Importance of mycotoxins

analysis in feed

Dr.Kedar Karki
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.

Mycotoxins toxicity
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.
Mycotoxicosis can cause:

 NUTRITIONAL  HEALTH
ASPECTS   ASPECTS 
 Feed consume    Mycotoxicosis

decrease. typical of every

mycotoxin.
Nutrient
absorption Immunosupressio
decrease. n: arise of other
pathologies.
MYCOTOXINS
CLASSIFICATION AND MODE
OF ACTION
 Lots of described
mycotoxins.

 Varied molecular weights

and structures: difficult
to classify.  Mycotoxins persist in
food chain.
  
 They keep associated to
fungus or substrate.

 Many of them are stable

to chemical/physical
treatments.
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
Classification according to
chemical structure
Chemical structure

 Chemical structure determines:

 Mycotoxin's mode of action.
 Mycotoxin's method of
detoxification.
Chemical structure and
mode of action
 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.
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
Hypothesis
 Mycotoxins with shared
chemical structure may
interact with the same
receptors an thus have an
alike biological effect.
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  Aflatoxin B1

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
MYCOTOXIN ANALYSIS IN
FEED MANUFACTURING

 Decision
making in raw
material
purchasing
Usual methods of analysis

 Thin layer  Immunoassay

Chromatography ELISA).
(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.
Maximum allowed
concentration
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
 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.
COMBINED EFFECTS
 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.
Additive effect

 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.
Additive effect
 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.
Synergic effect

 Aflatoxins + Ochratoxin A
Poultry: Weight decrease, mortality increase. Hepatotoxicity
and severe nephrotoxicity.

 Aflatoxins + Toxin T-2 Very 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).
Antagonistic effects

 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
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
BIAS OF THE ANALYTICAL
METHOD

 5. Concentration in sample out of detection limits

Limits depend on chosen analytical method.

6. Interferences with other substances present in

the sample.
TLC: false positive if sample contains certain natural
pigments.
TLC: false positive for B1 aflatoxin if sample contains
ethoxiquin.
ELISA: false negative for aflatoxins in sorghum
samples.

7. Errors in quantification.
Accuracy: is the degree of conformity of a measured or
calculated quantity to its actual (true) value.
Precision: characterises the degree of mutual
agreement among a series of individual measurements,
BIAS OF THE ANALYTICAL
METHOD

 PRESENCE OF NON-ANALYSED MYCOTOXINS

Not-analysed mycotoxins
In raw materials there could be mycotoxins whose are not
analysed:
1. WELL-KNOWN MYCOTOXINS Whose analysis is not
performed because of economic reasons, lack of validated
methods, presence unlikely, etc.
2. LITTLE-KNOWN MYCOTOXINS Mycotoxins whose
incidence and effects are little known.
THERE ARE MORE THAN 300 DESCRIBED MYCOTOXINS
Little known mycotoxins

 Stachybotrotoxicosis

Stachybotrys chartarum

Caused by H satratoxin, a
tricothecene 5 folds more toxic
than T2 toxin.
CONCLUSION

 In order to avoid
the
mycotoxicosis,
three factors must
be considered:
References
 Author: Paper
Presented at
Biovet
Symposium 2007
(Courtesy of
Biovet SA)