The assessment of mycotoxins contamination in feeds and adsorbents selection

Microscopic fungi are ubiquitous and have the ability to adapt to different habitat conditions. Some of them may develop products that are toxic to humans and animals, which are called mycotoxins (MT). This notion is due to their origin and effect in animals. From the point of the participation in metabolism of the fungi, they are referred to "secondary metabolites", i.e. substances that are formed during the metabolism, but do not play a role in their life. Not all fungi produce MT, even toxinogenic fungi can grow for several generations  not  producing MT, and then in some circumstances starting to produce them. The common in these substances they are produced by microscopic fungi (molds) and they are toxic for animals, however, MT are  the substances of various chemical structures.

Receiving MT by cultivation of moulds  in the laboratory, there has been  noted that the first 3-4 weeks an intense growth of the mycelium was detected, but in molds  there wasn’t  significant accumulation of MT, then, when the growth of mycelium was suspended, the start of the  active phase MT accumulation happened. Note that the MT synthesis was initiated while the growth of the mycelium was practically stopped. What did the growth of the mycelium limit? – Reduction of the nutrients availability. If the 3-weeks culture was transferred on fresh sterile substrate, the renewed growth of mycelium and only after 3-4 weeks of active growth has begun to show the formation of mycotoxins - this time was required for consumption of nutrients  and the emergence of their deficit. In the habitat fungi may also have deficiency of nutrients. Primarily in the environment along with the fungi there are bacteria which require the same basic nutrients as fungi. Such way between them happens a competition for nutrients. While nutrients are in sufficient amount, fungi and bacteria will not compete for the substrate, however under nutrients deficiency fungi begin to produce MT, which cause the death of bacteria.  Fungi produce toxins to kill competitors. From this point of view MT already are not secondary metabolites, as they provide "survival” of the fungi - they show a protective effect, and in a broad sense they are antibiotics for bacteria. Initiation of MT synthesis is controlled by biochemical regulation: under the limit of the available nutrients appears biochemical signal to switch on the mechanism of  MT formation. In the laboratory MT start to be formed on a sterile substrate (where there are no competitors) after reduction of the nutrient content below a certain critical level as a result of their use. In natural conditions, nutrients availability may be reduced while moisture is lacking, as it reduces nutrients solubility and availability, as well as the effect of other factors that create stress conditions for fungi growth, although they will not be associated with the presence of bacteria.

To completely eliminate the presence of MT in the raw feed material is not possible, therefore in many countries for  the most studied MT there were legally established Maximum Permissible Concentration (MPC)  of toxins, below which the raw materials or feeds can be used without restrictions. In the review of the scientific literature by studying the MT effect in animals it is necessary to pay attention to the sources of toxins, which can be principally two. Pure crystalline MT - they are used when setting the MPC, as well as in the study of the new mycotoxins. There is no another way, because researchers must be confident that the changes occurred in animals during the test is a result of specific MT and its dose. Pure mycotoxins are expensive and not always available for research, so reliable MPC values for large animals are much less than for laboratory animals, birds or piglets.

When the effect of mycotoxins is studied, especially while is determining the effectiveness of adsorbents, feeds contaminated MT under natural conditions are used often. With feeds contaminated naturally in the diet are contributed additional negative factors that the researchers can not control and in some cases do not even suspect their existence. The advantage of this option is - feeds contaminated MT naturally better reflect actual practice. The chemical composition of the contaminated feed is different from the composition of the feed, in which moulds did not grow. It occurs an independent influence in animals and excludes the possibility of objective findings. Research results in most cases are not consistent with those obtained by using pure MT. In this case in addition to toxins, available for analysis, may contain other unknown MT. An important factor to intensify the effect MT of natural origin, is the presence in the diet their unfinished products of biosynthesis, i.e. pretoxins. They also have toxicity, but the available control methods of the pretoxins content haven’t yet existed. Negative effect in animals of MT formed in feeds by natural way due to the presence of pretoxins is more obvious than equal doses of crystal MT.

The results obtained in experiments with the use of feeds contaminated with mycotoxins under natural conditions, are difficult to confirm by other researchers, as in the repeated experiments, the concentration of certain toxins and, respectively, their ratio in each case is unique and can not be duplicated even by the same researcher. The abovementioned differences lead to important practical conclusion: MT of natural origin will have in animals more pronounced negative effect compared with equal doses of toxins legislated as MPC.

