Diagnostic and control of mycotoxins-Interview with Dr. Carlos A. Mallmann

diagnostic control mycotoxins - Mycotoxins diagnostic and control _ Interview with Dr Carlos A Mallmann


We interviewed Dr Carlos A. Mallmann, a scientist with many years of experience in diagnostic and control of mycotoxins. He speaks about factors affecting the production of mycotoxins, different methods of diagnostic, the importance of appropriate sampling, and the procedure to approve mycotoxon adsorbers, among other topics. 

 

Doctor in Veterinary Medicine, Professor of the Federal University of Santa Maria (UFSM), Coordinator of the Laboratory of Mycotoxin Analysis (LAMIC). He also works as a technical, managerial, and quality control consultant for animal feed companies in Brazil and around the world, as well as for official organisations such as FAO/ONU and the Ministry of Agriculture of Brazil.

Dr. Mallmann has been a speaker in numerous international congresses in many countries (Argentina, Brasil, Bolivia, Mexico, USA, Portugal, Austria, Germany among others). He authored more than 100 publications in the last 10 years, including the book “Mycotoxins and mycotoxicosis in Swine”.

 

1. As an expert in the topic, what do you think are the main factors determining the production of mycotoxins in raw materials?

They are basically three: humidity, oxygen, and temperature. It is necessary that all three are present at the same time for fungi to grow. Therefore, if we can eliminate one of them, then fungi will not grow and, consequently, there will be no mycotoxins.

Normally, the approach is to reduce humidity because, for most cereals, the elimination of oxygen is much more difficult (it is possible but only in systems with controlled atmosphere, e.g. especial silos). In cooler countries, it is possible to control temperature below the ideal 25 ºC by means of ventilation, reducing fungal growth. However, some fungi do not grow at lower temperatures, but they can still produce toxins (in some cases even below 18 ºC).

Other factors to consider are insects, damage of grain during drying, mass of non-uniform grains, presence of invasive species, length of time the grain remains in the field once already ripe. The earlier the grain is collected and dried, the lower will be the amount of mycotoxin produced.

If those conditions occur, what are the actions to be implemented to avoid the production of mycotoxins?

The actions to be implemented are those allowing us to control any of the factors mentioned above, for example: drying the grains to control moisture, controlling the ambient temperature, and decontaminating the cereal when insects are present.

What are the most relevant factors to be considered when choosing a detoxifying agent? Is it its affinity, detoxifying capacity, selectivity, or efficacy against the mycotoxin to eliminate?

These aspects are related to the type of additives utilised in  mycotoxin control. Most of the products available in the market are not detoxifiers but adsorbents. They trap the secondary metabolite binding it electrochemically (e.g. Aflatoxin create a non-absorbable chelate, which is eliminated in faeces). The properties of this products are determined according to the mycotoxin. Sometimes, the product may have an in vitro affinity for a given mycotoxin of 99%, but its in-vivo efficiency is determined by the animal.Two thirds of products evaluated in vitro for Aflatoxin had an in vitro adsorption above 80%, however only half of them could protect the animal in vivo.

These factores detrmine the affinity, but the in vivo affinity, i.e. its stability. These factors change a lot, since most of this products are of natural origin, such as mineral sequestrants, enzymes, vegetal extracts, or plantas (algae). Their properties are modified by factors such as soil, vegetative state of the plant when the product was extracted, etc. Therefore, there are many factors to be controlled very well to establish the quality of the product. The in vitro evaluations are only to determine whether the characteristics of the product are mainteind throughout time, to see  whether the product that was initially evaluated in vivo, is the same being consumed in the market. To fulfil this objective, additional in vitro tests, other than mycotoxin fixation, are performed.

Regarding in vitro tests, the anti-mycotoxin product is mixed with gastric or intestinal solutions containing the mycotoxin, and incubated. Chromatography is used to determine the quantity of mycotoxin adsorbed to the product. Other tests include colorimetry (the colour of the product should be more or less constant), ash determination and pH measurement. These tests are performed to evaluate and classify the product in vitro.

