetoxy-scirpenol (MAS). Very few data are availa-ble on DAS and MAS toxicity and inci-dence. In a study on rats, Chi (1978) dem-onstrated that DAS is at least twice as toxic as T-2. According to Ademoyero (1991), MAS is 5 times less toxic than DAS on mouth lesions in broilers. Our incidence

data (Table 1) are similar to the one of Schollenberger (2006) as he also observed a higher incidence of MAS than DAS on 125 grains and by-products samples. Type A- and type B-trichothecenes are produced by different fungi strains such that there is no significant correlation between DON level and type A-trichothecenes level. Not all tri-chothecenes are well documented; more data are needed on metabolism, toxicity per specie and incidence. In addition, it is known that depending on climate and region, the profile of mycotoxins may vary significantly. Most of the quality control of trichothecenes in feed materials are based on DON test and decisions on the fate and use of those feed materials are based on a single contamination. Even if DON is often used as an indicator of polycontamination, it often occurs with other trichothecenes that can be more toxic even if they are pre-sent in a lower level. Type A-trichothecenes are also an important threat for animal performances, particularly in poultry, and they deserve special attention in Northern countries.

• In short: Type A-trichothecenes are also composed

of DAS and MAS but with a lower incidence than

T-2 and HT-2 whereas they are more toxic.

ConclusionMycotoxin analyses are often expensive and time consuming. It is good to have some indicators of polycontamination such as DON for some trichothecenes B and T-2/HT-2 for some trichothecenes A. However, in order to understand better the risk of the feed materials that are used in animal feeds and moreover the conse-quences that they may have on animal health and performance, a more extensive analysis is needed. AAF

100%

80%

60%

40%

20%

0%

ESTONIARUSSIA

UKRAINE

HUNGARYSPAIN

POLANDFRANCE

TURKEY

CZECH REPUBLIC

SLOVAKIA

% of

pos

itive

sam

ples

T-2 Toxin DON

Figure 2 - T-2 toxin and DON levels in feed samples in ten European countries.

Pennicillium. Ensiling is a forage preserva-tion method based on spontaneous lactic acid fermentation under anaerobic condi-tions that lowers the pH to a level at which Clostridia and most moulds growth are inhibited (Weinberg and Ashbell, 2003). This process of storage permits preserva-tion of materials with high level of mois-ture. On silages, visible moulds usually develop in the surface layers of the silage, where oxygen is present (Driehuis et al., 2008). But under poor storage conditions, mould can develop in the core of the silo, particularly if silage is exposed to air (eg: drier silages do not pack well; Muck, 1988). Last but not least, it is known that cleaning systems permit removal of broken kernels and thus significantly reduce the DON level, by 10-20% (Bennett et al., 1976). As corn silage and high moisture corn are poorly cleaned before storage, they present a higher risk of containing significant levels of DON than corn grain. It is often consid-ered that soya cannot be affected by DON as Fusarium poorly develops on soya plants. Nevertheless, in our study, 26.5% of the 34 soya samples analysed were positive in DON (Table 1). • In short: DON is the most frequent trichothecene

worldwide, mainly affecting corn. Corn silage

represents a higher risk than corn grain.

Deoxynivalenol derivativesThe major derivatives of DON are either formed by fungi (the acetylated derivatives: 3-acetyl deoxynivalenol (3ADON), 15-acetyl deoxynivalenol (15ADON), or bacteria (the de-epoxide derivatives of DON: DOM-1). The Fusarium fungi pro-duce more or less derivatives depending on their chemotype: the 3ADON chemo-type produces DON and primarily

