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summary

Co-occurrence, modified mycotoxins, emerging mycotoxins and interactions

Feed preservatives for mould and mycotoxins control are they resilient enough?

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Fumonisins the underrated mycotoxins in poultry, livestock and humans

Clinical and pathoanatomical effects of mycotoxins in animals

Biomonitoring mycotoxins in production animals perspectives & challenges Mycotoxins how do they get into aquafeed?

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An underestimated challenge for the aquafeed industry

Effects of the main mycotoxins on poultry production parameters

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Review of investigations regarding mycotoxin control in poultry throughout the years

The effects of mycotoxins on swine reproduction

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Mycotoxins effects and mechanisms of immune modulation in swine

Mycotoxins and disruption of vaccination efficacy in swine Mycotoxins and dairy cattle

Co-occurrence, MODIFIED MYCOTOXINS, EMERGING MYCOTOXINS and interactions

Mariano Gorrachategui Animal nutrition consultant and president of CESFAC

Co-occurrence of mycotoxins We know that a certain mycotoxin can be produced by several types of fungi and that the same toxigenic fungus can produce several mycotoxins. Moreover, nowadays there are different sources of raw materials. Cereals, plant protein crops, and oilseeds grow and are harvested in a diverse range of climatological conditions, determining the growth of the fungi and the toxins produced. The storage and transport conditions also affect the fungus and toxins that can be produced during that time.

Under these conditions, it’s easy to understand that the odds of finding only one mycotoxin contaminating raw materials or feed are slim. Thus, the term “cooccurrence” is increasingly being used when referring to the simultaneous presence of two or more mycotoxins in the same sample.

There are many publications in literature that highlight the co-occurrence of mycotoxins: In Germany, Goertz et al. (2010) found corn to be simultaneously contaminated with at least 14

Fusarium mycotoxins: Deoxynivalenol (DON) and its acetylated forms Zearalenone (ZEA) Moniliformin (MON) Beauvericin (BEA) Nivalenol (NIV)

Eniantins (ENNs)

Other studies have revealed the

Fumonisins (FBs)

simultaneous presence of several

HT2 Toxin

mycotoxins in samples from

Streit and col. (2013a) indicated in their global monitoring report that 72% of the samples of feed and raw materials were contaminated with more than one mycotoxin. The same authors (Streit et al.,

2013b) observed 83 samples of feed and raw materials to be contaminated with 7 to 69 mycotoxins per sample, having detected up to 169 different compounds.

European countries (Almeida et al.,

2011; Blajet-Kosicka et al., 2014; Driehuid et al., 2008; Labuda et al., 2005a, 2005b; Monbaliu et al., 2010), finding a high percentage of samples to be contaminated with trichothecenes (DON, AcDon, T2, HT2) and FBs at the same time, as well as with ZEA in many cases.

In Spain, the study published by Ibáñez-Bea et al. (2012)

DON 95% of the samples

regarding the simultaneous presence of AFB1, ZEA, and OTA in samples of barley harvested in 2007 and 2008, revealed:

AFB1 + ZEA + OTA 27% of the samples

The presence of the three mycotoxins in 27% of the samples The presence of AFB1, together

AFB1 + ZEA y/o OTA 43% of the samples

Mycotoxin contamination analysis in barley 2007-2008

AFB1 + OTA + DON 29% of the samples AFB1 + OTA + DON + ZEA 26% of the samples

with any one of the other two in 43% of the samples The same authors detected DON in 95% of the samples and two mycotoxins from this group in 43% of the samples.

More recent data (Biomin, 2017) from the analysis of 1.378 samples indicated that 94% of the samples contained more than 10 mycotoxins

Taken together, the results of these studies reveal that 96% of the samples contained three or more mycotoxins. The most frequent combinations are: AFB1, OTA, and DON in

and metabolites and that the average was of 28 mycotoxins. In another study, Raj et al. (2017) after screening 113 samples of maize from Serbia and Bosnia and Herzegovina for mycotoxin contamination, found 28% of the samples to be contaminated with more than one mycotoxin. In another study the authors reported 76% samples contaminated with one or more mycotoxins (Raj et al., 2019).

29% of the samples AFB1, OTA, DON, and ZEA

Regarding fodder, recently there has also been

en 26% of the samples

data published (Panasiuk et al., 2019).

Modified mycotoxins In addition to co-occurrence, there is another phenomenon, the presence of the so-called “masked mycotoxins”. This term was first coined by Gareis et al. (1990) in reference to some cases of mycotoxicosis in which, the clinical signs observed in the animals couldn’t be explained by the low content of the mycotoxins detected in the feed.

Masked mycotoxins are defined as “mycotoxins that are not detectable through standard routine analytical techniques”. Years later, Berthiller et al., (2013) and, particularly, Rychlik et al. (2016) introduced the terms following terms: “Matrix-associated mycotoxins” refer to mycotoxins that are associated with oligosaccharides and starch, and that are physically trapped or bound by covalent bonds, as in the case of Fumonisins. “Modified mycotoxins” including: “Biologically” modified mycotoxins, for example, by conjugation, with polar compounds, mainly β-glycoside, sulfate or even glutathione, and that would fall under the denomination of “masked”. “Chemically” modified mycotoxins, produced as a consequence of thermal processes or other kinds of processes that take place during the production of feed.

EFSA (2014a) refers to “modified mycotoxins” as all the forms that have been structurally modified in relation to their “parental compound” o the free mycotoxin.

Emerging mycotoxins The development of methods based on chromatographic techniques and mass spectrophotometry enables us to reliably detect smaller quantities of more and more compounds. Therefore, we are discovering new molecules that may have toxic effects and interactions that we know little about, making it important to study them in order to produce safe food. We are referring to the so-called “emerging mycotoxins”. Although this term hasn’t been clearly

Aspergillus metabolites such as sterigmatocystin (STE) and emodin (EMO). The Penicillium metabolite, mycophenolic acid (MPA).

Alternaria metabolites, that include more than 70 toxins. The best-known ones are alternariol (AOH), monomethyl alternariol ether (AME), altenuene (ALT), altertoxin (ATX) and tenuazonic acid (TeA) (GruberDorninger et al., 2017).

defined, in general, we refer to:

Fusarium metabolites such as eniantins (ENNs), beauvericin (BEA), moniliformin (MON), fusaproliferin (FP), fusidic acid (FA), culmorin (CUL), and butenolide (BUT).

This list is a non-exclusive list and, although these mycotoxins are not routinely screened for at the moment and they are not contemplated in animal feed legislation, there can be issues of toxicity or interactions between them.

Information relative to animal

What do we know about the toxicity of emerging mycotoxins?

they have not been linked to any transcendent pathologies in

ENNs & BEA

animals (Marín García, 2010). According to EFSA (2014b), there is a low probability that acute exposure to BEA and ENNs has adverse effects on the health of livestock and companion animals. It is also improbable to see adverse effects due to chronic exposure

ALTERNARIA METABOLITES

ENNs colonize cereals and can accumulate in grains. However,

sensitivity to Alternaria toxins (EFSA,

2011) is scarce and doesn’t allow to estimate the levels of tolerance for individual toxins and their combinations. There is only some information about toxicity in birds for evaluating the risk of these mycotoxins. EFSA concludes that it is improbable for AOH to pose a risk in broilers, but it is not entirely possible to exclude it as a risk for the species. Lack of toxicological data prevents us from drawing conclusions regarding other species.

in birds, although there isn’t enough information to assess them in other species. Regarding MON toxicity (EFSA, 2018), the available data on birds, pigs,

Interactions between mycotoxins

and minks indicates that exposure to MON via consumption of feed

MON

poses a low or insignificant risk for these species under current feeding practices. For the rest of the species, EFSA concludes that the risk is low

Sometimes, we observe symptoms that are hard to explain by the presence of only one or various mycotoxins, or by the amount of mycotoxins that is in the feed (Trenholm et al., 1983). These issues have been linked to the interactions between several mycotoxins, many of them that are not analytically determined.

or insignificant, but there isn’t enough information available on

This basic theory also explains the well-known fact that naturally

its toxicity to assess the risk.

contaminated food/feed is more toxic than the ones equally contaminated with purified mycotoxins (Trenholm et al., 1994).

Klaasen and Eaton (1991) classify the effects of the interactions between mycotoxins as: Less than additive Additive Synergic Enhanced Antagonistic

Synergic effects of mycotoxins In general, we know or sense that

Furthermore, after reviewing 112

Stoev et al. (2010) came to a

in most cases there are additive

publications on the toxicological

similar conclusion when studying

or synergic effects (Speijers and

interactions between mycotoxins,

nephropathies in poultry and

Speijers, 2004; Pedrosa, 2010). Many authors have highlighted this additivity, synergy or enhancement:

Grenier and Oswald (2011) found synergies and additivities associated with performance in most of the studies published. However, in relation to other parameters, especially biochemical ones, the results are more variable, ranging from synergic to antagonistic effects for the same combination of toxins.

pigs that could not be explained

Grenier et al. (2011) demonstrated in corn that the liver tumors initiated by AFB1 are exacerbated by the presence of FB1.

only by the content of OTA, under the limit recommended by the EU, finding the explanation in the simultaneous presence of OTA, FB1, and penicillic acid (PCA).

Phenomena such as the ones previously described justify the need to carry out multi-mycotoxins analysis in order to understand the effects that are seen in the field.

Antagonistic effects of mycotoxins Although most results reveal the

Thus, antagonism has been

Some publications reveal antagonistic

additive or synergic effects of

observed between:

effects associated with the

mycotoxins, it should be noted that antagonistic effects can also be seen (Koshinsky

and Khachatourians, 1992; Bernhoft et al., 2004).

DON and FB1 (Ficheux et al.,

2012; Wam et al., 2013a) DON and ZEA (Bensassi et al.,

simultaneous presence of three or more mycotoxins. For example, DON, NIV y FB1; NIV, ZEA, FB1 and DON, NIV, ZEA, and FB1 (Wam et al., 2013).

2014; Wam et al.,2013) DON and T2 (Thuvander et

Yang et al., (2017) demonstrated the

al., 1999; Ruíz et al., 2011)

antagonistic effect between some

DON and DAS (Thuvander et al., 1999)

modified DON toxins (acetylated

NIV and DON or FB1 (Wam et al., 2013) NIV and ZEA (Wam et al., 2013)

derivatives), such as 15-ADON + NIV and 15-ADON + FX.

BEA with DON or T2 (Ruiz et al., 2011)

Interactions between mycotoxins – In vitro vs In vivo Most of these studies have been

A clear example that highlights the

conducted in in vitro scenarios,

importance of the experimental design

with cell viability as the main

is the study carried out by Klaric et

parameter, although there

al. (2012) to asses the interactions

are other criteria, such as:

between OTA and citrinin (CIT)

Apoptosis and cell necrosis

in a model with PK15 kidney epithelial cells from pigs, determining

DNA damage

the final effect via cell viability,

Oxidative damage

apoptosis, necrosis, and genotoxicity,

Immunotoxicity Obviously, the combined toxic effects that are

obtaining the following results: Synergic effect on cell viability, apoptosis, and necrosis. Antagonistic effect for genotoxicity.

observed will depend on the experimental design:

Another factor that has complicated

1. Type of cells that are exposed

the interpretations up to now is that the response that is observed

2. Exposure time

in vivo does not always correlate

3. Dosage and relation

with what is seen in vitro.

between mycotoxins 4. Final points and tests used 5. Statistical aspects of the models

To illustrate this phenomenon, we can refer to the example of the interaction between DON and T-2.

In vitro studies reveal antagonism (EFSA, 2002), probably due to the competence for binding sites.

There are other published examples,

It is clear that the effects of the

such as the interaction between DON

combination of mycotoxins cannot

and ZEA, in which Swamy et al. (2002)

be predicted based solely on their

highlight their in vivo synergic effects in

individual effects and that, in

However, results from in vivo

piglets, while Ji et al. (2017) demonstrate

addition to additivities and synergies,

studies with mice (Schiefer et

their antagonistic effects in mice.

there can also be antagonisms.

al., 1986) demonstrate that the negative effects of DON in the animals are exacerbated in the presence of T-2, while in vivo studies with pigs (Friend et al., 1992) indicate that DON combined with T-2 present antagonism (with T-2 at half of the dose of DON).

Regarding the loss of growth that is

It is very difficult to predict these

sometimes observed in vivo in response

responses as they are dose,

to the presence of mycotoxins, Andretta

species, and toxin-dependent,

et al. (2015) point out that it is due to an increase in the energy required for the animals’ maintenance, which is supported by Pastorelli et al. (2012).

in addition to the variability due to methodology-related factors.

CONCLUSIONS

It goes without saying that the

Analysing a single mycotoxin may

The application of atoxic fungi

in vitro methodology must be standardized at an international level in order to expand our knowledge about the interactions between mycotoxins and to have access to comparable data.

not be enough to explain many

that grow on the crops, as well as

cases. However, excessive analytical

varieties that are resistant to the

information, if not correctly interpreted,

colonization of toxigenic fungi

isn’t a solution either, making it

or climate models that predict

important to carry out further studies.

the presence of mycotoxins are some of the methods that

Under these circumstances, without

are available to counteract the

These standards would only be

renouncing to other tools, the best

negative effects of mycotoxins

valid if they could predict the

way to reduce the risk is prevention

and significantly reduce the risk.

in vivo response in animals.

through good agricultural practices and risk analysis in the primary links of the food chain.

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FEED PRESERVATIVES FOR MOULD AND MYCOTOXIN CONTROL ARE THEY RESILIENT ENOUGH?

Prof. Naresh. Magan and Dr. Ángel. Medina Applied Mycology Group, Environment and Agrifood Theme, Cranfield University, Cranfield, MK43 0AL, U.K. Email: n.magan@cranfield.ac.uk

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The feed industry is increasing significantly because of the need for supplying the expanding demands for meat, especially in regions where economies are expanding rapidly, such as in Asia, especially China, and in South America, Brazil, and Argentina. Thus, the need for hay, mixed feeds and compound feed has become critically important.

However, when we examine the ingredients which are used, especially in Europe, a significant number of these are rejected at the borders because of mycotoxins, especially those originating from sub-tropical and tropical regions (RASFF, 2019).

This review covers aspects of the preservation of different animal feeds to reduce or minimise mould spoilage and mycotoxin contamination of such feeds. Often, because hay and mixed animal feeds have to be reasonably moist for consumption by animals, they are often treated with preservatives to obtain the necessary shortand medium-term safe storage. However, most of the preservatives are based on aliphatic acids and their salts.

We will address the use of such preservatives and their potential drawbacks, especially when using sub-optimal concentrations which may exacerbate mycotoxin contamination.

There has also been a lot of interest in finding alternative food/feed grade preservatives based on antioxidants and plant extracts, especially essential oils. However, many of these are not classed as GRAS compounds because the necessary toxicology has not been completed and in addition the cost-benefit analyses needs to be included to evaluate their potential for commercial use. These aspects will also be addressed.

213 13

ALIPHATIC ACIDS AND THEIR SALTS FOR CONTROLLING SPOILAGE AND MYCOTOXIGENIC MOULDS Animal feed consists of mixtures of ingredients including cereals, cereal by-products, cotton seed cake, groundnut cake, soya extracts, palm pulp, pulses and other additives depending on whether the product is destined for cattle, swine, or poultry.

The production of pelleted feed results in a relatively dry feed product that can be stored effectively in the short and medium term, provided they do not absorb moisture during the storage phase, due to poor management post-harvest.

This can occur because of ineffective partitioning of different types of commodities during transport or storage, since feed ingredients can re-absorb moisture. This is especially so if some of the ingredients are hygroscopic, which can allow the initiation of growth by spoilage and indeed mycotoxigenic moulds resulting in contamination with mycotoxins (Perreira et al., 2019).

Raw ingredients Humidity

Pelleted feed

Storage

Mycotoxigenic moulds

Economically, spoilage moulds and mycotoxin contamination are very important. In a 10-year study in the USA, it was shown that animal feed ingredients and forage samples contained significant amounts of mycotoxins. For example:

Aflatoxins (AFs) / 34% animal feed (>20 ppb) Deoxynivalenol (DON) / 60% dairy and poultry feed Zearalenone (ZEN) / 15-20% mixed feed T-2 toxin / 5% feed samples Fumonisins (FUMs) / 5-10% feed samples

Forages including concentrates, maize grain, soybean meal, cottonseed cake, corn silage, grass hay, small grain and grass silage have been surveyed for the presence of mycotoxins. These have all shown a significant increase over recent years in the percentage of samples of feed which are contaminated with different mycotoxins including those by Biomin, Alltech, Randox and Patent Co, amongst others (Biomin survey, 2019; Alltech survey, 2019; Randox survey, 2019, Patent Co 2019). Non-extruded feedstuffs, including hay and mixed feeds, are often treated with anti-mould preservatives, usually based on individual or mixtures of aliphatic acids or their salts. This can reduce the potential for initiation of mould spoilage and mycotoxin contamination. Most of the preservative mixtures used commercially are, however, fungistatic not fungicidal, and thus are not lethal to contaminating mould spores.

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Under-treated or non-treated pockets of feed, even very small ones, of a few grams, may provide initial foci for the initiation of germination of spoilage/mycotoxigenic mould spores if the feed moisture content is conducive to growth (Lord et al., 1981, 1983; Lacey et al., 1982). It is thus critical that, when preservatives are added, effective mixing of the preservative with the feed is achieved to ensure coverage of the feed with the right concentration to minimize opportunities for spoilage moulds/ mycotoxigenic mould initiation and toxin contamination from occurring during short- and medium-term storage.

Indeed, elegant studies by Lord et al. (1981) showed the importance of effective mixing of aliphatic acids or their salts with the feed to prevent undertreated or untreated pockets of the feed. They suggested that mould spoilage may be initiated as well as contamination with aflatoxins (AFs; class 1a carcinogens), other mycotoxins such as ochratoxin A (OTA) or type B trichothecenes, including DON, ZEN, and FUMs.

Studies of different poultry mix feeds used as starter and finisher blends were shown to have different mycobiota diversity depending on the season and the way the feed raw ingredients are processed. Often the predominant spoilage mould species were from the Aspergillus, Penicillium and Fusarium genera, responsible for mycotoxin contamination, especially in warmer regions of the world, e.g., South-East Asia (Alam et al., 2010 a,b). In Asia, poultry feed is produced by commercial feed mills as well as by local cooperatives. The feed mill owners purchase these ingredients in bulk in the production season and store them for processing into feed throughout the year. However, often in tropical and sub-tropical climatic regions, harvested raw commodities from the fields often have a relatively high moisture content, which makes them unsafe for even short-term storage without mould spoilage. Thus, without effective drying to <0.70 water activity, then intermediate moisture conditions will allow the development of dry loving (xerophilic) moulds mentioned previously, many of which are mycotoxigenic (Ilangantileke, 1987; Sanchis, 2000; Figure 1).

A number of commercial companies sell preservative formulations based on aliphatic acids including formic, propionic, or sorbic acids or mixtures thereof. Sometimes their salts are preferred because they have a reduced volatility and are more user friendly.

Figure 1. Diagrammatic profiles of growth/no growth limits for the three key mycotoxigenic genera in relation to the two key interaction abiotic factors.

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CONCENTRATION IS CRITICAL Of course, the formulation methods are confidential to each industrial producer. However, using the right concentration is critical to achieve effective control of spoilage and mycotoxigenic moulds. Table 1 shows difference in concentrations that can occur when mixing different ratios of aliphatic acids or their salts and their actual concentrations.

Table 1. Preservative concentrations of stock solutions (g in 100ml water) (from Marin et al., 1999).

Propionate in maize (%)

Commercial mixture

Sodium propionate

Propionic acid

0.03 0.05 0.07

6.4 10.3 14.1

5.0 8.0 11.0

3.9 6.2 8.6

Table 2 compares the effect of two commercial products and propionic acid on the Minimum Inhibitory Concentration (MIC) required for inhibiting growth of three important mycotoxigenic fungi on a maize-based medium (A.flavus, AFs; A.ochraceus/P. verrucosum, Ochratoxin A, OTA). On moist maize, 0.25% was necessary for similar control to be achieved of A. flavus and A. parasiticus.

Table 2. Comparison of minimum inhibitory concentration (%) required for propionic acid and two commercial formulations of aliphatic acids in vitro on maize-based media (Marin et al., 2000).

Product

A. flavus

A. ochraceus

P. verrucosum

Propionic acid

0.25

0.10

Lupromix NC

0.50

0.25

Lupro-grain

0.25

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Table 3 shows the effect of sorbic acid on different spoilage moulds, the pH and concentration range.

Table 3. Minimum Inhibitory concentration (MIC) of sorbic acid against different spoilage mould genera or specific species (adapted from Lück and Jager, 1997). Species

MIC (ppm)

Rhizopus species Penicillium species Penicillium digitatum Penicillium glaucum Aspergillus species Aspergillus flavus Aspergillus niger Fusarium species Cladosporium species

3.6 3.5 - 5.7 4.0 3.0 3.3 - 5.7 --2.5 - 4.0 3.0 5.0 - 7.0

1200 200 - 1000 200 - 1000 1000 - 2500 200 - 1000 1000 1000 - 5000 1000 1000 - 3000

Figure 2 compares the effect of using mixtures of propionic acid and its sodium salt on the growth of Fusarium section Liseola species (F. verticillioides, F. proliferatum) responsible for contamination of maize with FUMs (Marin et al., 2000). This clearly shows that the ratio of the mixture and, more importantly, the water availability of the maize significantly affected the colonization of maize by these species. Under wetter conditions there was practically no control of F. verticillioides, although there is some control of F. proliferatum, but only at 0.93-0.95 aw.

Figure 2. Effect of different ratios of propionic acid/sodium propionate on maize grain at 25 oC on growth rates (Marin et al., 2000).

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Table 4 shows the effect of different concentrations of propionic acid and aw levels on fumonisin B1 contamination of naturally contaminated maize or that with an additional inoculum of F. verticillioides after 4 weeks storage at 25oC. This shows that regardless of conditions, only at the highest concentration tested was there a significant decrease of contamination with this mycotoxin.

Table 4. Fumonisin B1 concentration (µg/ml) in natural maize treated with propionic acid concentrations and stored for 4 weeks at 25oC (from Marin et al., 2000). Statistically, Fusarium, Preser vative dose, and aw interactions: only Fusarium x Preser vative was statistically significant. Activity aw

Propionic acid (%)

Natural maize

Maize + Fusarium

0.85

0 0.05 0.10 0 0.05 0.10 0 0.05 0.10

9.27 4.96 9.98 5.65 7.34 8.04 9.46 3.61 6.58

3.71 11.15 5.36 4.78 5.72 3.38 8.57 6.42 5.97

0.90

0.95

Some studies have examined the changes in populations of different starter and finisher poultry feeds in Asian countries (Alam et al., 2012). This showed the dominance of the different mycobiota present in different feeds and the relative contamination with mycotoxins, especially aflatoxins in different seasons in different feeds (Table 5). The impact of calcium propionate to both starter and finisher feed were shown to have significant benefits, decreasing the fungal populations, especially of A. flavus and A. parasiticus, and contamination with aflatoxins (AFB1, AFB2, AFG1, AFG2; Alam et al., 2014a,b). It is important to reduce aflatoxin contamination in poultry feeds because of their significant impact in causing aflatoxicosis characterized by listlessness, anorexia with lowered growth rate, poor feed utilization, decreased weight gain, decreased egg weight and production, increased susceptibility to environmental and microbial stresses and increased mortality (Leeson, 1995; Wild, 2000). Thus, more effective mixtures of preservatives may provide benefits of either additive or synergistic effect on relative control, especially of these important mycotoxins, during short- and medium-term storage.

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Table 5. Seasonal occurrence of AFB1 (ng g-1) in poultry feeds and their ingredients collected from Peshawar (from Alam et al., 2012).

AFB1 concentration (ng g-1) Feed/ingredients

Dec-Feb

March-May

June-Aug

Sep-Nov

Mean

Maize Wheat Rice Cotton seed Starter ration Finisher ration Mean

31.07 25.10 13.71 35.61 158.79 183.45 74.62 C

37.05 28.16 42.12 53.29 173.04 187.74 86.90 A

32.74 20.81 46.84 55.70 134.41 191.65 80.36 B

26.37 19.34 41.85 22.70 147.68 152.00 68.32 D

31.81 E 23.35 F 36.13 D 41.83 C 153.48 B 178.71 A

Means in each row and column followed by same letters are not significantly different P≤0.05 There have been reports of the use of other acids such as adipic, ferulic, fumaric, tartaric and trans-cinnamic acid (plant extract) against mycotoxigenic moulds for feed applications. The effects have been variable with in some cases good efficacy in controlling growth by >50-75%. However, in many cases the effect on mycotoxin production was less effective, especially in cereals such as wheat and maize which are used in mixed feeds. Table 6 summarizes the effect of different acids on growth and mycotoxin production by different mycotoxigenic moulds.

Table 6. Summary of the effects of different acids in the aqueous or ethanolic form on the growth and mycotoxin production by different fungal species (adapted from Mylona, 2013).

Acid

Growth F. g

F. v.

F. l.

Mycotoxin A. f.

F. g

F. v.

F. l.

A. f.

Trans-cinnamic

Aqueous (0-200 ppm)

Ferulic Adipic Tartaric Fumaric

Effect was observed No effect observed/stimulation Further work required Not tested F.g, F. graminearum - Dexoynivalenol; F.v., F. verticillioides - Fumonisins; F.l., F. langsethiae - T-2/HT-2 toxins; A.f., A. flavus – aflatoxins.

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Previous studies have also examined the different chain lengths of a range of aliphatic acids for the control of spoilage moulds. Previous studies with moist hay have shown that the chain length of the aliphatic acid may influence the relative control of spoilage moulds in feed such as Paecilomyces variotii and Aspergillus glaucus group (=Eurotium species). Thus, fatty acids up to a chain length of C9-C10 were very effective for controlling spoilage moulds. However, anti-mould activity subsequently decreased with increasing chain length with solutions of dodecanoic acid (C12) unable to control growth at all.

Studies of moist barley (22, 28% m.c.) with recommended commercial doses of propionic acid showed that over a 6 months period A. flavus and AFs contamination was controlled. However, af ter 4-6 months the treatment was less effective against P. verrucosum resulting in an increase in OTA contamination (Skudamore et al., 2004). Thus, while one mycotoxigenic species was controlled, another was more resilient and increased the risk of OTA contamination during medium term storage, especially in the 22% moist barley.

Studies have also suggested that intermediate concentrations of such preservatives may result in a stimulation of the production of mycotoxins. Arroyo et al. (2000) showed that the production of OTA by P. verrucosum was stimulated by the decimal reduction of the recommended doses of calcium propionate or potassium sorbate.

Intermediate concentrations of propionic acid have also been shown to stimulate the production of AFs by A. flavus (Al Hilli and Smith, 1979).

Some spoilage moulds have been found to utilize lower concentrations of such preservatives as a carbon source and degrade them quite rapidly. This has been shown for both yeasts isolated from preservative-treated hay and by xerophilic mycotoxigenic moulds (Magan et al., 1986a,b; Mutasa et al., 1990; Schmid-Heydt et al., 2000).

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USE OF ANTIOXIDANTS, PLANT EXTRACTS AND BIOPRESERVATIVES There has been significant interest in the use of alternative compounds, especially antioxidants, plant extracts (both essential oils and their volatile components) and biopreservative inoculants to control mould spoilage of individual ingredients or mixed feeds for poultry, cattle, and swine. Other phenolic-derived antioxidants have been screened for their possible antimicrobial efficacy. Butylated hydroxyoluene (BHT), butylated hydroxyanisole (BHA), propylgallate (PG), 2-tert-butylhydroxybenzoic (TBHQ) and propyl parabens are among them.

They are GRAS compounds and can be used in food and feed.

ANTIOXIDANTS

In contrast, while many crude essential oils (ESOs) or their components (e.g., eugenol, thymol, cinnamaldehyde, vanillin, carvacrol, linalool) have been screened for controlling spoilage and mycotoxigenic moulds for feed applications many of these have not had the necessary toxicology tests for approved for use as antimicrobials.

This has been a driver for the development of biocontrol strategies as part of an IPM approach.

The use is biopreservation inoculants (especially yeasts) are thus attractive alternatives to try and preserve moist grain-based mixed feeds.

PARABENS

Biopreservatives have become more attractive in recent years as many countries, especially the EU, have banned a range of crop protection chemical groups because of safety and environmental concerns.

Originally, propyl parabens, esters of 4-hydroxybenzoic acid (PHB), were synthesized as a possible replacement of existing preservatives like salicylic and benzoic acids, effective only in the highly acid pH range. Although Lück and Jager (1997) maintained that one of the most important characteristics of parabens, together with a much higher antimicrobial action to that of phenols and organic acids, is their pH independent activity, some studies have reported a slight influence of pH on their activity (Thomson et al., 1993). Due to their high pKa value (8.5), parabens are chemical preservatives effective over a wider range of pH (3-8). Antimicrobial activity of parabens is related to the length of the ester group of the molecule. As additives, parabens have been extensively applied as alkali solutions or as ethanol or propyl glycol solutions in a range of food products, including pickled vegetables (Belitz and Grosch, 1999). However, few studies have examined their potential applications for use in feed.

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Kubo et al. (2001) compared the antifungal activity of three gallates, propyl (C3), octyl (C8) and dodecyl (C12) and found that only octyl gallate (OG) was the active compound against four different fungal genera with a MIC of 25ppm. These results did not however, take account of environmental conditions, especially aw and temperature, which have been shown to have an impact of efficacy. Table 7 shows more recent studies on the comparison of the e!cacy of OG and other antioxidants as well as salts of aliphatic acids and the ED50concentrations necessary for inhibiting AFB1 production by A. flavus. This clearly showed that OG was better than many of the other compounds screened.

Table 7. Effective dose for 50% control (ED50 values, ppm) of antioxidants and comparison with aliphatic acids for inhibition of AFB1 production by A. flavus when grown at different water availability conditions at 25°C for 10 days (Sultan, 2011).

GALLATES

Water activity level Treatment

0.95

0.92

0.89

BHA BHT OG PP SMP PS CP

190 ST 80 ST > 2000 ST >3000

200 > 500 35 180 > 2000 > 2000 510

ST 80 80 75 1600 ST 490

ST: stimulation of AFB production. BHA, butylated hydroxyl anisole; BHT, butylated hydroxyl toluene; OG, octyl gallate; PP, propyl paraben; SMP, sodium metabisulphite; PS, potassium sorbate; CP, calcium sorbate. 1

Subsequent studies were carried out by treating stored peanuts with different concentrations of OG and examining the effect on temporal populations changes of A. flavus (CFUs/g peanuts) over storage periods of 14 days at 25oC (Figure 3).

Figure 3. Temporal effect of different octyl gallate concentrations (OG, ppm) on populations of A. flavus isolated over 14 days in peanuts stored at 25°C. Bars indicate standard error of the means (from Sultan, 2011).

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The populations significantly increased, especially between 7 and 14 days of storage, suggesting that in situ application was not as effective as in vitro studies on peanut-based media. Statistically, single treatments, OG x Time, aw x Time interactions were all significant. However, there was no effect of the interactions of OG x aw and OG x Time x aw.

GALLATES

Storage time was the main significant effect followed by the storage aw conditions.

The peanuts were subsequently analyzed for AFB1 contamination levels. All the factors (OG, time, aw and their interactions) significantly influenced toxin production, except OG x aw interaction (P< 0.05). Storage time was the major significant effect followed by the initial aw of the peanuts entering storage (Sultan, 2011). The final A. flavus populations and the AFB1 contamination levels were significantly higher after 14 days of storage, indicating that even with these concentrations of this antioxidant, efficacy was not good enough relative to the legislative limits.

The use of such compounds thus requires careful consideration, especially where mixed feeds are considered where the fungal diversity can be wide and thus controlling specific mycotoxigenic species may allow other more tolerant species to become dominant. Mixtures of compounds may be one way forward provided that they can provide a synergistic effect rather than just an additive one in terms of mould spoilage control or toxin production in feeds, especially in mixtures of cereals, cotton seed meal, pulses and other additives.

ESSENTIAL OILS A significant body of work exists on the efficacy of plant essential oils (ESOs) or compounds extracted from mixtures of plant ESOs. The efficacy of ESOs was recently reviewed in relation to control of crude extracts as well as purified components of ESOs by Prakash et al. (2015a,b). However, while this examined their role for control of spoilage moulds and mycotoxin production in different agrifood chains the potential for use in feed was not prioritized.

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Screening of a range of ESOs has been carried out against different mycotoxigenic fungi which may contaminate feedstuffs with mycotoxins. Figure 4 shows an example where 25 ESOs were screened for efficacy and control of P. verrucosum, responsible for OTA contamination. This clearly showed that only a few had significant efficacy in controlling growth of this mycotoxigenic mould.

Figure 4. The effect of 25 different ESOs diluted 1:10 after 48 hours at 25ºC on the inhibition zones against P. verrucosum (OTA11; ochratoxin A producer) on a wheat-based medium at 25ºC. Bars indicate SEM. The circle indicates the best treatments (from Cairns et al., 2003). Figure 5 shows the example of cinnamon oil concentrations on the relative control of growth of a range of spoilage and mycotoxigenic fungi. This shows that only higher concentrations will have efficacy for complete control (MIC) values for a range of these fungi.

Figure 5. Effect of Cinnamon oil at different concentrations on growth of six different spoilage and mycotoxigenic species at 0.97 water activity and pH 4.5 at 25oC on a cereal-based medium. CH, Cladosporium herbarum; 453; P. verrucosum strain 453); PC, Penicillium corylophilum; PR, Penicillium roqueforti; ERE, Eurotium repens (=Aspergillus glaucus); AO, Aspergillus ochraceus (=A. westerdijkiae) (from Arroyo, 2000).

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Subsequent more detailed studies have examined the comparison of ESOs and, in some cases, antioxidants for control of specific mycotoxins for which legislative limits exist. Figure 6 shows the effect of 200 ppm of different ESOs and antioxidants on the control of DON in stored naturally contaminated moist wheat grain inoculated with F. culmorum. This shows that there was good efficacy at 0.93 and 0.95 aw. However, at 0.97 aw DON contamination could not be controlled.

Figure 6. Examples of the effect of different 200 ppm essential oils and comparison with two antioxidants on control of dexoynivalenol production by Fusarium culmorum in naturally contaminated stored wheat at different water activity levels for 30 days at 25oC. PP, Propyl paraben; Clove, Clove essential oil; Cin, Cinnamon essential oil; BHA, Butylhydroxy anisole; Bay, Bay leaf essential oil. (Hope, Aldred and Magan, unpublished data). More recent studies have examined the effect of different extracts of garlic (Propyl propane thiosulfonate (PTS), Propyl propane thiosulfinate (PTSO)), for use in food and feed application (Mylona et al., 2019). This focused on potential for control of Fusarium species in wheat, maize and oats (F. graminearum, DON; F. verticillioides, Fumonisn; F. langsethiae, T=2/HT-2 toxin).

In vitro studies showed that 200 ppm of either PTS or PTSO reduced fungal growth by 50-100% and mycotoxin production by >90% depending on species, mycotoxin and aw conditions on milled wheat, oats and maize respectively. PTS was generally more effective than PTSO. However, in situ studies showed that: DON and ZEN were decreased by 50% with 80 ppm PTSO.

Contrasting effects on T-2/HT-2 toxin contamination of oats were found, depending on aw, with PTS stimulating production under marginal conditions (0.93 aw), but at 0.95 aweffective control was achieved with 100 ppm. Treatment of stored maize inoculated with F. verticillioides resulted in a stimulation of total fumonisins in most treatments.

100 ppm of PTS reduced DON and ZEN production in wheat stored at 0.93 aw for 20 days, although contamination was still above the legislative limits.

14 25 25

These studies showed that where sub-optimal concentrations of these ESOs are used then sometimes stimulation of mycotoxins can occur. This suggests that care is needed because, unless MIC are used, the risk of mycotoxin contamination still exists.

BIOPRESERVATIVES

There has been particular interest in the use of inoculants for the preservation of feed grains or mixed feeds. This has included lactic acid bacteria (LABs) and yeasts. The approach here is to add the inoculants to moist cereals or mixed feed and then seal these to allow colonization and preservation by the production of naturally produced antimicrobial compounds, including bacteriocins (LABs), and colonization of the feed by inoculants (e.g. Pichia anomala) (Olstorpe et al., 2011).

Moist cereal or mixed feed Antimicrobial compounds

Sealed Colonization and preservation

Inoculant

A significant amount of work has been carried out on moist cereals as animal feed using yeasts.

15 26

For example, P. anomala was developed as an inoculant for the preservation of moist wheat and barley for feed used for cattle. The yeast was formulated and the effect of different formulations examined for both spoilage and mycotoxin control (Druverfors et al., 2002; Mokiou and Magan, 2008).

PICHIA ANOMALA

Figure 7 shows the effect of different formulations of P. anomala on the control of ochratoxin A production by P. verrucosum when stored for up to 30 days (Mokiou and Magan, 2008).

Figure 7. Effect of biopreservative formulations of P. anomala on Ochratoxin A contamination of stored moist wheat grain (0.93 aw) co-inoculated with Penicillium verrucosum for 30 days and 25oC. A: unmodified yeast biopreservative cells; B: proline-modified; C: proline+cottonseed flour+ skimmed milk in water; D: same as C plus isotonic solutions (from Mokiou and Magan, 2008). Subsequently, longer term studies were carried out in pilot scale storage silos with inoculation with P. anomala fresh cells, the best formulation identified. Figure 8 shows the long-term changes in fresh cells, formulated cells and that of P. roqueforti which can grow under microaerophilic conditions. The key period for control was achieved for 8-9 months but that subsequently the inoculant was less effective.