 

According to recently published data, climate change comports a change in the profile of fungal contamination in raw materials. Considering such changes, do you believe we should modify our current strategies to fight against mycotoxins?

The strategies shuld be constantly modified. They are dynamic. As new problems emerge we need to be able to find efficient answers to them. If we are, for example, in a drought area, plants suffer an important water stress, and we should proceed to water the crops. If we are in in areas with high humidity, were mould growth is favoured, it will be advisable to apply preventative fungicides on the plants. Therefore, there are different alternatives for different situations.

In Brazil, we are working in a holistic way, considering the entire food chain. This project became more intensive after the Brazilian government introduced strict limits for mycotoxins in raw materials. In the cereal productive chain we are working with seed companies with the objective of obtaining more resistant seeds, improving plant nutrition, performing a more rational use of agrochemicals (fungicides, insecticides and herbicides), and utilizing the systems of minimum tillage and crop rotation. Many times, the changes in seed quality are not enough to counteract the climate change. However the efforts continue and, when working   with more technified products, we have been able to find solutions and then apply them in the entire productive chain.

 

From a scientific point of view, do you consider that the advancements achieved so far, satisfy the current needs of detection and control of mycotoxins? Otherwise, are mycotoxins still winning the battle?

Nowadays, we have more knowledge, and in some situations, we have control. Such is the case of the utilization of anti-mycotoxin additives. However, there is still a lot to be done on the prevention side; this is the longest challenge we have.

Regarding mycotoxin detection, it have very much improved since the implementation of new sampling systems; sampling is actually the most complex aspect of detection.

In the latest projects we have been implemented the sampling in auger, which is simpler, practical and without any extra implementation cost. This method makes sampling 10 times more precise than the method previously in use, rendering more representative samples

Generally, less problems arise, althoough it is expected that when some problems are solved, some others will appear.

 

Regarding sampling techniques and tests for the detection of mycotoxins in grains and finished feed, what aspects would you remark as most relevant?

Sampling technique, depending on the mycotoxin, represents up to 80 or 90% of the final error. To reduce its impact, it is necessary to implement an automatised sampling system.

Sampling is the Achilles heel of the system of mycotoxin control.

Currently, in feed factories with auger transport system, sampling the feed is quite simple and wit zero cost. In newer plants that use a pneumatic feed transport system, it is necessary to invest in a piston system that can take a representative sample. The cost of this equipment and its maintenance is quite high. However, there is a general consensus on the need of improving sampling, and companies that want to invest can optimise the procedure. This is key to reliable results; if we have access to the best analytical method, but the sample is bad, then the result will not be good enough to make reasonable decisions.

What is your opinión regarding the currently available rapid tests, ELISA kits, in which the risk of error seems to have been substantially and significatively reduced?

Surely they are methods that can be used in the daily routine, as part of the reception of raw materials. They have been much improved for some mycotoxins, but they do not work well for others. Furthermore, they have problems for detecting some mycotoxins in compound feeds, although this is the main limitation of any of the analytical systems, even for HPLC. As a rule, what indicates the reliability of the results are the system certifications that the labs hold; for example, an ISO17025 accredited lab must fulfill a series of internationals rules and standards. Only in that way, a result is certain to be valid, and decisions can be made based on them.

The results produced by any lab that is not ISO accredited are, at least, questionable. Any analytical result for mycotoxins will be given in ppm or, in the case of milk, in ppt.

 

And regarding the control of mycotoxins in feed factories, what aspects could you mention regarding the fungal growth and mycotoxin contamination of the production plant?

This is the most difficult place to implement mycotoxin control.

A factory that follows rules regarding cleanliness and hygiene, will fulfil almost all the requirements for the preservation of raw materials, producing quality feeds from the mycotoxicologic point of view.

A critical point is to control the variation of clean water activity in the feed. This parameter guarantees the stability of the feed. If maintained in that way, there will be no problems of deterioration for presence of moulds, significant losses of ingredients due to peroxidation, or other processes leading to feed spoilage.

What would be the importance of implementing a HACCP program?

HACCP programs contemplate all these risks. Today, organized and approved factories implement a rigid control system with a constant quality control. If this does not occur, then the quality of its products can be questioned, and most probably those companies will not last long in the market.