3ADON while a 15ADON chemotype generates DON and primarily 15ADON. Scientists identified highly divergent pop-ulations of Fusarium producing more or less derivatives, which explains the varia-bility of contamination profile in type B-trichothecenes (Puri et al., 2010). In our study 15ADON is the most frequent derivative as shown by SCOOP, 2004. The 15ADON LD50 on mice was measured as 2.3 folds more toxic than DON (Forsell et al., 1987). In the pig model, Pinton et al., 2012, also concluded that 15ADON is more toxic for epithelial cells than DON. As shown for DON, in our study, high-moisture corn and silage are more often contaminated in 15ADON than corn grain (dry) (Table 1), but contrary to DON, barley is more contaminated in 15ADON than wheat. This is in accord-ance with Rishi et al. (2008) findings showing that all barley isolates had the 15ADON chemotype whereas it was only present in 62% of wheat isolates. The hydroxyl on carbon 3 plays a major role in DON toxicity, leading to the fact that 3 acetyl-DON is less toxic than DON. Following different studies (Danicke et al.,2008; Pinton et al., 2012; Kimura et al., 1998) it is considered that 3ADON is twice less toxic than DON. In all feed matrices, 3ADON is the least frequent derivative (SCOOP 2004; Table 1). Unlike 15ADON, 3ADON is more frequent in corn grain and barley than in high-mois-ture corn, silage and wheat (Table 1). The de-epoxidation of DON is performed by microbial strains under anaerobic condi-tions with fastidious nutritional demands and under specific conditions (pH). This de-epoxidation leads to the production of DOM-1 which is a detoxified metabolite of DON. This de-epoxy reaction mainly

occurs in the animal digestive system, the rumen being the most efficient organ, thanks to the presence of protozoa (Kiessling et al., 1984). A recent finding indicates that some of the anaerobic bac-terial strains de-epoxifying DON may be facultative aerobes (Karlovsky et al., 2011). Some of the feed samples analysed by Labocea, mainly corn-feed and cereals, contain low levels of DOM-1 that could be produced by this facultative aerobes bacteria or under unexpected aerobic conditions due to poor storage conditions (Table 1). On the contrary, grass-feed and soya do not contain any DOM-1.

• In short: DON derivatives can be detoxified into

forms that are safer for the animals but also into

forms that are more toxic for animal health and

performances.

Nivalenol (NIV) and its derivativesNivalenol is a mycotoxin produced main-ly by fungi Fusarium cerealis and Fusarium poae, and to a lesser extent Fusarium cul-morum and Fusarium graminearum (Eriksen, 2003). In contrast to DON, NIV occurs more frequently in years with dry and warm growing seasons (Pettersson, 1995). Nivalenol is more common in Europe, Australia and Asia than in America, where the occurrence of NIV is limited. Both mean levels and incidence of positive samples of NIV are lower than for DON (SCOOP, Eriksen and Alexander, 1998, Table 1). NIV is mainly present in corn-feed and to a lesser extent in wheat and barley and even less frequently in grass-feed (Table 1). The C-4 acetylated derivative of NIV, also called fusarenone X (FusX), is very rare in feed material (Table 1). According to different studies (NTP 2009), NIV is twice as toxic as DON

Type of Feed Number of

samples

Type A-Trichothecenes Type B-Trichothecenes

DAS MAS T-2 Toxin HT-2 Toxin T-2 tetraol DON DOM-1 15ADON 3ADON NIV Fus X

Limit of quantification (ppm) 0.010 0.010 0.010 0.010 0.020 0.010 0.010 0.010 0.010 0.010 0.010

All samples 1111 0.5% 25.2% 7.4% 34.6% 17.9% 85% 4% 57% 18% 58% 5%

Corn silage 337 0% 65% 4% 57% 38% 99% 3% 86% 18% 67% 0%

Corn grain 230 1% 10% 22% 33% 11% 84% 4% 73% 38% 67% 21%

High moisture grain 87 2% 33% 3% 51% 9% 100% 11% 99% 16% 74% 5%

Wheat 226 0% 1% 1% 12% 5% 92% 2% 19% 8% 52% 0%

Barley 98 0% 4% 9% 35% 17% 91% 7% 42% 21% 62% 0%

Grass silage 31 0% 6% 0% 6% 3% 19% 0% 3% 0% 16% 0%

Grass haylage 10 0% 6% 0% 6% 3% 19% 0% 3% 0% 16% 0%

Hay 58 0% 0% 5% 7% 10% 31% 0% 3% 0% 22% 2%

Soya 34 0% 0% 0% 9% 3% 26% 0% 3% 0% 6% 0%

Table 1 - Trends seen in different toxins in different types of feed. Feed analysis done by Labocea.

Eric Marengue in front of Labocea HPLC-MS machines, explaining the functioning of this state-of-the-art equipment able to detect low levels of mycotoxins in different feed or food matrices.

Sponsored articleSponsored article

www.AllAboutFeed.net www.AllAboutFeed.net6 ALLABOUTFEED 2015 ALLABOUTFEED 2015 7

Detection of mycotoxins is not always easy.

