mycotoxin's analysis in feed

Mycotoxins

• Mycotoxins are secondary metabolites produced by fungi present in feed.
• Mycotoxin's production depends on fungus specie and strand, plant specie, environmental moisture and temperature, presence of pests, etc.
• Mycotoxins cause damages in feed quality.
• Their incidence depends on geographical area and season.
• Mycotoxins are toxic: they produce mycotoxicosis and drop of performance.
• Their presence in feed can be reduced by applying Hazard Analysis and Critical Control Points.
  

Main factors that influence toxicity of mycotoxins are:

• Bioavailability.
• Combined effects between several mycotoxins.
• Amount of mycotoxins consumed. Continuous or intermittent ingestion of the contaminated feed.
• Animal weight, age, physiological and health status.
  
  
  

Mycotoxicosis

• Mycotoxicosis (1962, Forgacs and Carl): host's intoxication as a result of ingestion of a toxic substance of fungal origin.Some cases show evident symptoms that can be easily associated to mycotoxicosis.
• In the other hand, subclinical mycotoxicosis is only recognizable by drop of performance and health status.
• Susceptibility to mycotoxicosis depends on animal specie, age, sex and coexistence with other illness.

NUTRITIONAL ASPECTS		HEALTH ASPECTS
Feed consume decrease. Nutrient absorption decrease.		Mycotoxicosis typical of every mycotoxin. Immunosupression: arise of other pathologies.

MYCOTOXINS CLASSIFICATION AND MODE OF ACTION

Lots of described mycotoxins. Varied molecular weights and structures: difficult to classify.
They keep associated to fungus or substrate. Many of them are stable to chemical/physical treatments.		Mycotoxins persist in food chain.

Classification according to pathology


Hepatotoxins: sporidesmine, aflatoxins, luteoskirin, cycloclorotin, rubratoxins, sterigmatocistin.

Nephrotoxins: ochratoxin, citrinin.

Neurotoxins: penitrem A, patulin, fumonisins, citreoviridin.

Toxins of intestinal tract: trichotecenes, T-2 toxin, deoxynivalenol (Don, Vomitoxin), HT2 toxin, fusarenone.

Steroidal; strogenic (Zearalenone), D vitamin analogous

Haemorrhagic and circulatory toxins: Ergot alkaloids, aflatoxins.

Classification according to chemical structure

Chemical structure


Chemical structure determines:

1. Mycotoxin's mode of action.
2. Mycotoxin's method of detoxification.

Mode of action: specific biochemical interaction through which a substance produces its biological effect.

In order to achieve a biological effect, an interaction with a receptor is essential.






Chemical structure and mode of action

Chemical groups of the receptor must interact with chemical groups of the substance, that is, chemical structures in the binding point must be complementary.


KEY-LOCK MODEL








Mycotoxins with shared chemical structure may interact with the same receptors an thus have an alike biological effect.



Chemical structure and mode of action

Into practice T2 toxin, HT2 toxin, deoxynivalenol, nivalenol have a sesquiterpene group in their structure and they all have necrotic effects on mucous membranes.






Chemical structure and mode of action

B1, B2, G1, G2 aflatoxins, sterigmatocistin have cumarinic group in their structure and they all have haemorrhagic effects alike anticoagulant active principles used in human pharmacology: warfarine, acenocumarol. These active principles also show a cumarinic structure.



Aflatoxin B1



MYCOTOXIN ANALYSIS IN FEED MANUFACTURING


Decision making in raw material purchasing





Usual methods of analysis

• Thin layer Chromatography (TLC).
• Liquid Chromatography (HPLC).
• Gas Chromatography-Mass spectrometry (GC-MS).
• Immunoassay(ELISA).

Maximum allowed concentration


Gradually more countries legislate about mycotoxin limits in fodder and raw materials destined to animal nutrition.

Maximum concentrations are set depending on:

• Mycotoxin's toxicity
• Animal specie sensitivity and age
• Fungi load characteristic of plant specie
• Availability and limits of analysis method.

Maximum concentration for each mycotoxin depends on every country.


These limits can be the reference for raw material admittance or rejection in purchasing


Maximum allowed concentration








Maximum concentration depends on animal specie and age and on raw material or fodder.