 

Brazil is a pioneer in the implementation of the implementation of protocols to control the efficacy of mycotoxin adsorbents. What would be your main recommendations?

As we said before, the product should have an in vitro evaluation, followed by in vivo testing. The performance of in vivo tests is fundamental. For example, one of the best antibiotics we have is sat, but we cannot use it in vivo. This same occurs with mycotoxin adsorbents.

A producto can hae a 100% efectiveness in vitro, but that does not mean that product will work well in an animal model.

What would be your main recommendations for designing an in vivo trial to select the most effective mycotoxin adsorbent?

My recommendations would be to utilize at least 4 treatments: no.1: control without mycotoxin or additive; no.2: treatment without mycotoxin but with additive, to tests its innocuity; no. 3: treatment with mycotoxin but without additive, in a concentration high enough to produce clinical signs to be able to compare with treatment 4; no.4: mycotoxin with additive, with results to be situated between treatments no. 2 and no. 3.

. It is necessary to use mycotokxin doses higher than the ones normally encountered in feed.

If we use too low a dose, we will not have space between the intoxication state and the control to arrive to a statistically significant conclusion. Furthermore, we need to work with reduced number of animals and in completely controlled conditions, respecting welfare norms in order not to stress the animals.

What is the funcltion of LAMIC-UFSM  (Laboratory of Mycotoxicologic Analyses of the Federal University of Santa Maria, in the Centre of Rural Sciences, Department of Preventive Veterinary Medicine) on the approval of mycotoxin adsorbents and the evaluation of their safety limits for their use in animal production?

Lamic is a laboratory with more than 20 years’ experience on mycotoxin research.

We have a diagnostic section where, up to date, we have performed more than 1.2 million mycotoxin tests in samples from Brazil and other countries.

In 1996 the first mycotoxin adsorbent that worked in animals was found.

Since then, we have performed more than 300 in vivo tests, of which 40-43% have led to the approval of products. We evaluated the protective effects in different ways, for example considering the variation of productive parameters (Aflatoxin), morphology of organs (Zearalenone), enzymatic properties (alteration of total proteins in serum as a consequence of hepatotoxins), biological markers (alteration of sphingolipids by Fumonisin). We also used colorimetry, organ weight, etc. All the information generated is analysed to confirm the product can be utilized and that animals will be protected by it.

The objective of LAMIC is to screen all products we have in our database, which includes more than 1000 products present in the intenational market. So far, 300 of them have been tested in our facilities.

Approximately 10 years ago, we met with a group of experts of Brazil. We decided we needed to put in place some criteria to approve products, establishing certain minimum guarantees on the effectiveness of products. Evidently, some products were not approved over the years. However, many of those products are present in the international market.

In Brazil, adsorbent products are more controlled thanks to our history. Manufacturers know that we have established this type of evaluation. The procedures are available in LAMIC’s website for everyone to access it. We do this to democratize international information. It is there where we publish all the evaluations we have performed.

It is important to mention that, as most of the evaluated products are natural and have the risk of changing throughout time, the committee of experts decided that in vitro evaluations should be performed every 6 months, whilst an in vivo test should be performed every second year.

Since the final objective is to ensure the final consumer buys a product of good quality, able to solve problems as expected, the cost of reevaluations is lower than the cost of the first evaluation.

 

There are several strategies to control mycotoxicosis. One the one hand, we have the sequestration/elimination of toxins, and, on the other detoxification. What is your opinion regarding the advantages and disadvantages of each of these strategies?

Los secuestrantes actúan más en el origen del problema porque evitan que la toxina entre dentro del organismo animal. Esto nos ahorra bastantes energía porque no necesitamos detoxificar, aunque desafortunadamente los adsorbentes o secuestrantes no logran ser efectivos contra todo tipo de micotoxinas y, por lo tanto, la detoxificación tiene un papel bastante importante , protegiendo los órganos afectados.

Sequestering products act in the origin of the problem, avoiding the toxin from being ingested by the animal. This saves some work since we do not need to detoxify. However, adsorbents and sequestrants are not effective against all types of mycotoxins. Therefore, detoxification has an important role protecting the target organs from being affected.