Olmix has therefore developed a predictive

model of mycotoxin risk for dairy, swine and

poultry. Here we present the methodology used

to build the predictive model. The dairy model

will serve as an example.

By Maria Angeles Rodriguez (technical service manager, Olmix) and Julia Laurain

(technical service, Olmix)

Mycotoxins are toxic compounds produced by various fungal species that grow on various agricultural

commodities (Cullen and Newberne, 1994). The death of 100,000 turkey poults and other poultry in the UK just before Christmas in 1960 (Allcroft and Carnaghan, 1962) was traced to a toxic contaminant (later determined as afla-toxin) present in ground nut meal used in the diet. This incident illustrated the potential threat posed by mycotoxins. The toxicity of mycotoxins varies, rang-ing from hepatotoxic or even carcino-genic (aflatoxins) effects, to estrogenic (zearalenone), immunotoxic (patulin, trichothecenes, fumonisins), nephrotoxic (ochratoxin A) and neurotoxic (tremor-gens, ergot alkaloids) effects. In the field,

one of the most important effects of mycotoxins (mainly trichothecenes) is an alteration of feed conversion ratio and growth due to lower nutrient absorption (with or without feed intake reduction). The losses in performance, the increased incidence of disease and the reduced reproductive performance are of great economic impact in the field. As a con-sequence, it is very important to detect and protect animals from mycotoxin contamination in order to avoid this eco-nomic loss. Mycotoxin detection in ani-mal husbandry is not easy, as one of the characteristics of mycotoxins is their ability to compromise the immune response and consequently, to reduce resistance to infectious diseases. This is now widely considered to be the most important effect of mycotoxins, particu-larly in developing countries (FAO,

2001). This suppression of the immune function, even at levels that do not cause overt clinical mycotoxicosis, provokes symptoms that are common and can be due to other pathologies. So it is likely that farmers and technicians don’t think about mycotoxins as a primary cause of the problems that they are facing on the farm. In order to help them to detect mycotoxin contamina-tion, Olmix has developed a predictive model of mycotoxin risk for dairy, swine and poultry. This article presents the methodology used to build the pre-dictive model and the dairy model is presented as an example. Model based on risk factorsMycotoxins can be formed in the field pre-harvest (fusariotoxins: tri-chothecenes, fumonisins and zearale-nones) and/or under poor storage condi-tions, post-harvest (aflatoxins and ochra-toxins mainly). Depending on field and storage conditions, the occurrence of mycotoxins will be more or less impor-tant. The predictive model developed is based on risk factors for presence of mycotoxins in the feed, which were defined by a literature review (Table 1) and classified into three categories: agri-cultural practices, storage conditions of the feed and disorders observed on the animals. Each risk factor scored by “yes” is weighed by a coefficient. This coeffi-cient is defined by the degree of correla-tion observed between the risk factor and the level of mycotoxins in the feed as described in the literature (Table 1). The sum of coefficients for each category is itself weighed and used to calculate the probability of having a significant con-tamination by mycotoxins in the diet (%)

(Table 1). After literature review, the designed predictive model was tested in 18 farms in order to validate its accuracy. In each farm the predictive model was applied with the farmer and a sample of the diet was taken according to sampling recommendations for mycotoxin analysis (Whitaker et al., 2005). Each diet sample was analysed by multi-residues methods LC MS/MS (COFRAC 1-0632). In order to measure the accuracy of the predic-tive model (Figure 1), we calculated the correlation between the predictive model scores and the sum of fusariotox-ins (trichothecenes, zearalenone and fumonisines) via a calculation of the coefficient of determination (R2). With a small number of farms, an R2 of 0.70 was obtained, meaning that the correlation coefficient between the model and the chemical analysis is 0.83. The objective of the model is not to predict the value of contamination in the diet but to cal-culate a probability of significant or not occurrence of mycotoxins in the feed. Thus the choice of risk factors and their coefficient seems relevant. It was thus further published during the 3R (INRA Ruminant Congress). The model is freely available on the internet on Olmix web-site. Analysis on complete feedAccording to the correlation study, this predictive model is a relevant tool in order to identify situations at risk regard-ing mycotoxin contaminations. This tool is the starting point of mycotoxin diagno-sis (Figure 2). Nevertheless, the chemical analysis remains the most efficient tool to confirm mycotoxin contamination and to measure the accurate level of the different mycotoxins present in the feed. When the score obtained with the predictive model is over 50%, it is strongly recommended

to perform a mycotoxin analysis on the complete feed or TMR to confirm the presence of mycotoxins in the feed (Whitaker et al., 2005). In such case, it is advised to take several small subsam-ples from the animals’ feeders and mix them together to have at least 1 kg of a final sample to send for analysis. Most official analytical methods are chroma-tographic. Alternative strategies such as enzyme linked immunosorbent assay (ELISA) are also largely used as they are easy to implement, less expensive and quicker. Chromatographic methods are very reliable and can be used on any kind