Figure 8. Pilot scale study of formulated and fresh Pichia anomala cells over a 15 month stored period in moist wheat to control spoilage moulds, especially Penicillium roqueforti (from Druverfors, Mokiou, Magan and Schnurer, unplublished data).

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CONCLUSIONS Regardless of the preservatives used it is critical that nutritional value of the final feed, whether cereal-based, mixed feed or extruded pelleted feed, are conserved for ensuring that the nutritional value and necessary animal growth rates can be maintained.

The use of existing mixtures or these with alternative natural preservatives may be the main areas for development in the future. However, this must be with the proviso that effective coverage of the treated feed type can be achieved with the necessary recommended rates.

In addition, some of the preservative mixtures are not proving to be resilient enough, especially in cereal-based mixed feeds, when controlling spoilage and mycotoxigenic moulds.

With the impact of interacting climate-related abiotic factors becoming more important, the effect on the resident microbiome of commodities used for making different feeds may change significantly.

Preservation systems for animal feeds may also have to be modified to take into account of this as the dominant fungal populations may change and the mixtures of mycotoxins produced may also be modified (Medina et al., 2017).

The relative cost-benefit analyses of the use of ESOs, antioxidants alone or with aliphatic acids or their salts as mixtures needs to be compared for successful application in the feed industry. For example, studies by Aldred et al. (2008) compared a range of ESOs and antioxidants for controlling P. verrucosum and OTA in wheat stored under different conditions. Resveratrol was found to be superior to the others. However, the costs were prohibitive for the use of such compounds when compared to the aliphatic acids which are economically more cost effective, even if application rates and effective mixing and coverage is necessary for efficacy.

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REFERENCES Alam, S., Shah, H.U., Khan, H. & Magan, N. (2012). The effect of substrate, season and agroecological zone on mycoflora and aflatoxin contamination of poultry feed from Khyber Pakhtunkhwa, Pakistan. Mycopathologia 174, 341-349. Alam, S., Shah, H.U., Afzal, M. & Magan, N. (2014) Influence of calcium propionate, water activity and storage time on mould incidence and aflatoxins production in broiler starter ration. Animal Food Science and Technology 188, 137-144. Alam, S., Shah, H.U., Khan, N.A., Zeb, A., Shah, A.S. & Magan, N. (2014). Water availability and calcium propionate affect fungal populations and aflatoxins production in broiler finisher feed during storage. Food Additives and Contaminants Part A 31, 1896-1903. Aldred, D; Cairns-Fuller, V. & Magan, N. (2008). Environmental factors affect e!cacy of some essential oils and resveratrol to control growth and ochratoxin A production by Penicillium verrucosum and A. westerdijkiae on wheat grain. Journal of Stored Product Research 44, 341-346. Arroyo (2003) Natural antifungal systems for prevention of mould spoilage in bakery products. PhD Thesis, Institute of BioScience and Technology. Cranfield University, Cranfield. Beds. MK43 0AL, U.K. Belitz, H.D. & Grosch W. (1999). Food Chemistry. Springer, Berlin., Germany Cairns, R. & Magan, N. (2003). Impact of essential oils on growth and ochratoxin A production by Penicillium verrucosum and Aspergillus ochraceus on a wheat-based substrate. In Advances in Stored Product Protection. Eds. P.Credland, D.M. Armitage, C.H. Bell, P.M. Cogan. Cabi International, pp. 479-485. Druvefors, U., Jonsson, N., Boysen, M.E. & Schnurer, J. (2002). Efficacy of the biocontrol yeast Pichia anomala during long-term storage of moist feed grain under different oxygen and carbon dioxide regimens. FEMS Yeast Research 2, 389-394 Kubo, 1., Xiao, P. and Fujita, K. (2001). Anti-fungal activity of octyl gallate: structural criteria and mode of action. Bioorganic Medical Chemical Letters 11, 347-350. Lord, K.A., Gayley, G.R. and Lacey, J. (1981a). Laboratory application of preservatives to hay and the effects of irregular distribution on mould development. Animal Feed Science and Technology 6, 73-82. Lord, K.A., Lacey, J., Cayley, G.R. and Manlove, R. (1981b). Fatty acids as substrates and inhibitors of fungi from propionic acid treated hay. Transactions of the British Mycological Society 77, 41-45. Lacey, J., Lord, K.A., Cayley, G.R., Holden, M.R. and Sneath, R. W. (1983). Problems of testing novel chemicals for the preservation of damp hay. Animal Feed Science and Technology 8, 283-301. Liick, E. & Jager, M. (1997). Antimicrobial food additives, Characteristics, Uses and Effects. Spinger, Berlin & Barcelona. Magan, N. & Lacey, J. (1986a). Water relations and metabolism of propionate in two yeasts from hay. Journal of Applied Bacteriology 60, 169-173. Magan, N. & Lacey, N. (1986b). The effects of two ammonium propionate formulations on growth in vitro of Aspergillus species isolated from hay. Journal of Applied Bacteriology 60, 221-225.

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REFERENCES Marin, S., Sanchis, V., Ramos, A.J. & Magan, N. (1999). Control of growth and fumonisin B1 production by F.moniliforme and F.proliferatum isolates in maize grain with propionate formulations. Food Additives and Contaminants 16, 555-563. Marin, S., Magan, N., Abellana, M., Canela, R., Ramos, A.J. & Sanchis, V. (2000). Selective effect of propionates on maize mycoflora and impact on fumonisin B1 accumulation. Journal of Stored Product Research 36, 203-214. Medina, A., Akbar, A., Baazeem, A., Rodriguez, A. & Magan, N. (2017). Climate change, food security and mycotoxins: do we know enough? Fungal Biology Reviews 31, 143-154. Mutasa, E.S. & Magan, N. (1990). Utilization of potassium sorbate by tobacco spoilage fungi. Mycological Research 94, 965-970. Mokiou, S. & Magan, N. (2008). Physiological manipulation and formulation of the 379 biocontrol yeast Pichia anomala for control of Penicillium verrucosum and 380 ochratoxin contamination of moist grain. Biocontrol Science and Technology 18, 1063-1073. Mylona, K. (2013). Fusarium species in grains: dry matter losses, mycotoxin contamination and control strategies using ozone and chemical compounds. PhD thesis, Applied Mycology Group, Cranfield University, Cranfield. Bads. MK43 0AL, U.K. Mylona, K., Garcia-Cela, E., Sulyok, M., Medina, A. & Magan, N. (2019). Effects of two garlic extracts [Propyl propane thiosulfonate (PTS) and Propyl propane thiosulfinate (PTSO)] on growth and mycotoxin production by Fusarium species in vitro and in stored cereals. Toxins 11, 495. Doi:10.3390/toxins11090495. Olstorpe, M., Jacobsen, K., Passoth, V. & Schnurer, J. (2011). Controlling mould growth and mycotoxins in animal feed. In Protective Cultures, Antimicrobial Metabolites and Bacteriophages for Food and Beverage Biopreservation. Edt. C. Lacroix; Chapter 9, pp. 225-239. Woodhead Publishing Series in Food Science, Technology and Nutrition Prakash, B., Mishra, P.K., Kedia, A., Dwivedi, A.K. & Dubey, N.K. (2015a). E!cacy of some essential oil components as food preservatives against food contaminating moulds, aflatoxin B1 production and free radicle generation. Journal of Food Quality, 38, 231-239. Prakash, B., Kedia, A., Mishra, P.K., & Dubey, N.K. (2015b). Plant essential oils as food preservatives to control moulds, mycotoxin contamination and oxidative deterioration of agrifood commodities: potentials and challenges. Food Control 47, 381-391. Pereira, C.S., Cunha, S.C. & Fernandes, J.O. (2019). Prevalent Mycotoxins in Animal Feed: Occurrence and Analytical Methods. Toxins 11, 290; doi:10.3390/toxins11050290 Skudamore, K., Banks, J.N., Rizvi, R. & Jennings, P. (2004). Formation of ochratoxin A and aflatoxins following the use of propionic acid as a grain preservative for storing damp barley. Mycotoxin Research 20, 68-79. Sultan, Y. (2011). Biodiversity of mycotoxigenic Aspergillus species in Egyptian peanuts and strategies for minimizing aflatoxin contamination. PhD Thesis, Applied Mycology Group, Cranfield University, Cranfield, Beds. MK43 0AL, U.K. Thomson, D.P.; Metevia, L. & Vessel, T. (1993). Influence of pH alone and in combination with phenolic antioxidants on growth and germination of mycotoxigenic species of Fusarium and Penicillium. Journal of Food Protection, 56, 134-138.

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FUMONISINS THE UNDERRATED MYCOTOXINS in poultry, livestock and humans

Deepthi B V and M Y Sreenivasa Department of Studies in Microbiology, University of Mysore, India. Email: sreenivasamy@gmail.com; mys@microbiology.uni-mysore.ac.in

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Fumonisins are mycotoxins majorly produced by

Fusarium verticillioides and Fusarium proliferatum species and are predominantly associated with maize and maize-based poultry and animal feeds (Dass et al., 2008; Sreenivasa et al., 2011; Deepa et al., 2016).

Cereal grains and their by-products,

CONTAMINATION WITH FUMONISIN-PRODUCING FUSARIUM SPECIES

being major ingredients of feeds,

64.28%

represent an excellent substrate for

poultry feed

the growth and reproduction of fungi and have serious consequences on the safety of foods and feeds. In a mycological study carried

45.37% feed mixtures from Karnataka

25% animal feed

out by our laboratory, 45.37% of feed mixtures collected all over Karnataka (India) showed contamination with fumonisin-producing Fusarium species (Figure 1). Furthermore, the study revealed a frequency of 25% and 64.28% of Fusarium contamination in animal and poultry feeds, respectively.

Figure 1. Fusarium and other mycotoxigenic fungal species isolated from animal and poultry feed mixtures.

232

Per cent frecuency

On the other hand, other fungal genera such as Aspergillus flavus,

A. columnaris, A. candidus, A. niger, A. parasiticus, Penicillium,

Poultry feeds

Cladosporium, Rhizopus, Helminthosporium, Mucor, sterile hyphae, etc. showed a frequency of 50% and 82.1%

Animal feeds

in animal and poultry feeds, respectively (Figure 2).

10%

20%

30%

Other Fungi

Fumonisins are capable of inducing

40%

50%

60%

70%

80%

90%

Fusarium species

both acute and chronic toxic effects in poultry and livestock.

Figure 2. Per cent frequency of animal and poultry feeds showing contamination of Fusarium and other fungi.

These effects are dependent on: The type of mycotoxin The level and duration of exposure The animal species that is exposed and the age of the animal (D’mello et al., 1999) Acute toxicity generally leads to rapid onset and an obvious toxic response, while chronic toxicity is characterized by a low-dose toxin exposure for a long time period, leading to cancer and other

Tricarballylic esters

generally irreversible effects. Chemically, fumonisins are toxic, cancer-promoting secondary metabolites that have: A linear, 20-carbon long backbone An amine group at carbon atom 2 (C-2) Methyl groups at C-12 and C-16 Tricarballylic esters at C-14 and C-15

FUMONISIN

OH O

CH₃

H₃C CH₃

HO O HO

CH₃

Methyl groups

Amine group

333 33

Ab bsorption: FB1 is poorly absorbed

Fumonisins are divided into four groups based on structural differences: A, B,

in the gastrointestinal tract and is

C and P (Musser and Plattner, 1997).

extensively retained unmetabolized in tissues such as the liver and kidneys.

The most abundantly found and the most toxic fumonisin is fumonisin B1 (FB1):

Excretion: FB1 is eliminated in bile through some enterohepatic recirculation

Toxicity: FB1 is a Group 2B carcinogen

while exerting its toxicity. The toxin

(possibly carcinogenic to humans)

may also end up in the faeces and

(IARC, 2002). It has been associated

as trace contaminants in urine.

with hepatotoxic, neurotoxic, nephrotoxic and immune-suppressing effects in animals and in humans.

Fumonisin B1 mode of action The molecular aspects of FB1-induced

This leads to the intracellular accumulation

toxicity are poorly understood and the

of sphingoid bases, increased phosphate

downstream toxic cellular mechanisms of

adducts, and a reduced ceramide

FB1 have been deduced to be complex,

concentration resulting in growth

involving many molecular sites.

inhibitory effects, cytotoxicity, apoptosis, cell proliferation, carcinogenicity, DNA

1. Studies have ascribed FB1-induced toxicity to the structural similarity between fumonisins and the sphingoid bases (sphinganine and sphingosine) of sphingolipid layer in cell membrane. FB1 has a primary amino group at C-2 that competitively inhibits ceramide

damage (Riley et al., 2001; Voss et al., 2007) , disruption of normal cell cycling (Ramljak et al., 2000) , alteration of signalling by cAMP and protein kinase C (Huang et al., 1995) , and oxidative stress (Poersch et al., 2014; Abdellatef and Khalil, 2016; Deepthi et al., 2017).

synthase, resulting in the disruption of de novo biosynthesis of ceramide and thus deregulating the sphingolipid complex formation (Merrill et al.,

2001; Riley et al. 2001; Enongene et al. 2002; Riley and Voss, 2006).

434

Sphingolipids

2. It has been also reported that FB1 inhibits other intracellular enzymes such as protein phosphatases and arginosuccinate synthetase

(Jenkins et al., 2000).

2. Inhibition of intracellular enzymes

1. Ceramide synthase inhibition FB1

3. In addition, Domijan and Abramov

(2011) demonstrated that FB1 inhibits Complex I of the mitochondrial electron transport chain in the cell cultures of rat primary astrocytes and human

Ceramide synthase FB1-associated toxic cellular mechanisms

neuroblastoma (SH-SY5Y). This leads to a reduction in the rate of mitochondrial and cellular respiration, depolarization of mitochondrial membrane, over-production of reactive oxygen species (ROS) in mitochondria and deregulation of calcium signalling.

Accumulation of sphingoid bases

3. Inhibition of Complex I of the mitochondrial electron transport chain

Increased phosphate adducts Reduced ceramide concentration Reduced mitochondrial and cellular respiration Depolarization of the mitochondrial membrane Excess of ROS Deregulation of calcium signalling

5 35 35

Toxic effects of Fumonisin B1 Fumonisin B1 intoxication is evident in different domestic and laboratory animals and differences in sensitivity and clinical symptoms have been described (Figure 3).

Contaminated feed

Affects alimentary value & organoleptic features Decreased nutritive value of feed Decreased animal performance and productivity

FB1

Immune suppression Neurotoxicity Hepatotoxicity Nephrotoxicity Carcinogenicity

Figure 3. Toxic effects of FB1 in poultry and livestock.

The first syndrome attributed to fumonisin B1 was ELEM, equine leukoencephalomalacia in the 1980s, characterized by fatal necrotic lesions in the cerebrum of horses (Marasas et al., 1988). Fumonisin B1 induced cardiovascular dysfunction in horses with decreased heart rates, lower cardiac output, and right ventricular contractibility, which may be involved in the pathogenesis of the lesions in the central nervous system (Smith et al., 2002). FB1 toxicosis in pigs was characterized by pulmonary, cardiovascular and hepatic symptoms. Affected animals become anorexic, showing signs of encephalopathy, loss of body weight and hepatic nodular hyperplasia. The symptoms in swine have been referred to as Porcine Pulmonary Edema (PPE) and as “mystery swine disease” (Hollinger and Ekperigin, 1999).

636

For a long time, poultry has been considered to be less susceptible to fumonisins, probably because of the lack of strong clinical symptoms of impairment even with high contamination levels. The clinical features of the disease include diarrhea, weight loss, increased liver weight and poor performance.

Recent studies have focused on the subclinical effects of fumonisins and reveal that the intestinal tract of the birds is very sensitive to the exposure to fumonisins.

Dombrink-Kurtzman et al., (1992, 1993) demonstrated that FB1 and FB2 produced cytotoxic effects on lymphocytes of turkey, and morphologically changed the peritoneal macrophages, and diminished their viability and phagocytic potential. Chronic fumonisin-exposure in poultry has shown to have adverse effects on the immune system leading to increased pathogen susceptibility and lowered vaccinal response (Voss et al., 2007). Additionally, ingestion of fumonisins affects the expression of proteins related to pro- and anti-inflammatory responses in the intestinal tract of broilers (Grenier and Applegate., 2013).

Rats and mice have been used extensively for decades as a model organism to study human mycotoxicosis, especially with regard to the carcinogenic potential of mycotoxins.

Pozzi et al., (2000) have reported apoptosis in the liver cells, by studying the effects of prolonged oral administration of fumonisin B1 and aflatoxin B1 in rats. Theumer et al., (2002) have detected immunobiological alterations produced by the ingestion of FB1 in a model of experimental subchronic mycotoxicosis in rats.

737 37

Cattle, sheep, and goats are known to be less sensitive to fumonisins. However, a negative impact on the production of milk/wool, reproduction and growth can be noticed when animals are exposed to FB1 for longer periods of time. An in vitro study by Bernabucci et al. (2011) showed an increased production of malondialdehyde (MDA) in bovine peripheral blood mononuclear cells treated with 35 and 70 µg/mL of FB1. A study by Goel et al., (1994) showed an increase in Sa:So ratios in serum, liver, kidney and muscle of catfish fed with ≥10 mg FB1/kg feed after 12 weeks of treatment. An increase in serum enzymes, urea and creatinine were reported by Orsi et al. (2009) and Gbore et al. (2010) in rabbits administered oral doses of FB1. Epidemiological incidences of esophageal cancer in humans due to the consumption of fumonisin-contaminated food have been reported from various parts of the globe such as South Africa, Central America, Asia (Rheeder et al., 1992; Chelule et al., 2001; Marasas et al., 2004) and among the dark population in Charleston, South Carolina (Sydenham et al., 1991) .

Similar observations were also documented from China (Yoshizawa et al., 1994; Abnet et

al., 2001), Italy (Franceschi et al., 1990), and Brazil (Van der Westhuizen et al., 2003). An outbreak of FB1 -associated illness due to consumption of sorghum and maize contaminated with high levels of fumonisins was also reported from India characterized by acute onset of abdominal pain and diarrhea (Bhat and Krishnamachari, 1977). Consumption of FB1 contaminated maize has also been associated with neural tube defects (due to reduction in the uptake of folic acid via folate receptor) in human infants of the rural population in South Africa and Northern China (Marasas et al., 2004) .

8 38

Environmental stress factors related to mycotoxin occurrence The critical difficulty in assessing the risk of different mycotoxins to animal and human health is the multiple factors affecting fungal colonization and production of mycotoxins in foods or feeds.

Environmental conditions (physical factors) are often conducive to rapid spoilage of feeds by fungi and production of harmful mycotoxins resulting in a significant decrease in the quality of feeds. Abiotic factors or stress conditions determine

Optimum conditions for fungal growth are not necessarily optimum for toxin production..

the extent of fungal colonization and mycotoxin biosynthesis in crops, foods and feeds. They include: Relative humidity Temperature fluctuations (heat,

Under natural conditions, the combination

cold, chilling, freezing)

of two or more factors such as drought and salinity, relative humidity and temperature,

Salinity Drought Nutrients Light intensity

heat and salinity, extreme temperature and high light intensity, etc., may have an impact on fungal infestation. These environmental factors influence the fungal growth, metabolism and mycotoxin biosynthesis and are essential to understand

Ozone pH

the overall process and to prevent mycotoxin production and spoilage of food or feed.

Anaerobic stresses

(Wang et al., 2003; Mitchell et al., 2004; Agarwal and Grover, 2006; Hirel et al., 2007; Cavanagh et al., 2008; Munns and Tester, 2008; Chinnusamy and Zhu, 2009; Marin et al., 2010b; Mittler and Blumwald, 2010; Faneli et al., 2012)

9 39 39

Management of fumonisin contamination The negative effects of consuming

However, a wide gap still persists

Safe elimination of fumonisins from

food or feed contaminated with

globally when it comes to

feed is of paramount importance as

mycotoxins have gained much

implementing these practices.

the poultry/livestock sector suffers

attention in the public and scientific arena in the recent years. This has generated a hitherto unprecedented interest towards the development of new detoxification procedures.

from health-related issues and great Furthermore, mycotoxin

economic losses due to fumonisins.

contamination can be partially prevented by adapting proper processing methodologies and storage facilities of cereals, grains, food and feedstuffs.

Good Agricultural Practices (GAP) and Good Manufacturing Practices (GMP) are possible methods to minimize mycotoxin occurrence in field conditions.

However, detoxification of toxins cannot be fully achieved as their production is modulated by environmental factors.

10 40

Most physical and chemical strategies followed to reduce mycotoxin contamination have been shown to be rather ineffective or are difficult to implement into the production process (Pearson et al., 2004). Moreover, fungal resistance to chemical treatments has now become widespread (Davidson, 2001).

Therefore, a biological control method

The most commonly employed detoxification

would be an efficient

method in the poultry/livestock industry is the use of

alternative for the

mycotoxin binders (sequestering agents) in feed, but

management of

they are usually aflatoxin-binders and have much

fumonisins (Figure 4).

less affinity towards fumonisins or other mycotoxins.

Good Agricultural Practices (GAP) Growing disease resistant crops Insects and fungal treatments

AT HARVEST

Growing cultivars with geographic region and climatic conditions

Proper sanitation Maturity of the crop (yield)

PRE-HARVEST

Weather Climate conditions Moisture content

POST-HARVEST PROCESSING AND TRANSFORMATION Physical approaches – solvent extraction, thermal inactivation, photochemical, irradiation, toxin adsorbents/binders

STORAGE

Chemical approaches – acidic agents, alkalies, aldehydes, oxidizing agents, reducing agents

Drying

Biological approaches – plants extracts, antioxidants, essential oils, enzymatic degradation by microbial taxa, probiotic lactic acid bacteria

Proper sanitation Sorting

Figure 4. Mycotoxin management strategies in the poultry and livestock industries.

Shelling Grading

1141 41

Many species of bacteria and

Among the biocontrol agents,

The possible mechanisms

fungi such as Flavobacterium

probiotic lactic acid bacteria (LAB)

involved in the antifungal

aurantiacum, Corynebacterium rubrum, Candida lipolitica, Aspergillus niger, Trichoderma viride, Armillariella tabescens, Nuerospora species, Rhizopus species, Mucor species, etc., have been shown to enzymatically degrade mycotoxins (Bata and Lasztity, 1999; Ciegler et al., 1966).

represent a potent and interesting

efficiency of LAB include:

application as they are widely used in fermented food products and feed to extend the shelf life of food/feed. Moreover, lactic acid bacteria are GRAS “Generally Regarded as Safe” (USFDA, 2017) organisms having wide metabolic versatility and ability to produce a broad range of metabolic end products.

Production of organic acids

Competition for nutrients

Enzymes capable of degrading fumonisins have been isolated from a filamentous

Production of antagonistic compounds

saprophytic fungus growing on maize and the corresponding genes have been cloned and transferred in transgenic maize

ANTIFUNGAL PROPERTIES OF LAB

(Blackwell et al., 1999).

1. Isolation of fumonisin-degrading enzymes

Saprophytic filamentous fungus 2. Cloned DNA 3. Transfer to transgenic maize

1242

The inhibition of mycotoxin production by LAB is due to microbial competition, depletion of nutrients, low pH, and the production of heat-stable low-molecular-weight secondary metabolites (Batish et al., 1997;

Gourama and Bullerman, 1997; Laitila et al., 2002). The mechanism by which LAB detoxiffies mycotoxin remains to be elucidated. However, several reports suggest the binding nature of LAB to the mycotoxin moieties. Lactobacilli also produce antifungal metabolites

The majority of studies on mycotoxins are aflatoxin-oriented, with less attention being paid to Fumonisin B1 contamination in food/ feed and their toxicity in humans/ animals. Having a clear understanding of fumonisin interference

such as organic acids, hydrogen peroxide,

with the living system followed by the development

proteinaceous compounds, hydroxyl fatty acids and

of chronic disorders is of immense value.

phenolic compounds, offering valuable opportunities in food preservation as well as feed supplements or in

Furthermore, exploration of probiotic LAB as potent

veterinary medicine (Magnusson et al., 2003;

antifungal agents and as a tool to biodegrade fumonisins

Kecerova et al., 2004; Gerez et al., 2009; Bilkova et al., 2011; Cortes-Zavaleta et al., 2014; Deepthi et al., 2016).

will guard food/feeds from fungal infestation.

Bacteriocin-like substances and other low and medium molecular weight compounds produced by Lactobacillus have also shown antifungal properties (Rouse et al., 2008; Kos et al., 2011; Al Kassaa et al., 2014).

These probiotic species also act as biopreservatives of food/feed and also have additive effects on health, performance and production. Subsequently this leads to increased food/feed production, as well as improved food/feed quality and trade/economy conditions for the countries.

13 43 43

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Orsi, R.B., Dilkin, P., Xavier, J.G., Aquino, S., Rocha, L.O., Correa, B., 2009. Acute toxicity of a single gavage dose of fumonisin B1 in rabbits. Chemico-Biological interactions. 179, 351-355. Pearson, T., Wicklow, D., Pasikatan, M., 2004. Reduction of aflatoxin and fumonisin contamination in yellow corn by high-speed dual-wavelength sorting. Cereal Chem. 81, 490–498. Poersch, A.B., Trombetta, F., Braga, A.C.M., Boeira, S.P., Oliveira, M.S., Dilkin, P., Mallmann, C.A., Fighera, M.R., Royes, L.F.F., Oliveira, M.S., Furian, A.F., 2014. Involvement of oxidative stress in subacute toxicity induced by fumonisin B1 in broiler chicks. Vet. Microbiol. 1988, 6–11. Pozzi, C.R., Correa, B., Xavier, J.g., Direito, G.M., Orsi, R.B., matarazzo, S.V., 2000. Effects of prolonged oral administration of fumonisin B1 and aflatoxin B1 in rats. Mycopathologia. 151, 21-27. Ramljak, D., Calvert, R.J., Wiesenfeld, P.W., Diwan, B.A., Catipovic, B., Marasas, W.F.O., Victor, T.C., Anderson, L.M., Gelderblom, W.C.A., 2000. A potential mechanism for fumonisin B1-mediated hepatocarconigenesis: cyclin D1 stabilization associated with activation of Akt and inhibition of GSK-3β activity. Carcinogenesis. 21(8), 1537-1546. Rheeder, J.P., Marasas, W.F.O., Thiel, P.G., Sydenham, E.W., Shephard, G.S., van Schalkwyk, D.J., 1992. Fusarium moniliforme and fumonisins in corn in relation to human esophageal cancer in Transkei. Phytopathol. 82, 353-357. Riley, R.T., Enongene, E., Voss, K.A., Norred, W.P., Meredith, F.I., Sharma, R.P., Spitsbergen, J., Williams, D.E., Carlson, D.B., Merrill, A.H. Jr., 2001. Sphingolipid perturbations as mechanism for fumonisin carcinogenesis. Environ. Health Perspect. 109, 301-308. Riley, R.T., Voss, K.A., 2006. Differential sensitivity of rat kidney and liver to fumonisin toxicity:Organ-specific differences in toxin accumulation and sphingoid base metabolism. Toxicol. Sci. 92, 335-345. Rouse, S., Harnett, D., Vaughan, A., van Sinderen, D., 2008. Lactic acid bacteria with potential to eliminate fungal spoilage in foods. J. Appl. Microbiol. 104, 915-923. Smith, G.W., Constable, P.D., Foreman, J.H., Eppley, R.M., Waggoner, A.L., Tumbleson, M.E., Waschek, W.M., 2002. Cardiovascular changes associated with intravenous administration of fumonisin B1 in horses. Am. J. Vet. Res. 63(4), 538-545. M.Y. Sreenivasa, Regina Sharmila Dass, A. P. Charith Raj and G. R. Janardhana. 2011. Mycological evaluation of Maize grains produced in Karnataka (India) for the post harvest fungal contamination. World Applied Sciences Journal, 13(4), 688 – 692. Sydenham, E.W., Shephard, G.S., Thiel, P.G., Marasas, W.F.O., Stockenstrom, S., 1991. Fumonisin contamination of commercial corn-based human foodstuffs. J. Agric. Food Chem. 39, 2014-2018. Tabata, S., Kamimura, H., Ibe, A., Hashimoto, H., Tamura, Y. 1994. Degradation of aflatoxins by food additives. J. Food Prot. 57, 42–47. Tardieu, D., Bailly, J.D., Benard, G., Tran, T.S., Guerre, P., 2004. Toxicity of maize containing known levels of fumonisin B1 during force-feeding of ducks. Poult. Sci. 83, 1287–1293. Tardieu, D., Bailley, J.-D., Skiba, F., Metayer, J.-P., Grosjean, F., Guerre, P., 2007. Chronic toxicity of fumonisins in turkeys. Poult. Sci. 86, 1887-1893. Taubel, M., 2005. Isolierung und Charakterisierung von Mikroorganismen zur biologischen Inaktivierung von Fumonisinen. Doctoral Thesis. University of Natural Resources and Applied Life Sciences. Vienna, Austria. The International Agency for Research on Cancer (IARC). Monographs on the evaluation of carcinogenic risks to humans: Some traditional herbal medicines, some mycotoxins, naphthalene and styrene. 2002; 82. Theumer, M.G., Lopez, A.G., Masih, D.T., Chulze, S.N., Rubinstein, H.R., 2002. Immunobiological effects of fumonisin B1 in experimental subchronic mycotoxicosis in rats. Clin. Diagn. Lab Immunol. 9(1), 149-155. U.S. Food and Drug Administration, 2017. GRAS Notices. http://www.accessdata.fda. gov/scripts/fdcc/?set=GRASNotices (Accessed 08.02. 17). Van der Westhuizrn, L., Shephard, G.S., Scussel, V.M., Costa, L.L.F., Vismer, H.F., Rheeder, J.P., Marasas, W.F.O., 2003. Fumonisin contamination and Fusarium incidence in corn from Santa Catarina, Brazil. J. Agric. Food Chem. 51(18), 5574-5578. Voss, K.A., Smith, G.W., Haschek, W.M., 2007. Fumonisins: Toxicokinetics, mechanism of action and toxicity. Anim. Feed Sci. Technol. 137, 299–325.Waalwijk, C., van der Lee, T., de Vries, I., Hesselink, T., Arts, J., Kema, G.H.J., 2004. Synteny in toxigenic Fusarium species: the fumonisin gene cluster and the mating type region as examples. Eur. J. Plant Pathol. 110, 533-544. Wang, W., Vinocur, B., Altman, A., 2003. Plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance. Planta. 218, 1-14. Yoshizawa, T., Yamashita, A., Luo, Y., 1994. Fumonisin occurrence in corn from highand low-risk areas for human esophageal cancer in China. Appl. Environ. Microbiol. 60, 1626-1629.

16 46

CLINICAL and PATHO-ANATOMICAL EFFECTS of MYCOTOXINS in animals

R. K. Asrani and Rakesh Kumar Department of Veterinary Pathology, Dr G C Negi College of Veterinary and Animal Sciences, CSK Himachal Pradesh Agricultural University, Palampur, Himachal Pradesh, India

47 1

Mycotoxins are secondary harmful mold metabolites that produce significant detrimental health effects in human beings and animals1. These are low molecular weight compounds known to be harmful even at low concentrations2.

Approximately 25% of the crops, including cereal grains and nuts, are often presumed to be contaminated with fungus3.

The most frequently encountered harmful mycotoxins in foodstuffs and feed include aflatoxin B1 (AFB1), ochratoxin A (OTA), trichothecenes, HT-2 and T-2 toxins, fumonisin B1 (FB1), citrinin (CTN), zearalenone (ZEN) and ergot alkaloids. A predominately marked distribution of fumonisins, zearalenone and deoxynivalenol (DON) is documented globally .

Moisture content (20-25%)

Cereal crops may become contaminated in

Environmental temperature (22-30ºC) Composition of food items

the field or during harvesting, transport, processing or storage5, 6. The rate of contamination of crops with fungus is more frequently triggered by the rainy season7.

Relative air humidity (70-90%) Physical damage to cereals by pests pH

Factors facilitating the production of mycotoxins in contaminated products include8:

Presence of mold spores

48 2

Diseases in animals

BIOLOGICAL FACTORS

Aflatoxixosis (AFB1): liver damage, liver cancer

Susceptible crop

Zearalenone (ZEN): reproductive problems etc.

ENVIRONMENTAL FACTORS Humidity Temperature Moisture Physical damage

Promote fungal growth in crops/ silage/concentrate/ grass, etc.

MYCOTOXINS PRODUCTION

Contaminated feed/fodder intake by animals

Secretion in milk and milk products (AFM1), eggs, meat and meat products (sausages)

HARVESTING, STORAGE & PROCESSING Consumption by humans (Mycotoxicosis)

Improper moisture, maturity and temperature conditions

Figure 1. Mycotoxins production and their occurrence in the food chain.

Common routes of entry of

Mycotoxins are known to produce several

mycotoxins into the body are:

harmful effects in animals and human beings. Classification of these toxins can be made on

Direct consumption of contaminated

the basis of toxicity13 and clinical symptoms

products of plant origin (cereals, nuts,

related to the organs damaged14.

bread etc.) and products obtained from animals (meat and meat products, milk,

Highly toxic (lethal at 1-10 mg/Kg body weight) Trichothecenes Aflatoxin B1 Citreoviridin

offal’s, fermented sausages etc.)9, 10. Dermal contact and inhalation are not very common routes but can act as a potential mode of entry into the body11. Harmful toxic effects of mycotoxins depend on11,12:

Severely toxic (fatal even < 1 mg/ Kg body weight) Rubratoxin B Cyclochilorotine

MYCOTOXINS

Type of mycotoxin Dose introduced into the body Duration of exposure to the mycotoxins

Figure 2. Classification of mycotoxins on the basis of toxicity.

All other Mycotoxins (toxic > 10 mg/Kg body weight)

49 3

Heapatotoxicity (aflatoxins, rubratoxins, fumonisins, cyclochlorotine)

Nephrotoxicity (ochratoxins, citrinin, quinones, viomelein, xanthomegnin)

Figure 3. Classification of mycotoxins on the basis of clinical manifestations. Neurotoxicity (fumonisin, patulin, citreoviridin)

MYCOTOXINS Estrogenicity (zearalenone)

Photosensitivity (sporedesmins)

Cytotoxicity (trichothecenes)

Immunotoxicity (ochratoxins, trichothecenes)

Table 1. Summary of different Mycotoxins with their toxic effects

Name of the fungus

Mycotoxins

Acronyms

Common sources

Target organs

Pathological change/Disease condition

Species susceptibility

Mode of action

References

Aspergillus ochraceus, A. verrucosum, Penicillium nordicum Aspergillus flavus, A. parasiticus

Ochratoxin

OTA

Kidneys, liver

Renal damage and cancer, hepatotoxicity

Swine, poultry, quail, human beings

Protein synthesis inhibition, nucleic acid damage and lipid peroxidation

15, 16, 17

Aflatoxins

AFB1, AFG1, AFB2, AFG2, AFM1

Liver, gastrointestinal system

Liver damage, hepatocellular carcinoma (HCC)

Pigs, human, dogs, cats, poultry

DNA adducts formation, mutations, inhibition of protein synthesis

18, 19

Trichothecium spp., Stachybotrys sp., Fusarium sp.

Trichothecenes

T-2, DON

Coffee, cereal grains, peanuts, dried fruits, cocoa, wine, spices, grape juice Sorghum, soybeans, nuts, rice, corn, cotton seed, cocoa beans, barley, dried fruits, crude vegetable oil Rye, wheat, barley, millet, oats

Gastrointestinal system and skin

Skin and gastrointestinal disorders

Horse, pigs, poultry, cattle, human beings

20, 21, 22, 23, 24, 25

Fusarium verticillioides, F. avenaceum, F. Tricinctum Fusarium graminearum, F culmorum Fusarium cerealis, F. roseum, F. incarnatum Fusarium proliferatum, F. verticillioides

Moniliformin

MON

Wheat,maize, rice,oats

Heart

Heart problems, depression

Birds

Inhibition of mitochondrial translation, inhibition of protein synthesis, DNA fragmentation Inhibition of thiamine pyrophosphatase pyruvate dehydrogenase

Zearalenone

ZEN

Wheat and maize bran

Reproductive system

Abortions,estrus defects, malformation of genital organs

Pigs, bovines, human beings

Binding with estrogen receptors, blocking the secretion of steroid hormones and suppressing estrogenic responses

Fumonisins

FB1, FB2

Silage, corn, wheat, barley, rice

Brain, lungs, esophagus

Swine, human beings, horses

Ergocristine, ergotamine, ergocryptine, ergometrine

Ergot alkaloids

Rye, wheat, triticale, barley, millet, oats

Smooth muscle, nervous system

Inhibition of sphingolipid biosynthesis, inhibition of protein synthesis in eukaryotic cells Partial agonist and weak antagonist in smooth muscles of the body, including blood vessels, and antagonist in central nervous system

Claviceps purpurea

Pulmonary oedema in pigs, Leucoencephalomalacia in equines Dry gangrene, abortion, hallucinations

Cattle, horse, sheep, poultry, human beings

26, 27

30, 31, 32

50 4

Effects of AFLATOXINS exposure Episodes of aflatoxicosis are

The order of severity of the mutagenic, immunosuppressive

associated with the production

and carcinogenic effects of aflatoxins is:

of aflatoxins by common fungal species such as Aspergillus

flavus and A. parasiticus in contaminated food products33.