In animals consuming feeds containing toxin below the MPC, the last is inactivated by xenobiotic metabolizing system and, therefore, has no apparent effect on the organism. This system does not have a narrow specificity and inactivates in organism many foreign substances.  Xenobiotics or alien to the body substances include mycotoxins, and substances of anthropogenic origin: medications, synthetic antioxidants, colorants and some others (Park 1985; Sheweita 2000; P. Galtier et al., 2008).

Endogenous detoxification of certain mycotoxins occurs at different speed and it varies in animals of different species (Adav and Govindwar. 1997; Ramsdell and Eaton. 1990). The accumulation of scientific data has revealed that the consumption of feed contaminated by multiple MT  can enhance the negative effects of each MT on the organism  (Pedrosa and Borutova. 2011). This is because of the presence several MT in feed, they will be inactivated in the organism  by  the same system of endogenous detoxification, the effectiveness of which has a limit.

In practical conditions in feed there can be some MT at the different concentration capable to cause polymycotoxicosis. So, as a result of the analysis in compound feeds for broilers some mycotoxins were revealed: concentration of each of them didn't exceed MPC (tab. 1).

 

Table 1: The assessment of compound feeds for broilers on the mycotoxins content.

 

On the base of the current legislation, the above presented feeds can be fed without restrictions, so concentration of each MT is lower than MPC. The conclusion is not indisputable, but there are no other bases for the conclusion. On the estimation of MPC values it is possible to calculate the significance of each MT in parts of MPC which can be summarized, whether to estimate opportunities of the organism protection system. The sum of mycotoxins concentration in Starter consists 1,464, and in Grower - 4,091 mg/kg: a difference is 2,8 times. The increase in the sum of toxins in Grower is caused by the increased fumonizin content, but it remained lower than MPC. Summation of mycotoxins amounts in feed doesn't give the grounds for its objective assessment. However on the sum of MPC parts toxicity of feeds occurred identical and more than twice exceeded MPC. It means that it is impossible to call such feeds as a good-quality one.

It is possible to prevent loss in animal performance defining MT levels in raw materials and in case of necessity subsequent their exception from feed production. The majority of laboratories in the analysis of mycotoxins content use a method of the immunoenzyme analysis (IFA, ELISA). The method is characterized by low cost of the equipment and simplicity of procedure of the analysis. However it has a number of the shortcomings connected with situation that sets for IFA are limited for certain list of objects of researches and cross sensitivity  creates a problem. For the objects which haven't been specified in the instruction, influence of the nature of the sample on expected result hasn't studied and it can be so expressed that leads to artefacts. Besides, the analysis, which have been carried out with IFA-sets from different producers possess different sensitivity, and results of the analysis not always coincide on quantities. (Tuzhikova T. and Titov M. 2011). For the analysis of mycotoxins by  HPLC is used, however UF or fluorescent detectors which are often applied at this, limit its technical capabilities. Use of mass-spectrometer detectors allows expanding number of defined mycotoxins to 40 and more. The cost of mass-spectrometer detectors several times exceeds the cost of chromatographs, doing them inaccessible to the main part of laboratories. As a result in most cases the analysis is limited by definition of 5-6 mycotoxins that is obviously not enough. In scientific researches many various ways of MT inactivation in raw materials were tested, however behind laboratories or production tests offered methods were not received further distribution (Boudergue. et al. 2009).

Widespread method of mycotoxicosis prevention, was an use of adsorbents. For the first time as adsorbent (Hatch et al.1982) was applied activated coal, and then for prevention aflatoxicosis was tested hydrated sodium - calcium aluminosilicate (HSCAS) (Philips et al. 1988). Good results were received: the researchers inspired with success, decided to test HSCAS for T-2-toxicosis prevention, however their hopes didn't come true (Kubena et al. 1990). Since then hundreds tests were carried out, there were adsorbents on an organic basis, compositions of adsorbents became complicated, however a cardinal solution wasn't reached (Boudergue C. et al. 2009; Withlow L. 2006).