INTERPRETATION OF RESULTS


Interpretation of results


Considering the results obtained in the lab, decisions are made taking into account:

• Concentration of each mycotoxin (individual effect).
• Concentration of all mycotoxins analysed as a whole (combined effects).
• Possible bias of analysis.
• Presence of non-analysed mycotoxins.
  
  

COMBINED EFFECTS





There are more possibilities of finding combined effects in mycotoxins...

• With similar molecular structure.
• Synthesized by the same fungal strand or specie.
• Synthesized by the same fungal family.
  

The appearance of combined effects depends on:

• Concentration of each mycotoxin.
• Animal sensitivity (specie, age, health status).
  

Additive effect

Combined effect of A and B mycotoxins is equal to the addition of the effect of each mycotoxins.

• Aflatoxins + Deoxynivalenol
  Poultry: decrease in proventriculus weight, increase of DHL enzyme, indicator of tissue damage.
  
  
• Aflatoxins + Cyclopiazonic acid
  Poultry: growth decrease.
  Pigs: feed intake and growth decrease; inflammation and necrosis of the gastrointestinal tract.
  
  
• Aflatoxins + Diacetoxyscirpenol
  Pig: Weight and growth decrease, alteration of blood parameters that indicates hepathotoxicity.
  
  
• Aflatoxins + Moniliformin
  Poultry: weight and efficiency decrease. Increase of heart's relative weight. Biochemical parameters indicate nephro and hepatotoxicity.
  
  
• Fumonisin B1 + Diacetoxyscirpenol
  Turkey: Weight decrease. Hepatotoxicity.
  
  
• Fumonisin B1 + T-2 toxin
  Poultry: weight and efficiency decrease. Nephro and hepatotoxicity.
  
  
• Deoxynivalenol + Moliniformine
  Turkey: weight of kidney and heart increases. Tissular damage in myocardium.
  
  
• Citrinin + ochratoxin A
  Pig: nephrotoxicity. They affect transport of several molecules and protein synthesis.
  
  
• Fusaric
  Poultry: feed consumption and growth decrease. Nephro and cardiotoxicity.
  
  
• Ochratoxin A + T-2 toxin
  Poultry: Weight decrease, increase of kidney, liver, proventriculus and gizzard relative weight. Nephro and hepatotoxicity.
  

Synergic effect


Combined effect of A and B mycotoxins is higher than the addition of the effect of each mycotoxin.

• Aflatoxins + Ochratoxin A
  Poultry: Weight decrease, mortality increase. Hepatotoxicity and severe nephrotoxicity.
  
  
• Aflatoxins + Toxin T-2Very important because of its prevalence
  Poultry: weight decrease, increase of kidney, gizzard and heart relative weight; decrease of the medium corpuscular volume and of the potassium plasma levels.
  
  
• Deoxynivalenol + Fumonisin B1
  Pigs: great weight decrease.
  
  
• Deoxynivalenol + Zearalenone
  Pigs: theratogenesis in piglets.

Antagonistic effects


Combined effect of A and B mycotoxins is less than the addition of the effect of each mycotoxin. (but higher than the effect of each mycotoxin separately).

• Citrinin + ochratoxin A
  Poultry: the presence of these mycotoxins together reduces the toxic effects of the mycotoxins separately (growth and water consumption decrease).
  
  
• Aflatoxins + diacetoxyscirpenol
  Poultry: the presence of these myocotoxins together reduces the toxic effects of the mycotoxins separately (growth and feed consumption decrease).

BIAS OF THE ANALYTICAL METHOD


1. Not representative sampling
Sample not representative, because of the big sample size or the king of storage/container of the raw material.

2. Not validated analysis method
Analysis method should have been validated by a prestigious institution like International Organization for Standardization (ISO).

3. Low quality standards
Trouble to get mycotoxin standards in some countries.
% recovery of solid standards by dissolution <100%.
Standard solution not stable.

4. Procedure for sample extraction

4.1. Influence of the extraction method:



Recovery of B1 aflatoxin using different solvents (natural contamination, results in ppb)‏ FAO, 1994




Effect of time and extraction system for B1 aflatoxin (Natural contamination, results in ppb) using methanol-water 80:20 FAO, 1994