AFB1> AFG1> AFB2>AFG233 AFB1 is predicted to exhibit developmental defects along with immune system dysfynction38.

In 1960, in the UK, the first report of mortality caused by aflatoxins-contaminated groundnut meal in turkeys

Hepatotoxicity

Teratogenicity

Immunotoxicity

Mutagenicity

Carcinogenicity

AFB1

and poultry was reported34. The list of aflatoxins produced by

Figure 4. Harmful effects of AFB1

several fungal species includes AFB1, AFB2, AFG1, AFG2 and AFM1.

Species susceptibility to aflatoxins

Among all known aflatoxins, AFB1 is the most common and potent35.

All animal species are sensitive to aflatoxicosis, but outbreaks are usually encountered among pigs, cattle and sheep39.

Aflatoxins are very stable and are rarely destroyed after

The significant economic losses, including decline in growth rate

processing36. Additionally,

and productivity, are usually reported in farm animals depending

residues of aflatoxins

on individual susceptibility and the targeted species40, 41, 42.

are also reported to be excreted in milk, milk

Chronic exposure to AFB1 in farm animals can lead to various

products, meat and eggs33.

ailments, including liver dysfunction, compromised immune status and susceptibility to several diseases43,44,45,46,47,48.

AFB1 is well recognized for its hepatotoxic, teratogenic, immunotoxic and mutagenic

Some of the animal species, such as monkeys, chickens and

potential and is classified as group

mice have been found to be resistant to AFB149, whereas cattle,

1 carcinogen by International

horses and sheep are quite prone to AFB1-induced toxicity.

Agency for Cancer Research (IARC)37, as it causes hepatocellular

Younger animals have proven to be more

carcinoma in human beings.

susceptible than adult and older animals50. Among aquatic animals, trout have been observed to be the most sensitive to AFB1 toxicity51. Among poultry, the order of sensitivity is: ducks > turkeys> Japanese quail> chickens

51 5

AFB1

Metabolized by cytochrome P450 (CYP) in the liver to AFB1-8,9-exo-epoxide (AFBO), AFM1, aflatoxicol (AFL), AFB2a, AFQ1, AFP153

AFB1-8,9-exo-epoxide (toxic derivative and electrophilic in nature)

Binding with guanine residues of DNA and RNA in hepatocytes

Adducts induce mutations in DNA and inhibit DNA transcription and RNA translation54,55

Figure 5. Flow chart indicating mode of action of aflatoxin B1

Abdominal pain, vomiting and oedema can be observed in acute stages, whereas development of hepatocellular carcinoma is evident in later stages56.

AFB1 toxicity in ruminants leads to: Decline in ruminal motility Decline in the cellular digestion and fatty acid production Decline in feed efficiency and is secreted in milk as AFM1 after 12 h of consumption.

52 6

Aflatoxin M1

Aflatoxin M1 (AFM1) is a group 1 carcinogen

(IARC) formed through CYP1A2-dependent hydroxylation microbial biotransformation from AFB1. The nuclear adducts are formed

Aflatoxin B1 contaminated feed ingested by animals

Microbial biotransformation (CYP1A2-dependent hydroxylation) to AFM1

DNA adducts

Excretion in milk (DAIRY PRODUCTS) and urine Conjugation with glucuronic acid and excreted in bile

and secreted in milk and urine. The concentration of AFM1 in milk is influenced by several factors, such as duration of lactation and the milk yield of the animal57.

Table 2. Permissible limits of aflatoxins consumption58, 59. Agency

Maximum permissible limit of aflatoxins

AFM1 in milk as per US Food and Drug Administration (FDA)

0.5 μg/kg

Maximum permissible limit of AFM1 in milk and dairy products as per European Commission (EC)

50 ng/kg

Maximum permissible limit of AFB1 in dairy feed (FDA)

20 μg/kg

Health risk to infants and the human population

*An average intake of aflatoxins in human beings ranges between 10-200 ng/kg/day

Figure 6. Aflatoxin M1 in the food chain

Table 3. Clinical and patho-anatomical effects of aflatoxins.

Species

Clinical symptoms

Pathological changes

References

Humans

High fever, vomiting, tremors, hypoglycemia, coma and dark colored urine. Elevated levels of AST, ALT, ALP, creatinine, catalase, malondialdehyde (MDA) and declined values of total proteins, magnesium and reduced glutathione. Depression, anorexia, fever and ruminal contractions in a study conducted by Elgioushy et al.61. Acute aflatoxicosis in Hereford calves after the consumption of peanut hay containing 2230 µg AFB1/kg led to symptoms including icterus, photosensitization, diarrhoea, depression and anorexia by a study conducted by McKenzie et al.62. More sensitive to aflatoxicosis Contaminated feed consumption leads to reduced feed intake, declined growth and production status64.

Cerebral oedema, hemorrhages, fatty degeneration in liver and kidneys, encephalopathy, cirrhosis and hepatocellular carcinoma (HCC). Liver enlargement, distended gall bladder, congested intestine with congestion of the kidneys61. Proliferation of connective tissue involving portal triads in chronic toxicity of feed contaminated with aflatoxins63.

33, 60

Pregnant sows: distorted hepatic architecture, hemorrhages, distended sinusoids, cystic spaces in the liver, lymphoid depletion in lymph nodes and spleen65. Hyperacute cases: hepatic necrosis and hemorrhages. Acute toxicity cases: cellular infiltration, swollen hepatocytes with cholestasis. Subacute toxicity cases: vacuolar degeneration, cholestasis with profound bile duct hyperplasia66. Hydropic degeneration in the hepatocytes, necrotic changes, hyperemia, sinusoidal contraction with accumulation of ceroid pigments have been observed in macrophages from Merino rams treated with aflatoxins at the dose of 250 µg/day68. Hemorrhagic spots in muscles, atrophied spleen; enlarged, paler, fatty liver with hemorrhagic areas; distended gall bladder, nephropathy and thickened crop and proventricular mucosa. Microscopic fatty changes in hepatocytes, acinar arrangement of hepatocytes, lymphocytes and heterophilic aggregates with multiple areas of necrosis, hyperplastic changes in crop and proventricular mucosa70,71.

64, 65, 66

Cattle

Swine

Sheep

Decreased erythrocyte and leucocyte count with decreased values of hemoglobin and packed cell volume before initiation of clinical manifestations67.

Poultry

Feeding aflatoxins at a rate of 3.5 mg/kg of feed resulted in a marked decrease in body weight and growth performance with increased kidneys and liver weight69.

61, 62, 63

67, 68

69, 70, 71

53 7

Image 1. Gross pathological alterations associated with AFB1. Liver of a rabbit showing chronic hepatitis along with tumorous growth.

Image 2. Photomicrographs of pathological alterations associated with AFB1

a. Liver showing diffuse hemorrhages along with necrotic area in the hepatocytes along with hemosiderin deposition (H&E*66). b. Liver showing swollen hepatocytes with hydropic changes (H&E*66). c. Photomicrograph of liver showing portal fibrosis with bile duct hyperplasia (H&E*33). d. Liver showing peripheral shifting of nucleus giving a signet ring appearance indicating fatty changes in hepatocytes (H&E*66).

54 8

Effects of OCHRATOXIN A exposure Aspergillus ochraceus, Auplopus

This mycotoxin was first reported

Pigs and poultry more

carbonarius and Penicillium

in contaminated cornmeal

sensitive to OTA

verrucosum are the most common

it is considered to be the most

fungal species associated with

common and potent mycotoxin

the production of ochratoxins

produced by these fungi73.

and

induced toxicity. Ruminants are usually resistant, as OTA is degraded

in contaminated grains, raw and cooked food items and beverages

OTA is readily known for its

by ruminal microflora to

(coffee, beans, and wine).

nephrotoxic, carcinogenic,

less toxic metabolites

immunosuppressive, teratogenic

such as OTAα78.

Aspergillus ochraceus and

and genotoxicity in animals74,75,76.

Penicillium verrucosum are the

Additionally it has been found

Some researchers have shown

most potent moulds responsible

to produce hepatocellular

the release of OTA in breast

for the production of Ochratoxin

carcinoma as well, apart from

milk, which means it can act as

A (OTA) in tropical and

the nephrotoxic properties, in

a potent threat to the newborns

temperate regions, respectively.

a dose dependent manner .

through breastfeeding79.

Intake of OTA contaminated food items

Binding of OTA to blood albumin

Proximal convoluted tubule (PCT) (Target site)

Organic anion transporter (OAT) 1 and 3 help in the absorption of OTA in the interstitium and OAT 4 in the tubular lumen

Production of reactive metabolites that form adducts after reacting with DNA Alteration of the transmembrane potential of the mitochondrial membrane, causing the release of cytochrome c and apoptosis Inhibition of protein synthesis by competing with Phe-tRNA synthase

Figure 7. Flow chart indicating the mechanism of action of OTA in kidney tubular cells80 81 82.

559

Table 4. Tolerable limits of OTA. Agencies

Tolerable limits of OTA

Joint Expert Committee on Food Additives (JECFA)84

112 ng/kg body weight/week

Maximum limit of OTA (As per EU)85

3 μg/kg in processed cereal

European Food Safety Authority83

17 ng/kg body weight/day

*In one of the studies in Italy from Capei et al.83 has documented an 8% contamination of OTA in breakfast cereals and 50% contamination in sweet snacks with a contamination limit ranging between 2.9 – 8.6%. A daily intake of OTA at the dose of 1 mg/Kg body weight for 5-6 days can be harmful.

Table 5. Clinical and patho-anatomicaleffects of ochratoxins.

Species

Clinical symptoms

Pathological changes

Referencias

Humans

Weakness, brown discoloration of the skin and lumbar pain. Biochemical parameters such as glucose, gamma-glutamyl transferase and leucine aminopeptidase increase in urine, along with proteinuria with RBCS and WBCs in urine86.

86, 87, 88, 89, 90, 91

Swine

Bulgarian and Danish porcine nephropathy, reduced feed intake and weight gain92. Residues can be transported to human beings through pork or offals93.

Poultry, quail and rats

In poultry, they cause a decrease in egg production, decreased FCR, immunosuppression, developmental abnormalities, reduced feed consumption with an increased water intake49.

Acute renal failure , Balkan endemic nephropathy (BEN), Tunisian nephropathy88. BEN is a tubule-interstitial renal disease that leads to contracted kidneys in later stages86, which may also be followed by renal tumours89. Tubular and glomerular degenerations with fibrotic changes in interstitial tissue, necrosis, apoptosis and end stage kidney damage90,91. Swollen, pale, firm kidneys with gross lesions of fibrosis Microscopically, degenerative changes with fibrosis in the kidneys of pigs from abattoirs during random sampling were consistent finding in post weaning multisystemic wasting syndrome (PMWS), nephropathy syndrome (PDNS) and ochratoxicosis94. Diffuse tubular nephrosis and interstitial fibrosis95. OTA is found to majorly affect mitochondria in PCT cells and also causes degranulation of the rough endoplasmic reticulum (RER). The changes associated with damage of kidneys in quail include profound karyomegaly, cellular swelling, cytoplasmic vacoulations, margination of chromatin material96. Changes such as bile duct hyperplasia, necrosis of liver cells, vacuolar degenerations, dilation of central veins and sinusoids, along with mononuclear cell (MNCs) infiltration has also been reported in the liver by a research concluded by Patial et al.97. Abnormalities of the central nervous system (CNS) along with skeletal system defects were reported in rats when administered OTA during the gestation period98. 87

92, 93, 94, 95

49, 96, 97, 98

Image 3. Gross pathological alterations associated with OTA. a. Swollen and pale kidneys of Japanese quail (right) after administration of Ochratoxin A in diet in comparison to the kidneys on the left side b. Ruffled appearance of feathers in a Japanese quail after feeding Ochratoxin A.

56 10

Image 4. Photomicrograph of pathological alterations associated with OTA. Kidney showing fibrous tissue accumulation in the interstitial tissue causing atrophy of renal tubules in OTA toxicity (H&E*66).

Effects of FUMONISIN exposure Fumonisins are produced

Toxicity associated to fumonisins

Fumonisins are also reported

by fungal species such as

was firstly reported in 1980 as a

to cause leukoencephlomacia

Fusarium verticillioides and F. proliferatum, and they are frequently spotted on maize giving it a whitish appearance99.

cause of equine encephalomalacia

in horses, hepatocellular

(ELEM) and porcine pulmonary

carcinoma in rats and pulmonary

oedema (PPE) in the United States,

oedema in association with

and esophageal cancer in Africa.

hydrothorax in pigs103, whereas the IARC has also documented

The most common forms of

These mycotoxins cause

the carcinogenic potential of

fumonisins include fumonisin A and

neurotoxicity, hepatotoxicity,

fumonisins in human beings104.

fumonisin B (B1, B2, B3 and hydrolyzed

embryo toxicity and

B1), and among these fumonisin B1

nephrotoxicity in animals101,102.

is the most common and potent

100

As per JECFA, the maximum tolerable limit of FB on the basis of no-observable-effectlevel (NOEL) of 0.2 mg/kg The production of this

bw/day with a safety factor

mycotoxin is promoted when

100 is 2 μg/kg/day105.

moisture content is < 19%.

57 11

Mechanisms of action of Fumonisins102: Competitive inhibition of the ceramide synthase enzyme Oxidative stress and endoplasmic reticulum stress

Figure 8. Mechanisms of inhibition of sphingolipid metabolism.

Autophagy modulation Alteration of DNA methylation

Competitive inhibition of the ceramide synthase enzyme Required for sphingolipid biosynthesis

Disruption of sphingolipid metabolism

In liver, kidney and brain tissue

Inhibition of the acylation of sphingosine and sphinguanine

Inhibition of ceramide synthase

Table 6. Clinical and patho-anatomical effects of fumonisins. Species

Clinical symptoms

Pathological changes

Reference

Humans

Esophageal cancer106.

106, 107

Cattle

Optic nerve degeneration leading to blindness108.

Swine - Porcine pulmonary oedema (PPE)

Defective vision, staggering, drowsiness, weight loss, decreased feed intake, respiratory distress and cynosis110,111,112.

Horses - Equine leukoencephalomalacia (ELEM)/Moldy corn poisoning) Fish

Circling, head pressing, blindness, ataxia and depression110.

Liquefactive necrosis or softening of cerebral subcortical white matter with occasional areas of hemorrhages. Histological examination shows eosinophilic and swollen astrocytes in white matter of brain. Pregnant female mice treated with 2.5 or 10 mg/kg FB1 intraperitoneal injection show neural tube defects in fetuses107. Liver and kidney damage have been reported in calves treated with 1 mg FB1/kg body weight for 7 days109. Histological changes evidenced in cattle with blindness includes fibrosed septa, retinal degeneration, optic nerve degeneration and oedema of myelin108. Grossly, it is characterized by the presence of fluid in the thoracic cavity and airways with widened interlobular septa. Microscopic evaluation depicts widened interlobular septa with perivascular and peribronchiolar oedema along with MNCs infiltration. The right ventricle of the heart and pulmonary artery shows hypertrophy. Vacuolar changes and cellular swelling of hepatocytes is also evident111, 113. Focal to multifocal areas of necrosis of white matter. Degenerative changes in endothelial cells along with perivascular thrombosis. Oedema formation in neural tissue with neutrophilic infiltrations114.

Birds

Nervous symptoms115.

Decrease in body weight, increased serum biochemical markers in ducks116. Diarrhoea, increased gizzard, proventriculus and liver weight in broilers117.

Chronic exposure of one year old carp (Cyprinuscarpio. L) with a feed containing FB1 at the dose of 10 mg/kg body weight for 42 days showed nervous manifestations.Histological evaluation of brain tissue reflected degenerative changes, vacoulations, necrotic changes in the brain cells around periventricular area and capillaries115. Liver of affected quail showed necrotic hepatitis along with infiltration of heterophils and macrophages. Increased Kupffer cell activity, bile duct hyperplasia with increased granularity of cellular cytoplasm has also been evidenced118. Additionally, kidneys showed swelling of the tubular epithelial cells and glomerular tuft, obliteration of Bowman’s capsule, apoptotic changes and elevated mitotic activity118. Deshmukh et al.119 concluded progressive degenerative changes along with heterophilic infiltration in tubular epithelial cells and interstitial tissue of kidneys at the dose of 150 ppm for 21 days in quail. Hepatic necrosis, bile duct hyperplasia, thymic atrophy and rickets in broiler chicks affected with FB1 has also been observed in a study conducted by Ledoux et al.117.

108, 109

110, 111, 112, 113

110, 114

115

116, 117, 118, 119

58 12

Image 5. Gross pathological alterations associated with FB1. a. Enlarged liver of a Japanese quail after feeding Fumonisin (FB1) for 3 weeks at the dose of 300 ppm. b. Enlargement of liver (right side) with Fumonisin (FB1) toxicity in comparison to normal liver on the left side.

Image 6. Photomicrograph of pathological alterations associated with FB1. Liver of a Japanese quail reflecting necrotic changes along with heterophilic infiltration admixed with mononuclear cells after the administration of FB1 (H&E*330).

Effects of TRICHOTHECENES exposure Trichothecenes are toxic secondary

The main mycotoxins belonging to

metabolites produced by Fusarium

the trichothecene group include type

graminearum, Stachybotrys, Fusarium poae, Fusarium langsethiae, etc, often found contaminating wheat, maize, barley and oat kept in damp environmental conditions.

A (T-2) and type B toxins (DON), and their toxic potential is due to the presence of an epoxide ring122. These toxic metabolites are quite resistant to processing and are only destroyed at temperatures above

Production of these

260ºC for more than 30 min.

mycotoxins is often favored by ambient temperature (0-32oC) with humid conditions120, 121.

59 13

Harmful effects and tolerable limits of trichothecenes

In pigs, cattle, broilers and rats, trichothecenes

According to the EU, the maximum limit for the

damage the liver and stomach .

presence of DON in cattle feed is 5 mg/Kg feed,

123

whereas it is around 1 mg/Kg feed for calves. Therefore, trichothecenes toxicity in farm animals is often associated to symptoms such as vomiting,

In studies conducted by Ingalls129 and Cote et al.130

diarrhoea, anorexia, weight loss and death

no marked variation in the milk production was

124,125

Additionally, the malabsorption induced by

reported when DON is given at a rate of 14 mg/kg

trichothecenes in pigs, poultry and rats is often

for 3 weeks and 66 mg/kg for 5 days, respectively.

associated with necrosis of intestinal villi126,127. Based on the presence of ester-ether bonds between C-4 and C-15 at C-12 we can divide trichothecenes into 2 types: macrocyclic and non-macrocyclic. The nonThe maximum tolerable limits of

macrocyclic trichothecenes are enlisted in Table 7.

DON in most parts of the world are limited to 0.75 mg/kg in human diets and 1-5 mg/kg in animal rations128.

Table 7. Classification of trichothecenes.

A* Non-macrocyclic trichothecenes B

T2 toxin Diacetoxyscirpenol (DAS) Neosolaniol Nivalenol Deoxynivalenol (DON) Fusarenon-X

*T2 and DAS are used as bioweapons and are mainly produced by Fusarium poae and Fusarium langsethiae.

60 14

Inhibition of protein synthesis after binding to the 60S subunit of ribosome, leading to the inhibition of peptidyltransferase and inhibition of the initiation, elongation or termination steps in protein synthesis

Oxidative stress mediated DNA damage and apoptosis

TRICHOTHECENES

Figure 9. Clinical and patho-anatomical effects of trichothecenes20, 21, 22, 23.

Inhibition of mitochondrial translation

Table 8. Clinical and patho-anatomical effects of trichothecenes. Species

Clinical symptoms

Pathological changes

References

Humans

Type A trichothecenes can lead to alimentary toxic aleukia (ATA) in human beings causing severe leukopenia, vomiting and nervous symptoms131,132. Production losses, altered reproductive potential, decline in liver function and immunosuppression is seen in dairy cattle fed with silage and cereal grains contaminated with trichothecenes133. Calves affected with DON toxicity show icterus with altered liver enzymes134. Growth performance and feed intake in finishing pigs is severely influenced by DON toxicity138.

Skin rashes, necrotic stomatitis, hemorrhagic vaginitis and nervous system affections131.

131, 132

Postmortem evaluation of dead carcasses reveals congestion and hemorrhages in abomasum, splenomegaly and kidney damage. Histological investigation often reflects degenerative changes, cholestasis, bile duct hyperplasia, steatosis and infiltration of mononuclear cells (MNCs) especially macrophages and lymphocytes134. T-2 toxicity in cattle leads to absence of estrus, hemorrhagic and necrotic enteritis, decreased feed intake and reduced milk production135,136,137.

133, 134, 135, 136, 137

Piglets fed with DON (1.5-2.8 mg/kg feed for 4-5 weeks) were found to show gastrointestinal and hepatotoxic pathological alterations139, 140. DON interferes with reproductive potential of sows as is speculated to produce harmful effects on the ovaries and follicles141. Feed contaminated with T2 toxin in acute toxicity cases lead to myocarditis/cardiotoxicity, rumenitis with ulcerative abomasitis, anasarca, brain oedema and pancreatic necrosis142.

138, 139, 140, 141

Cattle

Swine

Sheep

Goats

Chronic exposure is often followed by declined reproductive potential, gastroenteritis, weight loss, myocarditis and pus in the oral cavity142. Growth retardation, lethargy, decrease platelet, Hb and total leucocyte count, decrease in the values of serum and tissue superoxide dismutase and catalase143.

Providing T-2 toxin contaminated feed (10-20ppm) in juvenile goats led to severe pathological alterations in the intestine and liver on ultra-structural evaluation. Apoptotic changes are markedly prominent in mesenteric lymph nodes, proximal convoluted tubules (PCTs) and distal convoluted tubules (DCTs) of the kidneys, enterocytes in the intestine and spleen with significant up-regulation of pro-apoptotic proteins, HSPs and cytokines143. The histological changes in the liver tissue of goats included centrilobular necrosis, sinusoidal congestion, peri-ductular connective tissue proliferation, bile duct hyperplasia and vacuolar degeneration143.

142

143

61 15

Effects of ZEARALENONE exposure The most common fungal species involved in the production of zearalenone (ZEN) include Fusarium

culmorum, F. cerealis and F. graminearum. This mycotoxin is commonly found in cereal grains in temperate regions with warm weather144, 145 and can remain stable at temperatures up to 150˚C146. The highest production of ZEN is reported at 25˚C with 16% moisture content147,148. Five major metabolites of ZEN include α-zearalenone (α-ZEN),

Pigs are speculated to be the most sensitive species for ZEN-induced

β-zearalenone (β-ZEN), α-zearalenol

reproductive disorders as compared to other animals157.

(α-ZAL), β-zearalenol (β-ZAL) and zearalenol (ZON), α-ZEN having the highest estrogenic activity

149, 150

Zearalenone is responsible for causing ear rot in maize and head blight in wheat and barley151, with immunotoxic, genotoxic, hepatotoxic and hematotoxic effects in animals, as well as significant nephrotoxic potential

About 80-85% of oral dose of ZEN is found to be efficiently absorbed in pigs158. The concentration of ZEN and α-ZEN in follicular fluid of swine is 38.9 and 17.6 pg/ml, respectively159. Very limited data is documented about the folliculogenesis in ovaries of domestic animals160, but ZEN shows affinity towards estrogen receptors in uterus, mammary gland, brain and bones, which reflects its estrogenic potential161.

with an ability to produce pituitary adenomas152,153,154,155. Additionally, ZEN is linked to reproductive disorders in animals and hyperestrogenic syndrome in human beings156.

Table 9. Tolerable limits of zearalenone (ZEN). Tolerable daily intake (TDI) in humans162 Cereals151

20-200 μg/kg Processed cereals

75 μg/kg

Unprocessed cereals

100-200 μg/kg

Unprocessed cereal snacks

50 μg/kg

Cereal foods

20 μg/kg

Regulatory limits (China) in wheat/corn/flour163

60 μg/kg

62 16

Figure 10. Mechanism of action of ZEN

Oral intake of ZEN

Absorption through the gastrointestinal tract

Converted to active metabolites (α-ZAL, β-ZAL) with the help of 3α and 3β hydroxysteroid hydrogenase

Binding to estrogen receptors in uterus, mammary gland, brain and bones, so reflecting its estrogenic potential

ZEN blocks the secretion of steroid hormones and suppresses the estrogenic response in the preovulatory phase

Table 10. Clinical and patho-anatomical effects of Zearalenone. Species

Clinical symptoms

Pathological changes

References

Humans

Hypoestrogen syndrome in human beings, acting as a stimulating factor for precocious puberty development in females164. Genotoxicity effect on the lymphocytes due to the formation of DNA adducts167,168. ZEN is excreted in the milk of cows fed with high doses of ZEN. Heifers fed with 99% pure ZEN at a rate of 250 mg/day showed a decline in the conception rate of 87-62%169. Weaned gilts affected with ZEN toxicity usually reflect ovarian atrophy, vulvar hypertrophy without any significant effects in uterus and mammary glands170,171. Hormone production and estrus cycle length is not altered in mares provided with oats contaminated with ZEN (2 mg/ Kg)160. ZEN leads to a decline in serum progesterone and testosterone concentration, reduced sperm count, increased incidence of infertility and decreased conception rate in pigs, cows, rats and mice168,174.

Endometrial hyperplasia, mammary tumors, adenocarcinoma and proliferative changes in women165,166.

164, 165, 166

Irregular estrus, infertility, abortion, retention of placenta, mastitis and metritis.

167, 168, 169

Decreased fertility, abnormal estrus cycle, abortion, vulvovaginits and reduced litter size172.

170, 171, 172

Ovarian follicular atresia173.

160, 173

Cystic mammary glands, hepatopathy and nephropathy, uterine fibrosis, persistent estrus, sterility, squamous metaplasia and, hyperplasia of the endometrial glands175,176.

168, 174, 175, 176

Cattle

Pigs Horses Rodents

63 17

Effects of MONILIFORMIN exposure Fungal sources involved in the production of monilformin (MON) include Fusarium moniliforme, F proliferatum, F. avenaceum, F. subglutinans, F. tricinctum and Pencillium melanoconidium177, 178, 179, 180. Contaminated cereal grains and plants used for silage

Inhibition of thiamine pyrophosphatase enzymes in tricarboxylic acid cycle

preparation are the major source of production of this mycotoxin. MON is cardiotoxic and hematotoxic , with acute toxicity 181

Altered oxidation of pyruvate and α-ketoglutarate

that is comparable to trichothecene toxicity (T2, HT-2)182, 183. Fatal outbreaks of MON are reported in animals, but experimental studies in birds and rats have shown potential pathological effects184,185,186.

Inhibition of synthesis of collagen type II and aggrecan causing catabolic effect on articular cartilages

Inhibition of pyruvate dehydrogenase

Figure 11. Mechanisms of action of MON187, 188.

64 18

Clinical and patho-anatomical effects of moniliformin

In birds and laboratory rodents, intestinal hemorrhages are seen in acute cases, whereas cardiac hemorrhages are typical lesions in sub-acute and chronic cases of MON toxicity189. In one of the sub-acute toxicity studies conducted by Jonsson et

al.190 reflected intestinal hemorrhages with pulmonary congestion in rats without other specific lesions in other organs. Cardiomyopathy depicted by necrotic and degenerative changes in the heart with hypertrophy of muscle fibers causing cardiac arrest in quail birds fed with MON at the dose of 100 ppm has also been documented in previous studies191.

Image 7. Gross pathological alterations associated with MON. Japanese quail showing rounding and dilation of heart (Right side) after feeding MON at the dose of 110- ppm for 3 weeks; Left side showing normal heart.

Image 8. Photomicrographs of pathological alterations associated MON. a. Heart of a Japanese quail showing hypertrophy of cardiac muscle fibers following MON administration (H& E*132). b. Glomerular tufts occupied by needle shaped uric acid crystals in MON toxicity (H&E*66).

65 19

MULTI-MYCOTOXIN toxicity

In field conditions, it is most common to find raw materials to be contaminated with one or more mycotoxins, with variations in the symptoms associated with exposure, as the combination of these toxins can involve different types of interactions, such as synergistic, additive or antagonistic effects as shown in Table 11.

Table 11. Combined toxic effects of various mycotoxins. Mycotoxin

Combination and type of interaction

Toxic effects produced

Ochratoxin (OTA)

Citrinin + FB1 (additive and synergistic)

Cytotoxicity to mononuclear cells

FB1 (additive and synergistic)

Nephrotoxicity, hepatotoxicity, genotoxicity and immunosuppression

ZEN (antagonistic)

Cytotoxicity161, 197

Citrinin (synergistic, antagonistic and additive)

194, 196

Trichothecenes (synergistic and additive)

Nephrotoxicity, immune organ depletion, gastrointestinal problems and fetal malformations194, 196 Nephrotoxicity, immunotoxicity and hepatotoxicity198, 199

AB1 (synergistic and antagonistic)

Nephrotoxicity, teratogenicity, hepatotoxicity and cardiotoxicity

196, 200

MON (Synergistic)

191, 201

ZEN (antagonistic and synergistic)

Cardiotoxicity, nephrotoxicity, hepatotoxicity, immunosuppression, respiratory distress191, 201 Cytotoxicity and immunostimulation202

Trichothecenes (antagonistic and synergistic)

203, 204

Neural tube defects, hepatotoxicity, esophageal cancer

203, 204

Trichothecenes + ZEA (synergistic, antagonistic and additive

Cytotoxicity, oxidative damage and blockade of synthesis of macromolecules204, 205

204, 205

Fumonisin B1 (FB1)

References 192

192 193, 194,

195, 196

196, 200

193, 194, 195, 196 161, 197

198, 199

202

*The combined harmful effects of different mycotoxins depend upon the absorption rate206

CONCLUSIONS Mycotoxins are very harmful

Although in many of the countries

In order to limit the production

metabolites known to contaminate

tolerable limits for various

of mycotoxins, several strategies

food items and are majorly

mycotoxins are standardized,

are proposed and followed time

implicated in several clinical

a wide range of developing

and again by various agencies and

and pathological impairments in

regions around the globe still

regulatory bodies. In the present

human beings and animals.

need a thorough establishment

scenario, to minimize the production

of such standards with a strict

of mycotoxins during processing of

follow-up to reduce the levels of

raw material and final food products

mycotoxins in the food chain.

for animal or human use the basic principles to be followed include:

It is of utmost concern to prevent fungal contamination of food Excessive levels of mycotoxins can cause health hazards to the animals directly and through animal products to human beings.

products by providing high quality crops or animal products with controlled storage, harvesting and distribution strategies.

Good Agricultural Practices (GAP) Good Manufacturing Practices (GMP) Hazard Analysis Critical Control Points System (HACCP)

Regular monitoring of food items, animal feed etc. by employing proper guidelines and safety standards definitely will help to limit the fungal contamination.

67 21

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BIOMONITORING MYCOTOXINS IN PRODUCTION ANIMALS Pe r s p e c t i ve s & C h alle n g e s

Professor Carlos Augusto Fernandes de Oliveira Department of Food Engineering, Faculty of Zootechnics and Food Engineering, University of São Paulo, Pirassununga, São Paulo, Brazil.

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Biomarkers of exposure have been extensively used for improving the assessment of exposure to dietary mycotoxins in humans. Recently, biomarkers have also been proposed for biomonitoring mycotoxins in production animals.

In this article, the main biomarkers used for animal biomonitoring of single or multiple mycotoxin exposure are presented, as well as the potential application of these biomarkers for diagnostic purposes and for evaluating the efficacy of chemo-protective interventions, such as mineral adsorbents.

Mycotoxins & Mycotoxicosis Mycotoxicosis is a disease

Importantly, while food &

associated with exposure to dietary

feedstuffs may contain individual

mycotoxins, causing immune

mycotoxins, contamination by

suppression and target organ

multiple mycotoxins in these

toxicity with lesions mainly in

products is quite common and

liver, kidneys, epithelial tissue

has become an important human

(skin and mucous membranes),

and animal health concern due

and central nervous system,

to the possible combined effects

depending on the type of toxin.

of different mycotoxins.

The main groups of toxigenic

Toxicity of some individual

fungi and their respective

mycotoxins may be

mycotoxins belong to the genus:

increased in a synergistic,

Aspergillus: A. flavus, A. parasiticus and A. nomius: aflatoxins

additive, or antagonistic way, when they occur as cocontaminants and are ingested by different animal species.

Fusarium: fumonisins, trichothecenes, moniliformins and zearalenone Aspergillus ochraceus: ochratoxins Penicillium: ochratoxins

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AFLATOXINS

OCHRATOXIN A

FUMONISINS

Among the mycotoxins affecting

Ochratoxin A (OTA) is an

28 structurally related fumonisins

farm animals, the aflatoxins

important nephrotoxic mycotoxin

have been isolated and identified,

are hepatotoxic, teratogenic

with immunotoxic, teratogenic,

although fumonisin B1 (FB1) is

and genotoxic compounds, also

carcinogenic and perhaps neurotoxic

the most predominant and toxic

classified as carcinogenic to humans

effects causing liver and kidney

form produced by the fungi.

(Group 1) by the International

cancer in numerous animal species.

Agency for Research on Cancer.

FB1 has been shown to be hepatotoxic, nephrotoxic and

The aflatoxins were identified

carcinogenic in several animal

in 1961, aflatoxin B1 (AFB1)

studies, also causing species-

being the main type of toxin

specific diseases including

produced by Aspergillus

porcine pulmonary edema and

under natural conditions.

equine leukoencephalomalacia.

DEOXYNIVALENOL Deoxynivalenol (DON), correspondingly named vomitoxin because of its emetic effects after ingestion, is another mycotoxin produced by Fusarium species, which belongs to the class B

ZEARALENONE

trichothecenes and often co-exist with ZEN in feed materials such

Zearalenone (ZEN) is a mycotoxin which binds competitively to estrogen receptors, leading to estrogenic abnormalities and generative syndromes, especially in pigs.

as corn, oats, barley, and wheat. The acute exposure to DON also causes abdominal pain, salivation, diarrhea, leukocytosis, and gastrointestinal hemorrhage

(Oliveira et al., 2014).

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The challenge of mycotoxicosis diagnosis Since the aflatoxins’ discovery in the early 1960s’, the assessment of negative effects of mycotoxins on production animals has been based on the observation of signs and symptoms of intoxication, including decreased performance parameters, combined with the mycotoxin contamination data in feed and/or ingredients.

EXPOSURE BIOMARKERS A biomarker of exposure refers to

The first mycotoxin biomarker used in

the quantification of the specific

production animals was aflatoxin M1

compound, its metabolites or

(AFM1) in milk of lactating animals

interaction products in a body

fed rations containing aflatoxin B1

compartment or fluid, which indicates (AFB1), as illustrated in Figure 1. the presence and magnitude of

In spite of several existing

exposure to the agent.

mycotoxicosis diagnosis criteria, these classical approaches are associated with important limitations such as the variability of individual susceptibility to mycotoxins and their heterogeneous distribution in feed.

Figure 1. . Metabolic pathway of aflatoxin B1 conversion into aflatoxin M1 in dairy cows

Liver metabolism

AFM1 excreted in milk = 0,3-6,2% AFB1 ingested (Veldman et al., 1992)

Ingestion

Aflatoxin B1 in the ration Aflatoxin M1 in milk Excretion in milk

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The available data on toxicokinetics of several mycotoxins in animal models indicate that exposure to mycotoxins can be accurately measured by biomarkers in several bio-specimens, especially in serum. Serum aflatoxin B1-lysine (AFB1-lys), a digest product of AFB1-albumin used for human biomonitoring of aflatoxin exposure has been confirmed as a specific biomarker of aflatoxicosis in broilers and piglets (Di Gregorio et al., 2017).

LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY (LC-MS/MS) In recent years, the liquid chromatography tandem mass spectrometry (LC-MS/MS) based on the multi-analyte approach has been successfully introduced in the field of mycotoxins analysis, opening new perspectives for the evaluation of suitable biomarkers for mycotoxins mixtures.

For fumonisin B1 (FB1), experimental studies indicate that plasma and urinary FB1 are good biomarkers of early exposure of pigs to low dietary FB1 levels, although plasma is recommended to assess prolonged exposure (>14 days) (Souto et al., 2017).

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Biomarkers for assessing mycotoxin exposure in production animals An ideal biomarker should be: Specific Quantifiable Detectable at low levels Obtained by non-invasive and inexpensive techniques These attributes should be determined for each potential toxicant based on its toxicokinetics, which refers to the study of absorption, distribution, metabolism/ biotransformation, and excretion (ADME) of toxicants in relation to

PHASES OF BIOTRANSFORMATION

time. Thus, depending on the toxicokinetics of a given

In all animal species, biotransformation occurs in two phases:

mycotoxin after ingestion, some

Phase I, is mainly based on hydrolysis, reduction, and

biomarkers could be approached

oxidation reactions.

to indicate the magnitude and level of its dietary exposure

Phase II, involves conjugation of the products formed

by means of quantification of

in Phase I.

its metabolites or interaction products in body fluids.