In the market the new adsorbents appeared their evolution had been described before (Krukov V.S., 2014). Each new generation of adsorbents resulted from a dissatisfaction with the previous products. Difficultly is how to estimate and choose reliable adsorbent. On the one hand it is caused by complexity of the problem which is due to quite changeable concentration of mycotoxins in feeds and it is impossible to choose by tests of various adsorbents the most suitable one on all the cause. On the other hand there are no reliable and available ways of a laboratory assessment of the binding efficiency mycotoxins adsorbents.

In advertising for an assessment of advantages of adsorbents, usually are given results of their tests in the experiments in vitro though numerous scientific investigations have proved not suitability of this method. In the studies in vitro it was established that various mineral adsorbents were able to prevent aflatoxicosis, but remained not effective in relation to action of other MT and it wasn't found communication between results received in vitro and in vivo (Plank et al. 1990; Huff et al. 1992; Lemke et al. 2001; Williams et al. 1994; Dwyer et al. 1997; Garcia et al 2003; Bueno et al. 2005).

The organic adsorbents produced on a basis glucomannans from yeast appeared after mineral adsorbents and gained bigger distribution in a type of their novelty and active advertizing. They were represented as original means for mycotoxicosis prevention as in some cases they really showed the advantage. Despite the proved binding of a number of mycotoxins by glucomannans in vitro, their action wasn't always accompanied by performance improvement (Freimund et al. 2003; Yiannikouris et al. 2004; Swamy et al. 2002; Swamy et al. 2004; Chowdhury and Smith 2004; Aravind et al. 2003; Bursian et al. 2004; Whitlow 2006). On the basis of application test methods in vitro it is possible to make only one correct conclusion with confidence: if the tested adsorbents don’t bind mycotoxins in vitro, there is nothing to expect from them in vivo.

The problem of the adsorbents assessment in vitro consists also that in the world scientific community there is no standard version the description of the method for adsorptive ability measurement.  In each case researchers independently establish concentration of toxins in the incubatory media, their list, amount of the adsorbent brought in the media, media composition and its ??, time of  incubation and other details which lead to receiving incomparable results.  Partly can be tracked on results of the following researches (Manafi et al.  2009, tab. 2).

Table 2: The binding of mycotoxins by commercial adsorbent in vitro, %

 

The values specified in the table allow to draw a number of conclusions:

Mycotoxin amount bound by adsorbent in the same conditions:

Above noted quantitative changes are specific only for particular conditions of the experiment and will be changed depending to the mycotoxins amount in the incubatory media and, respectively, their ratio and other factors. Thus, even in the in vitro conditions where concentration and the list of mycotoxins is accurately controlled, it is impossible to predict the volume of their binding. It can only be established experimentally in the form of ascertaining. Considering that in practical conditions an amount of mycotoxins in feeds and their concentration in each case are unique, it is impossible to do forecasts about efficiency of adsorbent on the basis of the researches in vitro.

At the level of adsorbents MT show the expressed antagonism as they compete for binding places. They compete and in an organism for approach to system of an endogenous detoxication - as a result - their action on  organism can mutually amplify. The last is very important for an assessment of practical effect manifestation of adsorbents, but this question remains so far poorly studied, as concerning separate mycotoxins, and adsorbents.

The widespread in vitro method used for measurement of adsorbents binding capacity  proceeds in the closed system (a test tube or other vessel with impenetrable walls). As a result concentration of mycotoxins and adsorbent in the media remains constant, and created conditions are absolutely not similar on what developed in a gastrointestinal tract with its difficult and dynamic structure. Changes only in ?? are obviously not enough to be claimed that there was simulated conditions of gastrointestinal tract.

At the large feed plants which follow ISO requirements, is created and supported a long-term database about quality of the raw materials delivered by certain suppliers. ISO requirements provide implementation of control critical points for quality control of raw materials according to which the place, a way and frequency of sampling, a method of the analysis of raw materials on the content of mycotoxins and preventive measures are established. Often the use of adsorbents is limited as mycotoxicosis in most cases proceeds in the hidden (subclinical) form and in the absence of clinical signs experts don't see reasons for justification mycotoxicosis prophylaxis. Broiler weight reduction at the end of fattening by 50 - 70 grams without visible reasons is possible to explain with anything (technology, raw materials change, high temperature and many other factors which difficult to control in conditions of production). Difference in the weight of one head between lots in 50 grams if average weight remains within norm, is rougher  frequent phenomenon, and always it is possible to find an excuse for explanation in weight reduction. Therefore subclinical toxicoses remain not visible though they bring the greatest damage to animal husbandry in world aspect.