Mycotoxin Risk Evaluator: A new predictive model

of feed matrices and mix of feed. Matrix effect or matrix interference commonly occurs in ELISA methods resulting in underestimations or overestimations in mycotoxin concentrations in complete feed or TMR samples. ELISA methods are relia-ble on single material matrices and not always recommended for complex matrices such as complete feed and TMR (Zheng et al., 2006). Whichever analytical methods is used, the sampling procedure remains the most critical point. ConclusionOnce it is known that mycotoxins are present, and since polycontamination is the common situation, the use of a wide spectrum toxin binder is the most helpful solution to stop or mitigate the problems on the farm. The Olmix Technical Service Team is at your disposal to help you choose the best solutions and dosage based on the type of contamination and problems present on your farm. AAF

Please note: Local regulations should be consulted

concerning the status of these products in the country

of destination. All information only for export outside

Europe, USA and Canada.

TROUBLES ON HERD

Lower health andperformances

Evaluation of mycotoxinsrisk with Evaluator Analyse feed Use a wide

spectrum binder

SUSPICION21 3 4CONFIRMATION SOLUTION

Figure 2 – Mycotoxin risk management involves different steps, including the risk evaluation.

0102030405060708090

100

0 0.5 1.51 2.5 3.52

R2 = 0.70225

3

Prob

abili

ty o

btai

ned

with

pre

dict

ive

mod

el (%

)

Fusariotoxins contamination TCT+ZEA+FUM (mg/kg)

Figure 1 – Relation between predictive model scores and the sum of fusariotoxins in the diet.

Risk Factor Coefficient References

Fiel

d

Corn is produced on single-crop farming field ++ Coulumbe, 1993

Corn fields are not ploughed +++ Gourdain et al, 2008

Fusarium moulds/diseases were observed on field +++ Obst et al, 1997

Silage was harvested late +++ Reyneri et Blandino, 2003

Grass silage was cropped after corn ++ Yi et al, 2002

Stor

age

Moulds are present (red, blue, white or black…) +++ AFSSA 2009

Troubles to press the silage (high DM, speed of harvesting) ++ Betina, 1989

Silage is warm or took a long time to cool down +++ Haggblom, 1990

Grass silage is not as clean as usual ++ Gareis et al, 1994

Silage front is consumed too slowly + Pelhate, 1987

The h

erd

Insufficient feed intake ++ AFSSA 2009

Lower milk production than the diet potential ++ Bamburg et Strong, 1971

Decreasing or unsatisfying body condition + Bonnet et al, 2002

Unsatisfaying coat condition + Columbe 1993

Low chewing activity +++ Fink-Gremmels, 2008

Significant increase in somatic cells or mastitis +++ Keese et al, 2009

Increase of lameness and leg troubles (swollen hooves, joints, dermatitis…) ++ Klang et al, 1978

Increase of metabolic and pathologic troubles (abomasum displacement, SARA, fatty

liver,metritis, jejunal hemorrhage…)

+++ Noller et al, 1979

Increased turnover (high % of heifers) +

Koroteleva et al, 2009

Too soft/liquid faeces +++

Riley et al,1998

Increase in milk urea +

Surai et Drovska, 2005

Weak calves (diarrhoea, stunted growth, oral and dermal lesions…) +

Trenholm et al, 1985

Fertility troubles (metritis, cysts, placenta retention...) ++

Withlow and Hagler, 1987

Low reproduction performance (heat detection, sucess at 1st AI…) +++

The above troubles started with the use of new forages or raw materials +++

Table 1 – Literature review summary of risk factors and sum of coefficients for each category.

Sponsored articleSponsored article

www.AllAboutFeed.net www.AllAboutFeed.net6 ALLABOUTFEED 2015 ALLABOUTFEED 2015 7

Detection of mycotoxins is not always easy.