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TOXICOKINETICS OF AFLATOXIN B1 For AFB1, the resulting

Cytochrome P450 enzymes in the

metabolites in Phase I include:

liver also transform AFB1 in

Hydroxylated products: AFM1 and aflatoxin Q1 Demethylated products:

AFB1-8,9-epoxide, its procarcinogen form, which is covalently bound to nucleic acids, mainly DNA (which yields the adduct AFB1-N7-guanine),

aflatoxin P1

and to serum albumin (which yields

A product from the reduction by

et al., 2017).

the adduct AFB1-lysine) (Di Gregorio

cytoplasmic enzymes: aflatoxicol All these compounds may be shed in urine, bile and feces (Oliveira et al.,

2014). The excretion rate of AFM1 in the milk of dairy cows ranges from 0,3 to 6,2% of the AFB1 ingested, depending on the lactation stage and volume of milk produced. In domestic poultry, the main products of AFB1 biotransformation are AFM1 and aflatoxicol, which may be found in eggs.

In this context, non-metabolized AFB1, its adducts, AFB1-lysine in blood serum and AFB1-N7guanine in urine, as well as its metabolite AFM1 in urine and milk, may be considered as validated biomarkers of AFB1 exposure.

AFM1 AFM1 & AFLATOXICOL

TOXICOKINETICS OF FUMONISINS Fumonisins are the most recently discovered group of mycotoxins. Since they were isolated in 1988, they have been associated with diseases such as equine encephalomalacia and pulmonary edema in pigs. The bioavailability of FB1 after ingestion in several animal species is

However, FB1 residues can be found in plasma and urine from pigs orally dosed with 3,1-9.,0 mg FB1/kg feed, with good correlations between the ingested FB1 and the residual levels in plasma or urine

(Souto et al., 2017).

usually lower than 6%. Moreover, FB1 has a short half-life (< 24 h) and less than 2% recovered in urine.

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TOXICOKINETICS OF ZEARALENONE Zearalenone is an estrogenic substance derived from resorcylic acid and produced by Fusarium species, such as F. roseum (F. graminearum), F. culmorum, and F. equisetum, among others. In mammals, zearalenone can be reduced to its hydroxy stereoisomer analogues, α-zearalenol and β-zearalenol (α- and β-ZOL). A glucuronic acid conjugate, preferentially in the 14-hidroxyl phenol group, may also be formed. In pigs, the plasma half-life of ZEA is 87 hours after the intravenous or oral routes. Piglets have shown an excretion rate of 37% of dietary ZEN

TOXICOKINETICS OF OCHRATOXIN

TOXICOKINETICS OF DON

in 24 hours (Gambacorta et al. 2013). Trichothecenes including DON may

The ochratoxin group comprises 7

be easily and quickly absorbed

related toxins, although only OTA has

in the gastrointestinal tract upon

been found as a natural contaminant

exposure. Studies in several animal

of grains.

species showed DON availability ranging from 50-60%, suggesting efficient absorption. One important DON metabolite is deepoxy-deoxynivalenol (DOM-1), produced by intestinal microorganisms in several animal species, especially in ruminants. Moreover, DON can be sulfonated or conjugated with glucuronic acid resulting in

After ingestion, OTA can remain in the serum linked to proteins and reach the kidneys, muscles, and liver. It has a high plasma-protein binding potential (up to 99%), with an estimated plasma halflife of 35 days (Dietrich et al.,

2005).

deoxynivalenol-glucoronide (DON-

It is also biotransformed by

GlcA), and excreted in urine.

cytochrome P450 enzymes to their less toxic hydroxyochratoxin

Both urinary DON and DONGlcA are considered validated

A metabolites, mainly ochratoxin alpha (OTα).

biomarkers for assessing the dietary exposure to DON (Nagl

et al, 2015).

Both OTα and OTA may be excreted in urine, although so far the urinary OTA has not been validated as a biomarker of dietary OTA.

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Application of Mycotoxin Biomarkers in Production Animals

For practical reasons, biomarkers of exposure to mycotoxins of interest in production animals are those that may be analyzed in plasma, whereas urine is the

DIFFERENTIAL DIAGNOSIS OF MYCOTOXICOSIS

most common specimen used for biomonitoring of mycotoxins in humans, as sample collection is

In production animals, the first

However, up to the present,

easily obtained in a non-invasive

important application of biomarkers

few studies have been carried

manner.

of exposure to mycotoxins would

out to determine dose-response

be the differential diagnosis

curves between symptoms of

of mycotoxicosis, as signs and

mycotoxicosis in production animals

symptoms of different mycotoxins

and biomarker concentrations.

are not evident and characteristic.

Probably the cost of analysis is

Thus, in an ideal condition,

an important obstacle for routine

preliminary diagnosis of a given

application of biomarkers in the

mycotoxicosis may be confirmed by

confirmation of mycotoxicosis

the levels of metabolites detected in

diagnosis, as it requires

plasma or urine of affected animals.

specialized laboratory equipment and highly qualified personnel to yield reliable results.

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MYCOTOXIN ADSORBENT EFFICIENCY ASSESSMENT A possible, more attractive application of biomarkers of exposure to mycotoxins in production animals, which may show good cost-benefit relationship, is the evaluation of efficiency of adsorbents used in large scale to prevent the toxic effects of mycotoxins.

However, experimental in vivo protocols are generally costly and

In this context, the use of

labor-intensive, as they involve

biomarkers to estimate the

the administration of toxins at

mycotoxin bioavailability of

different levels, with and without

adsorbent efficient in in vivo

the adsorbent, in order to assess

assays may reduce costs, besides

the effect on animal productivity.

being more practical, also helping the standardization of the

Besides, these assays

experimental trials, and making

The most important criterion for

generally require collection

it possible to assess the effect of

adsorbent assessment is the stability

of histopathological and

adsorbents in field conditions.

of the adsorbent-toxin bond in a

clinical data, among other

wide range of pH, as it is expected

issues that add to the cost

that it continues to function

and labor (European Food

throughout the gastrointestinal tract.

Safety Authority, 2010).

As adsorbents may show wide variation in composition and physical-chemical properties, it is necessary to assess their efficacy using in vitro and in vivo assays

(Di Gregorio et al., 2014).

87 10

APPLICATION OF AFM1 FOR ASSESSING ADSORBENT EFFICIENCY

The first biomarker used to evaluate adsorbent efficient probably was AFM1 in the milk of dairy cows. A good example of this kind of approach is the study by Diaz

et al. (2004), in which AFM1 concentration was reduced by 31-65% in the milk of cows fed diets containing four type of commercial adsorbents and 55 µg/kg AFB1.

APPLICATION OF AFB1-LYSINE FOR ASSESSING ADSORBENT EFFICIENCY

Carão (2016) and Di Gregorio et al. (2017) evaluated the efficiency of a commercial adsorbent based on HSCAS using the adduct AFB1-lysine in the serum of broilers and swine fed diets containing 500 and 1,100 µg/kg AFB1. In swine, HSCAS reduced serum levels of AFB1-lysine in 53-72% between days 7 and 21 of continuous exposure to contaminated feed. This

Edrington et al. (1996) used three types of adsorbents (Hydrated Sodium Calcium Aluminum Silicate (HSCAS), activated charcoal, and acidic HSCAS) in the feed of colostomized turkeys intoxicated with 0,75 mg/kg AFB1, and observed reduction of 52-72% in urinary excretion rates of AFM1 48 hours after the ingestion of the contaminated feed.

Higher precision due to exposure assessments at the individual level. Lower cost of in vivo studies because of lower required numbers of animals and laboratory analysis.

reduction was compatible with

The possibility of having

the protective effect of the

results in shorter time

adsorbent against the negative

(Di Gregorio et al., 2017).

effects of AFB1 in animals

(Di Gregorio et al., 2017). However, the use of the same commercial adsorbent in broilers did not show satisfactory results in decreasing AFB1 toxic effects (Carão, 2016), as there was no reduction in AFB1-lysine concentration in the serum of intoxicated birds.

An overview of the procedures for assessing the adsorbent’s efficiency using AFB1-lysine adduct is presented in Figure 2. However, an important limitation for the routine analysis of AFB1-lysine adduct is the requirement of reference standards, which

These results indicate that

are not commercially available

AFB1-lysine has potential as

(Jager et al., 2016) but may be synthesized in specialized laboratories (Sass et al., 2014).

an AFB1 specific biomarker

Figure 2. Overview of procedures for assessing the adsorbent’s efficiency using serum AFB1-lysine

ADVANTAGES ASSOCIATED WITH THE USE OF SERUM AFB1-LYSINE FOR EVALUATING ADSORBENTS FOR AFLATOXINS:

for evaluating the efficacy of chemo-protective interventions in pigs and broilers.

adduct in farm animals.

Analysis

AFB1-lysine

AFB1 Adsorbent

Liver P450 enzymes

AFB1-albumin in the blood

88 11

Biomarkers of Exposure to Multiple Mycotoxins in the Diet Recently, liquid chromatography-

Nowadays, liquid chromatography-

In this context, analytical

tandem mass spectrometry

methods for determination

(LC-MS / MS) aiming at multiple

(HPLC-MS), operating with an

of biomarkers of several

analytes was successfully

electrospray ionizing source (ESI) is

mycotoxins in pig plasma have

introduced in mycotoxin analysis,

unquestionably the most successful

been developed (Devreese et al.

including in the assessment of

analytical tool used in quantitative

adequate biomarkers for the

and qualitative determination of

evaluation of human exposure.

mycotoxins in natural samples.

2012). However, the influence of matrix effects is the major challenge in developing reliable quantitative multi-analyte methods. Therefore, considerable efforts to control matrix effects should be carried out to obtain accurate results, namely, the inclusion of a sample cleanup step (e.g. using QuEChERS) and the compensation of the signal suppression/enhancement through the usage of matrix matched standards.

The development of new techniques has brought important contributions for biomarkers of multiple mycotoxins and it allows for: The measurement of a more realistic data set on exposure, as in real conditions animals are exposed to a mixture of mycotoxins. The potential application of risk assessments for combined mycotoxin and the possible effects of their interaction. However, sample preparation continues to be a challenge for the development of methods of analysis for multi-mycotoxins due to matrix effects and the wide range of chemical properties of mycotoxins and their metabolites.

Particularly, the use of LC-MS/ MS leads to increased gains in sensitivity and analytical selectivity, once methods based on MS/MS use data on the molecular ion of a given analyte and of its product ions, providing a maximum confidence scale for the identification of a target analyte. Modern mass spectrometers are increasingly versatile in terms of possible combinations in a single device, different ionization sources, and different analyzers. The greatest advantage of existing equipment is that it enables more refined analytical development, making

Biomarkers are important tools in the evaluation of mycotoxin exposure as they make it possible to confirm the diagnostic of mycotoxicosis and identify individual animals that are at risk but do not show signs of intoxication.

it possible that a wider range of

The use of serum biomarkers

molecules are analyzed in a single

to estimate the mycotoxin

device.

bioavailability in in vivo adsorbent efficient assays looks promising to reduce the costs of these assays, especially for AFB1 and FB1, and possibly for DON. However, further validation studies are still required to provide physiologically based toxicokinetics of serum biomarkers of combined mycotoxins in production animals.

89 12

REFERENCES

Carão, A. C. P. Determinação de biomarcadores de aflatoxina B1e aplicabilidade na avaliação de adsorventes em frangos de corte. 2016.145 f. Tese (Doutorado) – Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Pirassununga, 2016. Devreese, M., De Baere, S., De Backer, P., Croubels, S., 2012. Quantitative determination of several toxicological important mycotoxins in pig plasma using multi-mycotoxin and analyte-specific high performance liquid chromatography-tandem mass spectrometric methods. J. Chromatogr. 1257, 74-80. Diaz D. E. et al. Aflatoxin binders II: reduction of aflatoxin M1 in milk by sequestering agents of cows consuming aflatoxin in feed . Mycopathologia, n. 157, p. 233-241, 2004. Dietrich, D.R., Heussner, A.H., and O’Brien, E. (2005). Ochratoxin A: comparative pharmacokinetics and toxicological implications (experimental and domestic animals and humans). Food Additives and Contaminants, 22, 45–52. Di Gregorio, M.C.; Jager, A.V.; Souto, P.C.M.C.; Costa, A.A.; Rottinghaus, G.E.; Passarelli, D.; Budiño, F.H.L.; Corassin, C.H.; Oliveira, C.A.F. (2017) Determination of serum aflatoxin B1-lysine to evaluate the efficacy of an aflatoxin adsorbing feed additive in pigs fed an aflatoxin B1-contaminated diet. Mycotoxin Research, 33:93-102. Di Gregorio, M.C.; Neeff, D.V.; Jager, A.V.; Corassin, C.H.; Carão, A.C.P.; Albuquerque, R.; Azevedo, A.C.; Oliveira, C.A.F. (2014) Mineral adsorbents for prevention of mycotoxins in animal feeds. Toxin Reviews, 33:1-11. Edrington, T. S. et al. Hydrated sodium calcium aluminosilicate (HSCAS), acidic HSCAS, and activated charcoal reduce urinary excretion of aflatoxin M, in turkey poults. Lack of effect by activated charcoal on aflatoxicosis. Toxicology Letters, n. 89, p. 115-122, 1996. European Food Safety Authority (2010) Statement on the establishment of guidelines for the assessment of additives from the functional group “substances for reduction of the contamination of feed by mycotoxins”. EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDA). EFSA J 8:1–8. Gambacorta, M.S, Visconti, M., Powers, S., Cossalter, A.M, Pinton, P., Oswald, I.P., 2013. Validation study on urinary biomarkers of exposure for aflatoxins B1, ochratoxin A, fumonisin B1, deoxynivalenol and zearalenone in piglets. World Mycotoxin J. 6, 299-308. Gonçalves, B.L.; Gonçalves, J.L.; Rosim, R.E.; Cappato, L.P.; Cruz, A.G.; Oliveira, C.A.F.; Corassin C.H. (2017) Effects of different sources of Saccaromyces cerevisae biomass on milk production, composition, and aflatoxin M1 excretion in milk from dairy cows fed aflatoxin B1. Journal of Dairy Science, 11:5701-5708. Jager, A. V., Tonin, F. G., Baptista, G. Z., Souto, P. C., & Oliveira, C. A. (2016). Assessment of aflatoxin exposure using serum and urinary biomarkers in São Paulo, Brazil: a pilot study. International Journal of Hygiene and Environmental Health, 219, 294-300. Nagl, V.; Schatzmayr, G. (2015). Deoxynivalenol and its masked forms in food and feed. Curr. Opin. Food Sci., 5, 43–49. Oliveira, C.A.F.; Corassin, C.H.; Corrêa, B.; Oswald, I.P. (2014) Animal Health: Mycotoxins. In: Encyclopedia of Agriculture and Food Systems, 2nd Edition. Oxford, UK: Elsevier Limited, pp. 358-377. Queiroz, O. C. M. et al. Effect of adding a mycotoxin-sequestering agent on milk aflatoxin M1 concentration and the performance and immune response of dairy cattle fed an aflatoxin B1-contaminated diet. Journal of Dairy Science, n. 95 , p. 5901-8, 2012. Sass, D.C.; Jager, A.V.; Tonin, F.G.; Rosim, R.E.; Constantino, M.G.; Oliveira, C.A.F. (2015) Synthesis and purification of the aflatoxin B1-lysine adduct. Toxin Reviews, 34:53-59. Souto, P.C.M.C.; Jager, A.V.; Tonin, F.G.; Petta, T.; Di Gregorio, M.C.; Cossalter, A.M.; Pinton, P.; Oswald, I.O.; Rottinghaus, G.E.; Oliveira, C.A.F. (2017) Determination of fumonisin B1 levels in body fluids and hair from piglets fed fumonisin B1–contaminated diets. Food and Chemical Toxicology, 108:1-9. Xiong, J. L. et al. Transfer of dietary aflatoxin B1 to milk aflatoxin M1 and effect of inclusion of adsorbent in the diet of dairy cows. J Dairy Sci, n. 98, p. 2545-2554, 2015.

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MYCOTOXINS HOW DO THEY GET INTO AQUAFEED?

Dr. Rui Alexandre Gonçalves Aquaculture and Mycotoxin Expert Aquaculture Business Developer – Lucta S.A. Innovation Division – Feed Additives UAB Research Park, Bellaterra, Barcelona, Spain

91 1

The awareness of mycotoxin-related

Mycotoxins are secondary metabolites

issues in the aquaculture industry has

produced by some molds (Hussein and Brasel, 2001). They are commonly reported to appear in agricultural commodities (pre-and/ or post-harvest), including finished feeds.

been increasing, accentuated by the replacement of marine ingredients (Gonçalves et al., 2018; Tacon et al., 2011).

Traditionally, the use of minor amounts

Chemically, mycotoxins have low molecular

of plant feedstuffs led to a general

weight, displaying a wide range of structures

perception that mycotoxins were not a

(Mallmann and Dilkin, 2007). This variability

relevant issue in aquaculture and that

is responsible for the diverse biological

the majority of mycotoxin-related issues

effects produced by mycotoxins.

would only arise due to poor storage conditions, i.e., aflatoxin contamination, but this is not entirely true.

CARCINOGENICITY ZEARALEONE

ESTROGENICITY AFLATOXIN B1

EXAMPLES OF MYCOTOXIN-INDUCED BIOLOGICAL EFFECTS

NEPHROTOXICITY NEUROTOXICITY

FUMONISIN B1

OCHRATOXIN A

92 2

Despite being identified as categorically undesirable for most animal species, the

Not all plant meals are the same!

occurrence of mycotoxins, at least in field conditions, is not completely preventable even when using good manufacturing practices.

As mycotoxins are mainly found in agricultural

The difficulty in understanding the risk of mycotoxin contamination in aquaculture finished feeds is related to the diversity of aquaculture species. For most species, the selection of plant meals depends on a combination of factors (Davis y Sookying, 2009; Gatlin et al., 2007; Krogdahl et al., 2010): Local market availability.

commodities, the tendency to replace animal-derived proteins, such as fish meal, with plant protein sources has increased the risk of mycotoxin contamination in aquaculture feeds.

Cost. The protein meal’s nutritional profile (anti-nutritional factor content and levels).

Generally, plant-based meals are known for their natural profile of anti-nutritional

However, depending on the species and

factors (ANF’s), such as cyanogens,

production region, evaluating mycotoxin

saponins, tannins, etc., that are harmful to

contamination may not be a common

fish and shrimp (Krogdahl et al., 2010).

practice in the aquaculture industry, so it becomes difficult to understand

Although there are processes that aid

the contamination risk of certain plant

in the removal or inactivation of many

commodities, especially the ones used locally.

of these ANF’s, the same does not apply to mycotoxins, as they are highly stable when subjected to processing conditions (e.g., high temperature and pressure) (Cheli et al., 2013).

93 3

Moreover, climate change and world commodities trade also contribute to the difficulty in predicting the risk of mycotoxin contamination in aquaculture finished feeds.

In some countries, mycotoxin contamination is considered a strictly seasonal issue. However, the increasing globalization of trade and incorporation of imported raw materials in aquafeeds exposes the industry to the risk of mycotoxins that may not be common for the region.

When plant meals get too expensive The increasing cost and sustainability concerns on the use of marine ingredients for aquaculture feeds have encouraged the use of plant proteins. However, recently, the cost of plant-based raw materials has begun to rise, partly due to the increased demand for human and livestock species consumption and production challenges associated with the sustainability of certain plant commodities. As a consequence, the volume and quality of affordable plant materials available for animal feed has fallen. Aquaculture feed manufacturers are now faced with either increasing the price of aquaculture feeds or trying to use alternative sources of plant meals.

Economic pressures can lead to the use of lower quality raw materials which may increase the risk of contamination with one or more mycotoxins.

Alternatively, by-products and processed ingredients could be used but they are known to have increased levels of mycotoxins since most of mycotoxins are not destroyed during commodity or feed processing (Gonçalves et al., 2017). These mycotoxins can also be redistributed and concentrated in certain milling fractions.

94 4

Consequences of limited availability of plant materials for aquafeed

Cost Volume & quality of raw materials

Aquaculture feed price

Inclusion of alternative sources of plant meals, by-products and processed ingredients

The lack of legislation regarding mycotoxin contamination for aquaculture species also leaves some room for aquafeed manufacturers to use feedstuffs that have been rejected by the livestock sector because of mycotoxin contamination and stricter regulation.

The contamination of aquafeeds and plant-based feedstuffs with mycotoxins is, in general, often neglected. Currently, there is a growing knowledge regarding mycotoxin contamination in aquafeeds and ingredients destined to be used in fish and shrimp feeds (Gonçalves et al., 2016; Gonçalves et al., 2017).

However, several gaps on how to improve and address mycotoxin risk management in aquaculture still remain.

95 5

Awareness of mycotoxin-related issues in the aquaculture industry The awareness of mycotoxin-related issues in the industry has grown as feed manufacturers

This was shown by Gonçalves et al.,

and producers realize the importance of

(2017), who reported that in Asian samples, soybean meal, wheat, wheat bran, corn, corn gluten meal, rapeseed/canola meal, and rice bran were mainly contaminated with Fusarium (ZEN, DON and FB).

mycotoxins and their potential to impact animal production. However, the idea that the majority of mycotoxin-related issues are a result of poor on-farm storage conditions leading to aflatoxin contamination is still deeply entrenched across the aquaculture industry.

The only exception was cottonseed meal, which was mainly contaminated with AF and Fusarium While it remains true that poor storage

toxins (ZEN and DON) in considerable amounts.

conditions can promote the growth of Aspergillus sp. and Penicillium sp.,ultimately leading to

Finished feed samples were also mainly

the production of aflatoxins and ochratoxin

contaminated with Fusarium mycotoxins,

A, the reality is that most of the mycotoxins

reflecting the use of plant meals.

found in finished feeds come from the raw materials used to produce them. Results shown by Gonçalves et al., (2017) confirm that mycotoxin contamination found in finished feeds is mostly related to the plant-based raw materials used in their formulation, since Fusarium fungi are generally found in field samples rather than in storage samples.

96 6

The mycotoxin risk of less typical commodities should not be ignored! As mentioned before, mycotoxins are mainly

Shrimp head constitutes 34 to 45% of

found in agricultural commodities. However,

the whole shrimp, being shrimp head

other commodities may also be contaminated

meal (SHM) an important by-product

with mycotoxins and should not be ignored.

of the shrimp industry. A possible SHM contamination with mycotoxins (at storage or by bio-accumulation) could represent

Theoretically, under suitable conditions, any

a big constraint for the industry..

commodity may be a good substrate for fungal growth and mycotoxin production. Shrimp meal can be manufactured by drying the material directly under the sun or in an In reality, little attention is given to other

oven (Hertrampf and Piedad-Pascual, 2000).

commodities besides agricultural commodities. Considering that production However, in the case of aquaculture,

is normally done in small

aquatic by-products (both from fisheries and

lots of sun-dried fish under

aquaculture) represent a significant inclusion

different conditions, a certain

level in aquafeed formulations. Therefore,

variation in the quality of the

their possible contribution to mycotoxin

commodity can be expected.

contamination should be also investigated.

For example, shrimp head meal is an important by-product of the shrimp industry.

While not being a typical product to analyze for the presence of mycotoxins, it is known that their presence is possible

This is especially true in the case of mycotoxins such as AF and OTA, as they are produced by Aspergillus sp. and Penicillium sp. species that proliferate under inadequate storage conditions.

97 7

While it is easy to understand the possible contamination of aquatic by-products with AF and OTA, as they are storage mycotoxins, it is harder to fully explain the presence of less common toxins produced by Fusarium molds that are generally associated with field conditions rather than storage.

However, Fegan and Spring (2007) reported several marine-derived samples from fishmeal to shrimp meal were contaminated with mycotoxins produced by Fusarium sp. (N=5, origin Asia; T-2=

The capacity of F. oxysporum or F. solani to produce toxins is unknown, but the possibility of having aquatic Fusarium strains producing these mycotoxins cannot be totally rejected and this hypothesis needs to be further investigated.

Another possibility for the presence of Fusarium toxins in these aquatic by-products may be due to their bioaccumulation and to the fact that they are not destroyed during processing.

60.186 ppb and ZEA= 72.036 ppb). However, the topic of mycotoxin Later on, Gonçalves et al. (2017) sourced

bioaccumulation in aquaculture products is

samples of dried fish and shrimp head

little documented (Gonçalves et al., 2020).

meal (SHM) contaminated with fumonisins (N=2; Origin: Thailand; FB1 + FB2; dried fish= 64 ppb; SHM= 24 ppb).

In reality, is difficult to fully understand the origin of Fusarium mycotoxins in marine-derived byproducts. Some Fusarium strains, namely F. oxysporum and F. solani,are known and well described as opportunistic pathogens for fish and shrimp (Hatai et al., 1986; Lightner, 1996; Ostland et al., 1987; Souheil et al., 1999).

98 8

Some take-home messages Despite the efforts to control fungal

Mycotoxins occurring in plant commodities

contamination, both in the field and in

and/or aquatic by-products are not destroyed

storage, extensive mycotoxin contamination

during most processing operations.

has been reported in commodities and finished feeds. The type and prevalence of mycotoxin contamination depend on:

On the contrary, processing affects the distribution of mycotoxins, concentrating them into fractions

The type of substrate (plant meal type

that are commonly used in animal feed (plant

and finished feed characteristics).

by-products; e.g., corn gluten meal, DDGS, etc.).

The geographical area. The fate of mycotoxins during cereal processing Seasonal and local weather

(sorting, cleaning, milling, and thermal processes) has

conditions during critical plant

been studied by several authors. However, their level in

growth stages or storage.

feedstuffs is variable and affected by several factors: The type of mycotoxins.

Besides agriculture commodities, the risk of mycotoxin contamination in other aquatic

The level and extent of fungal contamination.

by-products should not be ignored. Despite being less characterized when compared to

The complexity of the cereal-processing technology.

agricultural commodities, some scientific pieces of evidence point to these commodities as a possible source of mycotoxins.

Factors contributing to the presence or production of mycotoxins include environmental (temperature, humidity) and ecological conditions (insect attacks, physical plant damage, and general stress). However, these factors are oftentimes beyond human control.

It is recommended that aquafeed and aquaculture producers regularly monitor raw commodity feed ingredients and finished feeds for mycotoxin contamination, either through on-site rapid testing or through an external laboratory that may be equipped with more powerful detection equipment. In cases where feed quality has been compromised by mycotoxins, the use of a mycotoxin deactivator is advised.

99 9

MYCOTOXINS IN FEED An underestimated challenge for the aquafeed industry

Ram C. Bhujel, PhD Research Associate Professor Director, Aqua-Centre Asian Institute of Technology (AIT), Bangkok, Thailand Email: director@aqua-centre.org

100 1

More fish will be needed to feed the rapidly growing world population that is expected to double i.e., 15 billion by 2100. Aquatic food is the largest source of high-quality animal protein1.

An estimated 156.4 million mt aquatic food was consumed in 20182. More than half (82 mt million) came from aquaculture.

The required increase in supply for additional people needs to come from aquaculture (Figure 1). Annual aquaculture production has grown by over 500% from less than 15 million mt during 1986-95.

A rapidly growing aquaculture industry needs more feed which is the major input that accounts for up to 70-80% of the production cost.

Figure 1. Cage culture in rivers and lakes is a growing feedlot Aquaculture in Asia.

High cost and low feed quality are the major problems.

101 2

Mycotoxins – A hidden problem Feed quality depends mainly on the ingredients used and the level of mycotoxin contamination.

Mycotoxins are quite common in food and feed (Figures 2 and 3).

Figure 2. Grains used as ingredients for fish/animal feed.

Figure 3. Training on feed formulation using linear programming and manufacturing organized at Aqua-Centre, AIT, Thailand (for more information: info@aqua-centre.org).

Potential economic losses and human health hazards due to mycotoxins have not been completely interiorized.

There is a need to create awareness and make people understand the gravity of the problem.

102 3

The purpose of this paper is to highlight the importance of mycotoxins, their origins, and the methods of prevention and mitigation so that their occurrence and impacts can Figure 4. Feed with molds due to problem during storage.

be avoided or minimized.

What are mycotoxins? Mycotoxins are secondary mold by-products or metabolites with harmful effects. They are relatively small molecules but, as they are chemically stable, they are not destroyed by normal cooking or processing. Mycotoxins are produced by diverse groups of fungi from the genera Aspergillus, Fusarium and Penicilium3,4. Fungal reproduction occurs through the production of spores that are easily dispersed afar by winds. They can also attach on the bodies of insects, birds and rodents and be transported long distances. Fungal spores can infect plants at any stage.

In fact, a recent study showed that mycotoxins were detected in 60–80% of the samples of agricultural products contaminated annually, which is about three times higher than the figure (25%) earlier reported5.

103 4

The earliest history description of mycotoxins dates back to 1920 in the US: a yellow mold, Aspergillus flavus, was for the first time identified as a pathogen of maize6.

1942-1947

1920

The history of mycotoxins

Real concerns about mycotoxins began in Russia due to massive outbreaks of Alimentary Toxic Aleukia (ATA) in humans from 1942 to 1947, affecting up to 10% of the population in some districts7. This occurred when food was in short supply due to wars and people consumed the grains left in the field which were colonized by Fusarium sporotrichioides8.

1 960s

During the 1960s, over 500 cases of outbreaks called as “X-disease” in Turkey were reported linked to mycotoxins. Later on, a mycotoxin present in imported Brazilian peanut-meal9,10 was found to be linked with the similar disease in poultry in Brazil. The same “X-disease” seen in Turkey also appeared in the UK.

104 5

1920 Aspergillus flavus Maize

1960s X-disease Peanut-meal

1942-1947 Fusarium sporotrichoides (ATA) Grains

1960s X-disease Peanut-meal

Mycotoxins in aquaculture The earliest case of mycotoxicosis detected in fish may be the hatchery-reared rainbow trout (Oncorynchus

mykiss ) in the USA with cottonseed meal found to be responsible11. Feed is the major input for intensive farming, representing 50-70% of production cost. Fishmeal is considered to be the best ingredient for aquafeed but, due to sustainability issues, its use has been discouraged and attempts have been made to replace it with plant-based ingredients.

105 6

As a result, the risk of exposure to mycotoxins is higher because plant-based products have high chances of being contaminated by mycotoxins.

Many types of mycotoxins have been detected in fish feed12,13.

In most cases, more than one mycotoxin is simultaneously present in each batch of feed at unpredictable levels and combination patterns. Even if mycotoxins are present at a low level, long-term ingestion of such feed may be a cause of unexplained mortalities that occur on aquafarms. It is very difficult to identify the disease, even when mycotoxins are detected in fish’s body.

The major concern is that the mycotoxins may remain in the fish’s body e.g., liver, kidney and muscles, as residues and finally be ingested by humans14. Therefore, mycotoxins are emerging and an underestimated problem for the Aquaculture industry.

106 7

Types and sources of mycotoxins Over 300 mycotoxins have been identified. but only about 20 are known and produced naturally by fungi from the genera Aspergillus, Fusarium and Penicilium3,4. The most worrisome mycotoxins are aflatoxins, ochratoxin A and toxins produced by Fusarium molds. Aflatoxin was thought to be the most common in fish feed15-19. However, recent extensive survey of feeds and feed ingredients covering 79 countries conducted by Biomin in 2020 showed that Fusarium mycotoxins are the most prevalent ones. Deoxynivalenol (DON) was found in 65% of the samples, Fumonisin (FUM) in 64% and Zearalenone (ZEN) in 48% of the samples20. Table 1 shows the major types of mycotoxins and their sources.

Optimal temperature & water activity (aw)

Mycotoxins

Fungi source

Aflatoxins (B1, B2, G1, G2)

Aspergillus flavus Aspergillus parasiticus

T=33ºC aw=0,99

Peanuts, rice, corn, wheat, sorghum, cottonseed, copra, nuts, milk and milk products

Ocratoxin A

Aspergillus ochraceus Penicillium verrucosum Aspergillus carbonarius

T=15-30ºC aw=0,85-0,98

Cereal grain (wheat, barley, oats, corn), dry beans, moldy peanuts, cheese, coffee, resins, grapes, dried fruit, wine, cocoa

Zearalenone

Fusarium graminearum Fusarium culmorum Fusarium crookwellense

T=15-30ºC aw=0,98

Corn, wheat, moldy hay, pelleted commercial feed, waste systems

Fumonisin B1, B2, B3

Fusarium moniliforme Fusarium verticillioides Fusarium proliferatum

T=10-30ºC aw=0,93

Corn, Sorghum, asparagus

Deoxynivalenol

Fusarium graminearum Fusarium crookwellense Fusarium culmorum

T=15-25ºC aw=0,97-0,99

Wheat, Corn, barley

Patulin

Penicillium expansum

T=24ºC aw=0,99

Moldy feed, rotten apples, wheat straw residue

Citrinin

Penicillium expansum

T=24ºC aw=0,99

Cereal grain (wheat, barley, corn, rice)

Penicillic acid

Aspergillus ochraceus

T=15-30ºC aw=0,85-0,98

Stored corn, cereal grains, dried beans

Trichothecenes

Fusarium graminearum Fusarium sporotrichiodes; Fusarium poae

T=10-30ºC aw=>0,93

Corn, wheat, commercial cattle feed, mixed feeds, barley oats

Commodities

Table 1. shows the major types of mycotoxins and their sources. Sources 21,22,23,24

107

Based on the sources, fungi can be divided into four major categories:

SOIL-BORNE FUNGI Soil is the main source of fungi. Contamination of agricultural products or feed ingredients occurs during the production process in the field or during the handling, transportation and storage. Soil-borne “filamentous fungi” are the main source of mycotoxins25,26.

AIR-BORNE FUNGI Most fungi reproduce through spores that are very light and can be dispersed long distances by air or winds in the field, infecting plants at any stage.

Spores can enter together with grains into storage room and stay in vessel or equipment used for storing the grains, feeds and feed ingredients, growing when suitable conditions prevail.

SEED-BORNE FUNGI Many crops may get fungal infections from the seed if they are stored in bad conditions where fungi can remain and grow over long periods27.

ANIMAL-BORNE FUNGI Fungi spores can also attach themselves to the bodies of insects, birds and rodents, which allows them to be transported long distances.

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Factors affecting mycotoxin occurrence The two most important factors (Table 1) that affect the fungal life cycle are: TEMPERATURE WATER AVAILABILITY. Occurrence of different species of fungi may vary with geographic regions, seasons and weather conditions, and the production of mycotoxins depends on several factors during the whole process of production, harvesting, handling, transportation and storage:

Physical factors

Biological factors Plant variety, stress, insects and fungi spore load

Temperature, moisture and water activity, relative humidity, and mechanical damage of grains.

Chemical factors Oxygen, carbon dioxide, composition of substrate, pesticide and fungicides used.

Optimal conditions for each type of mycotoxin producing fungi are available23.

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Impacts of mycotoxins on fish Mycotoxins are dangerous food or feed contaminants with a high absorption rate. For example, bioavailability assays show that 85% of AFB1 may be absorbed by fish28. Presence of mycotoxins in feed critically reduces aquaculture productivity and profitability by reducing feed intake, causing abnormality and cancer, damaging gills and liver, reducing growth, increasing feed conversion ratio, suppressing immunity and increasing disease occurrence, causing toxicity and high mortality and reducing spawning frequency and fecundity. The impact of mycotoxins depends on various factors3,29:

Type and quantity of mycotoxins in the feed Feeding level Duration of exposure Fish species Sex, age, and health condition of the fish Nutritional status of the exposed species

Channel catfish and tilapia appear to be able to detoxify dietary aflatoxins more efficiently than other species11.

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In fact, up to 275 ppb and 250 ppb of aflatoxins had no effects on catfish and tilapia, respectively. However, higher doses i.e. >1,000 ppb (>1ppm) can cause damages. Similarly, for ochratoxin A (OTA) the dose of 2-4 ppm is likely to have negative effects in catfish, while DON has to be above 15 ppm in the diet to causes harm. T-2 toxin, at a dose above 0.625 ppm was associated with reduction in weight gain. In addition to catfish and tilapia, numerous studies have been carried out in trout and salmon, but studies are limited in other species. Therefore, further research is needed.

Effects of mycotoxins in humans The ultimate and most concerning consequences of mycotoxin contamination are their effects on human health. Various reports have demonstrated the presence of different amounts of mycotoxins and their residues in fish muscles, liver and kidney30-34, with the liver containing the highest amounts19,35,36.

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Mycotoxins enter the human body through the ingestion of fish, causing public health problems due to their genotoxic, carcinogenic, immunosuppressive and endocrine disrupting effects26.

Aflatoxin B1 targets the liver and it has genotoxic and carcinogenic effects. Fumonisin B1 disturbs the metabolism of sphingolipids. Deoxynivalenol and T-2 toxin inhibit protein synthesis in eukaryotic cells. Zearalenone is associated with reproductive problems. Ochratoxin A is known for its nephrotoxic properties.

Monitoring mycotoxins Approximately 100 countries covering 85% of the global population have specific regulations or detailed guidelines for mycotoxins in human food. However, for animal feed and feed ingredients regulations may not exist and, if they exist, they may not be strict.

Despite having policies and regulations, most countries lack monitoring of mycotoxins in food and agricultural products. Many people and animals get sick or die from unknown illnesses that may be associated to the presence of mycotoxins in food or feed.

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Soybean, rice, corn, wheat and their byproducts are heavily used in fish feed, and mycotoxins may develop during their production, processing and storage.

Aquatic animals are at high risk for exposure to mycotoxins if low-quality plant-based feed ingredients are used.