The accuracy of the analysis for mycotoxins content in feeds will be affected by the presented adsorbents as they will bind part of mycotoxins and underestimate results of a contamination (Gallo et al. 2010). Though there are guidelines that the presence in feeds adsorbents don't influence analysis accuracy (Kolossova et al, 2009) - it is impossible to agree with them as it is rejected by the mechanism of adsorbents effect and data of the table No. 2. The feeds sent to the analysis shouldn't contain adsorbents.

Adsorbents are improved all the time, - serious research teams are engaged in it. Transition from one generation of adsorbents to the following is caused by dissatisfaction with earlier products limited in their opportunities.  Only a few known manufacturers can spend funds for development of new, more perfect adsorbents. As a result in the market there are adsorbents according to properties relating to products of the first generation which arose still in the late eighties and modern, created in the last years possessing more powerful adsorptive properties in relation to mycotoxins. Experts in farm often come to a conclusion about need of adsorbents use and buy them from one producer, then on the basis of advertizing or on the experience, pass to other supplier, getting adsorbents, new to them. Over time they changed again and often come to incorrect conclusions, considered that adsorbents were useless and it was necessary to refuse them. If it was sincere decisions, they were caused by a lack of knowledge. The adsorbent assessment in the conditions of farm is influenced first of all by concentration of mycotoxins in feeds.

For the simplicity we will analyze probable two situations in practical conditions: 1 . Low contamination in feed: the sum of mycotoxins is about permissible concentration limit and 2. The extra contamination in feed: the sum of toxins is 3-5 times higher than permissible concentration limit. At a low contamination even weak adsorbent can be effective  and the conclusion will be true: it isn't necessary to pay for more powerful adsorbent. At the high content of mycotoxins, use of weak adsorbent will not provide protection of animals and  pay back the spent money, moreover expenses on veterinary preparations will increase, while performance is decreasing. Coming back to a problem of reliability of the current control of the mycotoxins content in feeds and considering that concentration of toxins in raw materials can vary widely  from minimum (MPC parts) to high ( up to several MPC) there is an increased risk of a contamination in feeds. The last is, especially, characteristic for feeds, fed to cows, as in silage or haylage, concentration of mycotoxins can differ during the short period of time several times. To prove an adsorbent choice in the conditions of farm it is necessary to carry out reliable monitoring the content of mycotoxins in feeds. For an assessment and comparison of adsorbents it is essential to create three groups of animals from which one receives a feed without adsorbent, another with that adsorbent which is applied in farm and the third – with new adsorbent. On the results of the test the experts can draw two conclusions: the first - to prove the need of use any adsorbent and the second: about advantage of this or that adsorbent. If there is no need to compare adsorbents, there is enough two groups. The conclusions got as a result of testing of adsorbent in certain farm will belong only to a specific situation – all difficulty of a choice of adsorbents consists in it, when even previous own experience not always helps. Considering high uncontrollable risk for contamination in feeds it is preferable to use the adsorbents providing reliable protection of animals from mycotoxins.

To define "reliability" of adsorbent, it is essential to know a supplier - serious companies acquaint potential clients with the production. It  speaks a much and gives rise to trust. Carefully treats promises of commercial experts. It is necessary to get acquainted with manuals on application of products – in them more reliable information and not to trust fair brochures and summaries of manuals – in them contents can be picked up tendentiously. Often firms refer to patents. Patents are published in the open press and links on certain "confidential know-how" aren't well-founded. The most reliable information is given in patents! For adoption of the reasonable decision sometimes it is essential to spend a plenty of time, but work of experts also consists in it. The choice of reliable adsorbent or continuous use of adsorbents is considerably expenses of current situation which is described above and specialists in nutrition not up to the end understand it.

It is possible to refer to habitual experience which took root for a long time into practice:  vitamins and trace elements are added in a feed without considering their contents  in raw materials.  All norms are considered as "the guaranteed norms of additives".

Probably, to adsorbents it is possible to apply concept: "the guaranteed additives for prevention mycotoxicosis", using adsorbents of the last generation.

 

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