Olmix has therefore developed a predictive

model of mycotoxin risk for dairy, swine and

poultry. Here we present the methodology used

to build the predictive model. The dairy model

will serve as an example.

By Maria Angeles Rodriguez (technical service manager, Olmix) and Julia Laurain

(technical service, Olmix)

Mycotoxins are toxic compounds produced by various fungal species that grow on various agricultural

commodities (Cullen and Newberne, 1994). The death of 100,000 turkey poults and other poultry in the UK just before Christmas in 1960 (Allcroft and Carnaghan, 1962) was traced to a toxic contaminant (later determined as afla-toxin) present in ground nut meal used in the diet. This incident illustrated the potential threat posed by mycotoxins. The toxicity of mycotoxins varies, rang-ing from hepatotoxic or even carcino-genic (aflatoxins) effects, to estrogenic (zearalenone), immunotoxic (patulin, trichothecenes, fumonisins), nephrotoxic (ochratoxin A) and neurotoxic (tremor-gens, ergot alkaloids) effects. In the field,

one of the most important effects of mycotoxins (mainly trichothecenes) is an alteration of feed conversion ratio and growth due to lower nutrient absorption (with or without feed intake reduction). The losses in performance, the increased incidence of disease and the reduced reproductive performance are of great economic impact in the field. As a con-sequence, it is very important to detect and protect animals from mycotoxin contamination in order to avoid this eco-nomic loss. Mycotoxin detection in ani-mal husbandry is not easy, as one of the characteristics of mycotoxins is their ability to compromise the immune response and consequently, to reduce resistance to infectious diseases. This is now widely considered to be the most important effect of mycotoxins, particu-larly in developing countries (FAO,

2001). This suppression of the immune function, even at levels that do not cause overt clinical mycotoxicosis, provokes symptoms that are common and can be due to other pathologies. So it is likely that farmers and technicians don’t think about mycotoxins as a primary cause of the problems that they are facing on the farm. In order to help them to detect mycotoxin contamina-tion, Olmix has developed a predictive model of mycotoxin risk for dairy, swine and poultry. This article presents the methodology used to build the pre-dictive model and the dairy model is presented as an example. Model based on risk factorsMycotoxins can be formed in the field pre-harvest (fusariotoxins: tri-chothecenes, fumonisins and zearale-nones) and/or under poor storage condi-tions, post-harvest (aflatoxins and ochra-toxins mainly). Depending on field and storage conditions, the occurrence of mycotoxins will be more or less impor-tant. The predictive model developed is based on risk factors for presence of mycotoxins in the feed, which were defined by a literature review (Table 1) and classified into three categories: agri-cultural practices, storage conditions of the feed and disorders observed on the animals. Each risk factor scored by “yes” is weighed by a coefficient. This coeffi-cient is defined by the degree of correla-tion observed between the risk factor and the level of mycotoxins in the feed as described in the literature (Table 1). The sum of coefficients for each category is itself weighed and used to calculate the probability of having a significant con-tamination by mycotoxins in the diet (%)

(Table 1). After literature review, the designed predictive model was tested in 18 farms in order to validate its accuracy. In each farm the predictive model was applied with the farmer and a sample of the diet was taken according to sampling recommendations for mycotoxin analysis (Whitaker et al., 2005). Each diet sample was analysed by multi-residues methods LC MS/MS (COFRAC 1-0632). In order to measure the accuracy of the predic-tive model (Figure 1), we calculated the correlation between the predictive model scores and the sum of fusariotox-ins (trichothecenes, zearalenone and fumonisines) via a calculation of the coefficient of determination (R2). With a small number of farms, an R2 of 0.70 was obtained, meaning that the correlation coefficient between the model and the chemical analysis is 0.83. The objective of the model is not to predict the value of contamination in the diet but to cal-culate a probability of significant or not occurrence of mycotoxins in the feed. Thus the choice of risk factors and their coefficient seems relevant. It was thus further published during the 3R (INRA Ruminant Congress). The model is freely available on the internet on Olmix web-site. Analysis on complete feedAccording to the correlation study, this predictive model is a relevant tool in order to identify situations at risk regard-ing mycotoxin contaminations. This tool is the starting point of mycotoxin diagno-sis (Figure 2). Nevertheless, the chemical analysis remains the most efficient tool to confirm mycotoxin contamination and to measure the accurate level of the different mycotoxins present in the feed. When the score obtained with the predictive model is over 50%, it is strongly recommended

to perform a mycotoxin analysis on the complete feed or TMR to confirm the presence of mycotoxins in the feed (Whitaker et al., 2005). In such case, it is advised to take several small subsam-ples from the animals’ feeders and mix them together to have at least 1 kg of a final sample to send for analysis. Most official analytical methods are chroma-tographic. Alternative strategies such as enzyme linked immunosorbent assay (ELISA) are also largely used as they are easy to implement, less expensive and quicker. Chromatographic methods are very reliable and can be used on any kind