Therefore, monitoring and assessment of the products is needed at different stages of the production chain. However, it is difficult to continuously monitor in many spots. A portable assay for detecting the presence of mycotoxins has proven to be a useful tool that allows for testing anywhere37,38. Ultimately, frequent testing is necessary to monitor production animals, especially when the probability of exposure to fungi and mycotoxins is high.

A combination of immunohistochemistry with genotoxicity assays has been suggested as an attractive biomonitoring tool in aquaculture16.

Other lines of research are focusing on a method consisting of the application of nanomaterial-based electrochemical biosensors which may lead to the development of a highly sensitive, reliable, sophisticated, rapid, and cost-effective sensing technique to monitor products in real-time39. Similarly, a dual DNA tweezers nanomachine has been developed for one-step simultaneous detection of AFB1 and OTA in food samples40.

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Any tool to assess the risk of mycotoxin exposure will surely be useful, as there are plenty of residues due to the inclusion of land-based feed ingredients. The risk is calculated through Bayesian models that determine the critical concentrations 5% (CC5) for different mycotoxins.

Based on the analysis of 97 commercial fish feeds, the most predominant mycotoxins in fish feed are deoxynivalenol, zearalenone, fumonisins and enniatins27. In the US, the Food and Drug Administration (FDA) has established advisory levels for other mycotoxins (e.g., DON and fumonisins) for the feed industry (Table 2) with the levels that are considered adequate to protect human and animal health. Each agricultural product has its own maximum limit, and the United States Department of Agriculture Agricultural Marketing Service (USDA-MS) and the Federal Grain Inspection Service (FGIS) have provided verified test kit names.

Their manufacturers, detection methods, range and the list of commodities for aflatoxins, fumonisins, deoxynivalenol, zearalenone and ochratoxins are available at FGIS42.

Maximum level of aflatoxins

Table 2. Information pertaining to the Food and Drug Administration (FDA) limits for aflatoxin levels applicable to human food and animal feed products41.

Intended use

20ppb

All foodstuffs for human consumption except milk (milk should be less than 0.5 ppb)

20 ppb

Corn, peanut products, cottonseed meal and other animal feed and feed ingredients intended for dairy animals. For animal species or uses not specified below, or when the intended use is not known

20 ppb

Corn, peanut products and other animal feeds and feed ingredients, excluding cottonseed meal, intended for immature animals

100 ppb

Corn and peanut products intended for breeding beef cattle, breeding swine or mature poultry

200 ppb

Corn or peanut products intended for finishing swine (100 lbs (45,35 kg) or more)

300 ppb

Corn and peanut products intended for finishing (i.e., feedlot) beef cattle

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HOW CAN WE ESTIMATE THE LEVEL OF MYCOTOXINS IN FEED? When feed manufacturers purchase ingredients, they should also ask for

If the information for each ingredient is available, the total amount of mycotoxins contributed to the total diet can be estimated by multiplying the amount of mycotoxins in the feedstuff by the ratio of feedstuff (%) in the total diet: Total diet Deoxynivalenol (DON) or Vomitoxin Level

Level of DON in feedstuff (%) x (Feedstuff (lbs. DM) Total Diet (lbs. DM))

INFORMATION NOT AVAILABLE

INFORMATION AVAILABLE

the test results.

Toxins

If information about mycotoxin levels in the ingredients is not available from suppliers, then feed manufacturers should arrange for testing so that they can test ingredients as well as feed. Table 3 provides the maximum level of mycotoxins in feed for different animals.

Dairy

Feedlot

Swine

Poultry

Trout

Catfish

Tilapia

Shrimp

2,000

0.5-1.0

0.2

Fumonisin (ppm)

100

T-2 Toxin (ppb)

100

500

100

2,500

600

Zearalenone (ppb)

400

5,000

300

5,000

5.000

700

4,000

2,000

1,000

500

750

Aflatoxins (ppb) Deoxynivalenol (ppm)

Ochratoxin A (ppb) Ergot Toxins (combined) - ppb

Table 3. Potentially harmful dietary limits for mycotoxins in total diet on a dry matter basis11 41,43. Note – empty cells mean data are not available

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The European Commission legislation also stipulates maximum admissible levels for mycotoxins. These levels are defined in the valid version of Commission Regulation (EC) No. 1881/2006 of 19 December 200644 setting maximum levels for certain contaminants in foodstuffs and in Directive 2002/32/EC of the European Parliament and of the Council of 7 May 2002 on undesirable substances in animal feed. Additionally, maximum levels have been established for citrinin in food supplements based on rice fermented with red yeast, Monascus purpureaus45. The European Commission also has the Recommendation 2012/154/EU of 15 March 2012 for the monitoring of the presence of ergot alkaloids in feed and food. In the feed sector, maximum levels are defined only for aflatoxin B1 and Ergot alkaloids indirectly via ergot, which indicates that more is needed.

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Sampling For mycotoxin analysis, sampling and preparing the samples are the key. Samples taken from a large volume of grains/feed ingredients or feed samples should represent the whole stock/lot, and multi-stage sampling is usually necessary.

Sample size, minimum volume and the steps for sampling are described in the Mycotoxin Handbook41.

Once samples are taken, they need to be handled correctly so that moisture never exceeds 20%.

At the same time, the EU also has its own methods for sampling and analysis for the official control of the levels of mycotoxins in foodstuffs under the Commission Regulation (EC) No 401/2006 of 23 February 200644.

Sampling protocols are also available from the Codex Alimentarius standard CODEX STAN 193-199546.

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Preventing mycotoxin occurrence Prevention of mycotoxins occurrence requires careful application of protocols throughout the entire value chain, including crop production, harvest, transport, distribution processing and storage as summarized in Table 4.

Major categories

Factors and descriptions

1. Crop production and harvesting

Select resistant varieties Rotate crops Control insect pests, birds, and rodents Use biofertilizers Harvest soonest possible Don’t leave in the field Clean properly Dry properly

2. Storage, transportation and distribution

Control temperature Control moisture/humidity Avoid mechanical injury Clean packaging Clean and dry room Control insects, birds, and rodents

3. Storage, transportation and distribution

Discard broken/damaged Control temperature Control moisture Test and monitor regularly Detect and discard infected ingredients Select and avoid inadequate ingredients

4. Quality monitoring system (testing)

System in place to test regularly Select right methods: Visual inspection Testing with equipment Standardize sampling size/method Standardize for selection/discarding

Table 4. Method of exclusion of mycotoxin occurrence in the food/feed value chain (Modified from47 and mycotoxinsite.com).

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Some plant varieties are naturally resistant to fungi. Identifying and selecting these varieties may be a good option to avoid mycotoxin occurrence. For example, improved corn varieties that are resistant to Aspergillus

flavus are available in the USA. Identifying those varieties and hybridizing may work.

In addition, selecting the genes or DNA loci responsible for resistance against certain fungus and inserting them into high yielding varieties should be future lines of research48.

Bt (Bacillus thuringiensis) technology has been applied in some crops to control insect pests so that chances of fungi production and spreading reduces considerably e.g., Bt corn or cotton11. As shown in Table 4, good agricultural practices such as crop rotation, soil tillage, and chemical and biological control of plant diseases are essential to minimize fungal growth.

Harvesting the products as quickly as possible, avoiding drying in the field, moving to better facilities as soon as possible, and drying on platforms raised above the ground can avoid mycotoxin occurrence.

Raw material suppliers should implement methods for quick testing to detect the presence of mycotoxins in cereal products in order to be able to accept or reject a batch. Unfortunately, these quick tests can only detect a few mycotoxins and the regulatory limits vary among different countries. Thus, the responsibility of mycotoxin management in aquafeeds comes to the feed producers.

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Potential treatments A number of ways and substances have been tested and used to reduce fungal growth or bind the mycotoxins to make them unavailable for absorption through the intestinal tract. There are some commercially available detoxification products and enzymes that appear to have a high selectivity for transforming mycotoxins into less toxic forms. However, this strategy is only effective when aquafeeds contain a single type of mycotoxin but, as mentioned previously, a cocktail of mycotoxins is likely to be present in the feeds, making it unfeasible to supplement with an enzyme for each potential toxin.

As a result, attention is shifting to more practical and universal solutions, such as mycotoxin binders. In general, organic adsorbents bind to a larger spectrum of mycotoxins than inorganic adsorbents.

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CONCLUSIONS AND RECOMMENDATIONS

Among the more than 300 known mycotoxins, only approximately 20 have been demonstrated to be harmful to animals and humans. However, more research on mycotoxins is needed. The negative consequences associated with mycotoxins have not been fully internalized by crop producers, aquaculture practitioners, feed manufacturers, fish farmers, researchers and policy makers. There is a need to create more awareness and make people understand the magnitude of the problem. More training, workshops and policy dialogues among stakeholders should be organized to transfer the knowledge and safe techniques of crop production practices, transportation, processing, storage and handling.

Furthermore, clear policies on mycotoxins, more laboratory facilities for testing, more trained human resources and clear guidelines for regular monitoring are required in each country or state.

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63. Vujčić, I. and Mašić, S.(2021). Preservation of hemp flour using high-energy ionizing radiation: The effect of gamma radiation on aflatoxin inactivation, microbiological properties, and nutritional values. Journal of Food Processing and Preservation. DOI: 10.1111/ jfpp.15314. 64. Martínez, M.P., Pereyra, M.L.G., Juri, M.G.F., Poloni, V., Cavaglieri, L. (2018). Probiotic characteristics and aflatoxin B1 binding ability of Debaryomyces hansenii and Kazaschtania exigua from rainbow trout environment. Aquaculture Research, 49 (4), 1588-1597. https://doi. org/10.1111/are.13614. 65. Zhou, H. (2016) Mixture of palygorskite and montmorillonite (Paly-Mont) and its adsorptive application for mycotoxins. Applied Clay Science, 131, pp. 140-143. DOI: 10.1016/j. clay.2016.03.012. 66. Abdelhamid, A.M., Salem, M.F.I., El-Shebly, A.A., Sultan, A.S.I. (2019). Is it possible to detoxify aflatoxic aquafeed? Egyptian Journal of Aquatic Biology and Fisheries, 23 (1), art. no. 5, pp. 47-63. DOI: 10.21608/ejabf.2019.25916. 67. El-Samawaty, A.E.-R.M.A., El-Wakil, D.A., Alamery, S., Mahmoud, M.M.H. (2021). Potency of plant extracts against Penicillium species isolated from different seeds and fruits in Saudi Arabia. Saudi Journal of Biological Sciences. DOI: 10.1016/j.sjbs.2021.02.074. 68. Avantaggiato, G., Solfrizzo, M., Visconti, A. 2005. ecent advances on the use of adsorbent materials for detoxification of Fusarium mycotoxins. Food Additives and Contaminants 22(4):379-88. DOI: 10.1080/02652030500058312. 69. GarcíaPérez, O.D., TapiaSalazar, M., NietoLópez, M.G. (2019). Effects of conjugated linoleic acid and curcumin on growth performance and oxidative stress enzymes in juvenile Pacific white shrimp (Litopenaeus vannamei) feed with aflatoxins. Aquac Res. 51, 1051– 1060. https://doi.org/10.1111/are.14451.

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EFFECTS OF THE MAIN MYCOTOXINS ON POULTRY PRODUCTION PARAMETERS

Milad Manafi Department of Animal Science, Faculty of Agricultural Sciences, Malayer University, Malayer, Iran. manafim@malayeru.ac.ir

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Chicken meat is healthy and can be produced anywhere. It is among the relatively cheapest animal-source proteins and is not subject to any type of religious restrictions (Manafi et al., 2019).

Food safety and security measures have to be taken up within the food chain from livestock feed to human food. Producing adequate and available amount of safe feed are important factors to be considered, otherwise, it may lead to food insecurity and malnutrition (Manafi et al., 2018a).

This technical paper reviews the incidence and toxic effects of major mycotoxin in poultry.

Contamination with mycotoxins is among the hidden-potential hazards having a variety of severe adverse health impacts (Manafi

and Khosravinia, 2013). To be more precise, mycotoxins are the secondary metabolites of different fungi (molds) species found on carbohydrate-rich feeds such as peanuts, cottonseed, corn, sorghum, and cereal grains (Manafi et al., 2014a).

There is a range of fungi that can produce different mycotoxins when put together in favorable situations of hot conditions without adequate drying and aeration (Manafi et al., 2012a).

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As FAO declared, nearly 25% of the world’s food crops

25%

crops contaminated with mycotoxins

and cereals produced annually are contaminated with mycotoxins, although this figure greatly underestimates the occurrence above the detectable levels (up to 60–80%) (Eskola et al., 2020).

This is to emphasize the importance of the world’s cereal production and distribution from two different angles: 1. The yield of production

As an expert, one should be aware of the variety and content levels of mycotoxins in the feedlot which

2. The quality of the product

they receive. Once known, farm nutritionists may think about how to

Aflatoxin contamination of feedstuffs has been

minimize those adverse effects.

reported to be of a wide range from 1 to 900μg/kg in commonly used ingredients as well as mixed feed samples in developing countries (Mohanamba et al., 2007). It is believed that, apart from acute Different countries have set down their permissible standard levels for importing and or providing the raw materials to feed their farm animals.

poisoning and severe liver damages and lesions, these metabolites could be a cause to increase the immune deficiencies and cancer risk in livestock and subsequently be carried on to humankind, leading to

This is crucial to know, as mycotoxin

genetic mutations when available in food

contamination starts right from the

for a long-run (Manafi et al., 2009).

production phase and is continued during harvest, transportation, and

When comes to poultry, currently aflatoxins,

storage, before reaching the final

ochratoxins, and T-2 toxins are considered

customer in every corner of the globe.

to be the most dangerous mycotoxins

Developing countries have very strict rules when it comes to this matter, but in certain countries, especially in Africa, a shortage

(Table 1) from food safety and regulatory viewpoints, as well as negatively affecting poultry production parameters (Eskola et al., 2018).

of raw material supply is affecting the permissible level (EUR-Lex., 2021).

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Table 1. Relative toxicity of different mycotoxins on different livestock species Toxin

Poultry

Ruminants

Swine

Aflatoxins

+++

T-2 toxins

+++

Ochratoxin

+++

Zearalenone

+++

Fumonisin

+++

Deoxynivalenol

Slight toxicity (+) Moderate toxicity (++) High toxicity (+++)

Aflatoxins Amongst several types of mycotoxins, aflatoxins are highly toxic, carcinogenic, and cause severe contamination (Manafi, 2012). Approximately 15 types of aflatoxins have

AFB1

been described, among which “B” and “G” families (aflatoxins B1, B2, G1, and G2) are particularly dangerous to livestock as they have been found in all major food crops, grains, and their derived products utilized for animal and poultry nutrition.

Among all aflatoxins, aflatoxin B1 (AFB1) is a key toxin that is tightly regulated and monitored in very small quantities (at minor pbb levels) in agricultural commodities to be used by the animal

(EUR-Lex., 2021).

These naturally occurring compounds are produced mainly by the fungi Aspergillus

flavus, Aspergillus parasiticus and Aspergillus nomius, leading to serious health consequences through contamination of a wide variety of food such as maize which can be used in animal/poultry feeding (Manafi et al., 2018b).

Aflatoxicosis, a disease that occurs when large doses of aflatoxins lead to acute poisoning, is life-threatening, usually due to liver damage to the liver. Other adverse effects of aflatoxins are:

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Poor performance Immunosuppression and Increased susceptibility to infections Increased the susceptibility to other diseases and mortality Drop in egg production and egg weight Decreased hatchability and hatchling weight Increased liver fat and decreased activity of several liver enzymes Changes in organ weights Reduction in serum protein levels Carcass bruising and poor pigmentation

Although the concentration level, dietary exposure period, sex, species, age,

(Manafi et al., 2009; Manafi et al., 2012b; Manafi et al., 2012c; Manafi et al., 2012d; Manafi et al., 2012e; Manafi et al., 2014a; Hedayati et al., 2014a; Manafi et al., 2014b; Hedayati et al., 2014b; Manafi et al., 2014c; Manafi et al., 2015a; Manafi et al., 2016; Manafi et al., 2018b; Manafi et al., 2018c; Manafi, 2018; Eskola et al., 2020).

breed, and health status of animals are different factors that affect the level of toxicity in poultry, all avian species, especially younger ones (chicks, goslings, ducklings, and turkey poults) are the most susceptible to AFB1 toxicity (Manafi

et al., 2012b; Manafi et al., 2014d).

FACTORS

AFB1

EFFECTS Poor performance

Concentration Dietary exposure period Sex Species Age Breed Health status

Egg production Egg weight Hatchability Hatching weight Liver fat Liver enzyme activity Changes in organ weights Protein serum levels

Figure 1. Factors and effects associated with aflatoxin exposure in poultry.

Carcass bruising Poor pigmentation Immunosuppresion Susceptibility to infections and other diseases Mortality

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Ochratoxins Ochratoxins are a group of naturally occurring

Increased relative weight of the liver, kidney,

foodborne mycotoxins found in a wide variety

spleen, pancreas, proventriculus, gizzard, heart

of agricultural goods worldwide, including staple food crops, cereal grains, dried fruits, and nuts. They are produced by some Aspergillus species (mainly A. ochraceus, A. carbonarius and A. niger) and some Penicillium species, especially P. verrucosum (Manafi et al., 2011). Ochratoxins represent three secondary metabolite forms (A, B, and C), among which Ochratoxin A is the most prevalent fungal toxin of its family. Ochratoxin A is known to have nephrotoxic, teratogenic, immunosuppressive, and hepatotoxic effects in many animal species.

Increased mortality Poor feed conversion efficiency Reduced relative weight of the Bursa of Fabricius

(Giambrone et al., 1985; Gibson et al., 1989; Scudamore, 2005; Martins et al., 2008; Manafi et al., 2009; Manafi et al., 2011) The effects of ochratoxin A in poultry have been found to be quite pronounced in younger animals (Dortant et al., 2001). In birds, the kidney is the main and primary organ affected, and a marked decrease in the percentage of circulating lymphocytes and

Its possible carcinogenic effects on

a significant increase in the percentage of

humans, which could be caused through

inflammatory cells (monocytes and heterophils)

the consumption of toxins accumulated

are also reported (Moura et al., 2004).

in animal meat, are also reported by scientists (Scudamore, 1996). Ochratoxin A also inhibits protein synthesis and lipid peroxidation. The latter could be related to oxidative damage which impairs the overall quality and safety of animals (Stander et al., 2000).

OTA

Poor performance Poor FCR

Consumption of ochratoxin A by poultry may lead to:

Age-specific pathological lesions Kidney, liver, spleen, pancreas, proventriculus, gizzard and heart weight

Decrease in farm productive performance Subcutaneous hemorrhage Immunosuppression Increased age-specific pathological lesions

Immunosuppression Mortality Weight of the Bursa of Fabricius % Circulating lymphocytes Inflammatory cells

Figure 2. Ochratoxinassociated effects in poultry.

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T-2 toxin Reported in many parts of the world, trichothecenes are produced as secondary metabolites (T-2 toxin being the earliest investigated and amongst the most toxic members of this family), mainly by fungi of the genus Fusarium of which the most important species are F. sporotrichioides, F.

langsethiae, F. acuminatum and F. poae, commonly found in various cereal crops (wheat, corn, barley, oats, rye, etc.) but also in soy meal (Manafi et al., 2015b). Routinely, the high-pressure liquid chromatography (HPLC) is used to detect the T-2 toxin in a wide range of feed and food.

Lipid peroxidation High fever

These compounds are generally very stable and are not degraded during

Muscle and skin necrosis, as well as

storage/milling and high temperatures

bacterial infections of the necrotic tissues

of cooking/processing of food. The toxicity and deleterious effects of T-2

Enlarged lymph nodes Delayed ovulation

toxin vary based on numerous factors, such as the administration route, the

Decreased sperm motility and increased

exposure time, the administered dosage,

sperm morphological abnormalities

and the age, sex, and overall health of the animal (Hossam et al., 2013).

Inhibition of protein, DNA, and RNA synthesis Cytotoxicity

T-2 toxin is believed to increase oxygen radical production hence resulting in direct cell injury (Manafi et al., 2012f).

Immunomodulation Lesions in the digestive tract, organs, and skin

Studies indicating that T-2-toxin ingestion in high doses by poultry through the contaminated grain, hay, and straw causes:

Neural disturbances and nervous disorders Declined performance and production parameters due to feeding refusal

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Bloody diarrhea, bone marrow, buccal lesions, serous hemorrhagic inflammation Dystrophy in liver, kidney, heart, brain and peripheral ganglia of the vegetative nervous system

(Kalantari et al., 1989; Zian et al., 2011; Kachuei et al., 2014; Krska et al., 2014; Drakulic et al., 2016; Yuan et al., 2016). Preliminary, the liver is one of the first target organs where the enzymes help metabolize drugs that pass through the liver. The decrease in its activity could lead to an increase of unmetabolized drugs in the plasma, which can pose a dangerous risk for the animal’s health (Goossens et al., 2013).

FACTORS Administration route Exposure time Dosage Age Sex Health status

The increased elevation of glutathione disulfide and 3-hydroxybutyrate suggests that the T-2 toxin promotes an anti-oxidative response in organ systems and helps with free radical generation.

T-2 toxin EFFECTS Muscle and skin necrosis Lesions in digestive tract and other organs

Delayed ovulation Sperm motility Sperm abnormalities Neural disturbances Nervous disorders

Inhibition of protein, DNA and RNA synthesis Dystrophy in liver, kidney, heart, brain and peripheral ganglia of the vegetative nervous system

Immunomodulation Cytotoxicity Bloody diahrrea Serous hemorrhagic inflammation Declined performance and production parameters due to feeding refusal

Figure 3. Factors and effects associated to T-2 toxin exposure in poultry.

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CONCLUSIONS Concentrations of mycotoxins in the feed are usually low and their immunosuppressive effects and secondary infections often make diagnosis difficult.

If at the onset of the disease, a change in the diet leads to health and performance improvements in animals, this may point to mycotoxin poisoning.

Although it is difficult to predict the effect of multiple toxins, certain studies confirm that presence of combined mycotoxins in animal feed show more severe impacts on livestock health and productivity.

Monitoring and controlling all the feed ingredients which are to be used for poultry diets is not always practical and could seriously compromise the world food supply, due to very large quantities, however, regular control of grain and feed samples is a valuable preventive measure and it is only accurate if representative samples are tested in the laboratory.

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Manafi, M., H.N.N. Murthy, M. Noor Ali and H.D. Narayana Swamy (2012e). Evaluation of Different Mycotoxin Binders on Broiler Breeders Induced with Aflatoxin B1 : Effects on Egg Quality Parameters. World Applied Sciences Journal. 17(3): 271-277. Manafi, M., B. Umakantha, K. Mohan and H.D. Narayana Swamy (2012f). Synergistic Effects of Two Commonly Contaminating Mycotoxins (Aflatoxin and T-2 Toxin) on Biochemical Parameters and Immune Status of Broiler Chickens. World Applied Sciences Journal. 17(3): 364-367. Manafi, M. and H. Khosravinia (2013). Effects of Aflatoxin on the Performance of Broiler Breeders and Its Alleviation by Herbal Mycotoxin Binder. Journal of Agricultural Science and Technology. 15: 55-63. Manafi, M., M. Hedayati and M. Yari (2014a). Aflatoxicosis and Herbal Detoxification: The Effectiveness of Thyme Essence on Performance Parameters and Antibody Titers of Commercial Broilers Fed Aflatoxin B1. Research in Zoology, 4(2): 43-50. Manafi, M., M. Hedayati and M. Yari (2014b). The Efficacy of Thyme Essence on Internal Organ Weights, Biochemical Traits and Mortality of Broilers Fed Aflatoxin B1. International Journal of Agriculture and Forestry, 4(4): 286-292. Manafi, M., M. Hedayati and M. Yari (2014c). Application of Rosemary (Rosmarinus officinalis L.) Essence on Chicks Fed Aflatoxin B1: Impacts on Internal Organ Weights, Biochemical Traits and Mortality. Research in Zoology, 4(1): 13-19. Manafi, M., M. Hedayati and M. Yari (2014d). Effectiveness of Rosemary (Rosmarinus officinalis L.) Essence on Performance and Immune Parameters of Broilers during Aflatoxicosis. Advances in Life Sciences, 4(3): 166-173. Manafi, M., H. Arak and Hedayati, M. 2015a. The effects of inclusion of various levels of aflatoxin B1 on performance, relative weights of internal organs and blood parameters of Japanese quail during the growing period (1-28 days). Animal Science Journal, 107: 33-40. Manafi, M., N. Pirany, M. Noor Ali, M. Hedayati, S. Khalaji, and M. Yari (2015b). Experimental pathology of T-2 toxicosis and mycoplasma infection on performance and hepatic functions of broiler chickens. Poultry Science, 94(7): 1483-1492.doi:10.3382/ps/pev115. Manafi, M. and M. Hedayati (2016). Combinational Effects of Thyme and Rosemary Ethanolic Extractions in Reducing the Effects of Aflatoxin B1 in Broilers. Animal Science Journal, 112: 105-116. Manafi, M., (2018). Toxicity of aflatoxin B1 on laying Japanese quails (Coturnix coturnix japonica). Journal of Applied Animal Research. 46(1): 953-959. Manafi, M., M. Hedayati and S. Mirzaie (2018a). Probiotic Bacillus Species and Saccharomyces boulardii improve performance, gut history and immunity in broiler chickens. South African Journal of Animal Science. 48(2): 379-389. Manafi, M., (2018b). Impact of Application of Natural Toxin Binder on Performance, Humoral Imumune Response, Cecal Microbial population and Changes in Small Intestine Morphology of Broilers Fed with Diet Contaminated with Aflatoxin B1. Journal of Veterinary Research. 73(3): 273-282. Manafi, M., M Hedayati, and H. Arak (2018c). The effect of concomitant use of ethanolic mixtures extractions of Thyme and Oregano on performance and morphology of grastrointestinal tracts in broilers fed contaminated feed with Aflatoxin B1. Animal Science Researches. 28(3): 1-16. Manafi, M., M. Hedayati, N. Pirany and Omede Apeh Akwu (2019). Comparison of performance and feed digestibility of the non-antibiotic feed supplement (Novacid) and an antibiotic growth promoter in broiler chickens. Poultry Science. 98(2): 904-911. Martins, H.M., M. Marques, I. Almeida, M. M. Guerra and F. Bernardo (2008). Mycotoxins in feedstuffs in Portugal: an overview. Mycotoxin Res. 24(1): 19-23. Mohanamba, T., Rao, M. R. and Habibi, S.M.M., 2007. Aflatoxin contamination in animal feeds. Indian Veterinary Journal, 84: 416. Moura, MA, Machado, CH, Porfírio, LC, and Freire, RB. (2004). Effects of ochratoxin a on broiler leukocytes. Brazilian Journal of Poultry Science, 6(3), 187-190. Scudamore KA (1996). Ochratoxin A in animal feed--effects of processing. Food Addit Contam. 13 Suppl: 39-42. Scudamore KA (2005). Prevention of ochratoxin A in commodities and likely effects of processing fractionation and animal feeds. Food Addit Contam. 22 Suppl 1:17-25. Stander MA, Bornscheneuer UT, Henke E, Steyr PS (2000). Screening of commercial hydrolases for degradation of Ochratoxin A. Journal of Agriculture and Food Chemists 48:5736-5739. Yuan Z, Matias FB, Yi JE, Wu J. (2016). T-2 toxin-induced cytotoxicity and damage on TM3 Leydig cells. Comp Biochem Physiol C Toxicol Pharmacol. 181–182. 47–54. Zain M.E. (2011). Impact of mycotoxins on humans and animals. J Saudi Chem Soc. 15:129–144.

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REVIEW OF INVESTIGATIONS REGARDING MYCOTOXIN CONTROL IN POULTRY THROUGHOUT THE YEARS

Milad Manafi Department of Animal Science, Faculty of Agricultural Sciences, Malayer University, Malayer, Iran. manafim@malayeru.ac.ir

140 1

Generally, different poultry species require an adequate supply of carbohydrates, proteins, fats/oils, vitamins, minerals, and water (Manafi et al., 2011).

Mycotoxins are recognized as secondary toxic metabolites mainly produced by various toxigenic fungal species of the Aspergillus, Fusarium,

and Penicillium (Egbontan et al., 2017).

However, poor raw material management and storage and high carbon and moisture rates in feedlots may lead to a final diet contaminated with fungi and mycotoxins (Döll and Dänicke, 2004).

The mycotoxin type, level and frequency of exposure (acute or chronic), body mass index, gender, concomitant health issues, and possible synergistic effects of other chemicals affect the manifestation of the disease (Manafi et al., 2009).

Increased incidence of leg malformations In poultry, the significance of these mycotoxins’ effects depends on their presence in food and feed above the regulatory limits.

Increased death rates and visceral hemorrhages Reduced villus height

Different symptoms in poultry and other animals have been well documented by many scientists:

Weight loss, anorexia and impaired feed conversion competency Immunosuppression and failed

(Alexandros and Jean, 2002; Döll et al., 2004; Jaynes et al., 2007; Marin et al., 2013; Mishra et al., 2013; Manafi et al., 2014; Manafi et al., 2015; Fowler et al., 2015; Manafi et al., 2016; Ji et al., 2016; Manafi, 2018; Manafi et al., 2019, Raj et al., 2021).

response to vaccination Low fertility

Furthermore, there is a high chance of mycotoxin carry-over (Figure 1) into

Drop in egg production and high

edible byproducts obtained from poultry fed

chances of egg blood spots

with contaminated feed. This phenomenon

Kidney enlargement Pale fatty liver and hepatitis Gizzard erosions and oral lesions

can lead to cancer, as some mycotoxins are recognized for their carcinogenicity by the World Health Organization’s (WHO) and International Agency for Research on Cancer (IARC) (Ganesan et al., 2021).

1412

OTA Contaminated feed

OTA accumulation in muscle fibrils

OTA transferred to layer chicken

Aspergillus sp.

OTA via bloodstream

Damaged enterocytes

OTA accumulation in ovaries

OTA deposition in chick

OTA carry-over to eggs

Figure 1. Schematic representation of Ochratoxin A (OTA) carry-over from feed to various parts of the chicken (adapted from Ganesan et al., 2021).

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Commonly, mycotoxin analysis methods include: Enzyme-linked immunosorbent assays (ELISA) High-performance liquid chromatography (HPLC) LC-MS/MS

The ELISA is a quick test with reliable accuracy in simple substrates, such as raw feed ingredients.

On the other hand, HPLC and LC-MS/MS-based analyses are relatively more accurate than rapid test kits and used for evaluating mycotoxin removers, especially in complex substrates, such as formulated feeds (Niderkorn et al., 2007)

Most of the mycotoxins are liposoluble compounds that can easily be absorbed from the site of exposure (gastrointestinal or respiratory tract) into the circulatory system, reaching the liver where they are metabolized by the microsomal system into active or detoxified metabolites and distributed throughout the organism (Haschek et al., 2002)

Through natural cellular processes of transcription and translation, mutations may manifest or even exacerbate the deregulation of cell growth (Manafi et al., 2012)

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MYCOTOXICOSIS CONTROL Increased efforts are being undertaken by

The ability of mycotoxin binders to remove

scientists to develop cost-effective and

toxins is determined by using in vitro and in

safe products to achieve decontamination

vivo assays with their specific pros and cons.

and remediation of different mycotoxins (preventive or curative) in feedstuffs

(Manafi et al., 2019). Preferably, the level of aflatoxins in feed should be zero. In any case, certain regulatory organizations have set their threshold as the maximum level for poultry.

For in vivo studies, to obtain consistent results specific bioassays should be conducted. In contrast, in vitro experiments are relatively easy to perform, and they can shorten the time and cost of experimentation. Keeping in mind that the ultimate goal of an in vitro study is to replace in vivo experiments in practice, the conditions of in vitro experiments should be tightly controlled and well-designed to obtain accurate research records (Hahn et al., 2015).

Currently, the most effective way of protecting animals against mycotoxicosis is the inclusion of adsorbents in the feed (Ditta et al., 2018)

On this basis, Physical and Organic methods to control mycotoxin in poultry feed are detailed hereunder with the emphasis on the previous carried out research experiments.

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Physical methods The extraction of mycotoxins from feedstuff is an effective tool, as these

Zeolites

secondary metabolites are highly soluble

Zeolites are crystalline

in organic solvents. The utilization of

aluminosilicate compounds.

mycotoxin-binding adsorbents is the most applied physical method for protecting animals against mycotoxicosis. Clays usually consist of two or more mineral-oxide layers and some of their particles can absorb moisture and expand. However, their efficiency depends on the chemical structure of the adsorbent and the mycotoxin, as it is important to ensure that the adsorbents do not remove essential nutrients from the diet (Manafi et al., 2012).

An improvement in 29-41% in body weight gain was reported in broilers exposed to 3.50 ppm of aflatoxin (AF) through dietary supplementation of commercial zeolite (Duarte and Smith, 2005). Many other scientists have tried to ameliorate the adverse effects of mycotoxins in different animals using zeolite (Daković

Zeolites, activated carbons, and hydrated sodium calcium aluminosilicate are among the most important clays used for controlling mycotoxicosis. Although these methods are comparatively expensive, their efficiency is partially efficient.

et al., 2005; Dhanasekaran et al., 2011; Rajendran et al., 2020; Raj et al., 2021).

Hydrated Sodium Calcium Aluminosilicate (HSCAS) HSCAS, a phyllosilicate derived from natural zeolite, is perhaps the most extensively investigated mycotoxin adsorbent.

Phillips et al. (1988) showed that HSCAS has a high affinity for AFB1 after screening 38 different adsorbents that were representative of the major chemical class of aluminas, silicas, and aluminosilicates. There is a wide range of efficacy observed in different studies incorporating HSCAS into the diets to reduce the toxicity of AF, OTA, and T-2 toxin in poultry (Huff et al., 1992; Jindal

et al., 1993; Kubena et al., 1998; Raju and Devegowda, 2000; Huwing et al, 2001; Girish and Devegowda, 2004; Duarte and Smith, 2005; Khatoon et al., 2018; Wei et al., 2019).

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Activated carbon Activated carbon is an insoluble powder formed through the pyrolysis of different kinds of organic materials and is quite effective for adsorbing OTA. Different studies have reported an improvement in the bodyweight broilers following the inclusion of activated charcoal in diets containing different mycotoxins

(Ramos and Hernández, 1997; Solfrizzo et al., 2001; Huwing et al., 2001; Duarte and Smith, 2005; Mgbeahuruike et al., 2018).

Bentonite Bentonite is a mineral clay with the unique characteristic of swelling to several times its original volume when placed in water. Due to their montmorillonite content, bentonites form thixotropic gels as a result of their ion exchange capabilities. The bentonite forms a complex with the toxin, preventing the absorption of mycotoxins, such as aflatoxins, across the intestinal epithelium (Duarte and Smith, 2005). Clay materials can bind to molecules of certain sizes and configurations and have been used effectively to decrease the effects of aflatoxin-contaminated diets in poultry. In this regard, there are plenty of publications reporting mycotoxin decontamination in poultry (Unsworth et

al., 1989; Smith and Ross, 1991; Hagler et al., 1992; Santurio et al., 1999; Rosa et al., 2001; Vieira, 2003; Eralsan et al., 2005; Eraslan et al., 2006; Murugesan et al., 2015; Mgbeahuruike et al., 2018).

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Organic methods In this category of mycotoxin control tools, the application of herbal-antioxidant agents, vitamins, algae, enzymes, nutritional manipulation, and biological methods will be reviewed.

Herbal-antioxidant agents The application of some plant-derived

There are several studies on the use of diverse herbal extracts against mycotoxicosis on different animals that could partially alleviate some negative effects:

extracts, such as turmeric (Curcuma longa) garlic (Allium sativum) and asafetida

Increased peroxides

(Ferula asafetida), have been shown to counteract aflatoxicosis in poultry through

Reduced antioxidant enzyme activities

their antioxidant activity by reducing the level of free radicals (Manafi et al., 2018).

Inhibited protein/DNA synthesis

The body’s antioxidant system mainly involves

Suppressed chromosomal aberration

reducing agents (tocopherol, ascorbic acid, glutathione, carotenoids), peroxidases (glutathione peroxidase, catalase), enzymes (peptidases, proteases, vitamin A), and superoxide dismutase (Renzulli et al., 2004). The most common functional chemical groups with radical scavenging properties are hydroxyl (phenolics), sulfhydryl (cysteine, glutathione), and amino groups (uric acid, spermine) (Lee et al., 2001).

Inhibited cytochrome P450 bioactivation of AFB1 Reduced antioxidant biomarkers (glutathione peroxidase and superoxide dismutase) Inhibited AFB1 mutagenicity and increased formation AFB1-DNA adducts (Iqbal et al., 1983; Nyandieka et al.,1990; Surai; 2001; Weiss, 2002; Gowda and Ledoux, 2008; Gowda et al., 2008; Dalleau et al., 2008; Ruan et al., 2019).

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Many other studies have stated the effects of different vitamins in animals when administered with mycotoxins (Nyandieka et

al., 1990; Coelho, 1996; Hoehler and Marquardt, 1996; Diaz and Smith, 2005; Kabak, 2009; Wayne, 2012; Murugesan et al., 2015).

Algae (Spirulina Platensis) The nutritional value of some algae like

Spirulina platensis is extremely high.