Both strategies are different, and sometimes are found in a same product. When we have a multimycotoxin product facing a contamination by a single mycotoxin, the active principle will be in low dose, not achieving sufficient efficiency against the mycotoxin in highest concentration.

For sure in the future, with more precise, faster diagnostic methods, the feed factory will select the required immediately after its diagnostic. In that way, the feed factory will have 3 or 4 products, which will be used based on a real need. For now, since the mycotoxins appear mixed, the utilization of mixed products are the only available alternative to protect the animals.

 

The effect of the mycotoxins with higher economic impact is immunosuppression. This translate in a higher susceptibility to suffer infectious diseases. Is it correct to say that the economic impact of immunosuppression is the one of major importance?

Immunosuppression can get to the point of immunological depression. It is known that animals exposed to diets contaminated with aflatoxins, for example, do not respond properly to vaccination, affecting the achievement of protective antibody titres against pathogens.

We could also add a second important point, regarding the energy consumption due to enzyme activity. When animals ingest feed contaminated with mycotoxins, animal enzymes normally used in other metabolic routes are activated to destroy/neutralize mycotoxins. The energy normally used for maintenance or production, is used for this unusual enzymatic activity. The consumption of enzymes, added to the damage to the immune system, results in a lower resistance of the animals to disease, as well as impairment of productive, reproductive, and sanitary parameters. All this translates into enormous economic loses.

What are the main effect of mycotoxins on animal health?

I will mention some cases in which the mycotoxin is not toxic per se, but their effects on the animal result on its death. For example, zearalenone in piglets produce splay-leg”, characterized by a paralysis of the hindlegs; since they cannot move, the animals die of inanition. Additionally, zearalenone produces a state of stress that leads the animals to cannibalism

Do you believe that, at farm level, mycotoxins are given enough attention?

Currently, at farm level, mycotoxins receive more attention that 20 years ago. In those days, people spoke about them but without giving them much relevance because there were more important problems. Today, with improved farm health and management, and with a more precise nutrition, mycotoxins have become a factor to control in order to maximise productivity and profits. Therefore, since mycotoxins are currently a hinder to productivity, they receive more attention.

What would be your recommendations in this link of the production chain?

The primordial recommendation is to buy mycotoxin-free feed. The supplier should guarantee that the product that we are going to feed to our animals is free of mycotoxins or that, at least, the mycotoxins are controlled by the addition of an anti-mycotoxin diet.

 

In the RASSFF (European Commissions’ Rapid Alert System for Food and Feed) of 2013, a total of 405 notifications of mycotoxin findings were reported. In order to minimise its effects, do you think it would be justified to produce genetically modified maize, resistent to mycotoxins?

Yes, it was justified years ago. However, the maize was not directly resistant to mycotoxins, but the genetic modification had effect on insects. They produce a contaminating material that increases 10 times (or more) the contamination by mycotoxins. The elimination of the insects reduced the contamination crops. But we already know that any new technology brings some consequence, and if misused produces new problems. As we know, that is what happened.

The use of transgenics led the producers to relax. They did not follow the recommendation of making lines in the crops with material attractive to the insects. so that they will get in contact with the modified maize, controlling the insect load. Instead, the insects, almost with no control, they became resistent to certain varieties of genetically modified maize. Now, due to the misuse of a technology that was perfect, we have genetically modified maize that once again began to have insect problems, and that needs to be sprayed with insecticides to maintain productivity. In essence, the use of transgenics is an alternative to reduce the cost of production and to lower mycotoxin production through the control of insects.

The biggest achievement we should pursue is the selection of maize resistent to mycotoxins through morphological properties, i.e. due to their intrinsic resistance. An example will be a modification so that a lipoxygenase breaks down linoleic acid to produce aldehydes, which have antifungal properties. In this way, we would have a greater protection of grains, achieving a higher inhibition of fungal growth with a consequent reduction in mycotoxin production.

 

 

 

 

 

 

 

This interview was originally published in nutriNews Spain, with the title «Entrevista al Dr. Carlos Alberto Mallmann»




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