Mycotoxin Risk Evaluator: A new predictive model

of feed matrices and mix of feed. Matrix effect or matrix interference commonly occurs in ELISA methods resulting in underestimations or overestimations in mycotoxin concentrations in complete feed or TMR samples. ELISA methods are relia-ble on single material matrices and not always recommended for complex matrices such as complete feed and TMR (Zheng et al., 2006). Whichever analytical methods is used, the sampling procedure remains the most critical point. ConclusionOnce it is known that mycotoxins are present, and since polycontamination is the common situation, the use of a wide spectrum toxin binder is the most helpful solution to stop or mitigate the problems on the farm. The Olmix Technical Service Team is at your disposal to help you choose the best solutions and dosage based on the type of contamination and problems present on your farm. AAF

Please note: Local regulations should be consulted

concerning the status of these products in the country

of destination. All information only for export outside

Europe, USA and Canada.

TROUBLES ON HERD

Lower health andperformances

Evaluation of mycotoxinsrisk with Evaluator Analyse feed Use a wide

spectrum binder

SUSPICION21 3 4CONFIRMATION SOLUTION

Figure 2 – Mycotoxin risk management involves different steps, including the risk evaluation.

0102030405060708090

100

0 0.5 1.51 2.5 3.52

R2 = 0.70225

3

Prob

abili

ty o

btai

ned

with

pre

dict

ive

mod

el (%

)

Fusariotoxins contamination TCT+ZEA+FUM (mg/kg)

Figure 1 – Relation between predictive model scores and the sum of fusariotoxins in the diet.

Risk Factor Coefficient References

Fiel

d

Corn is produced on single-crop farming field ++ Coulumbe, 1993

Corn fields are not ploughed +++ Gourdain et al, 2008

Fusarium moulds/diseases were observed on field +++ Obst et al, 1997

Silage was harvested late +++ Reyneri et Blandino, 2003

Grass silage was cropped after corn ++ Yi et al, 2002

Stor

age

Moulds are present (red, blue, white or black…) +++ AFSSA 2009

Troubles to press the silage (high DM, speed of harvesting) ++ Betina, 1989

Silage is warm or took a long time to cool down +++ Haggblom, 1990

Grass silage is not as clean as usual ++ Gareis et al, 1994

Silage front is consumed too slowly + Pelhate, 1987

The h

erd

Insufficient feed intake ++ AFSSA 2009

Lower milk production than the diet potential ++ Bamburg et Strong, 1971

Decreasing or unsatisfying body condition + Bonnet et al, 2002

Unsatisfaying coat condition + Columbe 1993

Low chewing activity +++ Fink-Gremmels, 2008

Significant increase in somatic cells or mastitis +++ Keese et al, 2009

Increase of lameness and leg troubles (swollen hooves, joints, dermatitis…) ++ Klang et al, 1978

Increase of metabolic and pathologic troubles (abomasum displacement, SARA, fatty

liver,metritis, jejunal hemorrhage…)

+++ Noller et al, 1979

Increased turnover (high % of heifers) +

Koroteleva et al, 2009

Too soft/liquid faeces +++

Riley et al,1998

Increase in milk urea +

Surai et Drovska, 2005

Weak calves (diarrhoea, stunted growth, oral and dermal lesions…) +

Trenholm et al, 1985

Fertility troubles (metritis, cysts, placenta retention...) ++

Withlow and Hagler, 1987

Low reproduction performance (heat detection, sucess at 1st AI…) +++

The above troubles started with the use of new forages or raw materials +++

Table 1 – Literature review summary of risk factors and sum of coefficients for each category.

January 2015

Contact details: OlmixZA du Haut du Bois56580 - BréhanFranceTel: +33 297 388 103Email: contact@olmix.comWebsite: www.olmix.com

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