Vitamins

Spirulina is rich in amino acids, vitamins,

There are reports of regeneration of α-tocopherol

elements (EFSA FEEDAP Panel, 2016).

gamma-linoleic acid, sugars, and trace

on reaction with other reducing agents like glutathione, urate (Kagan and Tyurina,

1998) and ascorbate (May et al., 1998). Chow (2001) stated that α-tocopherol is the most biologically active form, quickly scavenging peroxy radicals by forming a stable tocopheroxy radical and acting as a biological modifier.

It has also been reported that Spirulina platensis is effective against aflatoxicosis by Raju et al., (2004); Abdel-Wahhab and Aly (2005); Dal Bosco et al., (2008); Manafi et al., (2009); Manafi, (2011); Manafi et al., (2012) and Park et al., (2018).

Vitamin E pretreatment significantly lowered AF-induced lipid peroxidation

(Verma and Nair, 2004). Among vitamins, ascorbate (vitamin C) is very important due to its ability to scavenge superoxide, hydrogen peroxide, hydrogen radicals, hypochlorous acid, and singlet oxygen (Chow, 2001). Riboflavin also has a protective action against AFB1 induced DNA damage in rats (Gowda and Ledoux, 2008).

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Nutritional manipulations Enzymes Enzyme degradation reagents are used for the biodegradation of the toxic chemical structure of the mycotoxins into non-toxic metabolites by using microorganisms and their metabolites or specifically extracted components. They are believed to break the functional atomic group of the mycotoxin molecule, thereby rendering it non-toxic (Kabak et al., 2006). Some enzymes, such as carboxyesterase present in the microsomal fraction of the liver, esterase, and epoxidase are being tested for their practical applicability in field conditions (Pasteiner, 1997).

An increase in the dietary protein levels and supplementation of L-phenylalanine has been revealed to be effective against aflatoxicosis and ochratoxicosis. Additionally, increasing the supplementation of riboflavin, pyridoxine, folic acid and choline showed a protective effect against aflatoxicosis (Ehrich et al., 1986). Antioxidants, such as β-napthoflavone, vitamin C, and vitamin E offer protection against AF-induced genotoxicity in most in vitro studies

(Johri et al., 1990). Devegowda et al., (1998); Krska et al., (2008); Bryden, (2012), and Marin et al., (2013) have reported that additional supplementation of poultry diets with micronutrients supplementation can partially alleviate the adverse effects of mycotoxins.

The demonstrated efficacy of using enzymes against mycotoxicosis is welldocumented by a wide range of researchers

(Vekiru et al., 2010; Ji et al., 2016; Tso et al., 2019; Fruhauf et al., 2019).

Biological methods A rapid explosion in the feed industry has opened up new possibilities through the degradation of mycotoxins by microorganisms (Alexandros and Jean, 2002). Several yeasts, moulds, and bacterial strains possess the ability to destroy or transform mycotoxins successfully (Edlayne et al., 2009).

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B A C T E R I A L D E G R A D A T I O N

In this category, bacterial, protozoan, fungal and yeast degradations will be reviewed hereunder.

Several bacterial species have shown the ability to degrade AF (Kong et al., 2012).

Specifically, some acid-producing bacteria’s such as Lactobacillus plantarum and Lactobacillus acidophilus were found to detoxify AF, OTA, T-2 toxin, and zearalenone (ZEN) in different studies

(Linderfelser and Ceigler, 1970; Bata and Lásztity, 1999; Turbic et al., 2002; Lahtinen et al., 2004; Kusumaningtyas et al., 2006; Niderkorn et al., 2007; Wu et al., 2009; Kolossova and Stroka, 2009; Rawal and Roger, 2010; Ma et al., 2012; Mishra et al., 2013; Fan et al., 2013 and Fan et al., 2015).

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P R O T O Z O A N D E G R A D A T I O N

Protozoa are important agents for mycotoxin biodegradation in the rumen (Upadhaya et al., 2010). Most of the studies on the subject indicate that mycotoxin degradation was achieved primarily by ruminal protozoa (Jouany et

al., 2009). Besides Hussein and Brasel (2001) reported that up to 90% of T-2 toxin degradation was achieved by rumen protozoa. Tetrahymena pyriformis at a dose rate of 22x106 cells, detoxified AFB1 by converting it into its hydroxyl products to an extent of 5% in 24 hours and 67% in 48 hours (Robertson et al., 1970). Intact rumen fluid containing various protozoa was reported to metabolize T-2 toxin and OTA, while no effect on AF was noted (Kiessling et al., 1984). However, the fact that protozoa cannot be cultured in vitro limits further understanding of their ability to degrade mycotoxins (Chaucheyras-Durand

and Ossa, 2014; Newbold et al., 2015).

Studies involving culture-independent approaches based on analysis of particular genes and genomes potentially associated with mycotoxin degradation may help improve our understanding of the role of protozoa in the process (Garai et al., 2021).

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F U N G A L

An intracellular substance was found to be responsible for A. flavus and

A. parasiticus to degrade the formed toxins in a culture when their mycelium was subjected to fragmentation. Peroxidase enzymes produced by fungal mycelium, which can catalyze hydrogen peroxide into free radicals, react with

D E G R A D A T I O N

mycotoxins (Dvorak, 1989; Alexandros

and Jean, 2002; Edlayne et al., 2009).

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D E G R A D A T I O N

The advent of biotechnology in the last decade has opened a new pathway for tackling the problem of mycotoxicosis by using yeast extracts. Yeast is a rich source of numerous vitamins and certain species and strains can detoxify mycotoxins through their degradation (Biernasiak et al., 2006). Mannanoligosaccharide (MOS) derived from the cell wall of Saccharomyces cerevisiae appears to have a high affinity for a wide range of mycotoxins (Biernasiak et al., 2006).

B Y Y E A S T

It is believed that the glucomannan matrix of MOS preparation traps the mycotoxins in an irreversible way (Afzali y Devegowda, 1998). Besides, it is assumed that a small portion of modified-MOS might be taken up in the small intestine by M-cells, causing B-cell activation and subsequent activation of T-cells and macrophages, leading to an overall increase in the immune status of the birds (Savage et al., 1996). The mycotoxin binding ability of MOS has been demonstrated in various in vitro and in

vivo trials (Stanley et al., 1993; Devegowda et al., 1996; Afzali and Devegowda, 1998; Devegowda et al., 1998; Raju and Devegowda, 2000; Swamy et al., 2004; Yegani et al., 2006; Awaad et al., 2011; Fowler et al., 2015; Farooqui et al., 2019; Arif et al., 2020).

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CONCLUSIONS

There is a wide range of commercially available mycotoxin binders and antifungal agents that have varying potency effects at reducing the presence or eliminating the toxicity of mycotoxins in poultry feeds.

The development of a successful commercial mycotoxin binder calls for the incorporation of the best of these active ingredients at the required concentration to ensure an overall reduction of the harmful effects of the presence of mycotoxins in poultry diets.

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THE EFFECTS OF MYCOTOXINS ON SWINE REPRODUCTION

Panagiotis Tassis Assistant Professor of Swine Medicine and Reproduction Clinic of Farm Animals, School of Veterinary Medicine, Aristotle University of Thessaloníki Greece

161 1

The significance of reproductive performance for the outcome of swine production is undoubtable. Both sides, gilts/ sows and boars are the basis of proper and financially rational production in intensive swine farms globally. Genetic lines of hyperprolific sows, as well as high durability and increased performance boars are needed for increasing production demands and high-quality pork meat.

A variety of major issues, such as

Controlling the major viral or

reproductive management, selection of

bacterial swine pathogens affecting

gilts and boars, introduction of gilts to

the genital tract and reproductive

farm’s reproductive program,

performance, as well as proper

environmental conditions affecting

nutrition in terms of ingredient/

reproductive performance (e.g. heat

nutrient requirements and a proper

stress, etc.), along with maintenance of

feeding schedule during various

high health status and fulfillment of

stages of gestation and boar’s

specific nutritional requirements, should

life, are significant parts of proper

be taken care during the rearing of

reproduction management.

hyperprolific sows and boars.

As already suggested by various research efforts in the past 50 years, mycotoxins can also pose a significant threat to the health and reproductive e!ciency of swine.

162 2

Mycotoxins are secondary

The most important mycotoxin

significant for their devastating effects

metabolites of certain fungi

producing fungi belong to the genera

on pig production worldwide.

species that can be found in

Aspergillus, Penicillium, Fusarium, Alternaria and Claviceps3.

Other mycotoxins such as T-2

grains worldwide. They are produced before (fungi as plant

toxin, nivalenol or ergot alkaloids

pathogens) or after the harvest

Out of more than 500 mycotoxins, some

have been suggested as somewhat

of grains, or even during storage

are considered extremely significant

significant for swine, especially in

(fungi growing saprophytically).

for pig health and productivity.

particular geographical regions1,4.

In particular regions, the mycotoxin

Namely, aflatoxins (AF) B1, B2, G1 and

menace seems to be higher

G2, deoxynivalenol (DON), zearalenone

resulting in severe outbreaks

(ZEN), fumonisins (FB1, FB2, FB3) and

of intoxication (mycotoxicosis)

ochratoxin A (OTA) are considered

threatening humans and animals1. The significant impact of mycotoxins includes loss of human and animal life, increased health care and veterinary care costs, and reduced livestock production . 2

ZEN

Even though several mycotoxins have been suggested as capable of inducing or contributing to reproductive disorders (DON, ZEN, FB, T-2) in various species5, it is obvious that the most “interesting” in terms of reproductive performance is ZEN and its metabolic derivatives.

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In this article, we will try to synopsize effects of the most important mycotoxins ingested separately, on the two parts of reproduction, i.e. the female part (gilts, sows and the offspring) and the male part which includes the boars. Predominantly direct toxic effects of AF, OTA, DON, T-2, FB and ZEN are described, since presentation of indirect effects (e.g. increased susceptibility to infections due to impaired immunity that could affect reproduction, or effects of reduced protein synthesis or feed refusal on gestation and litter weight) is practically more or less extremely extensive. In vivo and

in vitro effects along with aspects related to the hormonal alteration of reproduction according to relative literature are also described.

Reproductive effects of Aflatoxins in swine Aflatoxins (AFs) are produced

The most common AFs are

mainly by Aspergillus flavus, A. parasiticus and A. nomius and are detected usually in maize, peanuts and cottonseed.

AFB1, AFB2, AFG1 and AFG2.

AFB1 is considered as an active hepatocarcinogen and is the most significant in terms of toxicity in swine6. The liver is considered the primary target organ of AFB1.

164 4

AFs, apart from being hepatotoxic, they have mutagenic, and possibly teratogenic effects in animals. They are categorized as Class 1 human carcinogens by the International Agency for Research on Cancer (IARC). AFs decrease the absorption of nutrients and reduce weight gain in pigs, while chronic exposure to low doses results in jaundice (pale-yellowish appearance of the liver) with hemorrhaging sites in the liver and variable levels of fibrosis and cirrhosis, diffused centrilobular necrosis and fat degeneration7. Few reports have connected AFs with

Sows are capable of normal gestation and reproduction when fed AF at levels between 500 and 700 ppb, whereas their piglets show growth retardation due to AF excretion in milk8,9.

reproduction in swine. Abortion is not

Reduced piglet birth weight has been reported after

expected at AF toxicosis cases7.

ingestion of 800 ppb AFB1 in feed of sows during the second half of gestation and the suckling period10.

In vitro detrimental effects on oocyte maturation through epigenetic modifications (increasing DNA methylation levels), induction of oxidative stress, excessive autophagy and apoptosis, have been presented after oocyte exposure to 50 μM AFB111.

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It has been further suggested that AFB1 can impair porcine early embryonic development (blastocyst formation was impaired with treatment of 1 nM AFB1) through oxidative stress (excessive reactive oxygen species), induce DNA damage, disrupt DNA damage repair process, while also induces apoptosis and consequently autophagy12. Possible adverse effects on boar reproductive efficiency have been suggested13, where lower sperm concentration, lower survival of spermatozoa, and a larger proportion of abnormal spermatozoa were found simultaneously with great levels of AFB1 in seminal plasma.

Reproductive effects of Ochratoxin A in swine Ochratoxin A (OTA) is produced by several Aspergillus and Penicillium species, such as Penicillium verrucosum A. ochraceus, A. westerdijkiae, A. steynii, A. carbonarius and A. niger14. Its primary target organs are the kidneys (nephrotoxicity), however it can induce several toxic effects such as teratogenic, embryotoxic, genotoxic, neurotoxic, immunosuppressive and carcinogenic14,15. OTA has been classified by the IARC as a possible human carcinogen (group 2B). In a previous study with boars (250 kg weight), which were given 0.08 μg/kg OTA orally for 6 weeks, reduction in sperm viability, initial forward motility, and motility were observed after 24 hours storage16. Moreover, it was suggested that OTA (fed to boars at

Additionally, OTA is able to negatively affect porcine oocyte maturation

high concentrations) might have the potential to affect

in vitro (reduced rate of porcine

sperm production and boar semen quality by inducing a

oocyte polar body extrusion) at levels

reduction of initial motility and longevity of spermatozoa17.

of exposure exceeding 5 μM18.

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Reproductive effects of Fumonisin B in swine Fumonisin Bs (FBs) are mycotoxins produced mainly by Fusarium verticillioides and F. proliferatum, commonly in maize. The predominant FB 1corresponds to 70% of FBs and is classified as a potential human carcinogen (Class 2B) by IARC19. Its main toxic mechanism is based on the disruption of sphingolipid biosynthesis, with inhibition of ceramide synthase that results in the accumulation of sphinganine and sphingosine. Acute intoxication of swine with high levels of FB (>100 ppm) is characterized by pulmonary edema7. Nevertheless, FBs have been well implicated in the impairment of immune response [e.g. modification of Th1/ Th2 (T-helper 1/T-helper 2) cytokine balance], as well as in significant effects in the gastrointestinal tract.

FB1 has been associated with the induction of intestinal barrier integrity alterations and its function/permeability [e.g. decreased transepithelial electrical resistance (TEER), reduced expression of occludin and E-cadherin in ileum], as well as with the modulation of digestive and absorptive processes (e.g. reduced aminopeptidase activity in jejunum) and reduction of intestinal defense during

In regard to reproduction, FBs have been also

pathogen exposure (increased

linked with delay in sexual maturity and

colonization and shedding

reproductive functionality alterations21.

of Escherichia coli)7,20.

Specifically, they have been suggested as responsible for the reduction of testicular and epididymal sperm reserves and daily sperm production in boars22,23. Additionally, detrimental effects on semen quality and motility after 6 months of exposure to FBs have been suggested23.

167 7

Furthermore in vitro, FB1 produced inhibitory effects on granulosa cell proliferation24. FB1 was found to influence the steroidogenic capacity of porcine granulosa cells (stimulation of progesterone production but no effect on estradiol production), as well as to inhibit their proliferation, thus it could compromise the normal follicle growth and oocyte survival in swine5,24,25.

In vivo, FB induced abortions 1–4 days after acute spontaneous toxicosis which was probably consequence of fetal anoxia due to severe pulmonary edema in the dam26, 27. Concentrations of 100 ppm FB1 fed to sows in the last 30 days of gestation did not induce pulmonary edema, abortions, or fetal abnormalities7.

Reproductive effects of Trichothecenes in swine Effects of Deoxynivalenol on swine reproduction

All trichothecenes are known to

DON toxicosis has been

affect reproductive performance

associated with gastrointestinal

in pigs. Deoxynivalenol (DON)

signs such as abdominal

belongs to the trichothecene

discomfort, diarrhea,

family of mycotoxins and it has

vomiting, anorexia and

been proved able to significantly

reduced weight gain.

inhibit protein synthesis.

DON heavily affects

Particular acetylated and

the intestinal barrier

modified forms of the parent

integrity and function,

toxin, such as 3-acetyl-DON

while can also modulate

(3-Ac-DON), 15-acetyl-DON

immune response7,20.

(15-Ac-DON) and DON-3glucoside [DON3G, main plant metabolite of DON], occur simultaneously in grains25.

168 8

Evidence of oocyte maturation and

DON has been associated

increased proliferation, but greater

embryo development impairment

with particular reproductive

concentration (3.4mM) have

along with the reduction of feed

effects, mainly through in vitro

the opposite effect], inhibition

intake are the main reasons behind

studies on porcine oocytes.

of progesterone and estradiol

the DON-induced detrimental reproductive effects in pigs.

In vivo, ingestion of DON contaminated feed by pregnant gilts, may result in reduced weight and body length of piglets28. A significant passage of DON through the placenta from exposed sows to fetuses that could potentially affect fetal function has been demonstrated29,30. Furthermore, several in vivo negative effects on fertility have been associated with consumption of combined DON and ZEN contaminated feed . 31

In vitro effects of DON include disturbance of porcine oocytes maturation through the induction of abnormalities of the meiotic spindles and by altering oocyte cytoplasmic maturation32,33,34. In addition to the DON-induced impairment of oocyte maturation, findings of autophagy/ apoptosis and epigenetic modifications in porcine oocytes have been presented . 35

production [induced by FSH plus Insulin-like growth factor I (IGF-I)] and CYP19A1 and CYP11A1 mRNA abundance5,36. Exposure of ovarian explants to 10 μM DON affected the process of follicular maturation with a decrease of the reserve pool of follicles, resulting in a significant decrease in the number of normal follicles, as well as an increase of pyknotic oocytes number in all stages of

Moreover, DON has been

follicular development37, whereas

associated with dose-dependent

treatment with 1 μM DON

effects on porcine granulosa

decreased the rate of polar body

cell proliferation [biphasic

extrusion in porcine oocytes18.

effect: lower concentration (0.034 mM) of DON results in

Taken together, it seems that DON can have a direct ovarian effect that could impact reproductive performance in swine.

169 9

Effects of T-2 toxin on swine reproduction Besides DON, which is the main representative of the trichothecenes group, T-2 toxin and its deacetylated form HT-2 toxin (type A trichotecenes) can be considered as quite significant for swine, according to recent studies. They are produced in crops (e.g. wheat, maize, barley) by various Fusarium species such as Fusarium sporotrichioides, F. poae and F. langsethiae, either in the field or during storage. Pigs are very susceptible animals towards their effects. HT-2 toxin is a natural contaminant in cereals but is also the main metabolite of T-2 toxin, thus T-2 toxin effects can be partially attributed to HT-2 toxin. T-2 toxin inhibits protein, RNA and DNA synthesis, inducing apoptosis and necrosis in particular cell types and has a detrimental effect on cell membrane integrity due to increased lipid peroxidation25.

In acute T-2 toxicosis cases, serous-haemorrhagic necroticulcerative inflammation of the digestive tract, vomiting, diarrhea, leukopenia (leukocyte apoptosis), hemorrhage, shock and death, oral/dermal irritation and immunosuppression can be observed. However, in chronic cases of mildly contaminated grains ingestion, growth retardation, weight gain suppression and feed refusal along with greater pro-inflammatory gene expression, are observed in pigs38.

170 10

Quite similarly to DON, the effects of type A trichothecenes on reproduction are mainly

It has been also suggested that the treatment of porcine oocytes with 50 nM HT-2 toxin and

demonstrated through in vitro studies,

greater concentrations significantly decreased the

since in vivo clinical reproductive disorders

rate of polar body extrusion18.

in pigs, that could be directly attributed to their effects have not been presented. According to a study39, T-2 toxin may be able to alter the growth of the granulosa cell layer as well as affecting steroidogenesis. T-2 toxin had potent inhibitory effects on IGF-I and FSH-induced steroid production in cultured porcine granulosa cells, since dosages of 1, 3, 30

Failure of oocyte maturation after HT-2 toxin treatment has also been suggested, since the toxin inhibited porcine oocyte polar body extrusion and cumulus cell expansion, while also disrupted meiotic spindle morphology and disturbed actin distribution40. Oxidative stress, apoptosis and autophagy in the treated oocytes were also among the findings of the previously mentioned study.

and 300 ng/mL inhibited estradiol production, but progesterone production was inhibited with a dose of 30 and 300 ng/mL. An inhibitory effect on cell number was observed at 3 ng T-2 toxin/mL.

Taken together, these studies confirm the potential of T-2 toxin and its metabolites to impair reproductive function in pigs.

Reproductive effects of Zearalenone in swine Zearalenone (ZEN) is a phenolic resorcylic acid lactone mycotoxin produced by several Fusarium species, especially F. graminearum and may undergo modification in plants, fungi and animals (prehepatic, hepatic and extrahepatic) by phase I and phase II metabolism. Major metabolites of ZEN include α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol, zearalanone (phase I), whereas conjugated forms with glucose, sulfate and glucuronic acid are the outcome of phase II25,41.

171 11

Pigs are very sensitive to ZEN, since the parent toxin is metabolized mainly to α-zearalenol in that species, which shows greater estrogenic potency than ZEN. ZEN toxicosis has also been associated with increased oxidative stress, reduction of nutrient digestibility and growth retardation. Toxic effects of ZEN on other tissues and systems outside the reproductive tract, such as liver and immune system have already been demonstrated42,43,44.

ZEN su!ciently resembles 17β-oestradiol that allows it to bind to estrogen receptors in various organs and induce estrogenic effects. Its effects depend on the dose, as well as on the time of administration in relation to estrous cycle5. It is considered the most important mycotoxin affecting swine reproduction and prepubertal gilts seem to be a very sensitive age group to the effects of the toxin. For more than 40 years, researchers have demonstrated the significant reproductive disorders that can be caused after ZEN ingestion in gilts and sows in

vivo (e.g. pseudopregnancy, diminished fertility, hyperestrogenism syndrome, reduced litter size). Placental passage of ZEN from sows to fetuses Moreover, in vitro studies have presented its

has been confirmed, as ZEN and its metabolites

negative effects on oocyte maturation and

have been detected in the bile of newborn piglets

porcine granulosa cells proliferation7,41.

from sows ingesting contaminated feed45.

172 12

As regard to the effects of ZEN on

Multiple impairment of semen quality and

Additionally, ZEN and

boars’ reproductive function and

kinetics, including decrease of sperm

α-zearalenol can reduce the

quality of semen, results of in vivo

viability and progressive motility46-48 can

ability of boar spermatozoa

studies have suggested reduced

be the outcome of boar semen in vitro

to bind to the zona

serum testosterone levels, testis

exposure to ZEN.

pellucida46 and affect sperm

weights and spermatogenesis, as

chromatin integrity48,49.

well as feminization and suppressed libido in young boars5. Further

in vitro studies on boar semen have suggested ZEN toxicity.

Hyperestrogenic effects in newborn piglets in Greek swine farms.

Due to the significance and extent of ZEN-toxicosis outcome on farm reproductive health and performance, a summary of the major reproductive effects of ZEN and its basic metabolites in swine is presented in Table 1.

173 13

Table 1. Summary of major ZEN, α- and β-zearalenol effects in gilts, sows and boars according to in vivo and in vitro studies5,7,25,40,45-50,52-62. IN VIVO EFFECTS Mycotoxin ZEN

Gilts

Sows

1–5 ppm: Edema and hyperemia of the uterus/ vulva. Could result in vaginal and rectal prolapse. Neonatal gilts: swelling of the vulva and mammary glands and edematous infiltration of the perineal region, ventral abdomen, and umbilicus. Edematic regions could also have exudative crusted inflammation. Possibly necrosis of the teats.

Boars

Hyperestrogenism syndrome: edema of the vulva and mammary gland, vulvo-vaginitis, enlargement of the uterus, retained corpora lutea, ovarian cysts, nymphomania or anoestrus, ovarian atrophy. Increase of weaning-to-estrus interval. 3–10 ppm can induce anestrus. Linear relation of anestrus length and ZEN concentration in feed.

Particular studies suggest presence of detrimental effects on puberty attainment, while others demonstrated absence.

Infertility, pseudopregnancy, reduced litter size. lower conception rate, increased numbers of repeat breeders, increased numbers of stillbirths.

Pseudopregnancy and prolonged estrous cycle in cyclic gilts prior to mating.

Birth of piglets with hyperestrogenism syndrome and splay-leg.

Preputial enlargement, possibly general loss of vigor and reproductive compromise. Young boars: reduced libido and decreased testicular size. At 9 ppm lower total and gel free volumes of semen with lower total motile sperm. Reduced serum testosterone levels, testis weights and spermatogenesis, as well as feminization in young boars. Mature boars: unaffected by concentrations < 200 ppm ZEN.

IN VITRO EFFECTS Mycotoxin ZEN

Oocytes/uterus/ovary (gilts and sows) Impairment of oocyte maturation. Reduction of polar body extrusion rates. Oocyte dies in the Graafian follicles. Signs of oestrus could be present but there is no ovulation. Suppressed pig oocyte progression through meiosis by inducing the malformation of meiotic spindles aneuploid embryos Interference with the initial chromatin status and maturation competence of oocytes. Reduction of healthy follicles quantity could result in premature oocyte depletion in adulthood.

Granulosa cells and steroid production (gilts and sows) Porcine granulosa cells: High concentrations (30–120 mM): apoptosis and impairment of porcine granulosa cells proliferation. Induction of disorders of the mitochondrial transmembrane potential and increase of the reactive oxygen species levels. Apoptosis or necrosis through the caspase-3/caspase-9 dependent mitochondrial pathway may induce atresia in porcine follicles. Increased expression of genes related to DNA damage and repair.

Hormonal balance (gilts and sows) Inhibition of FSH release and secretion (similarly to 17-β estradiol) depresses maturation of ovarian follicles during the preovulatory stage. May exhibit a luteotrophic property Prolongs corpus luteum life span to equal or longer than normal gestation period. Contradictory results on effects to LH. Absence of effect, or in prepubertal gilts at 3.2 ppm ZEN, decreased LH levels in serum.

Boars Decrease sperm viability and progressive motility. Significant detrimental effects on major kinetics parameters of boar semen (e.g. static, rapid motile spermatozoa, etc.). Reduces the ability of boar spermatozoa to bind to the zona pellucida. Affects sperm chromatin integrity.

Prepubertal gilts: > 2 ppm ZEN Increase of prolactin levels in serum.

Accelerated development of the ovaries (follicles) in post-weaning piglets promotes the autocrine action or expression of the ghrelin gene in piglet ovary. May affect oviduct tubal function (alterations in gene expression of tubal epithelial cells). Ovarian atrophy and changes in the endometrium (proliferation of uterine glands). Proliferation (hyperplasia) of the epithelial cell layer of the uterine and vaginal mucosa thickening and irregularity of the epithelium. Squamous metaplasia in uterus and hyperplasia of the endometrial glands. Increased genital organs size in gilts, along with hyperplasia of submucosal smooth muscles in the corpus uteri. Increased weight of the uteri and thickness of the myometrium and endometrium (greater growth hormone receptor expression in the uteri).

174 14

Table 1. Summary of major ZEN, α- and β-zearalenol effects in gilts, sows and boars according to in vivo and in vitro studies5,7,25,40,45-50,52-62.

IN VITRO EFFECTS Mycotoxin

Oocytes/uterus/ovary (gilts and sows)

Granulosa cells and steroid production (gilts and sows)

α-zearalenol

Reduced rate of oocyte maturation (7.5 μΜ).

Primarily increased progesterone (P4) production induced by FSH and insulin-like growth factor-I (IGF1). Biphasic dose–response with 0.094 mM increasing and 9.4 mM inhibiting FSH plus IGF-I-induced estradiol production or absence of effects at high ZEN concentration.

Hormonal balance (gilts and sows) No effects on LH.

Boars Reduced viability and motility of boar semen. Reduces the ability of boar spermatozoa to bind to the zona pellucida. Modified DNA integrity and structural stability at pM levels.

Increased progesterone and decreased estradiol production by porcine follicles = indicator of follicular atresia. Decreased abundance of CYP19A1 and CYP11A1 mRNA induced by FSH plus IGF-I. β-zearalenol

Reduced rate of oocyte maturation (30μΜ).

Increases the curvilinear velocity (VCL parameter). Modified DNA integrity and structural stability at nM levels

175 15

CONCLUSIONS

Diagnosing mycotoxin-induced

disorders, must be considered when

as the inclusion of proper

reproductive disorders in sows or

establishing differential diagnosis of

agents that would reduce

boars is definitely not an easy

reproductive inefficiency cases.

the level of mycotoxins available for absorption in

task. As presented in this review,

Feed analysis is of colossal

the extent of effects on the

importance along with evidence

reproductive system of swine is

of mycotoxins/metabolites

vast and includes a great variety

circulating in blood or detected

Proper management of sows

of direct and indirect mechanisms

in tissues and excreta.

(e.g. proper estrus detection,

Furthermore, the interactions of the abovementioned toxins in vivo and their final observed effects on sow and boar’s reproductive efficiency have not been fully elucidated yet and need further clarification.

of utmost importance51.

heat stress countermeasures)

of toxicity at the cellular and genomic levels.

the gastrointestinal tract are

Unfortunately, antidotes against

and regular semen viability

mycotoxins do not exist, thus, control

and kinetics analysis,

of such cases would need removal of

along with prevention of

contaminated feed or mixing with

infectious agents (e.g.

clear feed (usually at 1:10 rate), as

proper vaccination schedule

well as inclusion of agents that could

of the breeding stock), would

either adsorb or biotransform the

significantly assist on rapid

mycotoxins to non-toxic metabolites.

detection of abnormalities

Evidence so far suggests that

Moreover, clinical support should

ZEN and α-zearalenol are the

be given to splay-leg newborns

most important mycotoxins for

with signs of hyperestrogenism

swine reproduction and from a

that cannot receive the appropriate

clinical viewpoint they are probably

amount of colostrum/milk.

that could be associated with mycotoxicosis, thus proper treatment and prevention efforts would start timely.

the first to investigate in cases of reduced fertility on farm. However, such cases usually include concomitant DON ingestion via feed due to the “characteristically observed” combined mycotoxins contamination of pig feed.

In the majority of cases, removal of mycotoxins would lead to the improvement of fertility and reproductive parameters in due time through defense and repair mechanisms at cellular level50. However, time would be needed in order to achieve previous

From the diagnostic

reproductive rates again on farm.

standpoint, onset of reproductive signs right

The principle is that prevention of

after feed alterations

mycotoxicosis should be the basic tool.

on farm, as well as the absence of any significant impact of infectious agents or environmental or managerial factors that could induce reproductive

Regular feed screening (enrichment materials and other fiber sources can contain significant amounts of mycotoxins, thus they should be also included in the analysis), as well

Nevertheless, further intensive research efforts are needed on the field of reproductive failure due to mycotoxins ingestion, especially in terms of explaining underlying mechanisms associated with observed detrimental effects.

176 16

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179 19

MYCOTOXINS EFFECTS AND MECHANISMS of immune modulation IN SWINE

Assist. Prof. Panagiotis Tassis Assistant Professor of Swine Medicine and Reproduction, Clinic of Farm Animals, School of Veterinary Medicine, Aristotle University of Thessaloníki, Greece

180 1

Mycotoxin menace in grains worldwide Mycotoxins are secondary metabolites produced by fungi (genera Aspergillus, Penicillium, Fusarium,

Alternaria, and Claviceps) that can be found in grains (e.g., maize, wheat, barley) worldwide.

Studies have proven their variability in

Recent studies have suggested that up

distribution among regions and climate zones

to 80% of feed and food crops are

globally1. Among numerous mycotoxins, it has

contaminated with mycotoxins globally

been suggested that deoxynivalenol (DON),

(occurrence above the detectable

fumonisins (FBs, FB1-FB3), zearalenone (ZEN),

levels up to 60–80%), whereas

aflatoxins (AFs, mainly AFB1), ochratoxin A

co-contamination of grains with multiple

(OTA) and T-2 toxins are the most significant

mycotoxins is a common finding3.

for swine health and production . 2

A 10-year survey with samples from 100 countries reported that DON, FBs, and ZEN were most prevalent mycotoxins and were detected in 64%, 60%, and 45% of all samples, respectively. The

DON 64% samples 723 μg/kg

FBs 60% samples 388 μg/kg

median concentrations were 723 μg/ kg, 388 μg/kg and 55 μg/kg for FBs, DON and ZEN, respectively1. Pigs and poultry are very susceptible and sensitive to the effects of mycotoxins4. The effects of mycotoxins on pigs

ZEN 45% samples 55 μg/kg

Mycotoxin contamination in crops

are multiple and they depend on5: The type of mycotoxin The level and duration of exposure The age of the animal

181 2

ZEN

Ingestion of great dosage levels

Reproductive disorders Hyperestrogenism syndrome

can induce acute cases of mycotoxicosis with well-described clinical symptoms, such as2,4,6:

FBs Pulmonary edema

Reproductive disorders and hyperestrogenism syndrome in the case of ZEN

ACUTE MYCOTOXICOSIS

Vomiting and growth retardation in the case of DON

DON

Pulmonary edema after FBs ingestion

AF ↓Feed intake ↓Weight gain

Vomiting Growth retardation

Reduced feed intake and weight gain in acute AF cases Polydipsia, polyuria, and

OTA

reduced growth in OTA cases However, chronic consumption of

The chronic toxic effects of mycotoxins

low mycotoxins levels and the

in swine include hepatotoxicity,

induction of vague clinical symptoms

genotoxicity, nephrotoxicity,

seems more probable under ield

neurotoxicity, reprotoxicity,

conditions.

immunotoxicity as well as other effects

Polydipsia & Polyuria ↓Growth

such as neuroendocrine disorders6,7.

HEPATOTOXICITY

NEPHROTOXICITY

GENOTOXICITY

CHRONIC MYCOTOXICOSIS

NEUROTOXICITY

IMMUNOTOXICITY REPROTOXICITY

NEUROENDOCRINE DISORDERS

182 3

INNATE IMMUNE RESPONSE

The pig’s immune system

Phagocytic cells

Toll-like receptors (TLR)

Cytokines, chemokines, and proteins

Monitor pathogenassociated molecular patterns and induce signalling pathways

The pig’s immune system is the main defense mechanism against infectious and other agents. Its response is complicated, but the basic aspects of the immune system response are8: Inflammation Cellular response Humoral response Briefly, after engagement of the immune system (e.g. after contact with an infectious agent) a first defense multiple-mechanism takes action.

Antimicrobial protection

This mechanism includes the innate immune response with phagocytic cells

Recruit T cells

Activate acquired immune response

and the production of various cytokines, chemokines, and proteins that provide antimicrobial protection, recruit T cells through the inf ammatory process, and further activate the adaptive

Attack infectious agent-infected cells Cytokines

INNATE IMMUNITY

or acquired immune response. The innate system also includes natural killer (NK) cells that present

Natural Killer (NK) cells

a dual function including an innate response to attack infectious agent-infected cells and production of cytokines for assisting in the activation of acquired immunity9-11. Additionally, pattern recognition receptors, including Toll-like receptors (TLRs), participate in monitoring pathogen-associated molecular patterns and induce signaling pathways, that will enable activation of the immune system against infection11.

183 4

ACQUIRED IMMUNITY

ACQUIRED IMMUNE RESPONSE Further on, the adaptative

B cells & Antibodies

system uses B cells, T cells,

T cells

Cytokines

cytokines, and antibodies in order to provide pathogenspecific memory for protection from subsequent infections with the same pathogen.

Pathogen-specific memory

Taken together, the innate defense mechanisms that neither require previous exposure to antigen nor have an immunological “memory”, provide the first and almost immediate response to the infectious agent and control infection, while at the same time assist in the activation of the adaptive immune system, which has immunological “memory”, and will produce antibody and cell-mediated immune responses11.

The innate immune system First line of defense complement

system,

microbial ecosystem) and the swine

There are major parts of the

peptides),

innate immune system that act

toll--like receptors (TLR), type I

lymphoid system consisting of the

as the first line of defense or

interferons (IFNs), tumor necrosis

lymph nodes, lymphoid follicles,

“barriers” to different types of

factor-α (TNF-α), IL-6, and IL-8

tonsils, thymus, and spleen12,13.

infections (physical, chemical,

(proinflammatory cytokines) :

microbial), such as epithelial cells, bactericidal fatty acids, normal flora, and the mucus layer, as well as cells with phagocytic abilities such as granular leukocytes (neutrophils, basophils, mast cells,

Defend against pathogens

Additionally, an optimal microbiota14:

Control infections

Prevents colonization of the

Activate the cascade of

intestinal epithelium by pathogens

events of inflammation and

and penetration of the gut barrier

adaptive immunity response

Modulates the gut-associated lymphoid tissue (GALT)

and eosinophils), and mononuclear phagocytes (circulating blood

Parts that play a significant role

and systemic immunity

monocytes and tissue macrophages).

in this immune defense system

Influences gastrointestinal

and the response to pathogens

development

Natural killer cells (NK) and other parts of the innate immune system such as defensins (host defense

are the mucosal epithelium (e.g. intestinal and respiratory tract), the microbiome (intestinal

184 5

ELEMEMENTS OF THE IMMUNE DEFENSE SYSTEM

Mucosal epithelium (intestinal & respiratory tract)

Microbiome (intestinal microbial ecosystem) Phagocytic & NK cells, complement system, TLR, IFNs, proinflammatory cytokines

Lymphoid system (lymph nodes, lymphoid follicules, tonsils, thymus and spleen)

Defend against pathogens Control infections Activate the cascade of events of inflammation and adaptive immunity response

Effects of mycotoxins on the swine immune system The effects of the above-

Fusarium mycotoxins can either

mentioned mycotoxins in swine are

result in immunostimulatory

multiple and vary significantly.

or immunosuppressive effects, depending on the age of the host,

Considering that pigs ingest

exposure dose and duration7,16,

mycotoxin-contaminated feed,

whereas AFs and OTA induce

the gastrointestinal epithelial

immunosuppression17,18.

cell layer is the first site of contact and interaction14,15.

From that point and after, immunomodulation and a sequence of immunological reactions take place, and

Fusarium mycotoxins Immunostimulatory or immunosuppressive effects AFs & OTA Immunosuppression

they can be altered due to the mycotoxin’s effects at a molecular and cellular level.

185 6

The health and economic implication

Major part of the facts regarding

of the effects of mycotoxins on

the effects of mycotoxins on

the immune defense system of

vaccinal efficacy and disease

pigs is significant. Three major

susceptibility will be presented

outcomes have been described19-21:

in a separate review.

Increased susceptibility to infectious diseases Reactivation of chronic infections Decreased vaccination efficacy A sensitivity of the immune system to mycotoxin-induced immunosuppression has been suggested, due to the vulnerability of the continually proliferating and differentiating cells that take part in immune--mediated

TRICHOTHECENES

activities and regulate the communication between cellular and humoral components8. Moreover, it should be stated that

As regards the acetylated

DON effects on the H

immune system of the pig

O OH

effects of mycotoxin mixtures

O HO

forms of DON, there is evidence that at least 15-Ac-DON elicits similar general chronic toxicity as

in pig feed on the immune

DON and other Fusarium mycotoxins

DON, whereas the immunotoxicity

system can also increase

directly affect globulin synthesis

of 3-Ac-DON and 15-Ac-DON

variability of the outcome and

in the liver and compromise the

might be less expressed25.

cannot be easily predicted, since

immune response of pigs22.

they could have antagonistic,

As observed in vivo, trichothecenes

additive or synergistic

Type B trichothecenes, including

can be stimulatory in some

interaction and increase the

DON, have the capacity to up-

leukocyte models but inhibitory

impact of each mycotoxin20.

and down-regulate immune

in others; paradoxically, these

functions by disrupting intracellular

activities sometimes co-occur26.

The present review will focus on evidence regarding underlying mechanisms of mycotoxininduced immunomodulation.

signaling among leukocytes23. DON immunostimulatory or immunosuppressing effects depend on the dose, frequency

Special reference is given to

and duration of exposure24,

trials on pigs or porcine cell

whilst few research efforts have

lines, however evidence from

demonstrated the effects of

other farm animals, laboratory

3-Ac-DON, 15-Ac-DON and DON-

animals and cell lines are

3-glucoside on immune response.

also selectively presented.

Immune cells (macrophages, B and T lymphocytes and natural killer (NK) cells) are sensitive to DON, 3-Ac-DON and 15-Ac-DON, and dose-dependent immunostimulatory/ inflammatory or immunosuppressive effects can be observed7,23,24,27,28.

186 7

IMMUNOTOXIC EFFECTS

Differential inflammatory gene

Toxic effects of DON on farm

expression and DON-induced

animals have been extensively

apoptosis are mechanisms

reviewed25 with anorectic and

that play a signi icant role in

immune-modulatory effects being

those immune effects.

the most pronounced in pigs.

The most prominent molecular target

Feed refusal and reduced feed

of trichothecenes is the

intake after ingestion of DON-

60S ribosomal subunit suggesting

contaminated feed have been

that one underlying mechanism

associated with the hormonal

is translational inhibition29.

and immunotoxic effects of DON as changes in satiety hormones

However, it is known that

(e.g. cholecystokinin and peptide

trichothecenes and other translational

tyrosine tyrosine) and changes

inhibitors which bind to ribosomes can also rapidly activate mitogen-

of proinflammatory cytokines (e.g. IL-1β, IL-6, TNF-α) have

activated protein kinases

been observed to be related to

(MAPKs), eliciting expression of

DON-induced anorexia25.

inflammation-related genes as

Changes in proinflammatory cytokines

DON O

O OH

O HO

DON-contaminated feed

Changes in satiety hormones

Furthermore, it has been

pro-inflammatory cytokines23,24, and

suggested that DON

induce apoptosis in a process known as the “ribotoxic stress response”30,31.

predominantly affects vigorously proliferating cells such as intestinal epithelial cells (IEC), liver and immune cells, and

FEED REFUSAL AND REDUCED FEED INTAKE (ANOREXIA)

the order of system sensitivity to DON is immune >neuroendocrine>intestinal7,14. MAPKs modulate physiological processes including cell growth, differentiation, and apoptosis32 and are critical for signal transduction in the immune response33.

INFLAMMATORY GENE EXPRESIÓN AND DON-INDUCED APOPTOSIS

DON O

60S ribosomal subunit H

O OH

O HO

Translational inhhibition Expression of pro-inflammatory cytokines

Ribotoxic stress response (apoptosis)

187 8

As previously reported34, low

In the in vitro study (hepatocytes

The toxin had a biphasic

DON concentrations (up to

exposed to 500 or 2000nM

effect on the OVA-specific

840 μg/kg feed for 4 weeks)

DON with or without 1μg

lymphocyte proliferation,

do not affect piglet immune

lipopolysaccharides (LPS)/ml;

suggesting an up-regulation

responses for immunoglobulin

incubation for 48 hours) from

in the days after OVA

concentration, lymphocyte

Doll et al.41, it was suggested that DON has the potential to provoke and modulate the immunological reactions of porcine liver cells. The study provided evidence that:

immunization but a

However, in other studies DON has been shown to increase IgA concentration in blood, whereas nonspecific lymphocyte proliferation can be either increased or decreased19,35-38. On the other hand, Ferrari

et al .39 did not demonstrate significant immune effects after 6 weeks of oral DON exposure in pigs, confirming the variability of DON immune effects.

HEPATOTOXIC EFFECTS OF DON

IMMUNE EFFECTS OF DON IN PIGLETS

cytokine production.

according to previous investigations on pigs, other farm animals and humans there are usually only minor (up to 1.5-fold), insignificant, or no effects of DON on IgA.

pigs immunized with OVA, suggested an increase of anti-OVA IgG titers, after 42 days of exposure to a

mRNA expression of

DON contaminated diet.

TNF-α in hepatocytes.

Simultaneously, the

DON stimulated a dose-dependent induction

of LPS-induced IL-6 were significantly decreased. mRNA expression of the anti-inflammatory IL-10 was increased.

VACCINAL IMMUNE RESPONSE

As stated by Döll and Dänicke , 40

Another research effort on

synergistic for increased

Supernatant concentrations

al .37, DON (4 mg/kg feed for 14 days in weaned pigs) significantly increased the amount of DNA damage in lymphocytes by 28%.

weeks following.

DON and LPS were

of IL-6 mRNA.

According to Frankic et

down-regulation in the

expression of chemokines VACCINAL IMMUNE RESPONSE

proliferation, and

involved in inflammatory reactions [(IL-8, chemokine (C-X-C motif) ligand 20 (CXCL20), interferon-g (IFN-g)] were up-regulated. Deoxynivalenol also up-regulated the gene expression of

In a study of DON effects on

antioxidant glutathione

vaccinal immune responses

peroxidase 2 (GPX-2)

(2.2–2.5 mg DON/kg feed,

and down-regulated

weaned pigs for 9 weeks)19,

expression of genes

increased ovalbumin-specific

encoding enzymatic

(OVA) IgA and IgG were

antioxidants including

reported, whereas lymph nodes

GPX-3, GPX-4 and

from treated pigs had reduced

superoxide dismutase

expression of TGF-β and IFN-γ

3 (SOD-3), involved

mRNA, thus supporting the

in oxidative stress42.

possibility of DON-induced reduction of vaccinal response.

Reduced or delayed antibody response to thymus-dependent antigens was observed also in growing pigs fed DON-contaminated grains22,43.

188 9

induces suppressive effects

sensitive to trichothecenes26, 44.

on macrophage-involved

Macrophages are considered

processes, such as cytokine

to be cells with greater (10 to

secretion and phagocytosis, and

100-fold) sensitivity to DON

induces their apoptosis (through

when compared to fibroblasts,

p38 kinase activation), thus

Based on some hypothesis, increased sensitivity can be attributed to increased DON ability to enter macrophage cells or increased apoptosis of macrophages after

EFFECTS ON MACROPHAGES

High dose DON exposure

immune system appear

lymphocytes, IEC or astrocytes.

increase host susceptibility to pathogens and reduce activation of B and T lymphocytes (macrophages failing to act as antigen presenting cells)52-54.

porcine macrophages provide

JAK/STAT pathway

evidence for a lack of COX-2

4,45.

and IL-6 activation by DON Low dose DON exposure results in:

↓DON

in porcine macrophages, suggesting a distinct mode of action in this species36.

Macrophage stimulation and activation (human,

Modulation of dendritic

mouse, murine and porcine macrophages).

DON effects on the intestine of pigs are well reviewed by Pinton and Oswald56 suggesting multiple negative effects on integrity of the intestinal epithelium and barrier, as well as modulation of intestinal epithelium immune responsiveness, since type B trichothecenes can affect cytokine production by intestinal or immune cells and interfere with communication between epithelial cells and other intestinal immune cells.

However, studies in primary

DON-induced activation of

Cano et al.57, investigated in vitro effects of purified DON [porcine IPEC-1 and porcine jejunal explants (ex vivo model)], and suggested that DON can: Potentiate the expression of immune genes

cells (DC) function probably

Increase protein concentration

contributes to DON-induced

in differentiated IPEC-1 cells

Secretion of inflammatory

immunosuppressive

in a time-dependent manner

cytokines (IL-1β, IL-2, IL-4,

effects. In vitro and in vivo

IL-5, IL-6 and TNFα).

Cause an early intestinal

investigation revealed DON

inflammatory response

Expression of intracellular COX-2 and iNOS proteins (selective activation of ERK, NFκB and activator protein-1)24,36,46-48, nitric oxide synthase , and 49

numerous chemokines50,51.

EFFECTS ON DENDRITIC CELLS

EFFECTS ON MACROPHAGES

DON

The macrophage and innate

and DC interaction. Findings after feeding pigs

Disrupt the intestinal homeostasis

with 5.3 ppm DON in feed

Promote the intestinal immune

for 5–11 weeks (in vivo) or/

system towards a Th17 response

and after 100–800ng/mL DON treatment of monocyte-derived DC (in vitro), included55: Decreased endocytic activity Inhibition of IL-10 secretion Impairment of DC capacity for antigen-uptake

189

T-2 effects on the immune

The most prominent molecular

T-2 toxin exposure results in

system of the pig

target of trichothecenes

leukopenia and cell depletion

includes the 60S ribosomal

in lymphoid organs, significantly

According to EFSA58,

unit, where it prevents

impairing antibody production,

the domestic pig is

polypeptide chain initiation66.

reducing the proliferative

amongst the most sensitive species to the effects of T-2 and HT-2 toxins. T-2 toxin is reported to be immunotoxic, either by its cytotoxic, apoptotic or immunosuppressive attributes. Like other trichothecenes, T-2 toxin can be both immunosuppressive

PROTEIN SYNTHESIS INHIBITION

immunotoxic and haematotoxic

In vitro studies suggest that T-2 toxin interacts with the peptidyl transferase, which is an integral of the 60S ribosomal subunit, thus inhibiting the transpeptidation of peptide-bond formation, resulting in an inhibition of prolongation and termination of protein synthesis62,63.

response of lymphocytes and hindering the development of dendritic cells70. Moreover, it can disrupt DNA polymerases, terminal deoxynucleotidyl transferase, monoamine oxidase and several other proteins involved in the coagulation pathway71. A time- and dose-dependent DNA damaging effect of T-2

The toxic effects exerted by T-2

toxin could be demonstrated

depending on the dose

toxin and HT-2 toxin include the

using peripheral blood

and timing of exposure.

inhibition of protein synthesis

mononuclear cells from

(through binding and inactivation

pigs (incubation with 0.1-1

Effects of T-2 toxin on

of peptidyl-transferase activity

μM for 24 or 42 hours)72.

both humoral and cellular

at the transcription site),

and immunostimulatory

affecting also the synthesis of

immune response have been demonstrated in various studies . 59

Moreover, T-2 toxin

60,61

immunoglobulins and, in turn, the humoral immunity67-69.

Induces lipid peroxidation, affecting cell membrane integrity Causes cell depletion in lymphoid tissue Inhibits inflammatory cell function

Lipid peroxidation

Decreases humoral and cell-mediated immune responses, leading to an

TOXIN T2

increased susceptibility

Cell depletion in lymphoid tissue

to infection

Inhibition of prolongation & termination of protein synthesis

↓humoral & cellmediated immune responses

190 11

Dendritic cells, the most potent antigen-presenting cells (APCs) of the immune system, have

In a feeding study with pigs, (0.5-3.0 mg T-2/kg feed) immunosuppression was observed.

demonstrated sensitivity to trichothecene mycotoxins,

Pigs were immunized with horse globulin and synthesis of

and T-2 toxin disturbed their

antibodies towards this globulin was reduced, whereas

maturation process64.

a dose dependent depletion of lymphoid elements in the thymus and spleen, was also reported.

Moreover, in a previous in vitro study with primary porcine alveolar macrophages, pre-exposure of macrophages to 3 nM of T-2 toxin decreased the production of inflammatory mediators (IL-1β, TNF-α, nitric oxide) in response

Leukocyte counts and the portion of T lymphocytes were decreased in all exposure groups74. In pigs immunized with OVA, subclinical doses of T-2 toxin induced an early and transient increase of total IgA plasma concentration but a decrease in the anti-OVA IgG titer.

to LPS and the decrease of the

Pigs fed 1.324 or 2.102 mg T-2 toxin/kg exhibited reduced

pro-inflammatory response

anti-ovalbumin antibody production on day 21 without

was associated with a decrease

significant alteration to specific lymphocyte proliferation75.

of TLR mRNA expression. Thus, ingestion of low concentrations of T-2 toxin can affect the TLR activation by decreasing pattern recognition of pathogens and interfere with the start of inflammatory immune response against pathogens65. Acute T-2 toxicity (1.2 mg/kg body weight intravenously) has been characterized by emesis, posterior paresis, listlessness and lethargy, as well as severe damage to actively dividing cells

Frankic et al. 37 reported that, T-2 toxin (3 mg/kg feed for 14 days in weaned pigs) increased the amount of DNA damage in lymphocytes by 27% and decreased total serum IgG. Similarly to the effect on lymphocyte proliferation, low amounts of T-2 toxin were found to increase antibody levels, whereas high amounts were found to be immunosuppressive60, therefore increased susceptibility to infectious diseases can be observed (e.g. Mycobacterium, Staphylococcus, Listeria, Toxoplasma and Herpes simplex virus (HSV-1) – effects seen in rats, mice and chicken)58.

Li et al. 76 discussed that suppression of IFN-γ by T-2 toxin is probably one of the factors responsible for the decreased antiviral immunity in the presence of T-2 toxin. The suppression of IFN-γ may be due to increased IL-6 (interleukin 6) expression.

in bone marrow, lymph nodes, spleen, thymus and intestinal mucosa. However, within 24 hours, surviving pigs recovered and appeared normal60,73.

191 12

Interesting facts about the trichothecenes – immune system interaction In conclusion, macrophages,

1. Activation of mitogen-activated protein kinases (MAPKs)

IgA, and pro-in lammatory cytokines have a signifiicant role in the immunomodulatory effects of trichothecenes77. Underlying mechanisms of trichothecenes effects on the immune system, as suggested by reviews from Wu et al. 77-79 and Liao et al. 62, are:

DON

3. Induction of mitochondrial signaling pathways and apoptosis

O OH

2. Trigger endoplasmic reticulum stress and calcium-mediated signalling

O HO

UNDERLYING MOLECULAR MECHANISMS OF DON

4. Influence the pathway for protein synthesis in cells, like RNA synthesis, ribosome functioning and translation

1. Activation of mitogenactivated protein kinases (MAPKs) The most recognized theory is that DON and other ribosomebinding translational inhibitors can activate mitogen-activated protein kinases (MAPKs) through a mechanism known as “ribotoxic stress response” process30. MAPKs, which are crucial for signal transduction in the immune response, mediate transcriptional and post-transcriptional gene

rapidly triggers MAPKs signaling pathway (as well as NF-kB,

upregulation caused by DON.

and JAK/STAT pathways), eventually leading to cell apoptosis

DON binds to the

and the expression of pro-inflammatory cytokines80.

ribosome 28s peptidyl transferase locus, which

Additionally, trichotechenes regulate apoptosis-related

activates ribotoxic stress

signal molecules such as IL-6, IL-1β, and TNF-α.

response and induces the phosphorylation of protein kinase (PKR)

MAPK phosphorylation can be activated after

atopoeitic cell and hema

these toxins bind to the peptidyl of ribosomes to

kinase (Hck), then

regulate immune responses and apoptosis23,88.

192 13

Oxidative stress is an important

2. Trigger endoplasmic reticulum stress and calcium-mediated signalling

mechanism of trichothecene toxicity, since they disrupt the normal function

DON can also induce endoplasmic reticulum (ER) stress ,

of mitochondria and generate

as well as increasing ATF3 and DDIT3 (two major ER

free radicals, including ROS.

stress markers) protein expression within 3 hours . 81

These compounds induce lipid peroxidation, change

As it has been reported, the over-expression of ATF3 and

the antioxidant status of the

DDIT3 could result in cell cycle arrest and/or apoptosis82,83.

cells, and reduce the activity of antioxidant enzymes such

DON can induce the phosphorylation of protein kinases JNK in Jurkat event known to mediate apoptosis, within 3 hours after its exposure81. Activated caspase-12 can induce the activation of caspase-9 through the direct cleavage of caspase-9, which in turn induces the activation of caspase-3 and finally apoptosis takes place84.

3. Induction of mitochondrial signaling pathways and apoptosis

as glutathione-S-transferase OXIDATIVE STRESS

cells to trigger T-cell activation response and cleavage of caspase-3, an

(GST), superoxide dismutase (SOD), and catalase (CAT)77,88. DNA damage is also associated with the generation of ROS and lipid peroxidation. Some signaling pathways, including MAPK, JAK/ STAT, and NF-κB, are subsequently induced by oxidative stress, and the caspase-mediated apoptosis

DON also induces apoptosis by involving the mitochondrial intrinsic pathway through the following mechanisms

pathways are also activated89.

85,86

Opening of the mitochondrial permeability transition pore (mPTP) Loss of the mitochondrial transmembrane potential Increase of O2− (superoxide anion) Release of cytochrome C

Oxidative stress (generation of ROS as early as 30

Thus, mitochondrial dysfunction, pursuant release of cytochrome C into the cytoplasm and serial activation of caspases contribute to DON-induced apoptosis, which is possibly modulated by Bcl-2 family62.

min after exposure) is the mechanism by which T-2 toxin causes DNA damage and apoptosis90-92.

4. Influence the pathway for protein synthesis in cells, like RNA synthesis, ribosome functioning and translation DON can also upregulate microRNAs (miRNA) which are responsible for downregulation of selective genes and ribosome synthesis87. Additionally, trichothecenes significantly downregulate IFN-γ expression in pigs and mice, thereby reducing the host resistance to viruses and repairing ability19,93, whereas DON reduces IFN-b expression and promotes cell apoptosis48.

193

The immunomodulatory

The immunostimulatory effect

Trichothecenes have

effect of trichothecenes may

of trichothecenes may be partly

an “immune evasion”

be determined by a balance

mediated by autophagy

mechanism that suppresses

between cell-survival and

STAT3 pathway (which maintains the normal function of mitochondria) and promoted by the STAT1 pathway in the same cell94. DON initiates both a survival pathway (ERK/AKT/p90Rsk/ Bad) and a competing apoptotic pathway (p38/p53/ Bax/Mitochondria/Caspase-3)

inhibits the DON-induced apoptosis of intestinal epithelial cells by ameliorating the damage caused by oxidative stress,

IMMUNE EVASION

inhibited by the JNK1–

DON-induced autophagy IMMUNOSTIMULATION

IMMUNOMODULATION

apoptosis is simultaneously

host and vaccine-induced

According to Tang et al. , 96

death-signaling pathways26,88,94. T-2 toxin-induced cell

95,96

immune defenses. This mechanism interferes with anti-apoptotic genes, promoting oxidative stress-induced

thereby causing the cell

apoptosis93,97,98, allowing

stress response to fail.

the toxins to escape

According to Bin-Umer

et al.95, autophagy of damaged mitochondria (mitophagy) plays a key role in the resistance of cells to trichothecenes.

host resistance and immune repair78.

in RAW 264.7 macrophages26.

GENOTOXICITY ZEARALENONE

HEMATOTOXICITY

HEPATOXICITY

Due to its estrogenic properties, ZEN binds to estrogen receptors (ERs) and is typically associated with

ZEA

reproductive disorders in swine . 99

However, ZEN has also been known to exhibit hepatotoxicity, hematotoxicity, immunotoxicity and genotoxicity45,100.

BIND TO ESTROGEN RECEPTORS

Since, immune cells also express ERs such as ERα in NK cells, macrophages and T cells, as

NK cells, macrophages & T cells → ERα

well as ERβ in monocytes and B cells101, ZEN can also bind to such ERs and regulate a variety

Monocytes & B cells → ERβ

of metabolic pathways of the immune response100.

REPRODUCTIVE DISORDERS IMMUNOTOXICITY

194 15

ZEN not only activates immune response-related genes, but also interferes with the immune system of the spleen, changes the phenotypes of spleen lymphocytes, and even causes lymphocyte atrophy in mice or rats102,103. In addition, ZEN can induce immunosuppression by reducing immunoglobulins in serum and cytokines in lymphoid organs104. On the other hand, RNA sequencing on liver samples from piglets fed with ZEN and DON-contaminated feed, indicated an effect on the expression and network of immune-related transcripts105.

As regards ZEN effects on humoral immune response, a study performed with rats (5.0 mg/kg of ZEN for 36 days) revealed that ZEN alone (without immune challenge) can decrease the production of immunoglobulins106.

blood mononuclear cells (PBMC) of piglets also showed a decrease in immunoglobulin levels107. On the other hand, in an in vivo study by Swamy

et al.108, increased serum immunoglobulin concentrations (IgM and IgA were increased, but not IgG) were observed in pigs fed grains contaminated with DON, fusaric acid (FA), ZEN, and 15-acetyldeoxynivalenol (15-acetylDON). According to another in vivo study by the same group109 with different concentrations of the same mycotoxins in pigs, absence of effect of diet on the IgM and IgG antibody levels was reported.

The inconsistent effects of ZEN on humoral immune response could be related to receptor-specific effects,

In vitro experiments using Vero and Caco-2 cells suggested that ZEN induces cytotoxicity and oxidative damage in addition to its estrogenic potential111. CYTOTOXICITY AND OXIDATIVE DAMAGE

HUMORAL IMMUNE RESPONSE

In addition, an in vitro study with peripheral

Additionally, an in vitro study by Taranu et al.112, investigating the effects of ZEN (10 mM) on gene expression of porcine intestinal cells (IPEC-1), supported that even though such ZEN concentrations do not affect cell viability, 70% out of 190 differentially expressed genes were up-regulated. Genes coding for glutathione peroxidase enzymes (GPx6, GPx2, GPx1) were among those up-regulated, providing evidence for mycotoxins inducing oxidative damage, whereas increased expression of cytokines

since ZEN is an agonist toward ERα

involved in inflammation (e.g. TNF-α, IL-6,

and a mixed agonist-antagonist of

IL-8) and immune cell recruitment (e.g.

ERβ110, with possible full antagonism

IL-10) was also revealed, thus demonstrated

of the ERβ expressed by B cells103.

that ZEN modulates intestinal cell immune and/or cellular repair pathways.

195 16

Marin et al.113 investigated the effects of ZEN and its metabolites, α- zearalenol (α-ZEL), β-zearalenol (β-ZEL), and zearalanone (ZAN), on several neutrophil functions such as proliferation, cytokine synthesis and oxidative stress in a porcine polymorphonuclear (PMN) cells model.

β-ZEL also induced cell death, mainly by apoptosis rather than necrosis, whereas the other ZEN metabolites induced: Loss of mitochondrial membrane potential (MMP) Mitochondrial changes in Bcl-2 and Bax proteins

It was observed that the parental toxin

Cytoplasmic release of cytochrome c

was less toxic, whilst ZEN derivatives

and apoptosis-inducing factor (AIF)

induced a significant decrease of the

ZEN METABOLITES

IL-8 synthesis in swine PMNs. It was concluded that ZEN and its derivatives may have divergent effects on important parameters of swine innate immunity, such Explaining the observed alterations in

as cell viability, IL-8 and superoxide anion synthesis. In another study by the same group

macrophages, it was concluded that the

with PBMC, 5 and 10 μM of ZEN and ZAN

activation of p53, JNK or p38 kinase by

significantly decreased the TNF-α synthesis in

ZEN metabolites is the main upstream

the supernatant from the PBMC cell culture, and

signal required for the mitochondrial

10 μM of ZAN decreased also the IL-8 synthesis,

alteration of Bcl-2 (anti-apoptotic)/Bax

while ZEN and its metabolites at concentrations

(pro-apoptotic) signaling pathways and

higher than 5 μM also induced a significant

intracellular reactive oxygen species (ROS)

decrease in IgG, IgA or IgM concentration.

generation, while mitochondrial membrane

107

potential loss and nuclear translocation of Further in vitro evaluation of the toxicity of α-ZEL and β-ZEL on RAW264.7 macrophages114 showed that β-ZEL had a stronger inhibitory effect

apoptosis-inducing factor are the critical downstream events for ZEN metabolitemediated apoptosis in macrophages.

on the viability of macrophages than α-ZEL.

196 17

FUMONISIN B FB toxicosis, depending on contamination level and time of

FUMONISIN B

exposure, could result in porcine

Cardiovascular effects & Pulmonary oedema

pulmonary oedema syndrome due

Liver & Kidney toxicity

to cardiovascular toxic effects, as well as increased sphinganine/ sphingosine (Sa/So) ratio in serum

Disruption of lipid synthesis pathways

and tissues, liver and kidney toxicity, delay in sexual maturity and reproductive functionality alterations, impairment of innate and acquired immune response, histological lesions in internal organs, as well as alterations of brain physiology.

Reproductive disorders

Due to a structural resemblance with ceramide, fumonisins competitively inhibit ceramide synthases (CerS), a group of key enzymes in the biosynthesis of ceramide and more complex sphingolipids, resulting in the disruption of the de novo synthesis of ceramide as well as sphingolipid metabolism and, as a consequence, alterations in lipid pathways115.

FBs, especially B1 (FB1) influence the inflammatory response21,116. A reduced expression of cytokines (IL-6, IL-1β, IL-12p40 and IL-8) in spleen and a significant upregulation of IL-1β, IL-6, IFN-γ, and TNF-α

INFLAMMATION

in the small intestine of piglets fed with contaminated diets [either DON (3 mg/kg) or FB (6 mg/kg), or both for 35 days] was reported117. Following ingestion of 2.8 μM FB1/kg body weight (37–44 mg FB1/kg feed), a decreased expression of most of the cytokines was found in the different parts of the intestine segments after 14 days of exposure118. Moreover, 8 mg FB1/kg feed decreased the gene expression of Th2 cytokines IL-4, IL-6 and IL-10 in blood of pigs116,119. Some of the changes in the mRNA expression of IL1α, IL1β, IL6, IL8, TNFα and MCP-1 induced by FB or other Fusarium toxins could be also cytotoxicity-related120.

197 18

As regards the intestinal

In pigs exposed to FB1 and

morphology and function, FBs

vaccinated against Aujeszky's

have been associated with21:

disease virus (Suid Herpesvirus 1 [SuHV1]), the humoral

Intestinal villous

immune response was greatly

fusion and atrophy

disturbed, with a strong decrease in observed antibodies122.

Decrease of transepithelial electrical resistance (TEER), globet cell density, occludin

Similarly, in vivo exposure (28

and E-cadherin expression

days) of weanling piglets to feed contaminated with 8 mg FB1/

INTESTINAL FUNCTION

translocation to other organs and proliferation of intestinal opportunistic bacteria Significant negative effects have been demonstrated on intestinal immune system (1ppm FB oral exposure for 10 days, followed by Escherichia coli challenge), showing reduced intestinal expression of IL-12p40, impaired function of intestinal antigen presenting cells (APC), decreased upregulation of Major Histocompatibility Complex Class II molecule (MHC-II) and reduced T cell stimulatory capacity upon stimulation121.

HUMORAL IMMUNE RESPONSE

Greater bacterial

kg significantly decreased the expression of IL-4 mRNA by porcine whole blood cells and

According to in vitro and in

vivo experiments, FB1 modifies the Th1/Th2 (T-helper 1/T-helper 2) cytokine balance in pigs similar to an impaired humoral response116,119, as well as influencing the inflammatory response. Incubation of swine alveolar macrophages with FB1 led to a significant reduction of the number of viable cells and cell death by apoptosis124.

diminished the specific antibody

An in vivo experiment on pigs

titer after vaccination against Mycoplasma agalactiae 116.

[either DON (3 mg/kg) or FB

In a similar study with FB1 and vaccination against

Mycoplasma agalactiae, significantly decreased specific antibody levels after vaccination as well as the mRNA expression level of IL-10 was demonstrated119.

(6 mg/kg), or both for 35 days] demonstrated that IL-8, IL-1β, IL-6 and macrophage inflammatory protein-1β were significantly decreased in the spleen of piglets exposed to multi-contaminated diet (DON and FB), whereas animals that received only FB-contaminated feed

Another study reported

demonstrated a significant

decreased expression of IL-8

decrease in mRNA encoding

in the gut of pigs following

for IL-1b and IL-6125.

the oral administration of 0.5 mg/kg FB1, although other cytokines were unaffected123.

198 19

AFLATOXINS

HEPATOTOXICITY Aflatoxins have hepatotoxic, carcinogenic, and immunotoxic

AFLATOXIN B1

IMMUNOTOXICITY

properties, impairing both the innate and the acquired immune responses126. Ingestion of aflatoxins (140 and 280 ppb for 4 weeks) resulted in a biphasic effect on total white blood cell number, thus a low dose of AF (140 ppb) decreased the

CARCINOGENICITY

total number of white blood cells, whereas the high dose (280 ppb) had the opposite effect, while decreased proinflammatory (IL-1β, TNF-alpha) and increased anti-inflammatory

stimulation in vitro, whereas their

Findings of another investigation131

(IL-10) cytokine mRNA expression

ability to phagocytose red blood

on the involvement of AFB1 in Swine

was also observed127.

cells was not compromised.

Influenza Virus (SIV) replication in vitro and in vivo, supported that AFB1 exposure aggravates SIV replication, inflammation and lung damage by activating TLR4-NFkB signaling.

In that study, a reduced

Granulocytic cells showed

immune response induced by

a reduction of chemotactic

Mycoplasma agalactiae in the 280-ppb-treated group was also observed. Additionally, regarding the effects of AFB1 on the inflammation process, in vitro exposure of swine alveolar macrophage to this toxin has been shown to result in a timeand dose-dependent decreased viability and phagocytic activity of primary cultures cells124.

response to chemoattractant

Evaluation of 25-days old piglets blood samples, born from sows that received AFs through feed during gestation and lactation, demonstrated reduction of lymphoproliferative response to mitogens and failure of monocyte-derived macrophages to efficiently produce superoxide

bacteria factor and casein128. AFB1 interferes with the development of acquired immunity in swine following vaccination against erysipelas with bacterin

A study on porcine splenocytes132 provided evidence of underlying mechanisms implicated in AFB1-induced immunosuppression.

preparation (a suspension of

In that study, AFB1 inhibited

killed bacteria) of E. rhusiopathiae

the production of IL-2 when

and increases the severity of

exposed to porcine splenocytes,

infection with E. rhusiopathiae129.

leading to immunotoxicity in

On the other hand, in a pig model vaccinated with ovalbumin (OVA), AFB1 exposure had no major effect on humoral immunity (concentrations of total IgA, IgG and IgM and specific anti-OVA IgG), but impaired lymphocyte activation was reported130.

a dose-dependent manner. Moreover, AFB1 decreased the level of reduced glutathione (GSH) and increased lipid peroxidation in porcine splenocytes, which is accompanied by increased phosphorylation of ERK1/2.

anions after oxidative burst

199 20

Therefore, it was concluded that AFB1 inhibits anti-CD3induced lymphocyte proliferation

Furthermore, AFB1 impairs cell-mediated immunity, probably through dysregulation of the antigenpresenting capacity of dendritic cells133.

and IL-2 production by the

On the other hand, exposure to AF increases the T-cell

oxidative stress mediated ERK1/2

proliferation-inducing capacity of porcine monocyte-derived

MAPK signaling pathway.

dendritic cells, thus enhances presenting capacity of cells134.

OCHRATOXIN A

NEPHROTOXICITY OTA is a major nephrotoxic agent, whereas it has also liver

OCHRATOXIN A

IMMUNOTOXICITY

toxicity properties, as well as immunotoxic, neurotoxic and teratogenic properties21. Disturbance in humoral immune response was reported in an in vivo study with pigs (500 μg OTA/kg feed for 3 months)122, since a strong decrease in antibody titer was observed after

LIVER TOXICITY TERATOGENICITY

NEUROTOXICITY

HUMORAL IMMUNE RESPONSE

immunization against Morbus Aujeszky (Pseudorabies). Gilts fed OTA-contaminated

Decreased number and phagocytic activity of macrophages

feed had135: Reduced cutaneous basophil hypersensitivity response to phytohemagglutinin Reduced delayed hypersensitivity to tuberculin

Spontaneous occurrence of dose-related clinical

Salmonella choleraesuis infection occurred in piglets fed 1 and 3 mg OTA/kg feed dietary136. In a further experiment by the same group, piglets were vaccinated against S. choleraesuis, OTA ingestion (1mg OTA / kg feed) lead to spontaneous Brachyspira hyodysenteriae and Campylobacter coli infections which were associated

Decreased stimulation index

with OTA immunosuppression, showing delayed response

for lymphoblastogenesis

to antigen and reduced humoral response136.

Decreased IL-2 production

On the contrary, in a previous study135, OTA (2.5 mg

when lymphocytes

of OTA/kg feed for 35 days) had no effect on total

were stimulated with

and specific immunoglobulin concentrations.

concanavalin A

200 21

OTA also affects

CYTOKINE EXPRESSION

cytokine expression. An experiment on weaned pigs that ingested an OTA contaminated diet (181 ng/g of feed) has shown an increased level of TNF-α and IL-10 in plasma, with a decreased capacity to respond with cytokine expression in an ex vivo challenge with lipopolysaccharides (LPS)137.

There is evidence mainly from in

OTA can induce altered expression of genes involved in cellular

vitro studies, for effects of OTA on neutrophils and macrophages including oxidative stress, apoptosis, phosphorylation of the ERK1/2 and release of TNFα via NF-kB pathways138.

growth/proliferation, cell death/survival, and immune function

In a study that investigated the

miRNAs are involved in the MAPK signaling pathways138.

in the kidney, as well as altered expression of molecules involved in immune response and antioxidant self-defense in pig gut141,142. OTA can also modulate the expression of microRNAs, in kidney cells in vivo and in vitro, whilst many of the altered

toxicity of Penicillium mycotoxins on mitogen--induced lymphocyte

OTA also induces the phosphorylation of P38

proliferation, it was reported

and ERK1/2 in porcine splenocytes, leading to

that OTA was the most potent

nephrotoxicity and immunotoxicity, respectively143.

(50% inhibition at 1.3 mM) cell proliferation inhibitor139. Moreover, it has been reported that OTA induces the phosphorylation of P38 and ERK1/2 in porcine

According to proteomic approaches, enhanced expression of mitochondrial proteins involved in electron transport, protein synthesis, stress response and cell death and modulation of proteins involved in inflammation are factors related to OTA toxicity144,145.

alveolar macrophages, causing

Moreover, a decrease in TCR-induced T lymphocyte

immunotoxicity through an

viabilities in peripheral blood lymphocytes and

increase in Toll-like receptor 4

splenocytes (400, 800 μg/kg diet) was reported in

(TLR4)-mediated inflammatory

pigs146. Thus, it was suggested that nephrotoxicity

signaling pathway proteins

and immunotoxicity of OTA may involve ER stress,

and elevated intracellular

activation of MAPK signaling and autophagy143,146.

ROS production . 140

201 22

Concluding remarks and practical notes Fungi are proposed to be the

Such disruption in vaccine

greatest threat to animal and

immunity may lead to the

plant health among all the

occurrence of disease even in

taxonomic classes of pathogens147.

properly vaccinated groups. Such cases are of utmost importance

Research up to today has provided

when investigating effectiveness

clear evidence that mycotoxins

of on-farm vaccination programs.

affect the immune system of pigs. Furthermore, cases of feed The intestine is undoubtedly

contamination with mycotoxin

the key link between ingested

mixtures should be accounted for

mycotoxins and detrimental

as more probable than

effects on the animal.

contamination with only one

Negative effects of mycotoxins on the intestine (e.g. reduced

mycotoxin under field conditions. Effects of mycotoxin mixtures

barrier integrity) and immune

on the immune system of pigs

system mean that they can

have not been fully clarified yet.

play a critical role in the initiation, progression and duration of intestinal (and systemic) infections. Therefore, compromising the integrity of the intestine will also increase the likelihood of microbes or microbial

As regards the reduction of lymphocyte proliferation in in vivo studies, additivity has been suggested after co-exposure to AF and FB or OTA and T-2 toxin, and synergism after co-exposure to FB and DON151.

To further understand the complexity of interactions, in vivo co-exposure to FB and DON resulted in synergistic interaction on lymphocytes proliferation upon mitogenic stimulation, additive interaction on cytokines expression (IL-8; IL-1b, IL-6 and macrophage inflammatory protein 1b) and antagonistic interaction on levels of specific IgA and cytokine expression125.

products, or mycotoxins, entering circulation and inducing systemic disease148-150. The broad immunosuppressive effects of mycotoxins may decrease host resistance to infectious diseases20, whereas, vaccine immune response is also altered at mycotoxin doses that do not alter the global immune response75,116,130.

When such observations are combined with various immunomodulating properties of mycotoxins, it becomes obvious that vaccine responses, susceptibility to infections and use of antimicrobials, as well as productivity and financial outcome can be altered due to mycotoxins ingestion, under field conditions. Such issues should be considered when assessing the necessity of a regular preventive program for mycotoxins on farms.

202 23

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136. Stoev, S.D.; Goundasheva, D.; Mirtcheva, T.; Mantle, P.G. Susceptibility to secondary bacterial infections in growing pigs as an early response in ochratoxicosis. Experim. Toxicol. Pathol. 2000, 52, 287-296. doi: 10.1016/s0940-2993(00)80049-4. 137. Bernardini, C.; Grilli, E.; Duvigneau, J.C.; Zannoni, A.; Tugnoli, B.; Gentilini, F.; Bertuzzi, T.; Spinozzi, S.; Camborata, S.; Bacci, ML.; Piva, A.; Forni, M. Cellular stress marker alteration and inflammatory response in pigs fed with an ochratoxin contaminated diet. Res Vet Sci. 2014, 97, 244-50. 138. EFSA CONTAM Panel (EFSA Panel on Contaminants in the Food Chain), Schrenk, D.; Bodin, L.; Chipman, J.K.; del Mazo, J.; et al.. Scientific Opinion on the risk assessment of ochratoxin A in food. EFSA J. 2020, 18, 6113, 150 pp. https://doi.org/10.2903/ j.efsa.2020.6113. 139. Keblys, M.; Bernhoft, A.; Höfer, C.C.; Morrison, E.; Larsen, H.J.; Flåøyen, A. The effects of the Penicillium mycotoxins citrinin, cyclopiazonic acid, ochratoxin A, patulin, penicillic acid, and roquefortine C on in vitro proliferation of porcine lymphocytes. Mycopathologia. 2004, 158, 317-24. doi: 10.1007/s11046-005-5523-8. 140. Xu, H., Hao, S., Gan, F., Wang, H., Xu, J., Liu, D., Huang, K.,. In vitro immune toxicity of ochratoxin A in porcine alveolar macrophages: a role for the ROS-relative TLR4/MyD88 signaling pathway. Chem. Biol. Interact. 2017, 272, 107–116. 141. Marin, D.E.; Braicu, C.; Gras, M.A.; Pistol, G.C.; Petric, R.C.; Berindan Neagoe, I.; Palade, M.; Taranu, I. Low level of ochratoxin A affects genome-wide expression in kidney of pig. Toxicon, 2017a, 136, 67–77. 142. Marin, D.E.; Pistol, G.C.; Gras, M.A.; Palade, M.L.; Taranu, I. Comparative effect of ochratoxin A on inflammation and oxidative stress parameters in gut and kidney of piglets. Regulatory Toxicol. and Pharmacol. 2017b, 89, 224–231. 143. Gan, F.; Zhou, Y.J.; Hou, L.L.; Qian, G.; Chen, X.X.; Huang, K.H. Ochratoxin A induces nephrotoxicity and immunotoxicity through different MAPK signaling pathways in PK15 cells and porcine primary splenocytes. Chemosphere. 2017a, 182, 630–637. 144. Ferrante, M.C.; Bilancione, M.; Raso, G.M.; Esposito, E.; Iacono, A.; Zaccaroni, A.; Meli, R. Expression of COX-2 and hsp72 in peritoneal macrophages after an acute ochratoxin A treatment in mice. Life Sci. 2006, 79, 1242– 1247. 145. Shen, X.L.; Zhang, Y.; Xu, W.; Liang, R.; Zheng, J.; Luo, Y.; Wang, Y.; Huang, K. An iTRAQ-based mitoproteomics approach for profiling the nephrotoxicity mechanisms of ochratoxin A in HEK 293 cells. J. Proteomics, 2013, 78, 398–415. https://doi.org/10.1016/j. jprot.2012.10.010. 146. Gan F, Hou LL, Zhou YJ, Liu YH, Huang D, Chen XX and Huang KH, 2017b. Effects of ochratoxin A on ER stress, MAPK signaling pathway and autophagy of kidney and spleen in pigs. Environm. Toxicol. 2017b, 32, 2277–2286. 147. Fisher, M.; Henk, D.; Briggs, C.; Brownstein, J.S.; Madoff, L.C.;, McCraw, S.L.; Gurr, S.J. Emerging fungal threats to animal, plant and ecosystem health. Nature 2012, 484, 186–194. https://doi.org/10.1038/nature10947. 148. Basso, K.; Gomes, F.; Bracarense, A.P.L. Deoxynivanelol and fumonisin, alone or in combination, induce changes on intestinal junction complexes and in e-cadherin expression. Toxins 2013, 5, 2341–52. 149. Pinton, P.; Nougayrède, J-P.; Del Rio, J-C.; Moreno, C.; Marin, D.E.; Ferrier, L.; Bracarense, A.P.; Kolf-Clauw, M.; Oswald, I.P. The food contaminant deoxynivalenol, decreases intestinal barrier permeability and reduces claudin expression. Toxicol Appl Pharmacol. 2009, 237, 41–8. 150. Bracarense, A.F.L.; Lucioli, J.; Grenier, B.; Pacheco, G.D.; Moll, W.; Schatzmayr, G.; Oswald, I.P. Chronic ingestion of deoxynivalenol and fumonisin , alone or in interaction, induces morphological and immunological changes in the intestine of piglets. Br. J. Nutr., 2012, 107, 1776–86. doi:10.1017/S0007114511004946. 151. Grenier, B.; Oswald, I.P. Mycotoxin co-contamination of foods and feeds: metaanalysis of publications describing toxicological interactions. World Mycotoxin J 2011, 4, 285-313.

209 30

MYCOTOXINS AND DISRUPTION of vaccination efficacy IN SWINE

Assist. Prof. Panagiotis Tassis Assistant Professor of Swine Medicine and Reproduction, Clinic of Farm Animals, School of Veterinary Medicine, Aristotle University of Thessaloníki, Greece

210 1

Pig farm vaccination programs are a major

Therefore, proper vaccine selection

preventive tool for a wide range of diseases and

and proper implementation are

syndromes affecting swine. They have colossal importance in terms of herd health and productivity, as well as from a financial standpoint.

the basis for the construction of a herd immune status that will counteract antigenic pressure during different productive stages.

As already discussed in the respective literature, mycotoxins seem to play an important role in disrupting this

EFFECTS OF MYCOTOXINS ON THE SWINE IMMUNE SYSTEM

major preventive health tool1. In our previous technical article regarding the effects of major mycotoxins on the immune system of swine and

↓VACCINATION EFFICACY

cellular and molecular mechanisms involved, it had been reported that the health and economic impact of mycotoxins on the immune defense system of pigs is significant. Three major outcomes of these effects on the swine immune system, herd health and productivity have been described2: Increased susceptibility to infectious diseases

↑ SUSCEPTIBILITY TO INFECTIOUS DISEASES

REACTIVATION OF CHRONIC INFECTIONS

Reactivation of chronic infections Decreased vaccination efficacy The present article will focus on the main mycotoxins affecting swine and extensively contaminate crops worldwide3. The current knowledge on the effects of aflatoxins (AFs), fumonisins (FBs and mainly FB1) deoxynivalenol (DON), zearalenone (ZEN), ochratoxin A (OTA) and T-2 toxin, on the immune response after sensitization or vaccination in swine will be presented. Emphasis will be put on studies with pigs and vaccines against swine pathogens.

211 2

Swine vaccines and vaccinal immunity The use of veterinary vaccines in swine production is a disease prevention tool that has been used

Porcine Reproductive and Respiratory Syndrome (PRRS) Porcine Circovirus 2 -associated diseases (PCV2-AD)

by swine farmers worldwide in a

Aujeszky’s disease (PRV)

variety of production systems.

Parvovirus infection (PPV)

It is still implemented in every conventional pig production system. In the past few decades, facts in the field of novel vaccine production have changed rapidly. The scientific field of swine vaccine

Swine influenza (SIV) Enzootic pneumonia (Mycoplasma hyopneumoniae) Pleuropneumonia (App – Actinobacillus Pleuropneumoniae) Glasser’s disease (Haemophilus parasuis) Atrophic Rhinitis (Pasteurella multocida ± Bordetella bronchiseptica)

development is a rapidly evolving

Erysipelas (Erysipelothrix rhusiopathiae)

research and innovation field.

Leptospirosis (Leptospira spp.)

Major swine diseases that can be prevented or controlled at field level with the use of commercially available vaccines, as part of a veterinary health management programme, include:

Escherichia coli infections Clostridium spp. infections Ileitis (Lawsonia intracellularis) Classical Swine Fever

Salmonella spp. and others

In cases such as the recently introduced in the European region African Swine Fever, there aren’t any commercial vaccines available so far. However respective research and development efforts are under way4.

212

Apart from typical intramuscular vaccination

Swine vaccines are usually either “dead”

against one pathogen, innovations of vaccine

(inactivated) or “live” (attenuated) and

technology in the past decades have resulted

can be used in different production stages

in the production of intradermal vaccines,

in the breeding stock and/or in suckling,

intranasal vaccines, as well as vaccines against

weaned or growing pigs, depending on

more than one pathogen, reaching up to three

the vaccine, its pathogenic target and

swine pathogens in one vaccine up today.

suggested administration programme.

ADMINISTRATION ROUTE Intramuscular Intradermal Intranasal

NUMBER OF PATHOGENS 1 2 3

VACCINE CLASSIFICATION TYPE OF VACCINE Inactivated (“dead”) Attenuated (“live”)

PRODUCTION STAGE Breeding stock Suckling piglets Weaned piglets Growing pigs

Vaccination success will result in a timely and appropriate humoral response to counteract the specific pathogen in the most susceptible population and production stage at an acceptable extent.

2134

Vaccine success includes: Reduction of infected animals and susceptibility to infection against a specific pathogen Reduced animals as pathogencarriers (reduced pathogen spreading in terms of time and microbial load) Improved herd immunity levels

However, limited attention has been given to the impact of mycotoxin contaminated feed as a reason for vaccine failure under field conditions.

Vaccine failure is an issue of great concern due to the severe health and productivity impact on farms. Many reasons for such failure have been described so far, including environmental and management reasons, such as:

Nevertheless, recent research data demonstrates the ability of a number of mycotoxins to affect vaccine-induced humoral response with significant impact on health and productivity results.

Improper vaccine storage, handling and administration Incorrect timing of vaccination and others.

214 5

Effects of Trichothecenes on vaccinal immunity DON H

DON immunostimulatory

O OH

O HO

or immunosuppressing

DON

effects depend on the dose, frequency and duration of exposure5.

Apoptosis of lymphocytes

OH HO

O HO

It has been suggested that high doses of DON (greater than 10 μM), cause apoptosis of lymphocytes, resulting in immunosuppression, increased susceptibility to infection, reactivation of latent infections and reduced vaccine efficiency . 6

↓ Vaccine efficacy

Immunosuppression ↑ Susceptibility to infections

Reactivation of latent infections

After DON exposure, inhibition of immune response has been demonstrated after porcine parvovirus vaccination in rats8. Further three studies using ovalbumin (OVA) immunization suggested that DON affects anti-OVA

According to a previous review7, trichothecenes may generate an “immune evasion” environment that allows pathogens to escape host and vaccine immune defenses.

immunoglobulins response9-11. 1. In the first study (2.2–2.5 mg DON/kg feed, weaned pigs for 9 weeks), DON increased OVA-specific IgA and IgG, whilst

2. In a second study, 3.5 times greater

a biphasic effect of the toxin

levels of anti-OVA IgG titers in

on lymphocyte proliferation

comparison to control animals (3.5 mg

after antigen stimulation

DON/kg feed for 42 days) two weeks

(upregulation on 21st day post

after the first OVA immunization (day

exposure and down-regulation on

7 of the study) were demonstrated.

35th to 49th day post exposure) were reported, along with lower expression of both TGF-β and IFN-γ mRNA expression levels9.

215 6

3. In the latter study, seven days

Moreover, after feeding 1.8

after a second OVA immunization

or 4.7 mg DON/kg feed (pigs

(day 21 of the study) anti-OVA

over 25.3 kg body weight at the

IgG levels were similar

start of the trial) a significant

between groups (DON-fed

dose-dependent reduction in

animals vs. control animals).

secondary antibody response to

Anti-OVA IgA levels in that study were similar between the two

tetanus toxoid was present when compared to the control group14.

Quite similarly in the study of Gutzwiller et al.16, pigs were fed 3.2 mg DON and 0.06 mg ZEN, or 2.1 mg DON and 0.25 mg ZEN/ kg diet and received PPV vaccination (one-tenth of the recommended dose). In that study mycotoxin exposure

trial groups up to one week

Furthermore, after feeding a

did not affect antibody production,

prior to the end of the study

mixture of 1.0 mg DON /kg and

probably due to administration of

period, and then reduced in

250μg ZEN/kg contaminated

low vaccine dose and the short

the DON-treated animals .

feed and PRV double vaccination,

time interval between vaccination

PRV antibody titers were

and antibodies determination.

According to a recent study11 in

significantly decreased 14 days

which pigs were immunized with

after booster vaccination15.

OVA, PRV, swine fever and porcine circoviruses vaccines, ingestion of feed contaminated with 1.0 and 3.0 mg DON /kg feed reduced the concentration of serum porcine circoviruses antibody titer in pigs, whereas serum OVA antibody titer levels were not affected. Nevertheless, it should be mentioned that inhibition of IFN-γ (as suggested previously9) and Toll-like Receptors (TLR) expression, assists pathogens escaping host and vaccine immune defenses12. In a 28-day feeding study with 0.15, or 1.5, or 3 mg DON/kg feed13 and subsequent immunization with sheep red blood cells, delayed peak titers were observed (one week later) in DON-fed animals, when compared with control animals.

216 7

According to a series of studies

In an in vitro study (permissive

Moreover, in another study with

on the effects of DON on the

cells infected with PRRSV were

pigs, DON increased the severity

immune response against PRRS

treated with 140–280 ng/

of the viral infection in the

and PCV2 viruses, very interesting

ml DON) by the same research

presence of porcine circovirus

group , a similar observation was

type 2 (PCV2) virus20.

findings have been presented

17-20

Major findings included that ingestion of DON contaminated

reported i.e. replication of PRRSV was significantly inhibited.

Results showed that viremia and lung viral load tended

feed can decrease the immune

to be higher in animals

response against PRRSV

ingesting DON contaminated

and influence the course of PRRSV infection in pigs. In the study of Savard et al.17, piglets received DON- naturally DON vs PRRS & PCV2 VACCINATION

contaminated diets (2.5 and 3.5 mg/kg) and were then inoculated with PRRSV.

In vivo effects of DON ingestion supported a negative effect of the toxin on PRRSV-specific humoral responses (DON at 2.5 mg/kg significantly decreased PRRSV specific humoral responses), as well as amplification of PRRSVattributed negative effects such as those on weight gain, lung lesions, and mortality. However, such an effect did not associate with a significant increase in viral replication, since DON ingestion resulted in a decrease of PPRSV replication.

diet at 2.5 mg/kg (pigs were

In the latter study it was demonstrated that the reduction

inoculated with PCV2b virus).

of viral replication could be

However, DON had no

attributed to a DON-induced

significant effect on clinical

pro-inflammatory cytokine

manifestation of PCV2-AD.

environment that promoted activation of apoptosis, which is an

Authors of the latter study

important host defense mechanism,

supported that DON has neither in vitro nor in vivo clear potentiating effects in the development of PCV2 infection despite slight increases in viral replication.

as it interrupts viral replication and eliminates virus-infected cells12,21.

Furthermore, it has been proved that DON can decrease the replication of the attenuated PRRSV vaccine strain in

Nevertheless, it can be

vaccinated pigs and their antibody

concluded that DON exposure

response to the vaccine .

may hamper the acquisition

of vaccine-induced protective Such significant finding is

immune responses.

consistent with results from a study with DON-exposed mice vaccinated with inactivated PPV, which demonstrated disruption of the immune response to the vaccine through modulation of specific cytokines and chemokines22.

217 8

T-2 TOXIN

T-2 toxin is reported to

Pigs fed 1.324 or 2.102

be immunotoxic, through

mg T-2 toxin/kg exhibited

its cytotoxic, apoptotic

reduced anti-OVA antibody

or immunosuppressive

production on day 21 without

attributes .

significant alteration to specific

lymphocyte proliferation26. A study with necrotic enteritis B (NEB) vaccination of pigs that received 5 mg T-2/kg feed, resulted in significantly reduced NEB antibody

Apoptosis

levels in T-2-exposed animals24. Immunosuppression due to T-2 has

Cytotoxicity

been also observed in another feeding

Immunosuppression

study with pigs, (0.5-3.0 mg T-2/ kg feed), in which animals were immunized with horse globulin.

TOXIN T-2

Results suggested reduction of anti-horse globulin antibodies synthesis, whereas a dose

IMMUNOTOXICITY

dependent depletion of lymphoid elements in the thymus and spleen, was also reported25. After OVA immunization, subclinical doses of T-2 toxin induced an early and transient increase of total IgA plasma concentration but a decrease in the anti-OVA IgG titer.

218 9

Effects of Fumonisins on vaccinal immunity

FUMONISIN B1

Disruption of sphingolipid biosynthesis FBs competitively inhibit ceramide synthases (CerS), a group of key enzymes in the biosynthesis of ceramide and more complex sphingolipids, resulting in the

Accumulation of sphinganine & sphingosine

disruption of sphingolipid metabolism, whilst they have

Ceramide synthase

been also linked with impairment of innate and acquired immune response, including reduction of specific antibody response during vaccination27,28.

level of IL-10 were observed

Alterations were statistically

in a sex-related manner, thus

significant for animals

Previous studies have supported

proving the immunosuppressive

receiving FB-contaminated

that FB1 modifies the Th1/Th2

effects of the toxin.

feed, and more pronounced in animals that recived

(T-helper 1/T-helper 2) cytokine balance in pigs similar to an impaired humoral response27,29.

Such differences in the

the combined mycotoxins-

specific immune response

contaminated diet.

were observed only in male

In vivo exposure (28 days) of weanling piglets to feed contaminated with 8 mg FB1/ kg significantly decreased the expression of IL-4 mRNA (IL-4 is a Th2 cytokine involved in the humoral response) by porcine whole blood cells and diminished the specific antibody titer after vaccination against Mycoplasma agalactiae27.

pigs, but not female ones29. An increase of specific IgA was In a study by Grenier et al.30, pigs received a diet contaminated with

reported for animals receiving DON-contaminated diet, but not

either DON (3 mg/kg) or FB (6 mg/

for those that received combined

kg) or both toxins and immunized

DON and FB, possibly due to FB

twice with OVA.

interference at the intestinal level

Ingestion of diets contaminated with DON or FB individually

through its action on sphingolipids. At the same time, reduced

or in combination altered

lymphocyte proliferation upon

In a quite similar study with FB1

immunoglobulins production

OVA stimulation was demonstrated

(8mg FB1/kg feed), significantly

after OVA immunization

in the animals receiving any of the

decreased specific antibody

and reduced anti-OVA IgG

three contaminated diets (DON,

levels after vaccination against

plasma concentration.

FB, or combined DON and FB).

Mycoplasma agalactiae, as well as the mRNA expression

219 10

The humoral immune response

Both, F4-specific IgM and IgA

Quite similarly, pigs fed low

was significantly disturbed, with a

antibody secreting cells were

levels of FB-contaminated feed

strong decrease in antibodies levels

reduced after FB1-exposure

(2 mg FB1/kg contaminated

at days 21 and 35 after vaccination,

and the authors suggested

culture material/day for 5

in pigs exposed to 0.5 mg OTA/

that FB1 could interfere with

weeks) and vaccinated with PRV

kg feed and/or 10 mg FB1/kg feed

the induction phase of the

vaccine showed absence of an

for three months, and vaccinated

immune response through

FB-attributed significant effect

against Aujeszky’s disease (Suid

reduction of in vivo antigen

on PRV antibody titers34.

Herpesvirus 1 [SuHV1]) .

presenting cells maturation.

That antibody disruption

On the other hand, few particular

was detected in animals

studies have shown the absence of

fed both mycotoxins, either

FB-attributed significant effects on

alone or in combination.

immune response after vaccination. Nevertheless, the majority

In another study with piglets that

Exposure of piglets to FB1-

were orally exposed to a low dose of FB1 (1 mg FB1/kg body weight)

contaminated feed for up to 4 months (1, 5, and 10 mg FB1/kg feed) did

immunosuppressive properties,

for 10 days, a longer shedding of

not affect significantly their antibody

alters the cytokine profile

F4(+) enterotoxigenic Escherichia

titers against Aujeszky’s disease33.

and reduces the specific

coli (ETEC) following infection and lower induction of the antigenspecific immune response following oral immunization, were presented32.

of studies and respective findings support that FB1 has

antibody response built during a vaccination protocol.

220 11

Effects of Zearalenone on vaccinal immunity

ZEN

ZEN has significant estrogenic

(intraperitoneally), revealed that ZEN, with or without

potency in swine but has also been

immune challenge, can decrease immunoglobulins

suggested as an immunotoxic

in serum and cytokines in lymphoid organs36.

compound35. A study with pigs that received ZEN (dietary Few studies have investigated ZEN

levels of 1.1 to 3.2 mg/kg feed for 18 days) and

effects on humoral immune

a swine fever live vaccine, demonstrated that

response after vaccination with

specific antibody titers in the group treated with

a commercial vaccine or other

ZEN (2.0 and 3.2 mg/kg) were significantly lower

type of immunization in pigs.

18 days after immunization in comparison with the control group, in a dose-dependent manner.

Nevertheless, the effect of estrogens on the immune system

Levels of IgM showed a trend of decreasing

have received attention due to

linearly with increased levels of ZEN, indicating

their immunomodulatory activity

that ZEN (3.2 mg/ kg) inhibited humoral

on cell-mediated responses

immunity in piglets, whilst it was also suggested

and antibody production .

that ZEN may affect protein metabolism37.

A study performed with ZEN in rats

Additionally, it has been discussed that effects

(mycotoxin administered via gavage

of ZEN on humoral immune response could be

at dosages of 0, 1, 5, and 30 mg/

related to receptor-specific effects, since ZEN is

kg for 36 days) and subsequent

an agonist toward estrogen receptors α (ERα) and a

inactivated PPV vaccine administration

mixed agonist-antagonist of ERβ, with possible full antagonism of the ERβ expressed in B cells35.

221 12

Effects of Aflatoxins on vaccinal immunity

For more than half century,

Previous studies with pigs treated with

the detrimental effects of

AFs showed contradicting results.

AFLATOXIN B1

AFs on vaccinal response have been reported. It has been shown that AFB1 interferes with the development of acquired immunity in swine following erysipelas vaccination with bacterin preparation (a suspension of killed bacteria) of E. rhusiopathiae and increases the severity of infection with E. rhusiopathiae38. In a previous study that included

Joens et al.40 reported significantly lower hemagglutination titers against Treponema hyodysenteriae (name used at present: Brachyspira hyodysenteriae as swine dysentery causative agent), whilst AF ingestion did not alter humoral response of weanling pigs to sheep red blood cells41 or to Erysipelothrix rhusiopathiae42 in other studies.

In that later study, authors supported that AFB1 exposure does not result in significant modulation of the humoral immune response, whilst it can induce IgA increase but not at statistically significant levels.

ingestion of low doses of AFs (140 and 280 ppb for 4 weeks) a tendency

Moreover, after immunization of

towards reduced immune response

pigs with OVA and concurrent

Findings of another investigation in

against Mycoplasma agalactiae

AFB1 exposure (385 μg AFB1/

mice44 on the involvement of AFB1

(280-ppb-treated group)

kg feed; 867 μg AFB1/kg feed, or

in Swine Influenza Virus (SIV)

was observed39.

1807 μg AFB1/kg feed) absence of

replication in vitro and in vivo,

major effect on humoral immunity

supported that 10–40μg/kg of AFB1

(concentrations of total IgA, IgG

in vivo promotes SIV replication, inflammation and lung damage by activating TLR4-NFkB signaling.

and IgM and specific anti-OVA IgG), but impaired lymphocyte activation was reported43.

222 13

Effects of Ochratoxin A on vaccinal immunity

OCHRATOXIN A

OTA has a a well described signi icant nephrotoxic mode of action in swine, whilst it has been suggested as a compound that can affect immune response in swine.

As reported in the FB section, alterations in humoral immune response were reported also in an in

It has been reported that immunosuppression is the first expressed toxic effect of OTA that may become evident clinically

vivo study with pigs (500 μg OTA/ kg feed for 3 months with or without 10 mg FB1/ kg feed), in which a strong decrease in antibody titer was observed after immunization against Morbus Aujesky (PRV)31.

before nephropathy45.

A possible synergistic action of OTA and FB1 on immunosuppression in pigs could be discussed. In a study with 1 OTA/kg feed provided to swine for up to three weeks, animals were immunized against Salmonella choleraesuis haemorrhagic diarrhea45. Results proved OTA-attributed immunosuppression (reduced mean antibody titer on day 21 post immunization) and delayed response to immunization. Moreover, increased susceptibility to infectious agents (Brachyspira

hyodysenteriae and Campylobacter coli infections) was observed.

223

Remarks and conclusions as regards field conditions Taken together, a large number of studies have demonstrated the

Taking into account that a farm vaccination programme is of colossal importance in terms

negative effects of the previously

of disease prevention, the effects of mycotoxins

mentioned mycotoxins on the humoral

should be taken into consideration.

response after sensitization or vaccination. A vast majority of signifficant mycotoxins for swine have shown potential to induce a clear negative effect on immune response against various swine pathogens after vaccination in pigs.

However, it is important to remember that, under field

At the field level, diagnostic investigation of reduced vaccine efficacy cases could include feed mycotoxicological analysis, particularly when reduced vaccine efficacy is correlated with other clinical signs of mycotoxicosis or has occurred at a subsequent time interval after alterations in feed raw materials or feed production.

It should be highlighted that the presence of mycotoxins in the feed may lead to a breakdown in vaccinal immunity and to the occurrence of disease even in properly vaccinated flocks2.

conditions, such mycotoxins concurrently contaminate pig feed. Therefore, the immune system of pigs receives pressure from more than one mycotoxin, that could result in various interactions as regards immune response after vaccination.

224 15

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41. van Heugten, E., Spears, J.W., Coffey, M.T., Kegley, E.B., Qureshi, M.A. The effect of methionine and aflatoxin on immune function in weanling pigs. J. Anim. Sci. 1994, 72, 658–664. 42. Pananagala, V.S., Giambrone, J.J., Diener, U.L., Davis, N.D., Hoerr, F.J., Mitra, A., Schultz, R.D., Wilt, G.R. Effects of aflatoxin on the growth performance and immune responses of weanling swine. Am. J. Vet. Res. 1986, 47, 2062–2067. 43. Meissonnier, G.M.; Pinton, P.; Laffitte, J.; Cossalter, A.M.; Gong, Y.Y.; Wild, C.P.; Bertin, G.; Galtier, P.; Oswald, I.P. Immunotoxicity of aflatoxin B1: impairment of the cell-mediated response to vaccine antigen and modulation of cytokine expression. Toxicol Appl Pharmacol 2008b, 231, 142-149. 44. Sun, Y.; Su, J.; Liu, Z.; Liu, D.; Gan, F.; Chen, X.; Huang, K. Aflatoxin B1 Promotes Influenza Replication and Increases Virus Related Lung Damage via Activation of TLR4 Signaling. Front Immunol. 2018, 9, 2297. doi: 10.3389/fimmu.2018.02297. 45. Stoev, S.D.; Goundasheva, D.; Mirtcheva, T.; Mantle, P.G. Susceptibility to secondary bacterial infections in growing pigs as an early response in ochratoxicosis. Experim. Toxicol. Pathol. 2000, 52, 287-296. doi: 10.1016/s0940-2993(00)80049-4.

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MYCOTOXINS AND DAIRY CATTLE

María Rodríguez-Blanco, Sonia Marín, Vicente Sanchis, Antonio J. Ramos Applied Mycology Unit, Department of Food Technology, ETSEA-University of Lleida, UTPV-XaRTA, Agrotecnio, Spain antonio.ramos@udl.cat

228 1

Mycotoxins are low molecular weight secondary metabolites produced by certain genera of filamentous fungi under environmental conditions that are favourable for their synthesis (Bennett and Klich 2003).

Contamination of raw materials used for the formulation of feed with mycotoxins is a worldwide problem that causes significant economic losses. Furthermore, the intake of contaminated feed may lead to acute or chronic intoxication in the animals and may also contribute to its consumption

The main mycotoxins that can be

by humans, due to the possible

found contaminating feed and feed

transfer of these compounds to

materials are aflatoxins (AFs),

animal products such as milk,

deoxynivalenol (DON), fumonisins

meat or eggs (Fink-Gremmels

(FBs), ochratoxin A (OTA), T-2

2008a; Pinotti et al. 2016).

toxin and zearalenone (ZEN).

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Toxic effects of mycotoxins in dairy cows Ruminants are considered to be relatively resistant to mycotoxins, as ruminal microorganisms are able to degrade these compounds to less toxic, or even biologically inactive, compounds at normal exposure levels (Fink-Gremmels 2008b). However, it should be noted that the rumen’s degradation capacity can become saturated or affected by changes in the diet or as a result of metabolic diseases (Fink-Gremmels 2008b). Therefore, consumption of feed contaminated with these compounds can affect the health status of dairy cows. Some of the negative effects caused by mycotoxins in cattle are listed in Table 1.

Table 1. Main toxic effects in dairy cows derived from the consumption of feed contaminated with AFs, FBs, ZEN and DON.

MYCOTOXIN

TOXIC EFFECTS

AFLATOXINS

Impaired liver functions, reduced feed intake and milk production (Fink-Gremmels 2008b), decreased feed efficiency, immunosuppression, reduced reproductive performance (CAST 2003).

FUMONISINS

Reduced feed intake and milk production, mild hepatocellular lesions, immunocytotoxicity (Fink-Gremmels 2008b).

ZEARALENONE

Reproductive problems, infertility, reduced milk production and hyperestrogenism (Kallela and Ettala 1984).

DEOXYNIVALENOL

Reduced food intake and milk production (Jouany and Diaz 2005).

Liver alterations • AFs • FBs (mild hepatocellular lesions)

Immunological alterations • AFs (immunosuppression) • FBs (immunotoxicity)

Reproductive problems • AFs • ZEN (infertility and hyperestrogenism) Reduced feed intake • AFs • FBs • ZEN • DON

Reduced milk production • AFs • FBs • ZEN • DON

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Mycotoxins in feed for dairy cattle Dairy cows need fiber, proteins,

Among the materials included in

Silage is an important part of the

water, vitamins and minerals as

the formulation of dairy rations,

diet of dairy cows, as it usually

fundamental nutrients in daily

energy-rich components represent

represents a high percentage of the

diets. Furthermore, it is necessary

the main potential source of

final ration. These materials can be

to include a sufficient amount of

mycotoxins.

contaminated in the field, in post-

forage in their feed to maintain a functional ruminal microbiota. In addition, high amounts of energyrich components are needed, as they are essential to achieve high milk production and maintain the animal’s weight (Gonçalves et al.

2015). The great variety and variability of ingredients used in diets increases the risk of exposure to a wide range of different mycotoxins.

AFs, FBs, OTA, trichothecenes, and ergot alkaloids have been found contaminating some of these components such as cereals, soybeans, peanuts, or cottonseed. Fodder is the second source of mycotoxins, and preserved feeds such as silage, hay and straw are the third (Fink-Gremmels,

2008a).

harvest stages, as well as during storage.

Aspergillus, Fusarium, Alternaria and Penicillium are some of the filamentous fungi frequently found contaminating silage, so mycotoxins such as AFs, FBs, ZEN, trichothecenes, mycophenolic acid and roquefortine C can be detected in this raw material (Storm et al. 2008; Driehuis et al. 2008; Schmidt et al. 2015).

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Mycotoxin carry-over to milk In addition to the direct effects on animal health, one of the main problems associated with the presence of mycotoxins in animal feed is their possible carry-over to products derived from animals, such as milk.

AFM1 is detected in milk approximately 6 hours after consumption of the contaminated feed. Peaks of toxin can be detected 24 and 48 hours later if the intake of the feed continues and it disappears almost completely 72 hours after the withdrawal of the contaminated feed (Rodrigues 2014).

When dairy cows consume feed contaminated with aflatoxin B1 (AFB1), a part is broken down in the rumen to aflatoxicol, and another part reaches the liver where it is metabolized by liver enzymes through hydroxylation, hydration, demethylation and epoxidation.

Presence of AFM1 in milk after consumption of contaminated feed 6 hours

24 hours

48 hours

72 hours

The hydroxylation of AFB1 results in aflatoxin M1 (AFM1) and some of this compound is eventually excreted through the milk (Dhanasekaran et

First detection

Detection of peaks if intake continues

al. 2011).

Complete disappearence after removal of the contaminated feed

Liver metabolism

Hydroxylation

AFB1

Excretion of AFM1 in milk

232 5

The transfer rate from AFB1 in feed to AFM1 in milk can vary from 1-2% in low yielding cows to 6% in high yielding cows (Britzi et al. 2013; RodríguezBlanco et al. 2019).

Carry-over rate from AFB1 in feed to AFM1 in milk

1-2 %

Low yielding cows

High yielding cows

FACTORS THAT MAY AFFECT THE CARRY-OVER RATE

Lactation day (Veldman et al.

1992) Species Health status Ingestion and digestion rate Liver biotransformation capacity Integrity of the mammary alveolar cell membranes (FinkGremmels 2008a; Britzi et al. 2013)

233 6

Monitoring AFM1 in milk has increased, especially since this toxin was classified as a human carcinogen (Group 1) by the International Agency for Research on Cancer (IARC) (IARC, 2012). To minimize the exposure of AFB1 in humans, different countries have established regulations for maximum allowed concentrations of AFM1 in milk.

The European Commission and the Codex Alimentarius Commission (EC 2006; Codex

Alimentarius 2001) established a limit of: 50 ng/kg AFM1 in raw milk, heat-treated milk and milk for dairy product fabrication. 25 ng/kg AFM1 in infant milk and follow-on milk.

AS FOR THE TRANSFER OF OTHER TOXINS TO MILK, FEWER STUDIES HAVE BEEN CARRIED OUT

The European Food Safety Authority (EFSA) reported that the carry-over of FBs to milk is limited and does not contribute

In other countries such as the

significantly to total human exposure (EFSA 2005).

United States, the maximum

Similarly, as regards to ZEN, EFSA reported

limit is:

that the carry-over rate of this toxin to

500 ng/kg toxin in raw milk.

milk is very low (EFSA 2004). On the other hand, several studies have shown that DON is

25 ng/kg in baby milk products. The maximum limits of AFB1 established in feed for dairy animals (5 µg/kg) are aimed at reducing the presence of AFM1 in

transformed to de-epoxy-deoxynivalenol (DOM1) in the rumen (Coté et al. 1986; Seeling et al. 2006; Keese et al.

2008) and that the part that is not metabolized is excreted in the milk at a very low rate (Prelusky et al. 1984).

milk due to the transfer from feed to milk.

2347

Reduction of mycotoxin contamination Despite the limits for mycotoxins established in different countries to reduce the level of exposure to these compounds, their presence in food and feed is usually unavoidable. Mycotoxin contamination can occur

The implementation of good agricultural practices, good manufacturing practices, good hygiene practices and good storage practices is essential to reduce mycotoxin contamination.

at any point in the feed production chain, so strategies have been developed to prevent its occurrence as well as to eliminate them from contaminated products. However, they are very stable compounds and difficult to remove.

The problem of mycotoxins in feed

Physical treatments:

can be addressed from a preventive point of view, by avoiding post-

Some of the physical treatments that have been tested are

harvest mycotoxin production

thermal inactivation or the application of UV light (CAST 2003).

in crops, by controlling feed storage conditions, or once the

Chemical treatments:

contamination of the products

Among the chemical methods, treatments with acid/base

has occurred, by applying different

solutions or the use of additives have been successfully employed

technological strategies.

(CAST 2003). Although it should be noted that the use of chemical methods of mycotoxin detoxification is prohibited in the EU. Biological treatments: Moreover, biological methods based on the detoxifying action of microorganisms, such as yeasts, moulds, bacteria and algae, may represent in the future a more suitable alternative for the elimination of mycotoxins (EU 2018; EFSA 2013)

Another strategy, widely used in the field of animal nutrition, consists of adding adsorbent compounds to the feed, which bind to the toxins during its transit through the gastrointestinal tract, reducing its absorption, promoting its excretion or modifying its mechanism of action. Its use has already been authorized by the EU (EC, 2009).

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